ARTHRITIS & RHEUMATISM Vol. 58, No. 2, February 2008, pp S102–S109 DOI 10.1002/art.23053 © 2008, American College of Rheumatology The Development of Anticytokine Therapeutics for Rheumatic Diseases William P. Arend,1 with Mary B. Goldring2 The 5-year period of July 1995 through June 2000 saw considerable advancement in knowledge about the mechanisms of inflammation and tissue damage in many rheumatic diseases, particularly the important role of cytokines. Based upon this knowledge, the principles of rational drug design were applied to the development of new therapeutic approaches intended to block cytokine effects (1–3). Many of the most important articles that appeared in the literature during this period reporting both advances in basic science and the results of clinical trials in rheumatoid arthritis (RA) were published in Arthritis & Rheumatism (A&R). A classic article on anticytokine therapy, appearing in the May 1996 issue, is the most frequently cited basic research study published in A&R during this 5-year period. We selected this study—“Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNF-␣, anti-IL-1␣/␤, and IL-1Ra” by Joosten, Helsen, van de Loo, and van den Berg (4)—as a focal point in a brief review of the development of anticytokine therapeutics for rheumatic diseases, including the scientific background, subsequent developments in this area since publication of the article, and some comments on the broader implications for science, education, and practice in rheumatology. The senior author (WPA) was not only the Editor of A&R during this 5-year period, but was also a participant in this field of research. This review will not be comprehensive but will emphasize the evolution of concepts and ideas, including some personal comments on apparently serendipitous and paradoxical findings. Tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1) in rheumatic diseases The mechanisms whereby cells communicate with each other remained a mystery until the 1950s when studies began appearing that described biological activities in human or animal sera and in the supernatants of cells that mediated cell–cell communication. These biological activities were given different names based on their cellular sources or the functions they influenced, either systemically or on adjacent cells in culture. Later, it was discovered that many of these biological activities were due to single molecules with pleiotropic functions or that a specific biological activity could be attributed to more than one molecule. The first observation on TNF originated from experimental work in oncology on the mechanisms of “hemorrhagic necrosis” of transplanted tumors caused by material from gram-negative bacteria. In 1975 Old and colleagues reported that sera from BCG-infected mice contained a substance that caused necrosis of transplanted tumors in vivo or killed tumor cell lines in vitro (5). The conclusion was that this material was released from macrophages stimulated by bacterial products, accounting for the ability of activated macrophages to suppress transformed cells. Many investigators over the subsequent 30 years have described related biological activities in the supernatants of cultured cells called “cachectin” or other names. Finally in 1984, DNA cloning permitted identification of the proteins responsible for these antitumor activities as two closely related Supported by NIH grants AR-51749 to Dr. Arend and AG-022021 to Dr. Goldring. 1 William P. Arend, MD: University of Colorado School of Medicine, Denver (Editor, Arthritis & Rheumatism, 1995–2000); 2 Mary B. Goldring, PhD: Hospital for Special Surgery, Weill College of Medicine of Cornell University, New York, New York. Address correspondence to William P. Arend, MD, UCDHSC, Division of Rheumatology B115, 1775 N. Ursula Street, Aurora, CO 80045. E-mail: email@example.com. Submitted for publication August 22, 2007; accepted August 22, 2007. S102 ANTICYTOKINE THERAPEUTICS molecules: TNF␣ described by Goeddel and colleagues (6) and TNF␤, or lymphotoxin, described by Gray et al (7). Early work on fever laid the foundation for characterizing the molecule now known as IL-1. The concept that fever was due to products of tissue injury acting on cerebral regulatory centers was proposed as early as 1785 (discussed in ref. 8). Research in the 1940s on the possible existence of an endogenous pyrogen was plagued by the difficulty in separating the effects of exogenous bacterial materials, used to induce fever in experimental animals, from the release of a cellular product acting as an endogenous pyrogen. In 1953 Bennett and Beeson described the isolation of a possible endogenous pyrogen from rabbit polymorphonuclear leukocytes (PMNLs) (8,9). Atkins and Wood subsequently proved in 1955 the existence of endogenous pyrogen through serum transfer experiments in rabbits (10,11). These workers all hypothesized that fever seen in infection, inflammation, malignancy, and tissue injury might be due to the release of endogenous products from PMNLs and other cells. Over the next 30 years, numerous investigators described biological activities in serum and other body compartments, and in the supernatants of cultured cells, that induced seemingly unrelated responses in target cells. Some of these proteins were purified and characterized, including lymphocyte activating factor (12,13), endogenous pyrogen (14), and mononuclear cell factor (15). The consensus that they were all IL-1 was published in 1979 (16). This work culminated in the cloning in 1984 of two forms: IL-1␣ by Lomedico, Mizel, and colleagues (17), and IL-1␤ by Auron et al (18). Thus, the identification of TNF was based on research in cancer, whereas the scientific foundation of IL-1 was established by work in infectious diseases. The potential roles of these cytokines in cartilage destruction were first described by Fell and Jubb in this Journal in 1977, using the technique of organ culture developed previously by the Fell laboratory (19). A major issue in research on RA had been whether “the pannus destroys and invades the cartilage or whether the cartilage first undergoes some pathologic change that permits the ingrowth of the synovial tissue . . .” (19). These investigators observed that living pig cartilage in contact with synovium in a culture dish lost both collagen and proteoglycan; that dead cartilage lost some proteoglycan but less collagen; and most importantly, separation of the synovium from the cartilage in the same culture dish led to destruction of living cartilage only (Figure 1). They concluded that the synovium released a soluble material, S103 Figure 1. Synovial factor (catabolin) mediates degradation of cartilage proteoglycans and collagen. Culture of living pig synovium with dead cartilage produces little degradation of proteoglycans and no loss of cartilage. Culture of living pig synovium with living cartilage leads to significant degradation of both proteoglycans and collagen. This effect is still seen after separation of the synovium from the cartilage in the culture dish, suggesting the involvement of a soluble factor released from the synovium, termed catabolin. IL-1 ⫽ interleukin-1. Adapted from ref. 19. called catabolin, which stimulated the chondrocytes to release enzymes that destroyed the surrounding cartilage matrix. Catabolin was subsequently identified as pig IL-1 (20). The roles of cytokines in inflammation and tissue destruction in RA were first suggested by Dayer, Krane, and coworkers showing that purified IL-1 and TNF could stimulate the production of prostaglandins and collagenase by rheumatoid synovial cells (21,22). These observations and subsequent work by many investigators performed in the 1980s and early 1990s, in both culture models and animal models of RA, established the principle that TNF␣ and IL-1 originating in the synovium from patients with RA could lead to the production of inflammatory mediators and tissuedegrading enzymes (1–3). However, because of the apparent redundancy in the cytokine network, many prominent investigators remained skeptical that inhibition of any one cytokine would have a significant beneficial effect on human disease. As summarized in 1995, five criteria were met for the involvement of IL-1 and TNF␣ as mediators of joint tissue damage in RA (Table S104 Table 1. Criteria for the involvement of IL-1 and TNF␣ as mediators of joint tissue damage in rheumatoid arthritis* 1. These cytokines are present in the diseased tissue. 2. Synovial fluids containing these cytokines are injurious to normal cartilage in vitro. 3. Cartilage damage can be prevented by specific cytokine inhibitors or antagonists. 4. Recombinant IL-1 and TNF␣ produce damage to normal cartilage in vitro or in vivo. 5. High levels of these cytokines, or their messenger RNA, are present in the rheumatoid synovium at sites of active tissue destruction. 6. Progression of tissue damage in patients with rheumatoid arthritis should be prevented by treatment with inhibitors of IL-1 and TNF␣. * IL-1 ⫽ interleukin-1; TNF␣ ⫽ tumor necrosis factor ␣. Reproduced from ref. 2 and adapted, with permission, from Hollander AP. Criteria for identifying mediators of tissue damage in human autoimmune diseases. Autoimmunity 1991;9:171–6. 1) (2). The sixth criterion, the prevention of tissue damage in patients with RA by treatment with inhibitors of IL-1 and TNF␣, was being tested, based on parallel developments in work on cytokine inhibitors. Inhibitors of IL-1 and TNF␣ Given the ubiquitous presence of IL-1 and TNF␣ in inflammatory conditions and the pleiotropic nature of their effects, many investigators in the early 1980s hypothesized that natural regulators of their production or action must exist. Inhibitory activities against IL-1 in bioassays had been described by many investigators, who found these activities primarily in urine or in the supernatants of cultured cells (23,24). However, these IL-1 inhibitory activities remained uncharacterized, and in many cases they turned out to be due to substances interfering with the bioassays. At a symposium on IL-1 held in Ann Arbor in June 1985, two different laboratories reported specific inhibitory activities of IL-1 that were later determined to be caused by the same molecule, now termed IL-1 receptor antagonist (IL-1Ra). Continuing earlier work from many laboratories on excreted proteins, Dayer and coworkers described a specific inhibitory activity against IL-1 found in the urine of patients with fever or myelomonocytic leukemia (25). This laboratory subsequently described IL-1 inhibitory bioactivity in the serum and urine of patients with juvenile RA, suggesting its relevance to inflammatory diseases (26). Arend had previously spent a year (1980–1981) at the Strangeways Laboratory in Cambridge where he observed the continuing work on catabolin by Fell, who was still working into her 80s. At that time, he met the AREND AND GOLDRING coauthor of this editorial, who was a member of a team that identified IL-1–like activity in human synovial supernatants that stimulated plasminogen activator production by chondrocytes (27). Arend hypothesized that monocytes encountered adherent immune complexes as they entered an inflamed joint and may be stimulated to produce catabolin or IL-1. The Arend laboratory subsequently observed that monocytes cultured on adherent IgG released no detectable IL-1 bioactivity, but the addition of IL-1 revealed inhibitory bioactivity toward IL-1 in the supernatants (28). The ability of the semipurified material to specifically inhibit the binding of IL-1 to its receptors on target cells was first shown by Seckinger and Dayer for the IL-1 inhibitor in urine (29), followed by Arend and colleagues for the IL-1 inhibitor in the supernatants of monocytes cultured on adherent IgG (30). In the late 1980s, three different groups were racing to be the first to purify, clone the cDNA, and express recombinant IL-1Ra molecules (Figure 2). Earlier, Arend had given informal assistance to a team from Upjohn in Kalamazoo on the monocyte production of IL-1Ra and they proceeded independently. In 1987, Arend entered into a scientific collaboration with the biotechnology company Synergen in Boulder, with a team led by Thompson. Dayer formed a similar collaboration with investigators at Biogen in Geneva. Three different sources of IL-1Ra were employed: The Geneva team used frozen urine from AIDS patients collected in Boston and flown to Europe or urine from patients in Geneva with high fever. The Colorado investigators used supernatants from human monocytes cultured on a substrate of IgG. The Upjohn team used the supernatants of the human myelomonocytic cell line U937 cultured on IgG (Figure 2). The Colorado investigators were the first to be successful in this endeavor, followed by the team from Upjohn (31–33). Subsequent development and preclinical evaluation of IL-1Ra has been reviewed (34–36). A somewhat different endeavor was underway in the late 1980s to develop inhibitors of TNF␣. Based on observations that IL-1 inhibitory bioactivities were present in human urine, three different laboratories examined this source and found inhibitory activities against TNF␣: Seckinger et al (37), Olsson et al (38), and Engelmann et al (39). These TNF␣ inhibitory activities in urine were found to be due to the soluble extracellular portions of TNF receptors. Subsequently, investigators at Immunex developed a therapeutic agent containing the extracellular portions of two p75 TNF␣ receptors coupled to the Fc portion of human IgG1 (40). ANTICYTOKINE THERAPEUTICS S105 Classic article from A&R, 1996 Figure 2. Race to purify the protein, clone the cDNA, and express recombinant interleukin-1 receptor antagonist. Three different laboratory groups in Colorado, Kalamazoo, and Geneva were simultaneously working on this project in the late 1980s. The Colorado group was the first to be successful. The development of monoclonal antibodies against TNF␣ began in the laboratory of Vilcek in 1984, in collaboration with Centocor. In 1989, a murine anti-TNF␣ monoclonal antibody, termed A2, was developed (41). In the same year, a strong rationale for the inhibition of TNF␣ in RA was established through the studies of Brennan, Maini, and Feldman et al (42). They suggested that TNF␣ may be the main inducer of IL-1, since antibodies to TNF␣ reduced the production of IL-1 in cultured rheumatoid synovial cells. Based on the initial development of the monoclonal antibody A2, the construction and characterization of a mouse/human chimeric anti-TNF␣ antibody, termed cA2, was described in 1993 (43). This eventual therapeutic agent was capable of neutralizing TNF␣ in vitro, blocking TNF␣ binding to both types of receptors in vitro, and protecting against cachexia and lethality in TNF␣-transgenic mice (44). The subsequent development and preclinical evaluation of therapeutic agents inhibitory to TNF␣ has been reviewed (3,45). With the efficacy of blockade of TNF␣ or IL-1 in experimental animal models of RA, which was described in the early 1990s, questions were raised about the relative effects of these two therapeutic approaches on inflammation vs. tissue destruction. Early studies suggested that inhibition of TNF␣ was more antiinflammatory, whereas IL-1 blockade was more effective in preventing tissue destruction. The selected article from the van den Berg laboratory provides a complete analysis of various inhibitors of TNF␣ and IL-1 in collageninduced arthritis (CIA) in mice (4). Five important conclusions can be drawn from these studies: 1) Antibodies against TNF␣ prevented the onset of CIA, were mildly efficacious early in disease, and exhibited only weak effects in established CIA. 2) Antibodies to IL1␣/␤ or IL-1Ra completely prevented the onset of CIA and markedly suppressed established disease. 3) Blockade of TNF␣ did not necessarily eliminate the production of IL-1. 4) IL-1 inhibition in established CIA prevented destruction of cartilage more effectively than did blockade of TNF␣. 5) High continuous doses of IL-1Ra were necessary to prevent CIA and to treat established disease. The role of IL-1 and TNF␣ in inflammation and tissue destruction in CIA has been clarified in recent studies. Mice transgenic for human TNF␣ but lacking the genes for IL-1␣ and IL-1␤ developed inflammation but no destruction of cartilage or bone, whereas the opposite phenotype was found with mice lacking TNF␣ production but possessing intact genes for IL-1 (46). These results clearly establish that in this animal model of arthritis TNF␣ is primarily responsible for inflammation with cartilage and bone destruction mediated solely by IL-1. However, caution should be exercised in applying these findings to human disease. IL-1 inhibition is more beneficial in CIA than in other animal models such as antigen-induced arthritis. The doses, half-lives, and neutralizing capacities of the experimental therapeutic approaches may not have been comparable in the studies of Joosten et al (4). It is clear that no animal model of inflammatory arthritis is totally equivalent to the human disease of RA, in which disease mechanisms may be more complex than those operative in any animal model. Although anticytokine therapies in animal models have been useful to establish proof-of-principle, it is not a substitute for clinical trials in RA. S106 Development of IL-1 and TNF␣ inhibitory therapeutics since 1996 Treatment of sepsis syndrome was the primary interest of biotechnology companies supporting clinical trials with recombinant IL-1Ra or chimeric monoclonal antibodies to TNF␣. Sepsis syndrome is an acute disease that often leads to a rapid decline and death within 30 days. A not insignificant impetus for placing a high priority on targeting this disease was the financial incentive, since a successful new treatment for this lethal disease would return potentially early and vast profits. Despite extensive preclinical evidence that IL-1 and TNF␣ were important targets, clinical trials on treatment of sepsis syndrome with inhibitors of IL-1 or TNF␣ failed, forcing more than one biotechnology company out of business. Despite this setback, the development of anticytokine treatments for RA, other chronic rheumatic diseases, and inflammatory diseases of other organs was continued with considerable success. The clinical trials on TNF␣ blockade, using either chimeric or humanized monoclonal antibodies or using soluble receptors, gave positive results in one-half or more of RA patients, as summarized by Feldmann and colleagues (3,45). TNF␣ blockade with both approaches has also proven efficacious in spondylarthropathies and psoriatic arthritis, while treatment of inflammatory bowel disease with monoclonal antibodies to TNF␣ has been successful. Most importantly, blockade of TNF␣ prevents cartilage and bone damage, possibly even in patients who do not show a dramatic clinical response. Treatment of RA with IL-1Ra was only modestly beneficial, possibly due to its poor pharmacokinetics and the inability to achieve consistently high levels in the circulation even with daily injections (47). However, IL-1Ra results in a rapid and dramatic response in childhood or adult-onset Still’s disease and in a variety of autoinflammatory disorders characterized by over-production of IL-1 (48,49). Other cytokines in the pathogenesis of RA Considerable knowledge has been amassed over the past 10 years on pathogenic mechanisms in RA and the possible importance of additional cytokines beyond IL-1 and TNF␣. The chondrocyte is a complex cell that produces and responds to a variety of cytokines in its primary role in remodeling the cartilage matrix (50). All cells in the rheumatoid joint—including T and B lymphocytes, dendritic cells, macrophages, fibroblasts, and AREND AND GOLDRING Table 2. Some broader implications of the development of inhibitors of IL-1 and TNF␣* 1. Provided unique tools for basic, translational, and clinical research. 2. Permitted an elucidation of mechanisms of disease. 3. Used successfully for diseases outside of rheumatology. 4. Stimulated the development of new and novel anticytokine agents. 5. Changed the nature of postgraduate education and practice in rheumatology. * IL-1 ⫽ interleukin-1; TNF␣ ⫽ tumor necrosis factor ␣. mast cells in the synovial tissue as well as chondrocytes in the cartilage—are under the influence of a complex network of cytokines (51). A number of these molecules may promote inflammation including GM-CSF, IL-6, IL-12, IL-15, IL-17, IL-18, IL-23, IL-32, and IL-33. However, it remains unclear whether cytokines in the rheumatoid joint are organized in any hierarchical pattern and which molecules might be the best targets for clinical intervention (51). Broader implications for science and rheumatology The development of TNF␣ and IL-1 inhibitors has provided new therapeutic options for diseases where traditional approaches have often failed. This is the most important point for patients and physicians; we can now better relieve suffering and prevent disability from some severe diseases. In addition, the emergence of this new field has broader implications for science and for the practice of rheumatology (Table 2). Tools of basic and translational science. The development of specific inhibitors of IL-1 and TNF␣ has allowed scientists to explore more precisely the roles of these cytokines in systems in vitro, in normal physiology in vivo, and in animal models of disease. IL-1Ra is the first described naturally occurring molecule that functions as a specific competitor of receptor binding of a hormone-like molecule. Maintaining a local tissue balance between IL-1 and IL-1Ra is important in prevention of disease. Particular inbred strains of mice rendered genetically deficient in production of IL-1Ra spontaneously develop a chronic inflammatory arthritis resembling RA or inflammation of the arterial wall resembling vasculitis (52,53). An allelic polymorphism in the IL-1Ra gene is associated with a variety of human diseases, primarily of epithelial or endothelial cells, possibly through decreasing the production of an intracellular isoform of IL-1Ra and interrupting the balance with IL-1 (54). ANTICYTOKINE THERAPEUTICS Abnormalities in disease. The mechanism whereby TNF␣ may predispose to a disease process remains unclear. The results of initial studies indicated that the major action of TNF␣ blockers in vivo included down-regulation of the cytokine cascade, decreased trafficking of leukocytes into the joint, a reduction in local angiogenesis, and induced apoptosis in synovial fibroblasts (45). However, the results of recent studies indicate that anti-TNF␣ therapy restores deficient regulatory T cell function in RA (55,56). How excess TNF␣ may lead to abnormalities in regulatory T cell function in RA is under current study, although both IL-1 and TNF␣ may directly inhibit regulatory T cell function (57). Applications to other diseases. The availability of inhibitors of IL-1 and TNF␣ has led to a greater understanding of disease mechanisms in other organs including the brain, lungs, heart, kidneys, reproductive system, and endocrine system. For example, IL-1Ra is efficacious in treatment of type 2 diabetes mellitus and studies on type 1 diabetes are in progress (58). New anticytokine treatments. The success of antiIL-1 and TNF␣ agents in the treatment of rheumatic and other diseases has given considerable impetus to a search for better treatment approaches. Recent reviews have established some lessons learned over the past 10 years and possible directions in the development of new biologic therapies for RA (59,60). Among them are: 1) mechanisms of action of biologic therapies may differ from those derived from in vitro/ex vivo assays or preclinical studies; 2) Fc regions of monoclonal antibodies or Ig fusion proteins confer multiple functions on biologic agents, not all beneficial; 3) immunogenicity is a feature of all biologic agents; and 4) observed adverse effects of biologic therapies might not be entirely predictable. Recommendations for development of future biologic therapies are summarized in a recent review (60). Despite this complexity and uncertainty, several new approaches are being developed to block IL-1 including other biologics, inhibitors of IL-1 processing, and small molecule inhibitors (61). Based on the success of blocking IL-1 or TNF␣ in the treatment of disease, there is interest in the potential efficacy of inhibiting more upstream cytokines, including IL-15, IL-17, IL-18, IL-21, and IL-32 (59,62,63). Treatment of disease is also being explored through the use of inhibitors of signal transduction pathways induced by IL-1 or TNF␣. Postgraduate education and practice in rheumatology. The successful development of anticytokine therapies has not only benefited our patients but has been accompanied by changes in postgraduate education and practice. Physicians and trainees now routinely experi- S107 ence drug marketing masquerading as postgraduate education. The ACR faces ethical issues with regard to the potential financial influences of large companies, and the opportunity has emerged to enhance practice income through altering prescription patterns. In a sense, we have witnessed a “loss of innocence” in the field of rheumatology, joining other subspecialties such as oncology and cardiology, where such changes in education and practice are also occurring. Summary We have briefly reviewed the history of IL-1 and TNF␣ and the development of therapeutic agents that block these cytokines. 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