Cutting Edge: Involvement of the Immunoreceptor CD300c2 on Alveolar Macrophages in Bleomycin-Induced Lung Fibrosis

Yuta Nakazawa, Shigeo Ohtsuka, Chigusa Nakahashi-Oda and Akira Shibuya
J Immunol December 15, 2019, 203 (12) 3107-3111; DOI: https://doi.org/10.4049/jimmunol.1900890

Key Points

  • CD300c2 exacerbates BLM-induced pulmonary fibrosis.

  • CD300c2 on alveolar macrophages enhances neutrophil recruitment.

  • CD300c2 amplifies HMGB-1–mediated TLR4 signaling.

Abstract

Idiopathic pulmonary fibrosis is a chronic, progressive, and irreversible fibrotic lung disease. Although inflammation plays a central role in the pathogenesis of idiopathic pulmonary fibrosis, how inflammatory responses are regulated remains unclear. In this article, we show that mice deficient in the immunoreceptor CD300c2 (also called MAIR-II, LMIR2, and CLM-4) showed longer survival; less collagen deposition in the lung; lower levels of neutrophil chemoattractants, such as TNF-α, CXCL1, and CCL2; and fewer neutrophils in the bronchoalveolar fluid than wild-type mice after intratracheal administration of bleomycin (BLM). We also found that BLM administration induced the release of the danger-associated molecular pattern HMGB-1, which caused CD300c2-deficient alveolar macrophages, via TLR4, to produce lower levels of neutrophil chemoattractants than wild-type alveolar macrophages. Our findings demonstrate that CD300c2 contributes to BLM-induced inflammatory responses mediated by alveolar macrophages.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by progressive and irreversible destruction of the lung structure caused by abnormal scar formation (1). Recent studies suggest that robust inflammation is induced in the lung during both the initiation and fibrotic stages of IPF. In fact, accumulation of inflammatory cells, including neutrophils, monocytes, and lymphocytes, in the lung predicts a worse outcome (2). Proinflammatory cytokines that are involved in neutrophil chemotaxis and activation are elevated in tissue or bronchoalveolar lavage fluid (BALF) isolated from patients with IPF (3, 4) and from mice with bleomycin- (BLM) induced pulmonary fibrosis. However, how inflammation is regulated in pulmonary fibrosis remains poorly understood.

CD300c2 (also called MAIR-II, LMIR2, and CLM-4), which was encoded by AF2051705, is an activating Ig-like receptor expressed on macrophages, monocytes, and a subset of B cells (5–7). CD300c2 has a short cytoplasmic domain and can associate with both FcεRIγ (FcRγ-chain) and DAP12 adaptor proteins, which contain immune tyrosine-based activating motif (ITAM). Interestingly, CD300c2 transmits both an activating signal in myeloid cells and an inhibitory signal in B cells (8–10). MAIR-II–deficient (Cd300c2−/−) mice show significantly decreased monocyte migration in the septic model and less proinflammatory cytokine production in peritoneal macrophages (9, 10). In contrast, Cd300c2−/− B cells showed enhanced proliferation in response to anti-IgM and CpG (8). However, the role of CD300c2 in inflammatory responses in pulmonary fibrosis remains unclear.

In this study, we examined the role of CD300c2 in the inflammation associated with BLM-induced pulmonary fibrosis. We show that CD300c2 enhances high-mobility group box protein-1 (HMGB-1)–induced alveolar macrophage (AM) activation to produce neutrophil chemoattractants, resulting in augmented neutrophil accumulation and inflammation.

Materials and Methods

Mice

BALB/cAJcl mice (age: 8–12 wk) were purchased from Clea Japan (Tokyo, Japan). CD300c2-deficient (Cd300c2−/−) mice on the BALB/c background were bred in our laboratory (9). All procedures were done according to the guidelines of the animal ethics committee of the University of Tsukuba.

BLM-induced pulmonary fibrosis model and HMGB-1–induced inflammation

Mice were anesthetized with isoflurane and given 7.5 mg/kg BLM (Nippon Kayaku, Tokyo, Japan) in saline or saline alone via intratracheal injection. For HMGB-1–induced lung inflammation, mice were injected with 1 mg/kg HMGB-1 intratracheally, and their BALF was harvested 1 d later.

Depletion of AMs

To deplete AMs, mice were injected intratracheally once with clodronate liposomes or PBS liposomes 1 d before BLM administration. Clodronate liposomes were generated as previously described (10).

Abs and flow cytometry analyses

An mAb specific to mouse CD300c2 (TX52) was previously generated in our laboratory (7, 9). TX52 was biotinylated with a Biotinylation Kit (Sulfo-Osu; DOJIN, Tokyo, Japan). mAbs to mouse CD45.2 (104), CD11b (M1/70), CD11c (HL3), SiglecF (E50-2440), CD64 (X54-5/7.1), I-Ad (M5/114.15.2), Ly6G (1A8), CD4 (RM4-5), CD8 (53-6.7), B220 (RA3-6B2), and TNF-α (MP6-XT22) were purchased from BD Biosciences (San Jose, CA); an mAb against CD16/32 (2.4G2) was purchased from Tonbo Biosciences (San Diego, CA). For analyses by flow cytometry, cells were treated for 10 min with anti-CD16/32 mAb to prevent binding to FcγR prior to incubation with indicated Abs. Zombie Violet (BioLegend, San Diego, CA) was used to gate out dead cells. Cell analyses and sorting were performed by an LSR Fortessa cell analyzer and Aria III (BD Biosciences), respectively. Data were analyzed by FlowJo software program (Tree Star, San Carlos, CA). Cytometric bead array analyses for cytokine measurement were performed by an LSR Fortessa cell analyzer.

Cell preparation

To isolate BALF cells, BALF were harvested after washing the trachea with 1 ml of PBS five times through cannulation via a 20-gauge needle and a plastic tube. For induction of bone marrow–derived macrophages (BMDMs), bone marrow cells (5 × 105 per ml) were cultured for 5 d in RPMI medium containing 10% FBS and 40 ng/ml M-CSF (R&D Systems, Minneapolis, MN).

Measurement of cytokines

To measure cytokines in BALF, the trachea was cannulated by using a 20-gauge needle and a plastic tube and washed with 1 ml of PBS. TNF-α, CXCL1, IL-6, and CCL2 concentrations were determined by cytometric bead arrays (BD Biosciences), according to the manufacturer’s protocol. For intracellular cytokine staining, cells in BALF were harvested 1.5 h after 7.5 mg/kg BLM administration and cultured at 1 × 105 cells per 200 μl of RPMI medium containing 10% FBS and 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) for 3 h. Intracellular cytokine was stained by using Fix and Perm Kits (Thermo Fisher Scientific, Waltham, MA) and analyzed by flow cytometry.

Histology and immunohistochemistry

Twenty-eight days after 7.5 mg/kg BLM administration, lung tissues were harvested from anesthetized mice and fixed in 10% neutral buffered formalin. Fixed lungs were embedded in paraffin and stained with Masson trichrome stain. Histopathological evaluations of lung fibrosis were calculated by using established Ashcroft scoring methods (11). All images were acquired by using a BZ-X710 microscope (Keyence, Osaka, Japan). To quantify collagen deposition in lung tissue, whole tissue sections were scanned, and the Masson trichrome stain–positive area (collagen+ area) was automatically measured with hybrid cell counts software by using a BZ-X710 microscope (Keyence). For immunohistochemistry, lung samples were deparaffinized in xylene and rehydrated through graded concentrations of ethanol. To block endogenous HRP, tissue sections were incubated in 0.3% hydrogen peroxidase in methanol for 30 min at room temperature. For Ag retrieval, the specimens were preheated in AR6 buffer (PerkinElmer, Shelton, CT). Samples were incubated with anti–HMGB-1 polyclonal Ab (ab18256) (Abcam, Cambridge, U.K.) for 1 h at room temperature and then incubated with anti-rabbit IgG HRP (BioLegend). The HRP-conjugated dextran polymer system (PerkinElmer) was used for detection. After being washed with TBST, the sections were mounted with DAPI (Vector Laboratories, Burlingame, CA). Tissue sections were scanned by using laser-scanning confocal microscopy (FV10i FLUOVIEW) (Olympus, Waltham, MA).

Stimulation of AMs and BMDMs

AMs were harvested from naive wild-type (WT) and Cd300c2−/− mice. A total of 1 × 105 cells were seeded in 96-well plates and stimulated with 100 ng/ml HMGB-1 (Chondrex, Redmond, WA) in the presence of TAK-242 (Merck Millipore). A total of 1.5 h after stimulation, cells were collected with ISOGEN (Nippon Gene, Tokyo, Japan). For BMDM stimulation, 2 × 105 BMDMs were seeded into plates and stimulated with 250 ng/ml HMGB-1 overnight in the presence of TAK-242.

Quantitative real-time PCR analysis

Total RNA was extracted from AMs. Reverse transcription was performed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). Quantitative PCR was performed with Power SYBER Green PCR Master Mix (Applied Biosystems) by using an ABI 7500 sequence detector (Applied Biosystems). The PCR primers are as follows: tnf forward, 5′-CCTGTAGCCCACGTCGTAG-3′; tnf reverse, 5′-GGGAGTAGACAAGGTACAACCC-3′; cxcxl1 forward, 5′-CTGGGATTCACCTCAAGAACATC-3′; and cxcl1 reverse, 5′-CAGGGTCAAGGCAAGCCTC-3′. Normalization of quantitative real-time PCR was performed based on the gene encoding β-actin.

Statistics

Statistical analysis was performed by using the unpaired Student t test, one-way ANOVA, or two-way ANOVA, followed by Bonferroni multiple comparison test as a post hoc test. A p value <0.05 was considered statistically significant by using Prism software (GraphPad, San Diego, CA).

Results and Discussion

CD300c2-deficient mice are resistant to BLM-induced pulmonary fibrosis

To investigate the role of MAIR-II/CD300c2 in lung fibrosis, we intratracheally administered BLM to WT and Cd300c2−/− mice to induce pulmonary fibrosis. Whereas more than half of the WT mice died by 30 d after BLM administration, all of the Cd300c2−/− mice survived (Fig. 1A). Histological analysis demonstrated that WT mice exhibited greater collagen deposition in the lung compared with Cd300c2−/− mice on day 28 after BLM administration (Fig. 1B–D). These results suggest that CD300c2 exacerbates BLM-induced pulmonary fibrosis.

FIGURE 1.
FIGURE 1.

Cd300c2−/− mice are resistant to BLM-induced pulmonary fibrosis. WT and Cd300c2−/− mice were treated intratracheally with BLM or saline. (A) Survival curve of WT and Cd300c2−/− mice treated with BLM (n = 12 and 14, respectively) or saline (n = 5 in each group). (B) Masson trichrome staining (representative of two independent experiments). Scale bar, 100 μm. (C and D) Ashcroft score (C) and collagen depositions (D) of WT and Cd300c2−/− mice 28 d after administration of BLM [(C) n = 5 in each group; (D) n = 12 and 14, respectively] or saline [(C) n = 4 in each group; (D) n = 7 in each group]. Data were pooled from two independent experiments (A, C, and D). A Kaplan-Meier log-rank test was performed to determine significant differences between each group. Values were represented as the mean ± SEM. The p values were obtained from a two-way ANOVA followed by the Bonferroni posttest (C and D). *p < 0.05, ***p < 0.001.

CD300c2 enhances cytokine and chemokine production for neutrophil recruitment

Accumulating evidence from clinical and animal studies of lung fibrosis indicates that inflammatory responses precede and correlate with collagen deposition (1). We therefore analyzed the levels of proinflammatory cytokines and chemokines in BALF. Cd300c2−/− mice showed significantly lower levels of TNF-α than WT mice as well as CXCL1 and CCL2 in BALF just 1 and 3 d, respectively, after BLM administration (Fig. 2A). Consistent with these results, the number of neutrophils was significantly decreased in Cd300c2−/− mice at 1 and 3 d after BLM administration (Fig. 2B, 2C). These data indicate that CD300c2 enhances the production of neutrophil chemoattractants after BLM administration and exacerbates pulmonary fibrosis.

FIGURE 2.
FIGURE 2.

CD300c2 amplifies production of chemoattractants for neutrophils after BLM administration. (A) Cytokine concentrations in BALF of WT and Cd300c2−/− mice on days 0 (n = 5 in each group), 1 (n = 9 in each group), 3 (n = 8 and 10, respectively), 7 (n = 14 and 13, respectively), and 14 (n = 6 and 7, respectively) after BLM administration. (B) Gating strategy for flow cytometry analysis of BALF cells on day 3 after BLM administration. (C) Immune cell numbers in BALF of WT and Cd300c2−/− mice on day 0 (n = 5 in each group), 1 (n = 9 in each group), 3 (n = 9 and 10, respectively), 7 (n = 12 and 8, respectively), and 14 (n = 9 and 10, respectively) after BLM administration. Values were represented as the mean ± SEM. The p values were obtained from a two-way ANOVA followed by the Bonferroni posttest. All data were pooled from at least two independent experiments. *p < 0.05, ***p < 0.001. DC, dendritic cell; IM, interstitial macrophage; Lym, lymphocyte; Mo, monocyte; Neu, neutrophil.

CD300c2 on AMs is responsible for pulmonary fibrosis

To analyze how neutrophil numbers are reduced in the BALF of Cd300c2−/− mice after BLM administration, we analyzed the CD300c2 expression on immune cells in the BALF by using flow cytometry (12). We found that CD300c2 was expressed on AMs, interstitial macrophages, CD11b+ dendritic cells, and monocytes in BALF before and 3, 7, and 14 d after BLM administration, in which the expressions of CD300c2 were stable during the course (Supplemental Fig. 1). In contrast, CD11b− dendritic cells, eosinophils, neutrophils, or CD11b− CD11c− cells, including B and T lymphocytes, did not express CD300c2 (Supplemental Fig. 1). In patients with IPF, large amounts of neutrophil chemotactic factors are secreted by AMs, which correlates with the increased proportion of neutrophils in BALF (13). To clarify whether CD300c2 on AMs is involved in neutrophil accumulation, we intratracheally administered clodronate liposomes, which leads to the depletion of AMs, but not other myeloid cells, for more than 4 d after BLM administration (Supplemental Fig. 2A–C). AM depletion followed with BLM administration decreased BLM-induced lung fibrosis in WT mice to the comparable level of Cd300c2−/− mice (Fig. 3A, 3B). Moreover, depletion of AMs also decreased neutrophil accumulation in the BALF of WT mice to the comparable level of Cd300c2−/− mice at 1 d post-BLM administration (Fig. 3C). When we isolated AMs 2 h after BLM administration and examined the production of chemoattractants by using quantitative RT-PCR, we found that tnf and cxcl1 expression in Cd300c2−/− AMs were significantly decreased compared with that in WT AMs (Fig. 3D). Consistently, the proportion of TNF-α–expressing AMs in BALF was greater in WT mice than in Cd300c2−/− mice 4.5 h after BLM administration (Fig. 3E). These results indicate that CD300c2 on AMs accelerates pulmonary fibrosis by increasing the production of chemoattractants and recruitment of neutrophils.

FIGURE 3.
FIGURE 3.

CD300c2 on AMs augments neutrophil recruitment and lung fibrosis. (A–C) WT and Cd300c2−/− mice were once injected intratracheally with clodronate (Cl2MBP) or PBS liposomes 1 d before (on day −1) intratracheal administration of BLM. Masson trichrome staining (representative of two independent experiments) (A) and Ashcroft score (n = 5 in each Cl2MBP liposome group and n = 4 in each PBS liposome group) of lung tissues 28 d after BLM administration (B) and neutrophil numbers in BALF of WT (n = 8 in both groups) and Cd300c2−/− (n = 5 and 6 in Cl2MBP and PBS liposome groups, respectively) mice 1 d after BLM administration (C). Scale bar, 100 μm. (D) Quantitative RT-PCR analysis of tnf and cxcl1 in AMs from WT and Cd300c2−/− mice before and 2 h after administration of BLM (n = 4 in each group). (E) Flow cytometric analysis of TNF-α expression by WT and Cd300c2−/− AMs before (n = 4 in each group) and 4.5 h after (n = 6 and 5, respectively) BLM administration. Values were represented as the mean ± SEM. The p values were obtained from one-way ANOVA (B and C) and a two-way ANOVA, followed by the Bonferroni posttest (D and E). All data were pooled from at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., not significant.

CD300c2 enhances HMGB-1– and TLR4-mediated inflammatory responses

BLM is used as an anticancer drug because it induces cell death through reactive oxygen species production. These dead cells release several danger-associated molecular patterns, such as HMGB-1, from nuclei into the cytosol and extracellular spaces, causing sterile inflammation (14). Elevated HMGB-1 levels in BALF have been reported in IPF patients, and altered localization of HMGB-1 has been detected in bronchiolar epithelial cells after intratracheal BLM administration (15). Indeed, we observed that HMGB-1 was released from the nuclei into the cytosol of the bronchiolar epithelial cells of both WT and Cd300c2−/− mice 1 h after BLM administration, whereas HMGB-1 remained localized in the nuclei in PBS-treated mice (Fig. 4A). We therefore hypothesized that CD300c2 enhances the production of neutrophil chemoattractants from AMs induced by HMGB-1 stimulation after BLM administration. Intratracheal administration of HMGB-1, instead of BLM, to Cd300c2−/− mice led to less neutrophil accumulation and lower levels of TNF-α and CXCL1 in BALF than in WT mice 1 d after treatment (Fig. 4B, 4C). When we isolated AMs from the BALF of naive WT and Cd300c2−/− mice and stimulated the cells therein with HMGB-1, we found that the expression of tnf and cxcl1 was strongly upregulated in WT compared with Cd300c2−/− mouse-derived AMs (Fig. 4D). Because HMGB-1 binds to TLR4 on macrophages (16), we investigated whether CD300c2 amplifies TLR4-mediated signaling. When we inhibited TLR4-mediated signaling by using the specific TLR4 inhibitor TAK-242, BMDMs and AMs from WT and Cd300c2−/− mice produced comparable levels of TNF-α and tnf expression, respectively (Fig. 4E, 4F). Together these results indicate that CD300c2 amplifies the danger-associated molecular pattern–TLR4–mediated signal to produce the cytokines and chemokines that recruit neutrophils.

FIGURE 4.
FIGURE 4.

CD300c2 positively regulates HMGB-1– and TLR4-mediated inflammatory responses. (A) Fluorescence microscopy analyses of lung tissues stained with anti–HMGB-1 Ab from WT and Cd300c2−/− mice 1 h after BLM or PBS treatment. Scale bar, 10 μm. White arrows indicate the aberrant localization of HMGB-1 out of the nuclei. Images were representative of two independent experiments. (B and C) Neutrophil numbers (B) and concentrations of TNF-α and CXCL1 (C) in BALF from WT and Cd300c2−/− mice (n = 5 in each group) 1 d after intratracheal administration of HMGB-1. (D) Quantitative RT-PCR analyses of tnf and cxcl1 in AMs from WT and Cd300c2−/− mice, before (n = 3 and 4, respectively) and 1.5 h after stimulation with HMGB-1 (n = 4 and 5, respectively). (E) Concentrations of TNF-α in culture of WT and Cd300c2−/− BMDMs after stimulation with HMGB-1 for 18 h in the presence or absence of TLR4 inhibitor (TAK-242) (n = 5 in each group). (F) Quantitative RT-PCR analyses of tnf expression in WT and Cd300c2−/− AMs before (n = 3 in each group) and after stimulation with HMGB-1 in the presence (n = 5 and 6, respectively) or absence (n = 5 in each group) of TAK-242. Values were represented as the mean ± SEM. All p values were obtained from a two-way ANOVA, followed by the Bonferroni posttest. All data were pooled from at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

We previously reported that, in a sepsis model of cecum ligation and puncture, CD300c2 on inflammatory monocytes (iMo) associates with TLR4 and activates VLA-4 to induce iMo transmigration from the blood to the sites of infection and ameliorates inflammation because of enhanced phagocytosis of bacteria (10). In contrast, iMo seem not to be involved in the pathology of the BLM-induced nonsterile lung inflammation model because very low numbers of iMo were recruited into the lung after BLM treatment. Rather, CD300c2 on AMs exacerbated lung inflammation through enhanced production of chemoattractants for neutrophils that lead to sterile inflammation. Regulation of AM activation by targeting CD300c2 may be a potential therapeutic approach for lung fibrosis.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank S. Tochihara and W. Saito for secretarial assistance and F. Abe and R. Hirochika for technical assistance.

Footnotes

  • This work was supported by Japan Society for the Promotion of Science (KAKENHI) Grants 18H05022 and 16H06387 (to A.S.) and 19H03776 and 16H05350 (to C.N.-O.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AM
    alveolar macrophage
    BALF
    bronchoalveolar lavage fluid
    BLM
    bleomycin
    BMDM
    bone marrow–derived macrophage
    HMGB-1
    high-mobility group box protein-1
    iMo
    inflammatory monocyte
    IPF
    idiopathic pulmonary fibrosis
    WT
    wild-type.

  • Received July 25, 2019.
  • Accepted October 25, 2019.

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