Mitogen-induced Proliferative Responses Research Articles

Immunotoxic effects of gold and silver nanoparticles: Inhibition of mitogen-induced proliferative responses and viability of human and murine lymphocytes in vitro

Understanding the effects of nanoparticles (NP) on immune cell functions is essential in designing safe and effective NP-based in vivo drug delivery systems. The immunomodulatory potential of gold nanoparticles (GNP) and silver nanoparticles (SNP) was investigated in vitro using murine splenic and human peripheral blood lymphocytes (PBL) in terms of effects on viability and mitogen-induced proliferation. Hydrodynamic size and number of NP were characterized using NP tracking analysis (NTA); modal diameters of GNP and SNP were 28 (±1.5) and 66 (± 2.7) nm, respectively, with a unimodal distribution. Lymphocytes were incubated with GNP or SNP in the presence/absence of B- or T-cell mitogens and proliferative responses then determined using [3H]-thymidine incorporation. Concanavalin A (T-cell-specific) and lipopolysaccharide- (B-cell-specific) stimulated responses of murine splenic lymphocytes, as well as phytohemagglutinin (T-cell-specific) and pokeweed mitogen- (B-and T-cell specific) induced responses of human lymphocytes, were significantly inhibited by GNP (25–200 μg/ml) and SNP (12.5–50 μg/ml). However, [3H]-thymidine incorporation by unstimulated lymphocytes was unaffected in the presence of GNP or SNP. Viability of lymphocytes was determined using trypan blue dye exclusion and was significantly inhibited only at 200 μg GNP/ml and 25 or 50 μg SNP/ml. As mitogen responses are most useful to provide supportive mechanistic information on primary immunotoxicologic functional observations, and so far more comprehensive data (in vivo and in vitro) is still needed, the results nevertheless suggest to us that GNP and SNP might potentially be able to modulate immune responses by impacting on lymphocyte activation.

Synergism between phorbol myristate acetate and calcium ionophore in inducing proliferation of in vitro γ-irradiated murine lymphocytes.

The objective of this study was to analyze the in vitro effects of γ-irradiation (0-5 Gy) on lymphocyte proliferation in animals sensitive to radiation as BALB/c mice. Lymphocytes were irradiated and underwent different treatments: quiescent cells were cultured with calcium ionophore A23187 (5 min or 48 h) with or without phorbol myristate acetate (PMA); lymphocytes (control cells or incubated with A23187 and PMA) were also cultured with four mitogens that are specific to the different subpopulations to determine the degree of inhibition of the response to radiation. Results obtained indicated that in quiescent cells, A23187 and PMA treatment had a mitogenic effect, which peaked with long A23187 treatment (48 h); synergism was further demonstrated between both drugs and was enhanced with higher ionizing radiation doses. However, in both irradiated and non-irradiated mitogen-stimulated cells, A23187 (48 h) and PMA had a strong inhibitory effect on cell proliferation. In conclusion these results indicate that irradiated BALB/c mice lymphocytes respond to treatment with A23187 and PMA more actively than controls. Inhibition of the post-exposure mitogen-induced proliferative response and the synergic effect between A23187 and PMA also suggest altered PKC activation mechanisms in cell membranes. Comparing with previous studies with in vivo irradiated mice, the effects of IR in vitro were less intense.

Immune response to human embryonic stem cell‐derived cardiac progenitors and adipose‐derived stromal cells

Transplantation of allogeneic human embryonic stem cell-derived cardiac progenitors triggers an immune response. We assessed whether this response could be modulated by the concomitant use of adipose-derived stromal cells (ADSC). Peripheral blood mononuclear cells were collected from 40 patients with coronary artery disease (CAD) and nine healthy controls. Cardiac progenitors (CD15+ Mesp1+) were generated as already reported from the I6 cell line treated with bone morphogenetic protein (BMP)-2. Adipose-derived stromal cells were obtained from abdominal dermolipectomies. We assessed the proliferative response of peripheral lymphocytes from patients and controls to cardiac progenitors cultured on a monolayer of ADSC, to allogeneic lymphocytes in mixed lymphocyte culture and to the T cell mitogen phytohemaglutin A in presence or absence of ADSC. Cardiac progenitors cultured on a monolayer of ADSC triggered a proliferation of lymphocytes from both patients and controls albeit lower than that induced by allogeneic lymphocytes. When cultured alone, ADSC did not induce any proliferation of allogeneic lymphocytes. When added to cultures of lymphocytes, ADSC significantly inhibited the alloantigen or mitogen-induced proliferative response. Compared to healthy controls, lymphocytes from patients presenting CAD expressed a decreased proliferative capacity, in particular to mitogen-induced stimulation. Adipose-derived stromal cells express an immunomodulatory effect that limits both alloantigen and mitogen-induced lymphocyte responses. Furthermore, lymphocytes from patients with CAD are low responders to conventional stimuli, possibly because of their age and disease-associated treatment regimens. We propose that, in combination, these factors may limit the in vivo immunogenicity of cardiac progenitors co-implanted with ADSC in patients with CAD.

The primary mitogen (TCPOBOP)-induced hepatocyte proliferation is resistant to transforming growth factor- β-1 inhibition

Transforming growth factor (TGF)-β-1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. In this study, we compared the proliferative response in the liver between wild-type and transgenic mice, overexpressing active TGF-β-1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real-time polymerase chain reaction analysis of cell cycle-related genes. Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF-β-1 expression following the mitogenic treatment. TGF-β-1 does not inhibit the primary mitogen-induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.

Dichloromethane extract of Dicerocaryum senecioides leaves exhibits remarkable anti-inflammatory activity in human T-lymphocytes

The leaves of Dicerocaryum senecioides are used in South Africa as a traditional remedy for many ailments, including inflammatory disorders. The purpose of this study was therefore to evaluate the anti-inflammatory potential of a dichloromethane extract of D. senecioides leaves. Methanol extracts of the leaves were sub-fractionated with dichloromethane and the anti-inflammatory potential of this fraction investigated according to its effects on the mitogen-induced proliferative responses and cytokine profiles of isolated human blood mononuclear leucocytes (MNL). The cells were pre-treated with the extract (25–100 µg mL−1) followed by addition of the mitogen, phytohaemagglutinin (PHA, 5 µg mL−1 final), and measurement of lymphocyte activation and proliferation, using flow cytometric detection of up-regulation of expression of CD25 and incorporation of radiolabelled thymidine into newly synthesised DNA, respectively. Cytokine production by unstimulated and PHA-activated cells was measured using multiplex suspension bead array technology. Treatment of cells with the Dicerocaryum extract resulted in dose-related inhibition of PHA-activated lymphocyte proliferation and expression of CD25, as well as decreased production of Th1 (IFN-γ, TNF-α) and Th2 (IL-10) cytokines. These observations not only underscore the anti-inflammatory potential of components of Dicerocaryum leaves, but also provide a basis for future definitive studies.

S-Adenosylmethionine and Methylthioadenosine Inhibit Cellular FLICE Inhibitory Protein Expression and Induce Apoptosis in Colon Cancer Cells

S-Adenosylmethionine (SAMe) and its metabolite 5'-methylthioadenosine (MTA) inhibit mitogen-induced proliferative response in liver and colon cancer cells. SAMe and MTA are also proapoptotic in liver cancer cells by selectively inducing Bcl-x(S) expression. The aims of this work were to assess whether these agents are proapoptotic in colon cancer cells, and if so, to elucidate the molecular mechanisms. We found that both SAMe and MTA are proapoptotic in HT-29 and RKO cells in a dose- and time-dependent manner. Gene microarray uncovered down-regulation of cellular FLICE inhibitory protein (cFLIP). SAMe and MTA treatment led to a decrease in the mRNA and protein levels of both the long and short cFLIP isoforms. This required de novo RNA synthesis and was associated with activation of procaspase-8, Bid cleavage, and release of cytochrome c from the mitochondria. Inhibiting caspase 8 activity or overexpression of cFLIP protected against apoptosis, whereas supplementing with polyamines did not. SAMe and MTA treatment sensitized RKO cells to tumor necrosis factor alpha-related apoptosis-inducing ligand-induced apoptosis. Although SAMe and MTA are proapoptotic in colon cancer cells, they have no toxic effects in NCM460 cells, a normal colon epithelial cell line. In contrast to liver cancer cells, SAMe and MTA had no effect on Bcl-x(S) expression in colon cancer cells. In conclusion, SAMe and MTA are proapoptotic in colon cancer cells but not normal colon epithelial cells. One molecular mechanism identified is the inhibition of cFLIP expression. SAMe and MTA may be attractive agents in the chemoprevention and treatment of colon cancer.

Long-Term Feeding of the cis-9,trans-11 Isomer of Conjugated Linoleic Acid Reinforces the Specific Immune Response in Rats

Several effects on the immune system have been ascribed to the cis9,trans11 conjugated linoleic acid (CLA) isomer. We studied whether feeding a diet enriched with an 80:20 CLA isomer mix of cis9,trans11 and trans10,cis12 CLA from gestation to adulthood affects the capacity of adult rats to achieve a specific immune response. Pregnant Wistar rats were fed a 1% CLA diet or a control diet beginning on d 7 of gestation. Weaned pups received the same diet as dams until they were 15 wk old. Rats from both groups were immunized with ovalbumin (OVA) when they were 9 wk old. Dietary CLA enhanced splenocyte OVA-specific proliferation by ∼50% (P < 0.05) and decreased the mitogen-induced proliferative responses of these cells by ∼10–20% (P < 0.05). The diminished splenocyte proliferative response was accompanied by a lower interleukin-2 secretion (P < 0.05). Long-term CLA supplementation did not increase serum, spleen, or mesenteric lymph node production of OVA-specific antibodies (Ab) or the number of spleen anti-OVA Ab-secreting cells. Interestingly, dietary CLA increased intestinal anti-OVA IgA production by ∼75% (P < 0.05). In conclusion, a 1% CLA diet administered from gestation to adulthood enhanced specific systemic cell-mediated immunity as well as the mucosal IgA immune response, whereas it downregulated the polyclonal activation of the immune system. These data support the long-term effects of dietary cis9,trans11 CLA isomer on the immune system.

Prophylactic fenbendazole therapy does not affect the incidence and onset of type 1 diabetes in non-obese diabetic mice

Fenbendazole (FBZ) is a common, highly efficacious broad-spectrum anthelmintic drug used to treat and limit rodent pinworm infections. However, the effect of its prophylactic use on the immune response of rodents is largely undefined. The non-obese diabetic (NOD) mouse is a model commonly used to study type 1 diabetes (T1D). Parasitic infections will inhibit diabetes development in NOD mice; thus, in the presence of contamination, prophylactic treatment with anthelmintics must be considered to maintain experimental research. Herein, we investigated the prophylactic use of FBZ in NOD mice to determine its effect on the incidence and onset of diabetes, lymphocyte sub-populations and T cell proliferative responses. NOD mice were separated into control and treatment groups. The treatment group received a diet containing FBZ. Animals were monitored for the incidence and onset of T1D. At matched time points, diabetic and non-diabetic mice were killed and splenic lymphocytes analyzed for various cell sub-populations and mitogen-induced proliferative responses using flow cytometry. Treated and control mice were monitored >23 weeks with no detectable effects on the incidence or onset of diabetes. Moreover, no significant differences were detected in lymphocyte sub-populations and mitogen-induced CD4(+) and CD8(+) proliferative responses between control and treatment groups. These results suggest that prophylactic FBZ treatment does not significantly alter the incidence or onset of diabetes in NOD mice. The prophylactic use of FBZ, therefore, presents a viable approach for the prevention of pinworm infection in precious experimental animals with substantial scientific and economic benefits.

Hypoxia and hypoxia-inducible factor-1α promote growth factor-induced proliferation of human vascular smooth muscle cells

Hypoxia is thought to be a stimulus for the excessive proliferation of vascular smooth muscle cells (VSMC) that contributes to pulmonary hypertension, but the mechanisms involved are unknown. Here we tested whether hypoxia-inducible factor 1-alpha (HIF-1alpha), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O(2)) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), compared with those in normoxia (20% O(2)). Moderate hypoxia elicited increased cellular HIF-1alpha levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1alpha. Knockdown of HIF-1alpha or HIF-1beta levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or 20% O(2) but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1alpha similarly inhibited PDGF-stimulated proliferation, whereas HIF-2alpha knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1alpha expression also inhibited growth factor-induced expression of cyclin A. We conclude that HIF-1alpha promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.

Lack of Soluble TNF‐receptors in Women with Recurrent Spontaneous Abortion and Possibility for its Correction

Tumor necrosis factor (TNF) and soluble TNF receptors (sTNF-Rs) system related with Th1 and Th2 and activity of NF-kappaB/IkappaB regulatory system. This study was designed to compare sTNF-R1 and sTNF-R2 production (shedding) and levels of late activated CD8+ T-lymphocytes in non-pregnant (n = 30) and pregnant (n = 20) normal women and non-pregnant (n = 20) and pregnant (n = 30) RSA women. Effects of progesterone (natural structure) injections in RSA women were studied. Levels of sTNF-R1, sTNF-R2, TNF in peripheral blood serum were detected by enzyme-linked immunosorbent assay. Lymphocyte subsets were estimated by multicolor flow cytometry. NK cell cytotoxic activity of peripheral blood lymphocytes (PBL) in whole blood against K562 targets was determined using Europium-release cytotoxicity assay. Mitogen-induced proliferative response of PBL to PHA-P, Con A and PWM were determined by standard 3H-thymidine incorporation assay. Levels of soluble TNF-R1 and TNF-R2 in normal pregnancy were elevated when compared with non-pregnant normal women and pregnant RSA women. Levels of late activated CD8+ T-lymphocytes in normal pregnancy were decreased but no changes were detected in RSA women. After progesterone therapy (i.m. injections of 2.5% oil solution) in RSA women elevation of sTNF-R1 and sTNF-R2 to normal pregnancy ranges was observed. No changes in levels of late activated CD8+ T-lymphocytes after progesterone treatment were detected. Elevation of levels of sTNF-R1, sTNF-R2 and decrease of late activated cytotoxic T-lymphocytes are pronounce markers of normal human pregnancy. In RSA women there are no elevation of sTNF-R1 and sTNF-R2 levels during pregnancy. This deficiency may be restored by progesterone treatment.

Hypothalamic Control of Mitogen-Induced Proliferative Responses and Luteinizing Hormone-Releasing Hormone Levels in Thymus and Peripheral Blood of Rat Fetuses

The role of endogenous luteinizing hormone-releasing hormone (LHRH) in the development of concanavalin A (ConA)-induced proliferative responses was studied in rat fetuses. Preliminary treatment of fetuses in utero with either the LHRH receptor antagonist or anti-LHRH antibodies resulted in the suppression of ConA-induced proliferative responses of thymocytes. LHRH and LHRH-immunopositive cells, morphologically similar to thymocytes, were detected in intact fetal thymus. A significant content of LHRH was also found in the peripheral blood of fetuses. The LHRH content in thymus and plasma was similar in males and females. Surgical ablation of the hypothalamus resulted in 2-fold decreases in thymus and plasma levels of LHRH in 21-day-old fetuses compared to sham-operated fetuses. It was concluded that LHRH regulates mitogen-induced proliferative responses of thymocytes during prenatal ontogenesis in the rat. The main source of plasma LHRH at that period is the hypothalamus. Moreover, LHRH is synthesized in the fetal thymus. Thus, LHRH is suggested to have not only a central effect but also to be involved in autocrine or paracrine regulation of proliferative immune responses.

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide, which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were testedin vitro . Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli -stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600μmol l−1 NO−2) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.

Head kidney neutrophils of carp (Cyprinus carpio L.) are functionally modulated by the haemoflagellateTrypanoplasma borreli

In the present work responses of carp (Cyprinus carpio) head kidney-derived neutrophils to the blood parasite T. borreli, and the consequences of these responses for parasite survival and other host response mechanisms, were studied. In co-cultures of head kidney leucocytes (HKL) with viable and lysed T. borreli a prominent shape change of neutrophilic granulocytes towards increased size and complexity was observed. In addition, the longevity of neutrophils in vitro was prolonged in the presence of T. borreli antigens. In these cultures, neutrophils also exhibited an increased phagocytosis activity. An up regulation of the production of nitric oxide (NO) and reactive oxygen species (ROS) was observed in T. borreli- and mitogen-stimulated HKL cultures. However, addition of live, fluorescence-labelledT. borreli to previously stimulated HKL cultures, revealed neither killing nor phagocytosis of the parasite by activated neutrophils. Moreover, viable T. borreli, when added to HKL cultures of infected carp, reduced their phagocytosis activity and NO production. Supernatants of co-cultures between T. borreli and HKL also contained mediators, which suppressed a mitogen-induced proliferative response of peripheral blood leucocytes (PBL) in vitro. Thus, while T. borreli itself appeared not to be sensitive to responses of activated neutrophils, the flagellates interferes with the production of immunomodulatory signals of these cells, probably resulting in a partial immunosuppression, which may favour the parasite development in vivo.

In vitro and ex vivo effects of erythromycin A, azithromycin and josamycin on the splenic response to specific antigens and mitogens.

The immunomodulatory properties of antimicrobial agents and their clinical impact have been the focus of worldwide interest in recent years. In this study, the effects of different treatments with 14-, 15- and 16-membered ring macrolides on the mitogen-induced proliferative response of lymphocytes and the splenic response to immunization with sheep erythrocytes have been tested by in vitro and ex vivo assays in a murine experimental model. We observed that the in vivo administration of these antibiotics to mice induces a compensatory mechanism that abrogates the suppression observed by in vitro assays. Thus, physiological parameters may be important when testing the immunopharmacological effects of antibiotics.

Th1 and Th2 Cytokine Modulation by IL-10/IL-12 Imbalance in Autoimmune Haemolytic Anaemia (AIHA)

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN- n, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN- n compared with healthy controls ( p <0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN- n production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.

SIN-1, a nitric oxide donor, ameliorates experimental allergic encephalomyelitis in Lewis rats in the incipient phase: the importance of the time window.

NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on days 5-7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in PBS-treated control rats and was accompanied by reduced ED1(+) macrophages and CD4(+) T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-gamma production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on NO synthase.

Anti‐Interleukin‐6 Antibody Treatment Restores Cell‐Mediated Immune Function in Mice With Acute Ethanol Exposure Before Burn Trauma

Previous studies from this laboratory reported that suppression of cell-mediated immune function was coincident with elevated interleukin (IL)-6 production after acute ethanol exposure before burn trauma, compared with either insult alone. The goal of this study was to investigate whether treatment with an anti-IL-6 antibody could restore immunocompetence in mice subjected to burn trauma with previous exposure to alcohol, as assessed by delayed-type hypersensitivity (DTH) and mitogen-induced splenocyte proliferative responses. Mice given an ethanol treatment designed to reach a blood alcohol level of 100 mg/dl before a 15% total body surface area burn injury were treated with an anti-IL-6 antibody at 30 min and 24 hr postinjury. Burn/ethanol mice exhibited a 91% suppression of the DTH response ( < 0.01) and a 76% suppression of mitogen-induced splenocyte proliferation (p < 0.01) at 48 hr postinjury, along with increased levels of circulating and splenic macrophage-derived IL-6, compared with all other treatment groups. After anti-IL-6 antibody administration to burn/ethanol mice, there was a 25% (p < 0.05) and 63% (p < 0.01) recovery of the DTH and splenocyte proliferative responses, respectively. Addition of exogenous IL-6 to splenocyte cultures isolated from anti-IL-6 antibody-treated burn/ethanol mice resulted in a 70% inhibition of mitogen-induced proliferative responses (p < 0.03). These data confirm previous findings that burn in combination with acute ethanol exposure suppresses cell-mediated immune function compared with either insult alone. Furthermore, the ability of the anti-IL-6 antibody treatment to improve cellular immune responses in the burn/ethanol group suggests that blocking this cytokine may be beneficial for the ethanol-exposed, thermally injured individual.

483 Role of a1 and a2-adrenoreceptors in adrenergic regulation of mitogen-induced lymphocyte proliferative response

483 Role of a1 and a2-adrenoreceptors in adrenergic regulation of mitogen-induced lymphocyte proliferative response

Alterations of T cell distribution and functions in prenatally cypermethrin-exposed rats: possible involvement of catecholamines

The synthetic pyrethroid insecticide, cypermethrin (50 mg/Kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin-exposure induces a marked and long-lasting increase of adrenaline (A) and noradrenaline (NA) plasma concentrations. The enhancement of plasma catecholamine levels was accompanied by a marked increase of CD5 +, CD4 +, and CD8 + total T cell numbers in the peripheral blood, while in the spleen a reduction of all T cell subsets was observed. In addition, peripheral blood lymphocytes (PBL) from rats prenatally exposed to cypermethrin showed an enhanced capability to proliferate in response to different doses of Concanavalin A (ConA), or human recombinant interleukin-2 (hrIL-2), whereas an impaired proliferative response was observed in the spleen. The percent increase of NA, but not A plasma concentrations paralleles the immunomodulatory effects induced by cypermethrin neonatal exposure on T cell distribution and mitogen-induced proliferation in the peripheral blood and spleen. Collectively, our results suggest that the changes in mitogen-induced proliferative responses in the peripheral blood and spleen of prenatally cypermethrin-exposed rats may be attributable to pesticide-induced catecholamine release, which causes an increased output of CD5 +, CD4 +, and CD8 + T cells from the spleen to the peripheral blood, and a consequent lymphocytosis.

Integration of Rac-dependent Regulation of Cyclin D1 Transcription through a Nuclear Factor-κB-dependent Pathway

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.