Differential effect of hyperglycaemia on the immune response in an experimental model of diabetes in BALB/cByJ and C57Bl/6J mice: participation of oxidative stress.

Diabetes is associated with an increased risk of death from infectious disease. Hyperglycaemia has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. However, experimental evidence indicates individual susceptibility to develop complications of diabetes. In this context, the aim of this work was to study the immune response in a streptozotocin-induced type 1 diabetes in two mouse strains: BALB/cByJ and C57Bl/6J. The participation of hyperglycaemia and oxidative stress was also analysed. Diabetic BALB/cByJ mice showed a decrease in both the in-vivo and in-vitro immune responses, whereas diabetic C57Bl/6J mice had higher blood glucose but exhibited no impairment of the immune response. The influence of hyperglycaemia over the immune response was evaluated by preincubation of lymphocytes from normal mice in a high glucose-containing medium. T and B cells from BALB/cByJ mice showed a decrease in cell viability and mitogen-stimulated proliferation and an increase in apoptosis induction. An increase in oxidative stress was implicated in this deleterious effect. These parameters were not affected in the T and B lymphocytes from C57Bl/6J mice. In conclusion, BALB/cByJ mice were sensitive to the deleterious effect of hyperglycaemia, while C57BL/6J were resistant. Although an extrapolation of these results to clinical conditions must be handled with caution, these results highlight the need to contemplate the genetic background to establish models to study the deleterious effect of diabetes in order to understand phenotypical variations that are of clinical importance in the treatment of patients.

Possible involvement of stress hormones and hyperglycaemia in chronic mild stress-induced impairment of immune functions in diabetic mice.

Stress, an important aspect of modern life, has long been associated with an altered homeostatic state. Little is known about the effect of the life stress on the outcome of diabetes mellitus, especially related to the higher risk of infections. Here, we evaluate the effects of chronic mild stress (CMS) exposure on the evolution of type I diabetes induced by streptozotocin administration in BALB/c mice. Exposure of diabetic mice to CMS resulted in a significant reduction of survival and a sustained increase in blood glucose values. Concerning the immune response, chronic stress had a differential effect in mice with diabetes with respect to controls, showing a marked decrease in both T- and B-cell proliferation. No correlation was found between splenic catecholamine or circulating corticosterone levels and the proliferative response. However, a significant negative correlation was found between glucose levels and concanavalin A- and lipopolysaccharide-stimulated proliferative responses of T and B cells. A positive correlation between blood glucose and splenic catecholamine concentrations was found in diabetic mice but not in controls subjected to CMS. Hence, the present report shows that diabetic mice show a worse performance in immune function after stress exposure, pointing to the importance of considering life stress as a risk factor for patients with diabetes.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice.

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4(+)T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

Impaired immune responses in streptozotocin-induced type I diabetes in mice. Involvement of high glucose.

Diabetes is widely believed to predispose to serious infections. However, the mechanisms linking diabetes and immunosuppression are not well defined. One potential mediator of the altered defence mechanisms is hyperglycaemia. It has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. In this study we analyse the immune response in diabetes and the direct effect of hyperglycaemia on T and B lymphocyte reactivity. Diabetes induced an early decrease in IgG levels in the secondary response. However, both primary responses against a T-cell-dependent or independent antigen were affected after 6 months of diabetes induction. T- and B- cell proliferation was only decreased at this time. To gain insight into the potential mechanisms involved, we evaluated the influence of hyperglycaemia over the immune response. Pre-incubation of lymph node and spleen cells in a high glucose (HG) containing medium led to a significant time- and dose-dependent decrease in T- and B-cell proliferation. This effect was associated with the presence of HG-derived supernatants. Still viable cells after HG exposition were able to improve their proliferative response when cultured with the mitogen in a fresh standard medium. HG diminished cell viability, increased apoptosis and induced oxidative stress in lymphocytes. These results indicate that HG concentrations can directly affect lymphoid cell growth. An increase in oxidative stress would be implicated in this deleterious effect. The possibility that prolonged exposure to pathologically HG concentrations would result in the immunosuppressive state observed in diabetes is also discussed.

Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels.

Incubation of ex vivo cultured mature B cells in the presence of nitric oxide or nitric oxide-donor substances delays programmed cell death as determined by the appearance of DNA laddering in agarose gel electrophoresis or by flow-cytometry analysis of DNA. Nitric oxide also rescues B cells from antigen-induced apoptosis but fails to provide a co-stimulatory signal that converts the signal elicited by the antigen into a proliferative response. The protective effects of nitric oxide against programmed cell death can be reproduced by treatment of the cells with permeant analogues of cyclic GMP. Regarding the mechanisms by which nitric oxide prevents apoptosis in B cells, we have observed that nitric oxide release prevents the drop in the expression of the protooncogene bcl-2, both at the mRNA and protein levels, suggesting the existence of an unknown pathway that links nitric oxide signaling with Bcl-2 expression.

Alteration of neurotrophin and cytokine expression in lymphocytes as novel peripheral markers of spatial memory deficits induced by prenatal stress.

Much evidence has suggested that early life adversity can have a lasting effect on behavior. The aim of this study was to explore the impact of prenatal exposure to stress on cognition in adult life and how it impacts chronic stress situations. In addition, we investigated the participation of glucocorticoids, neurotrophins and cytokines in prenatal stress effects. For this purpose, pregnant mice were placed in a cylindrical restraint tube for 2h daily during the last week of pregnancy. Control pregnant females were left undisturbed during their entire pregnancy period. Object-in-place task results showed that adult female mice exposed to prenatal stress exhibited an impairment in spatial memory. However, in the alternation test this memory deficit was only found in prenatally stressed mice submitted to chronic stress. This alteration occurred in parallel with a decrease in BDNF, an increase in glucocorticoid receptors and an alteration of Th1/Th2 in the hippocampus. Interestingly, these changes were observed in peripheral lymph nodes as well. However, none of the mentioned changes were observed in adult male mice. These results indicate that lymphoid cells could be good candidates as peripheral markers of susceptibility to behavioral alterations associated with prenatal exposure to stress.

Participation of hypothalamic CB1 receptors in reproductive axis disruption during immune challenge.

Immune challenge inhibits reproductive function and endocannabinoids (eCB) modulate sexual hormones. However, no studies have been performed to assess whether the eCB system mediates the inhibition of hormones that control reproduction as a result of immune system activation during systemic infections. For that reason, we evaluated the participation of the hypothalamic cannabinoid receptor CB1 on the hypothalamic-pituitary-gonadal (HPG) axis activity in rats submitted to immune challenge. Male adult rats were treated i.c.v. administration with a CB1 antagonist/inverse agonist (AM251) (500 ng/5 µL), followed by an i.p. injection of lipopolysaccharide (LPS) (5 mg/kg) 15 minutes later. Plasmatic, hypothalamic and adenohypophyseal pro-inflammatory cytokines, hormones and neuropeptides were assessed 90 or 180 minutes post-LPS. The plasma concentration of tumour necrosis factor α and adenohypophyseal mRNA expression of Tnfα and Il1ß increased 90 and 180 minutes post i.p. administration of LPS. However, cytokine mRNA expression in the hypothalamus increased only 180 minutes post-LPS, suggesting an inflammatory delay in this organ. CB1 receptor blockade with AM251 increased LPS inflammatory effects, particularly in the hypothalamus. LPS also inhibited the HPG axis by decreasing gonadotrophin-releasing hormone hypothalamic content and plasma levels of luteinising hormone and testosterone. These disruptor effects were accompanied by decreased hypothalamic Kiss1 mRNA expression and prostaglandin E2 content, as well as by increased gonadotrophin-inhibitory hormone (Rfrp3) mRNA expression. All these disruptive effects were prevented by the presence of AM251. In summary, our results suggest that, in male rats, eCB mediate immune challenge-inhibitory effects on reproductive axis at least partially via hypothalamic CB1 activation. In addition, this receptor also participates in homeostasis recovery by modulating the inflammatory process taking place after LPS administration.

Haloperidol effect on intracellular signals system coupled to alpha1-adrenergic receptor in rat cerebral frontal cortex.

The induction of intracellular signals coupled to alpha1-adrenoceptor by haloperidol, were studied in rat cerebral frontal cortex. The neuroleptic exerts a biphasic effect on nitric oxide synthase (NOS), inhibiting the enzymatic activity at low concentrations (10(-9) M), while higher concentrations (10(-5) M) increased it. Protein kinase C (PKC) and phosphoinositol turnover (PIs) were involved in these actions, as haloperidol induced PKC translocation at low concentrations, and increased PIs turnover at high concentrations. All the effects of haloperidol were blocked by the alpha-adrenoceptor antagonist prazosin and the phospholipase C (PLC) inhibitor NCDC. The possibility that a cross-talk between both enzymatic pathways depending on the neuroleptic concentration used in rat cerebral frontal cortex, is also discussed.

Fluoxetine action on murine T-lymphocyte proliferation: participation of PKC activation and calcium mobilisation.

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 microg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 microg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 microg/mL Con A concentration, whereas PKC was degraded when 2 microg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.

Colchicine blocks beta adrenoceptor and class I antigen-specific interactions.

Previously we have demonstrated a molecular relationship between H-2 class I antigens and beta adrenoceptors from cardiac tissue. Here we show this type of interaction taking place with beta adrenoceptors from splenic cells and their purified membranes and the participation of cytoskeletal proteins in the phenomenon. Alloimmune, as well as anti-class I but not anti-class II, antibodies were able to inhibit in a competitive manner the binding of (-)-[3H]dihydroalprenolol to splenic lymphocytes and their purified membranes, and to increase cyclic AMP levels in intact cells as a consequence of beta adrenoceptor activation. Furthermore, colchicine (a microtubule disrupting drug), but not cytochalasin B (a microfilament disrupting drug), was able to abrogate alloimmune antibody inhibition over the beta radioligand binding to its receptor on both intact splenocytes and their membranes. Alloantibody actions were significantly diminished by peripheral protein solubilization in purified spleen cell membranes. These data pointed indirectly to the participation of a colchicine binding protein in class I antigen hormone-receptor associations.

Phorbol esters induce nitric oxide synthase and increase arginine influx in cultured peritoneal macrophages.

Incubation of peritoneal macrophages with beta-phorbol 12,13-dibutyrate promotes a time-dependent release of NO to the incubation medium. This effect was antagonized by LPS, a well known inducer of nitric oxide synthase (NOS) expression in macrophages, and was inhibited by NG-methyl-L-arginine and N omega-nitro-L-arginine. An increase in intracellular cGMP and NOS activity was observed in parallel with NO release. The induction of NOS was accompanied by a stimulation of arginine influx within the cell. These results suggest that activation of protein kinase C by phorbol esters is sufficient to promote NOS induction in macrophages.

Altered beta-adrenoceptor function associated to protein kinase C activation in hyperproliferative T lymphocytes.

beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.

Differential effects of fluoxetine on murine B-cell proliferation depending on the biochemical pathways triggered by distinct mitogens.

The effect of fluoxetine on mitogen-induced B-cell proliferation was studied. In particular, we analyzed the influence of fluoxetine on the signal transduction pathways triggered after stimulation with lipopolysaccharide (LPS) and anti-immunoglobulin M antibodies (anti-IgM). We showed that fluoxetine had a dual effect on anti-IgM-stimulated B-cell proliferation: at optimal anti-IgM concentration, fluoxetine inhibited proliferation, whereas at suboptimal anti-IgM concentration, the drug enhanced proliferation. Fluoxetine exerted only an inhibitory effect on LPS-induced B-cell proliferation. Calcium influx seemed to be involved in these effects.

Allogenic stimulus induces prostaglandin and thromboxane production in T lymphocytes.

We have examined the influence of an allogeneic stimulus on T lymphocyte prostanoid synthesis. PGE2 and TXB2 (the stable product of TXA2) were determined by radioimmunoassay. When T cells were derived from alloimmunized animals, the production of PGE2 and TXA2 was significantly higher than that of non-immunized cells. Moreover, T immune lymphocytes in the presence of the immunized alloantigen showed an increment in prostanoid production. We propose that the allogeneic stimulus provides a signal to the T lymphocytes for an increase in prostanoid synthesis.

Beta-adrenergic inhibitory effect of alloimmune antibody on isolated oviductal tract of mice.

The effects of IgG purified from BALB/c anti CF1 sera on the spontaneous contractions of isolated oviductal tract from CF1 mice were explored. Cumulative dose-response curves were constructed for the effect of immune IgG on nice oviductal tracts from proestrus, estrus, metestrus and diestrus, comparing them with those obtained with norepinephrine. Both the adrenergic agonist and the immune IgG produced a sustained inhibition of spontaneous motility during the whole sex cycle. Normal IgG was virtually devoid of activity. The sensitivity of CF1 mouse oviducts to the inhibitory actions of immune IgG and norepinephrine varied depending on the hormonal stage, i.e. it was higher in natural diestrus than in metestrus; it became smaller in proestrus and was minimal during estrus. The mechanism triggered involved a beta-adrenergic reaction that could be blocked by 10(-7) M (-)-propranolol and 10(-6) M butoxamine and potentiated by chemical sympathectomy of the mice with 6-hydroxydopamine. It is concluded that: (a) alloimmune antibody reacts with isolated oviductal tract of mice inducing functional changes; (b) this action could be associated with an activation of postsynaptic beta-adrenergic sites of the plasma membrane and (c) the different effectiveness of the immune IgG observed during the sex cycle appears to depend on the affinity of beta-adrenoreceptors to react with it.

Role of protein kinase C and cAMP in fluoxetine effects on human T-cell proliferation.

In this work, we studied the effect of fluoxetine on human T-lymphocyte proliferation using optimal and suboptimal concanavalin A concentrations. In particular, we analyzed the influence of fluoxetine on the kinases that are involved in intracellular signalling after stimulation with mitogens. We found that fluoxetine promoted the Ca2+ -mediated proteolysis of protein kinase C (PKC) and increased cyclic-AMP (cAMP) levels, thereby impairing lymphocyte proliferation, when optimal concanavalin A concentrations were used. In contrast, when suboptimal concanavalin A concentrations were used, fluoxetine only increased PKC translocation, without modifying cAMP levels, leading to T-cell proliferation. According to our results, fluoxetine has a dual effect on T-cell proliferation by modulating the PKC and protein kinase A pathways. This mechanism is thought to be mediated through Ca2+ mobilization.

Chronic stress influences the immune system through the thyroid axis.

The aim of the present work was to analyze the effect of chronic stress on thyroid axis and its influence on the immune response. For this purpose a murine model of chronic stress was developed to evaluate and to correlate thyroid hormone levels with humoral alloimmune response. Results show a reduction in serum levels of thyroid hormones, specially a significant decrease in serum levels of triiodotyronine (T3) in stressed animals. On the other hand, alloimmunization was not able to induce an early increment in T3 and thyroxine (T4) levels as it was previously reported in normal animals. In addition, lower titers of alloantibodies were obtained in animals under stress conditions as compared to normal mice. The sustitutive T4 treatment in stressed animals increased significantly alloantibody production as well as the early increment in thyroid hormones after antigenic challenge. These findings suggest that chronic stress induces an alteration of the function of thyroid axis that alters the immune response.

Involvement of the endogenous nitric oxide signalling system in bradykinin receptor activation in rat submandibular salivary gland.

Biochemical signalling events coupled to the bradykinin B(2)-receptor subtype, related to nitric oxide and prostaglandin E(2) generation were studied in rat submandibular gland. Bradykinin stimulation of the B(2)-receptor triggered activation of phosphoinositide turnover, translocation of protein kinase C, stimulation of nitric oxide synthase activity, increased production of cGMP and release of prostaglandin E(2). Bradykinin stimulation of nitric oxide synthase and cGMP production was blunted by agents able to interfere with calcium/calmodulin and phospholipase C activities, while a protein kinase C inhibitor was able to stimulate bradykinin action. Moreover, a specific B(2)-bradykinin antagonist of the reversible nitric oxide synthase inhibitor abrogated the bradykinin stimulation of nitric oxide synthase activity, cGMP accumulation and prostaglandin E(2) generation. Furthermore, a specific inhibitor of phospholipase A(2) blocked the bradykinin-induced prostaglandin E(2) release. These results suggest that apart, from the direct effect of bradykinin as an inducer of vasopermeability, it also appears to be a vasoactive chemical mediator that triggers, through release of prostaglandin E(2), a feedback mechanism that induces a protective adaptation of the gland, modulating the course of inflammation.

Stress induced cognitive deficit is differentially modulated in BALB/c and C57Bl/6 mice: correlation with Th1/Th2 balance after stress exposure.

This work shows a comparative study on the effects of chronic mild stress upon learning and memory and immunity, in BALB/c and C57BL/6 mice. Stressed BALB/c, but not C57Bl/6 mice, showed a poor learning performance, morphological alterations in the hippocampus with an increase in oxidative stress. A correlation between poor memory performance and the increase of the Th2/Th1 balance was found. Our results suggest that vulnerability to cognitive deficit associated with stress exposition could be related to a differential regulation of Th1/Th2 cytokine balance, suggesting a better learning performance for individuals that produce Th1 type cytokine after stress exposition.