CD11c(+)/CD11b(+) cells are critical for organic dust-elicited murine lung inflammation

Abstract

Organic dust exposure in the agricultural industry results in significant lung disease. Macrophage infiltrates are increased in the lungs after organic dust exposures, yet the phenotype and functional importance of these cells remain unclear. Using an established intranasal inhalation murine model of dust-induced lung inflammation, animals were treated once or daily for 3 weeks with swine confinement organic dust extract (DE). Repetitive DE treatment for 3 weeks resulted in significant increases in CD11c(+)/CD11b(+) macrophages in whole lung-associated tissue. These cells displayed increased costimulatory molecule (CD80 and CD86) expression, enhanced phagocytic ability, and an increased production of IL-6, CXCL1, and CXCL2. Similar findings were observed with the CD11c(+)/CD11b(+) macrophage infiltrate after repetitive exposure to peptidoglycan, a major DE component. To determine the functional importance of macrophages in mediating DE-induced airway inflammation, lung macrophages were selectively depleted using a well-established intranasal clodronate liposome depletion/suicide strategy. First, macrophage depletion by clodronate liposomes resulted in significant reductions in airway neutrophil influx and TNF-α and IL-6 production after a single exposure to DE. In contrast, after repetitive 3-week exposure to DE, airway lavage fluid and lung tissue neutrophils were significantly increased in clodronate liposome-treated mice compared with control mice. A histological examination of lung tissue demonstrated striking increases in alveolar and bronchiolar inflammation, as well as in the size and distribution of cellular aggregates in clodronate-liposome versus saline-liposome groups repetitively exposed to DE. These studies demonstrate that DE elicits activated CD11c(+)/CD11b(+) macrophages in the lung, which play a critical role in regulating the outcome of DE-induced airway inflammation.

Figure 1.

Repetitive organic dust extract (DE) exposure leads to the activation of CD11c⁺ lung macrophages. Lung-associated cells were collected from C57BL/6 mice after 3 weeks of repetitive intranasal exposure to DE or saline control, and stained for FACS. (A) Results represent total lung cells and absolute CD11c⁺ cells. #, number of. (B) Mean fluorescence intensity (MFI) of surface marker expression on gated CD11c⁺ cells is shown. Results represent the mean ± SEM (n = 6 mice/group), with statistical significance denoted by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 2.

DE treatment increases phagocytosis and cytokine production by CD11c⁺ lung cells. C57BL/6 mice were repetitively exposed to DE or saline for 3 weeks, and lung-associated cells were exposed ex vivo to FITC-labeled Saccharomyces cerevisiae zymosan-A bioparticles at 0 and 60 minutes to determine the phagocytic ability of gated CD11c⁺ lung macrophages. (A) A representative dot plot depicts particle uptake in gated CD11c⁺Ly-6G⁻ macrophages from DE-treated mice and saline control–treated mice as a rightward shift in fluorescence. (B) The phagocytic ability of macrophages is shown as fold change in mean MFI (± SEM) of the proportion of cells in the zymosan-exposed gated population at 60 minutes, compared with cells exposed for 0 minutes from DE and saline control mice (n = 6 mice/group). (C) CD11c⁺ macrophages isolated by FACS from DE-treated mice and saline-exposed mice were restimulated ex vivo with DE (1%) or saline (0% DE) for 24 hours, and cytokine/chemokine concentrations were measured in cell-free supernatant by ELISA. Results represent the mean ± SEM (n = 6–9 mice/group), with statistical significance between saline-and DE-treated denoted by asterisks (*P < 0.05 and ***P < 0.001).

Figure 3.

Repetitive intranasal challenge with peptidoglycan (PGN) increases activated CD11c⁺ lung macrophages. C57BL/6 mice were repetitively exposed to PGN (100 μg) or saline for 3 weeks, and lung-associated cells were enumerated and stained for FACS. (A) Results represent the means ± SEM (n = 6 mice/group) of total lung cells and CD11c⁺ lung macrophages. (B) The mean fluorescence intensity (MFI) of surface marker expression on gated CD11c⁺ cells is shown. Next, lung-associated cells were exposed ex vivo to FITC-labeled Saccharomyces cerevisiae zymosan-A bioparticles at 0 and 60 minutes, to determine the phagocytic ability of gated CD11c⁺ lung macrophages. (C) Results represent fold changes in MFI (proportion of cells in the gated population at 60 min, compared with cells exposed for 0 min), expressed as means ± SEM (n = 6 mice/group). Statistical significance is denoted by asterisks (*P < 0.05 and ***P < 0.001).

Figure 4.

Clodronate-encapsulated liposome treatment reduces lung alveolar macrophages. C57BL/6 mice were treated with clodronate liposomes (CL-LIP) and saline liposomes (SL-LIP), and exposed to saline or DE 48 hours later. Lung sections of all four mice per group were stained with hematoxylin and eosin, and lung macrophages in peripheral and central lung fields (10 total fields/section) were counted and averaged per individual mouse. (A) Results represent the means ± SEM of lung macrophage numbers per high-power field (HPF) in each group. (B) A representative 4- to 5-μm-thick section from each group is shown at ×40 magnification. Arrows indicate alveolar macrophages. Statistical significance is denoted by hatch marks (^###P < 0.001) between SL-LIP–treated and CL-LIP DE–treated mice.

Figure 5.

Alveolar macrophages (Macs) are important in mediating acute airway inflammatory response to DE. C57BL/6 mice were treated with clodronate liposomes (CL-LIP) or saline liposomes (SL-LIP) 2 days before a one-time DE challenge. Mice were subsequently challenged with DE or saline, and bronchoalveolar lavage fluid (BALF) was collected 5 hours after exposure. (A) Results represent the means ± SEM of total cells and cell differentials. (B) Results represent mean ± SEM of cytokine/chemokine concentrations quantitated in cell-free BALF. PBS-only controls are also shown (n = 4 mice per group). Statistical significance is denoted by asterisks (*P < 0.05 and *P < 0.01) between respective saline-treated and DE-treated groups. ^#P < 0.05 and ^##P < 0.01 indicate statistical significance between SL-LIP–treated and CL-LIP DE–treated mice.

Figure 6.

Repetitive DE-induced neutrophilic influx is increased when lung macrophages are depleted. C57BL/6 mice were treated with clodronate liposomes (CL-LIP) or saline liposomes (SL-LIP) beginning 2 days before the first DE challenge, and then every 3 to 4 days during the daily 3-week repetitive DE or saline exposure. (A) Results represent the means ± SEM (n = 4 mice/group) of the total cells and cell differentials recovered from the BALF of mice. Next, lung-associated cells were collected from mice, and total cells were enumerated and stained by FACS. (B) Results represent means ± SEM (n = 4 mice/group) of CD11c⁺ lung macrophages and neutrophils (percentage of cell type × total lung cell count). Statistical significance is denoted by asterisks (*P < 0.05, *P < 0.01, and ***P < 0.001) between respective saline-treated and DE-treated groups. Hatch marks (^#P < 0.05, ^##P < 0.01, and ^###P < 0.001) indicate statistical differences between SL-LIP + DE–treated and CL-LIP + DE–treated mice.

Figure 7.

Lung macrophages are important in mediating repetitive DE-induced lung inflammation. C57BL/6 mice were treated with clodronate liposomes (CL-LIP) or saline liposomes (SL-LIP) beginning 2 days before the first DE challenge, and then every 3 to 4 days during the daily 3-week repetitive DE or saline exposure. (A) Semiquantitative inflammatory scores (means ± SEM; n = 4 mice per group) of the degree and distribution of cellular aggregates, and of alveolar and bronchiolar lung inflammation, are shown. PBS-only control scores are also shown. (B) A representative 4- to 5-μm-thick, hematoxylin-and-eosin–stained section of one of four mice per treatment group is shown at ×10 magnification. All lung specimens were inflated to 10 cm H₂O pressure during fixation to avoid atelectasis artifacts. Statistical significance is denoted by asterisks (*P < 0.05, *P < 0.01, and ***P < 0.001) between respective saline-treated and DE-treated groups. ^#P < 0.05 and ^##P < 0.01 indicate statistical difference between SL-LIP + DE–treated and CL-LIP + DE–treated mice.