Clin. exp. Immunol. (1991) 86, 380-386

Transforming growth factor-fit in rheumatoid synovial membrane and cartilage/pannus junction C. Q. CHU, M. FIELD, E. ABNEY*, R. Q. H. ZHENG*, S. ALLARD, M. FELDMANN* & R. N. MAINI Clinical Immunology Division, Kennedy Institute of Rheumatology, and Charing Cross Sunley Research Centre, London, England (Acceptedfor publication 14 June 1991)

SUMMARY

Transforming growth factor (TGF)-,B has been shown to promote tissue repair and have immunosuppressive actions, and has been proposed to have a role in rheumatoid arthritis (RA). Using immunohistochemical techniques with rabbit F(ab')2 antibodies raised against recombinant human TGF-P1, we have detected TGF-,Bl in the synovial tissue and cartilage/pannusjunction (CPJ) from 18/ 18 patients with RA. TGF-,B1 was found predominantly in the thickened synovial lining layer in RA, but also detected in a perivascular pattern in the synovial interstitium as well as in occasional cells in the lymphoid aggregates. At the CPJ it was found both in cells at the distinct junction as well as in the transitional region of the diffuse fibroblastic zone. The cells staining for TGF-#1 were identified by double immunofluorescence staining as being from the monocyte/macrophage series as well as the type B synovial lining cells. TGF-,B1 was also detected in the synovial membrane sections from 4/4 patients with systemic lupus erythematosus/mixed connective tissue disease and 5/8 patients with osteoarthritis, in a similar distribution to that seen in RA, and in the lining layer of 1/7 normal synovial membranes. These results add to histological evidence confirming that TGF-fI is present in RA synovial cells and those from other arthritides. The distributions of TGF-, I in RA synovial membrane reflects its known actions, as it can be detected at the CPJ, where it could induce repair, and close to activated cells upon which it may exert an immunosuppressive action. Keywords transforming growth factor-#I rheumatoid arthritis synovial membrane cartilage/ pannus junction immunohistochemical technique

INTRODUCTION Transforming growth factor-beta (TGF-P) is a cytokine capable of inhibiting many aspects of immune reaction and promoting tissue repair. It is thus implicated in suppressing synovial inflammation in rheumatoid arthritis (RA), although it may also promote some aspects of inflammation (Wahl, McCartneyFrancis & Mergenhagen, 1989). Of the three members of the TGF-# family, TGF-#l has been the most extensively studied (Roberts & Sporn, 1988). TGF-fll can down-regulate B lymphocyte proliferation and differentiation (Kehrl et al., 1986a), and inhibits T cell proliferation by down-regulating IL-2 receptor expression (Kehrl et al., 1986b). In addition, TGF-#1 down-regulates macrophage HLA-DR expression (Czarniecki et al., 1988) and inhibits the production of IL-1, interferon-gamma (IFN-y) and tumour necrosis factor (TNF) by monocytes and macrophages in vitro (Espevik et al., 1987; Chantry et al., 1989). However, it

augments IL-6 production (Guerne, Carson & Lotz, 1990), and can increase IgA synthesis (Coffman, Lebman & Shrader, 1989).

TGF-P1 also stimulates fibroblast growth, promotes fibrosis, and stimulates endothelial cells to secrete angiogenic factors which promotes angiogenesis (Roberts et al., 1986). It also increases collagen synthesis by fibroblasts and prevents protease release (Roberts & Sporn, 1988) thereby increasing deposition of extracellular matrix and stimulating tissue repair following injury. TGF-fl has been detected in RA synovial effusion and synovial cells at both the protein and mRNA levels (Lafyatis et al., 1989; Fava et al., 1989; Brennan et al., 1990; Miossec et al., 1990), but data of cellular distribution of TGF-,B in the inflamed synovial tissue remains limited. In the present report we have localized TGF-fI both in the synovial membrane and at the cartilage/pannus junction (CPJ) in RA, and in the synovial membrane in other inflammatory arthritides, osteoarthritis (OA), and rarely in the normal synovial tissue. We have identified the phenotypes of cells producing TGF-#l in RA synovial membrane as being from monocyte/macrophage lineage, as well as the type B lining layer cells.

Correspondence: Dr C. Q. Chu, Division of Clinical Immunology, Mathilda and Terence Kennedy Institute of Rheumatology, 6 Bute Gardens, Hammersmith, London W6 7DW, UK.

380

381

Transforming growth factor-/Il in RA MATERIALS AND METHODS Preparation of samples Eighteen patients with definite or classical RA (Arnett et al., 1988) were included in the study. Synovial membrane tissue was obtained at arthroscopy or arthroplasty. Diseased CPJ tissue was obtained from 11 patients undergoing joint replacements and normal tissue was obtained from amputation specimens (donated by Dr M. Bayliss, Kennedy Institute of Rheumatology, London). Synovial membrane samples from patients with OA, systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) were all obtained at arthroscopy performed during the active phase of disease, usually with synovitis and effusion. All tissue samples were immediately snap-frozen in liquid nitrogen or hexane and stored at -70°C until required. Sections of 5 or 6 gtm were cut on a cryostat, and the sections were stored at -70°C for up to 6 months until used.

Anti- TGF-flJ antibody preparation Polyclonal rabbit anti-TGF-,B1 antiserum was raised against recombinant human TGF-,BI (Genentech, San Francisco, CA), and it acts as a neutralizing antibody (Chantry et al., 1989). For immunohistochemical staining the F(ab')2 fragments were prepared as described by Field et al. (1991) and Chu et al. (1991). The protein A purified IgG fraction of the antibodies was digested with pepsin (Sigma, Poole, UK). The undigested IgG and Fc fragments were removed using protein A linked Sepharose 4B (Pharmacia, Milton Keynes, UK). The anti-TGF/B1 F(ab')2 fragments were purified on a CNBr-activated Sepharose 4B affinity column coupled with recombinant human TGF-f31. F(ab')2 fragments were eluted with 3 M guanidine chloride (Sigma), concentrated and labelled with N-hydroxysuccinimidobiotin (Pierce Chemical Co., Rockford, IL). The activity and specificity of the F(ab')2 fragments were tested by ELISA against recombinant TGF-,B1 and other cytokines as described by Chu et al. (1991).

Immunohistochemical staining Sections were fixed in acetone/methanol (1:1) at -20'C and treated with 0 3% hydrogen peroxide in methanol to block endogenous peroxidase. Nonspecific binding was blocked by incubation of the sections with 20% normal goat serum. The sections were washed and incubated for 1 h with anti-TGF-,Bl F(ab')2 fragments linked to biotin at appropriate concentration (10 pg/ml) and binding detected with streptavidin-conjugated horseradish peroxidase (Amersham, Little Chalfont, UK) diluted in 2% normal goat serum. This was developed with 0-5 mg/ml diaminobenzidine (Sigma). Synovial membrane sections were counterstained with haematoxylin and CPJ sections with safranin-O and haematoxylin, and each was viewed by light microscopy (Leitz).

The following control antibodies sequential sections:

were

(1) Normal rabbit F(ab')2 fragments were prepared using a Sephacryl S-200 column from pepsin digested pre-immunized rabbit IgG fraction (Chu et al., 1991). The normal F(ab')2 fragments linked to biotin were incubated with the sections at an equivalent concentration. (2) Anti-TGF-PI F(ab')2 fragments were absorbed with TGF-f31 or TNF affinity column, and the drop-through from each column was tested for staining on sections. To identify cell phenotypes, double immunofluorescence staining was performed as described by Field et al., (1991). Briefly, the sections were treated as above, and binding detected with streptavidin-conjugated Texas-Red (Amersham). This was followed by incubation of the sections with monoclonal cell marker antibodies (see Table 1 for details). The binding was detected with anti-mouse IgG or IgM conjugated with fluorescein (Sigma). Fluorescein-conjugated F(ab')2 fragments of goat anti-human F(ab')2 immunoglobulin (ICN, Lisle, IL) were used to detect plasma cells containing immunoglobulins. The slides were viewed under ultra violet light (Leitz).

Quantification of cells The numbers of TGF-B I staining cells were graded by counting up to 500 cells in five high power fields in each area. To quantify the variety of the cells containing TGF-f31, the number of TGF,I-positive cells co-staining with the cell marker antibodies was expressed as a percentage of the total number of TGF-Ipositive cells. RESULTS

Antibody reactivity On ELISA, the anti-TGF-#l antibodies bound to recombinant TGF-,B1 in a dose-dependent manner, but not to recombinant TNF, IL-lo or IL-6 (Fig. 1).

TGF-fB localization in synovial membrane Abundant TGF-/JI -staining cells were detected in all of 18 RA synovial membrane sections examined. These cells were found throughout the sections, but often accumulated in the lining layer (Fig. 2a), in the inter-lymphoid aggregate area (Fig. 2c) and in a perivascular distribution (Fig. 2b). While fewer cells within the lymphoid aggregates were stained for TGF-#1 (Fig. 2c, Table 2), in the fibroblastic areas almost 80% of the fibroblast-like cells were shown to contain TGF-,BI (Fig. 2d). Seven normal synovial membrane sections were examined and one was shown to contain cells stained for TGF-,I, comprising 1% lining layer cells (Table 2). The intensity of staining was much less than that in RA sections.

Table 1. Monoclonal antibodies used in identification of phenotypes of TGF-1ll-staining cells

Antibody Leu-4 EBMII Mab67 RFDl

Specificity Pan T cells (anti-CD3) Macrophages/monocytes (anti-CD68) Type B synovial lining cells Dendritic cells (class II associated antigen)

used to stain the

Isotype

Source and references

IgG 1

Becton Dickinson, Mountain View, CA Dakopatts (Kelly et al., 1988) Dr N. Hogg (Stevens et al., 1990) Dr L. Poulter (Poulter et al., 1986)

IgGI

IgGl IgM

C. Q. Chu et al.

382

TGF-fI-staining cells were also found in the synovial membrane sections from patients with SLE/MCTD and OA patients. These positive cells were seen in a similar distribution pattern to that in RA sections, but in lower numbers (Table 2).

Table 2. TGF-Pll-containing cells in the synovial membrane in arthropathies

TGF-f3l localization in CPJ As described by Allard et al. (1987), two types of CPJ were seen after staining with haematoxylin, fast green and safranin-O. A distinct CPJ was characterized by a cellular and vascular pannus with a distinct margin demarcating the pannus and underlying cartilage. At this site pannus can often be seen invading the underlying cartilage. In distinct CPJ, TGF-f5-containing cells were detected throughout the pannus area, in particular at the interface of cartilage and pannus and at the site of cartilage erosion (Fig. 3a, b). About 90% of the pannus cells eroding into cartilage stained for TGF-3I. In indistinct CPJ a transitional fibroblastic zone (TFZ) consisting of fibrous tissue and fibroblast-like cells connects the pannus and cartilage with no evidence of cartilage invasion. This site of CPJ has been suggested to be an attempt at repair and in this site TGF-#fI was detected in over 80% fibroblast-like cells in the TFZ (Fig. 3c). None of the cells was shown to stain with anti-TGF-,BI antibody in seven normal synovium-cartilage junction sections (Table 2).

Diagnosis

Antibody specificity Normal rabbit F(ab')2 fragments showed no binding to TGF-,BI on ELISA and no staining was observed with these antibodies on synovial membrane sections or those of the CPJ (Fig. 3d). The TGF-P3I -staining cells were not observed when the sections were stained with recombinant TGF-,BI -absorbed antibody (Fig. 2e), but were positive when stained with the antibodies that had been passed through the TNF column (data not shown).

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Inter-lymphoid aggregate

18/18 4/4 5/8 1/7

74 (61-80) 40 (30-55) 25 5 (4-44) 1

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RA, Rheumatoid arthritis; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; OA, osteoarthritis; NP, not present. -, TGF-flI-staining cells not detectable.

Characterization of phenotypes of TGF-f3I positive cells in RA synovial membrane and CPJ Double staining of synovial membrane with monoclonal antibodies demonstrated that the majority (70%) of the cells containing TGF-#Il stained with the macrophage/monocyte cell marker CD68 in synovial tissue (Fig. 4a/b). In the lining layer this figure rose to 90% (Table 3). However, up to 7% of the cells stained with the MoAb 67, a marker of type B fibroblast derived synovial lining cells (Stevens et al., 1990). A few TGF-fllcontaining cells in the interstitium were also positive for the RFD1 antigen suggesting that some dendritic cells may make TGF-#1I in synovial tissue. An occasional T cell and no plasma cells stained for TGF-f I (Table 3). In the interstitium - 30% are unaccounted for, but morphometrically many fibroblast-like cells stained for TGF-f 1, indicating that they may also produce

TGF-f1. At the distinct CPJ, 50% of TGF-P 1-positive cells co-stained with anti-CD68 antibody, and 4% were RFDI positive (Table 3), but 40-50% were not identified with this panel of monoclonal antibodies. DISCUSSION The results of the present study add histological evidence to show that TGF-,13 is present in RA synovial tissue. The intracellular detection of the protein is consistent with the previous reports that TGF-fl is locally produced by synovial cells in RA (Fava et al., 1989; Lafyatis et al., 1989; Brennan et al., 1990; Miossec et al., 1990). Using the specific F(ab')2 antibody to overcome non-specific binding due to locally synthesized rheumatoid factors TGF-,ll-containing cells have been localized throughout the synovial tissue, but mainly in the lining layer, inter-lymphoid aggregate region and including the fibroblastic zone and perivascular area of synovial tissue, as well as at the CPJ. TGF-Il is found in RA synovial fluid both in the latent and bioactive forms (Fava et al., 1989; Brennan et al., 1990; Miossec et al., 1990) and may be released from the cells in the lining layer (Fig. 2a) of synovial membrane where it is mainly found. In this site TGF-#l may also be responsible for upregulation of fibroblast (type B lining layer cells) growth (Wilder et al., 1990) in a paracrine fashion. TGF-flI has also been shown to inhibit T cell activation (Kehrl et al., 1986b; Wahl et al., 1988)-the localization to the lymphoid aggregates in RA synovial mem-

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Fig. 2. Immunoperoxidase staining with biotinylated anti-TGF-jBl F(ab')2 fragments on RA synovial membrane sections. TGF-fll-staining cells were seen (a) in the synovial lining layers (SLL, open-headed arrows; most of the lining cells are positive in this section), (b) in a perivascular pattern, (c) within and outside a lymphoid aggregate (both indicated by small arrows), and (d) in a fibroblastic zone where the majority of cells are positive. TGF-#1 staining on synovial membrane was abolished by recombinant TGF-/31 linked to Sepharose 4B (e). Sections were counterstained with haematoxylin (original magnifications: a, b, c, e x 320; d x 400).

brane would suggest such a local function may be operating in RA, and TGF-f may account for the reduced presence of T cellderived cytokine proteins compared to mRNA levels (discussed by Feldmann et al., 1991). The finding that fibroblast-like cells also stain with these antibodies raises the possibility that TGF-,BI may also act in an autocrine fashion. Fibroblasts can respond to TGF-t3 1 by increasing synthesis of collagen and fibronectin (Ignotz & Massague, 1986; Postlethwaite et al., 1987; Raghow et al., 1987), down-regulating synthesis of destructive proteases and upregulating synthesis of their inhibitors (Edwards et al., 1987), thus promoting matrix component deposition. Chondrocytes can also respond to TGF-,B by increasing cellular glycosaminoglycan and proteoglycan synthesis, and

reducing proteoglycan catabolism (Morales & Roberts, 1988; Redini et al., 1988). The finding that normal chondrocytes stain with anti-TGF-f3I antibodies also implies an autocrine stimulation in this tissue, possibly regulating matrix deposition (Guerne et al., 1990) as has been suggested for other inflammatory cytokines (Shinmei et al., 1989; Chu et al., 1990). In RA, however, TGF-/31 can also be found in cells at the distinct CPJ, a site at which other pro-inflammatory cytokines such as TNF and IL-la can be readily detected (Chu et al., 1990). In this position TGF-f3i could be involved in inducing tissue repair by inhibiting protease release (Chandrasekhar & Harvey, 1988) as well as stimulating matrix deposition. This has been postulated as the mechanism of action of systemic TGF-f I treatment which reduced the formation of pannus and joint

384

C. Q. Chu et al.

Fig. 3. Immunoperoxidase staining of CPJ sections for TGF-B 1. TGF-J I -staining cells were localized in distinct CPJ (a) at the interface (indicated with open-headed arrows) of cartilage (C) and pannus (P), and (b) at the site of cartilage erosion (clusters of positive cells indicated with open-headed arrows). In the indistinct CPJ TGF-PlI-positive cells were localized to fibroblast-like cells (as indicated) in the TFZ (c). No cells stained with normal rabbit F(ab')2 fragments in the CPJ (d). Sections were counterstained with safranin-O and haematoxylin (Original magnifications: a, b x 200; c, d x 320).

Table 3. Phenotype characterization of TGF-fl -containing cells in RA synovial membrane and CPJ % of TGF-# I + ve cells

Cell phenotype T cells CD3

Lining layer

Interstitium

CPJ

0

1

0

0

0

0

90

70

50

7

0

0

05

3

4

fl/plasma cells Ig Macrophage/monocyte CD68 Fibroblast-like cells MoAb67 Dendritic cells RFDI

CPJ, Cartilage/pannus junction.

erosion in the streptococcal cell wall (SCW)-induced rat model of arthritis (Brandes et al., 1991). Interestingly, TGF-flI is also found at the diffuse fibroblastic zone, an area of CPJ where the presence of proteoglycan and collagen suggests that in this site there is an attempt to repair the joint damage in RA (Allard et al., 1987). Using double immunofluorescence techniques we have demonstrated that the TGF-flI -containing cells were mainly of the macrophage/monocyte lineage as 80-90% of them labelled with anti-CD68 antibody (EBM 11). In contrast, no MoAb 67 positive cells were found in the interstitium, but morphometrically many cells had a fibroblast-like shape and the majority of these cells in the fibroblastic areas ofthe RA synovial membrane contained TGF-,lB. They were not macrophage/monocytes or dendritic cells, implying that these cells were probably of fibroblastic origin. Although in vitro studies have suggested that lymphocytes such as T cell clones can produce TGF-,Bl (Grubeck-Leobenstein et al., 1989), we were unable to demonstrate significant numbers of T cells or plasma cells staining for TGF-fll by double immunofluorescence-a finding in agreement with Lafyatis et al. (1989). In the distinct CPJ, only 50% of the pannus TGF-f1 cells

385

Transforming growth factor-l] in RA

is seen in these immunohistochemical studies may not be in the mature active configuration. Analyses with antibodies raised against inactive TGF-31 are necessary to discriminate between these two proteins. Nevertheless, active TGF-l has been demonstrated in RA synovial fluid (Fava et al., 1989; Brennan et at., 1990), but may not be able to downregulate destructive cytokine production because the cells with which it comes in contact are already activated. Previous in vitro studies have demonstrated that it is necessary to add TGF-Pf1 to cultured cells before stimulation to exert a down-regulatory effect (Chantry et al., 1989), and therefore any active TGF-/31 may not exert an effect in the RA synovial fluid and tissue or CPJ, where the cells may already have been immunologically stimulated (Brennan et al., 1990). Local injection of TGF-#l into rat joints can cause synovitis (Fava et al., 1991). However, TGF-/31 has been shown to have powerful anti-inflammatory effects in vivo if administrated systemically. Treatment with TGF-fll greatly reduced infiltration of inflammatory cells into the tissues in experimental allergic encephalomyelitis model (Racke et al., 1991; Kuruvilla et al., 1991) and arthritides induced by collagen in mice (Kuruvilla et al., 1991) and by SCW in rats (Brandes et al., 1991). In addition, it reduced tissue destruction irn both the experimental arthritides suggesting that in vivo TGF-J can down-regulate the inflammatory process. In this study a large number of cells containing TGF-/31 in RA synovial membrane have been demonstrated in areas where TGF-fIl may be potentially active in controlling joint inflammation and destruction. Further analysis will be necessary to show whether cleavage to produce the active molecule may have modulating activity in RA. Fig. 4. Double immunofluorescence staining of RA synovial membrane sections. TGF-fll-positive cells (a) in the synovial lining layer (SLL) were also stained for (b) a macrophage/monocyte marker (CD68, detected with EBM 11 antibody) (original magnifications: x 320).

were EBM I I positive and a few (4%) expressed the dendritic cell marker RFDl. Therefore, about 50% of these cells were not characterized by the present techniques, and were not stained by MoAb 67. This suggests the possibility that the remainder are fibroblasts, as this area is rich in fibroblast-like cells by morphology (Kobayashi & Ziff, 1975), and many of the cells staining for TGF-f1i were of fibroblastic shape. TGF-fll was also detected in the synovial membranes from patients with SLE, MCTD and OA although in much reduced quantities (Table 2). This indicates that its presence is not specific to RA (Fava et al., 1989) and suggests that TGF-f I may also be involved in limiting synovial inflammation in these diseases which are not characterized by joint erosion. In RA, joint erosion is a characteristic disease feature and it has been postulated that the pro-inflammatory cytokines such as IL-I and TNF are involved in this process. However, the number of cells containing TGF-f 1 (74%) in the synovial lining layer outnumbers those containing TNF (37%), IL-6 (31 %) and IL-I (25%) (Chu et al., 1991; Field et al., 1991; Covington et al., 1989) and yet the balance of joint destruction could imply that TGF-flI was not present in sufficient quantity to fully inactivate the effects of these destructive cytokines. These apparently conflicting pieces of information may be explained by the fact that our antibodies recognize both the active and inactive forms of TGF-f.1 and hence the majority that

ACKNOWLEDGMENTS This work was supported by the Nuffield Foundation and the Arthritis and Rheumatism Council of Britain. Dr C. Q. Chu is supported by SinoBritish Friendship Scholarship Scheme.

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