A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient

A Novel Subset of Anti-Inflammatory CD138 + Macrophages Is Deficient in Mice with Experimental Lupus

This information is current as Shuhong Han, Haoyang Zhuang, Stepan Shumyak, Jingfan of October 2, 2021. Wu, Hui Li, Li-Jun Yang and Westley H. Reeves J Immunol 2017; 199:1261-1274; Prepublished online 10 July 2017; doi: 10.4049/jimmunol.1700099 http://www.jimmunol.org/content/199/4/1261 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Subset of Anti-Inﬂammatory CD138+ Macrophages Is Deﬁcient in Mice with Experimental Lupus

Shuhong Han,* Haoyang Zhuang,* Stepan Shumyak,* Jingfan Wu,* Hui Li,† Li-Jun Yang,† and Westley H. Reeves*

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MF). Impaired apoptotic cell uptake by MF also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MF. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predom- inantly of a novel subset of highly phagocytic MF resembling small peritoneal MF (SPM) that expressed CD138+ and the Downloaded from scavenger receptor Marco. Treatment with anti-Marco–neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MF. CD138+ MF expressed IL-10R, CD206, and CCR2 but little TNF-a or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MF. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear http://www.jimmunol.org/ apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation. The Journal of Immunology, 2017, 199: 1261–1274.

acrophages (Mf) play a key role in the noninﬂamma- receptors mediating uptake, and factors regulating the sorting of tory disposal of apoptotic cells (1). Monocyte-derived apoptotic cells after phagocytosis or the coupling of phagocytosis M Mf from systemic lupus erythematosus (SLE) patients to anti-inﬂammatory pathways (11–14). By overwhelming normal are poorly phagocytic (2) and patients accumulate apoptotic cells in clearance mechanisms, an increased rate of cell death also may their tissues (3–6). Dead cells also accumulate in tissues of mice promote lupus (15–19).

with pristane-induced lupus (6), but not in mice treated with min- We show impaired clearance of dead cells by lupus bone marrow by guest on October 2, 2021 eral oil (MO), an inflammatory hydrocarbon that does not cause (BM) Mf and report a novel subset of peritoneal syndecan-1 lupus. Impaired phagocytosis of apoptotic cells promotes murine (CD138)+ Mf with an anti-inflammatory phenotype that efficiently lupus (7–9). Although phagocytosis is usually noninflammatory takes up apoptotic cells in the peritoneum. This subset is deficient (8, 9), impaired phagocytosis of dead cells in lupus facilitates in mice with pristane-induced lupus, resulting in impaired apo- endosomal recognition of self-nucleic acids by TLR7 and TLR9, ptotic cell clearance and inflammation. resulting in proinflammatory cytokine production (10). The out- come of phagocytosis (pro- versus anti-inflammatory) depends on Materials and Methods the release of damage-associated molecular patterns by dying cells, Patients whether the cells are apoptotic or necrotic, the type of phagocyte, BM core biopsies were identified from the University of Florida (UF) Department of Pathology archives. SLE was classified using American *Division of Rheumatology and Clinical Immunology, Department of Medicine, College of Rheumatology criteria (20, 21). Biopsies from adults with acute University of Florida, Gainesville, FL 32610; and †Department of Pathology, Immu- myelogenous leukemia (AML) undergoing myeloablation with cytarabine nology and Laboratory Medicine, University of Florida, Gainesville, FL 32610 plus daunorubicin 14-d earlier and children with B cell acute lymphocytic leukemia (B-ALL) treated with vincristine, prednisone, anthracycline, plus ORCID: 0000-0001-5182-5369 (J.W.). cyclophosphamide and/or L-asparaginase 8-d earlier were de-identified and Received for publication January 23, 2017. Accepted for publication June 12, 2017. examined by H&E staining and immunohistochemistry (IHC). The patients This work was supported by National Institute of Arthritis and Musculoskeletal and were not treated with radiation and did not receive cytokines or growth Skin Diseases/National Institutes of Health Research Grant R01-AR44731 and a factors in the week before BM biopsy. Biopsies in which marrow cellu- grant from the Lupus Research Institute. larity dropped from 100% to ,5% following myeloablation were selected Address correspondence and reprint requests to Dr. Westley H. Reeves, Division of for further study (n = 4). BM biopsies from patients undergoing myeloa- Rheumatology and Clinical Immunology, University of Florida, PO Box 100221, blation were compared with biopsies from SLE patients (n = 6) and con- Gainesville, FL 32610-0221. E-mail address: whreeves@ufl.edu trols undergoing BM biopsy for staging of lymphoma who had no evidence Abbreviations used in this article: AML, acute myelogenous leukemia; BAL, bron- of BM involvement (n = 6). The UF Institutional Review Board approved choalveolar lavage; B-ALL, B cell acute lymphocytic leukemia; BM, bone marrow; these studies. CD138, syndecan-1; CD138+ Mf, CD11b+F4/80intCD138+Tim42 subset; IHC, immunohistochemistry; LPM, large peritoneal MF;Mf, macrophage; MFI, mean fluo- Immunohistochemistry rescence intensity; MHCII, MHC class II; MO, mineral oil; PDE4, phosphodiesterase type 4; PEC, peritoneal exudate cell; PKA, protein kinase A; Poly-I, polyinosinic BM core biopsies were fixed in 10% neutral buffered formalin and decalcified acid; Q-PCR, quantitative PCR; R, region; SLE, systemic lupus erythematosus; SPM, (6). Four-micrometer sections were deparaffinized and underwent heat- small peritoneal MF; UF, University of Florida. induced epitope retrieval before staining with anti–cleaved-caspase-3 (Cell Signaling), anti–TNF-a (Abcam), and anti-CD68 (Dako) Abs followed by Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 peroxidase- or alkaline phosphatase–conjugated goat secondary Abs (6). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700099 1262 CD138+ MF IN LUPUS

Reaction product was visualized using ultraView DAB (brown) or Alkaline anti-CD11b, anti-Ly6C, anti-CD138, and anti-Ly6G Abs; washed; and Phosphatase Red Kits (Ventana). Slides were counterstained with hematox- fixed as above. ylin. Numbers of activated caspase-3+ cells (red) that did not colocalize with To assess the role of Marco in apoptotic cell uptake, IgG anti-Marco MF (brown) were determined as the mean number of red+brown2 cells per neutralizing Ab (ED-31; AbD Serotec) or isotype control were injected 1003 field (4 fields per patient). i.p. into MO-treated mice (100 mg/mouse). Thirty minutes later, pHrodo Red–labeled apoptotic cells were injected and uptake was determined 1.5 h Mice later as above. The class A scavenger receptor inhibitor polyinosinic acid (Poly-I) (Sigma) was used to further confirm the role of Marco. Poly-I (200 Mice were maintained under specific pathogen-free conditions at the UF mg/mouse) or PBS was injected i.p and 30 min later, pHrodo Red–labeled Animal Facility. C57BL/6 (B6) mice (Jackson Laboratory) received 0.5 ml apoptotic BW5147 cells were injected. Uptake was measured by flow of pristane (Sigma), MO (C.B. Fleet), 100 ng LPS from Salmonella enterica cytometry 1.5 h later. serotype Minnesota (Sigma), or PBS i.p. At indicated times, peritoneal In vitro phagocytosis of unopsonized polystyrene beads (3.2-mm di- exudate cells (PEC) were collected by lavage. Cells were analyzed within ameter) was measured by flow cytometry. PEC were obtained 1 wk after 1 h. Bronchoalveolar lavage (BAL) was performed after euthanizing the MO treatment and cultured for 1 h with PE-labeled polystyrene beads at a mice. A small incision was cut in the trachea and 1-ml PBS was injected 10:1 bead/cell ratio (DakoCytomation). Cells then were stained with anti- using a 20-gauge plastic feeding tube (Instech Laboratories). Lung CD11b, -Ly6C, -Ly6G, and -CD138 Abs. The percentage of CD11b+ washings were analyzed within 1 h. Animal studies were approved by the 2 2 2 2 2 Ly6G Ly6C CD138+ and CD11b+Ly6G Ly6C CD138 cells that had UF Institutional Animal Care and Use Committee. taken up PE-labeled beads was determined by flow cytometry. Quantitative PCR CREB activation Quantitative PCR (Q-PCR) was performed as described (6) using RNA 6 Peritoneal cells from untreated mice were incubated with PBS or LPS (100 extracted from 10 peritoneal cells (RNA Isolation Kit; Qiagen). cDNA ng/ml) for 30 min. Total CREB and p-CREB staining was determined by was synthesized using the Superscript II First-Strand Synthesis Kit (Invi- + + 2 + 2 +

flow cytometry in the CD11b CD138 Tim4 , CD11b CD138 Tim4 , and Downloaded from trogen) and SYBR Green Q-PCR analysis was performed using an Opticon CD11b+CD1382Ly6Chi subsets. In some experiments, mice were treated II thermo cycler (Bio-Rad). Primer sequences were: Mertk forward 59- 9 9 with the cell-permeable adenylate cyclase inhibitor SQ22536 (24). Mice GAGACCTCCACACCTTCCTG-3 , reverse 5 -GAGCTGCCAAATCCC- received MO (0.5 ml i.p.) on day 1, and SQ22536 (250 mg/d i.p) (25) for TATGA-39; Sra1 forward 59-CAACATCACCAACGACCTCA-39, reverse 9 9 9 9 d starting on day 1. Peritoneal cells were analyzed by flow cytometry at 5 -TGTCTCCCTTTTCACCTTGG-3 ; Marco forward 5 -AGCCGATTT- day 9. TGACCAAGCTA-39, reverse, 59-GTGAGCAGGATCAGGTGGAT-39; Srb1 forward 59-CCGAGAGTCTGGCATTCAG-39, reverse 59-TGGGTTAG- Statistical analysis 9 9 GGTTCAGACCAA-3 ; Gata6 forward 5 -TACAAGAACACCAACA- http://www.jimmunol.org/ CAGTCC-39, reverse 59-GGCGTCAAGAGTGTTACAGATAC-39; Pparg Statistical analyses were performed using Prism 6.0 software (GraphPad forward 59-CAAGGTGCTCCAGAAGATGA-39, reverse 59-GTGAAGG- Software). Differences between groups were analyzed by unpaired Student CTCATGTCTGTCT-39; Runx3 forward 59-CGTGTAACACCAAGCA- t test (normally distributed data) or Mann–Whitney U test (non-normally CACC-39, reverse AAGGGGTTCAGGTCTGAGGA-39; Car4 forward distributed data). Data are expressed as mean 6 SD for normally distrib- ACCTCTGACCTCAGCCTTTA-39, reverse CCACAGCCAGTTCCTCA- uted data sets. Normality was determined by the D’Agostino and Pearson TATT-39; Alox15 forward 59-GCTTTCCATAGTCTGCTGTAGT-39, re- omnibus test. All tests were two sided and a p value ,0.05 was considered verse 59-TTTCCCAAGCATGGCTATTATTTAC-39; b-actin forward 59- significant. TGGAATCCTGTGGCATCCTGAAAC-39, reverse 59-TAAAACGCAGC- TCAGTAACAGTCCG-39. Results

Flow cytometry and cell sorting Dead cells accumulate in BM and lung tissue of mice with pristane- by guest on October 2, 2021 induced lupus and SLE patients, and TNF-a is produced locally (6, Flow cytometry was performed as described (6). Cells were incubated with 26). To see if these abnormalities are associated with a phago- anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary Ab or isotype controls. A total of 10,000–50,000 events per cytosis defect, we compared BM from SLE patients and controls. sample were acquired using an LSRII Flow Cytometer (BD Biosciences) Impaired clearance of dead cells by human lupus Mf and analyzed with FlowJo flow cytometry software (Tree Star). The following Abs were used: CD11b-Brilliant Violet 421, Tim4-PE, CD138- SLE BM contained cells staining with Abs against activated caspase- allophycocyanin, CD138-PE F4/80- Pacific Blue, Ly6G-allophycocyanin- 3, an apoptosis marker (Fig. 1A, middle). In contrast, caspase-3+ cells Cy7, Ly6C-allophycocyanin-Cy7, CD80-PerCp-Cy5, CD40-PerCP-Cy5, CD206-PerCP-Cy5, CCR2-FITC, CX3CR1-FITC, CD169-allophycocyanin, were largely absent in BM from a non-SLE control (Fig. 1A, left). CCR5-allophycocyanin, CD36-PE, CD36-AlexaFluor 488, and IL-10R–PE Wright staining also showed numerous dead cells in patients’ BM (BioLegend); Marco-FITC (Bio-Rad); Ly6C-FITC, CD86-FITC, CD11c- aspirates (Fig. 1A, right). FITC, I-A/I-E–PE, and CD93-PE (BD Bioscience); CREB-PE and To assess the clearance of dead cells, we studied patients un- Phospho-CREB-FITC (Cell Signaling); and CD11b-Pacific Blue, CD45- dergoing myeloablative therapy for AML and B-ALL, which FITC, TLR4-PE, and CD45-FITC (eBioscience). Buffers used in intracellular staining were from eBioscience. For cell sorting, PEC from un- causes massive death of BM cells (27). BM from a child with treated B6 mice were stained with anti-CD11b, -Tim4, and -CD138 Abs. B-ALL exhibited 100% cellularity prior to myeloablative therapy, CD11b+Tim4+ and CD11b+CD138+ cells were gated and sorted (FACSaria but 8-d later was hypocellular (,5% cellularity) with erythroid Cell Sorter). Forty-thousand cells per subset were collected and lysed regeneration (Fig. 1B). Large phagocytic cells, probably resident immediately for RNA extraction. BM Mf, were visualized in the hypocellular marrow by H&E Phagocytosis of apoptotic cells and polystyrene beads staining and IHC showed activated caspase-3–stained material Mouse thymus lymphoma cells (BW5147) were induced into apoptosis by within endosomes/phagosomes (Fig. 1B). Similar results were heat shock (45˚C, 10 min) and cultured for 4 h (22). The percentage of obtained using day-14 BM from AML patients (data not shown). apoptotic (annexin-V+) cells was routinely .80% by this technique. After Caspase-3+ material was restricted to endosomes or phagosomes inducing apoptosis, cells were washed twice and labeled with pHrodo Red of CD68+ Mf (Fig. 1C, top left), whereas in SLE patients acti- (Life Technologies) (23). Uptake of labeled apoptotic cells was assayed + 6 vated caspase-3 was found in intact cells located outside of CD68 in vitro by adding 10 apoptotic cells in AIM-V medium to each well of a f 12-well plate containing 106 adherent peritoneal cells. Plates were incu- BM M (Fig. 1C, right). Quantification of the number of caspase- + + + bated for 2.5 h at 37˚C, washed three times with ice-cold PBS, and stained 3 cells outside of CD68 Mf confirmed that dead (caspase-3 ) with Pacific Blue–conjugated rat anti-mouse CD11b Ab (30 min, 4˚C). cells were removed inefficiently in SLE BM versus BM from non- Cells were washed again, detached with 1-ml ice-cold PBS using a cell SLE patients undergoing myeloablative therapy (p , 0.01, Stu- scraper, and fixed with 1% paraformaldehyde before flow cytometry. 3 7 dent t test) (Fig. 1D). There was no significant difference in cas- For in vivo assays, 2 10 pHrodo Red–labeled apoptotic cells were + injected i.p. into pristane- or MO-treated mice (1 wk after treatment). After pase-3 cells in BM from myeloablated patients versus untreated 1.5 h, PEC were collected by lavage; stained with fluorescently labeled controls. Human lupus BM also stained prominently for TNF-a The Journal of Immunology 1263

FIGURE 1. IHC for dead cells in human BM. (A) Single IHC staining (left and middle) of BM from a patient with SLE versus a control with idiopathic thrombocytopenic purpura using anti-activated caspase-3 Abs (red). Right, BM aspirate smear from an SLE patient showing the presence of uncleared dead cells (arrows, Wright–Giemsa stain). (B)H&E staining of BM core biopsies from a patient with B-ALL prior to myeloablation (100% cellularity,left)and8dafterchemotherapy (,5% cellularity, middle). Arrows show MF that have taken up cellular debris. Right, IHC of day-8 BM using anti–activated caspase-3 Abs (red). Arrows show activated caspase-3+ material inside MF.(C) Double IHC of BM core biopsies from the B-ALL patient (postmyeloablation, left) and from an SLE patient (right) for activated caspase-3 (red) and CD68 Downloaded from (brown). Arrows (left) show caspase-3/CD68 double-positive cells. (D) Percentages of activated intact caspase-3–positive cells (IHC) located outside of MF in BM from patients undergoing myeloablation (ablated, n =4) versus SLE patients (n = 6) and controls (cont, http://www.jimmunol.org/ n = 6). p , 0.01, ablated versus SLE. p = NS, ablated versus controls (Mann–Whitney U test). (E) IHC of BM from the postmyeloablation B-ALL patient (left) and an SLE patient (right) for TNF-a (brown). H&E and IHC staining patterns shown are representative of four patients undergoing myeloablation, six patients with SLE, and six healthy controls. Original magniﬁcation 31000 for (A)to(C)and(E), right panel. Original magniﬁcation 3400 for by guest on October 2, 2021 (E), left panel.

(Fig. 1E, bottom right) (6), whereas TNF-a was undetectable in Because these cells disappear in certain forms of inflammation BM from B-ALL 8 d after myeloablation (Fig. 1E, bottom left). (29–31), we examined whether differences in their numbers might Thus, nonlupus BM has an enormous capacity to clear dead cells explain the impaired phagocytosis of apoptotic cells in pristane- but clearance is impaired and there is TNF-a production in BM versus MO-treated mice. However, CD11b+Tim4+-resident Mf from SLE patients (Fig. 1) and pristane-treated mice (6). To ex- disappeared 7 d after i.p. injection of either pristane or MO plore the mechanism, we assessed the clearance of dead cells in (Fig. 2C). Thus, although Tim4+-resident Mf are highly phago- pristane-induced lupus. cytic, a differential effect of pristane and MO on this subset was unlikely to explain impaired phagocytosis of apoptotic cells in Impaired clearance of dead cells in pristane-induced lupus pristane-treated mice. In vitro phagocytosis assays revealed that CD11b+ PEC from MO- + f treated mice took up 2.5-fold more apoptotic cells than CD11b+ Peritoneum contains a population of phagocytic CD138 M f PEC from pristane-treated mice (p , 0.01, Student t test) distinct from resident M (Fig. 2A). The mean fluorescence intensity (MFI) of pHrodo Red In addition to resident Mf, normal peritoneum contains BM- in CD11b+ cells also was lower in pristane- versus MO-treated derived Mf (32, 33). We found an unusual subset of CD11b+ mice. Because in vitro culture alters Mf function, in vivo phago- CD138+ cells in the peritoneum of mice treated with pristane or cytosis was measured by injecting pHrodo Red–labeled apoptotic MO (Fig. 3). These cells were more numerous in MO- versus cells into the peritoneum followed 1.5 h later by flow cytometry for pristane- or PBS-treated mice (Fig. 3A), and they expanded after internalized target cells. Consistent with the in vitro data, a higher MO (and to a lesser degree pristane) treatment (Fig. 3B). In con- proportion of CD11b+ cells from MO-treated mice took up apo- trast, CD11b+Ly6Chi (inflammatory) monocytes rapidly increased ptotic target cells and the pHrodo Red MFI was higher in com- in both pristane- and MO- treated mice, but decreased substantially parison with pristane-treated mice (Fig. 2B). We conclude that the over 14 d in MO-, but not pristane-, treated mice. At 1 wk, neu- uptake of dead cells is impaired in pristane-induced lupus, as in trophils increased to a similar degree in pristane- and MO-treated SLE patients. mice (data not shown). Thus, MO-treated mice exhibited an in- In nontreated mice, the peritoneum contains a population creasing predominance of CD11b+CD138+ cells, whereas in of large, strongly phagocytic CD11b+Tim4+-resident Mf (28). pristane-treated mice CD11b+Ly6Chi cells predominated. 1264 CD138+ MF IN LUPUS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Phagocytosis of apoptotic cells in the peritoneum. (A) In vitro phagocytosis of pHrodo Red–labeled apoptotic BW5147 cells by CD11b+ cells from pristane-treated versus MO-treated B6 mice. (B) In vivo phagocytosis of pHrodo Red–labeled apoptotic BW5147 cells by CD11b+ cells from pristane- treated versus MO-treated mice. (C) Peritoneal cells from mice treated 7 d earlier with pristane, MO, or PBS were stained with anti-CD11b and anti-Tim4, and analyzed by ﬂow cytometry. Percentages of peritoneal CD11b+Tim4+ cells in pristane, MO, and PBS-treated mice are shown on the right. *p , 0.05, **p , 0.01, ***p , 0.001 (two-tailed t test).

Although CD138 is usually thought of as a plasma cell marker, taken up via Marco-mediated phagocytosis, whereas smaller CD11b+CD138+ cells took up pHrodo Red–labeled apoptotic cells particles are endocytosed (34). CD11b+CD138+ cells took up the more actively in vivo than Tim4+ Mf from the same mouse beads more actively than CD11b+CD1382 cells (13.2% of cells (Fig. 3C). In addition, in vivo uptake of apoptotic targets by versus 5.4%, p , 0.001, Student t test) (data not shown), sug- CD11b+Tim42CD138+ cells was considerably higher than that of gesting that the high phagocytic activity of CD11b+CD138+ is not CD11b+Tim42CD1382 cells (p , 0.001, Student t test) (Fig. 3D). limited to uptake of apoptotic cells. However, the uptake of Flow cytometry of PEC from MO-treated mice revealed four polystyrene particles was less efﬁcient than uptake of apoptotic subsets of CD11b+ cells, which are best visualized in BALB/c cells (Figs. 2, 3). mice but also seen in B6 mice (Fig. 3E): CD11b+Ly6Chi (region Levels of Marco mRNA, encoding a class A scavenger receptor [R]1), CD11bloLy6Cneg (R2), CD11bhiLy6Clo/2 (R3), and CD11b+ involved in uptake of apoptotic cells (35), was increased in PEC Ly6Cint neutrophils, which also were Ly6G+ (data not shown). The from MO- versus pristane-treated mice (p , 0.005, Student t test). main subset expressing CD138 was R3, which also expressed the Expression of the class B scavenger receptor Srb1 also was in- Mf-restricted scavenger receptor Marco (Fig. 3E). CD138+ creased (p , 0.02). In contrast, levels of Mertk and Sra1 mRNA Marco+ cells (R3) were considerably more phagocytic for apo- were comparable (Fig. 3G). The role of Marco in clearing apoptotic cells than other subsets (Fig. 3F). ptotic cells was examined by treating mice with MO followed by In addition, we incubated peritoneal cells from 1-wk, MO- i.p. injection of anti-Marco neutralizing Abs or an isotype control treated mice for 1 h with PE-labeled polystyrene beads (3.2 mm, before measuring in vivo phagocytosis (Fig. 3H, left). Blocking bead/cell ratio of 10:1) and measured uptake by ﬂow cytometry. Marco inhibited apoptotic cell uptake, suggesting that Marco is Unopsonized polystyrene particles with a diameter .0.5 mm are involved in internalization. Peritoneal injection of the class A The Journal of Immunology 1265 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Depletion of CD11b+Tim4+-resident Mf and expansion of CD11b+CD138+ cells due to peritoneal inflammation. B6 mice were injected i.p. with pristane, MO, or saline (PBS) and peritoneal cells were collected at day 7 and stained with anti-CD11b, -Tim4, and -CD138 monoclonal Abs followed by flow cytometry. (A) Flow cytometry of peritoneal cells 1 wk after pristane, MO, or PBS treatment stained with anti-CD11b and -CD138 monoclonal Abs. (B) Percentages of CD11b+CD138+ cells and CD11b+Ly6Chi monocytes in the peritoneum 2, 7, and 14 d after i.p. injection with pristane or MO. (C)Invivo phagocytosis of pHrodo Red–labeled apoptotic cells by peritoneal cells from untreated mice. Cell subsets were defined by staining with anti-CD11b, -Tim4, and -CD138 Abs. Phagocytosis by the CD11b+CD138+ and CD11b+Tim4+subsets were compared. Graph at the right shows phagocytosis by the CD11b+ CD138+ and CD11b+Tim4+ subsets from individual mice. (D) In vivo phagocytosis of pHrodo Red–labeled apoptotic cells by peritoneal cells from MO- treated mice. Cells were stained with anti-CD11b, -Ly6G, and -CD138 Abs. Gating on the CD11b+Ly6G2 subset, phagocytosis of pHrodo Red–labeled apoptotic cells by CD138+ versus CD1382 cells was compared. (E) Flow cytometry of peritoneal cells 1 wk after MO treatment of BALB/c mice using anti- CD11b, -Ly6C, -CD138, -Ly6G, and -Marco Abs. Cell subsets were defined by surface markers: neutrophils (Neut) were CD11b+Ly6Cint; Ly6Chi monocytes (R1) were CD11b+Ly6Chi; Ly6Clo Mf (R2) were CD11b+Ly6C+/2; CD11b+CD138+ cells (R3) were CD11b+Ly6C+/2CD138+. Representative of five BALB/c mice (similar results were obtained in five B6 mice, data not shown). (F) In vivo phagocytosis of pHrodo Red– (Figure legend continues) 1266 CD138+ MF IN LUPUS scavenger receptor antagonist poly(I) also inhibited uptake of la- did not change after pristane or MO treatment (Fig. 5C). Analysis of beled targets by CD11b+ cells (Fig. 3H, right). Taken together, the cells collected by BAL from MO-treated mice revealed two pop- data suggest that impaired phagocytosis of apoptotic cells in ulations of F4/80+CD11b+ lung Mf:largeCD138loLy6Clo Mf (R2) pristane- versus MO-treated mice may reflect the relative numbers and smaller CD138hiLy6Chi Mf (R1) (Fig. 5D). Both expressed of highly phagocytic CD11b+CD138+ cells, and expression of Marco IL-10R, but expression was higher in R1. The large (R2) cells are and possibly other scavenger receptors by these cells promotes the likely to be alveolar Mf, whereas the small cells are similar to clearance of dead cells. In contrast to the disappearance of Tim4+- peritoneal CD138+ Mf (but expressed higher levels of Ly6C than resident Mf,CD11b+CD138+ cells expanded during inflammation. peritoneal CD138+ Mf). BAL from pristane- or PBS-treated mice Although CD11b+CD138+ cells have been reported (36), relatively contained few CD138+ Mf (data not shown). Thus, CD138+ Mf little is known about them. are not restricted to the peritoneum and may develop from CD1382 BM precursors upon migration to sites of inflammation. CD11b+CD138+ cells resemble small peritoneal MF + f The peritoneum contains two distinct subsets of Mf: resident large CD138 M are induced by LPS peritoneal Mf (LPM) and BM-derived small peritoneal Mf To see if the induction of CD138+ Mf is relevant to infection, mice (SPM). The relationship of the Tim4+ Mf and CD11b+CD138+ received LPS (100 ng i.p.) and PEC were analyzed by flow cy- cells in the peritoneum of untreated mice to LPM and SPM was tometry. At day 7 the peritoneum of LPS-treated mice contained examined by gating on CD11b+ cells and comparing surface- variable numbers of Ly6G+ neutrophils and 20–40% CD138+ Mf, staining characteristics of the Tim4+ versus CD11b+CD138+ cells but few (2–5%) Ly6Chi inflammatory monocytes (Fig. 6A, 6B). (Fig. 4A). Tim4+ Mf expressed more CX3CR1 than CD11b+CD138+ LPS depleted Tim+CD1382-resident peritoneal Mf within 1 d cells and expressed both CD169 (Siglec-1) and CD93, which were (Fig. 6C). At day 3, small numbers of Tim4+ cells were detected in Downloaded from nearly undetectable on CD11b+CD138+ cells (Fig. 4A). In con- MO-treated mice and larger numbers in LPS-treated mice. Un- trast, CD11b+CD138+ cells expressed more CCR2, CCR5, and expectedly, at day 7, many Tim4+ cells in LPS-treated, but not MHC class II (MHCII) than Tim4+ Mf and also expressed CD11c, MO-treated mice, were CD138+. At 14–28 d, MO-treated mice which was absent on Tim4+ Mf. The two subsets exhibited similar also had peritoneal Tim4+CD138+ cells (Fig. 6C). Tim42CD1382 forward scatter, but side scatter was lower in the CD11b+CD138+ cells were very poorly phagocytic for apoptotic cells; whereas + 2 + + 2 + subset (Fig. 4B). The surface-staining and forward-/side-scatter Tim4 CD138 , Tim4 CD138 , and Tim4 CD138 cells could all http://www.jimmunol.org/ properties of CD11b+CD138+ cells are similar to those reported take up apoptotic cells (data not shown). However, there was not a for SPM (33). significant difference in the ability of the three latter subsets to Peritoneal cells from untreated B6 mice were stained with Abs to take up apoptotic cells. F4/80, CD11b, Tim4, and CD138 (Fig. 4C). About 60% of peri- 2 CD138+Tim4 Mf have an anti-inflammatory phenotype toneal CD11b+ cells were Tim4+CD1382-resident Mf, whereas ∼10% were CD138+Tim42 and ∼30% were Tim42CD1382. CD138 is a transmembrane proteoglycan encoded by Sdc1 (40). Consistent with the high expression of F4/80 on resident perito- Although expressed mainly on epithelial, mesenchymal, and neal Mf (32), the Tim4+CD1382 cells were F4/80hi. In contrast, plasma cells, it also is found on monocytes (41). Peritoneal

int by guest on October 2, 2021 the CD138+Tim42 subset was F4/80int (Fig. 4C). Gene expression methylated BSA–induced F4/80 Mf express CD138 and may differed in flow-sorted peritoneal CD11b+F4/80hiCD1382Tim4+- promote the resolution of inflammation (42–44). Consistent with + resident Mf versus the CD11b+F4/80intCD138+Tim42 subset that possibility, IL-10R surface staining was higher on CD138 hi 2 (CD138+ Mf) from untreated mice (Fig. 4D). In comparison with Mf than on Ly6C monocytes or CD138 Mf in both pristane- + Tim4+ Mf, CD138+ Mf expressed little Gata6 (specific for res- and MO-treated mice (Fig. 7A, 7B), and CD138 Mf exhibited hi ident peritoneal Mf), Pparg (inducible in peritoneal Mf), Runx3, lower intracellular TNF-a staining than Ly6C monocytes + and Car4 (inducible in peritoneal Mf). Alox15 encodes 12/15- (Fig. 7C). CD138 Mf from untreated mice also expressed high lipoxygenase, an enzyme involved in noninflammatory clearance levels of the M2 Mf marker CD206 (mannose receptor) and 2 + of apoptotic cells by resident peritoneal Mf (13, 37) and CD138 Tim4 -resident Mf expressed lower levels (Fig. 7D). + resolution-phase Mf (38). It is upregulated by the uptake of ap- Thus, CD138 Mf have a phenotype (low TNF-a production, optotic cells (39). However, although high levels were found in the high IL-10R, and high CD206) suggestive of anti-inflammatory/ Tim4+ Mf subset, CD138+ Mf expressed little Alox15 mRNA resolution-phase Mf. + (Fig. 4D). In contrast, CD138+ Mf expressed higher levels of Like CD138 Mf, resolution-phase Mf express M2 markers, Marco mRNA than Tim4+ Mf. respond poorly to TLR ligands, and are highly phagocytic (39, 45). Because resolution-phase Mf markedly downregulate CD11b fol- + f CD138 M are not restricted to the peritoneum lowing the uptake of apoptotic cells in vitro (39), we examined Although present in the peritoneum, CD138+ Mf were also found CD11b expression on CD138+ Mf. The expression level of in spleen from MO-treated and (in low numbers) pristane-treated CD11b was lower on CD138+ Mf from MO- versus pristane- mice (Fig. 5A). They were absent in spleen from PBS-treated mice treated mice (Fig. 7E, left). Following 20-h coculture of CD138+ (Fig. 5A). Spleen contains CD138+ plasma cells, but the CD138+ Mf from MO-treated mice with apoptotic BW5147 cells (apo- CD11b+ population expressed CD36, F4/80, CD80, IL-10R, and ptotic targets/Mf ratio 5:1), CD11b fluorescence intensity sub- Ly6C (Fig. 5B); verifying that these were CD138+ Mf. CD138+ stantially decreased (Fig. 7E, right). Thus, like resolution-phase Mf were present at low levels in BM from PBS-treated mice and Mf, CD138+ Mf also downregulate CD11b after taking up ap-

labeled apoptotic cells by peritoneal cells from MO-treated BALB/c mice. After coculture for 1.5 h with labeled apoptotic targets, the cells were stained and gated as in (E). Uptake of pHrodo Red–labeled apoptotic cells by neutrophils (neut), and the R1, R2, and R3 populations was compared. (G) Levels of mRNA encoding phagocytosis-related genes in peritoneal cells from pristane-treated versus MO-treated mice. (H)InvivophagocytosisofpHrodoRed–labeled apoptotic BW5147 cells by peritoneal CD11b+ cells from MO-treated mice injected i.p. 1 wk later with rat anti-mouse Marco neutralizing Abs or isotype control (100 mg/mouse, left) or treated i.p. with Poly-I (200 mg/mouse) or saline (PBS, right). *p , 0.05, **p , 0.01, ***p , 0.001 (two-tailed t test). The Journal of Immunology 1267 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Comparison of the CD11b+CD138+ and CD11b+Tim4+subsets. (A) Peritoneal cells from untreated B6 mice were stained with F4/80 plus anti-CD11b, -CD138, and -Tim4 monoclonal Abs and gated on CD11b+ cells. CD11b+CD138+ and CD11b+Tim4+subsets were analyzed for surface marker expression (flow cytometry). Isotype control: filled curve; CD11b+CD138+ subset: dashed line; CD11b+Tim4+subset: solid line. (B) Forward (FSC, size) and side (SSC, complexity) scatter characteristics of the CD11b+CD138+ and CD11b+Tim4+subsets. (C) Peritoneal cells from untreated mice were stained with anti-CD11b, -Tim4, -CD138, and -F4/80 monoclonal Abs followed by flow cytometry, gating on CD11b+ cells. Percentages of CD11b+Tim4+CD1382 and CD11b+Tim42CD138+ cells (left), and fluorescence intensity of F4/80 staining (right) are shown (representative of 10 mice). (D) Peritoneal CD11b+ Tim4+ and CD11b+CD138+ cells from untreated mice were flow sorted and gene expression was compared by real-time PCR using mRNA isolated from each subset. *p , 0.05 (two-tailed t test). optotic cells. These data suggest that the improved phagocytic factor CREB induces an anti-inflammatory gene expression pro- capacity in CD138+ cells from MO-treated mice drives increased gram in Mf (46). As CD138 (Sdc1) is CREB regulated (41), we reprogramming to the CD11blow phenotype. investigated CREB activation in CD138+ Mf. Total CREB levels were similar in CD11b+CD138+Tim42 and CD11b+CD1382 + f Increased CREB activation in CD138 M Tim4+ Mf from untreated mice but activated CREB (p-CREB) The phenotype of resolution-phase Mf is controlled by cAMP (45) was increased in CD11b+CD138+Tim42 Mf (Fig. 8A). CD11b+ and the cAMP/protein kinase A (PKA)-activated transcription CD138+Tim42 Mf also expressed higher levels of p-CREB after 1268 CD138+ MF IN LUPUS

FIGURE 5. CD11b+CD138+ Mf subset is present in spleen, lung, and BM. (A) Spleen cells from PBS-, pristane-, and MO-treated B6 mice were stained Downloaded from with anti-CD11b, -CD138, and -Ly6G. Cell subsets were deﬁned by surface markers. Percentages of CD11b+CD138+ cells were compared in different treatment groups. (B)SpleenCD11b+CD138+ cells from MO-treated mice were analyzed for CD36, F4/80, CD80, IL-10R, http://www.jimmunol.org/ and Ly6C expression. (C) BM CD11b+ CD138+ cells were compared in different treatment groups. (D) Lung CD11b+ CD138+ cells from MO-treated mice were analyzed for CD138, F4/80, CD11b, Ly6C, and IL-10R surface staining (ﬂow cytometry). ***p , 0.001 (two-tailed t test). FSC, forward light scatter; R1, small CD138hiLy6ChiF4/80+ Mf; R2, large CD138loLy6CloF4/80+ alveolar Mf; by guest on October 2, 2021 SSC, side scatter.

LPS treatment (30 min in vitro). Similar to CD138+ Mf, CD11b+ significant effect on either the fluorescence intensity of CD138 CD1382Tim4+-resident Mf exhibited higher levels of p-CREB staining or the percentage of CD138+ Mf (data not shown). after LPS treatment. (Fig. 8B, left). Compared with Tim4+-resident Mf, CD138+ Mf from untreated mice expressed higher Discussion levels of p-CREB (Fig. 8B, right). Both phosphorylated and total Phagocytosis of apoptotic cells by SLE patients’ monocytes is CREB were higher in CD138+ Mf versus Ly6Chi monocytes impaired in vitro (2) and dead cells accumulate in their tissues (3– (Fig. 8C). CD11b+CD138+ peritoneal Mf from mice treated daily 5) and also in tissues of mice with pristane-induced lupus (6, 47). with the adenylate cyclase inhibitor SQ22536 had higher surface The present study suggests that the accumulation of dead cells Ly6C staining than controls and produced more TNF-a (intra- occurs in the setting of impaired clearance by Mf. Reduced cellular staining), suggesting that treatment promoted a proin- clearance of apoptotic cells in lupus mice was associated with flammatory phenotype (Fig. 8D). SQ22536 treatment had no low numbers of a novel subset of CD138+ Mf that was highly The Journal of Immunology 1269

FIGURE 6. CD11b+CD138+ Mf subset is induced by LPS. B6 mice were injected with LPS (100 ng i.p.) and peritoneal cells were analyzed by ﬂow

cytometry 0–7 d later for CD138 and Downloaded from CD11b (top four panels) or Ly6C and CD11b (bottom two panels). (A) Stain- ing of peritoneal cells from LPS-treated mice with anti-CD11b, anti-CD138, anti- Ly6C, and anti-Ly6G Abs. (B)CD138+ Mf in B6 mice before (day 0) and 7 d

after LPS treatment. (C)Flowcytometry http://www.jimmunol.org/ of peritoneal cells from PBS (0.5 ml i.p., toprow),LPS(100ngi.p.inPBS,middle row), and MO (0.5 ml i.p., bottom row) treated BALB/c mice with anti- CD138 and -Tim4 Abs (days 1–28). ***p , 0.001 (two-tailed t test). by guest on October 2, 2021

phagocytic for apoptotic cells and had an anti-inflammatory (Fig. 1), consistent with other evidence that apoptotic cells are phenotype. Phagocytosis was mediated partly by the scavenger cleared efficiently without inducing inflammation (52). In con- receptor Marco. CREB, which regulates CD138 expression and trast, dead cells accumulate in lupus BM and induce local TNF-a induces an anti-inflammatory transcriptional program, was acti- production (Fig. 1) (6). In pristane-induced lupus, phagocytosis of vated in these CD138+ Mf (41, 46, 48). apoptotic cells was impaired in vitro and in vivo (Fig. 2). This was not seen in mice treated with MO, an inflammatory but nonlupus- Impaired phagocytosis of dead cells in lupus inducing hydrocarbon oil. Defective apoptotic cell removal may Most tissues contain self-renewing resident Mf derived from contribute to the pathogenesis of lupus by activating innate embryonic precursors that seed the tissues before birth (49–51). immunity (7). During inflammation, resident Mf are transiently complemented f by short-lived recruited monocytes that differentiate in situ into Pristane impairs generation of M that clear apoptotic cells Mf (49). The accumulation of dead cells in patients’ tissues (3–6) CD11b+Tim4+-resident peritoneal Mf, which actively take up suggests that phagocytosis is abnormal, cell death is increased, or apoptotic cells, were reduced by .90% shortly after pristane or both. Cell death is increased in SLE patients (15–17) and mice MO treatment, consistent with prior studies showing that perito- with pristane-induced lupus (18, 19). But dead cells were effi- neal inflammation rapidly depletes resident peritoneal Mf (29, ciently cleared by BM Mf in nonlupus patients undergoing 53). This “Mf disappearance reaction” (31) is caused by reloc- myeloablative therapy without triggering TNF-a production alization of CD11b+Tim4+ (Gata6+)-resident peritoneal Mf to 1270 CD138+ MF IN LUPUS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. CD11b+CD138+ Mf subset has an anti-inflammatory phenotype. (A) IL-10R expression (MFI) in different cell subsets of peritoneal cells from pristane- and MO-treated B6 mice (1 wk after IP injection): Ly6Chi monocytes, Ly6ChiCD11b+; CD138+ Mf,Ly6Clo/2Ly6G2CD11b+ CD138+; CD1382 Mf, Ly6Clo/2Ly6G2CD11b+CD1382.(B) Quantification of IL-10R expression in Ly6Chi monocytes versus CD138+ Mf from pristane- and MO-treated mice. Gating for Mf subsets was done as in (A). (C) Comparison of intracellular TNF-a staining in CD11b+Ly6Chi monocytes (Ly6Chi) and the CD11b+CD138+ Mf subset (CD138+) from pristane- and MO-treated mice. (D) CD206 staining. Peritoneal cells from untreated B6 mice were stained with Abs against CD11b, Tim4, CD138, and CD206. CD206 staining was compared between the CD11b+CD138+ (dashed line) and CD11b+Tim4+subsets (solid line). Filled curve, isotype control. (E) CD11b expression is downregulated by uptake of apoptotic cells. Left, CD11b staining of CD138+ cells from pristane- versus MO-treated mice. Right, effect of apoptotic cells on CD11b expression on CD138+ Mf from MO-treated mice. Flow-sorted CD138+ Mf werecoculturedfor20hwithapoptotic BW5147 cells (apoptotic cells/Mf ratio of 5:1) and fluorescence intensity of CD138 was determined on the CD138+ Mf by flow cytometry. Shaded curve, isotype control. *p , 0.05, **p , 0.01, ****p , 0.0001 (two-tailed t test). The Journal of Immunology 1271

FIGURE 8. CREB activation. (A) Flow cytometry of total CREB and activated (p-CREB) staining. Peritoneal cells from untreated B6 mice were incubated with PBS or LPS (100 ng/ml) for 30 min. Total CREB and p-CREB staining was compared be- + + 2

tween the CD11b CD138 Tim4 (dashed Downloaded from line) and CD11b+CD1382Tim4+subsets (solid line). Filled curve, isotype control. (B) p-CREB levels in CD11b+CD138+ Tim42 and CD11b+CD1382Tim4+ cells from untreated mice. Data are representative of five separate experiments, total 20 hi mice. (C) Activated CREB in Ly6C mono- http://www.jimmunol.org/ cytes versus CD138+ Mf . Left, comparison of p-CREB staining by flow cytometry in Ly6Chi monocytes (dashed line) and CD138+ Mf (solid line) from MO-treated mice. Middle, p-CREB staining (MFI) of Ly6Chi monocytes versus CD138+ Mf. Right, total CREB staining (MFI) of Ly6Chi monocytes versus CD138+ Mf.(D) Effect of adenylate cyclase inhibition. Mice were treated with MO (0.5 ml i.p.) plus either SQ22536 (250 mg by guest on October 2, 2021 i.p. daily) or vehicle. At day 9, PEC were surface stained for CD11b, CD138, and Ly6C, and intracellularly stained for TNF-a. The percentage of CD11b+CD138+ cells positive for TNF-a (left) and the MFI of Ly6C staining (right) was determined by flow cytometry. *p , 0.05, **p , 0.01 (two-tailed t test).

omental milky spots (30). CD138+ Mf were more active than peritoneal myeloid subset at taking up apoptotic cells, and their CD11b+Tim4+ Mf at taking up apoptotic cells and were not de- relative deficiency may explain the decreased clearance of apoptotic pleted by peritoneal inflammation, but were affected differentially cells in pristane- versus MO-treated mice. CD138+ Mf were not by pristane and MO. In MO-treated mice, CD138+ Mf expanded unique to pristane- or MO-induced inflammation, as they appeared and Ly6Chi monocytes decreased, whereas high levels of Ly6Chi in the peritoneum 7 d after LPS treatment (Fig. 6). Thus, CD138+ monocytes and lower levels of CD138+ Mf were maintained in Mf may play a physiological role in resolving infectious as well as pristane-treated mice (Fig. 3). CD138+ Mf were the most active sterile inflammation, possibly by promoting clearance of dead cells. 1272 CD138+ MF IN LUPUS

Role of scavenger receptors in CD138+ Mf CD138+ Mf have an anti-inflammatory phenotype Impaired phagocytosis of apoptotic cells promotes murine lupus (7). Although an oversimplification (64), BM-derived Mf have been Two phagocytic processes remove dead cells: opsonin-dependent classified into two subtypes: M1 (classically activated, proin- (e.g., Fc receptors, complement receptors, and TAM receptors) flammatory) and M2 (alternatively activated, associated with reso- and opsonin-independent (including phosphatidylserine receptors, lution). M1/M2 polarization is influenced by transcription factors such as Tim4) processes (9). Opsonins such as MFG-E8, Gas6, and (64, 65) and epigenetic regulation (66). Mouse M1 Mf express protein S are involved in the recognition of apoptotic cells by iNOS (Nos2) and high levels of Ly6C, MHCII, and CCR2, and integrins and the TAM receptors (54–56). This plays a major role in produce TNF-a,IL-1b, and IL-12; whereas M2 Mf express argi- clearing apoptotic cells. In contrast, Marco and other scavenger nase 1 (Arg1), scavenger receptors, CX3CR1, and low levels of receptors recognize oxidized low-density lipoprotein on the surface Ly6C, and produce TGF-b and IL-10 (66). CD138+ Mf exhibited of apoptotic cells without a requirement for opsonization, promoting some features of both. Consistent with an M1 phenotype, they their clearance and inhibiting cytokine/chemokine responses (35, expressed high levels of MHCII and CCR2 and low levels of 2 57, 58). CD138+ Mf expressed the class A scavenger receptor CX3CR1 (Fig. 4). But they were Ly6C and expressed CD206 Marco and class B scavenger receptors SR-B1 and CD36. Treat- (Fig. 7), an M2c Mf marker expressed on BM-derived, but not ment with anti-Marco neutralizing Abs or the class A scavenger resident, M2-like Mf (63, 67, 68). Moreover, like other pro- + receptor antagonist Poly-I reduced phagocytosis of apoptotic cells resolving Mf (39), CD138 Mf downregulated CD11b, a com- by ∼1/3 (Fig. 3), suggesting that clearance involves Marco. CD138+ ponent of complement receptor 3, following coculture with 2 + f Mf also took up polystyrene particles more actively than CD138 apoptotic cells (Fig. 7E). In blood vessels, CD138 M are anti-

Mf. Consistent with the role of Marco in clearing apoptotic cells, inflammatory (36). A CREB and cAMP/PKA-regulated transcrip- Downloaded from phagocytosis of unopsonized particles also is Marco dependent (34). tional program controls Sdc1 (CD138), Klf4, Arg1, Il10,andother Marco promotes noninflammatory phagocytosis of apoptotic cells genes involved in M2 polarization (41, 46, 48). The increased levels + f and is expressed constitutively on marginal zone Mf and some tissue of p-CREB in CD138 M and high levels of IL-10R expression Mf. It is induced on other Mf subsets via TLR signaling (9, 51). A are consistent with an M2-like phenotype (Fig. 7). We speculate that hi lo Marco defect contributes to lupus in BXSB male mice (59), and the pattern of chemokine receptor expression (CCR2 CX3CR1 ) might allow anti-inflammatory CD138+ Mf to migrate to the same autoantibodies against Marco impair apoptotic cell uptake in murine hi hi http://www.jimmunol.org/ and human lupus (57, 60). Moreover, anti–DNA autoantibody pro- sites as proinflammatory Ly6C (CCR2 ) monocytes, promoting res- + f duction is increased in Marco-deficient mice following injection of olution of inflammation. Thus, CD138 M exhibit several features f apoptotic cells (57). Our data support the idea that Marco deficiency of resolution-phase M , including an M2-like surface phenotype promotes lupus. Expression of SR-B1 also was low in pristane- with some M1 markers, high phagocytic activity, and downregula- treated mice (Fig. 3). Because human SR-B1 recognizes apoptotic tion of CD11b following the uptake of apoptotic cells (39, 45). Finally, our studies suggest that the cAMP/PKA-regulated cells (61), it will be of interest to see if it plays a similar role in mice. transcription factor CREB may help maintain the M2-like or + CD138 Mf resemble SPM proresolving phenotype of CD138+ Mf. The increased level of p- +

f by guest on October 2, 2021 Normal peritoneum contains a major population of self-renewing CREB in CD138 M supports that possibility (Fig. 8). CREB resident LPM established during fetal life and a minor (∼10%) activation promotes M2 polarization (46, 69) and the phenotype of f population of BM-derived SPM (33). Gata6, a master transcriptional resolution-phase M is controlled by cAMP (45). Although regulator of resident peritoneal Mf (29, 30), was expressed at high CD138 expression is regulated by cAMP/PKA and CREB (41), levels by Tim4+CD1382 LPM, as expected (Fig. 4D). The near we did not see lower CD138 surface staining in mice treated with absence of Gata6 expression in Tim42CD138+ Mf and intermediate the adenylate cyclase inhibitor SQ22536 (data not shown). How- a F4/80 staining (Fig. 4C) are consistent with BM-derived SPM (32, ever, Ly6C staining and TNF- were increased, consistent with a 33). Their surface phenotype further supports the close relationship shift toward M1-like polarization (Fig. 8D). It remains unclear of CD138+ Mf to SPM (33). Both express high levels of MHCII whether CD138 is a component of a transcriptional program that and low or intermediate levels of F4/80, but not CD93 (AA4.1) dampens inflammatory responses (36), or if it is only a marker for f or Ly6C (Fig. 4). In contrast to LPM, CD138+ Mf (Figs. 2, 3) and a subset of anti-inflammatory/resolution-phase M . Inhibition of SPM (33) do not disappear from the peritoneum during inflam- cyclic nucleotide phosphodiesterase type 4 (PDE4), which hy- drolyzes cAMP, modulates disease progression in MRL/Mp–lpr/ mation. However, the kinetics of appearance/disappearance of lpr lupus mice (70) and the PDE4 inhibitor apremilast is beneficial F4/80hiCD11bhi (LPM-like) and F4/80intCD11bint (SPM-like) cells may in human discoid lupus (71). It will be of interest in the future to vary depending on the model of peritoneal inflammation used (62). see if PDE4 inhibitors promote expansion of the CD138+ Mf In contrast to previous reports, our studies suggest that CD138+ subset via CREB activation. Mf or SPM are not restricted to the peritoneum, as they were detected in the spleen and lungs of pristane- and MO-treated mice (Fig. 5). They did not change in the BM after treatment, sug- Disclosures gesting that CD138+ Mf arise peripherally by differentiation of The authors have no financial conflicts of interest. CD1382 BM precursors. Ly6Chi monocytes can give rise to SPM during inflammation, suggesting that Ly6C+ cells expressing low References + + levels of CD138 (Fig. 3E) and possibly the CD138 Ly6C lung 1. Serhan, C. N., and J. Savill. 2005. Resolution of inflammation: the beginning Mf (Fig. 5D) are an intermediate stage in the differentiation of programs the end. Nat. Immunol. 6: 1191–1197. CD138+Ly6C2 Mf from CD1382Ly6Chi precursors. 2. Herrmann, M., R. E. Voll, O. M. Zoller, M. Hagenhofer, B. B. Ponner, and + + J. R. Kalden. 1998. Impaired phagocytosis of apoptotic cell material by A population of CD138 Tim4 Mf was found in LPS-treated monocyte-derived macrophages from patients with systemic lupus eryth- mice (Fig. 6C) and similar cells appeared later on in MO-treated ematosus. Arthritis Rheum. 41: 1241–1250. mice. These cells resemble a subset of alternatively activated resi- 3. Baumann, I., W. Kolowos, R. E. Voll, B. Manger, U. Gaipl, W. L. Neuhuber, f T. Kirchner, J. R. Kalden, and M. Herrmann. 2002. Impaired uptake of apoptotic dent M (63). However, further studies are needed to confirm that cells into tingible body macrophages in germinal centers of patients with sys- they are not BM derived. temic lupus erythematosus. Arthritis Rheum. 46: 191–201. The Journal of Immunology 1273

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