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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Fibroblasts are the predominant cell type responsible for the synthesis of collagen and other matrix elements in normal and fibrotic lungs. We have previously reported that human lung fibroblasts are heterogeneous in C1q binding and that subpopulations differing in C1q binding can be isolated and subcultured. We have investigated the distribution of receptors for C1q-collagen domain (cC1q-R) and globular domain (gC1q-R) in adult human lung fibroblasts. Fibroblasts were isolated from cultures of adult human lung explants in medium containing fresh- or heated plasma-derived human sera and separated by FACS-cell sorting into populations binding to C1q with high- (HF) and low- (LF) fluorescence. The cC1q-R was obtained from fibroblast membrane preparations by affinity chromatography through an anti-cC1q-R antibody column and its distribution was determined by Western analysis. The presence of gC1q-R was determined by immunoblots using an anti-gC1q-R antibody raised against a synthetic peptide. The results showed that a 54 kD protein crossreacting with anti-cC1q-R antibody was produced by LF cells, but it was barely detectable in HF cultures. Immunostaining with anti-cC1q-R antibody revealed that most of the cells in LF cultures were positive while the HF cells were negative. A 38 kD protein recognized by anti-gC1q-R antibody was produced by lung fibroblasts; however, no differences were detected in its distribution between LF and HF cultures. SDS-polyacrylamide gel electrophoresis of membrane proteins binding to an affinity column of C1q-globular fragment showed that the HF cultures contain a ~ 51 kD protein, which was a minor component in LF membranes. These data show that cC1q-R is expressed predominantly by a population of human lung fibroblasts, while the 38 kD gC1q-R is produced by all cells. Another 51 kD protein appears to be produced by a separate population of fibroblasts which does not express cC1q-R. Our results indicate that two lung fibroblast subtypes may be distinguished based on production of the 54 kD putative cC1q-R and another 51 kD protein which binds to C1q-globular domain.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Fibroblasts are the major cell type responsible for the synthesis of matrix elements in soft connective tissues and in
the lung they constitute 35 to 40% of the cells in the interstitium. These cells remain largely quiescent under healthy
conditions; however, during inflammation and wound healing they are activated to proliferate and synthesize collagen in response to platelet-derived growth factor (PDGF),
transforming growth factor-
(TGF-) and other mediators present in the local environment (1). Recent studies
have demonstrated that fibroblasts manifest differences in
their interaction with and response to inflammatory mediators and that cultures of cells derived from fibrotic and
inflamed tissues differ in growth, collagen synthesis, and
other functional properties (4). These observations indicate that fibroblasts are heterogeneous and that specific
interactions between certain fibroblast subpopulations
and environmental factors may contribute to selection of
specific subpopulations in diseased tissues (12). Observations from our and other laboratories have provided evidence that lung fibroblasts are also heterogeneous. For
example, two morphologically distinct interstitial fibroblast subtypes differing in the presence of lipid have been
identified (8, 9), and cells isolated from normal and fibrotic lungs differ in proliferation and collagen synthesis
characteristics (15). Pulmonary fibroblasts also manifest diversity in cell surface binding to Thy-1 antigen, type
I and III collagens and C1q, and these differences have
been exploited to separate subpopulations of fibroblasts of
lungs and other tissues (10, 18, 19).
The polypeptide subunits of the C1q component of C1 complement consist of discrete collagen (cC1q) and globular (gC1q) amino acid sequence domains, and fibroblasts and other cells bind to the C1q through specific cell surface receptors for the collagen (cC1q-R) and globular (gC1q-R) domains. The cC1q-R receptor, which also binds to collagen-like sequences of lung surfactant protein, participates in immune-complex formation, complement lysis, chemotaxis, phagocytosis and other immune functions (20). Bordin and coworkers showed that different subpopulations of human gingival fibroblasts bind to cC1q and gC1q domains (26, 27). We have previously demonstrated that fibroblast cultures isolated from human lung explants in media containing fresh human serum or complement-inactivated plasma-derived human serum consist of cells binding to C1q and FITC-anti-C1q antibody with low- (LF) and higher fluorescence (HF) (10, 28). The LF and HF fibroblasts corresponded to cells expressing cC1q-R and gC1q-R, respectively (10, 26, 27). We have extended these observations and investigated the distribution of these C1q receptors in human lung fibroblasts, and provide evidence that these receptors may serve to distinguish two lung fibroblast subpopulations in culture.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials
Human complement C1q was obtained from Quidel Corp., San Diego, CA and from Sigma Chemical Co., St. Louis, MO. Mono- and polyclonal antibodies to the cC1q-R were obtained as a generous gift from Dr. Berhane Ghebrehewit, Department of Pathology, SUNY, Stony Brook, NY. The preparation and properties of these antibodies have been described previously (29, 30). Antibody to the gC1q-R-receptor was raised by immunizing rabbits with a synthetic amino-terminal peptide (31). Briefly, a synthetic peptide HTDGDKAFVDFLSDEIKEE was conjugated with keyhole-limpet hemocyanin with 0.2% glutaraldehyde (32) and rabbits were immunized with 20 µg of antigen mixed with 200 µl of incomplete Freund's adjuvant. After four injections at 2-wk intervals, rabbits were tested for antibody production. A final injection was given after 4 wk and animals were bled. The antiserum was purified by ammonium sulfate precipitation and DEAE-cellulose chromatography (32). 125I was obtained from Amersham Corp., and ethyleneglycolbis(succinimidyl succinate) (EGS) and Iodobeads were purchased from Pierce, Rockford, IL. Fresh and heated plasma-derived human sera were prepared as described previously (10). Bradford-protein assay kit was purchased from BioRad, Hercules, CA, and Centricon 10 from Amicon Inc., Beverley, MA.
Cell Culture
Human lung fibroblasts were isolated from explants of pulmonary parenchyma obtained from lung specimens of adult patients undergoing thoracotomy for clinically relevant reasons as described previously in accordance with the approval from the institution's human subjects review committee (10). Primary and subsequent cultures were maintained in Dulbecco-Vogt medium containing 10% of either fresh human serum or heated plasma-derived human serum (10). High- (HF) and low- (LF) fluorescence populations were enriched by cell-sorting using a fluorescence-activated cell sorter (FACS) machine as described elsewhere (10).
Preparation of cC1q and gC1q Fragments and Iodination
C1q was digested with pepsin at 4°C or with purified bacterial collagenase to obtain collagen tail- and globular-fragments of C1q, respectively (28, 33). Iodination of C1q and C1q fragments was done using Iodobeads following the protocol recommended by the manufacturer. Briefly, 125I was activated by incubating with Iodobead for 15 minutes and then added to 5-100 µg protein; after 30 min incubation, iodinated proteins were separated from unincorporated label using a P2 column.
Partial Purification of cC1q-R
Fibroblasts were incubated with serum-free medium containing 5 µCi/ml [35S]-methionine for overnight and lysed in 20 mM NaH2PO4, pH 7.2 buffer containing 0.15 M NaCl, 1 mM each of phenylmethanesulfonylfluoride, and N-ethylmaleimide, 25 mM EDTA, 0.5 µg/ml pepstatin and leupeptin and 0.1% Triton-X 100. The lysate was centrifuged first at 450 × g and then at 20,000 × g for 15 min. The 20,000 × g pellet containing membranes was loaded on a Sepharose 6B column and proteins not retained were applied to a column of anti-cC1q-R receptor antibody coupled to CNBr-activated Sepharose 4B. The column was washed with the buffer to remove unbound proteins and bound proteins recovered with 0.1 M glycine, pH 2.5. The bound fraction was immediately neutralized and concentrated using a Centricon 10. We have previously shown that a 54 kD radioactive protein which crossreacts with anti-cC1q-R antibody can be separated by this procedure from metabolically labeled cells (28).
Separation of Proteins Binding to C1q Globular Domain
Membranes were prepared from [35S]-methionine labeled cells as above in 10 mM Na2HPO4 pH 7.4 buffer containing 20 mM NaCl, 25 mM EDTA, 1 mM each of N-ethylmaleimide and PMSF, 0.5 µg/ml each of leupeptin and pepstatin A and 1.0% Triton X-100. The preparation was pre-run through a Sepharose-6B column and proteins not retained were applied on a column of C1q-globular fragment coupled to CNBr-activated Sepharose 6B. Unbound proteins were removed and bound proteins were eluted with 0.1 M glycine, pH 3.0. The latter was neutralized and concentrated using a Centricon 10. This ligand-affinity procedure separates a labeled 51 kD protein from membrane preparations of metabolically labeled cells, and it does not crossreact with anti-cC1q-R-antibody (28).
Crosslinking
Crosslinking of ligands to receptors was done using either monolayer cultures or membrane preparations. Fibroblast cultures were first incubated in serum-free medium for 18- 24 h prior to crosslinking. Medium was removed and the cells or membrane preparations were incubated with 125I ligands in 10 mM Na2HPO4, pH 7.2, containing 75 mM NaCl. Control incubations contained a 20-fold excess of nonradioactive ligands. After incubation at 4°C for 2 h, a solution EGS in dimethylsulfoxide was added to a final concentration of 2 mM and the mixture was incubated with mixing for an additional 15 min at 20°C. To the preparation an equal volume of 2× sample buffer was added and then subjected to Na-dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. To cleave the crosslink, EGS-treated samples were incubated with 1.0 ml of 1M hydroxylamine at 37°C for 6 h, concentrated through Centricon 10, lyophilized and then dissolved in SDS-sample buffer.
Protein Concentrations
Protein concentrations were determined using BioRad Bradford-protein assay kit.
SDS-Polyacrylamide Gel Electrophoresis
SDS-polyacrylamide gel electrophoresis of crosslinked proteins was carried out on 3-20% gradient gel slabs, while for staining and Western analysis proteins were separated in 7.5% gels (28).
Immunostaining of Fibroblasts
Cells were grown in Lab-Tek chamber slides and incubated in serum-free medium for 18 h prior to immunostaining. The cells were fixed in 2% paraformaldehyde at 4°C for 30 min, and then incubated with a mono- or polyclonal anti-cC1q-R antibody, DEAE-cellulose purified rabbit-anti-gC1q-R antiserum or non-immune serum. Staining was visualized by indirect immunofluorescence with FITC-conjugated secondary antibody.
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Results |
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Abstract
Introduction
Materials & Methods
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Discussion
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Binding of cC1q and gC1q Fragments
Previously we observed by Scatchard analysis that C1q binds
to human lung fibroblasts with two Kas of 1.68-3.50 × 108
M
1 and 1.03-1.5 × 109 M1 (28). These values correspond
to the Kas of the receptors for cC1q (cC1q-R) and gC1q
(gC1q-R), respectively (26, 27). To determine if human
lung fibroblasts indeed bind to these C1q domains, cultures of lung fibroblasts were incubated with 125I-labeled
cC1q or gC1q fragments for 2 h at 4°C and then crosslinked with EGS. SDS-polyacrylamide gel electrophoresis
and fluorography showed that a labeled protein band was
present near the origin in cells incubated with 125I-cC1q fragment (Figure 1a, lane 1), but not after cleaving the crosslink with hydroxylamine (lane 2). This band was present in incubations containing unlabeled gC1q fragment (Figure
1a, lane 3), but absent in those containing unlabelled cC1q
fragment (lane 4). A similar radioactive high molecular
weight protein band was also present in cells incubated
with 125I-globular fragment and EGS; formation of this
band was inhibited by unlabeled gC1q fragment (Figure
1b, lane 2), but not by unlabeled cC1q fragment (Figure
1b, lane 1). The high molecular weight labeled complex
was not present in samples not crosslinked with EGS (data not shown). These data indicated that human lung fibroblasts bind to 125I-labeled collagen- and globular-fragments
of C1q, that the binding is specific and that these fragments
bind to separate receptors. The size of the crosslinked material (~ 450,000), indicated that binding most likely occurs with aggregates of the C1q fragments.
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Detection of cC1q-R in LF and HF Human Lung Fibroblasts
We previously isolated two lung fibroblast subpopulations which manifest either high (HF) or lower (LF) C1q-fluorescence by cell sorting (10, 28). To determine if cultures of these cells produce cC1q-R and differ in its production, approximately 2.5 × 106 LF and HF cells of comparable passage number derived from the same biopsy were incubated in serum-free medium for 24 h, membranes prepared and subjected to immuno-affinity chromatography as described in the methods. This procedure separates proteins binding to anti-cC1q-R antibody, and SDS-polyacrylamide gel electrophoresis revealed that bound fraction contained a 54 kD polypeptide (54 ± 4 in 6 different experiments) which was metabolically labeled and crossreacted with anti-cC1q-R antibody (Figure 2a, b). Then, the production of this protein by LF and HF fibroblasts was compared. Fractions were obtained from LF and HF fibroblasts and subjected to Western analysis. The results showed that the bound fraction from LF-cells contained a 54 kD component which crossreacted with anti-cC1q-R antibody (Figure 2c, lane 1). However, this band was barely detectable in HF-cells (Figure 2c, lane 2).
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These data demonstrated that while a putative cC1q-R is produced by LF lung fibroblasts, it was barely detectable in HF cultures. In order to determine if this difference is due to low levels of cC1q-R expression by all HF cells or due to the presence of less cC1q-R producing cells, both LF and HF cell-pairs obtained from two different lung specimens were grown in Lab-Tek slides, incubated with anti-cC1q-R antibodies and staining visualized by indirect immunofluorescence. LF and HF gingival fibroblasts were also stained for control. Results showed that the majority of LF cells manifested positive immunofluorescence (Figure 3a, b, c), while HF cells were largely negative (Figure 3d, e, f).
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Detection of gC1q-R in HF and LF Cells
Several cell types produce a 33 kD gC1q-R which binds to the globular region of C1q (31, 34, 35). To determine whether LF and HF cultures produced this protein and if they manifested differences in its production, membrane preparations obtained from equal number of LF and HF cultures were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and subjected to Western analysis using anti-gC1q-R-antibody prepared as described in MATERIALS AND METHODS. Both fibroblast cultures contained a prominent protein band migrating with 38 kD (38 ± 2 kD in five different experiments) which was recognized by the antibody; however, no significant differences were noted among the LF and HF cell strains in the intensity of this band in Western analyses (Figure 4). Both cultures were also immunostained with anti-gC1q-R antibody, and, as expected, all cells in these cultures were positive (Figure 5).
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The above data showed that HF and LF lung fibroblasts did not differ in the production of 38 kD gC1q-R species. However, their FACS-C1q binding profile was different and indicated the presence of high affinity C1q-globular domain binding by the HF cells (10, 11, 28). We examined whether this is because of the presence of other proteins binding to the gC1q domain. Membrane proteins were subjected to ligand-affinity chromatography to separate proteins binding to gC1q fragment as described in MATERIALS AND METHODS, and bound proteins were separated by SDS-polyacrylamide gel electrophoresis. Silver staining showed that at least four protein bands were present in the bound fraction from HF cells (Figure 6a, lane 2). One of these was a 51 kD (51 ± 4 kD, n = 3) protein, which was not detectable in the LF-cultures even though twice the number of cells were used for fractionation (Figure 6, lane 1, 2). The other components were present in both cultures. Autoradiography of metabolically labeled preparations showed that the 51 kD component was the major radioactive species (Figure 6b). In order to examine whether the 51 kD component and 38 kD gC1q-R are related, fraction bound to the gC1q-column was subjected to Western analysis using the anti-gC1q-R antibody; however, the 51 kD component was not recognized by the antibody (data not shown). These results indicated that the 51 kD protein is different from the 38 kD gC1q-R and that it is produced by HF lung fibroblasts.
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Discussion |
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Abstract
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Materials & Methods
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Discussion
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We have shown that human lung fibroblast membrane preparations contain proteins which crossreact with antibodies to the cC1q-R and gC1q-R. The former is a 54 kD protein and this size is comparable to that reported for the cC1q-R from other cell types, including endothelial cells and platelets (20, 21, 36). The putative cC1q-R receptor was present in membranes of LF cells. However it was present in much lower concentration in HF fibroblasts, indicating that the cC1q-R is produced predominantly by LF-type fibroblasts. This conclusion is supported by immunostaining data which showed that most LF cells are positive while HF cultures were largely negative (Figure 3). Human lung fibroblasts also produce a 38 kD protein which crossreacts with an antibody raised against a synthetic peptide of gC1q-R; this indicates that the lung fibroblast gC1q-R is larger than the 33 kD molecule reported for other cells (31, 34, 35). Western analysis and immunostaining data demonstrate that it is produced by both LF and HF lung fibroblasts in similar quantities. However, previous observations have shown that these fibroblasts differ in C1q-FACS fluorescence and that the HF cells, not LF cells, manifest higher fluorescence characteristic of gC1q binding (10, 11, 27, 28). The apparent inconsistency between these two observations can be explained by the presence of other proteins that bind to the gC1q. One such protein could be the 51 kD component which is produced by the HF, not LF, fibroblasts (Figure 6). This protein appears to be different from the cC1q-R and 38 kD gC1q-R because it does not crossreact with antibodies to cC1q-R (28), and 38 kD gC1q-R (data not shown). These data indicate that the globular domain of C1q binds with at least two proteins with molecular sizes 38 kD and 51 kD. Presence of multiple proteins binding to both collagen and globular C1q domains has also been reported in other cells (22, 25, 35). The 38 kD species is detectable as a minor component in bound fractions of affinity-gC1q chromatography of both LF and HF cells (Figure 6a, lanes 1, 2). However, while the 38 kD species is produced by all fibroblasts, the 51 kD component appears to be produced by only LF cells.
Western analysis showed that the 38 and 51 kD gC1q-binding proteins are apparently unrelated, however, we have not attempted to confirm if they are indeed different, or to determine whether the latter is an actual receptor located on the cell surface.
The above results offer additional support for the presence of two distinct fibroblast subtypes in the human lung.
Although we performed no experiments, the presence of
such subtypes can be expected to be associated with normal and diseased tissue phenotypes. For example, the
cC1q-R is believed to play a role in cellular-humoral immune network and mediate immune functions such as phagocytosis and cell killing, binding of immune complexes to
cells and secretion of IgG and IL-1, and in cell proliferation response (20, 39). This receptor is expressed by
many inflammatory and connective tissue cell types, and
fibroblasts with the cC1q-R differ from those expressing
gC1q-R in rates of proliferation, collagen synthesis, and
response to cytokines and growth actors (10, 11). The latter fibroblast type has properties of wound healing cells
(11, 14) and it retains high collagen synthesis rates even in
the presence of IFN-
(10). Such differential response of
lung fibroblast types to cytokines has also been reported
by others (13, 18). Thus, cells with low levels of proliferation and protein synthesis rates, such as the cC1q-R cells,
are likely to be responsible for normal tissue homeostasis.
During inflammation and wound healing these cells may
be replaced by the gC1q-R type, which are characterized by higher proliferation and protein synthesis rates. Less or
a lack of susceptibility of collagen synthesis by these cells
to regulatory molecules such as IFN- could lead to a fibrotic response to injury. Such a possibility is supported by
the presence of high C1q-binding (HF-type) fibroblasts in
inflamed and early scleroderma lesions (27, 40). If this is
the case, the subtypes of fibroblasts described above may
provide useful markers in staging clinical course of human
pulmonary fibrosis and have prognostic significance.
The relationship between expression of C1q-R types and other cellular activities such as collagen synthesis is not known; nevertheless our results indicate that antibodies to C1q-receptors may be useful to identify at least the LF- and HF-type fibroblasts. Of the C1q-R types, the cC1q-R is a logical choice as a marker for LF cells as it does not appear to be expressed by the HF fibroblast type. In contrast, the 33-38 kD gC1q-R species appears unsuitable for this purpose because it is expressed by all fibroblasts (Figures 4, 5), and many cell types including fibroblasts co-express this receptor as well as the cC1q-R (31). The 51 kD molecule on the other hand appears suitable for distinguishing the HF cells because it is produced by HF, not by LF, fibroblasts. We do not know whether this molecule is produced by other cell types; nevertheless, experiments utilizing this protein and the cC1q-R to identify fibroblast subtypes must include additional cell-type specific antibodies to confirm fibroblast phenotype; this is because many cells, including inflammatory cells, may produce these receptors (20).
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Footnotes |
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Address correspondence to: A. Sampath Narayanan, Department of Pathology, Box 357470, University of Washington, Seattle, WA 98195.
(Received in original form August 2, 1996 and in revised form November 18, 1996).
Acknowledgments: This work was supported by NIH grant HL 39854. The authors acknowledge Steve Olson for technical assistance and Ms. Shoshana Maslan for help in generating anti-gC1q-R antibody. They also thank Dr. Ghebrehewit for his generous gift of mono- and polyclonal antibodies to cC1q-R.
Abbreviations
cC1q, collagen domain of the C1q;
cC1q-R, receptor for collagen-domain of C1q;
EGS, ethyleneglycolbis(succinimidyl succinate);
gC1q, globular domain of the C1q;
gC1q-R, receptor for globular domain of C1q;
FACS, fluorescence-activated cell sorter;
HF, high fluorescence fibroblasts;
IFN-, interferon-;
kD, kilodaltons;
LF, low fluorescence fibroblasts;
SDS, Na-dodecyl sulfate.
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References |
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Materials & Methods
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Discussion
References
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