Increase in granzyme B+ lymphocytes and soluble granzyme B in bronchoalveolar lavage of allergen challenged patients with atopic asthma

Abstract

Asthma has been linked to a chronic, T-cell-mediated bronchial inflammation. Because other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with elevated granzyme B (grB) expression we tested the hypothesis that atopic asthma might be associated with elevated grB levels in the bronchoalveolar compartment. Therefore we performed intracellular grB staining in lymphocytes from bronchoalveolar lavage (BAL) collected 42 h after segmental allergen provocation (SAP) in allergic patients with bronchial asthma. There was a significant increase in CD3(+), CD8(+), and CD16/56(+) lymphocytes expressing grB in BAL 42 h after SAP as compared to saline challenged controls. However, compared to peripheral blood the percentages of these lymphocyte subsets detected as grB(+) in BAL remained significantly lower. Measurement of extracellular grB in BAL fluids by a particle immunoassay revealed significantly elevated grB levels in the allergen challenged bronchoalveolar compartment 42 h following SAP in six of the eight patients (range, <1.0-348.1 pg/ml) as compared to saline challenged controls (range, <1.0-70.5 pg/ml). We conclude that total cell numbers of grB(+) lymphocyte subsets increase 42 h after SAP in the lower respiratory tract. In addition there is evidence to suggest that grB is released into the airways of asthmatic patients. This suggests a role for grB in the pathophysiological processes following SAP but its definitive role in allergic bronchial asthma needs to be established.

Fig. 1

Distribution of grB in peripheral blood lymphocytes (PBL) and BAL lymphocytes after SAP as indicated by flow cytometric analysis. Percentages are the mean ± SEM of eight different allergic asthmatic patients. GrB+, □ GrB−. (a) Percentages of grB⁺ total lymphocytes in BAL and peripheral blood. Percentages of (b) CD16/56⁺, (c) CD8-dim⁺, (d) CD8-bright⁺ and (e) CD3⁺ lymphocytes expressing grB in BAL samples and peripheral blood. PBL 0 h, PBL before SAP; PBL 42 h, PBL 42 h after SAP; BAL C 10 min, BAL lymphocytes from saline challenged lung segments 10 min after SAP; BAL C 42 h, BAL lymphocytes from saline challenged lung segments 42 h after SAP; BAL P 10 min, BAL lymphocytes from allergen challenged lung segments 10 min after SAP; BAL P 42 h, BAL lymphocytes from allergen challenged lung segment 42 h after SAP.

Fig. 2

Concentrations of extracellular (a) grB and (b) IL-5 in BAL fluid from eight different asthmatic patients 42 h after SAP. Six of eight patients showed significantly higher grB and IL-5 concentrations in the allergen challenged lung segment compared to saline challenged control. BAL C 42 h, BAL fluid from saline challenged lung segments 42 h after SAP; BAL P 42 h, BAL fluid from allergen challenged lung segment 42 h after SAP.