The generation of Th memory in neonates versus adults: prolonged primary Th2 effector function and impaired development of Th1 memory effector function in murine neonates

Immunization during the neonatal period often results in Th2-biased secondary responses. To understand the regulation of this phenomenon, we have examined all phases of Th development, from the generation of primary effectors to the duration of the primary effector stage to the production of memory effector function. First, we had previously reported that although primary responses in the neonatal lymph nodes are mature, mixed Th1/Th2-like, primary responses in the spleens of the same animals are exclusively Th2-like. To determine whether Th2-dominant secondary responses are due to the Th2-polarized primary function in the spleen, neonates were splenectomized before immunization. Even in the absence of primary neonatal splenic responses, the secondary responses of neonates were Th2 dominant. Thus, the overwhelmingly Th2 primary responses in the neonatal spleen are not required to generate Th2-dominant memory in the lymph nodes. Second, we have compared the kinetics of the primary response phase in neonates and adults. In adults, Ag-specific Th2 function disappeared rapidly from both the lymph nodes and spleen. In contrast, primary Th2 function persisted out to 5 wk in both neonatal organs. Third, the generation of Th memory responses was examined in animals initially immunized as neonates and in adults. These experiments demonstrated that neonates are selectively impaired in the development of Th1 memory effector function. Together, these results indicate that neonates are biased to Th2 function at all phases of an immune response.     

Balance of Th1 and Th17 effector and peripheral regulatory T cells

Transfer of antigen-specific T cells into antigen-expressing lymphopenic recipients leads to the sequential generation of Th1 and Th17 effector and protective CD25⁺FoxP3⁺ regulatory cells in the periphery with surprisingly different kinetics. Such an experimental model is potentially valuable for defining the stimuli that regulate lineage decision and plasticity of various T cell effectors and peripheral regulatory T cells. Our studies have shown that IL-17 production occurs rapidly and declines within the first week with the appearance of IFN-γ producing T cells. Regulatory T cells appear during the recovery phase of the disease. The factors that mediate this complex differentiation originating from a starting naïve T cell population remain to be defined.     

Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.     

Developmental control of integrin expression regulates Th2 effector homing

Integrin CD18, a component of the LFA-1 complex that also includes CD11a, is essential for Th2, but not Th1, cell homing, but the explanation for this phenomenon remains obscure. In this study, we investigate the mechanism by which Th2 effector responses require the LFA-1 complex. CD11a-deficient T cells showed normal in vitro differentiation and function. However, Th2 cell-dependent allergic lung disease was markedly reduced in CD11a null mice and wild-type mice given LFA-1 inhibitors, whereas control of infection with Leishmania major, a Th1-dependent response, was enhanced. In both disease models, recruitment of IL-4-, but not IFN-gamma-secreting cells to relevant organs was impaired, as was adhesion of Th2 cells in vitro. These diverse findings were explained by the markedly reduced expression of CD29, an alternate homing integrin, on Th2, but not Th1, cells, which precludes Th2 homing in the absence of CD11a. Thus, murine Th1 and Th2 cells use distinct integrins for homing, suggesting novel opportunities for integrin-based therapeutic intervention in diverse human ailments influenced by Th2 cells.     

Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice

The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.     

Inhibitory effects of calcitonin on adenylate cyclase activity in different rat brain areas

We have investigated the effect of calcitonin (CT) on adenylate cyclase in membranes from different rat brain areas. Salmon calcitonin (sCT) dose-dependently inhibited the enzyme activity in midbrain, hypothalamus, medulla, pons and caudate nucleus, but was ineffective in adenohypophysis. The inhibitory effect was enhanced by GTP. Comparison of calcitonins of different origin indicated that sCT was the most potent in inhibiting the enzyme in hypothalamic membranes, eel CT (eCT) was slightly less potent, and human CT (hCT) was ineffective. Chronic I.C.V. pretreatment with sCT did not modify the subsequent in vitro sensitivity of adenylate cyclase to sCT. It is concluded that some of CNS actions of CT might involve modulation of intracellular cAMP levels.     

Homocarnosinase activity in the rat brain areas kidneys under different states of hyperbarooxygenation

The homocarnosinase activity in different brain areas and kidneys of the normal rats and under different conditions of hyperbarooxygenation are determined. The highest activity of this enzyme is observed in cerebellum. The high homocarnosinase activity is typical of kidneys as well. The action of oxygen in a dose of 0.425 MPa for 60 min (in the absence of convulsions) increases the homocarnosinase activity in the cerebral hemispheres by 18.6%, in the midbrain by 18.6%, in midbrain and diencephalon--by 56.5%, and in the medulla oblongata--by 40.6%. The homocarnosinase activity in the cerebellum decreases by 16.7%, in kidneys--by 18.5%. At the convulsive stage of oxygen intoxication caused by the effect of 0.7 MPa dose of oxygen the homocarnosinase activity in cerebral hemispheres rises by 158.5%, in the midbrain and diencephalon--by 141.5%, in the medulla oblongata by--161.1%. Under the same conditions homocarnosinase activity in the cerebellum is unchanged as compared with the control.     

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.     

Modulation of HIV-1 replication by a novel RhoA effector activity

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Galpha13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.     

Caspase-dependent Cdk activity is a requisite effector of apoptotic death events

The caspase-dependent activation of cyclin-dependent kinases (Cdks) in varied cell types in response to disparate suicidal stimuli has prompted our examination of the role of Cdks in cell death. We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants (DN-Cdks) and Cdk inhibitory genes. Here we demonstrate that Cdk2 activity is necessary for death-associated chromatin condensation and other manifestations of apoptotic death, including cell shrinkage and the loss of adhesion to substrate. Susceptibility to the induction of the cell death pathway, including the activation of the caspase cascade, is unimpaired in cells in which Cdk2 activity is inhibited. The direct visualization of active caspase activity in these cells confirms that death-associated Cdk2 acts downstream of the caspase cascade. Cdk inhibition also does not prevent the loss of mitochondrial membrane potential and membrane phospholipid asymmetry, which may be direct consequences of caspase activity, and dissociates these events from apoptotic condensation. Our data suggest that caspase activity is necessary, but not sufficient, for the full physiological cell death program and that a requisite function of the proteolytic caspase cascade is the activation of effector Cdks.     

Regulatory T cell responses develop in parallel to Th responses and control the magnitude and phenotype of the Th effector population

Host survival during schistosomiasis requires the development of a tightly regulated and Th2-polarized immune response against parasite egg Ags. In this system, Th1 response suppression has been thought to be enforced through the production of IL-10 by Th2 cells and natural T regulatory (Treg) cells. By comparing Th responses in schistosome egg-injected mice that lack IL-10, IL-4, and/or Treg cells, we have been able to build a detailed picture of the relative contributions of Treg cells, Th2 cells, and IL-10 to regulation of the egg-induced response. Our data indicate that eggs induce a marked Treg cell response, evident as the extensive proliferation of Foxp3(+) cells that is proportionally as great as the response occurring within the Th compartment. Furthermore, we show that Treg cells prevent Th1 response development and limit the magnitude of the Th2 response. Although Treg cells are able to produce IL-10 after egg injection, we found no evidence for a role for IL-10 in Treg-mediated suppression of Th cell responses, nor did we find evidence for an inhibitory effect of Th2 cells on Th1 response development. Thus, the magnitude and phenotype of the egg-induced effector Th response are controlled by a parallel response within the Treg population.     

Pyruvate dehydrogenase fuels a critical citrate pool that is essential for Th17 cell effector functions

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Absence of the complement anaphylatoxin C3a receptor suppresses Th2 effector functions in a murine model of pulmonary allergy

Asthma is a chronic inflammatory disease of the lung resulting in airway obstruction. The airway inflammation of asthma is strongly linked to Th2 lymphocytes and their cytokines, particularly IL-4, IL-5, and IL-13, which regulate airway hyperresponsiveness, eosinophil activation, mucus production, and IgE secretion. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, our previous reports have demonstrated that complement contributes to bronchial hyperreactivity, recruitment of airway eosinophils, IL-4 production, and IgE responses in a mouse model of pulmonary allergy. To define the complement activation fragments that mediate these effects, we assessed the role of the complement anaphylatoxin C3a in a mouse model of pulmonary allergy by challenging C3aR-deficient mice intranasally with a mixed Ag preparation of Aspergillus fumigatus cell culture filtrate and OVA. Analysis by plethysmography after challenge revealed an attenuation in airway hyperresponsiveness in C3aR-deficient mice relative to wild-type mice. C3aR-deficient mice also had an 88% decrease in airway eosinophils and a 59% reduction in lung IL-4-producing cells. Consistent with the reduced numbers of IL-4-producing cells, C3aR-deficient mice had diminished bronchoalveolar lavage levels of the Th2 cytokines, IL-5 and IL-13. C3aR knockout mice also exhibited decreases in IgE titers as well as reduced mucus production. Collectively, these data highlight the importance of complement activation, the C3a anaphylatoxin, and its receptor during Th2 development in this experimental model and implicate these molecules as possible therapeutic targets in diseases such as asthma.     

Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells

Th cells can be subdivided into IFN-gamma-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H(+)K(+)-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.     

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.