Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in pregnant C57BL/6 mice infected with L. major at different gestational periods.

BACKGROUND AND OBJECTIVE: To assess if gestational factors affect the resistance of C57BL/6 mice to L major infection, this study determined the levels of IL-4 and IFN-gamma in popliteal lymph node cells of pregnant C57BL/6 mice infected with L. major at 16 hours, 5 days-, 10 days- and 15 days- post plug by PCR, ELISA and BIOASSAY. DESIGN/SETTING: Experimental. RESULTS: Infected pregnant C57BL/6 mice developed larger cutaneous footpad lesions compared with non-pregnant C57BL/6 mice (that showed signs of resolution 7-10 weeks after infection). But, the lesions in infected pregnant C57BL/6 mice and infected non-pregnant C57BL/6 mice were not as large as in susceptible BALB/c mice. The mean litter weight was also reduced in pregnant infected C57BL/6 mice particularly in the groups infected at later stages of pregnancy (day 10- and day 15-post plug). The levels of both IL-4 and IFN-gamma increased with gestation in pregnant infected C57BL/6 mice compared with pregnant non-infected group, while only IL-4 was raised in pregnant infected mice compared with infected non pregnant mice. CONCLUSIONS: It may be concluded that increased IL-4 in pregnant infected C57BL/6 mice caused the transient susceptibility to L major infection while reduced litter weight was associated with increased IFN-gamma. These effects were pronounced in C57BI/6 mice infected with L major in late pregnancy.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Deficient T cell fate specification in mice with an induced inactivation of Notch1.

Notch proteins are cell surface receptors that mediate developmental cell specification events. To explore the function of murine Notch1, an essential portion of the gene was flanked with loxP sites and inactivation induced via interferon-regulated Cre recombinase. Mice with a neonatally induced loss of Notch1 function were transiently growth retarded and had a severe deficiency in thymocyte development. Competitive repopulation of lethally irradiated wild-type hosts with wild-type- and Notch1-deficient bone marrow revealed a cell autonomous blockage in T cell development at an early stage, before expression of T cell lineage markers. Notch1-deficient bone marrow did, however, contribute normally to all other hematopoietic lineages. These findings suggest that Notch1 plays an obligatory and selective role in T cell lineage induction.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production.

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor beta (IL-2R beta; CD122) and the polymorphic antigen NK1.1. These "NK-like" T cells are distinguished by their highly skewed V alpha and V beta repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1+ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2R beta+ T cells with a skewed V beta repertoire characteristic of "NK-like" T cells. Furthermore lack of NK1.1 expression on IL-2R beta+ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2R beta+ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that "NK-like" T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.

Combined effects of interleukin-3 and interleukin-11 on hematopoiesis in irradiated mice

The survival rate and recovery of peripheral blood cells and platelets were studied in Balb/c mice subjected to different single doses of whole-body irradiation and treated with a combination of interleukin-3 (IL-3) and interleukin-11 (IL-11). In a first group of 20 mice, 7.5 Gy irradiation, immediately followed by 2 and 5 days therapy of IL-3 and IL-11, respectively, increased the survival rate to 82% compared to 20% in untreated controls. In a second group of mice irradiated with 7 Gy, we observed significantly higher platelet, white blood cell (WBC), and red blood cell (RBC) counts after treatment with both cytokines, as compared to IL-3 or IL-11 alone or untreated controls. In addition, the survival rate of the mice with the combined therapy was also increased to 84%, compared to 48% in untreated controls. Irradiation (8.5 Gy) gave 100% mortality for the control mice, and therapy with combined IL-3 plus IL-11 had only a marginal effect. Interestingly, syngeneic bone marrow transplantation (BMT) alone, performed 16 hours after irradiation, increased the survival rate to 70%, while BMT combined with administration of IL-3 plus IL-11 increased it to 97%. Furthermore, BMT combined with cytokine administration could partially prevent the severe WBC and RBC depletion observed in mice treated with BMT alone and promoted a more rapid recovery of platelets and RBC. These data show that the combination of IL-3 and IL-11 has a radioprotective effect and can enhance recovery of platelets, WBC, and RBC in irradiated mice. Combined IL-3 plus IL-11 therapy may be clinically useful in myelodepression, especially in platelet depletion related to radiation therapy or chemotherapy, or after bone marrow transplantation.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormone-releasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormone-receptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3 +/- 1.5 ng/ml was observed compared with 1.04 +/- 1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5 +/- 9.7 ng/ml and a higher growth hormone release of 163 +/- 46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormonereleasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormonereceptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Host immune response in immunodeficient mice against infection with Cryptosporidium parvum

Immunantwort von immundefizienten Mäusen gegenüber Infektionen mit Cryptosporidium parvum. Cryptosporidium parvum ist ein intrazellulärer, protozoischer Krankheitserreger, der im immunkompromittierten Wirt zu lebensbedrohender Enteritis führen kann. CD4+ T-Zellen und Interferon (IFN)-γ spielen wesentliche Rollen bei der Wirtsimmunantwort gegen die Infektion. Dennoch sind die Effektormechanismen, die zur Resistenz führen nur wenig verstanden. In dieser Studie wurde die Immunantwort von IFN-γ- und Interleukin (IL)-12-Defektmäusen parallel zu Wildtypmäusen analysiert. Die Ergebnisse identifizierten IFN-γ als Schlüsselzytokin bei der natürlichen und erworbenen Immunität während der Erst- und Folgeinfektion mit C. parvum. Tumornekrosefaktor (TNF)-α ist möglicherweise ein Induktor der frühen IFN-γ-Antwort in IL-12 Knockout-Mäusen. Weiterhin tragen offenbar sowohl Th1- als auch Th2-Zytokine zur Überwindung der Primärinfektion bei, die ersten mehr als die letztgenannten. Zytokingene waren am Ort der Infektion (Ileum) dramatisch verändert, nicht aber in den lokalen Lymphknoten und der Milz. Nach Folgeinfektion ergab sich in Abwesenheit von IFN-γ eine signifikante Erhöhung der Th2-Zytokine IL-5 and IL-13. Die Ergebnisse zeigten weiterhin, dass das Th1-Zytokin IL-18 zur Resistenz gegenüber C. parvum beiträgt, möglicherweise durch verschiedene Immunfunktionen, wie der Regulation von Serum-IFN-γ während der Infektion und/oder der Erhaltung der Homeostase der Th1/Th2-Zytokine durch Regulation der Th2-Zytokine. Weiterhin zeigten diese Untersuchungen den Transfer von Resistenz gegenüber C. parvum von infizierten auf naïve Mäuse mittels stimulierter intraepithelialer Lymphozyten und CD4+ T-Zellen. Diese Ergebnisse weisen auf die Gegenwart von C. parvum-spezifischen CD4+ T-Zellen in anderen lymphatischen Geweben neben der Darmmukosa hin. Eine Stimulation der Spendertiere durch Infektion war notwendig für eine übertragbare schützende Immunität. Dennoch konnte die übertragene Immunität nicht die Infektion der Empfängertiere vollständig verhindern; eine Verdopplung der Spenderzellen führte zu keinem besseren Ergebnis. Weiterhin ergab der Transfer von CD4+ und CD8+ T-Zellen (Pan-T-Zellen) keinen erhöhten Schutz der naiven Empfängertiere als der alleinige Transfer von CD4+ T-Zellen. Dies weist auf die fehlende Bedeutung der CD8+ T-Zellen beim Schutz vor C. parvum-Infektion hin.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck (Hck) and p58c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2 and β3 integrins. To determine if the defective function of hck−/−fgr−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck−/−fgr−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

Normal cardiovascular development in mice deficient for 16 genes in 550 kb of the velocardiofacial/ DiGeorge syndrome region

Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of “cat eye” and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.