Impaired alveolar macrophage response to Haemophilus antigens in chronic obstructive lung disease

Abstract

Rationale: Interactions of nontypeable Haemophilus influenzae (NTHI) with macrophages are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-macrophage inflammation are poorly understood. Outer membrane protein (OMP) P6 and lipooligosaccharide (LOS) of NTHI are potent immunomodulators. We theorized that alveolar macrophages in COPD possess fundamental immune defects that permit NTHI to evade host responses.

Objective: To test this hypothesis, we obtained human alveolar and blood macrophages from exsmokers with COPD, exsmokers without COPD, and nonsmokers.

Methods: Alveolar and blood macrophages from each donor were incubated with purified LOS and OMP P6 and with OMP P2 and the total outer membrane preparation (0.1-1 microg/ml).

Measurements: Supernatants (24 h) were assayed for IL-1beta, TNF-alpha, IL-10, IL-12, and IL-8 by multianalyte multiplexed flow cytometry.

Results: Comparative induction of COPD and non-COPD alveolar macrophages by LOS and OMP P6 revealed diminished IL-8, TNF-alpha, and IL-1beta responses of COPD alveolar macrophages (p < or = 0.03 for each). COPD alveolar macrophages also had diminished responses to total outer membrane (p < or = 0.03 for each). In contrast, COPD blood macrophages had no significant differences among donor groups in IL-8, TNF-alpha, or IL-1beta responsiveness to NTHI antigens. Diminished IL-12 responses of COPD blood macrophages to NTHI antigens, compared with nonsmokers, could not be independently dissociated from group differences in age and pack-years.

Conclusions: These findings support a paradigm of defective immune responsiveness of alveolar macrophages, but not blood macrophages, in COPD.

Figure 1.

Lipooligosaccharide (LOS) and outer membrane protein (OMP) P6 induction of human alveolar and blood macrophage TNF-α. Alveolar (left column) and blood macrophages (right column) were obtained from former smokers with chronic obstructive pulmonary disease (COPD) (Group 1, light gray shading), former smokers without COPD (Group 2, dark gray shading), and nonsmokers (Group 3, stippled shading). Cells were incubated with LOS (top graphs) and OMP P6 (lower graphs) each at 0.1 μg/ml. Supernatant TNF-α concentration was measured at 24 h. Results are shown as box plots for each group. Each box encompasses the 25th to 75th interquartile range, with the horizontal line in each box representing median values. Each vertical bar encompasses the 10th to 90th percentile ranges. Values correspond with data given in Table 2.

Figure 2.

LOS and OMP P6 induction of human alveolar and blood macrophage IL-8. Alveolar (left column) and blood macrophages (right column) were obtained from former smokers with COPD (Group 1, light gray shading), former smokers without COPD (Group 2, dark gray shading), and nonsmokers (Group 3, stippled shading). Supernatant concentrations for TNF-α, elicited by LOS (top graphs) and OMP P6 (lower graphs) at 0.1 μg/ml, are as detailed in Figure 1. Data are represented by box plots for each group as detailed in Figure 1. Values correspond with data given in Table 3.

Figure 3.

LOS and OMP P6 induction of human alveolar and blood macrophage IL-1β. Alveolar (left column) and blood macrophages (right column) were obtained from former smokers with COPD (Group 1), former smokers without COPD (Group 2), and nonsmokers (Group 3). Shading denoting each individual group and supernatant concentrations for IL-1β elicited by OMP P6 (top graphs) and LOS (lower graphs) at 1 μg/ml are as detailed in Figure 1. Data are represented by box plots for each group as detailed in Figure 1. Values correspond with data given in Table 4. ^λ Multivariate regression analysis showed age as an independent determinant of OMP P6 induction of IL-1β from alveolar macrophages.

Figure 4.

LOS and OMP P6 induction of human alveolar and blood macrophage IL-12. Alveolar (left column) and blood macrophages (right column) were obtained from former smokers with COPD (Group 1), former smokers without COPD (Group 2), and nonsmokers (Group 3). Shading denoting each individual group and supernatant concentrations for IL-12 elicited by LOS (top graphs) and OMP P6 (lower graphs) at 0.1 μg/ml are as detailed in Figure 1. Data are represented by box plots for each group as detailed in Figure 1. Values correspond with data given in Table 5. ^λ Multivariate regression analysis showed cumulative pack-years to be an independent determinant of LOS induction of IL-12 and age as an independent determinant of OMP P6 induction of IL-12 from blood macrophages only. No demographic variables were independent determinants for alveolar macrophage IL-12 responses.

Figure 5.

LOS and OMP P6 induction of human alveolar and blood macrophage IL-10. Alveolar (left column) and blood macrophages (right column) were obtained from former smokers with COPD (Group 1), former smokers without COPD (Group 2), and nonsmokers (Group 3). Shading denoting each individual group and supernatant concentrations for IL-10 elicited by LOS (top graphs) and OMP P6 (lower graphs) at 1 μg/ml are as detailed in Figure 1. Data are represented by box plots for each group as detailed in Figure 1. Values correspond with data given in Table 6.