A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance

In the present study, we analyzed the cytokine network among TNFalpha, IL-1beta, IL-8, and IL-1 receptor antagonist (IL-1Ra) in a rabbit experimental model of acute gout. The production of TNFalpha in synovial fluids reached the peak at 2 hours after the intra-articular injection of monosodium urate (MSU) crystals. The production of IL-1beta and IL-8 reached the first peak at 2 hours and the second peak at 9 and 12 hours, respectively. The production of endogenous IL-1Ra reached the peak at 9 hours. The source of TNFalpha and the first phase of IL-8 was synovial cells, whereas infiltrating leukocytes were the source of the second phase of IL-8 and also of IL-1beta and IL-1Ra. The production of TNFalpha was not altered by either anti-lL-8 IgG or IL-1Ra. The first IL-1beta peak was reduced only with a combination of anti-TNFalpha mAb and anti-lL-8 IgG, whereas the second peak was significantly reduced by either inhibitor. The first IL-8 peak was not altered with anti-TNFalpha mAb or IL-1 Ra, whereas the second IL-8 peak was reduced with IL-1Ra. Anti-TNFalpha mAb or anti-lL-8 IgG significantly reduced the peak level of endogenous IL-1Ra. These cytokine inhibitors also attenuated the maximal leukocyte accumulation at 9 hours, but not the initial phase, which occurred within 2 hours. These results provide evidence that IL-8 and TNFalpha were responsible for the production of IL-1beta and IL-1Ra, and that IL-1beta was responsible for the second phase of IL-1beta and IL-8 production. Our data also suggest that the initial and the maximal phases of leukocyte influx are differently regulated. Finally, the intravenous injection of colchicine inhibited neutrophil infiltration without affecting the production of TNFalpha or the first peak of IL-8, suggesting that colchicine inhibits MSU crystal-induced arthritis by directly inhibiting the migration of neutrophils.     

Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor alpha and interleukin-1 in rheumatoid arthritis

OBJECTIVE: To investigate the regulation of expression of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA), in order to determine whether new blood vessel formation could be a potential therapeutic target in RA. METHODS: Dissociated RA synovial membrane cells were cultured in the presence of cytokine inhibitors, or under hypoxic conditions. Serum VEGF levels were serially measured in RA patients enrolled in clinical trials of anti-tumor necrosis factor alpha (anti-TNFalpha) monoclonal antibody treatment. RESULTS: Combined neutralization of TNFalpha and interleukin-1 (IL-1) in RA synovial membrane cultures reduced VEGF release by 45% (P < 0.05 versus control), although blockade of either TNFalpha or IL-1 activities alone resulted in only small inhibitory effects. In addition, release of VEGF from RA synovial membrane cells was selectively up-regulated by hypoxia. Serum VEGF levels were significantly elevated in RA patients relative to control subjects, and correlated with disease activity. Treatment of RA patients with anti-TNFalpha significantly decreased serum VEGF, and this effect was enhanced by cotreatment with methotrexate. CONCLUSION: Inhibition of TNFalpha and IL-1 activity in vivo could reduce the drive to new blood vessel formation, and hence pannus mass, adding to other therapeutic effects of anti-TNFalpha therapy in RA.     

Interleukin 8 and monocyte chemoattractant protein-1 in patients with juvenile rheumatoid arthritis. Relation to onset types, disease activity, and synovial fluid leukocytes

OBJECTIVE: To measure serum and synovial fluid (SF) levels of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in patients with juvenile rheumatoid arthritis (JRA) and to compare them with adult rheumatoid factor-positive rheumatoid arthritis (RA). METHODS: IL-8 and MCP-1 were measured by immunoassay (1) in sera obtained from 55 children with JRA and from 16 adults with RA, and (2) in SF obtained from 30 children with JRA and 11 adults with RA. RESULTS: Patients with active systemic JRA had serum levels of IL-8 and MCP-1 higher than in controls (p<0.01) and in patients with active polyarticular or pauciarticular JRA (p<0.05). In patients with RA serum MCP-1 levels were higher than in patients with the 3 JRA onset types, while no difference was found for IL-8 levels. Patients with systemic JRA and with current systemic features had serum levels of IL-8 and MCP-1 higher (p = 0.03 and p = 0.04, respectively) than patients in which systemic features had subsided. No significant differences in SF IL-8 or MCP-1 levels were found among the 3 JRA onset types or adults with RA. In patients with JRA SF leukocyte counts were correlated with SF IL-8 levels (p = 0.002), but not with MCP-1 levels. Moreover, SF levels of both IL-8 and MCP-1 were correlated with those of IL-1beta (p<0.001) and IL-6 (p<0.01), but not with those of TNF-alpha. CONCLUSION: Elevated serum levels of IL-8 and MCP-1 in patients with systemic JRA with current systemic features at sampling suggest systemic production of the 2 chemokines during systemic phases of the disease. Similar SF levels of IL-8 and MCP-1 among the 3 JRA onset-types and RA suggest comparable local production of the 2 chemokines.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA). METHODS: We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot. RESULTS: SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils. CONCLUSION: These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.     

Human retinal pigment epithelial cell interleukin-8 and monocyte chemotactic protein-1 modulation by T-lymphocyte products

PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.     

Endothelial activation in monosodium urate monohydrate crystal-induced inflammation: in vitro and in vivo studies on the roles of tumor necrosis factor alpha and interleukin-1

OBJECTIVE: There is relatively little direct evidence for the roles of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in activating endothelium in vivo. The aim of this study was to use in vitro and in vivo models to investigate the contribution of these cytokines to both E-selectin expression and the recruitment of polymorphonuclear cells (PMN) in monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: MSU crystals were incubated with freshly isolated mononuclear cells, after which the harvested supernatants were tested for their ability to induce E-selectin expression during coculture with human umbilical vein endothelial cells. Subsequent experiments were performed with the addition of neutralizing anticytokine antibodies/antisera. The role of TNF alpha was then studied in an MSU crystal-induced monarthritis model, in the presence or absence of anti-TNF alpha (5 mg/kg intravenously). 99mtechnetium (99mTc)-labeled PMN cells and (111)indium (111In)-labeled anti-E-selectin monoclonal antibody (MAb) 1.2B6 were intravenously administered 4 hours after intraarticular injection to quantify PMN recruitment and E-selectin expression in inflamed joints. RESULTS: MSU crystals were a potent stimulus for IL-1 and TNF alpha production by monocytes in vitro, and these cytokines fully accounted for MSU crystal-stimulated, monocyte-mediated endothelial activation. In the MSU crystal-induced monarthritis model, TNF alpha blockade was very effective in suppressing both E-selectin expression and PMN emigration into the inflamed joints, as judged by gamma-camera image analysis and postmortem tissue counting following the intravenous injection of 99mTc-PMN and 111In-anti-E-selectin MAb. CONCLUSION: IL-1 and TNF alpha appear to be the only factors released by monocytes following incubation with MSU crystals, which induce E-selectin expression in vitro. Anti-TNF alpha is effective in suppressing endothelial activation and PMN recruitment in vivo E-selectin imaging can be used to assess the endothelial response to therapy and may prove useful for clinical studies.     

Important immunoregulatory role of interleukin-11 in the inflammatory process in rheumatoid arthritis

OBJECTIVE: To investigate the possible immunoregulatory role of interleukin-11 (IL-11) in rheumatoid arthritis (RA). METHODS: IL-11 protein was assayed in RA tissue, and the effect of exogenous IL-11 on neutralization of endogenous IL-11 was investigated with respect to tumor necrosis factor alpha (TNFalpha), matrix metalloproteinase (MMP), and tissue inhibitor of metalloproteinases (TIMP) production. RESULTS: IL-11 was found in RA synovial membranes, synovial fluids, and blood sera. Blockade of endogenous IL-11 resulted in a 2-fold increase in TNFalpha levels, which increased to 22-fold if endogenous IL-10 was also blocked. Addition of exogenous IL-11 inhibited spontaneous TNFalpha production in RA synovium only in the presence of soluble IL-11 receptor. However, exogenous IL-11 directly inhibited spontaneous MMP-1 and MMP-3 production, and up-regulated TIMP-1 in RA synovial tissue. CONCLUSION: IL-11 has important endogenous immunoregulatory effects in RA synovium, which suggests that exogenous IL-11 may have therapeutic activity in RA.     

Therapeutic actions of cyclosporine and anti-tumor necrosis factor alpha in collagen-induced arthritis and the effect of combination therapy

OBJECTIVE: To define the mechanisms of action of 2 novel drugs, cyclosporine and anti-tumor necrosis factor alpha (TNFalpha), in collagen-induced arthritis and to determine the effect of combination therapy. METHODS: Type II collagen-immunized DBA/1 mice with established arthritis were treated with cyclosporine alone, anti-TNFalpha alone, cyclosporine plus anti-TNFalpha, or saline. RESULTS: Cyclosporine was found to ameliorate arthritis, suppress interferon-gamma (IFNgamma) production by CD4+ T cells, and reduce TNFalpha expression in arthritic joints. However, cyclosporine did not directly inhibit TNFalpha production by macrophages, indicating that the decrease in TNFalpha expression observed in vivo was probably an indirect consequence of the reduction in type 1 T helper cell activity. Anti-TNFalpha also reduced IFNgamma production by T cells, indicating that TNFalpha is involved in the cellular immune response to collagen. Combined treatment with cyclosporine plus anti-TNFalpha had an additive therapeutic effect. CONCLUSION: Although cyclosporine and anti-TNFalpha target different points in the inflammatory pathway, there is an overlap in the consequences of their actions in vivo.     

Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes--regulation by IFN-gamma and IL-4

This study analyzes the effects of the T cell cytokines IL-4 and IFN-gamma on the spontaneous and stimulated production of IL-8, MCP-1, IL-1 receptor antagonist (IL-1ra), and PGE by synoviocytes from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells from both sources constitutively released IL-8 and MCP-1, but no IL-1ra or PGE. Stimulation with IL-1 beta or TNF-alpha massively increased chemokine production and induced the generation of PGE and low amounts of IL-1ra. The constitutive or cytokine-stimulated release of IL-8 was inhibited by IFN-gamma, but not by IL-4. The constitutive or IL-1 beta-stimulated release of MCP-1, by contrast, was markedly enhanced by IL-4 and IFN-gamma. Both cytokines, however, had only borderline effects on the release stimulated by TNF-alpha. The yield of IL-1ra was strongly enhanced by IFN-gamma in all cases, whereas the effect of IL-4 was pronounced only in IL-1 beta-stimulated OA synoviocytes. IL-4, on the other hand, markedly decreased the release of PGE, which was less susceptible to IFN-gamma. The observed effects on chemokines, IL-1ra expression, and PGE release by synoviocytes suggest that IFN-gamma and IL-4 are important regulatory elements in the inflamed synovium and may exert anti-inflammatory effects.     

Identification of two closely related genes, inducible and noninducible carbonyl reductases in the rat ovary

OBJECTIVE: To investigate the potential role of cytokines in psoriatic arthritis (PsA) by assessing the profiles of the proinflammatory cytokines in synovial fluid (SF) of PsA in comparison with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, and IL-8 were measured in SF using ELISA. RESULTS: Levels of TNF-alpha, IL-1beta, and IL-8 were significantly higher in PsA SF than in OA SF, although lower than in RA SF. No difference was detected in the IL-6 levels between PsA and RA SF, both of which were much higher than in OA SF. CONCLUSION: The pattern of expression of proinflammatory cytokines seen in PsA is similar to that in RA. Since PsA is also a destructive arthropathy, cytokines, in particular TNF-alpha and IL-1, may be principle factors in joint destruction.     

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.     

Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: immunochemical and in-situ hybridization analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1 beta) (0.2-20 ng ml-1), tumor necrosis factor (TNF-alpha) (0.2-20 ng ml-1) or interferon-gamma (IFN-gamma) (10-1000 U ml-1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr. ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1 beta or TNF-alpha, but cell associated IL-8 was detectable only after high dose (20 ng ml-1) IL-1 beta stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1 beta > TNF-alpha > IFN-gamma stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (> 60%) and monocyte (53-57%) chemotactic activity, respectively. Using in situ hybridization IL-8 mRNA was readily detected within IL-1 beta > TNF-alpha stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1 beta > TNF-alpha > or IFN-gamma stimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1 beta (20 ng ml-1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-beta, TNF-alpha, or IFN-gamma. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.     

Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system

A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB.     

Inflammatory infiltrate and interleukin-8 expression in the synovium of psoriatic arthritis--an immunohistochemical and mRNA analysis

Psoriatic arthritis is an inflammatory arthropathy that ultimately can lead to joint destruction. In this study, we investigated the immunophenotypes of the inflammatory cells and the expression of interleukin-8 (IL-8), which is the hallmark chemoattractant cytokine of psoriasis in synovial membranes from patients exhibiting active psoriatic synovitis (n = 9). The tissue samples were examined by immunohistochemistry, Western blot analysis and in situ hybridisation. The inflammatory infiltrate consisted predominantly of CD3+ T lymphocytes, with a higher proportion of CD4+ than CD8+ T lymphocytes in six cases. CD3+ T lymphocytes were focally distributed near small blood vessels and the enlarged synovial intima. CD1+ interdigitating reticulum cells were not detected. CD22+ B lymphocytes and plasma cells were found in small aggregates without KiM4+ follicular dendritic cells. KiM8+ macrophages were located in the synovial intima and were distributed in a diffuse pattern near the synovial lining cells. CD15+ neutrophil granulocytes were detected in four cases. They were preferentially located in the vicinity of blood vessels and the synovial intima. IL-8 was found at a high level in the synovial lining cells and to a lesser extent in cells located in the perivascular areas. Immunofluorescence double staining showed IL-8 to be expressed in KiM8+ multinucleated giant cells, KiM8+ macrophages and CD3+ T lymphocytes. IL-8 receptor A was demonstrated in the synovial lining and in macrophages and lymphocytes. IL-8 was detected by immunoblot analysis of the synovial tissue at 8.4 kD. Employing in situ hybridisation, IL-8 mRNA was strongly and preferentially expressed in the synovial intima, as well as in macrophages and lymphocytes. The immunophenotype of the psoriatic arthritis inflammatory cells shows great similarity to the inflammatory infiltrate found in the synovial tissue of patients with rheumatoid arthritis. The preferential expression of IL-8 and IL-8 mRNA in the enlarged synovial intima and in lymphocytes and macrophages suggests that IL-8 exerts its action through activated mononuclear cells and T lymphocytes. It seems to play a role in regulating leucocyte traffic into the enlarged synovial intima and may contribute to the aggressive synovitis of patients with psoriatic arthritis.     

Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent

Tumor necrosis factor (TNF) alpha has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNFalpha antibody clinical trials. Although signaling pathways leading to TNFalpha expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNFalpha synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system in vitro we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IkappaB alpha, the natural inhibitor of NF-kappaB, that induction of TNFalpha in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNFalpha from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-kappaB pathway is an essential step for TNFalpha synthesis in synovial macrophages and demonstrating that NF-kappaB should be an effective therapeutic target in this disease.     

Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient

OBJECTIVE: To investigate the production of proinflammatory cytokines and expression of cell adhesion molecules in the rheumatoid nodule. METHODS: Cytokine content (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], and IL-1 receptor antagonist [IL-1Ra]), at the messenger RNA (mRNA) and protein levels, and cell adhesion molecule expression were studied in 16 rheumatoid nodules and 6 synovial membranes. RESULTS: Macrophages in the rheumatoid nodules contained TNFalpha, IL-1beta, and IL-1Ra mRNA and protein, particularly in perivascular cells of the stroma and in the palisading layer. All cell adhesion molecules studied were expressed in both the rheumatoid nodules and synovial membranes, with increased expression of E-selectin in the rheumatoid nodule compared with the synovial membrane, and with the absence of vascular cell adhesion molecule 1 expression on cells of the palisading layer in the rheumatoid nodule. CONCLUSION: The presence of similar proinflammatory cytokines and cell adhesion molecules in the rheumatoid nodule and synovial membrane suggests that similar pathogenic processes result in the chronic inflammation and tissue destruction in these lesions.     

Intermittent parathyroid hormone administration stimulates bone formation in the mandibles of aged ovariectomized rats

OBJECTIVE: To assess the effect of various antirheumatic drugs on cytokine, cytokine inhibitor, and prostaglandin E (PGE) production by normal blood mononuclear cells (MNC) and rheumatoid arthritis (RA) synovial fibroblasts in vitro. METHODS: MNC from healthy donors and RA synovial fibroblasts were preincubated with or without prostaglandin E2 (PGE2), indomethacin, dexamethasone, gold sodium thiomalate (GSTM), methotrexate (MTX), and cyclosporin A (CyA), and then cultured in the absence or presence of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) for 48 h. We characterized cytokines such as IL-1 beta, IL-8, monocyte chemoattractant protein-1 (MCP-1), and cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra) and soluble TNF receptors (sTNFR p55 + p75) as well as PGE in the cell-free culture supernatants. RESULTS: In MNC and synovial fibroblast cultures dexamethasone, GSTM, and PGE2 most markedly downregulated spontaneous and/or cytokine stimulated production of IL-1 beta, IL-14a, IL-8, and MCP-1, whereas sTNFR shedding was not affected. In contrast, MTX and CyA had only marginal or no effects on mediator release, whereas indomethacin inhibited only PGE production. CONCLUSION: Among several antirheumatic drugs examined, dexamethasone and GSTM exhibited the most potent inhibitory effects on inflammatory cytokine and cytokine inhibitor production by blood mononuclear cells and synovial fibroblasts. These drugs may exert their antiinflammatory actions by unspecific suppression of monocyte and fibroblast secretory function.