Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma

Abstract

The role and origin of alveolar macrophages (AMs) in asthma are incompletely defined. We sought to clarify these issues in the context of acute allergic lung inflammation using house dust mite and OVA murine models. Use of liposomal clodronate to deplete resident AMs (rAMs) resulted in increased levels of inflammatory cytokines and eosinophil numbers in lavage fluid and augmented the histopathologic evidence of lung inflammation, suggesting a suppressive role for rAMs. Lung digests of asthmatic mice revealed an increased percentage of Ly6C(high)/CD11b(pos) inflammatory monocytes. Clodronate depletion of circulating monocytes, by contrast, resulted in an attenuation of allergic inflammation. A CD45.1/CD45.2 chimera model demonstrated that recruitment at least partially contributes to the AM pool in irradiated nonasthmatic mice, but its contribution was no greater in asthma. Ki-67 staining of AMs supported a role for local proliferation, which was increased in asthma. Our data demonstrate that rAMs dampen, whereas circulating monocytes promote, early events in allergic lung inflammation. Moreover, maintenance of the AM pool in the early stages of asthmatic inflammation depends on local proliferation, but not recruitment.

Figure 1. Augmented allergic lung inflammation in the murine HDM model after depletion of rAMs

(A) Schematic representation of experimental protocol employing oropharyngeal (o.p.) administration of HDM and i.t. administration of liposomal clodronate (CL). (B) BALF numbers of total cells, macrophages, eosinophils, and neutrophils in mice subjected to HDM-induced asthma, treated with liposomal clodronate to deplete rAMs or control (empty) liposomes. Mice were sacrificed at day 9 of the protocol. Total cells from BALF were counted by light microscopy; differential cell count after Wright-Giemsa staining was performed on 300 cells. (C–H) BALF levels of cytokines were measured by ELISA in asthmatic mice subjected to depletion of rAMs. Data presented in all panels are expressed as the mean ± SEM from 4 experiments with 2–3 mice per group per experiment.* p<0.05, ** p<0.01, *** p<0.001. (I) Representative lung sections stained with H&E (10× magnification) to visualize peribronchial leukocyte infiltration and thickened smooth muscle tissue (arrow). (J) Quantitation of inflammation area (micron² per micron of basement membrane) in H&E-stained lung of asthmatic mice treated with i.t. liposomal clodronate or empty liposomes (control). Data presented in panel J are expressed as the mean ± SEM from 10 bronchi from each of 3 mice per group.* p<0.05

Figure 2. Depletion of rAMs reduces TGF-β levels but does not affect T reg percentage in the lungs

(A) TGF-β levels were measured in BALF by ELISA in asthmatic mice subjected to depletion of rAMs. Data are expressed as the mean ± SEM from 4 experiments with 2–3 mice per group.* p<0.05. (B) Lungs from asthmatic mice treated with i.t. liposomal clodronate or empty liposomes (control) were lavaged, perfused and processed to obtain single cell suspensions as described in Materials and Methods. Gates were set to remove debris, set on low SSC, and Fixable Viability Dye eFluor® 450 staining was used to eliminate dead cell contamination. Representative dot-plot shows CD4 and Foxp3 staining, with statistical analysis of CD4^posFoxp3^pos cell population. (C) Mice were treated as described in panel B and data are expressed as the mean ± SEM from 3 experiments, each utilizing 3 mice.

Figure 3. Recruitment of Ly6C^high monocytes in asthma

(A) Lungs from asthmatic mice treated with i.t. liposomal clodronate or empty liposomes (control) were lavaged, perfused and processed to obtain single cell suspensions as described in Materials and Methods. Gates were set to remove debris, set on low SSC, and Fixable Viability Dye eFluor® 450 staining (viability dye) was used to eliminate dead cell contamination. Representative dot-plots show the Ly6C^highCD11b^pos population denoted within the gates. (B) Statistical analysis of Ly6C^highCD11b^pos cell populations in lung digests following i.t. administration of liposomal clodronate vs. control liposomes. Data presented as the mean ± SEM from 4 experiments, each utilizing 2–3 mice per group. *** p<0.001. (C) Lung digests from mice treated with i.v. clodronate or control liposomes were processed for staining and gated as described above. Representative dot-plots show the Ly6C^highCD11b^pos population denoted within the gates. (D) Statistical analysis of Ly6C^highCD11b^pos cell populations in lung digests following i.v.administration of liposomal clodronate vs. control liposomes. Data presented as the mean ± SEM from 3 experiments, each utilizing 2–3 mice per group. *** p<0.001.

Figure 4. Circulating precursor cells do not contribute to BALF AM accumulation in asthma as revealed by chimeric mice

(A) Gating strategy for identification of AMs in asthma. Recipient CD45.2 mice were sub-lethally irradiated and reconstituted with bone marrow cells from CD45.1 donor mice. 5 weeks after BMT, mice were subjected to HDM protocol and at day 9 CD45.2 mice were sacrificed and BALF was collected for flow cytometric analysis of the percentage of CD45.1-positive donor cells. AMs were gated based on their higher FSC, followed by negative selection for cell death using Fixable eFluor® 450 (viability dye), and CD11c^pos and high autofluorescence (AF)^high. (B) Percentage of donor (CD45.1) cells among BALF AMs in asthmatic mice. AMs from BALF of CD45.2 mice exposed to asthma (-/HDM) or that underwent irradiation and CD45.1 reconstitution alone (BMT/-) or were subsequently subjected additionally to the HDM protocol (BMT/HDM) were gated as described in panels (A) and the percentage of CD45.1-positive donor AMs was calculated. Representative dot-plots are presented and the donor and recipient populations are indicated by separate gates. (C) Quantitative analysis of CD45.1^pos population of AMs (recruited AMs) in BALF. Data are presented as the mean ± SEM from 2 experiments, each utilizing 5 mice per group.

Figure 5. Depletion of circulating monocytes attenuates allergic lung inflammation

(A) Schematic representation of the protocol for i.v. administration of liposomal clodronate relative to HDM challenge. Asthmatic mice treated with i.v. clodronate (CL) or empty liposomes (control) were sacrificed at day 9 of the protocol. (B) Total BALF cells, eosinophils, macrophages and neutrophils were determined by counting under light microscopy; differential cell count after Wright-Giemsa staining was performed on 300 cells. (C–I) Cytokines in BALF of asthmatic mice were assessed by ELISA following depletion of circulating monocytes. Data presented in all panels are expressed as the mean ± SEM from 4 experiments, each utilizing 2–3 mice per group.* p<0.05, ** p<0.01. (J) Representative lung sections stained with H&E (10× magnification) to visualize peribronchial leukocyte infiltration and thickened smooth muscle tissue (arrow). (K) Quantitation of inflammation area (micron² per micron of basement membrane) in H&E-stained lung of asthmatic mice treated with i.v. liposomal clodronate or empty liposomes (control). Data presented in panel J are expressed as the mean ± SEM from 10 bronchi from each of 3 experiments with 2 mice per group.* p<0.05

Figure 6. Proliferation of AMs isolated from control and asthmatic mice

(A) Gating strategy for identification of AMs in asthma. Mice received HDM or PBS as described in Materials and Methods. At day 9 of the protocol mice were sacrificed and BALF and lung digests were collected. AMs were defined as CD45^pos, Ly6G^neg, SiglecF^pos and CD11c^pos. (B and C) Proliferation of AMs isolated from BALF (B) and from lung digests of previously lavaged lungs (C) from asthmatic mice compared to PBS-treated controls. AMs were gated as described in panel (A) and stained with Ki-67 or isotype controls. Percent of Ki-67^pos AMs was calculated relative to isotype controls. (D and E) Quantitative analysis of Ki-67^pos proportion of AMs from BALF (D) and (E) lung digests of previously lavaged lungs. Data are presented as the mean ± SEM from 1 experiment utilizing 5 mice per group. * p<0.05