novial T cells4 and FLS.16 These cells, together with im- migrating activated leukocytes, produce a "second wave" of inflammatory cytokines that include, among ...
American Journal of Pathology, Vol. 152, No. 3, March 1998 Copyright X American Societyfor Investigative Pathology
Commentary Production of Interleukin-6 by Synovial Fibroblasts in Rheumatoid Arthritis
Heinz Baumann* and Irving Kushnert From the Department of Molecular and Cellular Biology,* Roswell Park Cancer Institute, Buffalo, New York and Metro Health MIedical Center and Case Western Reserve University,t
Cleveland, Ohio
Persistent inflammation, as exemplified by rheumatoid arthritis (RA), draws the attention of the afflicted patient, the treating physician, and the investigator to three basic questions: what causes the disease, what maintains the inflammatory response, and what treatment will provide relief or recovery? RA is a systemic illness, the major manifestation of which is a destructive inflammatory process of the peripheral synovial joints,1 which shows the same physiological and biochemical features that characterize inflammatory processes in other body tissues.2 Whereas inflammation may in some instances be caused by infection or local tissue injury. In the case of RA, an autoimmune reaction leads to progressive recruitment of inflammatory cells (monocytes, T and B cells, and neutrophils) from the circulation to the synovium.3 Interactions between the immigrating leukocytes and resident synoviocytes, which include monocyte-like, fibroblast-like (FLS), and dendritic cells, establish a local milieu of cytokines, chemokines, and other inflammatory mediators and metabolites,-12 which provides stimulatory and proliferative signals to synovial cells and promotes the degradation of cartilage and bone.13'14 RA can also elicit a systemic acute phase reaction resulting in increased concentrations of acute-phase plasma proteins that may influence the inflammatory process.2'15 During the last decade, the roles of many cytokines in control of specific aspects of the inflammatory reaction have been defined, particularly in regard to their engagement in intercellular communication cascades and in determining disease progression. Tumor necrosis factor (TNF)-a and interleukin (IL)-13, as initiating inflammatory cytokines derived from activated monocytes, act on synovial T cells4 and FLS.16 These cells, together with immigrating activated leukocytes, produce a "second wave" of inflammatory cytokines that include, among others, IL-6, leukemia-inhibitory factor, oncostatin M,17 gran-
ulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and to a variable extent,
the "anti-inflammatory" cytokines IL-10, transforming growth factor-f, and IL-4.18,19 High concentrations of IL-6 in RA synovial fluid correlate well with the severity of disease. As IL-6 in the circulation of RA patients is also elevated, it has been suggested that this cytokine serves as a marker for RA activity. Fibroblast-like synovial cells have been identified as significant producers of IL-6 by in situ immunocytochemistry and mRNA hybridization and by biochemical analysis of primary cultures of synoviocytes. Expression of IL-6 by these cells in culture is enhanced by treatment with TNF-a, IL-1i, prostaglandin E2, and various activators of the intracellular signaling pathways acting through protein kinase A (PKA) or protein kinase C (PKC), suggesting that the IL-6 gene is a target for multiple intracellular regulatory mechanisms. Insight into the mechanisms regulating these pathways has been obtained from molecular characterization of the IL-6 gene promoter.20-23 Several DNA sequences within the 5' flanking region of the IL-6 gene have been identified that serve as binding sites of transcription factors that are specifically activated by cytokines and that mediate transcriptional induction of IL-6. These regulatory elements, which have been localized to the following positions relative to the transcription initiation site, include from -283 to -277, the interaction site for the activating protein-1 (AP-1), from -173 to -151, the enhancer for regulation by PKA and PKC, from -158 to -145, the binding site for CCAAT/enhancerbinding proteins-,B, and from -73 to -64, the binding site for nuclear factor (NF)-KB. The promoter region containing the last binding site proved to be most critical for producing transcriptional induction by TNF-a and IL-ip as a result of the activating effects of these cytokines on NF-KB in synovial fibroblasts and many other cell types. Among the various NF-KB isoforms, the dimer of p65 is a particularly effective inducer of IL-6.24 Indeed, markedly elevated DNA binding activity of NF-KB is observed in RA synovial tissue.25'26 Recently, a binding site for the Supported by the National Institutes of Health Grants CA 26122 (H. Baumann) and AG 02467 (I. Kushner). Accepted for publication January 15, 1998. Address of corresponding author: Dr. Heinz Baumann, Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263-0001.
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ubiquitous transcriptional repressor protein C-promoterbinding factor 1 (CBF1) has been detected at position -68 to -61 of the IL-6 promoter immediately adjacent to the NF-KB binding site.27 Functional analyses of the effects of CBF1 on IL-6 promoter activity by transfection experiments in tissue culture cells have indicated that CBF1 does not affect basal level expression. Rather, it reduces induced IL-6 gene transcription when both CCAAT/enhancer-binding proteins-,8 and NF-KB are coactivated and bound to their respective interaction sites at the promoter, a circumstance that is expected following TNF-a or IL-1, treatment. Hence, a role of CBF1 in moderating IL-6 gene transcription following activation by cytokines has been presumed. Substantial inhibition of IL-6 gene expression by dexamethasone in various cell types has long been recognized.28 This inhibitory action depends on the steroidactivated glucocorticoid receptor (GR) in fibroblastic cells and appears to be accomplished by the physical interaction of GR with the p65 subunit of NF-KB, thereby reducing the level of active NF-KB available for IL-6 gene regulation.29'30 Alternative modes of inhibition of IL-6 gene transcription, such as by binding of GR directly to specific GR response elements on the IL-6 promoter31 or by increasing the expression of the inhibitory subunit IKBa,32'33 do not seem to apply for fibroblastic cells.30 Earlier, however, Tobler et a134 provided a somewhat different model that proposes that dexamethasone destabilizes IL-6 mRNA in fibroblasts or alternatively prevents IL-1i -35 or TNF-a-36 mediated prolongation of IL-6 mRNA half-life. Although the inhibition of cytokine-induced IL-6 expression by dexamethasone is similarly detected in synoviocytes and monocytes, inhibition by IL-4 or IL-10, as achieved in monocytes by enhanced mRNA turnover and prevention of NF-KB activation, respectively,37 has not been convincingly demonstrated in FLS cells.38-40 The combination of inflammatory leukocytes, activated synovial cells, and conditioned extracellular synovial milieu is thought of by many to determine RA-associated damage and destruction of cartilage and bone tissue. The question arises whether the persistently high IL-6 concentration in RA synovial fluid serves as a critical participant in this process. In tissue culture, IL-6 does not significantly affect proliferation or gene function in FLS,41 suggesting minimal if any autocrine response probably due to the absence of the ligand-binding subunit for IL-6, IL-6Ra, in these cells. However, in the presence of soluble IL-6Ra (slL-6R, the extracellular domain of IL-6Ra that has also been detected in synovial fluid),42'43 IL-6 forms a complex (IL-6-slL-6R) that is capable of interacting with gp130, the signal transducing subunit of the receptor for IL-6 and related cytokines, on FLS. Engagement of gp130 triggers a proliferative response of synovial fibroblasts,42 which surprisingly is not observed in dermal fibroblasts.44 A similar mechanism for activation of osteoclasts by IL-6*slL-6R has been observed.43 Moreover, the high level of IL-6 in RA synovial tissue is expected to act on the local lymphocytes and dendritic cells that, in contrast to FLS, do express IL-6Ra. Such IL-6-activated
cells, in part, would exacerbate the inflammatory and tissue destructive activity of the synovial cell population. With the RA-dependent increase of circulating IL-6 that may originate from the site of inflammation, hepatic synthesis of acute phase plasma proteins is stimulated.2 There are reasons to believe that some of these positive acute phase proteins may augment the inflammatory process, whereas others may modulate it. Complement components clearly play pro-inflammatory roles. In contrast, the increase of several antiproteases in the circulation is viewed as beneficial, as they neutralize extracellular proteolysis at sites of inflammation.15 Similarly, recent studies indicate that C-reactive protein transgenic mice, which express high levels of C-reactive protein in the circulation, are more resistant to endotoxemia,45 suggesting an anti-inflammatory effect of C-reactive protein. In addition, RA or many other inflammatory processes through enhanced production of inflammation-associated cytokines may also cause cachexia46'47 and impaired growth through an IL-6-mediated reduction of insulin-like growth factor-1.48 In view of the pathophysiological role of IL-6 and the recognition that RA synoviocytes contribute to increased IL-6 production, a number of important questions become obvious. Is elevated production of IL-6 by RA FLS a consequence of persistent stimulation by inducing cytokines from inflammatory leukocytes, in particular TNFa'? An answer to this question is provided by the study of Miyazawa et al49 published in this issue. These authors recognized that the commonly used primary cultures of RA synovial cells consist of a mixture of cell types and that paracrine cell communication might contribute to the observed increased IL-6 expression in such cultures.50-53 Hence, to assess IL-6 expression in RA FLS under defined culture conditions, the authors studied clonal cell lines of RA FLS. Clonal lines that might differ in the level of IL-6 expression would be useful in identifying molecular mechanisms responsible for differences in IL-6 expression. Miyazawa et a149 demonstrated that clonal FLS lines could be obtained from primary RA synovial cell cultures but intriguingly not from osteoarthritic synovial cell cultures. Approximately one half of the clonal RA FLS lines exhibited high IL-6 production, whereas the other half were low IL-6 producers, indicating that elevated IL-6 expression is maintained in the former cells over an extended in vitro culture period and is not dependent on an inducing agent(s) produced by other cell types. These results are also in agreement with previous studies that demonstrated constitutive expression of IL-6 by longterm cultures of RA FLS.52'53 The authors have also demonstrated that production of IL-6 by FLS lines is not a result of autocrine stimulation through endogenous IL-1 or TNF-a. However, present and previous cell culture studies as yet have not ruled out the possibility that IL-6 production is, in part, because of deregulated IL-6 expression that arises during the in vitro culture period. Inappropriate activation of IL-6 expression in primary cell cultures is not unprecedented, as has been shown by the induction of the IL-6 gene in primary rat hepatocytes54'55 and transformed hepatoma cells,56 whereas adult liver parenchymal cells in situ do not express IL-6.
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The established FLS lines with high and low IL-6 expression offered Miyazawa et al49 the opportunity to determine which differences in transcription factor activities correlated with levels of IL-6 expression. The authors found that the high IL-6-producing FLS cell lines supported greater transcriptional activity from the IL-6 promoter than the low IL-6-producing lines. High IL-6 gene expression specifically correlated with elevated levels of the DNA-binding activity of NF- KB and CBF1. The involvement of these two factors in IL-6 gene transcription was tested indirectly by analyzing the regulation of IL-6 promoters in which the binding site for NF-KB, CBF1, or both was mutated. As each mutation reduced gene transcription, the authors conclude that probably both NF-KB and CBF1 act as IL-6 gene activators and that their constitutively elevated DNA-binding activity may account for the high IL-6 expression seen in some of the RA FLS lines. Finally, Miyazawa et al49 proposed that a fraction of fibroblastic cells in RA synovial tissue constitutively transcribe IL-6 thus contributing to the characteristically high IL-6 production seen in both the synovium and in RA synoviocyte cultures and, critically, that this high IL-6 expression is independent of inducing agents. As is inevitable when any study that provides new and significant findings appears, this report raises a set of new questions that address broad issues ranging from physiology to molecular biology. For example, does the relative number of high IL-6-producing FLS lines that are recovered by the limiting dilution culture technique represent the ratio of constitutively activated fibroblastic cells in the RA synovial tissue? Although clonal FLS lines with elevated IL-6 production were recovered, is the level of IL-6 expression in these cells equal to the maximal level of IL-6 expression in cells within the RA synovial tissue? Do high IL-6-producing RA FLS lines still respond to TNF-a or IL-13 by additional enhanced expression of IL-6? As elevated DNA binding activities of NF-KB and CBF1 were found and felt to be responsible for IL-6 gene induction, what are the molecular and cellular mechanisms that lead to enhanced activity of these transcription factors? Also, the contradictory attribution of CBF1, as an inducer49 or as a suppressor of IL-6 gene expression,27 needs to be resolved. Finally, what influence would constitutive IL-6-production by RA FLS have on the efficacy of treatment? An important issue is to identify whether elevated IL-6 expression in RA FLS is still sensitive to inhibition by dexamethasone or to the moderating influence of those anti-inflammatory cytokines, immunomodulators,57 and antirheumatic drugs58 that act directly on synovial fibroblasts. Considering that the action of these agents affects, at least in some cases, signal transduction pathways and gene transcription, it is conceivable that modifications in the control mechanisms of IL-6 gene expression in RA FLS act downstream of the inhibitory pathways and hence are resistant to these drugs' effect. If this proves to be the case and the relative number of constitutively-activated, drug-resistant FLS cells in synovial tissue is substantial, then exciting new approaches to the treatment of RA present themselves.
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