Proliferation Of Murine Spleen Cells Research Articles

Immunosuppression induced by Salmonella involves inhibition of tyrosine phosphorylation in murine T lymphocytes.

It is well known that facultative intracellular pathogens such as Salmonella suppress the host immune system. In the present study we attempted to clarify the mechanism responsible for the suppression of T-cell proliferation in mice infected with Salmonella typhimurium. The proliferation of murine spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were infected with S. typhimurium, but not with Escherichia coli. The suppression of T-cell proliferation did not necessarily parallel the level of interleukin-2 (IL-2) secretion, and was not restored by treatment with a calcium ionophore, indomethacin or IL-2. Only phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C (PKC), caused a slight recovery of cell proliferation with an augmentation of IL-2 secretion. Furthermore, Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 76-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited by S. typhimurium infection. Also, the inhibition of tyrosine phosphorylation was not restored by treatment with PMA. These results suggest that the suppression of T-cell proliferation induced by Salmonella infection may be regulated by inhibition of tyrosine phosphorylation in T-cells, although the inhibition is not associated with PKC activation and subsequent IL-2 secretion of T cells.

Inhibition of mitogen-induced proliferation of spleen lymphocytes is correlated with the induction of cell-mediated immunity in Salmonella infection in mice

The proliferation of murine spleen cells stimulated by a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were immunized with either the viable cells or the sonicate of Salmonella typhimurium but not of Escherichia coli. The suppression of T-cell proliferation caused by the sonicate of S. typhimurium was completely restored by addition of phorbol 12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Western blots using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-,94-,76-,68- and 57-kDa proteins in murine splenic T-cells was inhibited in the mice immunized with the viable cells but not the sonicate of S. typhimurium. These results suggest that the inhibition caused by the sonicate involves suppression of PKC activity, whilst that produced by viable cells involves down-regulation of tyrosine phosphorylation, and both inhibitions correlate with the induction of cell-mediated immunity in mice, as evidenced by the induction of delayed-type hypersensitivity reactions.

Inhibition of mitogen-induced proliferation of spleen lymphocytes is correlated with the induction of cell-mediated immunity in Salmonella infection in mice.

The proliferation of murine spleen cells stimulated by a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were immunized with either the viable cells or the sonicate of Salmonella typhimurium but not of Escherichia coli. The suppression of T-cell proliferation caused by the sonicate of S. typhimurium was completely restored by addition of phorbol 12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Western blots using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 76-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited in the mice immunized with the viable cells but not the sonicate of S. typhimurium. These results suggest that the inhibition caused by the sonicate involves suppression of PKC activity, whilst that produced by viable cells involves down-regulation of tyrosine phosphorylation, and both inhibitions correlate with the induction of cell-mediated immunity in mice, as evidenced by the induction of delayed-type hypersensitivity reactions.

Proliferative Responses of Peripheral Blood Lymphocytes of Mice and Humans Stimulated with Recombinant Interleukin 2

In vitro proliferative responses of human and murine peripheral blood lymphocytes (PBL) were studied. The cells were stimulated with recombinant interleukin 2 (IL-2) or with a mixture of phorbol-13-myristate-12-acetate with either IL-2 or calcium ionophores. Proliferation of human PBL could be induced by doses of human IL-2 or phorbol that were much lower than those required to produce responses of murine cells. However, the magnitude of the responses of human PBL was definitely lower than that of the responses of murine PBL. Murine IL-2 did not induce proliferation of human PBL, but it synergized with phorbol. The responses of murine PBL reflected the previously described strain differences in responses of spleen cells that were shown to be under complex polygenic control. The proliferation of murine spleen cells could be induced with human, as well as murine IL-2, and both species of IL-2 synergized with phorbol. While no clear-cut low and high responders could be identified among normal healthy human subjects, 2 individuals produced consistently low responses to IL-2 and 1 individual displayed low responses to phorbol and calcium ionophore A23187. Available data suggest, but do not allow a firm conclusion, that proliferative responses of human PBL are under genetic control, perhaps as complex as that reported for mice.

The influence of synthetic peptide from retroviral transmembrane protein p15E on murine spleen cell proliferation and bone marrow hemopoietic precursor colony formation

The retroviral transmembrane p15E peptide is known to suppress a wide variety of immune cell functions, suggesting a role for immunosuppression associated with retroviral infection. The 10-amino acid sequence from the highly conserved portion of p15E (CKS-10) is capable of reproducing this inhibitory activity. In this study we set out to determine the influence of this decapeptide on murine spleen cell mitogen-induced proliferation and hematopoietic granulocyte-macrophage and erythroid precursor colony formation in vitro. A dose- and time-dependent suppression of spleen cell blastogenic response was produced by the CKS-10 peptide. When bone marrow cells were incubated with decapeptide, the significant decrease of CFU-GM colony number was also dose-dependent. In contrast, the same doses of CKS-10 peptide which induced a most significant inhibition of CFU-GM colony formation caused a marked increase of BFU-E colonies. A most pronounced effect of the peptide on bone marrow hematopoietic progenitor activity was produced by prolonged exposure to the peptide. Given the results of this study, it seems likely that, in addition to the cytopathic effect of retroviruses on the lymphocytes, viral peptide-mediated hematopoiesis disorders may also play an important role in the pathogenesis of immunodeficiency associated with retroviral infections.

Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host.

Characterization of T‐ and B‐cell epitopes of a simian retrovirus (SRV‐2) envelope protein

Synthetic envelope peptides of a simian retrovirus (SRV-2) were used to define both T- and B-cell epitopes of the envelope protein. The SRV-2 peptide 100-106 specifically blocks rhesus anti-SRV-2 neutralizing antibody activity, and a peptide 100-106 keyhole limpet hemocyanin conjugate induces a strong antipeptide antibody response. SRV-2 peptide 100-106 and 233-249 induces good T-cell proliferation of murine spleen cells immunized with the SRV-2 virus. Thus, SRV-2 envelope peptide 100-106 represents both a T- and B-cell epitope, and peptide 233-249 a T-cell epitope.

Effects of heptaminol AMP amidate(HAA) on cyclic AMP level and mitogen-induced proliferation of murine spleen cells.

Heptaminol AMP amidate (HAA), a nucleotide derivative, was found to elevate the intracellular cyclic AMP (cAMP) level of cultured mice spleen cells in a time- and dose-dependent manner. Theophylline and imidazole, when added to the spleen cell culture simultaneously with HAA, respectively caused a further rise and a fall of the cAMP level increased by HAA alone. When comparatively higher doses of the T cell mitogen concanavalin A (Con A) were used in the culture, Con A-induced cell proliferation was mildly inhibited in the culture of spleen cells pooled from HAA administered mice in comparison to the culture of spleen cells pooled from saline treated mice. On the other hand, when another T cell mitogen phytohemagglutinin P (PHA) was used in different concentrations in the culture, there was a trend of enhanced cell proliferation in the culture of spleen cells pooled from HAA administered mice in comparison to the responses in the culture of spleen cells pooled from saline treated mice. The present results supported the previous findings that HAA-mediated immunopotentiation was closely related with a cAMP level elevating property of HAA, and the compound also enhanced the function of helper T cells.

Effects of Heptaminol AMP Amidate (HAA) on Cyclic AMP Level and Mitogen-lnduced Proliferation of Murine Spleen Cells

Heptaminol AMP amidate (HAA), a nucleotide derivative, was found to elevate the intracellular cyclic AMP (cAMP) level of cultured mice spleen cells in a time- and dose-dependent manner. Theophylline and imidazole, when added to the spleen cell culture simultaneously with HAA, respectively caused a further rise and a fall of the cAMP level increased by HAA alone. When comparatively higher doses of the T cell mitogen concanavalin A (Con A) were used in the culture. Con A-induced cell proliferation was mildly inhibited in the culture of spleen cells pooled from HAA administered mice in comparison to the culture of spleen cells pooled from saline treated mice. On the other hand, when another T cell mitogen phyto-hemagglutinin P (PHA) was used in different concentrations in the culture, there was a trend of enhanced cell proliferation in the culture of spleen cells pooled from HAA administered mice in comparison to the responses in the culture of spleen cells pooled from saline treated mice. The present results supported the previous findings that HAA-mediated immunopotentiation was closely related with a cAMP level elevating property of HAA, and the compound also enhanced the function of helper T cells.

In vitro Proliferation of Murine Spleen Cells: Genetic Control of Proliferative Responses Induced by Phorbol Ester and Calcium Ionophore A23187

Proliferative responses of normal (not immunized intentionally) spleen cells from inbred strains of mice to co-stimulation with phorbol ester (PMA) and calcium ionophore A23187 were studied. Striking differences in the magnitude of the responses of spleen cells and splenic T cells from various strains were observed. It appeared that these differences reflected mainly differences in the inducibility of the expression of the gene for the a chain of the IL 2 receptor (IL 2R) by phorbol ester. Formal genetic analysis suggested that the differences in response to phorbol ester and calcium ionophore are controlled by two independent genes with the alleles controlling good response being dominant. The differences in the inducibility of the IL 2R gene seemed to be controlled by alleles of a single gene. At least one of the putative genes may be a regulatory element affecting the gene for the a chain of IL 2R. The results may have a practical significance for devising more efficient procedure(s) to generate LAK cells used for tumor immunotherapy.

In vitro proliferation of murine spleen cells: I. Strain variation of response to medium from cultures of EL-4 cells.

Proliferative responses of murine lymphoid cells were elicited in vitro with supernatant fluid from cultures of EL-4 thymoma cells stimulated with phorbol ester. It was demonstrated that such responses depend on IL-2 contained in the supernatant fluid, but reflect co-stimulation with IL-2 and phorbol ester. Striking differences in the magnitude of proliferative responses of spleen, splenic T lymphocytes, thymus and bone marrow cells from various strains were observed. Three classes of responders could be identified. The differences in responsiveness, at least in part, reflected differences in the frequency of responsive cells and were genetically controlled by codominant alleles of two independent somatic genes.

In vitro proliferation of murine spleen cells. Strain variation of proliferative responses induced by recombinant IL-2.

Proliferative responses of normal (not immunized intentionally) spleen cells from inbred mouse strains to human recombinant interleukin 2 (L-2) were studied. It was shown that inbred strains vary in their responses to IL-2. The differences were observed for various doses of IL-2 and at various times after stimulation. Experimental data suggested that the strain differences in the magnitude of proliferative responses may result from an interplay of several interrelated factors such as differences in the frequency of the IL-2-responsive cells, differences in inducibility of the expression of the gene for the alpha-chain of the IL-2R, and genetic control of the responses. The latter was found to be exerted by two independent genes with the dominant alleles determining good responses to IL-2. The magnitude of the responses to IL-2 correlated with the magnitude of responses to concanavalin A, but seemed to have no effect on the interferon production or on the primary response to sheep red blood cells. The described phenomenon may have a practical significance in studies aimed at improving tumor immunotherapy with lymphokine-activated killer cells.

In vitro Proliferation of Murine Spleen Cells: Inhibition by Monoclonal Antibodies to L3T4 and Lyt-2 T Cell Markers or Intracellular Cyclic Adenosine Monophosphate

Proliferation of normal (not immunized intentionally) murine spleen cells was elicited with concanavalin A, supernatant fluid from cultures of EL-4 cells, human recombinant interleukin 2 (IL-2), or a mixture of phorbol ester and calcium ionophore A23187. IL-2-induced proliferation was inhibited by membrane-permeable dibutyryl cyclic adenosine monophosphate (cAMP) or by the adenylate cyclase activator forskolin. Consistent with these observations was the finding that stimulation with IL-2 decreased and forskolin increased the intracellular content of cAMP. IL-2-induced proliferation, as well as that induced by concanavalin A or phorbol-ionophore mixture, was inhibited by monoclonal antibodies specific for L3T4 or Lyt-2 cell surface markers. This inhibition was observed even when antibodies were added several hours after exposure of cells to IL-2. Notably, antibodies did not alter the intracellular content of cAMP. Thus, the experimental data failed to establish a functional linkage between the inhibitory effect of antibodies and the regulatory effect of the adenylate cyclase system. However, our results provide a rational basis for the postulation that antibodies, upon binding to their corresponding ligands, generate a negative signal that interferes with IL-2-induced proliferation. Therefore, L3T4 and Lyt-2 molecules appear to play an important role in the regulation of lymphocyte proliferation.

Suppression by delta-9-tetrahydrocannabinol of interleukin 2-induced lymphocyte proliferation and lymphokine-activated killer cell activity

The major psychoactive marijuana component, delta-9-tetrahydrocannabinol (THC), suppressed proliferation of murine spleen cells stimulated with recombinant human interleukin 2 (IL-2) and also suppressed the appearance of the lymphokine-activated killer (LAK) cell phenomenon in IL-2-treated spleen cell preparations. Cell function was depressed in a dose-dependent manner with as little as 2.5 μg/ml THC (8μM). In addition, spleen cells previously stimulated in culture with IL-2 and then incubated with THC for 4 h prior to target cell addition, displayed suppressed cytolytic activity against both YAC-1 and EL-4 tumor targets. Killing of EL 4 cells was suppressd at lower drug doses than the killing of YAC-1 targets. These results suggest that THC can suppress several important functions of IL-2 including clonal expansion of lymphocytes, expansion of killer cell populations and stimulation of killer cell cytotoxic activity.

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis.

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.

The HTLV-III envelope protein contains a hexapeptide homologous to a region of interleukin-2 that binds to the interleukin-2 receptor

A region of human interleukin-2 (IL-2) which was predicted to be a contact point with its receptor was used to locate a homologous region in the envelope protein of human T-lymphotropic retrovirus (HTLV-III). This homologous six amino acid peptide from the carboxy (C)-terminus of the HTLV-III envelope protein was found to inhibit the biological activity of human IL-2 in a murine spleen cell proliferation assay. When conjugated to a carrier protein, this peptide inhibited the binding of radiolabelled IL-2 to its receptor. The biological activity of the peptide was antagonized by a six amino acid peptide fragment of the IL-2 receptor which was predicted to be the contact point on the receptor that corresponded to the binding region of IL-2. The HTLV-III peptide also inhibited the binding of radiolabelled IL-2 to polyclonal anti-IL-2 antiserum. These data support the previous assignment of contact points between IL-2 and its receptor. They also suggest two possible mechanisms of immunosuppression during acquired immunodeficiency syndrome (AIDS). One involves direct competition of the envelope protein or its fragments with IL-2 for binding to the IL-2 receptor. The other involves antibodies to the envelope protein which crossreact with and neutralize IL-2.

Immunomodulating activities of ethylene-2,2'-bis(dithio)bis(ethanol) and related compounds. Effects on murine lymphocyte proliferation and functions in vitro

HEDS (2-hydroxyethyl disulfide) and structurally related compounds were found to affect lymphocyte proliferation and functions in vitro. HEDS, and ADA 202–718 (ethylene-2-2'-bis(dithio)bis(ethanol)), were shown to stimulate the proliferation of murine spleen cells. The growth of populations of murine T-cells (thymocytes) was not stimulated. HEDS and ADA 202–718 enhanced the allogeneic response in the mixed lymphocyte reaction and stimulated the formation of antibody producing cells in a primary humoral immune response. A similar stimulatory effect was observed in a secondary humoral response towards a T-cell specific antigen, DNP-keyhole limpet hemocyanine. Neither ADA 202–718 nor HEDS exhibited γ-interferon inducing ability, when tested on quiescent Balb/c spleen cell cultures in absence of antigen. However, HEDS, and especially ADA 202–718 potentiated the allogen-induced γ-interferon production and release in the mixed lymphocyte reaction. Both HEDS and ADA 202–718 seemed to induce or stimulate the release of an interleukin-1-like activity as well as interleukin-2 in Balb/c spleen cells. No obvious effect, however, was seen with either compound on maturation or immune-phagocytic activity of bone-marrow derived macrophages.

Regulation of Fc fragment-induced murine spleen cell proliferation.

Murine splenic lymphocytes proliferate in response to supernatant material derived from Fc fragment-pulsed splenic adherent cells. The stimulatory supernatant results from the interaction of Fc fragments with adherent cells or adherent cell supernate. Isolation of the stimulatory material in the supernate by Sephadex chromatography revealed that the mitogenic component was a cleavage product of Fc with a mol wt of approximately 14,000. The spleen cell type responsible for the generation of mitogenic Fc subfragments appears to be a macrophage. Unstimulated macrophages release an active supernate without being exposed to Fc fragments. The supernate of unstimulated macrophages apparently contain an enzyme which is capable of cleaving Fc fragments into the 14,000-mol wt mitogenic molecules. The spleen cell population induced to proliferate in response to the adherent cell supernate is present in T-cell depleted and Sephadex G-10 filtered cell preparations. Depletion of cells bearing immunoglobulin on their surfaces results in a reduced proliferative response to the mitogenic supernatant material indicating that it is probably a B cell.