Kita, Hirohito

Abstract Allergic asthma is commonly thought to result from dysregulated airway inflammatory responses to ubiquitous environmental antigens mediated by CD4+ T cells polarized to a Th2 or Th17 cell. However, the mechanisms involved in the development of these T-cell responses remain unknown. This study examines the effects of IL-1 family cytokines, such as IL-33 and IL-1β, on the development of antigen-specific Th2 and Th17 cells in the airway. We administered IL-1 family cytokines and model antigens, such as ovalbumin, into the airways of naive BALB/c mice, and examined the cellular and humoral immune responses. To investigate the immunologic mechanisms, we used IL-4 green fluorescent protein reporter mice and mice deficient in the Il4 gene. Innocuous antigens, such as endotoxin-free ovalbumin and short ragweed extract, did not sensitize naive mice when administered through the airways. However, when mice were exposed to the same antigens with IL-1β or IL-33, they developed IgE antibodies. In particular, IL-33 induced robust and long-lasting Th2 cells that produced a large quantity of IL-5 and IL-13 and asthma-like airway pathology. IL-1β induced Th17 cells. In naive, nonsensitized animals, IL-33 stimulated endogenous IL-4 expression by CD4+ T cells, which was critical for the polarization of CD4+ T cells to the Th2 type. In the absence of IL-4, mice developed Th17 cells and neutrophilic airway inflammation. In conclusion, IL-1 family cytokines possess a potent adjuvant activity to promote both Th2 and Th17 cells to innocuous airborne antigens, and they may play fundamental roles in the immunopathology of asthma.

Abstract Epithelial cells at mucosal surfaces are integral components of innate and adaptive immunity. IL-25 is reportedly produced by epithelial cells and likely plays vital roles in regulating type-2 immune responses. However, little is known regarding the mechanisms that control production and extracellular releases of IL-25. We hypothesized that proteases from the multiple allergens may induce IL-25 production in airway epithelial cells. In this study, we found that IL-25 is constitutively produced and detectable in cytoplasm of resting normal human bronchial epithelial (NHBE) cells. When exposed to airborne allergens such as house dust mite (HDM), stored IL-25 was released rapidly to the extracellular space. IL-25 release was not accompanied by cell death, suggesting involvement of active secretory mechanism(s). HDM also enhanced IL-25 mRNA transcription, which was dependent on their protease activities. Furthermore, activation of NHBE cells with authentic proteases, such as trypsin and papain, or with a peptide agonist for protease-activated receptor 2 was sufficient to enhance IL-25 mRNA transcription and protein. Protease-driven increase in mRNA transcription and allergen-driven extracellular release of IL-25 protein was also observed in primary nasal epithelial cells from healthy individuals. These findings suggest that IL-25 production by airway epithelial cells is regulated by the transcription and protein release levels and that allergen proteases likely play pivotal roles in both biological processes.

Abstract Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. We hypothesized that cytokine(s) produced in the disease sites are implicated in the pathophysiology of CEP. We studied peripheral blood and bronchoalveolar lavage fluids (BALF) obtained from two lung segments of a patient with CEP. Seventy times more eosinophils were found in the BALF from an involved lung segment (showing patchy opacification on a chest roentgenogram) than from an uninvolved segment. The eosinophil-active cytokines interleukin-5 (IL-5), IL-6, and IL-10 were strikingly elevated in the BALF from the involved lung segment, whereas no or minimal levels of these cytokines were detectable in the BALF from the uninvolved segment or serum, respectively. Leukocytes in the involved lung segment, but not those in peripheral blood, expressed messenger ribonucleic acid (mRNA) for IL-5, IL-6, and IL-10. In contrast, IL-2, IL-3, IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) were not detected in any sample. These findings suggest that increased production of several cytokines, such as IL-5, IL-6, and IL-10, in the involved lung segment, but not in the uninvolved lung segment or peripheral blood, is a critical pathophysiologic feature of CEP.

Abstract Segmental bronchoprovocation (SBP) with allergen was used in an attempt to study eosinophils recruited to the airway 24 h after challenge. Unexpectedly, in the first four patients, neutrophils (rather than eosinophils) were recruited in the bronchoalveolar lavage (BAL) fluids, and we hypothesized that the allergen extracts were contaminated with endotoxin. The extracts used for challenge in the first four patients tested positive for bacterial endotoxin in a limulus amebocyte lysate assay. Rechallenge of one patient from the first group with a comparable dose of an endotoxin-free extract and SBP with endotoxin-free extract in five additional patients resulted in preferential recruitment of eosinophils rather than neutrophils. The number of neutrophils recovered from the challenged segments in the patients challenged with endotoxin-free extract was significantly less than that observed in the first four patients. Taken together, these observations suggest that neutrophil recruitment in the 24-h BAL fluids from the first four patients was probably due to endotoxin contamination of the allergen extract. We caution investigators that endotoxin contamination of allergen extract may alter the cellular inflammation during the late airway response following allergen challenge.

Abstract The mechanism of airway eosinophilia during antigen-induced inflammation was investigated by measurement of eosinophil-active cytokines utilizing an eosinophil survival assay. In the first study, 4 patients with allergic rhinitis underwent segmental bronchoprovocation (SBP) with low, medium, and high doses of ragweed extract instilled into different bronchial subsegments; bronchoalveolar lavage (BAL) fluids were collected from each segment 12 min and 48 h after challenge. Eosinophil granule proteins and eosinophil survival activity were significantly elevated in the 48-h (late-phase) BAL fluids from these segments. Correlations were observed between the concentrations of eosinophil granule proteins and eosinophil survival activity (rs = 0.717 to 0.880, p < 0.001) in BAL fluids. Eosinophil survival activity was completely neutralized by anti-IL-5 monoclonal antibody in five of the seven 48-h samples tested representing three of the 4 patients. In the two remaining samples, eosinophil survival activity was only partially neutralized by either anti-IL-5 antibody or anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) but was completely neutralized by anti-IL-5 and anti-GM-CSF in combination. Subsequently, in the second study, 10 patients with allergic rhinitis were challenged by SBP with ragweed extract. Eosinophil survival activity was significantly elevated in the 48-h BAL fluids; this activity was partially neutralized by anti-IL-5 antibody about (48%) and completely neutralized by the combination of anti-IL-5 and anti-GM-CSF antibodies. These findings suggest that the eosinophil survival activity in the late inflammatory lesions following SBP with allergen is mainly associated with IL-5, with small contributions from GM-CSF. Thus, IL-5 is the predominant eosinophil-active cytokine present in BAL fluids during allergen-induced late-phase inflammation and may play a key role in the pathophysiology of allergen-induced, eosinophil-predominant airway inflammation.

Abstract Psychological stress can lead to asthma exacerbations in some patients. It is our hypothesis that the stress effect can occur through an enhancement of allergic inflammatory response. To investigate this possibility, airway antigen challenge was evaluated in 20 college students with mild asthma during both a low-stress phase (midsemester or two weeks postfinal examination) and a stress phase (final examination week). Subjects completed questionnaires to assess psychological state and underwent inhaled antigen challenge. Sputum samples were collected before challenge, and six and 24 hours and seven days postchallenge. Leukocytes were counted and eosinophil-derived neurotoxin (EDN) was measured in sputum supernates. Sputum cells were cultured and stimulated ex vivo with phytohemagglutinin (10 μ g/ml), and culture supernates were assayed for interleukin-5 (IL-5) and interferon- γ by enzyme-linked immunosorbent assay. Sputum eosinophils and EDN levels significantly increased at six and 24 hours postchallenge and were enhanced during the stress phase (p < 0.01). IL-5 generation by sputum cells was also increased at 24 hours during stress and correlated with airway eosinophils (rs = 0.65, p < 0.05). Students' anxiety and depression scores were significantly higher during the examination period. Our findings suggest that stress associated with final examinations can act as a cofactor to increase eosinophilic airway inflammation to antigen challenge and thus may enhance asthma severity.

Abstract A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P < 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-γ. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.

Abstract Migration of eosinophils through the basement membrane barrier is an important step for their infiltration into tissues. To investigate the mechanism of transmigration, we used a chamber fitted with a Matrigel membrane as a model of the basement membrane. In this model, eosinophils treated with cytokines or chemotactic factors alone did not transmigrate from the upper to the lower chamber. However, platelet-activating factor (PAF) strongly induced transmigration of eosinophils stimulated by interleukin (IL)-5, indicating that both a cytokine and a chemotactic factor are required for eosinophil migration through Matrigel. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 also stimulated eosinophil transmigration in the presence of PAF. Of seven eosinophil chemotactic factors tested, leukotriene B4, C5a, RANTES, macrophage inflammatory protein-1α, and IL-8 did not induce significant eosinophil transmigration. Only PAF and eotaxin induced transmigration of eosinophils through Matrigel in the presence of IL-5; PAF was more potent than eotaxin at the optimal concentration. In contrast, PAF, eotaxin, and RANTES all potently induced migration of eosinophils through bare membrane in the absence of IL-5. Finally, eosinophil migration through Matrigel was markedly reduced by a combination of anti-CD18 and anti-CD29 monoclonal antibodies, suggesting that it is mediated by β1- and β2-integrin adhesion molecules. Our findings demonstrate that eosinophil transmigration through a basement membrane model requires both a specific chemoattractant, such as PAF, and an eosinophil-activating cytokine, such as IL-5. This synergistic effect is likely important in the tissue accumulation of activated eosinophils in allergic and other eosinophil-associated diseases.

Abstract In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel®-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel® layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel® layer. Matrigel® transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel®. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.