Expression and responsiveness of human interleukin-18 receptor (IL-18R) on hematopoietic cell lines.

Interleukin-18 (IL-18) is a new inflammatory cytokine sharing biological functions with IL-12. The human IL-18 receptor (IL-18R) was recently identified and was found to be expressed on normal peripheral blood lymphocytes. To further characterize IL-18R, we analyzed IL-18R expression using a series of human hematopoietic cell lines selected from various cell lineages. We found the IL-18R expression on cells of T and B lineages as expected from analysis on normal cells. The IL-18R expression, however, was found not to be restricted to any specific maturation stages of T and B cells. In addition, we detected IL-18R expression in myeloid, monocytoid, erythroid and megakaryocytic cell lines, indicating that normal counterparts of these cell lineages could express IL-18R and participate in in vivo reactions caused by IL-18. Biochemical studies showed that IL-18R proteins exist as heterogeneous molecules ranging from 60 to 110 kDa. Deglycosylation experiments indicated that the heterogeneity could not be explained only by a difference in glycosylation. We also found that tumor necrosis factor-alpha (TNF-alpha) modulated the IL-18R expression, which implies an important in vivo effect of TNF-alpha on IL-18-induced reaction. Analyzing the responsiveness of IL-18R, we found that only KG-1 responded to IL-18 stimulation. This suggests that certain inhibitory mechanisms of IL-18 responsive genes are involved in the all IL-18R-positive cell lines except KG-1.

Identification of a vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human hematopoietic cell line NALM-16.

An antagonistic activity against vascular endothelial growth factor (VEGF) was identified in the culture supernatants of certain human hematopoietic cell lines and the antagonistic protein was purified from NALM-16 (B cell) culture supernatant. Amino acid sequencing of the N-terminus and Western blot analysis confirmed that the antagonist was identical to a soluble truncated form of Flt-1 (sFlt-1). Seventeen of 52 leukemia and lymphoma cell lines investigated expressed sFlt-1 mRNA, and 16 of the sFlt-1 expressing cells also expressed VEGF and membrane-bound Flt-1 (mFlt-1). This report is the first showing that sFlt-1 can be produced by malignant hematopoietic cells, suggesting that the production of VEGF antagonist by hematopoietic cells may play some role in the regulation of VEGF activity in normal and malignant hematopoietic cell proliferation.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters. An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines.

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10(5)-10(6) in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Effects of exogenous insoluble glucan primer on insoluble glucan synthesis by Streptococcus mutans.

The effects of exogenous glucans on WIG synthesis by S. mutans were investigated by turbidimetric measurement. The rapid synthesis of WIG by an enzyme preparation from PS-14 culture fluid proceeded after a lag period of several min. The addition of exogenous WIG, which was previously prepared using the PS-14 enzyme, caused a marked reduction of the lag period with little change in the reaction rate. The length of lag period was affected by the concentrations of WIG primer, enzyme and salts, and the reaction pH. The WIGs from other strains and the periodate-treated WIGs also exhibited effects similar to the PS-14 glucan. In contrast, dextran, starch, dextrin, laminarin, cellulose, chitin, and inulin were without any effect. These results suggest that such lag-reducing effect is specific to glucans containing a significant amount of alpha-(1 leads to 3) linkages.

IFN-gamma-dependent and -independent mechanisms in adverse effects caused by concomitant administration of IL-18 and IL-12.

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1beta. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-gamma level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-alpha was not induced. The involvement of IFN-gamma in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-gamma treatment of wild-type mice alleviated it. When IFN-gamma-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-gamma-dependent mechanisms but also IFN-gamma-independent processes.

Identification of IFN-gamma-producing cells in IL-12/IL-18-treated mice.

Both IL-12 and IL-18 have been characterized as effective IFN-gamma-inducing cytokines. Concomitant treatment with IL-12 and IL-18 has been shown to synergistically induce IFN-gamma and may be an effective therapy for treating cancer, allergy, and infectious diseases. To understand the mechanisms underlying the strong induction of IFN-gamma by IL-12/IL-18 in mice, we focused our studies on the IFN-gamma-producing cells in various lymphoid organs and tissues and utilized the intracellular cytokine staining method to detect such cells in situ. After combined treatment with IL-12 and IL-18, IFN-gamma-positive cells in C57BL/6 mice were detected in the liver (12.18%), spleen (0.68%), bone marrow (1.80%), and peritoneum (2.12%), but not in the thymus or lymph nodes (<0.05 and <0.08%, respectively). A two-color staining method revealed that the majority of IFN-gamma-producing cells in the liver were NK1.1(+) cells, while those in the spleen were mostly CD3(+) cells, and to a lesser degree NK1.1(+) cells. Both CD4(+) and CD8(+) cells in the liver and in the spleen produced IFN-gamma. The CD19(+) B cell population was not definitely shown to produce IFN-gamma in our induction experiments. NKT cells, which are a subpopulation of NK1. 1(+) CD3(+) cells, were diminished in the liver and did not seem to contribute to IFN-gamma production arising from IL-12/IL-18 treatment. Further in vitro experiments confirmed the responsiveness of hepatic mononuclear cells to IL-12/IL-18 stimulation. This study is the first to show the IFN-gamma-producing mechanisms of IL-12/IL-18 treatment at the phenotypic level.