Leukotrienes (LTs) are 5-lipoxygenase metabolites derived from arachidonic acid and play important roles in many inflammation and allergic disorders. They are classified to two groups, LTB₄ and peptide-LTs. Both groups are thought to show their biological activities through their cellsurface receptors, but no LT receptor had been purified to homogeneity or cDNA-cloned. Recently, we cloned a cDNA for a LTB₄ receptor from HL 60 cells. The open reading frame of the cDNA encodes a protein of 352 amino acids and is predicted to contain seven membrane-spanning domains. In CHO cells stably expressing the LTB₄ receptor, LTB₄ elicited many signal transductions such as increase in intracellular calcium, InsP₃ accumulation, and inhibition of adenylyl cyclase. The CHO cells revealed a marked chemotactic response toward nM order of LTB₄. Next, we analyzed the signal transduction mechanisms through LTD₄ receptors using human monocytic leukemia THP-1 cells. When these cells were stimulated with LTD₄, intracellular calcium concentration was increased and MAP kinase was activated severalfold. This activation was mediated a protein kinase Cα-Raf-1 dependent pathway. A chemotactic response of THP-1 cells toward LTD₄ was observed. LT receptors transduce these signals through both PTX-sensitive and-insensitive G proteins.

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The anaerobic oral bacterium Porphyromonas gingivalis is thought to be a major etiologic agent of progressive periodontal disease. Recently, two major cysteine proteinases responsible for trypsine-like activity from the organism were purified from the culture supernatant of various P. gingivalis strains, termed “Arg-gingipain (RGP) and Lys-gingipain (KGP) ”. The secretory RGP and KGP were shown to be associated with periodontal tissue breakdown and disruption of the normal host defense mechanisms. To further clarify their virulence, RGP-and KGP deficient mutants were constructed by gene disruption using suicide plasmid systems. The RGP-null mutant showed a remarkable loss of both the suppressive activity of PMN functions and the hemagglutinating activity observed with the wild-type strain, suggesting that RGP is responsible for these activities of the organism. The RGPnull mutant also showed the defect in fimbriation and processing of prefimbrilin and the 75-kDa surface protein precursor, suggesting that RGP is involved in processing and translocation of certain cell surface proteins of the organism. On the other hand, the KGP-deficient mutant, like the wild type strain, strongly retained the abilities to suppress the PMN bactericidal activity and to hemagglutinate, but it could not form the black pigmentation of colonies when grown in blood agar plate. P. gingivalis is known to stockpile heme from hemoglobin in blood agar, resulting in the black pigmentation. Therefore, KGP appeared to be involved in the adsorption and degradation of hemoglobin and the heme accumulation in the organism.

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To analyze human diseases at molecular level and to promote gene therapy strategy, we developed HVJ-liposome gene delivery system. By the transfer of TGF-β gene to rat glomerulus using the system, glomerulosclerotic changes characterized by the accumulation of extracellular matrix were induced in rat kidney. To suppress TGF-β by antisense oligonucleotides in experimental glomerulonephritic rats, glomerulosclerotic changes were inhibited. Therefore, TGF-β appears to be responsible for the pathogenesis of glomerulosclerotic changes. Toward the gene therapy of glomerulosclerosis, decorin gene was introduced into nephritic rat muscle. Decorin was expressed in muscle and accumulated in glomerulus. By decorin gene transfer, TGF-β expression was suppressed in nephritic rat kidney and a number of TGF-β positive glomerulus was reduced to 40% compared with the rat treated with CAT gene. The accumulation of extracellular matrix proteins was decreased. These results indicate that decorin gene transfer to muscle will be promising for gene therauv of glomerulosclerosis.

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To elucidate the mechanism of switching from neutrophils accumulation in the early phase to chronic inflammation characterized by the predominant accumulation of mononuclear cells in tuberculous inflammation, expression of the chemokines was examined. Neutrophils stimulated with lipopolysaccharide (LPS), M. tuberculosis (MT), or purified protein derivative (PPD) expressed both IL-8 and MIP-1α, however, the kinetics of expression did not differ. The viability of neutrophils was much reduced in response to mycobacterial derivatives with TNFα. Apoptosis seemed to cause the low viability of neutrophils, which might rapidly remove neutrophils from tuberculous inflammatory sites.
Monocytes stimulated with M. tuberculosis or PPD expressed IL 8, MIP-1α and MCP 1. The expression of MCP-1 appeared in the later phase than those of IL-8 and MIP-1α. However monocytes stimulated with LPS could not induce production of MCP-1 strongly. These kinetics of chemokines induced in inflammatory cells by tuberculous derivatives may contribute to formation of tuberculous inflammation. In addition killing mycobacterium in macrophages depended on activation of Thl cellsmacrophages response, which was associated with at least partially IL 12 production.

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Cytokines are diffusible and soluble factors with pleiotropic actions. However, heparan sulfate proteoglycan on either the cell surface or matrices promotes the accumulation of heparin-binding cytokines, such as chemokines, at high concentration by binding them on the appropriate location where they encounter with their target cells. We here propose that this paradigm is particularly relevant to the stimulation of integrin-mediated adhesion of circulating leukocytes and leukemic cells to endothelium induced by chemokines through their binding to heparan sulfate proteoglycan, which is relevant not only to the regulation of inflammation and leukocyte trafficking but also to the process of leukemic cell infiltration and tumor metastasis.

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Nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) play an important role in the induction of pro-inflammatory factors such as cytokines and cell adhesion molecules, which could be involved in the pathogenesis of inflammatory diseases. In vitro study using cultured cells have suggested that these transcription factors are possible molecular targets for the anti-inflammatory action of steroids. However, the precise molecular mechanisms of the therapeutic effects of steroids on inflammatory diseases in vivo are not yet fully understood. Here, we show the suppressive effects of steroids on NF-κB and AP-1 activation in rat experimental glomerulonephritis induced by nephrotoxic serum (NTS), and discuss the recent approaches for the development of novel anti-inflammatory agents controlling NF-κB and AP-1 activation.

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It is well known that eosinophils increase in peripheral blood, sputum and lung tissues of patients suffering from bronchial asthma. Because eosinophil contains various cytotoxic proteins in the granule, and has abilities to produce bronchoconstrictive mediators such as peptide-leukotrienes, it has been suggested that the leukocyte involves in the occurrence of LAR as well as bronchial hyperresponsiveness, characteristic features of the disease in a serious case. In our basic experiments in vivo using a guinea pig asthmatic model, occurrence of LAR, and considerable increases of eosinophil number in the peripheral blood and the bronchoalveoli coincided with each other 5-7 hr after the antigen inhalation challenge. Significant elevation of eosinophil peroxidase activity in the lung homogenate was also observed 5 hr after the challenge. It can be considered that the increased eosinophils in the circulation, which might be recruited by cytokines such as interleukin-5 from the rapidly mobilizable bone marrow pool of eosinophils, infiltrated into the lung tissue. We also describe postulated mechanisms about eosinophil infiltration into tissues, which was recently reported, or obtained in our laboratory.

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We investigated whether endotoxin (LPS) -induced desensitization develops to mouse dermal microvascular permeability after systemic administration of a single low-dose LPS to male mice. Plasma extravasation was determined by Pontamine sky blue leakage at the site of the skin where LPS and mediators were injected s.c. The dye leakage induced by LPS, 5-hydroxytryptamine (5-HT), platelet-activating factor, substance P or histamine was significantly decreased about 60-80% in LPS-primed mice, indicating the development of homologous and heterogeneous tolerance. Tumor necrosis afctor (TNF) -α and interleukin (IL) -1α but not IL-6 induced the tolerance to LPS, and anti-TNF-α antibody and anti-IL-lα antibody reduced hyporesponsiveness to 5-HT. Homologous tolerance was not seen in adrenalectomized mice. When mice were pretreated with both LPS and N^G-nitro-L-arginine methyl ester, a nitric oxide (NO) synthase inhibitor, the hyporesponsiveness to LPS and 5-HT disappeared. These results suggest that tolerance develops to the increase in microvascular permeability elicited by LPS or some inflammatory mediators by LPS pretreatment and that endogenous cytokines, glucocorticoids and NO are necessary for induction of the tolerance. The tolerance induced by LPS may provide a potential basis for the treatment of septic shock.

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