Interleukin-6 (IL-6) levels are known to be increased in patients with rheumatoid arthritis (RA). Tocilizumab, a monoclonal antibody to the IL-6 receptor (IL-6R), reduces disease activity in RA, although its mechanisms of action remain unclear. Since IL-6 regulates cytokine production by CD4 T cells during activation, we investigated whether treatment with tocilizumab altered the phenotype and cytokine production by CD4 T cells in patients with rheumatoid arthritis. We show here that tocilizumab treatment does not change the production of cytokines by naïve CD4 T cells. However, tocilizumab treatment causes a selective decrease of IL-21 production by memory/activated CD4 T cells. Since IL-21 is known to promote plasma cell differentiation, we examined the effect of tocilizumab on the production of autoantibodies. We show that there is a decrease in the levels of IgG4 anti-CCP antibodies, but there is no effect on IgG1 anti-CCP antibodies. In addition, we show that IL-21 is a powerful inducer of IgG4 production by B cells. Thus, IL-6 contributes to the presence of IgG4-specific anti-CCP autoantibodies in RA patients, likely through its effect on IL-21 production by CD4 T cells, and IL-6R blockade down-regulates this pathway.

In addition to immune cells, airway epithelial cells can contribute to and shape the immune response in the lung by secreting specific cytokines. IL-6 is a key factor in determining the effector fate of CD4+ T cells. Here we show that under basal conditions, the IL-6 gene is already highly expressed in lung epithelial cells, but not in immune cells resident in the lung. However, upon exposure of the lungs to fungal allergens, the direct contact of β-glucans present in the fungus cell wall with lung epithelial cells is sufficient to trigger the rapid synthesis and secretion of IL-6 protein. This posttranscriptional regulation of IL-6 in response to fungal extracts is mediated by the p38 mitogen-activated protein kinase pathway. The inhalation of β-glucans with a nonallergenic antigen is sufficient to provide an adjuvant effect that leads to mucous hyperplasia in the airways. Thus, β-glucans may constitute a common determinant of the fungal and plant-derived allergens responsible for some of the pathological features in allergic asthma.

Influenza virus infection is considered a major worldwide public health problem. Seasonal infections with the most common influenza virus strains (e.g. H1N1) can usually be resolved, but they still cause a high rate of mortality. The factors that influence the outcome of the infection remain unclear. Here we show that deficiency of IL-6 or IL-6 receptor is sufficient for normally sublethal doses of H1N1 influenza A virus to cause death in mice. IL-6 is necessary for the resolution of influenza infection by protecting neutrophils from virus-induced death in the lung and by promoting neutrophil-mediated viral clearance. Loss of IL-6 results in persistence of influenza virus in the lung leading to pronounced lung damage and, ultimately, death. Thus, we demonstrate that IL-6 is a vital innate immune cytokine in providing protection against influenza A infection. Genetic or environmental factors that impair IL-6 production or signalling could increase mortality to influenza virus infection.

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.

The incidence and severity of chronic lung diseases is growing and affects between 100 and 150 million people worldwide and is associated with a significant rate of mortality. Unfortunately, the initial cause that triggers most chronic lung diseases remains unknown and current available therapies only ameliorate, but do not cure the disease. Thus, there is a need for identification of new targets and development of novel therapies especially for those most severely affected. IL-6, like other inflammatory cytokines, has been shown to be elevated in different lung diseases, but it was considered a byproduct of ongoing inflammation in the lung. However, recent studies support a dissociation of IL-6 from inflammation in the lung and suggest that this cytokine plays an active role in pathogenesis of asthma and, in all likelihood, COPD. IL-6 may therefore be a germane target for treatment of these and other chronic lung disease. Here, we provide an overview of the studies in mouse models and human patients that provide support for the involvement of IL-6 in lung diseases.

Allergic asthma is caused by inhaled allergens and it is characterized by airway eosinophilia as well as mucus hypersecretion which can lead to airflow obstruction. Despite the association of increased IL-6 levels with human atopic asthma, the contribution of IL-6 to the development of allergic airway inflammation triggered by inhaled allergens remains unclear. In this study, we examined the role of IL-6 in a mouse model of allergic airway inflammation induced by direct airway exposure to extracts of Aspergillus fumigatus, a common allergen in humans. We show here that inhaled A. fumigatus extracts rapidly triggers the production of IL-6 in the airways. IL-6 appears to be dispensable for the recruitment of eosinophils to the lung during the development of allergic airway inflammation. However, IL-6 is essential for mucus hypersecretion by airway epithelial cells triggered in response to inhaled A. fumigatus antigens. Impaired mucus production caused by IL-6 deficiency correlates with a severe reduction in the levels of IL-13, a major inducer of mucin glycoproteins. Thus, IL-6 is a key regulator of specific hallmark features of allergic airway inflammation, and it could be a potential target for pulmonary diseases that are associated with goblet cell metaplasia and mucus hypersecretion.

Bcl2-modifying factor (Bmf) is a member of the BH3-only group of proapoptotic proteins. To test the role of Bmf in vivo, we constructed mice with a series of mutated Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation by the c-Jun NH2-terminal kinase (JNK) on Ser74, or mimic Bmf phosphorylation on Ser74. We report that the loss of Bmf causes defects in uterovaginal development, including an imperforate vagina and hydrometrocolpos. We also show that the phosphorylation of Bmf on Ser74 can contribute to a moderate increase in levels of Bmf activity. Studies of compound mutants with the related gene Bim demonstrated that Bim and Bmf exhibit partially redundant functions in vivo. Thus, developmental ablation of interdigital webbing on mouse paws and normal lymphocyte homeostasis require the cooperative activity of Bim and Bmf.

Background

Asthma is a chronic inflammatory disease of the airway that is characterized by a Th2-type of immune response with increasing evidence for involvement of Th17 cells. The role of IL-6 in promoting effector T cell subsets suggest that IL-6 may play a functional role in asthma. Classically IL-6 has been viewed as an inflammatory marker, along with TNFα and IL-1β, rather than as regulatory cytokine.

Objective

To investigate the potential relationship between IL-6 and other proinflammatory cytokines, Th2/Th17 cytokines and lung function in allergic asthma, and thus evaluate the potential role of IL-6 in this disease.

Methods

Cytokine levels in induced sputum and lung function were measured in 16 healthy control and 18 mild-moderate allergic asthmatic subjects.

Results

The levels of the proinflammatory biomarkers TNFα and IL-1β were not different between the control and asthmatic group. In contrast, IL-6 levels were specifically elevated in asthmatic subjects compared with healthy controls (p < 0.01). Hierarchical regression analysis in the total study cohort indicates that the relationship between asthma and lung function could be mediated by IL-6. Among Th2 cytokines only IL-13 (p < 0.05) was also elevated in the asthmatic group, and positively correlated with IL-6 levels (rS = 0.53, p < 0.05).

Conclusions

In mild-moderate asthma, IL-6 dissociates from other proinflammatory biomarkers, but correlates with IL-13 levels. Furthermore, IL-6 may contribute to impaired lung function in allergic asthma.

Cytokines have long been known to profoundly influence the adaptive immune response by determining CD4 T cell differentiation. Although IL-6 has been initially characterized as a B cell growth factor and inducer of antibody production research from our lab and others has revealed over the last years that IL-6 also plays a significant role in CD4 T cell differentiation. This review highlights the variety of ways in which IL-6 affects CD4 effector functions and how this may contribute to different types of diseases.

IP3 receptors (IP3Rs) regulate the release of Ca++ from intracellular stores in response to IP3. Little is known about the regulation of IP3R expression and their role during the activation of CD4 T cells. In this study we show that mouse naïve CD4 T cells express IP3R1, IP3R2 and IP3R3, but gene expression of IP3R3, primarily, is downregulated upon activation due to loss of the Ets-1 transcription factor. Downregulation of IP3R expression in activated CD4 T cells is associated with the failure of T cell receptor ligation to trigger Ca++ release in these cells. We also show that downregulation of specific IP3Rs in activated CD4 T cells correlates with the requirement of IP3R-mediated Ca++ release only for the induction, but not for the maintenance of IL-2 and IFNγ expression. Interestingly, while inhibition of IP3R function early during activation blocks IL-2 and IFNγ production, it promotes the production of IL-17 by CD4 T cells. Thus, IP3Rs play a key role in the activation and differentiation of CD4 T cells. The immunosuppressive effect of pharmacological blockers of these receptors may be complicated by promoting the development of inflammatory CD4 T cells.

SUMMARY

Allergic airway disease is characterized by eosinophilic inflammation, mucus hypersecretion and increased airway resistance. Fungal antigens are ubiquitous within the environment and are well know triggers of allergic disease. Bacterial products are also frequently encountered within the environment and may alter the immune response to certain antigens. The consequence of simultaneous exposure to bacterial and fungal products on the lung adaptive immune response has not been explored. Here we show that oropharyngeal aspiration of fungal lysates (Candida albicans, Aspergillus fumigatus) promotes airway eosinophilia, secretion of Th2 cytokines and mucus cell metaplasia. In contrast, oropharyngeal exposure to bacterial lysates (Pseudomonas aeruginosa) promotes airway inflammation characterized by neutrophils, Th1 cytokine secretion and no mucus production. More importantly, administration of bacterial lysates together with fungal lysates deviates the adaptive immune response to a Th1 type associated with neutrophilia and diminished mucus production. The immunomodulatory effect that bacterial lysates have on the response to fungi is TLR4-independent but MyD88 dependent. Thus, different types of microbial products within the airway can alter the host's adaptive immune response, and potentially impact the development of allergic airway disease to environmental fungal antigens.

Interleukin (IL) 6 is a proinflammtory cytokine produced by antigen-presenting cells and nonhematopoietic cells in response to external stimuli. It was initially identified as a B cell growth factor and inducer of plasma cell differentiation in vitro and plays an important role in antibody production and class switching in vivo. However, it is not clear whether IL-6 directly affects B cells or acts through other mechanisms. We show that IL-6 is sufficient and necessary to induce IL-21 production by naive and memory CD4+ T cells upon T cell receptor stimulation. IL-21 production by CD4+ T cells is required for IL-6 to promote B cell antibody production in vitro. Moreover, administration of IL-6 with inactive influenza virus enhances virus-specific antibody production, and importantly, this effect is dependent on IL-21. Thus, IL-6 promotes antibody production by promoting the B cell helper capabilities of CD4+ T cells through increased IL-21 production. IL-6 could therefore be a potential coadjuvant to enhance humoral immunity.

IL-6 trans-signaling via the soluble IL-6R (sIL-6R) plays an important role in the progression of several autoimmune diseases and cancer by providing IL-6-responsiveness to cells lacking IL-6R. However, the potential sources of sIL-6R are less understood. In this study we show that sIL-6R is produced by both naïve and memory CD4 T cells upon TCR activation. The production of sIL-6R by activated CD4 T cells is mediated by shedding of the membrane-bound IL-6R and this process correlates with the expression of the metalloproteinase ADAM17 in these cells. In contrast to CD4 T cells, CD8 T cells do not express ADAM17 and their production of sIL-6R is negligible. Thus, during an immune response CD4 T cells are an important source of sIL-6R. Production of sIL-6R by autoreactive CD4 T cells may contribute to their role in the development of autoimmune disease by conferring IL-6-responsiveness to cells lacking IL-6R such as synoviocytes.

Structural polymorphisms (L263P, M313V and S331P) in the third intracellular loop of the murine histamine receptor H1 (H1R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis (EAE) and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H1RS) whereas the L-M-S haplotype is associated with less severe disease (H1RR). Here we show that selective reexpression of the H1RS allele in T cells fully complements EAE susceptibility and the production of disease associated cytokines while selective reexpression of the H1RR allele does not. Mechanistically, we show that the two H1R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H1RR allele being retained within the endoplasmic reticulum (ER). Moreover, we show that all three residues (L-M-S) comprising the H1RR haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G-protein coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.

This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.

Mammalian cells respond to UV radiation by signaling cascades leading to activation of transcription factors, such as activated protein 1, NFκB, and p53, a process known as the “UV response.” Nuclear factor of activated T cells (NFAT) was first identified as an inducible nuclear factor in immune response and subsequently found to be expressed in other tissues and cells. To date, however, the regulation and function of NFAT in tissues and cells, other than the immune system, are not well understood. In this study, we demonstrate that UV radiation activates NFAT-dependent transcription through a calcium-dependent mechanism in mouse epidermal JB6 cell lines, as well as in the skin of NFAT-luciferase reporter transgenic mice. Exposure of JB6 cells to UV radiation leads to the transactivation of NFAT in a dose-dependent manner. A23187 had a synergistic effect with UV for NFAT induction, whereas pretreatment of cells with nifedipine, a calcium channel blocker, dramatically impaired the NFAT activity induced by either UV or UV plus A23187. Calcium-dependent activation of NFAT by UV was further confirmed by an in vivo study using NFAT-luciferase reporter transgenic mice. These results demonstrated that UV radiation is a strong activator for skin NFAT transactivation through calcium-dependent pathways, suggesting that NFAT activation may be a part of the UV response.

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.

Glycogen synthase kinase 3β (GSK3β) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3β activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3β by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3β substrate β-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3β by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of β-catenin. p38 MAPK-mediated phosphorylation of GSK3β occurs primarily in the brain and thymocytes. Activation of β-catenin-mediated signaling through GSK3β inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.

Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell–mediated models of organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-γ production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-γ expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-γ production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.

Background

Silicosis features foci of inflammation where macrophages and lymphocytes precede and accompany fibroblast proliferation, alveolar epithelial hyperplasia, and increased deposition of connective tissue matrix material. In the mouse following silica inhalation there is recruitment of natural killer-, B-, and CD4+ and CD8+ lymphocytes to the alveolar spaces, enlargement of bronchial-associated lymphoid tissues (BALT), and aggregation of lymphocytes surrounding small airways and blood vessels. A substantial fraction of the recruited lung lymphocytes produce interferon-γ (IFN-γ), and IFN-γ gene-deleted mice develop less silicosis than wild-type mice. Interleukin-12 (IL-12) is an important pathway for driving the adaptive immune response towards a TH1-like phenotype. We hypothesized that IL-12 might stimulate lymphocyte activation and the up-regulation of IFN-γ, and consequently be an essential mediator for silicosis.

Results

C57Bl/6 wild-type (WT) and IL-12 deficient (IL-12 KO) mice were exposed to sham-air or crystobalite silica (61 mg/m3) by inhalation for 5 hours/day for 12 days and then studied from 1 to 112 days after exposure. Mice exposed to sham-air had normal lung histology at all time points. WT mice exposed to titanium dioxide (72 mg/m3) showed pulmonary macrophage recruitment but no increase in lung collagen. Both WT and IL-12 KO mice exposed to silica showed similar progressive lung pathology, increased wet lung weight and increased total lung collagen (hydroxyproline). IL-12 p35 mRNA was not increased in either strain after silica exposure; IL-12 p40 mRNA was up-regulated after silica in WT mice and constitutively absent in the IL-12 KO mice. IL-18 mRNA was not increased after silica exposure. The expression of IL-15 (an important driver for innate immunity, Natural Killer cell activation, and IFN-γ production) was abundant in air-exposed mice and was increased slightly in the lungs of mice with silicosis.

Conclusion

The axis of IL-12 driving IFN-γ production is not essential for the full manifestations of silicosis in mice exposed to a crystobalite silica aerosol.

IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13–induced inflammation and alveolar remodeling. There were associated decreases in IL-13–induced chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of α1-antitrypsin. IL-13–induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13–induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13–induced diseases and disorders.