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Cholesterol Accumulation Is Associated with Lysosomal Dysfunction and Autophagic Stress in NpcC/C Mouse Brain

【摘要】  Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1C/C mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1C/C mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1C/C mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC.
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Niemann-Pick type C disease (NPC) is a fatal neurodegenerative disorder that mainly affects children. The pathological hallmark of NPC is the massive accumulation of cholesterol and other lipids in late endosomes and lysosomes.1,2 In most cases (approximately 95%), the disease is caused by mutations in the NPC1 gene3 and the remainders by mutations in the NPC2 gene; both genes encode proteins that play important roles in intracellular cholesterol transport.4-6 Neurodegeneration in NPC shares a number of pathological features with those observed in Alzheimer??s disease (AD), although the structures most affected in NPC are cerebellum and brainstem. Both diseases exhibit cholesterol metabolism impairment,7-10 endosomal/lysosomal dysfunction,5,8,11 and tauopathies.12-15 Interestingly, the neurofibrillary tangles, the most common form of tauopathies, found in NPC are indistinguishable from those found in AD brains.12,14 In both diseases, levels of free cholesterol are positively correlated with the incidence of intraneuronal neurofibrillary tangles.16,17
Several lines of evidence have established lysosomal dysfunction as an early-onset neuropathological feature of AD. Levels of lysosomal cathepsin D in neurons are increased in AD vulnerable regions before the onset of major pathology.18 Cathepsin D up-regulation correlates on a cell-by-cell basis with other markers of early-stage AD, including decreased levels of the synaptic vesicular protein synaptophysin and increased levels of intraneuronal neurofibrillary tangles.19,20 Experimentally induced lysosomal dysfunction is associated with rapid formation of neurofibrillary tangles in hippocampal slices cultured from apolipoprotein E knockout mice.21 Cytoplasmic presence of cathepsin D can induce release of cytochrome c from mitochondria and activation of proapoptotic factors, which leads to caspase-dependent apoptosis, also referred to as type 1 programmed cell death.22-24
Lysosomes also participate in type 2 programmed cell death, referred to as autophagic cell death, which is defined by the presence of autophagic morphology.25,26 Neuronal death with features of autophagy has been observed during normal development27 and in pathological conditions, such as in AD28,29 and in Parkinson??s disease.30 On the other hand, neuroprotective function of autophagy has also been implicated in certain neurodegenerative diseases, such as Huntington??s disease. A recent study reported the existence of autophagic features in Purkinje cells in Npc1C/C mice.31 To investigate further the roles of autophagy-lysosome system in neurodegeneration in NPC, the present study determined levels and localization of the lysosomal enzyme cathepsin D and of autophagic activity and the potential association of autophagic-lysosomal dysfunction with accumulation of cholesterol and neurodegeneration in brains of Npc1C/C mice.

【关键词】  cholesterol accumulation associated lysosomal dysfunction autophagic

Materials and Methods

Breeding pairs of BALB/cNctr-npc1NIH mice heterozygous for Npc1 (+/C) were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in our animal facility in accordance with National Institutes of Health guidelines and protocols approved by the Institutional Animal Care and Use Committee with care to minimize distress to the animals. Mouse breeding and genotyping were performed as previously described.32 Animals were sacrificed at postnatal weeks 1, 2, 4, and 8 (four to eight animals for each age group) under deep anesthesia (100 mg/kg sodium pentobarbital) by perfusion for immunohistochemical and histological studies or by decapitation for biochemical analyses. For histological studies, animals were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed and incubated with 15% sucrose followed by 30% sucrose before being sectioned at 25 µm with a microtome. Coronal sections were stored in a cryoprotective solution at C20??C before being processed for immunohistochemical studies.

Subcellular Fractionation

Brains from Npc1C/C and their wild-type littermates were dissected in ice-cold artificial cerebrospinal fluid and homogenized in homogenization buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO); homogenates were centrifuged for 10 minutes at 1500 x g. The sucrose concentration of the collected postnuclear supernatant was adjusted to 40.6% by the slow addition of 62% sucrose in homogenization buffer-EDTA. Postnuclear supernatant was then carefully overloaded with 1.5 ml of 35% and 1.0 ml of 25% sucrose in homogenization buffer-EDTA, and the samples were centrifuged in an SW 55 rotor (Beckman Instruments, Inc., Palo Alto, CA) at 14,000 x g for 90 minutes at 4??C. Subcellular fractions were collected from the top of the tube. The late endosome/lysosome-enriched fraction was localized in the upper interface, containing 25% sucrose and homogenization buffer, and the early endosome-enriched fraction in the middle interface containing 35 and 25% sucrose. The lower interface containing 40.6 to 35% sucrose was enriched in plasma membranes and other heavy membrane compartments.

Western Blots

Electrophoresis and immunoblotting were performed following conventional procedures. In brief, after protein concentration was determined, proteins (40 to 60 µg) of postnuclear supernatant from different brain regions or of other subcellular fractions were denatured by boiling for 5 minutes in a sample buffer (2% sodium dodecyl sulfate, 50 mmol/L Tris-HCl pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue) and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (12%), after which proteins were transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with primary antibodies for 12 to 16 hours at 4??C; immunoreactivity was visualized by using enhanced chemiluminescence (ECL Plus kit and reagents; Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies used included anti-cathepsin D (1:1000; EMD Biosciences, San Diego, CA), anti-cathepsin B (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-rab7 (1:1000; Santa Cruz Biotechnology), anti-ubiquitin (1:500; Zymed, Carlsbad, CA), and anti-LC3 serum (gift from T. Yoshimori, National Institute of Genetics, Mishima, Shizuoka, Japan33 ). Levels of different bands were analyzed by using the National Institutes of Health Image program (Bethesda, MD). Statistical significance was determined by two-tailed Student??s t-test.

Activity Assay of Cathepsins B and D

Whole homogenates of brainstem, cerebellum, or hippocampus from Npc1C/C and wild-type mice were used to analyze the activity of cathepsins B and D using fluorogenic immunocapture activity assay kits (EMD Biosciences) according to the kit instructions.

Sagittal sections from cerebellum and coronal sections from the rest of the brain of animals from different ages were simultaneously processed for immunostaining. Immunohistochemistry was performed using the avidin-biotin horseradish peroxidase complex method. In brief, free-floating sections were first incubated in 10% normal horse serum (for monoclonal antibodies) or 3% normal goat serum (for polyclonal antibodies) diluted in PBS with 0.1% Triton X-100 for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4??C. Antibodies used were anti-cathepsin D (1:500; EMD Biosciences) and anti-cathepsin B (1:100; Santa Cruz Biotechnology). After three washes in PBS, sections were incubated with corresponding biotinylated secondary antibodies (1:400; Vector Laboratories, Burlingame, CA) in 5% normal horse serum or 1.5% normal goat serum solution for 2 to 3 hours, then in avidin-biotin horseradish peroxidase complex diluted in PBS for 45 minutes. Peroxidase reaction was performed with 3,3'-diaminobenzidine tetrahydrochloride (0.05% in 50 mmol/L Tris-HCl buffer, pH 7.4) as chromogen and 0.03% H2O2 as oxidant. Free-floating sections were mounted on precoated slides (SuperPlus; Fisher Scientific International Inc.) and air-dried. Sections were then dehydrated in graded ethanol and finally covered with Permount (Fisher Scientific).

Double-labeling immunohistochemistry was done with sections first incubated with primary antibodies , then with corresponding secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594. Both secondary antibodies were purchased from Molecular Probes, Eugene, OR.

Filipin Staining

Filipin has been demonstrated to specifically stain free cholesterol because treatment with cholesterol oxidase results in a complete loss of fluorescence.34 Brain tissue sections were washed with phosphate-buffered saline and incubated in the dark with 125 µg/ml filipin in PBS for 3 hours under agitation at room temperature. After washing in PBS, some sections were further processed for immunostaining with anti-calbindin or -LC3 (1:3000; Abgent, San Diego, CA) antibodies and corresponding secondary antibodies conjugated with Alexa Fluor 594.

Images of immunostained sections from different brain regions were visualized using a Zeiss microscope (Axioskop 2 Mot Plus) and digitized via a Zeiss digital photo camera (AxioCam Hrc) and the Axiovision program, version 3.1 (Zeiss), was used to capture and save digitized images. Digitized images were then assembled in Photoshop (version 7; Adobe Systems, Mountain View, CA) with only the brightness adjusted to match other panels in a given figure. Images of double fluorescent labeled sections were acquired by using a Nikon confocal microscope (Nikon TE 2000U with D-Eclipse C1 system; Melville, NY).

Electron Microscopy Analysis

Electron microscopy analysis was performed as previously described.35 In brief, animals were perfused with an ice-cold solution of 0.1 mol/L phosphate buffer, pH 7.4, containing 1.5% paraformaldehyde and 1.5% glutaraldehyde. Cerebellum blocks were transferred to 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, at 4??C for 24 hours, rinsed overnight in the phosphate buffer, postfixed with 1% osmium tetroxide in phosphate buffer for 2 hours, followed by dehydration and embedded in epoxy resin. Ultrathin sections were prepared using a Reichert ultramicrotome, contrasted with uranyl acetate and lead citrate, examined under a Philips CM120 transmission electron microscope at 80 kV.

Increased Levels of Cathepsin D in Brains of Npc1C/C Mice during Postnatal Development

Cathepsin D is synthesized as an inactive 52- to 53-kd proenzyme; cathepsin D activation produces a 48-kd (single chain) intermediate and mature forms at 34 and 14 kd (heavy and light chains, respectively).36,37 Immunoblotting studies using anti-cathepsin D antibodies revealed an early-onset increase in levels of cathepsin D (both single chain and heavy chain) in all brain regions tested. At 2 weeks postnatal, levels of single chain cathepsin D (Figure 1A , arrows) in brainstem, cerebellum, cerebral cortex, and hippocampus of Npc1C/C mice were 274 ?? 7%, 190 ?? 5%, 176 ?? 12%, and 199 ?? 5% of those measured in Npc1+/+ mice, respectively (means ?? SEM, n = 5, P < 0.001; Figure 1B ). Levels of single chain-cathepsin D remained elevated at 4 weeks with further increase being only evident in cerebellum (Figure 1B) . Changes in heavy chain-cathepsin D (Figure 1A , C) were similar to those observed for the single chain isoform (Figure 1, A and B) .

Figure 1. Cathepsin D levels in brain of Npc1+/+ and Npc1C/C mice during postnatal development. A: Representative images of blots of samples from 2-week-old animals labeled with anti-cathepsin D antibodies. Arrows correspond to the "single chain" isoform of cathepsin D, whereas lines indicate the "heavy form" of cathepsin D (see Results for details). B and C: Quantitative results for levels of single chain cathepsin D and heavy chain cathepsin D isoforms, respectively. Data are presented as percentage of values from Npc1+/+ mice and are means ?? SEM. n = 5; *P < 0.05, and **P < 0.01.

Immunohistochemical results revealed significant increases in cathepsin D immunoreactivity throughout the brain in 1-week-old Npc1C/C mice. In contrast to the pattern observed in wild-type mice, numerous darkly labeled cells were found in cerebellum of Npc1C/C mice, and most of them were located in white matter (compare Figure 2, B with A), suggesting that they were glial cells. High magnification examination showed that cathepsin D immunoreactivity was also moderately increased in Purkinje cells in Npc1C/C (Figure 2D) compared with wild-type mice (Figure 2C) . The number of cathepsin D-immunoreactive cells was also increased in the ventral posterior nuclei of the thalamus at 1 week (Figure 2F) compared with that in Npc1+/+ mice (Figure 2E) , and it was further increased by 4 weeks. By 8 weeks, the ventral posterior nuclei of the thalamus were filled with anti-cathepsin D-immunopositive cells (Figure 2H) . Higher magnification images showed that although cathepsin D-immunoreactive products existed in small granules that were scattered in cell bodies in the ventral posterior nuclei of the thalamus of Npc1+/+ mice (Figure 2I) , those in Npc1C/C mice were present in larger punctates that often clustered together (Figure 2J) . Furthermore, higher cytoplasmic levels of cathepsin D immunoreactivity were observed around these punctates. Immunohistochemical and immunoblotting results showed that levels of another lysosomal hydrolase, cathepsin B, were also increased in mutant mice as compared with wild-type mice (Figure 2, KCM) . Cathepsin D activity in homogenates from brainstem of 4-week-old Npc1C/C mice was about 2.4 times higher than that from Npc1+/+ mice (n = 3 for Npc1+/+ and n = 4 for Npc1C/C mice; P < 0.01). Cathepsin B activity also increased in samples from hippocampus (295 ?? 8%; P < 0.01) and cerebellum (187 ?? 3%; P < 0.01) of 8-week-old Npc1C/C mice (n = 5) compared with Npc1+/+ mice (n = 5).

Figure 2. Distribution of cathepsin D and B in cerebellum and thalamus of Npc1C/C mice during postnatal development. Cerebellar (ACD) and thalamic (ECL) tissue sections were prepared from Npc1+/+ (A, C, E, G, I, and K) and Npc1C/C (B, D, F, H, J, and L) mice at postnatal week 1 (ACF), 4 (K and L), and 8 (GCJ) and were immunostained with anti-cathepsin D (ACJ) or anti-cathepsin B (K and L) antibodies. Higher magnification images show that in Npc1+/+ mice (I) cathepsin D immunoreactive products are mainly located in small-sized granules, whereas in Npc1C/C mice they are present in larger puncta and their surrounding cytoplasmic structures (J). M shows immunoblots of cathepsin B-labeled samples from brainstem of 4-week-old mice. ml, molecular layer; pl, Purkinje layer; gl, granular layer; VPT, ventral posterior nucleus of thalamus. Scale bar = 50 µm (A and B); 12.5 µm (CCH and K and L).

Double immunofluorescence staining was used to determine the cellular and subcellular localization of cathepsin D in mutant mice. At 4 weeks, cathepsin D-immunoreactive granules were observed in cell bodies of calbindin-immunopositive Purkinje cells in Npc1C/C mice (Figure 3A , the asterisk in bottom panels), whereas very few cathepsin D positive granules were found in the cerebellum of Npc1+/+ mice (Figure 3A , top panels). In the cerebellum of 4-week-old Npc1C/C mice, cathepsin D immunoreactivity was also found in reactive microglia identified with antibodies against the macrophage marker F4/80 antigen (data not shown). By 8 weeks, cathepsin D-labeled granules accumulated mainly in the apical processes of small cells dispersed among Purkinje cells in wild-type mice; from their position and morphology, these cells resembled Bergman glia (Figure 3B , arrows). Cathepsin D immunoreactivity in Bergmann glia in Npc1C/C mice was similar to that in wild-type mice (Figure 3B) . In the cerebellum of 8-week-old Npc1C/C mice, cathepsin D immunoreactivity was also observed in F4/80-labeled reactive microglia (Figure 3B , mg); at this postnatal age, microglia became larger and rounder and invaded both the Purkinje cell layer and the molecular layer.

Figure 3. Cellular localization of cathepsin D in cerebellar cortex at 4 and 8 weeks postnatal. A: Double immunofluorescence staining using antibodies against cathepsin D (red) and calbindin (green) in cerebellum of 4-week-old Npc1+/+ (top panels) and Npc1C/C (bottom panels) mice. DAPI (blue) was included in the mounting medium to label nuclei. B: Double immunofluorescence staining using antibodies against cathepsin D (red) and F4/80 (green; a marker for microglia) in cerebellum of 8-week-old Npc1+/+ (top panels) and Npc1C/C (bottom panels) mice. *, Purkinje cells; arrows, Bergmann glia; mg, microglia. Scale bar = 10 µm.

Abnormal Subcellular Protein Distribution in Brains of Npc1C/C Mice

The subcellular localization of various proteins was determined by combining subcellular fractionation and immunoblotting analysis. Cathepsin D levels were markedly higher in the late endosomal/lysosomal fractions in mutant compared with wild-type mice (Figure 4 , top panel). Levels of the small GTP-binding protein Rab7, which participates in the maturation of autophagic vacuoles,38,39 were higher in the late endosomal/lysosomal fractions but lower in the early endosomal fractions in Npc1C/C compared with wild-type mice. As a close link between autophagy and protein ubiquitination has previously been reported,40,41 levels of ubiquitinated proteins in different subcellular fractions were determined by immunoblotting using anti-ubiquitin antibodies. Proteins residing in the late endosomal/lysosomal fractions were highly ubiquitinated (Figure 4 , bottom panel). Ubiquitin immunoreactive products were smeared from the top to the middle part of the gel resulting in a typical staining pattern that generally implies polyubiquitination.

Figure 4. Abnormal protein distribution and ubiquitination in brain of Npc1C/C mice. Immunoblots of samples from different fractions labeled with anti-cathepsin D and pro-cathepsin D (arrow in top panel), -rab7 (middle panel), or -ubiquitin (ubi, bottom panel) antibodies. Note the marked increase in cathepsin D in fraction 1 in samples from Npc1C/C mice. Note also the marked increase in levels of ubiquitinated proteins in endosomal/lysosomal fraction in the mutant mice. Interface 1 contains membrane from late endosome/lysosomes, interface 2 contains mainly early endosomes, and interface 3 contains other membrane structures. Western blots of subcellular fractions are representative of two experiments; each included four animals from each genotype. The results were very similar in both experiments.

Increased Levels of the Mammalian Autophagic Protein LC3-II in Brains of Npc1C/C Mice

Lysosomal dysfunction perturbs normal protein degradation and amino acid recycling, which could result in a state of "cellular amino acid starvation," the most common cause of autophagy. To determine the status of autophagic activity in brains of Npc1C/C mice, levels of the microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of the yeast autophagic protein Atg8, were assessed in various brain regions by immunoblotting. Like Atg8, LC3 is modified via a ubiquitination-like system33,42 ; LC3 is first cleaved in its carboxyl terminal and becomes LC3-I, which is further modified by Atg7 and Atg3 into a membrane-bound form, LC3-II.42 Modification of LC3 is essential for the formation of autophagosomes; thus LC3-II has been widely used as an autophagosomal marker.33 Although brain levels of LC3-I in mutant mice did not significantly differ from that in wild-type mice, levels of LC3-II in 2-week-old mutant mice were significantly higher than those in wild-type mice (Figure 5) . This difference was even greater in 4-week-old animals. Interestingly, elevation of LC3-II was more prominent in areas that are more sensitive to NPC-type injury. The LC3-II/LC3-I ratio exhibited similar changes as those of LC3-II in brain of Npc1C/C mice compared with those in wild-type mice, further confirming that only LC3-II was altered.

Figure 5. LC3-II levels in brains of Npc1C/C mice during postnatal development. A: Representative images of blots labeled with anti-LC3 serum. Levels of LC3-II (B) and the ratio of LC3-II/LC3-I (C) are higher in mutant mice, especially in brainstem (BS) and cerebellum (CB); moderate increases were observed in cortex (CX) and hippocampus (Hipp). Data are presented as percentage of values from Npc1+/+ mice and are means ?? SEM. n = 4; *P < 0.05, and **P < 0.01.

Combined calbindin immunofluorescence and filipin staining confirmed that at postnatal week 4, accumulation of cholesterol in the cerebellum was mainly in Purkinje cells (Figure 6A , arrows in bottom panels). Combined LC3 immunofluorescence and filipin staining showed that although there was virtually no LC3 immunopositive granules in wild-type mice at 4 weeks, occasional LC3-immunopositive clusters were found in the apical dendrites of Purkinje cells filled with filipin-labeled free cholesterol in Npc1C/C mice (data not shown). Interestingly, by 8 weeks LC3 immunopositive granules were found scattered in the soma of Purkinje cells in wild-type mice (Figure 6B , top panels), whereas in mutant mice, smaller LC3 granules aggregated with cholesterol clusters at one pole of Purkinje cells (Figure 6B , asterisks in bottom panels) or in some densely packed small cells. Colocalization of LC3 with cholesterol was confirmed by orthogonal three-dimensional analysis of individual Purkinje cells (Figure 6B) .

Figure 6. Cholesterol accumulation and sequestration of LC3 in Purkinje cells in Npc1C/C mice. A: Localization of filipin-stained free cholesterol (blue) in calbindin-immunopositive Purkinje cells (red) in cerebellum from 4-week-old Npc1+/+ (top) and Npc1C/C (bottom) mice. Note the accumulation of cholesterol in Purkinje cells in mutant mice but not in wild-type mice. Anti-NeuN (green) was used to label neurons. B: Combined LC3 immunostaining (red) with filipin staining (blue) in 8-week-old Npc1+/+ (top) and Npc1C/C (bottom) mice. Top panels show LC3-positive puncta present in Purkinje cells of 8-week-old Npc1+/+ mice; inset is a higher magnification image showing the subcellular distribution of LC3 puncta. Bottom panels show three-dimensional colocalization of LC3 with cholesterol in Purkinje cells of an 8-week-old mutant mouse. Scale bar = 10 µm.

Ultrastructural Changes in Brains of Npc1C/C Mice

As ultrastructural examination by transmission electron microscopy remains the most convincing and standard method to detect autophagy,43 the morphology of intracellular inclusions in brain of Npc1C/C mice was further evaluated by electron microscopy. Purkinje cells in the cerebellum of 6-week-old wild-type mice had a centrally located nucleus (Figure 7A) with stacks of perinuclear Golgi complex (Figure 7, A and B) , polyribosomes, rough endoplasmic reticulum and mitochondria that were distributed relatively evenly in the cytoplasm (Figure 7) . Spherical or oval-shaped lysosomes (Figure 7, A, C, and D) were also observed in the cytoplasm of Purkinje cells in wild-type mice. Electron microscopy analysis of Purkinje cells from 6-week-old Npc1C/C mice revealed a different feature: numerous intracellular inclusion bodies with different sizes and shapes accumulated in one side of the cell body and pushed a kidney-shaped nucleus to the other side (Figure 8A) and a cluster of endoplasmic reticulum and mitochondria aggregated along the indent side of the nucleus. High-magnification images showed that these inclusion bodies were mostly membranous vacuoles with double membranes (arrowheads) or multilamellated electron-dense material (Figure 8 , arrows). In addition, abnormal multivesicular profiles (Figure 8) similar to the polymembranous cytoplasmic bodies described in human NPC disease were also common. Interestingly, lysosome-like structures with homogeneous filling of moderate levels of electron-dense materials, as those observed in wild-type mice, seemed to disappear in mutant mice. Aggregation of membranous vacuoles was also observed in myelinated Purkinje cell axons that were located among granule cells; these vacuoles clustered with mitochondria and formed axonal spheroids (Figure 9B) . Finally, membranous vacuoles were also observed in endothelia in capillaries located among parallel fibers and numerous synapses (compare Figure 10, B to A ).

Figure 7. Ultrastructure of Purkinje cells in Npc1+/+ mice. A: A Purkinje cell in the vicinity of granule cells in a 6-week-old Npc1+/+ mouse. B: Stacks of Golgi apparatus are present in a Purkinje cell. C and D: Lysosome-like structures exist in Purkinje cells. ER, endoplasmic reticulum; G, Golgi apparatus; L, lysosome; M, mitochondria; Ng, nucleus of granule cell; Npc, nucleus of Purkinje cell. Scale bars = 2 µm (A); 1 µm (BCD).

Figure 8. Ultrastructure of Purkinje cells in Npc1C/C mice. A: A Purkinje cell in a 6-week-old Npc1C/C mouse. Numerous vacuoles (arrowheads) of different sizes with various levels of electron-dense materials are present in the cytoplasm. B: Stacks of Golgi apparatus are clustered in the cytoplasm. CCH: Morphology of various membranous vacuoles. Some of them are with double membranes (arrowheads), whereas others have multilamellated structures (arrows). Scale bars = 2 µm (A); 1 µm (BCH).

Figure 9. Axonal pathology in Npc1C/C mice. A: A myelinated axon of a Purkinje cell axon exists in the vicinity of two granule cells in a 6-week-old wild-type mouse. B: An axonal spheroid in a myelinated Purkinje cell axon is located among granule cells in a 6-week-old Npc1C/C mouse. Note that a cluster of mitochondria is surrounded by vacuoles accumulated within the spheroid. Scale bars = 2 µm.

Figure 10. Capillary pathology in cerebellum of Npc1C/C mice. A capillary is surrounded by parallel fibers and synapses in the cerebellum of a 6-week-old wild-type (A) or a mutant (B) mouse. Note the membranous inclusions in the endothelial cell in the mutant mouse. Insets show synapses. Scale bars = 2 µm.

Early-Onset Lysosomal Abnormality May Contribute to Neurodegeneration in Npc1C/C Mice

Results from the present study indicated that abnormal levels of the lysosomal enzyme cathepsin D occurred early during postnatal development in Npc1C/C mouse brain. Increases in levels of both single chain and heavy chain cathepsin D isoforms were clearly detected by immunoblotting in all brain areas examined at 2 weeks postnatal. Both isoforms possess catalytic activities,36,37 suggesting that cathepsin D activity might be increased in these areas in mutant mice. Indeed, enzymatic assay confirmed that cathepsin D activity was increased in the brainstem. Likewise, activity of another lysosomal hydrolase, cathepsin B, was also increased in the brains of mutant mice. However, the highest increases in cathepsin D levels were observed in the brainstem and cerebellum, two regions that exhibit early and marked neurodegeneration. Immunohistochemistry analyses indicated that increases in cathepsin D occurred both in neurons and in glial cells; dense cathepsin D immunoreactivity was observed in the soma of Purkinje cells of Npc1C/C mice by 2 weeks postnatal, whereas clear glial localization was prominent in several brain regions at 4 weeks, results which are in agreement with those reported in an earlier study44 and with the early-onset inflammatory response we previously reported.32 Enhanced cathepsin D immunostaining occurred mainly in brain structures exhibiting both accumulation of intracellular free cholesterol and neurodegeneration. Double immunofluorescence analysis showed that enhanced cathepsin D was present in both neurons and glia at 4 weeks but mainly in glial elements by 8 weeks postnatal.

Increases in number of secondary lysosomes and changes in levels of lysosomal enzymes have previously been associated with brain aging and age-related neurodegeneration.45 In particular, increased cathepsin D levels occur in AD brains before the onset of major pathology, and this was correlated on a cell-by-cell basis with decreases in synaptic proteins and with the presence of one of the disease??s hallmarks, neurofibrillary tangles.19,20 Furthermore, pharmacological suppression of cathepsins B and L resulted in increase in cathepsin D,46,47 lysosomal proliferation, formation of meganeurites (axon swellings that were often located proximal to the cell body) in cultured rat hippocampal slices,35,48 and in tau hyperphosphorylation and generation of neurofibrillary tangles in cultured hippocampal slices from apolipoprotein E-deficient mice.21 Furthermore, the compromise of lysosomal membrane and subsequent leakage of cathepsin D into cytoplasm are early events in amyloid ß peptide treatment-induced cell death in cultured hippocampal neurons.49 These results have supported the hypothesis that lysosomal dysfunction contributes to AD-type neuropathologies. A recent study from Nixon??s laboratory29 showed that extensive macroautophagy might contribute to changes in lysosomal function in AD. Thus our results further expand the previously noted similarities in neuropathological mechanisms between NPC and AD and suggest therefore that lysosomal dysfunction may contribute to NPC type neurodegeneration.

Abnormal Autophagic Activity and Protein Ubiquitination in Brains of Npc1C/C Mice

Impairment in cholesterol transport induced by NPC1 mutations is associated with abnormal vesicle trafficking and redistribution of presenilin,50 glycosphingolipids,51 rab7,52,53 and annexin II,54 although the underlying mechanism has remained elusive. Immunoblotting analysis revealed an early-onset increase in levels of LC3-II, a widely used marker for autophagy. Levels of this protein were particularly high in the brainstem and cerebellum, two brain regions exhibiting the severest neuronal death, which suggests that autophagy may contribute to neurodegeneration. Increases in autophagic stress were further confirmed by ultrastructural detection of autophagosome-like vacuoles that were prominent in brains of Npc1C/C animals but uncommon in wild-type mice, findings that are consistent with results from a recent report.31 We previously showed that levels of inactive GSK-3ß were markedly increased in Npc1C/C mouse brains and this increase was closely associated with inactivation of nuclear factor B signaling in brains of Npc1C/C mice during early development.55 Sequestration of GSK-3ß by autophagy may contribute to inhibition of this kinase, which also explains the predominant lysosomal location of the enzyme.55 Relocation of GSK-3 to lysosomes could also result from enhanced chaperone-mediated autophagy, a possibility that needs to be further explored. It has previously been reported that, although GSK-3ß was located predominantly in the cytosol of SH-SY5Y cells, its active form was disproportionately higher in nuclei and mitochondria.56 The active form of another kinase, extracellular signal-regulated kinase 1/2, was also localized in the mitochondria and autophagosomes in Lewy body disease.57 It is conceivable that changes in subcellular localization of these kinases could alter their activities; however, further experiments are needed to clarify this issue. Interestingly, proteins in the late endosomal/lysosomal fractions of brains from Npc1C/C mice were also highly ubiquitinated. Protein ubiquitination, a type of post-translational protein modification, is generally used to deliver targeted proteins for degradation through the proteasome.58 In addition to this "classic" route for protein breakdown, ubiquitination of membrane integral proteins with one ubiquitin (monoubiquitination) directs these proteins to multivesicular bodies then to lysosomes for degradation.59 However, lysosome-degraded proteins are thought to be deubiquitinated before import into the lysosome. Immunoblotting results showed that proteins accumulated in late endosomal/lysosomal fractions were mostly polyubiquitinated; these proteins should be degraded in proteasomes located in the cytoplasm. How these proteins are delivered to lysosomes is not clear. Mutation of NPC1 proteins not only leads to accumulation of cholesterol in late endosomal/lysosomal compartments but also results in abnormal distribution of proteases along the endocytic pathways60 as well as leakage of cathepsins into the cytoplasm, which would lead to lysosomal dysfunction and incomplete digestion of "cargos." As a compensative response, autophagic activity would be increased. However, due to lysosomal dysfunction, LC3 and other proteins in autophagolysosomes cannot be as efficiently degraded as under normal conditions,61,62 which leads to further increase in accumulation of undigested proteins.

Cholesterol Accumulation-Associated Autophagic-Lysosomal Dysfunction May Contribute to Neurodegeneration in Npc1C/C Mice

Several lines of evidence have indicated that autophagic cell death is involved in cell death that normally occurs during postnatal development in the nervous system27 and in several neuronal degenerative diseases and animal models of these diseases.28-30 Expression of - synuclein with the same mutations as those found in early-onset Parkinson??s disease in a cultured cell line induced massive accumulation of autophagic vacuoles and impairment of the ubiquitin-proteasome system.63 Ultrastructural examination revealed that both apoptotic and autophagic features were present in degenerating neurons of the substantia nigra in Parkinson??s disease patients.30 In the case of AD, an earlier study reported the existence of active caspase 3 in autophagic vacuoles, which led the authors to propose that autophagy might be neuroprotective in AD.28 However, a more recent study by Nixon and colleagues29 demonstrated that autophagic vacuoles were abundant in degenerating neurites and were specifically colocalized with neurofibrillary tangles in perikarya. These findings support the involvement of autophagy in neurodegeneration. In vitro experiments showed that trophic factor withdrawal induced Purkinje cell death with increased autophagy,64 whereas autophagy inhibition prevented both increased vacuolation and loss of Purkinje cells. In vivo evidence supporting a role of autophagy in neurodegeneration also came from studies of lurcher mice. Selective Purkinje cell death in lurcher mice is caused by mutations in the 2 glutamate receptor (GluR2).65 Additional experiments demonstrated that mutations in GluR2 resulted in enhanced autophagy, possibly by interactions between the mutated receptors and the autophagic protein Beclin1.65,66 These results provided strong evidence for a direct link between autophagy and Purkinje cell death in lurcher mice. As autophagy is generally followed by the fusion of lysosomes with autophagosomes and formation of autophagolysosomes, in which the autophagic components are degraded.67 The beneficial or detrimental effect of autophagy may depend on the functional status of compartments downstream of the autophagic pathway. Defects in completing autophagy could result in accumulation of autophagosomes and autophagolysosomes, which could impair cell function. Furthermore, accumulation of autophagolysosomes could feedback on lysosomal function and induce lysosomal membrane permeabilization and translocation of cathepsins to the cytosol, a process implicated in cell death induced by various insults.49,68 Release of cathepsins from lysosomes into cytosol could also initiate caspase-dependent apoptosis via activation of proapoptotic factors such as Bax, Bid, and caspases, as in the case of staurosporine-induced cell death.22-24 Cleavage of the microtubule associated protein tau by cytosolic cathepsin D has been proposed to participate in AD pathogenesis and transport failure due to impairment in microtubule formation is postulated to contribute to accumulation of autophagosomes/autophagolysosomes in AD brain.29

In addition to demonstrating enhanced autophagic activity in brains of Npc1C/C mice, results from the present study showed an abnormal subcellular distribution of LC3-labeled autophagosomes. In contrast to the notion that autophagy is a reaction to starvation, appreciable amounts of autophagosomes existed in Purkinje cells of 8-week-old wild-type mice, suggesting that autophagy may contribute to the maintenance of normal morphology and function of neurons. This notion is supported by the recent discoveries that knocking out two critical proteins of the autophagy machinery, Atg5 and Atg7, resulted in massive neurodegeneration.69,70 In Purkinje cells of Npc1C/C mice, LC3-labeled autophagosomes aggregated and colocalized with cholesterol clusters, indicating an abnormal autophagy-lysosome system. It is conceivable that cholesterol accumulation in the endosomal/lysosomal system impairs autophagosome fusion with lysosomes. It is also possible that accumulated cholesterol "traps" the autophagy machinery and other proteins in late compartments of the endocytic pathway, thereby impairing cell function. The fact that Purkinje cell in mutant mice seemed to lack classic lysosomes as observed in wild-type mice indicates an abnormal autophagic-lysosomal system in the Npc1C/C mice. The hypothesis that lysosomal dysfunction redirects autophagy toward cell death is supported by the finding that inhibition of lysosome fusion with autophagic vacuoles in starved cells induced an early-onset autophagic cell death followed by classic apoptosis.71,72

In summary, the present study presented evidence that increases in brain levels of lysosomal cathepsins B and D occurred early during postnatal development in brains of Npc1C/C mice, in particular in areas that exhibited early-onset neurodegeneration. Changes in lysosomal function were accompanied with relocation of ubiquitinated proteins in endosomes/lysosomes. Redistribution of these proteins may result from enhanced autophagic activity, which was demonstrated by immunoblotting and immunofluorescence analysis of LC3 and ultrastructural detection of autophagic vacuoles. These results provide the first evidence that accumulation of cholesterol alters autophagy-lysosome function and diverts this system toward neurodegeneration in NPC.

We thank Kevin Lee, Carman Rivera, Cynthia Chan, and Clara Yu for excellent technical assistance, and Dr. T. Yoshimori, National Institute of Genetics, Japan, for anti-LC3 antibodies.

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作者单位:From the Department of Basic Medical Sciences,* College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, California; the Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, California; the Electron Microscopy Core Laboratory, Sh

日期:2008年5月29日 - 来自[2007年第169卷第9期]栏目

Macrophage-Specific Expression of Human Lysosomal Acid Lipase Corrects Inflammation and Pathogenic Phenotypes in lalC/C Mice

【摘要】  Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in the cell. The downstream metabolites of these compounds serve as hormonal ligands for nuclear receptors and transcription factors. Genetic ablation of the lal gene in the mouse caused malformation of macrophages and inflammation-triggered multiple pathogenic phenotypes in multiple organs. To assess the relationship between macrophages and lalC/C pathogenic phenotypes, a macrophage-specific doxycycline-inducible transgenic system was generated to induce human LAL (hLAL) expression in the lalC/C genetic background under control of the 7.2-kb c-fms promoter/intron2 regulatory sequence. Doxycycline-induced hLAL expression in macrophages significantly ameliorated aberrant gene expression, inflammatory cell (neutrophil) influx, and pathogenesis in multiple organs. These studies strongly support that neutral lipid metabolism in macrophages contributes to organ inflammation and pathogenesis.
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Macrophages produce and secrete cytokines, chemokines, and growth factors that influence gene expression, cell proliferation/differentiation, and apoptosis in organ tissues through paracrine and autocrine mechanisms, making them vitally important to the physiological functions of multiple organs. Many diseases are tightly associated with malformation and malfunction of macrophages within organs. Design and establishment of a system to specifically express "genes of interest" in macrophages in a temporal/spatial fashion will greatly assist in understanding the molecular mechanisms of macrophage differentiation/maturation and the functional roles of macrophages in disease formation. The tetracycline-inducible transgenic mouse system has proven to be very effective in inducing genes of interest in a temporal and spatial manner. In this system, "activator" transgenic mice bear the reverse tetracycline-responsive transactivator (rtta) fusion protein under control of a cell type-specific promoter. The rtta expression is restricted to specific cell types in transgenic mice. In a separate transgenic mouse line, the gene of interest is under control of the tet operator DNA-binding sequence that is linked to a minimal promoter. After crossbreeding, expression of the gene of interest can be precisely controlled by addition or removal of tetracycline or doxycycline in double transgenic mice. This strategy has been proven to be very effective in many organ systems, including the lung.1,2
To design a tetracycline-controlled macrophage-specific system, the key is to select a macrophage-specific promoter. It has been reported previously that the c-fms gene, which encodes the receptor for macrophage colony-stimulating factor (CSF-1), is selectively expressed in macrophage and trophoblast cell lineages. In a transgenic mouse study, 7.2-kb of the 5'-flanking regulatory sequence and the downstream intron 2 of the c-fms gene have been defined to direct specific expression of the green fluorescent protein (GFP) reporter gene in the same locations as the endogenous gene, therefore providing an ideal DNA sequence to direct gene expression in mononuclear phagocyte lineage during embryonic development and in bone marrow, peripheral blood, and all adult tissues.3
Cholesteryl esters and triglycerides are important components in neutral lipids, which can be hydrolyzed by lysosomal acid lipase (LAL) in the lysosome of cells to generate free cholesterol and free fatty acids. After LAL cleaves these lipids, they exit the lysosome and enter the cytosol. An lalC/C mouse model has been generated by gene targeting.4 lalC/C mice developed pathogenic phenotypes in multiple organs, including lung, liver, spleen, adrenal glands, and small intestine.4-6 These pathogenic phenotypes are caused by abnormal gene expression. For example, Affymetrix GeneChip microarray analysis of lalC/C mice showed aberrant gene expression for inflammatory cytokines/chemokines, endopeptidases (eg, matrix metalloproteinases), apoptosis inhibitors, transcription factors, and oncogenes in the lung.5,7 These gene profile changes correlated well with pulmonary phenotype progression in the lalC/C lung in an age-dependent manner. It is known that many metabolic derivatives of free cholesterols and free fatty acids serve as hormonal ligands for nuclear receptors and transcription factors that have profound and diverse functions in gene regulation, cell proliferation, differentiation, and apoptosis. In a rescue study, treatment of peroxisome proliferator-activated receptor (PPAR) ligands (downstream metabolites of LAL) significantly improved lalC/C pulmonary inflammation and aberrant gene expression, suggesting that inflammation-triggered pathogenesis during LAL deficiency is partially caused by inactivation of PPAR because of a lack of ligand production. Because abnormal appearance of macrophages is the major manifestation of lalC/C mice within multiple organs, we hypothesize that a deficiency of LAL greatly affects the normal functions of macrophages and leads to various pathogenic phenotypes.
To test this hypothesis, doxycycline-controlled c-fms 7.2-kb promoter/intron2-rtTA and (tetO)7-CMV-hLAL transgenic mouse lines were generated. After crossbreeding the double transgenic lines into lalC/C mice, expression of hLAL in macrophages significantly ameliorated tissue inflammation, pathogenic phenotypes, and aberrant gene expression in several organs, supporting a concept that cholesteryl ester and triglyceride metabolism is essential for the normal biological function of macrophages.

【关键词】  macrophage-specific expression lysosomal corrects inflammation pathogenic phenotypes

Materials and Methods

Animal Care

All scientific protocols involving the use of animals in this study have been approved by the Institution Animal Care and Usage Committee and followed guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association. Protocols involving the use of recombinant DNA or biohazardous materials followed guidelines established by the National Institutes of Health. Animals were housed in an Institution Animal Care and Usage Committee-approved facility. Animals were regularly screened for common respiratory pathogens and murine viral hepatitis. Experiments involving animal sacrifice used CO2 narcosis to minimize animal discomfort.

Generation of Transgenic Mice

To generate c-fms-rtTA transgenic mice, the cDNA fragment of rtTA was amplified by polymerase chain reaction (PCR) using a downstream primer (5'-AAGGAAAAAAGCGGCCGCGTACATTGAGCAACTGAC-3') and an up-stream primer (AAGGAGGGCCCGCCACCATGTCTAGATTAGATAAA-3'). The PCR product was digested with ApaI/NotI and subcloned into the p7.2MCS construct containing the 7.2-kb c-fms promoter/intron2 fragment. The c-fms-rtTA expression cassette containing the 7.2-kb c-fms promoter/intron2 fragment, the rtTA cDNA, and the SV40 polyadenylation signaling sequence was microinjected into eggs of FVB/N mice by the Transgenic Core Facility at the University of Cincinnati, College of Medicine. Founder lines were identified by a pair of primers covering the 7.2-kb c-fms promoter/intron2 sequence (5'-TGATTGAAGGGTCCAGACTCATTC-3') and an rtTA cDNA coding region sequence (5'-AGTGTAGGCTGCTCTACACCAAGC-3').

To generate the (tetO)7-CMV-hLAL transgenic mouse line, hLAL cDNA was amplified by PCR using a downstream primer (5'-AAGGAAAAAGCGGCCGCTTATCACTTGTCATCGTCGTCCTTGTAGTCCTGATATTTCCTCATTAG-3') that contains a Flag sequence and an upstream primer (5'-CTAGACGCGTGCCACCATGAAAATGCGGTTCTTGG-3'). The PCR product was digested with MluI/NotI and subcloned downstream of the CMV minimal promoter linked to seven Tet-responsive elements (7x TRE) at the MluI and NotI sites in the pTRE2 vector (BD Bioscience-Clontec, Mountain View, CA). The expression cassette containing the CMV promoter, the hLAL cDNA, and the ß-globin polyadenylation signaling sequence was excised and purified for microinjection into FVB/N mice. Founder lines were identified by a pair of primers covering the pTRE2 vector sequence (5'-ACGCCATCCACGCTGTTTTG-3') and the hLAL cDNA sequence (5'-AGACAACTGGTTTGGGACCTTTG-3').

Isolation of Macrophages from Bronchoalveolar Lavage Fluid (BALF) for Antibody Array Analysis

BALF was collected by perfusing each lung of wild-type or lalC/C mice with 1-ml aliquots of 0.9% sodium chloride and withdrawing back the fluids three times. BALFs from eight mice were combined and centrifuged for 5 minutes at 1000 rpm and 4??C to collect macrophage pellets. Purified macrophages were lysed for protein preparation. After determination of protein concentrations, expression levels of cytokines/chemokines were determined with the Mouse Inflammation Antibody Array System from RayBio Cytokine Antibody Array service (RayBiotech, Inc., Norcross, GA). Fold changes of cytokines and chemokines between lalC/C and wild-type macrophages were analyzed by the software service from the same company. The intensity of each signal was scanned and normalized with backgrounds.

Isolation of Total RNA from Whole Lung Tissue for Reverse Transcriptase (RT)-PCR Assay

Whole lung tissues were dissected from doxycycline-treated (4-month) or untreated c-fms-rtTA/(tetO)7-CMV-hLAL double mice or c-fms-rtTA/(tetO)7-CMV-hLAL/lalC/C triple mice after mice were anesthetized by intraperitoneal injection. Whole lung tissues were homogenized in RLT lysis buffer for total RNA purification as recommended by the manufacturer (Qiagen, Valencia, CA). Total RNAs were purified using the Qiagen total RNA purification kit as recommended by the manufacturer (Qiagen).

For semiquantitative RT-PCR assay, total RNAs were used to detect mRNA expression by a SuperScript One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA). Primers for RT-PCR amplification of Api6, MafB, MMP-9, MMP-12, Spi-C, and GAPDH genes were described previously.7 Primers for tumor necrosis factor- (TNF-), interleukin (IL)-1ß, IL-6, CCL7, and CXCL1 were purchased from SuperArray Bioscience (Frederick, MD). Primers for RT-PCR amplification of hLAL were an upstream primer corresponding to the hLAL cDNA sequence (5'-CACATTCTCCTGCTGGAACTTCTG-3') and a downstream primer corresponding to the pTRE vector sequence (5'-CCTGAAAACTTTGCCCCCTC-3').

Tissue Lipid Extraction and Determination of Cholesteryl Ester and Triglyceride Concentrations

Total tissue lipids were extracted from the liver and small intestine by the Folch method.4 Concentrations of cholesteryl esters and triglycerides were determined as previously described.6,8

Liver Extraction and Lysosomal Acid Lipase Activity Assays

Liver tissues were homogenized, sonicated, and extracted in tissue lysis buffer (100 mmol/L NaPO4, pH 6.8, 1 mmol/L ethylenediamine tetraacetic acid, 10 mmol/L dithiothreitol, 0.5% NP-40, and 0.02% sodium azide) followed by centrifugation at 4??C, 10,000 x g for 15 minutes. Aliquots of the supernatant were stored at C20??C until assayed. Protein concentration of the liver extract was determined by the BCA assay following the protocol from manufacturer (Pierce, Rockford, IL). hLAL activities were estimated with the fluorogenic substrate 4-methyl-umbelliferyl-oleate (4-MUO). The assay was performed in a microtiter plate at 37??C at pH 5.5 using a 4-methylumbelliferone (4-MU) standard curve. In brief, 4-MUO in hexane (100 mg/ml) was diluted 1 to 100 in 4% Triton X-100. The assay used 50 µl of the diluted substrate, 25 µl of diluted liver extract, and 125 µl of assay buffer (0.2 mol/L Na2Ac and 0.01% Tween 80, pH 5.5) at 37??C for 30 minutes. The reaction was stopped by adding 100 µl of 0.75 mol/L Tris, pH 8.0. The resulting fluorescence signal was detected at excitation 360 nm and emission 460 nm. The relative fluorescence units from each sample were directly compared with that of the standard curve. One unit of activity is defined as producing 1 µmol/L 4-MU per minute. All assays were conducted in duplicate, and the results were the mean of three experiments. Assays were linear within the time frame used, and less than 10% of substrates were cleaved.

Oil Red-O Staining

Frozen tissue sections were prepared from liver and intestine after a standard cryostat procedure. Tissue section slides were stained with Oil Red-O solution (0.5% in propylene glycol) in a 60??C oven for 10 minutes and placed in 85% propylene glycol for 1 minute; slides were counterstained in hematoxylin.

Tissues from liver, intestine, and lung were collected after mice were anesthetized. For lung tissue preparation, lungs were inflation-fixed with 4% paraformaldehyde in phosphate-buffered saline overnight at 4??C. All tissues were washed with phosphate-buffered saline and dehydrated by a series of increasing ethanol concentrations, followed by paraffin embedding. Sections were incubated with rat anti-mouse Ly6G antibody (1:500; BD Biosciences PharMingen) or rabbit anti-mouse macrophage antibody (1:200; Accurate Chemical & Scientific Co., Westbury, NY) as the primary antibody. Tissue sections were washed and treated with biotinylated secondary antibodies. To visualize signals, interactions were detected with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer??s instructions.

Inflammatory Cytokine/Chemokine Expression in Macrophages of Wild-Type and lalC/C Mice

Abnormal appearance and accumulation of macrophages in multiple organs suggests that they play a major role in contributing to pathogenesis in lalC/C mice. These pathogenic phenotypes are associated with inflammation. To establish that macrophages are a primary source for mediating inflammation in lalC/C mice, wild-type and lalC/C macrophages were isolated from BALF. Macrophages were lysed by using cell lysis buffer (from RayBiotech). The expression levels of various cytokines and chemokines in macrophages were measured by inflammatory antibody array analysis (Table 1) . Expression levels of many cytokines and chemokines were changed (most were up-regulated) in lalC/C macrophages in comparison with wild-type macrophages. This strongly suggests that the LAL activity in macrophages controls production of pro-inflammatory cytokines and chemokines, and malfunction of macrophages has a primary effect in lalC/C pathogenesis in various organs.

Table 1. Aberrant Expression of Cytokines/Chemokines in lalC/C Bronchoalveolar Macrophages in Antibody Array Analysis

Generation of c-fms-rtTA Transgenic Mice

To test the primary role of macrophages in lalC/C pathogenesis, we designed a system to specifically express hLAL in macrophages. This is achieved by subcloning the cDNA fragment of rtTA into the p7.2MCS construct that contains the 7.2-kb c-fms promoter/intron2 fragment (Figure 1A) . The c-fms-rtTA expression cassette DNA containing the 7.2-kb c-fms promoter/intron2 fragment, the rtTA cDNA, and the SV40 polyadenylation signaling sequence was isolated and microinjected into eggs of FVB/N mice. Seven positive clones were identified after screening by PCR using a pair of primers covering the 7.2-kb c-fms promoter/intron2 sequence and the rtTA cDNA sequence (Figure 1B) . The clones are designated as c-fms-rtTA transgenic founder lines. Among these founders, only two founders showed positive transgene F1 generations. Founder 77 was chosen for further characterization of rtTA expression in the macrophage lineage compartment. Alveolar macrophages, peritoneal macrophages, and bone marrow cells were isolated from this founder line for mRNA purification. A pair of primers corresponding to the rtTA cDNA sequence was used for detection of rtTA expression by RT-PCR. Both macrophages and bone marrow cells showed rtTA expression (Figure 1C) . As a control, no expression was detected in isolated AT II cells (data not shown).

Figure 1. Generation of c-fms rtTA transgenic mice. A: Illustration of c-fms-rtTA transgene design. B: PCR genotyping of c-fms-rtTA transgenic founders. MW, molecular weight marker; NC, negative control from tail DNAs of a negative pup; PC, positive control using the c-fms-rtTA construct as a DNA template. C: Expression of rtTA mRNA in peritoneal macrophages, lung macrophages, and bone marrow cells of c-fms-rtTA transgenic mice.

Generation of (tetO)7-CMV-hLAL Transgenic Mice

In parallel, the cDNA fragment of hLAL was subcloned into the pTRE construct that contains seven copies of the tet operator DNA binding sequence linked to a minimal CMV promoter and ß-globin polyA signals (Figure 2A) . The expression cassette of the (tetO)7-CMV-hLAL construct was microinjected into eggs of FVB/N mice. Six positive clones were obtained by PCR using a pair of primers covering the hLAL cDNA and pTRE sequences (Figure 2B) . These clones were designated as (tetO)7-CMV-hLAL founder lines. All founders showed positive F1 generations. Founder 556 was crossbred with the c-fms Founder 77 line to obtain double transgenic mice (Figure 2C) . After treatment of c-fms-rtTA/(tetO)7-CMV-hLAL double transgenic mice with doxycycline-treated food for 1 month, macrophages (from peritoneal and lung lavage fluid) and bone marrow cells showed induced hLAL mRNA expression (Figure 2D) . Increased hLAL mRNA expression level was also observed in liver, lung, and intestinal tissues that contain macrophages (Figure 2E) .

Figure 2. Generation of (tetO)7-CMV-LAL transgenic mice. A: Illustration of (tetO)7-CMV-hLAL transgene design. B: PCR genotyping of (tetO)7-CMV-hLAL transgenic founders. MW, molecular weight marker; NC, negative control from tail DNAs of a negative pup; PC, positive control using (tetO)7-CMV-hLAL construct as a DNA template. C: PCR genotyping of c-fms-rtTA/(tetO)7-CMV-hLAL double transgenic mice. MW, molecular weight marker. D: Expression of hLAL mRNA in bronchio-alveolar macrophages (BAL Mac), peritoneal macrophages (P Mac), and bone marrow (BM) cells. E: Expression of hLAL mRNA in lung, intestine, and liver of c-fms-rtTA/(tetO)7-CMV-hLAL double transgenic mice after doxycycline (Dox) treatment (+). Untreated (C) double transgenic mice were the control.

Macrophage-Specific Expression of hLAL Reduced Neutral Lipid Accumulation in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

LAL deficiency in mice caused massive neutral lipid accumulation in Kupffer cells in the liver, lamina propria (inside of epithelia villia), and the base of villia in the intestine and in macrophages and alveolar type II cells in the lung.4-6,8 To test whether deficiency of LAL in macrophages is responsible for lipid accumulation, c-fms-rtTA/(tetO)7-CMV-hLAL double transgenic mice were introduced into the lalC/C background by crossbreeding to generate c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice. Without doxycycline treatment, neutral lipid accumulation was still observed in the liver and the intestine of c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice, similar to lalC/C mice (Figures 3 and 4 ; identified in the asterisk area). However, expression of hLAL in macrophages induced by doxycycline treatment significantly reduced neutral lipid accumulation in the same liver and intestinal areas of triple lalC/C mice by Oil-Red-O staining (Figures 3 and 4) . This clearly indicates the effectiveness of doxycycline-induced hLAL activity in degrading neutral lipids in triple lalC/C mice.

Figure 3. Macrophage-specific expression of hLAL reduced neutral lipid accumulation in the liver of c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Frozen sections of liver were stained with Oil Red-O. Massive neutral lipid accumulation (red color) was detected in Kupffer cells (macrophages) in livers of lalC/C (B) and c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice (C) without doxycycline treatment as indicated by asterisks. D: Doxycycline treatment significantly reduced Oil Red-O staining in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice. Wild-type lal+/+ mice (A) were the negative control for Oil Red-O staining.

Macrophage-Specific Expression of hLAL Restored the LAL Activity in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

To monitor changes of neutral lipid degradation at the biochemical level in addition to Oil-Red-O staining, concentrations of cholesteryl esters and triglycerides were measured in doxycycline-treated and untreated triple mice. In agreement with Figures 3 and 4 , concentrations of cholesteryl esters and triglycerides were significantly higher in single lalC/C and triple lalC/C mice without doxycycline treatment. Expression of hLAL in macrophages by doxycycline treatment significantly decreased concentrations of cholesteryl esters and triglycerides (Figure 5A) . A direct measurement of the LAL enzymatic activity using the fluorogenic substrate 4-MUO was also performed. Proteins were isolated from the livers of doxycycline-treated and untreated c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice. Wild-type and lalC/C mice were used as controls. Although doxycycline-untreated triple mice showed a similar LAL activity with that in lalC/C mice, treatment of doxycycline partially restored the LAL activity (Figure 5B) . Taken together, the neutral lipid metabolism was partially recovered by expression of hLAL in macrophages of c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice.

Figure 5. Macrophage-specific expression of hLAL restored the LAL activity in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice. A: Concentrations of cholesteryl esters (left) and triglycerides (right) in the liver and small intestine of wild-type mice, lalC/C mice, untreated (Dox off) triple lalC/C mice, and doxycycline-treated (Dox on) triple lalC/C mice. B: The liver LAL activity in wild-type mice, lalC/C mice, untreated (Dox off) triple lalC/C mice, and doxycycline-treated (Dox on) triple lalC/C mice were measured using the fluorogenic substrate, 4-MUO in vitro.

Macrophage-Specific Expression of hLAL Reduced Neutrophil Infiltration in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

Neutrophil infiltration is a major inflammatory manifestation in lalC/C mice.5,7 Triple lalC/C mice were treated with doxycycline for 3 months to induce hLAL expression in macrophages. The liver, intestine, and lung were examined for assessment of neutrophil infiltration because these organs displayed dramatically pathogenic phenotypes during LAL deficiency.4-6,8 As demonstrated in Figure 6 , all three organs from triple lalC/C mice without doxycycline treatment exhibited significant neutrophil infiltration versus wild-type mice. After doxycycline induction in triple lalC/C mice, expression of LAL in macrophages significantly attenuated neutrophil influx into liver, intestine, and lung tissues. These results indicate that LAL activity in macrophages plays an essential role in controlling tissue inflammation in various organs during LAL deficiency.

Figure 6. Macrophage-specific expression of hLAL reduced neutrophil infiltration in c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Paraffin embedded liver, intestine, and lung tissue sections from wild-type (lal+/+) and untreated (CDox) or doxycycline-treated (+Dox) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice were immunostained with Ly6G antibody. Small black dots in various sections represent stained neutrophils.

Macrophage-Specific Expression of hLAL Reduced Liver Pathogenesis in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

We reported previously that LAL deficiency in mice caused severely pathogenic phenotypes and enlargement in the size of the liver in association with foamy macrophage formation (Kupffer cells).4,6,8 To specifically address the functional role of macrophages in liver pathogenic processes, c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice with or without doxycycline treatment were examined. At the histological level, triple lalC/C mice without doxycycline treatment displayed massive foamy Kupffer cell formation (Figure 7C) , similar to lalC/C mice (Figure 7B) . After 3 months of doxycycline treatment beginning from 1 month of age, the size of foamy Kupffer cells was significantly reduced (Figure 7D) . It appears that lipid-filled hepatocytes remained unchanged in treated triple lalC/C mice, indicating cell type-specific hLAL rescue in the liver. This is confirmed by immunohistochemical staining using macrophage-specific antibody, in which positively stained and lipid-stored macrophages were significantly reduced in the livers of treated triple lalC/C mice (Figure 7, ECH) . These results suggest that LAL activity in macrophages contribute to lalC/C pathogenesis in the liver.

Figure 7. Macrophage-specific expression of hLAL reduced liver pathogenesis in c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Histological (ACD) and immunohistochemical (ECH) comparison of liver sections from untreated (C and G) and doxycycline-treated (D and H) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Wild-type (A and E) and lalC/C (B and F) mice were used as negative and positive controls, respectively, for foamy Kupffer cell formation. Kupffer cells are indicated by arrows.

Macrophage-Specific Expression of hLAL Reduced Intestine Pathogenesis in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

To address the functional role of macrophages in the intestinal pathogenic process, a histological study of intestines from c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice was performed. Similar to lalC/C mice (Figure 8B) , macrophage accumulation was observed in the lamina propria (inside of epithelia villia) and the base of villia in doxycycline-untreated triple lalC/C mice (Figure 8C , identified in the asterisk area). Doxycycline treatment significantly reduced both the size and the number of macrophages in triple lalC/C mice (Figure 8D) .

Figure 8. Macrophage-specific expression of hLAL reduced intestine pathogenesis in c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Histological comparison of intestine sections from untreated (C) and doxycycline-treated (D) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Wild-type (A) and lalC/C (B) mice were negative and positive controls.

Macrophage-Specific Expression of hLAL Reduced Lung Pathogenesis in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

In the lung, alveolar space enlargement (emphysema) was closely associated with macrophage proliferation in lalC/C mice.5 Extracellular membrane degrading enzymes such as MMP-9 and MMP-12 were highly expressed in the lalC/C lung during pulmonary pathogenesis. To assess the functional role of macrophages in this pulmonary pathogenic process, histological analyses of c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice with or without doxycycline treatment were performed. In untreated triple mice (Figure 9C) , the alveolar space area was significantly larger than that seen in the wild-type lung (Figure 9A) , similar to that seen in the single lalC/C lung (Figure 9B) . Expression of hLAL in macrophages for 5 months significantly reversed emphysema in doxycycline-treated triple lalC/C mice (Figure 9D) . A quantitative analysis revealed a reduced ratio between the alveolar airspace area and the parenchyma area in the lalC/C lung after macrophage hLAL expression (Figure 9E) . In the immunohistochemical staining study using a macrophage-specific antibody, positively stained macrophages were significantly reduced in the lung of doxycycline-treated versus untreated triple lalC/C mice (Figure 9F) . Therefore, LAL activity in macrophages is essential for maintaining normal alveolar structure and function in the lung.

Figure 9. Macrophage-specific expression of hLAL reduced pulmonary pathogenesis in c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. ACD: Histological comparison of lung sections from untreated (C) and doxycycline-treated (D) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Wild-type (A) and lalC/C (B) mice were controls. E: Morphometric analysis of alveolar space in wild-type (lal+/+) mice, lalC/C mice, and untreated (CDox) and doxycycline-treated (+Dox) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Differences between ratios of airspace/parenchyma were analyzed by analysis of variance. P < 0.05. Values are means ?? SD; n = 3 (mice). F: Immunohistochemical staining comparison of lung sections from untreated and doxycycline-treated c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice using an antibody specifically against macrophage surface antigens. Positively stained cells from various microscopic fields of each sample were counted. Differences between untreated (CDox) and doxycycline-treated (+Dox) samples were analyzed by analysis of variance. P < 0.05. Values are means ?? SD; n = 3 (mice).

Macrophage-Specific Expression of hLAL Reduced Aberrant Gene Expression in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C Triple Mice

Pathogenic phenotypes are caused by aberrant gene expression in lalC/C mice. Gene profile changes have previously been assessed in the lalC/C lung by Affymetrix GeneChip microarray analysis. This analysis identified aberrant expression of many genes during LAL deficiency. These gene products include cytokines/chemokines, MMPs, apoptosis inhibitors, transcription factors, and oncogenes.5,7 Using these molecules as biomarkers to assess how expression of hLAL in macrophages corrects aberrant gene expression in the lung, liver, and intestine, mRNA expression levels were examined by RT-PCR. As demonstrated in Figure 10A , Api6, MMP-9, MMP-12, MafB, and Spi-C were highly overexpressed in the lung, liver, and intestine of c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice, in a manner similar to single lalC/C mice.7 Expression of hLAL by doxycycline treatment in macrophages significantly reduced the expression levels of these molecules in triple lalC/C mice. This indicates that these molecules contribute to pathogenic progression in multiple organs during LAL deficiency. A group of cytokines and chemokines were also up-regulated in lungs of lalC/C mice as we previously reported.5 A set of cytokines/chemokines in this group (TNF-, CCL7, and CXCL1) showed significant reduction in triple lalC/C mice after doxycycline treatment in the lung, liver, and intestine. Another set of cytokines/chemokines in this group (IL-1ß and IL-6) only showed changes in the lung but not in the intestine and liver, after doxycycline treatment (Figure 10B) . This observation suggests that pathogenesis in different organs share common genes and pathways with certain distinctions during LAL deficiency.

Figure 10. Macrophage-specific expression of hLAL reduced aberrant gene expression in c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. A: RT-PCR analysis of Api-6, MMP-9, MMP-12, Spi-C, MafB, and GAPDH in lung, intestine, and liver of wild-type (lane 1), untreated (lane 2), and doxycycline-treated (lane 3) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. B: RT-PCR analysis of TNF-, IL-1ß, IL-6, CCL7, and CXCL1 in lung, intestine, and liver of wild-type (lane 1), untreated (lane 2), and doxycycline-treated (lane 3) c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice.

It is intriguing that many seemingly unrelated disease phenotypes of various organs coexist in lalC/C mice. Some common cellular and molecular mechanisms must exist to link these pathogenic processes together. Based on previous observations, abnormal macrophages were observed in multiple organs in lalC/C mice, including the liver, intestine, and lung.4-8 Therefore, macrophages play a central role in pathogenic progression during LAL deficiency. Aberrant expression levels of multiple inflammatory cytokines and chemokines were monitored in association with lalC/C macrophages (Table 1) . These inflammatory molecules can change the local microenvironment by binding to the residential cells to affect gene expression (Figure 10) and cause pathogenic phenotypes in various organs. Establishment of an in vivo macrophage-specific controllable system is essential to elucidate and dissect molecular, cellular, and physiological mechanisms in lalC/C mice. In this report, we generated a c-fms 7.2-kb promoter/intron2-rtTA transgenic line, in which rtTA expression is restricted to macrophages as previously reported.3 In the c-fms 7.2-kb promoter/intron2-directed GFP transgenic system, GFP has been detected in Kupffer cells of the liver, lamina propria of the small intestine, and bronchio-alveolar macrophages of the lung. This expression pattern overlaps with abnormal macrophage malformation in lalC/C mice as determined by Oil Red-O staining of neutral lipids.4-6,8 Macrophages in these areas appear foamy and lipid filled. Therefore, the c-fms 7.2-kb promoter/intron2 DNA sequence is ideal for directing LAL expression in vivo to correct macrophage-mediated pathogenic phenotypes in lalC/C mice. After crossbreeding these mice with the (tetO)7-CMV-hLAL transgenic mouse line, the induced expression level of hLAL was readily detectable by RT-PCR in macrophages and in bone marrow cells (Figure 2D) , as well as in liver, intestine, and lung tissues that contain macrophages (Figure 2E) . The LAL enzymatic activity was restored with correction of neutral lipid metabolism (Figure 5) .

After crossbreeding c-fms-rtTA/(tetO)7-CMV-hLAL double transgenic mice with lalC/C mice, expression of hLAL in lalC/C macrophages by doxycyline treatment significantly reduced neutral lipid accumulation in the liver and intestine as assessed by Oil Red-O staining (Figures 3 and 4) in association with attenuated inflammatory neutrophil infiltration. Neutrophil infiltration has been observed in the liver, intestine, and lung of lalC/C mice (Figure 6) , suggesting that inflammation is a major trigger for pathogenic progression in various lalC/C organs. In general, inflammation starts with stimulation of vascular endothelial cells by pro-inflammatory cytokines and chemokines generated by either regional macrophages or residential cells.9 Cytokines and chemokines regulate leukocyte trafficking, homing, and migration. The process begins with activating intracellular signaling cascades in endothelial cells, which influences expression of an array of inflammation-associated genes, including a variety of adhesion molecules. Increased expression of adhesion molecules facilitates migration and adhesion of phagocytic neutrophils to vascular endothelium at vessel sites adjacent to inflammatory sites. Neutrophils migrate through vessel walls to infiltrate inflammatory tissues. Activated neutrophils produce both free radicals (eg, superoxide) and proteinases (eg, neutrophil elastase and MMPs) causing tissue damage. Correction of neutrophil infiltration and pathogeneses in multiple organs by restoring LAL production in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice suggests that macrophages play an important role in controlling inflammation-triggered pathogenesis through production and secretion of cytokines and chemokines. This has been confirmed by RT-PCR analysis in the lung, liver, and intestine (Figure 10B) . Although migrating macrophages are a major source and site for production of cytokines and chemokines, partial correction of pathogenic phenotypes in c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice suggests that other residential cell types in these organs also contribute to aberrant production of inflammatory cytokines and chemokines leading to tissue pathogenesis. This has been confirmed by the observation that TNF- and IL-6 were up-regulated in the lung but not in macrophages (Table 1) . Interactions between macrophages and residential cells are important for tissue pathogenic development in lalC/C mice.

Based on previous and current observations, it appears that neutral lipid metabolism is essential for controlling malformation and malfunction of macrophages caused by aberrant gene expression in lalC/C mice. Deficiency of LAL in macrophages blocks the normal synthesis of various hormonal ligands for nuclear receptors such as PPAR and other transcription factors such as nuclear factor E2 p45-related factor 2 (Nrf2).10-13 Treatment with PPAR ligands significantly attenuated lalC/C pulmonary inflammation and aberrant gene expression in the lungs.7 In addition to PPAR, there is emerging evidence indicating that other lipid mediators such as Nrf2 contribute to cyclooxygenase product function in inflammation.13 Expression of hLAL in macrophages of c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice restored the production of ligands for nuclear receptors and lipid-mediating transcription factors that suppress synthesis and secretion of pro-inflammatory cytokines and chemokines. As a consequence, pathogenic phenotypes were ameliorated in various lalC/C organs. Recently, biochemical pathways were identified that use polyunsaturated fatty acids to generate resolvins and protectins that can expedite inflammatory resolution.14 These mediators might be regulated and enhanced by hLAL. Expression of hLAL in macrophages may restore these pathways.

In addition to the cytokines, chemokines, and lipid-mediated nuclear receptors/transcription factors mentioned above, other common molecules also contribute to lalC/C pathogenic processes in various organs. While studying the relationship between aberrant gene expression and pathogenic progression in the lalC/C lung, a set of genes was identified with aberrant expression, including Api6, MMP-9, MMP-12, MafB, and Spi-C.7 To see whether aberrant expression of these genes occurs in other organs, RT-PCR analysis was performed. Interestingly, Api6, MMP-9, MMP-12, MafB, and Spi-C were all up-regulated in the liver and intestine in addition to the lung. In c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice, doxycycline treatment significantly reduced expression of these molecules in all tested organs (Figure 10A) . Api6 has been shown to be an apoptotic inhibitor with a molecular weight of 37 kd (331 amino acids) and belongs to the scavenger receptor cysteine-rich superfamily.15,16 Transcription factor MafB belongs to the maf proto-oncogene family and is expressed in a wide variety of tissues and encodes a 311-amino acid protein containing a typical bZip motif in its carboxy-terminal region.17,18 Spi-C belongs to the Ets family of transcription factors.19 Ets proteins comprise a large family of transcription factors involved in a variety of cellular processes. The MMPs are a group of zinc-dependent endopeptidases including collagenases, gelatinases, and stromelysins.20 These molecules cause a profound impact on tissue structure by degrading components of the extracellular matrix.21,22 Although it is unclear how the LAL activity in macrophages affects pathogenic expression of these molecules at this time, it is obvious that these molecules are crucial to the pathogenic progress in various lalC/C organs. Study of these molecules will provide significant insight into the molecular mechanisms and linkages between various pathogenic phenotypes in lalC/C mice.

Figure 4. Macrophage-specific expression of hLAL reduced neutral lipid accumulation in the intestine of c-fms-rtTA/(tetO)7-CMV-LAL;lalC/C triple mice. Frozen sections of small intestine were stained with Oil Red-O. Massive neutral lipid accumulation (red color) was detected in the lamina propria and the base of villi in the intestine of lalC/C (B) and c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice (C) without doxycycline treatment as indicated by asterisks. D: Doxycycline treatment significantly reduced Oil Red-O staining in the c-fms-rtTA/(tetO)7-CMV-hLAL;lalC/C triple mice. Wild-type lal+/+ mice (A) were the negative control for Oil Red-O staining.

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作者单位:From The Center for Immunobiology,* Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana; the Division of Human Genetics, Cincinnati Children??s Hospital Medical Center, Cincinnati, Ohio; and the Institute for Molecular Bioscience, University

日期:2008年5月29日 - 来自[2006年第168卷第9期]栏目

Lysosomal Cysteine Proteases in Atherosclerosis

From the Department of Molecular and Cell Biology (J.L., J.-S.S., W.-H.X.), School of Life Science, University of Science and Technology of China, Hefei, Anhui, China; and Donald W. Reynolds Cardiovascular Clinical Research Center (G.K.S., P.L., G.-P.S.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.

ABSTRACT

Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S–deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor–null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.

Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis.

Key Words: atherosclerosis ? cysteine proteases ? cathepsins ? cystatin ? cytokines

Introduction

The extracellular matrix (ECM) of the vascular wall, largely elastin and collagen, subserves many functions essential for vessel homeostasis. These macromolecules serve as an adhesive substrate for vascular endothelial cells (ECs) and smooth muscle cells (SMCs), furnish survival signals to resident cells, bind and retain lipoproteins, and provide a reservoir for growth factors.1 Elastin and collagen also contribute to the strength, resiliency, and structural integrity of the vascular wall.1,2 Normal tissues exhibit strict regulation of the expression and turnover of ECM. However, ECM damage or remodeling in conditions such as rheumatoid arthritis,3 malignant tumors,4 aortic aneurysm,5,6 and atherosclerosis7,8 contributes to the formation, progression, and clinical expressions of these disorders.

Atherosclerosis is an inflammatory disease whose complex developments encompass 3 main stages: initiation, progression, and complication.8 Throughout these stages, several pathologic events involve proteolysis, including initial translocations of mononuclear leukocytes from the vascular lumen through the basement membrane into the subendothelium; progressive migration of SMCs through the elastic laminas from the media into the intima; and finally, disruption of the arterial wall—outward in aneurysmal disease and luminally in athero-occlusive disease.

Many studies have documented augmented elastase, collagenase, and gelatinase activities within atherosclerotic lesions. Among the responsible proteolytic enzymes, matrix metalloproteinases (MMPs) and serine proteases have garnered the most attention.9–15 Increased expression of MMP-1, -2, -3, -7, -8, -9, and -13 and of metalloelastase-12 (MMP-12) occurs in macrophages bordering the lipid core adjacent to the fibrous cap and in macrophages and SMCs in the shoulder regions of well-developed atherosclerotic plaques, sites prone to rupture.7,12,13,16 Studies in animals have permitted assessment of the contribution of these proteases to arterial remodeling. Administration of small-molecule MMP inhibitors delayed SMC migration and intima formation in injured rat arteries.17 Overexpression of tissue inhibitors of MMPs (TIMPs) reduced atheroma progression18 or intimal thickening in veins.19 However, introduction of an MMP-1 transgene driven by a macrophage promoter into atherosclerosis-prone, apolipoprotein E (Apo E)–deficient mice surprisingly reduced atherosclerotic lesions20 for reasons that remain uncertain. Human atherosclerotic lesions overexpress both urokinase- and tissue-type plasminogen activators.21,22 Deficiency of urokinase-type plasminogen activator protected against medial elastic destruction and atherosclerotic aneurysm formation in Apo E–null mice, probably via impaired plasmin-mediated elastolytic MMP activation.23 In contrast, transgenic expression of rabbit urokinase-type plasminogen activator enlarged neointimal lesions and caused degradation of elastic laminas in cholesterol-fed rabbits.24 However, plasminogen deficiency accentuated atherogenesis in atherosclerosis-prone, Apo E–null mice.25 Recent findings have described expression of neutrophil elastase, a serine proteinase, in the macrophage-rich shoulders of human atherosclerotic lesions and its regulated secretion from monocytes by the inflammatory mediator CD40 ligand.26 In addition, serine proteases can activate MMPs secreted from SMCs and degrade fibrin within advanced plaques.27,28 Thus, different families of proteolytic enzymes may participate in atherogenesis.

Cathepsins of the cysteine protease family localize in lysosomes and endosomes and function there to degrade unwanted intracellular or endocytosed proteins. In the early 1990s, we recognized few of these proteases and knew little of their physiologic or pathologic functions. More recently, modern molecular biology permitted the characterization of a series of novel cathepsins, many widely expressed in humans and animals. However, the recognition of inducible cathepsins such as S, K, C, V, and W led to the unraveling of their roles in inflammatory and/or autoimmune diseases such as cancer, rheumatoid arthritis, osteoporosis, hyperkeratosis/periodontitis, and atherosclerosis.29 Most strikingly, we now recognize that these lysosomal cysteine proteases can function outside lysosomes or endosomes, although these intracellular organelles still contain the bulk of these enzymes. Detection of active cysteine proteases in culture media of SMCs, ECs, and macrophages significantly broadened our understanding of their potential roles in arterial pathobiology. This review will discuss some of the recent findings in this field and highlight the significance of lysosomal cysteine proteases in arterial ECM remodeling and atherogenesis.

Regulated Expression of Cysteine Proteases and Their Endogenous Inhibitor, Cystatin C

Overexpression of Lysosomal Cysteine Proteases and Deficiency of Cystatin C in Atherosclerosis

Atherosclerotic lesions contain much more cathepsin S and K mRNAs and proteins than do normal arteries. Either protease can degrade elastin and collagen.29,30 Cathepsin K, one of the most potent mammalian collagenases, participates in joint and bone collagen metabolism. Deficiency of this protease impairs bone growth in both humans and animals.31,32 Immunohistochemical studies demonstrated expression of cathepsins S and K mainly in macrophages in the shoulder regions of atheromata, in SMCs of the fibrous cap, and at sites of internal elastic laminae fragmentation.33 These findings suggest involvement of these proteases in degradation of elastic laminae in the vascular wall, which may facilitate SMC migration, and destabilization of atherosclerotic plaque by degrading collagen of the fibrous caps.34 Also, the ECs lining the lumen of the vessel (not shown) as well as those in the plaque microvessels (Figure 1) express cathepsin S, indicating a role for this protease in neovascularization, a process implicated in plaque growth and complication.35 In contrast, healthy human aortae contain no immunoreactive cathepsin S or K, although Northern blot analyses of healthy aortas have shown low levels of cathepsin K mRNA.33 Western blot analysis and elastase assay of tissue extracts from human atherosclerotic lesions demonstrated increased levels of active forms of cathepsins S and K with significantly elevated elastolytic activity, sensitive to the cysteine protease inhibitor E64d or the cathepsin S selective inhibitor morpholinurea leucine-homophenylalanine-vinylsulfone-phenyl (LHVS),33 suggesting an important contribution of cysteine proteases in arterial elastic tissue remodeling.

Figure 1. Colocalization of cathepsin S and cystatin C with microvascular endothelium. Frozen sections of human carotid lesions were double immunostained with rabbit anti-human cathepsin S (1:300)33 or cystatin C (1:1500, Vortex) polyclonal antibodies followed by biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, Calif). After application of the avidin/biotin blocking kit, anti-CD31 monoclonal antibody (1:30, Dako) for ECs was added. Subsequently, biotinylated horse anti-mouse secondary antibody was applied, followed by streptavidin-fluorescein isothiocyanate (FITC, Amersham). Nuclei were counterstained with 0.5 μg/mL bis-benzimide H33258 (Calbiochem) in PBS. Both cathepsin S (red, left; x400) and cystatin C (red, right; x400) colocalized with endothelial marker CD31 (green). Orange color on the top panels indicates microvascular ECs expressing both antigens. Lumen of the microvessel is indicated.

Atherosclerotic lesions in Apo E–deficient mice showed a similar increase in expression of lysosomal cathepsins S, L, and B.36 As described in human atherosclerotic lesions,33 cathepsin S localized mainly in intimal SMCs and macrophages and medial SMCs. Nonatherosclerotic arteries showed no expression of cathepsin S.36 This expression pattern affirms an association of these proteases with atherogenesis in humans. Consistent with this hypothesis, human atherosclerotic lesions have relatively low levels of cystatin C, an endogenous inhibitor of these cathepsins, whereas normal arteries express abundant cystatin C in medial SMCs and ECs.37 This expression profile suggests an imbalance between cysteine proteases and their inhibitors that favors arterial ECM breakdown. Similar loss of this counterbalance was observed in atherosclerotic lesions from Apo E–deficient mice. In mouse aortic atherosclerotic lesions, immunostaining for cystatin C was also much less intense than that in normal mouse aortas (Sukhova and Shi, unpublished data). Such inverse regulation of vascular proteases and protease inhibitors appears much less prominent in the case of MMPs and their tissue inhibitors (TIMPs). In human atherosclerotic lesions, TIMP expression changes little or increases compared with that in control vessels.38–40 Murine atherosclerotic lesion development showed similar expression patterns.36 Increased MMP-9 expression in Apo E–deficient, murine atherosclerotic lesions accompanies increased TIMP-1 levels, whereas expression of cysteine proteases and their inhibitor cystatin C remains inversely regulated.

Expression and Secretion of Lysosomal Cysteine Proteases and Cystatin C In Vitro

Macrophages, SMCs, and ECs account for most of the cysteine protease expression in human atherosclerotic arteries. To study the regulation of cysteine protease expression in these cells, we isolated human monocytes, vascular SMCs, and vascular ECs and then examined cysteine protease expression under different conditions. Although human blood monocytes express negligible levels of cathepsin K, maturation of monocytes into macrophages during incubation in 40% fetal calf serum markedly augments cathepsin K expression and elastolytic activity.41 These human monocyte-derived macrophages also express and secrete cathepsins S, L, and B.42,43 Similar to medial SMCs in the normal vessel wall, cultured SMCs do not express cathepsin S or K under basal conditions. However, incubation of these cells with inflammatory cytokines such as interleukin-1? (10 ng/mL), tumor necrosis factor- (10 ng/mL), and interferon (IFN)- (400 U/mL) for 24 hours significantly induces the expression and secretion of cathepsins S and K at both the mRNA and protein levels.33 Furthermore, increased cathepsin expression occurs in tandem with enhanced elastase activities, largely sensitive to E64, establishing a predominant contribution to the elastolytic activity of cysteine proteases in inflamed SMCs. More important, culture media from IFN-–stimulated SMCs contained active cathepsin S detected by both active-site labeling of de novo synthesized enzymes and by degradation of water-insoluble elastin.33 These findings indicate release of cathepsin S from SMCs and interaction with ECM proteins, a process that likely occurs in atherosclerotic lesion development during SMC migration and further neointima formation.

In contrast, cystatin C, the endogenous inhibitor of the cysteine proteases, shows distinct regulation by different cytokines.37 For example, IFN- or interleukin-1? increases cathepsin S expression 10- to 20-fold in SMCs but does not affect cystatin C expression. Tumor necrosis factor- induces cathepsin S expression in SMCs but reduces cystatin C expression in vascular ECs.37 We obtained similar results with the angiogenic stimulus basic fibroblast growth factor, which augments cathepsin S expression by >10-fold in ECs but also reduces cystatin C expression.35 Grainger et al44 detected significantly lower blood levels of transforming growth factor-?1 in patients with atherosclerosis, but transforming growth factor-?1 increases SMC cystatin C secretion by a posttranscriptional mechanism.37 The Table lists the effects of various cytokines and angiogenic factors on cathepsin S and cystatin C expression and secretion in these cells. These observations indicate that some inflammatory factors differentially regulate the expression and secretion of lysosomal cysteine proteases and their endogenous inhibitors in arterial cells.

Effect of Cytokines and Angiogenic Factors on Cathepsin S and Cystatin C Expression and Secretion

Proteolytic Activities of Extracellular Lysosomal Cysteine Proteases

Lysosomal cysteine proteases are synthesized and targeted to the acidic compartments, lysosomes and endosomes, via either the mannose-6-phosphate receptor–dependent pathway45,46 or the mannose-6-phosphate receptor–independent pathway,47 where they are activated to degrade their substrates. These compartments provide cysteine protease cathepsins with the optimal pH for their activity.29 Although cathepsin cDNA sequences do not appear to encode a conventional secretory signal peptide,48 several groups including our own have demonstrated the existence and activity (collagenolytic and/or elastolytic) of these proteases in media conditioned by SMCs, ECs, and monocyte-derived macrophages.33,37,42,43 Therefore, several questions remain. These include how these proteases are released and how they retain their activity once they have left the cells, because most of these proteases have a very narrow pH optimum (pH 4 to 6). For instance, cathepsins K and L lose their activity at neutral pH.49 Although cathepsin S does exceptionally retain some activity at neutral pH,50 it is questionable whether this partial activity explains all of the cysteine protease–dependent ECM degradation observed in vitro. Hence, in the recent proposal of a "focal contact" hypothesis, Punturieri et al43 used human monocyte-derived macrophages to demonstrate the formation of a localized acidic environment in a zone of contact that excludes the surrounding extracellular milieux. Such focal contact permits lysosomal or endosomal cysteine proteases to degrade extracellular elastin efficiently. After incubating cultures for 24 hours, they detected an acidic interface between the cell surface and elastin filaments by use of a fluorescent pH indicator. Punturieri and colleagues further demonstrated by use of bafilomycin, an inhibitor of acidification of both intracellular and extracellular compartments that inhibited monocyte-derived macrophage elastase activity, that these extracellular acidic milieux resulted from increased expression of vacuole-type H+-ATPase. These findings provide a reasonable explanation for ECM degradation by cysteine proteases released from macrophages.

Monocyte adhesion, migration, and differentiation into macrophages play essential roles in the pathogenesis of the atherosclerotic lesion. These cells may also use extracellular cysteine proteases to assist their migration during atherogenesis. We recently demonstrated in vitro that cathepsin S–deficient monocytes cannot migrate through artificial membranes containing SMCs, collagen mixtures, and an endothelial monolayer.51 This finding suggests a critical involvement of cysteine proteases in blood-borne cell transmigration from peripheral blood into the vessel wall. Therefore, it is reasonable to hypothesize that these monocytes/macrophages may degrade ECM via the pathways of vacuole-type H+-ATPase expression, extracellular acidic milieu formation, and extracellular collagen degradation. Validation of this hypothesis will require further investigation.

It is therefore conceivable that SMCs and ECs use the same focal contact mechanism to dissolve ECM as do macrophages.33,35 Medial SMCs contain immunoreactive cathepsins S, K, and L in atherosclerotic human and animal vessel walls, notably near sites of internal elastic lamina fragmentation33,51 (see below). These SMCs likely also form an extracellular acidic microenvironment that allows extracellular cathepsin S and possibly other elastolytic cathepsins (eg, K and L) to break down the elastic barriers. Several lines of evidence support this hypothesis. Stimulation of cultured human or murine SMCs with IFN- induced expression of elastolytic cathepsins and increased extracellular degradation of water-insoluble elastin. The cathepsin inhibitor E64d blocks much of this activity.33,51 Furthermore, SMCs lacking cathepsin S showed a significantly reduced ability to degrade extracellular elastic fibers. Although we do not have direct evidence of formation of a cell-substrate acidic interface, we observed attachment of SMCs to exogenous water-insoluble elastin fibers in culture (Figure 2). Nevertheless, it remains to be confirmed whether SMCs act as monocyte-derived macrophages do and form acidic focal contact compartments that facilitate degradation of extracellular elastin in culture and within human and animal vascular walls.

Figure 2. Focal contact of SMCs and elastin fibers. Human saphenous vein SMCs were cultured in monolayers. Water-insoluble elastin fibers were seeded on top of the cell monolayer (left). By 24 hours of culture, SMCs had formed direct focal contacts with elastin fibers (right).

Lysosomal Cysteine Proteases and Atherogenesis

Increased expression of cysteine proteases cathepsins S, K, B, H, and L and decreased expression of their endogenous inhibitor cystatin C in human atherosclerotic lesions suggested the involvement of cysteine proteases in atherogenesis. Recent findings from in vitro and in vivo experiments indicate that these cysteine proteases may participate in the main stages of atherogenesis.

Initiation

Atherogenesis initially involves leukocyte recruitment from the circulation by adhesion to the endothelium, followed by penetration through the endothelial layer and arterial basement membrane.8 Current understanding implicates specific adhesion molecules expressed on the surface of vascular ECs, eg, vascular cell adhesion molecule-1 (VCAM-1), and chemoattractant molecules, such as macrophage chemoattractant protein-1 (MCP-1), in this process.8 Deficiency or impaired function of these molecules significantly reduces atherogenesis in animals.52–54 We currently do not know whether cysteine proteases play any role in regulating MCP-1 or VCAM-1 expression or leukocyte adhesion. Data from studies of cathepsin S–knockout mice illustrated a significant reduction of these molecules in sera of mice initiated for atherosclerosis with a high-cholesterol diet.51 Therefore, cathepsin S may act like MMPs and release adhesion molecules from the surface of ECs.55 Alternatively, cathepsin S may indirectly influence the production of adhesion molecules by affecting the -T lymphocyte population,56 another possible source of VCAM production.57 On the other hand, the reduced levels of MCP-1 and VCAM-1 observed in cathepsin S–deficient mice may result from lower serum lipid levels58 or decreased lesional monocyte content59 and may not directly depend on cathepsin S activity.

After adhesion and transmigration through the endothelial layer and basement membrane, monocytes become macrophages, proliferate, and become lipid-laden foam cells. The arterial subendothelial basement membrane contains type IV collagen, laminin, and fibronectin.60,61 Migration of blood-borne leukocytes requires degradation of these ECM components, as does microvessel ingrowth from vasa vasorum of the adventitia and tunica media.62,63 Macrophages derived from human monocytes express and secrete substantial amounts of active cathepsins K, L, and S, which can degrade these subendothelial basement membrane components.64–67 The mononuclear cells may well use these proteolytic enzymes to aid their transmigration. Indeed, the subendothelial basement membrane in early atherosclerotic lesions (fatty streaks) contains large amounts of immunoreactive cathepsins S and K.33 To test for a role for cysteine proteases in monocyte transmigration, we recently used an in vitro preparation comprising layers of SMCs and a mixture of collagen types I and IV overlaid by a monolayer of ECs.51 Human monocytes seeded on top of this structure migrated through the collagen matrix into the SMC layer, where they matured into macrophages. Interestingly, treatment of human monocytes with a low concentration (8 nmol/L) of LHVS (a condition that selectively inhibits cathepsin S) or a high concentration (1 μmol/L) of LHVS (that also inhibits other cysteine proteases) significantly reduced monocyte translocation (Shi et al, unpublished data). Cathepsin S–null monocytes yielded similar results.51 More than 90% of cathepsin S–null monocytes remained atop the endothelial monolayer, whereas the majority of wild-type monocytes migrated into the SMC layer. These observations suggest involvement of cathepsin S and possibly other cysteine proteases in leukocyte transmigration.

Progression

During the progression of atherosclerosis, lipid-laden SMCs and macrophages accumulate along with T lymphocytes. These cells release inflammatory cytokines, and SMCs in particular elaborate ECM constituents. In addition to the transmigration of leukocytes from the lumen, SMC migration from the media also occurs. Medial SMC migration and neointimal formation are part of the main pathologic events during progression of atherosclerosis. This SMC migration requires traversal of the internal elastic laminas. Although the elastic laminae may contain fenestrae, dissolution of these elastic barriers may aid the migration of SMCs to the intima. In vitro experiments demonstrated that elastin peptides in a modified Boyden chamber halted aortic SMC migration.68 Furthermore, in SMCs, intact elastin regulates actin stress fiber organization, proliferation, and migration via a heterotrimeric G protein–coupled pathway.69 Thus, understanding atherogenesis requires identification of the proteases that mediate elastolysis. The ability of human monocyte-derived macrophages to release elastolytic cysteine proteases led us to hypothesize that SMCs may also use cysteine proteases for their migration through arterial elastin. We validated this hypothesis by showing reduced elastolytic activity in experiments in vitro with cysteine protease inhibitors with human SMCs and aortic SMCs isolated from cathepsin S–deficient mice.33,51 In vivo experiments further supported this hypothesis: studies of atherosclerosis-prone LDL receptor–deficient mice that consumed an atherogenic diet for 8, 12, and 26 weeks showed that SMCs at sites of elastin fragmentation expressed cathepsin S similar to those in human lesions. Mice lacking both the LDL receptor and cathepsin S on the same diet showed substantially less fragmentation of the arterial internal elastic lamina, reduced intimal SMC accumulation and collagen production, and significantly smaller atherosclerotic lesions. Indirect evidence comes from recent observations of Apo E–deficient mice. In these atherosclerosis-prone mice, deficiency of the endogenous cysteine protease inhibitor cystatin C increased aortic elastolysis mediated by SMC cysteine protease and consequently enhanced medial degradation of elastic laminae (Shi and Sukhova, manuscript in preparation).

Oxidized LDL (oxLDL) may favor formation of foam cells and necrotic or apoptotic lipid cores, hallmarks of both human and murine atherogenesis. Several experiments have implicated cysteine proteases in these pathologic processes. The lysosomal cysteine proteases cathepsins B and L may process caspases and induce apoptosis70–72 and localize in the cytoplasm and nuclei of apoptotic (caspase-3–positive) macrophages in human atheromata.73 Inhibition of cathepsins B and L with E64 or Z-Phe-Arg-FMK protects mononuclear cells from oxysterol-induced cell death. Thus, cytotoxic oxLDL and associated oxysterols in atheromata may cause lysosomal destabilization and release of cathepsins, fostering the apoptotic death of phagocytic cells and contributing to further atherosclerotic lesion evolution, including lipid core formation.73,74 This proapoptotic mechanism may also apply to other cells in atheromata, such as SMCs and ECs. In cathepsin S–deficient, LDL receptor–null atherosclerotic mice, we detected not only significantly less accumulation of lipid in lesions and lower serum levels of LDL or total cholesterol51 but also lower titers of autoantibody to both malondialdehyde-oxLDL and copper-oxLDL epitopes (Shi et al, unpublished data). Because some proteases have been linked to major histocompatibility class II–associated antigen processing and presentation, cathepsin S, and possibly other cathepsins, may participate in the adaptive humoral and cellular immune responses that operate during atherosclerosis.75–77

Neovascularization provides a portal for leukocyte trafficking6 and entry of plasma constituents, including lipoproteins, and occurs during progression of atherosclerotic lesions.78 This process requires lysis of the ECM to pave the way for neovessel formation.79 Although the microvessels of various human malignant tumors express cysteine proteases, direct evidence for involvement of these enzymes in neovascularization emerged only recently from studies of cathepsin S–deficient mice.35 Healing skin wounds in cathepsin S–null mice contained far fewer microvessels. Defining the contribution of impaired neovascularization due to loss of cathepsin S activity to reduced atherosclerosis in cathepsin S–null mice is difficult. However, in human atherosclerotic lesions, areas of microvascularization contain both cathepsin S and its inhibitor cystatin C (Figure 1), in contrast to other regions of plaque that contain less cystatin C.37 Therefore, cathepsin S and possibly other cysteine proteases may also regulate the growth of lesion microvessels during atherogenesis.

Complications

Thrombotic complications of atherosclerosis often involve plaque rupture. Lesions that have ruptured typically have a thin fibrous cap, prominent lipid deposition, and macrophage accumulation. Such thrombotic complications actually cause most of the acute manifestations of atherosclerosis, such as myocardial infarction or stroke.7,8 Indeed, thrombus formation usually results from physical disruption of atherosclerotic plaque and appears related to the level of collagen in the lesion’s fibrous cap. Therefore, the collagen content of the fibrous cap critically influences plaque stability and may depend in turn on the expression of interstitial collagen-degrading proteases.

Vulnerable human atherosclerotic plaques overexpress several MMP collagenases capable of degrading interstitial collagen types I and III.79 For example, macrophages in plaques contain abundant MMP-1, -8, and -13 and colocalize with sites of collagen degradation in situ. Macrophages appear to furnish the bulk of cysteine proteases in atheromata. Indeed, Chen et al80 used near-infrared imaging to detect cathepsin B activity in vivo in atheromatous lesions of Apo E–deficient mice. Increased expression of cathepsin B in atheromatous plaques colocalized with the macrophage marker Mac-3. We made similar observations in sections of human atherosclerotic lesions with cathepsins S, K, and L, all of which can degrade collagen.29,30 Cathepsins S and K were originally isolated from human macrophages, and inflammatory cytokines increase cathepsin S secretion from macrophages (the Table).81 In human atherosclerotic plaques, macrophages and fibrous cap SMCs express all 3 collagenolytic cathepsins33 (Shi, unpublished data). However, the possible involvement of cysteine proteases in plaque rupture requires further examination. Fibrous cap thickness is directly associated with plaque vulnerability. Cathepsin S/LDL receptor double-deficient mice have decreased SMC and collagen contents in the lesions and a reduction in the size of the fibrous cap compared with those in control LDL receptor–null mice. Such a reduction in fibrous cap size could be the result of a decrease in the number of SMCs in lesions, which may increase plaque vulnerability.34 Cystatin C/Apo E double-deficient mice consistently had increased lesional SMC and collagen contents and better developed fibrous caps (Sukhova et al, unpublished data), characteristics of stable plaques in humans. Thus, cysteine proteases could play dual roles. Medial SMCs release cathepsins to allow them to pass through the elastica and accumulate in the neointima, where they produce collagens and reinforce the fibrous caps.34 On the other hand, SMCs in the fibrous cap can also produce cathepsins,33 which are collagenolytic.29,30 These cathepsins may cause weakening of the fibrous cap and lead to plaque rupture, although this conjecture requires experimental validation.

To date, no direct experiment has tested for a role of cysteine proteases in thrombosis during atherogenic complications. Data from our laboratory and collaborators indicate that cathepsin S regulates thrombotic responses to arterial injury.82 After photochemical carotid artery injury, the time to the development of occlusive thrombosis decreased in cathepsin S–null mice. The accelerated thrombotic response to arterial injury and the shortening of plasma clotting times accompanied an increased activity of coagulation factors VIII, IX, and X and plasma von Willebrand factor, as measured by 1-stage clotting-based assays in cathepsin S–deficient mice. These results suggest that cathepsin S has antithrombotic properties. However, the mechanism by which cathepsin S affects thrombosis and whether its antithrombotic properties impact atherogenesis remain undetermined.

Conclusions

Numerous observations now support key functions of cysteine protease cathepsins in atherogenesis (Figure 3). Cathepsins may pave the way for entry of blood-borne monocytes and lymphocytes into the arterial vessel wall by degrading the subendothelial basement membrane and for the migration of SMCs through elastic laminae to enter the intima. Several cell types, including medial SMCs, neointimal SMCs, macrophages, and neointimal microvascular ECs, express cathepsins in atheromatous lesions. Exposure to inflammatory cytokines or growth factors released by transmigrated monocytes, T lymphocytes, or ECM degradation products may enhance the expression or release of cathepsins in these cells (Figure 3 and the Table), a hypothesis supported by observations from studies of cultured cells. Thus, the inflammatory process at the core of atherogenesis is linked tightly with proteolysis due to cathepsins. Furthermore, cathepsin function in thrombosis82 or even immunity76,83 also may contribute to the pathogenesis of atherosclerosis, although we lack direct evidence for these functions. Several recent reports related primarily to the remodeling of the ECM do establish an important role for cysteine proteases in atherogenesis in genetically altered mice. We therefore propose that multiple proteases, including MMPs and serine proteases, work in concert during the initiation, progression, and complication of atherosclerotic plaques.

Figure 3. Potential roles of cysteine protease cathepsins in atherogenesis. The diagram depicts a cross section of an atherosclerotic lesion. Immunohistologic analysis demonstrated localization of cysteine protease cathepsins in the subendothelium (regulating blood-borne leukocyte transmigration), in SMCs in the fibrous cap (influencing plaque stability), at sites of internal elastic lamina fragmentation (controlling SMC migration), in macrophages and foam cells at the lipid core (degrading matrix proteins and affecting cell apoptosis), and in ECs of the vessel wall and microvessels (involving neovascularization). Inflammatory cytokines released from infiltrated lymphocytes, growth factors from degradation of matrix protein (eg, collagens), or even oxLDL may regulate cathepsin expression during atherogenesis. Both macrophages and SMCs take up modified LDL and become foam cells, which become the major components of the lipid core.

Acknowledgments

This work was funded in part by grants to G.-P.S. from the National Heart, Lung, and Blood Institute (HL 60942, HL67283) and the American Heart Association (0355130Y), to G.K.S. from the National Heart, Lung, and Blood Institute (HL 67249), and to P.L. from the National Heart, Lung, and Blood Institute (HL-56985), and by a grant from the Donald W. Reynolds Foundation. We also thank Karen Williams for her editorial expertise.

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日期:2007年5月18日 - 来自[2004年第24卷第8期]栏目

Reduction of Atherosclerotic Plaques by Lysosomal Acid Lipase Supplementation

From the Division and Program in Human Genetics (H.D., G.A.G.) and Division of Pathology and Laboratory Medicine (D.P.W.), Children’s Hospital Research Foundation, Cincinnati, OH, 45229; and Genzyme Corporation (S.S., N.W., M.L.), Cambridge, MA.

Correspondence to Gregory A. Grabowski, MD, Professor and Director, Division and Program in Human Genetics, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail greg.grabowski@cchmc.org

    Abstract

Objective— Proof of principle is presented for targeted enzyme supplementation by using lysosomal acid lipase to decrease aortic and coronary wall lipid accumulation in a mouse model of atherosclerosis.

Methods and Results— Mice with LDL receptor deficiency were placed on an atherogenic diet and developed predictable aortic and coronary atheroma. -Mannosyl-terminated human lysosomal acid lipase (phLAL) was produced in Pichia pastoris, purified, and administered intravenously to such mice with either early or late lesions. phLAL injections reduced plasma, hepatic, and splenic cholesteryl esters and triglycerides in affected mice. phLAL was detected in hepatic Kupffer cells and in atheromatous foam cells. Repeated enzyme injections were well tolerated, with no obvious adverse effects. In addition, the coronary and aortic atheromatous lesions were (1) eliminated in their early stages and (2) quantitatively and qualitatively reduced in their advanced stages.

Conclusion— These results support the potential utility of lysosomal acid lipase supplementation for the treatment of atherosclerosis, a leading cause of mortality and morbidity in Westernized nations.

Key Words: lysosomal acid lipase ? atherosclerosis ? lesion ? mice

    Introduction

Atherosclerosis is the number 1 cause of mortality and morbidity in the developed countries. A number of interventions or preventions delay the consequences of atherosclerosis, eg, low cholesterol diet and exercise, HMG-CoA reductase inhibitors, and coronary artery bypass. However, they are not suitable for all patients, and few have been shown to promote regression of lesions. Therefore, new approaches are needed for the treatment and prevention of atherosclerosis. Several stages characterize the progression of atherosclerotic lesions.1,2 The earliest lesion is the "fatty streak," an aggregation of lipid-rich macrophages and T-lymphocytes within the intimae layer of an artery. The fatty streaks evolve into intermediate lesions that have a layer of foamy macrophages and smooth muscle cells. This develops into complex and occlusive lesions, as well as fibrous plaques. These plaques have a dense cap of connective tissue, with embedded smooth muscle cells overlaying a core of lipid and necrotic debris. Macrophages are present at all lesional stages with excessive cholesterol and cholesteryl esters in lysosomes. Continuing development of atherosclerotic plaques requires progressive macrophage processing of cholesteryl esters in and through the lysosomes. Perpetuation and maturation of the plaques depends on additional complex inflammatory and scarring processes. Lysosomal acid lipase (LAL) is the only hydrolase for cleavage of cholesteryl esters delivered to the lysosomes.3

The receptors that mediate cholesteryl ester uptake into macrophage include those for LDL and oxidized LDL (oxLDL), ie, LDL receptor (LDL-R), the scavenger receptors type AI and AII (SR-AI and SR-AII),4–6 the scavenger receptor type B1 (SR-BI),7–9 CD36,10 and the LDL receptor related protein (LRP).11 All of these, except SR-BI, direct associated lipids to the lysosome.12–15 Furthermore, modification of LDL by oxidation, aggregation, and glycation occurs in the atherosclerotic lesions by histochemical and biochemical studies.16–20 The delivery of oxidized LDL to lysosomes of macrophages decreases cholesteryl ester hydrolysis, apolipoprotein degradation, and cholesterol to acyl-CoA/cholesterol acyltransferase (ACAT) activity.21–25 Aggregated LDL induces cholesteryl ester accumulation in cultured macrophages.18,20,26,27 Also, LDL particles, modified by advanced glycation, promote cholesterol accumulation in cultured macrophages.19,28 Consequently, enhancement of LAL activity in macrophages could provide a means to decrease accumulated, pathologic cholesteryl esters and triglycerides (TGs) that are causally related to atherosclerosis.

LAL is an essential enzyme for the cleavage of cholesteryl esters and TGs delivered to the lysosomes. The biochemical phenotype of the LAL knockout mouse indicates that other lipases cannot compensate for the loss of LAL activity in the lysosomes.3 The endogenously synthesized mature, soluble glycoprotein has 372 amino acids, is trafficked to the lysosome by the mannose-6-phosphate receptor, and has active site properties similar to those of other lipases.29,30 LAL contributes to cholesterol and fatty acid homeostasis by several mechanisms. Once cholesteryl esters are cleaved by LAL, free cholesterol exits the lysosome and participates in the modulation of cholesterol and fatty acid metabolism via the sterol regulatory element-binding protein (SREBP) system.31–34 Free cholesterol, a product of LAL hydrolysis of CE, leaves the lysosome and leads to downregulation of endogenous cellular cholesterol synthesis and LDL receptor-mediated uptake through the cholesterol sensing mechanisms of the cell. The free fatty acids generated by LAL and their metabolites released from the lysosome could be ligands for the peroxisome proliferated-activator receptor- (PPAR), a master regulator of lipogenesis, macrophage maturation, anti-inflammation, and glucose homeostasis in the body.13,35–38 Deficiency of LAL via targeted gene disruption leads to a multisystemic disease that includes the phenotype of human Wolman disease, aberrant cholesterol and TG metabolism, and macrophage proliferation.39 These diverse effects support the important and central role of LAL in the control of cholesterol and fat metabolism and macrophage proliferation, as well as a potential role in the treatment of atherosclerosis.

Expression of the human LAL (hLAL) in Pichia pastoris (phLAL) systems produced active hLAL that was targeted to macrophage lysosomes.39 Intravenous injections of phLAL into LAL-deficient mice (lal-/-) corrected the lipid storage phenotype in multiple tissues.39 Here, phLAL was administered to the mouse model of atherosclerosis, the LDL-receptor knockout (ldlr-/-) mice on a high fat/high cholesterol diet (HFCD). Repeated doses of phLAL resulted in almost complete elimination of early stage lesions and significant improvement in the quality and quantity of advanced lesions.

    Methods

Animal Models

Homozygous ldlr-/- and apoE-/- mice in a C57BL/6 background were from The Jackson Laboratory (Bar Harbor, Maine). Homozygous lal-/- mice were created in this laboratory.3 Cross breeding of lal-/- and apoE-/- mice generated double knockout of lal-/-;apoE-/- mice. Mice were genotyped by PCR using tail DNA.3 The ldlr-/- mice were fed a high fat/high cholesterol diet (HFCD, 7.5% cocoa butter fat, 1.25% cholesterol, TD86257, Harlan Tekland) from the age of 1.5 months. All animals had ad libitum access to food and water. The animal experiments were performed according to National Institutes of Health guidelines and were approved by IACUC at Cincinnati Children’s Hospital Research Foundation. phLAL was produced in Pichia pastoris as previously described.39

Study Design

Experiments were designed to evaluate the efficacy of LAL enzyme supplementation therapy to treat the athero/arterial-sclerosis at the early foam cell stage (group A) and at the advanced lesional stages (group B). The group A mice received phLAL (1.5 U/injection, n=8) after 1 month of HFCD, ie, beginning at 2.5 months of age. The group B mice received phLAL (6 U/injection, n=7) after 2 months of HFCD, ie, beginning at 3.5 months of age. The control mice for each group were injected with PBS (n=4 to 5). Mice in both groups started HFCD at 1.5 months of age and continued HFCD for the entire study period. PBS or phLAL injections (100 μL) were done every third day for 10 injections. Mice were harvested 2 days after the 10th injection. Tissues were processed for histologic, immunohistochemical, and biochemical analyses. phLAL was 96% pure on silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Plasma Lipids, Lipoprotein Profile, and Tissue Lipids

Blood was collected from the inferior vena cava (IVC) of anesthetized mice (ketamine, acepromazine, and xylazine, 0.2 mL, intraperitoneal injection) after an overnight fast. Plasma was collected (5000g, 10 minutes, 4°C) and stored (4°C and -20°C). Plasma free cholesterol, cholesteryl esters, and TG concentrations were tested by using colorimetric assays (COD-PAP kit for free cholesterol, Wako Chemicals GmbH; TGs/GB kit and cholesterol/HP kit were from Boehringer, GmbH). Plasma from group B mice was pooled (200 μL) and subjected to fast protein liquid chromatography by using 2 Superose 6 HR columns (Pharmacia Biotech Inc.).39,40 Cholesterol concentrations were determined in each fraction (0.5 mL; cholesterol/HP kits). Total lipids were extracted from liver and spleen by the Folch method.3,41 TG concentrations were estimated as described.39,42,43 Total tissue cholesterol concentrations were estimated using O-phthalaldehyde.39,43,44

Tissue Preparation and Morphometric Analyses of Atherosclerotic Lesions

After blood collection under anesthesia, livers, spleens, and small intestines were removed and processed for histologic and lipid analyses. The hearts were perfused through the left ventricle with PBS followed by 10 mL of 4% paraformaldehyde. The aortas were isolated under microscopy and photographed. For qualitative analysis, hearts were split in half along the aortas, embedded in paraffin with the open aorta upward, and sectioned at 4-μm thickness from midsagittal to parasagittal regions of the heart. Aortic arches were cut to 5 fragments at same relative positions, embedded in paraffin with distal end upward, and cross-sectioned at 4-μm thickness from the distal end of each segment of the aorta. Samples were mounted on slides and stained with hematoxylin/eosin (H&E), elastic Van Gieson, or Gomori’s trichrome. For quantitative analyses, 40 to 60 sections (7 μm) were collected from each heart with same orientation and stained with H&E. Lesional area was defined by vessel wall involvement on H&E staining. Images of every fifth sections were captured with a digital camera and analyzed by blinded readers by using computerized morphometric (MetaMorph) software. Briefly, images at the same magnification were captured, and a grid (19x19 lines) divided the image into 400 U. The area covered by lesional macrophages, collagen positive extracellular matrix and necrotic core, and the vessel wall were numbered and manually counted. The areas were converted into μM2 by using an integrated size marker in the MetaMorph program. Staining by Gomori’s trichrome and elastic Van Gieson were used to identify the collagen positive areas, macrophage cell areas, and elastic vessel wall areas. The lesional area was defined from the first elastin positive vessel wall section. Extensive sections and H&E staining (160 to 210 sections, 7 μm) were also performed for one representative heart in control group and 2 representative heart in treated group A. The positive coronary arterial lesions were scored by a blinded reader. The severity of the coronary lesion was scored by a percentage of the sections that had atherosclerotic lesions in arterial vessels and by an estimation of vessel area that was blocked by the lesion as defined in the legend for Table I (available online at http://atvb.ahajournals.org).

Immunohistochemical Staining

Immunohistochemical analyses were performed with frozen or paraffin-embedded aortic valve sections by using rabbit anti-hLAL antibody (1:500) or rat anti-mouse Mac-3 monoclonal antibody (PharMingen). Biotinylated conjugated anti-rabbit or anti-rat IgG (Vector) were used as secondary antibody, respectively.45 The signal was detected by using VECTASTAIN ABC kit (Vector) and counter-stained with Nuclear Fast Red.

Statistical Analysis

All data are expressed as mean±SEM. A 2-tailed unpaired t test was used to compare tissue lipid concentration, lesion area between PBS and phLAL treated mice. A P<0.05 was considered significant.

    Results

Reduction of Plasma and Tissue Lipids by phLAL

Two groups were studied: group A received a lower dose (1.5 U phLAL /injection, 60 U/kg), beginning at 2.5 months, and group B received higher doses (6 U phLAL/injection, 240 U/kg), beginning at 3.5 months. phLAL was given every third day for a total of 10 injections as tail vein boluses. Age-matched control mice received PBS injections. Both groups and their controls received HFCD beginning at 1.5 months, and the diet was continued throughout the study. Compared with wild-type mice, the plasma free cholesterol and cholesteryl esters were increased 9.1-fold and 4.7-fold, respectively, in ldlr-/- mice on HFCD (Table I). In group A, phLAL reduced plasma free cholesterol (29.8%, P=0.089) and cholesteryl esters (52.1%, P=0.0025) relative to control mice. In group B, phLAL decreased plasma free cholesterol (27.6%, P=0.0135), cholesteryl esters (28.6%, P=0.0107), and plasma TGs (46.2%, P=0.0045) in ldlr-/- mice compared with their controls.

In control ldlr-/- mice receiving HFCD, cholesterol and (TGs) levels in liver and spleen were increased relative to those of wild-type mice receiving chow diet (Table I). Decreases in total hepatic (43.2%, P=0.002) or splenic (52.6%, P=0.0351) cholesteryl esters were observed in group B mice. Total hepatic TGs were reduced by 34.9% and 62.2% in groups A and B, respectively (P=0.002 and P=0.0218). Total splenic TGs were reduced by 46.6% and 70% in groups A and B, respectively (P=0.0183 and P=0.0589).

Effects of phLAL Injections on Lipoprotein Profiles

The plasma lipoprotein profiles were compared between control and phLAL treated mice in group B. A large amount of VLDL cholesterol accumulated in ldlr-/- mice receiving HFCD for 3 months. Treatment with phLAL did not alter the VLDL cholesterol, but there was a small consistent decrease in IDL/LDL cholesterol in lipoprotein profile.

Reduction in Atherosclerotic Lesions in the Aorta and Aortic Valves

To evaluate the gross effects of phLAL injections on the progression of atherosclerotic lesions, whole mounts of aortic arches were examined by transillumination. In group A, all (3/3) control mice showed extensive lesions of the arch and branch points for the major vessels, eg, brachiocephalic arteries. The lesions were particularly prominent at the lesser curvature of the arch and the subclavian branch points. Treatment of phLAL at lower dose (1.5 U/injection) had little effect on the extent of the lesions in aorta of ldlr-/- mice (data not shown).

With high dose-treated mice (group B), the lesional area was reduced in the lesser curvature of aortic arch. The lesions at the bifurcation points of major vessels were similar in the test and control mice (Figure 1). Lesional reductions in isolated aortas from 5 enzyme treated mice were somewhat variable (Figure 1B and 1C), but this variation did not affect the significance of lesional reduction by phLAL treatment.

   Figure 1. Effect of phLAL on aortic lesions: Representative aortic arches were isolated from typical controls after 10 injections of PBS (A) and 2 group B mice, T1 (B) and T2 (C), that are typical of this group after receiving 10 injections of phLAL These were then photographed under transillumination (bright field). The dark lesions in the arch and the branch points of the brachiocephalic trunk are atherosclerotic plaques. Magnification, 25x.

Histologic analyses of atherosclerotic lesions of group A mice are in Figure 2 (typical results). Five of 8 group A mice were processed for aortic valve foam cell accumulations and these showed major reductions [from ++++ (30% to 40% of aortic valve areas, AVAs), to ++ (10% to 19% of AVA); 2/5, or 0 (<10% of AVA, 2/5)] (Figure 2B). In control mice the aortic valves showed large atherosclerotic lesions with large numbers of foam cells (Figure 2A).

   Figure 2. Histology of typical groups A and B and control mice. Representative H&E (100x) aortic valve sections of group A mice—control (A) and phLAL-treated (B)—and group B mice—control (C) and phLAL-treated (D). The asterisks indicate necrotic zones next to disrupted medial layers. The arrow indicates a fibrous cap, and arrowheads highlight cholesterol crystals. Reduced foamy cells were evident in the lesions of the aortic valve lesions in group A (B) or in group B (D) mice.

In control mice for group B, the atherosclerotic lesions at 4.5 months were very complex with fibrous caps, necrotic cores, and cholesterol crystals (Figure 2C). In comparison, phLAL-treated mice showed reductions, but not elimination, of atherosclerotic lesions in the aortic valve (Figure 2D). The number of foam cells was greatly decreased, but the necrotic core and cholesterol crystals remained. Quantitative analyses of the lesional areas were conducted by using serial sections of the aortic valve from the control and group B (n=5 in each). Every fifth section (10 μm) was mounted and stained for a total of 15 sections from each heart. This covered 750 μm around the aortic valves. The lesional areas on the aortic valves were quantified by using computerized morphometric software (MetaMorph). Significant reductions (P=0.0115) in lesional area were found in phLAL-treated mice compared with controls. The mean lesional area in phLAL-treated mice was similar to the pretreatment values (Figure 3).

   Figure 3. Quantitative analyses of aortic valve lesional areas. Lesion areas of ldlr-- mice on HFCD before receiving (C0) either PBS (C) or phLAL (T) injections. The lesion area was defined from the first elastin positive area. C and T mice each received 10 injections of PBS or phLAL (6 U/injection) and were analyzed 2 days after injection 10. n=5 for each group, P=0.0115 for C vs T.

Effects of phLAL Treatment on Coronary Artery Lesions

Control mice for groups A and B had extensive multifocal lesions in the coronary arteries. All had heavy infiltration with foamy macrophages and plaques extending a considerable distance into the coronary arteries. In one case, the main branch of the left coronary artery was completely obliterated with an advanced lesion containing cholesterol crystals and inflammatory cells Figure I, available online at http://atvb.ahajournals.org). In comparison, 7/8 of the phLAL treated mice in group A had normal coronary vessels Figure I). One enzyme treated mouse in group A had foamy cells in a small intramuscular coronary vessel. The other coronary arteries in this mouse did not have lesions. These results were confirmed in a more quantitative assessment of the coronary arterial lesions as obtained by sequential H & E sections (total=210 sections; 10 μm thickness) of the hearts from a control and 2 treated group A mice. The control mouse had multiple severe plaques (filled 80% to 100% area of coronary artery lumen) in the coronary arteries in 48% of the sections. One treated mouse had completely normal coronary arteries, and another treated mouse had mild lesions (filled 7% to 24% area of artery lumen) in the coronary arteries in 10% of the sections.

Reduction in the Complexity of the Lesions in phLAL-Treated ldlr-/- Mice

The effects of phLAL on the complexity of atherosclerotic lesions in group B mice were evaluated by histological analyses of lesional elastin and collagen staining. The aortas from group B control and treated mice were sectioned into 5 fragments (3/5 segment positions) (Figure 1A). Sections from distal end of fragment 1 were stained for elastin and collagen. Compared with the control mice, the group B treated mice had the lesional areas that were much smaller and with less extracellular matrix content, ie, collagen, and less vessel wall dilation (Figure II, available online at http://atvb.ahajournals. org). The lesional areas and collagen contents in group B mice were similar to those isolated from the mice before the initiation of enzyme administration. The macrophage area and collagen positive area were quantitatively analyzed by MetaMorph software (Figure IIG). The phLAL-treated mice had less area of macrophages and collagen-rich extracellular matrix. The atherosclerotic lesions from control mice were highly complex, with fibrous caps (Figure 4 A and 4B, arrow), and large amounts of collagen (Figure 4D and 4E, blue). The clear areas within these advanced, complicated lesions had lipid or/and cholesterol crystal accumulation and necrotic cores (Figure 4A, 4B, 4D, and 4E, asterisk). Collagen-rich fibrosis was present in aortic valves of control mice (Figure 4B and 4E, arrowhead). In the group B treated mice the lesions were less complex, with lesser degrees of macrophage infiltration and fibrous caps were lacking (Figure 4C and 4F). The deposition of excess extracellular matrix also was much less in treated mice (Figure 4E versus 4F). The macrophage area, collagen positive area, and necrotic core were quantitatively analyzed by using MetaMorph software (Figure 5).

   Figure 4. Reduced the foam cells in atherosclerotic lesion in aortic valve of group B treated mice. Sections of aortic valvular lesions were stained for elastin (A through C, black) and collagen (D through F, blue). C0, ldlr-- mice prior to injection at age 3.5 months. C, PBS (10 injections) control mice. T indicates phLAL-treated (10 injections) mice. *Area of necrotic core. Fibrous caps are indicated with arrow, and thrombosis is indicated with arrowhead. Notice the reduction the number of foam macrophages in phLAL treated samples. Magnification, 100x.

   Figure 5. Quantitative analysis of areas in the lesion of aortic valve. The methods are detailed in Methods. The macrophage and collagen positive areas were defined by van Gieson and Gomori’s trichrome stains.

Macrophage Targeting of phLAL to the Atherosclerotic Lesions

Endogenous LAL protein in ldlr-/- mice could confound these analyses. Thus, ldlr-/-;lal-/- mice were developed and placed on a HFCD. However, lal-/-;ldlr-/- mice died within a few days of HFCD initiation (data not shown). The alternative of apoE-/-;lal-/- mice was developed and used. These mice had no detectible LAL protein and developed spontaneous atherosclerotic lesions without dietary challenge (Figure 6A). By immunohistochemical staining, phLAL protein was taken up into the foam cells of atherosclerotic lesions (Figure 6C). Serial sections were stained with the macrophage marker, Mac-3 (Figure 6D), or oil red O (Figure 6B). The results show uptake of phLAL protein into foamy macrophages and surface endothelial cells.

   Figure 6. Localization of phLAL in foam cells of atherosclerotic lesion. Sections of the lesions at the aortic lesions of lal--;apoE-- mice stained with H&E (A), oil red O (B, red droplets indicates lipids), anti-hLAL antibody (C, brown), or macrophage marker, anti-Mac-3 (D, brown). The tissues were harvested 4 hours after injection of phLAL (24 U). LAL-positive cells were positive for Mac-3 (arrows). Not all macrophages have uptake of hLAL enzyme. Magnification, 400x.

Survival and Antibody Development

All ldlr-/- mice in group A survived. Fifteen of 17 ldlr-/- mice in group B survived. In group B, 1 mouse died after 2 injections and another after 5 injections of phLAL. There was no obvious cause for the death. No other adverse effects were noted in either treated or untreated groups. All ldlr-/- mice injected with phLAL developed anti-phLAL antibodies as assessed by Western blot analyses. One developed a high titer (1:32 000) antibody (data not shown). The earliest development of anti-phLAL antibody was after 5 injections (15 days). These antibodies were not inhibitory toward activity. The antibodies were directed against the LAL protein because they reacted similarly with the deglycosylated and the glycosylated proteins.

    Discussion

This study demonstrates that enzyme supplementation with phLAL had significant effects on the atherosclerotic lesions in ldlr-/- mice receiving HFCD. The lesions were diminished or absent in treated mice compared with the extensive and very severe lesions in the untreated cohorts. This was most evident during early lesional development. For preexisting advanced lesions, treatment of LAL reduced the lesional area and the macrophage component and, thus, appeared to prevent progression of and stabilized the preexisting lesions. The majority of the enzyme was localized to the liver macrophages, Kupffer cells, by immunohistochemical staining after intravenous injection.39 Biological activity of phLAL was assessed previously in lal-/- mice.39 Such in vivo activity also was evident from the reductions in tissue CEs and TGs in lal-/- and HFCD-fed ldlr-/- mice. Injection of phLAL in the ldlr-/- mice also led to significant reductions of plasma CE in group A mice, and of plasma free cholesterol, CE, and TG in group B mice. This suggested the dose-dependent systemic effect of phLAL. The reduction of plasma lipids by phLAL might be mediated by a decrease of lipoprotein production or an increase of lipoprotein uptake in these ldlr-/- mice. Based on an effect of phLAL in reduction of tissue lipid in the liver, we speculate that plasma lipid reductions are mediated though a decrease of lipoprotein production in these ldlr-/- mice.

At least 3 mechanisms could account for the lesional reduction by phLAL administration. First, LAL enters the lesional foam cells and hydrolyzes the stored CEs and TGs.46 Electron microscopic analyses of lesions confirmed that much of the accumulated lipid in foam cells occurred within large, lipid-filled lysosome.47 Most of the cholesterol in atherosclerotic lesions is esterified with linoleate rather than oleate,48,49 which indicates a LDL source rather than a product of ACAT.50 Here macrophages in the atherosclerotic lesions of apoE-/-;lal-/- mice were positive for injected phLAL. By incubation of phLAL enzyme with cultured mouse macrophages, J-774E cells, hLAL is colocalized with the lysosomal marker, lysosomal associated membrane protein 1 (LAMP-1)39. This suggests an important protective effect of LAL by locally reducing the accumulated CEs and TGs in the foamy macrophages. Second, LAL promotes lysosomal egress of free cholesterol that modulates cellular lipid biosynthesis mediated by the SREBPs. This would lead to increased egress of free cholesterol from lysosomes that would inhibit the endogenous synthesis of cholesterol and fatty acid through the SREBP system.34 Most administered phLAL localized to macrophages of the liver and spleen where it hydrolyzed CEs and TGs and reduced tissue lipids in ldlr-/- mice. Third, LAL cleaves oxLDL and generates 9-hydroxyocatadecadienoic acid (9-HODE) and 13-HODE that are ligands for PPAR.13 The ligand activated PPAR could have beneficial effects on the reduction of atherosclerotic lesion by its anti-inflammatory activity.51–54 LAL activity is required to produce ligands for PPAR from oxLDL13 that can contribute to in vivo PPAR ligand generation. Because synthetic ligands for PPAR, eg, rosiglitazone, reduced dietary induced atherosclerotic lesions in male ldlr-/- mice.54 The natural ligands generated by LAL lipid hydrolysis might be expected to have similar effects on reducing atherosclerotic lesions by activation of PPAR and its anti-inflammatory function.54 Here, quantitative analyses showed that hLAL administration in group B mice reduced macrophages in the lesions, but also reduced the areas of extension of collagen positive advanced plaques, cholesterol crystal-rich necrotic cores, and the vessel wall dilation (Figure 5). This suggested that LAL also could have anti inflammatory effects. Alternatively, ligand activated PPAR could stimulate the gene expression of LXR, with downstream stimulation of ABCA1 expression.55,56 The outcome of this cascade of gene regulation is to promote cholesterol efflux from macrophages in the lesion. Peritoneal macrophages isolated from lal-/- mice have lower cholesterol efflux than those from lal+/+ mice (data not shown). These data suggest that LAL derived ligands could promote cholesterol efflux from the cells through the ABCA1 transporter. Finally, recent studies have shown the role of free cholesterol in promoting apoptosis in cultured macrophages.57,58 It is not clear whether the free cholesterol has same role in lesional macrophages in vivo. The remaining question here is the balance between the production of free cholesterol by LAL that potentially could be toxic and a cause of macrophage cell death and the production of free fatty acid by LAL that could be a ligand for PPAR to activate the cholesterol efflux through LXR and ABCAI pathway and anti-inflammatory activity of PPAR.

Why does supplemental LAL work? Alternatively, at what level of substrate presentation to the lysosome does endogenous LAL activity become insufficient to maintain normal CE and TG flux through this organelle? Studies in atherosclerotic rabbit model indicated that the density of lysosomes was decreased and lysosomal cholesteryl esters was increased in lesional smooth muscle cells and endothelial cells compared with that in normal vessel walls.47 These data suggested that LAL activity in lesional cells was/is relatively decreased compared with that in nonlesional vessel walls. Also, LAL activity tends to decrease with age.59 Additional factors include the acetylation and oxidation of LDL leading to modified LDL uptake by unregulated scavenger receptors. Because oxLDL can inactivate lysosomal proteases and cause poor degradation of oxLDL in mouse peritoneal macrophages,60 one might speculate that oxLDL could potentially inactivate lysosomal lipases as well. Indeed, oxLDL CE are resistant to lysosomal hydrolysis in human macrophage THP-1 cells.21 These data suggest that oxLDL could accentuate lysosomal lipid accumulation in foam cells due to direct effects on LAL. As a result, LAL supplementation could compensate for this loss of endogenous LAL activity and reestablish the lysosomal flux of lipids.

    Acknowledgments

 

We thank Jaya Mishra and Bradley B. Jarrold for their excellent technical assistance, Lisa McMillin for the histology analyses, and Chris Woods for the color photography. This work was partially supported by grants from Genzyme Corporation (G.A.G) and from NIH DK 54930 (H.D). Additional support was provided by the Cincinnati Children’s Hospital Research Foundation.

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日期:2007年5月18日 - 来自[2004年第24卷第1期]栏目

Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis

    1 Department of Cell Biology
    2 Section of Immunobiology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, CT 06520
    3 Cancer Institute, New York University School of Medicine, New York, NY, 10016

    T cells recognize protein antigens as short peptides processed and displayed by antigen-presenting cells. However, the mechanism of peptide selection is incompletely understood, and, consequently, the differences in the immunogenicity of protein antigens remain largely unpredictable and difficult to manipulate. In this paper we show that the susceptibility of protein antigens to lysosomal proteolysis plays an important role in determining immunogenicity in vivo. We compared the immunogenicity of proteins with the same sequence (same T cell epitopes) and structure (same B cell epitopes) but with different susceptibilities to lysosomal proteolysis. After immunizing mice with each of the proteins adsorbed onto aluminum hydroxide as adjuvant, we measured serum IgG responses as a physiological measure of the antigen's ability to be presented on major histocompatibility complex class II molecules and to prime CD4+ T cells in vivo. For two unrelated model antigens (RNase and horseradish peroxidase), we found that only the less digestible forms were immunogenic, inducing far more efficient T cell priming and antibody responses. These findings suggest that stability to lysosomal proteolysis may be an important factor in determining immunogenicity, with potential implications for vaccine design.

    To be recognized by T lymphocytes, protein antigens must be converted into short peptides bound to MHC molecules, which are displayed on the surface of APCs. The ability of APCs to generate peptide–MHC complexes is, therefore, essential to the initiation of the immune response (1, 2). Although the interaction between peptides and MHC class II molecules and the ability of the T cell repertoire to generate antigen receptors of cognate specificity have been extensively studied, it remains difficult to predict or to manipulate the extraction of peptide ligands from protein antigens (3, 4). As a consequence, the differences in immunogenicity between protein antigens are poorly understood and, therefore, approaches to induce antigen-specific immunity remain largely empirical (5, 6).

    Antigenic peptides are produced by lysosomal proteolysis, and, thus, efficient lysosomal degradation is often assumed to favor production of ligands for MHC class II molecules. This notion derives mostly from in vitro experiments. For instance, blocking lysosomal function with protease or acidification inhibitors decreased antigen presentation (7–11). Enhancing lysosomal proteolysis by the presence of protease-specific cleavage sites (12) or by destabilizing proteins also favored presentation to T cell hybridomas (13–15). However, these in vitro studies did not evaluate the role of lysosomal proteolysis on immunogenicity in vivo.

    We decided to take a direct and physiological approach to investigating the relationship between antigen proteolysis and immunity in vivo. We chose not to use pharmacological or genetic approaches that could potentially have multiple effects on APCs. We studied instead the immunogenicity of proteins with the same sequence (same T cell epitopes) and structure (same B cell epitopes) but with different susceptibilities to lysosomal proteolysis. We found that less digestible forms of otherwise identical antigens are more immunogenic, inducing more efficient T cell priming and antibody responses.

    RESULTS AND DISCUSSION

    We began by comparing bovine pancreatic ribonuclease (RNase-A), a compact stable protein, with its variant RNase-S, in which a single peptide bond is cleaved (between Ala20 and Ser21) (16, 17). Although both RNase-A and RNase-S are otherwise structurally and enzymatically identical (16, 17) (Fig. 1 A), RNase-S was far more susceptible to lysosomal proteolysis both in vitro by lysosomal extracts and after internalization by bone marrow–derived DCs (BM-DCs); this difference was maintained after antigen adsorption onto an adjuvant such as aluminum hydroxide (Alum; Fig. 1, B and C). We next asked if the differential susceptibilities to proteolysis of these model antigens affected their capacity to induce IgG responses as a physiological in vivo measure of their ability to be presented on MHC class II molecules and to prime CD4+ T cells in vivo. After injecting each of the proteins adsorbed onto Alum into mice, the stable form of RNase (RNase-A) was found to induce much higher (>10,000-fold) IgG titers than did the unstable form (RNase-S; Fig. 2 A).

    We next examined antigen uptake by APCs in vivo by FACS analysis. DCs in the draining lymph nodes contained comparable amounts of RNase-A and RNase-S 2.5 h after intradermal injection (Fig. 1 D). Moreover, differential antibody responses were also observed when BM-DCs loaded ex vivo with the same amount of RNase-A or RNase-S were adoptively transferred into naive recipient mice (Fig. 2 D). Collectively these results rule out that the differences in immunogenicity between the two proteins could result from differential access to APCs.

    That both forms of RNase indeed share the same B cell epitopes was further emphasized by the fact that the small amount of anti-RNase IgG elicited after injection of high doses of RNase-S also reacted with RNase-A, and vice versa (unpublished data). Moreover, both forms of RNase induced comparable soluble IgM responses (2–4-fold difference, as opposed to >10,000-fold difference in IgG responses; Fig. 2, A and C). This indicated that, despite being recognized similarly by B cells, RNase-S was not adequately presented to CD4+ T cells.

    To further show that the rapid lysosomal degradation of RNase-S limited MHC class II presentation and T cell priming in vivo, we analyzed the antibody responses to defined B cell epitopes introduced on RNase-A or RNase-S. The IgG responses to haptens such as FITC and DNP were strong only when they were coupled to the stable protein carrier (RNase-A; Fig. 2 B and not depicted). When low doses of antigens were injected with stronger adjuvants (incomplete or complete Freund's), RNase-S was still less immunogenic than RNase-A (unpublished data). The differences in immunogenicity of the model antigens were maintained over a broad dose range (1–1,000 μg) and after multiple injections of antigen (unpublished data), emphasizing that the different immunogenicity of RNase-A and RNase-S most likely reflects their efficiency of presentation on MHC class II molecules.

    Indeed, cellular immune responses were also stronger to the antigen more resistant to lysosomal proteolysis (RNase-A). Primarily, immunization with RNase-A but not RNase-S elicited robust T cell recall responses in vitro (Fig. 2, E and F). Also, the delayed-type hypersensitivity response in mice immunized with the stable form (RNase-A) was substantial, whereas it was undetectable in mice immunized with RNase-S (Fig. 2 G). In addition, the IgG responses to RNase-A and to FITC coupled to RNase-A exhibited the hallmarks of conventional CD4-dependent T cell responses, as no anti-RNase or anti-FITC IgGs were detected in CD4- or MHC class II–deficient mice (Fig. 3 A). The same difference in immunogenicity between RNase-A and RNase-S was observed in different mouse strains (Fig. 3 B), indicating that the increased immunogenicity of RNase-A was not associated with individual MHC class II haplotypes. Similarly, the differential immunogenicity of RNase-A and RNase-S was independent of the route of injection (intraperitoneal, intradermal, or intramuscular; Fig. 2 A and Fig. 3 C) and, consequently, of the population of APCs that initially encounter the antigens.

    We extended the analysis to another antigen with a different structure and T cell epitopes. For this purpose, we compared the immunogenicity of horseradish peroxidase (HRP) and its variant apo-HRP, from which the calcium and heme group have been removed, leaving the intact but destabilized polypeptide chain of HRP. Both forms of HRP have the same amino acid sequence and were not covalently modified; therefore, they possess the same T cell epitopes. They also retain intact their four intramolecular disulfide bonds, maintaining similar three-dimensional structures and antigenicity, as reflected by the fact that polyclonal antibodies from several species recognized HRP and apo-HRP identically under native conditions (Fig. 4 A). However, the two forms of HRP, soluble or adsorbed onto Alum, differed markedly in their susceptibility to lysosomal proteolysis, with apo-HRP being more readily digested by DC lysosomal proteases in vitro and ex vivo after internalization by BM-DCs (Fig. 4, B and C).

    Immunization experiments in mice showed essentially the same pattern of immunogenicity as with stable and unstable forms of RNase: the stable form of HRP (intact HRP) induced stronger T cell priming (Fig. 4, D and E) and IgG responses (Fig. 4 F) than the unstable form (apo-HRP). Similarly, the IgG responses to a hapten (FITC) were more robust when it was presented on the protein backbone of the stable form (HRP; Fig. 4 G). As with RNase, these differences were maintained over a 1,000-fold dose range (unpublished data). Both forms of HRP also produced similar soluble IgM responses (Fig. 4 H), and all antisera raised against HRP fully recognized apo-HRP (unpublished data), indicating that both forms of HRP shared the same IgG epitopes, but that the rapidly degraded form was weakly immunogenic. If the poor priming of T cells by proteins that are rapidly degraded (RNase-S and apo-HRP) was primarily caused by enhanced susceptibility to lysosomal degradation, stabilizing them to proteolysis should enhance their capacity to induce IgG responses. To test this possibility, we generated inter- and intramolecular cross-linked forms of RNase-S or apo-HRP by fixation with aldehydes (18, 19), resulting in molecules that became more resistant to lysosomal proteolysis in vitro (Fig. 5 A and Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20052442/DC1). The stabilization to lysosomal proteolysis of RNase-S and apo-HRP by fixation was also observed in intact cells after internalization into BM-DCs (Fig. 5 B and Fig. S1 B). In both cases, aldehyde-mediated stabilization largely restored the ability of these otherwise poorly immunogenic proteins to induce IgG responses to RNase (Fig. 5 C) and HRP (Fig. 5 E), as well as to coupled haptens (FITC; Fig. 5, D and F).

    One contributing factor to the increased ability of more stable antigens to elicit immune responses is that the restricted susceptibility to lysosomal proteolysis favored the production of peptide–MHC class II complexes by DCs, at least in vitro (Fig. 2 E and Fig. 4 D). In addition, and just as important in an in vivo setting, we found that the increased stability to lysosomal proteolysis also favored the retention of antigens captured by DCs to lymphoid organs. 16 h after a single intradermal injection, the stable forms of RNase (Alexa 488–RNase-A) could be detected in CD11c+ DCs in the draining lymph nodes (Fig. 1 D). In contrast, the rapidly degraded form (Alexa 647–RNase-S) was barely detectable under the same conditions (Fig. 1 D). Combined with the fact that differential immunogenicity was observed by adoptively transferring DCs containing either RNase-A or RNase-S (Fig. 2 D), these results strongly suggest that at least one effect of decreased susceptibility to proteolysis is to facilitate intracellular antigen survival in DCs, which would allow for a sustained provision of MHC–peptide complexes.

    Our findings show that, in contrast to a prevailing view derived mostly from in vitro experiments, the immunogenicity of protein antigens in vivo can be enhanced by reducing their susceptibility to lysosomal proteolysis. This may therefore be an important factor contributing to the largely unexplained differences among proteins in their abilities to elicit immune responses. Although this feature may vary for different antigens, the fact that we have obtained identical results for two entirely unrelated proteins, and for haptens coupled to them, suggests that it represents a general principle. It is noteworthy that we did not find major differences in the presentation of defined epitopes from RNase-A and RNase-S to CD4+ T cell hybridomas in vitro (unpublished data), although such differences were observed in vivo and using primary T cells, emphasizing the importance of in vivo studies to examine protein immunogenicity. It would be interesting to examine the impact of protein digestibility on the activation of naive versus memory T cells.

    The susceptibility to lysosomal proteolysis may not only enhance the preservation of CD4+ T cell epitopes themselves but also clearly increases the persistence of the proteins from which those epitopes are extracted. DCs can take many hours or days to traverse from the periphery to lymphoid organs where they engage their cognate T cells to initiate immune responses (20); therefore, antigen persistence provides a source of antigen for sustained processing and presentation by DCs within (or en route to) secondary lymphoid organs (21, 22) and, potentially, presentation of intact antigens to B cells (23, 24). Although the production of peptide–MHC class II complexes from a given antigen may not always be favored by restricting digestion, the enhanced dissemination and persistence of stable antigens (or antigens in APCs of restricted lysosomal proteolysis) should consistently contribute to immunogenicity. It is interesting to speculate that, as part of their mechanism of action, some adjuvants and carrier proteins could act at least in part by protecting the antigens they carry against lysosomal destruction. For example, coupling peptides to large, poorly digestible carriers would in essence convert labile peptides into relatively stable proteins.

    The susceptibility of exogenous antigens to lysosomal proteolysis may also affect their ability to elicit CD8+ T cell responses during cross-presentation. Less digestible antigens may have a greater chance of surviving the digestive environment inside lysosomes to gain access to the cystosol for presentation on MHC class I molecules. This in fact may contribute to the enhancement of antigen cross-presentation observed in the presence of chloroquine (25). In addition, the comparative efficiency of DCs relative to macrophages in cross presentation (26) may at least in part reflect the relative inefficiency with which DCs degrade endocytosed antigens (27).

    Our results provide direct support for the concept that the limited proteolytic capacity of DCs plays an important role in vivo in augmenting their efficiency as APCs by enhancing not only peptide–MHC class II production but also antigen dissemination and persistence in vivo (27). This may have implications for vaccine design, particularly for the elicitation of MHC class II–dependent antibody responses. Chemical modifications or the use of carriers that enhance antigen resistance to lysosomal proteolysis may enhance antigen immunogenicity. This approach would capitalize on one of the key biological properties of DCs to further enhance their antigen-presenting functions.

    MATERIALS AND METHODS

    Mice and cells.

    C57BL/6, C3H/HeJ, and B6D2F1 mice were purchased from The Jackson Laboratory. CD4–/– and MHC class II–/– mice on the C57BL/6 background were purchased from Taconic. All mice were males and were used at 6–12 wk of age. The Institutional Animal Care and Use Committee at Yale University approved all animal protocols. BM-DCs were grown as previously described (28). Lysosomal extracts of DCs were prepared as previously described (27).

    Model antigens.

    RNase-A and RNase-S (Sigma-Aldrich) were characterized as previously described (29). Apo-HRP was prepared as previously described (30). FITC (Sigma-Aldrich), Alexa 488, and Alexa 647 (Invitrogen) derivatives were prepared according to the manufacturer's recommendations. For fixation with aldehydes, 2 mg/ml of antigens were incubated in PBS in the presence of 10 mM paraformaldehyde and 2 mM glutaraldehyde on ice for 30 min. Reactions were stopped by addition of 50 mM glycine, followed by a 10-min centrifugation at 10,000 g to remove aggregates and a subsequent desalting into PBS. Recognition of model antigens by polyclonal antibodies was conducted by ELISA or by immunodiffusion in gels.

    Immunizations.

    Mice were immunized by intraperitoneal, intradermal, or intramuscular injection of 1–1,000 μg of the different antigens adsorbed onto Alum adjuvant (Imject Alum; Pierce Chemical Co.) twice at a 2-wk interval. Alternatively, mice were immunized by a single injection with 300,000 CD11c+ BM-DCs (loaded with 0.5 mg/ml of antigens for 2 h) in the footpad. Sera were collected before immunization and 10 d after the last injection.

    ELISA.

    Sera were titrated by using plates (Maxisorp; Nunc) coated with RNase-A, HRP, or FITC-BSA (5 μg/ml each). Antibodies were detected using alkaline phosphatase–conjugated donkey antibodies against mouse IgM or IgG (Jackson ImmunoResearch Laboratories), as well as with 4-methylumbelliferyl phosphate (Sigma-Aldrich).

    Delayed-type hypersensitivity response.

    After immunization, the mice were challenged by injection of 1 μg of antigen into one ear, while the other ear received PBS. The ear thickness was measured 24 h later using a micrometer (Ultra-Mic; Fowler).

    In vitro degradation assays.

    Antigen degradation assays were done as previously described (27).

    Ex vivo degradation assays.

    BM-DCs were loaded with 0.5 mg/ml of antigens for 1 h and incubated at 37°C for the times indicated in the figures. The presence of the proteins and cell surface expression of CD11c were monitored by FACS.

    In vivo degradation assays.

    Alexa 488–RNase-A and Alexa 647–RNase-S (20 μg each) were simultaneously injected intradermally. At the times indicated in the figures, the draining lymph nodes were removed, and cells were dissociated by treatment with Blendzyme 2 (Roche). The presence of the proteins and cell surface expression of CD11c were monitored by FACS.

    Antigen processing and presentation assays.

    Splenocytes from immunized mice were incubated with the doses of antigens indicated in the figures for 48 h. T cell responses were evaluated by measuring T cell proliferation, as estimated by [3H]thymidine incorporation.

    Online supplemental material.

    Fig. S1 shows that fixation of apo-HRP with aldehydes increases its resistance to lysosomal proteolysis in vitro and enhances its survival ex vivo. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20052442/DC1.

    Acknowledgments

    We are grateful to Tracy Ferguson and Craig Burton for expert help.

    This work was supported by the National Institutes of Health (I. Mellman), the American Heart Association (E.S. Trombetta), and the Ludwig Institute for Cancer Research (I. Mellman and E.S. Trombetta).

    The authors have no conflicting financial interests.

    Submitted: 6 December 2005

    Accepted: 21 July 2006

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