Mark Schuyler

Changes in T-Lymphocyte Subpopulations after Antigenic Bronchial Provocation in Asthmatics

To determine whether inhaled agents can alter T-cell subsets in the peripheral blood of patients with bronchial asthma, we tested six asymptomatic asthmatics who were sensitive to mixed grass (positive skin test) with mixed grass extract, methacholine, and an antigen to which they were not sensitized (negative skin test). Levels of OKT4 cells (helper T lymphocytes) were reduced in the peripheral blood immediately after the challenge with mixed grass extract, and remained low for at least 72 hours. Levels of Ia-positive (activated) T cells were increased 48 hours after the challenge. No changes were observed in any of these T-cell subpopulations after challenge with methacholine or after the inhalation of an equal amount of an antigen to which the subjects were not sensitized. These results suggest that the selective loss of circulating helper T cells and an increase in activated T cells after an asthmatic attack induced by antigenic inhalation may serve as an indicator of immune-mediated bronchoconstriction.

Characterization of Myeloid and Plasmacytoid Dendritic Cells in Human Lung

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c+CD11c+CD14−HLA-DR+ cells and comprised ∼2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123+CD11c−CD14−HLA-DR+ cells and comprised ∼1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c+CD14− lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c+CD14+ lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-α than MDC-enriched cells. MDC-enriched cells were either CD123+ or CD123−, but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-α upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.

Classification of hypersensitivity pneumonitis: a hypothesis.

Background: Regardless of the causative antigen, hypersensitivity pneumonitis (HP) is usually classified as ‘acute’, ‘subacute’ or ‘chronic’. Considerable confusion still surrounds this classification because there are no widely accepted criteria to distinguish the various stages. The objective of this study wasto determine whether the current classification of HP truly reflects categories of patients with distinct clinical features. Methods: Data obtained from a large prospective multicenter cohort study (the HP Study) were used to divide a cohort of patients with HP into a limited number of categories (clusters) with maximally differing clinical patterns, without prejudgment. The results of this cluster analysis were compared with the current classification of HP (acute, subacute or chronic). Results: 168 patients were included in the analysis. A 2-cluster solution best fitted the data. Patients in cluster 1 (41 patients) had more recurrent systemic symptoms (chills and body aches) and normal chest radiographs than those in cluster 2 (127 patients) who showed significantly more clubbing, hypoxemia, restrictive patterns on pulmonary function tests and fibrosis on high-resolution computed tomography (HRCT). All p values were <0.0001, using Fisher’s exact test. Nodular opacities were seen on HRCT as often in cluster 1 as in cluster 2. There was considerable disagreement between the current classification of HP and the results of our analysis. Conclusion: The current classification of acute, subacute and chronic HP is not supported by our analysis. Subacute HP is particularly difficult to define.

The Regulation of Pulmonary Immunity

Publisher Summary Thechapter describes the cells and structures of the lung that participate in pulmonary immunity and how the lung responds to challenges fromforeign antigens, with particular emphasis on animal models that have been developed to explore these issues. Some ligands-receptor interactions are specific while others are not, and it is the particular pattern of surface molecules and secreted factors expressed by interacting immune cells that determines the type of immune response that develops during central processing. The cells that are the major initiators and regulators of immunity in the lung include macrophages, dendritic cells (DCs), and lymphocytes, each expressing surface molecules and secretory products that depend on perturbations in the environments. Immune cells and structures of the lung and lung immunity to noninfectious particulate and soluble antigens are discussed. Several models for regulation of pulmonary immunity such as models for immunity in lung infections, models for hypersensitivity lung disease, models for lung transplantation, and graft versus host are also presented. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immune-suppressed hosts, and to suppress manifestations of immunologically mediated lung disease.

IFN-γ, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-γ levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-γ was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-γ but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-γ is crucial for reducing MCM during prolonged exposures to allergen. IFN-γ but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-γ was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.

Histamine Release from the Basophils of Control and Asthmatic Subjects and a Comparison of Gene Expression between “Releaser” and “Nonreleaser” Basophils

Most human blood basophils respond to FcεRI cross-linking by releasing histamine and other inflammatory mediators. Basophils that do not degranulate after anti-IgE challenge, known as “nonreleaser” basophils, characteristically have no or barely detectable levels of the Syk tyrosine kinase. The true incidence of the nonreleaser phenotype, its relationship (if any) to allergic asthma, and its molecular mechanism are not well understood. In this study, we report statistical analyses of degranulation assays performed in 68 control and 61 asthmatic subjects that establish higher basal and anti-IgE-stimulated basophil degranulation among the asthmatics. Remarkably, 28% of the control group and 13% of the asthmatic group were nonreleasers for all or part of our 4-year long study and cycling between the releaser and nonreleaser phenotypes occurred at least once in blood basophils from 8 (of 8) asthmatic and 16 (of 23) control donors. Microarray analysis showed that basal gene expression was generally lower in nonreleaser than releaser basophils. In releaser cells, FcεRI cross-linking up-regulated >200 genes, including genes encoding receptors (the FcεRI α and β subunits, the histamine 4 receptor, the chemokine (C-C motif) receptor 1), signaling proteins (Lyn), chemokines (IL-8, RANTES, MIP-1α, and MIP-1β) and transcription factors (early growth response-1, early growth response-3, and AP-1). FcεRI cross-linking induced fewer, and quite distinct, transcriptional responses in nonreleaser cells. We conclude that “nonreleaser” and “cycler” basophils represent a distinct and reversible natural phenotype. Although histamine is more readily released from basophils isolated from asthmatics than controls, the presence of nonreleaser basophils does not rule out the diagnosis of asthma.

Low serum adiponectin predicts future risk for asthma in women

RATIONALE Our previous cross-sectional study showed that serum adiponectin is inversely associated with asthma among women. However, it is not known if serum adiponectin predicts future development of asthma or if asthma affects subsequent serum adiponectin concentrations among women. OBJECTIVES To determine longitudinal association between serum adiponectin and incident asthma among women. METHODS We used data from examinations at Years 10, 15, and 20 of the Coronary Artery Risk Development in Young Adults (CARDIA) cohort. In our primary analysis, the association of CARDIA Year 15 serum adiponectin concentration with Year 20 incident asthma was evaluated. In our secondary analysis, the converse direction, that is, the association of CARDIA Year 10 prevalent asthma with Year 15 serum adiponectin, was evaluated, using logistic regression techniques. MEASUREMENTS AND MAIN RESULTS Our primary analysis included 1,450 women, mostly premenopausal. Multivariable analyses demonstrated that the lowest tertile of Year 15 serum adiponectin concentration (<7 mg/L) predicted significantly higher risk for incident asthma at Year 20 among women (odds ratio, 2.07; 95% confidence interval, 1.05, 4.10), and particularly among current smokers (interaction P = 0.051). Further, low serum adiponectin was more important than body mass index in predicting the risk for incident asthma among women. We also showed that the converse relationship was not true; that is, Year 10 prevalent asthma did not predict Year 15 serum adiponectin concentrations in women. CONCLUSIONS Serum adiponectin affects future risk for asthma in women and not vice versa. Measures that raise systemic adiponectin concentrations may lead to newer ways to prevent asthma among women, particularly among those who smoke.

Reduced Fc RI-Mediated Release of Asthma-Promoting Cytokines and Chemokines from Human Basophils during Omalizumab Therapy

Background: Treating asthmatics with the humanized IgE-scavenging antibody, omalizumab (rhuMAb-E25, Xolair®), reduces airways inflammation and asthma symptoms. Previously, omalizumab was shown to cause a dramatic and reversible loss of cell surface high-affinity IgE receptors, FcΕRI, from the peripheral blood basophils of asthmatics. The consequences of receptor loss for the FcΕRI-mediated synthesis and release of cytokines implicated in allergic asthma have not been examined. Methods: Fifteen asthmatic volunteers each received omalizumab for 12 weeks. Peripheral blood basophils were isolated before, during, 2 weeks after and 6 months after omalizumab. Basophils were assayed for the basal and anti-IgE-stimulated release of cytokines, chemokines and histamine. Pooled data were analyzed by repeated measures ANOVA and by paired t tests. Results: Anti-IgE-stimulated human basophils synthesize and release Th2 cytokines (IL-4, IL-13) and chemokines (IL-8, RANTES). The anti-IgE-stimulated release of IL-4, IL-13 and IL-8 was reduced during omalizumab treatment and returned to pretreatment levels after omalizumab withdrawal. Omalizumab did not alter basophil histamine levels or basal and anti-IgE-stimulated histamine release. Conclusions: Omalizumab may reduce asthma symptoms in part by suppressing the FcΕRI-mediated production by basophils of Th2 cytokines and selected chemokines. Anti-IgE-stimulated basophil cytokine synthesis appears more sensitive than histamine release to the loss of FcΕRI caused by omalizumab treatment.

Experimental murine hypersensitivity pneumonitis.

To establish a model of experimental hypersensitivity pneumonitis (EHP) in mice and to examine the influence of genetic background on the pulmonary inflammatory response to Micropolyspora faeni, we determined the responses of C57BL/6, SJL/J, and C3H/HeJ mice to intratracheal (i.t.) injections of M. faeni. Recipient animals received lymph node cells (LNC), peritoneal exudate cells (PEC), and spleen cells (SC) from sensitized mice cultured in vitro with M. faeni. Controls included serum containing anti-M. faeni antibody; uncultured SC from M. faeni-sensitized donors, and M. faeni-cultured SC from ovalbumin (OA)-sensitized donors. Recipients were challenged i.t. with M. faeni or normal saline 48 hr after the cell or serum transfer. We developed a model of EHP in mice. Increasing amounts of i.t. M. faeni were associated with increasing extent of pulmonary inflammation with no difference between the mouse strains. There was substantial increase of the extent of pulmonary abnormalities in the animals receiving cultured SC. The number of transferred cells and the M. faeni concentration correlated with the extent of pulmonary histologic abnormalities. Cultured PEC and LNC could transfer EHP in C3H/HeJ mice only. Serum containing anti-M. faeni antibody, cultured SC from OA-sensitized donors, and noncultured SC from sensitized donors could not transfer EHP. We conclude that it is possible to adoptively transfer EHP.

Cigarette Smoke Suppresses Bik to Cause Epithelial Cell Hyperplasia and Mucous Cell Metaplasia

RATIONALE Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. OBJECTIVES To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. METHODS We screened for dysregulated expression of the Bcl-2 family members. MEASUREMENTS AND MAIN RESULTS We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. CONCLUSIONS These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.