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Clinicalrheumatology, 1995, 14, Suppl. N ~2

Cytokines in Rheumatoid Arthritis F.A. H O U S S I A U Summary An ever,growing number of cytokines that play a critical role as soluble mediators of immune and inflammatory responses are being described. Not surprisingly, most of them have been detected in SF or serum of patients with RA, However, given the numerous interactions within the cytokine network- e.g. agonistic and antagonistic properties and natural inhihitors - one should beware of over-simplistic viewsi the more so as extrapolation from in vitro and animal mod' els is always a challenge! ~ o m a clinical viewpoint, more work is required before measurements of eytqMnes in RA be used as activity indices and- more importantly ' as prognostic markers. Key words

Cytokines, Rheumatoid Arthritis, T Cells.

INTRODUCTION Cytokines are low-molecUlar weight glycoproteins produced by activated lymphocytes, fibroblasts, mast cells and various other connective tissue cells: Their production is tightly regulated by antigens or non'specific activators such as endotoxins. Although most cytokines are structurally unrelated to each other, some families have been defined, such as interleukins (IL-1 to IL-13), interferons (c~, [3, y), cytotoxins (TNFa, TNFI3/LT), colony stimulating factors (G-CSF, M-CSE GM-CSF, MultiCSF, steel factor), growth factors (EGF, TGF, FGF, PDGF) and Chemokines (IL-8, MIP, MCR NAP)! Table I outlines the main structural and functional characteristics of some Of these mole6uiesl Cytokines play a critical role in numerous host defence mechanisms such as T- and B-cell immune responses, inflammation, tissue damage, tissue repair and hematopoiesis. Pleiotropism and redundancy are the hallmarks ~df the cytokine network. On the one hand, a single cytokine elicit numerous biological activities on many different target cells through specific ligand-receptor interactions. On the other hand, different cytokines share some activities with each other. The existence of synergies, antagonisms, and natural iuhibitors of cytokines further complicates an already sophisticated picture. The aim of this short review is to summarize - i.e. to over-simplify - what every rheumatologist should know about the potential involvement of cytokines in the pathogenesis of rheumatoid arthritis (RA), rather than to present an extensive review on the topic. We would like to briefly address the following issues:

RheumatologyUnit, Universityof Louvain,B-1200Brussels,Belgium.

- Which cytokines are detected in RA synovial fluid (SF)? - Do these cytokines play a pathogenic or protective role, if any? - Are Circulating cytokine measurements useful as activity and/or prognostic markers? - Could cytokines be used as therapeutic tools in RA?

1. Which cytokines are detected in RA SF?

Not Surprisingly, almost every single cyt0kine has been detected in the SF of RA patients, using one or another method of detection at the level of the protein o r the messenger RNA. AS a general rule, macrophage: or synovi0cyte-derived cyt0kines, Such as IL-1, TNF(z, IL-6, IL-8 and TGF[3, are readily detected in SF, whereas T cell-derived cytokines, such as IL-2, IL-4, IFNy or TNF~, are present in much lower concentrations, usually below 50 pg/ml (Table II) (1). Since cytokines are usually active in very low concentrations, these findings, confirmed at the R N A level on synovial fluid ceils or synovial tissue, should not be misinterpreted as an argument against a potential role of T cells in the pathogenesis of rheumatoid arthritis: the low amounts of T cellderived cytokines detected might indeed be quite sufficient to trigger immune or inflammatory responses. The pattern of cytokine expression observed in RA SF is not disease-specific. Thus, we found elevated IL-6 titers not only in RA SF but also in samples from patients suffering from ankylosing spondylitis or psoriatic arthritis (2). Incidentally, IL-6 titers measured in SF from osteoarthritic patients, albeit two orders of magnitude lower, were not negligible, indicating that dysregulated cytokine expression also exists in conditions considered to be non-inflammatory in nature.

Cytokines in RA

Table I: Cytokines: main characteri;ticg* Cytokine

Structure

Source

Biological targets Immune system

ttematopoiesis

Others

neutrophils

hepatocytes CNS

IL 1{2./13

monomer 153/159 A A 25 kDa

macrophages endothelmm fibroblasts .._

T cells B cells mast cells macrophages

I L-2

monomer 133 AA 15.5 kDa

T cells

T cells B cells NK cells

IL-3

monomer 133 AA • 2(1 kDa

T cells masl cells

mast cells

IL-4

monomer 129 AA 17 kDa

T cells mrs{ cells

B cells T cells mast cells m acrophages

Ik-5

homodimer 115 AA 40 kDa

T cells mast cells

B cells

eosinophils

II,-6

monomer 184 AA 26 kDa

macrophages T cells fibroblasts

T cells B cells

megakaryocytes pluripotent precursors

IL-7

monomer 152 AA -+ 2{) kDa

bone-marrow stromal cells

T cells

lymphoid progenitors

I L-8

monomer 72 AA • 8 kDa

macrophages endothelium fibroblasts

1L-9

monomer 126 AA • 25 kDa

T cells

mast cells B cells T cells

IL-10

monomer 160 AA • 18 kDa

macrophages B cells T cells

macrophages mast cells B cells T cells

lk- 11

monomer 178 AA 23 kDa

bone-marrow stromal cells

IL- 12

heterodimer p40:3.06 A A 40 kDa p35:197 AA 35 kDa

inacrophages B cells

NK cells T cells

IIA3

monomer 132 AA 17 kDa

T cells

B cells mast cells

M-CSF

homodimer 223/158 AA

fibroblasls macrophages endothelium

macrophage lineage

G-CSF

monomer 174 AA 20 kDa

macrophages fibroblasts endothelium

granulocyte lineage

GM-CSF

monomer 127 AA 22 kDa

macrophages T cells mast cells endothelium

granulocyte lineage macrophage lineage

IFN 7

homodimer 143 AA 48 kDa

T cells NK cells

macrophages B cells T ceils NK ceils

TNF~.

trimer 157 AA 52 kDa

macrophages endothelium fibroblasts

macrophages T cells

hepatocytes endothelium CN S

TNF[3

trimer 171 AA 75 kDa

T cells

macrophages

hepatocytes endothelium CNS

LlF/Hi/da

monomer I79 AA 40/60 kDa

bone-marrow stromal cells fibrnblasts T cells macrophages

all lineages

hepatocytes

neutrophils (chemotaxis)

erythroid progenitors

immature neurones

megakaryocytes pluripotent progenitors

hepatocytes

pluripotent progenitors

megakaryocytes pluripoten{ precursors

placenta

hepatocytes embryonic stem cells

"2 informations regarding the structure relate to the human protein. The list of cellular sources and of biological activities are not exhaustive.

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F.A. Houssiau

Table II: Cytokines in SF of RA patients Cytokine IL-6 IL-8 TGF[~ GM-CSF TNFc~ IL-1 IL-2 IL-4 IFNy TNFI3

Origin macrophages, fibroblasts macrophages, fibroblasts macrophages, fibroblasts fibroblasts macrophages macrophages T ceils T cells T cells T cells

Concentration 10-50 ng/ml 10-50 ng/ml 10-50 ng/ml 10-1000 pg/ml 100-1000 pg/ml 10-100 pg/ml < 50 pg/ml < 50 pg/ml < 50 pg/ml < 50 pg/ml

2. Potential pathogenic or protective role?

It is commonly believed that IL-1 and TNF~ behave as the "villains", while IL-6 and TGF~ would play some protective role. These assumptions stem from experimental data obtained in animal models of arthritis and in vitro cultures of chondrocytes and synoviocytes. Whether these findings are truly relevant in vivo - i.e. in the joint of the RA patient - is still somewhat speculative and indeed most difficult to ascertain given the already mentioned numerous interactions operating within the cytokine network. IL-1 injection into rat joints previously treated with a streptococcal cell wall peptidoglycan polysaccharide complex causes arthritis (3). Moreover, addition of IL-1 to chondrocyte cultures inhibits - at least to some extent proteoglycan and collagen syntheses, stimulates the production of PGE 2 and of metalloproteinases such as collagenase and stromelysin, and down-regulates the tissue inhibitor of metalloproteinases (TIMP). Moreover, IL-1 is a potent osteoclast-activating factor leading to bone resorption. These purported pro-inflammatoryproperties of IL-1 (4) are, however, opposed in vivo by the {elease of a natural inhibitor of IL-1, produced by macrophages and designated IL-1 receptor antagonist (IL1-RA). The molecule binds to the IL-1 receptor in a competitive way but its interaction with the receptor is not followed by signal transduction (5). Interestingly, strikingly elevated titers of IL-1-RA have been detected in the SF of RA patients (6). Similarly, elevated levels of TNF~ have been detected in the SF of RA patients (7). The recent finding that TNF~ transgenic mice develop arthritis (8), together with the potent pro-inflammatory activities of the cytokine in chondrocyte or synoviocyte cultures, indicates that over-expression of TNFc~ might be deleterious for the rheumatoid joint. Again, these findings should be tempered by the description of a natural inhibitor of TNFoL namely a soluble form of the TNF~ receptor capable of binding to the ligand and thereby preventing its interaction with membrane-bound receptors (9).

In contrast to IL-1 and TNFo~, IL-6 and TGF[3 are usually considered as "good boys": IL-6 does not induce collagenase nor inhibit proteoglycan synthesis but stimulates TIMP production (10). While IL-6 is probably not responsible for much of the cartilage destruction seen in RA, the cytokine might however be involved in bone resorption (11). TGF[3 displays anti-inflammatory effects in various models of arthritis by reducing the intensity of the inflammatory reaction and by opposing bone and cartilage destruction (12). More specifically, TGF[3 downregulates IL-1 receptor expression (13) and upregulates the production of IL-1-RA monocytes (14). Besides their activities on cartilage, synovium and bone, cytokines such as IL-1, TNFc~ and IL-6 are all potent inducers of the production of acute phase reactants by hepatocytes and thereby mediate the acute phase response seen in RA.

3. Circulating cytokine titers as activity or prognostic markers?

The clinical usefulness of measuring circulating cytokine activities in patients suffering from RA is still an unresolved yet important issue. Some studies have found a correlation between circulating levels of one or another cytokine and disease activity. Thus, plasma IL-113 titers were found to correlate with the Ritchie articular index in RA patients (15,16). Similarly, serum IL-6 titers were found to parallel those of acute phase reactants (2). More surprisingly, serum levels of the soluble form of the IL-2 receptor, a purported marker of T cell activation, do usually not correlate with disease activity in RA patients (17). Although such correlations - or absence of correlations - are of great interest from a physiopathological point of view, these studies will not convince any clinical rheumatologist to switch from the sedimentation rate to an expensive cytokine immunoassay as a disease activity index. Beforehand, the clinicians need to know whether cytokine measurements would be of any help in identifying the very patients with a poorer articular outcome and/or those responsive to a given therapy. Should this question be answered positively, the conservative world of rheumatology would certainly embrace these sophisticated tests, provided their costs would become fair. Thus far, there is not sufficient data available on cytokine titers during therapy with DMARDs. A recent study has demonstrated that serum IL-6 levels are downregulated during treatment with methotrexate, whereas TNF~ titers are not (18). Given the purported protective role ascribed to IL-6 and the potential pro-inflammatory properties of TNFot, these findings contrast somehow with the dramatic clinical effectiveness of the drug. This example illustrates that much more studies will be

Cytokines in RA

r e q u i r e d b e f o r e cytokine m e a s u r e m e n t s be used in r h e u m a t o l o g y practice.

4. Cytokines as therapeutic tools? T h e o r e t i c a l l y , the r e c e n t availability of r e c o m b i n a n t I L - 1 - R A should p r o v i d e us with a n e w class of " a n t i - i n f l a m m a t o r y " agents c a p a b l e of b l o c k i n g specifically the d e l e t e r i o u s effects of IL-1 o n synovial tissue. At p r e s e n t , this i n t e r e s t i n g a p p r o a c h is h o w e v e r u n p r a c t i c a l given the huge a m o u n t s of I L - 1 - R A n e e d e d to block c o m p l e t e ly the in vitro a n d in vivo biological activities of IL-1 (19). A large excess of I L - 1 - R A is i n d e e d r e q u i r e d - at the synovial level - to p r e v e n t the b i n d i n g of only a few m o l e cules of IL-1 to its r e c e p t o r , that are sufficient to trigger a biological r e s p o n s e (spare r e c e p t o r effect of IL-1).

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A n t i - c y t o k i n e or a n t i - c y t o k i n e r e c e p t o r m o n o c l o n a l a n t i b o d i e s are b e i n g e v a l u a t e d in selected cases of autoi m m u n e diseases in o r d e r to m o d u l a t e o v e r - e x p r e s s i o n of o n e or a n o t h e r cytokine. A first u n c o n t r o l l e d r e p o r t i n d i c a t e s t h a t i n j e c t i o n s of anti-TNFc~ m o n o c l o n a l antib o d i e s in R A p a t i e n t s were followed by a significant red u c t i o n in the swollen j o i n t count, lasting for at least 8 weeks, which was a c c o m p a n i e d by a drop in C-reactive p r o t e i n titers (20). P l a c e b o - c n t r o l l e d trials are obviously n e e d e d to c o n f i r m these b e n e f i c i a l clinical effects. W h e t h e r IL-10, a n i n h i b i t o r of IL-1 p r o d u c t i o n by m o n o c y t e s a n d an i n d u c e r of I L 1 - R A synthesis (21), will b e of clinical i n t e r e s t as a n t i - i n f l a m m a t o r y m o l e c u l e in a u t o - i m m u n e diseases in g e n e r a l a n d in R A p a t i e n t s in p a r t i c u l a r is c u r r e n t l y u n d e r investigation.

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13. Dubois, C.M., Ruscetti, EW., Palaszynski, E.W., Falk, L.A., Oppenheim, J.J., Keller, J.R. Transforming growth factor [3 is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action. J Exp Med 1990, 172, 737-744. 14. Turner, M., Chantry, D., Katsikis, P., Berger, A., Brennan, F.M., Fetdmann, M. Induction of the interleukin 1 receptor antagonist protein by transforming growth factor [5.Eur J Immunol 1991, 21, 1635-1639. 15. Eastgate, J.A., Symons,J.A., Wood, N.C., Grinlinton, EM., di Giovine, F.S., Duff, G,W. Correlation of plasma interleukin l lcvels with disease activity in rheumatoid arthritis. Lancet 1988, ii, 706709. 16. di Giovine, ES., Symons, J.A., Wilson, G., Duff, G.W. Interleukins and arthritis. In: Autoimmune connective tissue diseases, 1st ed., Eds.: Khamashta, M.A., Font, J., Hughes, G.R.V., Barcelona, Ediciones Doyma, 1993, 11-26. 17. Keystone, E.C., Snow, K.M., Bombardier, C., Chang, C.H., Nelson, D.L., Rubin, L.A. Elevated soluble IL-2 receptor levels in the sera and synovial fluids of patients with rheumatoid arthritis. Arthritis Rheum 1988, 31,844-849. 18. Barrera, R, Boerbooms, A.M.T., Janssen, E.M., Sauerwein, R.W., Gallati, H., Mulder, J., de Boo, T., Demacker, RN.M., van de Putte, L.B.A., van der Meer, J.W.M. Circulating soluble tumor necrosis factor receptors, interleukin-2 receptors, tumor necrosis factor c~, and interleukin-6 levels in rheumatoid arthritis. Longitudinal evaluation during methotrexate and azathioprine therapy. Arthritis Rheum 1993, 36, 1070-1079. 19. Dinarello, C.A., Thompson, R.C., Blocking IL-I: interleukin 1 receptor antagonist in vivo and in vitro. Immunol Today 1991, 12, 404-410. 20. Eliott, M.J., Maini, R.N., Feldmann, M., Long-Fox, A., Charles, R, Katsikis, R, Brennan, M.J., Walker, J., Bijl, H., Ghrayeb, J., Woody, J.N. Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor a. Arthritis Rheum 1993, 36, 1681-1690. 21. Moore, K.W., O'Garra, A., de Waal Malafyt, R., Vieira, R, Mosmann, TR. Interleukin-10. Ann Rev Immunol 1993, 11,165-190. Correspondence to: EA. HOUSSIAU, Rheumatology Unit, UCL 5390, Avenue Hippocrate 10, B-1200 Bruxellcs, BELGIUM.