Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Analysis of the cytokine interaction among interleukin-1beta, interleukin-8, and interleukin-1 receptor antagonist in the rabbit ovulatory process

OBJECTIVE: To elucidate the regulation and involvement of interleukin (IL)-1beta, IL-8, and IL-1 receptor antagonist in the hCG-induced rabbit ovulatory process. DESIGN: Randomized, controlled animal study. SETTING: University research laboratory. ANIMAL(S): Mature female New Zealand white rabbits. INTERVENTION(S): After i.v. administration of 100 IU of hCG to rabbits, ovarian levels of IL-1beta. IL-8, and IL-1 receptor antagonist were determined at indicated times by ELISA. Anti IL-1beta, anti-lL-8, or anti-IL-1 receptor antagonist antiserum was given i.v. 30 minutes before hCG injection. MAIN OUTCOME MEASURE(S): Effects of each antiserum on the levels of the other cytokines and neutrophil accumulation, assessed by myeloperoxidase activity, were determined. Ovulation rate (rate of ruptured follicles) was also evaluated. RESULT(S): The maximal level of IL-8 was detected at 4 hours. which preceded that of IL-1beta and IL-1 receptor antagonist, detected at 6 hours after hCG injection. Administration of anti-IL-1beta antiserum resulted in a statistically significant reduction of the peak levels of IL-8 and IL-1 receptor antagonist. Administration of anti-IL-8 antiserum reduced the accumulation of IL-1beta and IL-1 receptor antagonist. Anti-IL-1 receptor antagonist antiserum significantly augmented the accumulation of IL-1beta and IL-8. Myeloperoxidase activity was reduced by anti-IL-8 antiserum. Anti-IL-1beta and anti-lL-8 antiserum reduced the hCG-induced ovulation rate, but a synergistic effect was not evident when these antisera were injected simultaneously. Anti-IL-1 receptor antagonist antiserum had no apparent effect on ovulatory efficiency. CONCLUSION(S): IL-1beta, IL-8, and IL-1 receptor antagonist may affect the accumulation of related cytokines in ovaries and may be involved in ovulation.     

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.     

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.     

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.     

Studying the mental health of a nation: a preliminary report on a population survey in Singapore

OBJECTIVE: Requirements for cytokines and adhesion molecules for peritoneal neutrophil recruitment during glycogen-induced peritonitis in rats were systematically defined. SUBJECTS: Male Long Evans rats (275-300 g). METHODS: Four hours after intraperitoneal injection of 25 mg oyster glycogen, neutrophilic exudates were harvested. Effects of blocking reagents (injected intravenously) to rat E-, L- and P-selectins, beta1 (VLA-4) and beta2 integrins (LFA-1 and Mac-1), ICAM-1, and the cytokines TNFalpha, IL- and IL-8 were assessed. RESULTS: Administration of synthetic sialyl Lewis(x) oligosaccharide reduced neutrophil recruitment to the peritoneum by 26%. Antibody to E-selectin reduced neutrophil accumulation by 71%, while anti-L-selectin reduced neutrophil accumulation by 59%, and anti-P-selectin was without an effect. Similar patterns of inhibition were found when selectin-Ig chimeras were employed. Antibodies to LFA-1 (CD11a), Mac-1 (CD11b) or CD18 reduced neutrophil accumulation by 62 percent, 59 percent and 86%, respectively, while anti-VLA-4 was without effect. Anti-ICAM-1 reduced cell influx by 65%. IL-1 receptor antagonist and antibodies to IL-1 and human IL-8 reduced neutrophil accumulation by 43alpha, 40% and 62 percent, respectively. Unexpectedly, blockade of TNFalpha had no effect. CONCLUSIONS: These studies identify requirements for selectins, beta2 integrins, IL-1 and a rat chemokine(s) similar to human IL-8 for neutrophil recruitment during glycogen-induced peritonitis. The lack of participation of VLA-4, P-selectin and TNFalpha suggests organ-specific cytokine and adhesion molecule requirements for neutrophil recruitment.     

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.     

Bridging town and gown: building research partnerships between community-based professional providers and academia

Interleukin 1 (IL-1) is postulated to function in maintaining homeostasis, however, over-action of this cytokine may lead to disruption of homeostasis due to it's wide spectrum of activities. To understand the endogenous regulation of this cytokine, we examined the existence of IL-1 receptor antagonist (IL-1Ra) in tissues from healthy rabbits. IL-1Ra was constitutively produced in all tissues examined (lung, liver, spleen, thymus, caecum, skin, kidney, heart, and brain), as estimated by ELISA. Immunoprecipitation, RT-PCR and immunohistochemical studies indicated that all tissues produced secreted form of IL-1Ra (sIL-1Ra), whereas thymus, caecum, skin and kidney produced both sIL-1Ra and intracellular of IL-1Ra. All tissue IL-1Ra purified using anti-IL-1Ra IgG affinity chromatography had inhibitory activity on the IL-1-induced thymocyte proliferative response, and the activity was totally abolished by anti-IL-1Ra mAb. No IL-1 activity was detected in any tissues except skin and heart, however, after preincubation of the samples with anti-IL-1Ra, the activity was first visible in the tissues. Under these conditions, IL-1 activity in skin and heart was enhanced to 170% and 280%, respectively. Taken together, we conclude that tissue IL-1Ra is involved in health maintenance by masking co-existing IL-1 activity present in tissues.     

Monomeric IgG2 enhances Ig production and proliferation in human B cells

The effect of purified polyclonal human IgG subclasses on B-cell responses was studied using the human IgA-producing B-cell line GM-1056. IgG2 at concentrations of 0.01-1 microgram/mL enhanced both IgA production and proliferation, while IgG1, IgG3, and IgG4 each failed to do so at tested concentrations between 0.001 and 10 micrograms/mL. This enhancement was Fc gamma R mediated, since IgG2 Fc fragments enhanced IgA production and proliferation to the same extent as did the whole IgG2 molecule, whereas F(ab')2 fragments did not. However, in contrast to monomeric IgG2, aggregated IgG2, which was expected to bind Fc gamma RII on B cells, affected neither IgA production nor proliferation. Similarly, anti-CDw32 mAb (2E1, anti-Fc gamma RII), anti-CD 64 mAb (32.2 anti-Fc gamma RI), and anti-CD16 mAb (Leu 11a, anti-Fc gamma RIII) mAb each failed to stimulate GM-1056 cells, and more importantly did not block IgG2-induced stimulation. Of various cytokines tested, including IFN-alpha, IFN-gamma, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, IL-6 alone augmented IgG2-induced enhancement of IgA production and proliferation. Moreover, the IL-6 effect was lost following preabsorption with anti-IL-6 antibody but not following preabsorption with control antibody. IgG2 also enhanced Ig production and proliferation in tonsillar large activated B cells, while IgG1, IgG3 and IgG4 each failed to do so. In contrast, IgG2 had no effect on Ig production and proliferation in tonsillar small resting B cells or SAC-stimulated small B cells. IgG2-induced enhancement of Ig production and proliferation in large B cells was not blocked by 2E1, 32.2, or Leu 11a, while enhancement was augmented in a specific fashion by IL-6. These results indicate that monomeric IgG2 specifically enhances B cell responses via an Fc gamma R receptor distinct from Fc gamma RI, Fc gamma RII, and Fc gamma RIII, and that IL-6 may play a role in augmenting this response.     

Antiinflammatory properties of hepatic acute phase proteins: preferential induction of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis by human peripheral blood mononuclear cells

This study was undertaken to determine whether acute phase proteins (APP) induce the synthesis of interleukin 1 beta (IL-1 beta) and its specific antagonist, IL-1 receptor antagonist (IL-1Ra), in human peripheral blood mononuclear cells (PBMC). PBMC from healthy volunteers were incubated with C-reactive protein (CRP), alpha 1-antitrypsin (alpha 1-AT), or alpha 1-acid glycoprotein (AGP), and the levels of IL-1 beta and IL-1Ra produced were measured by specific radioimmunoassay. To evaluate the effects of alpha 1-AT further, a synthetic pentapeptide FVYLI corresponding to the minimal binding sequence for the serpine-enzyme complex receptor was also evaluated. PBMC incubated for 24 h with CRP, alpha 1-AT, or the pentapeptide FVYLI synthesized large quantities of IL-1Ra, 5-10-fold greater than the amount of IL-1 beta produced by these cells. AGP induced significantly less IL-1Ra than the other APP tested. These effects were shown to be specific, in that polyclonal antibodies against CRP, alpha 1-AT, and AGP eliminated the cytokine production induced by these respective proteins. CRP, alpha 1-AT, FVYLI, and AGP were synergistic with low concentrations of endotoxin in the induction of both IL-1Ra and IL-1 beta synthesis. We suggest that the preferential induction of IL-1Ra by APP may contribute to their antiinflammatory effects and provide an important regulatory signal for the acute phase response.     

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 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-mu antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-alpha (anti-TNF-alpha) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-alpha, but not other cytokines including IL-1 beta, IL-3, IL-5, IL-6, interferon-alpha (IFN-alpha) or IFN-gamma, inhibited IL-4-mediated growth, and inhibition by TNF-alpha was blocked by anti-TNF-alpha Ab but not by control IgG. IL-4 had no effect on TNF-alpha binding by B cells while it decreased TNF-alpha production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-alpha by B cells, however, it enhanced TNF-alpha production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-alpha production.     

In vitro role of IL-1 in heightened IgG, anti-DNA, and nephritogenic idiotype production by B cells derived from the murine MRL/lpr lupus model

The MRL/lpr murine model resembles human lupus both in its serologic and immunopathologic features, and is characterized by high-level IgG and autoantibody production. The precise mechanisms for this B cell hyperactivity are poorly understood. This study explored the role of IL-1 in determining high-level IgG and autoantibody production in the MRL/lpr murine lupus model by blocking IL-1 activity with a recombinant IL-1 receptor antagonist (IL-1Ra). IgG and autoantibody production (anti-DNA ab and Id-H130 activity) by B cells derived from MRL/lpr mice was significantly suppressed by treating B cell cultures with IL-1Ra. In contrast, IgG and autoantibody production by B cells derived from young MRL/lpr, MLR/++, or normal C3H/HeJ mice showed virtually no suppression with IL-1Ra. Collectively, these findings indicate that IL-1 may be an important factor in determining the heightened production of IgG, anti-DNA, and id-H130 antibody production in lupus-prone MRL/lpr mice. Furthermore, heightened IL-1 activity appears to be influenced by both age and the presence of the lpr mutation.     

Effects of sumatriptan on coronary flow and left ventricular function in the isolated perfused guinea pig heart

IBD is associated with an increased activation of intestinal immune cells, which causes overproduction of proinflammatory cytokines such as IL-1beta. IL-1beta is implicated in mediating the sustained inflammatory response. IL-1 receptor antagonist (IL-1Ra), the naturally occurring inhibitor of IL-1, has been shown to have beneficial effects in experimental models of colitis. In this study we investigated the hypothesis that an imbalance between IL-1 and IL-1Ra exists in IBD by measuring their secretion by explant cultures of colonic biopsies. Freshly homogenized biopsies from involved tissue in IBD patients exhibited significantly lower IL-1Ra/IL-1beta ratios than control and uninvolved IBD mucosal tissue. Using explant cultures, in vitro production of IL-1beta and IL-1Ra increased progressively during the 4-18-h culture periods. IL-1beta secretion was higher in supernatants from involved Crohn's disease (CD) and ulcerative colitis tissue compared with control tissue, and IL-1beta levels increased with severity of inflammation. IL-1Ra secretion was not elevated in involved IBD samples, but significantly higher levels were released when moderate to severely involved tissue samples were compared with noninflammatory controls. Similar to freshly homogenized tissue, explant studies showed that the IL-1Ra/IL-1beta ratios were significantly decreased in involved IBD tissue, but not in uninvolved CD or inflammatory control specimens. These data support the hypothesis of an imbalance between IL-1beta and IL-1Ra in IBD.     

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.     

The association of peripheral monocyte derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist, and tumor necrosis factor-alpha with osteoarthritis in the elderly

OBJECTIVE: To examine the association of peripheral blood mononuclear cell (PBMC) derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor alpha (TNF-alpha), and radiographic osteoarthritis (OA) in the elderly. METHODS: A total of 703 subjects (436 women, 267 men, mean age 78.5+/-4.5 yrs) had both knee and hand radiographs, and cytokines were measured during the 22nd biennial examination of the Framingham Cohort. PBMC derived IL-1beta , IL-1Ra, and TNF-alpha production was assessed using a non-cross reacting polyclonal radioimmunoassay. Knee OA was defined as a score of > 2 using a modified Kellgren and Lawrence scale. The presence of osteophytes and joint space narrowing were scored separately on a 0-3 scale, in which disease was defined a priori as a score > 0 for each feature. Sex-specific odds ratios were calculated for knee OA after adjusting for weight, history of knee injury, and use of estrogen and nonsteroidal antiinflammatory drugs. RESULT: No uniform associations were found for IL-1beta or IL-1Ra in men, or for TNF-alpha production and radiographic OA in either sex. We found possible associations for the highest levels of IL-1beta production and the presence of knee osteophytes [OR=2.0 (1.2-3.5)] and joint space narrowing [OR=1.7 (1.1-2.8)] in women. Our data suggested a possible protective effect for IL-1Ra production and hand OA in women [OR=0.6 (0.4-1.0)]. CONCLUSION: We found no consistent association of PBMC cytokine production and radiographic OA. However, women with the highest production of IL-1beta and IL-1Ra had respectively higher rates of knee OA and lower rates of hand OA than expected.