Scand. J. Immunol. 35, 603-610. 1992
T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Thl Subset A. M. M M I L T E N B U R G , J. M. VAN LAAR. R. DE KUIPF.R. M, R. DA HA* & F. C. BREEDVELD IX'pLtnmenls of Rheumatology and 'Nephrology. University Hospiial. Leiden. The Netherlands
Millcnburg AMM. \an l.aar JM. dc Kiiipcr R. Daha MR. Brccdvcld \C T Cells Ckincd friim Huniiin Rhcumaunii Synovial Membriine lunciionaliy Represent Ihe Thl Subset. Scand J Immunol 1992:35:603 10 The presenee ofactivaled T cells in Ihe synovia! membrane of palients wilh rheumatoid arthritis (RA) suggests a role for these cells in the piithogencsis of ihc disease, Rceenl evidence indieates ihat humiin T cells inav fall into I'unL-linnal categories depcndciU i>n iheir cytokine prolile and cyltHoxic capacity. The human Thl subset is cylolylie and produces high levels oflFN-gamma whereas tlicTh2 type olT cell produces IL-4. inorder to investigate whether Thl orTh2 type eells iire presenl in iheinllammalorv synovial membrane in RA. a panel ol"synovial membrane derived T-eell clones («=I9) was generated and studied funciionally. Anii-CD.'* induced eytoloxicity assays were performed In demonstrate thecy lotoxle poieniial olclones. ExeepI for two, all clones were cytoiylie in Ihis lest. Clone eells were activated Io initiale eylokine production and assessment oflhecylokine leveis showed thai all clones produced large amounts of IFN-gamma (IX out of 14 clones: over 5(1.(11111 pg ml) wliereas IL-4 was absent or present in minimal amounts (17 oul ol" ly clones: less ihan llHXJ pg ml). The production o["U.-l, 11.-2 and IL-h i\as variable. The runetional cKaraeterislics of the clones studied indicate Ihal Ihey may resemble ihe Thl subtype of T ceils. Our dala suggest a relalion betvveen Thl-type lunelions and Ihe ehronie inliammation eharaeterislic of RA. Dr A. M. M. Mitivuhwfi. Uninr.silv Hospilid. DepanmnU of Rtwuintiintufiv. Building I. C2-Q. PO So.x 9600. 2300 RC Leiden. The Neihertuiids
The majority ofthe inflammatory cells invading the synovial tissue of rheumatoid arthritis (RA) palienls has been tlcmonslrated to be of T-cell origin |l-3]. A large proportion of these T cells may be activated, as has been shown using monoclonal antibodies (MoAb) directed against T-cell activation molecules such as hutiian leucocyte antigen (HLA)-DR or the interlcukin-2 (iL2) receptor (0025) (4. 5], Some authors reported A prcdt)minancc of CD4-positivc cells [3| with phcnotypic characteristics of hctpcr-induccr T cells [6j, whereas others found activated CD4 as well as CD8 cells in lymphocyte populations derived from synovia! tissue [5], The presence of activated T cells within the rheumatoid joint strongly suggests a role for these cells in the palhogenesisof RA. Human T-cell clones may. as has been reported for muHne T cells [7]. be categorized as Thl- or Th2-type cells depending
on their functional characteristics [8]. ThI-iypc clones differ from Th2-lypc clones in their cytotoxic potential (Thl»Th2) [8. 9) and their cytokine profile. Thl-type clones producing predominantly lL-2 and IKN-gamma and Th2-type clones producing IL.-4 and IL-5 [8. 10. 11]. Using the anti-CD3-induced cytotoxicily status and the cytokine (IL-I. -2. -4. -6. and IFN-gamma) profile of a panel of clones (/j= 19) obtained from Ihe synovial tissue of a patient with RA as criteria fur Thl or Th2. we investigated whether Thl- or Th2-type eells predominated in this T-eell clone panel. The relevance of our findings in relation to ihc pathogenesis of RA will be discussed.
MATERIALS AND M E T H O D S Paiieni. Synovial membrane samples were obtained 603
604
.4. M. M. Miltenhur^ ft al.
during a lhcr;ipL'ulii: surgical procedure. The synovium was derived from a palieni I female, 5K years) who had been diagnosed as having seroposilive classical rheumaloid arthritis 14 years earlier, Al the lime of ihc operation she was being treated with hydro\ychloroquine KN) mg daily. Celt eullure procedures. Fragments of synovial lissue were placed in 24-well lissue culture plates (Costar 3524, Cambridge. MA. USA) in Istove's modified Dulbecco's medium (IMDM) supplemented with antibiotics (penicillin IOO iU,ml.-sireptomycin KH)/ig/ml; Boehringer Mannheim GmbH. Mannheim. Germany), fetal calf serum (FCS, IO"v,) and 5"^;. vol vol T-cel! growth factor (TCGF) 112], Afler approximately one week, growing cells were separated from the lissue fragments and cloned by limiling dilution. Cells were cloned at 0.5 cellswell in a feeder mixlure consisting of irradiated (.ltXK) rad) allogeneic penpheral blood mononuclear cells (PBMC) (Kf ml). OKT3 aseites (10 • dilution). and 5"I. vol vol TCGF (modified according to Ref. 13). Thecloningelllciencyof thepRK-edure was21".> in this particular experiment. The probability of ihe ecll populations lo be clonal is >95"';i. Clones were expanded using ihe same feeder cell mixture, at a cell concentration of 2 x l C responder cells/well in 24-well plates. Restimulation was performed every 10 14 days and in between ihc clones were maintained in culture in the presence of 5'l^i vol/vol TCGF. Imnmnophenoivping. Phcnotypic analysis of the clones was performed using standard methodology [ 12|, Briefly. 1 *. KJ^ cells per sample were ineubaled with the MoAb indicated for at least 30 min on ice. After two washes with iee-cold phosphate-bulVered saline conlaining 1"'" wt vol bovine serum albumin (PBS-BSA). cells were incubated with lluorescein ist)thuKyanaten markers on C D 4 ' and C D 8 ' cells from synovial tluid, synovial tissue and peripheral blood of patients with inflammatory arthritidcs, Scand J Immunol 1989:29:631 9. 6 Moore K, Walters MT. Jones DB cf i;/ An inimunohistological study of C D 4 ' lymphocyte subsets within inllammalory lesions with special rcferenee lo rheumaloid arthritis and intlanimatory bowel disease. Immunology 1988:65:457 63, 7 Mosmann TR. Cherwinsky H, Bond MW. Gicdiin MA. Coffman RL Iwo lypes of murine helper T cell clone. I, IX-iiniiion according to profiles of lymphokine activities and secreted proteins. J Immunol 19X6:136:2.14X 57, 5 Romapnani S. Human Tnl and T||2 subsets: doubt no more, immuntil Today 1991:12:256 7. 9 Rotlcvccl FTM. Kokkeiink I. van Licr RAW 4' T cell clones, J Immunol 1989:143:907 12, Cirubcck-Locbcnstcin B. Turner M. Pinch K ci ut. CD4" T-ce!i clones from aulomimune ihyroid lissue cannot be classified aceording lo iheir lymphokine production, Scand J Immunol 1990:32:433 40, Haanen JBACJ. Waal Malelijt de R. Res PCM el at. Seleclion of a hunutn T helper type I-like T eell subsel b> mycobacleriu, J Exp Med 1441:174:583 'J2. Yssel H. Shanafell MC. Soderbcrg C. Schneider PV. Anzola J. Pell?(i, Borrelia burgdorferiactivalesaT helper lypc l-hke T cell subset in I.yme arihrilis, J ExpMc'd I44l:174:.5y3 601, Del Prcte G1-. De Carii M. Ricei M. Ronuignaiii S, Helper activity lor iiiimunogltibulin synthesis o f T helper type I (Thl) and Th2 human T cell clones: ihe heip of Thl clones is limited by iheir eytolylic capacity J I x p Med 1941:174:804" 13Cardell's. Sander B. Mollcr G Helper Intcrleukins are produced hj both CD4 and COK splenic T cells after milogen stimulation. Fur J Immunol 1991:21:2445 500, KogaT.Wand-WiirtienbergerA.de BruynJ. Munk ME. Schoel B, Kaufmann SHE, T cells against a baeierial heat shock protein recognize stressed maerophages Scienee 14X4:245:1112 15, Iwamoio M. Koike T. Nakashima K. Saio K. Kalo Y, Interleukm-I: a regulator of cliondrocyle proliferalion, Imnumol Lett 14X4:21:151 6, Dodge CiR. Poole AR, Immunohistochemical detection and immunochcmical analysis of type II collagen degradation in human normal, rheumaloid. and osteoarlhniie articular carlilages and in exphmis of bovine articular cartilage eultured with inlerleukin I, J Clin Invest I489;X3:647 61.
Received 20 N.nember 1441 Accepted in ^evl^ed form 21 Januar\ 1442
T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Thl Subset A. M. M M I L T E N B U R G , J. M. VAN LAAR. R. DE KUIPF.R. M, R. DA HA* & F. C. BREEDVELD IX'pLtnmenls of Rheumatology and 'Nephrology. University Hospiial. Leiden. The Netherlands
Millcnburg AMM. \an l.aar JM. dc Kiiipcr R. Daha MR. Brccdvcld \C T Cells Ckincd friim Huniiin Rhcumaunii Synovial Membriine lunciionaliy Represent Ihe Thl Subset. Scand J Immunol 1992:35:603 10 The presenee ofactivaled T cells in Ihe synovia! membrane of palients wilh rheumatoid arthritis (RA) suggests a role for these cells in the piithogencsis of ihc disease, Rceenl evidence indieates ihat humiin T cells inav fall into I'unL-linnal categories depcndciU i>n iheir cytokine prolile and cyltHoxic capacity. The human Thl subset is cylolylie and produces high levels oflFN-gamma whereas tlicTh2 type olT cell produces IL-4. inorder to investigate whether Thl orTh2 type eells iire presenl in iheinllammalorv synovial membrane in RA. a panel ol"synovial membrane derived T-eell clones («=I9) was generated and studied funciionally. Anii-CD.'* induced eytoloxicity assays were performed In demonstrate thecy lotoxle poieniial olclones. ExeepI for two, all clones were cytoiylie in Ihis lest. Clone eells were activated Io initiale eylokine production and assessment oflhecylokine leveis showed thai all clones produced large amounts of IFN-gamma (IX out of 14 clones: over 5(1.(11111 pg ml) wliereas IL-4 was absent or present in minimal amounts (17 oul ol" ly clones: less ihan llHXJ pg ml). The production o["U.-l, 11.-2 and IL-h i\as variable. The runetional cKaraeterislics of the clones studied indicate Ihal Ihey may resemble ihe Thl subtype of T ceils. Our dala suggest a relalion betvveen Thl-type lunelions and Ihe ehronie inliammation eharaeterislic of RA. Dr A. M. M. Mitivuhwfi. Uninr.silv Hospilid. DepanmnU of Rtwuintiintufiv. Building I. C2-Q. PO So.x 9600. 2300 RC Leiden. The Neihertuiids
The majority ofthe inflammatory cells invading the synovial tissue of rheumatoid arthritis (RA) palienls has been tlcmonslrated to be of T-cell origin |l-3]. A large proportion of these T cells may be activated, as has been shown using monoclonal antibodies (MoAb) directed against T-cell activation molecules such as hutiian leucocyte antigen (HLA)-DR or the interlcukin-2 (iL2) receptor (0025) (4. 5], Some authors reported A prcdt)minancc of CD4-positivc cells [3| with phcnotypic characteristics of hctpcr-induccr T cells [6j, whereas others found activated CD4 as well as CD8 cells in lymphocyte populations derived from synovia! tissue [5], The presence of activated T cells within the rheumatoid joint strongly suggests a role for these cells in the palhogenesisof RA. Human T-cell clones may. as has been reported for muHne T cells [7]. be categorized as Thl- or Th2-type cells depending
on their functional characteristics [8]. ThI-iypc clones differ from Th2-lypc clones in their cytotoxic potential (Thl»Th2) [8. 9) and their cytokine profile. Thl-type clones producing predominantly lL-2 and IKN-gamma and Th2-type clones producing IL.-4 and IL-5 [8. 10. 11]. Using the anti-CD3-induced cytotoxicily status and the cytokine (IL-I. -2. -4. -6. and IFN-gamma) profile of a panel of clones (/j= 19) obtained from Ihe synovial tissue of a patient with RA as criteria fur Thl or Th2. we investigated whether Thl- or Th2-type eells predominated in this T-eell clone panel. The relevance of our findings in relation to ihc pathogenesis of RA will be discussed.
MATERIALS AND M E T H O D S Paiieni. Synovial membrane samples were obtained 603
604
.4. M. M. Miltenhur^ ft al.
during a lhcr;ipL'ulii: surgical procedure. The synovium was derived from a palieni I female, 5K years) who had been diagnosed as having seroposilive classical rheumaloid arthritis 14 years earlier, Al the lime of ihc operation she was being treated with hydro\ychloroquine KN) mg daily. Celt eullure procedures. Fragments of synovial lissue were placed in 24-well lissue culture plates (Costar 3524, Cambridge. MA. USA) in Istove's modified Dulbecco's medium (IMDM) supplemented with antibiotics (penicillin IOO iU,ml.-sireptomycin KH)/ig/ml; Boehringer Mannheim GmbH. Mannheim. Germany), fetal calf serum (FCS, IO"v,) and 5"^;. vol vol T-cel! growth factor (TCGF) 112], Afler approximately one week, growing cells were separated from the lissue fragments and cloned by limiling dilution. Cells were cloned at 0.5 cellswell in a feeder mixlure consisting of irradiated (.ltXK) rad) allogeneic penpheral blood mononuclear cells (PBMC) (Kf ml). OKT3 aseites (10 • dilution). and 5"I. vol vol TCGF (modified according to Ref. 13). Thecloningelllciencyof thepRK-edure was21".> in this particular experiment. The probability of ihe ecll populations lo be clonal is >95"';i. Clones were expanded using ihe same feeder cell mixture, at a cell concentration of 2 x l C responder cells/well in 24-well plates. Restimulation was performed every 10 14 days and in between ihc clones were maintained in culture in the presence of 5'l^i vol/vol TCGF. Imnmnophenoivping. Phcnotypic analysis of the clones was performed using standard methodology [ 12|, Briefly. 1 *. KJ^ cells per sample were ineubaled with the MoAb indicated for at least 30 min on ice. After two washes with iee-cold phosphate-bulVered saline conlaining 1"'" wt vol bovine serum albumin (PBS-BSA). cells were incubated with lluorescein ist)thuKyanaten markers on C D 4 ' and C D 8 ' cells from synovial tluid, synovial tissue and peripheral blood of patients with inflammatory arthritidcs, Scand J Immunol 1989:29:631 9. 6 Moore K, Walters MT. Jones DB cf i;/ An inimunohistological study of C D 4 ' lymphocyte subsets within inllammalory lesions with special rcferenee lo rheumaloid arthritis and intlanimatory bowel disease. Immunology 1988:65:457 63, 7 Mosmann TR. Cherwinsky H, Bond MW. Gicdiin MA. Coffman RL Iwo lypes of murine helper T cell clone. I, IX-iiniiion according to profiles of lymphokine activities and secreted proteins. J Immunol 19X6:136:2.14X 57, 5 Romapnani S. Human Tnl and T||2 subsets: doubt no more, immuntil Today 1991:12:256 7. 9 Rotlcvccl FTM. Kokkeiink I. van Licr RAW 4' T cell clones, J Immunol 1989:143:907 12, Cirubcck-Locbcnstcin B. Turner M. Pinch K ci ut. CD4" T-ce!i clones from aulomimune ihyroid lissue cannot be classified aceording lo iheir lymphokine production, Scand J Immunol 1990:32:433 40, Haanen JBACJ. Waal Malelijt de R. Res PCM el at. Seleclion of a hunutn T helper type I-like T eell subsel b> mycobacleriu, J Exp Med 1441:174:583 'J2. Yssel H. Shanafell MC. Soderbcrg C. Schneider PV. Anzola J. Pell?(i, Borrelia burgdorferiactivalesaT helper lypc l-hke T cell subset in I.yme arihrilis, J ExpMc'd I44l:174:.5y3 601, Del Prcte G1-. De Carii M. Ricei M. Ronuignaiii S, Helper activity lor iiiimunogltibulin synthesis o f T helper type I (Thl) and Th2 human T cell clones: ihe heip of Thl clones is limited by iheir eytolylic capacity J I x p Med 1941:174:804" 13Cardell's. Sander B. Mollcr G Helper Intcrleukins are produced hj both CD4 and COK splenic T cells after milogen stimulation. Fur J Immunol 1991:21:2445 500, KogaT.Wand-WiirtienbergerA.de BruynJ. Munk ME. Schoel B, Kaufmann SHE, T cells against a baeierial heat shock protein recognize stressed maerophages Scienee 14X4:245:1112 15, Iwamoio M. Koike T. Nakashima K. Saio K. Kalo Y, Interleukm-I: a regulator of cliondrocyle proliferalion, Imnumol Lett 14X4:21:151 6, Dodge CiR. Poole AR, Immunohistochemical detection and immunochcmical analysis of type II collagen degradation in human normal, rheumaloid. and osteoarlhniie articular carlilages and in exphmis of bovine articular cartilage eultured with inlerleukin I, J Clin Invest I489;X3:647 61.
Received 20 N.nember 1441 Accepted in ^evl^ed form 21 Januar\ 1442
