IL-33 drives airway hyper-responsiveness through IL-13-mediated mast cell: airway smooth muscle crosstalk.

BACKGROUND: Mast cell localization within the airway smooth muscle (ASM)-bundle plays an important role in the development of airway hyper-responsiveness (AHR). Genomewide association studies implicate the 'alarmin' IL-33 in asthma, but its role in mast cell-ASM interactions is unknown. OBJECTIVES: We examined the expression and functional role of IL-33 in bronchial biopsies of patients with and without asthma, ex vivo ASM, mast cells, cocultured cells and in a mouse model system. METHODS: IL-33 protein expression was assessed in human bronchial tissue from 9 healthy controls, and 18 mild-to-moderate and 12 severe asthmatic patients by immunohistochemistry. IL-33 and ST2 mRNA and protein expression in human-derived ASM, epithelial and mast cells were assessed by qPCR, immunofluorescence and/or flow cytometry and ELISA. Functional assays were used to assess calcium signalling, wound repair, proliferation, apoptosis and contraction. AHR and inflammation were assessed in a mouse model. RESULTS: Bronchial epithelium and ASM expressed IL-33 with the latter in asthma correlating with AHR. ASM and mast cells expressed intracellular IL-33 and ST2. IL-33 stimulated mast cell IL-13 and histamine secretion independent of FcεR1 cross-linking and directly promoted ASM wound repair. Coculture of mast cells with ASM activated by IL-33 increased agonist-induced ASM contraction, and in vivo IL-33 induced AHR in a mouse cytokine installation model; both effects were IL-13 dependent. CONCLUSION: IL-33 directly promotes mast cell activation and ASM wound repair but indirectly promotes ASM contraction via upregulation of mast cell-derived IL-13. This suggests that IL-33 may present an important target to modulate mast cell-ASM crosstalk in asthma.

T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen, under the control of OX40L and independent of innate-like lymphocytes.

Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4(+) Th cells induces full Th2 immunity. Further, CD4(+) Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4(+) Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.

Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood.

The origins of allergic asthma, particularly in infancy, remain obscure. Respiratory viral infections and allergen sensitization in early life have been associated with asthma in young children. However, a causal link has not been established. We investigated whether an influenza A infection in early life alters immune responses to house dust mite (HDM) and promotes an asthmatic phenotype later in life. Neonatal (8-day-old) mice were infected with influenza virus and 7 days later, exposed to HDM for 3 weeks. Unlike adults, neonatal mice exposed to HDM exhibited negligible immune responsiveness to HDM, but not to influenza A. HDM responsiveness in adults was associated with distinct Ly6c+ CD11b+ inflammatory dendritic cell and CD8α+ plasmacytoid (pDC) populations that were absent in HDM-exposed infant mice, suggesting an important role in HDM-mediated inflammation. Remarkably, HDM hyporesponsiveness was overcome when exposure occurred concurrently with an acute influenza infection; young mice now displayed robust allergen-specific immunity, allergic inflammation, and lung remodeling. Remodeling persisted into early adulthood, even after prolonged discontinuation of allergen exposure and was associated with marked impairment of lung function. Our data demonstrate that allergen exposure coincident with acute viral infection in early life subverts constitutive allergen hyporesponsiveness and imprints an asthmatic phenotype in adulthood.

A mouse GM-CSF receptor antibody attenuates neutrophilia in mice exposed to cigarette smoke.

We investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subchronic exposure model of cigarette smoke (CS)-induced inflammation using antibodies directed against GM-CSF or the GM-CSF receptor (GM-CSFR) α-chain. CS-induced mononuclear and neutrophilic inflammation following 4 days of CS exposure in BALB/c mice was assessed in bronchoalveolar lavage (BAL) fluid. An increase in mature dendritic cells (DCs) (CD11c+ and major histocompatibility complex II+) and Gr-1-high neutrophils was also observed by flow cytometric analysis of whole-lung tissue. Daily i.p. injection of 400 µg GM-CSF or GM-CSFR antibody prior to daily smoke exposure attenuated the accumulation of neutrophils within the BAL by 60%. A reduction in mature DCs was also observed. Anti-GM-CSFR antibody administration did not have an effect on the percentage of lung T-cells; however, a significant decrease in activated CD69+ CD8+ T-cells was observed. Anti-GM-CSFR antibody administration decreased the mRNA and protein expression of interleukin-12 p40 and matrix metalloproteinase 12. Taken together, intervention with this receptor antibody implicates the GM-CSF pathway as an important mediator of smoke-induced inflammation.

Macrophage inflammatory protein-1alpha uses a novel receptor for primitive hemopoietic cell inhibition.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the chemokine family of proinflammatory mediators. In addition to its inflammatory roles, MIP-1alpha has been shown to be active as an inhibitor of primitive hemopoietic cell proliferation. Indeed, a dysfunction in this inhibitory process has been postulated to contribute to leukemogenesis. Research has been aimed at characterizing the receptor involved in cellular inhibition by MIP-1alpha. This study demonstrates that of all the beta-chemokines tested, only MIP-1alpha is capable of inhibiting primitive hemopoietic cell proliferation. Because no MIP-1alpha-specific receptors have been identified, this suggests that inhibition is mediated by an uncharacterized receptor. Further evidence for the involvement of a novel receptor in this process is the equivalent potencies of MIP-1alphaS and MIP-1alphaP variants of human MIP-1alpha and the fact that primitive cells from bone marrow derived from individual MIP-1alpha receptor null mice display a full response to MIP-1alpha inhibition.

A role for the C3a anaphylatoxin receptor in the effector phase of asthma.

Asthma is a chronic inflammatory disease of the airways and lung mucosa with a strong correlation to atopy and acquired (IgE) immunity. However, many features of bronchial asthma, such as smooth muscle contraction, mucus secretion and recruitment of inflammatory cells, are consistent with the actions of complement anaphylatoxins, in particular C3a and C5a. Complement activation forms a central core of innate immune defence against mucosal bacteria, viruses, fungi, helminths and other pathogens. As a system of 'pattern-recognition molecules', foreign surface antigens and immune complexes lead to a proteolytic cascade culminating in a lytic membrane attack. The anaphylatoxins C3a and C5a are liberated as activation byproducts and are potent pro-inflammatory mediators that bind to specific cell surface receptors and cause leukocyte activation, smooth muscle contraction and vascular permeability. Here we show that in a murine model of allergic airway disease, genetic deletion of the C3a receptor protects against the changes in lung physiology seen after allergen challenge. Furthermore, human asthmatics develop significant levels of ligand C3a following intra-pulmonary deposition of allergen, but not saline. We propose that, in addition to acquired immune responses, the innate immune system and complement (C3a in particular) are involved in the pathogenesis of asthma.

Lymph node trafficking and antigen presentation by endobronchial eosinophils.

Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell-rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3(-/-) and CCR3(+/+) eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86-dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4(+) T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4(+) T cells.

Structure and function of the murine chemokine receptor CXCR3.

The gene encoding the murine homologue of human CXCR3 exists in a single copy consisting of two exons with an intron interrupting the coding sequence between nucleotides 10 and 11. The deduced amino acid sequence is 86% identical to the predicted human sequence. Murine CXCR3 mRNA is detectable in bone marrow cells cultured in the presence of IL-2 but not unstimulated cells. It is also detectable at low abundance in normal mouse spleen, lymph node, mammary gland, and thymus. Transfection of murine CXCR3 in murine pre-B lymphocyte line (CXCR3++/L1.2) conferred binding of the ligands IP10, ITAC and Mig with K(D)'s of 1.35 +/- 0.56, 1.41 +/- 0.20, and 11.65 +/- 0.90 nM, respectively. Lower affinity binding was observed for several beta or CC chemokines (eotaxin, MCP-3, MIP3alpha and SLC/6Ckine/Exodus 2). ITAC, IP10 and Mig induced chemotaxis with an order of potency ITAC > IP10 = Mig. The chemokines also increased intracellular calcium concentration and were variably desensitized to repeated agonist stimulation. The hierarchy for cross- desensitization was ITAC > Mig > IP10. Thus, while Mig, ITAC and IP10 all act on the same receptor for binding and agonist stimulation, they may interact with different receptor conformational isoforms to produce divergent responses.

Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis.

To study the biologic role of migration inhibitory factor (MIF), a pleiotropic cytokine, we generated a mouse strain lacking MIF by gene targeting in embryonic stem cells. Analysis of the role of MIF during sepsis showed that MIF-/- mice were resistant to the lethal effects of high dose bacterial lipopolysaccharide (LPS), or Staphylococcus aureus enterotoxin B (SEB) with D-galactosamine and had lower plasma levels of tumor necrosis factor alpha (TNF-alpha) than did wild-type mice, but normal levels of interleukin (IL)-6 and IL-10. When stimulated with LPS and interferon gamma, macrophages from MIF-/- mice showed diminished production of TNF-alpha, normal IL-6 and IL-12, and increased production of nitric oxide. MIF-/- animals cleared gram-negative bacteria Pseudomonas aeruginosa instilled into the trachea better than did wild-type mice and had diminished neutrophil accumulation in their bronchoalveolar fluid compared to the wild-type mice. Thioglycollate elicited peritoneal exudates in uninfected MIF-/- mice, but showed normal neutrophil accumulation. Finally, the findings of enhanced resistance to P. aeruginosa and resistance to endotoxin-induced lethal shock suggest that the counteraction or neutralization of MIF may serve as an adjunct therapy in sepsis.

Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo.

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12-24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation.

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralleled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer and attractive target for therapeutic intervention.

Formoterol on airway smooth muscle and human lung mast cells: a comparison with salbutamol and salmeterol.

Formoterol, like salbutamol and salmeterol, relaxed isolated preparations of guinea-pig trachea and human bronchus, and inhibited antigen-induced mediator release from human lung fragments in a concentration-related fashion. In each case, these actions were mediated through beta 2-adrenoceptors, with formoterol being 50-120-fold more potent than salbutamol, and 2-27-fold more potent than salmeterol. The duration of action of formoterol was longer than that of salbutamol in all preparations, but was markedly shorter than that of salmeterol, whose actions persisted for many hours despite continuous or extensive washing of the tissues. In conscious guinea-pigs, inhaled formoterol, salbutamol and salmeterol all caused dose-related inhibition of histamine-induced bronchoconstriction. Formoterol was again more potent (10-20-fold) than either salbutamol or salmeterol. However, while the actions of a threshold-effective dose of formoterol persisted for less than 3 h, somewhat longer than those of salbutamol (< 1.5 h), an equivalent dose of salmeterol was active for at least 6 h. Therefore, while formoterol is a potent beta 2-adrenoceptor agonist in vitro and in vivo, and is consistently longer-acting than salbutamol, its duration of action is markedly shorter than that of salmeterol.