Interleukin 27 attenuates collagen-induced arthritis.

OBJECTIVE: To investigate the potential role of interleukin (IL) 27 in rheumatoid arthritis (RA) by examining the expression of IL27 in the articular joints of patients with RA and the effect of recombinant IL27 in vivo in a murine model of collagen-induced arthritis (CIA). METHODS: Synovial membranes from patients with RA were examined for the presence of IL27 by immunohistochemistry and by western blot. Mice developing CIA were treated with IL27 and the ensuing disease progression and immunological profile determined. The effect of IL27 on T-cell response in vitro was also ascertained. RESULTS: IL27 was clearly detected in the RA synovial membranes. Short-term administration of IL27 at the onset of the disease significantly attenuated disease severity compared with untreated controls. Histological examination showed that while untreated mice developed severe cellular infiltration in the joints, synovial hyperplasia and joint erosion, this pathology was profoundly reduced in IL27-treated animals. Treatment of mice with IL27 also decreased the amounts of serum IL6 and collagen-specific IgG2a. Spleen and lymph node cells from the IL27-treated mice produced significantly less interferon gamma and IL17 than cells from the control mice when cultured with collagen in vitro. CONCLUSION: These results demonstrate that IL27 may be a potential therapeutic agent against RA at the onset of the disease.

Role of nitric oxide in the regulation of T cell functions.

There is a close relation between T helper (Th) 1 cells and nitric oxide in disease. Thus it is possible that a reciprocal regulatory mechanism exists between them. This paper briefly describes the experimental studies which have helped elucidate the mechanism by which nitric oxide selectively enhances Th 1 cell proliferation and the potential effect of nitric oxide on regulatory T (Treg) cells. On the basis of the results the authors propose that nitric oxide represents an additional signal for the induction of T cell subset response, contributing to the increasingly complex network of immune regulation essential for health and disease.

The Sushi domain of soluble IL-15 receptor alpha is essential for binding IL-15 and inhibiting inflammatory and allogenic responses in vitro and in vivo.

IL-15 is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 functions through the trimeric IL-15R complex, which consists of a high affinity binding alpha-chain and the common IL-2R beta- and gamma-chains. Characterization of IL-15/IL-15R interactions may facilitate the development of improved IL-15 antagonists for therapeutic interventions. We previously constructed soluble murine IL-15Ralpha (sIL-15Ralpha) by deleting the cytoplasmic and transmembrane domains. To localize the functional domain of IL-15Ralpha, we have now constructed various truncated versions of sIL-15Ralpha. The shortest region retaining IL-15 binding activity is a 65-aa sequence spanning the Sushi domain of IL-15Ralpha. Sushi domains, common motifs in protein-protein interactions, contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern. Amino acid substitution of the first or fourth cysteine in sIL-15Ralpha completely abolished its IL-15 binding activity. This also abrogated the ability of sIL-15Ralpha to neutralize IL-15-induced proinflammatory cytokine production and anti-apoptotic response in vitro. Furthermore, the mutant sIL-15Ralpha lost its ability to inhibit carrageenan-induced local inflammation and allogenic cell-induced T cell proliferation and cytokine production in vivo. Thus, the Sushi domain is critical for the functional activity of sIL-15Ralpha.

IL-18 not required for IRBP peptide-induced EAU: studies in gene-deficient mice.

PURPOSE: Interleukin (IL)-18 has been described as a proinflammatory cytokine in rheumatoid arthritis and bacterial infectious diseases. The present study was designed to determine the role of IL-18 in a model of ocular experimental autoimmune uveitis (EAU). The initial studies were conducted to detect the expression of IL-18 in normal mouse eye tissue, and the later studies investigated induction of EAU in mice with an IL-18(-/-) phenotype. METHODS: IL-18 detection was performed by using 5-bromo-4-chloro-3-indoyl-ss--D-galactopyranoside (X-Gal) staining on frozen sections of eyes from mice (129/CD1, DBA1, and Balb/c), either of normal phenotype (+/+) or of deficiency (+/-, -/-) in the IL-18 gene which had been replaced by introduced genes including LacZ under the control of an IL-18 promotor. Severity of EAU was assessed in DBA1 and 129/CD1 wild-type (WT) or IL-18 knockout (KO) mice after immunization with the uveitogenic antigen: interphotoreceptor retinal binding protein (IRBP) peptide 161-180. Lymphocyte proliferation and cytokine production were also measured in WT and IL-18 KO DBA1 mice 15 days after immunization. RESULTS: IL-18 is constitutively expressed in the epithelial cells in iris, ciliary body, and retina. EAU-resistant mice (129/CD1) with an IL-18(-/-) phenotype remained resistant after immunization with IRBP peptide (P161-180). However, EAU-susceptible mice (DBA1) exhibited disease with similar histologic characteristics, despite a generalized reduction of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on an IL-18(-/-) phenotype. DBA1 IL-18(-/-) also demonstrated reduced IL-10 production. CONCLUSIONS: The IL-18 gene is not necessary for the initiation or pathogenesis of EAU induced by IRBP peptide 161-180. IL-18 is expressed in the epithelial cells in iris, ciliary body, and retina in the eyes, but its role in the eye remains undetermined.

Effects of nitric oxide on the induction and differentiation of Th1 cells.

We have previously shown that mice lacking inducible NO synthase are markedly more susceptible to Leishmania major infection but developed a significantly enhanced Th1 cell response compared with wild-type mice. Furthermore, at high concentrations, NO inhibited IL-12 synthesis by activated macrophages, thereby indirectly suppressing the expansion of Th1 cells. We report here that at low concentrations, NO selectively enhanced the induction of Th1 cells and had no effect on Th2 cells. NO exerted this effect in synergy with IL-12 during Th1 cell differentiation and had no effect on fully committed Th1 cells. NO appears to affect CD4(+) T cells directly and not at the antigen-presenting cells. These results therefore provide an additional pathway by which NO modulates the immune response and contributes to the homeostasis of the immune system.

Altered immune responses and susceptibility to Leishmania major and Staphylococcus aureus infection in IL-18-deficient mice.

IL-18, formerly designated IFN-inducing factor, is a novel cytokine produced by activated macrophages. It synergizes with IL-12 in the induction of the development of Th1 cells and NK cells. To define the biological role of IL-18 in vivo, we have constructed a strain of mice lacking IL-18. Homozygous IL-18 knockout (-/-) mice are viable, fertile, and without evident histopathologic abnormalities. However, in contrast to the heterozygous (+/-) or wild-type (+/+) mice, which are highly resistant to the infection of the protozoan parasite Leishmania major, the IL-18-/- mice are uniformly susceptible. The infected IL-18-/- mice produced significantly lower levels of IFN-gamma and larger amounts of IL-4 compared with similarly infected +/- and +/+ mice. In contrast, when infected with the extracellular Gram-positive bacteria Staphylococcus aureus, the IL-18-/- mice developed markedly less septicemia than similarly infected wild-type (+/+) mice. However, the mutant mice developed significantly more severe septic arthritis than the control wild-type mice. This was accompanied by a reduction in the levels of Ag-induced splenic T cell proliferation, decreased IFN-gamma and TNF-alpha synthesis, but increased IL-4 production by the mutant mice compared with the wild-type mice. These results therefore provide direct evidence that IL-18 is not only essential for the host defense against intracellular infection, but it also plays a critical role in regulating the synthesis of inflammatory cytokines, and therefore could be an important target for therapeutic intervention.

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages.

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2-deficient mice infected with Leishmania major in vivo or stimulated with IFN-gamma or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL-12 than those from heterozygous or wild-type mice. A macrophage cell line, J774, produced significant amounts of IL-12 following activation with LPS, or LPS plus IFN-gamma. This could be markedly enhanced by the NOS inhibitor L-NG monomethyl arginine (L-NMMA), but profoundly inhibited by the NO-generating compound S-nitroso-N-acetyl-penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L-NMMA decreased TNF-alpha synthesis by LPS-activated J774 cells. The differential effect of NO on IL-12 and TNF-alpha is at the transcriptional level and is activation dependent. Since IL-12 is a major inducer of Th1 cells which produce IFN-gamma that can activate macrophages to produce IL-12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

A comparison of three methods for production of human hybridomas secreting autoantibodies.

Human hybridomas that secrete monoclonal antibodies (MAbs) in a stable manner are technically difficult to obtain. The problems limiting their production are the low numbers of sensitized B cells in peripheral blood, the limited choice of techniques for B cell immortalization, the limited number of suitable human myeloma or lymphoblastoid fusion partners, and the inability to immunize humans with most antigens. In order to circumvent these problems, we have compared the efficiency of different methods for production of B cell lines secreting human MAbs against the nuclear antigens dsDNA, ssDNA, and Sm/RNP from patients with the autoimmune disease systemic lupus erythematosus (SLE). We have tested various combinations of the following procedures: (1) EBV infection for immortalization of activated B lymphocytes, (2) activation of human resting B lymphocytes by anti-CD40, and (3) direct fusion of lymphocytes with a myeloma cell line using PBL or splenocytes from SLE patients. The methodological aspects of this investigation include optimization of the CD40 system and the generation of human hybridomas specific for nuclear antigens by fusion between sensitized lymphocytes and the human/mouse heteromyeloma cell line CBF7. The most efficient method for producing stable, IgG autoantibody-secreting human hybridomas was fusion of lymphocytes with cell line CBF7; human spleen was the best source of lymphocytes.

In vitro sensitization for human monoclonal antibody production.

We have found that cell adhesion prior to in vitro antigenic stimulation enriched the B-cell population and diminished the CD8 lymphocyte subset. These changes in lymphocyte proportions were favourable for increasing the percentage of antibody-producing cells after culturing in vitro followed by Epstein-Barr virus (EBV) infection. The immunodepletion of leukocytes by methyl esters did not yield satisfying results under analogous culture conditions. In vitro primary antigenic stimulations with the addition of IL-2 (25 U/ml medium) and low amounts of interferon-gamma provided better recruitment of antibody-producing cells and higher binding activity of antibodies to sperm than secondary antigenic stimulation. IL-6 did not positively influence the EBV-transformed cell lines.

The cytokines effect on EBV-immortalized human B cells producing antisperm antibody.

We have analyzed the antisperm antibody production of autoimmunized male subjects using Epstein-Barr virus (EBV) immortalization of B lymphocytes. We evaluated the influence of several in vitro culture variants applied prior to EBV infection on the frequence of antibody-producing cells and affinity of secreted antibodies. The following variants were applied: a) polyclonal antigenic stimulation of lymphocytes with PWM, b) PWM (pokeweed mitogen) + IL-2 + interferon gamma and c) PWM + IL-2 + interferon gamma + sperm antigenic extract. The variants where the cytokines were added did not increase the frequency of EBV-infected antibody-producing cells as comparing to EBV infection previously amplified by the use of polyclonal activator. Furthermore the cytokine activation either in combination with mitogen or in vitro secondary antigenic sensitization (prior to EBV transformation) did not seem to have beneficial effect on affinity of antibodies produced by EBV-infected cells in comparison to straight EBV infection. On the other hand, the attempt to promote an immunoglobulin secretion (IgM) by previously obtained human-human antisperm hybridomas by adding of IL-2 was quite successful.

The binding of SRBC by human peripheral blood monocytes.

In a population of E-rosette forming human peripheral blood cells, about 20% of the rosettes were formed by monocytes. About 60% of the peripheral blood monocytes bound SRBC in a standard E-rosette test.

Depressed PHA--induced lymphocyte transformation in women taking oral contraceptives.

The transformation of lymphocytes in response to the mitogen phytohemagglutinin (PHA) was determined in 30 women taking oral contraceptives (O.C.V.) and in 10 women of an age-matched control group. The group taking oral contraceptives demonstrated significant depression of their response to PHA as compared to the control group.