Indirect IL-4 pathway in type 1 immunity.

Recall Ag-specific IL-4 was detected in the spleen and in the blood, but not in lymph nodes of mice in which polarized type 1 immunity was induced. This IL-4 was not produced by T cells, but soluble factors secreted by the recall Ag-activated T cells, including IL-3, triggered cells of the innate immune system, primarily mast cells, to secrete IL-4. This notion has profound implications for immunodiagnostics: the detection of apparently recall Ag-specific IL-4 does not necessarily reflect the presence of Th2 or Th0 memory T cells with long-term cytokine commitment as is of interest for assessing adoptive immunity. We found that in vivo the indirect IL-4 pathway did not suffice to trigger IgE isotype switching, but promoted IgG1 production and inhibited type 1 T cell differentiation. Therefore, the indirect IL-4 pathway can explain partial type 2 immune response phenotypes in vivo in face of unipolar Th1 T cell immunity. The representation of mast cells in different tissues may explain why immune responses in certain organs are more type 2 biased. Therefore, the indirect pathway of IL-4 production represents a novel type of interaction between the innate and the adoptive immune system that can contribute to the outcome of host defense and immune pathology.     

Generation and regulation of human Th1-biased immune responses in vivo: a critical role for IL-4 and IL-10

Tissue damage in many human autoimmune diseases is mediated by activated autoantigen-specific Th1 cells. Delineation of the regulatory mechanisms controlling a Th1-biased human immune reaction and its pathologic potential is, therefore, a critical step in the understanding of autoimmune diseases. In this study, we introduce a novel means to investigate human Th1-biased immune responses in vivo. Intraperitoneal injection of human mononuclear cells into immunodeficient mice generates a xenogeneic Th1-biased human immune response characterized by systemic inflammation and leukocytic infiltrates with a granuloma-like architecture in the liver, and the perigastrointestinal and perirenal fatty tissue. Th1 cell activation was dependent on the presence of APCs and could be blocked by cyclosporine. Importantly, neutralization of endogenously produced IL-4 and IL-10 markedly exaggerated the immune response, whereas exogenous IL-4 and IL-10 inhibited systemic Th1 immunity. Thus, the model described in this paper presents a useful means to analyze the regulation of human immune reactions in an in vivo situation. The results suggest that both IL-4 and IL-10 contribute to controlling the development of a human Th1-biased immune reaction.     

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1-/-mice infected with influenza virus (H1 N1)

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI).The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo.IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation.We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice.Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6,TNF-α,G-CSF,KC,and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1-/-mice in comparison with that of wild type infected mice.The adaptive immune response against the H1N1 virus in IL-1R1-/-mice was impaired with downregulated anti-viral Th1 cell,CD8+ cell,and antibody functions,which contributes to attenuated viral clearance.Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1-/-mice compared with that in WT infected mice.Moreover,the infected IL-1R1-/-mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung.Together,these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury,particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.     

Inhibition of Kit expression by IL-4 and IL-10 in murine mast cells: role of STAT6 and phosphatidylinositol 3'-kinase.

The c-kit protooncogene encodes a receptor tyrosine kinase that is known to play a critical role in hemopoiesis and is essential for mast cell growth, differentiation, and cytokine production. Studies have shown that the Th2 cytokine IL-4 can down-regulate Kit expression on human and murine mast cells, but the mechanism of this down-regulation has remained unresolved. Using mouse bone marrow-derived mast cells, we demonstrate that IL-4-mediated Kit down-regulation requires STAT6 expression and phosphotidylinositide-3'-kinase activation. We also find that the Th2 cytokine IL-10 potently down-regulates Kit expression. IL-4 enhances IL-10-mediated inhibition in a manner that is STAT6 independent and phosphotidylinositide-3'-kinase dependent. Both IL-4- and IL-10-mediated Kit down-regulation were coupled with little or no change in c-kit mRNA levels, no significant change in Kit protein stability, but decreased total Kit protein expression. Inhibition of Kit expression by IL-4 and IL-10 resulted in a loss of Kit-mediated signaling, as evidenced by reduced IL-13 and TNF-alpha mRNA induction after stem cell factor stimulation. These data offer a role for STAT6 and phosphotidylinositide-3'-kinase in IL-4-mediated Kit down-regulation, coupled with the novel observation that IL-10 is a potent inhibitor of Kit expression and function. Regulating Kit expression and signaling may be essential to controlling mast cell-mediated inflammatory responses.     

IL-1, IL-6, and PDGF mRNA expression in alveolar cells following stimulation with a tobacco-derived antigen.

To test the hypothesis that inflammatory cytokine production might be an early event in the development of the disease associated with smoking, we used alveolar cells from healthy nonsmokers stimulated with TGP as a model system. TGP, a phenol-rich glycoprotein which is present in tobacco leaves and cigarette smoke condensate, activates the immune system. It stimulates polyclonal B cell differentiation, induces primarily an IgE response, and activates human leukocytes to produce IL-1. Using in situ nucleic acid hybridization we show that the steady-state levels of IL-1 alpha, IL-1 beta, IL-6, platelet-derived growth factor (PDGF)-A, and PDGF-B mRNAs are consistently elevated in the alveolar cells of all donors following TGP stimulation. The kinetics of mRNA expression suggest that IL-1 alpha and IL-1 beta mRNAs are independently regulated in alveolar cells, while the regulation of PDGF-A and PDGF-B mRNA seems to be similar. The activated cells also synthesize elevated levels of IL-1 and IL-6. These findings lend support to the suggestion that some clinical consequences of smoking might be initiated and enhanced by the production of inflammatory cytokines. Moreover, IL-6 could also activate a polyclonal B cell response, which could lead to the synthesis of autoantibodies and thus cause immune-mediated tissue injury.     

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.     

Murine interleukin-11 (IL-11) is expressed at high levels in the hippocampus and expression is developmentally regulated in the testis

IL-11, derived from a bone marrow stromal cell line, has pleiotropic effects on both hematopoietic cells and nonhematopoietic cells. However, no previous studies have systematically addressed expression of IL-11 in primary tissues in vivo and the relationship of IL-11 tissue specific gene expression and function of IL-11 is not clear. In the present study, we examined constitutive IL-11 expression in various murine adult tissues in vivo. IL-11 mRNA is expressed in a wide range of normal tissues (including hematopoietic organs) at levels only detected by RT-PCR. IL-11 protein was detected in brain and testis by Western blot analysis. The in vivo cellular distribution of IL-11 expression was examined by in situ hybridization. In brain, IL-11 message is distributed in granular layer dentate gyrus and pyramidal cell layers of hippocampus. IL-11 is also expressed in anterior horn cells and lateral column neuronal cells of the spinal cord. In testis, Il-11 mRNA is expressed in round spermatids at stage VI-IX seminiferous tubules. IL-11 expression in testis is restricted to developing spermatogonia and is developmentally regulated, since no expression is seen in mice genetically deficient in germ cells and in mice prior to sexual maturation. These expression data correlate with functional data demonstrating that IL-11 stimulates proliferation in vitro of a hippocampus neuronal progenitor cell line and administration of IL-11 in vivo accelerates recovery of spermatogenesis after cytotoxic therapy. These studies suggest that IL-11 may be an important regulator in neural and testicular function. © 1996 Wiley-Liss, Inc.     

Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells.

We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha-induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3-E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli.     

Calcitonin gene-related peptide indirectly inhibits IL-7 responses in pre-B cells by induction of IL-6 and TNF-alpha in bone marrow

Calcitonin gene-related peptide (CGRP) has inflammatory and immunoregulatory properties. CGRP directly inhibits IL-7 induced proliferation in developing B cells and also induces soluble factors that inhibit IL-7 responses. We identified 2 cytokines, IL-6 and TNF-alpha, induced by CGRP, that inhibit IL-7 pre-B cell responses. CGRP induction of IL-6 and TNF-alpha mRNA in long-term bone marrow cultures is transient and IL-6 or TNF-alpha inhibit IL-7 induced colony formation by 60%. When added with CGRP, colony formation is completely inhibited. TNF-alpha directly inhibits IL-7 responses in B220(+)/IgM(-) cells whereas IL-6 inhibits only colony formation with whole bone marrow. This suggests that the effect of IL-6 is mediated by other cells in the bone marrow. These results suggest that the indirect effect of CGRP on IL-7 depends in part on induction of IL-6 and TNF-alpha.     

Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells.

The death of hemopoietic cells on withdrawal of CSF occurs by a mechanism known as apoptosis characterized by the early degradation of chromatin into oligonucleosome-length fragments. Insulin-like growth factor I plays a pivotal role in the regulation of somatic cell growth as a mediator of growth hormone action. Animals with low levels of circulating IGF-I are more vulnerable to infections and have diminished immune responses. To analyze the possibility of a regulatory role of IGF-I on hemopoiesis and determine its mechanism of action, we have studied the effect of this growth factor on the survival and proliferation of two IL-3-dependent hemopoietic cell lines and in IL-3-responsive primary cultures of bone marrow-derived mast cells. In IL-3-depleted cultures, IGF-I prevented DNA fragmentation and apoptotic cell death. Insulin at high concentration had a weak protective action and IGF-II was inactive in suppressing apoptosis in these IL-3-dependent hemopoietic cells. Cell proliferation was also stimulated by IGF-I in the absence of other hemopoietic growth factors although it was a weak mitogen when compared with IL-3. These results indicate that circulating or locally produced IGF-I may promote survival of both the steady state hemopoietic precursor population and cytokine-producing cells and could therefore regulate hemopoiesis acting in a concerted manner with other CSF.     

Secretory leukocyte protease inhibitor reduces inflammation and alveolar bone resorption in LPS-induced periodontitis in rats and in MC3T3-E1 preosteoblasts

In periodontitis, alveolar bone resorption is induced by excessive host immune and inflammatory response against bacterial infection. Secretory leukocyte protease inhibitor (SLPI) has anti-bacterial and anti-inflammatory activity in inflammatory responses. SLPI inhibits joint inflammation and bone destruction, but the function of SLPI in periodontitis is unclear. Therefore, this study investigated whether SLPI inhibits the inflammatory response and alveolar bone resorption in LPS-induced periodontitis of rats. Micro-computed tomography and histological analysis showed that SLPI inhibited alveolar bone resorption by LPS-induced periodontitis. Immunohistochemistry revealed that SLPI decreased tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) expression in periodontitis tissue, and decreased mRNA and protein expression of TNF-α and IL-1β in LPS-stimulated MC3T3-E1 cells. The results indicated that SLPI reduced alveolar bone resorption in LPS-induced periodontitis and inhibited inflammatory cytokine, such as TNF-α and IL-1β, expression in LPS-stimulated MC3T3-E1 preosteoblasts. Therefore, SLPI could be a regulatory molecule by inhibiting alveolar bone resorption through the reduction of inflammatory cytokines, and inducing osteoblast activation for bone formation.     

Intracellular Delivery of Lipopolysaccharide Induces Effective Th1-Immune Responses Independent of IL-12

Lipopolysaccharide (LPS) is responsible for many of the inflammatory responses and pathogenic effects of Gram-negative bacteria, however, it also induces protective immune responses. LPS induces the production of inflammatory cytokines such as TNF-α, IL-6, and IL-12 from dendritic cells (DCs) and macrophages. It is thought that IL-12 is required for one of the protective immune responses induced by LPS, the T helper 1 (Th1)-immune response, which include the production of IFN-γ from Th1cells and IgG2c class switching. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) does not induce the production of inflammatory cytokines from DCs, but enhances Th1-immune responses via type-I IFNs, independent of IL-12. Collectively, our results strongly suggest that LPS-liposomes can effectively induce Th1-immune responses without inducing unnecessary inflammation, and may be useful as an immune adjuvant to induce protective immunity.     

IL-1beta-mediated proinflammatory responses are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.     

Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis

Dendritic cells (DCs) retrovirally transduced with IL-4 have recently been shown to inhibit murine collagen-induced arthritis and associated Th1 immune responses in vivo, but the mechanisms that underly these effects are not yet understood. In this report we demonstrate that IL-4-transduced DCs loaded with antigen led to lower T cell production of IFN-gamma, increased production of IL-4, and an attenuated, delayed type hypersensitivity response. We hypothesized that the ability of such DCs to regulate the Th1 immune response in vivo depends in part on their capacity to produce IL-12 and IL-23. Quantitative mRNA analysis revealed that IL-4-transduced DCs stimulated with CD40 ligand expressed higher levels of IL-12p35 mRNA, but lower levels of mRNA for IL-23p19 and the common subunit p40 found in both IL-12 and IL-23, compared with control DCs. These results, which indicate that expression of the IL-12 and IL-23 subunits is differentially regulated in IL-4-transduced DCs, were confirmed by ELISA of the IL-12 and IL-23 heterodimers. Thus, therapeutic suppression of Th1 -mediated autoimmunity (as recently shown in murine collagen-induced arthritis) and induction of Th2 responses in vivo by IL-4-transduced DCs occurs despite their potential to produce increased levels of IL-12, but could reflect, in part, decreased production of IL-23.     

Caspase-1-processed IL-1 family cytokines play a vital role in driving innate IL-17

The interleukin (IL)-1 cyokine family plays a vital role in inflammatory responses during infection and in autoimmune diseases. The pro-inflammatory cytokines, IL-1β and IL-18 are members of the IL-1 family that require cleavage by caspase-1 in the inflammasome to generate the mature active cytokines. Cells of the innate immune system, including γδ T cells and invariant natural killer T (iNKT) cells respond rapidly to invading pathogens by producing inflammatory cytokines, such as IFN-γ and IL-17. IL-1β or IL-18 in combination with IL-23 can induce IL-17 production by γδ T cells without T cell receptor (TCR) engagement. IL-1β and IL-23 can also synergize to induce IL-17 production by iNKT cells. Furthermore, CD4+ αβ effector memory T cells secrete IL-17 in response to IL-23 in combination with either IL-1β or IL-18, in the absence of any TCR stimulation. The early IL-17 produced by innate cells induces recruitment of neutrophils to the site of infection, stimulates local epithelial cells to secrete anti-microbial proteins, such as lipocalins and calgranulins, induces production of structural proteins important in tight junction stability, and promotes production of matrix metalloproteinases. Caspase-1 processed IL-1 family cytokines therefore play a vital role in the innate immune response and induction of IL-17 from innate immune cells which functions to fight infections and promote autoimmunity.     

A potent adjuvant monophosphoryl lipid A triggers various immune responses, but not secretion of IL-1beta or activation of caspase-1

Lipid A, the membrane anchor portion of LPS, is responsible for the endotoxin activity of LPS and induces many inflammatory responses in macrophages. Monophosphoryl lipid A (MPL), a lipid A derivative lacking a phosphate residue, induces potent immune responses with low toxicity. To elucidate the mechanism underlying the low toxicity of MPL, we examined the effects of MPL on the secretion of proinflammatory cytokines by mouse peritoneal macrophages, a murine macrophage-like cell line (RAW 264.7), and a human macrophage-like cell line (THP-1). MPL enhanced the secretion of TNF-alpha, but not that of IL-1beta, whereas Escherichia coli-type lipid A (natural source-derived and chemically synthesized lipid A) enhanced the secretion of both cytokines. Although MPL enhanced the levels of IL-1beta mRNA and IL-1beta precursor protein to levels similar to those induced by lipid A, IL-1beta precursor processing in MPL-treated cells was much lower than that in E. coli-type lipid A-treated ones. Moreover, MPL, unlike E. coli-type lipid A, failed to induce activation of caspase-1, which catalyzes IL-1beta precursor processing. These results suggest that an immune response without activation of caspase-1 or secretion of IL-1beta results in the low toxicity of this adjuvant.     

Suppression of IL-2-induced T cell proliferation and phosphorylation of STAT3 and STAT5 by tumor-derived TGF beta is reversed by IL-15.

IL-2 responses are susceptible to suppression by TGFbeta, a cytokine widely implicated in suppression of inflammatory responses and secreted by many different tumor cell types. There have been conflicting reports regarding inhibition of IL-2-induced STAT3 and STAT5 phosphorylation by TGFbeta and subsequent suppression of immune responses. Using TGFbeta-producing multiple myeloma tumor cells we demonstrate that tumor-derived TGFbeta can block IL-2-induced proliferation and STAT3 and STAT5 phosphorylation in T cells. High affinity IL-2R expression was required for the suppression of IL-2 responses as a novel CD25(-) T cell line proliferated and phosphorylated STAT3 when cultured with tumor cells or rTGFbeta1. Activating T cells with IL-15, which does not use the high affinity IL-2R, completely restored the ability of T cells to phosphorylate STAT3 and STAT5 when cultured with tumor cells. IL-15-treated T cells proliferated normally when cocultured with tumor cells or rTGFbeta1, whereas IL-2 responses were consistently inhibited. Preincubation with IL-15 also restored the ability of T cells to respond to IL-2 by phosphorylating STAT3 and STAT5, and proliferating normally in the presence of tumor cells. IL-2 pretreatment did not restore T cell function. IL-15 also restored T cell responses by T cells from multiple myeloma patients, and against freshly isolated bone marrow tumor samples. Thus, activation of T cells by IL-15 renders T cells resistant to suppression by TGFbeta1-producing tumor cells and rTGFbeta1. This finding may be exploited in the design of new immunotherapy approaches that will rely on T cells avoiding tumor-induced suppression.