Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque BENEDIKT WEITKAMP,1 PAUL CULLEN,1 GABRIELE PLENZ, HORST ROBENEK, AND ¨ RGEN RAUTERBERG2 JU Institut fu¨r Arterioskleroseforschung, 48149 Mu¨nster, Germany ABSTRACT Type VIII collagen is a short-chain collagen that is present in increased amounts in atherosclerotic lesions. Although the physiological function of this matrix protein is unclear, recent data suggest an important role in tissue remodeling. Type VIII collagen in the atherosclerotic lesion is mainly derived from smooth muscle cells. We now show that macrophages in the atherosclerotic vessel wall and monocytes in adjacent mural thrombi also express type VIII collagen. We demonstrated this using a novel combined fluorescence technique that simultaneously stains, within the same tissue section, specific RNAs by in situ hybridization and proteins by indirect immunofluorescence. In culture, human monocyte/macrophages expressed type VIII collagen at all time points from 1 h to 3 wk after isolation. Western blotting and immunoprecipitation also revealed secretion of type VIII collagen into the medium of 14-day-old macrophages. Because this is the first report of secretion of a collagen by macrophages, we tested the effect of lipopolysaccharide (LPS) and interferon g, substances that stimulate macrophages to secrete lytic enzymes, on macrophage expression of type VIII collagen. LPS and interferon g decreased expression of type VIII collagen. By contrast, secretion of matrix metalloproteinase 1 (MMP 1) was increased, indicating a switch from a collagenproducing to a degradative phenotype. Double in situ hybridization studies of expression of type VIII collagen and MMP 1 in human coronary arteries showed that in regions important for plaque stability, the ratio of MMP 1 RNA to macrophage type VIII collagen RNA varies widely, indicating that the transition from one phenotype to the other that we observed in vitro may also occur in vivo.—Weitkamp, B., Cullen, P., Plenz, G., Robenek, H., Rauterberg, J. Human macrophages synthesize type VIII collagen in vitro and in the atherosclerotic plaque. FASEB J. 13, 1445–1457 (1999)

Key Words: double in situ hybridization z atherosclerosis z matrix metalloproteinase z fluorescence staining

The invasion of blood monocytes and their differentiation into tissue macrophages is a central step in inflammation and wound repair. Macrophages syn-

0892-6638/99/0013-1445/$02.25 © FASEB

thesize numerous lytic enzymes for degradation of tissue debris that is subsequently removed and neutralized by phagocytosis and intracellular digestion. They also ingest and destroy bacteria, and probably virally infected or malignant cells. Macrophages also secrete a wide variety of cytokines, growth factors, and chemotactic agents, attracting and activating cells in their vicinity. They are one of the main cell types involved in T cell activation via major histocompatibility complex class II-mediated antigen presentation. Macrophages and macrophage-derived foam cells also play a key role in the process of atherosclerosis and are found in the atherosclerotic plaque at all stages of its development (1). Recent studies have highlighted the importance of macrophages located in the ‘collar’ of the atherosclerotic plaque for the pathology of the lesion (2, 3). Secretory products of such macrophages, particularly matrix metalloproteinases such as collagenase, have been shown to solubilize extracellular matrix and hence to contribute to destabilization and rupture of the fibrous cap (4 –11). While indirect macrophage stimulation of matrix formation by mesenchymal cells via secretion of cytokines such as transforming growth factor b (TGF-b)3 is known (12), the direct generation of new extracellular matrix, an important event in tissue remodeling, has rarely been described for these cells. Nevertheless, reports exist of the gener1

These authors both contributed equally to this work. Correspondence: Institut fu¨r Arterioskleroseforschung, Domagkstrasse 3, 48149 Mu¨nster, Germany. E-mail: cullen@ uni-muenster.de 3 Abbreviations: APAAP, alkaline phosphatase, anti-alkaline phosphatase; CD, cluster of differentiation; DIG, digoxin; EDTA, ethylene diamine tetraacetic acid; ELF, enzyme-linked fluorescence; FACS, fluorescence-activated cell scanning; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IgG, immunoglobulin G; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RPMI, Roswell Park Memorial Institute; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SMC, smooth muscle cell; SSC, standard saline citrate; TGF, transforming growth factor; TRITC, tetramethyl rhodamine B isothiocyanate; UTP, uridyl triphosphate. 2

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ation of fibronectin that not only functions as a component of the extracellular matrix, but has also been implicated in phagocytosis (‘opsonic protein’) (13–16). Macrophages have also been shown to produce proteoglycans (17) and components of the basement membrane such as laminin (18). Finally, macrophages in the atherosclerotic lesion have been shown to synthesize osteopontin, which may play a role in vessel wall calcification (19, 20). Thus it is possible that in addition to their degradative function, macrophages may synthesize components of the extracellular matrix of the arterial wall. Type VIII and type X collagen constitute a group of short-chain collagens (21). Type VIII collagen was initially described as a product of endothelial cells, but has since been found in many types of tissue (22–26). Its expression appears to be enhanced in abnormal tissues such as cancers (27), in atherosclerotic arterial media (28), and in transplanted organs showing signs of vasculopathy. Thus, type VIII collagen may play a role in tissue remodeling and repair. Type X collagen is a specific product of hypertrophic chondrocytes but has never been detected in other mesenchymal tissues, including macrophages (29 – 32). We (unpublished observations) and others (28, 33) have recently shown that in the vessel wall, type VIII collagen is generally expressed by smooth muscle cells. Whereas in normal arteries the expression of type VIII collagen is very low, expression in atherosclerotic arteries is increased in areas showing infiltrates of monocytes/macrophages. In these areas, the predominant cell type expressing type VIII collagen is the smooth muscle cell. However, not all type VIII collagen-expressing cells in atherosclerotic arteries were shown to bear smooth muscle cell or endothelial cell markers. In this report, we show that a proportion of the type VIII collagen-expressing cells in the human atherosclerotic lesion can be identified as monocytes/macrophages by using lineage-specific cell markers. We also demonstrate that cultivated human monocytes produce type VIII collagen and that this ability is maintained over several weeks in culture, when cells take on the phenotype of differentiated tissue macrophages.

Cell culture Peripheral blood monocytes were isolated from volunteers by monocytapheresis, followed by elutriation and countercurrent centrifugation as described previously (34). The monocytapheresis procedure was approved by the local Hospital Ethics Committee. Purity of isolated monocytes was greater than 95% as revealed by fluorescence-activated cell scanning (FACS). For all experiments, monocytes were maintained in a

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Antibodies used for cell characterization For characterization of monocyte/macrophages, a mouse monoclonal antibody to the heterocomplex MRP8-MRP14 (clone 27E10; BMA, Augst, Switzerland), a marker of proinflammatory monocytes (here referred to as ‘antibody 27E10’) (35), and the mouse monoclonal antibody 25F9 (clone 25F9, BMA), which recognizes human late stage macrophages (36), were used. Endothelial cells were detected using rabbit polyclonal anti-von Willebrand factor antibody (Dako, Hamburg, Germany). Smooth muscle cells (SMC) were detected using a mouse monoclonal antibody to SMC myosin (clone hSM-V; Sigma, Deisenhofen, Germany). Mature macrophages were detected using a mouse monoclonal antibody to the marker of mature macrophages cluster of differentiation (CD) 68 (Dako). Expression of type VIII collagen in monocyte/macrophages in culture To investigate the time course of type VIII collagen expression, monocyte/macrophages were maintained for 1 h, 24 h, 7 days, and 14 days in Labtec chamber slides (Nunc, Wiesbaden, Germany). Thereafter, the cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4, for 10 min at room temperature. Immunocytochemistry was performed using the alkaline phosphatase, anti-alkaline phosphatase (APAAP) method as described by Cordell et al. (37). Type VIII collagen was detected with a mouse monoclonal antibody (clone 8C, Medac, Hamburg, Germany). For controls, ‘non-immune’ mouse immunoglobulin G (IgG) (10 mg/ml; Dako) was used in place of anti-type VIII collagen antibody. To characterize the deposition pattern of type VIII collagen, immunofluorescence studies were performed with a two-step indirect labeling procedure using anti-mouse IgG F(ab)2 fragments conjugated with cyanin 3 (Dianova, Hamburg, Germany). Images were generated by digital microscopy using an Axiophot II microscope (Zeiss, Oberkochen, Germany), a charged-coupled device camera, and KS-300 software (Kontron, Neufahrn, Germany). To permeabilize the cells, slides containing 14-day-old macrophages were treated with a 1% concentration of the non-ionic detergent Nonidet P-40 (Calbiochem, Bad Soden, Germany) in PBS for 3 min before incubation of with anti type VIII collagen antibody. Labeling and immunoprecipitation of newly synthesized type VIII collagen

MATERIALS AND METHODS

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5% CO2 atmosphere at 37°C in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 20% pooled human serum (PAA Laboratories, Co¨lbe, Germany), 1 mM sodium pyruvate, 20 mM glutamine, essential amino acids, 100 IU/ml penicillin, and 100 ng/ml streptomycin (all from Life Technologies, Inc.).

Fourteen-day-old macrophages were used for studies of biosynthesis of collagen type VIII. Macrophages were preincubated for 1 h in serum-free RPMI 1640 medium without methionine and cysteine and supplemented with 50 mg/ml sodium ascorbate. Metabolic labeling of cells with Trans 35 S-label (10 mCi/ml; ICN Biochemicals, Eschwege, Germany) was carried out for 20 h in serum-free RPMI 1640 supplemented with 50 mg/ml sodium ascorbate at an isotope activity of 50 mCi/ml of culture medium, from which the

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appropriate amino acids had been deleted. Immediately after collection, phenylmethyl-sulfonyl fluoride (diluted from a 1003 stock in absolute ethanol, 1 mM end concentration) and ethylene diamine tetraacetic acid (EDTA; 5 mM end concentration) were added to the medium, which was then centrifuged to remove debris and stored at 280°C. Radiolabeled type VIII collagen was immunoprecipitated from 1.2 ml medium at 4°C as follows. The medium was preadsorbed with 50 ml of 50% (v/v) gelatin-Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden) for 30 min to remove fibronectin. Samples were than incubated with 3 ml monoclonal anti-type VIII collagen antibody (clone 6A2; Medac) or a ‘non-immune’ mouse IgG (10 mg/ml; Dako). The amount of antibody that results in maximum precipitation of type VIII collagen was determined by titration assay. After 1 h, 50 ml of 50% (v/v) protein G-Sepharose (Pharmacia LKB Biotechnology) was added and incubation was continued for 1 h. Immunoprecipitates were collected by centrifugation, washed three times in 500 mM NaCl, 1% Nonidet P-40, 50 mM Tris pH 8.0, 5 mM EDTA, and three times in the same buffer with the NaCl concentration adjusted to 150 mM. Before resuspension in 30 ml reducing Laemmli sample buffer (38), the immunoprecipitates were washed once in 50 mM Tris pH 6.8. Samples were heated for 10 min at 80°C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% acrylamide concentration) and autoradiography. Dried gels were evaluated on a Fuji BAS-1500 scanner (raytest; Sprockho¨vel, Germany) after exposure for 12 h to Fuji BAS phosphorimaging plates.

with secondary peroxidase-conjugated goat anti-mouse antibody. Specific primary antibody immunoreactivity was detected by enhanced chemiluminescence with a sensitive charged-coupled device camera (raytest). Effect of immune mediators on type VIII collagen production in vitro Monocyte/macrophages were cultivated for 14 days in Labtec chamber slides (Nunc) and incubated for 24 h in the presence of 10 ng/ml lipopolysaccharide (LPS) alone (Sigma), 5 ng/ml recombinant human interferon g alone (Pharma Biotechnologie, Hannover, Germany), 100 ng/ml LPS alone or 10 ng/ml LPS plus 5 ng/ml interferon g. Thereafter, the cells were fixed and examined using the monoclonal antibodies 27E10, 25F9, and anti-type VIII collagen as described above. Patients, samples and tissue preparation Human coronary artery specimens were obtained from the explanted hearts of patients undergoing cardiac transplantation. The protocol used for this study was also approved by the Hospital Ethics Committee. Immediately on removal of the heart, coronary artery samples were placed in cryoprotective medium (Cambridge Instruments, Nussloch, Germany) on cork discs and snap-frozen in liquid nitrogen for cryosectioning. The frozen samples were stored at 280°C until needed.

SDS-PAGE SDS-PAGE was carried out using 7.5% polyacrylamide minigels and the Laemmli buffer system (38). Molecular mass determination was carried out with the following globular protein standards: glyceraldehyde-3-phosphate dehydrogenase (36 kDa), ovalbumin (45 kDa), glutamate dehydrogenase (55 kDa), albumin (66 kDa), fructose-6-phosphate kinase (84 kDa), phosphorylase b (97 kDa), b-galactosidase (116 kDa), and myosin (205 kDa) obtained from Sigma. Westem blots Fourteen-day-old human macrophages were preincubated for 24 h in serum-free medium (RPMI 1640) and for another 24 h in serum-free medium supplemented with 50 mg/ml sodium ascorbate and effectors, as indicated in the figure legends. Cell layers were used for RNA analysis. EDTA and phenylmethysulfonyl fluoride were added to the medium and debris was removed by centrifugation. Prior to lyophilization, samples were dialyzed for 48 h against 0.2 M ammonium carbonate, pH 8.2, containing 5 mM EDTA. Adsorptive losses of type VIII collagen were minimized by treating centrifuge tubes, collection tubes, storage tubes, and dialysis membrane with 100 mg/ml bovine serum albumin in PBS containing 0.05% Tween 20, pH 7.4. Lyophilized protein from 5 ml samples of medium was dissolved in 200 ml reducing Laemmli sample buffer and heated at 80°C for 10 min prior to electrophoresis; 30 ml was loaded per lane. Gels were electroblotted on immobilizing polyvinylide fluoride membranes (Immobilon; Millipore, Eschbach, Germany) with a semi-dry electroblotting apparatus (39). Molecular weight markers were stained with Coomassie brilliant blue. For immunostaining, the membrane was blocked with 3% bovine serum albumin in PBS-Tween for 2 h, incubated with anti-type VIII collagen (clone 6A2) or anti-MMP 1 (clone 41–1E5, ICN) antibodies for 1 h, washed with PBS-Tween, and incubated

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Probes and labeling procedure for in situ hybridization and Northern blotting The following were used for in situ hybridization or Northern blot analysis: probes transcribed from the cDNA clones pBSIIa1Col8, complementary to the mRNA of the a1 chain of human type VIII collagen (a generous gift from Dr. E. Poeschl, University of Erlangen, Nu¨rnberg, Germany); D5/2 human Coll (a gracious gift of Dr. Peter Angel, Deutsches Krebsforschungszentrum, Heidelberg, Germany), complementary to the mRNA for the catalytic fragment of human matrix metalloproteinase 1 (MMP1, also termed collagenase); and cG3PDH (Clontech, Heidelberg, Germany), complementary to the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. In vitro transcription was performed according to the manufacturer’s protocol using digoxigenin (DIG) -labeled-UTP (uridyl triphosphate) (Boehringer, Mannheim, Germany). Northern blot analysis Total RNA was isolated according to Chirgwin et al. (40). For Northern blot analysis, 5 or 10 mg total RNA was fractionated by electrophoresis under denaturating conditions on a 1.1% agarose/formaldehyde gel using standard methods. Northern blot analysis was performed as described previously (41) with the following modifications: membranes were hybridized for 16 h at 72°C in hybridization solution (50% formamide, 5 3 standard saline citrate buffer (SSC; 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0), 0.1% laurylsarcosine, 0.2% SDS, 2% blocking reagent (Boehringer) containing the respective probe (50 ng/ml). Blots were incubated with alkaline phosphatase-labeled sheep anti-DIG F(ab)2 fragments (Boehringer) and detection was performed according to manufacturer’s instructions (Boehringer) using the chemiluminogenic alkaline phosphate substrate CSPD (Tropix/

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Serva, Heidelberg, Germany). Densitometric analysis was performed with the Personal Densitometer and Imagequant software (Molecular Dynamics, Sunnyvale, Calif.). In situ hybridization In situ hybridization was performed (42) with the modifications described below and in ref 43. Five micrometer cryostat sections were dried for 3 min at 50°C and for a further 30 min at room temperature. After dehydration in a graded ethanol series, sections were fixed for 10 min in 4% paraformaldehyde in PBS and washed in PBS. Hybridization was carried out for 16 h at 50°C with 400 ng/ml DIG-labeled cRNA in hybridization buffer containing 2 3 SSC, 1 3 Denhardt’s solution (Sigma), 10% dextran sulfate, 0.5 mg/ml yeast tRNA, 1 mg/ml denatured and sheared herring sperm DNA, and 50% deionized formamide. RNA-RNA hybrids were detected with alkaline phosphatase conjugated anti-DIG-F(ab)2 fragments and the enzyme-labeled fluorescence (ELF) alkaline phosphatase substrate (Mobitech, Go¨ttingen, Germany) usually in combination with immunofluorescence staining of the same section, as described below. Double in situ hybridization Double in situ hybridization was carried out as described above except that two probes labeled either with biotin or DIG were added to the hybridization solution. The RNA probe for type VIII collagen was transcribed in the presence of biotin-16-UTP and the RNA probe for MMP 1 in the presence of DIG-11-UTP (Boehringer), according to manufacturer’s instructions. DIG-RNA-RNA hybrids were detected by immunofluorescence staining with anti-DIG-alkaline phosphatase antibody and the ELF system as described above. Biotin-RNA-RNA hybrids were detected with streptavidinconjugated horseradish peroxidase and the tyramidine signal amplification system TSA-Direct (NEN, Boston, Mass.) according to manufacturer’s instructions. In situ hybridization combined with double indirect immunofluorescence staining using antibodies generated in different species After in situ hybridization, immunofluorescent detection of monocytes and endothelial cells were carried out on the same

Figure 1. Expression pattern of type VIII collagen in advanced atherosclerotic lesions [Stary types IV and V (51)] of human coronary arteries demonstrated by combined fluorescence staining of RNA and protein on the same tissue section. a, b) Double indirect immunofluorescence staining for smooth muscle cells and macrophages combined with in situ hybridization for the a1 chain of type VIII collagen in the same cross section of a type IV lesion. Images were captured with a CCD camera. Smooth muscle-specific myosin is shown in blue, the macrophage marker CD68 is shown in red, and type VIII collagen RNA is shown in green. Panel b shows a detail of panel a. The arrow in panel b indicates a macrophage that stains positive for type VIII

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collagen RNA, as revealed by the yellow color that results from the overlapping of the green collagen and red macrophage. The arrowhead indicates a smooth muscle cell-expressing type VIII collagen. Note that most of the macrophages located in the shoulder and lipid core region of the plaque strongly express type VIII collagen. c) Indirect immunofluorescence staining and in situ hybridization for the a1 chain of type VIII collagen of a type V lesion (fibroatheroma). The digitized image shows a detail of the plaque shoulder region. Type VIII collagen protein is shown as red and RNA as green. Note the area in the plaque shoulder that shows enhanced staining for type VIII collagen protein and type VIII collagen mRNA. Staining of a serial section revealed that this region consists predominantly of macrophages (result not shown). The section also indicates the widespread presence of smaller amounts of type VIII collagen in regions where no collagen mRNA can be detected. This is probably due to deposition of type VIII collagen at an earlier stage in the life of this lesion. The green color lining the lumen of the vessel most likely indicates expression of type VIII collagen by endothelial cells. Bar in panel a indicates 1 mm; bars in panel b, c, 200 mm.

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section. The sections were incubated simultaneously for 1 h with alkaline phosphatase-labeled anti-DIG F(ab)2 fragments from sheep, mouse monoclonal antibodies 27E10, and polyclonal anti-von Willebrand factor antibodies from rabbit in ELF blocking buffer. The bound cell-specific primary antibodies were detected simultaneously by incubation for 1 h with lissamine rhodamine-conjugated goat F(ab)2 fragments directed against mouse IgG, and fluorescein isothiocyanate (FITC) -conjugated goat F(ab)2 fragments directed against rabbit IgG (Dianova), all in ELF blocking buffer (Molecular Probes, Eugene, Oreg.). The alkaline phosphatase-labeled anti-DIG antibodies bound to the RNA-RNA hybrids were visualized with the ELF alkaline phosphatase substrate (Molecular Probes) according to the manufacturers instructions. Sections were embedded in fluorescence mounting medium (Dako), examined with an epifluorescence microscope (Leitz, Wetzlar, Germany), and photographed for color slides (Fujichrome 400, Fuji Industries, Osaka, Japan). In situ hybridization combined with double indirect immunofluorescence staining using antibodies generated in the same species The detection of CD 68-bearing macrophages and of smooth muscle cells was performed in combination with in situ hybridization as follows. After hybridization with a DIGlabeled RNA probe to type VIII collagen mRNA, sections were blocked with ELF blocking buffer (Molecular Probes) and incubated with mouse anti-CD 68 monoclonal antibody and sheep polyclonal alkaline phosphatase-conjugated sheep antiDIG F(ab)2 fragments for 1 h. Sections were then incubated with biotin-conjugated goat anti-mouse IgG antibodies (Sigma) for 3 h. After washing and incubation with ExtrAvidin-TRITC (Sigma) for 1 h, the second monoclonal antibody against smooth muscle myosin (Sigma) was applied to the section and visualized with an FITC-conjugated goat F(ab)2 fragment directed against mouse IgG (Dianova). The monoclonal antibody applied first is blocked by the avidin– biotin complex and therefore is not available for binding by the FITC-conjugated anti-mouse IgG (second) antibody. Visualization of RNA-RNA hybrids, embedding, and examination were performed as described above.

RESULTS Type VIII collagen is produced by macrophages in the atherosclerotic plaque and in mural thrombus We (unpublished observations) and others (28) have found that the atherosclerotic plaque usually contains cells staining positively for type VIII collagen protein and mRNA, and that the majority of these are smooth muscle cells. The expression of type VIII collagen was often accompanied by the presence of cell infiltrates that stain positively for CD68, a general marker for cells of the monocyte-macrophage lineage. Some of these cells also stained positively with the monoclonal antibody 27E10, which has been shown to bind to monocyte infiltrates in inflamed tissues. A combination of in situ hybridization using a probe specific for the a1 component of type VIII collagen mRNA and immunostaining using anti-

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Figure 2. Expression pattern of type VIII collagen of a thrombus overlying an area of transplant vasculopathy in a renal artery of a rejected transplant kidney. a, b) Double indirect immunofluorescence staining for activated monocytes using an antibody directed toward the MRP8/14 heterocomplex of two calcium binding proteins and endothelial cells using an antibody directed toward Von Willebrand factor, combined with in situ hybridization for type VIII collagen of the same tissue section. Nuclei were stained with Hoechst dye. The conventional photographic images shows that some monocytes of the thrombus (red staining in panel b) express type VIII collagen (indicated by arrows in panel a). In addition, endothelial cells show a positive staining for type VIII collagen. c) Controls for immunostaining using ‘non-immune’ mouse and rabbit IgGs as primary antibodies and for in situ hybridization using a sense probe for type VIII collagen.

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Figure 3. Indirect immunofluorescence staining of type VIII collagen (secondary antibody labeled with Cy3, shown here as false color green) in 14-day-old human macrophages before (b) and after (c) partial lysis of cell membranes by treatment with the nonionic detergent NP40. In the nondetergent-treated cells with intact cell membranes, type VIII collagen is distributed evenly in punctate fashion on the cell surface (b, arrows). After permeablization of cell membranes by detergent, additional cytoplasmic staining and punctate deposition of collagen on the culture dish at the site of detached cells (c, arrowhead) is visible. Panel a shows control stained with ‘non-immune’ mouse IgG.

CD68 antibodies revealed that many of the type VIII collagen-producing cells were positive for this marker, indicating that not only smooth muscle cells, but also cells of the monocyte-macrophage lineage produce type VIII collagen in vivo (Fig. 1a, b). Heavy staining for both type VIII collagen mRNA and protein was detected in the macrophage-rich plaque shoulder, a region of the plaque that consists almost exclusively of macrophages (Fig. 1c). The identity of the cells in this section was confirmed by staining with macrophage specific antibody 25F9 and anti-CD68 in serial sections lying directly adjacent to the one in Fig. 1c (data not shown). This indicates that in some regions of the plaque, macrophages may be virtually the only contributor of type VIII collagen. We also performed in situ hybridization combined with double indirect immunofluorescence staining in sections of mural thrombi overlying areas of atherosclerosis or transplant vasculopathy, and found cells that stained positively for type VIII collagen mRNA. Some of these cells were also positive for 27E10, indicating their monocytic nature (Fig. 2). Type VIII collagen is produced by human monocyte-derived macrophages in vitro Immunofluorescence staining of 14-day-old cultured macrophages with intact cell membranes showed that type VIII collagen was localized in a punctate pattern distributed evenly on the cell surface (Fig. 3b). After permeabilization of cell membranes by application of detergent, additional cytoplasmic staining was visible. Moreover, in areas where the cell body had been removed after detergent treatment, a

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punctate deposition of type VIII collagen was visible on the surface of the culture dish (Fig. 3c). This punctate deposition probably mainly represents secreted collagen in the main, but may also be due in part to cell fragments adhering to the cell culture dish. Human monocyte-macrophages in culture stained positively for type VIII collagen protein at all times from 1 h to 3 wk after isolation (Fig. 4a— d; data for the 3 wk time point not shown). During this time the morphology of the cells varied substantially, as can be seen. Expression of the marker recognized by the antibody 27E10 was strong at the start (Fig. 4e) of culture and diminished thereafter (Fig. 4f, g), disappearing after ;2 wk (Fig. 4h). By contrast, the marker for mature macrophages, 25F9, only began to appear at 1 wk (Fig. 4k) and was expressed more strongly after 2 wk in culture (Fig. 4l). Upon Northern analysis (Fig. 5), type VIII collagen mRNA appeared as two bands above and below the 28S RNA band as described previously (33). Type VIII collagen mRNA was detected at all time points up to 3 wk (Fig. 5). Effect of macrophage activation by LPS on the expression of type VIII collagen To study the effect of macrophage activation on the expression of type VIII collagen, 14-day-old monocyte-derived macrophages were incubated in the presence of 10 or 100 ng/ml LPS and analyzed by Northern blots. The level of type VIII collagen mRNA was reduced by ;50% in the presence of 10 ng/ml LPS and by ;70% in the presence of 100 ng/ml LPS (Fig. 6). Incubation of the cells in the presence of 5 ng/ml interferon g, which is known to prime macrophages for activation by LPS, also

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Figure 4. Time course of type VIII collagen expression by cultured macrophages demonstrated by immunostaining. Human monocyte-macrophages were maintained in culture for 1 h, 24 h, 1 wk, and 2 wk (columns from left to right), and incubated with a monoclonal antibody directed against type VIII collagen (a– d), the anti-monocyte monoclonal antibody 27E10 (e– h), or the anti-macrophage monoclonal antibody 25F9 (I–l ) as described in the Methods. Primary antibodies were visualized using the APAAP method. Whereas staining for type VIII collagen did not change appreciably over this time (a– d ), staining with antibody 27E10 disappeared at 2 wk (h); staining with antibody 25F9 appeared at 1 wk (k) and increased thereafter (l ). Appropriate negative controls are shown in the bottom row (m–p).

reduced the level of type VIII collagen by ;50% (Fig. 6). The level of type VIII collagen was reduced by ;95% in the presence of 10 ng/ml LPS when 5 ng/ml interferon g was also added to the culture medium (Fig. 6). By contrast, incubation of the cells with 10 ng/ml LPS produced a

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1.4-fold increase in the level of MMP 1 mRNA, and incubation with 5 ng/ml interferon g and 10 ng/ml LPS a 1.8-fold increase in the level of MMP 1 mRNA, indicating opposite regulation of collagen type VIII and MMP 1 during macrophage activation (Fig. 6).

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Figure 5. Time course of type VIII collagen RNA expression by cultured macrophages. Northern blot analysis with a riboprobe directed toward the a 1 chain of type VIII collagen. 10 mg total RNA isolated from human monocyte-macrophages after 12 h (lane 1), 1 wk (lane 2), 2 wk (lane 3), and 3 wk (lane 4) in culture were loaded on the gel. Note that type VIII collagen RNA was detected at every time point.

Secretion of type VIII collagen and MMP 1 by macrophages in vitro The secretion of type VIII collagen and MMP 1 into the culture medium by 14-day-old macrophages was assessed by Western blotting and immunoprecipitation. The influence of LPS, interferon g, and LPS 1 interferon g on synthesis of MMP 1 and type VIII collagen was analyzed by Western blotting (Fig. 7a). Immunostaining with anti-type VIII collagen antibody revealed a major protein of 64 kDa and a minor one of 58 kDa, as estimated using globular standards. The 64 kDa protein corresponds to an anti-type VIII collagen-positive protein from extracts of Descemet’s membrane and is of the same size as one of the anti-type VIII collagen positive proteins synthesized by corneal epithelial cells as described by Sawada et al. (44), who also found multiple forms of immunoreactive protein in these cells. In smooth muscle cells, a 61 kDa protein detected on Western blotting under reducing conditions has also been reported (28). Using Western blots, we were unable to detect higher molecular weight forms of type VIII collagens in the media of cultured macrophages (data not shown), indicating the absence of cross-linking with dimer and trimer formation. Immunoprecipitation with anti-type VIII collagen antibody revealed a protein of an apparent size of 90 kDa, using globular protein standards (Fig. 7b), which is the size of the undigested monomeric form of type VIII collagen (44). The difference in size between the forms detected by Western blot and that detected by im-

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Figure 6. Influence of LPS, and interferon g on the expression of type VIII collagen RNA and MMP 1 by macrophages. a) Northern blot of total RNA isolated from 14-day-old monocyte-derived macrophages incubated in the absence (lane 2) or presence of 10 ng/ml (lane 3) or 100 ng/ml (lane 4) of lipopolysaccharide alone, 5 ng interferon g alone (lane 5), or 10 ng/ml lipopolysaccharide 1 5 ng/ml interferon g (lane 6). 10 mg RNA was loaded onto each lane. Lane 1 shows a positive control loaded with 5 mg total RNA from human fibroblasts. The upper panel was probed with type VIII collagen riboprobe and the lower panel with a riboprobe for MMP 1. b) Densitometric analysis of panel a. The numbers of the columns indicate the lanes of the gel as described in panel a. Errors resulting from loading different amounts of total RNA were corrected by normalizing each signal to the corresponding signal of GAPDH.

munoprecipitation may be a result of either degradation of the larger species during dialysis prior to SDS-PAGE or posttranslational processing. Type VIII collagen is known to be extremely sensitive to degradation by proteases (45), suggesting that the former hypothesis is the more likely. Secretion of type VIII collagen is decreased in the presence of 100 ng/ml LPS alone or 10 ng/ml LPS combined with 5 ng/ml interferon g (Fig. 7a). Secretion of total MMP 1 was similar in the control and in the presence of LPS alone or interferon g alone (Fig. 7a). By contrast, secretion of MMP 1 was strongly increased in

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the presence of LPS combined with interferon g (Fig. 7a); in this case, MMP 1 occurs mainly in its active form, represented by the 42 and 46 kDa double band; in the other lanes, the proenzyme (57 kDa) and the active collagenase are present in comparable amounts. Synthesis of type VIII collagen and MMP 1 by macrophages in the atherosclerotic plaque

Figure 7. Expression and secretion of type VIII collagen and MMP 1 (collagenase) by cultured human macrophages. a) Western blots of cell culture medium stained with anti-type VIII collagen antibody (upper panel) and anti-MMP 1 antibody (lower panel). The gel was loaded as shown with extract of bovine Descemet’s membrane and medium that had been conditioned by macrophages incubated for 24 h in the presence of 10 ng/ml LPS, 10 ng/ml LPS 1 5 ng/ml interferon g, and 5 ng/ml interferon g. The control consisted of macrophages incubated in RPMI medium without either LPS or interferon g. A major band of 64 kDa apparent molecular mass by comparison with a standard of globular proteins was stained with anti-type VIII collagen antibody. Immunostaining of MMP 1 revealed three proteins with apparent molecular masses of 57 kDa, 47 kDa, and 42 kDa. The two lower molecular mass proteins correspond to the active forms of MMP 1; the 57 kDa form corresponds to pro-MMP 1. The small arrows indicate a double band corresponding to the active forms of MMP 1. No higher molecular weight forms of type VIII collagen were found, indicating the absence of cross-linking with dimer and trimer formation. b) Autoradiogram of metabolically labeled proteins from medium conditioned by macrophages incubated without serum in the presence of 35S-labeled methionine and cysteine after immunoprecipitation with anti-type VIII collagen or non-

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To study the expression of type VIII collagen and MMP 1 by macrophages in the atherosclerotic human coronary artery, we performed double in situ hybridization studies using a biotin-labeled probe complementary to the a1 chain of type VIII collagen and a DIG-labeled probe complementary to the mRNA of MMP 1. The identity of the cells was confirmed in these studies by immunofluorescence staining of serial sections with macrophage-specific antibodies. Figure 8 shows a macrophage-rich ‘shoulder’ region of a human atherosclerotic plaque. Nearly all cells in these regions were identified as macrophages by positive staining with an antibody that recognizes late-stage macrophages (Fig. 8c) and anti-CD68 antibody (Fig. 8d). The double in situ hybridization of a serial section (Fig. 8a, b) shows that macrophages express type VIII collagen and MMP 1 in the atherosclerotic plaque with a broad spectrum of different ratios. The expression pattern of MMP 1 and type VIII collagen in the base of a fibrous plaque is shown in Fig. 9. The digitized image demonstrated that most cells that contribute to MMP 1 expression in this region are nonsmooth muscle cells, but probably macrophages, as indicated by staining of serial sections (result not shown). These nonsmooth muscle cells also express type VIII collagen. In addition, type VIII collagen was expressed by smooth muscle cells of the plaque base.

DISCUSSION The results presented here clearly show that type VIII collagen is synthesized and secreted by human macrophages in vitro and synthesized by macrophages within the atherosclerotic plaque in vivo. The punctate nature in which type VIII collagen is deposited extracellularly in vitro (Fig. 3c) suggests that this protein may play a role in anchoring macrophages

immune mouse IgG (control), analysis by SDS-PAGE, and autoradiography. A protein with an apparent mass of 90 kDa as determined by globular standards was precipitated with anti-type VIII collagen antibody. Acrylamide concentration: 7.5%.

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Figure 8. Expression pattern of type VIII collagen and MMP 1 of type V lesions of a coronary artery demonstrated by double in situ hybridization. a, b) Conventional image of a cross section hybridized with a ribirobe for type VIII collagen, shown in red, and a riboprobe for MMP 1 shown in yellow. Panel b shows a detail of panel a. Arrows point to cells that only stain positive for type VIII collagen RNA, and the arrowheads point to cells that express both proteins in varying ratios (yellow to white). c, d) Serial sections stained with antibody 25F9, a marker of late-stage tissue macrophages (c), and macrophage marker CD68 (d) demonstrated that this region consists predominately of macrophages. Nuclei were stained with Hoechst dye (blue). Size bar in panel a indicates 1 mm; size bar in panels b— d indicates 250 mm.

within the extracellular matrix. Type VIII collagen protein and mRNA were already detectable in adherent monocytes 1 h after isolation from the peripheral blood. Production of type I collagen by macrophages was excluded by Northern blot analysis and by immunocytochemical demonstration of the absence of intracellular procollagen type I (results not shown). After 8 to 10 days in culture, human monocytes take on the characteristics of mature macrophages (46). We found that when cultivated in 20% pooled human serum, monocyte/macrophages retained the ability to synthesize type VIII collagen for up to 3 wk,

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the longest time point examined. During this period, the cells showed marked alterations in their morphology, as previously described. They greatly increased in size and showed a tendency to fuse with formation of polynucleated cells (47). There were also pronounced changes in the expression of marker proteins. For example, the protein detected by the monoclonal antibody 25F9 (36) was induced during this time, whereas expression of the protein complex detected by the monoclonal antibody 27E10 (35) diminished and was virtually undetectable at 3 wk Activation of macrophages by endotoxin

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Figure 9. Double in situ hybridization for MMP 1 and type VIII collagen combined with immunofluorescence staining for smooth muscle cells of a type V lesion of a human coronary artery (other vessel, shown in Fig. 8). The digitized image shows a region of the plaque base. Type VIII collagen RNA is shown in red, MMP 1 RNA in green, and smooth muscle cell-specific myosin in blue. The yellow color is produced when red and green overlap. Smooth muscle cells of the plaque base only express type VIII collagen. Nonmuscle cells of the fibrous region of the plaque express both MMP 1 and type VIII collagen. Staining of a serial section with the macrophage marker CD68 indicates that these cells are probably macrophages.

is known to increase their secretion of degradative enzymes, an effect that is amplified in the presence of interferon g (48). Accordingly, in our 14-day-old macrophages, endotoxin and interferon g increased the in vitro expression of MMP 1 (Figs. 6 and 7). However, the expression of type VIII collagen was decreased under these conditions. This may indicate that endotoxin switches macrophages from a more synthetic to a more degradative phenotype. Comparison of the results shown in Figs. 6 and 7 indicate that the secretion of type VIII collagen does not exactly parallel changes in type VIII collagen mRNA levels. This suggests differences between the kinetics of type VIII collagen mRNA levels and type VIII collagen secretion and processing. Within the atherosclerotic plaque, macrophages are known not to form a homogeneous population. Rather, differences in the degree of activation are known to exist among different parts of the lesion (1). This notion is also supported by our finding that not all those cells in the plaque bearing macrophage markers were also positive for type VIII collagen. Indeed, type VIII collagen-producing and nonproducing macrophages were often found directly adjacent to each other. A similar observation was made of 27E10 positive-staining monocytes that were detected either in association with the endothelium or within mural thrombi; not all monocytes expressed type VIII collagen, and type VIII collagen-expressing

TYPE VIII COLLAGEN IN MACROPHAGES

cells and nonexpressing cells were often located in close proximity to each other. Double in situ hybridization experiments using probes for type VIII collagen and MMP 1 indicated that the expression and secretion of these proteins by macrophages within the atherosclerotic plaque are not a simple mechanism of converse regulation, as we were able to show in vitro using LPS and interferon g. Some macrophages within the lesion express only type VIII collagen, others express only MMP 1, and others express both type VIII collagen and MMP 1. The simultaneous synthesis and secretion of a matrix protein and a degradative enzyme by the same cell may appear contradictory, but this is not necessarily the case. Extracellular proteolysis is a finely regulated process that is incompletely understood and involves multiple proteases and restriction of protease activity to small regions. One can imagine, for example, that a cell moving through the matrix might produce a protease at the leading edge while producing matrix protein at the trailing edge. The balance between a degradative and a synthetic phenotype of macrophages, if this exists in vivo, probably depends on multiple factors such as autocrine or paracrine control and cell–matrix interactions. What are the implications of the finding that monocytes and macrophages produce type VIII collagen? Type VIII collagen, like its close homologue, type X collagen, forms a 3-dimensional network structure (48, 49). Thus type VIII collagen is important in stabilizing Descemet’s membrane within the eye (47), whereas type X collagen is thought to play an important role in tissue stabilization in the epiphyseal transition zone between cartilage and bone. Type VIII collagen has also been shown to be expressed in tissues undergoing active remodeling. For example, it is expressed during neointima formation after injury to the endothelium (33, 50). The cells producing type VIII collagen in such processes have mainly been shown to be either fibroblasts or smooth muscle cells (28, 33, 44, 50). However, the first cells to enter such tissues are usually monocytes, and their ability to produce type VIII collagen may be important for the stabilization of provisional tissue prior to the ingrowth of other connective tissue cell types. This may indicate that macrophages use type VIII collagen as a provisional scaffold, particularly under circumstances when other matrix-producing cells (e.g., smooth muscle cells) are absent. It is of note that expression of macrophage type VIII collagen is greatest in critical regions of the plaque (e.g., the shoulder), which are thought by many to be of central importance in plaque fissuring, plaque rupture, and acute occlusion of the coronary arteries. It is possible that the production of type VIII

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collagen by macrophages in the plaque counterbalances macrophage production of degradative enzymes. Thus, plaque stability may depend on a balance between these two macrophage functions. Such an interaction, if it exists, is likely to be complex. From our results and those of others, it would appear that the phenotype of macrophages may vary widely within the same lesion, and even within the same area of a lesion. Liptay et al. (12) have shown that collagen production by smooth muscle cells is not stimulated in the vicinity of macrophage-derived foam cells, whereas nonfoamy macrophages exert a stimulatory effect (12). Thus, the overall characteristics of a plaque may depend not only on the number of macrophages within the lesion, but also on their degree of activation and localization. This work was supported by grants Cu 31/2–1 to P.C. and Ra 255/7–3 to J.R. from the Deutsche Forschungsgemeinschaft. We thank Karin Tegelkamp, Silke Kummer, Brigitta Milskemper, and Ingrid Otto for excellent technical assistance. We also thank Dr. Susanne Mohr for technical advice and assistance and Marianne Opalka for expert photographic work. We are grateful to Renate Berfeld and Christiane Jordan for their help with the monocytapheresis and to Dr. Mario Deng and Prof. Hans H. Scheld for providing us with arterial tissue.

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