Barbara A. Araneo

The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells

We have investigated the ability of dehydroepiandrosterone (DHEA) to alter the production of interleukin-2 (IL-2) and to bind to a specific binding complex in antiCD3 epsilon activated T cells. Binding activity correlated with the presence of a specific DHEA binding complex in the cytosol and nuclei of DHEA-responsive T-cell hybridomas, as well as in CD4+ and CD8+ cells isolated from peripheral lymph nodes of normal mice. Scatchard analysis determined that intact lymphocytes and cytosolic fractions contained high affinity binding for [3H]DHEA (approx. 2.6 nM) with 1000-7000 binding sites existing per cell. Five of the T-cell hybridomas tested both responded to DHEA treatment with increased production of IL-2 and also contained specific high affinity [3H]DHEA binding. Four additional T-cell hybridomas were found to contain no specific [3H]DHEA binding and were also unresponsive to DHEA influences on IL-2 production. Sucrose density gradients demonstrated a 3-4s [3H]DHEA binding complex in high salt and a 7-8s binding complex in low salt. Specific binding was inhibited by preincubation of the cytosol fractions with either trypsin or chymotrypsin, or by heating to 60 degrees C for 1 h (less than 15% of control). [3H]DHEA binding was unaffected by preincubation of the cytosol fractions with ribonuclease, deoxyribonuclease, or phospholipase A. The DHEA-protein complexes bound to DNA-cellulose with the amount of binding being slightly increased by preincubation at 25 degrees C as compared to 4 degrees C. As expected, [3H]DHEA binding was inhibited by the addition of unlabeled DHEA, but was also modestly inhibited by dihydrotestosterone and cortisol. Binding of DHEA was unaffected by progesterone, dexamethasone, estradiol, androsterone, DHEAS, and beta-etiocholanolone at all concentrations tested. DHEA was incapable of inhibiting the binding of [3H]DHT to the androgen receptor or [3H]dexamethasone to the glucocorticoid receptor. Collectively, these findings suggest that murine T cells contain a specific DHEA receptor. We believe that DHEA is a steroid hormone that is directly involved in the regulation of IL-2 production by both normal and some T-cell hybridomas.

Decidual cell biosynthesis of interleukin-6: regulation by inflammatory cytokines.

Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues. We have evaluated the biosynthesis of the inflammatory cytokine, interleukin-6 (IL-6), by human decidua and its regulation by other cytokines essential to the inflammatory process. We found that decidual cells secrete small amounts of IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum. Interleukin 1 (alpha and beta) and tumor necrosis factor (TNF) all induced a significant concentration-dependent stimulation of IL-6 production by decidual cells. Treatment of decidual cells with actinomycin D or cycloheximide abrogated the increase in IL-6 production induced by IL-1 beta. Northern blot analysis of cultured decidual cells revealed an increase in IL-6 messenger RNA (mRNA) over time in response to IL-1 beta. These data indicate that IL-1 beta stimulates an increase in IL-6 mRNA and protein production, reflecting either direct gene activation or stabilization of IL-6 mRNA. The concentration range tested (0.1 to 10 ng/mL) of each cytokine is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues. Our data suggest that IL-6 is produced by human decidua in response to inflammation and, in conjunction with other inflammatory mediators, may play a role in the pathophysiology of preterm labor due to infection.

Adaptive immune responses during murine pregnancy: Pregnancy-induced regulation of lymphokine production by activated T lymphocytes

OBJECTIVE We hypothesized that the lymphokine production by splenocytes and decidual lymphocytes would be altered because of changes in immunoregulation during pregnancy. STUDY DESIGN Splenocytes and decidual lymphocytes were isolated from syngeneic and allogeneic pregnant mice at different times of gestation. The lymphocytes (10(7) cells/ml) were stimulated with anti-CD3 antibody, and culture supernatants were assayed for several lymphokines, including interleukin-2, interferon-gamma, interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Statistical analysis was by analysis of variance or paired t test. RESULTS Activated splenocytes produced significantly less interleukin-2 and more interleukin-4, interleukin-6, and interleukin-3 as murine pregnancy advanced. Production of interferon-gamma and granulocyte macrophage colony-stimulating factor by activated splenocytes peaked in the first 8 to 14 days of pregnancy. Stimulated decidual lymphocytes produced modest amounts of interleukin-6, granulocyte-macrophage colony-stimulating factor, and interleukin-3 during pregnancy but no interleukin-2, interferon-gamma, or interleukin-4. Similar results were found for both syngeneic and allogeneic matings. CONCLUSIONS Our findings indicate that splenocyte lymphokine production favors interleukin-4 production over interleukin-2 production. This finding suggests that antibody production would be enhanced and cytotoxic cellular immune responses inhibited during pregnancy. These changes occurred regardless of mating partner, suggesting that the specific antigenic stimulus during normal pregnancy does not regulate lymphokine production. Activated splenocytes and decidual lymphocytes were found to differ in their capacity to produce lymphokines, indicating that the decidua constitutes a distinct and unique immunologic microenvironment.

The Development of Effective Vaccine Adjuvants Employing Natural Regulators of T-Cell Lymphokine Production in Vivoa

Steroid hormones are important regulators of gene function in vivo. A number of naturally occurring species of steroid hormones are able to qualitatively and quantitatively influence the production of lymphokines by activated T cells in vitro. Similar mechanisms are probably also occurring naturally in vivo and could explain why mucosal and nonmucosal lymphoid organs harbor T cells having unique potentials for lymphokine production. It was established that the topical application of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to normal mice changed the pattern of lymphokines produced by activated T cells isolated from the draining peripheral lymph nodes. The hormone-treated T cells produced a pattern of lymphokines similar to that normally found in Peyers patches. Subcutaneous vaccination with a protein antigen, in a site afferent to 1,25(OH)2D3-manipulated lymph nodes, resulted in an enhanced serum antibody response and was uniquely capable of also stimulating a common mucosal immune response to the antigen as well. Common mucosal immunity was confirmed by demonstrating the presence of antigen-specific IgA and IgG responses in a number of mucosal secretions and by further establishing that antibody-secreting plasma cells had migrated to the lungs and small intestines of the hormone-treated and vaccinated animals. Additional experiments established that common mucosal immunity could also be induced in aged animals as long as the immune system of the vaccinated animals was functioning normally. This was accomplished by providing the aged animals with a dietary supplement of dehydroepiandrosterone sulfate (DHEAS). Previous studies by us have documented that aged animals provided with replacement levels of DHEAS, a natural steroid hormone whose endogenous production declines with advancing age, are able to mount normal systemic humoral and cellular immune response following subcutaneous vaccination with a variety of protein and polysaccharide antigens. The combination of supplemental DHEAS therapy with topical 1,25(OH)2D3 treatment at the time of vaccination provided the conditions needed to generate mucosal and systemic immune responses to inactivated influenza virus antigen by old animals.

Natural regulators of T-cell lymphokine production in vivo.

Summary: The mammalian immune system possesses the intrinsic capacity to evoke a wide variety of functionally distinct effector mechanisms following stimulation by a particular antigenic substance. Such diversity in available responses is absolutely essential to the immunocompetent host, which must continually deal with a diverse set of potential pathogens within its everchanging environment. The development of appropriate types of immune responses, therefore, represents a highly dynamic process that requires that an equivalent consideration be given to a large array of components, any one of which is capable of modulating the final outcome. While the nature and complexity of the antigen(s), plus the intracellular or extracellular mode of presentation, provide specificity and some selection to the developing process, the route of antigen entry, as well as the physiological status of the host at the time of antigen insult, also contribute significantly to the formation of any immune response. The overall objective of this article is to introduce the concept that platelet-derived growth factor (PDGF) (either preformed or synthesized in response to stimulation), plus a number of steroid hormones (some of which are end-organ metabolized at local tissue sites), can all play significant roles in the genesis of immunologic responses in vivo.

Anti-Interleukin-6 Antibodies Inhibit Herpes Simplex Virus Reactivation

Herpes simplex viruses (HSVs) infect epithelial cells, become localized in neurons, and can reactivate in response to a variety of stimuli, including ultraviolet light and hyperthermia. The sequence of gene activation during viral replication is known, but the molecular linkage between exogenous stimuli and HSV reactivation has not been determined. It was hypothesized that interleukin (IL)-6 acts as a signal between exogenous stimuli and neurons, stimulating HSV reactivation from latency. Mouse corneas were infected with HSV-1, and ocular reactivation was induced 5-7 weeks later by thermal stress or corneal exposure to ultraviolet light. Anti-IL-6 monoclonal antibodies were administered to the latently infected mice 8-12 h before the reactivation stimulus. Treatment with anti-IL-6 antibodies resulted in significantly lower frequencies of ocular reactivation compared with those in mice treated with a control immunoglobulin. These results support the hypothesis that IL-6 plays a role in HSV reactivation from latency.

Th1Th2-like immunity and resistance to herpes simplex labialis

To investigate potential immunologic mechanisms of resistance to recurrent herpes simplex labialis, we assayed serum antibody titers and cultured peripheral blood mononuclear cell (PBMC) cytokine production among patients with a history of frequent episodes (H+S+), herpes simplex virus (HSV)-seropositive individuals without a history of herpes labialis (H-S+) and HSV-seronegative persons (H-S-). In addition, H+S+ patients were exposed to experimental ultraviolet radiation (UVR) on the lips and the immunologic assay results compared among those who developed experimental lesions and those who did not. H+S+ patients were found to have higher median serum titers of HSV antibody and trends to lower levels of HSV-specific PBMC IFN-gamma and IL-2 than H-S+ control patients (123 vs 66, P = 0.04; 424 vs 548 pg/ml, P = 0.08; 14 vs 26 pg/ml, P = 0.14, respectively). Correlation of the results with the occurrence of experimental lesions showed the inverse: the subgroup of H+S+ patients with UVR-induced lesions had lower titers of antibody and trends to higher levels of IFN-gamma and IL-2 than H+S+ patients who could not be induced (93 vs 149, P = 0.02; 501 vs 347 pg/ml, P = NS; 26 vs 11 pg/ml, P = NS, respectively). The size and duration of UVR-induced lesions showed positive correlations with IFN-gamma and IL-2 levels (r = 0.60-0.67, P = 0.02-0.04). Although the small number of patients limited the power of this study, the overall pattern of the findings suggests that a Th1-like cytokine response (IFN-gamma and IL-2 production) may be associated with resistance to naturally occurring episodes of herpes labialis. The development and severity of experimental UVR-induced herpes labialis appears to be regulated differently and may involve an immunopathologic mechanism.

Dehydroepiandrosterone protects the microcirculation of muscle flaps from ischemia-reperfusion injury by reducing the expression of adhesion molecules.

Adhesion molecules contribute to ischemia-reperfusion injury by increasing the endothelial adhesion and extravasation of leukocytes. Scientific evidence suggests that presurgical treatment with dehydroepiandrosterone may protect the microvasculature against this damage, but the exact mechanism is not known. The purpose of this study was to investigate the effects of presurgical dehydroepiandrosterone treatment on microcirculatory hemodynamic parameters and the expression of adhesion molecules in a rat cremaster muscle flap model. Twenty male rats were randomly assigned to three experimental groups. In group I (n = 5), the muscle flaps did not receive presurgical treatment. In group II (n = 6), propylene glycol (30 mg/kg), the vehicle for dehydroepiandrosterone, was injected intravenously before ischemia was induced. In group III (n = 9), dehydroepiandrosterone (30 mg/kg) was injected intravenously before ischemia was induced. All flaps were subjected to 6 hours of ischemia and 90 minutes of reperfusion. Microcirculatory variables (functional capillary density, red blood cell velocity in the main flap arteriole, and numbers of rolling, sticking, and transmigrating leukocytes), blood levels of three adhesion molecules (L-selectin, Mac-1 integrin, and CD44), and the numbers of leukocytes expressing those molecules were analyzed. Analysis of the microcirculatory parameters revealed that dehydroepiandrosterone treatment before ischemia had significant preservative effects on the red blood cell velocity and functional capillary density 30 and 90 minutes after reperfusion, compared with the control and vehicle-treated groups. Leukocyte-endothelial cell interactions were also affected by dehydroepiandrosterone treatment, as reflected by significant decreases in the numbers of sticking and transmigrating leukocytes 30 and 90 minutes after reperfusion. In dehydroepiandrosterone-treated animals, leukocytes exhibited lower levels of expression of adhesion molecules after the onset of ischemia, compared with the control groups. In this study, intravenous dehydroepiandrosterone administration reduced the activation of leukocytes and improved red blood cell velocity and capillary perfusion in the muscle flap microcirculation during ischemia-reperfusion injury. This protective effect was most likely the result of delayed expression of Mac-1 integrin, L-selectin, and CD44 molecules on leukocytes.

Dehydroepiandrosterone protects muscle flap microcirculatory hemodynamics from ischemia/reperfusion injury : An experimental in vivo study

This study evaluated the potential for dehydroepiandrosterone (DHEA) to protect skeletal muscle from reperfusion injury using intravital microscopic observations of isolated rat cremaster muscle flaps. The flaps were subjected to warm ischemia followed by reperfusion in three groups of rats. In group 1 (control, n = 14), muscle flaps were subjected to 6 hours of ischemia and then evaluated after either 90 minutes (n = 8) or 24 hours (n = 6) of reperfusion. Group 2 animals (propylene glycol pretreatment, n = 8) were pretreated with a propylene glycol vehicle, then underwent 6 hours of ischemia and were evaluated after 90 minutes reperfusion. Group 3 animals (DHEA pretreatment, n = 12) were pretreated with DHEA dissolved in propylene glycol, subjected to 6 hours of ischemia, and then evaluated after either 90 minutes (n = 6) or 24 hours (n = 6) of reperfusion. Red blood cell velocity in the flaps main arteriole, functional capillary density, venular constriction index (the ratio of internal to external diameter of postcapillary venules), and microemboli formation were measured. Muscle samples were evaluated by electron microscopy. Control animals showed a 61% reduction in red blood cell velocity (p < 0.05) accompanied by a 69% reduction in functional capillary density (p < .05) acutely and total cessation of flow by 24 hours. No differences between control and propylene glycol treated animals were noted. In DHEA-pretreated animals, reflow occurred in 100% of the flaps, there was a temporary 39% reduction (p < 0.05) in functional capillary density, and all flaps remained viable at 24 hours. In this study, DHEA pretreatment markedly improved muscle flap microcirculatory hemodynamics and protected flaps against ischemia/reperfusion injury.

Thymic modulation of IL-2 and IL-4 synthesis by peripheral T cells

In this paper we provide several lines of evidence to support the hypothesis that the thymus can exert regulatory influences on the functional capabilities of mature recirculating T cells. Our studies demonstrate that while the IL-2-producing potential of T cells that repopulate the secondary lymphoid organs of lethally irradiated and stem cell-reconstituted mice is significantly reduced compared to that of T cells harvested from normal mice, the amount of IL-4 produced by the T cells of these experimental animals is equivalent to, or greater than, the amount produced by T cells from control animals. In addition, we determined that the amount of biologically active IL-2 and IL-4 secreted by T cells harvested from lethally irradiated animals who reconstitute their hematopoietic and immune systems under the influence of nonirradiated thymic epithelial grafts is essentially identical to the amount produced by T cells harvested from nonirradiated control animals. Collectively, these findings suggest that: (1) the alterations observed in the lymphokine-producing potential of T cells harvested from lethally irradiated and stem cell-reconstituted mice is not due to a direct effect of ionizing radiation on the T lymphocytes themselves, and (2) the exposure of the epithelial cells of the thymus to ionizing radiation during marrow-ablative regimens abrogates or modifies a component of thymic function which can influence the lymphokine-secreting potential of recirculating T cells. Further evidence for thymic involvement in the regulation of lymphokine production by peripheral T cells comes from our finding of a post-thymectomy time-dependent reduction in the capacity of T cells from animals to produce IL-2. Coincident with this reduction, T cells harvested from peripheral lymphoid organs of thymectomized animals demonstrated an augmentation in their IL-4-producing capabilities. The finding that treatment of thymectomized animals with the androgen steroid hormone dehydroepiandrosterone reestablished a normal IL-2-producing potential by their T cells makes it unlikely that the reduced capacity to produce IL-2 was secondary to a loss in fresh thymic emigrants.