In situ hybridization analysis of synovial and systemic cytokine messenger RNA expression in superantigen-mediated Staphylococcus aureus arthritis

Objective. To investigate patterns of synovial and systemic cytokine messenger RNA (mRNA) expression in mice with superantigen-mediated Staphylococcus aureus arthritis. Methods. Mice were inoculated intravenously with 1 × 10⁷ colony-forming units of toxic shock syndrome toxin–1–producing S aureus LS-1. Synovial tissues and spleens were obtained at varying time intervals after bacterial inoculation, and examined for mRNA expression of interleukin-1β (IL-1β), IL-4, IL-10, IL-12, tumor necrosis factor α (TNFα), TNFβ, interferon-γ (IFNγ), transforming growth factor β, and perforin, by an in situ hybridization technique. Results. In situ hybridization revealed early synovial up-regulation of TNFα and IL-1β mRNA expression. Peak frequencies of these proinflammatory cytokines were observed at the second and third week of the infection. Expression of T cell-derived cytokine mRNAs was detected later, and in a relatively low frequency. Notably, induction and peak numbers of Th2 cytokine (IL-4 and IL-10) mRNA expression preceded Th1 cytokine (IFNγ and TNFβ) mRNAs. In comparison with synovial tissues, peak spleen cytokine mRNA expression of IL-1β, TNFα, TNFβ, IL-12, and IFNγ occurred earlier, but displayed a clearly lower magnitude of expression. Conclusion. These findings demonstrate synovial and systemic up-regulation of cytokine mRNA expression during S aureus arthritis, indicating that both monocyte/macrophage and T cell–derived products are involved in the pathogenesis of this disease.     

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro

Objective. To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet–endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovascular human umbilical vein endothelial cells (HUVE). Methods. Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor α (TNF α). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. Results. SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF α treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E-selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF α treatment and then declined to near basal levels by 24–48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF α-stimulated endothelial cells correlated with their respective messenger RNA levels. Conclusion. Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.     

Role of cytokines in inflammatory synovitis

This study was undertaken in an effort to understand the role of cytokines in T lymphocyte trafficking into inflamed synovium and in the potential enhancement of antigen presentation by human synovial fibroblasts. We found that interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and interferon-γ (IFNγ) each increased the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) on human synovial fibroblasts in a dose- and time-dependent manner. Maximal ICAM-1 expression occurred within 8 hours of induction, with the following order of efficacy: IFNγ > TNFα > IL-1β. The number of cells bearing the ICAM-1 antigen also increased, from a basal level of ∼30% to more than 83% after cytokine induction (for all 3 cytokines). ICAM-1 expression rapidly decreased following cytokine removal. The expression of lymphocyte function-associated antigen 3 was also examined, but it was not changed by any of the 3 cytokines. Class I major histocompatibility complex antigen expression was increased modestly by all 3 cytokines, and expression was maximal by 24 hours after treatment. Only IFNγ induced HLA class II antigen expression, and this expression persisted for up to 6 days following removal of the lymphokine. IL-6 and granulocyte-macrophage colony-stimulating factor had no effect on any of the parameters examined. Our data support an interactive role for inflammatory cytokines and the expression of adhesion ligands and HLA antigens by human synovial fibroblasts in the pathogenesis of synovial inflammation in rheumatoid arthritis.     

Up-regulation by tumor necrosis factor α of intercellular adhesion molecule 1 expression and function in synovial fibroblasts and its inhibition by glucocorticoids

Objective. To examine the regulation of the intercellular adhesion molecule 1 (ICAM-1) gene in cultured human synovial fibroblasts in response to tumor necrosis factor α (TNFα), and investigate its modulation by the synthetic glucocorticoid, dexamethasone. Methods. Cell surface expression of ICAM-1 was determined by flow cytometry, enzyme immunoassay, and immunoprecipitation. ICAM-1 messenger RNA (mRNA) levels were monitored by Northern blot. ICAM-1 function was determined by measuring the adhesion of monocytes to synovial fibroblasts. Results. ICAM-1 expression on unstimulated cells was weak but was rapidly enhanced in both a time- and dose-dependent manner following exposure to TNFα. Treatment of the cells with TNFα also resulted in both a time- and dose-dependent increase in steady-state ICAM-1 mRNA levels, as determined by Northern blot. The increased expression of ICAM-1 was inhibited by cycloheximide and actinomycin D. Cultured synovial fibroblasts from patients with rheumatoid and nonrheumatoid arthropathies responded similarly to TNFα. Adhesion studies demonstrated that ICAM-1 is involved in the adherence of peripheral blood monocytes to TNFα-activated synovial fibroblasts. In addition, dexamethasone inhibited TNFα-induced surface expression of ICAM-1, accumulation of ICAM-1 mRNA, and adhesion of monocytes to TNFα-activated synovial fibroblasts. Conclusion. These combined results provide further evidence of an important role of ICAM-1 in inflammatory synovitis, as well as a potentially novel site of antiinflammatory action of glucocorticoids.     

Crucial role of interleukin-10/interleukin-12 balance in the regulation of the type 2 T helper cytokine response in reactive arthritis

Objective. To investigate whether a predominant type 1 T helper (Thl) or Th2 cytokine pattern is present in the joints of patients with reactive arthritis (ReA), and whether the cytokine pattern can be modulated by cytokines or anticytokines. Methods. Eleven patients with ReA following infection with either Chlamydia trachomatis, Yersinia enterocolitica, or Salmonella enteritidis were investigated for the presence of Th1/Th2 cytokines in the joints. Release of the bacteria-specific cytokines interferon-γ (IFNγ), tumor necrosis factor α (TNFα), interleukin-10 (IL-10), and IL-4 was measured in synovial fluid mononuclear cells (SFMC) using enzyme-linked immunosorbent assay and polymerase chain reaction. In the synovial membrane, secretion of IFNγ and IL-4 was determined by immunohistologic analysis. Cytokine regulation was studied by adding cytokines and anticytokines to the cultures. Results. Upon stimulation with specific bacteria, SFMC secreted low amounts of IFNγ and TNFα, but high amounts of IL-10. IL-10 was responsible for the suppression of IFNγ and TNFα, as judged by the effect of adding either anti-IL-10 antibodies or exogenous IL-10 to these cultures. The addition of neutralizing anti-IL-12 to the cultures completely abolished the effects of anti-IL-10, suggesting that inhibition of the Th1-like cytokines by IL-10 is mediated through suppression of IL-12 synthesis. Exogenous IL-12 clearly enhanced IFNγ and TNFα secretion. In the synovial membrane, a higher number of cells were positive for the Th2 cytokine IL-4, compared with the amount of IFNγ-secreting cells. Conclusion. These data indicate that a Th2 cytokine pattern predominates in the joints of patients with ReA. Since Thl cytokines are necessary for the elimination of ReA-associated bacteria, Th2 cytokines might contribute to bacterial persistence in the joint. Therefore, the IL-10/IL-12 balance appears to be crucial for regulation of the cytokine pattern in the joints of patients with ReA.     

TNFα and IFNγ potentiate IL-1β induced mitogen activated protein kinase activity in rat pancreatic islets of Langerhans

Aims/hypothesis. Interleukin-1 beta (IL-1β) in synergy with tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) is cytotoxic to pancreatic beta cells. Mitogen-activated protein kinase (MAPK) activity that is induced by interleukin-1 beta has been suggested to signal nitric oxide-dependent as well as nitric oxide-independent beta-cell destructive pathways. The aim of this study was to investigate if TNFα and IFNγ signal through mitogen-activated protein kinases in isolated rat islets of Langerhans and if they potentiate mitogen-activated protein kinase activity induced by IL-1β.¶Methods. Islets of Langerhans were isolated from 5- to 7-day-old Wistar rats and precultured for 7 days before stimulation with IL-1β, TNFα and/or IFNγ for 20 min followed by lysis. Kinase activity was measured with a whole cell lysate kinase assay and after immunoprecipitation of the kinase using immunocomplex kinase assay.¶Results. Exposure to IL-1β or TNFα significantly increased mitogen-activated protein kinase activity, whereas IFNγ tended to decrease extracellular-signal-regulated kinase activity. Further, TNFα and IFNγ were found to synergistically increase mitogen-activated protein kinase activity induced by IL-1β.¶Conclusion/interpretation. We hypothesise that the synergistic effect of IL-1β, TNFα and IFNγ in the functional inhibition and induction of cell death in pancreatic beta cells is signalled through a synergistic activation of mitogen-activated protein kinase activity [Diabetologia (2000) 43: 1389–1396].     

Restricted cytokine expression in rheumatoid arthritis

Objective. To determine the cytokine profile of the phenotypically activated T cell in rheumatoid arthritis (RA) synovium. Methods. Interleukin-2 (IL-2), IL-2 receptor (IL-2R), IL-6, IL-4, and interferon-γ (IFNγ) gene expression was examined in T cells from freshly isolated synovial fluids (SF) and synovial tissues (ST) from patients with RA. Estimates of baseline expression were determined using unstimulated peripheral blood (PB) T cells from healthy individuals. The corresponding positive controls were phytohemagglutinin-activated tonsil T cells. Results. In studies of paired PB and SF T cell samples from 17 RA patients, IL-2 messenger RNA (mRNA) levels in only 1 PB and 3 SF samples were more than 2 standard deviations above the mean of levels in unstimulated PB from healthy donors. Similarly, only 5 PB and 7 SF samples exhibited elevated IL-2R mRNA levels. IFNγ gene expression was not detected in any of the paired RA PB or SF samples. Fractionated T cells from 12 RA ST were screened with similar results: Only 1 of 12 samples exhibited IL-2 mRNA levels more than 2 standard deviations above levels in baseline controls. IL-2R mRNA levels were low or not detected, and IFNγ mRNA was absent. Subsequent studies showed that IL-4 and IL-6 gene expression levels were also low in RA tissues compared with tonsil T cell–positive controls. Conclusion. These data provide evidence for restricted cytokine expression in the T cell population in RA tissues.     

T lymphocyte adhesion to human synovial fibroblasts. Role of cytokines and the interaction between intercellular adhesion molecule 1 and CD11a/CD18.

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN gamma treatment resulted in the largest increase in adhesion, followed by TNF alpha and IL-1 beta. Combinations of IFN gamma + TNF alpha and IFN gamma + IL-1 beta had a synergistic effect on intercellular adhesion molecule 1 (ICAM-1) expression and adhesion. The increase in cellular adhesion induced by cytokines correlated with the up-regulation of the number of cells expressing ICAM-1 and the density of antigen/cell. There was no synergistic effect on leukocyte function-associated antigen 3 (LFA-3) or on HLA class I or class II antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18. These findings suggest the existence of ICAM-1--independent and CD11/CD18-independent adhesion mechanisms. Anti-LFA-3 was completely ineffective as an inhibitor of adhesion. There was no additive or synergistic advantage of using combinations of antibodies to increase the level of inhibition, i.e., anti--ICAM-1 + anti-LFA-3, anti-ICAM-1 + anti-CD18, or anti-ICAM-1 + anti-LFA-1 (CD11a). Our data indicate that proinflammatory cytokines may play a prominent role in the formation and exacerbation of synovial hyperplasia, by regulating the recruitment and retention of T lymphocytes via the up-regulation of adhesion molecules on synovial fibroblasts.     

Effects of Th1 and Th2 cytokines on cytokine production and ICAM-1 expression on synovial fibroblasts.

OBJECTIVES--To investigate the influence of the Th1 and Th2 lymphokines interleukins (IL)-4 and IL-13, interferon gamma (IFN gamma), and several monokines on the adhesion of mononuclear cells to synovial fibroblasts and intercellular adhesion molecule-1 (ICAM-1) expression and cytokine production of synovial fibroblasts in patients with osteoarthritis. METHODS--Synovial fibroblasts were isolated from patients with osteoarthritis and stimulated with IL-1 beta, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor alpha (TNF alpha), and IFN gamma. Subsequently, we determined the production of IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, IFN alpha and TNF alpha, and the expression of ICAM-1 lymphocyte function associated antigen 3 (LFA-3), BB7, and major histocompatibility complex class II molecules on these cells. Furthermore, the adhesion of freshly isolated mononuclear cells from the peripheral blood was tested using a colourimetric cell-cell adhesion assay. RESULTS--Only production of IL-6 and the expression of ICAM-1 were observed. IL-1 beta and TNF alpha were the most potent stimulatory mediators of both cytokine production and ICAM-1 expression. IL-4 and IL-13 had differential effects as they upregulated cytokine production but downregulated IFN gamma induced ICAM-1 expression. In functional adhesion assays, TNF alpha, IL-1 alpha and, to a lesser extent, IFN gamma led to increased adhesion of mononuclear cells, whereas IL-4 and IL-13 had no effect. CONCLUSIONS--Our data indicate that Th1 and Th2 lymphokines can modulate the function (cytokine production and expression of adhesion molecules) of synovial fibroblasts.     

Cytokeratin Expression and Hyaluronic Acid Production in Cultures of Human Synovial Microvascular Endothelial Cells: Influence of Cytokines and Growth Factors

Objectives: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. Methods: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeaus agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. Results: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K4.62 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1α (IL-1α) or acidic fibroblast growth factor (aFGF), but not tumor necrosis factor (TNFα), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200–800 ng/10⁵ cells/24 h, which could be upregulated when the cells were incubated with either IL-1α or aFGF. HA production was not significantly increased when HSE cells were incubated with TNFα, IL-4 or interferon-γ. Conclusions: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.     

IL-29 and IFNα differ in their ability to modulate IL-12 production by TLR-activated human macrophages and exhibit differential regulation of the IFNγ receptor expression

The interferon-λ (IFNλ) family of cytokines, consisting of interleukin-28A (IFNλ2), IL-28B (IFNλ3), and IL-29 (IFNλ1), have been extensively studied for their antiviral activities. However, little is known about the effect of IFNλ on antigen-presenting cells. In the present study, we show for the first time that IL-29 can increase Toll-like receptor (TLR)-induced IL-12p40 production by human monocyte-derived macrophages. In contrast, IL-29 did not affect monocytes or monocyte-derived dendritic cells (DCs) because of restricted IL-28 receptor α chain expression by macrophages. Furthermore, IL-29-treated macrophages were more responsive to IFNγ, because IL-29 enhanced IFNγ-induced IL-12p40 and tumor necrosis factor (TNF) production by macrophages on R848 stimulation. However, IFNα suppressed IFNγ-induced IL-12p40 and tumor necrosis factor TNF production by human macrophages. The differential effects of IL-29 and IFNα on the responsiveness of macrophages to IFNγ could not be explained by an effect on TLR7 or TLR8 mRNA expression or by altered IL-10 signaling. However, we demonstrated that IL-29 up-regulated, whereas IFNα down-regulated, the surface expression of the IFNγ receptor 1 chain on macrophages, thereby resulting in differential responsiveness of TLR-challenged macrophages to IFNγ. Our findings on the differences between IFNα and IL-29 in modulating TLR-induced cytokine production by macrophages may contribute to understanding the role of IFNs in regulating immunity to pathogens.     

Inhibition of lymphocyte adhesion to cytokine-activated synovial fibroblasts by glucocorticoids involves the attenuation of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 gene expression

Objective. The aim of this study was to evaluate the ability of glucocorticoids to inhibit lymphocyte adhesion to human synovial fibroblasts. Methods. Adhesion of lymphocytes to cultured synovial fibroblasts was measured by counting the number of cells bound to fibroblasts. Surface expression of intercellular adhesion molecule 1 (ICAM-1) was measured by enzyme-linked immunosorbent assay, while vascular cell adhesion molecule 1 (VCAM-1) surface expression was measured by flow cytometry. ICAM-1 and VCAM-1 messenger RNA (mRNA) levels were assessed by Northern blot analysis. Results. Stimulation of synovial fibroblasts by the proinflammatory cytokines tumor necrosis factor α, interleukin-1β, and interferon-γ resulted in a dose-dependent increase in lymphocyte adhesion to synovial fibroblasts. This response was inhibited by preincubation of the cells with the synthetic glucocorticoid dexa-methasone. Since lymphocyte adhesion to synovial fibroblasts is known to be mediated by VCAM-1 and ICAM-1, we examined the modulation of VCAM-1 and ICAM-1 expression in these cells. All 3 cytokines stimulated VCAM-1 and ICAM-1 surface and mRNA expression. Dexamethasone inhibited both VCAM-1 and ICAM-1 surface and mRNA expression in a dose-dependent manner, which correlated with the inhibition of lymphocyte adhesion. Conclusion. Taken together, these results suggest that glucocorticoids may reduce inflammatory responses at extravascular sites by inhibiting the expression of these adhesion molecules, thereby reducing the adhesion of lymphocytes to connective tissue cells.     

Interferon γ modulates production of interleukin 1 and tumor necrosis factor by murine Kupffer cells

ABSTRACT— When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). In this study, we examined the effect of in vitro Kupffer cell activation by recombinant murine interferon γ (IFNγ) on IL-1 and TNF secretion. IFNγ enhanced TNF production in the presence or absence of lipopolysaccharide (LPS), but suppressed IL-1 production by Kupffer cells. Because IFNγ also stimulated prostaglandin E₂ (PGE₂) production, the effect of indomethacin, which is an inhibitor of cyclooxygenase and which inhibits PGE₂ biosynthesis, on IL-1 and TNF production by Kupffer cells was examined. As a result, indomethacin enhanced TNF production by Kupffer cells, but had no effect on IL-1 synthesis. These results suggested that IFNγ modulates the production of IL-1 and TNF by Kupffer cells through different mechanisms.     

Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood,synovial fluid,and synovial membrane in rheumatoid arthritis

Objective. To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments. Methods. Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1β (IL-1β), IL-8, and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E₂ (PGE₂) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFα, IL-8, IL-1β, and the IL-1 receptor antagonist protein (IL-1Ra). Results. MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFα in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFα in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1β and IL-1Ra or synovial fluid IL-1β and PGE₂ was found. Conclusion. MP therapy rapidly reduces IL-8 and TNFα levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFα expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.     

Interleukin-1, Interleukin-2, Interleukin-4, Interleukin-6, Tumor Necrosis Factor α, and Interferon-γ Levels in Sera from Patients With Scleroderma

Objective. To determine whether interleukin-lα (IL-1α), IL-1β, IL-2, IL-4, interferon-γ (IFNγ), IL-6, and tumor necrosis factor α (TNFα) are detected more frequently in sera from scleroderma patients than in sera from controls. Methods. Serum concentrations of these cytokines were measured in 78 scleroderma patients and 73 controls, using enzyme-linked immunosorbent assay, radioimmunoassay, and bioassay techniques. Results. IL-2, IL-4, and IL-6 were each detected more frequently in sera from scleroderma patients than in sera from controls. TNFα and IL-1α were found with equal frequency in patient and control sera. IL-1β and IFNγ were not detected in any sera. Conclusion. IL-2, IL-4, and IL-6 may be among the cytokines that contribute to the disease process in scleroderma patients. To our knowledge, this is the first report of elevated serum IL-4 levels in human disease.