Inhaled endotoxin induces a systemic neutrophil response without affecting cardiovascular measures in a randomized cross-over exposure study.

OBJECTIVE: The gram-negative bacterial cell wall component endotoxin (lipopolysaccharide, LPS) is a key component of particulate matter (PM). PM exposure is associated with cardiovascular morbidity and mortality. However, the contribution of individual components of PM to acute and chronic cardiovascular measures is not clear. This study examines whether systemic inflammation induced by LPS inhalation causes acute changes in cardiovascular physiology measures. MATERIALS AND METHODS: In this double blinded, placebo-controlled crossover study, fifteen adult volunteers underwent inhalation exposure to 20,000 EU Clinical Center Reference Endotoxin (CCRE). Peripheral blood and induced sputum neutrophils were obtained at baseline and six hours post-exposure. Blood pressure, measures of left ventricular function (ejection fraction (LVEF) and global longitudinal strain (LVGLS)), and indices of endothelial function (flow mediated dilation (FMD) and velocity time integral during hyperemia (VTIhyp)) were measured before and after treatment. Wilcoxon sign-rank tests and linear mixed models were used for statistical analysis. RESULTS: In comparison with normal saline, LPS inhalation resulted in significant increases in peripheral blood and sputum neutrophils but was not associated with significant alterations in blood pressure, LVGLS, LVEF, FMD, or VTIhyp. DISCUSSION AND CONCLUSIONS: In healthy adults, systemic inflammation after LPS inhalation was not associated with acute changes in cardiovascular physiology. Larger studies are needed to investigate the effects of other PM components on inflammation induced cardiovascular dysfunction.

IL-1 receptor antagonist attenuates proinflammatory responses to rhinovirus in airway epithelium.

BACKGROUND: Rhinoviruses (RVs) are the most common trigger for asthma exacerbations, and there are currently no targeted therapies for viral-induced asthma exacerbations. RV infection causes neutrophilic inflammation, which is often resistant to effects of glucocorticoids. IL-1 receptor antagonist (IL-1RA) treatment reduces neutrophilic inflammation in humans challenged with inhaled endotoxin and thus may have therapeutic potential for RV-induced asthma exacerbations. OBJECTIVE: We sought to test the hypothesis that IL-1RA treatment of airway epithelium reduces RV-mediated proinflammatory cytokine production, which is important for neutrophil recruitment. METHODS: Human bronchial epithelial cells from deceased donors without prior pulmonary disease were cultured at air-liquid interface and treated with IL-13 to approximate an asthmatic inflammatory milieu. Human bronchial epithelial cells were infected with human RV-16 with or without IL-1RA treatment. RESULTS: RV infection promoted the release of IL-1α and the neutrophil-attractant cytokines IL-6, IL-8, and CXCL10. Proinflammatory cytokine secretion was significantly reduced by IL-1RA treatment without significant change in IFN-ß release or RV titer. In addition, IL-1RA reduced MUC5B expression after RV infection without impacting MUC5AC. CONCLUSIONS: These data suggest that IL-1RA treatment significantly reduced proinflammatory cytokines while preserving the antiviral response. These results provide evidence for further investigation of IL-1RA as a novel targeted therapy against neutrophil-attractant cytokine release in RV-induced airway inflammatory responses.

Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness.

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.

Age-Associated Changes in the Respiratory Epithelial Response to Influenza Infection.

Older adults suffer a disproportionate burden of influenza-related morbidity and mortality typically attributed to defects in the aging immune system collectively known as immunosenescence. While the age-related decline in the adaptive immune system has been well characterized, little is known about how aging affects the principal site of influenza infection-the nasal epithelium. In human nasal epithelial cell cultures (hNECs) from older adults, we found similar or increased levels of cytokines during influenza infection compared with hNECs from younger individuals. However, hNECs from older individuals demonstrated decreased mRNA expression for several key proteins that affect clearance of infected cells, including MHC-I and transporter associated with antigen presentation (TAP). These findings were confirmed at the level of protein expression. In vivo studies corroborated the in vitro differences in MHC-I and TAP gene expression and also revealed important decreases in the expression of key influenza-specific antiviral mediators MX1 and IFITM1. Furthermore, epithelial cell-cytotoxic T lymphocyte co-cultures demonstrate that CTL cytotoxic activity is dose-dependent on MHC-I antigen presentation. Taken together, these results indicate that aging is associated with important changes in the nasal epithelium, including antigen presentation and antiviral pathways, which may contribute to increased severity of disease in older adults through impaired clearance of infected cells.

Mast Cell Deficiency Limits the Development of Chronic Rhinosinusitis in Mice.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common chronic diseases in adults in both developing and developed countries. The etiology and pathogenesis of CRS remain poorly understood, and the disease is refractory to therapy in many patients. Mast cell activation has been demonstrated in the sinonasal mucosa of patients with CRS; however, the specific contribution of mast cells to the development and pathogenesis of this disease has not been established. OBJECTIVE: The objective of this study was to investigate the role of mast cells in the development of CRS. METHODS: C57BL/6 wild-type and C57BL/6-Kit(W-sh/W-sh) mast cell-deficient mice were immunized by intraperitoneal allergen injection and subsequent chronic low dose intranasal allergen challenges. The sinonasal phenotypes of these groups were then evaluated and compared to saline-treated controls using radiologic, histologic, and immunologic methods. RESULTS: Wild-type mice exposed to chronic intranasal allergen developed many features seen in human CRS, including mucosal thickening, cystic changes, polyp development, eosinophilia, goblet cell hyperplasia, and mast cell activation. In contrast, sinonasal pathology was significantly attenuated in mast cell-deficient mice subjected to the same chronic allergen protocol. Specifically, tissue eosinophilia and goblet cell hyperplasia were reduced by approximately 50% compared to wild-type levels. Surprisingly, none of the mast cell-deficient mice subjected to chronic allergen challenge developed cystic changes or polypoid changes in the nose or sinuses. CONCLUSIONS: These data identify a critical role for mast cells in the development of many features of a mouse model of eosinophilic CRS, suggesting that therapeutic strategies targeting mast cells be examined in humans afflicted with this disease.

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures.

Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNß, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.

Gs-coupled adenosine receptors differentially limit antigen-induced mast cell activation.

Mast cell activation results in the immediate release of proinflammatory mediators prestored in cytoplasmic granules, as well as initiation of lipid mediator production and cytokine synthesis by these resident tissue leukocytes. Allergen-induced mast cell activation is central to the pathogenesis of asthma and other allergic diseases. Presently, most pharmacological agents for the treatment of allergic disease target receptors for inflammatory mediators. Many of these mediators, such as histamine, are released by mast cells. Targeting pathways that limit antigen-induced mast cell activation may have greater therapeutic efficacy by inhibiting the synthesis and release of many proinflammatory mediators produced in the mast cell. In vitro studies using cultured human and mouse mast cells, and studies of mice lacking A(2B) receptors, suggest that adenosine receptors, specifically the G(s)-coupled A(2A) and A(2B) receptors, might provide such a target. Here, using a panel of mice lacking various combinations of adenosine receptors, and mast cells derived from these animals, we show that adenosine receptor agonists provide an effective means of inhibition of mast cell degranulation and induction of cytokine production both in vitro and in vivo. We identify A(2B) as the primary receptor limiting mast cell degranulation, whereas the combined activity of A(2A) and A(2B) is required for the inhibition of cytokine synthesis.

IL-4 amplifies the pro-inflammatory effect of adenosine in human mast cells by changing expression levels of adenosine receptors.

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A(2B)siRNA and selective A(2B) receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A(2B) and reduced expression of A(2A) adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine.

Human embryonic stem cells: a source of mast cells for the study of allergic and inflammatory diseases.

Human mast cells are tissue resident cells with a principal role in allergic disorders. Cross-linking of the high-affinity receptor for immunoglobulin E (FcepsilonRI) results in release of inflammatory mediators initiating the clinical symptoms of allergy and anaphylaxis. Much of our knowledge regarding the mechanisms of mast cell activation comes from studies of mouse bone marrow-derived mast cells. However, clear differences have been identified between human and mouse mast cells. Studies of human mast cells are hampered by the limited sources available for their isolation, the resistance of these cells to genetic manipulation, and differences between cultures established from different persons. To address this limitation, we developed a simple coculture-free method for obtaining mast cells from human embryonic stem cells (hES). These hES-derived mast cells respond to antigen by releasing mast cell mediators. Moreover, the cells can be generated in numbers sufficient for studies of the pathways involved in their effector functions. Genetically modified mast cells, such as GFP-expressing cells, can be obtained by introduction and selection for modification in hES cells before differentiation. This direct coculture-free differentiation of hES cells represents a new and unique model to analyze the function and development of human mast cells.

Exacerbation of allergic inflammation in mice exposed to diesel exhaust particles prior to viral infection.

BACKGROUND: Viral infections and exposure to oxidant air pollutants are two of the most important inducers of asthma exacerbation. Our previous studies have demonstrated that exposure to diesel exhaust increases the susceptibility to influenza virus infections both in epithelial cells in vitro and in mice in vivo. Therefore, we examined whether in the setting of allergic asthma, exposure to oxidant air pollutants enhances the susceptibility to respiratory virus infections, which in turn leads to increased virus-induced exacerbation of asthma. Ovalbumin-sensitized (OVA) male C57BL/6 mice were instilled with diesel exhaust particles (DEP) or saline and 24 hours later infected with influenza A/PR/8. Animals were sacrificed 24 hours post-infection and analyzed for markers of lung injury, allergic inflammation, and pro-inflammatory cytokine production. RESULTS: Exposure to DEP or infection with influenza alone had no significant effects on markers of injury or allergic inflammation. However, OVA-sensitized mice that were exposed to DEP and subsequently infected with influenza showed increased levels of eosinophils in lung lavage and tissue. In addition Th2-type cytokines, such as IL-4 and IL-13, and markers of eosinophil chemotaxis, such as CCL11 and CCR3, were increased in OVA-sensitized mice exposed to DEP prior to infection with influenza. These mice also showed increased levels of IL-1alpha, but not IL-10, RANTES, and MCP-1 in lung homogenates. CONCLUSION: These data suggest that in the setting of allergic asthma, exposure to diesel exhaust could enhance virus-induced exacerbation of allergic inflammation.

Adenosine induces airway hyperresponsiveness through activation of A3 receptors on mast cells.

BACKGROUND: The mechanisms responsible for the development of airway hyperresponsiveness in asthma are poorly understood. Adenosine levels are high in the lungs of patients with asthma, but a role for adenosine in the development of this cardinal feature of asthma has not been previously reported. OBJECTIVE: To determine the capacity of adenosine to induce airway hyperresponsiveness, and to investigate the mechanisms behind these effects of adenosine on airway physiology. METHODS: Wild-type C57BL/6 mice were exposed to aerosolized adenosine analog adenosine-5' N-ethylcarboxamide (NECA), and subsequent hyperresponsiveness to methacholine was investigated by measuring airway mechanics after anesthesia and tracheostomy. Similar experiments were conducted with A(1)-deficient, A(3)-deficient, and mast cell-deficient mice, as well as with mast cell-deficient mice engrafted with wild-type (wt) or A(3)(-/-) mast cells. The effect of NECA on methacholine-induced tension development in ex vivo tracheal rings was also examined. RESULTS: Exposure of wt mice to NECA resulted in the robust induction of airway hyperresponsiveness. NECA failed to induce hyperresponsiveness to methacholine in tracheal ring preps ex vivo, and NECA-induced airway hyperresponsiveness in vivo was not affected by the genetic inactivation of the A(1) adenosine receptor. In contrast, NECA-induced airway hyperresponsiveness was abolished in A(3) adenosine receptor-deficient mice and in mice deficient in mast cells. Reconstitution of mast cell-deficient mice with wt mast cells restored hyperresponsiveness, whereas reconstitution with A(3) receptor-deficient mast cells did not. CONCLUSION: Adenosine induces airway hyperresponsiveness indirectly by activating A(3) receptors on mast cells.

Involvement of A1 adenosine receptors and neural pathways in adenosine-induced bronchoconstriction in mice.

High levels of adenosine can be measured from the lungs of asthmatics, and it is well recognized that aerosolized 5'AMP, the precursor of adenosine, elicits robust bronchoconstriction in patients with this disease. Characterization of mice with elevated adenosine levels secondary to the loss of adenosine deaminase (ADA) expression, the primary metabolic enzyme for adenosine, further support a role for this ubiquitous mediator in the pathogenesis of asthma. To begin to identify pathways by which adenosine can alter airway tone, we examined adenosine-induced bronchoconstriction in four mouse lines, each lacking one of the receptors for this nucleoside. We show, using direct measures of airway mechanics, that adenosine can increase airway resistance and that this increase in resistance is mediated by binding the A(1) receptor. Further examination of this response using pharmacologically, surgically, and genetically manipulated mice supports a model in which adenosine-induced bronchoconstriction occurs indirectly through the activation of sensory neurons.

Retinoid-related orphan receptor gamma controls immunoglobulin production and Th1/Th2 cytokine balance in the adaptive immune response to allergen.

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORgamma, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORgamma-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORgamma, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-gamma and IL-10 production was markedly greater in splenocytes from RORgamma-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORgamma-deficient animals in vivo. These data reveal a critical role for RORgamma in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.

Enhanced mast cell activation in mice deficient in the A2b adenosine receptor.

Antigen-mediated cross-linking of IgE bound to mast cells via the high affinity receptor for IgE triggers a signaling cascade that results in the release of intracellular calcium stores, followed by an influx of extracellular calcium. The collective increase in intracellular calcium is critical to the release of the granular contents of the mast cell, which include the mediators of acute anaphylaxis. We show that the sensitivity of the mast cell to antigen-mediated degranulation through this pathway can be dramatically influenced by the A2b adenosine receptor. Loss of this Gs-coupled receptor on mouse bone marrow-derived mast cells results in decreased basal levels of cyclic AMP and an excessive influx of extracellular calcium through store-operated calcium channels following antigen activation. Mice lacking the A2b receptor display increased sensitivity to IgE-mediated anaphylaxis. Collectively, these findings show that the A2b adenosine receptor functions as a critical regulator of signaling pathways within the mast cell, which act in concert to limit the magnitude of mast cell responsiveness when antigen is encountered.

Pulmonary immune responses to Propionibacterium acnes in C57BL/6 and BALB/c mice.

Propionibacterium acnes (PA) is a gram-positive anaerobic bacterium implicated as a putative etiologic agent of sarcoidosis. To characterize the pulmonary immune response to PA, C57BL/6 and BALB/c mice were intraperitoneally sensitized and intratracheally challenged with heat-killed bacteria. C57BL/6 mice challenged with PA developed a cellular immune response characterized by elevations in Th1 cytokines/chemokines, increased numbers of lymphocytes and macrophages in lung lavage fluid, and peribronchovascular granulomatous inflammation composed of T- and B-lymphocytes and epithelioid histiocytes. T-lymphocytes in the lung lavage fluid showed a marked CD4+ cell predominance. In contrast, C57BL/6 mice challenged with Staphylococcus epidermidis (SE), another gram-positive commensal of human skin, and BALB/c mice challenged with PA, showed only a modest induction of Th1 cytokines, less pulmonary inflammation, and no granulomatous changes in the lung. Enhancement of Toll-like receptor expression was seen in PA-exposed C57BL/6 mice within 24 h after exposure, suggesting that induction of innate immunity by PA contributes to the robust, polarized Th1 immune response elicited by this bacterium. These findings suggest that PA-induced pulmonary inflammation may be a useful model for testing the contributions of both bacterial and host factors in the development, maintenance, and resolution of granulomatous inflammation in the lung.