Mycophenolic acid, an inhibitor of IMP dehydrogenase that is also an immunosuppressive agent, suppresses the cytokine-induced nitric oxide production in mouse and rat vascular endothelial cells.

Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase and de novo GTP biosynthesis, also has immunosuppressive activity. The effect of MPA on nitric oxide (NO) production by rodent brain vascular endothelial cells in culture was investigated. MPA inhibited NO production by mouse and rat brain endothelial cells that had been stimulated with a combination of interferon-gamma and tumor necrosis factor-alpha. The 50% inhibitory concentration (EC50) was in the range of 0.5-1.0 microM. However, MPA had no effect on basal NO production in mouse brain vascular endothelial cells. Brequinar, an inhibitor of de novo pyrimidine synthesis, had no effect on NO production in cytokine stimulated endothelial cells. Guanosine, which can act as a salvage pathway precursor for GTP biosynthesis, reversed the inhibitory effect of MPA in a dose-dependent fashion. We suggest that inducible NO synthase activity is dependent on GTP level and can be blocked by curtailing IMP dehydrogenase activity.

Some antioxidants inhibit, in a co-ordinate fashion, the production of tumor necrosis factor-alpha, IL-beta, and IL-6 by human peripheral blood mononuclear cells.

Some antioxidants, including butylated hydroxyanisole (BHA), tetrahydropapaveroline (THP), nordihydroguiauretic acid, and 10,11-dihydroxyaporphine (DHA), were found to be potent inhibitors of the production of tumor necrosis factor (TNF)-alpha, IL-1 beta, and IL-6 by human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS) (IC50s in the low micromolar range). Inhibition of cytokine production was gene selective and not due to general effects on protein synthesis. Inhibition of cytokine production by PBMC was observed also when other inducers were used (staphylococci, silica, zymosan). Much higher concentrations of other antioxidants--including ascorbic acid, trolox, alpha-tocopherol, butylated hydroxytoluene, and the 5-lipoxygenase inhibitor zileuton--did not affect the production of these cytokines. The active compounds did not inhibit IL-1-induced production of IL-6 in fibroblasts, showing the cell selectivity of the effect. Antioxidant-mediated inhibition of cytokine production was correlated with low levels of the corresponding messenger RNAs. Nuclear run-on experiments showed that THP inhibited transcription of the IL-1 beta gene. THP decreased the concentration of the transcription factors NF-kappa B and AP-1 detected in nuclear extracts of PBMC cultured in the presence or absence of LPS. THP and DHA markedly decreased the levels of TNF-alpha and IL-1 beta in the circulation of mice following LPS injection. Thus antioxidants vary widely in potency as inhibitors of the activation of transcription factors and of the transcription of genes for pro-inflammatory cytokines. Coordinate inhibition of the transcription of genes for inflammatory cytokines could provide a strategy for therapy of diseases with inflammatory pathogenesis and for septic shock.

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity.

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.

A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties.

A fragment of rat transforming growth factor alpha (TGF alpha) comprising the third disulfide loop (residues 34-43) was selected as a potential antigenic and receptor binding region. Immunization of rabbits with a peptide conjugate resulted in antibodies which were specific for both the peptide and rat TGF alpha, but not for the homologous epidermal growth factor (EGF). The synthetic decapeptide exhibited low affinity for EGF receptors on human cells. Affinity was increased 100x to 0.2% of EGF or TGF alpha binding by blocking the peptide ends. The blocked decapeptide had no mitogenic activity but prevented the mitogenic effect of EGF and TGF alpha on fibroblasts. This decapeptide is an antagonist and contains an important receptor binding region of TGF alpha.

The absence of antibodies to type II collagen in established adjuvant arthritis in rats.

Utilizing an adjuvant arthritis model in rats, we examined humoral immunity to collagen and inflammation in animals with active disease and during drug therapy. Humoral immunity to types I or II collagen was not detected in the sera of rats with advanced adjuvant arthritis; this was in marked contrast to rats with type II collagen-induced arthritis which possessed serum antibodies to native and denatured type II collagen. Hind paw edema and bone pathology were monitored as parameters of inflammation. A new investigational drug, Wy-41,770, was most effective in reducing all of these aspects of inflammatory disease while indomethacin, methylprednisolone, and D-penicillamine caused a less significant diminution of only some of these parameters of inflammation. Antibodies to collagen were not detected in the sera of rats treated with the drugs under study. These data demonstrate that adjuvant arthritis can occur in rats in the absence of antibodies to types I or II collagen.

Immune response to connective tissue components of the basement membrane.

The immune response to connective tissue components of basement membrane (type IV collagen and laminin) and to interstitial collagen (type I) has been examined in human and murine systems. We also examined the role that immunologic sensitization to autologous connective tissue components might play in inducing an inflammatory response resulting in pathologic sequelae. Mice receiving a single subcutaneous injection of 5 micrograms type IV or type I murine collagens, or murine laminin in complete Freund's adjuvant mount a delayed-type hypersensitivity response characterized by a mononuclear cell infiltrate when challenged in the footpad with the sensitizing antigen. Cell-mediated immunity to these connective tissue antigens can be transferred to normal syngeneic mice with sensitized T-lymphocytes. In addition, repeated immunizations with these homologous connective tissue components elicit antibody responses in mice. Our data demonstrate the immunogenic nature of types IV and I collagen, and of laminin in a syngeneic murine model. We have demonstrated autoantibodies to the basement membrane and interstitial collagens in the sera of patients with scleroderma (systemic sclerosis); ELISA ratios correlate directly with the extent of pulmonary fibrosis in these patients. Anti-type IV collagen autoantibodies were found to be primarily IgM and anti-type I collagen antibodies, primarily IgG. An antibody response to autologous connective tissue antigens could lead to complement activation, immune complex formation, and deposition of the complexes along vascular endothelium with recruitment of blood monocytes in situ, mirroring the early scleroderma lesion (perivascular mononuclear cell filtrates). In vitro we examined the role of human peripheral blood mononuclear cells in the activation of fibroblasts. Adherent human blood monocytes release mediators which stimulate fibroblast proliferation and collagen deposition. A model is presented for the induction of immunity to autologous connective tissue components, leading to mononuclear cell inflammation, fibroblast activation and fibrosis. Selective immunity to basement membrane collagens may influence the clinical expression of diffuse connective tissue syndromes such as scleroderma (systemic sclerosis).