Graciela Cremaschi

Altered Expression of Autonomic Neurotransmitter Receptors and Proliferative Responses in Lymphocytes from a Chronic Mild Stress Model of Depression: Effects of Fluoxetine

We studied beta-adrenergic and muscarinic cholinergic receptor (MR) expression and proliferative response in lymphocytes from animals under chronic mild stress (CMS) model of depression (CMS animals). Animals were subjected to CMS (periods of food or water deprivation, changes in lighting conditions, tilted cage, etc.) for 12 weeks. CMS lymphocytes showed an altered mitogen-induced proliferation. CMS-B and -T lymphocytes showed an increment on beta-adrenoceptor number and on intracellular responses to a beta-agonist. CMS-T cells showed higher MR expression and lower cGMP responses than normal lymphocytes. MR were not detectable in normal B cells while CMS-B cells showed both MR expression and cGMP response. Beta and muscarinic stimulation influenced lymphocyte proliferative responses, in accordance with cAMP and cGMP responses. After 12 weeks of the CMS procedure, animals were treated with fluoxetine while the CMS procedure continued. Fluoxetine treatment reverted the alterations induced by CMS. These findings suggest a possible mechanism for the immune alterations found in depressive disorders and for the effect of fluoxetine treatment on immune response.

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.

Altered lymphocyte catecholamine reactivity in mice subjected to chronic mild stress

There is considerable evidence that the sympathetic nervous system influences the immune response via activation and modulation of beta(2)-adrenergic receptors (beta(2)R). Furthermore, it has been suggested that stress has effects on the sympathetic nervous system. In the present study, we analyzed the influence of catecholamines on the reactivity of lymphocytes from mice exposed to a chronic mild stress (CMS) model of depression (CMS-animals). The effects of the CMS treatment on catecholamine and corticosterone levels and on beta(2)R lymphoid expression were also assessed. For this purpose, animals were subjected to CMS for 8 weeks. Results showed that catecholamines (epinephrine and norepinephrine) exert an inhibitory effect on mitogen-induced normal T-cell proliferation and a stimulatory effect on normal B-cell proliferation in response to selective B lymphocyte mitogens. Specific beta- and beta(2)-antagonists abolished these effects. Lymphocytes from mice subjected to CMS had an increased response to catecholamine-mediated inhibition or enhancement of proliferation in T and B cells, respectively. Moreover, a significant increase in beta(2)R density was observed in animals under CMS compared to normal animals. This was accompanied by an increment in cyclic AMP production after beta-adrenergic stimulation. On the other hand, neither catecholamine levels, determined in both urine and spleen samples, nor serum corticosterone levels showed significant variation between normal and CMS-animals. These findings demonstrate that chronic stress is associated with an increased sympathetic influence on the immune response and may suggest a mechanism through which chronic stress alters immunity.

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.

DOWN-REGULATION OF BETA -ADRENERGIC RECEPTORS INDUCED BY MITOGEN ACTIVATION OF INTRACELLULAR SIGNALING EVENTS IN LYMPHOCYTES

The expression of β‐adrenergic receptors on murine lymphocytes stimulated with concanavalin A was studied. A decrease in β‐adrenoceptor number on T lymphocytes and a diminished response to specific agonist stimulation at the peak of proliferation was found. The blockade of cell proliferation by tyrosine kinases or protein kinase C inhibitors reversed the decrease in β‐adrenoceptor number. PMA plus ionophore or interleukin‐2 but not PMA alone were able to induce β‐adrenoceptor downregulation accompanying cellular proliferation. These results showed that the intracellular signals triggered during lymphocyte activation are involved in β‐adrenoceptor down‐regulation and it would represent the loss of a mechanism that exerts negative neuroimmune control of cellular proliferation.

β-Adrenoceptor distribution in murine lymphoid cell lines

beta-Adrenergic receptors (R) on several tumor lymphoid cell lines were characterized both directly by beta radioligand binding of 125iodo-cyanopindolol (125I-CYP) to intact cells and membranes, and functionally by assessing hormone-dependent changes in cyclic 3,5 adenosine monophosphate (cAMP) levels on intact cells and measuring adenylate cyclase (a.c.) activity on membranes. Only two lymphoid cell types, BW 5147 (a T cell derived lymphoma cell line) and TIB 221 (a B cell derived line) displayed significant amounts of beta-adrenergic R by 125I-CYP specific binding. Despite this, no stimulation of the a.c. activity was found in the presence of beta-adrenergic agonists in these cells in comparison with native lymphocytes or cells of the well-known S49 cell line used as a positive control. beta-Adrenoceptor specific uncoupling was confirmed by aluminum tetrafluoride (AlFl4) direct activation of the a.c. system in the beta R-bearing cell membranes and by an increase in cAMP production induced by PGE1, another hormone that activates the a.c. Structural characterization of beta-adrenoceptors by photoaffinity-labeling demonstrates that uncoupling was not due to a structural alteration of the beta-adrenergic R expressed in these lymphoma cell lines, as these R gave similar results as native or S49 cells. It can be concluded that functional beta-adrenoceptors are absent in these lymphoma cells. The possible implication of alternative transmission pathways and original neuroendocrine control in tumor lymphoid cells is 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.

Involvement of endogenous nitric oxide signalling system in brain muscarinic acetylcholine receptor activation

Summary. Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10−9 M to 10−7 M lead to NOS activation while higher doses (>10−6 M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways.

Protein kinase C-dependent NF-κB activation is altered in T cells by chronic stress

Abstract.Chronic stress has been associated with impaired immune function. In this work we studied the effect of chronic mild stress (CMS) exposure on the early intracellular pathways involved in T cells after stimulation with mitogen. We found that mitogen stimulation of T lymphocytes from CMS-exposed mice resulted in a reduction of the intracellular [Ca2+] rise, an impairment of growth-promoting protein kinase C (PKC) activation, a lower NF-κB activation and an increase in the inhibitory cAMP-protein kinase A (PKA) pathway activity with respect to those found in control lymphocytes. However, T cell activation with the direct PKC activator phorbol 12-myristate 13-acetate plus calcium ionophore led to a similar proliferative response in both CMS and control lymphocytes, indicating that signals downstream of PKC would not be affected by stress. In summary, our results show that chronic stress induced an alteration in T cell early transduction signals that result in an impairment of the proliferative response.

Differential expression of protein kinase C isoenzymes related to high nitric oxide synthase activity in a T lymphoma cell line

Protein kinase C (PKC) is critical for T lymphocyte activation and proliferation, while nitric oxide synthase (NOS) may function both as an activator or inhibitor of T cell apoptosis. Both enzymatic activities were studied in T lymphoma cells in comparison to normal and activated T lymphocytes. Here we show a higher translocation of PKC in BW5147 lymphoma cells than in mitogen-stimulated T lymphocytes. Tumor cells overexpressed PKC zeta isoform, while high levels of the PKC beta isotype were found in mitogen-stimulated T lymphocytes. Moreover, tumoral T cells showed high NOS activity, almost undetectable in normal or stimulated T lymphocytes. PKC and NOS inhibitors or the intracellular delivery of an anti-PKC zeta antibody diminished both NO production and proliferation in tumor cells. These results suggest that atypical PKC zeta isoform expression and its association with NOS activity regulation would participate in the multistep process leading to BW5147 cell malignant transformation.