p38 kinase inhibitors for the treatment of arthritis and osteoporosis: thienyl, furyl, and pyrrolyl ureas.

Inhibitors of the MAP kinase p38 are potentially useful for the treatment for osteoporosis, arthritis, and other inflammatory diseases. A series of thienyl, furyl, and pyrrolyl ureas has been identified as potent p38 inhibitors, displaying in vitro activity in the nanomolar range.

Discovery of a new class of p38 kinase inhibitors.

The MAP kinase p38 has been implicated in cytokine signaling, and its inhibitors are potentially useful for the treatment of arthritis and osteoporosis. Novel small-molecule inhibitors of p38 kinase were derived from a combinatorial chemistry effort and exhibit activity in the nanomolar range. Very steep structure-activity relationships are observed within this class.

1-Phenyl-5-pyrazolyl ureas: potent and selective p38 kinase inhibitors.

Inhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-dichlorophenyl ureas were identified as small-molecule inhibitors of p38 by a combinatorial chemistry effort. Optimization for cellular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exemplified by the 1-phenyl-5-pyrazolyl urea 7 (IC50 = 13 nM).

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Inhibition of ceramide pathway does not affect ability of TNF-alpha to activate nuclear factor-kappa B.

TNF-alpha is a multifunctional cytokine that has been shown to activate a number of intracellular second messenger pathways. Recent studies demonstrate that the sphingomyelinase-ceramide pathway plays a potential role in the activation of nuclear factor-kappa B (NF-kappa B) by TNF-alpha. The following experiments both confirm that the addition of ceramide to cells can activate NF-kappa B and demonstrate that a 48-h pretreatment with phorbol 12-myristate (PMA) results in the loss of the ceramide-induced NF-kappa B response. In parallel experiments, in which SW480 cells were pretreated with PMA, TNF-alpha provided a signal resulting in the nuclear translocation of NF-kappa B that was similar to untreated cells. These data combined suggest that additional pathways exist that TNF-alpha can use for the activation of NF-kappa B. Supplementary data demonstrates that TNF-alpha, ceramide, and PMA activate a human cytomegalovirus-(HCMV) beta gal construct (promoter is responsive to NF-kappa B) that was stably transfected into the TNF receptor-bearing tumor cell line, SW480. PMA pretreatment of these cells resulted in a significant decrease in both the PMA and ceramide generated responses, 6% and 0% of controls, respectively. However, the response generated by TNF-alpha was not inhibited significantly (96% of control cells). This data suggests that although ceramide and 1,2-diacylglycerol (DAG) pathways may contribute to TNF-alpha activation of NF-kappa B, impedance of these pathways does not block TNF-alpha from activating NF-kappa B nor induction of the functional activation of the NF-kappa B responsive reporter construct, HCMV.

Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines.

Human activated T cells adhere to synovial fibroblast-like cells in vitro. The present study was conducted to investigate the consequences of T cell-synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence-activated cell sorter (FACS) analysis were used to analyze the induction of VCAM-1 and ICAM-1 expression in T cell-synovial fibroblast cocultures. VCAM-1 and ICAM-1 expression could be induced in synovial fibroblast-like cells by 2 h. PCR amplification showed that both forms of VCAM-1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up-regulation of VCAM-1 and ICAM-1 was confined to synovial fibroblasts; T cells did not express VCAM-1 or increased ICAM-1. In contrast to the T cell-synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up-regulation of ICAM-1 but not VCAM-1, suggesting tissue-specific regulation of VCAM-1. The T cell-synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon-gamma, and interleukin-6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti-TNF could significantly inhibit VCAM-1 and ICAM-1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM-1 and ICAM-1 failed to be up-regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell- and synoviocyte-specific cytokines and suggest a cell contact-mediated and T cell-initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM-1/ICAM-1 by synovial fibroblasts in the rheumatoid synovium.

Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody.

Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. A10G10 is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of A10G10 was raised and affinity purified over an A10G10 column. The resultant anti-idiotypic antibody recognized not only A10G10 but also both TNF receptors. It showed TNF agonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.

Chromosome assignments of the human TNF p55 and p75 receptor genes.

At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.

A synthetic mycobacterial heat shock peptide prevents adjuvant arthritis but not proteoglycan-induced synovitis in the rat.

Intradermal immunization of female Lewis rats with 100 micrograms of a nine-amino acid synthetic peptide corresponding to the arthritic T cell-reactive epitope of mycobacterial heat shock protein, three weeks prior to induction of adjuvant arthritis, produced inhibition of day 16 ankle swelling and histologic score. Intraarticular injection of 10 micrograms of bovine articular cartilage proteoglycan monomer emulsified in heavy mineral oil into normal Lewis rat stifle joints produced several hallmarks of chronic synovitis at day 16. Pre-treatment with nonapeptide did not inhibit proteoglycan-induced synovitis. These results indicate that tolerance to the critical epitope of heat shock protein does not abrogate the ability of proteoglycan to induce synovitis in rats.

Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine with immunosuppressive effects on T cells in vitro. Experimental allergic encephalomyelitis is an archetypal T cell-mediated autoimmune demyelinating disease of the central nervous system that often serves as a model for multiple sclerosis. In vivo administration of TGF-beta 1 into SJL mice was successful in reducing the incidence of clinical disease and the histologic severity of inflammation and demyelination in the brain and spinal cord. Immunohistochemical studies performed on control animals showed that TGF-beta-1, -2, and -3 were present in inflammatory perivascular lesions in the brain. The use of a naturally occurring cytokine with immunoregulatory functions in the treatment of an autoimmune disease is novel. However, potential long term complications of such therapy must be addressed before its use in human autoimmune disease such as multiple sclerosis.

Serotonin-activated signal transduction via serotonin receptors on Jurkat cells.

Serotonin (5-hydroxytryptamine) (5HT) a neurotransmitter and vasoactive amine, is a major storage product of platelets that are released at sites of inflammation. Several different subtypes of serotonin receptors have been defined. 5HT receptors have been divided into three major families based on molecular, biochemical, and pharmacologic properties. Binding of serotonin to the 5HT1 family results in inhibition of adenylate cyclase whereas binding to the 5HT2 family results in stimulation of phosphatidylinositol turnover and mobilization of intracellular Ca2+. 5HT has been shown to have effects on lymphoid cells. The question of whether human T lymphocytes express receptors for 5HT and transduce signals through 5HT receptors has not been adequately addressed. As a model system, Jurkat cells (a transformed human T lymphocyte line) were examined to determine if they expressed 5HT receptors and whether 5HT stimulated an increase in inositol phosphates or affected adenylate cyclase activity. The results show that Jurkat cells bind 5HT with an average dissociation constant of 90 nM and that 5HT stimulates an increase in inositol phosphate and intracellular Ca2+ levels. These results link the 5HT receptor on Jurkat cells to the 5HT2 family; however, studies with 5HT receptor agonists and antagonists failed to clearly classify the 5HT receptor on Jurkat cells as a known member of the 5HT2 family.

Alteration of bronchoalveolar lavage cell populations following bleomycin treatment in mice.

Clinical use of the antitumor antibiotic bleomycin (BLM) is associated with the development of interstitial pulmonary fibrosis. Shortly after acute lung damage caused by intratracheal (it) administration of BLM to experimental animals there is an influx of inflammatory cells which are believed to modulate the process of fibrosis. This study was undertaken to determine what subpopulations of lymphocytes were in the bronchoalveolar lavage (BAL) cell population of C57BL/6J mice at various times after a single it dose of BLM and to determine whether BAL T-lymphocytes were activated after BLM treatment. The BAL lymphocyte population was analyzed by differential cell analysis and flow cytometry utilizing monoclonal antibodies specific for lymphocyte subpopulations. The majority of lymphocytes in the BAL of control and BLM-treated mice were T-lymphocytes, with less than 10% being B-cells. During the first 7 days after BLM the number of Lyt-2+ T-cells exceeded L3T4+ T-cells while in control mice the reverse was observed. The percentage of BAL lymphocytes expressing the IL-2 receptor did not change significantly at 3 and 7 days after BLM treatment, but was significantly decreased at Day 14. In contrast, the total number of lymphocytes expressing the IL-2 receptor was increased at all time points investigated. These results demonstrated that the majority of lymphocytes in the BAL were T-cells and that while the percentage of activated lymphocytes did not increase following BLM treatment, the absolute number did and this increased number of activated lymphocytes may be important in the disease process.

Effect of cytotoxic monoclonal antibody depletion of T-lymphocyte subpopulations on bleomycin-induced lung damage in C57BL/6J mice.

Treatment of cancer patients with the antitumor antibiotic bleomycin (BLM) is associated with lung damage which can progress to pulmonary fibrosis. Shortly after intratracheal (it) administration of BLM to experimental animals there is an influx of inflammatory cells into the lung. These inflammatory cells, consisting primarily of polymorphonuclear cells, monocytes and lymphocytes, are believed to modulate the pathogenesis of pulmonary fibrosis. The objective of the present study was to determine the role of specific T-lymphocyte subpopulations in this disease process following a single it administration of BLM to C57BL/6J mice. Specific in vivo T-lymphocyte subpopulation depletion was accomplished by multiple intraperitoneal administrations of cytotoxic monoclonal antibodies to mice prior to and following BLM administration. Acute lung damage was assessed by measuring levels of angiotensin-converting enzyme and total protein in the bronchoalveolar lavage fluid 7 days after BLM treatment while chronic fibrosis was assessed by total lung hydroxyproline 28 days after BLM. Although we were able to deplete lymph nodes and BAL of specific T-lymphocyte subpopulations we were unable to detect a difference in the extent or severity of either the acute or chronic stage of BLM-induced lung damage. These results suggest that BLM lung disease progresses unabated in C57BL/6J mice despite virtually complete depletion of either L3T4+ or Lyt-2+ T-lymphocytes. Although a greater than 80% decrease in Thy-1.2+ T-lymphocytes was accomplished, there was a residual population of Thy-1.2+ lymphocytes resistant to the cytotoxic antibody. It is possible, therefore, that this population of cells does play a role in the development of BLM lung disease.

Chronic therapy with recombinant tumor necrosis factor-alpha in autoimmune NZB/NZW F1 mice.

We studied the effects of recombinant murine tumor necrosis factor-alpha (TNF-alpha) on autoimmune disease in lupus-prone NZB/NZW F1 (B/W) mice. Treatment with TNF-alpha, begun after the onset of clinical disease, improved survival relative to control mice: at age 10 months, 92% of mice treated with TNF-alpha were alive compared with 42% of control mice (P less than 0.05). Administration of TNF-alpha delayed the progression of renal disease, but sustained therapy did not prevent the eventual development of severe nephritis. Despite the improvement in survival, treatment with TNF-alpha did not inhibit anti-dsDNA antibody production. However, it accelerated T lymphocytopenia and abolished natural killer cell activity. These observations suggest that TNF-alpha may retard murine lupus in B/W mice through effects on cellular rather than humoral mechanisms. Our findings also indicate that the beneficial effects of TNF-alpha cannot be sustained indefinitely by chronic therapy.

Genomic cloning and heterologous expression of human differentiation-stimulating factor.

Differentiation-stimulating factor (D-factor) purified from mouse Ehrlich ascites cells was sequenced partially and found to be almost identical to leukemia inhibitory factor (LIF) from mouse Krebs II ascites cells. In comparison to LIF, D-factor had an additional amino-terminal serine residue. Using synthetic oligonucleotide probes designed from the murine D-factor sequence, we cloned the human gene encoding D-factor. A partial D-factor cDNA was cloned from COS-1 cells transfected with the human D-factor gene under the control of a heterologous promoter. We used this cDNA to construct a vector for direct expression of the protein in Escherichia coli. A mammalian cell expression vector was constructed using the signal sequence of interferon-alpha A linked to the D-factor cDNA. Both forms of recombinant human D-factor were active on the murine myeloid leukemia cell line M1 in a dose- and time-dependent manner for the inhibition of [3H]thymidine incorporation, and also induced phagocytosis, Fc receptor expression, and prostaglandin E2 synthesis by M1 cells.

Tumor necrosis factor-alpha as a proliferative signal for an IL-2-dependent T cell line: strict species specificity of action.

The ability of TNF-alpha to stimulate T cell proliferation was examined. We demonstrate that murine rTNF-alpha induces the proliferation of CT6, a murine T cell line previously thought to be responsive only to IL-2. This activity appears to be the result of the direct action of murine rTNF-alpha on the CT6 cells because neither 1) murine IL-2 or murine IL-4, lymphokines also capable of inducing CT6 proliferation, were detected in culture supernatants from murine rTNF-alpha-treated CT6 cells nor 2) did antibodies specific for IL-2 or IL-4 inhibit murine rTNF-alpha-induced CT6 proliferation. Unlike many of the activities displayed by TNF-alpha, its ability to induce CT6 cell proliferation shows strict species specificity as indicated by the failure of human rTNF-alpha to stimulate these cells. Flow cytometric analysis and binding of radiolabeled TNF-alpha have indicated that receptors for TNF-alpha on these cells are specific for murine TNF-alpha. The ability of murine rTNF-alpha to induce the proliferation of certain T cell lines further indicates that this molecule plays an important role in regulation of T cell-mediated immune responses.

Suppression of murine collagen-induced arthritis with monoclonal anti-Ia antibodies and augmentation with IFN-gamma.

Collagen-induced arthritis (CIA) is an experimental model in which a specific immune response to type II collagen (CII) is associated with the development of inflammatory arthritis. In this study, we evaluated the effects of early and delayed treatments with anti-Ia mAb and IFN-gamma on murine CIA. Administration of anti-Ia mAb at the time of immunization with CII decreased the incidence and delayed the onset of arthritis, whereas anti-Ia treatments begun 2 wk after immunization had no effect upon either arthritis incidence or onset. Neither treatment protocol resulted in a significant decrease in antibody titer or proliferative response to CII. Because IFN-gamma increases Ia expression in a variety of cells, we determined its effect on arthritis incidence and onset. When IFN-gamma treatments were begun at the time of immunization the incidence of arthritis was increased and arthritis onset was more rapid. Treatment with IFN-gamma did not result in an increase in anti-CII antibody levels. These results support the importance of Ia expression in the induction of murine collagen-induced arthritis, and suggest that suppression with anti-Ia antibodies and augmentation with IFN-gamma are not the result of changes in the humoral response to CII, but may be due to local effects within the target organ.

In vivo modulation of murine collagen induced arthritis.

The effects of in vivo modulation of murine collagen induced arthritis with monoclonal anti-CD4 antibodies, monoclonal anti-Ia antibodies, and gamma interferon are reviewed. We detail the mechanism of action of monoclonal anti-CD4 antibody on humoral and cell mediated immune responses and discuss the implications for designing therapeutic strategies. To further explore the induction of collagen induced arthritis, a syngeneic cell transfer system using collagen primed T lymphocytes is described. This cell transfer system provides an opportunity to study the role of CD4 positive T lymphocytes in arthritis induction during a short, defined time period.

The involvement of human tumor necrosis factors-alpha and -beta in the mixed lymphocyte reaction.

The influence of recombinant human TNF-alpha and -beta (rHuTNF-alpha and -beta) in a human mixed lymphocyte reaction (MLR) was investigated. The addition of 1000 U/ml of either cytokine at the initiation of culture caused up to a sixfold increase in [3H]thymidine incorporation by responder cells. Furthermore, it was found that endogenous HuTNF-alpha is produced after allogeneic cell interaction and can be detected in the MLR supernatant within 1 h of culture initiation. The results also show that, in the absence of exogenous HuTNF-alpha, antibodies to rHuTNF-alpha can cause a significant inhibition of the MLR. These observations indicate the importance of TNF-alpha in allogeneic cell interaction and raise considerations for the use of antibodies, or other antagonists, to TNF-alpha as regulators of disease states associated with cell-mediated immune reactions.

Transforming growth factor-beta 1 (TGF-beta 1) and recombinant human tumor necrosis factor-alpha reciprocally regulate the generation of lymphokine-activated killer cell activity. Comparison between natural porcine platelet-derived TGF-beta 1 and TGF-beta 2, and recombinant human TGF-beta 1.

We have investigated the ability of porcine-platelet-derived transforming growth factor-beta 1 (TGF-beta 1) to inhibit the generation of lymphokine-activated killer (LAK) cells by human rIL-2. The results demonstrate that TGF-beta 1, in a dose-related manner, significantly inhibits rIL-2-induced LAK cell activity against Daudi and COLO target cells and, to a lesser degree, against K-562 cells. Maximal inhibition was obtained by the addition of TGF-beta 1 at the time of culture initiation and, to a lesser degree, on day 1. Only minimal inhibition was obtained when TGF-beta 1 addition was delayed until day 2 of culture or when added directly into the LAK cell assay. Additional studies demonstrated that porcine platelet-derived TGF-beta 2 and human rTGF-beta 1 inhibited LAK cell generation similar to that obtained with TGF-beta 1. The inhibition of LAK cell activity by TGF-beta 1 was reversed by the addition of human rTNF-alpha at the initiation of culture. In addition, rTNF-alpha synergized with suboptimal levels of rIL-2 in the generation of LAK activity. After stimulation with rIL-2, LAK cells produced significant levels of IFN-gamma, TNF-alpha, and TNF-beta. TGF-beta 1 inhibited the production of these cytokines in a dose-related manner. The results extend the previous known activities for human rTNF-alpha and TGF-beta 1 and further demonstrate the reciprocal relationship between these two molecules in the regulation of certain immune functions.