Annette M. Colavita, MD, Annette T. Hastie, PhD, Ali I

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Kinetics of IL-10 production after segmental antigen challenge of atopic asthmatic subjects Annette M. Colavita, MD, Annette T. Hastie, PhD, Ali I. Musani, MD, Rodolfo M. Pascual, MD, Alan J. Reinach, MD, H.Todd Lustine, MD, Steve A. Galati, BS, James G. Zangrilli, MD, James E. Fish, MD, Stephen P. Peters, MD, PhD Journal of Allergy and Clinical Immunology Volume 106, Issue 5, Pages 880-886 (November 2000) DOI: 10.1067/mai.2000.110475 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 IL-10 levels in the BAL fluid of allergic asthmatic and control subjects. Asthmatic subjects were divided into two groups, those with both early and late (dual) reactions (AD , n = 8) and those with only a single early reaction after whole-lung antigen challenge (AS , n = 4). The control group (C) consisted of eight subjects. Data are presented as modified box plots with extremes of the boxes representing the 25th and 75th percentiles, respectively, and the horizontal line in the box representing the median. When all days were considered together, a Kruskal-Wallis ANOVA on ranks revealed a significant difference between groups (P < .001) with a pairwise multiple comparison procedure by Dunn’s methods revealing significant differences between the AD and AS (P < .05) and AD and control (P < .05) groups. When individual days were analyzed with these same procedures, the AD group was significantly different from the control group on days 1, 2, and 9 (shown by asterisk ). Journal of Allergy and Clinical Immunology 2000 106, 880-886DOI: (10.1067/mai.2000.110475) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 IL-10 levels in macrophages of allergic and control subjects. Adherent cells from both asthmatics (AAD group, n = 8) and controls (n = 3) were studied for the ex vivo production of IL-10. When all days were considered, a Mann-Whitney rank sum test revealed a significant difference between groups (P = .036). A significant difference between AAD and controls was noted for day 16 (P = .038, t test) and a trend for day 1 (P = .07, t test). Journal of Allergy and Clinical Immunology 2000 106, 880-886DOI: (10.1067/mai.2000.110475) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Effect of dexamethasone on IL-10 production by asthmatic macrophages. The effect of in vitro dexamethasone treatment (1 μmol/L) on the spontaneous secretion of IL-10 from asthmatic adherent BAL cells (AAD, n = 8) was studied. Wilcoxon signed rank test revealed no difference between treatments when all data were considered (P = .922), and pairwise comparisons (paired t tests) for each day also revealed no significant differences. Journal of Allergy and Clinical Immunology 2000 106, 880-886DOI: (10.1067/mai.2000.110475) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Cytospins immunostained with anti-human IL-10. BAL leukocyte cytospin preparations from an allergic asthmatic subject (A) and a control subject (B) at baseline (day 1) and 24 hours (day 2), 1 week (day 9), and 2 weeks (day 16) after SAC were stained with monoclonal anti-human IL-10. Note that some small cells having bilobar nuclei (eosinophils) stain intensely for IL-10 (arrowheads). All micrographs were printed at the same magnification (marker = 100 μm), except for the magnified insert in A (day 2), which suggests that stain is cytoplasmic rather than plasma membrane bound. Journal of Allergy and Clinical Immunology 2000 106, 880-886DOI: (10.1067/mai.2000.110475) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Quantitation of IL-10 staining intensity of BAL leukocytes according to leukocyte size. Samples from 4 asthmatics and 4 controls were analyzed. A, A trend for greater IL-10 stain intensity was observed in the asthmatic subjects 24 hours after SAC (day 2) compared with the control group (P = .088, t test) for cells in the 126 to 250 μm2 size range. This size category contains both eosinophils (Eos) and neutrophils (Neu), but the majority of the cells in this size category were eosinophils in asthmatics at the 24-hour (day 2) and 1-week (day 9) time points. This is shown in the tabulated data from the differential cell counts for controls and for asthmatics beneath their respective bars. Absolute cell numbers (cells × 104/mL) for eosinophils are also given. B, IL-10 stain intensity was found to be greater in the asthmatic group compared with the control group (P = .021, t test) on day 2 for cells in the size range >250 μm2. This size category contains predominantly macrophages (M φ), as determined by the differential cell counts and absolute numbers for the asthmatic and control subjects given beneath their respective bars. Journal of Allergy and Clinical Immunology 2000 106, 880-886DOI: (10.1067/mai.2000.110475) Copyright © 2000 Mosby, Inc. Terms and Conditions