Toshimasa Nitta

Mitogenicity of bacterial lipopolysaccharide on the T lymphocyte population bearing the γδT cell receptor

In vitro [3H]-thymidine (TdR) uptake in T lymphocyte populations, taken and purified from proteose peptone-induced peritoneal exudate cells (PEC), spleens and thymuses of C3H/HeN mice, were investigated by stimulation with bacterial lipopolysaccharide (LPS). The T cells taken from PEC (PEC-T) were purified by passing them through a nylon fiber column (Nfc) and a G-10 column. They were then treated with anti-lak, anti-Mac-1 and anti-LR-1 antibodies plus complement (C). The PEC-T cells showed an increased [ 3H]-TdR uptake in response to LPS, while the splenic T cells and thymic T cells that had been purified and treated by a similar procedure with minor modifications, poorly responded to LPS and did not respond to LPS, respectively. The response of PEC-T to LPS was abolished when the PEC-T were pretreated with anti-Thy-1 or anti-γδ antibody plus C, but not αβ, T cell receptor (TCR) antibody plus C. The thymic T cells did not show any increase of [ H]-TdR uptake in response to LPS or anti-αβ or γδTCR antibody. However, obvious uptake did occur when the cells were stimulated with LPS and anti-γδTCR antibody, but not with anti-αβTCR antibody. IL-1 did not substitute for LPS in the response. These results suggest that LPS has a mitogenic ability to respond to a T cell population bearing γδTCR in PEC, and to a γδTCR-stimulated cell population in thymic cells. The difference in response to LPS between PEC-γδT and thymic γδT cells was discussed.

Role of Interleukin 2 on Enhancement of Concanavalin A‐Induced Human Peripheral Blood Lymphocyte Proliferation by Murine B Cell Mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol‐extracted water soluble adjuvant (Bu‐WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP‐Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures.

Antigen-Specific Proliferative Response of Peritoneal Exudate Lymphocytes Primed with Antigen and Bacterial Lipopolysaccharide: The Roles of Ia+ Accessory Cells and IL-2

In vitro antigen‐specific proliferation was investigated in a lymphocyte population that had been taken from the peritoneal exudate cells (PEC) of C3H/HeN mice (Iak) primed in vivo with both bacterial lipopolysaccharide (LPS) and horse red blood cells (HRBC) and had been purified by passage through a nylon fiber column (Nfc). The proliferative response of the Nfc‐passed lymphocytes primed with HRBC and LPS [T(HRBC + LPS) cells] depended on the dose of antigen in the cultures, and the response was higher than that of cells prepared from mice primed with HRBC alone [T(HRBC) cells]. No response was seen in the cells prepared from the LPS‐primed mice [T(LPS) cells] or normal mice [T(N) cells]. The response of the T(HRBC) cells was abolished by previous treatment of the cells with anti‐Iak antibody and complement (C), whereas the response of the T(HRBC + LPS) cells was retained after the same treatment, indicating that the Ia– T(HRBC + LPS) cells can proliferate in response to antigen in spite of Ia+ accessory cell‐depletion. Supernatants from the cultures of Ia– T(HRBC + LPS) cells in the presence of HRBC showed abundant IL‐2 activity, while those of Ia– T(HRBC) cells did not. The IL‐2 should be produced by the L3T4 cell population in T(HRBC + LPS) cells in response to antigen, since the previous treatment of the cells with anti‐L3T4 antibody and C abrogated the production. On the other hand, the Ia– T(HRBC + LPS) cells as well as the Ia– T(LPS) cells could respond to IL‐2 dose‐dependently when recombinant IL‐2 was added into the cultures, but the response of Ia– T(HRBC) cells to IL‐2 was very weak. The cell population responding to IL‐2 in the T(HRBC + LPS) cells as well as T(LPS) cells must be AsGM1‐positive or natural killer (NK) cells, since previous treatment of the cells with anti‐AsGM1 antibody and C abrogates the response. Together these results suggest that L3T4 lymphocytes capable of producing IL‐2 in response to HRBC antigen without Ia+ accessory cells are generated in the PEC of the mice after priming with LPS and antigen together, and the IL‐2 produced by the L3T4 lymphocytes induces the proliferation of the LPS‐primed AsGM1+ cells.

In Vitro Proliferative Response and Polyclonal Antibody Production in Spleen Cells of Immunologically Defective CBA/N and C3H/HeJ Mice by Water-Soluble Adjuvant (Bu-WSA) Extracted from Bacterionema matruchotii

Mitogenicity and the polyclonal plaque forming cell(PFC)‐inducing property of a water soluble‐adjuvant extracted from Bacterionema matruchotii by butanol (Bu‐WSA) were examined in vitro in the spleen cells of hybrid (CBA/N female × BALB/c male) F1 mice and C3H strain of mice. The hybrid F1 male cells which expressed a CBA/N‐defect were unable to respond to Bu‐WSA, when assessed by the incorporation of [3H] thymidine into the cells and the generation of anti‐trinitrophenyl (TNP)‐PFC or autoantibody PFC defined by the anti‐bromel‐ain‐treated mouse erythrocyte PFC assay. However, hybrid F1 female cells with normal traits responded to Bu‐WSA. Cultured spleen cells of bacterial lipopolysaccharide (LPS)‐nonresponsive C3H/HeJ mice responded to Bu‐WSA as in the case of cells of LPS‐responsive C3H/He mice, and the [3H]thymidine‐uptakes and the numbers of PFC in these culture cells increased. Re‐extraction of Bu‐WSA by phenol did not affect its activities, while the activity of butanol‐extracted LPS on C3H/HeJ cells decreased after re‐extraction by the same procedure with phenol.

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii: IV. Synergistic Effects of Bu-WSA on Concanavalin A-Induced Proliferative Response of Human Peripheral Blood Lymphocytes

Butanol‐extracted water‐soluble adjuvant (Bu‐WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub‐ and/or supra‐optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu‐WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu‐WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu‐WSA, Con A and a mixture of Bu‐WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell‐cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu‐WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T‐cell response with a B‐cell helper effect. Moreover, Con A and Bu‐WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.

In vivo priming of mice with antigen and lipid A bestows proliferative ability on peritoneal exudate T cells in response to antigen or anti-αβ TCR antibody in the absence of macrophages in vitro:

Requirement of macrophages for the in vivo proliferative response of the peritoneal exudate T cells of mice that had been primed in vivo with antigen and lipid A was investigated. Two weeks in advance, C3H/HeN mice (la k) were primed with horse red blood cells (HRBC) and synthetic lipid A. The highly purified T cells [T(HRBC+lipid A)] proliferated in response to HRBC or anti-αβ T cell receptor (TCR) antibody. However, T cells [T(HRBC)] that had been prepared from the mice primed with SRBC did not respond to HRBC and anti-αβ TCR antibody. When T(HRBC) were co-cultured in the presence of HRBC or anti-αβ TCR antibody with an adherent cell population (macrophages) prepared from PEC, the T(HRBC) proliferated. Previous treatment of T(HRBC+lipid A) with anti-CD45 (180 kDa) antibody plus C abolished the response, while the same pretreatment of T(HRBC) did not abolish the response in the presence of macrophages. The previous treatment of these cells with CD45 (220 kDa) antibody plus C eliminated their proliferative response. These results suggest that T(HRBC+lipid A) are different from T(HRBC) on macrophage-dependency, and T(HRBC+lipid A) expressing CD45 (180 kDa) on the membrane are the proliferative cells when their αβ T cell receptors (TCR) are stimulated with the antigen or anti-TCR antibody.

Mitogenic activity of a water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii. III. Mechanisms of inactivation of mitogenesis by antiimmunoglobulin serum.

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.

In vivo priming of mice with antigen and lipid A bestows proliferative ability on peritoneal exudate T cells in response to antigen or anti-αβ TCR antibody in the absence of macrophages in vitro: accessory function of NK cells

Highly purified T cells of mice that have been primed in vivo with horse red blood cells (HRBC) and lipid A, but not with HRBC alone, can proliferate in vitro in response to HRBC or anti-αβ T cell receptor (TCR) antibody without macrophages. The mechanism of proliferation without macrophages was investigated. Mice were primed with HRBC and lipid A together or with HRBC alone 10 days in advance, and the peritoneal exudate cells (PEC) were obtained. Purified T cells were obtained by treating the PEC by the following series of procedures: nylon fiber column-passage, antibody (anti-Ia, anti-Mac-1, and LR-1) treatments with complement (C), and a G-10 column-passage. Cell proliferation was assessed by [3H]-thymidine (TdR) incorporation into the cultured T cells in response to HRBC or anti-αβ TCR antibody. The proliferation of T cells [T(HRBC+lipid A)] that had been prepared from the PEC of the mice primed with HRBC and lipid A increased dose-dependently to these stimulants, but T cells [T(HRBC)] prepared from PEC of mice primed with HRBC showed little or no proliferation in response to them. The expression pattern of a memory cell marker, CD44, on the cell surface of T(HRBC+lipid A) was obviously different from that on T(HRBC). The proliferation of T(HRBC+lipid A) was suppressed when the cells were cultured in the wells coated with hyaluronate, a ligand for CD44, or cultured after a previous treatment with anti-CD44 (IgG2a) and anti-IgG2a antibodies. In contrast, the proliferation of T(HRBC) was up-regulated in culture under the same conditions. Proliferative responses of T(HRBC+lipid A) to anti-αβ TCR antibody were enhanced in the wells coated with anti-CD28 antibody. These findings indicate that the proliferation of T(HRBC+lipid A) was not only supported by a stimulation of TCR but also regulated by another stimulation via ligands such as CD44 and CD28. The proliferation of T(HRBC+lipid A) was abolished when the cells were pretreated with AsGM1 antibody or anti-CD80 antibody and C. These findings indicate that the in vivo priming of mice with HRBC and lipid A generates a memory T cell population that is capable of proliferating without help of macrophages, but with NK cells, when stimulated their TCR.

Mitogenic activity of bacterial lipopolysaccharide on the .GAMMA..DELTA. T lymphocyte.

In vitro proliferative responses of T lymphocytes to bacterial lipopolysaccharides (LPS) were examined by determining the uptake of tritiated thymidine (3H-TdR) into cells. T lymphocyte populations were taken and purified from proteose peptoneinduced peritoneal exudate cell (PEG), spleen and thymus of C3H/HeN or C3H/HeJ mice. LPS were prepared from Salmonella typhimurium, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum by the phenolwater proceduce.The T lymphocytes taken from PEG (PEG-T) of C3H/HeN mice showed an increased 3H-TdR uptaken in response to LPS from S. typhimurium (S. t-LPS), while PEG-T of G3H/HeJ, splenic T and thymic T lymphocyte of C3H/HeN mice did not respond to S. t-LPS. Other LPS also have a mitogenic ability to PEG -T lymphocytes too, while those of S. t-LPS and P. i-LPS were higher than that of F. n-LPS or P. g-LPS. The response of PEG from C3H/HeN mice to S. t-LPS was abolished, when the PEG-T were pretreated with anti-Thy-1 orγδTCR antibody plus complement (c), but not anti-αβTCR antibody plus C. The thymic T lymphocytes did not show any increase of 3H-TdR uptake in response to LPS or anti-γδ TCR antibody. However, obvious uptake did occur when the cells were stimulated with LPS and anti-γδ TCR antibody together.The results suggest that LPS has a mitogenic ability to a T lymphocytes population bearing γδ TCR in PEG that had been stimulated previously by proteose peptone.

Synergistic Effect of Endotoxin with Concanavalin a on DNA Synthesis in Lymphocytes and the Role of Interleukins 1 and 2

Bacterial lipopolysaccharide (LPS) extracted from the cell walls of gram-negative bacteria has a number of effects on the cells of the immune system (see 21 for review). The effects of LPS on macrophages (Mo) and B lymphocytes are very prominent. Mo activated by LPS increase their phagocytic ability (20,21), pinocytosis (5,30), oxidative metabolism (15,25), synthesis of cellular proteins including lysosomal enzymes (19,20), secretion of collagenase (36) and arginase (4), tumor cytotoxicity (33), microbicidal activity (27), interleukin (IL) 1 (18), colony-stimulating factor, and interferon (21). When murine splenic B lymphocytes are stimulated by LPS, about one-third of the B lymphocytes initiates DNA synthesis (13), and subsequently, these activated B lymphocytes produce antibodies polyclonally (21). However, its effect on human peripheral B lymphocytes is not obvious (21,26,29), unless these cells are cultured for long periods of time (seven to nine days) with prescreened lots of fresh human serum (17). On the contrary, LPS has no obvious mitogenic effect on T lymphocytes, except on a very small percentage of cells (35), and it is incapable of initiating T lymphocytes to produce lymphokines. However, if T lymphocytes are activated by T cell stimulants, the T lymphocytes seem to be able to accept the stimulus of LPS. The combination of some kinds of phytomitogens with LPS can synergistically enhance the blastogenic responses of thymocytes in mice (7,24,32), spleen lymhocytes of rats (8) or peripheral blood T lymphocytes of humans (12,29) as measured by increase[3H]thymidine ([3H]TdR)uptakes.