DAP12 is required for macrophage recruitment to the lung in response to cigarette smoke and chemotaxis toward CCL2

Abstract

DAP12 is an adapter protein that associates with several receptors in macrophages. Little is known about the biological role of DAP12 in alveolar macrophages. In genome-wide profiling, we previously found that two DAP12-associated receptors, myeloid DAP12-associated lectin-1 and triggering receptor expressed on myeloid cells 2 (TREM2), were highly induced in alveolar macrophages from habitual smokers. Here, we found that transcript levels for these receptors in alveolar macrophages increased with packs per day of cigarettes smoked and expression of TREM2 protein was increased in lung macrophages of former smokers with emphysema compared with that in controls. In vitro, cigarette smoke directly induced expression of myeloid DAP12-associated lectin-1 and TREM2 and activation of DAP12 signaling in mouse macrophages. To determine whether DAP12 plays a role in cigarette smoke-induced pulmonary inflammation, we exposed wild-type and DAP12-deficient mice to chronic cigarette smoke and found significant reduction in recruitment of alveolar macrophages in DAP12-deficient mice. Because cigarette smoking induces the macrophage chemoattractant CCL2, we tested the chemotactic ability of DAP12-deficient macrophages and found abrogation of chemotaxis toward CCL2 in vitro. Airway administration of CCL2 also resulted in a significant reduction of macrophage recruitment to the lungs of DAP12-deficient mice compared with that in controls. DAP12 was also required for normal macrophage migration in a "scratch" assay. Reconstitution studies revealed that phosphorylation of the DAP12 ITAM was required for normal migration in vitro and association with TREM2 was sufficient for normal migration. These findings indicate that DAP12, possibly through association with TREM2, contributes to alveolar macrophage chemotaxis and recruitment to the lung and may mediate macrophage accumulation in lung diseases such as emphysema.

FIGURE 1

Habitual cigarette smoking leads to a dose-related increase in expression of DAP12-associated receptors in alveolar macrophages. A and B, Alveolar macrophages were isolated by bronchoscopy and purified by flow cytometry (12). Total RNA was isolated and mRNA transcript levels for CLEC5A (MDL-1) and TREM2 were measured by quantitative RT-PCR. Transcript levels for both receptors were increased in smokers’ macrophages compared with those of nonsmokers (A, B, left panel) and increased in proportion to the numbers of packs per day smoked (A, B, right panel). *p < 0.0001; *p < 0.001 using nonparametric trend test, which tests stepwise increase in each group. Data are expressed as normalized transcript copy number; bars represent means. n = 15 for NS and 12 for S. C, Lung tissue sections were stained for TREM2 protein using anti-TREM2 Ab or isotype-matched control Ig, and macrophage staining intensity was independently scored by two blinded investigators. Data are expressed as staining intensity ± SE. n = 8 healthy subjects and 27 former smokers with COPD. We did not find significant associations of staining intensity with COPD severity (as reported by the Lung Tissue Research Consortium). *p < 0.005. NS, nonsmokers; S, smokers.

FIGURE 2

Constituents of cigarette smoke increased MDL-1 and TREM2 surface expression on macrophages and phosphorylated DAP12 in vitro. RAW264.7 cells were exposed to either control media or 50 μg/ml cigarette smoke-conditioned media for 24 h. A and B, Cells were analyzed by flow cytometry for (A) MDL-1 and (B) TREM2 surface expression. Data are displayed as flow cytometric histogram plots with gray line representing isotype-matched control Ab, black filled histogram representing MDL-1 or TREM2 Ab, and black line representing smoke media plus MDL-1 or TREM2 Ab. Data are representative of three independent experiments. C and D, RAW264.7 cells expressing an HA epitope-tagged DAP12 were exposed to C media or 250 μg/ml CS-conditioned media for 60 min. Lysates were immunoprecipitated with anti-HA Ab, and immunoprecipitates were analyzed by SDS-PAGE. Membranes were blotted with PY, then stripped and reblotted with (C) anti-DAP12 Ab as a loading control or (D) anti-SYK Ab (showing increased coimmunoprecipitation of SYK with phospho-DAP12). Data are representative of three independent experiments. C, control; CS, cigarette smoke; PY, phosphotyrosine Ab.

FIGURE 3

DAP12 is required for normal recruitment of macrophages to the lung in response to cigarette smoke. Wild-type and DAP12-deficient (Tyrobp^−/−) mice were exposed to 3 mo of SHS, and numbers of BAL cells were quantified (n = 16 per group). Data are expressed as cell counts per milliliter of lavage ± SE. SHS, secondhand cigarette smoke.

FIGURE 4

Abnormal chemotaxis and recruitment of DAP12-deficient macrophages to CCL2 in vitro and in vivo. A, Chemotaxis of wild-type and DAP12-deficient BMMs was assessed using Transwells containing culture medium with or without CCL2. Data are expressed as mean ± SE of nine replicates for each condition (three independent experiments, each done in triplicate). *p < 0.001 compared with wild-type and DAP12-deficient controls; ϕp < 0.001 compared with wild-type cells exposed to chemoattractants and p > 0.8 compared with DAP12-deficient control. B, Wild-type and DAP12-deficient mice (Tyrobp^−/−) were given CCL2 intranasally over 2 wk, and numbers of BAL cells were quantified (n = 6–7 per group). Data are expressed as cell counts per milliliter of lavage ± SE.

FIGURE 5

DAP12-deficient BMMs demonstrated significantly reduced migration in an in vitro scratch assay. A and B, Macrophages were cultured to confluence and incubated with or without pertussis toxin as described in Materials and Methods. Macrophage monolayers were “wounded” by scratching with the tip of a pipette. Serial images of cells were taken immediately (time 0) and at the indicated times after “wounding.” Images of the scratch area are shown (A), and macrophage migration into the scratch area is quantified (B). Data in B are expressed as numbers of macrophages migrating into scratch area (defined as counts at given time point minus counts at time 0). Data are representative of three independent experiments. p < 0.001 compared with other conditions at same time point adjusted for multiple comparisons. Ptx, pertussis toxin-treated; WT, wild type.

FIGURE 6

Reconstitution of DAP12-deficient macrophages with DAP12 mutants reveals a role for TREM2 and phosphorylation of DAP12 ITAM in migration in vitro. Wild-type and DAP12-deficient macrophages were transduced with retroviruses containing a vector with one of the following mutants: empty vector (cont), wild-type DAP12 (+DAP12), DAP12 Y65,76F tyrosine ITAM mutations (Y-F), DAP12 D52A transmembrane mutation (TM), and TREM2/DAP12 chimera containing a DAP12 transmembrane mutation (TREM2 TM). Migration of cells was quantified in the scratch assay. Data are expressed as numbers of macrophages migrating into scratch area (defined as counts at given time points minus counts at time 0). Transduction efficiency was determined by flow cytometry to be ~60–70% for each condition. Data are representative of three independent experiments. *p < 0.0001 compared with Tyrobp^−/− control, Y-F and TM and p value was not statistically significantly different compared with Tyrobp^−/− +DAP12 at the same time point adjusted for multiple comparisons; **p not statistically significant compared with Tyrobp^−/− +DAP12.