Additive effects of suboptimal doses of estrogen and cortisone on the suppression of T lymphocyte dependent inflammatory responses in mice

Many immune-mediated inflammatory diseases are treated with corticosteroids. This type of treatment is, however, often afflicted with side-effects such as osteoporosis and atherosclerosis. During the last decades also sex steroids, such as estrogens, have been shown to have immunoregulatory properties. In this report we studied the effect of combined treatment with suboptimal doses of dexamethasone and estradiol on T lymphocyte mediated delayed type hypersensitivity (DTH), granulocyte-mediated inflammatory responses, immunoglobulin production and antigen specific antibody responses in mice. The results show that the two hormones display additive effects on suppression of DTH. In contrast, such additive effects were not observed in granulocyte-mediated inflammation. B lymphocyte activity, measured by immunoglobulin production and antigen-specific antibody responses, were increased after exposure to estradiol and suppressed by dexamethasone. In mice treated with both hormones the up regulation of B lymphocytes was still evident. The results could indicate the potential to use combinations of corticosteroids and estrogen in the treatment of T lymphocyte dependent rheumatic diseases such as rheumatoid arthritis (RA). In addition, the B lymphocyte stimulation by estrogen in cortisone exposed mice stimulate to future studies in humans if estrogen containing contraceptives or post menopausal hormone treatment could have triggering effects in patients with immune complex mediated diseases also when they are on corticosteroid treatment.accepted by I. Ahnfelt-Rønne     

Nonspecific suppression of primary antibody responses and presence of plastic-adherent suppressor cells in Toxoplasma gondii-infected mice.

The effect of Toxoplasma infection on primary antibody responses to both T-dependent and T-independent antigens was examined in mice. Drastic suppression of primary responses to sheep erythrocytes (SRBC) occurred when mice were immunized 7 days after infection. The suppression was observed in both 2-mercaptoethanol-sensitive and -resistant hemagglutinin responses. Anti-dinitrophenol (DNP) immunoglobulin E and G1 responses to DNP-conjugated keyhole limpet hemocyanin were also suppressed by infection. It was suggested that the suppressive effect is nonspecific for the antigens and immunoglobulin classes produced. Anti-DNP responses to DNP-Ficoll, a T-independent antigen, were suppressed by infection, but the suppressive effect was weaker than that on the responses to SRBC. This suggests that both T and B cells are suppressed by infection. In vitro responses of infected mouse spleen cells to SRBC and DNP-Ficoll confirmed the results of in vivo experiments. In addition, the presence of plastic-adherent suppressor cells was demonstrated in the spleen cells of infected mice, which suppressed the ability of normal mouse spleen cells to mount an SRBC-specific plaque-forming cell response. These plastic-adherent suppressor cells appeared to be a major cause of nonspecific suppression of primary antibody responses in Toxoplasma-infected mice.     

Serum-mediated suppression of lymphocyte transformation responses in coccidioidomycosis.

Lymphocyte transformation (LT) responses to coccidioidin (CDN) and spherulin were suppressed in 11 (73%) of 15 patients with active coccidioidomycosis when their mononuclear cells were assayed in autologous serum as compared to serum from healthy, CDN skin test-positive subjects. Suppressed LT responses were specific for Coccidioides immitis antigens in 7 (64%) of the 11 patients. Immunoaffinity chromatography of patient sera with Staphylococcus protein A adsorbed the suppressor component(s) and thereby established that suppression was attributed to immunoglobulin G, either alone or complexed with antigen. The possibility that suppression was mediated by immune complexes was examined by adding complexes formed in vivo or in vitro to mononuclear cell cultures of healthy CDN-reactive persons before LT assays. Although complexes prepared in this manner were reactive in an enzyme-linked immunosorbent assay designed to detect Coccidioides antigen-specific immune complexes, no suppression of LT responses was observed. We conclude that serum-mediated suppression of LT responses in coccidioidomycosis is attributed to monomeric and not immune-complexed immunoglobulin G antibody.     

Cortisol mediated suppression of salmonid lymphocyte responses in vitro

The suppressive activity of cortisol on the in vitro induction of coho salmon (Oncorhynchus kisutch) B cell activation was examined. Suppression was observed with splenic and pronephric (anterior kidney) derived lymphocytes. The kinetics of cortisol-induced suppression revealed distinct differences in the sensitivity of splenic and pronephric lymphocytes. Pronephric lymphocytes were only sensitive to cortisol early in the induction of the antibody response, whereas the splenic cells were sensitive to cortisol throughout the culture period. Addition of supernatants from antigen stimulated pronephric cultures completely restored the ability of pronephric lymphocytes to produce an antibody response, suggesting that this glucocorticoid-suppression may be mediated by inhibition of lymphokine production.     

Immune depression in trypanosome-infected mice. I. Depressed T lymphocyte responses.

Using a wide range of experimental conditions, several kinds of T lymphocyte responses in spleen cell populations from trypanosome-infected mice were studied. Lymphocyte stimulation after culture with the mitogen concanavalin A or with histoincompatible cells differing at H-2 or minor lymphocyte-stimulating loci was reduced or abolished in spleen cells from infected mice when compared with responses of spleen cells from uninfected controls. In addition, cytotoxic lymphocytes were not generated in mixed lymphocyte cultures which contained spleen cells from infected animals. Allogeneic skin grafting experiments performed with normal and infected mice showed that a decreased T lymphocyte response also occurs in vivo. The depressed immune responses were not simply due to low numbers of T lymphocytes in spleens of infected animals, but reflected a generalized immune depression which was not antigen-specific.     

Nonspecific suppression of initiation of memory cells in Toxoplasma gondii-infected mice.

The effect of Toxoplasma infection on initiation and expression of memory cells to dinitrophenol (DNP)-conjugated protein antigens in humoral immune responses was studied in mice. Marked suppression in the initiation of memory cells to DNP-conjugated keyhole limpet hemocyanin occurred in the acute phase of infection. However, once the memory cells were induced before infection, expression of the memory cells was not affected. Moreover, the suppression of priming occurred on both T and B cells. The suppressive effect was observed in all immunoglobulin classes tested, i.e., immunoglobulin M (IgM), IgG1, IgG2, and IgE, regardless of the four kinds of DNP-conjugated protein antigens. This nonspecific suppression could be induced only by living toxoplasmas, by either the peroral or parenteral route, but not by lysed organisms. A transfer of normal spleen cells could not restore the ability of infected mice to initiate memory cells. Furthermore, the suppressive effect of the infected mice was not removed by 400 R of gamma-irradiation. These results suggested that irradiation-resistant suppressor cells cause nonspecific suppression of the initiation of memory cells in Toxoplasma-infected mice.     

Tumor-induced changes in T cell mitogen responses in rats: suppression of spleen and blood lymphocyte responses and enhancement of thymocyte responses.

Concanavalin A (Con A) and phytohemagglutinin (PHA) responses of spleen and blood lymphocytes from tumor-bearing (TB) rats were found to be markedly depressed in 4 different models employing tumors of spontaneous origin. Removal of phagocytic cells from both spleen and blood lymphocyte suspensions led to a complete restoration of the responses, indicating that the decreased responses were not due to intrinsic defects in the lymphocytes. The reduction was shown to be due to the inhibitory effect of an increase in the percentage of phagocytic cells. In addition, TB induced an atrophy of the thymus and a decrease in the number of thymic lymphocytes, mainly due to severe lymphocyte depletion in the cortex. The cells that remained in the thymus exhibited increased responsiveness to PHA and Con A as compared to thymus cells from normal rats. Similar results were found in hydrocortisone acetate-treated rats, suggesting that TB leads to a decrease in nonresponsive, cortical corticosteroid-sensitive thymocytes.     

Specific suppression of antibody responses by soluble protein-specific, class II-restricted cytolytic T lymphocyte clones

Antigenic stimulation with exogenous soluble proteins induces cytotoxic T lymphocytes (CTL) recognizing antigenic peptides presented on class II major histocompatibility complex (MHC) molecules. Such CTL have been shown to lyse preferentially B cells expressing immunoglobulin receptors reactive with the relevant antigens, presumably because such B cells can efficiently trap and present the antigen. Therefore, possible involvement of soluble protein antigen-specific CTL in specific suppression of antibody responses has been hypothesised. In this report, keyhole limpet hemocyanin and ovalbumin-specific, class II-restricted CD4+ CTL clones established from lymph nodes of immunized mice were examined for their suppressive activities on antibody production. When these CTL clones were added to in vitro secondary cultures, genetically restricted, carrier-specific suppression of anti-2,4,6-trinitrophenyl antibody production was observed. These data therefore demonstrate that CTL directed toward soluble antigens are capable of mediating specific suppression of antibody responses. Furthermore, the antibody response of MHC-heterozygous F1 lymphocytes was almost completely suppressed by a CTL clone restricted to one parental class II MHC antigen, indicating that the mechanism of suppression by these CTL is distinct from that by classical suppressor T cells.     

T cell-mediated immunity to oncornavirus-induced tumors III. Specific and nonspecific suppression in tumor-bearing mice

A strong cell-mediated immune response against Friend, Moloney, Rauscher virus-induced (FMR) cell surface antigens has been demonstrated previously in mice which reject oncornavirus-induced tumors. In order to identify an eventual suppressor mechanism in animals with progressively growing tumors, experiments were initiated in C 57 BL/6 mice bearing either a murine sarcoma virus (MSV) tumor or Moloney virus-induced lymphoma (MBL2). Progressive tumor growth was induced (a) in viremic animals first infected with Moloney murine leukemia virus (M.MuLV), then inoculated as adult with MSV; (b) in nonviremic animals injected with MBL2 lymphoma cells. In the absence of tumor cells, viremia induces specific tolerance for which there is no evidence for suppressor cells. In tumor-bearing mice, specific suppressor T cells are detected which are able to inhibit the generation of anti-FMR cytolytic T lymphocytes in vitro and enhance the tumor growth in vivo. In addition to the specific suppressor T cells, a nonspecific suppressive activity mediated by metastatic T lymphoma cells is demonstrated in the spleens of lymphoma-bearing animals. The respective role of the virus and tumor cells in the induction of tolerance to M.MuLV-induced antigens, and their relationship to other components of the specific cell-mediated immune response is discussed.     

双阴性调节性T细胞在免疫抑制中的作用及其机制

αβTCR+CD3+CD4-CD8-双阴性调节性T细胞(DN Treg细胞）被证实具有通过对效应性T细胞的直接杀伤作用从而抑制免疫应答的能力。DN Treg细胞与特异性抗原接触后增加其调节活性,该过程部分通过其对抗原呈递细胞表面MHC抗原肽复合物的识别作用介导。DN Treg细胞与靶细胞接触后可通过Fas和Fas配体之间的交互作用介导其杀伤效应。对DN Treg细胞功能特性、分子表达方式及其激活机制的深入研究将有助于探索临床新的治疗手段。     

Murine cytomegalovirus-induced suppression of antigen-specific cytotoxic T lymphocyte maturation

Antigen-specific cytotoxic T lymphocyte (CTL) maturation was inhibited in mice acutely infected with murine cytomegalovirus (MCMV). When immunization with Simian virus 40 (SV40) either preceded or followed infection with MCMV by 1 day, the frequency of SV40-specific CTL precursors among lymph node cells (LNC) was significantly reduced compared to noninfected mice. Replication of the herpesvirus in LNC could not be detected; however, MCMV rendered noninfectious by heat treatment was not suppressive to CTL development. Lymph node cells from nonimmunized, MCMV-infected mice contained a cell(s) present in low frequency which suppressed in vitro maturation of SV40 CTL in immune lymph nodes from mice not infected with MCMV. These suppressor cells did not affect the antigen- and interleukin-2-dependent growth nor cytotoxic activity of a mature, SV40-specific CTL clone. These results indicate that MCMV interferes with immunoregulatory functions required for development of antigen-specific CTL precursors to mature effector CTL. The immunosuppression is mediated at least in part by the MCMV-induced suppressor cell(s).     

Plexin-A4 negatively regulates T lymphocyte responses

Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4-/-) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4-/- mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4-/- mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4-/- T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1(Sema-)) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A-/-) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.     

Kinetics of T lymphocyte responses to persistent antigens

Long term sequential study of immune responses in the same individuals is difficult from the time commitment required and the problem of maintaining enough subjects to provide for comparative analysis. We closely studied one hundred women with silicone mammary devices through cross sectional analysis up to 26 years post implantation and a similar sample of women to 6 years post explantation. The T cell index, calculated from tritiated thymidine incorporation during lymphoblast transformation, rose to a post implant peak at 10.5-12.0 years, falling progressively over the next 14.0-15.5 years to values indicative of probable immune quiescence. Post explantation, the index rose over the first 3 years and then sharply declined to within the range for unexposed controls. The shape of these time curves contains considerable information referent cell dynamics for both stimulatory and inhibitory factors and for demonstrating net group effects, appropriate to analysis in the cross sectional perspective. When a subset of four women was studied frequently and sequentially up to 8 years, an internal oscillatory pattern emerged, focusing attention on both the stimulatory and the inhibitory aspects of long term clonal expansion. IL-2 has stimulatory and inhibitory properties at different levels of production and is considered a prime candidate as the essential cytokine. The equations have details, however, which require exploration beyond any such provisional conclusion. The analytic process was aided by normalization of oscillatory data to eliminate subject variability and by Pareto optimization to assess the trend shown by normalization. Pareto analysis revealed two minimally coordinated oscillations, one over time and the other along net clonal expansion or decline of the siloxane specific T lymphocyte clone. The segments of the time related oscillation greatly exceeded the reaction times of cytokines currently known to be active in T cell regulation. Although the ultimate controlling factor(s) may be cytokine or chemokine combinations, the data are compatible with some more basic regulatory factor(s) of cell integrity, including limits on the number of cell divisions which can be sustained in long term immunopathic lesions, among other processes.     

T lymphocyte function in protein deprived mice.

The thymus-dependent (T-dependent) limb of the immune response in protein malnourished mice was examined by using several tests of T cell function, both in vivo within the intact animal, and after removal of lymphocytes from the protein deprived host. The capacity to provide help for IgG antibody responses and the DTH response to antigen were diminished after short-term (3 weeks) deprivation. However, both these responses were normal in mice maintained on a protein deficient diet for 11 weeks. The depressed DTH responses of protein deprived mice were due, at least in part, to a failure to mount the inflammatory phase of the response. Finally, using a graft-versus-host reaction (GvHR) as an index of T lymphocyte function, we found that spleen cells from malnourished donors were fully capable of inducing the splenic and intestinal changes associated with a GvHR in unirradiated F1 animals. Overall, these results suggest that T cell function is not irreversibly damaged by protein deprivation. However we propose that cell-mediated immune responses are influenced indirectly by the restrictive environment which interferes with cell migration, and by the impaired capacity of protein-deprived animals to mount a non-specific inflammatory reaction.     

Nonspecific and Candida-specific immune responses in mice suppressed by chronic administration of anti-mu

CBA/J mice were immunosuppressed by repeated administration of goat antibody specific for mu chain of immunoglobulin M (IgM) and tested for nonspecific and Candida albicans-specific immune responses. Immunosuppression was demonstrated by a dramatic reduction in the number of antibody-forming cells in the spleens of anti-mu-treated mice when immunized with sheep erythrocytes, by greatly reduced in vitro responsiveness of both spleen and lymph node lymphocytes from anti-mu-treated mice to lipopolysaccharide, and by a large reduction in the number of splenic IgM-positive cells. T cell function, on the other hand, appeared to be relatively unaltered in anti-mu-treated animals, in the cytotoxic T lymphocyte activity against an allogeneic target was similar in splenocyte cultures from anti-mu- and mock-treated animals, and splenic and lymph node lymphocytes proliferated in response to concanavalin A in a lymphocyte stimulation assay. Moreover, Candida-specific delayed hypersensitivity to two different Candida antigens, one cell wall-derived (GP) and the other cell membrane-derived (BEX), was of comparable intensity in immunosuppressed and normal animals. When anti-mu- and mock-treated mice were immunized by the cutaneous inoculation of viable C. albicans blastospores and then challenged intravenously to assess the development of protective immunity, only mock-treated animals, male and female, had significant (p less than or equal to 0.05) protective responses demonstrable by reduction in the number of colony-forming units cultured from their kidneys 28 days after intravenous challenge. If consideration was given to the number of animals which had cleared Candida completely from the kidney, however, there appeared to be protective responses operative in the female anti-mu-treated animals as well. Neither anti-mu-treated males nor females, when immunized and challenged with C. albicans, produced Candida-specific antibody detectable by counterimmunoelectrophoresis, whereas all immunized and challenged mock-treated animals produced antibody. The data are consistent with the hypothesis that anti-mu treatment has little effect on multiple cellular immune functions, including those specific for C. albicans, and the combination of antibody, cell-mediated immunity and innate defenses are responsible for solid systemic defense against the fungus.     

Antigen-specific suppression of cytotoxic T cell responses in mice. I. Suppressor T cells are not cytotoxic cells.

The main conclusion from these experiments is that the antigen-specific suppressor T cell of mice which inhibits the induction of cytotoxic T lymphocytes is not itself a cytotoxic T cell. This conclusion is supported by two main observations: first, a certain cell number from first-step cultures which was suppressive in the presence of a high dose of antigen actually helped the cytotoxic response at a lower antigen dose. This observation is difficult to reconcile with the hypothesis that suppression is due to the killing of the stimulator or the responder cells in the second-step culture by cytotoxic T cells. Second, cells from first-step cultures of cortisone-treated mice displayed cytotoxic activity but had no suppressive effect on the generation of killer cells. It was further demonstrated that these cells failed to influence in any way the suppressive effect, however weak, of cells from first-step cultures of normal spleen. We therefore favor the view that the suppression observed in this system is due to a regulatory signal which occurs as a result of the ability of both inhibitory cells and responder cells to recognize and respond to allogeneic determinants expressed on the surface of stimulator cells. The suppressor T cells described here act by linked associative recognition of antigen. That is, suppressor T cells only inhibit the induction of a precursor cytotoxic T cell in the presence of an antigen to which both the precursor cell and the suppressor cell can bind. In this sense, suppressors act in a manner analogous to helper T cells in T-B cell cooperation; carrier-specific helper T cells only enhance an anti-hapten B cell response in the presence of hapten-carrier conjugates. Similarly, alloantigen a (carrier)-specific suppressor T cells only inhibit alloantigen b (hapten)-specific cytotoxic responses in the presence of (a × b)F₁ stimulator cells (hapten-carrier conjugate), not in the presence of a mixture of parental stimulator cells (a + b).     

Nonspecific esterase staining of lymphocyte subsets in leprosy

A comparative study of lymphocyte subpopulations from leprosy patients were carried out using nonspecific esterase (ANAE) staining and monoclonal antibodies. Regardless of the methodology used, an intense reduction in the total number of the T helper/inducer subset and an inversion of the helper/suppressor ration could be observed. These alterations were more evident with the progress of the disease from the tuberculoid (TL) to the lepromatous (LL) pole. Thus ANAE may be considered an economical and reliable method to be used in the study of the immune status of leprosy patients.     

Nonspecific influx of cytotoxic T cells into influenza virus-infected lungs of mice

The influx of cytotoxic T cells into A/WSN influenza virus-infected mouse lungs was investigated by adoptive transfer with [¹²⁵I]5-iodo-2-deoxyuridine ([¹²⁵I]UdR)-labeled syngeneic cells. More A/WSN virus-immune secondary effector T cells were localized in the A/WSN virus-infected lungs than in the uninfected lungs, the ratios being in the range 2.5–5.0 Nonimmune control cells, in contrast, showed no significant difference in the localization pattern in infected compared to uninfected lungs. Virus-immune T cells of different antigenic specificities, i.e., Sendai or Semliki Forest virus-immune secondary effector T cells, however, also localized more in A/WSN virus-infected than in uninfected lungs, but the heterologous virus-immune T cells were retained in the A/WSN virus-infected lungs for a shorter time than A/WSN virus-immune secondary effector T cells. The work suggests mechanisms other than antigenic specificity may be important in the localization of immune T cells in virus-infected lungs.