A cell-free extract of Salmonella typhimurium inhibits mitogen-induced proliferation of murine splenic T lymphocytes

Abstract In this study, a cell-free extract of Salmonella inhibited T cell mitogen-induced proliferation of spleen cells from non-immunized mice. The proliferation of murine spleen cells stimulated with a T cell mitogen, such as phytohaemagglutinin (PHA) or concanavalin A (ConA) was suppressed significantly when the cells were treated with a sonicate of S. typhimurium , but not of E. coli . The agent(s) responsible for the suppressive effect existed mainly in the soluble fraction of S. typhimurium , whereas the membrane fraction possessed minimal activity. The T cell proliferation suppression paralleled the level of interleukin-2 (IL-2) secretion. Addition of phorbol 12-myristate-13 acetate (PMA) to the cultures restored IL-2 secretion to normal levels, although proliferation remained suppressed and was not reversed by treatment with recombinant IL-2. These results suggest that the suppression of T cell proliferation induced by a soluble Salmonella fraction is associated with inhibition of IL-2 secretion and the response of T cells to IL-2 and the former effect is dependent upon the inhibition of the stimulatory activity of protein kinase C on IL-2 secretion. This type of suppression may explain a mechanism of immunosuppression induced by murine typhoid fever.     

Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E

Abstract Syringomycin E (SR-E) is low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.     

Dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation of rat lymphocytes and have found that a serum-free tissue culture medium supports significant cell division. When the compound dextran sulfate (DxS), which itself is not mitogenic, is added to LPS-stimulated cultures, significant augmentation of proliferation results. DxS will significantly enhance anti-immunoglobulin-induced proliferation as well. An intact T-cell compartment is not required for DxS-mediated enhancement of LPS-induced cell division, in that lymphocytes derived from athymic rats proliferate and respond to the influence of DxS to the same degree as euthymic derived lymphocytes. A "DxS-free" supernatant from DxS-stimulated spleen cell cultures is able to substitute for DxS in all stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity. However, purified recombinant IL-1 does not cause enhanced proliferation when added to LPS-stimulated cultures as is seen with DxS or the "DxS-free" supernatant.     

Interstitial stem cell proliferation in hydra: evidence for strain-specific regulatory signals.

We have examined the growth behavior of small numbers of interstitial stem cells transplanted into tissue of genetically unrelated strains of Hydra magnipapillata. We show that such stem cells, which are at low density following transplantation, proliferate more rapidly than the stem cells of the host, which are at normal density. The rapid proliferation is similar to the proliferation rate of stem cells transplanted into interstitial cell free tissue. The results suggest that stem cells transplanted into heterotypic tissue are unable to "sense" the presence of host stem cells and to adopt their growth rate to that of the surrounding cells. Thus, the feedback signal which negatively regulates stem cell growth as a function of stem cell density must be strain specific.     

Purification of a product from Salmonella typhimurium with the ability to inhibit mitogen-induced proliferation of murine splenic T-lymphocytes

Abstract We attempted to purify a substance that inhibits mitogen-induced proliferation of murine splenic T-lymphocytes from Salmonella typhimurium . The soluble fraction of a suspension of bacteria disrupted by sonication was chromatographed serially on Mono Q HR, Superdex 200 HR and HiLoad Superdex 75 p.g. columns. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis analysis revealed that the purified active substance migrated as a single band corresponding to a molecular mass of 87 kDa. We designated the purified substance S. typhimurium -derived inhibitor of T-cell proliferation (STI). which, at 0.2 μg/ml and above, inhibited proliferation and augmented CD25 expression of phytohemagglutinin-stimulated murine splenic lymphocytes. These findings suggested that the immunosuppression induced by Salmonella infection may be attributable to STI.     

A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes

Mitogen-stimulated DNA synthesis in human peripheral blood lymphocytes is dependent on extracellular Na. DNA synthesis was similarly inhibited in:

1. 1. Cells that were suspended in hypotonic media containing decreased extracellular Na.

2. 2. Cells that were suspended in media containing decreased Na and equimolar replacement with choline.

3. 3. Cells that were suspended in media containing decreased Na and equiosmolar replacement with mannitol.

A decreased PHA-induced DNA synthesis was observed at day 3 even when lymphocytes were exposed to low Na for only the first 3 h and then returned to normal levels of Na. Our studies of protein synthesis indicate that the effect of lowered extracellular Na on DNA synthesis and cell division is not due to an initial inhibition of overall protein synthesis. These data suggest that reduced external Na has a significant effect on some specific early event(s) (3 h) in lymphocyte mitogenesis.     

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.     

Role of adherent cells in dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation in the rat and have found that the addition of the compound Dextran sulfate (DxS), which itself is not mitogenic, to LPS stimulated cultures results in significant enhancement of cell division. A "DxS-free" supernatant from DxS stimulated spleen cell cultures is able to substitute for DxS in stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity, however, the addition of purified recombinant IL-1 to LPS-stimulated cultures does not result in augmentation of proliferation. A DxS-free supernatant from DxS stimulated adherent cells is also able to substitute for DxS in stimulatory activity. The active molecule(s) present in the adherent cell-derived DxS-free culture supernatant appears to be distinct from classical IL-1.     

N-acetyl-beta-endorphin1-31 antagonizes the suppressive effect of beta-endorphin1-31 on murine splenocyte proliferation via a naloxone-resistant receptor.

High affinity binding sites for beta-endorphin1-31 (beta-EP) have been observed on transformed mononuclear cells such as the human U937 monocyte-like cell line and the murine EL4-thymoma line, and on normal murine splenocytes. Binding of beta-EP at these sites is resistant to competition by naloxone and other opiate receptor ligands but sensitive to N-acetyl-beta-endorphin1-31 (N-Ac), cations and GTP-gamma-sulfate. Thus, the following studies were done to determine the functional significance of binding beta-EP and N-Ac. beta-EP suppressed phytohemagglutinin (PHA)-stimulated [3H]thymidine uptake in a dose-dependent, naloxone-insensitive fashion. beta-Endorphin1-27, (des)-tyrosine beta-endorphin2-31, or N-Ac failed to duplicate the suppressive effect of beta-EP. However, N-Ac, which is equipotent to beta-EP at displacing 125I-beta-EP bound to murine splenocytes or U937 cells, antagonized the suppressive effect of beta-EP. Taken together with previous binding studies, the present observations suggest that beta-EP effects receptor-mediated responses on normal immunocytes that do not depend on the activation of neuronal-like opiate receptors which are naloxone-sensitive. N-Ac, which shows minimal binding to such brain opiate receptors, is a potent functional antagonist of the naloxone-insensitive immunocyte receptor for beta-EP.     

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.     

Polysaccharides isolated from <Emphasis Type=Italic>Phellinus baumii</Emphasis> stimulate murine splenocyte proliferation and inhibit the lipopolysaccharide-induced nitric oxide production in RAW264.7 murine macrophages

Polysaccharides isolated from Phellinus baumii (PBP) significantly enhanced both lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation. However, PBP (12.5–100 μg ml⁻¹) significantly suppressed the LPS-induced nitric oxide (NO) production in RAW264.7 cells in a concentration-dependent manner. The maximal inhibition of PBP on NO production was 37.5% at 100 μg ml⁻¹. These results provide useful in vitro information to explain the immunostimulating activity and anti-inflammatory activity of PBP.     

Rheumatoid factor blocks regulatory Fc signals

Immunosuppressed cultures of murine spleen cells, partly deprived of T cells and antigen-stimulated, can be reconstituted to near full activity in their antibody-forming cell response with murine rheumatoid factors (RF). The dose of RF required for recovery of 50% of the reconstitutable immune response was 10-100 ng and reconstitution was blocked by intact murine IgG added to the cultures. IgG subclass specificity of RF was demonstrated; RF specific for IgG2a was more potent than RF specific for IgG1 in reconstituting the response. Synergy was observed between RF added at culture initiation and late-acting B-cell differentiation factors. The greater the degree of T-cell deprivation, the more stringent the conditions needed for reconstitution. Suitable conditions for reconstitution with profound T-cell depletion included the limited reconstitution by specific RF, the synergistic action of RF with late-acting T-cell-replacing supernatants, and multiple additions of a number of RFs to the cultures on Days 0, 1, and 2. RF was also shown to block Fc-dependent immunosuppression by added antigen-antibody complexes. These results are interpreted as favoring the hypothesis put forward previously that the normal production of RF acts to reduce T-cell dependency by preventing negative Fc signal transmission by immune complexes on the B-cell surface. Abnormal production of RF may be a primary destabilizer of the immune responses leading to autoimmunity.     

Phosphorylation of specific polypeptides in isolated murine splenocyte nuclei which is controlled by cyclic nucleotides

Murine splenocyte nuclei were phosphorylated with a less than 10(-5) M concentration of [gamma-32P]ATP at 0 degrees C and the phosphorylated nuclear proteins were analyzed by SDS-polyacrylamide gel slab electrophoresis and Sephadex gel filtration column chromatography. Two polypeptides of 10K and 11K daltons were predominantly phosphorylated. These polypeptides were likely linked by a disulfide bond to form a nonhistone protein of 21K daltons. Both phosphoserine and phosphothreonine were detected in the hydrolysate of the 10.5K dalton polypeptide, while phosphoserine was predominant in the 10K dalton polypeptide. Maximal activation of phosphorylation by cAMP of both polypeptides was shown at a concentration of 10(-6) M. On the contrary, cGMP activated phosphorylation of the 10K dalton polypeptide at 10(-8) M and at 10(-4) M. The phosphorylation of the 10.5K polypeptide was not activated by 10(-4) M cGMP and suppression of the phosphorylation was seen in both polypeptide chains by cAMP at higher concentrations.     

Scent-marking displays provide honest signals of health and infection

Males of many species produce scent marks and other olfactorysignals that function to intimidate rivals and attract females.It has been suggested that scent marks provide an honest, cheat-proofdisplay of an individual's health and condition. Here we reportseveral findings that address this hypothesis in wild-derivedhouse mice (Mus musculus domesticus). (1) We exposed males tofemale odor, which induces an increase in testosterone, andfound that sexual stimulation significantly increased the males'scent-marking and the attractiveness of their scent marks tofemales. (2) We challenged sexually stimulated males with anonreplicating strain of bacteria (Salmonella enterica C5TS)to activate immunity and found that this significantly decreasedthe males' scent-marking and the attractiveness of their scentmarks to females. (3) We collected scent marks from infectedand sham-infected males when they were sexually stimulated ornot, and we found that females could significantly discriminatethe scent marks of infected versus control males, but only whenthe males were sexually stimulated. Taken together, our resultsindicate that male mice modulate their scent-marking displaydepending on their health and perceived mating opportunities.Increased scent marking enhances males' attractiveness to females,scent marks provide an honest indicator of bacterial infection(and perhaps immune activation), and females are able to assessthe health of males more easily when males mark at a high rate.     

Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

Inhibition of mitogen-induced lymphocyte proliferation correlated to anticomplementary activity in sera from melanoma patients

Summary Sera from 98 melanoma patients and 90 normal donors were analyzed for antibody (Ab) to melanoma extracts, melanoma-associated antigen (Ag) and anticomplementary (Ac) activities by the microcomplement consumption technique. Sera were also tested for their ability to inhibit mitogen(phytohemagglutinin: PHA)-ininduced blastogenesis of normal lymphocytes. The results of the complement consumption assays were correlated with the results of inhibition of PHA-induced blastogenesis. Of 98 melanoma sera, 22% were Ab-positive, 30% were Ag-positive, and 44% were Ac-positive, in contrast to only 6% Ab-positive, no Ag-positive, and 7% Ac-positive in 90 normal sera. Fifty-nine percent of melanoma sera were inhibitory to PHA-induced blastogenesis, as against 12% of normal donors' sera. Ac-positive melanoma sera were significantly more inhibitory than Acnegative melanoma sera. The inhibitory activity of Acpositive sera was potentiated by the simultaneous presence of detectable Ag activity and was diminished by detectable Ab activity. Presence of Ab or Ag activity alone did not correlate with the inhibitory activity of the melanoma sera. Increasing incidence of Ac activity and ability to inhibit PHA-induced blastogenesis was observed with increasing tumor burden or advancing clinical stage. Sera from patients who displayed a delayed cutaneous hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) were less inhibitory to PHA-induced blastogenesis than the sera from patients who did not respond to this contact allergen. Thus, Ac activity may be one of the circulating factors responsible for the immunosuppressive effect of cancer patients' sera.