Fibroblast expression of collagen integrin receptors

British Journal of Dermatology 1999; 141: 218±223.

Fibroblast expression of collagen integrin receptors a1b1 and a2b1 is not changed in systemic scleroderma K.HERZHOFF, S.SOLLBERG, C.HUERKAMP, T.KRIEG AND B.ECKES Department of Dermatology, University of Cologne, Joseph Stelzmann Str. 9, D-50924 Cologne, Germany Accepted for publication 28 February 1999

Summary

The skin of patients with systemic scleroderma (SSc) is characterized by excessive extracellular matrix deposition in the dermis. As collagens represent the major structural component, we used ¯uorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of ®broblasts derived from involved skin areas. In contrast to previous reports, no differences in the expression of a1, a2 or b1 integrin subunits, which constitute the major collagen receptors on ®broblasts, were detected on SSc ®broblasts as compared with normal control ®broblasts. Variation of cell culture conditions, e.g. passage number (from 2 to 10), seeding density, cell cycle or serum concentration, did not change this result. These observations indicate that any abnormal response of SSc ®broblasts to their matrix environment is not controlled at the level of receptor expression. Key words: cell±matrix interaction, collagen receptors, extracellular matrix, ®broblast, ®brosis, integrin expression, skin, systemic scleroderma

Systemic scleroderma (SSc) is a generalized disorder characterized by excessive deposition of extracellular matrix (ECM). The pathophysiology is still unclear, but involves alterations of the immune response and vascular changes in early stages of the disease.1±3 Increased matrix deposition has been shown to be due to enhanced gene transcription4±8 and to enhanced stability of mRNA.9,10 We have previously shown that SSc ®broblasts, in comparison with normal control ®broblasts, respond differently to their extracellular environment, indicating an altered feedback.10±12 Fibroblasts interact with their surrounding matrix by way of speci®c receptors, mainly belonging to the b1 family of integrins. As collagens represent the major structural component of ECM, we studied the expression of ®brillar collagen-binding integrins a1b1 and a2b1.13 Using antibody perturbation studies, our previous work showed that a1b1 and a2b1 appear to ful®l distinctly different functions:14 a2b1 is responsible for the mechanical contraction of collagen lattices and concomitant remodelling of collagen ®brils, as well as for the induction of matrix metalloproteinase-1, whereas a1b1 is involved in the downregulation of collagen I in three-dimensional collagen matrices. Correspondence: Dr B.Eckes. E-mail: [email protected]

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Experimental data on the expression of these two receptors on SSc ®broblasts are limited and in part contradictory. Using immunohistochemistry, Gruschwitz et al. detected similar expression of subunits a1 and a2 in early and late stages of SSc and normal skin.15 Sollberg et al. observed elevated b1 integrin epitopes in SSc skin relative to normal skin.16 We reported decreased a2 mRNA levels in SSc ®broblast strains that fall into the `high collagen producer' category.17 This study, however, did not address surface expression of the respective receptor subunit. Furthermore, surface iodination of SSc and control ®broblasts, followed by immunoprecipitation of b1 chains, revealed decreased a1 levels (coimmunoprecipitated) in nine of 11 strains.18 The present study investigated whether the abnormal response of SSc ®broblasts to contact with ®brillar collagen is due to defective matrix recognition by the respective receptors, or due to altered signalling. As quantitative data from previous reports on the expression of integrins on SSc ®broblasts are limited, we performed a systematic in vitro study to determine surface expression levels of the collagen receptors a1b1 and a2b1 on primary dermal ®broblast strains from patients with SSc, using a variety of culture conditions with modulatory potential. q 1999 British Association of Dermatologists

COLLAGEN RECEPTORS ON SSc FIBROBLASTS

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Table 1. Systemic sclerosis (SSc) patient clinical data and demographics

Patient

Sex/age (years)

SSc type and duration of disease (years)

Extracutaneous manifestations

Antinuclear antibody titre/other autoantibodies

M/60 M/47 F/70 M/38 F/57 M/51 M/59 F/46

limited, 30 limited, 12 imited, 13 diffuse, 3 diffuse, 5 diffuse, 6 limited, 4 limited, 3±4

L L A A, L G, L, A G, L, A ± L

1 : < 1280/negative 1 : 1280/Scl-70 1 : 1280/anticentromere 1 : 20,400/Scl-70, histone 1 : 5120/negative 1 : 320/negative 1 : 1280/Scl-70 1 : 1280/SSA, SSB

1 2 3 4 5 6 7 8

L, lung; A, arthralgia; G, gastrointestinal tract (oesophagus).

Materials and methods Subjects Eight patients with limited (n 5) or diffuse SSc (n 3) with cutaneous disease, who were diagnosed according to the classi®cation of LeRoy et al.19 were studied (Table 1). All ful®lled the American College of Rheumatology (formerly the American Rheumatism Association) criteria for the classi®cation of SSc,20 and none was receiving D-penicillamine, corticosteroids or immunosuppressive agents. Antinuclear antibodies, demonstrated by immuno¯uorescence, were present in the sera of all patients. Antitopoisomerase I (Scl-70) antibodies were present in three patients (patients 2, 4 and 7), anticentromere (CEN-B) antibodies in one patient (patient 3), and SSA/SSB antibodies also in one patient (patient 8). Biopsies were taken from involved skin areas after informed consent for routine histology and for ®broblast explant cultures. As controls, normal skin biopsies were taken from individuals undergoing surgery for other reasons. Cell culture Fibroblast cultures were established by outgrowth from skin biopsies of SSc patients or healthy donors. The cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM; Gibco, Eggenstein, Germany), supplemented with 10% fetal calf serum (FCS; PAA, Linz, Austria), glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL) and sodium ascorbate (50 mg/mL) and grown in the moist atmosphere of a CO2 incubator (5% CO2) at 37 8C. Cells were subcultured by trypsinization [0´1% trypsin, 0´02% ethylenediamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS)], reseeded at a ratio of 1 : 2 (6 ´ 103 cells/cm2) and used

in passages 1±10. For some experiments, cells were incubated with transforming growth factor (TGF)-b1 (Peprotech, Rocky Hill, NJ, U.S.A.). Cells were seeded at 1 ´ 104 cells/cm2 and grown for 12 h in the presence of serum, then for 24 h in the absence of serum, followed by a 24-h incubation with 10 ng/mL TGF-b1 in serumfree DMEM. Fluorescence-activated cell sorter analysis Trypsinized cells were resuspended (on ice) at a density of 3 ´ 105 cells/mL in PBS in the presence or absence (negative control) of primary antibodies (TS 2/7: 1 mg/ mL; P1E6: 0´2 mg/mL; 4B4: 1 : 25) in a ®nal volume of 50 mL. All steps were performed on ice or at 4 8C. Following incubation for 10 min, cells were centrifuged (700 g, Eppendorf Centrifuge 5415C), washed in 500 mL of 0´5% bovine serum albumin (BSA) in PBS (Serva, Heidelberg, Germany) and resuspended in 50 mL BSA/PBS containing the secondary antibody (described below, at 0´05 mg/mL). Following 15 min in the dark, cells were washed again with 500 mL BSA/ PBS and ®nally resuspended in 400 mL BSA/PBS for ¯uorescence-activated cell sorter (FACScan) analysis (Becton Dickinson, San Jose, CA, U.S.A.). Five thousand events were measured. Relative ¯uorescence intensity was calculated on the basis of mean ¯uorescence DF F1.MoAb/2.MoAb F2.MoAb, where 1.MoAb and 2.MoAb represent the ®rst and second monoclonal antibodies, respectively. Antibodies All antibodies used recognize the extracellular domains of each integrin subunit chain. MoAb TS 2/721 recognizes the a1 subunit of integrin a1b1 (T Cell Diagnostics, distributed by Endogen, Cambridge, MA, U.S.A.).

q 1999 British Association of Dermatologists, British Journal of Dermatology, 141, 218±223

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MoAb P1E622 detects the a2 chain (Telios Pharmaceutical, San Diego, CA, U.S.A.). MoAb 4B423 is directed against the b1 integrin chain (Coulter Corp., Hialeah, FL, U.S.A.). As secondary antibodies, MoAb X56 (rat antimouse IgG1, phycoerythrin-conjugated antibody, and goat antimouse IgG, ¯uorescein isothiocyanateconjugated polyclonal antibody (both from Becton Dickinson) were used.

105 cells/cm2) vs. subcon¯uent (5´6 ´ 104 cells/cm2) monolayer cultures (Fig. 2B) with cells in passage 6 or lower. Peaks indicated homogeneous a1 expression that was unaffected by the variations introduced, with no evidence for subpopulations with aberrant a1 levels. Further, ®broblasts were serum-starved for 24 h (Fig. 2C,

Results Eight strains of SSc and ®ve of control ®broblasts were investigated by FACScan analysis using speci®c antibodies against the integrin subunits a1, a2 and b1 to investigate and quantify surface expression of the collagen receptors a1b1 and a2b1. In view of our previous results which imply that regulation of collagen levels is mediated by a1b1 integrins, the expression levels of the a1 subunit were of particular interest. All strains tested revealed the same ¯uorescence pattern, showing one uniform peak of a1 integrin expression at the identical position in control and SSc ®broblasts (Fig. 1A). This result clearly indicates that no signi®cant fraction of SSc ®broblasts displays an aberrant a1 surface expression. Quanti®cation of the scans (Fig. 1B) illustrates that surface expression of a1, a2 and b1 in SSc ®broblasts does not differ from controls. Moreover, it shows that levels of a1 and a2 are comparable, whereas b1 levels are 30% higher. This observation was expected, as b1 dimerizes with further a subunit chains (e.g. a5, a6) and is therefore needed in greater amounts. To determine whether variations in cell culture conditions may modulate integrin expression, in particular that of a1, we undertook a systematic analysis to determine whether particular culture conditions might result in the generation of subpopulations with altered a1 expression (Fig. 2). To this end, control ®broblasts from healthy skin were analysed for a1 expression as a function of time in culture (at passage numbers 4, 6 and 10) (Fig. 2A), or in con¯uent (1´7 ´

Figure 1. (A) Surface expression of a1 integrin subunit on normal skin (Co) or scleroderma (SSc) ®broblasts analysed by ¯uorescence-activated cell sorter (FACScan). Dotted lines represent non-speci®c binding of the secondary antibody, used as control. The graphs are representative of experiments using ®broblasts from seven different donors, each analysed at least twice. (B) Surface expression of a1, a2 and b1 integrin subunits on normal (clear bars) or SSc ®broblasts (®lled bars), analysed by FACScan. Relative ¯uorescence was calculated on the basis of mean ¯uorescence, as described in Materials and methods (n 8 for SSc; n 7 for control). q 1999 British Association of Dermatologists, British Journal of Dermatology, 141, 218±223


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Figure 2. Surface expression of a1 integrin subunit on normal skin ®broblasts is not modulated by variations in cell culture. All experiments were performed at least twice using ®broblasts derived from ®ve different donors. (A) Fibroblasts derived from one donor, analysed in passage numbers (p.) 4, 6 or 10. (B) Fibroblasts seeded either as con¯uent (1´7 ´ 105 cells/cm2) or subcon¯uent (5´6 ´ 104 cells/cm2) monolayers. (C) Fibroblasts were serum-starved for 24 h (thin line) and compared with ®broblasts maintained in Dulbecco's modi®ed Eagle's medium/10% fetal calf serum (bold line). (D) Fibroblasts were detached from culture plates with trypsin/ethylenediamine tetraacetic acid (0´1%/0´02%) in phosphate-buffered saline for either 7 min (solid line) or 20 min (dotted line). Note that for experiments described in (A) and (B), the secondary antibody used was ¯uorescein isothiocyanate-conjugated goat antimouse IgG, whereas in (C) and (D), phycoerythrin-conjugated monoclonal antibody X56 was used.

thin line) and compared with ®broblasts maintained in DMEM supplemented by 10% FCS (Fig. 2C, bold line). Both curves are superimposed, indicating that a1 expression is not affected. Lastly, a1 expression was analysed as a function of duration of trypsin treatment needed to detach cultured ®broblasts for harvesting for subsequent antibody incubation and FACScan analysis (Fig. 2D). Incubation of cells (®broblasts in passage numbers below 6) for 7 min (solid line) or 20 min (dotted line) with trypsin/EDTA (0´1%/0´02% in PBS) produced superimposed scans, implying unaltered a1 expression levels.

In summary, no cell culture condition used could be identi®ed which affected a1 surface expression, indicating that a1 expression is very stable and that, if an a1-diminished subpopulation can be produced artefactually or really exists, the number of cells therein is too low to be puri®ed from cultured ®broblasts using sensitive ¯uorescent cytometric techniques. In addition, we examined whether activity of TGF-b1, which has been reported to be involved in the induction of ®brosis,24 can modify integrin expression. SSc or control ®broblasts were incubated in the presence or absence of 10 ng/mL TGF-b1 for 24 h. No differences between control and


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SSc ®broblasts regarding surface expression of a1, a2 or b1 were detected (data not shown).

Discussion This study was undertaken with the aim of investigating on a quantitative basis the expression of collagen receptor subunits a1, a2 and b1 integrins. On ®broblasts, these receptors (a1b1 and a2b1) are the major structures for recognition of collagen and for relaying signals which direct synthesis and degradation of collagen. We were interested in determining whether the altered response of SSc ®broblasts to their surrounding ECM might be due to an altered cell surface expression level of these integrins or due to functional differences. This study focused in particular on the a1b1 receptors for two reasons: ®rst, Ivarsson et al.18 had postulated decreased levels of a1b1 on SSc ®broblasts; second, previous work from our group has shown that in normal dermal ®broblasts, a1b1 is involved in the signalling that controls synthesis of collagen I in response to a three-dimensional collagenous environment.14 As in situ hybridization studies in SSc skin revealed a marked heterogeneity of ®broblasts with respect to their collagen gene expression,25 and as several groups had demonstrated the existence of ®broblast subpopulations in SSc,26,27 we reasoned that decreased a1 expression on SSc ®broblasts might represent a suitable marker for a subpopulation of SSc ®broblasts with high levels of collagen synthesis. So far, no marker for such an activated subpopulation in SSc has been described, and markers specifying these cells would enhance our understanding of the defective regulatory mechanisms leading to the development of skin ®brosis. The results presented here, however, clearly indicate that, using FACScan analysis, no a1-depleted ®broblast subpopulation can be detected in cells cultured from SSc skin, i.e. there is no difference between SSc and control ®broblasts regarding a1 integrin expression at the cell surface. Modulation of cell culture conditions, including serum deprivation, did not affect a1 expression, indicating that expression of this receptor subunit is stable. In addition, TGF-b1, reported to be a ®brogenic agent, did not exert its ®brogenic effects at the level of modulating collagen receptor expression at the surface of SSc ®broblasts as compared with control ®broblasts. Thus, aberrant ®broblast±collagen interaction in SSc is not due to altered expression of a1, a2 or b1 integrin subunits. As this conclusion derives from in vitro experiments, one might argue that integrin expression may

change in culture, and different expression levels may have been seen in ®broblasts isolated directly from SSc skin. To address this, however, because of dif®culties with respect to the cell number required for proper analysis, would require an unjusti®ably large biopsy. A possible explanation for the abnormal response of SSc ®broblasts to their ECM would be the possibility that the a1 subunits on SSc ®broblasts, although expressed at levels identical to controls, assume an activity state which differs from that on control ®broblasts. There is abundant evidence showing that integrin receptors assume different conformational states,28 which are controlled either by external ligand or by the cytoskeletal network and intracellular signalling molecules. These states determine the avidity/af®nity for external ligands and ultimately the activity of the receptor. Therefore, we cannot be certain at this point whether identical amounts of expressed a1 subunits indeed re¯ect identical activity of a1b1 on SSc and control ®broblasts. Last, and in line with the previous hypothesis, a1b1mediated signalling in SSc ®broblasts may be defective downstream of receptor±ligand interaction. Reports on a1b1-induced signalling pathways are scarce: work has been initiated to identify molecules which participate in this signalling cascade. We thus hope to help elucidate whether a1b1 is instrumental in the abnormal response of SSc ®broblasts to their environment, and if so, at which level the defect is seen.

Acknowledgments We thank Dr Karin Hartmann (Cologne), Gloria ChiRosso and Dr Victor Koteliansky (Biogen, Boston, MA, U.S.A.) for discussion and help with the FACScan analysis, and Gabi HuÈppe and Renate Knaup (Cologne) for excellent technical assistance. This study was supported in part by the Deutsche Forschungsgemeinschaft (KR 558) and the European Community (BIO4-CT960036).

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