Central corticotropin-releasing hormone reduces cellular immunity.

Corticotropin-releasing hormone (CRH) acts within the brain to elicit changes in neuroendocrine, autonomic, and behavioral activity similar to those observed after stress. A reduction of splenic natural killer (NK) activity has also been described following the central administration of CRH. In this study, we examined whether other in vitro measures of cellular immunity, including peripheral and splenic NK activity, lymphocyte responses to mitogen stimulation, and numbers of splenic T and NK cell subpopulations, are altered following CRH. Synthetic rat CRH (1.0 microgram) microinjected into the lateral ventricle reduced splenic and peripheral blood NK activity, lymphocyte responses to mitogenic stimulation, and percentage of splenic NK cell numbers. Numbers of splenic lymphocytes and T cell subpopulations were not altered by central CRH. These findings suggest that central CRH acts to reduce a number of in vitro cellular immune measures similar to the effects of inescapable stress.     

The role of central corticotropin-releasing hormone in the anorexic and endocrine effects of the bacterial T cell superantigen, Staphylococcal enterotoxin A

Bacterial superantigens, such as the staphylococcal enterotoxins, exert a strong capacity for in vivo stimulation of T cell proliferation and cytokine production. Previously, staphylococcal enterotoxin A (SEA) was shown to induce an anorexic effect under novel contextual conditions of testing, and produced an increase in plasma ACTH and corticosterone levels in C57BL/6J mice. In the present study, the role of corticotropin releasing hormone (CRH) in promoting these effects of SEA was addressed via intracerebroventricular (icv) administration of alpha-helical CRH(9-41) ((alpha)hCRH), a non-selective CRH receptor antagonist, and astressin-2B, a selective CRH receptor 2 antagonist. The efficacy of (alpha)hCRH and astressin-2B in blocking anorexic responses to CRH and urocortin under the current conditions of testing was first confirmed. Subsequently, it was found that (alpha)hCRH (20 microg icv), but not astressin-2B (10 and 25 microg icv), significantly attenuated the anorexia induced by SEA. This suggested that central CRH is involved in mediating the anorexia induced by SEA, but potentially through CRH receptor 1. Additional results revealed that plasma ACTH stimulation in response to SEA was not significantly attenuated by either antagonist administered icv. However, the plasma corticosterone elevation showed a modest, but significant, attenuation in SEA challenged mice given (alpha)hCRH. These data suggest a possible influence of central CRH on adrenocorticoid activity subsequent to SEA challenge. More importantly, it appears that central activation of CRH receptors is a consequence of SEA challenge, and this likely contributes to its anorexic effects.     

Involvement of central, but not placental corticotropin releasing hormone (CRH) in heat stress induced immunosuppression during pregnancy

To clarify whether corticotropin releasing hormone (CRH) and beta-endorphin (betaEP) system mediate maternal immunosuppression in pregnant rats exposed to heat through central or placental pathway, we examined the effects of intravenous (iv) (100 or 500 microg) or intracerebroventricular (icv) (5 microg) administration of CRH receptor antagonist alpha-helical CRH (9-41) on splenic natural killer cell activity (NKCA) as well as betaEP in blood, pituitary lobes, and placenta in pregnant rats at 15 to 16 days gestation. Two-way ANOVA revealed that heat reduced NKCA and elevated blood and pituitary betaEP but did not change placental betaEP. Iv administered 500 microg and icv administered alpha-helical CRH reversed the reduced NKCA and the elevated pituitary betaEP, while iv administration of 100 microg alpha-helical CRH did not. The increased blood betaEP was reversed by iv 100 and 500 microg alpha-helical CRH and icv administration. Both iv and icv administrations reduced placental betaEP independent of heat exposure. Thus, the response of placental betaEP to iv administration of alpha-helical CRH seemed to be stronger than that of pituitary betaEP. These results indicate that alpha-helical CRH which acts on pituitary betaEP antagonizes heat-induced immunosuppression during pregnancy, suggesting that immunosuppression produced by heat stress during pregnancy is mediated by the central CRH system. The placental CRH-betaEP system seems unlikely to be involved in the immunosuppression. Physiologic roles of placental CRH and opioid system should be clarified by future in vitro experiments using placenta specimen including placental immunocyte.     

Neonatal immune challenge affects the regulation of estrus cyclicity and feeding behavior in female rats

A single immune challenge with lipopolysaccharide (LPS) in the neonatal period has a long-lasting influence on immune response. Using female Sprague–Dawley rats, we examined whether neonatal LPS challenge influences the life-long neuroendocrine sensitivity of reproductive function and feeding behavior to LPS, and whether stress-related neuropeptides and their receptors are involved in neonatal LPS-induced physiological change. On day 10 after birth, all pups were injected with LPS (100 μg/kg, i.p.) or saline. Then, in Experiment 1, LPS (100 μg/kg, i.p.) or saline was injected at diestrous in adulthood, and the length of the estrous cycle, 24 h food intake and body weight change were recorded. In Experiment 2, the mRNA expression levels of corticotropin-releasing hormone (CRH), urocortin (UCN), urocortin 2 (UCN2), CRH receptor type 1 (CRH-R1) and CRH receptor type 2 (CRH-R2) in the hypothalamus were measured using real-time PCR. LPS injection in adulthood prolonged the estrous cycle in neonatal LPS-injected rats. LPS injection in adulthood decreased food intake and body weight in both neonatal LPS- and saline-injected rats, more so in the latter. Basal expressions of UCN2 and CRH-R2 mRNA were higher in neonatal LPS-injected rats than in saline-injected rats. These findings indicate that neonatal immune challenge influences the anti-stress regulation of the estrous cycle and feeding behavior in adulthood. Increased expression of UCN2 and CRH-R2 might enhance the sensitivity of the estrous cycle in suppressing the effects of LPS.     

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation

We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we show that this antibody detects an epitope associated with lymphocytes and that it triggers a proliferative response of the cells. BIP-4 epitope can be detected on both human peripheral blood and murine splenic mononuclear cells. Surface immuno-globulin-negative (i.e. resting T) lymphocytes are neither bound by the antibody nor proliferate to it. Proliferative response exerted in 7-day cultures of murine splenic mononuclear cells by recombinant interleukin-2 was blocked by BIP-4 antibody. We conclude that the epitope shared by a type of brain sodium channel protein and lymphocyte surface is involved in some, yet unrecognized, step of immune cell activation.     

Activation of CRHR2 exerts an inhibitory effect on the expression of collapsin response mediator protein 3 in hippocampal neurons

Corticotropin-releasing hormone (CRH) family peptides as well as their receptors have been shown to exhibit various functions in hippocampus. However, effects of CRH receptors activation on collapsin response mediator protein 3 (CRMP3), the key protein for dendrite outgrowth and cell apoptosis, remain unclear. In the present study, we determined the effects of CRHR1 and CRHR2 on CRMP3 expression in cultured hippocampal neurons. CRH and urocortin II (UCNII) dose-dependently suppressed CRMP3 mRNA and protein expression. The inhibitory effect on CRMP3 expression was completely reversed by CRHR2 antagonist but not by CRHR1 antagonist. Investigations on the signaling pathways of UCNII showed that CRHR2 mediated UCNII-induced increase in phosphorylated phospholipase C (PLC)-β3 expression. Blocking PLC activity with U73122 and PKC with G?6976 completely prevented UCNII-inhibited CRMP3 expression. Our results suggest that CRHR2 activation decrease CRMP3 expression in hippocampal neurons via a mechanism that is dependent on PLC/PKC signaling pathways.     

Brain Angiotensin II Modulates Sympathoadrenal and Hypothalamic Pituitary Adrenocortical Activation during Stress

Angiotensin II (Ang II) type-1 (AT₁) receptors are present in areas of the brain controlling autonomic nervous activity and the hypothalamic-pituitary-adrenal (HPA) axis, including CRH cells in the hypothalamic paraventricular nucleus (PVN). To determine whether brain AT₁ receptors are involved in the activation of the HPA axis and sympathetic system during stress, we studied the effects of acute immobilization stress on plasma catecholamines, ACTH and corticosterone, and mRNA levels of CRH and CRH receptors (CRH-R) in the PVN in rats under central AT₁ receptor blockade by the selective antagonist, Losartan. While basal levels of epinephrine, norepinephrine and dopamine in plasma were unaffected 30 min after icv injection of Losartan (10 μg), the increases after 5 and 20 min stress were blunted in Losartan treated rats (P<0.05 for norepinephrine, and P<0.01 for epinephrine and dopamine, vs controls). Basal or stress-stimulated plasma ACTH and corticosterone levels were unaffected by icv Losartan treatment. Using in situ hybridization studies, basal levels of CRH mRNA and CRH-R mRNA in the PVN were unchanged after icv Losartan. While Losartan had no effect on the increases in CRH-R mRNA levels 2 or 3 h after 1 h immobilization, it prevented the increases in CRH mRNA. The blunted plasma catecholamine responses after central AT₁ receptor blockade indicate that endogenous Ang II in the brain is required for sympathoadrenal activation during immobilization stress. While Ang II appears not to be involved in the acute secretory response of the HPA axis, it may play a role in regulating CRH expression in the PVN.     

In vivo administration of L-dopa or dopamine decreases the number of splenic IFN gamma-producing cells

Although dopamine receptors are present on peripheral immune system cells, relatively little is known about the functional role that dopamine may play in immune responses. The purpose of this study was to determine the effects of chronic treatment with L-dopa and dopamine on murine lymphocyte proliferation and cytokine production/release. In vivo treatment with L-dopa resulted in an increase in the proliferative response of splenic lymphocytes to ConA. Spleen cell supernatant concentrations of IL-4 and IFN gamma were not significantly altered following treatment with L-dopa for 5 days. However, the number of IFN gamma-, but not IL-4-producing cells was significantly inhibited by L-dopa. This effect was blocked by co-treatment with a dopamine antagonist. The effect on IFN gamma was replicated by infusion of dopamine. The results suggest that dopamine may have a direct role in regulating immune responses through down-regulation of IFN gamma.     

Long-term effects on feeding and body weight after stimulation of forebrain or hindbrain CRH receptors with urocortin

Research on the contribution of CRH receptor stimulation to energy homeostasis has focused on forebrain substrates. In this study, we explored the effects of caudal brainstem administration of the CRH receptor agonist, urocortin, on food intake and body weight, and on plasma glucose and corticosterone (CORT) in non-deprived rats. Urocortin (0, 0.3, 1, 3 microg) delivered, respectively, to the fourth and lateral ventricles yielded substantial suppression of food intake measured 2, 4 and 24 h later. A significant but more modest anorexia was observed between 24 and 48 h after injection. Intake responses did not differ between the injection sites, but body weight loss measured 24 h after lateral-i.c.v. injection was substantially greater than that after fourth-i.c.v. injection. Fourth-i.c.v. urocortin administration (3 microg) produced substantial elevations in plasma glucose and CORT that were not distinguishable in magnitude and duration from responses to lateral-i.c.v. delivery. Unilateral microinjection of urocortin into the dorsal vagal complex significantly reduced 24-h food intake at a dose (0.1 microg) that was subthreshold for the response to ventricular administration, suggesting that fourth-i.c.v. effects are mediated in part by stimulation of CRH receptors in this region of the caudal brainstem. The results indicate that similar effects can be obtained from stimulation of anatomically disparate populations of CRH receptors, and that interactions between forebrain and hindbrain structures should be considered in the evaluation of CRH contributions to food intake and body weight control.     

Urocortin and inflammation: confounding effects of hypotension on measures of inflammation

Urocortin, a newly isolated 40-amino-acid mammalian peptide homologous to corticotropin-releasing hormone (CRH), activates both CRH type 1 and 2 receptors, but may be an endogenous ligand for CRH receptor type 2. Urocortin given systemically inhibited heat-induced paw edema in the rat, and was therefore ascribed anti-inflammatory properties. We examined the effects of urocortin in the carrageenin-induced subcutaneous inflammation model. Rats were treated with urocortin 200 (n = 6) or 20 nmol/kg (n = 6); inflammatory exudates were reduced by approximately 30% compared to controls (n = 7) at both doses. However, since subcutaneous urocortin has been shown to reduce arterial blood pressure, we tested the hypothesis that its antiedema and antiextravasatory effects were secondary to arterial hypotension. Therefore, we examined the parallel effects of urocortin- and hydralazine-induced hypotension on acute inflammation induced by carrageenin in the rat. Rats were treated with subcutaneous carrageenin and control injections (n = 8), carrageenin and urocortin (20 nmol/kg, n = 9), or carrageenin and intraperitoneal hydralazine (10 mg/kg, n = 8). Mean arterial blood pressure was measured hourly for 7 h in 12 animals, and after 2 h, the nadir of treatment, in a further 13 animals. Rats were then sacrificed, and the inflammatory exudate volume and leukocyte count were measured. Mean exudate volumes were reduced from 4.8 +/- 0.5 ml (controls) to 2.4 +/- 0.3 ml (p = 0.004) and 2.9 +/- 0.6 ml (p = 0.007) in urocortin- and hydralazine-treated animals, respectively. Urocortin and hydralazine both produced a significant fall in blood pressure compared to controls, with mean arterial pressure 2 h after carrageenin injection falling to 51.0 +/- 4.1 (p < 0.001) and 34.6 +/- 4.6 (p < 0.001) vs. 92.9 +/- 3.7 mm Hg in controls, respectively. A significant positive correlation was noted between blood pressure and inflammatory exudate volume (r = 0. 52, p = 0.007). As both hydralazine and urocortin lowered blood pressure and inflammatory exudate volume, we suggest that the anti-inflammatory effects of urocortin and related neuropeptides may be nonspecific, acting through hypotension rather than through direct anti-inflammatory mechanisms. The use of inflammatory models which rely on extravasation may be inappropriate for the study of substances that produce hypotension.     

Corticosterone-independent alteration of lymphocyte mitogenic function by amphetamine.

Amphetamine, a neural stimulatory agent with acute effects mimicking those of stress, is shown here to elevate plasma corticosterone levels and suppress spleen and peripheral blood lymphocyte (PBL) mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) when administered to rats. Pretreatment of the rats with propranolol, a nonselective beta-adrenergic receptor antagonist, totally prevented the amphetamine-induced suppression of lymphocyte mitogenic reactivity to Con A and PHA in the spleen and to PHA in the peripheral blood; however, the PBL mitogenic response to Con A was only partially restored. Although the amphetamine-induced alterations in immune function were prevented by propranolol pretreatment, the elevated plasma corticosterone response was not. This suggests that corticosterone is not modulating the mitogenic activity of splenic lymphocytes or PHA-reactive PBLs. On the other hand, Con A-reactive PBLs may be affected by corticosterone and/or other mechanisms, which may include the catecholamines.     

Forced swim stress activates rat hippocampal serotonergic neurotransmission involving a corticotropin-releasing hormone receptor-dependent mechanism

Serotonin is important for adequate coping with stress. Aberrant serotonin function is implicated in the aetiology of major depression and anxiety disorders. Dysregulation of the hypothalamic-pituitary-adrenocortical axis, involving elevated corticotropin-releasing hormone (CRH) activity, also plays a role in these stress-related illnesses. Here we studied the effects of stress on hippocampal serotonin and the role of the CRH system using in vivo microdialysis. First, rats were subjected to a forced swim stress, resulting in a dramatic increase in hippocampal serotonin (1500% of baseline), which was associated with the occurrence of diving behaviour. The diving-associated increase in serotonin depended on activation of CRH receptors, as it was antagonized by intracerebroventricular pretreatment with D-Phe-CRH12-41. Secondly, the effects of intracerebroventricular administration of CRH and urocortin (0.03-1.0 microg) were studied. Both CRH and urocortin caused a dose-dependent rise in hippocampal serotonin (maximally 350% of baseline) and 5-hydroxyindoleacetic acid levels, suggesting the involvement of CRH receptor type 1. Because the effects of urocortin were prolonged, CRH receptor type 2 could play a role in a later phase of the neurotransmitter response. Experiments using adrenalectomized rats showed that CRH-induced serotonin changes were adrenally independent. These data suggest that the raphe-hippocampal serotonin system is able to mount, CRH receptor-dependent, responses to specific stressful situations that surpass the usually observed maximal increases of about 300% of baseline during stress and enhanced vigilance.     

Immunomodulatory effects of footshock in the rat

Intermittent inescapable footshock (IIFS) treatment, administered to 3-month-old male rats, resulted in analgesia as well as discrete immunological and endocrine changes. The splenic lymphocyte proliferative response to concanavalin A (ConA) and phytohemagglutinin (PHA) was decreased by 20% and 41%, respectively. The primary IgM plaque forming cell (PFC) response to sheep red blood cells (SRBC), however, was not altered by IIFS administered either immediately or 24 h after injection of SRBC. IIFS also produced a significant (20-fold) increase in plasma corticosterone (CORT) as compared to non-shocked controls. The shock-induced suppression of splenic lymphocyte mitogenic response to PHA was blocked by 10 mg/kg naltrexone (NTX) administered immediately before IIFS. NTX alone had no effect on this mitogen response. However, NTX significantly attenuated the shock-induced rise in CORT even though NTX alone significantly elevated CORT in the non-shocked controls. These data suggest that IIFS alters cellular immune response but not the primary IgM PFC response (a measure of humoral immune function) and that the immunomodulatory effects may involve both an opioid and a corticosteroid component with respect to alterations in the splenic lymphocyte mitogenic response to PHA.     

Hypothalamic-Pituitary-Adrenal Activation Following Endotoxin Administration in the Developing Rat: A CRH-Mediated Effect

The present studies assessed hypothalamic-pituitary-adrenal (HPA) responses following immune activation with endotoxin (ip) in three-day old Long Evans rats. Marked plasma corticosterone (B), adrenocorticotrophic hormone (ACTH) responses and biphasic fluctuations in plasma glucose were maximal at a dose of 0.05 mg/kg. HPA responses peaked between 3–5 h following immune challenge and plasma ACTH and B responses were greater in female than in male rat pups. Plasma levels of corticosterone binding globulin (CBG) were reduced in males and substantially increased in females during the peak HPA response. Changes in plasma glucose were biphasic with slight increases when ACTH and B levels were maximal, but hypoglycemia was evident once plasma B levels returned to resting values. Endotoxin challenge reduced median eminence corticotropin-releasing hormone (CRH) levels at times corresponding with elevated HPA activity, and prior icv injection of the CRH antagonist, a-helical CRH, significantly attenuated elevations in plasma ACTH and B. In addition, α-helical CRH pretreatment completely blocked endotoxin-induced changes in plasma CBG in both males and females. These findings support the view that endotoxin-induced HPA activation in the neonate may occur via CRH.     

Corticotropin-releasing hormone immunoreactivity in human T and B cells and macrophages: colocalization with arginine vasopressin

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are expressed in cells of the immune system where they exert immunomodulatory roles, but these neuropeptides are poorly characterized in human immune tissues. The aim of this study was to determine concentrations and distribution of CRH and AVP in nonactivated human peripheral blood mononuclear cells (PBMC). PBMC from normal human subjects were separated into enriched subpopulations of T and B cells and monocytes/macrophages by a magnetic bead/monoclonal antibody technique. CRH and AVP were measured in cell extracts by radioimmunoassay (RIA). CRH-immunoreactivity (ir) ranged 0.24-0.8 fmol/million cells (n = 6 subjects) in T cell extracts, 0.4-2.7 fmol/million cells (n = 4) in B cells and 0.63-2.16 fmol/million cells (n = 4) in macrophages. AVP-ir ranged 0.2-0.95 fmol/million cells in T cell extracts, <0.1-0.8 fmol/million cells in B cells and 0.14-3.19 fmol/million cells in macrophages. Reversed-phase high-performance liquid chromatography (HPLC) of T and B cell extracts revealed a peak of CRH-ir which coeluted with synthetic CRH-41; this peak was not present in macrophages. A second peak of CRH-ir which eluted in a more hydrophobic position was observed in extracts of T and B cells and macrophages. This unidentified form of CRH-ir is the predominant form of CRH-ir in nonactivated human PBMC. This is the first study to demonstrate that CRH-ir and AVP-ir are colocalized within human T cells, B cells and monocytes/macrophages. We have confirmed observations of a variant form of CRH-ir in human PBMC and show that this is the predominant form in macrophages and B cells whereas CRH-ir, which coelutes with CRH(1-41) on HPLC, is present in significant amounts only in T cells. These data also confirm that CRH-ir in human PBMC is not urocortin because the antiserum used in the CRH RIA does not bind to urocortin.     

Role of CRH in Glucopenia-lnduced Adrenomedullary Activation in Rats

Acute glucoprivation profoundly stimulates hypothalamic-pituitary-adrenocortical (HPA) and adrenomedullary outflows. Whether these responses reflect a single central mechanism regulated by corticotropin-releasing hormone (CRH) has been unclear. This study examined the role of endogenous CRH in HPA and adrenomedullary responses to hypoglycemia in Sprague-Dawley rats, by using anti-CRH immune serum or a CRH antagonist (α-helical h/r CRH_9–41, and in Lewis rats, a strain characterized by deficient hypothalamic CRH responses during stress. In conscious Sprague-Dawley rats with indwelling arterial and venous cannulas, insulin (0.3 U/kg) was injected iv, and responses of serum glucose concentrations and plasma levels of corticotropin (ACTH) and catechols (including epinephrine, EPI; norepinephrine, NE; dihydroxyphenylalanine, DOPA; dihydroxyphenylglycol, DHPG; and dihydroxyphenylacetic acid, DOPAC) were assessed, with or without pretreatment with anti-CRH immune serum (0.5 or 1.0ml iv or 10μI icv) or α-helical h/r CRH_9–41 (130 nmol iv or 13 nmol icv). Responses to insulin (1.0 U/kg iv) were also measured in conscious juvenile Lewis and Fischer 344/N rats. Insulin-induced hypoglycemia markedly increased plasma levels of EPI and ACTH in all groups. Pretreatment iv with 1.0ml of anti-CRH immune serum blocked the ACTH response to insulin but failed to attenuate the EPI response, α-helical h/r CRH_{9_41}, whether given iv or icv, failed to alter ACTH or EPI responses to insulin, although the antagonist did block EPI responses to icv CRH. Hypoglycemia elicited similar increments in ACTH levels in Lewis rats and Fischer 344/N control rats; and although Lewis rats had lower baseline EPI and smaller responses of NE, DHPG, DOPA, and DOPAC levels, the groups did not differ in proportionate increments in EPI levels. The results indicate that the ACTH response to hypoglycemia depends on availability of CRH outside the blood-brain barrier—presumably in the pituitary gland. The findings with icv α-helical h/r CRH_{9_41} can be explained by failure of the antagonist to reach effective concentrations at central sites of action of endogenous CRH, or by mechanisms other than CRH release determining the adrenomedullary response to hypoglycemia. Lewis rats seem to have less adrenomedullary secretion at baseline and smaller responses of NE synthesis and release during hypoglycemia than do Fischer 344/N rats. Neurochemical evidence for differential adrenomedullary and sympathoneural responses during hypoglycemia in all three rat strains is inconsistent with Cannon's view of a functionally unitary sympathoadrenal system. Since Lewis and Fischer 344/N rats had similar proportionate responses of both ACTH and EPI levels during hypoglycemia, either Lewis rats have deficient CRH responses to some stressors but not to others, or else pituitary-adrenocortical and adrenomedullary responses in this setting depend on mechanisms other than CRH release in the brain. Both explanations are inconsistent with the doctrine of non-specificity, the main tenet of Selye's stress theory.     

Brain vasopressin is involved in stress-induced suppression of immune function in the rat

The possibility that vasopressin (VP) is involved in stress-induced suppression of immune function was examined in rats. Intermittent electrical footshock for 60 min suppressed the proliferative response of splenic T cells to the mitogen concanavalin A as well as natural killer (NK) cytotoxicity, and the former change was partially, and the latter was completely, blocked by intracerebroventricular (i.c.v.) preadministration of a V1 receptor antagonist. The footshock-induced suppression of the T cell proliferative response was completely abolished by coadministration of a corticotropin-releasing hormone (CRH) receptor antagonist and the V1 receptor antagonist. The i.c.v. administration of VP suppressed the proliferative response of splenic T cells and NK cytotoxicity in an adrenal-independent manner. These effects were completely reversed by i.c.v. preadministration of the V1 receptor antagonist. These results suggest that brain VP, in conjunction with CRH, suppresses immune function through the V1 receptor in rats under stress.     

Opposite effects of mild and severe stress on in vitro activation of rat peripheral blood lymphocytes.

The effects of short-term handling and different durations of immobilization on serum levels of catecholamines, ACTH, prolactin, and corticosterone and in vitro functions of lymphocytes were examined in rats. The results show that changes in the immune response of peripheral blood lymphocytes (PBL) depend on the intensity of the stressor: Short (1 min) handling of cannulated rats induced an enhanced stimulation of PBL to respond to T and B cell mitogens, whereas immobilization of the same animals led to suppression, dependent on the time this stressor was applied. The decrease in the mitogen reactivity of PBL after 120 min of immobilization was reversible within 24 h, and could be largely prevented by adrenalectomy, confirming that factors released by this gland are mainly responsible for immunosuppression. In contrast to PBL, spleen cells showed an enhanced mitogen response to immobilization and adrenalectomy, indicating that the immune response is differently regulated in the various compartments of the immune system. Possible correlations of the various effects with changes in stress hormone levels are discussed.