Delayed Microbial Maturation Durably Exacerbates Th17-driven Asthma in Mice.

Microbial maturation disrupted by early-life dysbiosis has been linked with increased asthma risk and severity; however, the immunological mechanisms underpinning this connection are poorly understood. We sought to understand how delaying microbial maturation drives worsened asthma outcomes later in life and its long-term durability. Drinking water was supplemented with antibiotics on Postnatal Days 10-20. To assess the immediate and long-term effects of delaying microbial maturation on experimental asthma, we initiated house dust mite exposure when bacterial diversity was either at a minimum or had recovered. Airway hyperresponsiveness, histology, pulmonary leukocyte recruitment, flow cytometric analysis of cytokine-producing lymphocytes, and assessment of serum IgG1 (Immunoglobulin G1) and IgE (Immunoglobulin E) concentrations were performed. RT-PCR was used to measure IL-13 (Interleukin 13)-induced gene expression in sequentially sorted mesenchymal, epithelial, endothelial, and leukocyte cell populations from the lung. Delayed microbial maturation increased allergen-driven airway hyperresponsiveness and Th17 frequency compared with allergen-exposed control mice, even when allergen exposure began after bacterial diversity recovered. Blockade of IL-17A (Interleukin 17A) reversed the airway hyperresponsiveness phenotype. In addition, allergen exposure in animals that experienced delayed microbial maturation showed signs of synergistic signaling between IL-13 and IL-17A in the pulmonary mesenchymal compartment. Delaying microbial maturation in neonates promotes the development of more severe asthma by increasing Th17 frequency, even if allergen exposure is initiated weeks after microbial diversity is normalized. In addition, IL-17A-aggravated asthma is associated with increased expression of IL-13-induced genes in mesenchymal, but not epithelial cells.

Modulation of IL-4/IL-13 cytokine signaling in the context of allergic disease.

Aberrant activation of CD4 TH2 cells and excessive production of TH2 cytokines such as IL-4 and IL-13 have been implicated in the pathogenesis of allergic diseases. Generally, IL-4 and IL-13 utilize Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways for induction of inflammatory gene expression and the effector functions associated with disease pathology in many allergic diseases. However, it is increasingly clear that JAK/STAT pathways activated by IL-4/IL-13 can themselves be modulated in the presence of other intracellular signaling programs, thereby changing the overall tone and/or magnitude of IL-4/IL-13 signaling. Apart from direct activation of the canonic JAK/STAT pathways, IL-4 and IL-13 also induce proinflammatory gene expression and effector functions through activation of additional signaling cascades. These alternative signaling cascades contribute to several specific aspects of IL-4/IL-13-associated cellular and molecular responses. A more complete understanding of IL-4/IL-13 signaling pathways, including the precise conditions under which noncanonic signaling pathways are activated, and the impact of these pathways on cellular- and host-level responses, will better allow us to design agents that target specific pathologic outcomes or tailor therapies for the treatment of uncommon disease endotypes.

Prenatal antibiotics exposure does not influence experimental allergic asthma in mice.

Changes in microbiome (dysbiosis) contribute to severity of allergic asthma. Preexisting epidemiological studies in humans correlate perinatal dysbiosis with increased long-term asthma severity. However, these studies cannot discriminate between prenatal and postnatal effects of dysbiosis and suffer from a high variability of dysbiotic causes ranging from antibiotic treatment, delivery by caesarian section to early-life breastfeeding practices. Given that maternal antibiotic exposure in mice increases the risk of newborn bacterial pneumonia in offspring, we hypothesized that prenatal maternal antibiotic-induced dysbiosis induces long-term immunological effects in the offspring that also increase long-term asthma severity. Therefore, dams were exposed to antibiotics (gentamycin, ampicillin, vancomycin) from embryonic day 15 until birth. Six weeks later, asthma was induced in the offspring by repeated applications of house dust mite extract. Airway function, cytokine production, pulmonary cell composition and distribution were assessed. Our study revealed that prenatally induced dysbiosis in mice led to an increase in pulmonary Th17+ non-conventional T cells with limited functional effect on airway resistance, pro-asthmatic Th2/Th17 cytokine production, pulmonary localization and cell-cell contacts. These data indicate that dysbiosis-related immune-modulation with long-term effects on asthma development occurs to a lesser extent prenatally and will allow to focus future studies on more decisive postnatal timeframes.

Asthma and posttraumatic stress disorder (PTSD): Emerging links, potential models and mechanisms.

Posttraumatic stress disorder (PTSD) is a highly prevalent, debilitating mental health condition. A better understanding of contributory neurobiological mechanisms will lead to effective treatments, improving quality of life for patients. Given that not all trauma-exposed individuals develop PTSD, identification of pre-trauma susceptibility factors that can modulate posttraumatic outcomes is important. Recent clinical evidence supports a strong link between inflammatory conditions and PTSD. A particularly strong association has been reported between asthma and PTSD prevalence and severity. Unlike many other PTSD-comorbid inflammatory conditions, asthma often develops in children, sensitizing them to subsequent posttraumatic pathology throughout their lifetime. Currently, there is a significant need to understand the neurobiology, shared mechanisms, and inflammatory mediators that may contribute to comorbid asthma and PTSD. Here, we provide a translational perspective of asthma and PTSD risk and comorbidity, focusing on clinical associations, relevant rodent paradigms and potential mechanisms that may translate asthma-associated inflammation to PTSD development.

Combined administration of anti-IL-13 and anti-IL-17A at individually sub-therapeutic doses limits asthma-like symptoms in a mouse model of Th2/Th17 high asthma.

BACKGROUND: Recent studies have demonstrated that Th2 responses have the ability to antagonize Th17 responses. In mouse models of allergic asthma, blockade of Th2-effector cytokines results in elaboration of Th17 responses and associated increases in pulmonary neutrophilia. While these can be controlled by simultaneous blockade of Th17-associated effector cytokines, clinical trials of anti-IL-17/IL-17RA blocking therapies have demonstrated increased of risk of bacterial and fungal infections. Identification of minimally effective doses of cytokine-blocking therapies with the goal of reducing the potential emergence of infection-related complications is a translationally relevant goal. OBJECTIVE: In the current report, we examine whether combined blockade of IL-13 and IL-17A, at individually sub-therapeutic levels, can limit the development of allergic asthma while sparing expression of IL-17A-associated anti-microbial effectors. METHODS: House dust mite was given intratracheally to A/J mice. Anti-IL-13 and anti-IL-17A antibodies were administered individually, or concomitantly at sub-therapeutic doses. Airway hyper-reactivity, lung inflammation, magnitude of Th2- and Th17-associated cytokine production and expression of IL-13- and IL-17A-induced genes in the lungs was assessed. RESULTS: Initial dosing studies identified sub-therapeutic levels of IL-13 and IL-17A blocking mAbs that have a limited effect on asthma parameters and do not impair responses to microbial products or infection. Subsequent studies demonstrated that combined sub-therapeutic dosing with IL-13 and IL-17A blocking mAbs resulted in significant improvement in airway hyperresponsiveness (AHR) and expression of IL-13-induced gene expression. Importantly, these doses neither exacerbated nor inhibited production of Th17-associated cytokines, or IL-17A-associated gene expression. CONCLUSION: This study suggests that combining blockade of individual Th2 and Th17 effector cytokines, even at individually sub-therapeutic levels, may be sufficient to limit disease development while preserving important anti-microbial pathways. Such a strategy may therefore have reduced potential for adverse events associated with blockade of these pathways.

l-Citrulline Metabolism in Mice Augments CD4+ T Cell Proliferation and Cytokine Production In Vitro, and Accumulation in the Mycobacteria-Infected Lung.

Activation, recruitment, and effector function of T lymphocytes are essential for control of mycobacterial infection. These processes are tightly regulated in T cells by the availability of l-arginine within the microenvironment. In turn, mycobacterial infection dampens T cell responsiveness through arginase induction in myeloid cells, promoting sequestration of l-arginine within the local milieu. Here, we show T cells can replenish intracellular l-arginine through metabolism of l-citrulline to mediate inflammatory function, allowing anti-mycobacterial T cells to overcome arginase-mediated suppression. Furthermore, T cell l-citrulline metabolism is necessary for accumulation of CD4+ T cells at the site of infection, suggesting this metabolic pathway is involved during anti-mycobacterial T cell immunity in vivo. Together, these findings establish a contribution for l-arginine synthesis by T cells during mycobacterial infection, and implicate l-citrulline as a potential immuno-nutrient to modulate host immunity.

Type I interferons regulate susceptibility to inflammation-induced preterm birth.

Preterm birth (PTB) is a leading worldwide cause of morbidity and mortality in infants. Maternal inflammation induced by microbial infection is a critical predisposing factor for PTB. However, biological processes associated with competency of pathogens, including viruses, to induce PTB or sensitize for secondary bacterial infection-driven PTB are unknown. We show that pathogen/pathogen-associated molecular pattern-driven activation of type I IFN/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of PTB. Similarly, treatment with recombinant type I IFNs recapitulated such effects by exacerbating proinflammatory cytokine production and reducing the dose of secondary inflammatory challenge required for induction of PTB. Inflammatory challenge-driven induction of PTB was eliminated by defects in type I IFN, TLR, or IL-6 responsiveness, whereas the sequence of type I IFN sensing by IFNAR on hematopoietic cells was essential for regulation of proinflammatory cytokine production. Importantly, we also show that type I IFN priming effects are conserved from mice to nonhuman primates and humans, and expression of both type I IFNs and proinflammatory cytokines is upregulated in human PTB. Thus, activation of the type I IFN/IFNAR axis in pregnancy primes for inflammation-driven PTB and provides an actionable biomarker and therapeutic target for mitigating PTB risk.

IL-17A enhances IL-13 activity by enhancing IL-13-induced signal transducer and activator of transcription 6 activation.

BACKGROUND: Increased IL-17A production has been associated with more severe asthma; however, the mechanisms whereby IL-17A can contribute to IL-13-driven pathology in asthmatic patients remain unclear. OBJECTIVE: We sought to gain mechanistic insight into how IL-17A can influence IL-13-driven responses. METHODS: The effect of IL-17A on IL-13-induced airway hyperresponsiveness, gene expression, mucus hypersecretion, and airway inflammation was assessed by using in vivo models of IL-13-induced lung pathology and in vitro culture of murine fibroblast cell lines and primary fibroblasts and human epithelial cell lines or primary human epithelial cells exposed to IL-13, IL-17A, or both. RESULTS: Compared with mice given intratracheal IL-13 alone, those exposed to IL-13 and IL-17A had augmented airway hyperresponsiveness, mucus production, airway inflammation, and IL-13-induced gene expression. In vitro, IL-17A enhanced IL-13-induced gene expression in asthma-relevant murine and human cells. In contrast to the exacerbating influence of IL-17A on IL-13-induced responses, coexposure to IL-13 inhibited IL-17A-driven antimicrobial gene expression in vivo and in vitro. Mechanistically, in both primary human and murine cells, the IL-17A-driven increase in IL-13-induced gene expression was associated with enhanced IL-13-driven signal transducer and activator of transcription 6 activation. CONCLUSIONS: Our data suggest that IL-17A contributes to asthma pathophysiology by increasing the capacity of IL-13 to activate intracellular signaling pathways, such as signal transducer and activator of transcription 6. These data represent the first mechanistic explanation of how IL-17A can directly contribute to the pathogenesis of IL-13-driven pathology.

Intratracheal myriocin enhances allergen-induced Th2 inflammation and airway hyper-responsiveness.

INTRODUCTION: Ceramide is the central substrate of sphingolipid metabolism and plays a key role in cellular signal transduction pathways, regulating apoptosis, differentiation, and chemotaxis. Alterations in airway ceramide levels are observed in multiple pulmonary diseases and recent human genetic association studies have linked dysregulation of sphingolipid regulatory genes with asthma pathogenesis. METHODS: Utilizing myriocin, a potent inhibitor of sphingolipid synthesis, we evaluated the immune regulatory role of de novo ceramide generation in vitro and in vivo. Intratracheal myriocin was administered alone or during house dust mite sensitization (HDM) of BALB/C mice and airway hyper-responsiveness (AHR) was evaluated by invasive plethysmography followed by bronchial lavage (BAL) cytology and cytokine quantification. RESULTS: Myriocin inhibits and HDM exposure activates de novo ceramide synthesis in bone marrow-derived dendritic cells. Mice receiving intratracheal myriocin developed a mild airway neutrophilic infiltrate without inducing a significant increase in AHR. CXCL1 was elevated in the BAL fluid of myriocin-treated mice while the neutrophilic chemotactic factors anaphylatoxin C5a, leukotriene B4, and IL-17 were unaffected. HDM treatment combined with myriocin led to a dramatic enhancement of AHR (63% increase over HDM alone, p < 0.001) and increased granulocyte pulmonary infiltrates versus HDM or myriocin alone. Elevated Th2 T cell counts and Th2 cytokines/chemokines (IL5, IL13, CCL17) were observed in mice treated with combined HDM/myriocin compared to HDM alone. Myriocin-treated pulmonary CD11c+ cells stimulated with HDM secreted significantly more CXCL1 than cells stimulated with HDM alone while HDM stimulated airway epithelial cells showed no change in CXCL1 secretion following myriocin treatment. CONCLUSIONS: Intratracheal myriocin, likely acting via ceramide synthesis inhibition, enhances allergen-induced airway inflammation, granulocyte and Th2 lymphocyte recruitment, and allergen-induced AHR. Sphingolipid pathways may represent novel targets for possible future anti-inflammatory asthma medications.

Differential control of CD4(+) T-cell subsets by the PD-1/PD-L1 axis in a mouse model of allergic asthma.

Studies examining the role of PD-1 family members in allergic asthma have yielded conflicting results. Using a mouse model of allergic asthma, we demonstrate that blockade of PD-1/PD-L1 has distinct influences on different CD4(+) T-cell subsets. PD-1/PD-L1 blockade enhances airway hyperreactivity (AHR), not by altering the magnitude of the underlying Th2-type immune response, but by allowing the development of a concomitant Th17-type immune response. Supporting differential CD4(+) T-cell responsiveness to PD-1-mediated inhibition, naïve PD-1(-/-) mice displayed elevated Th1 and Th17 levels, but diminished Th2 cytokine levels, and ligation of PD-1 in WT cells limited cytokine production by in vitro polarized Th1 and Th17 cells, but slightly enhanced cytokine production by in vitro polarized Th2 cells. Furthermore, PD-1 ligation enhanced Th2 cytokine production by naïve T cells cultured under nonpolarizing conditions. These data demonstrate that different CD4(+) T-cell subsets respond differentially to PD-1 ligation and may explain some of the variable results observed in control of allergic asthma by the PD-1 family members. As the PD-1/PD-L1 axis limits asthma severity by constraining Th17 cell activity, this suggests that severe allergic asthma may be associated with a defective PD-1/PD-L1 regulatory axis in some individuals.

ATF3 is a novel regulator of mouse neutrophil migration.

Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3(-/-) neutrophil recruitment to wild-type lungs. In vitro, ATF3(-/-) neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3(-/-) neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3(-/-) and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

Differential colonization with segmented filamentous bacteria and Lactobacillus murinus do not drive divergent development of diet-induced obesity in C57BL/6 mice.

Alterations in the gut microbiota have been proposed to modify the development and maintenance of obesity and its sequelae. Definition of underlying mechanisms has lagged, although the ability of commensal gut microbes to drive pathways involved in inflammation and metabolism has generated compelling, testable hypotheses. We studied C57BL/6 mice from two vendors that differ in their obesogenic response and in their colonization by specific members of the gut microbiota having well-described roles in regulating gut immune responses. We confirmed the presence of robust differences in weight gain in mice from these different vendors during high fat diet stress. However, neither specific, highly divergent members of the gut microbiota (Lactobacillus murinus, segmented filamentous bacteria) nor the horizontally transmissible gut microbiota were found to be responsible. Constitutive differences in locomotor activity were observed, however. These data underscore the importance of selecting appropriate controls in this widely used model of human obesity.

The TLR4 antagonist Eritoran protects mice from lethal influenza infection.

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)-a potent, well-tolerated, synthetic TLR4 antagonist-blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes.

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-κB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cγ2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IκBα, which we previously reported leads to NF-κB p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase Cγ2 and protein kinase Cα/ß(II), which we have previously reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.

Epigenetic regulation of beta2-adrenergic receptor expression in T(H)1 and T(H)2 cells.

We showed previously that murine naive CD4(+) T cells and T(H)1 cell clones express the beta2-adrenergic receptor (ß(2)AR), while T(H)2 cell clones do not. We report here that naive CD4(+) T cells that differentiated for 1-5 days under T(H)1 driving conditions increased ß(2)AR gene expression, while cells cultured under T(H)2 driving conditions decrease ß(2)AR gene expression. Chromatin immunoprecipitation revealed that the increase in ß(2)AR gene expression in T(H)1 cells is mediated by an increase in histone 3 (H3) and H4 acetylation, as well as an increase in histone 3 lysine 4 (H3K4) methylation. Conversely, the decrease in ß(2)AR gene expression in T(H)2 cells is mediated by a decrease in H3 and H4 acetylation and a decrease in H3K4 methylation, as well as an increase H3K9 and H3K27 methylation. The histone changes could be detected as early as 3 days of differentiating conditions. Genomic bisulfite sequencing showed that the level of methylated CpG dinucleotides within the promoter of the ß(2)AR gene was increased in T(H)2 cells as compared to naive and T(H)1 cells. Collectively, these results suggest that epigenetic mechanisms mediate maintenance and repression, respectively, of the ß(2)AR gene expression in T(H)1- and T(H)2-driven cells, providing a potential mechanism by which the level of ß(2)AR expression might be modulated pharmacologically within immune cells and other cell types in which the expression profile may change during a disease process.

Hematopoietic protein tyrosine phosphatase mediates beta2-adrenergic receptor-induced regulation of p38 mitogen-activated protein kinase in B lymphocytes.

Stimulation of the beta(2)-adrenergic receptor (beta(2)AR) on a CD40L/interleukin-4-activated B lymphocyte increases the level of immunoglobulin E (IgE) in a protein kinase A (PKA)- and p38 mitogen-activated protein kinase (MAPK)-dependent manner. However, the mechanism by which beta(2)AR stimulation mediates the increase in the level of p38 MAPK activation has remained unclear. Here we show that the beta(2)AR-induced increase in p38 MAPK activation occurred via a hematopoietic protein tyrosine phosphatase (HePTP)-mediated cross talk between PKA and p38 MAPK. beta(2)AR agonists, cAMP-elevating agents, and PKA inhibitors were used to show that beta(2)AR stimulation resulted in a PKA-dependent increase in p38 MAPK phosphorylation. Pharmacological agents and gene-deficient mice revealed that p38 MAPK phosphorylation was regulated by the G-stimulatory (Gs)/cAMP/PKA pathway independently of the G-inhibitory or beta-arrestin-2 pathways. Coimmunoprecipitation and Western blot analysis showed that HePTP was phosphorylated in a PKA-dependent manner, which inactivated HePTP and allowed for increased free p38 MAPK to be phosphorylated by the MAPK cascade that was activated by CD40L. HePTP short hairpin RNA confirmed that HePTP played a role in regulating the level of p38 MAPK phosphorylation in a B cell. Thus, beta(2)AR stimulation on a B cell phosphorylates and inactivates HePTP in a Gs/cAMP/PKA-dependent manner to release bound p38 MAPK, making more available for phosphorylation and subsequent IgE regulation.

The activation of natural killer cell effector functions by cetuximab-coated, epidermal growth factor receptor positive tumor cells is enhanced by cytokines.

PURPOSE: Natural killer (NK) cells express an activating Fc receptor (FcgammaRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell-stimulatory cytokines. EXPERIMENTAL DESIGN: Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-gamma and T cell-recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. RESULTS: Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-gamma production than was observed with either agent alone. NK cell-derived IFN-gamma significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1alpha, and RANTES) that could direct the migration of naive and activated T cells. IL-2, IL-12, and IL-21 enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21 enhanced the effects of cetuximab in a murine tumor model. CONCLUSIONS: These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell-stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.

The level of IgE produced by a B cell is regulated by norepinephrine in a p38 MAPK- and CD23-dependent manner.

Although the causes of asthma vary, the severity of the disease correlates with the level of IgE produced. In this study we show that mice produced less IgE when they were depleted of the neurotransmitter norepinephrine (NE) before the administration of Ag. The suppression was prevented when a beta2-adrenergic receptor (beta2AR)-selective agonist was administered, suggesting that NE stimulated the beta2AR to regulate the level of an IgE response in vivo. Although the cell targeted by NE to produce this effect in vivo is unknown, we show in vitro that the level of IgE increased on a per cell basis without an effect on class switch recombination when NE stimulated the beta2AR on a B cell directly. The beta2AR-induced increase in IgE depended on p38 MAPK but not protein kinase A activation, was due to an increased rate of mature IgE mRNA transcription, and was lost when beta2AR-deficient B cells were used. Also, CD23 transcription was increased in a p38 MAPK-dependent manner and resulted in an increased level of soluble CD23 (sCD23). The beta2AR-induced increase in sCD23 was associated with IgE up-regulation and possibly interacted with CD21/CD19. Using B cells from respective knockout mice, data showed that the beta2AR-induced increase in IgE depended on B cell expression of CD23, CD21, and CD19. These findings suggest that at least one mechanism by which endogenous B cell activity in vivo is regulated by NE involves stimulation of the beta2AR on the B cell alone to increase the level of IgE produced in a p38 MAPK- and sCD23-dependent manner.