Upregulation of leukocyte immunoglobulin-like receptor B4 on interstitial macrophages in COPD; their possible protective role against emphysema formation

This study included 51 patients who received surgery for lung cancer in Ishinomaki Red Cross Hospital and Tohoku University Hospital. Patients with respiratory disease other than COPD were excluded. The diagnosis of COPD was determined based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (https://​goldcopd.​org). The patients were divided into three groups: non-smokers, non-COPD smokers, and COPD patients. We used the Goddard score for the assessment of low attenuation areas (LAA) [22]. This study was approved by the Ethics Committee at Tohoku University School of Medicine (2017–1-352). Written informed consent was obtained from all patients.

We prepared human single-cell suspension as previously described with some modification [23‐26]. Minced lung tissues were incubated with Hanks Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA, USA) containing 1.5 mg/ml Collagenase A (Sigma-Aldrich, St. Louis, MO) and 2000 KU/ml DNase I (Sigma-Aldrich) at 37 °C for 45 min, then minced again with scissors and incubated at 37 °C for 45 min. Single-cell suspensions were filtered with a 100 µm cell strainer (BD biosciences) twice and red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Thermo Fisher Scientific). Cells were resuspended in RPMI 1640 medium (containing l-glutamine and 25 mM HEPES; Thermo Fisher Scientific) with 5% fetal bovine serum and 2% penicillin–streptomycin-amphotericin B suspension (100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml, amphotericin B; FUJIFILM Wako Chemicals, Osaka, Japan) and filtered twice with 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Cell numbers were calculated using trypan blue staining and analyzed by flow cytometry.

C57BL/6 mice were purchased from Charles River Laboratories Japan (Yokohama, Japan) and 7 to 10-week-old female mice were used. LILRB4-deficient mice (gp49B^−/−) with the B6 background were previously established [27]. All mice were maintained and bred in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, under specific pathogen-free conditions. All animal protocols were reviewed and approved by the Animal Studies Committee of Tohoku University.

A mouse model of elastase-induced emphysema was prepared as previously described [26]. Briefly, mice were anesthetized with isoflurane temporarily and were given an intranasal instillation of 3 units porcine pancreatic elastase (FUJIFILM Wako Chemicals) in 50 µl of PBS or 50 µl of PBS alone. Analysis of macrophages was conducted on day 7 and histological examination and CT scan were conducted on day 21.

Mice were injected intraperitoneally with triple mixed anesthesia of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). After euthanization by cutting the aorta, we inserted a 20-gauge needle into the trachea. Bronchoalveolar lavage fluid (BALF) was collected by washing the lungs three times with 1 ml of PBS. BALF was centrifuged at 300 rpm for 5 min and the supernatant was used for cytokine analysis. Cells were resuspended in PBS and cell counts were determined by a hemocytometer. Cell fractionation was calculated by cytospin slides stained by Diff-Quick method.

Mouse single-cell suspensions were prepared as previously described with some modification [28]. Lungs chopped with scissors were incubated at 37 °C for 45 min in RPMI solution containing 50 µg/ml Liberase TM (Roche, Basel, Switzerland) and 10 µg/ml DNase I (Roche). The lung tissue was passed through a 40 µm cell strainer. After centrifugation, the cell pellets were resuspended in ACK lysis buffer (Thermo-Fischer Scientific) and incubated to remove red blood cells. The samples were washed with PBS and resuspended in the staining buffer for flow cytometric analysis.

Flow cytometry was performed as previously described with some modification [24, 29]. LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) was added to single-cell suspensions and incubated at 4 °C for 30 min. After resuspension in FACS buffer containing PBS with 0.1% sodium azide and 2% FBS, human FcR Blocking Reagent (Miltenyi Biotec) in the case of human and anti-CD16/32 mouse antibody in the case of mouse were added to prevent non-specific staining, then incubated 4 °C for 5 min. Based on previous reports [30‐32], we defined human alveolar macrophages as FSC^highCD45⁺CD206⁺CD14⁻ cells, human interstitial macrophages as FSC^midCD45⁺CD206⁺CD14⁺ cells, mouse alveolar macrophages as CD45⁺Ly6G⁻CD64⁺CD24⁻CD11b^intCD11c⁺ cells and mouse interstitial macrophages as CD45⁺Ly6G⁻CD64⁺CD24⁻CD11b⁺CD11c⁻ cells. Data were collected by LSR Fortessa (BD Bioscience) and analyzed by FCS express 6 software (De Novo Software, Glendale, CA). Cell sorting was performed using FACS Aria II (BD Biosciences). We utilized fluorescence minus one controls to distinguish the positive population from the negative one.

Brilliant Violet 421-conjugated anti-human CD45 (HI30), APC-conjugated anti-human CD3 (OKT3), APC-conjugated anti-human CD19 (HIB19), FITC-conjugated anti-human CD14 (M5E2), CD11c-conjugated anti-human CD11c (3.9), PE-Cy7-conjugated anti-human HLA-DR (LN3), PerCP-Cy5.5-conjugated anti-human CD56 (5.1H11), PE-conjugated anti-human LILRB4 (ZM4.1), Pacific blue-conjugated anti-mouse CD45 (30-F11), Brilliant violet 510-conjugated anti-mouse CD11b (M1/70), PerCP-Cy5.5-conjugated anti-mouse CD11c (N418), FITC-conjugated anti-mouse Ly6G (1A8), PE-Cy7-conjugated anti-mouse CD64 (X54-5/7.1), APC/Fire 750-conjugated anti-mouse CD24 (M1/69), APC-conjugated anti-mouse IA/IE (M5/114.15.2), PE-conjugated anti-mouse LILRB4 (H1.1), PE-conjugated American Hamster IgG control antibody (HKT888) were purchased from Biolegend. APC conjugated anti-human CD206 (19.2), APC-conjugated mouse IgG₁κ control antibody (P3.6.2.8.1), PE-conjugated mouse IgG₁κ control antibody (P3.6.2.8.1) were purchased from eBiosciences.

The lung was refluxed by injecting PBS from the right ventricle. The 10% neutral buffered formalin was injected from the trachea at a pressure of 30 cmH₂O and the lung was fixed for 24 h. We entrusted the production of paraffin-embedded sections to Experimental Animal pathology Platform Section, Tohoku University. Emphysematous changes were evaluated by the mean liner intercept (MLI) [33].

Under 2% isoflurane anesthesia, mouse chest CT scans were performed using an X-ray CT system for laboratory animals (LaTheta LCT-200; Hitachi Aloka Medical Ltd., Tokyo, Japan). Calibration was carried out according to the manufacturer’s protocol. The CT value of air was set to -1000 Housefield Units (HU), and water was set to 0 HU. Data were converted to DICOM files and analyzed by LaTheta software (version 3.22) and Image J software (National Institutes of Health, Frederick, MD). The quantitative evaluation of emphysema was performed using the percentage of low attenuation area, which was defined as the area from − 871 to − 610 HU [34].

RNA was extracted from sorting cells using RNeasy Micro Kit (Qiagen, Valencia, CA) and from lung tissue using RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized by the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Quantitative PCR was conducted on StepOne Plus (Thermo Fisher Scientific) using SYBR Premix Ex Taq (TaKaRa, Kusatsu, Japan).

The levels of mRNA expression were evaluated by the comparative CT method and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control gene. The reference sample was one of the PBS-treated control samples or wild-type control samples. Primer sets were as follows: for mouse IL-10: forward, 5′-GCCAGAGCCACATGCTCCTA-3′, and reverse, 5′-GATAAGGCTTGGCAACCCAAGTAA-3′; for mouse IL-1β: forward, 5′-TCCAGGATGAGGACATGAGCAC-3′, and reverse, 5′-GAACGTCACACACCAGCAGGTTA-3′; for mouse Mmp12: forward, 5′-CTCTAGCCAGCACATGACTCCAA-3′, and reverse, 5′-CTGATGTGAAATGAGCCACACAAC-3′; for mouse TNF-α: forward, 5′-ACTCCAGGCGGTGCCTATGT-3′, and reverse, 5′-GTGAGGGTCTGGGCCATAGAA-3′; for mouse GAPDH: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, and reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′.

Because which types of leukocytes expressed LILRB4 in human lung were not clarified, we first examined the expression of LILRB4 on each type of leukocyte in single cell suspensions of normal lung tissue from the patients who received pneumonectomy for lung cancer. Among leukocytes, LILRB4 was clearly expressed on dendritic cells, monocytes, alveolar macrophages (AMs) and interstitial macrophages (IMs) (Fig. 1a–c). We then tried to examine whether the expression of LILRB4 was changed according to the smoking status and prevalence of COPD. We focused on monocytes and macrophages because it has been reported that these cells, especially lung macrophages, are involved in the pathogenesis of COPD [1]. We also had to exclude dendritic cells from further analyses because they were a relatively rare cell population and we could not harvest enough cells from every lung tissue sample for analyses. We compared LILRB4 expression on monocytes, AMs and IMs among non-smokers, non-COPD smokers, and COPD patients. The clinical characteristics of the human studies are shown in Table 1.

The percentage of LILRB4-positive cells in IMs of the COPD patients was significantly higher than in those of non-smokers and non-COPD smokers (Figs. 1d and 2a). On the other hand, there were no significant differences in the percentages of LILRB4-positive cells in lung AMs and monocytes between the patient groups (Fig. 2a).

We next analyzed whether the percentage of LILRB4-positive cells in IMs was correlated with the patients’ characteristics including lung function and severity of emphysematous lesions. The percentage of LILRB4-positive cells in IMs was significantly associated with the FEV₁/FVC ratio and percent predicted FEV₁ (%FEV₁) (Fig. 2b). The percent predicted DLCO/VA (%DLCO/VA) had a significant negative correlation with the percentage of LILRB4-positive cells in IMs (Fig. 2b). We then used the Goddard score to examine the severity of emphysematous lesions by analyzing low attenuation areas on CT images and their correlation with the existence of LILRB4-positive IMs. The severity of emphysematous lesions was well correlated with the percentage of LILRB4-positive cells in IMs (Fig. 2c). The percentage of LILRB4-positive cells in IMs did not significantly correlate with the smoking amount or age (Fig. 2c). These results suggested that the severity of emphysematous lesions was correlated with the accumulation of LILRB4-positive IMs.

The correlation between the severity of emphysematous lesions and the accumulation of LILRB4-positive IMs in human lungs suggested that LILRB4 could have a role in the pathogenesis of emphysema. We then decided to elucidate whether LILRB4 was involved in the pathogenesis of emphysematous lesions. To answer this question, we utilized LILRB4-deficient (LILRB4^−/−) mice and analyzed an elastase-induced emphysema mouse model. We first examined the LILRB4 expression on leukocytes in a mouse lung single cell suspension prepared by enzymatic digestion. In the mouse lungs, LILRB4 was also expressed on neutrophils, natural killer cells, and eosinophils, in addition to macrophages, monocytes and dendritic cells (Fig. 3a, b). We distinguished IMs from AMs by cell surface markers as previously reported [31] and examined the expression of LILRB4 on mouse IMs, finding that IMs expressing LILRB4 also existed in mouse lungs (Fig. 3a, c). We further examined the change of expression of LILRB4 on IMs in an elastase-induced emphysema mouse model. As in the lungs of human COPD patients, the percentage of LILRB4-positive cells in IMs significantly increased after the administration of elastase (Fig. 3c).

We then examined the emphysema formation and the accumulation of inflammatory cells in the airway of the elastase-induced emphysema model. We measured both the mean linear intercept (MLI) by histopathological analysis and the percentage of the low attenuation area (LAA) in the lung field by CT at 21 days after the elastase administration. We found that both indicators, MLI and LAA, were significantly higher in LILRB4^−/− mice than to those in wild-type mice, indicating that the emphysematous lesion was exacerbated by the deficiency of LILRB4 (Fig. 4a–d). These data suggested that LILRB4 may have a protective function against emphysema formation in the elastase-induced mouse model.

We also examined inflammatory cell accumulation in the airway because it was possible that the exacerbated emphysema in LILRB4^−/− mice might have been due to the exacerbated inflammation induced by elastase. We performed BAL on day 7 and examined the number of the cells in BAL fluids. The numbers of total cells, as well as macrophages and neutrophils, were significantly elevated in the BAL fluid by elastase administration in both the wild-type and LILRB4^−/− mice. However, there were no significant differences in the cell counts between the wild-type and LILRB4^−/− mice (Fig. 5a–c). We further examined the levels of mRNA expression from pro-inflammatory cytokines (IL-1β and TNF-α) and an anti-inflammatory cytokine (IL-10) in the whole lungs of an emphysema model. The levels of IL-1β and TNF-α, but not those of IL-10, were significantly increased by the administration of elastase (Fig. 6a). In an elastase-induced emphysema model, the level of IL-10, but not those of inflammatory cytokines, was higher in LILRB4^−/− mice compared with wild-type mice (Fig. 6b). We also examined isolated lung IMs to determine the levels of cytokine mRNA because it has been reported that lung IM is a major producer of IL-10 [35]. Isolated IMs from elastase administered LILRB4^−/− mice showed similar levels of IL-10 and TNF-α, although they showed a higher level of IL-1β (Fig. 6c). These data suggested that the exacerbation of emphysema in LILRB4^−/− mice was not due to the aggravation of inflammatory cell accumulation and inflammatory cytokine production.

To explore the cause of the exacerbated emphysematous lesions in LILRB4^−/− mice, we focused on matrix metalloprotease 12 (MMP-12). MMP-12 is a protease associated with emphysema that is elevated in the sputum and BAL of COPD patients [36‐39]. In animal models, emphysema is not induced by smoking stimulation in MMP-12 knockout mice [40]. A recent investigation has revealed that IM rather than AM is the major producer of MMP-12 in lungs [41]. We examined the expression level of MMP-12 mRNA in whole lungs. The expression level of MMP-12 mRNA in the whole lung of LILRB4^−/− mice in the emphysema model was significantly higher than that in wild-type mice (Fig. 7a). We then isolated IMs from digested lungs of the emphysema model and examined the levels of MMP-12 mRNA. LILRB4^−/− IMs had significantly higher levels of MMP-12 mRNA than wild-type IMs (Fig. 7b). These results suggested that the deficiency of LILRB4 enhanced the upregulation of MMP-12 by IMs in the lungs of the emphysema model, which may have caused the exacerbation of emphysematous lesions.