Control B6 Mice Research Articles

Astrocyte Leptin Receptor (ObR) and Leptin Transport in Adult-Onset Obese Mice

The agouti viable yellow (A vy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in A vy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in A vy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, (125)I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young A vy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, A vy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) A vy and B6 mice. Higher ObRb mRNA was seen in the A vy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the A vy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the A vy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

Interferon-γ Induces Chronic Active Myocarditis and Cardiomyopathy in Transgenic Mice

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-alpha is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine interferon (IFN)-gamma remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-gamma transgenic mice, which constitutively express IFN-gamma in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-gamma mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4(+) and CD8(+) T cells but also Mac2(+) (galectin 3(+)) macrophages and CD11c(+) dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-gamma overexpression. IFN-gamma-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-alpha. Myotoxic IFN-gamma effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-gamma effect. The precise mechanism of IFN-gamma cardiotoxicity remains to be elucidated.

Genetic Effects on Bone Mechanotransduction in Congenic Mice Harboring Bone Size and Strength Quantitative Trait Loci

The degree to which bone tissue responds to mechanical loading events is partially under genetic control. We assess the contribution of three genetic loci (QTLs linked to bone geometry and strength)--located on mouse Chrs. 1, 8, and 13--to mechanically stimulated bone formation, through in vivo skeletal loading of congenic strains. Bone size was not consistently associated with mechano-responsiveness, indicating that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific. We showed previously that C57BL/6J (B6) mice are more responsive to mechanical stimulation than C3H/HeJ (C3H) mice and that B6 mice harboring a 40-Mb region of distal C3H Chromosome (Chr.) 4 are more responsive to mechanical stimulation than are fully B6 mice. Here, we assess the contribution of three more genetic loci--located on mouse Chrs. 1, 8, and 1--to mechanically stimulated bone formation. Three congenic mouse strains were created in which a region of mouse Chr. 1 (approximately 64 cM; 150 Mb), Chr. 8 (approximately 45 cM; 86 Mb), or Chr. 13 (approximately 24 cM; 42 Mb) was moved from C3H stock to a B6 background through selective breeding over nine generations. The regions moved to the B6 background correspond to three of several quantitative trait loci (QTLs) identified for bone size and strength. The resulting congenic mice were 99% B6, with the remaining genomic DNA comprised of the Chr. 1, 8, or 13 QTLs of interest. Male and female congenic (1T, 8T, and 13B) and B6 control mice were subjected to in vivo loading of the right ulna at one of three different load magnitudes. A separate set of animals from each group had strain gauges applied at the ulnar midshaft to estimate strain at each loading level. Loading was conducted once per day for 3 days (60 cycles/d; 2 Hz). Fluorochrome labels were injected intraperitoneally 4 and 11 days after loading began. Using quantitative histomorphometry, bone formation rates were measured in loaded (right) and control (left) ulnas. All male congenic mice exhibited significantly reduced mechano-responsiveness compared with male B6 controls, but the same comparison among females yielded no difference from controls, with the exception of the 1T congenics, which showed increased responsiveness to loading. Among the congenic strains, smaller bone size was not consistently associated with reduced mechano-responsiveness. Our results indicate that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific. Our data might explain why different individuals can engage in similar exercise protocols yet experience different results in terms of bone mass accrual.

CD4+CD25+ regulatory T cells attenuate hypersensitivity pneumonitis by suppressing IFN-γ secreting T cells (B25)

Abstract Hypersensitivity pneumonitis(HP) is developed after repeated inhalation of finely dispersed antigens. The pathogenesis of HP is complex and not completely understood. Currently, HP is mainly caused by Th1 immune response. CD4+CD25+ T cells play critical roles in preventing the development of various immune diseases. Therefore, we postulated that CD4+CD25+ T cells might contribute to attenuating HP. To address this postulation, we investigated functional roles of CD4+CD25+ T cells in Saccharopolyspora rectivirgula (SR)-induced HP. CD4+CD25+ T cells infiltrated the lung tissue in B6 mice during the development of SR-induced HP. In CD4+CD25+ T cell-depleted B6 mice using PC61 mAb, HP was worse in terms of total cell number, SR-specific IgG in serum and bronchoalveolar lavage fluid (BALF) and SR-specific T cell proliferation than those in control B6 mice. The adoptive transfer of CD4+CD25+ T cells attenuated SR-induced HP in mice depleted CD4+CD25+ T cell. CD4+CD25+ T cell depleted mice showed elevated IFN-γ production and reduced IL-4 production in the lung during HP. Moreover, CD4+CD25+ T cell suppressed IFN-γ-producing CD4+ and CD8+ T cells in BALF with direct contact manner rather than indirect mechanism. Taken together, these results suggest that CD4+CD25+ regulatory T cell play a protective role in SR-induced HP by suppressing IFN-γ-producing CD4+ and CD8+ T cell, which induced Th1 phenotype during the pathogenesis of HP. The enhancement of CD4+CD25+ regulatory T cell function may be useful therapeutic modality for HP.

Intrinsic B cell defect results in abnormal TD antigen response in lupus-prone mice (130.17)

Abstract The response to TD antigen NP-KLH was compared between the lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice and control B6 mice. The amount of anti-NP IgG Abs in TC mice was significantly decreased as compared to B6 mice. In contrast, total anti-IgG Abs and anti-dsDNA Abs were increased compared to B6 mice, indicating that the decreased was specific for the immunization. This corresponds to the known poor immunization response of lupus patients. In addition, germinal center (GC) NP-specific B cells were decreased in TC mice compared to B6 mice, although there was no difference in the total number of GC B cells between the two strains. Co-transfer of purified CD43- B and CD4+ T cells from either B6 or TC mice showed that a TC intrinsic B cell defect was involved. B6.Rag−/− mice transferred with non-T BM cells from immunized TC mice lost anti-NP antibody production unlike transfer with BM cells from B6 mice. In contrast, there was similar Ab production between the two strains when transferred with non-T splenocytes, indicating that long-lived plasma cells in TC mice accumulated in spleen, instead of migrating to BM. In conclusion, lupus-prone mice show a deficient response to NP-KLH, in which NP-specific B cells differentiate differently from autoantigen-specific B cells. This deficiency is due at least in part to a defect in B cell maturation and plasma cell migration. SLE susceptibility loci involved in these defects will be mapped in future.

NK cell phenotypes in Foxk1 deficient mice (82.13)

Abstract Foxk1 is a member of the forkhead/winged-helix transcription factor family. Foxk1−/− mice have a gross deficit of natural killer (NK) cells, roughly three-fold, in the spleen, peripheral blood and bone marrow. Both mature and immature NK cells are lacking in the affected tissues. NK cells are the only major lymphocyte compartment affected. Bone marrow chimera experiments between Foxk1−/− and B6 (control) mice have shown that the defect is NK cell intrinsic. The remaining NK cells in Foxk1−/− are functionally defective directly ex vivo as determined by YAC-1 killing. However, following in vitro culture with either IL-2 or IL-15, the Foxk1−/− NK cells regain lytic function. To further analyze the cytotoxic defect of Foxk1−/− NK cells, we have examined the expression of components of the lytic pathway. Foxk1−/− NK cells express lower amounts of Perforin, but increased amounts of FasL, compared to B6 mice. These levels of cytotoxic effector molecules still do not provide us with an answer as to why Foxk1−/− NK cells have a defect in lytic function. To examine the NK cell number deficit in Foxk1−/− mice we have used flow cytometry to assess the various developmental stages of NK cells in Foxk1−/− and B6 mice and have found that Foxk1−/− mice had altered cell surface marker expression at the earliest progenitor stage. Gene expression profiles of early precursors from B6 and Foxk1−/− mice are being examined by microarray analysis.

NK cells promote peritoneal xenograft rejection through an IFN‐γ‐dependent mechanism

Natural killer (NK) cells have emerged as major players in anti-viral and anti-tumour immune responses. Like cytotoxic T lymphocytes (CTL), they express perforin and are potent secretors of gamma-interferon (IFN-gamma). However, there is conflicting evidence about their role in mediating rejection of xenogeneic tissue. A pig-to-mouse peritoneal cell model of xenotransplantation was used to investigate the effect of NK deficiency on xenograft recovery and the possible mechanisms behind this NK-mediated graft rejection. gamma c(-/-)RAG(-/-) mice were used as a model of NK deficiency. Additionally, NK cells were depleted in RAG(-/-) mice using anti-asialo GM1. The contributions of IFN-gamma, perforin and NKT cells were studied using knock-out mice that were depleted in vivo of T cells. Mice were injected with 10(7) pig cells intraperitoneally and peritoneal fluid was assessed 5 days later for xenograft recovery and phenotypic analysis. The requirement for NK cells for xenograft rejection was also assessed using luciferase-transfected porcine cells in a renal subcapsular model of transplantation. Pig cell recovery was enhanced in both gamma c(-/-)RAG(-/-) and NK-depleted RAG(-/-) mice when compared with RAG(-/-) control mice. IFN-gamma(-/-) mice depleted of T cells also demonstrated superior graft survival compared with their B6 counterparts. However, there were minimal graft survival differences between Pfp(-/-) and B6 control mice. Similarly, a deficiency in NKT cells did not improve pig xenograft recovery from the peritoneum of these mice. Therefore, we conclude that NK cells, but not NKT cells, are important mediators of xenograft rejection in the peritoneal cavity, and that their role may be unmasked in the absence of T cells. The mechanism for this xenorejection appears to involve IFN-gamma but is perforin independent.

Comparative study between Hfe‐/‐ and β2m‐/‐ mice: progression with age of iron status and liver pathology

Hepatic iron overload in hemochomatosis patients can be highly variable but in general it develops in older patients. The purpose of this study was to compare development of iron load in of beta2m-/- and Hfe-/- mice paying special attention to liver pathology in older age groups. Liver iron content of beta2m-/-, Hfe-/- and control B6 mice of different ages (varying from 3 weeks to 18 months) was examined. Additional parameters (haematology indices, histopathology, lipid content and ferritin expression) were also studied in 18-month-old mice. The beta2m-/- strain presents higher hepatic iron content, hepatocyte nuclear iron inclusions, mitochondria abnormalities. In addition, hepatic steatosis was a common observation in this strain. In the liver of Hfe-/- mice, large mononuclear infiltrates positive for ferritin staining were commonly observed. The steatosis commonly observed the beta2m-/- mice may be a reflection of its higher hepatic iron content. The large hepatic mononuclear cell infiltrates seen in Hfe-/- stained for ferritin, may point to the iron sequestration capacity of lymphocytes and contribute to the clarification of the differences found in the progression of hepatic iron overload and steatosis in older animals from the two strains.

Involvement of lymphocytes in dextran sulfate sodium-induced experimental colitis

To investigate the roles of lymphocytes in the development of dextran sulfate sodium-induced colitis. Using various doses of dextran sulfate sodium (DSS), we induced colitis in wild-type B6 control and Rag-1 knockout (H-2b haplotype) mice, and evaluated the colitis in terms of symptomatic and histologic parameters, such as weight loss, survival, severity of diarrhea, shortage of colon length and histological changes. Symptomatic parameters were checked daily and histological changes were scored. Although development of colitis in Rag-1 knockout mice treated with high dose (5%) of DSS was comparable to that in B6 control mice, colitis progression was much more tolerable in Rag-1 knockout mice compared to than in B6 mice treated with low dose (1.5%) DSS. Symptomatic parameters as well as histopathologic changes were improved in Rag-1 knockout mice. These results indicate that the presence of lymphocytes contributes to colitis progression at low dose of DSS stimulation. Lymphocytes may play roles as an aggravating factor in DSS-induced colitis.

Multiple Genetic Loci From CAST/EiJ Chromosome 1 Affect vBMD Either Positively or Negatively in a C57BL/6J Background

Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively. BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1). After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines. Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (-6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = -0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb. Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.

Fetal Tolerance to Maternal Antigens Improves the Outcome of Allogeneic Bone Marrow Transplantation by a CD4+CD25+ T Cell-Dependent Mechanism.

The lack of donor availability is a major limitation to the widespread use of allogeneic HSCT and therefore it would be beneficial to identify permissible HLA mismatches. The maternal and fetal antigens which are transmitted through the bi-directional transplacental passage during pregnancy may induce tolerance to noninherited maternal antigens (NIMAs) in offspring and to inherited paternal antigens (IPAs) in the mother. Using mouse models of BMT, we investigated whether fetal-maternal tolerance could affect the outcome of allogeneic BMT. To generate NIMA-exposed mice, we used the F1 x P backcross breeding schema. A B6 (H-2b) male and a B6D2F1 (H-2b/d) female were mated to generate H-2b/b offspring that was exposed to NIMA-H-2d in utero. To generate IPA-exposed mice, a female B6 mouse was mated with a male B6D2F1 mouse and was exposed to IPA-H-2d from her H-2b/d offspring. First, to examine the influence of the NIMA/IPA exposure on the alloreactive T cell responses to the corresponding antigens in vitro, CD4+ T cells isolated from these mice were cultured with B6D2F1 stimulators. Proliferation and IFN-gamma production of T cells from NIMA-exposed mice in response to NIMAs were significantly reduced in comparison to those from the controls, but T cells from IPA-exposed mice did not reduce reactivity to IPAs. We then performed allogeneic BMT from NIMA-exposed donors. Lethally irradiated B6D2F1 mice were transplanted with 5x106 TCD BM from the control B6 mice together with 2x106 T cells from either 8 week-old NIMA-exposed or control B6 donors. GVHD mortality was significantly less in recipients of a NIMA-exposed donor than in those of a control donor (28% vs 84% on day 80, p<0.005). When transplanted to third-party B6C3F1 (H-2b/k) recipients, no protective NIMA effect was observed, thus demonstrating the antigen-specificity of the NIMA-effects. In contrast, BMT from an IPA-exposed donor did not reduce GVHD in comparison to BMT from a control donor. We then studied the effects of NIMA complementary transplantation on both subsequent T cell reconstitution and graft-versus-leukemia (GVL) effect. T cell reconstitution was significantly improved in NIMA-recipients in comparison to that in the allogeneic controls 40 days after BMT (p<0.05). The number of double positive thymocytes was also significantly higher in the recipients of NIMA-exposed donors than in those of the controls (p<0.05). In GVL experiments, 2.5x104 host-type P815 leukemia cells (H-2d), that were uniformly lethal to the recipients of syngeneic BMT by day 20 after BMT, were injected to recipients of allogeneic BMT on day 0. Allogeneic BMT from a control donor and a NIMA-exposed donor significantly delayed the leukemia relapse (p<0.005), thus demonstrating that NIMA complementary transplantation did not abrogate GVL effect. Lastly, we examined whether CD4+CD25+ T cells mediate the NIMA effect. The tolerogenic NIMA effect was completely abolished by the depletion of CD4+CD25+ cells from the donor inoculums, thus suggesting the involvement of CD4+CD25+ regulatory T cells in the tolerogenic NIMA effects. Our findings have profound implications on clinical HSCT, they suggest that the use of a NIMA-mismatched donor in the absence of a HLA-identical donor may be a potentially effective treatment strategy.

Natural Killer T (NKT) Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis by Producing Interferon-γ

Pulmonary fibrosis is a progressive illness characterized by interstitial fibrosis. Although the precise mechanism for pulmonary fibrosis is not completely understood, an immune response involving interferon (IFN)-gamma appears to play a role. Therefore, we examined the functional roles of natural killer T (NKT) cells, which produce IFN-gamma and interleukin-4 on activation, in bleomycin-induced pulmonary fibrosis. In NKT cell-deficient mice, pulmonary fibrosis was worse in terms of histology, hydroxyproline levels, and mortality than in control mice. The transforming growth factor (TGF)-beta1 levels were higher in the lung after injecting bleomycin, and blockade of TGF-beta1 by neutralizing monoclonal antibody attenuated the pulmonary fibrosis in CD1d-/- mice. In contrast, the production of IFN-gamma was reduced in lungs from CD1d-/- mice. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice increased IFN-gamma and reduced TGF-beta1 production, attenuating pulmonary fibrosis. An in vitro assay demonstrated that IFN-gamma was involved in suppressing TGF-beta1 production in cells collected from bronchoalveolar lavage. The adoptive transfer of NKT cells from IFN-gamma-/- mice did not reverse pulmonary fibrosis or TGF-beta1 production in lungs of CD1d-/- mice whereas NKT cells from B6 control mice attenuated fibrosis and reduced TGF-beta1 production. In conclusion, IFN-gamma-producing NKT cells play a novel anti-fibrotic role in pulmonary fibrosis by regulating TGF-beta1 production.

Antibody response is required for protection from Theiler's virus-induced encephalitis in C57BL/6 mice in the absence of CD8 + T cells

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that β 2M-deficient C57BL/6 mice lacking functional CD8 + T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice (μMT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected μMT and control B6 mice, the levels of CD4 + T cells were higher in the CNS of μMT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in μMT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated μMT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8 + T cells.

The Immunologic Function of 1B2+ Double Negative (CD4−CD8−) T Cells in the 2C Transgenic Mouse 1

2C mice bearing the cytotoxic TCR for class I L(d) on a C57BL/6 (B6) background have a preponderance of 1B2+CD8+ T cells directed against L(d). These naive CD8+ T cells are not directly cytotoxic without prior in vivo or in vitro activation. However, after in vitro sensitization, they become highly cytotoxic and will acutely and specifically reject a tolerant L(d+) BALB/c heart graft. Anti-lymphocyte serum (ALS) treatment eliminates CD4+ and CD8+ cells and a large double negative (CD4-CD8-) 1B2+ non-cytotoxic transgenic cell population remains. The immunological function of this unique peripheral population of T cells is investigated in the 2C transgenic mouse. To determine the activation characteristics of the 2C CD4-CD8- T cells, 2C peripheral T cells were analyzed for 1B2+, CD8+, and CD4+ marker by FACS before and 48-h after 0.5 cc ALS i.p. Similarly, in vitro, the response of these 2C CD4-CD8- T cells remaining after deletion of mature CD4+ and CD8+ T cells with ALS plus complement were evaluated by mixed lymphocyte culture and cytotoxic T lymphocyte after 7 days culture with BALB/c, IL-2, or BALB/c + IL-2. Parallel experiments were performed with control non-transgenic B6 mice. Following in vitro culture with BALB/c + IL-2, 2C CD4-CD8- T cells were injected into B6 mice with a tolerant BALB/c heart (tolerization via anti-CD4 mAb and intrathymic BALB/c) to determine their immunogenicity. While peripheral T cells in control B6 mice have <5% CD4-CD8- cells, transgenic 2C mice have a significantly increased percentage at 29 to 35% (P < 0.01). After the deletion of CD4+ and CD8+ T cells with either in vivo or in vitro ALS, 2C CD4-CD8- T cells increased to 96 to 99%. After 7-day culture, the 2C CD4-CD8- T cells decreased again to 33 to 38%. Simultaneously, 2C CD8+ T cells decreased from 56 to 62% to 0.1 to 3% after ALS treatment, but again increased to 61 to 70% after in vitro culture. Untreated 2C cells responded to IL-2 or BALB/c antigen equally well. However, after ALS treatment, CD4-CD8- T cells responded to IL-2 and IL-2 plus antigen, but not BALB/c antigen alone. Finally, CD4-CD8- T cells cultured for 7 days with BALB/c + IL-2 rejected the tolerant BALB/c heart in 5.3 +/- 0.3 days. In the periphery of transgenic 2C mice is a unique CD4-CD8- population of T cells bearing the transgenic specific marker 1B2. These non-cytotoxic cells can be optimally stimulated to develop marked specific L(d) cytotoxicity in parallel with the expression of the CD8+ epitope.

The yellow agouti mutation alters some but not all responses to diet and exercise.

Effects of ectopic expression of the agouti signaling protein were studied on responses to diet restriction and exercise in C57BL/6J (B6) mice and obese B6 mice congenic for the yellow agouti mutation [B6.Cg-Ay (Ay)]. Adult male Ay mice were either kept sedentary or exercised on a running wheel and fed ad libitum or diet restricted until weight matched to ad libitum-fed B6 control mice. Body composition, plasma lipids, leptin, and adiponectin were measured. mRNA levels for leptin, adiponectin, lipoprotein lipase, and pyruvate dehydrogenase kinase 4 were measured in a visceral (epididymal) and a subcutaneous (femoral) fat depot by real-time polymerase chain reaction. Correlations among traits exhibited one of three patterns: similar lines for B6 and Ay mice, different slopes for B6 and Ay mice, and/or different intercepts for B6 and Ay mice. Correlations involving plasma leptin, mesenteric and epididymal adipose weights, or low-density lipoprotein-cholesterol were most likely to have different slopes and/or intercepts in B6 and Ay mice. mRNA levels for leptin, Acrp30, pyruvate dehydrogenase kinase 4, and lipoprotein lipase in epididymal adipose tissue were not correlated with corresponding levels in femoral adipose tissue. The agouti protein interferes with leptin signaling at melanocortin receptors in the hypothalamus of Ay mice. Our results are consistent with the hypothesis that the melanocortin portion of the leptin-signaling pathway mediates effects primarily on certain fat depots and on some, but not all, components of cholesterol homeostasis.

Oral and portal venous tolerance in the interferon-γ knockout (GKO) mouse 1

Background The role of the proinflammatory cytokine interferon-γ (IFN-γ) in the development of oral or portal venous tolerance is poorly defined. By using knockout mice for IFN-γ (GKO mice), we investigated the effect of both oral and portal venous administration of alloantigen on the systemic delayed type hypersensitivity (DTH) response to alloantigen rechallenge. Materials and methods C57BL/6 (B6) control and GKO mice (also on a B6 background) were given either saline or BALB/c spleen cells (25 million) by oral gavage (PO) or by injection into the portal vein (PV) on day 0. The injection of 10 million BALB/c spleen cells subcutaneously into the dorsal flanks was performed after 7 days followed by footpad injection of 10 million BALB/c spleen cells on day 14. Specific footpad swelling was measured 24 h later using a micrometer. Splenocyte responsiveness was measured by in vitro mixed lymphocyte culture and cytotoxic T lymphocyte assay. Cytokine production of interleukin (IL)-2, IL-4, IL-10, and IFN-γ was measured by enzyme-linked immunosorbent assay. Results Although B6 mice given PO or PV saline demonstrated a DTH response of 0.47 ± 0.04 mm and 0.49 ± 0.05 mm, respectively, in GKO mice, a greater DTH response of 0.72 ± 0.08 mm and 0.75 ± 0.05 mm was measured after either PO or PV saline ( P < 0.05, P < 0.05, respectively). In vitro MLC and CTL confirmed the heightened DTH response to BALB/c observed in GKO versus B6. Despite this heightened response in control GKO mice, the DTH response was completely suppressed in both GKO and B6 mice, after both PO and PV BALB/c feedings ( P < 0.001, P < 0.001, respectively, in both GKO and B6 mice). The unchanged brisk response to third-party C3H/HeJ demonstrated antigenic specificity. Conclusions Although DTH responsiveness to alloantigen is increased in the absence of IFN-γ, both oral and portal venous alloantigen-specific tolerance can still be established in GKO mice similar to that in control B6 mice. The achievement of a tolerogenic immune response by both oral and portal venous routes indicates that lack of IFN-γ does not preclude the induction of tolerance in this murine model.

Oral and portal venous tolerance in the IFN-gamma knockout (GKO) mouse

We investigated the effect of both oral and portal venous administration of alloantigen on the systemic delayed type hyper-sensitivity (DTH) response to alloantigen rechallenge in GKO mice. C57BL/6 (B6) control and GKO mice were given either saline or BALB/c spleen cells (SC) by oral gavage (PO) or by injection into the portal vein (PV) on day 0. Injection of BALB/c SC SQ was performed after 7 days followed by footpad injection of BALB/c SC on day 14. Footpad swelling was measured 24 hours later using a micrometer. While B6 mice given PO or PV saline demonstrated a DTH response of 0.47 ± 0.04 mm and 0.49 ± 0.05 mm, respectively, in GKO mice, a greater DTH response of 0.72 ± 0.08 mm and 0.75 ± 0.05 mm was measured after either PO or PV saline (p < 0.05, p < 0.05, respectively). Despite this heightened response in control GKO mice, the DTH response was completely suppressed in both GKO and B6 mice, following both PO and PV BALB/c feedings (p < 0.001, p < 0.001, respectively in both GKO and B6). The unchanged brisk response to third party C3H/HeJ demonstrated antigenic specificity. While DTH responsiveness to alloantigen is increased in the absence of IFN-γ both oral and portal venous alloantigen-specific tolerance can still be established similar to that in control B6 mice. Achievement of a tolerogenic immune response by both oral and portal venous routes indicates that IFN-γ does not influence tolerance induction. TABLE—ABSTRACT P26MouseAntigen (route)SQ antigenFootpad swelling (mm)B6Saline (PO)BALB/c0.47 ± 0.04B6BALB/c (PO)BALB/c0.02 ± 0.01B6BALB/c (PO)C3H/HeJ0.52 ± 0.09B6Saline (PV)BALB/c0.49 ± 0.05B6BALB/c (PV)BALB/c0.01 ± 0.01B6BALB/c (PV)C3H/HeJ0.44 ± 0.06GKOSaline (PO)BALB/c0.72 ± 0.08GKOBALB/c (PO)BALB/c0.02 ± 0.00GKOBALB/c (PO)C3H/HeJ0.77 ± 0.11GKOSaline (PV)BALB/c0.75 ± 0.05GKOBALB/c (PV)BALB/c0.03 ± 0.02GKOBALB/c (PV)C3H/HeJ0.70 ± 0.05

Restraint stress and anti-tumor immune response in mice.

Psychological stress modulates the immune system through the hypothalamic-pituitary-adrenal axis, the sympatho-adrenomedullary axis and the opioid system. According to literature data, restraint stress increases the immune cell apoptosis, decreases the spleen and thymus cell content, the natural killer (NK) activity in the spleen, and it compromises the anti-tumor immune response in mice. We immobilized mice in two consecutive nights, and then determined the cell number, apoptosis, NK cell content, NK activity and the level of cytokine mRNAs (TNF-beta, TNF-alpha, IL-4, IL-5, IL-1alpha, IFN-gamma, IL-2, IL-6, IL-1beta and IL-3) in the thymus and spleen. No consistent changes were detected in any of the immune parameters either in C57Bl/6 or in DBA/2 mice. Stressed or control B6 mice were injected with B16 melanoma cells immediately after the immobilization or one week later. No significant differences were found in the growth of primary tumors and lung metastases in stressed and control animals. Taken together, our mice, kept in a general-purpose non-SPF animal house, seemed to be refractory to the stress-induced immunomodulation. Our interpretation is that stress-induced immunomodulation can occur only in mice isolated from any background stressors, or rather natural stimuli, throughout their life.

NK cell tolerance in mixed allogeneic chimeras.

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.

Essential role of extrathymic T cells in protection against malaria.

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1(-)TCR(int) cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 x 10(3) per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1(-)TCR(int) cells (IL-2Rbeta(+)TCRalphabeta(+)). Unconventional CD8(+) NKT cells (V(alpha)14(-)) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.