Number Of Splenocytes Research Articles

Splenic Atrophy in Experimental Stroke Is Accompanied by Increased Regulatory T Cells and Circulating Macrophages

Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. In the current study, we evaluated effects on spleen and blood cells 4 days after stroke induction. Surprisingly, there was a less inflammatory cytokine profile in the middle cerebral artery occlusion-affected right brain hemisphere at 96 h compared with earlier time points. Moreover, our results demonstrate that stroke leads to splenic atrophy characterized by a reduction in organ size, a drastic loss of splenocyte numbers, and induction of annexin V+ and TUNEL+ cells within the spleen that are in the late stages of apoptosis. The consequence of this process was to reduce T cell proliferation responses and secretion of inflammatory cytokines, resulting in a state of profound immunosuppression. These changes produced a drastic reduction in B cell numbers in spleen and blood, and a novel increase in CD4+FoxP3+ regulatory T cells. Moreover, we detected a striking increase in the percentage of nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke-affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia provides a powerful negative signal to the peripheral immune system that ultimately induces a drastic state of immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ regulatory T cells.

Splenic γδ T Cells Regulated by CD4+T Cells Are Required To Control ChronicPlasmodium chabaudiMalaria in the B-Cell-Deficient Mouse

Little is known about the function and regulation of splenic gammadelta T cells during chronic Plasmodium chabaudi malaria. The splenic gammadelta T-cell population continues to expand, reaching levels equal to 4 times the number of splenocytes in an uninfected mouse. Splenic gammadelta T cells from J(H)-/- mice with chronic malaria expressed Vgamma1+ or Vdelta4+ in the same ratio as uninfected controls with Vgamma1 cells dominating, but the Vgamma2 ratio declined about twofold. Gammadelta T cells from G8 mice specific for the TL antigen increased only 2-fold in number, compared with 10-fold in BALB/c controls, but G8 gammadelta T cells failed to express the B220 activation marker. Elimination of the parasite by drug treatment caused a slow depletion in the number of splenic gammadelta, CD4+, and CD8+ T cells. Following challenge, drug-cured J(H)-/- mice exhibited nearly identical parasitemia time courses as naïve controls. Depletion of either CD4+ T cells or gammadelta T cells from chronically infected J(H)-/- mice by monoclonal antibody treatment resulted in an immediate and significant (P < 0.05) exacerbation of parasitemia coupled with a marked decrease in splenic gammadelta T-cell numbers. The number of CD4+ T cells, in contrast, did not decrease in mice after anti-T-cell receptor gammadelta treatment. The results indicate that cell-mediated immunity against blood-stage malarial parasites during chronic malaria (i) requires the continued presence of blood-stage parasites to remain functional, (ii) is dependent upon both gammadelta T cells and CD4+ T cells, and (iii) lacks immunological memory.

Reactive oxygen species augment B-cell-activating factor expression

B-cell-activating factor (BAFF) plays a role in mature B-cell generation and maintenance. Lipopolysaccharide (LPS) activates toll-like receptor 4 (TLR4)-dependent signal transduction and induces ROS production. Here, we investigated BAFF production regulated by reactive oxygen species (ROS). BAFF expression was augmented by LPS stimulation and by serum deprivation that induced ROS production. BAFF expression was inhibited by treatment with various antioxidants including N-acetyl -L-cysteine (NAC). We also investigated BAFF expression in vivo using peroxiredoxin II (PrxII)-deficient mouse spleen cells. PrxII is a member of the antioxidant enzyme family that protects cells from oxidative damage. Constitutive production of endogenous ROS was detected in spleen cells lacking PrxII. Serum BAFF protein level and BAFF transcript expression in splenocytes were significantly higher in PrxII –/– mice than wildtype mice. A higher BAFF level is consistent with the higher total number of splenocytes and B220 +cells. Results were supported by NF-κB activation as judged by reduced IκBα degradation and increased nuclear translocation of p65/RelA with LPS stimulation, serum deprivation, and PrxII deletion. Data suggest that TLR4-mediated BAFF expression was increased by ROS and it was inhibited by PrxII controlling ROS production.

Delayed occurrence of H- ras12V-induced hepatocellular carcinoma with long-term treatment with cinnamaldehydes

Cinnamaldehyde from the bark of Cinnamomum cassia has been reported to have antitumor activity mediated by the inhibition of farnesyl transferase. We assessed in vivo the chemo-preventive effect of cinnamaldehydes on H- ras12V-induced hepatocellular carcinoma formation. A mouse model of hepatocellular carcinoma was established by using the transgene of mutated H- ras12V under the regulation of albumin enhancer/promoter. When treated with cinnamaldehyde for 10 weeks, hepatic tumor development was delayed with 2′-benzoyloxycinnamaldehyde (BCA) compared with control hepatocellular carcinoma formation. The effect of 2′-hydroxycinnamaldehyde (HCA) was comparable. The number of lesions and the size of each lesion were significantly reduced by BCA. Cell proliferation in the lesion was detected by incorporation of 5-bromo-2′-deoxyuridine (BrdU). BCA increased the number of splenocytes, concanavalin A-stimulated splenocyte proliferation and the infiltration of lymphocytes into liver. Data suggest that the delayed hepatic tumor development observed with BCA could be mediated by a long-term immunostimulating effect on T cells.

TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-γ knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.

T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene

Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3 + T cells) was significantly higher in Prx II −/− mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II −/− mice was also higher than wild type. Differentiation of Prx II −/− mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II −/− bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).

Modification of gamma radiation induced response of peritoneal macrophages and splenocytes by Hippophae rhamnoides (RH-3) in mice

Alcoholic extract of Hippophae rhamnoides, RH-3, reported to render >80% survival against lethal whole body Co-60-gamma irradiation (10 Gy) in mice, was investigated for its immunostimulatory effects. In comparison with un-irradiated control, whole body irradiation did not reduce peritoneal macrophage counts at 24 h post-irradiation. RH-3 treatment (30 mg kg(-1) body weight) alone or 30 min before whole-body irradiation enhanced viable counts of macrophages significantly (P< or =0.05) compared with both un-irradiated control and irradiated groups. Whole-body irradiation reduced the number of viable splenocytes significantly (P<0.05) compared with un-irradiated control at 24 h post-irradiation. RH-3 treatment alone or before whole-body irradiation appreciably countered radiation-induced decrease in splenocyte count. 3H-thymidine uptake method revealed that whole-body irradiation reduced splenocyte proliferation significantly (159 +/- 45 counts min(-1)/10(6) cells; P< or =0.05) in comparison with control (607 +/- 142 counts min(-1)) at 24 h after irradiation but RH3 treatment before irradiation reduced the steep decrease and maintained it as 444+/-153 counts min(-1). After whole-body irradiation, the ratio of spleen weight/mouse weight decreased to 1.5 +/- 04 compared with 2.9 +/- 0.32 in un-irradiated control at 24 h post-irradiation. Similarly, total protein content in splenocytes also decreased to 48 +/- 6 microg/10(6) cells in comparison with 368 +/- 16 microg/10(6) cells of un-irradiated control. RH-3 treatment before irradiation countered radiation-induced decrease in both spleen weight/mouse weight ratio (4.0 +/- 0.35) and total protein content (360 +/- 13 mug/10(6) splenocytes). In the supernatant of peritoneal macrophage cultures exposed to 2 Gy Co-60-gamma radiation ex-vivo, the total nitrite content was enhanced significantly (P<0.05) to 5.72 +/- 0.09 microM in comparison with un-irradiated control (1.64 +/- 0.09 microM). RH-3 treatment (30 microg mL(-1)) before irradiation reduced total nitrite significantly (0.93 +/- 0.3; P< or =0.05) in comparison with irradiated control group. At 24 h after whole body irradiation, the CD4+/CD8+ ratio reduced to 1.5 in comparison with un-irradiated control (1.9) but RH-3 treatment before irradiation restored the ratio to 2.1. These findings explicitly reveal the immunostimulatory activity of RH-3, which may play an important role in the manifestation of its radioprotective efficacy.

Effect of electric foot shock and psychological stress on activities of murine splenic natural killer and lymphokine-activated killer cells, cytotoxic T lymphocytes, natural killer receptors and mRNA transcripts for granzymes and perforin

To explore the mechanism of stress-induced inhibition of natural killer (NK) activity, female C57BL/6 mice were stimulated by electric foot shock and psychological stress for 7 days consecutively. The shocked mice received scrambled, uncontrollable, inescapable 0.6 mA electric shocks in a communication box 120 times during 60 min. The mice in the psychological stress group were put into the communication box without electric foot shock. The plasma corticosterone level in both stressed groups was significantly higher than that in controls on days 1, 3, 5 and 7 and showed the highest level on day 3 in the foot shock stress. According to these results, therefore, we investigated the effect of stress on immunological function on day 3, and measured body weight, weight of the spleen, number of splenocytes, splenic NK, lymphokine-activated killer (LAK) and cytotoxic T lymphocyte (CTL) activities, NK receptors, and mRNA transcripts for granzymes A and B and perforin in splenocytes. The NK, LAK and CTL activities, and NK receptors in mice with both types of stress were significantly decreased compared to those of the control mice, but the decreases were greater in the foot-shocked mice than in the psychological-stress mice. The mRNA transcripts for granzyme A and perforin were significantly decreased only in the foot-shocked mice. On the other hand, the foot-shock stress increased granzyme B. The above findings suggest that stress induced inhibition of NK, LAK and CTL activities partially via affecting NK receptors, granzymes and perforin.

Influences of DTC and zinc supplementation on the cellular response restoration in restrained mice

The studies were conducted on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to restraint stress the mice were treated with sodium diethyldithiocarbamate (DTC) i.p. at a dose of 20 mg/kg five times at 48 h intervals. DTC was used per se or with zinc ions interaction, by adding zinc sulfate to drinking water at a dose of 72 microgram/mouse daily. The results obtained in the study show that restraint stress causes involution of lymphatic organs, decreased the percentage of immature (CD4+CD8+) and, mature (CD4+) thymocytes and CD4+), CD8+ and CD19+ splenocytes and proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The restraint stress decreased also interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from E. coli. Pretreatment with DTC counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymocytes, splenocytes and IL-1 production, accelerated regeneration of thymus and spleen, shorter suppressive action of restraint stress on the percentage of CD4+CD8+thymocytes and in total normalisation of the CD4+thymocytes and splenocytes. DTC administered prior to restraint stress augmented the proliferative response of thymocytes to two mitogens. The immunocorrecting action of DTC is enhanced by zinc supplementation, expressed in the increased percentage of CD4+thymocytes and splenocytes, CD19+splenocytes, proliferative activity of thymocytes stimulated with PHA and IL-1 production. The obtained results show that DTC administration can be supplemented with zinc in order to restore the immune system impaired by stress.

Trichloroethylene Does Not Accelerate Autoimmune Diabetes in NOD Mice

Pre-existing or contributing risk factors, including genetic predisposition and environmental influences, are largely thought to play a crucial (though ill-elucidated) role in the development of autoimmunity. Trichloroethylene (TCE) is a widely used organic solvent, which has been suspected of increasing the prevalence of autoimmune diseases, e.g., lupus, following environmental contamination. Although few epidemiological data are available, several studies reported an accelerated and more severe disease in TCE-exposed autoimmunity-prone MRL+/+ mice. To test whether TCE can exert similar deleterious effects on organ-specific autoimmune diseases, non obese diabetic (NOD) mice were given 5 mg/ml TCE via the drinking water for 12 weeks. TCE administration induced a decrease in CD44+ splenic T-cells and CD45RBhigh, CD54+ blood and splenic T-cells. Conversely, the number of CD45RBlow splenocytes was increased. Interestingly, the progressive increase in serum TNF-α and IFN-γ levels normally seen with age in these mice was inhibited by TCE. There was also a relative lower incidence of histological changes in the pancreas of TCE-exposed NOD mice than in unexposed mice. Contrary to what has been found in systemic models of autoimmunity, TCE did not accelerate the diabetes of NOD mice and may have a protective effect. This finding suggests that comparative studies using different genetically related autoimmune-prone models are needed to investigate the role of xenobiotics in the precipitation of autoimmunity, particularly in sensitive populations.

Altered Splenocyte Function in Aged C57BL/6 Mice Prenatally Exposed to Diethylstilbestrol

The linkage between in utero exposure to diethylstilbestrol (DES) and the manifestation of a variety of reproductive disorders and possibly immune alterations in adults (i.e., human and mice) is suggestive of a fetal basis of adult disease. While the long-term adverse consequences of prenatal DES-exposure on reproductive disorders are well known, there is a paucity of data with regard to immune outcome. We hypothesize that prenatal DES-exposure “imprints” the immune system, altering the response to subsequent exposure to DES in adult mice. In this pilot study, C57BL/6 mice were prenatally exposed to DES or vehicle only (oil) and then exposed to DES at 1 year of age. Potential alterations in the spleen were then examined. Female DES-exposed mice (DESprenatal/DESadult) or femaleDES had higher serum levels of interferon-gamma (IFNγ) in response to administration of an IFNγ -inducer (soluble proteins-derived from Toxoplasma gondii), compared to female controls, which received oil during prenatal life (Oilprenatal/DESadult). Splenic lymphocytes from female DESprenatal/DESadult mice, when activated with Concanavalin A (ConA), also secreted higher levels of IFNγ compared to female controls (Oilprenatal/DESadult) when examined at 14-months of age. This increase in IFNγ in prenatal DES-exposed mice is not due to enhanced numbers of splenocytes or increased relative percentages of CD4+ or CD8+ cells. ConA-activated T-cells from female DESprenatal/DESadult had increased expression of the co-stimulatory molecule, CD28. These above immune changes were not evident in the males prenatally exposed to DES. Prenatal DES exposure also did not induce autoimmunity in non-autoimmune C57BL/6 mice. Overall, results from these prefatory studies suggest that prenatal DES exposure may have long-term immune alterations, which become evident following a secondary exposure to DES in adult life.

T Cells from Presensitized Donors Fail to Cause Graft-versus-Host Disease in a Pig-to-Mouse Xenotransplantation Model

The ability of T cells from pigs, the most suitable donors for clinical xenotransplantation, to induce graft-versus-host disease (GVHD) and to facilitate hematopoietic cell engraftment in highly disparate xenogeneic recipients remains unclear. In this article, the authors address these questions in a presensitized pig-to-mouse transplantation model using porcine cytokine-transgenic mice. Swine donors were presensitized by mouse skin grafting and boosted by the injection of mouse cells after the skin graft was rejected. Porcine peripheral blood mononuclear cells (PBMC) and splenocytes were collected at various times after mouse skin grafting, and their potential to induce GVHD and to facilitate donor hematopoietic cell engraftment in conditioned murine recipients was evaluated. Presensitization of donor pigs resulted in marked enhancement of anti-mouse responses, as reflected in augmented anti-mouse mixed lymphocyte responses, cell-mediated cytotoxicity, and antibody production. However, injection of high numbers of PBMC and splenocytes (>1 x 10(8)) with bone marrow cells from the presensitized pigs failed to induce detectable GVHD or long-term chimerism in mice that were treated with depleting anti-T-cell and natural killer cell antibodies, cobra venom factor, medronate-liposomes, and 4 Gy of whole-body and 7 Gy of thymic irradiation. Histologic analysis revealed no mononuclear cell infiltration or GVHD-associated lesions in the liver, intestine, lungs, or skin of the mouse recipients. Furthermore, the recipient mice had no detectable T cells or anti-pig immunoglobulin G antibodies in the blood by 6 weeks after injection of porcine cells. These results demonstrate that porcine T-cell function is severely impaired in the xenogeneic murine microenvironment.

Immunosuppressive and autoimmune effects of thimerosal in mice

The possible health effects of the organic mercury compound thimerosal (ethylmercurithiosalicylate), which is rapidly metabolized to ethylmercury (EtHg), have recently been much debated and the effect of this compound on the immune system is largely unknown. We therefore studied the effect of thimerosal by treating A.SW (H-2 s ) mice, susceptible to induction of autoimmunity by heavy metals, with 10 mg thimerosal/L drinking water (internal dose ca 590 μg Hg/kg body weight/day) for up to 30 days. The lymph node expression of IL-2 and IL-15 mRNA was increased after 2 days, and of IL-4 and IFN-γ mRNA after 6 and 14 days. During the first 14 days treatment, the number of splenocytes, including T and B cells as well as Ig-secreting cells decreased. A strong immunostimulation superseded after 30 days treatment with increase in splenic weight, number of splenocytes including T and B cells and Ig-secreting cells, and Th2- as well as Th-1-dependent serum immunoglobulins. Antinucleolar antibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin, and systemic immune-complex deposits developed. The H-2 s strains SJL and B10.S also responded to thimerosal treatment with ANoA. The A.TL and B10.TL strain, sharing background genes with the A.SW and B10.S strain, respectively, but with a different H-2 haplotype ( t1), did not develop ANoA, linking the susceptibility to H-2. Thimerosal-treated H-2 s mice homozygous for the nu mutation (SJL- nu/nu), or lacking the T-cell co-stimulatory molecule CD28 (B10.S–CD28 −/−), did not develop ANoA, which showed that the autoimmune response is T-cell dependent. Using H-2 s strains with targeted mutations, we found that IFN-γ and IL-6, but not IL-4, is important for induction of ANoA by thimerosal. The maximum added renal concentration of thimerosal (EtHg) and inorganic mercury occurred after 14 days treatment and was 81 μg Hg/g. EtHg made up 59% and inorganic mercury 41% of the renal mercury. In conclusion, the organic mercury compound thimerosal (EtHg) has initial immunosuppressive effects similar to those of MeHg. However, in contrast to MeHg, thimerosal treatment leads in genetically susceptible mice to a second phase with strong immunostimulation and autoimmunity, which is T-cell dependent, H-2 linked and may at least partly be due to the inorganic mercury derived from the metabolism of ethyl mercury.

Host Environment Dictates the Outcome Following Transfer of Graft-Versus-Host Reactive Effector/Memory T Cells.

Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). Identical DLI transferred immediately following lethal irradiation lead to severe GVHD. In contrast to freshly irradiated mice developing GVHD, established MC receiving DLI show delayed kinetics of effector/memory T cell (TE/M) expansion and a distinct distribution with no accumulation of cells in the gut. Lethal irradiation is associated with tissue injury, altered chemokine expression and cytokine dysregulation, and resolution of these changes is important in limiting GVHD following delayed DLI. In an attempt to define the respective roles of ‘intrinsic' T cell versus ‘extrinsic' environmental factors in shaping the character of the GVH response elicited, TE/M developing following GVHD induction were transferred to established MC. Conversely, TE/M developing following delayed DLI to MC were transferred to lethally-irradiated 2° recipients. In the first series of experiments, we transferred 1 x 107 C57BL/6 (B6) splenocytes and 5 x 106 B6 T-cell depleted bone marrow (TCD BM) to lethally-irradiated BALB/c recipients. On day 4 following BMT, when GVH-reactive T cell activation and expansion was maximal, 3 x 106 nylon wool-passaged TE/M derived from recipient spleens (equivalent to 1 recipient spleen to 1 secondary recipient) were transferred to established B6 → BALB/c MC or to lethally irradiated BALB/c 2° recipients. The latter mice received B6 TCD BM to prevent death from aplasia. Secondary established MC recipients were resistant to GVHD whereas the lethally-irradiated 2° recipients developed severe, lethal GVHD (median survival time-MST- mixed chimeras >100d versus irradiated mice 52d, p<0.0001). Histological studies confirmed that 4/5 lethally-irradiated 2° recipients developed colitis versus 0/4 MC. TE/M derived from GVHD mice did not increase donor chimerism in 2° MC recipients. In reciprocal experiments, we transferred 2.5 x 107 B6 CD45.1 splenocytes to established B6 CD45.2 → B6D2F1 MC. This number of splenocytes represents the dose required to convert 100% of MC to full donor chimerism without induction of lethal GVHD. On day 12 post-DLI, when GVH-reactive T-cell expansion was maximal, a cohort of recipient mice was sacrificed and splenocytes harvested. On the basis of 1 recipient spleen to 1 secondary recipient, 2.7 x 107 nylon wool-passaged lymphocytes were transferred to 2° freshly-irradiated B6D2F1 recipients together with B6 TCD BM. In parallel, we depleted host-derived cells by sorting for DLI-derived CD45.1+ lymphocytes and transferred these cells on the basis of 1 recipient to 1 2° recipient (7 x 106 lymphocytes) to a second cohort of lethally-irradiated recipients. After a lag-period of about 20 days, 2° recipients of non-fractionated cells lost >10% weight and developed clinical signs of mild GVHD, but then showed almost complete recovery by day 60 post-BMT. In sharp contrast, after a similar lag period, 2° recipients of CD45.1+-selected lymphocytes developed severe lethal GVHD (MST CD45.1+ cells 55d vs >100d non-fractionated cells, p<0.0005). In conclusion, these findings suggest that the host environment strongly influences the capacity of TE/M to induce GVHD. Furthermore, host-derived cells pre-existing in or developing following delayed DLI, reduce the potential of TE/M to induce GVHD.

Dietary methoxychlor exposure modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in F0 and F1 generations of Sprague Dawley rats

Methoxychlor, a chlorinated hydrocarbon pesticide, is a persistent environmental contaminant that has been identified in human reproductive tissues. Methoxychlor has been shown to be estrogenic in both in vivo and in vitro studies. As an endocrine disrupter, it may have the potential to adversely affect endocrine, reproductive, and immune systems in animals. The present study evaluated methoxychlor's immunotoxic potential in F0 (dams) and F1 generations of Sprague Dawley rats exposed to an isoflavone-free diet containing methoxychlor at concentrations of 10, 100, and 1000 ppm. In dams, exposure to methoxychlor from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the NK activity (1000 ppm) and the percentages of T cells (1000 ppm), helper T cells (1000 ppm) and macrophages (100 and 1000 ppm). In contrast, a decrease in the numbers of splenocytes and B cells was observed at the 100 and 1000 ppm concentrations. In F1 males, exposure to methoxychlor gestationally, lactationally and through feed from postnatal day 22-64 (78 days total exposure) produced an increase in the spleen IgM antibody-forming cell response to sheep red blood cells (100 and 1000 ppm) and the activity of NK cells (1000 ppm). However, there was a decrease in the terminal body weight (1000 ppm), spleen weight (1000 ppm), thymus weight (100 and 1000 ppm), and the numbers of splenocytes (1000 ppm), B cells (100 and 1000 ppm), cytotoxic T cells (1000 ppm) and NK cells (100 and 1000 ppm). In F1 females, exposure to methoxychlor produced a decrease in the terminal body weight (1000 ppm) and the percentages of cytotoxic T cells (10, 100 and 1000 ppm). These results demonstrate that developmental and adult dietary exposure to methoxychlor modulates immune responses in Sprague Dawley rats. Immunological changes were more pronounced in the F1 generation male rats that were exposed during gestation and postpartum, when compared to the F0 and F1 generation females. Increases in antibody-forming cell response and NK cell activity, and altered spleen cell subpopulation numbers were observed in the F1 generation male rats, without similar changes to the F1 generation females.

Pretransplant portal venous adoptive transfer in conjunction with sirolimus prolongs renal allograft survival in non-human primates

Abstract Introduction: Portal venous inoculation of alloantigen has been demonstrated to induce hyporesponsiveness in multiple rodent models and has been suggested as a preferred method of donor specific transfusion (DST) for human tolerance protocols. Portal infusion of donor splenocytes and/or bone marrow has also been shown to induce tolerance to skin transplants across complete MHC barriers in mice. The objective of this study was to determine if portal DST had a demonstrable graft-sparing effect in a non-human primate renal allograft model. Methods: Outbred rhesus monkeys were used as donor-recipient pairs based on genetic non-identity at MHC and pretransplant MLR responsiveness. Perioperative DST was administered portally using splenocytes or bone marrow cells on day -7. A similar number of splenocytes or bone marrow cells were infused via a peripheral vein on day -2. On day 0, a kidney transplant was performed. Between the first exposure to alloantigen and the time of kidney transplant, the monkeys were maintained on FK506 1 mg/kg PO BID or sirolimus 1 mg/kg PO qD. No immunosuppression was used thereafter. Results: Histological examination of the transplant kidney at the time of euthanasia was consistent with acute cellular rejection. Group 4 had a significantly longer survival than either group 3 (p = 0.037), group 5 (p = 0.025) or group 6 (p = 0.001). GroupPV infusionIV infusionImmuno- suppressantSurvivalMean survival1SplenocyteSplenocyteFK5065, 1392SplenocyteBone marrowFK5065, 149.53Bone marrowBone marrowFK5064, 12, 14, 1511.254Bone marrowBone marrowSirolimus14, 26, 28, 40275——Sirolimus7, 7, 776———5, 5, 6, 7, 8, 86.5 Conclusions: Portal venous donor bone marrow inoculation with sirolimus adjuvant therapy resulted in prolonged renal allograft survival in this non-human primate model. Additional study is under way to determine humoral immune response and transcriptional analysis of the liver in response to portal inoculation.

A mouse model of pemphigus vulgaris by adoptive transfer of naive splenocytes from desmoglein 3 knockout mice.

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by antidesmoglein3 (anti-Dsg3) IgG autoantibodies. Recently, we developed a PV mouse model by adoptive transfer of splenocytes from recombinant Dsg3-immunized Dsg3(-/-) mice to Rag2(-/-) immunodeficient mice that expressed Dsg3. We determined whether the adoptive transfer of naive splenocytes from nonimmunized Dsg3(-/-) mice induces the anti-Dsg3 IgG production and the PV phenoytpe in recipient mice. We adoptively transferred naive Dsg3(-/-) splenocytes into Rag2(-/-) mice and compared their PV phenoytpe with those mice receiving immunized Dsg3(-/-) splenocytes. The numbers of splenocytes and their subpopulations required for anti-Dsg3 IgG production were examined. Mice that received naive Dsg3(-/-) splenocytes produced anti-Dsg3 IgG, which bound to keratinocyte cell surfaces in vivo, and developed the PV phenotype, including oral erosions with suprabasilar acantholysis. Antibody production and the appearance of the PV phenotype were delayed by approximately 2 weeks in mice that received naive splenocytes compared with mice that received immunized splenocytes. However, once the PV phenotypes developed, there were no apparent differences in disease severity between the two models. Interestingly, the anti-Dsg3 IgG titres were significantly lower in mice that received naive splenocytes than in mice that received immunized splenocytes, suggesting that the former antibodies were more potent than the latter. The frequency of anti-Dsg3 IgG production depended on the number of transferred naive splenocytes. Both CD4+ T cells and B220+ B cells from naive Dsg3(-/-) mice were essential for the production of anti-Dsg3 IgG antibodies. Dsg3-specific naive lymphocytes in Dsg3(-/-) mice can be primed and activated by the endogenous Dsg3 in recipient mice to produce pathogenic anti-Dsg3 IgG without active immunization. This approach using naive lymphocytes provides a unique model to dissect immunological mechanisms of tolerance against peripheral autoimmune targets.

LOSS OF DONOR ISLET GRAFTS IN DIABETIC NOD MICE WITH A LOW LEVEL OF DONOR CHIMERISM IS MEDIATED BY AUTOIMMUNITY

P1207 Aims: A substantial level of donor lymphocyte chimerism is required to protect donor-specific islet grafts in the mouse model with autoimmune diabetes. Donor islet grafts can be rejected in diabetic NOD mice with a low level of donor lymphocyte chimerism. In this study, we investigated whether donor islet graft rejection in diabetic NOD mice with a low level of donor lymphocyte chimerism is mediated by autoimmune or alloimmune response. Methods: Allogeneic BALB/c (H-2d) splenocytes were injected into diabetic NOD mice (H-2g7) at day -3, followed by cyclophosphamide at day -1. BALB/c bone marrow and islets were transplanted simultaneously at day 0. Anti-CD154 mAb and rapamycin were given from day 0 to day 14. Mixed chimerism was measured by flow cytometric analysis. Islet graft function was monitored by measuring blood glucose. After donor islet grafts were rejected, anti-islet autoantigens-specific autoimmune response was determined by adoptive transferring splenocytes from chimeric NOD mice that had rejected donor-specific islet grafts into NOD.scid mice. Alloimmune response was tested by MLR for lymphocyte proliferation and ELISPOT for INF-γ producing cells in vitro.. Results: Mixed chimerism was achieved in all recipient mice. At 8 weeks post-bone marrow transplantation, the level of donor-derived CD3+ T cells was 11.4±7.9%. After donor-specific islet grafts were rejected, 5x106 splenocytes from these chimeric NOD mice with at least over 1% donor CD3+ T cells were adoptive transferred into each NOD.scid mouse. 12 of 15 NOD.scid mice developed diabetes 12 weeks after adoptive transfer. All 8 NOD.scid mice that received splenocytes from control diabetic NOD mice also developed diabetes 5 weeks after adoptive transfer. Proliferation of splenocytes from chimeric NOD mice against donor BALB/c stimulators was markedly reduced, as compared with proliferation of splenocytes from naïve NOD mice (P<0.01). However, the response of chimeric mice to third-party C3H/He was not significantly different from the response of naïve NOD mice. The number of IFN-γ secreting splenocytes significantly increased in the splenocytes of control NOD mice under the stimulation of BALB/c mouse splenocytes. In contrast, the level of IFN-γ-secreting splenocytes in the chimeric NOD mice did not increase and it was the same as the background level under the stimulation of BALB/c mouse splenocytes (P<0.01). The number of IFN-γ-secreting splenocytes in both the chimeric NOD mice and control NOD mice significantly increased when third-party (C3H/He) splenocytes were used as the stimulators, as compared with the naïve splenocytes as the stimulators. Conclusions: These data demonstrate that islet antigen-specific autoreactive T cells exist in diabetic NOD mice with donor lymphocyte chimerism and they are responsible for islet graft loss.

Protective effect of (+)-catechin against 5-fluorouracil-induced myelosuppression in mice.

The object of this study was to investigate the efficacy of (+)-catechin, which was isolated from Actinidia arguta Planch (Actinidiaceae), as a bone marrow cell proliferation-promoting compound against the hematotoxicity of 5-fluorouracil (5-FU) in mice. Intraperitoneally injected (+)-catechin (1 and 10 mg/kg per day) accelerated the recovery of the number of white blood cells (WBC) and platelets (PLT) but did not affect the number of circulating red blood cells (RBC). (+)-Catechin also augmented the number of myelocytes and splenocytes. Dual color flow cytometric analysis revealed that (+)-catechin reversed the reduction of the population of leukocytes (CD11b+ monocytes, Gr-1+ granulocytes and CD3+ T and CD45RA+ B lymphocytes) in whole blood, spleen and bone marrow caused by 5-FU. (+)-Catechin (1 and 10 mg/kg per day) showed remarkable recovery of Gr-1+ cells in all three types of tissues and of CD11b+ cells in the bone marrow cells. These findings suggest that (+)-catechin selectively enhances the recovery of the population of granulocytes reduced by 5-FU in mice.

Role of macrophages in a mouse model of postoperative MRSA enteritis

Background We have established a mouse model for fatal postoperative enteritis due to Staphylococcus aureus to analyze mechanisms of bacterial translocation and determine reasons for the lethality of this infection. In the present study the role of macrophages was ascertained in protection against S. aureus induced enteritis. Materials and methods Mice were pretreated with cyclophosphamide (CY), an immunosuppressant, and then infected directly into the jejunum with methicillin-resistant S. aureus (MRSA) isolated from a patient. In other groups of mice liposome-encapsulated dichloromethylene diphosphate (Cl 2MDP) was administered to deplete macrophages in vivo. Other experimental groups received lipoteichoic acid (LTA), which was used to inhibit the ability of macrophages to bind MRSA, and additionally, to analyze dependence of bacterial clearance on the macrophage scavenger receptor. The ability of macrophages to bind MRSA was compared with survival rates in this mouse model of fatal postoperative enteritis. Results Injection of liposome-encapsulated Cl 2MDP decreased survival rate of mice infected intraintestinally with MRSA in a dose-dependent manner. Cyclophosphamide also decreased survival rate of MRSA-infected mice and was found to correlate with its ability to decrease the number of macrophages in the spleen. Intravenous LTA administration did not affect total splenocyte numbers or the number of splenic macrophages but decreased the ability of macrophages to bind MRSA and adversely affected survival of mice infected with MRSA. Conclusions Macrophages play a critical role in protection against MRSA administered directly into the jejunum. LTA recognition sites (probably type A scavenger receptors) on macrophages are required for binding and phagocytosing MRSA.