Inhibition of suppressor cell generation by mouse serum in culture

Normal C57BL/6 mouse spleen cells cultured for five days in the presence of fetal calf serum (FCS-induced suppressor generation culture) were shown, in mixing experiments, to suppress the primary humoral response of freshly explanted C57BL/6 spleen cells against sheep erythrocytes (SRBC). This suppressor cell generation was largely dependent on the FCS concentration in the suppressor generation culture. Ten or 5% FCS effectively supported the generation of suppressor cells, but 1% FCS only marginally supported it. When mouse serum (MS) from normal C57BL/6 mice was added to the suppressor generation culture, it inhibited the generation of the suppressor cells. Sera from allogeneic mice and athymic nude mice were also effective. The effect of MS was resistant to heat treatment (56 degrees C, 30 min). The inhibitory activity of MS was not dialyzable, and concentrated into the fraction which was not precipitated by 50% saturation of ammonium sulfate and which was eluted at a concentration of about 0.2 M NaCl from a DEAE-Sephadex A-50 column. The active fraction of MS also effectively inhibited the growth of Ehrlich tumor cells in culture. Further, MS also inhibited the generation of Con A-induced suppressor cells. The inhibition by MS of FCS-induced suppressor generation was eliminated by an interleukin 2-containing preparation.     

Inhibition of Fas-mediated apoptotic cell death of murine T lymphocytes in a mouse model of immunosenescence in linkage to deterioration in cell membrane raft function

We previously developed a transgenic mouse line into which a rabbit protein kinase Calpha (PKCalpha) gene fused to a human CD2 promoter/enhancer was introduced, and we found that immunosenescence was facilitated in these transgenic mice. In this study, we found that along with age-dependent increase in the level of protein expression of PKCalpha and its translocation to the membrane, activated T cells became less sensitive to apoptosis-inducing anti-Fas antibody. The capacity of T cells to express Fas antigen on their surfaces in response to anti-CD3 and interleukin-2 was impaired in PKCalpha-transgenic mice of relatively advanced age, although background Fas expression levels on T cells from those mice were high. We then found that out of proportion to a high level of cell surface Fas expression the density of cholera toxin B (CTx)-binding raft elements decreased in PKCalpha-transgenic mice of relatively advanced age and to a lesser extent in normal mice of advanced age. Correspondingly, the expression level of raft-associating Lck was decreased in these mice. These findings suggest for the first time that immunosenescence of T cells involves a decrease in density of cell surface CTx-binding raft elements, which might underlie a deterioration in T-cell signal pathway for either cell death or cell activation.     

Inhibition of immediate-type allergic reactions by Prunella vulgaris in a murine model.

We studied the effect of aqueous extract of Prunella vulgaris (Labiatae) (PVAE) on immediate-type allergic reactions. PVAE (0.005 to 1 g/kg) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/ 80 in rats. When PVAE was given as pretreatment, at concentrations ranging from 0.005 to 1 g/kg, the serum histamine levels induced by compound 48/ 80 were reduced in a dose-dependent manner. PVAE (0.001 to 1 g/kg) inhibited the passive cutaneous anaphylaxis activated by anti-dinitrophenyl (DNP) IgE antibody dose dependently. PVAE also inhibited the histamine release induced by compound 48/80 or anti-DNP IgE from the rat peritoneal mast cells (RPMC). The level of cyclic AMP in RPMC, when PVAE was added, significantly increased, compared with that of normal control. Moreover, PVAE (0.01 and 0.1 mg/ml) had a significant inhibitory effect on anti-DNP IgE-mediated tumor necrosis factor-alpha production from RPMC. These results indicate that PVAE inhibits immediate-type allergic reactions in rats.     

Inhibition of renal alkaline phosphatase by cimetidine

Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.     

Inhibition of murine L cell interferon action by heparin

Summary Heparin added together with murine L cell interferon inhibited the development of antiviral activity in mouse L cells. When added after interferon treatment, heparin had no effect on antiviral activity. There was also no inhibition of interferon action in L cells treated with heparin before addition of interferon. On the other hand, heparin did not inhibit antiviral activities of human interferon and . Since murine L cell interferon, but not human interferon and , binds to a heparin affinity column and can be eluted with a solution of high salt, it is presumed that murine L cell interferon and heparin must interact with each other. The apparent interaction of heparin with murine L cell interferon was prevented by protamine, a drug that neutralizes heparin. Dextran sulfate inhibited murine L cell interferon action, but dextran and chondroitin sulfate A did not. These results suggests that heparin inhibited murine L cell interferon action by the binding via sulfate groups on its molecules. Heparin also inhibited antiviral activity of murine L cell interferon in mice infected with herpes simplex virus (+GC Miyama strain).With 6 Figures     

Inhibition of murine cytotoxic T cell responses by progesterone

In the present study we evaluated the effect of sex hormone on the generation of murine cytotoxic T cell responses. We show that the in vitro CTL response was strongly inhibited by progesterone but not by E1, E2 or testosterone. Our experiments attempting to understand the mechanism of the hormone action on CTL development have revealed that the ability of the cells to generate helper signals was not affected. This was demonstrated by the fact that neither IL-2 production nor IL-2 receptor expression was altered by the hormone. Rather, it appears that the capacity of the cells to respond to the signals and to become cytotoxic was modified. Furthermore, we show that the hormone mediated an inhibition of CTL development in thymocyte cultures externally supplemented with all the required helper factors. These results strongly suggest that progesterone has a direct effect on the differentiation of cytotoxic effector cells.     

Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides

Depending on sequence and backbone structure, DNA can inhibit as well as stimulate immune responses. As previously shown, single-base phosphorothioate (Ps) oligodeoxynucleotides (ODN) can inhibit murine macrophage activation. To determine whether these compounds can also affect dendritic cells (DC), the effects of 30-mer Ps ODN (SdA, SdT, SdG, and SdC) on DC activation were assessed in an in vitro system. With DC preparations obtained from murine bone marrow cultured in granulocyte macrophage-colony stimulating factor, the Ps ODN blocked the production of interleukin-12 and nitric oxide induced by bacterial DNA, an immunostimulatory cytosine phosphate guanosine dinucleotide (CpG) ODN and lipopolysaccharide (LPS). Furthermore, these compounds inhibited up-regulation of costimulatory molecules CD40 and CD86 as well as major histocompatibility complex-II molecules, indicating an effect on DC maturation. Although the Ps ODN limited uptake of CpG ODN as assessed by flow cytometry, the Ps ODN did not affect LPS uptake, suggesting that these compounds inhibit DC responses by effects on downstream signaling pathways. Together, these observations extend the range of action of inhibitory ODN to DC and suggest a role of these compounds as immunomodulatory agents.     

Abrogation of suppressor cell function by inhibitors of prostaglandin synthesis

The weekly intraperitoneal injection of rat erythrocytes into mice induces both a stable autoimmune state, as judged by the appearance of anti-mouse erythrocyte autoantibody and suppressor T cells capable of regulating this response; the latter being demonstrable only in a subsequent transfer system. This autoimmune response and the parallel anti-rat erythrocyte response were both insensitive to exogenous prostaglandin E2 (PGE2). The administration of prostaglandin synthetase inhibitors (indomethacin or aspirin) to mice undergoing immunization with rat erythrocytes had no effect on the anti-rat response, yet mildly exacerbated the onset of the autoimmune state and potently inhibited the generation of suppressor cells. Furthermore the administration of these drugs to recipients of suppressor cells virtually abrogated suppressor cell activity. These observations imply that both the generation and effector function of these suppressor cells may be modulated by prostaglandin synthetase inhibitors while at the same time T helper and B cell functions remain unimpaired.     

Inhibition of T suppressor cell function by local administration of an active cyclophosphamide derivative at the sensitization site.

In previous work, we have shown that local administration of active cyclophosphamide (CY) derivatives, or other cytostatic drugs, at the sensitization site induced a similar immunopotentiation to that of systemic CY. Since it is well documented that CY can inhibit suppressor cells, it was proposed that immunoenhancement by locally administered drugs might be based on a similar principle. The objective of the present study was to test this hypothesis, using an experimental model of Ts mediated suppression of delayed type hypersensitivity to sheep erythrocytes. In this model, mice are made tolerant to sheep erythrocytes by i.v. injection of a high dose of sheep erythrocytes. Local treatment of sheep erythrocytes-tolerant mice with the active CY derivative Z7557 at the site of attempted sensitization reversed suppression in a dose-dependent manner. Local treatment with the cytostatic drug etoposide (VP-16) and systemic CY treatment were also effective. In transfer experiments, the function of afferently acting suppressor cells was blocked by local treatment with Z7557 or systemic CY. These data support the concept of anti-suppressor cell activity of locally administered cytostatic drugs. As with CY, the pharmacological basis of this effect remains to be determined.     

Sex-related splenocyte function in a murine model of allergic asthma

Background The prevalence and severity of asthma are higher among boys than girls, but the ratios are reversed after puberty. These observations strongly suggest that sex hormones have a role in the pathogenesis of the disease. However, the mechanisms underlying the gender differences in asthma are not fully understood.
Objective The aim of this study was to investigate sex differences in allergic inflammation in terms of immune function.
Methods Male and female C57BL/6 mice were sensitized and challenged with ovalbumin (OVA). OVA-specific IgE in serum and airway inflammation were compared between sexes. Splenocytes from OVA-sensitized male or female donor mice were transferred to male or female naïve recipient mice. Subsequently, the recipient mice were challenged, followed by the evaluation of OVA-specific IgE and airway inflammation. Cytokines secreted from splenocytes of the sensitized mice were measured.
Results The levels of OVA-specific IgE and the allergen-induced airway inflammation were higher in female than in the male mice. The contents of T-helper type 2 (Th2) cytokines, IL-4, IL-5 and IL-13, in the bronchoalveolar lavage fluid from female mice were higher than those from male mice. The airway inflammation in female recipients transferred with splenocytes from female donors was more severe than that in any other combination of recipients and donors. Splenocytes from the sensitized female mice produced more of the Th2 cytokine, IL-5, than those from the sensitized male mice upon stimulation with OVA.
Conclusion Our findings suggest that the sex difference in allergic airway inflammation may be attributable to the sex difference in not only the hormonal environment but also in the immune cells themselves.     

Endogenous glucocorticoids promote the expansion of myeloid-derived suppressor cells in a murine model of trauma

Stress-dose of glucocorticoid has been demonstrated to be beneficial for trauma patients in clinical studies. Recently, a heterogeneous population of myeloid cells with immunosuppressive activity named myeloid-derived suppressor cells (MDSCs) has been found to accumulate in the trauma host and can be induced by glucocorticoids in vitro. In order to explore the effect of endogenous glucocorticoids on MDSCs under trauma conditions, we blocked the glucocorticoid signal in a murine trauma model using the antagonist of the glucocorticoid receptor RU486 (mifepristone). We found for the first time that RU486 not only blunted MDSC expansion induced by trauma in the spleen, peripheral blood and bone marrow especially at 6 h after traumatic stress but also decreased the survival rate from 100 to 20% in traumatic mice within 7 days. Moreover, neither MDSCs producing arginase-1 nor the morphological characterization of trauma-induced MDSCs was affected by the blockage of the glucocorticoid receptor. Our results suggest that endogenous glucocorticoids may promote MDSCs expansion in a murine trauma model and MDSCs may be beneficial for the trauma host.     

Cimetidine abrogates suppressor T cell function in vitro

Cimetidine increased the [3H] thymidine incorporation of normal human mononuclear cells in culture both when unstimulated or when under the stimulus of phytohemagglutinin or pokeweed mitogen (PWM). It also increased their supernatant immunoglobulin production under PWM stimulus. These effects were higher when the cells were preincubated with cimetidine than when it was added simultaneously. To determine if this effect of cimetidine reflects an abrogation of suppression we studied concanavalin-A-induced suppressor function of normal mononuclear cells using both [3H] thymidine incorporation and immunoglobulin synthesis as indicator systems and found that preincubation with cimetidine caused significant decrease in suppressor cell function in both systems.     

Seasonal variation in suppressor T cell subsets and non-specific suppressor cell function in hay fever sufferers

The helper/suppressor T cell ratio, as defined by monoclonal antibodies, was significantly higher in hay fever sufferers compared with controls (P less than 0.05), but only during or shortly after the pollen season. This was due to a reduction in the suppressor subset, which returned to control values in the winter. There was no significant difference in the non-specific concanavalin A-induced suppressor cell function compared with controls. The mean summer value was significantly lower than the winter value (P less than 0.05), but we cannot be sure that this was not the result of changes in laboratory conditions. No relationship was found between T cell subsets or suppressor cell function and total or specific IgE levels, or between T cell subsets and suppressor cell function. Our findings suggest that in hay fever, reduction in suppressor cell numbers and function is a secondary phenomenon.     

Inhibition of NK and K cell function by phenothiazines

Several phenothiazines have been shown to inhibit NK cell-mediated cytotoxicity (NKC) and antibody-dependent cell-mediated cytotoxicity (ADCC) effected by human peripheral blood lymphocytes. Of those tested, chlorpromazine was found to inhibit at the lowest concentration (5 microM) with a 90% inhibition at 10 microM for NKC and 70% inhibition for ADCC. Pre-incubation of either effector or target cells with chlorpromazine did not provide any evidence of inhibition in subsequent cytotoxicity assays. The data for chlorpromazine in particular may be of clinical significance. In vivo inhibition would presumably require the presence of the drug since preincubation did not produce inhibition of NK or K cell function.     

Regulation of accessory cell function by retinoids in murine immune responses

This study examines the effects of in-vivo immune regulation by vitamin A acetate (VAA) and 13-cis-retinoic acid (13-CRA) on in-vitro accessory cell function. Mice were fed a control diet, or diet containing VAA or 13-CRA, and monitored by body weight gains and diet consumptions at weekly intervals. At 4, 7 and 12 weeks mice were killed, differential blood counts performed and accessory cells isolated from lymphomedullary tissues. Histology confirmed that the chief feature of the lymphomedullary organs of the VAA-fed animals was an expansion of the splenic marginal zone and the paracortical region of the lymph nodes. There was an increase in the number of accessory cells present, and this included both dendritic cells and macrophages. The accessory cell function of these cells was also increased, as evidenced by both alloproliferative and allocytotoxic responses in vitro. In 13-CRA-fed animals the effects were similar to those seen with VAA, but were less pronounced. We suggest that the primary effects of these compounds on in-vivo immunoregulation could be due to their promotion of accessory cell function.     

Pathogenesis of mucous cell metaplasia in a murine asthma model

Increased mucus production in asthma is an important cause of airflow obstruction during severe exacerbations. To better understand the changes in airway epithelium that lead to increased mucus production, ovalbumin-sensitized and -challenged mice were used. The phenotype of the epithelium was dramatically altered, resulting in increased numbers of mucous cells, predominantly in the proximal airways. However, the total numbers of epithelial cells per unit area of basement membrane did not change. A 75% decrease in Clara cells and a 25% decrease in ciliated cells were completely compensated for by an increase in mucous cells. Consequently, by day 22, 70% of the total epithelial cell population in the proximal airways was mucous cells. Electron microscopy illustrated that Clara cells were undergoing metaplasia to mucous cells. Conversely, epithelial proliferation, detected with 5-chloro-2-deoxyuridine immunohistochemistry, was most marked in the distal airways. Using ethidium homodimer cell labeling to evaluate necrosis and terminal dUTP nick-end labeling immunohistochemistry to evaluate apoptosis, this proliferation was accompanied by negligible cell death. In conclusion, epithelial cell death did not appear to be the stimulus driving epithelial proliferation and the increase in mucous cell numbers was primarily a result of Clara cell metaplasia.