Characterization of specific proteases associated with ...

JOURNAL OF CELLULAR PHYSIOLOGY 151:37%385 (1992)

Characterization of Specific Proteases Associated With the Surface of Human Skin Fibroblasts, and Their Modulation in Pathology FRANCOISE RAYNAUD,* BRIGITTE BAUVOIS, PASCALE CERBAUD, AND D A N IELE EVAIN-BRION Laboratoire cle Physiopathologie du DPveloppernent, C N R S , U R A 1337, Ecole Norrnale Superieure (F.R., P.C., D.E.-B.); and Laboraloire des Cytokines et Interf@rons,Unite INSERM 196, Institut Curie (/3.B.), 75230 Paris Cedex 05, France Human skin fibroblasts were probed for cell surface protease activity. One activity removing dipeptides from the NH2-terminal end of Gly-Pro-pNA was specifically inhibited by di-isopropyl-fluorophosphate (DFP), phenylrnethanesulphonyl fluoride (PMSF), and diprotin A, and thus was identified as dipeptidyl peptidase IV (DPP IV). A group of bestatin-sensitive N-exoaminopeptidase activities was also characterized when Ah-, Leu-, and Arg-pNA were used as chromogenic substrates. Using human monoclonal antibodies antiLCD 13 and anti-CD 26 that recognized, respectively, an N-Ala-aminopeptidase and DPP IV, it was found that human dermal fibroblasts expressed the CD 13 and C D 26 antigen on their surface. In addition, both peptidases were specifically immunoprecipitated by monoclonal antibodies anti-CD 13 and anti-CD 26 from plasma membranes. Cell surface proteolytic activities were also investigated in human fibroblasts derived from dermatological and rheumatic diseases (i.e., psoriasis, rheumatoid arthritis, and lichen planus). It was found that these fibroblasts also expressed both types of proteinases initially identified on normal skin fibroblasts and that the levels of Ala-aminopeptidase activities were similar in all cases. In contrast, the levels of Arg-, Leu-exoaminopeptidase, and DPP IV activities were significantly higher (up to 6.6-fold) in the three pathological fibroblast populations than in their normal counterparts. These proteolytic enzymes, therefore, can potentially serve as markers in dermatological diseases. Taken together, our results suggest that skin fibroblast-derived proteinases associated with both serine and N-aminopeptidase activities may play an important role by participating in the extracellular events associated with fibroblast behaviour. D 1992 WiIey-Liss, Inc.

Increasing numbers of in vitro and in vivo experiments have demonstrated that the dermis, considered for a long time as a passive mechanical support system, plays a key role in controlling the growth and differentiation of the epidermis. In the dermis, fibroblasts function to synthesize and degrade the extracellular matrix, as well as to regulate epidermization by remodeling the network of collagen fibers and by secreting diffusible factors that promote epidermal cell growth (Finch et al., 1989; Coulomb et al., 1989). In in vitro model systems, fibroblasts have been shown to regulate keratinocyte proliferation and differentiation (Bohnert et al., 1986).It also has become clear that fibroblasts play a crucial role in epithelial metaplasia and cancer (Beresford, 1988).In psoriasis, a generalized inflammatory disease with both cutaneaous and rheumatic symptoms, psoriatic fibroblasts induce in vitro the hyperproliferation of normal keratinocytes, suggesting that the primary defect in psoriasis skin may reside in dermal fibroblasts (Saiag et al., 1985). It is now well established that proteolytic activities are present on the surface of various cell types. The 0 1992 WILEY-LISS, INC.

physiological roles of these membrane-bound proteinases, however, remain poorly understood. During embryogenesis, wound healing, and tumor metastasis, cell invasion is considered to require basement membrane penetration and the turnover and destruction of tissue matrices through the action of specific cell surface proteinases. Through their specific binding and their degradation of extracellular components, it appears that cell surface proteases could induce most of these biological processes, including cellular proliferation and differentiation, chemotaxis, cell adhesion and invasion, antigen presentation, and cell-mediated cytotoxicity (reviewed in Bauvois et al., 1991). Most of the proteinases previously described that are produced by fibroblasts are metalloproteinases such a s collagenase. These metalloproteinases are secreted in a

Received February 20,1991; accepted November 25,1991. * To whom reprint requestsicorrespondence should be addressed.

HUMAN SKIN FIBROBLASTS ECTOPROTEASES

latent form and require activation for proteolytic activity. They contain zinc ion and are inhibited by chelating agents and by specific tissue inhibitors (Matrisian, 1990). These metalloproteinases have been implicated in the pathophysiology of diseases such as rheumatoid arthritis. As far as cell surface proteases are concerned, few endopeptidases and exopeptidases have been characterized. Endopeptidases cleave bonds from the ends of polypeptide chains, whereas exopeptidases cleave bonds within one or two residues from the aminoterminal ends. The three well-characterized cell surface proteases are the neutral endopeptidase NEP, also called enkephalinase, the dipeptidyl peptidase IV (DPP IV), and the N-aminopeptidase. The neutral endopeptidase (EC 3.4.24.11), which has been recently identified as the CD 10 antigen (also called CALLA for common acute lymphoid leukemia antigen) is expressed by a wide variety of cells, including fibroblasts (Letarte et al., 1988; Shipp et al., 1988). It is a zinc metalloenzyme which cleaves at the aminoterminal side of hydrophobic residues in peptides such as neuropeptides, enkephalins, interleukin 1, and the chemotactic peptide Met-Leu-Phe (Almenoff et al., 1981; Mumford et al., 1981; Malfroy et al., 1978; Pierart et al., 1988; Connelly et al., 1985). Dipeptidyl aminopeptidase IV (EC.34 15.5) which is identical to CD 26 in the immune system, is a serine exopeptidase which specifically removes the dipeptides of the Gly-Pro type present in collagens (for a review, see Kreisel et al., 1980). DPP IVI CD 26 appears to be expressed by a variety of tissues, including human and murine fibroblasts (Saison et al., 1983; Bauvois, 1988). The N-aminopeptidase (EC 3.4.11.2), which is also a n exopeptidase, is identical to the CD13 antigen (Look et al., 1989) and appears to be expressed on the surface of many cells and tissues other than fibroblasts, such as hematopoietic cells, the intestinal and renal tubular epithelia, and the synaptic membranes of the central nervous system (Kenny and Maroux, 1982; Matsas et al., 1985; Noren et al., 1986; Semenza, 1986). Hence, Ala-aminopeptidase activity was demonstrated for CD 13 molecules specifically immunoprecipitated from the surface of CD 13-positive myeloid cells (Ashmum and Look, 1990). Aminopeptidase N is thought to participate in the degradation of regulatory peptides such as enkephalins (Turner et al., 1985), Recently, it has been suggested that the CD23 antigen is released in soluble form following a n autocatalytic process (Delespesse, 1989). CD 23 is identical to the low-affinity receptor for IgE (FceRII), which is expressed onto a variety of cells, including epidermal Langherhans cells (Lawrence et al., 1975; GonzalezMolina and Spielberg, 1976; Delespesse et al. 1989). Very little work has been done regarding surface peptidases in pathologic conditions. Therefore, we report in this study the biochemical characterization of several peptidases detected at the surface of normal dermal fibroblasts and from dermal fibroblasts in patients with immunological disorders including psoriasis, rheumatoid arthritis, and lichen planus. Up-modulation of the hydrolyzing activities correlated with pathologic fibroblasts in psoriasis and in the dermatological and rheumatic pathologies which can be confused with this disease, suggesting that these ectoenzymes have a role as effector functions at inflammatory dermal sites.

379

MATERIALS AND METHODS Reagents Leu-p-nitroanilide (pNA), Ala-pNA, Gly-Pro-pNA, Arg-Pro-pNA, Val-Ala-pNA, Arg-pNA, Pro-pNA, (Benzoy1:BZ) BZ-DL-Arg-pNA, Gly-Arg-pNA, ZBZGly-GlyLeu-pNA, PMSF, DFP, aprotinin, antipain, leupeptin, PZmercaptoethanol, N-ethylmaleimide, pepstatin, bestatin, and diprotin A were purchased from Sigma Chemical Co (St. Louis, MO). Trypsin and EDTA were purchased from Gibco (Grand Island, NY). DMEM (Dulbecco's Medium Eagle Modified) and fetal calf serum were purchased from Seromed (Berlin, Federal Republic of Germany). The monoclonal antibodies antiCD13 (My7, mIgG1) and antiCDZ6 (Tal, mIgG1) were purchased from Coulter Electronics (Krefeld, Federal Republic of Germany). The isotype control mIgGl was purschased from Sigma. FITC-conjugated goat antimouse Ig was from Immunotech (Luminy, France). Cell cultures Human fibroblasts were isolated from fragments of skin taken during surgery in the trunk area in normal subjects (10 to 65 years old). Fragments from lesional skin of psoriatic patients and patients with lichen planus and from the skin of patients with rheumatoid arthritis were also taken (1child. 10 years old: 5 adults, 40 years old; 1 elderly, 65 years old). Each patient was sex and age matched with one normal control subject. The psoriatic patients and the patient with lichen planus had not been treated. The patient with rheumatoid arthritis had been treated with 5 mg per day of oral prednisone. The skin fragments were rinsed twice with 5 ml of warm DMEM containing 50 Uiml penicillin and 50 piml streptomycin. The washed skin sample was then minced and incubated for 1 h with 2.5 mgiml trypsin in 10 ml of DMEM. The trypsinized skin fragments were pelleted by low-speed centrifugation (400g for 10 m i d and then incubated for 1h a t 37°C in DMEM containing 1mgiml collagenase with shaking in rotary incubator (New Brunswick). After a second low speed of centrifugation, the cells were incubated for 12 h in DMEM medium with 0.5 mgiml collagenase. A cell pellet was obtained by centrifugation a t 400g for 10 min and was resuspended in 5 ml of DMEM supplemented by 10% decompleted fetal calf serum, 50 Uiml penicillin, and 50 p/ml streptomycin and plated onto a 60 mm cell culture dish. Cells were grown in a 5%C 0 2 humidified atmosphere at 37°C and the culture medium was changed every 3 days. At confluency, cells were then cultured in 100 mm culture dish. The cells were used from the 3rd to 6th passage. Cells were collected after a brief treatment with a 0.05% trypsin solution containing 0.02% EDTA. immediately followed by washes in DMEM supplemented with 10% decomplemented fetal calf serum and centrifugation. The cell pellet was then washed in serum-free DMEM and finally washed with peptidase buffer. This method was prefered to t h a t using scraping with a rubber policeman, because it preserved cell viability much more efficiently (less than 10% dead cells obtained with trypsin treatment compared with 4 3 4 4 % with cell scraping). Cell viability was quantified with trypan blue staining.

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RAYNAUD ET AL

Peptidase assays The presence of peptidases was assessed with a number of chromogenic substrates. Leu-pNA, Ala-pNA, Gly-Pro-pNA, Val-Ala-pNA, Arg-pNA, Pro-pNA, benzoyl (BZ)-DL-Arg-pNA, Gly-Arg-pNA, Arg-Pro-pNA were dissolved in dimethyl sulfoxide. DPP IV activity was assayed on Gly-Pro-pNA. Aminopeptidases activities were assayed on Leu-pNA, Ala-pNA, Arg-pNA. Dipeptidyl-aminopeptidase activities were assayed on Gly-Arg-pNA and Val-Ala-pNA. Proteolytic activity was determined from the amount of pNA formed (increase in absorbance a t 405 nm) and quantified by reference to a standard curve prepared with pNA. Routinely, lo5 cells were incubated for 15 min, 30 min, 1h, and 2 h at 37°C with 0.5 mgiml substrate in 100 mM Hepes buffer pH 7.6 containing 0.12 M Nacl, 5mM KC1, 1.2 mM MgS04, 8 mM glucose, and 10 mgiml BSA peptidase buffer. The final volume of incubation mixture was 0.1 ml. A cell-free control mixture was tested in parallel. The reaction was stopped by the addition of 800p.1of 1 M sodium acetate-acetic acid pH 4.5. The cells were pelleted a t 1,OOOg for 10 min. Results were expressed as nanomoles of p-nitroanilide (pNA) formed per hour per lo5 cells at 37°C (each point representing three independent assays).To test the effect of potential inhibitors, the remaining activity was expressed as the percentage of the control activity without inhibitor. Viability of cells measured a t each time of the assay was >95%. Fluorescence labeling All staining steps were done in phosphate buffer saline (PBS) with 1% BSA and were carried out in 96microwell plates on ice. Cells (lo5) were centrifuged to form a pellet and mixed with the appropriate concentration of antibody under saturating conditions (anti-CD 26 or anti-CD 13)and incubated. After 30 min incubation, the cells were washed twice. The secondstage antibody was a FITC-conjugated goat anti-mouse Ig (30 m i d . After incubation the cells were washed twice and fixed with 4% paraformaldehyde in PBS. Cell fluorescent intensity was measured using a FACScan fluorescence activated cell analyzer (Becton-Dickinson, Mountainview, CAI. For each sample, data from 5,000 cells were collected. Fluorescence data were expressed in relative fluorescence intensity. Plasma membrane preparation Plasma membranes were purified from freshly harvested cells either by trypsinization (0.05%Trypsin) or by scraping. No difference in peptidase activities were observed between these two procedures. Routinely scraped cells were used. The cells were collected by centrifugation at 400g for 10 min at 4"C, the supernatant was removed, and the cell pellet was resuspended in 3 ml of buffer A (3 mM Imidazole, 250 mM Sucrose) a t 4"C, washed once in buffer A, and finally was homogenized with a Dounce homogenizer 30 times on ice, and then centrifuged a t 1,200g. The resulting supernatant was collected, and then centrifuged at 100,OOOg in a TL 100 Beckmann for 30 min a t 4°C. The final plasma membrane pellet was solubilized in the peptidase buffer (described above) containing 1% Nonidet P 40 a t

TABLE 1. Characterization of enzyme activities expressed by normal human fibroblasts' Chromogenic substrate Ala-pNA Leu-pNA Arg-pNA Gly-Pro-pNA Val-Ala-pNA GIy-Arg-pNA Pro-pNA N-Cbz-Gly-Gly-Leu-pNA Arg-Pro-pNA Bz-DL-Arg-pNA

Proteolytic activity nmol/h/50 pg substrate/105 cells

50.4 k 11.3 24.7 F 5.6 13.3 f 2.4 8.6 f 2.1 2.6 i 0.7 1.3 0.7 1 0.5 0 0

+

(n = 6 ) (n = 5) (n = 7) (n = 6 ) (n = 5) (n = 3) (n = 2) (n = 2) (n = 1) (n = 2)

'Fibroblasts (1O6/ml)wereincubated as describedin Materials and Methods with0.5 mg/ml of the various chromogenic substrates for 1 h at 37°C. (n =) indicates the number of primary cultures tested. Results are expressed as mean t SEM.

a concentration of 1 mg/ml of protein without proteolytic inhibitors.

Immunoprecipitation Plasma membranes were preincubated in the presence of anti-CD13 o r anti-CD26 monoclonal antibodies or their respective irrelevant isotypes (3 pg per assay) in 15Opl of buffer B (150 mM NaC1, 1%Triton, 50 mM Tris) for 2 h a t 4°C with shaking. Protein G Plus was then added (Clinisciences-15 p1 per pg of antibody), and was followed by 1 additional h of shaking a t 4°C. After centrifugation at 4°C for 3 min a t 7,80Og, subsequent rinsing (4 times with 500 pl of the buffer B without SDS, and 2% aprotinin, and 2 times with the peptidase buffer), and after a centrifugation (7,8OOg, 3 min) the final supernatant was discarded from the pellet. Then, the enzymatic assays were performed on the pellet, which was resuspended in peptidase buffer and incubated with 1mgiml of the chromogenic substrates for 1h a t 37°C.

RESULTS Substrate specificity of peptidase activities associated with human dermal fibroblasts The possible presence of peptidase activities on human dermal fibroblasts was investigated using various chromogenic substrates. Results are presented in Table 1. Intact fibroblasts exhibited high aminopeptidase activities toward Ala-pNA, Leu-pNA, Arg-pNA (13.3 * 2.4 nmolih/50 pg substrate/105cells). The undetectable protease activity of the cells with N-Cbz-GlyGly-Leu-pNA, Val-Ala-pNA, BZ-DLArg-pNA, and GlyArg-pNA demonstrated that Ala-, Leu-, and Arg-pNA hydrolysis was not due t o endopeptidases, but to specific exopeptidases. Table 1 also shows that Gly-PropNA hydrolysis activity was associated with dermal fibroblasts. The latter was previously reported to be the preferential substrate for the serine exopeptidase dipeptidyl aminopeptidase IV (DPP IV) (Kreisel et al., 1982). The lower levels of activity observed with PropNA, Arg-Pro-pNA, and Gly-Arg-pNA indicated the preference for Gly-Pro-pNA as the substrate, typical of DPP IV (Table 1). In addition, as shown in Table 2, when compared with whole cells, the enrichment factors of peptidase activities expressed by plasma membranes were rang-

HUMAN SKIN FIBROBLAST'S ECTOPROTEASES

TABLE 2. Characterization of enzyme activities expressed by alasma membranes of human fibroblasts'

Chromogenic substrate Ala-pNA Leu-pNA Arg-pNA Gly-Pro-pNA

Proteolytic activity nmol/h/50 jtg substrate/pg of protein 13.3 f 1.8 11.3 f 2.0 8.00 ?c 2.5 3.1 f 1.2

(n = 4) (n = 4) (n = 4) (n = 4)

Enrichment factor 19 33 44 26

'Fibroblast membranes (100 pg/ml) were incubated as described in Materials and Methods with0.5mg/mlofthevarious chromogenicsubstratesfor 1hat37"C.(n=) indicates the number of primary cultures tested. Results are expressed as mean SEM. Enrichment factorwas calculated by the comparison ofvalues summarizedin Table 1, i.e. the enzymatic activities obtained in whole cells (for lo5 cells, 70 pg o f protein) and these activities obtained in plasma membranes.

*

381

the cell pellets, which were then resuspended in peptidase assay buffer, were incubated for a n additional of 2 h. Hydrolysis of the different substrates by the supernatant in the absence of cells did not change with time (< 10% activity). Furthermore, a s shown in Figure 1, cell pellets did not release a significant amount of pNA (< 10% at 180 min). These results clearly demonstrate that the release and/or secretion of enzymatic activities by intact fibroblasts was not responsible for the degradation of synthetic substrates by cells.

Kinetics of Ala-, Leu-, Arg-exoaminopeptidase, and DPP IV activities The rates of hydrolysis of Ala-pNA, Leu-pNa, ArgpNA, and Gly-Pro-pNA by the whole dermal fibroblasts ing from 19-fold to 44-fold, indicating that these activi- were dependent on cell concentration with a linear relaties were in the majority located in the plasma mem- tionship up to 12 x lo6 cellsiml (data not shown). The brane preparation. In contrast, a residual activity was different peptidase activities exhibited typical saturafound in the soluble fraction (mean: 12.456,range:6% to tion with increasing amounts of pNA substrate (from 15%,for the four chromogenic substrate tested, data not 0.1 to 5 mgiml). For all the tested substrates, the enzymatic activity was linear up to 1 mgiml (Fig. 2). Their shown). respective K,,, and V,, values were estimated from Effects of protease inhibitors linear Lineweaver-Burk plots. K, values were relaranged Bestatin, which is known t o specifically inhibit N-exo- tively close 0.63-2.2 mM, and values of V,, aminopeptidase activities (Harbeson and Rich, 1988) from 2.5 to 22 nmolesih (data not shown). inactivated almost totally Arg- and Leu-pNA hydrolyImmunological detection, and sis ( S 6%of remaining activity, Table 3) and to a lesser by anti-CD23and immunoprecipitation degree Ala-pNA hydrolysis (25% of remaining activity, anti-CD26 monoclonal antibodies Table 3). The disulfide reducing agent p-2 mercaptoethWe investigated the binding of monoclonal antibodanol (2-ME), potently inhibited Ala-pNA hydrolysis (7% of remaining activity), as well as that of Arg- and ies to human dermal fibroblasts, and reported to be Leu-pNA ( S 37% of remaining activity, Table 3). None respectively clustered in CD13 and CD26. Both monoof the other potential inhibitors tested had any signifi- clonal antibodies bind these cells (> 70%, Figure 3). In cant inhibitory effect on these activities (Table 2). Gly- addition, the experiments using anti-CD13 and antiPro-pNA hydrolysis was strongly inhibited by the CD26 monoclonal antibodies indicate that DPP IV and serine protease inhibitors PMSF and DFP (Table 3). Ala-exoaminopeptidase activity levels recovered in the Diprotin A (Ile-Pro-Ile) is specifically used to inhibit plasma membrane fraction were specifically immunoDPP IV activity (Umezawa at al., 1984). As shown in precipitated with these antibodies. No significant pepTable 3, the exclusive inhibition of Gly-Pro-pNA hy- tidase activity was found when the irrelevant control drolysis by diprotin A confirmed the identity of DPP IV. isotype was used for the immunoprecipitation (Table 4). Unexpectedly, a significant inhibition of Gly-Pro-pNA hydrolysis was observed by a n known inhibitor for Modulation of peptidase activities in pathologies some aspartic proteases, pepstatin. All the other potenWhether or not the protease activities may be regutial inhibitors tested were ineffective (Table 3). lated with cell density was investigated. No significant difference with the four peptidases tested was observed Evidence for cell surface peptidase activities between sparse growing and very confluent, arrested The rates of hydrolysis of Ala-pNA, Leu-pNA, Arg- cells. Table 5 indicates the capacities of primary human pNA, and Gly-Pro-pNA by intact human skin fibro- fibroblast cultures belonging to three different patholoblasts were time-dependent with a linear relationship gies (psoriasis, lichen planus, and rheumatoid arthriup to 3h (Fig. 1A). The detection of the reaction product tis) to express the protease activities initially detected pNA in the extracellular medium without any lag time on normal human fibroblasts. All of the populations suggested the presence of cell surface peptidases. In expressed the N-aminopeptidases and DPP IV. When order to rule out the possible role of any endopeptidasc compared with normal fibroblasts, higher levels of activity released by lysed cells, we have checked Leu-, Arg-aminopeptidase, and DPP IV activities were (Fig. 1B) that less than 5% dead cells occurred during detected on all fibroblast cell populations derived from the 3 h of incubation. Moreover, the possible phagocyto- the dermis of patients with lichen planus, rheumatoid sis of chromogenic substrates, their intracellular degra- arthritis, and psoriasis. In contrast, the levels of Aladation, and the subsequent release of pNA or the secre- aminopeptidase activities were similar in all three tion of intracellular enzymes also had to be excluded. types of cells to those expressed by the normal counterTo rule out these possibilities, the following experi- part fibroblasts. ments were done: Fibroblasts were incubated with the DISCUSSION chromogenic substrates at 37°C for 1h and then centrifuged a t 400g for 5 min (arrow in the Fig. 1A). The In the present study, we have characterized several supernatant containing the chromogenic substrate and types of peptidases present on the surface of human

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TABLE 3. Effects of protease inhibitors on exoaminopeptidase activities detected on normal human fibroblasts' Compound

Concentration (final)

DDPIV

-

100

1 mM 1 mM 1 mM 1 U/ml 100 pM

26 47 25 84

Control Diprotin A PMSF DFP Aprotinin Leupeptin 2-ME N-ethylmaleimide Bestatin Antipain Pepstatin EDTA

Remaining activity (%) Arg-peptidase Ala-peptidase 100 74

2 mM 1 mM 100 W M

110 46 68

1 FM

10 m M

100 77 79 80 92 97

64 106 96 96 34 92 1 GO 127 106

100 89 73 63

1%

Leu-peptidase 100 84 69 107 96 96 37 96 6 106

7 88 25 130 113 98

105

108

'Fibroblasts (106/mI) were incubated with 0.5 mg/ml chromogenic substrate for 1h a t 37OC in the absence (control) or the presence of the potential inhibitor to be tested. Results of one representative experiment are shown.

1 105 1 9 4

5

w

-

W

" 0

0 100. 200 Incubation time (min)

;b5, ;y. ;b* 0

151 10

1%

200

Incubation tlme (min)

0

151 10

IoQ

200

Incubation time (min)

0

100

200

Incubation time (min)

Fig. 1. Kinetic studies ofhydrolysis of Ala-pNA, Leu-pNA, Arg-pNA, and Gly-Pro-pNA by intact fibroblasts. A: Incubations were performed as described in Materials and Methods at 37°C with a final concentration of lo6 cellsiml and 0.5 mg/ml chromogenic substrate. Arrows indicate that in a set of tubes, the cell suspensions were centrifuged after 60 min of incubation; the supernatant containing the chromogenic substrate and the correspondingcell pellets resuspended in pepti-

dase medium were incubated for an additional of 2 h. ( H 1 nmol pNA formed by intact fibroblasts; (0-1 nmol pNA formed by supernatant-resulting from preincubation of cells with the chromogenic substrate; ( m d ) , nmol pNA released by cells preincubated 60 min with chromogenic substrate. In parallel IB), the percenlage of lysed cells was determined for each time point (c-0).

dermal fibroblasts. These cells were shown to possess a t least three N-aminopeptidase activities and a serine protease. N-aminopeptidase activities were demonstrated by the exclusive bestatin inhibitor profile and specificities toward Arg-, Ala-, and Leu-pNA substrates. In addition, all these different forms of N-aminopeptidase activities were inhibited by monoclonal antibodies to CD13. The CD13 molecules have previously been shown to express Ala-aminopeptidase activity (Look et al., 1989). Whether Leu- and Arg-aminopeptidase activities are also expressed by the CD13 antigen or are represented by other molecular entities

clustered in CD13 needs to be considered. Experiments are in progress to examine this point in detail. Identification of the serine protease a s dipeptidyl peptidase IV derived from the selective action of the fibroblasts on Gly-Pro-pNA and the selective sensitivity of the activity to DFP, PMSP, and diprotin A. DPP IV is identical to the CD26 activation marker present on the surface of human T lymphocytes, activated B cells, and macrophages (Sannes, 1983; Scholtz et al., 1985). In the experiments described here, human dermal fibroblasts expressed the marker, as evidenced by immunofluorescent labeling with anti-CDB6 antibod-

-

383

IlUMAN SKIN FIEROELAST’S ECTOPROTEASES

87

I

TABLE 4. Immunoprecipitation-peptidase activities (nmol/h/100 fig substrate/5 UP ulasma membrane protein)’

Ala-aminopeptidase DPP IV

IgGl

Anti-CD13

Anti-CD26

0 (0%) 0.3 (12%)

6.4 -

2.6

-

‘DPPIV and Ala-exoaminopeptidase activities were assayed after immunoprecipitation of plasma membranes of normal fibroblasts with monoclonal antibodies (anti-CD13 = My7, anti-CD 26 =Tal,mIgGl a s isntype).(%) relative activitywhen compared with immunoprecipitation activity obtained with the anti-CD 13 and anti-CD 26.

with Mr of 105 kDa. The lack of inhibition by this monoclonal antibody can reflect the reported heterogeneity of DPP IV isolated from different tissues with 0 4 2 3 4 5 reported submit Mrs of 105 kDa from T cells (monomer), 110-120 kDa from rat liver (homodimer; Elovson, Substrate concentration (mglml) 1980; Kreisel et al., 1982), 120 kDa in placenta membranes (homotrimer; Mizutani et al., 1985), 100 kDa in Fig. 2. Substrate concentration dependence of the peptidase activities exhibited by intact normal human fibroblasts. Fibroblasts (lofi/ mouse fibroblasts (homodimer; Bauvois, 1988), 125 ml, primary cultures) were incubated with different concentrations of kDa and 135 kDa in human fibroblasts (heterodimer; four chromogenic substrates (Ala-, Leu-, Arg-, Gly-Pro-pNA)for 1 h a t Saison et al., 1983). 37°C. 0-0Ala-pNA; 0Leu-pNA; -0 Arg-pNA; D-W Gly-ProAll the four peptidases detected at the surface of derpNA. mal fibroblasts have common characteristics: (1)they are shown to be surface associated (Fig. 1)and (2) they express a Km in the mM range similar to that previously described for aminopeptidase activity of human lymphocytes (Amoscato, 1989; K, = 0.4mM for AlapNA affinity compared with the value of 0.95 mM for U the affinity expressed by dermal fibroblasts) and for W tn mouse fibroblasts (Bauvois, 1988; K, = 0.63 mM for 5 Gly-Pro-pNA affinity compared with the value of 0.63 3 mM value obtained here). z We next investigated these four ectopeptidase activi-I A ties on human fibroblasts derived from dermatological W and rheumatic diseases. In the first dermatological dis0 ease studied, lichen planus-like eruptions are commonly W seen as a manifestation of graft versus host rejection, > being therefore interpretatcd a s an immunological disi= ease (Saurat et al., 1975). Psoriasis and rheumatoid arthritis are diseases that are both associated with W inflammatory and immunological characteristics U (Valdimarsson et al., 1986;Harris, 1990). In contrast to Ala-N-aminopeptidase activity, the levels of Leu- and Arg-activities were enhanced (from 1.5- up to 6.6-fold). With regard to DPP IV, in all patients tested, we observed a significant increase in activity and this increase was even more pronounced on fibroblasts obFLUORESCENCE INTENSITY tained from patients with lichen planus (up to 4.5-fold). To our knowledge, only one previous paper reported Fig. 3. Expression of CD 13 (top) and CD 26 (bottom) antigens on abnormal enhanced level of DPP IV activity in a pathohuman fibroblasts. CD 13 and CI) 26 expression was determined by immunofluorescence studies, a s dcscribed in Materials and Methods. logical condition; in hepatic carcinoma enhanced level Black areas indicate negative controls (IgGl). Data are representative DPP IV was considered to be a marker in the category of one typical experiment. of carcinoembryonic antigens (Kojima et al., 1987). Although the field of membrane bound proteases is currently under intense investigation, little is known ies. However, this monoclonal antibody did not inhibit about the respective roles of proteases located on the the Gly-Pro hydrolyzing activity expressed by dermal surface of cells. Aminopeptidase N E D 13 is thought to fibroblasts. We suggest that the monoclonal antibody participate in the degradation of regulatory peptides anti-CDZ6 used in this work does not recognize the such a s enkephalins, neurokinin A, endorphins, and hydrolyzing site fragment of CD26 molecule present on opioid peptides (Turner et al., 1987). DPP I V E D 26 is human dermal fibroblasts. This monoclonal antibody a n exopeptidase which cleaves Gly-Pro bonds in the anti-CD26 was produced from human activated T cells synthetic substrate Gly-Pro-pNA and has been shown and precipitated a T-cell membrane bound antigen to activate precursor forms of substance P and of

U

5

RAYNAUD ET AL.

384

TABLE 5. N-aminopeptidases and DPP IV activities expressed by human normal and pathological fibroblasts'

Chromogenic substrate Ala-pNA Leu-pNA Arg-pNA G1y-Pro-pNA

a Normal 50.4 f 11.3 24.7 f 5.6 13.3 5 2.4 8.6 f 2.1

Peptidase activity (nmol/h/50 pg/105 cells + SEM) b C d Lichen planus Rheumatoid arthritis Psoriasis 79 49 87 43

+

70 9.2 53 k 5.0 28 f 8.0 20.5 f 10.5

65.5 f 16.6 30.8 f 9.1 23.2 k 2.1 18.2 2.4

+

'Fibroblasts (lOh/ml) were tested for peptidase activitiesin the same conditions as those described in Table 1. We tested one for b. two for c, and three for d primary cultures (for a, see Table 1).

promelittin (Heymann and Mentlein, 1978; Kreil et al., 1980). Collagens contain a high proportion of repeating Gly-Pro-X aminoacid triplets. Some are located in nonhelical peptide chains (Becker et al., 1975), whereas others, such as Gly-Pro-Met and Gly-Pro-Arg, are present at the first and third triplets in the helical region of both a1 and a2 chains (Fietzek and Kuhn, 1976). Using gelatin-containing SDS gels, it has recently been shown that murine thymocyte DPP IV exhibits endoproteolytic activity toward gelatin, therefore indicating that DPP IV can exhibit both endo- and exoproteolytic activities (Bauvois et al., 1991). Taken together, these data clearly show t h a t regulatory proteases such as N-aminopeptidases associated with CD 13 and DPP IV have wider substrate specificity than originally suspected. Experiments are now in progress in our laboratory to ascertain the affinity of the four peptidase activities detected on normal and pathological human dermal fibroblasts on different physiological substrates. Several proteins, such as fibronectin, laminin, and collagens, contribute to the structural integrity of the extracellular matrix and may be involved in fibroblast attachment, migration, growth control, and wound healing. Cell surface receptors for fibronectin and laminin participating in cell attachment have been reported in several fibroblastic cell types (Yamada, 1988; Akiyama et al., 1989; Peltonen et al, 1989). Fibroblastcollagen interactions may occur via fibronectin or laminin, which act a s mediator proteins by forming bridges between specific membrane receptors and collagen molecules (Kleinman e t al., 1981; Woodley et al., 1983; Yamada, 1983; Charonis et al., 1985). However, adhesion mechanisms independent of such bridging proteins have also been demonstrated with respect t o the adhesion of some fibroblastic cell lines to collagen (Grinnell and Minter, 1978; Harper and Juliano, 1981; Aumailley and Timpl, 1986; Bauvois and Roth, 19871, suggesting that receptor-ligand interactions operate in cellcollagen adhesion phenomena. In addition to the VLAproteins which have been implicated in cell-matrix adhesion functions (Humphries, 1990), DPP IV has been suggested a s participating in cell adhesiveness by binding to extracellular matrix collagen type I and fibronectin (Bauvois, 1988: Hanski e t al., 1988; Piazza et al., 1989). Further experiments are required to substantiate the role of DPP IV on the surface of dermal fibroblasts in promoting cell adhesion and migration onto extracellular macromolecules.

In conclusion, although human fibroblasts are capable of secreting a number of metalloproteases, our results clearly show that several degrading activities are also associated with the outer surface of these cells. The increased levels of the serine protease DPP IV and of the Leu- and Arg-N-exoaminopeptidase activities in three diseases associated with a n immunological pathogenesis suggest a potential role for these proteases in the modulation of matrix catabolism and/or circulating factors.

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