In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease.

In contrast to intact BALB/c mice, BALB/c mice rendered deficient in Vbeta4+ CD4+ T cells develop a Th1 response to infection with Leishmania major and are resistant. Vbeta4-deficient BALB/c mice are unable to generate the early IL-4 transcription occurring in Vbeta4 Valpha8 CD4+ T cells of BALB/c mice within 1 day of infection. Here we demonstrate that treatment of Vbeta4-deficient BALB/c mice with IL-4 during the first 64 h after infection instructs Th2 cell development and susceptibility to infection. The demonstrated inability of IL-4 to reverse the resistant phenotype of BALB/c mice treated with anti-CD4 mAb the day before infection suggest that these effects of IL-4 require its interaction with CD4+ T cells. In contrast to draining lymph node cells from BALB/c mice, cells from Vbeta4-deficient BALB/c mice remain responsive to IL-12 following infection. Strikingly, administration of IL-4 to Vbeta4-deficient BALB/c mice renders their lymph node cells unresponsive to IL-12 by down-regulating IL-12R beta2-chain expression. This study directly demonstrates that in BALB/c mice IL-4 is necessary and sufficient to initiate the molecular events steering Th2 cell maturation and susceptibility to L. major.

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions.

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells.

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.

Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor.

To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice.

The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O2.-). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O2.- is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II-induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD+/+ and ecSOD-/- mice. After 6 days of angiotensin II-treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD-/- than ecSOD+/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD-/- mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD+/+ mice. Compared with sham-operated ecSOD+/+ mice, sham-operated ecSOD-/- mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O2.-, as measured by lucigenin chemiluminescence, was higher in ecSOD-/- compared with ecSOD+/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD-/-, as well as from clipped ecSOD+/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD-/- mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O2.-, controlling blood pressure and vascular function in angiotensin II-dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Functional plasticity of the LACK-reactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice.

Early production of IL-4 by LACK-reactive Vbeta4-Valpha8 CD4(+) T cells instructs aberrant Th2 cell development and susceptibility to Leishmania major in BALB / c mice. This was demonstrated using Vbeta4(+)-deficient BALB / c mice as a result of chronic infection with MMTV (SIM), a mouse mammary tumor virus expressing a Vbeta4-specific superantigen. The early IL-4 response was absent in these mice which develop a Th1 response to L. major. Here, we studied the functional plasticity of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells using BALB/ c mice inoculated with L. major shortly after infection with MMTV (SIM), i. e. before deletion of Vbeta4(+) cells. These mice fail to produce the early IL-4 response to L. major and instead exhibit an IFN-gamma response that occurs within LACK-reactive Vbeta4-Valpha8 CD4(+) T cells. Neutralization of IFN-gamma restores the production of IL-4 by these cells. These data suggest that the functional properties of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells are not irreversibly fixed.