A Critical Role for ABCG1 in Macrophage Inflammation and Lung Homeostasis Allison J. Wojcik, Marcus D. Skaflen, Suseela Srinivasan and Catherine C. Hedrick This information is current as of September 27, 2021. J Immunol 2008; 180:4273-4282; ; doi: 10.4049/jimmunol.180.6.4273 http://www.jimmunol.org/content/180/6/4273 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Critical Role for ABCG1 in Macrophage Inflammation and Lung Homeostasis1

Allison J. Wojcik,*† Marcus D. Skaflen,† Suseela Srinivasan,† and Catherine C. Hedrick2*†‡

ATP-binding cassette transporter G1 (ABCG1) effluxes cholesterol from macrophages and plays an important role in pulmonary -lipid homeostasis. We hypothesize that macrophages from Abcg1؊/؊ mice have increased inflammatory activity, thereby promot ing acceleration of pulmonary disease. We herein demonstrate increased numbers of inflammatory cytokines and infiltrating neutrophils, eosinophils, dendritic cells, T cells, and B cells into lungs of Abcg1؊/؊ mice before the onset of severe lipidosis. We further investigated the role of macrophages in causing pulmonary disease by performing bone marrow transplantations using B6 and Abcg1؊/؊ bone marrow. We found that it was the macrophage, and not pneumocyte type II cells or other nonhematopoietic cells in the lung, that appeared to be the primary cell type involved in the onset of both pulmonary lipidosis and inflammation in ؊/؊ ؊/؊ the Abcg1 mice. Additionally, our results demonstrate that Abcg1 macrophages had elevated proinflammatory cytokine Downloaded from production, increased apoptotic cell clearance, and were themselves more prone to apoptosis and necrosis. However, they were quickly repopulated by monocytes that were recruited to Abcg1؊/؊ lungs. In conclusion, we have shown that ABCG1 deletion in macrophages causes a striking inflammatory phenotype and initiates onset of pulmonary lipidosis in mice. Thus, our studies reveal a critical role for macrophage ABCG1 in lung inflammation and homeostasis. The Journal of Immunology, 2008, 180: 4273–4282.

acrophages are involved in maintaining cholesterol Alveolar pulmonary surfactant is composed of phospholipids http://www.jimmunol.org/ homeostasis and regulating inflammation in the lung. and surfactant proteins (SP)3 and is essential for maintenance of M There are different populations of macrophages that normal lung function (11). Surfactant associated with SP-B or play distinct roles in the lung. Alveolar macrophages are localized SP-C functions to maintain a low surface tension for alveolar in- in the alveolar space, and tissue macrophages are located within flation and normal gas exchange in the lung (12). Surfactant as- the alveolar and airway walls (1, 2). Alveolar macrophages release sociated with SP-A or SP-D is involved in innate immunity and the more proinflammatory mediators and demonstrate increased clearance of apoptotic cells (13, 14). Saturated phosphatidylcho- phagocytosis, cytotoxicity, and release of reactive oxygen species line, the most abundant phospholipid in surfactant, is synthesized compared with tissue macrophages (3). Tissue macrophages are and secreted by type II cells. Surfactant is taken up equally by both by guest on September 27, 2021 more specialized in immune responses and immunoregulation, as alveolar macrophages and type II cells. Alveolar macrophages ca- seen by their increased Ag presentation and secretion of IL-1 and tabolize all surfactant, but type II cells both degrade and recycle IL-6 (4–6). surfactant (15). There are many pulmonary diseases associated Macrophages play a crucial role in innate and acquired immu- with altered surfactant homeostasis and ATP-binding cassette nity, defense against pathogens, and in the clearance of inhaled transporters may play a role in some of them (16). particles. Alveolar macrophages are also important in initiating the inflammatory response in the lung. In response to danger, alveolar ATP-binding cassette transporter G1 (ABCG1) is a transmem- macrophages produce various proinflammatory mediators to or- brane transporter that mediates cholesterol efflux from macro- chestrate the inflammatory response (3, 7). This leads to the re- phages (17–23). Recently, a role for ABCG1 in pulmonary func- cruitment of numerous inflammatory cells, including monocytes, tion was discovered (24). The lung phenotype of older (8 mo of Ϫ/Ϫ Ϫ/Ϫ dendritic cells, eosinophils, neutrophils, B cells, and T cells (3, 7, age) Abcg1 mice is similar to that observed for Abca1 mice 8). These cells contribute to the immune response and eventually (25). The deletion of ABCG1 or ABCA1 leads to progressive lipid apoptose once the inflammation is resolved. The alveolar macro- accumulation and cellular accumulation in the lung (24). In the phages clear the apoptotic cells and produce antiinflammatory me- present study, we tested whether ABCG1 deficiency in macro- diators (9, 10). phages was causal in the onset of pulmonary inflammation and lipidosis in these mice. Surprisingly, we found that ABCG1 ex- pression in macrophages is an important regulator of pulmonary *Department of Pharmacology, †The Robert M. Berne Cardiovascular Research inflammation. ABCG1-deficient macrophages are highly proin- Center, and ‡Department of Internal Medicine, University of Virginia, Charlottes- flammatory and cause increased trafficking of inflammatory cells ville, VA 22908 to the lung. Abcg1Ϫ/Ϫ macrophages have enhanced ability to en- Received for publication September 26, 2007. Accepted for publication January 10, 2008. gulf apoptotic cells, accumulate lipid, and become apoptotic. Thus, The costs of publication of this article were defrayed in part by the payment of page we show a novel role for ABCG1 in the inflammatory response. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant P0I HL55798 (to C.C.H.). 3 Abbreviations used in this paper: SP, surfactant protein; ABCG1, ATP-binding cas- 2 Address correspondence and reprint requests to Dr. Catherine C. Hedrick, Cardio- sette transporter G1; BAL, bronchoalveolar lavage; ER, endoplasmic reticulum; vascular Research Center, University of Virginia, 415 Lane Road, Building MR-5, 7AAD, 7-aminoactinomycin D; Siglec-F, sialic acid-binding Ig-like lectin F. Room G123, P.O. Box 801394, Charlottesville, VA 22908. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 4274 ABCG1 AND LUNG INFLAMMATION

Materials and Methods Reagents Oil Red O and collagenase D were purchased from Sigma-Aldrich. ABCG1 Ab was purchased from Novus Biologicals. HRP-conjugated anti-mouse and anti-rabbit secondary Abs came from Amersham Biosciences. Fluorochrome- labeled Abs were purchased from BD Pharmingen or eBioscience and used according to the manufacturers’ protocols. NuPAGE 4–12% denaturing gels and TAMRA dye came from Invitrogen. The mouse 23-plex suspension bead arrays were purchased from Bio-Rad. Mice C57BL/6J (stock no. 000664) were purchased from The Jackson Laboratory, and Abcg1Ϫ/Ϫ/lacZ knock-in mice on a C57BL/6J background were purchased from Deltagen. Cx3cr1ϩ/GFP mice on a C57BL/6J background were a gift from Dr. Steffen Jung (The Weizmann Institute of Science, Rehovot, Israel). Mice were fed a standard rodent chow diet and housed in microisolator cages in a pathogen-free facility. All experiments followed University of Virginia Ani- mal Care and Use Committee guidelines, and approval for use of rodents was obtained from the University of Virginia. Histology Downloaded from Mice were euthanized with isoflurane, and the tracheas were exposed and cannulated. For frozen lung sections, OTC compound (Sakura Finetek USA) was injected into the lungs, the trachea was tied off, and the lungs were removed and frozen on dry ice. Lungs were sectioned into 10-␮m sections and stained with Oil Red O for lipid accumulation. Images were taken at ϫ40 and ϫ200. For morphometric and lung structure analysis,

lungs were fixed with paraformaldehyde at 30 cm inflation pressure and http://www.jimmunol.org/ paraffin-embedded. The tissue sections were stained with H&E and images were taken at ϫ200. To measure alveolization, five random tissue sections were taken from age-matched mice. An alveolization scoring system was FIGURE 1. Lipid accumulates in the lungs of Abcg1Ϫ/Ϫ mice. A–F, used to calculate mean linear intercept, as previously described (26). Lungs were perfused and frozen for histology. Sections were stained for lipid using Oil Red O. Representative images at ϫ200 are shown: (A) Bronchoalveolar lavage (BAL) Ϫ Ϫ 3-wk-old B6, (B) 7-wk-old B6, (C) 8-mo-old B6, (D) 3-wk-old Abcg1 / , Mice at 7 wk of age were euthanized with isoflurane, and the tracheas were (E) 7-wk-old Abcg1Ϫ/Ϫ, and (F) 8-mo-old Abcg1Ϫ/Ϫ. G–L, Lungs were exposed and cannulated. The lungs were flushed three times with 1-ml fixed with paraformaldehyde and paraffin-embedded. Sections were stained aliquots of BAL buffer (TBS and 10 mM EDTA). The aliquots were combined with H&E and images were taken at ϫ200: (G) 3-wk-old B6, (H) 7-wk-old and centrifuged (200 ϫ g, 5 min) to separate the supernatant (surfactant and Ϫ/Ϫ Ϫ/Ϫ B6, (I) 8-mo-old B6, (J) 3-wk-old Abcg1 ,(K) 7-wk-old Abcg1 , and by guest on September 27, 2021 soluble proteins) and cells. The cellular fraction was used for flow cytometry (L) 8-mo-old Abcg1Ϫ/Ϫ. M, Number of alveolar wall intercepts (n ϭ 1 or plated to purify the macrophage population. The supernatant was kept at mouse, five slides per mouse, and four random measurements per slide). Ϫ80°C until used for cytokine or surfactant measurements. Data were analyzed by two-way ANOVA with Bonferroni posttest. F test .p Ͻ 0.05 ,ء Cytokine measurements for age and interaction were significant as Cytokines and chemokines were measured in BAL fluid, plasma, and alveolar macrophage culture media using a Bio-Plex Mouse 23-plex suspension bead array Bone marrow transplantations (Bio-Rad) according to the instructions of the manufacturer. IL-12p40, IL-12p70, Ϫ/Ϫ IL-17, G-CSF, GM-CSF, IFN-␥, MIP-1␣, RANTES, IL-1␣, IL-1␤, TNF-␣, IL-2, B6 or Abcg1 mice were lethally irradiated (650 rad, 2 repetitions) and Ϫ/Ϫ ϫ 6 IL-3, IL-5, IL-6, and IL-10 were detected using high-sensitivity settings. injected with either B6 or Abcg1 bone marrow (4 10 cells). The mice were allowed to recover for 9 wk to allow for repopulation of resident alveolar Lung tissue digestion macrophages with donor cells (27). To test for reconstitution, bone marrow- derived macrophages were tested for ABCG1 expression by immunoblotting. Mice were euthanized with isoflurane. The heart was exposed, the aorta was severed, and the lungs were perfused gently with 5 ml of cold PBS Monocyte trafficking studies through the right ventricle using a syringe fitted with a 25-gauge needle. ϩ/GFP The lungs were excised, minced, and digested for 30 min at 37°C with Bone marrow was harvested from tibias of Cx3cr1 mice. The cells collagenase D (200 U/ml). The suspensions were passed through 70-␮m were washed and RBCs were depleted with ammonium chloride lysing cell strainers (BD Biosciences). The cells were washed with TBS, and buffer. The bone marrow cells were injected into the tail veins of B6 or Ϫ/Ϫ RBCs were depleted with ammonium-chloride lysing buffer. Samples were Abcg1 mice. After 48 h, the lungs and spleen were removed for flow used for flow cytometry experiments as described below. cytometry analysis. Flow cytometry Annexin V and 7-aminoactinomycin D (7AAD) staining Single-cell suspensions of BAL cells, lung tissue digest, or blood were surface- BAL cells were isolated and stained with annexin V (Invitrogen Life Tech- stained for 20 min at 4°C with the following Abs: anti-CD8 (clone 53-6.7), nologies) and 7ADD (BD Biosciences) according to the manufacturers’ anti-CD4 (clone RM4-5), anti-CD3 (clone 145-2C11), anti-CD19, anti-CD11b protocols. (clone M1/70), anti-7/4 (clone MCA771FA), anti-CD11c (clone N418), anti- sialic acid-binding Ig-like lectin F (Siglec-F) (clone E50-2440), MHC class II TUNEL staining (clone M5/114.15.2), anti-CD115 (clone AFS98), and anti-Nk1.1 (clone BAL macrophages were isolated, plated for 1 h, washed to remove non- PK136). After 20 min, cells were washed and analyzed at the University of adherent cells, and stained for DNA strand breaks with TUNEL according Virginia Flow Cytometry Core using a BD FACSCalibur instrument (BD Bio- to the instructions of the manufacturer (Roche). Cells were visualized by sciences), collecting data on the entire sample. Analysis was performed using fluorescence microscopy, and images were taken at ϫ100. FlowJo software. Calculations of percentages were based on live cells as de- termined by FSC/SSC analysis. To determine total cell counts, flow cytometry Apoptotic cell clearance assay counting beads (Invitrogen) were used and the actual number of cells was calculated based on the number of beads in the sample and the number of Jurkat cells were made apoptotic by UV cross-linking at 100 mjoules. beads counted by the flow cytometer. Apoptotic Jurkat cells were labeled with TAMRA dye (Invitrogen Life The Journal of Immunology 4275

FIGURE 2. Increased expression of proinflammatory cytokines in BAL fluid and alveolar macrophages of Abcg1Ϫ/Ϫ mice. Cytokine and chemo- kine protein expression was measured using a cytokine bead array (Bio-Rad). Values shown are means Ϯ SEM. Sig- nificantly higher than control by un- ;p Ͻ 0.05; $, p Ͻ 0.005 ,ء :paired t test and #, p Ͻ 0.0001. A, BAL fluid was analyzed from B6 (n ϭ 6) and Abcg1Ϫ/Ϫ 7-wk-old (n ϭ 6) mice. B, Alveolar macrophages were isolated from B6 (n ϭ 10) and Abcg1Ϫ/Ϫ (n ϭ 10) mice and plated for 24 h. The su- Downloaded from pernatant was collected and analyzed. C, Plasma was analyzed from B6 (n ϭ 9) and Abcg1Ϫ/Ϫ (n ϭ 8) mice. http://www.jimmunol.org/

Technologies), incubated for2hat37°C, and washed extensively. BAL parisons between groups and tests of interactions were made assuming a macrophages and apoptotic Jurkat cells were then incubated together for 45 two-factor analysis, with the interaction term testing each main effect and with min at 37°C to allow engulfment of apoptotic cells by the macrophages to the residual error testing the interaction. All comparisons were made using occur. At the end of the incubation period, macrophages were washed exten- Fisher’s least significant difference procedure, so that multiple comparisons sively to eliminate any unengulfed cells, and engulfment was quantified using were made at the 0.05 level only if the overall F test from the ANOVA was by guest on September 27, 2021 flow cytometry following the methods of Kiss et al. (28). Results are shown as significant at p Ͻ 0.05. the percentage of macrophages that engulfed apoptotic Jurkat cells. Statistical analysis Results Lipid accumulates in the lungs of weanling Abcg1Ϫ/Ϫ mice Data for all experiments were analyzed by unpaired Student’s t test (2 groups) or ANOVA (Ͼ2 groups) using the Statview 6.0 software program To determine how early a disruption in lipid homeostasis occurs in Ϫ Ϫ (SAS Institute). Data are graphically represented as means Ϯ SEM. Com- Abcg1 / mice, we examined Oil Red O staining of lung tissue in

FIGURE 3. Increased infiltration of inflammatory cells into the alveolar space of Abcg1Ϫ/Ϫ mice. BAL cells from B6 (n ϭ 4) and Abcg1Ϫ/Ϫ mice (n ϭ 4) were collected, washed, and stained with fluorophore-conjugated Abs. Samples were run on a FACSCalibur flow cytometer and analyzed using FlowJo software. Representative plots are shown in A–F:(A) T cells were identified as CD3high and CD8high or (B) CD3high and CD4high,(C) B cells were identified as CD19high,(D) neutro- phils were identified as CD11bhighGr- 1high7/4high cells, (E) eosinophils were identified as Siglec-FhighCD11bhigh CD11clow cells, and (F) dendritic cells were identified as Siglec-FlowCD11chigh MHCIIhigh cells. G, Plot of average number of cells per group from three experiments. Values shown are means Ϯ SEM. Significantly higher p Ͻ ,ء :than control by unpaired t test 0.05; $, p Ͻ 0.005; and #, p Ͻ 0.0001. 4276 ABCG1 AND LUNG INFLAMMATION Downloaded from

FIGURE 4. No change in the number of macrophages in the alveolar space or lung tissue of Abcg1Ϫ/Ϫ mice. Alveolar and tissue macrophages were collected from B6 (n ϭ 5) and Abcg1Ϫ/Ϫ mice (n ϭ 6), washed, and stained with fluorophore-conjugated Abs. Samples were run on a FACSCalibur flow cytometer and analyzed using FlowJo software. Representative plots are shown in A and B:(A) Gating for live cells using forward vs side scatter and 7AAD low high high staining on lung tissue samples, and (B) tissue macrophages were identified as 7AAD CD11c Siglec-F and plotted FSC vs SSC to show granularity. http://www.jimmunol.org/ Gating for alveolar macrophages was performed the same as shown for tissue macrophages. Alveolar macrophages were identified as 7AADlowCD11chighSiglec-Fhigh and plotted FSC vs SSC to show granularity. C, Plot of average number of macrophages per group from three experiments. Values shown are means Ϯ SEM. There were no significant changes between B6 and Abcg1Ϫ/Ϫ mice. D, Alveolar macrophages were isolated by BAL from B6 and Abcg1Ϫ/Ϫ mice and plated. Cells were fixed and stained with Oil Red O. Images were taken at ϫ200.

3-wk-old, 7-wk-old, and 8-mo-old C57BL/6J (B6) and Abcg1Ϫ/Ϫ macrophage is the inflammatory cell promoting disease in the mice. We found that 3-wk-old weanling Abcg1Ϫ/Ϫ mice accumulated lungs of Abcg1Ϫ/Ϫ mice, we measured cytokine levels in isolated lipid at the edge of the lung lobe (Fig. 1D). This was not observed alveolar macrophage cell supernatants. We found a significant el- by guest on September 27, 2021 in 3-wk-old B6 mice (Fig. 1A). At 7 wk of age, there was no evation in the inflammatory cytokines IL-6, IL-1␤, IL-1␣, IL-12, additional increase in lipid accumulation in Abcg1Ϫ/Ϫ mice (Fig. and KC and a decrease in antiinflammatory IL-10 (Fig. 2B). Sur- 1, B and E). However, at 8 mo of age, as previously reported by prisingly, chemokine and cytokine levels in Abcg1Ϫ/Ϫ plasma Edwards and colleagues (24), Abcg1Ϫ/Ϫ mice had severe lipid ac- were normal, except for the chemokine KC (Fig. 2C). cumulation in all parts of the lung that was not observed in B6 mice (Fig. 1, C and F). Moreover, we found significant alveolar enlargement in 8-mo-old Abcg1Ϫ/Ϫ mice compared with B6 mice (Fig. 1, I, L, and M). However, there was no change in lung struc- ture or alveolar space in 3-wk-old and 7-wk-old Abcg1Ϫ/Ϫ mice (Fig. 1, J and K), with lung structure being similar to that of age- matched B6 control mice (Fig. 1, G and H). Thus, lipid accumu- lation in Abcg1Ϫ/Ϫ mice begins at a very early age and becomes progressively more severe. Next, we evaluated the type of pulmonary surfactant that was accumulating in the lungs of Abcg1Ϫ/Ϫ mice. We chose to study 7-wk-old mice for all remaining experiments. SP-A, SP-B, SP-C, and SP-D were measured in the lung tissue of 7-wk-old B6 and Abcg1Ϫ/Ϫ mice. SP-A, SP-B, and SP-C appeared normal in Abcg1Ϫ/Ϫ mice at 7 wk of age, yet SP-D was greatly increased in 7-wk-old Abcg1Ϫ/Ϫ mice (data not shown). SP-D is involved in regulating the immune response in the lung (13, 14), and therefore these data suggest that lungs of Abcg1Ϫ/Ϫ mice are inflamed.

Ϫ Ϫ Increased inflammation in the lungs of Abcg1 / mice FIGURE 5. ABCG1 expression on macrophages regulates pulmonary Ϫ/Ϫ Ϫ/Ϫ lipidosis. B6 and Abcg1 mice were irradiated and reconstituted with B6 To investigate the inflammatory state of young Abcg1 mice Ϫ Ϫ or Abcg1 / bone marrow. After 9 wk, lungs were perfused and frozen for before onset of pulmonary lipidosis, we measured cytokine and histology. Sections were stained for lipid using Oil Red O. Images were chemokine levels in the BAL fluid, plasma, and alveolar macro- taken at ϫ400 magnification. Representative images are shown in A–D:(A) Ϫ/Ϫ Ϫ/Ϫ phages of 7-wk-old B6 and Abcg1 mice. Abcg1 mice had B6 bone marrow into B6 recipients, (B) B6 bone marrow into Abcg1Ϫ/Ϫ significant increases in levels of cytokines in BAL fluid compared recipients, (C) Abcg1Ϫ/Ϫ bone marrow into B6 recipients, and (D) with B6 mice (Fig. 2A). Because we had hypothesized that the Abcg1Ϫ/Ϫ bone marrow into Abcg1Ϫ/Ϫ recipients. The Journal of Immunology 4277

tified as CD11bhighSiglec-FhighCD11clow (Fig. 3E). For neutrophil analysis, we first identified CD11bhigh cells (not shown), and sub- sequently gated on Gr-1high7/4high. Because there were fewer CD11bhigh cells in a noninflamed lung, the neutrophil plot for B6 mice appears different from that for the Abcg1Ϫ/Ϫ mice, which contains CD11bhigh eosinophils and more CD11bhigh macro- phages. Resident macrophages appeared to have low to high CD11b expression, which is why there is another small CD11bhigh cell population shown in Fig. 3D. For eosinophil analysis, we first gated on CD11clow cells and then identified the CD11bhighSiglec- Fhigh, as shown in Fig. 3E. Dendritic cells were identified as Siglec- FlowCD11chigh (Fig. 3F). First, we measured inflammatory cells re- cruited into the alveolar space of the lung by analyzing the BAL. In normal, noninflamed lungs, these cells are very low in number. How- FIGURE 6. Increased proinflammatory cytokine expression in bone ever, in the Abcg1Ϫ/Ϫ mice at 7 wk of age, we found dramatic in- Ϫ/Ϫ marrow transplant mice receiving Abcg1 bone marrow. B6 and creases in populations of CD8ϩ ϩ - Ϫ/Ϫ Ϫ/Ϫ and CD4 T cells, B cells, eosino Abcg1 mice were irradiated and reconstituted with B6 or Abcg1 phils, neutrophils, and dendritic cells in the BAL fluid (Fig. 3G). bone marrow (BM) (n ϭ 5/group). Cytokine and chemokine protein ex- Next, we examined whether more inflammatory cells resided in pression was measured in BAL fluid from bone marrow transplant mice Downloaded from using a cytokine bead array (Bio-Rad). Values shown are means Ϯ SEM. the lung tissue as well. In contrast to the alveolar space, there were p Ͻ 0.05; $, p Ͻ 0.005; and resident neutrophils, eosinophils, dendritic cells, T cells, and B ,ء :Significance was calculated using ANOVA #, p Ͻ 0.0001. cells in the tissue of noninflamed lungs (data not shown). Inflam- matory cells were identified in the digested lung tissue of 7-wk-old B6 and Abcg1Ϫ/Ϫ mice using the same cell-surface markers men- Ϫ Ϫ ϩ Given the change in lung cytokine levels, we sought to deter- tioned above. Abcg1 / mice had a 1.3-fold increase in CD8 T Ϫ Ϫ mine which inflammatory cells were infiltrating the lungs. Inflam- cells (Abcg1 / 1.46 Ϯ 0.07 ϫ 105 vs B6 1.06 Ϯ 0.11 ϫ 105 http://www.jimmunol.org/ matory cells were identified in the alveolar space and lung tissue of cells), a 1.5-fold increase in CD4ϩ T cells (Abcg1Ϫ/Ϫ 2.13 Ϯ 7-wk-old B6 and Abcg1Ϫ/Ϫ mice by flow cytometry. T cells were 0.14 ϫ 105 vs B6 1.44 Ϯ 0.14 ϫ 105 cells), a 1.7-fold increase in identified as CD3high and CD8high or CD4high (Fig. 3, A and B). B B cells (Abcg1Ϫ/Ϫ 7.89 Ϯ 1.1 ϫ 105 vs B6 4.59 Ϯ 0.54 105 cells), cells were defined as CD19high (Fig. 3C). Neutrophils were defined a 2.1-fold increase in neutrophils (Abcg1Ϫ/Ϫ 3.56 Ϯ 0.11 ϫ 105 vs as CD11bhighGr-1high7/4high (Fig. 3D), and eosinophils were iden- B6 1.63 Ϯ 0.29 ϫ 105 cells), a 12-fold increase in eosinophils by guest on September 27, 2021

FIGURE 7. Increased inflammatory cell infiltration in bone marrow transplant mice receiving Abcg1Ϫ/Ϫ bone marrow. B6 and Abcg1Ϫ/Ϫ mice were irradiated and reconstituted with B6 or Abcg1Ϫ/Ϫ bone marrow (BM) (n ϭ 5/group). BAL cells from bone marrow transplant mice were collected, washed, and stained with fluorophore-conjugated Abs. Samples were run on a FACSCalibur flow cytometer and analyzed using FlowJo software. Representative plots are shown in A–D:(A) macrophages were identified as CD11chigh Siglec-Fhigh,(B) neutrophils were identified as CD11bhigh Gr-1high7/4high cells, (C) eosinophils were identified as Siglec-FhighCD11bhighCD11clow cells, and (D) dendritic cells were identified as Siglec-FlowCD11chighMHC class IIhigh cells. E, Plot of average number of cells per group. Values shown are means Ϯ SEM. Significance was cal- .p Ͻ 0.05; and #, p Ͻ 0.0001 ,ء :culated using ANOVA 4278 ABCG1 AND LUNG INFLAMMATION

(Abcg1Ϫ/Ϫ 3.26 Ϯ 0.59 ϫ 104 vs B6 0.26 Ϯ 0.15 ϫ 104 cells), and a 2.6-fold increase in dendritic cells (Abcg1Ϫ/Ϫ 4.24 Ϯ 0.58 ϫ 105 vs B6 1.63 Ϯ 0.41 ϫ 105 cells) compared with B6 mice. Thus, there is a dramatic infiltration of inflammatory cells to both the alveolar space and lung tissue of young Abcg1Ϫ/Ϫ mice before onset of severe lipidosis. Abcg1Ϫ/Ϫ mice show no change in macrophage content of lungs We next investigated the alveolar and tissue macrophage content in the lungs of 7-wk-old B6 and Abcg1Ϫ/Ϫ mice using flow cy- tometry. We used both forward vs side scatter and 7AAD staining to gate on live cells (Fig. 4A). Macrophages were defined as Siglec- FhighCD11chigh (Fig. 4B). Surprisingly, in contrast to other inflam- matory cell populations, there were no significant differences in the total numbers of alveolar macrophages or tissue macrophages be- tween the two mouse groups (see Fig. 4C). However, there was a striking difference in the appearance of the macrophages when evaluating forward vs side scatter. Both tissue and alveolar Ϫ Ϫ Abcg1 / macrophages had a high degree of side scatter, which is Downloaded from indicative of granularity (Fig. 4B). Lipid accumulation in macro- phages would make them appear more granular, as seen in Fig. 4B. Indeed, we found that alveolar macrophages from Abcg1Ϫ/Ϫ mice were larger in size and had severe lipid accumulation, whereas the B6 alveolar macrophages were lipid-free (Fig. 4D). http://www.jimmunol.org/ ABCG1 expression in macrophages regulates development of pulmonary lipidosis and inflammation

To determine the role of macrophages in causing pulmonary FIGURE 8. Increased monocyte recruitment and macrophage differen- lipidosis in this mouse model, we performed bone marrow trans- tiation in the lungs of Abcg1Ϫ/Ϫ mice. A, Blood was taken from B6 (n ϭ Ϫ/Ϫ plantations using B6 and Abcg1 bone marrow. After 9 wk of 6) and Abcg1Ϫ/Ϫ mice (n ϭ 6). Cells were stained with CD115 and CD11b reconstitution, lungs from mice were harvested to examine inflam- to identify monocyte populations. Samples were run on a FACSCalibur mation and onset of pulmonary lipidosis. Surprisingly, we found flow cytometer and analyzed using FlowJo software. Representative plots Ϫ Ϫ that bone marrow-derived cells appeared to be solely responsible are shown of total CD11bhighCD115high monocytes from B6 and Abcg1 / for onset of lipidosis (Fig. 5). Moreover, the presence of ABCG1 mice. B, Plot of the average number of cells per group from two experi- by guest on September 27, 2021 ments. Values shown are means Ϯ SEM. Significantly higher than control on bone marrow-derived cells prevented onset of lipidosis in Ϫ/Ϫ Ϫ Ϫ by unpaired t test: $, p Ͻ 0.005. C,B6(n ϭ 6) and Abcg1 mice (n ϭ Abcg1 / mice (Fig. 5B), while absence of ABCG1 expression in 6) were given tail vein injections of bone marrow isolated from bone marrow-derived cells caused onset of lipidosis in wild- Cx3cr1GFP/ϩ mice. After 48 h, the lungs and spleens were harvested and type B6 mice (Fig. 5C). Because ABCG1 is highly expressed on stained with CD11c and Siglec-F to identify macrophages, CD11b to iden- macrophages and plays a significant role in reverse cholesterol trans- tify monocytes, and Nk1.1 to identify NK cells. Samples were run on a port (22), we anticipate the macrophage to be the bone marrow-de- FACSCalibur flow cytometer and analyzed using FlowJo software. Rep- rived cell causing pulmonary disease. Thus, these data strongly sug- resentative plots of GFPϩ newly recruited monocytes (GFPhighCD11bhigh gest that the macrophage, and not pneumocyte type II cells or other Nk1.1low) to the lung and spleen and newly differentiated macrophages nonhematopoietic cells in the lung, appears to be the primary cell type (GFPhighCD11bhighNk1.1lowSiglec-FhighCD11chigh) are shown. D, Plot of involved in onset of pulmonary lipidosis in Abcg1Ϫ/Ϫ mice. average number of cells per group from two experiments. Values shown Ϯ We next examined inflammatory cytokine levels in the BAL are means SEM. Significantly higher than control by unpaired t test: .p Ͻ 0.05; and #, p Ͻ 0.0001 ,ء fluid of the bone marrow-transplanted mice, and noted some in- teresting findings. First, B6 mice reconstituted with Abcg1Ϫ/Ϫ bone marrow had significantly higher levels of cytokines com- pared with B6 mice reconstituted with B6 bone marrow (Fig. 6). Ϫ/Ϫ Furthermore, Abcg1Ϫ/Ϫ mice reconstituted with B6 bone marrow transplantation with Abcg1 bone marrow. Overall, inflam- had significantly lower levels of IL-12p40 and KC, with no de- matory cytokine production in the lung was drastically higher in tectable TNF-␣ production, compared with Abcg1Ϫ/Ϫ mice recon- mice with macrophages lacking ABCG1 expression, again sup- stituted with Abcg1Ϫ/Ϫ bone marrow (Fig. 6). We unexpectedly porting the notion that ABCG1 expression in macrophages reg- found that transplantation of Abcg1Ϫ/Ϫ bone marrow into B6 mice ulates lung inflammation. increased cytokine levels more strongly than did transplantation of We also found that macrophage accumulation in the BAL was Ϫ Ϫ Abcg1Ϫ/Ϫ bone marrow into Abcg1Ϫ/Ϫ mice. We are unsure as to significantly lower in B6 mice receiving Abcg1 / bone marrow, why this occurs; however, one likely possibility is that the but we found a significant increase in alveolar macrophage accu- Ϫ Ϫ Abcg1Ϫ/Ϫ recipient mice have adapted to their global loss of mulation in Abcg1 / mice reconstituted with B6 bone marrow ABCG1 and are somewhat sensitized to the chronic cytokine el- (Fig. 7A). There was no significant difference in the number of evation that occurs when Abcg1Ϫ/Ϫ marrow is introduced. The macrophages in B6 mice reconstituted with B6 marrow and Ϫ Ϫ Ϫ Ϫ B6 control recipients have not, of course, encountered the Abcg1 / mice reconstituted with Abcg1 / marrow (Fig. 7E). chronic cytokine elevation that occurs in the global ABCG1- The results of our bone marrow transplantation studies suggest that deficient mouse, so the nonhematopoietic cells in the B6 recip- both the environment of the lung and macrophage ABCG1 expres- ient mice may have a heightened inflammatory response after sion affect accumulation of macrophages in the lung. The Journal of Immunology 4279 Downloaded from http://www.jimmunol.org/

FIGURE 9. Abcg1Ϫ/Ϫ alveolar macrophages are more apoptotic. A, Alveolar macrophages were isolated from the alveolar space of B6 and Abcg1Ϫ/Ϫ mice and stained with Siglec-F and CD11c to identify macrophages and annexin V and 7AAD for apoptosis. Samples were run on a FACSCalibur flow cytometer and analyzed using FlowJo software. Macrophages were gated as CD11chighSiglec-Fhigh. Representative plots are shown of alveolar macrophages to measure early apoptosis (7AADlowannexin Vhigh), late apoptosis (7AADhighannexin Vhigh), necrotic (7AADhighannexin Vlow), and live cells (7AADlow annexin Vlow). Average percentages are shown from B6 (n ϭ 8) and Abcg1Ϫ/Ϫ (n ϭ 10) mice. Values shown are means Ϯ SEM. Significantly higher than p Ͻ 0.05; and $, p Ͻ 0.005. B, TUNEL staining on B6 and Abcg1Ϫ/Ϫ alveolar macrophages isolated by BAL. Representative ,ء :control by unpaired t test ϫ images are shown. Images were taken at 100. by guest on September 27, 2021

The absence of ABCG1 in bone marrow-derived cells also sig- of 7-wk-old B6 and Abcg1Ϫ/Ϫ mice. Monocytes were identified as nificantly contributed to the accumulation of neutrophils, eosino- CD115highCD11bhigh cells by flow cytometry (Fig. 8A). Surpris- phils, and dendritic cells in the alveolar space (Fig. 7, B–E). Both ingly, Abcg1Ϫ/Ϫ mice had a 2-fold increase in the number of cir- B6 and Abcg1Ϫ/Ϫ recipient mice reconstituted with B6 bone mar- culating monocytes compared with B6 mice (Fig. 8B). This result row had few neutrophils (Fig. 7B) and eosinophils (Fig. 7C)inthe suggests that inflammation in the lung caused increased circulating alveolar space, as expected in mice with noninflamed lungs. How- monocytes in Abcg1Ϫ/Ϫ mice. We next used mice expressing GFP ever, both B6 and Abcg1Ϫ/Ϫ recipient mice reconstituted with in monocytes to measure the number of newly recruited monocytes Abcg1Ϫ/Ϫ bone marrow had significant infiltration of neutrophils to the lung. Cx3cr1GFP/ϩ mice are heterozygous mice that express (Fig. 7B) and eosinophils (Fig. 7C) into the alveolar space. There GFP only in Cx3cr1-expressing cells (29). Specifically, mono- was also approximately a 2-fold increase in the number of den- cytes, dendritic cells, and NK cells are Cx3cr1high and have been dritic cells in the alveolar space of B6 mice reconstituted with shown to traffic normally in heterozygous Cx3cr1GFP/ϩ mice (29). Abcg1Ϫ/Ϫ bone marrow compared with B6 mice reconstituted with For these experiments, we injected bone marrow from B6 bone marrow (Fig. 7D). We also observed a trend toward a Cx3cr1GFP/ϩ mice on a B6 background into 7-wk-old B6 and reduction in dendritic cell infiltration when Abcg1Ϫ/Ϫ mice were Abcg1Ϫ/Ϫ recipient mice. After 48 h, the lungs and spleen were reconstituted with B6 bone marrow (Fig. 7D). Thus, these results analyzed for the presence of newly recruited monocytes and newly demonstrate that the absence of ABCG1 in bone marrow-derived differentiated macrophages. The spleen was used as a control to cells leads to inflammation and recruitment of inflammatory cells determine whether the monocyte recruitment was specific to the into the alveolar space. lung. Newly recruited monocytes were identified as GFPhigh high low Ϫ/Ϫ CD11b Nk1.1 , and newly differentiated macrophages were Increased monocyte trafficking to the lungs of Abcg1 mice identified as GFPhighCD11bhighNk1.1lowSiglec-FhighCD11chigh We were struck by the fact that there appeared to be no change in (Fig. 8C). Abcg1Ϫ/Ϫ mice had a 3.4-fold increase in the number of the number of macrophages in the lungs of Abcg1Ϫ/Ϫ mice, yet the newly recruited monocytes to the lung (Fig. 8D). Furthermore, macrophages appeared lipid-filled and appeared to be of primary there was a 45-fold increase in the number of newly differentiated importance in disease onset in the Abcg1Ϫ/Ϫ mice. These findings macrophages in the lungs of Abcg1Ϫ/Ϫ mice (Fig. 8D). However, suggest that there is either reduced trafficking of monocytes to the there was no change in the number of monocytes recruited to the lung in Abcg1Ϫ/Ϫ mice or increased macrophage apoptosis occur- spleen (Fig. 8D). The results from this experiment demonstrate ring in the lungs of Abcg1Ϫ/Ϫ mice. To examine monocyte traf- increased monocyte recruitment and increased macrophage differ- ficking, we first quantified the number of monocytes in the blood entiation to the lungs in Abcg1Ϫ/Ϫ mice. 4280 ABCG1 AND LUNG INFLAMMATION

phages were isolated from the alveolar space of 7-wk-old B6 and Abcg1Ϫ/Ϫ mice, incubated with fluorescently-labeled apoptotic Jurkat cells, and measured by flow cytometry for engulfment of apoptotic Jurkat cells. Abcg1Ϫ/Ϫ alveolar macrophages had a 2-fold increase in the percentage of macrophages that engulfed apoptotic cells compared with B6 macrophages (Fig. 10A). More- over, we found that Abcg1Ϫ/Ϫ macrophages were activated, having increased expression of the activation markers CD11b and MHC class II (Fig. 10B). Taken together, our studies indicate that Abcg1Ϫ/Ϫ macrophages are activated and show increased ability to engulf apoptotic cells. However, these macrophages also become apoptotic, and subsequently necrotic, most likely due to increased lipid accumulation in response to lack of ABCG1. These dying macrophages appear to be quickly repopulated in the Abcg1Ϫ/Ϫ lung by newly recruited blood monocytes, as we observed in our monocyte trafficking studies.

Discussion

In this study, we have demonstrated that ABCG1 expression in Downloaded from macrophages plays a critical role in pulmonary inflammation and lipidosis. Abcg1Ϫ/Ϫ mice had increased proinflammatory cytokine levels in their lungs, leading to the recruitment of neutrophils, eosinophils, B cells, T cells, and dendritic cells (Figs. 2 and 3). FIGURE 10. Abcg1Ϫ/Ϫ macrophages have increased apoptotic cell Furthermore, we show for the first time that the absence of ABCG1 clearance and expression of activation markers. A, Alveolar macrophages expression in the macrophages was responsible for the lung in- http://www.jimmunol.org/ from B6 (blue lines) and Abcg1Ϫ/Ϫ mice (pink lines) were isolated by BAL flammation and lipid accumulation (Figs. 5–7). Our results reveal and plated. Jurkat cells were stained with TAMRA dye and made apoptotic a novel role for ABCG1 in regulating macrophage inflammation by UV crosslinking. Apoptotic Jurkat cells were incubated with the alve- and lung homeostasis. olar macrophages for 45 min. After washing, macrophages were removed Our most exciting finding was the role of the macrophage in the from the plate and run on a FACSCalibur flow cytometer. A representative development of pulmonary disease. We performed bone marrow plot from one experiment is shown. Note the increase in apoptotic cell engulfment by the Abcg1Ϫ/Ϫ macrophages (pink lines). Also shown is a transplant studies to look at the contribution of bone marrow-de- plot of the percentage of cells per group from two experiments (n ϭ 7 per rived cells vs non-bone marrow-derived cells to the development group). Values shown are means Ϯ SEM. Significantly higher than control of pulmonary disease. We anticipated that the macrophage is the p Ͻ 0.05. B, Alveolar and tissue macrophages were bone marrow-derived cell responsible for pulmonary disease, be- by guest on September 27, 2021 ,ء :by unpaired t test collected from B6 (blue lines) and Abcg1Ϫ/Ϫ mice (pink lines), washed, cause macrophages both initiate the immune response in the lung and stained with fluorophore-conjugated Abs. Samples were run on a and are involved in reverse cholesterol transport (3, 7, 22). We FACSCalibur flow cytometer and analyzed using FlowJo software. Mac- showed that both B6 and Abcg1Ϫ/Ϫ mice lacking ABCG1 expres- high high rophages were identified as Siglec-F CD11c cells as shown in Fig. 9. sion in macrophages accumulated lipid in their lungs (Fig. 5) and Macrophage activation was measured by increased expression of CD11b had massive lung inflammation (Figs. 6 and7). Moreover, the pres- and MHC class II. Representative plots from one experiment are shown Ϫ Ϫ ence of ABCG1 on macrophages in Abcg1 / mice was able to (n ϭ 6/group). Note the increase in MHC class II and CD11b expression in the Abcg1Ϫ/Ϫ macrophages (pink lines). rescue both the lipidosis (Fig. 5) and the inflammatory phenotype (Figs. 6 and 7). Therefore, we rescued the pulmonary disease in Abcg1Ϫ/Ϫ mice by reconstituting mice with wild-type B6 bone Increased macrophage apoptosis and apoptotic cell clearance Ϫ/Ϫ marrow, thereby restoring ABCG1 expression in macrophages in Abcg1 mice (Figs. 6 and 7). One question that remains is which process occurs We ruled out reductions in monocyte trafficking to the lung in first, macrophage lipid accumulation or inflammation. It will be of Abcg1Ϫ/Ϫ mice with the GFP-labeled monocyte studies, so we interest to determine whether the accumulation of lipid inside next examined macrophage apoptosis. Apoptosis and necrosis of Abcg1Ϫ/Ϫ macrophages causes the macrophage to become in- alveolar macrophages from the alveolar space of 7-wk-old B6 and flamed, or whether the absence of ABCG1 in macrophages triggers Abcg1Ϫ/Ϫ mice were analyzed by flow cytometry using annexin V inflammation, leading to the excess production of surfactant and and 7AAD staining (Fig. 9A). Macrophages were identified as Si- pulmonary lipidosis. This question will be addressed in future glec-FhighCD11chigh (Fig. 9A). There were significant elevations in studies. the percentage of early apoptotic (annexin Vhigh 7AADlow) and The lipid phenotype in the lungs of Abcg1Ϫ/Ϫ mice has been late apoptotic (annexin Vhigh7AADhigh) macrophages in Abcg1Ϫ/Ϫ described by others and shown to resemble various respiratory mice (Fig. 9A). There was also a 4.6-fold increase in the percent- syndromes. Abcg1Ϫ/Ϫ lungs at Ͼ6 mo of age displayed cellular age of necrotic (annexin Vlow7AADhigh) macrophages in the al- accumulation, macrophage and pneumocyte type II cell hypertro- veolar space of Abcg1Ϫ/Ϫ mice (Fig. 9A). TUNEL staining of phy, increased levels of surfactant, and massive lipid accumulation alveolar macrophages revealed increased numbers of TUNELϩ (24). There are several pulmonary diseases that are associated with cells in Abcg1Ϫ/Ϫ mice, again supporting the notion of increased a disruption in lipid homeostasis and inflammation, including al- macrophage apoptosis. Taken together, these data indicate that veolar proteinosis, respiratory distress syndrome, and Niemann- there is increased alveolar macrophage apoptosis occurring in Pick disease (30–32). Niemann-Pick disease is a lipid storage dis- Abcg1Ϫ/Ϫ mice. ease that results in lipid accumulation in various organs, such as Finally, we measured the ability of B6 and Abcg1Ϫ/Ϫ alveolar spleen, liver, lung, and brain, similar to that observed in Abcg1Ϫ/Ϫ macrophages to clear apoptotic cells in vitro. Alveolar macro- mice. Progressive lung disease is one manifestation of the disease, The Journal of Immunology 4281 characterized by the presence of “foamy” macrophages leading to phages engulf many apoptotic cells (Fig. 10), yet cannot properly chronic inflammation (33). Pulmonary alveolar proteinosis is a dis- efflux the cholesterol derived from these apoptotic cells, despite ease that is also similar to the lung disease found in Abcg1Ϫ/Ϫ up-regulation of ABCA1 (24). Furthermore, the apoptosis of a cell mice. Patients with pulmonary alveolar proteinosis are deficient in is considered an antiinflammatory process, but necrosis is proin- the macrophage growth factor GM-CSF, due to the presence of flammatory. We measured increases in both apoptosis and necrosis circulating GM-CSF-neutralizing Abs (34). Pulmonary alveolar by Abcg1Ϫ/Ϫ alveolar macrophages (Fig. 9). There may be differ- proteinosis is characterized by impaired surfactant clearance, lipid ent factors leading to the apoptotic and necrotic pathways in accumulation in alveolar macrophages, and increased cholesterol Abcg1Ϫ/Ϫ macrophages, which could contribute to or dampen pul- metabolites in the lung (35, 36). Recently, patients and mice with monary inflammation. pulmonary alveolar proteinosis have been shown to lack ABCG1 The effect of macrophage ABCG1 expression on atherosclerotic in alveolar macrophages (37), due to the absence of GM-CSF sig- lesions is highly controversial. Because ABCG1 is important in naling, thus illustrating a critical link between ABCG1 and lung macrophage reverse cholesterol transport and inflammation, we homeostasis. would expect that the absence of ABCG1 would increase athero- ABCG1 has been suggested to play a critical role in maintaining sclerosis development. However, two groups have found a slight lipid homeostasis in a variety of organs and is highly expressed in decrease in atherosclerotic lesion size in mice lacking ABCG1 in the ileum, liver, lung, spleen, and kidney (17). Abcg1Ϫ/Ϫ mice bone marrow-derived cells (40, 45). Yet, Van Eck and colleagues accumulate lipid in their liver and lungs, but not the spleen, kidney, have shown that both macrophage-specific deletion of ABCG1 and or ileum, when fed a high-fat/high-cholesterol diet (17). Surpris- total body deletion of ABCG1 moderately increase atherosclerotic Ϫ Ϫ ingly, Abcg1 / mice have reduced adipose cell size, and the mice lesion size (46, 47). Thus, despite the pulmonary inflammation and Downloaded from are protected against diet-induced obesity, suggesting a role for macrophage activation observed in Abcg1Ϫ/Ϫ mice, the role of ABCG1 in regulating energy balance (38). Furthermore, ABCG1, macrophage ABCG1 in atherosclerosis is not clear. One hypoth- along with ABCG4, has been demonstrated to regulate cholesterol esis is that the increased engulfment of apoptotic cells by transport in the brain (39). Edwards and colleagues have shown Abcg1Ϫ/Ϫ macrophages may be important in the aortic wall for that ABCG1 and ABCG4 mediate intracellular vesicular transport atherosclerosis (48). Another possible explanation is that only or-

of sterols within both neurons and astrocytes (39). Thus, there is gans containing an abundance of macrophages, such as the lung, http://www.jimmunol.org/ still much to be revealed about the function of ABCG1 in these spleen, and liver, are affected by the lack of ABCG1. The envi- organs. ronment of the lung in particular appears to be easily disrupted by In our studies, a surprising observation was that there was no changes in ABCG1 expression. This could be due to increased change in macrophage content in Abcg1Ϫ/Ϫ mice, even though the surfactant lipid or the abundance of resident inflammatory cells in lungs were noticeably inflamed and accumulating lipid (Fig. 4). the lung tissue that can quickly respond to inflammation. All fac- We hypothesized that more macrophages would be present in the tors considered, it is still puzzling that the lack of ABCG1 in mac- lungs of Abcg1Ϫ/Ϫ mice to engulf apoptotic cells and clear sur- rophages does not significantly increase inflammation in the aortic factant lipids. In the bone marrow transplant studies, we found that wall and in turn affect atherosclerotic lesion development. B6 mice given Abcg1Ϫ/Ϫ bone marrow had fewer macrophages in In conclusion, we propose that ABCG1 plays a critical role in by guest on September 27, 2021 their lungs, despite the increased inflammation and lipid accumu- maintaining lung homeostasis. We show that ABCG1 deletion in lation (Fig. 7). These data suggest that Abcg1Ϫ/Ϫ monocytes are macrophages causes a striking inflammatory phenotype and con- not properly trafficking to the lung and differentiating into macro- tributes to onset of pulmonary lipidosis in mice. These studies phages. Further studies are needed to determine how the absence suggest a novel role for ABCG1 in inflammation; understanding of ABCG1 in monocytes affects their ability to traffic to inflamed this role may be important for developing therapeutic approaches tissue. for lipid and inflammatory diseases. Our data clearly demonstrate that macrophages contribute to pulmonary disease, the mechanism by which, to some extent, is Acknowledgments explained after measuring macrophage inflammation, apoptosis, We thank Kodi Ravichandran (University of Virginia) for helpful advice and apoptotic cell clearance. Abcg1Ϫ/Ϫ alveolar macrophages are regarding engulfment studies, Joanne Lannigan, Director of the University clearly more activated, having increased production of proinflam- of Virginia Flow Cytometry Core Facility, for advice with flow cytometry matory cytokines and decreased IL-10 production (Fig. 2). Our analysis, Robert M. Strieter (University of Virginia) for technical advice on Ϫ Ϫ lung airspace measurements, and Klaus Ley (La Jolla Institute for Allergy studies also show that Abcg1 / alveolar macrophages are more and Immunology) and Sun-sang J. Sung (University of Virgina) for helpful prone to apoptosis (Fig. 9). These data are in concordance with discussions. what was previously published by Edwards and colleagues, who showed that Abcg1Ϫ/Ϫ peritoneal macrophages were more apopto- Disclosures tic (40). Oxysterols that are formed within the macrophages as a The authors have no financial conflicts of interest. consequence of cholesterol accumulation may cause the macro- phages to be more prone to apoptosis (41). When free cholesterol References accumulates in the macrophage, the ratio of free cholesterol to 1. Lehnert, B. E. 1992. Pulmonary and thoracic macrophage subpopulations and clearance of particles from the lung. Environ. Health Perspect. 97: 17–46. phospholipids is disturbed in the endoplasmic reticulum (ER) 2. Brain, J. D. 1992. Mechanisms, measurement, and significance of lung macro- membrane (42). The “stiffening” of the ER membrane bilayer phage function. Environ. Health Perspect. 97: 5–10. leads to ER protein dysfunction, ER stress, and the unfolded pro- 3. Zhang, P., W. R. Summer, G. J. Bagby, and S. Nelson. 2000. Innate immunity and pulmonary host defense. Immunol. Rev. 173: 39–51. tein response, resulting in apoptosis of the cell (42). Furthermore, 4. Barnes, P. J. 2004. Alveolar macrophages as orchestrators of COPD. COPD 1: ABCG1 has been shown to protect against 7-ketocholesterol- and 59–70. 7␤-hydroxycholesterol-induced cell death by effluxing these oxys- 5. Laskin, D. L., B. Weinberger, and J. D. Laskin. 2001. Functional heterogeneity in liver and lung macrophages. J. Leukocyte Biol. 70: 163–170. terols from macrophages to prevent oxysterol-induced cytotoxicity 6. Fathi, M., A. Johansson, M. Lundborg, L. Orre, C. M. Skold, and P. Camner. (43, 44). Therefore, one possibility is that Abcg1Ϫ/Ϫ alveolar mac- 2001. Functional and morphological differences between human alveolar and interstitial macrophages. Exp. Mol. Pathol. 70: 77–82. rophages are more prone to apoptosis due to the toxic effects of J. Thorac. Imaging Ϫ Ϫ 7. Twigg, H. L., III. 1998. Pulmonary host defenses. 13: lipid accumulation. Another explanation is that Abcg1 / macro- 221–233. 4282 ABCG1 AND LUNG INFLAMMATION

8. Crapo, J. D., B. E. Barry, P. Gehr, M. Bachofen, and E. R. Weibel. 1982. Cell targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell. number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. Biol. 20: 4106–4114. 126: 332–337. 30. Bullard, J. E., S. E. Wert, J. A. Whitsett, M. Dean, and L. M. Nogee. 2005. 9. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and ABCA3 mutations associated with pediatric interstitial lung disease. P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro Am. J. Respir. Crit. Care Med. 172: 1026–1031. inhibit proinflammatory cytokine production through autocrine/paracrine mech- 31. Garmany, T. H., M. A. Moxley, F. V. White, M. Dean, W. M. Hull, ␤ anisms involving TGF- , PGE2, and PAF. J. Clin. Invest. 101: 890–898. J. A. Whitsett, L. M. Nogee, and A. Hamvas. 2006. Surfactant composition and 10. Huynh, M. L., V. A. Fadok, and P. M. Henson. 2002. Phosphatidylserine-depen- function in patients with ABCA3 mutations. Pediatr. Res. 59: 801–805. ␤ dent ingestion of apoptotic cells promotes TGF- 1 secretion and the resolution of 32. Shulenin, S., L. M. Nogee, T. Annilo, S. E. Wert, J. A. Whitsett, and M. Dean. inflammation. J. Clin. Invest. 109: 41–50. 2004. ABCA3 gene mutations in newborns with fatal surfactant deficiency. 11. Goerke, J. 1998. Pulmonary surfactant: functions and molecular composition. N. Engl. J. Med. 350: 1296–1303. Biochim. Biophys. Acta 1408: 79–89. 33. Minai, O. A., E. J. Sullivan, and J. K. Stoller. 2000. Pulmonary involvement in 12. Perez-Gil, J. 2001. Lipid-protein interactions of hydrophobic proteins SP-B and Niemann-Pick disease: case report and literature review. Respir. Med. 94: SP-C in lung surfactant assembly and dynamics. Pediatr. Pathol. Mol. Med. 20: 1241–1251. 445–469. 34. Kitamura, T., N. Tanaka, J. Watanabe, K. Uchida, S. Kanegasaki, Y. Yamada, 13. McCormack, F. X., and J. A. Whitsett. 2002. The pulmonary collectins, SP-A and and K. Nakata. 1999. Idiopathic pulmonary alveolar proteinosis as an autoim- SP-D, orchestrate innate immunity in the lung. J. Clin. Invest. 109: 707–712. mune disease with neutralizing antibody against granulocyte/macrophage colony- 14. Vandivier, R. W., C. A. Ogden, V. A. Fadok, P. R. Hoffmann, K. K. Brown, stimulating factor. J. Exp. Med. 190: 875–880. M. Botto, M. J. Walport, J. H. Fisher, P. M. Henson, and K. E. Greene. 2002. Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in 35. Presneill, J. J., K. Nakata, Y. Inoue, and J. F. Seymour. 2004. Pulmonary alveolar vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. proteinosis. Clin. Chest Med. 25: 593–613, viii. J. Immunol. 169: 3978–3986. 36. Seymour, J. F., and J. J. Presneill. 2004. Pulmonary alveolar proteinosis: what is 15. Ikegami, M. 2006. Surfactant catabolism. Respirology 11(Suppl.): S24–S27. the role of GM-CSF in disease pathogenesis and treatment? Treat. Respir. Med. 16. van der Deen, M., E. G. de Vries, W. Timens, R. J. Scheper, H. Timmer-Bosscha, 3: 229–234. and D. S. Postma. 2005. ATP-binding cassette (ABC) transporters in normal and 37. Thomassen, M. J., B. P. Barna, A. G. Malur, T. L. Bonfield, C. F. Farver, pathological lung. Respir. Res. 6: 59. A. Malur, H. Dalrymple, M. S. Kavuru, and M. Febbraio. 2007. ABCG1 is de- Downloaded from 17. Kennedy, M. A., G. C. Barrera, K. Nakamura, A. Baldan, P. Tarr, M. C. Fishbein, ficient in alveolar macrophages of GM-CSF knock-out mice and patients with J. Frank, O. L. Francone, and P. A. Edwards. 2005. ABCG1 has a critical role in pulmonary alveolar proteinosis. J. Lipid Res. 48: 2762–2768. mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. 38. Buchmann, J., C. Meyer, S. Neschen, R. Augustin, K. Schmolz, R. Kluge, Cell Metab. 1: 121–131. H. Al-Hasani, H. Jurgens, K. Eulenberg, R. Wehr, et al. 2007. Ablation of the 18. Klucken, J., C. Buchler, E. Orso, W. E. Kaminski, M. Porsch-Ozcurumez, cholesterol transporter adenosine triphosphate-binding cassette transporter G1 re- G. Liebisch, M. Kapinsky, W. Diederich, W. Drobnik, M. Dean, et al. 2000. duces adipose cell size and protects against diet-induced obesity. Endocrinology ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regu- 148: 1561–1573.

lator of macrophage cholesterol and phospholipid transport. Proc. Natl. Acad. 39. Tarr, P. T., and P. A. Edwards. 2008. ABCG1 and ABCG4 are co-expressed in http://www.jimmunol.org/ Sci. USA 97: 817–822. neurons and astrocytes of the CNS and regulate cholesterol homeostasis through 19. Oram, J. F., and A. M. Vaughan. 2006. ATP-binding cassette cholesterol trans- SREBP-2. J. Lipid Res. 49: 169–82. porters and cardiovascular disease. Circ. Res. 99: 1031–1043. 40. Baldan, A., L. Pei, R. Lee, P. Tarr, R. K. Tangirala, M. M. Weinstein, J. Frank, 20. Smith, J. D. 2006. Insight into ABCG1-mediated cholesterol efflux. Arterioscler. A. C. Li, P. Tontonoz, and P. A. Edwards. 2006. Impaired development of ath- Thromb. Vasc. Biol. 26: 1198–1200. erosclerosis in hyperlipidemic LdlrϪ/Ϫ and ApoEϪ/Ϫ mice transplanted with 21. Vaughan, A. M., and J. F. Oram. 2005. ABCG1 redistributes cell cholesterol to Abcg1Ϫ/Ϫ bone marrow. Arterioscler. Thromb. Vasc. Biol. 26: 2301–2307. domains removable by high density lipoprotein but not by lipid-depleted apoli- 41. Lusis, A. J. 2000. Atherosclerosis. Nature 407: 233–234. poproteins. J. Biol. Chem. 280: 30150–30157. 42. Maxfield, F. R., and I. Tabas. 2005. Role of cholesterol and lipid organization in 22. Wang, N., D. Lan, W. Chen, F. Matsuura, and A. R. Tall. 2004. ATP-binding disease. Nature 438: 612–621. cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density 43. Engel, T., F. Kannenberg, M. Fobker, J. R. Nofer, G. Bode, A. Lueken, lipoproteins. Proc. Natl. Acad. Sci. USA 101: 9774–9779. G. Assmann, and U. Seedorf. 2007. Expression of ATP binding cassette-trans- 23. Wang, X., H. L. Collins, M. Ranalletta, I. V. Fuki, J. T. Billheimer, by guest on September 27, 2021 porter ABCG1 prevents cell death by transporting cytotoxic 7␤-hydroxycholes- G. H. Rothblat, A. R. Tall, and D. J. Rader. 2007. Macrophage ABCA1 and terol. FEBS Lett. 581: 1673–1680. ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J. Clin. Invest. 117: 2216–2224. 44. Terasaka, N., N. Wang, L. Yvan-Charvet, and A. R. Tall. 2007. High-density 24. Baldan, A., P. Tarr, C. S. Vales, J. Frank, T. K. Shimotake, S. Hawgood, and lipoprotein protects macrophages from oxidized low-density lipoprotein-induced P. A. Edwards. 2006. Deletion of the transmembrane transporter ABCG1 results apoptosis by promoting efflux of 7-ketocholesterol via ABCG1. Proc. Natl. Acad. in progressive pulmonary lipidosis. J. Biol. Chem. 281: 29401–29410. Sci. USA 104: 15093–15098. 25. Bates, S. R., J. Q. Tao, H. L. Collins, O. L. Francone, and G. H. Rothblat. 2005. 45. Ranalletta, M., N. Wang, S. Han, L. Yvan-Charvet, C. Welch, and A. R. Tall. 2006. Decreased atherosclerosis in low-density lipoprotein receptor knockout Pulmonary abnormalities due to ABCA1 deficiency in mice. Am. J. Physiol. 289: Ϫ/Ϫ L980–L989. mice transplanted with Abcg1 bone marrow. Arterioscler. Thromb. Vasc. 26. Londhe, V. A., J. A. Belperio, M. P. Keane, M. D. Burdick, Y. Y. Xue, and Biol. 26: 2308–2315. R. M. Strieter. 2005. CXCR2/CXCR2 ligand biological axis impairs alveologen- 46. Out, R., M. Hoekstra, R. B. Hildebrand, J. K. Kruit, I. Meurs, Z. Li, F. Kuipers, esis during dsRNA-induced lung inflammation in mice. Pediatr. Res. 58: T. J. Van Berkel, and M. Van Eck. 2006. Macrophage ABCG1 deletion disrupts 919–926. lipid homeostasis in alveolar macrophages and moderately influences atheroscle- 27. Matute-Bello, G., J. S. Lee, C. W. Frevert, W. C. Liles, S. Sutlief, K. Ballman, rotic lesion development in LDL receptor-deficient mice. Arterioscler. Thromb. V. Wong, A. Selk, and T. R. Martin. 2004. Optimal timing to repopulation of Vasc. Biol. 26: 2295–2300. resident alveolar macrophages with donor cells following total body irradiation 47. Out, R., M. Hoekstra, I. Meurs, P. de Vos, J. Kuiper, M. Van Eck, and and bone marrow transplantation in mice. J. Immunol. Methods 292: 25–34. T. J. Van Berkel. 2007. Total body ABCG1 expression protects against early 28. Kiss, R. S., M. R. Elliott, Z. Ma, Y. L. Marcel, and K. S. Ravichandran. 2006. atherosclerotic lesion development in mice. Arterioscler. Thromb. Vasc. Biol. 27: Apoptotic cells induce a phosphatidylserine-dependent homeostatic response 594–599. from phagocytes. Curr. Biol. 16: 2252–2258. 48. Tabas, I. 2005. Consequences and therapeutic implications of macrophage apo- 29. Jung, S., J. Aliberti, P. Graemmel, M. J. Sunshine, G. W. Kreutzberg, A. Sher, ptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency. and D. R. Littman. 2000. Analysis of fractalkine receptor CX3CR1 function by Arterioscler. Thromb. Vasc. Biol. 25: 2255–2264.