Role of cytokines in inflammatory synovitis. The coordinate regulation of intercellular adhesion molecule 1 and HLA class I and class II antigens in rheumatoid synovial fibroblasts.

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-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN gamma) 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: IFN gamma greater than TNF alpha greater than IL-1 beta. The number of cells bearing the ICAM-1 antigen also increased, from a basal level of approximately 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 IFN gamma 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.     

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.     

Interferon-gamma and interleukin-4 differentially regulate ICAM-1 and VCAM-1 expression on human lung fibroblasts.

The expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and more specifically vascular adhesion molecule-1 (VCAM-1) on lung fibroblasts may be important for migration of inflammatory cells through the submucosa to the airway lumen in the asthmatic inflammatory response. This study aimed to assess which cytokines are regulating ICAM-1 and VCAM-1 expression on human lung fibroblasts. For this purpose, confluent fibroblast cultures (derived from lung tissue from a nonasthmatic donor) were stimulated for 4 h with interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, interferon (IFN)gamma, IL-4, IL-5 or transforming growth factor (TGF)beta. IL-1beta (optimal concentration (OC) 1 U x mL(-1)) and TNFalpha (OC 100 U x mL(-1)) both increased ICAM-1 and VCAM-1 expression. IFNgamma (OC 2 U x mL(-1)) increased only ICAM-1 expression and IL-4 (OC 5 ng x mL(-1)) increased only VCAM-1 expression, whereas IL-5 (20 ng x mL(-1)) and TGFbeta (10 ng x mL(-1)) did not influence ICAM-1 or VCAM-1 expression. ICAM-1 expression reached a plateau at 8-12 h after cytokine stimulation and remained constant for at least 24 h. VCAM-1 showed a transient increased expression within 24 h after IL-1beta and TNFalpha stimulation. In contrast, VCAM-1 expression did not decrease after maximal expression at 4 h upon IL-4 stimulation. It is concluded that the Helper-1T-cell, type cytokine interferon gamma and the Helper-2 T-cell type cytokine interleukin-4 differentially regulate intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on human lung fibroblasts. The proinflammatory cytokines interleukin-1beta and tumour necrosis factor alpha increase both intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, without differential regulation of the expression of these adhesion molecules.     

Proinflammatory cytokine production and cartilage damage due to rheumatoid synovial T helper-1 activation is inhibited by interleukin-4.

OBJECTIVES--To investigate the role of T helper-1 cell (Th1) activation in the induction of proinflammatory cytokine production and cartilage damage by rheumatoid arthritis (RA) synovial fluid mononuclear cells (SFMNC) and the subsequent possible beneficial role of the T helper-2 cell (Th2) cytokine interleukin-4 (IL-4) in the inhibition of this process. METHODS--SFMNC were stimulated with bacterial antigen (hsp60) to activate Th1 cells. Th1 and Th2 specific cytokine profiles (interferon gamma (IFN gamma) and IL-4) and proinflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) in the conditioned media were analysed. In addition, the conditioned media were tested for their ability to induce cartilage damage. The same parameters were measured in the presence of IL-4. RESULTS--Stimulation of SFMNC with bacterial antigen resulted in an increase in IFN gamma, IL-1, and TNF alpha production which was accompanied by the induction of cartilage damage. Th1 activation could be inhibited by IL-4 as shown by a reduction of IFN gamma. This was accompanied by a decrease in IL-1 and TNF alpha production and inhibition of cartilage damage. CONCLUSIONS--Th1 activation is a possible mechanism by which inflammation in RA joints is enhanced. The Th2 cytokine IL-4 inhibits this Th1 activity and may diminish inflammation and induction of cartilage damage in RA joints.     

Interleukin 7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis

BACKGROUND: A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA. OBJECTIVE: To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA. METHODS: In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNF alpha), IL1 beta, interferon gamma (IFN gamma), and IL4 was measured by enzyme linked immunosorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of IL12 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied. RESULTS: IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFN gamma and TNF alpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on IL12, indicating that IL7 also stimulates accessory cell function, leading to T cell activation. CONCLUSION: IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNF alpha mediated proinflammatory responses in patients with RA.     

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.     

Systemic sclerosis Th2 cells inhibit collagen production by dermal fibroblasts via membrane-associated tumor necrosis factor alpha

OBJECTIVE: In systemic sclerosis (SSc; scleroderma), T cells infiltrate organs undergoing fibrotic changes and may participate in dysregulated production of collagen by fibroblasts. The objective of this study was to functionally characterize T cells infiltrating skin lesions in early SSc and investigate their capacity to affect production of type I collagen and interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) by dermal fibroblasts. METHODS: Four-color cytometric analysis was used to characterize subset distribution and production of interferon-gamma (IFN gamma) and interleukin-4 (IL-4) in T cell lines generated from the skin of patients with SSc. T cell clones were generated, and their capacity to modulate collagen and MMP-1 production by fibroblasts derived from patients with SSc and from normal individuals was assessed. Neutralizing reagents were used to identify T cell mediators involved in fibroblast modulation. RESULTS: The skin of individuals with early-stage SSc contained T cells preferentially producing high levels of IL-4. Cloned CD4+ Th2-like cells inhibited collagen production by normal fibroblasts. Th2 cell-dependent inhibition was, at least in part, contact-dependent, was essentially mediated by tumor necrosis factor alpha (TNF alpha), and was dominant over the enhancement induced by profibrotic IL-4 and transforming growth factor beta cytokines. The simultaneous induction of MMP-1 production confirmed the specificity of these observations. To be inhibitory, Th2 cells required activation by CD3 ligation. Th2 cells were less potent than were Th1 cells in inhibiting collagen production by normal fibroblasts via cell-to-cell interaction, and SSc fibroblasts were resistant to inhibition. CONCLUSION: These findings indicate that, despite their production of IL-4, Th2 cells reduce type I collagen synthesis by dermal fibroblasts because of the dominant effect of TNF alpha, and suggest that strategies based on TNF alpha blockade aimed at controlling fibrosis in SSc may be unwise.     

Tumour necrosis factors (alpha, beta) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication

Cytokines such as tumour necrosis factor (TNF) can induce HIV-1 production in T-cell tumour lines. However, it is not known if the same occurs in freshly isolated mononuclear cells, nor is it known if the virus can itself regulate cellular cytokine production. In this paper we report that HIV-1 induces peripheral blood mononuclear cells (PBMC) and CD4+ T lymphocytes to secrete TNF alpha, TNF beta and interferon-gamma (IFN gamma), three cytokines having multifunctional activities and complex physiological roles. We also show that separate addition of exogenous recombinant (r) TNF alpha or rTNF beta or rIFN gamma increases HIV-1-induced syncytium formation in both PBMC and CD4+ cells by up to 10,000-fold, with TNF alpha being most potent in this regard. Finally, we show that syncytium formation induced by diverse HIV-1 isolates and LAV-2 is inhibited without the addition of exogenous r-cytokines by the respective anti-cytokine antibodies. Our study therefore demonstrates that efficient HIV replication in primary mononuclear cells is associated with the ability of the virus to induce TNF and IFN gamma secretion.     

Interleukin 4 increases human synovial cell expression of VCAM-1 and T cell binding.

OBJECTIVE--The effects were studied of interleukin 4 (IL-4) on T cell-synovial cell adhesion and on the expression of adhesion molecules on the surface of synovial fibroblast-like cells. METHODS--The adhesion of T cells toward the synovial cells were measured by 51chromium-labelled adhesion assay. The expression of adhesion molecules on synovial cells were analysed by flowcytometry. RESULTS--Stimulation of synovial cells with IL-4 increased T cell-synovial cells adhesion in a time- and dose-dependent manner. IL-4 considerably enhanced the expression of VCAM-1 on the surface of synovial cells, but not the expression of ICAM-1 and ELAM-1. The combination of IL-1 beta and IL-4 had no effect on the expression of ICAM-1 or VCAM-1 on the surface of synovial cells. The increased adhesion of T cells to IL-4 stimulated synovial cells was inhibited significantly by adding anti-VCAM-1 or anti-CD29 monoclonal antibody. Furthermore, anti-VLA-4 alpha or the combination of anti-VLA-4 alpha and anti-VCAM-1 antibodies blocked completely T-cell binding to IL-4 stimulated synovial cells. CONCLUSIONS--These results suggest that the increased adhesion of T cells to IL-4-stimulated synovial cells is mediated by VLA-4/VCAM-1 pathway.     

Interleukin-18 enhances monocyte tumor necrosis factor alpha and interleukin-1beta production induced by direct contact with T lymphocytes: implications in rheumatoid arthritis

OBJECTIVE: At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up-regulation of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha). We examined the regulatory effects of IL-18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA). METHODS: Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)-stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFalpha, IL-1beta, IL-10, and IL-18 were measured by enzyme-linked immunosorbent assay. Expression of adhesion molecules, IL-18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF-kappaB p65, phosphorylated IkappaBalpha, and phosphatidylinositol 3-kinase (PI 3-kinase) p110 was analyzed by Western blotting. RESULTS: IL-18 dose-dependently enhanced the production of IL-1beta and TNFalpha, but not IL-10, by monocytes following contact with RA synovial T cells or PHA-prestimulated T cells. NF-kappaB inhibitors N-acetyl-L-cysteine and Bay 11-7085 and PI 3-kinase inhibitor LY294002 inhibited the enhancing effects of IL-18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF-kappaB in the nucleus, phosphorylated IkappaB, and PI 3-kinase were confirmed in monocytes cocultured with PHA-prestimulated T cells, and the levels were further increased by stimulation with IL-18. Neutralizing antibody to IL-18 inhibited monocyte activation induced by direct contact with PHA-prestimulated T cells. Via cell-cell contact, PHA-prestimulated T cells increased autocrine production of IL-18 by monocytes, which was mediated by activation of the NF-kappaB and PI 3-kinase pathways, and up-regulated the expression of the IL-18 receptor in monocytes. IL-18 up-regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on monocytes. Blocking the binding of the TNF receptors VCAM-1 or ICAM-1 on monocytes to their ligands on stimulated T cells suppressed the IL-18-enhanced production of TNFalpha and IL-1beta in monocytes induced by contact with PHA-prestimulated T cells. CONCLUSION: IL-18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF-kappaB and PI 3-kinase pathways. IL-18 up-regulates the expression of the TNF receptors VCAM-1 and ICAM-1 on monocytes, which mediate the enhancing effects of IL-18 on T cell-monocyte contact.     

Cytokines in experimental autoimmune vasculitis: evidence for a Th2 type response.

OBJECTIVE: To investigate the pathogenic role of cytokines in the development of experimental autoimmune vasculitis. METHODS: BALB/c mice were immunized with human IgG-ANCA from a patient with WG. Control mice were immunized with normal human IgG. Levels of mouse IgG-ANCA and other autoantibodies were determined. The mice lungs and kidneys were examined for the development of vasculitis. Levels of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, interferon gamma (IFN gamma) and TNF alpha were determined by ELISA two weeks after immunization of the mice. RESULTS: Mice immunized with human IgG-ANCA developed anti-human IgG-ANCA (= Ab2) and anti-anti-human IgG-ANCA (mouse IgG-ANCA = Ab3), while the controls did not develop these antibodies. The mice that were immunized with human IgG-ANCA developed perivascular mononuclear cell infiltrates in the lungs, suggesting vasculitis. Levels of IL-4, IL-6 and TNF alpha but not IL-1 beta, IL-2 and IFN gamma were significantly elevated in the mice 2 weeks after immunization with IgG-ANCA. CONCLUSION: Our results suggest a pathogenic role for IL-4, IL-6 and TNF alpha in the initiation phase of autoimmune vasculitis. This suggests that a Th2 type immune response is responsible for the initiation of experimental autoimmune lung vasculitis, similar to Wegener's granulomatosis in humans.     

Interleukin-4 (IL-4) inhibits secretion of IL-1 beta, tumor necrosis factor alpha, and IL-6 by human monocytes

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin-4 (IL-4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL-1, tumor necrosis factor alpha (TNF alpha), and IL-6. To elucidate which cytokines produced by monocytes are controlled by IL-4, we tested the effect of IL-4 on the secretion of IL-1 alpha, IL-1 beta, TNF alpha, and IL-6 induced by LPS or IFN gamma. IL-4 was found to inhibit the secretion of IL-1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL-6 was found to be reduced 70% to 85% in the presence of IL-4, whereas there was no effect on the secretion of IL-1 alpha (IL-1 alpha is mainly cell-associated). Time-course experiments demonstrate that IL-4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL-1 beta and TNF alpha was specifically induced by IL-4 because anti-IL-4 antiserum completely restored normal monokine production. These data suggest that IL-4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.     

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.     

Pioglitazone, a peroxisome proliferator-activated receptor gamma activator, ameliorates experimental autoimmune myocarditis by modulating Th1/Th2 balance

OBJECTIVES: The aim of this study is to investigate the effect of Peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand on experimental autoimmune myocarditis (EAM). BACKGROUND: Rat EAM model resembles the giant cell myocarditis in human. Recent studies have suggested that Th1 cytokines are involved in the initiation and progression of EAM, whereas Th2 cytokines are associated with the remission. PPAR gamma, which is a member of nuclear hormone receptor superfamily, has been known to affect not only glucose homeostasis but also immune responses, by regulating the Th1/Th2 balance. METHODS: Lewis rats were immunized with cardiac myosin and divided into three groups: Group N, normal control rats (n = 16); Group E, EAM rats (n = 17); and Group P, EAM rats treated with a PPAR gamma activator pioglitazone (n = 20). RESULTS: Cardiac dysfunction and remodeling were inhibited in Group P compared to Group E. Heart weight/body weight ratio and the degree of inflammation and fibrosis were significantly lower in Group P than in Group E. The mRNA levels of macrophage inflammatory protein-1alpha (MIP-1alpha), which plays an important role in the recruitment of inflammatory cells in the early stage of EAM, were upregulated in the heart of Group E, but not in the heart of Group P. Furthermore, the treatment with pioglitazone decreased the expression levels of proinflamatory cytokine (TNF alpha and IL-1beta) genes and Th1 cytokine (IFN-gamma) genes, and increased the expression levels of Th2 cytokine (IL-4) gene. CONCLUSIONS: PPAR gamma ligands may have beneficial effects on myocarditis by inhibiting MIP-1alpha expression and modulating the Th1/Th2 balance.     

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium

Objective. To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. Methods. Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. Results. Treatment of HSE with interleukin-1α (IL-1α) or tumor necrosis factor α (TNFα) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-γ (IFNγ) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFNγ and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFNγ also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNFβ/lymphotoxin. Immunoprecipitation of TNFα + IFNγ–stimulated, ¹²⁵I-labeled HSE cells with anti–ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFNγ alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. Conclusion. These studies indicate that IFNγ plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.     

Cytokine augmentation of HIV-1 LTR-driven gene expression in neural cells.

The induction of human immunodeficiency virus type 1 (HIV-1) gene expression by cytokines was investigated in cells of central nervous system origin. These were human neuroblastoma, glioblastoma, and astrocytoma cell lines, a murine oligodendroglioma and primary murine astrocyte cultures. The cytokines used were tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), IL-6, and interferons alpha and gamma (IFN alpha, gamma). Transient transfection of cells with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) showed significant augmentation following treatment by particular cytokines. TNF alpha was found to augment HIV LTR-directed CAT activity in all cell types. IL-1 beta also activated the HIV LTR reporter gene in glioblastoma, astrocytoma, and astrocyte cells. IL-6 enhanced HIV gene expression in one example only, the primary astrocyte cultures. The interferons generally suppressed expression from the LTR except IFN gamma which produced a twofold rise in the murine glial cells and IFN alpha augmenting expression in one neuroblastoma cell line. No synergy was observed between pairs of activating cytokines tested. The HIV tat gene product was found to be functional in all cells, cotransfection of a tat expression vector transactivating expression from the LTR, with varying degrees of efficiency. In some cell lines the combination of an activating cytokine and tat resulted in an enhancement above that obtained by cotransfection of tat alone. In others, the level of CAT activity did not significantly change. Analysis of nuclear extracts from cytokine-treated cells further implicated the involvement of NFKB in the induction of HIV-1 gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)