Ronald L. Wilder

The Pathophysiologic Roles of Interleukin-6 in Human Disease

Dr. Dimitris A. Papanicolaou (Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health [NIH], Bethesda, Maryland): During inflammation, the inflammatory cytokines tumor necrosis factor-, interleukin-1, and interleukin-6 are secreted, in that order [1, 2]. Interleukin-6 then inhibits the secretion of tumor necrosis factor- and interleukin-1 [3], activates the production of acute-phase reactants from the liver [4], and stimulates the hypothalamic-pituitary-adrenal axis [5] to help control the inflammation. In this sense, interleukin-6 is both a proinflammatory and an anti-inflammatory cytokine. It is produced not only by immune and immune accessory cells (such as monocytes, macrophages, lymphocytes, endothelial cells, fibroblasts, mast cells, astrocytes, and microglia) but also by many nonimmune cells and organs (such as osteoblasts, bone marrow stromal cells, keratinocytes, synoviocytes, chondrocytes, intestinal epithelial cells, Leydig cells of the testis, folliculostellate cells of the pituitary, endometrial stromal cells, trophoblasts, and vascular smooth-muscle cells) [4, 6-12]. What makes interleukin-6 particularly interesting to physicians is its marked pleiotropy and its involvement not only in inflammation but in the regulation of endocrine and metabolic functions. Its diverse actions are summarized in the Table 1 [13]. Table 1. Actions of Interleukin-6 Molecular Biology of Interleukin-6 Located on the short arm of chromosome 7, the interleukin-6 gene consists of 5 exons and 4 introns and has a fairly complex transcriptional regulation [14]. The interleukin-6 promoter has recognition sites for transcription factors NF-IL6 (C/EBP ), which belongs to the C/EBP family, and NF-B, which is a major mediator of inflammatory stimuli [15, 16] (Figure 1). Figure 1. Transcriptional regulation of the interleukin-6 (IL-6) promoter. Interleukin-6 exerts its broad range of action through the interleukin-6 receptor, a single-pass transmembrane receptor not directly involved in signal transduction. Instead, activation of the receptor by interleukin-6 induces homodimerization of another transmembrane receptor, gp130, which initiates the transduction cascade [13]. The interleukin-6 receptor has a second soluble form that consists of the extracellular domain of the membrane receptor. Interleukin-6 also activates gp130 through this soluble form, even on cells that lack the interleukin-6 receptor on their membranes [17, 18]. For example, interleukin-6 can cause cardiac hypertrophy through gp130, even though cardiac myocytes lack the interleukin-6 receptor. The gp130 receptor is shared by many cytokines and growth factors for signal transduction, including interleukin-11, oncostatin-M, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotropin-1, and leptin [13] (Figure 2). Figure 2. Pleiotropy of interleukin-6 (IL-6) action. Endocrine and Metabolic Actions of Interleukin-6 As Figure 3 shows, interleukin-6 has a broad array of actions on the endocrine and metabolic systems. Figure 3. Regulation of the secretion and endocrine actions of interleukin-6 (IL-6). Hypothalamic-Pituitary-Adrenal Axis Animal studies have shown that interleukin-6 acutely activates the hypothalamic-pituitary-adrenal axis by acting primarily on the corticotropin-releasing hormone neuron. Specifically, a blockade of corticotropin-releasing hormone inhibits the effects of exogenous interleukin-6 on the hypothalamic-pituitary-adrenal axis in rats [19]. Subcutaneous administration of interleukin-6 to normal human volunteers resulted in elevated plasma levels of adrenocorticotropin hormone (ACTH) and then an increase in plasma levels of cortisol [20]. The plasma level of cortisol peaked after the plasma level of ACTH peaked; this indicates that, at least in this acute setting, cortisols response to interleukin-6 administration is mediated by release of ACTH [21]. Interleukin-6 seems to be one of the most potent stimuli of the hypothalamic-pituitary-adrenal axis in humans. Subcutaneous administration of interleukin-6 once a day for 7 days resulted in remarkable enlargement of the adrenal glands similar to that seen after prolonged activation of the adrenal glands by ACTH (as in Cushing disease or ectopic ACTH production) [22]. In animals and humans, glucocorticoids inhibit production of interleukin-6 in vitro and in vivo [23, 24]. In a recent study [25], administration of hydrocortisone or dexamethasone attenuated exercise-induced elevation of plasma levels of interleukin-6. Conversely, correction of hypercortisolism by surgical removal of a corticotroph adenoma when plasma levels of cortisol were undetectable increased plasma levels of interleukin-6 more than fourfold in patients with Cushing disease [26]. Therefore, interleukin-6 stimulates the hypothalamic-pituitary-adrenal axis and cortisol exerts negative feedback on secretion of interleukin-6. Interleukin-6 thus functions as a hormone in the traditional sense: It participates in a feedback loop of the hypothalamic-pituitary-adrenal axis. Thermogenesis and Basal Metabolic Rate Several cytokines, especially interleukin-1, are pyrogenic in humans and animals [27]. Administration of interleukin-6 causes elevations in body temperature and resting metabolic rate in humans [20]. In animals, the fact that nonsteroidal anti-inflammatory agents can inhibit the thermogenic effect of interleukin-6 suggests that this effect may be mediated by prostanoids [28]. The Steroid Withdrawal Syndrome The concept of the steroid withdrawal syndrome was introduced in 1965 by Amatruda and colleagues [29]. The syndrome is characterized by fever; headache; nausea; fatigue; malaise; somnolence; anorexia; and, less commonly, flu-like symptoms, such as arthralgias and myalgias. These symptoms occur during an abrupt reduction in levels of circulating cortisol and have been seen in patients who became severely hypocortisolemic when they underwent curative transsphenoidal surgery for Cushing disease. At that time, plasma levels of interleukin-6 were greatly elevated [26]. Normal volunteers and patients who received interleukin-6 had similar symptoms; this suggests that interleukin-6 participates in the pathogenesis of the steroid withdrawal syndrome [20, 21, 30]. Vasopressin and the Syndrome of Inappropriate Secretion of Antidiuretic Hormone The release of arginine vasopressin by the posterior pituitary is controlled by changes in intravascular volume and by osmotic stimuli. The syndrome of the inappropriate secretion of antidiuretic hormone occurs in the absence of serum hyperosmolarity or hypovolemia and can be caused by several conditions, including certain types of trauma, infections (meningitis and pneumonia), and inflammation [31]. During the syndrome, production of inflammatory cytokines (including interleukin-6) increases. Because high doses of interleukin-6 increase plasma levels of vasopressin in humans [32], endogenous interleukin-6 may also participate in the pathogenesis of this syndrome. Interleukin-6 as a Stress Hormone Because it innervates many immune organs, such as the spleen and the thymus, the autonomic nervous system interacts directly with the immune system [33, 34]. Stress or administration of adrenaline to animals elevates levels of endogenous interleukin-6, but pretreatment with a -adrenergic antagonist abolishes this effect. These effects suggest that interleukin-6 secretion is stimulated through -adrenergic receptors [35, 36]. In a recent study [37], administering adrenaline to humans increased plasma levels of interleukin-6. In normal volunteers, treadmill exercise also increased levels of plasma interleukin-6. In addition, peak plasma levels of catecholamines were positively correlated with plasma levels of interleukin-6 [25]. These data indicate that interleukin-6 is secreted during stress, probably through a -adrenergic receptor mechanism, and that it participates in the stress response. Lipid Metabolism Normal volunteers had precipitous reductions in serum total cholesterol levels, apolipoprotein B levels (this reflects low-density lipoprotein cholesterol), and triglyceride levels within 24 hours of interleukin-6 administration [38]. During sustained elevation of plasma catecholamine levels (such as that which occurs immediately after myocardial infarction), serum lipid levels are temporarily reduced, rendering serum cholesterol measurements misleading [39]. Whether catecholamine-stimulated endogenous interleukin-6 contributes to the transient decrease in serum lipid concentrations observed in conditions with increased sympathoneural discharge requires further study. Thyroid Axis and the Euthyroid Sick Syndrome Exogenous interleukin-6 decreased the secretion of thyroid-stimulating hormone in animals in vivo [5], and interleukin-6 was recently shown to be associated with a decrease in serum levels of thyroid-stimulating hormone and triiodothyronine in humans within 4 hours of administration. Interleukin-6 seemed to have a more lasting effect on triiodothyronine levels; the decrease persisted for at least 24 hours after a single injection of interleukin-6 [20, 21]. Thus, interleukin-6 was associated with changes in thyroid function test results similar to those seen in the euthyroid sick syndrome, a condition of physiologic hypothyroidism that occurs during nonthyroidal illness, apparently in an attempt by the organism to conserve energy. Depending on the severity and duration of the illness, it ranges from an isolated decrease in serum triiodothyronine levels in mild cases to a decrease in serum levels of free thyroxine and, finally, to subnormal thyroid-stimulating hormone levels in more severe cases [40]. Interleukin-6 levels are frequently elevated in conditions that are associated with the euthyroid sick syndrome (such as infection or inflammation, major trauma or surgery, and prolonged stays in the intensive care unit) and

Glucocorticoid Therapy for Immune-Mediated Diseases: Basic and Clinical Correlates

Dr. Dimitrios T. Boumpas (Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], National Institutes of Health [NIH], Bethesda, Maryland): Since 1949, when Hench and colleagues first introduced cortisone for the treatment of rheumatoid arthritis, glucocorticoids have revolutionized the treatment of immunologically mediated diseases. Although substantial complications associated with glucocorticoids have tempered enthusiasm for their use, they have remained the cornerstone of therapy for virtually all immunologically mediated diseases. In recent years, an explosion of new information has occurred relevant to both basic and clinical aspects of glucocorticoid therapy. We describe the molecular mechanisms, sites of action, and effects of glucocorticoids on various cells involved in inflammatory and immunologically mediated reactions. Treatment principles are also provided with examples of specific glucocorticoid regimens in prototypical conditions. We also review selective complications of glucocorticoid therapy and discuss recent information about their pathogenesis and management. Mechanisms of Action Dr. George P. Chrousos (Chief, Pediatric Endocrinology Section, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland): Glucocorticoids exert most of their effects through specific, ubiquitously distributed intracellular receptors [1]. The classic model of glucocorticoid action was described more than two decades ago and is briefly updated here (Figure 1, panel A). Glucocorticoids circulate in blood, is either in the free form or in association with cortisol-binding globulin. The free form of the steroid can readily diffuse through the plasma membrane and can bind with high affinity to cytoplasmic glucocorticoid receptors (the role of receptors primarily residing in the nucleus is controversial). The formation of the ligand-receptor complex is followed by its activation (that is, translocation into the nucleus and binding to what are called acceptor sites). The bound complex modulates transcription of specific genes that encode proteins responsible for the action of glucocorticoids. Figure 1. Mechanisms of glucocorticoid action. Panel A. Panel B. Glucocorticoid Receptors In 1985, the complementary DNA of the human glucocorticoid receptor was cloned [2]; it contains three main functional domains Figure 1, panel B): first, the DNA-binding domain in the center of the molecule that recognizes specific sequences of the DNA called hormone-responsive elements; second, the ligand-binding domain in the carboxyl terminal region that interacts with the specific steroid; and third, the immunogenic domain in the amino terminal region. The nonactivated glucocorticoid receptor resides in the cytosol in the form of a hetero-oligomer with other highly conserved proteins [3]. This molecular complex comprises receptor, heat-shock proteins, and immunophilin (Appendix Table 1) [4]. The binding of the receptor to the heat-shock protein 90 facilitates its interaction with the ligand [5]. When the ligand binds, the receptor dissociates from the rest of the hetero-oligomer and translocates into the nucleus. Before or after the translocation, the receptor forms homodimers through sequences present in the DNA and ligand-binding domains [6]. Appendix Table 1. Glossary of Genetic Terms Gene Regulation After specific interaction with pore-associated proteins, the hormone-receptor complexes enter the nucleus through the nuclear pores [7]. The interaction is facilitated by two nuclear localization sequences in the receptor, both in the ligand-binding domain. Inside the nucleus, the hormone-receptor complexes bind to specific glucocorticoid responsive elements within DNA [8]. The complexes modulate the transcription rates of the corresponding glucocorticoid-responsive genes [9], apparently by stabilizing the initiation complex, composed of RNA polymerase II and its ancillary factors A through F. The hormone-receptor complex may interact directly with factor IIB [10], but it also interacts with other nuclear proteins to produce the conditions necessary for effective transcription [11]. These proteins may be able to relax the DNA away from the nucleosome and thus make it easier for the polymerase to exert its effects. In addition, glucocorticoid receptors may interact with DNA-binding proteins that are associated with different regulatory elements of the DNA [12, 13]. At least two such proteins have been described: One is the glucocorticoid modulatory element-binding protein and the other is the CACCC-box-binding protein. Both of these transcription factors potentiate the modulatory effects of glucocorticoids after transcription of specific genes. Transcription appears to be important in the regulation of genes involved in growth and inflammation. Glucocorticoid response elements can act both positively and negatively on transcription, depending on the gene on which the complex acts [14, 15]. One major way by which glucocorticoids exert down-modulatory effects on transcription is through noncovalent interaction of the activated hormone-receptor complex with the c-Jun/c-Fos heterodimer [16-18], which binds to the activator protein (AP)-1 site of genes of several growth factors and cytokines. The glucocorticoid-receptor complex prevents the c-Jun/c-Fos heterodimer from stimulating the transcription of these genes. Another mechanism by which glucocorticoids may suppress gene transcription is by an interaction between the hormone-receptor complex and glucocorticoid response elements that are in close proximity to responsive elements for other transcription factors [19]. Thus, the promoter region of the glycoprotein hormone- subunit, which is stimulated by cyclic AMP through the cyclic AMP-responsive element, contains a glucocorticoid response element in close proximity, so that when the receptor dimer binds to its own element, it hinders the cyclic AMP-binding protein from exerting its stimulatory effect on that gene. Post-Transcriptional Effects In addition to modulating transcription, glucocorticoids also have effects on later cellular events, including RNA translation, protein synthesis, and secretion. They can alter the stability of specific messenger RNAs of several cytokines and other proteins, thereby altering the intracellular steady-state levels of these molecules [20, 21]. This may occur through modulation of transcription of still unknown proteins that bind RNA and alter its translation and degradation rates. Also, glucocorticoids influence the secretion rates of specific proteins through mechanisms that have not yet been defined. Finally, the receptor itself has guanylate cyclase activity, and glucocorticoids can rapidly alter the electrical potential of some cells [22, 23]. Anti-inflammatory and Immunosuppressive Effects Dr. Dimitrios T. Boumpas: Although the cause and pathogenesis of many immunologically mediated diseases are not completely understood, it is known that the localization of leukocytes at sites of inflammation, their subsequent activation, and the generation of secretory products contribute to tissue damage, as shown in Figures 2 and 3 [24-26]. Glucocorticoids inhibit the access of leukocytes to inflammatory sites, interfere with their function and the function of fibroblasts and endothelial cells at those sites, and suppress the production and the effects of humoral factors. In general, leukocyte traffic is more susceptible to alteration by glucocorticoids than is cellular function; in turn, cellular immunity is more susceptible than humoral immunity to these agents. Figure 2. Models of the pathogenesis of inflammation and immune injury. Panel A. Panel B. Figure 3. Cellular adhesion molecules. Even though the effects of glucocorticoids on the different types of inflammatory cells will be discussed separately, each cell type is actually involved in complex interactions with other cells. Glucocorticoids affect many, if not all, the cells and tissues of the body, thus provoking a wide range of changes that involve several cell types concurrently. Effects on Nonlymphoid Inflammatory Cells Dr. Ronald L. Wilder (Chief, Inflammatory Joint Diseases Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland): Glucocorticoids are among the most potent anti-inflammatory agents available in clinical medicine. Pharmacologic doses of glucocorticoids dramatically inhibit exudation of plasma and accumulation of leukocytes at sites of inflammation. Several factors influence the magnitude of these effects, including the dose and route of administration of the glucocorticoids used, as well as the type and differentiation state of the target cell population [27]. Several host variables also modify the anti-inflammatory response to glucocorticoids. For example, some persons (those with active systemic lupus erythematosus) appear to have an accelerated rate of glucocorticoid catabolism [28]. Various levels of target tissue resistance may exist in some patients with systemic lupus erythematosus and rheumatoid arthritis [29]. These factors, alone or in combination, may explain the observation that different patients and diseases have variable therapeutic responses to glucocorticoids [30, 31]. Macrophages Glucocorticoids antagonize macrophage differentiation and inhibit many of their functions [27]. These agents 1) depress myelopoiesis and inhibit expression of class II major histocompatibility complex antigens induced by interferon-; 2) block the release of numerous cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-; 3) depress production and release of proinflammatory prostaglandins and leukotrienes; and 4) depress tumoricidal and microbicidal activities of activated macrophages. Neutrophils The major effect of glucocorticoids on neutrophil

Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1 beta, phorbol ester, and corticosteroids.

High levels of immunoreactive cyclooxygenase (Cox; prostaglandin H synthase) are present in synovia from patients with rheumatoid arthritis (RA). We now show that the recently identified inducible isoform of Cox, Cox-2, is expressed in synovia from patients with RA. To further explore modulation of the Cox isoforms in RA synovial tissues, we examined the expression and modulation of Cox-1 and -2 in rheumatoid synovial explant cultures and cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Immunoprecipitation of in vitro labeled proteins and Western blot analysis demonstrated the presence of both Cox-1 and -2 under basal conditions in freshly explanted rheumatoid synovial tissues. De novo synthesis of Cox-2 polypeptide was enhanced by IL-1 beta or PMA, and dramatically suppressed by dexamethasone (dex). Cox-1 expression, under the same conditions, showed only minor variation. Since mRNA for Cox-2 is highly unstable, we examined the regulation of Cox-2 transcripts in cultured rheumatoid synoviocytes. Under basal conditions both Cox-1 and -2 mRNAs were present at low levels, but Cox-2 mRNA was markedly increased by treatment with IL-1 beta or PMA. dex markedly suppressed the induction of Cox-2 mRNA. In sharp contrast, Cox-1 transcripts were not modulated by IL-1 beta or dex. These data suggest that modulation of Cox-2 expression by IL-1 beta and corticosteroids may be an important component of the inflammatory process in synovial tissues from patients with RA.

Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin‐1: a potential mechanism for inflammatory angiogenesis

Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin‐1 (IL‐1) induce angiogenesis by yet undefined mechanisms. We demonstrate that PGE2 and IL‐1 induces the expression of vascular endothelial growth factor (VEGF), a selective angiogenic factor by rheumatoid synovial fibroblast cells. Transcripts for the EP1 and EP2, subtypes of PGE receptors are expressed in synovial fibroblasts. Activators of protein kinase A pathway stimulated the expression of VEGF whereas down‐regulation of protein kinase C did not influence the PGE effect, suggesting that signalling from the EP2 receptor via the protein kinase A pathway is important. The induction of VEGF expression by PGE2 and interleukin‐1α a may be an important mechanism in inflammatory angiogenesis.

The Stress Response and the Regulation of Inflammatory Disease

The molecular and biochemical bases for interactions between the immune and central nervous systems are described. Immune cytokines not only activate immune function but also recruit central stress-responsive neurotransmitter systems in the modulation of the immune response and in the activation of behaviors that may be adaptive during injury or inflammation. Peripherally generated cytokines, such as interleukin-1, signal hypothalamic corticotropin-releasing hormone (CRH) neurons to activate pituitary-adrenal counter-regulation of inflammation through the potent antiinflammatory effects of glucocorticoids. Corticotropin-releasing hormone not only activates the pituitary-adrenal axis but also sets in motion a coordinated series of behavioral and physiologic responses, suggesting that the central nervous system may coordinate both behavioral and immunologic adaptation during stressful situations. The pathophysiologic perturbation of this feedback loop, through various mechanisms, results in the development of inflammatory syndromes, such as rheumatoid arthritis, and behavioral syndromes, such as depression. Thus, diseases characterized by both inflammatory and emotional disturbances may derive from common alterations in specific central nervous system pathways (for example, the CRH system). In addition, disruptions of this communication by genetic, infectious, toxic, or pharmacologic means can influence the susceptibility to disorders associated with both behavioral and inflammatory components and potentially alter their natural history. These concepts suggest that neuropharmacologic agents that stimulate hypothalamic CRH might potentially be adjunctive therapy for illnesses traditionally viewed as inflammatory or autoimmune.

A Genomewide Screen in Multiplex Rheumatoid Arthritis Families Suggests Genetic Overlap with Other Autoimmune Diseases

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder with a complex genetic component. We report the first major genomewide screen of multiplex families with RA gathered in the United States. The North American Rheumatoid Arthritis Consortium, using well-defined clinical criteria, has collected 257 families containing 301 affected sibling pairs with RA. A genome screen for allele sharing was performed, using 379 microsatellite markers. A nonparametric analysis using SIBPAL confirmed linkage of the HLA locus to RA (P < .00005), with lambdaHLA = 1.79. However, the analysis also revealed a number of non-HLA loci on chromosomes 1 (D1S235), 4 (D4S1647), 12 (D12S373), 16 (D16S403), and 17 (D17S1301), with evidence for linkage at a significance level of P<.005. Analysis of X-linked markers using the MLOD method from ASPEX also suggests linkage to the telomeric marker DXS6807. Stratifying the families into white or seropositive subgroups revealed some additional markers that showed improvement in significance over the full data set. Several of the regions that showed evidence for nominal significance (P < .05) in our data set had previously been implicated in RA (D16S516 and D17S1301) or in other diseases of an autoimmune nature, including systemic lupus erythematosus (D1S235), inflammatory bowel disease (D4S1647, D5S1462, and D16S516), multiple sclerosis (D12S1052), and ankylosing spondylitis (D16S516). Therefore, genes in the HLA complex play a major role in RA susceptibility, but several other regions also contribute significantly to overall genetic risk.

In vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis.

Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids.

Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.

Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with 20% serum or in basal media supplemented with platelet-derived growth factor (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal growth factor (EGF), interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) did not support growth, although EGF enhanced PDGF-dependent growth. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony growth. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic fibroblast-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of fibroblast-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine growth factors, such as PDGF and TGF-beta.

Corticotropin releasing hormone related behavioral and neuroendocrine responses to stress in Lewis and Fischer rats

We have recently shown that susceptibility to streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats is related to a lack of glucocorticoid restraint of inflammation while the relative SCW arthritis resistance in histocompatible Fischer (F344/N) rats is related to their greater hypothalamic-pituitary-adrenal (HPA) axis response. The difference in pituitary-adrenal responsiveness results from decreased inflammatory mediator-induced hypothalamic corticotropin-releasing hormone (CRH) biosynthesis and secretion in LEW/N rats. Because CRH not only activates the pituitary-adrenal axis, but also is associated with behavioral responses that are adaptive during stressful situations, we wished to determine if the differential LEW/N and F344/N CRH responsiveness to inflammatory mediators could also be associated with differences in neuroendocrine and behavioral responses to physical and emotional stressors. In this study, LEW/N rats exhibited significant differences compared to F344/N rats, in plasma adrenocorticotropin hormone (ACTH) and corticosterone responses during exposure to an open field, swim stress, restraint or ether. Furthermore, hypothalamic paraventricular CRH mRNA expression was also significantly lower in LEW/N compared to F344/N rats after restraint. These differences in neuroendocrine responses were associated with differences in behavioral responses in LEW/N compared to F344/N rats in the open field. Outbred HSD rats, which have intermediate and overlapping arthritis susceptibility compared to LEW/N and F344/N rats, exhibited intermediate and overlapping plasma corticosterone and behavioral responses to stressful stimuli compared to the two inbred strains. These data suggest that the differences in CRH responses in these strains may contribute to the behavioral and neuroendocrine differences we have observed. Therefore these strains may provide a useful animal model for studying the relationship between behavior, neuroendocrine and inflammatory responses.

Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes.

Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5′-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue’s metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.