The histocompatibility restrictions on macrophage T-helper cell interaction determine the histocompatibility restrictions on T-helper cell B-cell interaction.

To study the histocompatibility restriction between macrophages and helper T cells, carrier primed guinea pig T cells were positively selected in vitro with antigenpulsed macrophages for 7 days and the selected T cells were then mixed with hapten-primed B cells and stimulated with antigen in a modified Mishell-Dutton system. Helper T cells could only be selected with syngeneic, but not allogeneic, antigen-pulsed macrophages and would then collaborate only with syngeneic, but not allogeneic, hapten-primed spleen cells. When F1 T cells were selected with antigen-pulsed parental macrophages they would only collaborate with B cells of the same parental strain as the macrophages used in the selection culture. These results are strongly in support of the view that the primed T cell is activated by carrier determinants of the nominal antigen in association with Ia antigens on macrophages and the helper T cell, in turn, activates B cells which bear the same Ia antigens and determinants of the nominal antigen bound to immunoglobulin receptors on their surface. In addition, in experiments with antigens the response to which is controlled by I-linked genes, we demonstrated that primed (responder X nonresponder)F1 T cells would only collaborate with B cells of the responder parent. The defect appeared to be at the level of the B cell in that the addition to the cultures of antigen-presenting cells of the responder type did not restore the ability of F1 T cells to collaborate with non-responder B cells.

1-BP inhibits NF-kappaB activity and Bcl-xL expression in astrocytes in vitro and reduces Bcl-xL expression in the brains of rats in vivo.

1-Bromopropane (1-BP) has been widely used as a substitute for chlorofluorocarbon that destroys the ozone layer. Although the central neurotoxicity of 1-BP has been recently reported, a molecular mechanism is not clear. In particular, the effects on cells in brain have not been fully analyzed. Here, we studied the effects of 1-BP on the activation of transcription factors involved in anti-apoptotic function or cell survival in astrocytes. Astrocytoma cell lines, U251, U373 and VM, or murine primary astrocytes were used for in vitro assay. DNA binding activities of NF-kappaB in these cells induced by interleukin (IL)-1 or LPS were inhibited by 1-BP. Consequently, the treatment of U251 cells with 1-BP resulted in suppression of NF-kappaB reporter activity. Furthermore, 1-BP blocked IkappaBalpha degradation, which is important for NF-kappaB activation. In addition, the level of Bcl-xL mRNA, which is known as an anti-apoptotic gene, were reduced in U251 treated with 1-BP or in the brain from rat exposed to 1-BP (400 ppm, 12 weeks). These results suggest that subchronic inhalation exposure to 1-BP vapor may affect the Bcl-xL expression in astrocytes.

Calcium dependency in the growth of adult T-cell leukemia cells in vitro.

The effects of calcium, calcium antagonists, and calmodulin inhibitors on the growth of adult T-cell leukemia (ATL) cells were studied in vitro. Fresh ATL cells from patients and established ATL cell lines did not grow well in a low-calcium (less than 0.01 mM) medium. However, their growth was enhanced by the addition of calcium to the medium in a dose-dependent manner. The maximum response was induced at 4 mM calcium, which was higher than that of the normal serum calcium level, 2.5 mM. Other leukemia cells, except ATL, grew well in the low-calcium medium, and their growth was not enhanced by the addition of calcium. Calcium antagonists and calmodulin inhibitors inhibited the growth of ATL cells at the concentration of 10(-5)-10(-7) M, while they did not inhibit the growth of other leukemia cells. Furthermore, the expression of interleukin 2 receptors (Tac antigens) on ATL cells was also enhanced by calcium and was inhibited by calcium antagonists and calmodulin inhibitors. In accordance with these results, the increase of the extracellular calcium concentration resulted in the increase of the intracellular calcium concentration in ATL cells, but not in other leukemia cells. These results suggest that calcium and calmodulin play a critical role in regulating the growth of ATL cells.

Autocrine stimulation of interleukin 1 alpha in the growth of adult human T-cell leukemia cells.

In a previous study, we reported that adult T-cell leukemia (ATL) cells produce interleukin 1 (IL1)-like factors that stimulate murine thymocyte proliferation, the production of interleukin 2 (IL2), and the expression of IL2 receptors (IL2R) on normal human T-cells in the presence of concanavalin A. In this communication, we studied the effect of IL1 on the growth of ATL cells in vitro. When ATL cells freshly obtained from patients were cultured with recombinant (r) human IL1 alpha, IL1 beta, or IL1-like factors produced by ATL cell lines, the growth of ATL cells was stimulated in a concentration-dependent manner. Maximum stimulation was observed at a concentration of 50-100 units/ml of IL1. The expression of IL2R on ATL cells was also enhanced by IL1, but the production of IL2 was not induced. These effects of rIL1 alpha or beta were specifically inhibited by anti-IL1 alpha or anti-IL1 beta antibody. Furthermore, the spontaneous growth of ATL cells was also inhibited by anti-IL1 alpha antibody, but not by anti-IL1 beta antibody. ATL cells exhibited enhanced expression of IL1 receptors on their surface as detected by the binding of 125I-labeled rIL1 alpha. These results suggest that IL1 alpha produced by ATL cells stimulates the growth of ATL cells by an autocrine mechanism.

Calcium dependency of the production of interleukin 1 and the expression of interleukin 1 receptors of human adult T-cell leukemia cells in vitro.

The effect of calcium on the production of interleukin 1 (IL 1) and the expression of IL 1 receptors (R) of adult T-cell leukemia (ATL) cells was studied in vitro. ATL cells freshly obtained from patients and ATL cell lines produced limited amounts of IL 1 by culturing in a low-calcium concentration of medium (less than 0.01 mM). However, the production of IL 1 was enhanced by the addition of calcium chloride to the medium in a concentration-dependent manner and reached the maximum at the higher calcium concentration (3-4 mM) than at the standard calcium concentration of medium (1.26 mM). The production of IL 1 from ATL cells was further enhanced by calcium ionophore. Furthermore, the expression of IL 1R on ATL cells was augmented in proportion to the extracellular calcium concentration and calcium ionophore. In accordance with the change of the extracellular calcium concentration, the intracellular calcium concentration of ATL cells detected by Fura 2 was changed. However, this calcium dependency was not observed in the human T-cell leukemia virus I-negative acute T-cell leukemia cells. These results suggest that calcium plays a critical role in the regulation of the production of IL 1 and the expression of IL 1R on ATL cells.

Immunosuppressive factors from adult T-cell leukemia cells.

The mechanism of immunodeficiency in adult T-cell leukemia (ATL) patients was studied in vitro. Peripheral blood lymphocytes from ATL patients and ATL cell lines such as Hut 102, MT 1, and MT 2 were not activated to proliferate by the stimulation with concanavalin A and suppressed normal lymphocyte proliferative responses induced with concanavalin A when cultured together. The sera from ATL patients and the culture supernatants from ATL cells and ATL cell lines also suppressed normal lymphocyte proliferative responses induced with concanavalin A. By Sephacryl S-200 column chromatography, the suppressive factors were fractionated as a single peak with the molecular weights of 50,000 to 70,000. The suppressive factors were unstable to acid treatment but stable to the treatment with base, heat, freezing-thawing, and trypsin. The factors suppressed the production of interleukin 2 by T-cells and the responsiveness of T-cells to interleukin 2, but not the expression of interleukin 2 receptors on T-cells and the production of interleukin 1 by monocytes. These results suggest that the immunosuppressive factors produced by ATL cells have some roles in the induction of immunodeficient states in ATL patients.

Interleukin 1 alpha acts as an autocrine growth stimulator for human gastric carcinoma cells.

The expression and effect of interleukin 1 alpha (IL-1 alpha) were examined in human gastric carcinoma cell lines to determine if IL-1 alpha acts as a growth stimulator for these cells. Six of 8 gastric carcinoma cell lines expressed IL-1 alpha mRNA at various levels. Among them, TMK-1 and MKN-7 cells secreted IL-1 alpha into the culture fluid, in an especially large amount by MKN-7 cells. Scatchard plot analysis of IL-1 alpha binding revealed that TMK-1 cells had only one type of high-affinity receptors, whereas MKN-7 cells had high- and low-affinity receptors. Cell growth and DNA synthesis of TMK-1 and MKN-7 cells were stimulated by IL-1 alpha, and those of MKN-7 were inhibited by addition of anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor, or transforming growth factor alpha. On the other hand, IL-1 alpha increased the mRNA expression for transforming growth factor alpha and epidermal growth factor receptor. These findings indicate that IL-1 alpha is an autocrine growth stimulator for gastric carcinoma cells and the interaction with epidermal growth factor/transforming growth factor alpha/receptor system should be involved in the growth modulation by IL-1 alpha.

Production of bone-resorbing activity corresponding to interleukin-1 alpha by adult T-cell leukemia cells in humans.

The physicochemical properties and relationship of bone-resorbing activity and interleukin 1 (IL-1) produced by adult T-cell leukemia (ATL) cells and cell line were studied in vitro. The culture supernatant of ATL cell line, MT2, and peripheral blood lymphocytes freshly obtained from ATL patients had both IL-1 activity detected by the stimulation of murine thymocyte-proliferative responses and bone-resorbing activity detected by the stimulation of 45Ca release from prelabeled murine fetal bones. By Sephacryl S-200 column chromatography, both activities were eluted as a single peak at approximately Mr 15,000. By the chromatofocusing technique, the isoelectric point values of both activities were estimated as pH 4.8 and 5.2. Furthermore, both activities were absorbed with rabbit anti-IL-1 alpha antiserum, but not with anti-IL-1 beta antiserum. These results suggest that ATL cells and cell line produce bone-resorbing activity which corresponds to IL-1 alpha and that this IL-1 alpha is one of the most important causes of hypercalcemia in ATL patients.

CD44 stimulation induces integrin-mediated adhesion of colon cancer cell lines to endothelial cells by up-regulation of integrins and c-Met and activation of integrins.

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins lymphocyte function-associated antigen (LFA) 1 and very late antigen 4 play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers including colorectal cancer do not express suitable adhesion receptors, LFA-1 and very late antigen 4. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using colorectal carcinoma cell lines. Our results showed the following novel features of CD44 on the cells: (a) colon cancer cells express high levels of CD44; (b) stimulation of cancer cells by CD44 cross-linking or fragmented hyaluronan markedly induces the expression of LFA-1s, some of which reveal an activation epitope on the cells; (c) CD44 cross-linking induces F-actin polymerization in the cell cortex; (d) fragmented hyaluronan induces up-regulation of the activation epitope of LFA-1, which is mediated through protein kinase C; (e) stimulation of CD44 augments the LFA-1-mediated adhesion of cancer cells to endothelial cells and intercellular adhesion molecule 1-transfected cells and facilitates transendothelial migration; (f) stimulation of CD44 also induces expression of the hepatocyte growth factor (HGF) receptor c-Met on cancer cells; and (g) HGF further amplifies the LFA-1-mediated adhesion of cells prestimulated by CD44-derived signaling. Our results indicated that stimulation by CD44 induces "outside-in signaling," which consists of a direct pathway via CD44 and an alternate pathway through the induction of c-Met expression via HGF. Such stimuli augment the expression and trigger the function of integrins via "inside-out signaling" in colon cancer cells, which leads to amplification of integrin-mediated adhesion to the vessel wall and subsequent transendothelial migration.

Hyperbaric oxygenation reduces the cytostatic activity and transcription of nitric oxide synthetase gene of mouse peritoneal macrophages.

The effects of hyperbaric oxygenation (HBO) and hyperbaric air (HBA) on the cytostatic activity, peroxynitrite synthesis and transcription of the inducible nitric oxide synthetase (iNOS) gene of murine peritoneal macrophages were studied in vitro. Exposure of mice to HBO or HBA significantly reduced the cytostatic activity, peroxynitrite synthesis and transcription of iNOS mRNA of their macrophages stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). These results indicate that HBO and HBA treatments of mice reduce the cytostatic activity of peritoneal macrophages by reducing iNOS mRNA synthesis.

Suppressive effect of interleukin-4 on the differentiation of M1 and HL60 myeloid leukemic cells.

The effect of interleukin-4 (IL-4) on the proliferation and differentiation of myeloid leukemic cell lines was studied in vitro. A culture of murine myeloid cell line, M1, with lipopolysaccharide (LPS) from Escherichia coli, induced differentiation into macrophages that expressed Fc receptors and phagocytic activity. IL-4 did not induce the differentiation of M1 cells but inhibited the differentiation of M1 cells induced with LPS. On the other hand, LPS arrested the proliferation of M1 cells. IL-4 had no effect on the proliferation of M1 cells but restored the LPS-induced arrest of the proliferation of M1 cells. IL-1, IL-6 and tumor necrosis factor alpha (TNF-alpha) also induced the differentiation of M1 cells into macrophages and arrested proliferation. IL-4 suppressed the IL-1-, IL-6-, and TNF-induced differentiation of M1 cells and restored the arrested proliferation with IL-1, IL-6, and TNF. Similar results were obtained with human myeloid cell line HL60. These results suggest that IL-4 has a suppressive effect on the differentiation of myeloid cells into macrophages.

Autocrine stimulation of interleukin-1 in the growth of human thyroid carcinoma cell line NIM 1.

We reported first in this study that human thyroid cell line NIM 1 established from a patient with papillary adenocarcinoma of the thyroid associated with hypercalcemia and peripheral neutrocytosis produced interleukin (IL)-1 alpha and IL-1 beta in the culture supernatant and cell lysate as detected by murine thymocyte proliferative response and enzyme-linked immunosorbent assay. Production of IL-1 alpha and IL-1 beta was further confirmed by the demonstration of IL-1 alpha and IL-1 beta messenger ribonucleic acid expression with Northern blot hybridization analysis. The in vitro growth of NIM 1 cells was inhibited by the addition of anti-IL-1 alpha and IL-1 beta antibody. The growth of NIM 1 cells was further enhanced by the addition of recombinant human IL-1 alpha and IL-1 beta, whereas this enhancement was also inhibited by the addition of anti-IL-1 antibody. IL-1 receptors were expressed on NIM 1 cells. These results suggest that IL-1 plays a regulatory role in the growth of NIM 1 cells by an autocrine mechanism.

Macrophage-colony stimulating factor inhibits the growth of human ovarian cancer cells in vitro.

The effect of macrophage-colony stimulating factor (M-CSF), which regulates the growth and differentiation of haematopoietic progenitor cells on the growth of ovarian cancer cells was investigated in three ovarian cancer cell lines in vitro. The spontaneous growth of these cells was significantly inhibited by the addition of M-CSF in a concentration-dependent manner over 96 h of culturing. The maximum response was obtained with 10 ng/ml (3857 U/ml) of M-CSF by counting the viable cell number using the trypan blue exclusion assay. [(3)H]-thymidine incorporation by these cells was also suppressed following a 96-h incubation with M-CSF. The inhibitory effect of M-CSF was reversed by the addition of anti-M-CSF monoclonal antibody. Flow cytometric analysis revealed that the treated ovarian cancer cells arrested at the G0/G1 phase of the cell cycle. These cells expressed M-CSF receptors on their surface as detected by Scatchard plot analysis using (125)I-labelled M-CSF. These results indicate that M-CSF has an antitumour activity for ovarian cancer cells and suggest that it can be applied for the treatment of this disease.

Different inducing activities for cytotoxic T cells by heterogeneous thymocyte-activating factors from SV40-transformed human embryo fibroblasts.

Thymocyte-activating factors are produced by human embryo fibroblasts and their production is enhanced by SV40-induced transformation (Okai, Gotoh and Yamashita, Immununol. Lett. (1985), 9, 153-159). The activities for thymocyte DNA synthesis in the culture medium from SV40-transformed cells are separated into the two fractions by a DEAE Sephadex A-25 column chromatography eluting at 150 and 500 mM NaCl. Each fraction contains the heterogeneous molecular weight activities as judged by a Sephadex G-100 column chromatography. In the fraction eluted at 150 mM NaCl, the inducing activities for cytotoxic T cells by the factors are dependent upon their molecular weights; the larger factors exhibit much higher cytotoxic activity than that of the smaller factors. In contrast, the heterogeneous molecular weight factors eluted at 500 mM NaCl do not show the remarkable difference of their biological activities. In addition, the inducing activities by thymocyte-activating factors are also dependent upon their charge properties. These different inducing activities for cytotoxic T cells by thymocyte-activating factors is discussed from the aspect of the immunological regulation.

Thymocyte activating factors from SV40-transformed human embryo fibroblasts.

A novel factor was found in the medium conditioned by SV40-transformed human embryo fibroblasts, which stimulate concanavalin A-induced thymocyte DNA synthetic response. This activity was estimated to be 10-15 kD and divided into two activities by ion exchange chromatography. One of them is a protein molecule and the other is a glycoprotein. In addition, these activities are not derived from the growth factors reported previously such as interleukin 2 (Morgan, R., Ruscetti, F. and Gallo, R. C. (1976) Science 193, 1007-1008) and transforming growth factor (De Larco, J. E. and Todaro, G. J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4001-4005).

B cell mitogenic activity of house dust mite, Dermatophagoides farinae, antigens.

The effect of mite antigens on murine and human lymphocytes was studied in vitro. Antigens prepared from Dermatophagoides farinae feces and bodies stimulated normal murine spleen cells to proliferate in a dose-dependent manner. The responder cells are B cells, because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement, but not with anti-Thy 1 antibody and complement. Furthermore, nylon column-purified T cells did not respond. The stimulation of B cells with mite antigens was not due to the contamination of lipopolysaccharide, a representative B cell mitogen, because C3H/HeJ spleen cells which are low responders to lipopolysaccharide could respond to mite antigens. These antigens induced not only proliferative response of murine B cells, but also immunoglobulin production. By gel-filtration column chromatography, the active fractions were eluted around the molecular weight of 150-155 kDa. Furthermore, mite antigens also stimulated human B cells to proliferate and to produce immunoglobulin. All these results suggest that mite antigens are a potent B cell mitogen and this activity might concern the induction of allergic reaction.

Interleukin-1alpha regulates G1 cell cycle progression and arrest in thyroid carcinoma cell lines NIM1 and NPA.

This study provides the first report that the same cytokine (interleukin-1 (IL-1)) can induce opposite effects on cyclin-dependent kinases (Cdks) and Cdk inhibitors (Cdkis) in the G1 phase even in the same type of cancer cells (papillary thyroid carcinoma cells). Cell cycle analysis revealed an increase in NIM1 cells and a decrease in NPA cells in the S and G2+M phases after treatment with IL-1alpha. The addition of IL-1alpha to NIM1 cells reduced the expression of p16 and p21 protein and induced the expression of Cdk2 and Cdk4 protein, which leads to the phosphorylation of retinoblastoma protein. The addition of IL-1alpha to NPA cells induced the expression of p27 protein and reduced the expression of Cdk2 protein, which leads to induction of p107 protein expression. It is of interest that p21 protein expression was not observed in NPA cells. These results suggest that several Cdks and Cdkis play a regulatory role in the G1 cell cycle progression and arrest induced by IL-1alpha in thyroid carcinoma cell lines.

Growth stimulatory effect of interleukin (IL)-1 alpha produced by oral squamous carcinoma cell lines.

The expression of IL-1 alpha and its effect on the cell growth were examined in six human oral squamous carcinoma cell lines. All the cell lines expressed IL-1 alpha mRNA and protein at various levels. Particularly, HSC-2 and HSC-3 cells showed high level of the mRNA expression and secreted large amounts of IL-1 alpha into the culture fluid. Scatchard plot analysis of IL-1 alpha binding revealed that HSC-2 cells had high-and low-affinity receptors, whereas IL-1 alpha receptors on HSC-3 cells were of undetectable level. The cell growth of HSC-2 and HSC-3 cells was stimulated by IL-1 alpha and inhibited by anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha). On the other hand, IL-1 alpha promoted the mRNA expression of TGF-alpha and EGF receptor. These findings indicate that IL-1 alpha acts as an autocrine growth stimulator for oral squamous carcinoma cells in vitro and its interaction with EGF/TGF-alpha/receptor system may play a role in this enhanced growth by IL-1 alpha.

Concomitant transcriptional activation of nitric oxide synthase and heme oxygenase genes during nitric oxide-mediated macrophage cytostasis.

During in vitro activation of mouse peritoneal macrophages with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), their synthesis of peroxynitrite and their cytostatic activity against mouse lymphocytic leukemia (L1210) cells were examined. The activation of the genes for nitric oxide synthase (iNOS) and heme oxygenase (HO-1) was also determined during the activation of the macrophages. Results showed that activation of peroxynitrite synthesis in macrophages was accompanied by the transcriptional activation of iNOS and HO-1 genes. Both genes seem to be activated simultaneously upon activation of the macrophages. Simultaneous activation of iNOS and HO-1 genes may be important because degradation of heme by HO-1 is one of the most important reaction that produces CO in higher organisms, and nitric oxide (NO) and carbon monoxide (CO) can react with heme-containing guanylate cyclase.

Immunosuppressive factor produced by a B cell line derived from an adult T cell leukemia patient.

The immunosuppressive activity of culture supernatants from human T cell leukemia virus type I (HTLV-I)-infected cell lines was examined in vitro. Culture supernatants of both a HTLV-I-infected B cell line, IWS, established from an adult T cell leukemia (ATL) patient and a T cell line, MT-2, suppressed lymphocyte proliferative responses to stimulation with the mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). The immunosuppressive factor was not cytotoxic for lymphocytes and did not inhibit the spontaneous growth of ATL cells. It inhibited interleukin-2 (IL-2) production by PHA-stimulated T cells and it arrested PHA-stimulated T cells at the G0/G1 phase of the cell cycle and inhibited entry into the S phase. Furthermore, the factor significantly inhibited the expression of CD3, CD4, and IL-2 receptor (IL-2R) alpha-chain (CD25) on PHA-stimulated T cells. These results suggest that the immunosuppressive factors produced by HTLV-I-infected cell lines might function in the regulation of normal lymphocyte proliferative responses, and that they could play some role in the induction of the immunodeficient condition observed in ATL patients.