Mitogenic Response Of Splenic Lymphocytes Research Articles

Effect of L-Cycloserine on Cellular Responses Mediated by Macrophages and T Cells

In this study, we examined the immunoregulatory roles of L-cycloserine (L-CS), a sphingolipid metabolism regulator with inhibitory activity of serine palmitoyltransferase (SPT), on immune responses mediated by monocytes/macrophages and T cells. Mitogenic responses of splenic lymphocytes induced by LPS, PHA, and Con A were very strongly suppressed by L-CS with IC(50) values ranging from 0.5 to 1 muM. In contrast, this compound less strongly blocked IL-2-induced CD8+ CTLL-2 cell proliferation with an IC(50) value of 540 muM. Interestingly, L-CS enhanced the number of IL-4-producing helper T cells, indicating the favored induction of Th2 condition. Although tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production was not altered under 10% FCS condition, U937 cell-cell adhesion as well as the surface level of adhesion molecules (CD29 and CD98) were significantly suppressed by L-CS. In particular, reduced serum level (5%) under L-CS treatment strongly enhanced the production of TNF-alpha and the inhibitory potency of NO production and cell adhesion. Finally, sphingolipids (D-sphingosine and DL-dihydrosphingosine) did not remarkably abrogate L-CS-mediated T cell proliferation. Therefore our data suggest that de novo sphingolipid metabolism may represent an important aspect of immunomodulatory activities mediated by T cells and macrophages/monocytes, depending on serum level.

Immunomodulatory and cytoprotective role of RP-1 in gamma-irradiated mice.

RP-1 has been reported to provide protection against lethal gamma-irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 +/- 0.224 and 4.9 +/- 0.057 mM Fe2+) as compared to control (6.29 +/- 0.733 mM Fe2+) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G2 delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G2 delay and elicited significant (p < 0.05) recovery in S-phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.

Ventromedial hypothalamus suppresses splenic lymphocyte activity through sympathetic innervation

The ventromedial hypothalamic nucleus (VMH) has been proposed to be a locus intimately associated with sympathetic facilitation in peripheral tissues. To investigate a possible role of the VMH in controlling peripheral immune functions, we examined the effects of VMH stimulation on proliferative activity of lymphocytes isolated from the spleen in rats. Electrical stimulation of the VMH (2 s at every 30 s for 30 min) caused a remarkable decrease in the mitogenic response of splenic lymphocytes to Concanavalin A, whereas the stimulation of other hypothalamic regions such as the paraventricular nucleus did not show any significant effects. The suppressive effect of VMH stimulation was not influenced after adrenalectomy, suggesting a minor role of the adrenocortical system. In contrast, pretreatments with a ganglionic blocking agent (chlorisondamine) and a β-adrenergic receptor antagonist (propranolol) completely reversed the suppressive effect. Surgical severing of the sympathetic nerves entering the spleen also reversed the effect of VMH stimulation. Together with the previous finding that VMH stimulation activates the splenic sympathetic nerves, it is concluded that the VMH suppreses the proliferative activity of splenic lymphocytes through the activation of sympathetic nerves via the β-adrenergic pathway.

Long-term intake of a low-casein diet is associated with higher relative NK cell cytotoxic activity in F344 rats.

Earlier studies in our laboratory showed that animals exposed to aflatoxin B1 (AFB1) develop fewer gamma-glutamyl transpeptidase-positive preneoplastic foci and tumors when fed 6% dietary casein than when fed 22% casein during promotion; mechanisms underlying this effect have not been elucidated. We examined natural killer (NK) cell activity, mitogenic responses, and lymphocyte surface antigen profiles in male Fischer 344 rats dosed with AFB1 or dosing vehicle alone and then fed 6% or 22% casein isocaloric diets for one year. Mean body weights and food intake did not differ significantly among the groups during the study. NK cells purified from peripheral blood of rats fed 6% casein mediated higher specific lysis (p < 0.0001) against YAC-1 target cells than cells obtained from animals fed 22% casein. Mitogenic responses of splenic lymphocytes to concanavalin A and lymphocyte subpopulations, identified by flow cytometry, did not differ significantly among dietary groups. Hepatic tumors were detected in 27% of the 22% casein AFB1-treated group and in 6% of animals in the 6% casein AFB1-treated group. The association between long-term intake of a 6% casein diet and higher relative NK cell cytotoxic activity suggests a potentially important mechanism that may help protect against the development of hepatocellular tumors. Further study of this mechanism as a causal factor in limiting tumor development is required.

Dietary Curcuma xanthorrhiza Roxb. increases mitogenic responses of splenic lymphocytes in rats, and alters populations of the lymphocytes in mice.

Rhizomes of Curcuma xanthorrhiza Roxb. (C. xanthorrhiza), a medicinal plant in Indonesia, has been shown to exert diverse physiological functions. Hitherto, a little attention has been paid to its effect on immune functions. This study was carried out to determine the effect of this medicinal plant on mitogenic response of splenic lymphocytes in rats and population of splenic lymphocytes and macrophages and peripheral blood macrophages in mice. Mitogenic responses of splenocytes to phytohemagglutinin, concanavalin A, and pokeweed mitogens were examined in rat fed C. xanthorrhiza for 3 weeks. The medicinal plant increased the blastogenesis to these mitogens. Flow cytometric analysis was carried out for mice fed the medicinal plant for 3 to 5 weeks. C. xanthorrhiza increased the proportion of the splenic T cells throughout the experimental period, but exerted a variable effect on B cells and T cell subsets, that is, elevations of B cells at 3 weeks and of Th cells at 4 weeks without any elevation of Ts cells. The effect of this medicinal plant on a proportion of macrophages from the spleen and peripheral blood was not consistent. Thus, the present study suggests that C. xanthorrhiza contains some principle(s) activating T and B cell-mediated immune functions.

Role of colony-stimulating factor-1 in macrophage activation in tumor-bearing mice.

We previously reported a dramatically increased number of macrophages in tumor-bearing mice. In this study, we investigated the involvement of CSF in that phenomenon. CSF-1 responding cells as macrophages precursors increased significantly in number in the spleens of tumor-bearing mice as compared with those in normal mice. Splenic cells and sera from the tumor-bearing mice respectively expressed CSF-1 in mRNA and serum protein levels, but failed to express the other CSF (granulocyte-macrophage-CSF or IL-3). Nonadherent splenic mononuclear cells (< 0.5% macrophages) from normal mice proliferated and differentiated into mature macrophages in culture within 7 days with recombinant mouse CSF-1 (rCSF-1). Both macrophages harvested from tumor-bearing mice and those activated in vitro with rCSF-1 expressed mostly Mac-1, -2 (and -3) Ag, showed yeast phagocytosis, produced IL-1 but not IL-2 or IL-3, and displayed potent cytotoxicity against NK cell resistant Meth-A tumor cells. These macrophages also expressed lipocortin I mRNA and secreted lipocortin I protein, and suppressed mitogenic responses of splenic lymphocytes. rCSF-1-activated macrophages derived from nonadherent splenic cells expressed both CSF-1 and CSF-1 receptor (c-fms) mRNA. Administration of rCSF-1 into normal mice induced hemopoietic and immunologic alternations similar to those observed in tumor-bearing mice. These results suggest that CSF-1 is involved in the dramatic increase of macrophages in tumor-bearing mice, possibly through an autocrine or paracrine loop.

Effect of retinoic acid on tumor‐mediated immunologic alterations in mice bearing a variant of the ß16 melanoma

This investigation examined the effect of retinoic acid on tumor progression and immunological status of mice bearing the B16-F10 melanoma (previously selected for high lung-colonizing capacity). Tumor cells were implanted s.c. in syngeneic C57BL/6 mice, half of which were treated with beta-all trans retinoic acid (RA). Although RA failed to exhibit direct toxicity on this variant at the concentration used, the immunologic aberrations induced by the tumors were diminished by i.p. RA administration (at 45 micrograms twice/week for 3 weeks). In mice bearing B16-F10 tumors, tumor burdens were decreased from 2.9% of body weight to 1.6%. The mitogenic responses of splenic lymphocytes to concanavalin A (ConA) were increased in tumor-bearing mice following this RA treatment. The presence of these tumor cells decreased the absolute number of CD4- and CD8-positive splenic lymphocytes. Following RA treatment, the CD8-positive population was increased in tumor-bearing mice, while the CD4+ population was not significantly altered. Since previous studies indicated that plasma membrane fragments (or vesicles) could alter lymphocyte distributions and proliferative capacities, the in vitro shedding of membrane fragments from B16-F10 tumor cells was assayed and observed to be decreased after continuous treatment of cultures with 10(-6) M RA for 21 days. Membrane shedding from B16-F10 cells was inhibited by 48.5% following RA treatment. Based on these in vivo and in vitro results, we suggest that RA treatment may diminish tumor growth by decreasing tumor-induced immunosuppressive events.

Augmentation of immune responses by a muramyl dipeptide analog, MDP-Lys(L18).

The effects of N2-(N-acetyl-muramyl-L-alanyl-D-isoglutamyl)-N6-stearoyl-L-lysine (MDP-Lys(L18], a muramyl dipeptide (MDP) analog, on the immune responses in mice were studied. MDP-Lys(L18) augmented the mitogenic responses of splenic lymphocytes to phytohemagglutinin (PHA) and lipopolysaccharide (LPS) at 0.1-10 micrograms/ml, and antibody formation to sheep red blood cell (SRBC) in normal and immunosuppressed mice, and to dinitrophenyl (DNP)-Ficoll. In addition, MDP-Lys(L18) potentiated polyclonal B cell activation both in vivo and in vitro. It was also found that MDP-Lys(L18) augmented the cellular immune responses, such as mixed lymphocyte reaction (MLR) and delayed type hypersensitivity (DTH). These effects of MDP-Lys(L18) were more potent than those of MDP. These findings may be attributed to the interleukin 1 (IL-1)-inducing activity of MDP-Lys(L18).

Characterization of immunologic and neuropathologic abnormalities in wasted mice.

Mice bearing the autosomal recessive gene "wasted" (wst/wst) have been reported to develop low or absent secretory immune responses, abnormal DNA repair mechanisms, and uncoordinated body movements. We have performed a detailed immunologic and neuropathologic investigation of the disease. Con A and LPS mitogenic responses of splenic lymphocytes as well as total serum Ig levels were equivalent in wst/wst and undiseased control littermates (wst/+ and +/+). Amounts of serum IgM, IgG2a, IgG2b, IgG3, and IgA, however, were reduced in wst/wst animals. FACS analyses of Ig+ and isotype-specific B cell populations revealed similar percentages of Ig+, mu +, gamma +, and alpha + cells in wst/wst and control mice. However, the percentage of "very bright" Ig+ cells as well as "very bright" heavy and light chain-specific B cell subpopulations was increased at least 10-fold in wst/wst B cells as compared with littermate controls. In addition, studies of Ig-specific mRNA accumulation in these animals revealed significant decreases in all isotypes except gamma 3 in spleens of wst/wst mice as compared with littermate controls. Neuropathologic studies in wst/wst mice showed prominent vacuolar degeneration of neurons within anterior horns of the spinal cord and the motor nuclei of the brain stem. No abnormalities were noted in Purkinje cells of the cerebellum nor within myelin sheaths. The abnormalities in the nervous system resembled human motor neuron disease rather than ataxia-telangiectasia as had been reported previously. The immunologic studies suggest that splenic B cells from wst/wst mice have a defect in the ability to "switch" from membrane to secreted Ig. In addition, this mutation provides a mechanism to study regulatory interactions between the immune and nervous systems.

The antigenic and mitogenic response of murine T and B lymphocytes to soluble proteins of Listeria monocytogenes.

Solubilized constituents from Listeria monocytogenes were fractionated by various techniques including isopycnic gradient centrifugation, molecular sieve chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Fractionated material was tested in vitro for mitogenic and antigenic activity by quantitating the proliferation of splenic lymphocytes and the interleukin production by peritoneal T cells. Fractionation by isopycnic gradient centrifugation revealed both antigenic and mitogenic material fractionating with the protein at a density of 1.3 g/ml. This characteristic density, together with the reduction of activity with trypsin treatment, defined the material as protein. This material was termed soluble listerial proteins (SLP). Fractionation of SLP by molecular sieve chromatography using Sephacryl 200 (S-200) revealed predominant antigenic and mitogenic activity in proteins of greater than 100,000 m.w. In contrast, fractionation of SLP by preparative SDS-PAGE (nonreducing conditions) showed activity in groups of proteins with m.w. of less than 76,000. This difference (S-200 vs SDS-PAGE) may indicate an aggregation or subunit composition which is disrupted by SDS. When fractionated by SDS-PAGE, antigens which induced macrophage-dependent interleukin production by Listeria-immune T cells were observed over a broad range of molecular sizes. Major groups of antigenic proteins were observed at 57,000 to 76,000 m.w., approximately 40,000 and less than 25,000 m.w. Mitogenic activity (spleen cell proliferation) was associated with a more restricted group of proteins with major peaks at 57,000 and 40,000 m.w., with some weak activity in proteins less than 20,000 and greater than 64,000 m.w. Experiments involving T or B lymphocyte-depleted spleen cells and spleen cells from athymic mice revealed that the mitogenic response of splenic lymphocytes to SLP was predominantly B cell-mediated. Thus, we have defined groups of listerial proteins with potent antigenic activity with respect to T lymphocyte activation and as mitogenic activity for B cells.

Differences in Mitogenic Responses of Murine T Cells to Two Distinct Phytohemagglutinin (Pha) Subcomponents

Two distinct mitogenic subcomponents of phytohemagglutinin (PHA)--leucoagglutinin (LA) and "purified" PHA--apparently stimulated different subpopulations of murine T cells. In the DBA/2J strain, the mitogenic responses of splenic lymphocytes to LA reached maximal levels after 24 to 36 hr exposure and almost completely disappeared by 48 hr, whereas maximal responses to PHA were maintained after 48 hr incubation. The levels of LA-responding T cells were highest in DBA/2J spleens at 5 weeks of age but markedly declined by 9 weeks of age, whereas thymic levels of LA-responding T cells reached a maximum at 9 weeks of age and remained maximal past 15 weeks of age. PHA-responding cells, in contrast, reached maximal levels in both the spleen and thymus of DBA/2J mice at 9 weeks of age. In C57BL/6J mice, splenic and thymic lymphocytes responded similarly to both components, except that the response of splenic lymphocytes to PHA reached a maximum after shorter incubation time and declined sooner than their response to LA. The mitogenic responses of C57BL/6J thymocytes to both components were already at their peak by 5 weeks of age and almost totally disappeared by 9 weeks, whereas the responses of splenic lymphocytes were maximal at 9-15 weeks of age. The responses of DBA/2J splenocytes to LA was significantly augmented by PHA, but LA markedly suppressed the proliferative responses to PHA.

African hamster lymphocytes as a model for immunologic studies

The mitogenic response of splenic lymphocytes from Mystromys albicaudatus was studied to evaluate the potential of this animal as a model for immunologic research. In response to phytochemagglutinin and concanavalin A, splenic cells from Mystromys, unlike those from mouse strains, incorporate [3H] thymidine optimally in microculture at 10(5) to 2 X 10(5) cells per microculture. Maximum magnitude of incorporation in response to the same doses of mitogen used in mouse splenic lymphocyte microculture is greater than 10(5) cpm. Moreover, this high incorporation at low cell concentration has been observed in cultures from animals ranging from six to 24 months of age. Splenic cells from Mystromys give little or no incorporation with either LPS or PPD in doses mitogenic to mouse lymphocytes. These features of mitogenic response in Mystromys lymphocyte cultures suggest several useful applications to studies of mechanisms of mitogenesis.

Promotion of replication in lymphoid cells by specific thiols and disulfides in vitro. Effects on mouse lymphoma cells in comparison with splenic lymphocytes.

Numerous lines of mouse lymphoid tumors (13 of 22 tested) showed, with increased sensitivity, a property of normal mouse splenic lymphocytes, the potential for growth promotion in vitro by specific thiols added to standard culture media. For lymphoma L1210 (V), structure activity relationships were examined; 9 of 30 thiols promoted growth; the most active was alpha-thioglycerol, effective at 0.2 microM. Thiols became oxidized under conditions of tissue culture and had half-lives of less than 8 h. Disulfides of active thiols promoted growth of lymphoma cells. The mitogenic response of splenic lymphocytes to lectins was increased by thiols-disulfides which promoted the growth of lymphoma cells, but the response varied with the mitogen preparation used and under some conditions thiols-disulfides were inhibitory.