Mechanisms of corticosteroid action on lymphocyte subpopulations. IV. Effects of in vitro hydrocortisone on naturally occuring and mitogen-induced suppressor cells in man.

The effects of in vitro hydrocortisone (OHC) on human peripheral blood (PB) suppressor cell function were investigated. Two types of suppressor cells were studied: (i) the naturally occurring PB suppressor cell seen in 10% of normal people whose lymphocytes do not respond to in vitro PWM stimulation with direct anti-SRBC PFC responses, and (ii) Con A-generated suppressor cells. The addition of OHC to PWM-stimulated cultures from nonresponders reconstituted the PFC response in two of three individuals. The addition of OHC to allogenic cocultures of nonresponder and responder lymphocytes completely inhibited the ability of the naturally occurring suppressor cell of the nonresponder cultures to inhibit the PFC responses of normal responders. Preincubating the nonresponder cultures in 10^?5M OHC for 30 min followed by washing did not inhibit suppressor function, whereas readdition of OHC to cocultures did inhibit nonresponder suppressor cell function. The addition of up to 10^?4M OHC to previously generated Con A-activated suppressor cell-fresh cell cocultures in vitro did not prevent or inhibit mitogen-activated suppressor cell function. However, preincubation of PB cells for 6 hr prior to the addition of Con A prevented the generation of suppressor cells and in two of eight experiments generated a population of cells which were in and of themselves mitogenic for autologous fresh PB. Thus, the function of naturally occurring suppressor cells as well as the induction but not the function of Con A-activated suppressor cells is sensitive to pharmacologic levels of OHC. The effect of OHC on naturally occurring suppressor cell function or on the generation of suppressor cells by Con A did not involve cell lysis, but rather was a reversible phenomenon requiring the continued presence of OHC in culture.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. V. Effects of in vivo hydrocortisone on the circulatory kinetics and function of naturally occuring and mitogen-induced suppressor cells in man.

The present study evaluated the effect of in vivo administration of hydrocortisone (OHC) to normal subjects on the kinetics and function of naturally occurring and mitogen-induced suppressor cells (SC). It was demonstrated that in vivo OHC abrogates the function of naturally occurring SC and converts normal nonresponder peripheral blood (PB) cells to responder status. However, 4 hr following OHC administration at the point of maximal lymphocytopenia, the cells remaining in the circulation could still be activated by Con A to suppress. In addition, unstimulated cells obtained 4 hr after OHC administration markedly enhanced PFC responses when cocultured with fresh autologous cells. It was demonstrated that this enhanced PFC response was independent of 48-hr incubation, monocyte depletion, or changes in B-cell numbers and most likely represented a manifestation of in vivo OHC-induced changes in the circulatory kinetics of immunoregulatory cell populations.     

Oxytocin action: Lack of correlation between receptor number and tissue responsiveness

Brattleboro rats exhibit diabetes insipidus (DI) because of a genetic autosomal recessive defect in the synthesis of vasopressin; oxytocin is synthesized normally. Preliminary work suggests that elevated circulating oxytocin levels may compensate for the absence of vasopressin. To evaluate the consequences of presumed elevations of oxytocin levels, oxytocin binding and tissue responsiveness have been measured in the uterus and epididymal fat cells of homozygous-DI (HoDI) and heterozygous-DI (HeDI) animals and Sprague-Dawley and Long-Evans controls. Surprisingly, whereas membranes from HoDI rat uteri exhibited an 85% reduction in oxytocin binding, the biological response (contraction) to oxytocin was indistinguishable from the uteri of HeDI or Sprague-Dawley animals. The uterine response to carbachol was also normal in HoDI rats. In contrast, in adipocytes from HoDI animals, the biological response to oxytocin (glucose oxidation) was abolished, whereas the binding of oxytocin was normal; insulin-stimulated glucose oxidation was, however, normal. These results indicate that receptor binding, while critical to hormone action, is not the sole determining factor. With oxytocin action, postreceptor mechanisms are most important in determining oxytocin responsiveness.     

Lack of correlation between in vitro corticosteroid effect on hemopoietic colony formation and response to corticosteroid therapy in aplastic anemia

We performed hemopoietic colony culture assays in 15 patients with aplastic anemia (AA) in order to test the effect of hydrocortisone (HC) on late erythroid colony (CFU-e) formation of the patients' marrow and to correlate the in vitro culture results with the clinical response to corticosteroid therapy. HC enhanced CFU-e growth in four patients. All four patients failed to respond to corticosteroid, but three improved with with androgens. The addition of HC did not increase CFU-e colony formation in 11 patients. However, two of them responded to corticosteroid therapy. Among the nine patients showing no HC effect in vitro, two subsequently improved with androgens and one each with anti-thymocyte globulin and anti-lymphocyte globulin. The results suggest that the in vitro corticosteroid effect may not necessarily correlate with responsiveness to corticosteroid therapy.     

Lack of correlation between two markers of lymphocyte differentiation: 5' nucleotidase activity and T lymphocyte colony formation

Ecto 5' nucleotidase (5' NT) activity and T lymphocyte colony formation (TLCF) are both reputed to be markers for lymphocyte maturation. In order to determine whether these two expressions of lymphocyte activity are related, we compared 5' NT activity with the TLC forming capacity of mononuclear cells from three study groups: normal adults, cord blood, and patients with immunodeficiencies. Despite individual examples of correlation between these two measurements, there was poor overall correlation in any of the groups studied. Although both measurements may reflect maturation of certain cellular activities, these are unlikely to be related.     

Lack of correlation between hormonal effects on cyclic AMP and glycogenolysis in rat liver.

Portions of liver were obtained by biopsy from rats infused with various concentrations of glucagon or epinephrine and analyzed for cyclic AMP, glycogen, phosphorylase activity, and glycogen synthetase I activity. The response of tissue cyclic AMP to glucagon or epinephrine was far less sensitive than other metabolic parameters; at certain lower doses of glucagon or epinephrine, glycogen decomposed without a simultaneous increase in the hepatic level of cyclic AMP. It is probable that hormonal activation of adenylate cyclase results in an increase of cyclic AMP only in its small “active” pool without detectable changes in its much larger inactive or bound pool. Though the active cyclic AMP is expected to be released into the circulation or to be labeled with [³H]adenine in preference to the inactive nucleotide, neither the increase of cyclic AMP in the vena cava in vivo nor the incorporation of [³H]adenine into tissue cyclic AMP in liver slices in vitro exhibited more sensitivity to glucagon than the hepatic level of cyclic AMP as a whole. Thus, it remains to be settled whether cyclic AMP is compartmentalized in the cell or plays no essential role in the stimulation of hepatic glycogenolysis induced by small doses of hormones.     

Human lymphocyte subpopulations: the effect of pregnancy.

Peripheral blood lymphocytes from pregnant and nonpregnant females were studied for the presence of the following surface receptors: (i) Receptor for heat-aggregated human IgG (AggIgG); (ii) Receptor(s) for complement components; (iii) Receptor for sheep red blood cells (SRBC). Absolute numbers of lymphocytes with receptors for complement and those with receptors for SRBC were present in equal numbers in both groups. However, in the pregnant females there was a significantly lower number of lymphocytes with receptors for AggIgG. It is hypothesized that some of the immunological changes which occur during pregnancy may be mediated partly through changes in the total numbers of certain lymphocyte subpopulations.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

Selective effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and corticosteroid on in vitro lymphocyte maturation

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the corticosteroid dexamethasone have potent effects on lymphocyte function, although the effects of the former have not been well characterized. In the present studies murine B cell maturation was used as a model system to examine and compare the effects of TCDD and dexamethasone on cell function. Immunosuppression by TCDD and dexamethasone is mediated by binding to specific intracellular R referred to as the Ah and glucocorticoid R, respectively. Although both compounds were comparable in their ability to inhibit antibody responses to the T-independent antigen TNP-LPS, the events responsible for suppression were found to be distinct. Dexamethasone, although affecting multiple stages of B cell maturation, had its primary effect very early, manifested by inhibition of the phosphoinositide signal transduction pathway. This was evidenced by a decrease in accumulation of inositol phosphate and surface Ia antigen expression as well as an inability to enter the cell cycle after stimulation with anti-Ig. In contrast, neither early signaling events nor proliferation were affected in B cells treated with TCDD. However, TCDD inhibited Ig secretion after stimulation of B cells with T cell-replacing factor, suggesting that TCDD modulates the differentiation of B cells into plasma cells. These differential results were confirmed by monitoring the expression of surface antigens that occur on B cells, including Ia, 7D4, and PC.2, during this maturational process. Whereas dexamethasone inhibited the expression of surface antigens that occur early in maturation (Ia and 7D4), TCDD blocked only the expression of the plasma cell marker PC.2. Although TCDD altered later stages of the B cell cycle, the presence of TCDD was required at the time of initial activation to be effective, suggesting that TCDD may interfere with early cell programming.     

Lack of species specificity in the action of immunosuppressive factors of tumor cells and absence of competition between them and recombinant interleukin-2 and phytohemagglutinin for lymphocyte membrane receptors

We studied some possible mechanisms of action of immunosuppressor factors (ISF) produced by tumor cells on lymphocyte proliferation. ISF of murine tumor cell lines inhibited the mitogen induced proliferation of murine splenocytes as well as human mononuclear blood cells. Normal human mononuclear blood cells or concanavalin A-activated murine spleen cells preincubated with phytohemagglutinin (PHA) or interleukin 2 (IL-2) respectively, were strongly suppressed by ISF in response to these activators. When preincubated with splenocytes or blood cells for 2 h at 4 degrees C following washing, ISF suppressed the lymphocyte proliferation as effectively as when being with cells during all period of cultivation. ISF inhibited mitogen-induced lymphocyte proliferation at low dilutions. There was no competition for lymphocyte membrane receptors between these functionally heterogenic kinds of ISF. Collectively, these results show that ISF acted when being attached to some lymphocyte membrane receptors.     

Cellular immunity in allograft rejection: role of lymphocyte subpopulations and T-cell subsets in rat cardiac allograft rejection

In this study, cellular requirements for rejection are examined by the use of adoptive transfer assays in the ACI to Lewis cardiac allograft model. The findings show that adoptive transfer of 1 x 10(8) spleen cells (SpL), 5 x 10(7) T-cells, and 2 x 10(7) helper T-cells (W3/25+) obtained from normal, nonsensitized donors restores acute ACI graft rejection in sublethally irradiated (750 rad) Lewis recipients. In contrast, reconstitution with 2 x 10(7) cytotoxic T-cells (0X8+) does not restore first-set graft rejection. Reconstitution of the irradiated recipients with either W3/25+ or 0X8+ T-cells obtained from specifically sensitized syngeneic donors resulted in acute rejection. The W3/25+ T-cell subset was significantly more potent (P less than 0.01) in effecting rejection on a per-cell basis. Adoptive transfer of SpL, T-cells, and 0X8+ T-cells obtained from sensitized rats led to accelerated cardiac allograft rejection in the naive secondary recipients while W3/25+ T-cells did not. This study suggests that although the W3/25+ T-cells alone have the capacity to initiate first-set graft rejection, both W3/25+ and 0X8+ subsets appear to be critical to the completion of rejection of heart allografts. We also examined the capacity of adoptively transferred B-cells from sensitized donors to influence graft rejection. Our findings suggest that while B-cells fail to restore the capacity for graft rejection in irradiated recipients, they can, however, present MHC antigens to the secondary naive host thus causing allosensitization which results in accelerated rejection of a subsequent graft.     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.     

Characterization of cytotoxic human lymphocyte subpopulations: the role of Fc-receptor-carrying cells.

Natural cytotoxicity against tumor-derived monolayer and lymphoblastoid cell lines was found to be most marked in the non-T non-B subpopulation of human lymphocytes. A high proportion of cells in this population were Fc receptor positive. This receptor seems to be important in exerting the cytotoxic effect. The T-cell fraction had a weak cytotoxic activity for which Fc-positive T cells were responsible. Steps in the cell separation procedure such as EA resetting and ammonium chloride treatment impaired cytotoxic activity. However, the cells recovered to a large degree during the 48-hr assay. Cells incubated at 37 °C prior to the functional test regained part of the activity and could thus be used in a short-term, 8-hr assay. Within a certain effector cell range, the dose-response curves were linear. Thus, it was possible to express the efficiency of a certain fraction in lytic units (the cells which cause 50% lysis of the target cell). Considering the representativeness of the cell type in the total population, the non-T non-B cells were calculated to be responsible for 24% of the total activity though their proportion was only 7%.     

Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study

The intracellular distribution of the glucocorticosteroid and progesterone receptors (GR and PR, respectively) was studied immunohistochemically. In control adrenalectomized (Adx) rat liver, immunostaining of paraffin sections revealed GR in cell nuclei, with a wide range of intensity between individuals. Following dexamethasone (Dex) treatment, the nuclear staining was uniformly high in all animals; the cytoplasmic staining was always weak and remained unchanged after Dex treatment. In frozen sections, the GR immunoreactivity in cell nuclei was weak in the absence and very strong in the presence of Dex, while no GR-specific cytoplasmic staining was observed. In frozen sections fixed in vapor of formaldehyde to avoid any artifactual redistribution of the receptor, some GR immunostaining was observed in the cytoplasm and the nucleus. In contrast, in paraffin as well as in frozen sections of chick oviduct, fixed by immersion or in vapor, PR was exclusively nuclear, including in the absence of progesterone, and the intensity of immunostaining was not modified by progesterone treatment. In order to verify if loss of nuclear receptors during tissue preparation could explain the differences in nuclear immunostaining observed between hormone-free and hormone-occupied GR, and between GR and PR, frozen sections of Adx rat liver and chick oviduct were preincubated at 4 degrees C in buffer solutions before the fixation procedure. It was found that hormone-free GR diffused out of the nucleus faster than hormone-occupied GR nuclei, and that nuclear GR diffused faster than nuclear PR. Based on these results, we propose that, during the fixation procedure, the fraction of nuclear GR which diffuses out of the nucleus is much smaller in the presence than in the absence of Dex. This lesser loss of nuclear GR after Dex treatment results in an increase of immunostaining after hormonal administration, which might have been erroneously interpreted as a sign of translocation from cytoplasm to nucleus. That the nuclear PR detection is not modified by progesterone treatment may be explained by its reduced diffusibility as compared to nuclear GR. This hypothesis does not rule out the existence of some cytoplasmic GR, whose significance remains unclear, but it offers a unified mechanism of action for all steroid hormone receptors. In the case of glucocorticosteroids, as already proposed for estradiol and progesterone, no step of cytoplasm to nucleus translocation would be required for hormone action, and transformation-activation would occur in the nucleus, resulting in tighter binding of the hormone receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the adherence of human lymphocytes to measles-infected cells by prostaglandin E1: a differential effect on lymphocyte subpopulations

Prostaglandins of the E series (PGE) may serve as important regulators of human immune responsiveness. The present study was designed to examine the possibility that PGE may effect human lymphocyte function by the modulation of surface receptors. The presence of surface binding sites on human lymphocytes for measles virus antigens was studied using a rosette adherence assay. We observed that the addition of PGE1 increased the proportion of measles-infected cells (Hela-Kll) with adherent lymphocytes (75% increase at 3 x 10(-6) M PGE1). PGE was observed to enhance the adherence of purified normal peripheral T cells (87%) and T lymphoid cells (Molt 3) (27%). In contrast, no significant change in normal peripheral B cell or B lymphoid cell (Raji) adherence was observed with the addition of PGE. These results are consistent with a selective modulation of surface measles virus binding sites by PGE1 on T and not B lymphoid cells.