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

Transforming growth factor-fit in rheumatoid synovial membrane andcartilage/pannus junction

C. Q. CHU, M. FIELD, E. ABNEY*, R. Q. H. ZHENG*, S. ALLARD, M. FELDMANN* & R. N. MAINIClinical 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 haveimmunosuppressive actions, and has been proposed to have a role in rheumatoid arthritis (RA). Usingimmunohistochemical techniques with rabbit F(ab')2 antibodies raised against recombinant humanTGF-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 thelymphoid aggregates. At the CPJ it was found both in cells at the distinct junction as well as in thetransitional region of the diffuse fibroblastic zone. The cells staining for TGF-#1 were identified bydouble immunofluorescence staining as being from the monocyte/macrophage series as well as the typeB synovial lining cells. TGF-,B1 was also detected in the synovial membrane sections from 4/4 patientswith systemic lupus erythematosus/mixed connective tissue disease and 5/8 patients with osteoarthri-tis, in a similar distribution to that seen in RA, and in the lining layer of 1/7 normal synovialmembranes. These results add to histological evidence confirming that TGF-fI is present in RAsynovial cells and those from other arthritides. The distributions ofTGF-, I in RA synovial membranereflects its known actions, as it can be detected at the CPJ, where it could induce repair, and close toactivated 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 capableof inhibiting many aspects of immune reaction and promotingtissue repair. It is thus implicated in suppressing synovialinflammation in rheumatoid arthritis (RA), although it mayalso promote some aspects ofinflammation (Wahl, McCartney-Francis & Mergenhagen, 1989). Of the three members of theTGF-# family, TGF-#l has been the most extensively studied(Roberts & Sporn, 1988).

TGF-fll can down-regulate B lymphocyte proliferation anddifferentiation (Kehrl et al., 1986a), and inhibits T cell prolifer-ation by down-regulating IL-2 receptor expression (Kehrl et al.,1986b). In addition, TGF-#1 down-regulates macrophageHLA-DR expression (Czarniecki et al., 1988) and inhibits theproduction of IL-1, interferon-gamma (IFN-y) and tumournecrosis factor (TNF) by monocytes and macrophages in vitro(Espevik et al., 1987; Chantry et al., 1989). However, it

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

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

TGF-P1 also stimulates fibroblast growth, promotes fibro-sis, and stimulates endothelial cells to secrete angiogenic factorswhich promotes angiogenesis (Roberts et al., 1986). It alsoincreases collagen synthesis by fibroblasts and prevents proteaserelease (Roberts & Sporn, 1988) thereby increasing depositionof extracellular matrix and stimulating tissue repair followinginjury.

TGF-fl has been detected in RA synovial effusion andsynovial cells at both the protein and mRNA levels (Lafyatis etal., 1989; Fava et al., 1989; Brennan et al., 1990; Miossec et al.,1990), but data ofcellular distribution ofTGF-,B in the inflamedsynovial tissue remains limited. In the present report we havelocalized TGF-fI both in the synovial membrane and at thecartilage/pannus junction (CPJ) in RA, and in the synovialmembrane in other inflammatory arthritides, osteoarthritis(OA), and rarely in the normal synovial tissue. We haveidentified the phenotypes of cells producing TGF-#l in RAsynovial membrane as being from monocyte/macrophagelineage, as well as the type B lining layer cells.

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Transforming growth factor-/Il in RA

MATERIALS AND METHODS

Preparation ofsamplesEighteen patients with definite or classical RA (Arnett et al.,1988) were included in the study. Synovial membrane tissue wasobtained at arthroscopy or arthroplasty. Diseased CPJ tissuewas obtained from 11 patients undergoing joint replacementsand normal tissue was obtained from amputation specimens(donated by Dr M. Bayliss, Kennedy Institute of Rheumato-logy, London). Synovial membrane samples from patients withOA, systemic lupus erythematosus (SLE) and mixed connectivetissue disease (MCTD) were all obtained at arthroscopyperformed during the active phase of disease, usually withsynovitis and effusion. All tissue samples were immediatelysnap-frozen in liquid nitrogen or hexane and stored at -70°Cuntil required. Sections of 5 or 6 gtm were cut on a cryostat, andthe sections were stored at -70°C for up to 6 months until used.

Anti- TGF-flJ antibody preparationPolyclonal rabbit anti-TGF-,B1 antiserum was raised againstrecombinant human TGF-,BI (Genentech, San Francisco, CA),and it acts as a neutralizing antibody (Chantry et al., 1989). Forimmunohistochemical staining the F(ab')2 fragments were pre-

pared 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 IgGand Fc fragments were removed using protein A linkedSepharose 4B (Pharmacia, Milton Keynes, UK). The anti-TGF-/B1 F(ab')2 fragments were purified on a CNBr-activatedSepharose 4B affinity column coupled with recombinant humanTGF-f31. F(ab')2 fragments were eluted with 3 M guanidinechloride (Sigma), concentrated and labelled with N-hydroxysuc-cinimidobiotin (Pierce Chemical Co., Rockford, IL). Theactivity and specificity of the F(ab')2 fragments were tested byELISA against recombinant TGF-,B1 and other cytokines as

described by Chu et al. (1991).

Immunohistochemical stainingSections were fixed in acetone/methanol (1:1) at -20'C andtreated with 0 3% hydrogen peroxide in methanol to blockendogenous peroxidase. Nonspecific binding was blocked byincubation of the sections with 20% normal goat serum. Thesections were washed and incubated for 1 h with anti-TGF-,BlF(ab')2 fragments linked to biotin at appropriate concentration(10 pg/ml) and binding detected with streptavidin-conjugatedhorseradish peroxidase (Amersham, Little Chalfont, UK)diluted in 2% normal goat serum. This was developed with 0-5mg/ml diaminobenzidine (Sigma). Synovial membrane sectionswere counterstained with haematoxylin and CPJ sections withsafranin-O and haematoxylin, and each was viewed by lightmicroscopy (Leitz).

The following control antibodies were used to stain thesequential sections:

(1) Normal rabbit F(ab')2 fragments were prepared using a

Sephacryl S-200 column from pepsin digested pre-immunizedrabbit IgG fraction (Chu et al., 1991). The normal F(ab')2fragments linked to biotin were incubated with the sections at anequivalent concentration.

(2) Anti-TGF-PI F(ab')2 fragments were absorbed withTGF-f31 or TNF affinity column, and the drop-through fromeach column was tested for staining on sections.

To identify cell phenotypes, double immunofluorescencestaining was performed as described by Field et al., (1991).Briefly, the sections were treated as above, and binding detectedwith streptavidin-conjugated Texas-Red (Amersham). This wasfollowed by incubation of the sections with monoclonal cellmarker antibodies (see Table 1 for details). The binding was

detected with anti-mouse IgG or IgM conjugated with fluores-cein (Sigma). Fluorescein-conjugated F(ab')2 fragments of goatanti-human F(ab')2 immunoglobulin (ICN, Lisle, IL) were usedto detect plasma cells containing immunoglobulins. The slideswere viewed under ultra violet light (Leitz).

Quantification of cellsThe numbers ofTGF-B I staining cells were graded by countingup to 500 cells in five high power fields in each area. To quantifythe variety of the cells containing TGF-f31, the number ofTGF-,I-positive cells co-staining with the cell marker antibodies wasexpressed as a percentage of the total number of TGF-I-positive cells.

RESULTS

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

TGF-fB localization in synovial membraneAbundant TGF-/JI -staining cells were detected in all of 18 RAsynovial membrane sections examined. These cells were foundthroughout the sections, but often accumulated in the lininglayer (Fig. 2a), in the inter-lymphoid aggregate area (Fig. 2c)and in a perivascular distribution (Fig. 2b). While fewer cellswithin the lymphoid aggregates were stained for TGF-#1 (Fig.2c, Table 2), in the fibroblastic areas almost 80% of thefibroblast-like cells were shown to contain TGF-,BI (Fig. 2d).

Seven normal synovial membrane sections were examinedand one was shown to contain cells stained for TGF-,I,comprising 1% lining layer cells (Table 2). The intensity ofstaining was much less than that in RA sections.

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

Antibody Specificity Isotype Source and references

Leu-4 Pan T cells (anti-CD3) IgG 1 Becton Dickinson, Mountain View, CAEBMII Macrophages/monocytes (anti-CD68) IgGI Dakopatts (Kelly et al., 1988)Mab67 Type B synovial lining cells IgGl Dr N. Hogg (Stevens et al., 1990)RFDl Dendritic cells (class II associated antigen) IgM Dr L. Poulter (Poulter et al., 1986)

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C. Q. Chu et al.

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

TGF-f3l localization in CPJAs described by Allard et al. (1987), two types of CPJ were seenafter staining with haematoxylin, fast green and safranin-O. Adistinct CPJ was characterized by a cellular and vascular pannuswith a distinct margin demarcating the pannus and underlyingcartilage. At this site pannus can often be seen invading theunderlying cartilage. In distinct CPJ, TGF-f5-containing cellswere detected throughout the pannus area, in particular at theinterface of cartilage and pannus and at the site of cartilageerosion (Fig. 3a, b). About 90% of the pannus cells eroding intocartilage stained for TGF-3I.

In indistinct CPJ a transitional fibroblastic zone (TFZ)consisting of fibrous tissue and fibroblast-like cells connects thepannus and cartilage with no evidence of cartilage invasion.This site of CPJ has been suggested to be an attempt at repairand in this site TGF-#fI was detected in over 80% fibroblast-likecells in the TFZ (Fig. 3c). None of the cells was shown to stainwith anti-TGF-,BI antibody in seven normal synovium-cartilagejunction sections (Table 2).

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

3

2E

0

0

1:10 1:20 1:40 1:80 1:160 1: 320 [:640 1:1280 I-2560

Antibody dilution

Fig. 1. Anti-TGF-jlI antibody ELISA. Recombinant human cytokines

were coated on microtitre plates and the antibody binding was detected

with avidin-conjugated alkaline phosphatase. This shows biotinylated

anti-TGF-fll F(ab')2 fragments found to TGF-fll in a dose-dependent

manner, but not to TNF, IL-loc or IL-6. A, anti-TGF-f51 +TGF-fll;A,&

anti-TGF-fllI +TNF; 0, anti-TGF-flI +IL-6; 0, anti-TGF-f3+ IL-lIa.

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

TGF-jlI +ve cells in synovium (% and range)

No. +ve Lining Lymphoid Inter-lymphoidDiagnosis samples layer aggregate aggregate

RA 18/18 74 (61-80) 4-5 (3-7-5) 49 (40-56)MCTD/SLE 4/4 40 (30-55) NP 33 (25-41)OA 5/8 25 5 (4-44) 2 (0-6) 7 (0-27)Normal 1/7 1 NP

RA, Rheumatoid arthritis; MCTD, mixed connective tissue disease;SLE, systemic lupus erythematosus; OA, osteoarthritis; NP, not present.

-, TGF-flI-staining cells not detectable.

Characterization ofphenotypes of TGF-f3I positive cells in RAsynovial membrane and CPJDouble staining of synovial membrane with monoclonal anti-bodies demonstrated that the majority (70%) of the cellscontaining TGF-#Il stained with the macrophage/monocyte cellmarker CD68 in synovial tissue (Fig. 4a/b). In the lining layerthis figure rose to 90% (Table 3). However, up to 7% of the cellsstained with the MoAb 67, a marker of type B fibroblast derivedsynovial lining cells (Stevens et al., 1990). A few TGF-fll-containing cells in the interstitium were also positive for theRFD1 antigen suggesting that some dendritic cells may makeTGF-#1I in synovial tissue. An occasional T cell and no plasmacells stained for TGF-f I (Table 3). In the interstitium - 30% areunaccounted for, but morphometrically many fibroblast-likecells stained for TGF-f 1, indicating that they may also produceTGF-f1.

At the distinct CPJ, 50% ofTGF-P 1-positive cells co-stainedwith anti-CD68 antibody, and 4% were RFDI positive (Table3), but 40-50% were not identified with this panel of mono-clonal antibodies.

DISCUSSION

The results of the present study add histological evidence toshow that TGF-,13 is present in RA synovial tissue. Theintracellular detection of the protein is consistent with theprevious reports that TGF-fl is locally produced by synovialcells in RA (Fava et al., 1989; Lafyatis et al., 1989; Brennan etal., 1990; Miossec et al., 1990). Using the specific F(ab')2antibody to overcome non-specific binding due to locallysynthesized rheumatoid factors TGF-,ll-containing cells havebeen localized throughout the synovial tissue, but mainly in thelining layer, inter-lymphoid aggregate region and including thefibroblastic zone and perivascular area of synovial tissue, as wellas at the CPJ.

TGF-Il is found in RA synovial fluid both in the latent andbioactive forms (Fava et al., 1989; Brennan et al., 1990; Miossecet al., 1990) and may be released from the cells in the lining layer(Fig. 2a) of synovial membrane where it is mainly found. In thissite TGF-#l may also be responsible for upregulation offibroblast (type B lining layer cells) growth (Wilder et al., 1990)in a paracrine fashion. TGF-flI has also been shown to inhibit Tcell activation (Kehrl et al., 1986b; Wahl et al., 1988)-thelocalization to the lymphoid aggregates in RA synovial mem-

382

Transforming growth factor-#I in RA

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brane would suggest such a local function may be operating inRA, and TGF-f may account for the reduced presence ofT cell-derived cytokine proteins compared to mRNA levels (discussedby Feldmann et al., 1991).

The finding that fibroblast-like cells also stain with theseantibodies raises the possibility that TGF-,BI may also act in anautocrine fashion. Fibroblasts can respond to TGF-t3 1 byincreasing synthesis of collagen and fibronectin (Ignotz &Massague, 1986; Postlethwaite et al., 1987; Raghow et al., 1987),down-regulating synthesis of destructive proteases and up-

regulating 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

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

reducing proteoglycan catabolism (Morales & Roberts, 1988;Redini et al., 1988). The finding that normal chondrocytes stainwith anti-TGF-f3I antibodies also implies an autocrine stimula-tion in this tissue, possibly regulating matrix deposition (Guerneet al., 1990) as has been suggested for other inflammatorycytokines (Shinmei et al., 1989; Chu et al., 1990).

In RA, however, TGF-/31 can also be found in cells at thedistinct CPJ, a site at which other pro-inflammatory cytokinessuch as TNF and IL-la can be readily detected (Chu et al.,1990). In this position TGF-f3i could be involved in inducingtissue repair by inhibiting protease release (Chandrasekhar &Harvey, 1988) as well as stimulating matrix deposition. This hasbeen postulated as the mechanism of action of systemic TGF-f Itreatment which reduced the formation of pannus and joint

383

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Fig. 3. Immunoperoxidase staining ofCPJ 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 cellsindicated with open-headed arrows). In the indistinct CPJ TGF-PlI-positive cells were localized to fibroblast-like cells (as indicated) inthe TFZ (c). No cells stained with normal rabbit F(ab')2 fragments in the CPJ (d). Sections were counterstained with safranin-O andhaematoxylin (Original magnifications: a, b x 200; c, d x 320).

Table 3. Phenotype characterization of TGF-fl -containing cells in RAsynovial membrane and CPJ

% of TGF-# I + ve cells

Cell phenotype Lining layer Interstitium CPJ

T cellsCD3 0 1 0

fl/plasma cellsIg 0 0 0

Macrophage/monocyteCD68 90 70 50

Fibroblast-like cellsMoAb67 7 0 0

Dendritic cellsRFDI 0 5 3 4

CPJ, Cartilage/pannus junction.

erosion in the streptococcal cell wall (SCW)-induced rat modelof arthritis (Brandes et al., 1991). Interestingly, TGF-flI is alsofound at the diffuse fibroblastic zone, an area of CPJ where thepresence of proteoglycan and collagen suggests that in this sitethere is an attempt to repair the joint damage in RA (Allard etal., 1987).

Using double immunofluorescence techniques we havedemonstrated that the TGF-flI -containing cells were mainly ofthe macrophage/monocyte lineage as 80-90% of them labelledwith anti-CD68 antibody (EBM11). In contrast, no MoAb 67positive cells were found in the interstitium, but morphometri-cally many cells had a fibroblast-like shape and the majority ofthese cells in the fibroblastic areas ofthe RA synovial membranecontained TGF-,lB. They were not macrophage/monocytes ordendritic cells, implying that these cells were probably offibroblastic origin. Although in vitro studies have suggested thatlymphocytes such as T cell clones can produce TGF-,Bl(Grubeck-Leobenstein et al., 1989), we were unable to demon-strate significant numbers ofT cells or plasma cells staining forTGF-fll by double immunofluorescence-a finding in agree-ment with Lafyatis et al. (1989).

In the distinct CPJ, only 50% of the pannus TGF-f1 cells

384

Transforming growth factor-l] in RA 385

Fig. 4. Double immunofluorescence staining ofRA synovial membranesections. 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 cellmarker RFDl. Therefore, about 50% of these cells were notcharacterized by the present techniques, and were not stained byMoAb 67. This suggests the possibility that the remainder arefibroblasts, as this area is rich in fibroblast-like cells bymorphology (Kobayashi & Ziff, 1975), and many of the cellsstaining for TGF-f1i were of fibroblastic shape.

TGF-fll was also detected in the synovial membranes frompatients with SLE, MCTD and OA although in much reducedquantities (Table 2). This indicates that its presence is notspecific to RA (Fava et al., 1989) and suggests that TGF-f I mayalso be involved in limiting synovial inflammation in thesediseases which are not characterized by joint erosion.

In RA, joint erosion is a characteristic disease feature and ithas been postulated that the pro-inflammatory cytokines suchas IL-I and TNF are involved in this process. However, thenumber of cells containing TGF-f 1 (74%) in the synovial lininglayer outnumbers those containing TNF (37%), IL-6 (31 %) andIL-I (25%) (Chu et al., 1991; Field et al., 1991; Covington et al.,1989) and yet the balance ofjoint destruction could imply thatTGF-flI was not present in sufficient quantity to fully inactivatethe effects of these destructive cytokines.

These apparently conflicting pieces of information may beexplained by the fact that our antibodies recognize both theactive and inactive forms ofTGF-f.1 and hence the majority that

is seen in these immunohistochemical studies may not be in themature active configuration. Analyses with antibodies raisedagainst inactive TGF-31 are necessary to discriminate betweenthese two proteins. Nevertheless, active TGF-l has beendemonstrated in RA synovial fluid (Fava et al., 1989; Brennan etat., 1990), but may not be able to downregulate destructivecytokine production because the cells with which it comes incontact are already activated. Previous in vitro studies havedemonstrated that it is necessary to add TGF-Pf1 to culturedcells before stimulation to exert a down-regulatory effect(Chantry et al., 1989), and therefore any active TGF-/31 may notexert an effect in the RA synovial fluid and tissue or CPJ, wherethe cells may already have been immunologically stimulated(Brennan et al., 1990).

Local injection ofTGF-#l into rat joints can cause synovitis(Fava et al., 1991). However, TGF-/31 has been shown to havepowerful anti-inflammatory effects in vivo if administratedsystemically. Treatment with TGF-fll greatly reduced infiltra-tion of inflammatory cells into the tissues in experimentalallergic encephalomyelitis model (Racke et al., 1991; Kuruvillaet 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 theexperimental arthritides suggesting that in vivo TGF-J candown-regulate the inflammatory process.

In this study a large number of cells containing TGF-/31 inRA synovial membrane have been demonstrated in areas whereTGF-fIl may be potentially active in controlling joint inflamma-tion and destruction. Further analysis will be necessary to showwhether cleavage to produce the active molecule may havemodulating activity in RA.

ACKNOWLEDGMENTSThis work was supported by the Nuffield Foundation and the Arthritisand Rheumatism Council of Britain. Dr C. Q. Chu is supported by Sino-British Friendship Scholarship Scheme.

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