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(Journal of Leukocyte Biology. 2001;69:575-582.)
© 2001 by Society for Leukocyte Biology

The collagenous domain of class A scavenger receptors is involved in macrophage adhesion to collagens

Brian B. Gowen*, Thomas K. Borg{dagger}, Abdul Ghaffar* and Eugene P. Mayer*

Departments of
* Microbiology and Immunology, and
{dagger} Developmental Biology and Anatomy, University of South Carolina, School of Medicine, Columbia, South Carolina

Correspondence: Eugene P. Mayer, Department of Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC 29208. E-mail: mayer{at}med.sc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Class A macrophage scavenger receptors (MSRs) have a remarkably broad ligand specificity and are well-known for their roles in atherogenesis and host defense. Recently, we demonstrated that these receptors also recognize and mediate adhesion to denatured forms of type I collagen. In this study, the involvement of the collagenous domain of MSRs in binding to denatured type I collagen was investigated. Transient expression of full-length, native type II MSR in COS-1 cells conferred adhesion to denatured type I collagens, whereas expression of a truncated receptor lacking the distal portion of the collagenous domain did not. Further, a synthetic peptide derived from the collagenous domain was effective in abrogating M{phi} adhesion to denatured forms of type I collagen. We also addressed collagen-type specificity by examining MSR affinity for type III and type IV collagens. As with type I collagen, M{phi}s adhered only to denatured forms of type III collagen. Moreover, the adhesion was mediated by MSRs. In contrast, adhesion to denatured type IV collagen was not shown to be MSR-dependent, but adhesion to the native form was. MSR-mediated adhesion to types III and IV collagens was also shown to be dependent on the collagenous domain. Taken together, these data strongly suggest that the collagenous domain is involved in MSR-mediated adhesion to denatured forms of types I and III collagens and native, but not denatured, type IV collagen.

Key Words: artherogenesis • extracellular matrix • nonfibrillar • monoclonal antibodies


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood monocytes, recruited to sites of infection and inflammation, migrate through the vascular endothelium and into various tissues via an elaborate series of interactions with various collagens and other extracellular matrix proteins. Once in the underlying tissues, monocytes differentiate and develop into macrophages (M{phi}s), which are essential to proper immune function and wound resolution [1 2 3 4 ]. However, the accumulation of M{phi}s at inflammatory sites can also be detrimental in certain cases. In atherosclerosis, which is characterized as a chronic inflammatory disorder, the infiltration and persistent presence of M{phi}s are believed to play a central role in the development and progression of the disease [5 , 6 ]. In addition to releasing various factors that can influence atherosclerotic plaque formation and stability, M{phi}s are largely responsible for the unregulated deposition of cholesterol in atherosclerotic lesions by the uptake of various forms of low-density lipoprotein (LDL) via their class A scavenger receptors.

One of the remarkable characteristics of class A M{phi} scavenger receptors (MSRs) is that they exhibit an exceptionally broad ligand specificity. In addition to their well-characterized affinity for modified LDL [7 8 9 ], they also bind to an assortment of macromolecules (sulfated polysaccharides and polyribonucleotides) [7 ], bacterial cell-wall constituents (lipopolysaccharide and lipotechoic acids) [10 , 11 ], advanced glycation end (AGE) products [12 , 13 ], crocidolite asbestos [14 ], and ß-amyloid fibrils [15 , 16 ]. Moreover, MSRs have been shown to mediate cell adhesion to apoptotic thymocytes [17 , 18 ], activated B lymphocytes [19 ], and serum-coated, tissue-culture plastic [20 ], but the respective ligands in each of these cases are unknown. MSRs on human monocytes/M{phi}s have also been shown to bind AGE-modified matrix proteins [21 ]. We have demonstrated recently that class A type II scavenger receptors are involved in M{phi} adhesion to denatured forms of the major extracellular matrix (ECM) component, type I collagen [22 ]. Because modified or denatured forms of collagens are commonly found in acute, as well as chronic-inflammatory, conditions, the ability of MSRs to mediate adhesion to these collagens may influence M{phi} homing and localization to regions of inflammation.

Class A MSRs from human, bovine, rabbit, and mouse have been cloned, and their structure and ligand specificity are well-characterized [23 24 25 26 ]. The receptors consist of six domains, which are well-conserved across species [27 ]. Type I and type II receptors are products of alternate mRNA splicing and have a short N-terminal cytoplasmic tail, a single membrane-spanning domain, and a brief spacer region, followed by an {alpha}-helical, coiled-coil and a collagen-like (collagenous) domain. At the C-terminal end, immediately following the collagenous domain, type I MSRs contain a cysteine-rich domain of 110 amino acids, which are substituted in the type II receptors by a short stretch of 6–17 amino acids, depending on the species [27 ]. In spite of their abbreviated C-terminal region, the type II receptors display strikingly similar affinity and ligand-binding specificity to that of the type I receptors [14 , 24 , 28 ].

Although the collagenous domain of MSRs is widely accepted as the ligand-binding region for soluble ligands such as modified LDL and other polyanions, there is evidence that other regions of the receptors may also be involved in adhesion. A mouse monoclonal antibody (mAb), designated 2F8, which recognizes type I and type II class A MSRs, inhibits serum-dependent, divalent cation-independent M{phi} adhesion to tissue-culture plastic (TCP) [20 ]. It is important that the epitope for the 2F8 mAb is contained in the {alpha}-helical, coiled-coil domain of the receptors [29 ], suggesting that this region may be involved in ligand recognition and binding to some undefined serum component coating the plastic. These antibodies were also effective in abrogating the uptake of modified LDL by RAW 264.7 M{phi}s [20 ], an interaction that is presumably mediated through the collagenous domain. Thus, it appears that a cooperative multi-domain interaction may be involved in ligand-binding by MSRs.

We have shown previously that various MSR ligands, as well as the anti-MSR 2F8 mAb, could abrogate the adhesion of M{phi}s to denatured type I collagens [22 ]. In this study, we examined which region of MSRs, the {alpha}-helical, coiled-coil and/or the collagenous domain, was involved in binding to denatured type I collagen. Further, we investigated the ability of MSRs to mediate adhesion to various forms of types III and IV collagens to determine their collagen-type specificity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and antibodies
Bovine dermal type I collagen and mouse type IV collagen were obtained from Becton Dickinson (Bedford, MA). Bovine dermal type III collagen was purchased from Chemicon (Temecula, CA). Synthetic decapeptide, Gly-Pro-Lys-Gly-Gln-Lys-Gly-Glu-Lys-Gly (GPKGQKGEKG), derived from the collagenous domain of the murine MSR corresponding to residues (337–346), and the control decapeptide, Glu-Glu-Arg-Ile-Gln-Ser-Ile-Ser-Asn-Ser (EERIQSISNS), derived from the {alpha}-helical, coiled-coil domain (residues 127–136), were synthesized by Sigma Genosys (Woodlands, TX). Poly-L-lysine was obtained from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase (HRP)- and fluorescein isothiocyanate (FITC)-conjugated anti-rat secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). mAbs directed against murine MSR (clone 2F8) were obtained from Serotec (Raleigh, NC).

Cells
The RAW 264.7 murine M{phi} and the COS-1 African green monkey kidney cell lines were obtained from the American Type Tissue Collection (Rockville, MD). The RAW 264.7 M{phi}s were maintained in RPMI 1640 medium (CellGro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), penicillin-streptomycin (100 IU penicillin and 100 µg streptomycin/ml), and 2 mM L-glutamine, obtained from Gibco BRL (Grand Island, NY). For COS-1 cell cultures, Dulbecco’s modified Eagle’s medium (DMEM; CellGro) was used in place of RPMI 1640.

Coating of collagens
For coating native collagen matrices, types I, III, and IV collagens were diluted in phosphate-buffered saline (PBS), pH 7.2, dispensed into 96-well, tissue-culture-treated microtiter plates (100 µl/well), and incubated overnight at 4°C. At neutral pH, types I and III collagens polymerize forming fibrillar collagen matrices. The native type IV collagen coating consists of a nonfibrillar network of collagen. Collagens were coated at final concentrations of 50 µg/ml. To achieve a monomeric (nonfibrillar or non-native) coating, collagens were diluted in 0.02 N glacial acetic acid, in place of PBS. For heat-denaturation of collagens, the proteins were diluted in PBS and then incubated for 15 min in a 100°C water bath. After incubation, the heat-denatured collagens were placed on ice briefly and coated onto 96-well microtiter plates as described above. Preliminary experiments with wells coated with various doses of heat-denatured collagen indicated that complete coating was achieved with as little as 10 µg/ml protein (unpublished results).

Adhesion assays
Adhesion was measured by assaying for the level of the endogenous lysosomal enzyme, hexosaminidase, as originally described by Landegren [30 ]. Briefly, 5.0–7.5 x 104 cells were cultured in coated 96-well microtiter plates for 1 h at 37°C and 5% CO2. Unbound M{phi}s were removed by washing three times with PBS as follows: Plates were inverted, and medium/PBS was removed by a swift downward motion of the plates. Following the last wash, the plates were placed (inverted) into a centrifuge and briefly spun until they reached a relative centrifugal force of 300 g, at which time centrifugation was ceased immediately. For COS-1 transient-transfection studies, the cells were cultured in the presence of 5 mM ethylenediaminetetraacetate (EDTA), and washing was performed by aspiration to gently remove nonadherent cells. After washing, 50 µl hexosaminidase substrate (3.75 mM {rho}-nitrophenyl-N-acetyl-ß-D-glucosaminide, 0.25% Triton X-100, 0.05 M citrate buffer, pH 5) was added to each well. After 1.5–3 h in the presence of substrate, hexosaminidase activity was stopped, and color was developed by adding 75 µl/well of stop/development buffer (5 mM EDTA, 50 mM glycine, pH 10.4). Plates were read at 405 nm on a Benchmark microtiter plate reader (Bio-Rad, Hercules, CA). Unless otherwise indicated, experiments were conducted in serum-free medium, in replicates of three or four, and the experimental groups were compared by Student’s t- test. All experiments were performed a minimum of three times.

Blocking of adhesion to the various conformations of collagens with anti-MSR mAbs was performed as follows: Cells were preincubated at 4°C with the specified antibodies for 30 min and then cultured on the coated collagens. Adhesion was determined after a 1-h incubation period, also in the presence of antibodies. For the collagenous domain peptide studies, coated collagens were treated for 10 min at room temperature with peptides or poly-L-lysine diluted in RPMI prior to the addition of cells. Adhesion was determined after 1 h in culture at 37°C, also in the presence of peptides or poly-L-lysine, as well as 1 mg/ml bovine serum albumin (BSA).

Plasmid constructs
Plasmids containing the full-length murine type II MSR or a truncated version of the receptor were generated by polymerase chain reaction (PCR). The sense primer (5'-CGC CGA GCG GCC GCG CTG TCT TCT TTA CCA GC-3') and anti-sense primer (5'-CGC CGG TCT AGA TTA TAC TGA TCT TGA TCC GC-3') for the full-length receptor have been described previously [7 ]. For the truncated receptor, an anti-sense primer (5'-CGC CGG TCT AGA CTA TTA TAC CTG AGC ACC AGG TGG ACC AGT-3') was designed to insert a Gln-Val and two stop codons after amino acid 298 in the collagenous domain of the receptor. All primers contained additional, unpaired nucleotides (overhangs) that contained a Not I or Xba I cut site to facilitate subcloning of cDNAs. Synthetic oligonucleotide primers were ordered from Integrated DNA Technologies (Coralville, IA).

Full-length and truncated cDNAs were amplified by PCR from RAW 264.7 murine M{phi} cDNAs and subsequently cloned using the PCR-Script Cam cloning kit (Stratagene, La Jolla, CA) following the manufacturer’s specifications. Included in the PCRs was thermostable cloned Pfu DNA polymerase (Stratagene) at a ratio of 1:5 with Taq DNA polymerase (Promega, Madison, WI) for enhanced proofreading and fidelity of cDNA synthesis. The resulting cDNAs were blunt-end-cloned into pPCR-Script Cam and verified by sequence analysis. Plasmids containing the full-length or truncated receptor cDNA inserts were cut with Not I and Xba I, and the gel-purified inserts were subcloned into the mammalian expression vector pcDNA3.1/ZEO (Invitrogen, Carlsbad, CA). These constructs were used for transient transfection into COS-1 cells.

Transfections and analysis of MSR expression
Transient transfections using GenePORTER (Gene Therapy Systems, San Diego, CA) were performed following the manufacturer’s recommendations with minor modifications. Briefly, 0.5 x 106 COS-1 cells were passaged the day before transfection into 60 mm tissue-culture plates. The following day, the media was aspirated, and 2 ml DMEM containing GenePORTER-plasmid DNA complexes (35 µl GenePORTER and 7 µg plasmid DNA) was added to each plate. Four hours after the addition of the complexes, an equal volume of DMEM containing 20% FBS, 2x penicillin-streptomycin (200 IU penicillin and 200 µg streptomycin/ml), and 4 mM L-glutamine was added. The following day, the cells were passaged into poly-L-lysine-coated, 60-mm tissue-culture dishes, and the media was replaced after overnight culturing. Cells were detached by a 15-min treatment with PBS containing 0.5 mM EDTA (37°C and 5% CO2) followed by gentle pipetting to dislodge the cells. Experiments using the COS-1 transfectants were performed routinely 72 h post-transfection. Total MSR protein levels and cell-surface expression were assessed by Western blot analysis and immunofluorescence, respectively. Blots were developed with the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Cell-surface expression of MSR was examined by staining COS-1 transfectants with 2F8 mAbs followed by secondary FITC-conjugated anti-rat antibodies. Flow cytometric analysis of MSR cell-surface expression was performed using an EPICS XL-MCL flow cytometer (Coulter Corporation, Miami, FL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The collagenous domain of class A MSRs has been shown previously to contain the ligand-binding region for modified LDL [7 , 9 ]. To examine whether the collagenous domain is also involved in the recognition of and adhesion to denatured type I collagen, expression vectors that would give rise to truncated or native type II class A MSRs were generated. The truncated receptors lacked critical lysine residues shown to be important for acetylated-LDL recognition [9 ] and contained only a fraction (1/3) of the tripeptide collagen-like repeats present on native receptors (Fig. 1 ). Expression vectors containing the cDNAs for the native and truncated type II MSRs were used for transient transfection of COS cells. These cells were ideal for these studies because they adhere poorly to collagens in the absence of Ca++ and Mg++ (required for ß-integrin binding), and they have been used successfully by others for the expression of functional MSR constructs [7 , 9 ]. The Ca++- and Mg++-free conditions did not affect MSR-mediated adhesion to collagens (unpublished results). Three days post-transfection, COS-1 cells were cultured on native and denatured type I collagen matrices. As shown in Figure 2A , COS-1 cells transfected with native MSR constructs adhered significantly to denatured conformations of type I collagen (nonfibrillar or heat-denatured). Compared with control cells transfected with the empty vector alone, those expressing the native receptors adhered 2.4- and 2.1-fold better to nonfibrillar and heat-denatured type I collagens, respectively (Fig. 2A) . In contrast, cells expressing the truncated MSR transiently failed to adhere significantly to denatured collagens above the background adhesion demonstrated by the cells transfected with the empty vector (Fig. 2A) . The observed increase in adhesion was inhibitable specifically by the 2F8 mAb, specific for murine-trimeric MSRs, whereas treatment with isotype-matched control antibodies had no effect on adhesion (Fig. 2B) . These data are consistent with our previous finding that MSRs mediate M{phi} adhesion to denatured conformations of type I collagen but not the native, fibrillar form [22 ]. Moreover, these results suggest that the collagenous domain is necessary for MSR adhesion to denatured forms of type I collagen.



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  		Figure 1. Diagram of full-length and truncated class A MSRs. The extracellular structure of the receptors includes an {alpha}-helical, coiled-coil domain, a collagen-like (collagenous) domain, and a cysteine-rich domain that is present only on type I receptors. The truncated MSR is missing two-thirds of the collagenous domain (truncated at 8th Gly-X-Y tripeptide repeat; indicated by arrow) including critical lysine residues (underlined) required for acetylated-LDL binding.

 


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  		Figure 2. Adhesion of COS-1 cells, transiently expressing truncated or full-length native class A type II MSRs, to type I collagen. COS-1 cells transiently transfected with the empty expression vector, truncated, or native MSR constructs were harvested 72 h after transfection and plated (5.0x104 cells/well) onto (A) fibrillar and denatured type I collagen matrices. Values are represented as % of total COS-1 transfectants plated (control). (B) Adhesion of COS-1 native MSR transfectants treated with isotype-matched control mAbs or anti-MSR 2F8 mAbs prior to and during culturing on denatured type I collagen matrices. Values are represented as % of untreated control COS-1 cells transiently expressing native MSRs. Adhesion was measured in the presence of 5 mM EDTA and determined as described in Materials and Methods. The data represent the mean and standard deviation of three replicate wells. Significant differences between cells transfected with (A) truncated or native MSR and (B) cells treated with control isotype-matched or 2F8 mAbs were determined by Student’s t-test; *, P < 0.05.

 
Acton et al. [7 ] and others [9 ] have shown that truncated and full-length bovine MSRs are expressed equally well in COS cells. To confirm that our murine constructs were being expressed comparably, proteins were extracted from the transfectants for analysis by Western blot. As shown in Figure 3A , lanes 3 and 4, two bands per lane were resolved, which is consistent with previous findings for the precursor (~171 kD and 188 kD) and fully processed (~197 kD and 214 kD) forms for the truncated and native type II MSRs, respectively [7 ]. The levels of expression of the fully processed, mature, truncated receptor were slightly greater in all transfections analyzed. As expected, no bands were detected in COS-1 cells transfected with the empty expression vector (Fig. 3A , lane 2). In Figure 3A , lane 1, MSR from RAW 264.7 M{phi} protein extract was slightly larger (~218 kD) than the native transfected type II MSR (lane 4), presumably because of differences in post-translational modifications. Although similar protein levels of MSRs were demonstrated by Western blotting, the lack of truncated receptor-transfectant adhesion to denatured type I collagen could still be explained by differences in cell- surface expression. To quantitate the relative levels of expression, flow cytometric analysis was performed on the native and truncated MSR transfectants using the 2F8 mAb. As shown in Figure 3B and 3C , no appreciable differences in the levels of surface expression were evident. Taken together, these findings show that the loss of a portion of the collagenous domain does not alter the expression of truncated MSRs significantly. Thus, the inability of cells expressing the truncated receptors to adhere to denatured forms of type I collagen suggests that the collagenous domain is critical to this interaction.



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  		Figure 3. MSR expression analysis by Western blot and immunofluorescence. (A) Western blot analysis of proteins extracted from COS-1 cells transiently transfected with MSR constructs. Proteins were separated under nonreducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 5% gels, transferred onto polyvinylidene difluoride (PVDF) membranes, and probed with mouse MSR-specific, 2F8 mAbs. Lane 1, RAW 264.7 M{phi} extract. Lanes 2, 3, and 4, COS-1 proteins extracted from empty vector, truncated, and native MSR transfectants, respectively. In lanes 3 and 4, precursor (shorter arrows) and mature (longer arrows) forms of truncated (shaded arrows) and native (black arrows) type II MSRs are resolved. The reduced molecular weight of the truncated receptor is consistent with the abbreviated collagenous domain. (B and C) Flow cytometric analysis of COS-1 cells transiently transfected with MSR constructs. Cells were harvested 72 h after transfection, stained for MSR expression using 2F8 mAbs, followed by FITC-conjugated secondary antibodies, and analyzed for green fluorescence. Shown in B are COS-1 cells transiently expressing the native, full-length MSR (33.6% positives) and in C are cells expressing the truncated receptor (30.5% positives). Percent-positives are based on staining above the background fluorescence (±2%) of cells transfected with the empty vector alone (shaded graph).

 
El Khoury et al. [21 ] have shown that class A scavenger receptor-mediated adhesion to a known ligand (AGE-modified proteins) was inhibited by a synthetic peptide analogous to the ligand-biding region of the collagenous domain [21 ]. To test whether a similar peptide would also block adhesion to denatured forms of type I collagen, the matrices were treated with a synthetic peptide of 10 amino acids derived from the collagenous domain. Because the collagenous domain is a coiled-coil conformation, a control peptide derived from the {alpha}-helical, coiled-coil region of the MSR was used to control for possible conformational inhibitory effects. Following pretreatment, M{phi}s were cultured on the denatured collagen matrices for 1 h, also in the presence of the respective peptides. As shown in Figure 4 , the collagenous domain peptide inhibited M{phi} adhesion to heat-denatured type I significantly (50%) and to a much lesser extent, nonfibrillar collagen (13%), and the control peptide had no effect. To verify the specificity of the peptides, matrices to which M{phi} adhesion is MSR-independent were also treated, and no inhibition was observed (unpublished results). Because lysine residues appear to be critical for adhesion, and the fact that the overall charge of the inhibitory peptide was +2 and that of the control peptide was -1, poly-L-lysine (+35) was also tested to control for nonspecific inhibition. Pretreatment of matrices with poly-L-lysine had no effect on M{phi} adhesion to denatured forms of type I collagen (Fig. 4) . Combined, these data also suggest that the region contained within the peptide is involved in adhesion to heat-denatured type I collagen, although it is unclear whether it is necessary for adhesion to the nonfibrillar form.



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  		Figure 4. Treatment of denatured forms of type I collagen with peptides derived from the collagenous domain of MSRs. Denatured type I collagen matrices were treated for 10 min at room temperature (RT) with collagenous domain peptides, poly-L-lysine, or a control peptide from the {alpha}-helical, coiled-coil region of the MSR. RAW 264.7 M{phi}s (7.5x104 cells/well), suspended in RPMI 1640 medium containing 5 mg/ml peptides or poly-L-lysine and 1 mg/ml BSA, were cultured on the treated nonfibrillar or heat-denatured type I collagen, and adhesion was determined as described in Materials and Methods. Values are represented as % adhesion of M{phi}s cultured on untreated, denatured type I collagens. Data represent the mean and standard deviation of three replicate wells. Significant differences between cells plated on control or collagenous domain, peptide-treated collagen matrices were determined by Student’s t-test; *, P < 0.05.

 
To investigate whether adhesion to denatured collagen was type-specific, M{phi}s were exposed to other collagen types. Type III, like type I, is a fibril-forming interstitial collagen that is found in various tissues. Type IV is a network-forming collagen that is a major constituent of the basal lamina. When M{phi}s were cultured on type III collagen, similar results to those observed on type I collagen were obtained. As seen in Figure 5A , M{phi}s adhered selectively only to denatured forms of type III collagen, and this adhesion was inhibitable by 2F8 mAbs (Fig. 5B) . When M{phi}s were cultured on native and denatured conformations of type IV collagen, they were able to adhere very well to all forms (Fig. 5A) . However, only adhesion to native type IV collagen was blocked markedly (70%) by anti-MSR antibodies, although a slight inhibition (14%) was noted on heat-denatured collagen in most experiments (Fig. 5C) . It is interesting that in contrast to the results for type I [22 ] and type III collagens, M{phi} adhesion to altered or damaged type IV collagen did not appear to be mediated by MSRs because only modest inhibition, at best, resulted from treatment with anti-MSR 2F8 mAbs (Fig. 5C) . Taken together, these findings suggest that M{phi}s adhere to denatured forms of type I and type III collagens via their MSRs. In contrast, recognition of type IV collagen seems to be somewhat different in that M{phi}s were capable of adhering to all forms, however only adhesion to the native matrix was dependent on MSRs.



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  		Figure 5. RAW 264.7 M{phi} adhesion to type III and type IV collagens. M{phi}s (7.5x104 cells/well) were cultured on (A) fibrillar, nonfibrillar, or heat-denatured type III collagens and native, non-native, or heat-denatured type IV collagens, and adhesion was determined as described in Materials and Methods. Values are represented as % adhesion of control M{phi}s cultured on TCP. (B and C) Blocking MSR-mediated adhesion to denatured type III collagens and native and denatured type IV collagens. Anti-MSR mAb inhibition of adhesion to (B) type III and (C) type IV collagens. M{phi}s were incubated with anti-MSR mAb, 2F8 (10 µg/ml), for 30 min prior to culturing on (B) nonfibrillar and heat-denatured type III collagens or (C) native, non-native, and heat-denatured type IV collagens. As a negative control, M{phi}s were incubated with equal concentrations of isotype-matched mAbs. Data represent the mean and standard deviation of four replicate wells. Significant differences between cells treated with 2F8 or isotype-matched mAbs were determined by Student’s t-test; *, P < 0.05.

 
As with type I collagen, only COS cells transfected with full-length, native MSRs adhered to denatured type III and native type IV collagens, and the adhesion was inhibited by the anti-MSR 2F8 mAb specifically (unpublished results), suggesting the involvement of the collagenous domain in this interaction. To further address this issue, the ability of the collagenous domain peptide to block M{phi} adhesion to denatured type III and native type IV collagens was also investigated. As evident on type I collagen (Fig. 4) , adhesion to heat-denatured type III collagen was blocked (~50%) by treatment of the matrix with collagenous domain peptides, whereas no inhibition resulted from treatment with the {alpha}-helical, coiled-coil, domain-control peptide (Fig. 6 ). This finding suggested that adhesion to this form of denatured type III collagen is also dependent on the region contained within the peptide. In contrast, only a small amount (15%) of the MSR- mediated adhesion to the nonfibrillar conformation of type III collagen could be inhibited by the same peptide treatment (Fig. 6) . As seen on nonfibrillar type I and type III collagens, MSR- mediated adhesion to native type IV collagen was abrogated only weakly (11%) by treatment with the collagenous domain peptides (Fig. 6) . As expected, MSR-independent adhesion of M{phi}s to heat-denatured type IV collagen was not affected by treatment with peptides (Fig. 6) . Further, pretreatment of the matrices with poly-L-lysine also had no effect on adhesion (unpublished results). The inability of the collagenous domain peptide to reduce MSR-mediated adhesion prominently to native type IV or nonfibrillar type III collagen suggests that other residues not included in the peptide may also be involved in adhesion to these collagen types and conformations.



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  		Figure 6. Treatment of types III and IV collagens with collagenous domain peptides. Denatured type III collagens and native and heat-denatured type IV collagen matrices were treated as described in Figure 4 . RAW 264.7 M{phi}s were plated on the treated matrices, and adhesion was determined also as indicated in Figure 4 . Values are represented as % adhesion of M{phi}s cultured on respective, untreated matrices. Data represent the mean and standard deviation of three replicate wells. Significant differences between cells plated on control or collagenous domain, peptide-treated collagen matrices were determined by Student’s t-test; *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The distal portion of the collagenous domain of MSRs is widely accepted to contain the ligand-binding region for modified LDL and other ligands [29 ]. In this study, we showed that MSR-mediated adhesion to denatured forms of type I collagen also seems to be dependent on this region, because COS-1 cells transiently expressing truncated receptors failed to adhere to these matrix proteins, and cells expressing the full-length native receptors adhered remarkably well. Thus, the loss of adhesion as a result of removal of the MSR collagenous domain suggests that this region is involved in adhesion to denatured forms type I collagen. Despite these findings, these data do not eliminate the possibility that other regions including the {alpha}-helical, coiled-coil domain may contribute to adhesion. In fact, the adhesion of COS-1 cells transiently expressing native MSRs was inhibited by treatment with 2F8 mAbs specifically. Because the conformational epitope for this antibody is contained within the {alpha}-helical domain [20 , 29 ], it is possible that this region may also influence ligand-binding in cooperation with the collagenous domain. Evidence for such a collaborative interaction between these domains exists in the form of electron microscopy studies that revealed a jackknife conformation in which the collagenous domain hinges back in close proximity to the {alpha}-helical domain [31 ]. Deletion of a major portion of the collagenous domain or binding of antibodies/ligands could disrupt the cooperation between these two domains, thus altering its ligand-binding characteristics. Alternatively, 2F8 mAb-binding to the {alpha}-helical domain may disrupt denatured type I collagen recognition allosterically by impeding the interaction of collagen ligands with contact points contained in the collagenous domain.

To examine further the involvement of the collagenous domain in adherence to denatured forms of type I collagen, a different method for blocking MSR-mediated adhesion was used. In this approach, synthetic peptides derived from the proposed ligand-binding portion of the collagenous domain were used in an attempt to saturate MSR-binding sites. Because the inhibitory peptides may form triple-helical, coiled-coil conformations and are particularly rich in lysine residues, controls included peptides derived from the {alpha}-helical, coiled-coil domain and poly-L-lysine. Although the treatment of nonfibrillar and heat-denatured type I collagen matrices with the collagenous domain peptides was able to reduce M{phi} adhesion significantly to both forms of denatured collagen, only the inhibition observed on heat-denatured matrix was substantial. Thus, although the lysine-rich stretch of residues encompassed in the peptides may be particularly important in mediating adherence to heat-denatured collagen, presumably other portions of the collagenous domain may also be involved in adhesion to nonfibrillar type I collagen. Evidence that a large portion of the collagenous domain is necessary for MSR-binding of Ac-LDL has been demonstrated in a recent study by Andersson and Freeman [32 ]. Alternatively, it is also conceivable that the affinity of the collagenous domain peptides for nonfibrillar collagen is lower than that for the heat-denatured conformation. The affinity of synthetic collagen-like peptides for MSR ligands has been shown to be substantially weaker than that of actual receptors [33 ]. Although these peptide-based studies suggest that the portion of the collagenous domain included in the inhibitory peptide is involved in MSR-mediated binding to various forms of collagen, we cannot eliminate the possibility that the activity of the peptides may be a result of their overall charge. However, we do not suspect this to be the case, because others have clearly demonstrated the importance of this region in ligand-binding.

The copious amounts of type I collagen found throughout the body are an indication of the overall importance of this ECM component. Nevertheless, other fibrillar, as well as network-forming collagens, can have a significant influence on M{phi} behavior and function in specific regions and tissues of the body. To address the issue of collagen-type specificity, M{phi} adhesion to type III and type IV collagens and the involvement of MSRs were investigated. Type I and type III collagens commonly associate to form fibrils in tissues [34 , 35 ]. When cultured on various forms of type III collagen, M{phi}s and COS-1 transfectants were found to display similar MSR/collagenous domain-dependent adhesion characteristics to those observed on type I collagen. Thus, MSR-mediated adhesion to denatured type I collagen is not specific to this collagen type, because adhesion to type III collagen appears to be very similar in nature. It is likely that MSRs are also involved in M{phi} adhesion to other fibril-forming collagens.

Another prominent body collagen is type IV, a network-forming collagen found primarily in basement membranes. When M{phi}s were exposed to native and denatured forms of type IV collagen, they adhered well to all conformations. However, it is interesting that only adhesion to native type IV collagen was determined to be mediated primarily by MSRs, because 2F8 mAb treatment was ineffective in blocking adhesion to non-native and heat-denatured forms of type IV collagen. As with nonfibrillar forms of types I and III collagens, treatment with collagenous domain peptides had a small but significant effect on M{phi} adhesion to native type IV collagen, suggesting a similar mechanism of adhesion to these types and conformations of collagens. It is possible that MSRs play a secondary role to that of the ß integrins or other nonintegrin receptors in mediating adhesion of M{phi}s to denatured type IV collagens, which would explain why attempts to block adhesion with a variety of scavenger-receptor antagonists have been unsuccessful. The identification of receptor(s) involved in M{phi} adhesion to denatured type IV collagens is currently under investigation.

In contrast to our findings, a previous study by El Khoury and colleagues [21 ] demonstrated that human monocytes and monocyte-derived M{phi}s failed to adhere to native type IV collagen but did adhere to its AGE-modified counterpart. Moreover, the human promonocyte cell line, U937, only adhered to the AGE-modified collagen when transfected with bovine type I or type II scavenger receptors [21 ]. The fact that these cells were adhering to the AGE adducts was not surprising, because other studies have also demonstrated MSR affinity for AGE modifications [12 , 13 ]. However, the lack of adhesion to unmodified, native, type IV collagen is not consistent with our data using the RAW 264.7 murine M{phi} cell line and COS-1 cells transiently expressing murine class A type II scavenger receptors. Although there is no definitive explanation for these inconsistencies, they may be a result of a variety of factors, including species variations in M{phi} recognition of type IV collagen, differences associated with the cells that were transfected (COS-1 vs. U937), or perhaps the sources of the type IV collagen. The latter is likely to be the case, because others have also observed differences in cell adhesion to type IV collagen preparations from different commercial sources (I. Corachi, personal communication).

Adhesion of RAW 264.7 M{phi}s expressing type II scavenger receptors was greater than that of COS-1 cells also expressing full-length receptors. Clearly, this is, in part, because of the percentage of COS-1 cells expressing MSRs, which ranged from 30–65%. Additionally, there may be several other factors contributing to the weaker attachment of COS-1 transfectants. First, there are differences in the post-translational machinery between M{phi}s and COS-1 cells, as indicated by the slight difference in molecular weight of respective scavenger receptors synthesized in these cell types (Fig. 3A , lanes 1 and 4). Reduced adhesion may also be a result of overburdening the COS-cell machinery, resulting in the under-glycosylation of the scavenger receptors. Alternatively, COS-1 cells may lack accessory molecules or co-receptors present on M{phi}s, which serve to strengthen the interaction of MSRs with their ligands. Differences in the adhesive properties of distinct cells expressing the same receptor have been documented [36 , 37 ]. For example, expression of type I or type II MSRs in Chinese hamster ovary cells does not confer an adhesive phenotype [36 ], and expression of the same receptors in human embryonic kidney cells does [37 ]. Currently, we are investigating possible candidate co-receptors or accessory molecules that may mediate adhesion-to-collagen ligands or may interact with scavenger receptors during ligand-binding.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from the South Carolina Cancer Center and by the South Carolina Consortium for Cardiovascular Disease and Stroke.

Received May 8, 2000; revised November 15, 2000; accepted November 16, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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