Induced sputum and bronchoalveolar lavage as tools for evaluating the effects of inhaled corticosteroids in patients with asthma.

Changes in airway inflammation can be studied with bronchoalveolar lavage, but the widespread use of this procedure is limited by its invasiveness. The aim of this study was to evaluate the usefulness of induced sputum as a non-invasive alternative to bronchoalveolar lavage for studying changes in airway inflammation in patients with asthma. Thirty patients were treated for 12 weeks with an inhaled corticosteroid (fluticasone propionate (FP), 250 microg twice daily) or a short-acting beta-agonist (salbutamol (Sb), 400 microg twice daily) in a double-blind, double-dummy, randomized parallel group study. Sputum induction with hypertonic saline solution was performed twice before treatment and after 4, 8, 10, and 11 weeks of treatment. Bronchoalveolar lavage fluid divided into two pools (first 60 mL portion as bronchoalveolar lavage/bronchial wash (BAL/BW) and subsequent 80 mL as bronchoalveoalar lavage (BAL)) was obtained before and after 12 weeks of treatment. Changes in cell differentials and plasma-protein leakage (alpha2-macroglobulin, albumin, and their ratio (relative coefficient of excretion, RCE)) were analyzed in induced sputum and were compared with changes in BAL/BW and BAL. During treatment with FP, the PC20histamine (interpolated concentration of histamine that caused a fall in FEV1 of 20% of the baseline value) increased (P < .0001), and the percentage of eosinophils (P = .004), levels of (alpha2-macroglobulin (P = .09) and RCE (P = .007) decreased in sputum. These changes were different from those in the Sb group (PC20histamine P< .0001, eosinophils P= .004, alpha2-macroglobulin P= .003, RCE P = .01), in which alpha2-macroglobulin showed a significant increase (P = .015). Changes in the percentage of eosinophils and in the levels of alpha2-macroglobulin in sputum were associated with changes in the PC20histamine (Rs = -0.59, P = .007 and Rs = -0.47, P = .03, respectively). These correlations did not reach significance in BAL/BW and BAL fluid. The statistical power to detect changes in induced sputum was higher for the percentage of eosinophils and similar for plasma protein leakage as compared with analysis of BAL/BW and BAL fluid. We conclude that the analysis of induced sputum is a useful, non-invasive alternative to bronchoalveolar lavage for assessing the effects of antiinflammatory drugs in asthma.

Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid.

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-gamma, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4 h with 1 ng/ml PMA and 1 microg/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN-gamma positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4+ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-gamma+ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-gamma+. We conclude that this method allows detection of intracellular cytokine proteins in both CD4+ and CD8+ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.

A double-blind study on the effect of inhaled corticosteroids on plasma protein exudation in asthma.

Plasma protein exudation into the airways is an important pathophysiological event in asthma. The effect of 12 wk of treatment with inhaled fluticasone propionate (FP; 250 microgram twice a day) or salbutamol (Sb; 400 microgram twice a day) on plasma protein leakage was compared in a double-blind, randomized parallel-group study of 30 patients with asthma. Primary outcomes were plasma protein leakage and size selectivity of the blood-airway lumen barrier, cell differentials in BAL fluid, and bronchial responsiveness to histamine (PC20histamine). Two independent procedures to account for the effect of variable dilution of BAL on the levels of albumin (Alb) and alpha2-macroglobulin (A2M) in BAL fluid consisted of correction based on urea levels and on the application of the relative coefficient of excretion [RCE = ([A2M] in BAL fluid/[A2M] in serum)/([Alb] in BAL fluid/[Alb] in serum)]. In the FP group a significant decrease was found in the A2M level and the RCE, and in the percentage of eosinophils in BAL fluid. The PC20histamine increased significantly (mean increase, 2.4 doubling doses), whereas PC20histamine decreased in the Sb group. Differences between groups were significant except for the decrease in eosinophils. We conclude that 12 wk of FP (250 microgram twice a day) decreased the permeability of the blood-airway lumen barrier, in particular for high molecular weight proteins.