Resident alveolar macrophages suppress while 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 utilizing house dust mite and ovalbumin 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 histopathologic evidence of lung inflammation, suggesting a suppressive role of 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 non-asthmatic 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, while 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.

INTRODUCTION

Asthma is a major global health problem affecting both children and adults [1]. Alveolar macrophages (AMs) are key orchestrators of pulmonary immune responses [2], and under steady state conditions they account for 95% of the leukocytes in the lower respiratory tract [3]. Nevertheless, as compared to dendritic cells, T cells, eosinophils, and mast cells, there are very few studies examining the role of AMs in this condition, and they have been appropriately termed “the forgotten cell in asthma” [4]. Resident AMs (rAMs) can actively suppress T cell proliferation induced by antigen [5] and downregulate the antigen presentation capacity of pulmonary dendritic cells [6]. By virtue of these various immune-inhibitory functions together with their ability to protect against airway hyperresponsivness [7], rAMs might be expected to dampen immune responses and help to preserve physiologic functions of the lung. Indeed, depletion of rAMs by intrapulmonary administration of liposomal clodronate under steady sate conditions was found to increase the subsequent immune response to an intrapulmonary antigen [8].

AM depletion by this method has, however, yielded conflicting results in murine models of allergic asthma, with studies suggesting both pathogenic [9, 10] and suppressive [11, 12] roles for these cells. Inflammatory responses in the lung can lead to recruitment of monocytes with a pro-inflammatory phenotype [13], but the possibility that recruited AMs contribute to allergic lung inflammation in these models was not considered. Because intratracheal (i.t.) clodronate fails to discriminate between resident and recruited AMs and between differentially polarized subsets of AMs, it therefore has the potential to deplete potentially suppressive as well as pathogenic populations.

Here we employed both i.t. and intravenous (i.v.) liposomal clodronate to selectively deplete rAM and circulating monocyte populations, respectively. We demonstrate that rAMs are protective in asthma while recruited inflammatory monocytes – putative precursors of AMs – are pathogenic. We also show that during the early stages of allergic inflammation, maintenance of the AM pool depends on local proliferation rather than on recruitment of circulating precursor cells.

MATERIALS AND METHODS

Animals

Wild type (WT) C57BL/6 (Ly5.1; CD45.2) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). B6Ly5.2 (CD45.1) mice were purchased from the National Cancer Institute Frederick Cancer Research Facility (Frederick, MD). The University of Michigan Committee on Use and Care of Animals approved these experiments.

Asthma protocols

OVA-induced acute allergic inflammation was elicited as previously described by intraperitoneal sensitization with 20 µg of OVA (Sigma-Aldrich, St. Louis, MO) mixed with 2 mg of alum (Thermo Fisher Scientific, Waltham, MA) [14] on day 0 followed by two nebulizer-delivered airway challenges with 1 % OVA on days 7 and 8 [15]. House dust mite (HDM)-induced inflammation utilized extracts of Dermatophagoides pteronyssinus mites (Greer, Lenoir, NC) crushed with a mortar and pestle. Mice were anaesthetized and 100 µg of HDM extract suspended in PBS in a volume of 50 µl was given by oropharyngeal administration on days 0, 7 and 8. For both models, lung samples were collected at day 9. These acute allergic inflammation protocols resulted in induction of Th2 cytokines and eosinophilic accumulation measured in bronchoalveolar lavage fluid (BALF), with eosinophilic inflammation verified by H&E staining of slides prepared from lung sections.

Depletion of rAMs and blood monocytes

Liposomal clodronate (Cl₂MDP-L) was a gift from Roche Diagnostics GmbH, Mannheim, Germany. Encapsulation of clodronate was performed in the laboratory of Dr. Nico van Rooijen according to a previously established method [16].To deplete rAMs, liposomes encapsulating clodronate (or empty liposomes encapsulating PBS as control) were given i.t. in a volume of 50 µl 2 days before the first airway challenge; this timing was based on kinetic data presented in Results. To deplete blood monocytes, liposomal clodronate was given i.v. in a volume of 200 µl according to previously described protocols [17, 18] 8 h before each airway challenge. Control mice received an equal volume of empty control liposomes.

Bone marrow transplantation

Bone marrow was harvested from CD45.1 donor mice and infused by tail vein injection into sub-lethally irradiated CD45.2 recipients, using a protocol described previously [19]. Ablation of recipient-derived hematopoietic stem cells was achieved by the administration of a fractionated 7-Gy dose of total body irradiation from an x-ray orthovoltage source. Maximal immune reconstitution was achieved 5 weeks following the infusion of 5 × 10⁶ whole bone marrow cells into total body irradiated recipients, and mice were subjected to asthma protocols at this time point post-transplantation. The percentage of donor-derived leukocytes was ~85% in the spleen and blood. Specificity of CD45.1 and CD45.2 antibodies was verified in both mouse strains (CD45.1 and CD45.2). CD45.1 versus CD45.2 expression profiles of AMs after appropriate gating were analyzed on a FACSCanto II flow cytometer (BD Biosciences, San Diego, CA).

Isolation of cells from BALF

Lung lavage was performed according to a previously described protocol [20] employing 800 μl aliquots of sterile PBS/2 mM EDTA (pH 7.2) until a BALF volume of 3 ml was recovered. Total BAL cells were suspended in PBS/2 mM EDTA/0.5% FCS, counted, and prepared for flow cytometric staining. Aliquots were taken to prepare cytospins for H&E staining and differential counting.

Isolation of cells from lung digests

Single cell suspensions from lung digests were prepared as described in detail previously [20] . Briefly, lungs were perfused with PBS, then removed and transferred into Petri dishes containing 0.7 mg/ml collagenase A (Roche Diagnostic, Indianapolis, IN) and 50 μg/ml DNAse I (Roche) in RPMI-1640 medium. Lungs were minced and cut into small pieces, agitated on a shaker (30 min, room temperature) and then incubated at 37°C for 30 min in a humidified atmosphere containing 5% CO₂. Cell aggregates were dispersed by repeated passage through a syringe, and filtered through a 40 μm cell strainer (BD Biosciences, San Jose, CA) in order to obtain a single cell suspension. Erythrolysis was performed with 10 ml 0.8% ammonium chloride lysis buffer, and cells were subsequently washed, suspended in PBS/2 mM EDTA/0.5% FCS, counted, and subjected to flow cytometric staining.

Lung histology

Inflammation was determined from H&E-stained slides prepared from paraffin-embedded sections of the lungs. The sections were chosen randomly and the reviewer was blinded to the treatments. Since infiltration of mononuclear cells and eosinophils was most severe in large airways, inflammation was quantified from 10 bronchi per mouse and was reported as inflammation area (micron² per micron of bronchial basement membrane as a correction for size of the bronchi).

Flow cytometry

Staining for flow cytometric analysis was performed on cells resuspended in PBS/2 mM EDTA/0.5% FCS. Fc receptor-mediated and nonspecific antibody binding was blocked by addition of excess CD16/CD32 (BD Pharmingen, San Jose, CA). Staining for cell surface receptors was performed at 4°C in the dark for 15 min with previously established dilutions. Intracellular staining for proliferation and Foxp3 staining employed a Foxp3 Staining kit (eBioscience, San Diego, CA), according to manufacturer’s instructions; for each experiment, dead cells were eliminated using Fixable Viability Dye eFluor® 450 from eBioscience.

The following monoclonal antibodies were used at appropriate dilutions for staining: CD11c-PerCP/Cy5.5, and CD11c-Pacific Blue (N418, Biolegend, San Diego, CA); CD11b-FITC (M1/70, BD Pharminogen); Siglec F-PE (E50-2440, BD Pharminogen); Ly6G-PE-Cy7 (1A8; BioLegend); CD11b-APC, CD11b-APC-Cy7, CD11b-PE/Cy7, CD11b-PerC/PCy5.5 (M1/70, Biolegend); Ly6C-APC, Ly6C-PE/Cy7 (HK1.4, Biolegend); F4/80-APC (BM8, eBioscience); CD4-FITC (GK1.5, Biolegend); CD45.1-PE (A20, BD Pharmingen); CD45.2-FITC (104, BD Pharmingen); Ki-67-APC (SolA15, eBioscience); Foxp3-PE (FJK-16s, eBioscience). A FACSCanto II flow cytometer from BD Biosciences was used for flow cytometric characterization of cell populations in chimera experiments and a LSR II was used for Ki-67 proliferation determination. Data was analyzed with 7.6.4 FlowJo software (Tree Star, Ashland, OR).

ELISA

The quantification of murine IL-4, IL-5, IL-10, IL-13, IL-17, TGF-β, and IFN-γ protein from BALF was performed by commercially available ELISA kits following the instructions of the manufacturer (R&D Systems, Minneapolis, MN).

Statistical analysis

Data are presented as mean ± SEM. Statistical significance was analyzed using the GraphPad Prism 5.0 statistical program (GraphPad Software, La Jolla, CA). Comparisons between two experimental groups were performed using the Student’s t test. A p value < 0.05 was considered statistically significant.

RESULTS

rAMs dampen allergic lung inflammation

To investigate the role of rAMs in allergic lung inflammation, we used a well-characterized depletion model [11]. I.t. administration of liposomal clodronate to naïve mice resulted in ~80% depletion of rAMs that was evident on day 2 and that lasted through day 4 post-administration (Supplemental Fig. 1A). Such treatment in naïve mice resulted in minimal accumulation in BALF of leukocytes other than AMs, as determined by cytospin differential counts (data not shown); neutrophils, eosinophils, and lymphocytes were highest on day 1 post-liposomal clodronate and together accounted for 6% of total BALF cells (data not shown). BALF levels of Th2 cytokines (IL-4, IL-5, IL-13), IFN-γ, and IL-17 were all either undetectable or detectable at <20 pg/ml, and were not different from those measured in mice that received control liposomes (data not shown).

Application of this same rAM depletion protocol to mice challenged with HDM, as indicated on the scheme in Fig. 1A, resulted in increased numbers of eosinophils, macrophages, and total cells in BALF; neutrophils accounted for <3% of total cells in HDM-challenged lungs and did not change with liposomal clodronate (Fig. 1B). As compared to mice treated with empty liposomes, liposomal clodronate-treated mice exhibited significantly increased BALF levels of IL-5, IL-10 and IL-13, and non-significant upward trends in levels of IL-4, IFN-γ, and IL-17 (Fig. 1C–H). Interestingly, in contrast to the pattern observed with all other measured cytokines, asthmatic mice subjected to rAM depletion exhibited decreased TGF-β levels in BALF (Fig. 2A). This finding suggested the possibility that TGF-β was itself produced by rAMs. In this regard, TGF-β elaborated by lung tissue macrophages was recently demonstrated to lead to the generation of T regs, suggesting a new mechanism for dampening asthmatic lung inflammation [21]. However, we observed no reduction in the percentage of T regs among lung cells after rAM depletion, suggesting that the mechanism by which rAMs exert their inhibitory actions is independent of an effect on T reg frequency (Fig. 2B, C).

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.

H&E staining of lung sections of rAM-depleted mice revealed an increase in the area of inflammation, mainly localized around large airways and characterized by infiltration of predominantly mononuclear cells and eosinophils (Fig. 1I–J). PAS staining revealed no significant increase in PAS positive cells in mice subjected to rAM depletion (data not shown). Increased lung inflammation following rAM depletion, as indicated by BALF cytokine levels and cell counts, was confirmed in the OVA model of allergic airway inflammation (Supplemental Fig. 1B–F). Together, data from both of these models support the conclusion that rAMs constrain allergic airway inflammation.

Depletion of rAMs increases recruitment of Ly6C^pos monocytes in allergic lung inflammation

An increase in total BALF AM numbers in association with the potentiation of allergic inflammation following i.t. clodronate in allergen-challenged mice (Fig. 1B) is consistent with our previous finding of AM accumulation in allergic asthma [14]. Expansion of the AM pool could occur by increased recruitment of circulating monocytes and/or increased local proliferation. We addressed the first possibility by measuring Ly6C^highCD11b^pos monocytes – putative precursors of AMs during inflammatory responses [22] – in lung tissue digests of mice with HDM-induced asthma. Depletion of rAMs by i.t. liposomal clodronate resulted in a significantly higher percentage of Ly6C^highCD11b^pos cells in lung tissue (Fig. 3A, B), suggesting a contribution of circulating Ly6C^high monocytes to increased lung inflammation.

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.

I.v. clodronate has been shown to deplete circulating monocytes within 4 h [18] and for a duration of up to 24 h [17, 18]. Upon i.v. clodronate administration no leukocytes other than AMs were detected in BALF of antigen-unchallenged mice, as determined by cytospin differential counts (data not shown). BALF levels of Th2 cytokines, as well as IFN-γ and IL-17, were undetectable in these animals (data not shown). I.v. administration of liposomal clodronate 8 h prior to airway challenge with HDM (Fig. 5A) resulted in almost complete elimination of Ly6C^high monocytes from the lung tissue digests (Fig. 3C, D), establishing the importance of recruitment of Ly6C^high monocytes in allergic asthma.

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

Circulating precursor cells do not contribute to the AM pool in early events in asthma

Having documented monocyte recruitment to the lungs using i.v. clodronate above, we sought to assess the contribution of the circulating progenitor cells to the AM pool by utilizing CD45.1 (donor)-CD45.2 (recipient) chimeras. Chimeric CD45.2 mice were challenged with HDM and the percentages of donor (CD45.1) versus recipient (CD45.2) AMs in BALF, identified as CD11c^pos and autofluorescence (AF)^high, were determined at day 9. BALF at day 9 of our protocol contained two main leukocyte populations, namely eosinophils and AMs, as evidenced by Wright-Giemsa staining of BALF cytospins. AMs were gated based on FSC and SSC to remove cell debris, negative staining for cell viability dye, and positivity for CD11c and AF^high (Fig. 4A), and analyzed for their expression of CD45.1 and CD45.2 markers (Fig. 4B). The percentage of AMs in BALF of donor origin (CD45.1^pos) was similar between asthmatic mice and irradiated non-asthmatic controls (Fig. 4C). This suggests that although recruitment of Ly6C^pos cells to the lung occurs, it does not contribute to accumulation of cells identified as AMs during the early events of asthma, as also demonstrated previously for AMs under steady-state conditions [23].

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.

Depletion of circulating monocytes attenuates allergic lung inflammation

Having established that recruitment of circulating monocytes may not contribute to the accumulation of AMs in asthma, we next sought to interrogate their biologic role in allergic inflammation by using i.v. clodronate depletion before each airway challenge with HDM (Fig. 5A). This resulted in attenuated parameters of allergic asthmatic inflammation, including decreased cytokine levels, including TGF-β (Fig. 5C–I), decreased eosinophil numbers (Fig. 5B), and decreased histologic evidence of inflammatory cell infiltration (Fig. 5J–K). Although levels of IL-13 and IFN-γ were higher in Fig. 5 than in Fig. 1, these data derived from different experiments; when compared directly within the same experiment, administration of control liposomes i.v. vs. i.t. resulted in cytokine levels that were similar and comparable to those in Fig. 1 (data not shown).

Blood monocyte depletion did not affect numbers of total cells, neutrophils, or AMs (Fig. 5B) in BALF, verifying the observations from chimeric mice that recruited monocytes did not contribute to the maintenance of the AM pool. In the OVA model (Supplemental Fig. 2A), depletion of circulating monocytes caused a significant decrease in BALF eosinophil numbers and total leukocytes (Supplemental Fig. 2B), and caused a non-significant reduction in cytokines (Supplemental Fig. 2C–E). H&E staining revealed visible and significant attenuation of inflammatory cell infiltration in mice receiving i.v. liposomal clodronate (Supplemental Fig. 2F and 2G). Taken together, these findings demonstrate for the first time the pro-inflammatory and pathogenic role of circulating monocytes, in opposition to the inhibitory role of rAMs, in allergic lung inflammation.

Proliferation of AMs in allergic asthma

Although tissue macrophages, including AMs, have long been thought to derive predominantly from precursor blood monocytes [24, 25], recent studies have challenged this paradigm by identifying a crucial role for local proliferation in the maintenance of tissue macrophage numbers during both steady-state [26–28] and Th2 inflammatory [29] conditions. In fact, in a helminth infection model of Th2 inflammation, macrophage accumulation in the peritoneum was exclusively attributable to local proliferation [29]. Although AM proliferation has been demonstrated in response to environmental toxins [30, 31] and cytokines [32–34] and in the context of pulmonary sarcoidosis [35], it has never been reported in experimental models of allergic asthma. We utilized staining of the proliferation-associated nuclear protein Ki-67 to assess proliferation of AMs, defined as CD11c^pos SiglecF^pos cells [23], from PBS- or HDM-challenged mice (Fig. 6A). The utility of this index of proliferation was verified by experiments demonstrating over 95% Ki-67 positivity during log-phase growth of monocytic leukemia U937 cells (data not shown). The proportion of proliferating AMs in BALF from control mice was ~10% (Fig. 6B), consistent with a contribution of local proliferation to the pool of rAMs during steady-state conditions. The proportion of proliferating AMs in BALF from asthmatic mice was not significantly different from that from control mice (Fig. 6D). However, when we analyzed AMs from lung digests (Fig. 6C) we did observe a significant increase in the proportion of proliferating cells in asthmatic as compared to control mice (Fig. 6E). This difference from AMs recovered in BALF might reflect the fact that lung digests contained mostly non-lavageable AMs, since most lavageable cells would have been removed by repeated lavage performed prior to digestion. Non-lavageable AMs are presumed to be strongly adherent to epithelial cells, and perhaps more activated [36]. A higher percentage of Ki-67 positivity was also noted in non-lavageable (Fig. 6C) than in lavageable (Fig. 6B) AMs from control mice, which supports the likelihood of intrinsic differences between these two subpopulations. These results suggest a contribution of local proliferation to maintenance of the AM pool during the early inflammatory events in allergic asthma.

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

DISCUSSION

Here we have utilized both HDM and OVA mouse models to interrogate the roles of macrophages in allergic inflammation in the lung. Our first key finding was that depletion of rAMs increased allergic lung inflammation, verifying the protective role of rAMs in models of allergic asthma. In distinct contrast, for the first time we have demonstrated the pathogenic role of circulating monocytes in early allergic inflammation. Finally, we have also for the first time delineated the contributions of recruitment and local proliferation to maintenance of the AM pool in allergic asthma.

In considering the potential mechanisms by which rAMs mediate protection against allergic inflammation, we profiled BALF levels of potentially anti-inflammatory cytokines. While levels of one such inhibitory cytokine, IL-10, were increased following rAM depletion, levels of another, TGF-β, were decreased. Activated AMs secrete TGF-β [37] and a recent study suggested that lung tissue macrophages promote airway tolerance by elaborating TGF-β, which in turn induces T regs [21]. We quantified the frequency of T regs in the lungs of allergen-challenged mice but observed no reduction following rAM depletion. These data suggest that rAMs suppress allergic inflammation by a mechanism independent of T reg generation. Although TGF-β can inhibit both Th1 and Th2 immune responses independent of T reg induction [38, 39], the fact that TGF-β levels in BALF were reduced following elimination of circulating monocytes – an intervention that led to attenuation of asthma – argues for a mechanism not involving TGF-β. A number of years ago, elimination of rAMs was found to up-regulate the capacity of lung DCs to present antigen to T cells [6], and suppressive effects of rAMs on DC-mediated allergic inflammation were recently described in a rat model [40]. The mechanisms underlying rAM-mediated inhibition of DC functions remain to be determined, but this phenomenon may represent an important means of immune control in the lung in a broad variety of circumstances also beyond asthma.

While our findings are consistent with a body of literature demonstrating that depletion of rAMs prior to lung challenge increases pulmonary immune responses [8, 11, 12, 41], contrary results have been reported in studies in which depletion was performed on AMs previously polarized towards an M2 phenotype [10] or during allergic inflammation [9]. These observations suggest that allergic inflammation results in an alteration of AM phenotype characterized by a loss of inhibitory function. The fact that the development of allergic asthma was favored when rAMs were depleted from rats that were allergy-resistant, but not from those that were allergy-susceptible [7], emphasizes the important braking function of AMs on pulmonary immune responses. Interestingly, antigen sensitization per se was sufficient to abrogate this braking function of AM [42], further demonstrating the exquisite plasticity of these cells.

In the present study we also sought to understand how maintenance of the AM pool is controlled in asthma. Although AMs have been demonstrated to proliferate in response to environmental factors [30] or endogenous molecules [32, 33], recent studies have focused on macrophage proliferation in the context of Th2 disease [29]. To address the importance of recruitment in maintenance of the AM pool we utilized a chimera model, since differentiation of recruited and resident macrophages based on markers can be challenging to interpret. These experiments showed that irradiated and subsequently antigen-exposed mice had no greater proportion of circulating precursor cells within their AM pool than did irradiated controls. However, Ki-67 staining demonstrated a role for local proliferation in maintenance of the AM pool. Interestingly, this increase in proliferation of AMs was demonstrable only in cells obtained from lung digests that are enriched in non-lavageable AMs, and not in lavaged AMs. The resistance to recovery of non-lavageable AMs may reflect a greater degree of adherence, owing either to a greater degree of activation and/or preferential localization in inflamed areas of lung. Possible phenotypic differences between lavageable and non-lavageable subpopulations of AMs will require further investigation in future studies. The factor(s) responsible for driving AM proliferation in this context remain to be determined, but candidates might include M-CSF, GM-CSF and IL-3 [32, 33, 43].

In contrast to rAMs, depletion of circulating monocytes revealed their pathogenic role in allergic inflammation, mainly by affecting lung eosinophilia and cytokine levels. Candidate molecules responsible for recruiting monocytes may include chemokines such as CCL2 or CCL5 [44], or the cytokine IL-17, which has previously been implicated in AM accumulation [45]. As noted previously, the fact that BALF TGF-β levels decreased upon monocyte depletion in parallel with those of all other measured cytokines argues against the importance of TGF-β as an inhibitory factor in our asthma model. A pathogenic role of monocytes might be explained by previous observations identifying circulating monocytes from asthmatics as a source of IL-5 [46–48], which is an eosinophil chemoattractant. Finally although our data fail to support that circulating monocytes serve as precursors for AMs, previous studies have shown that blood monocytes can be precursors for lung dendritic cells in both steady-state conditions [49] and in models of allergic asthma [50] and infectious lung disease [51, 52]. Indeed, the importance of monocytes as precursors of CD11b^pos dendritic cells in initiating (by antigen presentation) and maintaining (by chemokine generation) HDM-induced allergic inflammation was recently demonstrated [53]. Therefore in our study we cannot exclude the possibility that increased Ly6C^high monocytes present in the lung upon rAM depletion could cause augmented allergic responses by their potential to differentiate into lung dendritic cells.

Although both asthma models deployed in our study yielded very similar results when rAMs were eliminated, there were some differences upon monocyte depletion. In the HDM model the decrease in lung cytokine levels in mice treated with clodronate i.v. was very substantial, while in the OVA model there was merely a trend towards their diminution. Apart from the obvious difference in the allergen itself, this could reflect a difference in the route of allergen sensitization. We speculate that antigen sensitization in the lung may be associated with a more pronounced role for monocyte recruitment. Whether blockade of AM proliferation would yield similar results as inhibition of recruitment remains an intriguing question. It is alternatively possible that AM proliferation and monocyte recruitment promote different aspects of asthmatic lung inflammation.

Another important issue arising from our findings is how proliferation and possibly recruitment affect AM polarization. In a skin allergy model, Ly6C^pos inflammatory monocytes recruited to a Th2 tissue environment acquired an M2 phenotype and exerted anti-inflammatory functions [54], while CCR2-deficient mice which exhibit impaired recruitment of monocytes had exacerbated inflammation [54]. Interestingly CCR2-deficient mice exhibited enhanced allergic airway responses in two different murine models [55, 56]. Monocytes recruited to the lung have been demonstrated to lose their pro-inflammatory properties with time [57]. Herein we have looked exclusively at early inflammatory responses and we cannot exclude the possibility that depletion of monocytes at later stages of allergic inflammation could yield different results. Alternatively, CCL2-CCR2 signaling may influence other relevant phenomena besides merely recruitment. Defining the relative contributions of monocyte recruitment and AM proliferation in chronic asthma models and investigating the polarization status of recruited versus locally proliferating macrophages will shed additional light on the roles of AMs not only in asthma, but in other forms of allergic lung inflammation.