Type I Interferon Modulates Monocyte Recruitment and Maturation in Chronic Inflammation
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.090328
ISSN1525-2191
AutoresPui Y. Lee, Yi Li, Yutaro Kumagai, Yuan Xu, Jason S. Weinstein, Erinn S. Kellner, Dina C. Nacionales, Edward J. Butfiloski, Nico van Rooijen, Shizuo Akira, Eric S. Sobel, Minoru Satoh, Westley H. Reeves,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoChronic inflammation is characterized by continuous recruitment and activation of immune cells such as monocytes in response to a persistent stimulus. Production of proinflammatory mediators by monocytes leads to tissue damage and perpetuates the inflammatory response. However, the mechanism(s) responsible for the sustained influx of monocytes in chronic inflammation are not well defined. In chronic peritonitis induced by pristane, the persistent recruitment of Ly6Chi inflammatory monocytes into the peritoneum was abolished in type I interferon (IFN-I) receptor-deficient mice but was unaffected by the absence of IFN-γ, tumor necrosis factor-α, interleukin-6, or interleukin-1. IFN-I signaling stimulated the production of chemokines (CCL2, CCL7, and CCL12) that recruited Ly6Chi monocytes via interactions with the chemokine receptor CCR2. Interestingly, after 2,6,10,14-tetramethylpentadecane treatment, the rapid turnover of inflammatory monocytes in the inflamed peritoneum was associated with a lack of differentiation into Ly6Clo monocytes/macrophages, a more mature subset with enhanced phagocytic capacity. In contrast, Ly6Chi monocytes differentiated normally into Ly6Clo cells in IFN-I receptor-deficient mice. The effects of IFN-I were specific for monocytes as granulocyte migration was unaffected in the absence of IFN-I signaling. Taken together, our findings reveal a novel role of IFN-I in promoting the recruitment of inflammatory monocytes via the chemokine receptor CCR2. Continuous monocyte recruitment and the lack of terminal differentiation induced by IFN-I may help sustain the chronic inflammatory response. Chronic inflammation is characterized by continuous recruitment and activation of immune cells such as monocytes in response to a persistent stimulus. Production of proinflammatory mediators by monocytes leads to tissue damage and perpetuates the inflammatory response. However, the mechanism(s) responsible for the sustained influx of monocytes in chronic inflammation are not well defined. In chronic peritonitis induced by pristane, the persistent recruitment of Ly6Chi inflammatory monocytes into the peritoneum was abolished in type I interferon (IFN-I) receptor-deficient mice but was unaffected by the absence of IFN-γ, tumor necrosis factor-α, interleukin-6, or interleukin-1. IFN-I signaling stimulated the production of chemokines (CCL2, CCL7, and CCL12) that recruited Ly6Chi monocytes via interactions with the chemokine receptor CCR2. Interestingly, after 2,6,10,14-tetramethylpentadecane treatment, the rapid turnover of inflammatory monocytes in the inflamed peritoneum was associated with a lack of differentiation into Ly6Clo monocytes/macrophages, a more mature subset with enhanced phagocytic capacity. In contrast, Ly6Chi monocytes differentiated normally into Ly6Clo cells in IFN-I receptor-deficient mice. The effects of IFN-I were specific for monocytes as granulocyte migration was unaffected in the absence of IFN-I signaling. Taken together, our findings reveal a novel role of IFN-I in promoting the recruitment of inflammatory monocytes via the chemokine receptor CCR2. Continuous monocyte recruitment and the lack of terminal differentiation induced by IFN-I may help sustain the chronic inflammatory response. Chronic inflammation is a pathological condition characterized by unremitting production of cytokines and other mediators in response to persistent microbial infection or chemical agents.1Kumar V Abbas AK Fausto N Robbins SL Cotran RS Acute and chronic inflammation.in: Kumar V Abbas A Fausto N Robbins & Cotran Pathologic Basis of Disease. 7th ed. Elsevier Health Sciences, Philadephia2004: 50Google Scholar In autoimmune disorders such as systemic lupus erythematosus (SLE), endogenous antigens also may play an important role in perpetuating a chronic inflammatory state.2Herrmann M Voll RE Zoller OM Hagenhofer M Ponner BB Kalden JR Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus.Arthritis Rheum. 1998; 41: 1241-1250Crossref PubMed Scopus (716) Google Scholar Regardless of the cause, a common feature of chronic inflammation is the persistent homing of circulating monocytes to the site of injury.1Kumar V Abbas AK Fausto N Robbins SL Cotran RS Acute and chronic inflammation.in: Kumar V Abbas A Fausto N Robbins & Cotran Pathologic Basis of Disease. 7th ed. Elsevier Health Sciences, Philadephia2004: 50Google Scholar Two major subsets of blood monocytes have been identified in mice. Inflammatory monocytes (also called Ly6Chi monocytes), characterized by surface expression of Ly6C and the chemokine receptor CCR2, are released from the bone marrow and actively recruited to inflamed tissue.3Geissmann F Jung S Littman DR Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2634) Google Scholar Under steady-state conditions, Ly6Chi monocytes mature in the circulation into residential (Ly6Clo) monocytes, which express CX3CR1 and infiltrate noninflamed tissues where they differentiate into residential tissue macrophages.3Geissmann F Jung S Littman DR Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2634) Google Scholar, 4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar On their recruitment to sites of injury, inflammatory monocytes also can differentiate into macrophages with enhanced phagocytic activity or into dendritic cells, which capture antigens for presentation to lymphocytes.5Serbina NV Pamer EG Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.Nat Immunol. 2006; 7: 311-317Crossref PubMed Scopus (1219) Google Scholar, 6Ginhoux F Tacke F Angeli V Bogunovic M Loubeau M Dai XM Stanley ER Randolph GJ Merad M Langerhans cells arise from monocytes in vivo.Nat Immunol. 2006; 7: 265-273Crossref PubMed Scopus (552) Google Scholar, 7Shortman K Liu YJ Mouse and human dendritic cell subtypes.Nat Rev Immunol. 2002; 2: 151-161Crossref PubMed Scopus (1919) Google Scholar, 8Swirski FK Libby P Aikawa E Alcaide P Luscinskas FW Weissleder R Pittet MJ Ly-6C monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata.J Clin Invest. 2007; 117: 195-205Crossref PubMed Scopus (1004) Google Scholar Tissue monocytes/macrophages also can amplify the immune response by secreting inflammatory cytokines and chemokines to recruit additional immune cells. In chronic inflammation, however, persistent production of these inflammatory mediators can lead to tissue damage, resulting in cycles of tissue destruction and repair. The mechanism(s) responsible for the continuous recruitment of monocytes in chronic inflammation are not well defined. Intraperitoneal administration of 2,6,10,14 tetramethylpentadecane (TMPD; also known as pristane) potently induces chronic inflammation in mice. The influx of mononuclear and polymorphonuclear leukocytes to the peritoneal cavity persists for months after injection of this hydrocarbon oil.9Cancro M Potter M The requirement of an adherent cell substratum for the growth of developing plasmacytoma cells in vivo.J Exp Med. 1976; 144: 1554-1567Crossref PubMed Scopus (74) Google Scholar The chronic inflammatory state also promotes tumorigenesis, as TMPD was found to induce plasmacytomas more than three decades ago.10Anderson PN Potter M Induction of plasma cell tumours in BALB-c mice with 2,6,10,14-tetramethylpentadecane (pristane).Nature. 1969; 222: 994-995Crossref PubMed Scopus (153) Google Scholar TMPD is used to stimulate monoclonal antibody production by hybridoma cells injected i.p., an effect of chronic interleukin (IL)-6 production in response to the oil.11Hoogenraad N Helman T Hoogenraad J The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production.J Immunol Methods. 1983; 61: 317-320Crossref PubMed Scopus (99) Google Scholar TMPD treatment stimulates the development of ectopic lymphoid tissue, a feature often associated with chronic inflammatory states.9Cancro M Potter M The requirement of an adherent cell substratum for the growth of developing plasmacytoma cells in vivo.J Exp Med. 1976; 144: 1554-1567Crossref PubMed Scopus (74) Google Scholar Ectopic lymphoid tissue may play a key role in the induction of lupus-like autoimmune disease (autoantibodies, glomerulonephritis, arthritis, and pulmonary vasculitis) in TMPD-treated mice.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Chowdhary VR Grande JP Luthra HS David CS Characterization of haemorrhagic pulmonary capillaritis: another manifestation of pristane-induced lupus.Rheumatology (Oxford). 2007; 46: 1405-1410Crossref PubMed Scopus (64) Google Scholar How TMPD causes chronic inflammation is not well understood, but a pathological role of several cytokines has been suggested. IL-6 is essential for generating plasmacytomas,14Richards HB Satoh M Shaheen VM Yoshida H Reeves WH Induction of B cell autoimmunity by pristine.Curr Top Microbiol Immunol. 1999; 246: 387-392; discussion 393PubMed Google Scholar whereas IL-12 is required for the development of TMPD-induced glomerulonephritis.15Calvani N Satoh M Croker BP Reeves WH Richards HB Nephritogenic autoantibodies but absence of nephritis in Il-12p35-deficient mice with pristane-induced lupus.Kidney Int. 2003; 64: 897-905Crossref PubMed Scopus (47) Google Scholar Recently, it was found that type I interferons (IFN-I, including IFN-α and IFN-β) and type II interferon (IFN-γ) both contribute to autoantibody production in TMPD-treated mice.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Richards HB Satoh M Jennette JC Croker BP Yoshida H Reeves WH Interferon-γ is required for lupus nephritis in mice treated with the hydrocarbon oil pristane.Kidney Int. 2001; 60: 2173-2180Crossref PubMed Scopus (112) Google Scholar Using TMPD-induced chronic peritonitis as a model, we examined the mechanism(s) underlying the chronic recruitment of monocytes and granulocytes to the inflamed peritoneum. These studies were approved by the institutional animal care and use committee. Wild-type C57BL/6, IL-6−/−, TNF-α−/−, CCR2−/−, and IL-1R−/− mice (all on a C57BL/6 background), C3H/HeJ, C3H/HeOuJ, BALB/c, and BALB/c.IFNγ−/− mice were from The Jackson Laboratory (Bar Harbor, ME). 129Sv/Ev type I interferon receptor α-chain-deficient (IFNAR−/−) mice and wild-type controls (129Sv/Ev), originally from B&K Universal Limited (Grimston, Aldbrough, UK), were bred in our facility. MyD88−/− and IFNAR−/− mice (both on a C57BL/6 background) and littermate controls were bred and maintained in a specific pathogen-free facility at Osaka University. All other mice were maintained in a specific pathogen-free facility at the University of Florida. Mice (8 to 10 weeks old) received TMPD (0.5 ml i.p., Sigma-Aldrich, St. Louis, MO), 4% thioglycollate (BD Biosciences, San Jose, CA), recombinant mouse IFN-α5 (3 × 104 units in 200 μl of PBS, PBL InterferonSource, Piscataway, NJ), or PBS. Peripheral blood and peritoneal exudate cells (PECs) were isolated as described previously.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Scumpia PO McAuliffe PF O'Malley KA Ungaro R Uchida T Matsumoto T Remick DG Clare-Salzler MJ Moldawer LL Efron PA CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis.J Immunol. 2005; 175: 3282-3286PubMed Google Scholar For morphological analysis, 5 × 104 sorted cells were cytocentrifuged onto glass slides (Fisher Scientific, Pittsburgh, PA) and stained using a Hema3 kit (Fisher). Q-PCR was performed as described.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar In brief, total RNA was extracted from 106 peritoneal cells using TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using a SuperScript II First-Strand Synthesis Kit (Invitrogen). Q-PCR was performed using the SYBR Green Core Reagent Kit (Applied Biosystems, Foster City, CA) with an Opticon II thermocycler (MJ Research, Waltham, MA). Amplification conditions were 95°C for 10 minutes, followed by 45 cycles of 94°C for 15 seconds, 60°C for 25 seconds, and 72°C for 25 seconds. After the final extension (72°C for 10 minutes), a melting curve analysis was performed to ensure specificity of the products. Primers used in this study have been described,18Lee PY Weinstein JS Nacionales DC Scumpia PO Li Y Butfiloski E van Rooijen N Moldawer L Satoh M Reeves WH A novel type I IFN-producing cell subset in murine lupus.J Immunol. 2008; 180: 5101-5108PubMed Google Scholar, 19Lee PY Kumagai Y Li Y Takeuchi O Yoshida H Weinstein J Kellner ES Nacionales D Barker T Kelly-Scumpia K van Rooijen N Kumar H Kawai T Satoh M Akira S Reeves WH TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus.J Exp Med. 2008; 205: 2995-3006Crossref PubMed Scopus (184) Google Scholar except for the following: CCL7 (forward 5′-GATCTCTGCCACGCTTCTGT-3′ and reverse 5′-ATAGCCTCCTCGACCCACTT-3′); CCL12 (forward 5′-GTCCTCAGGTATTGGCTGGA-3′ and reverse 5′-CACTGGCTGCTTGTGATTCT-3′); CX3CL1 (forward 5′-CGCGTTCTTCCATTTGTGTA-3′ and reverse 5′-AGCTGATAGCGGATGAGCAA-3′); CXCL 1 (forward 5′-GCTGGGATTCACCTCAAGAA-3′ and reverse 5′-TCTCCGTTACTTGGGGACAC-3′); arginase-1 (forward 5′-GCACTGAGGAAAGCTGGTCT-3′ and reverse 5′-GACCGTGGGTTCTTCACAAT-3′); TGF-β (forward 5′-AGCCCGAAGCGGACTACTAT-3′ and reverse 5′-TTCCACATGTTGCTCCACAC-3′) cathepsin S (forward 5′-ACCTACCAAGTGGGCATGAA-3′ and reverse 5′-TGTCAGGCAATGTCCGATTA); cathepsin L (forward 5′-CCATCCGTCTCTCCAGTTCT-3′ and reverse 5′-ATAGCCATAGCCCACCAACA-3′); MMP-2 (forward 5′-CAGAACACCATCGAGACCAT-3′ and reverse 5′-CCAGGTCAGGTGTGTAACCA-3′); and MMP-9 (forward 5′-CATGCACTGGGCTTAGATCA-3′ and reverse 5′-GGCTTAGAGCCACGACCATA-3′). All antibodies were purchased from BD Biosciences with the exception of anti-F4/80-FITC, anti-Sca-I-PE, avidin-APC (eBioscience, San Diego, CA), and anti-CD11b-Pacific blue (Caltag Laboratories, Burlingame, CA). Cell staining was performed as described.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar CCR2 expression on monocytes was assessed using rat anti-mouse CCR2 antibody (clone MC-21, a kind gift from Dr. Matthias Mack, Regensburg University Medical Center, Regensburg, Germany) and rat IgG2b isotype control (BD Biosciences) as described.20Mack M Cihak J Simonis C Luckow B Proudfoot AE Plachy J Bruhl H Frink M Anders HJ Vielhauer V Pfirstinger J Stangassinger M Schlondorff D Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.J Immunol. 2001; 166: 4697-4704PubMed Google Scholar Fifty thousand events per sample were acquired using a CyAn ADP flow cytometer (Beckman Coulter, Hialeah, FL) and analyzed with FCS Express 3 (De Novo Software, Los Angeles, CA). Clodronate-liposomes and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD)-liposomes were produced as described previously.4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar, 21Van Rooijen N Sanders A Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1510) Google Scholar Clodronate was a gift from Roche Diagnostics. To label Ly6Clo monocytes, 150 μl of DiD-liposomes were injected i.v. into TMPD-treated mice. To label Ly6Chi monocytes, clodronate-liposomes (200 μl) were injected i.v. 24 hours before the injection of DiD-liposomes.4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar Cells were analyzed 24 or 72 hours after labeling. PECs resuspended in complete Dulbecco’s modified Eagle’s medium (containing 10% fetal bovine serum, 10 mmol/L HEPES, glutamine, and penicillin/streptomycin) were seeded on 96-well cell culture plates (105 cells/well). Cells were stimulated with the indicated doses of peptidoglycan, R848 (Invivogen, San Diego, CA), or lipopolysaccharide (from Salmonella typhimurium, Sigma-Aldrich) and incubated at 37°C in a 5% CO2 atmosphere for 24 hours before the supernatant was collected. IL-12, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and IL-6 enzyme-linked immunosorbent assays (BD Biosciences) were performed following the manufacturer’s instructions. Optical density was converted to concentration using standard curves based on recombinant cytokines analyzed by a four-parameter logistic equation (Softmax Pro 4.3, Molecular Devices Corporation, Sunnyvale, CA). For in vivo phagocytosis, DiD-liposomes (50 μl) or apoptotic BW5147 lymphocytes (2 × 105 cells in 200 μl of PBS) were injected i.p. in mice treated with TMPD 2 weeks earlier, and PECs were isolated and analyzed by flow cytometry 1 hour later. DiD-labeled apoptotic BW5147 cells (>70% annexin V+) were generated by heat shock as described previously.19Lee PY Kumagai Y Li Y Takeuchi O Yoshida H Weinstein J Kellner ES Nacionales D Barker T Kelly-Scumpia K van Rooijen N Kumar H Kawai T Satoh M Akira S Reeves WH TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus.J Exp Med. 2008; 205: 2995-3006Crossref PubMed Scopus (184) Google Scholar For in vitro phagocytosis assays, bone marrow-derived macrophages (106 cells/well) were generated as described19Lee PY Kumagai Y Li Y Takeuchi O Yoshida H Weinstein J Kellner ES Nacionales D Barker T Kelly-Scumpia K van Rooijen N Kumar H Kawai T Satoh M Akira S Reeves WH TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus.J Exp Med. 2008; 205: 2995-3006Crossref PubMed Scopus (184) Google Scholar and incubated with or without IFN-β (0.5 or 5 ng/ml) for the final 24 hours of culture. DiD-liposomes (10 μl/well) or fluorescein isothiocyanate-labeled microbeads (10:1 beads/cell ratio, Invitrogen) were added for 30 minutes at 37°C (in PBS with 0.5% bovine serum albumin). Cells were washed three times and analyzed by flow cytometry. For continuous variables, differences between groups were analyzed by an unpaired Student’s t-test. Data are presented as means ± SE All tests were two-sided. P < 0.05 was considered significant. Statistical analyses were performed using Prism 4.0 (GraphPad Software, San Diego, CA). To study the chronic inflammatory response to i.p. TMPD, we first analyzed the peritoneal cell infiltrate at various time points. In untreated mice, peritoneal lavage yielded mainly B220+CD11b+ B1 lymphocytes (Figure 1A, circle; also CD19+, not shown) and B220−CD11b+ myeloid cells (Figure 1A, box) that included granulocytes and monocytes/macrophages. Ly6G+ granulocytes comprised only a small portion of the myeloid population (CD11b+B220− gate; Figure 1A, right). Thus, the B220−CD11b+ cells were predominantly monocytes/macrophages. Consistent with other studies,4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar, 22Cook AD Braine EL Hamilton JA The phenotype of inflammatory macrophages is stimulus dependent: implications for the nature of the inflammatory response.J Immunol. 2003; 171: 4816-4823Crossref PubMed Scopus (87) Google Scholar residential peritoneal macrophages did not exhibit surface expression of Ly6C (CD11b+B220−Ly6G− gate; Figure 1A, right), a marker highly expressed on inflammatory monocytes. A strikingly different pattern was observed 18 hours after TMPD treatment (Figure 1B). A dramatic expansion of CD11b+B220− PECs and a reduction in both B1 lymphocytes and conventional B cells were observed (Figure 1B, left). Granulocytes were the predominant population at this time point, outnumbering monocytes/macrophages by ∼2 to 1. Unlike residential peritoneal macrophages, the majority of monocytes elicited by TMPD expressed high levels of Ly6C, suggestive of an inflammatory phenotype. The chronic nature of the response to TMPD was confirmed by the presence of granulocytes and Ly6Chi monocytes in the peritoneal cavity 2 weeks and even 2 months after the initial injection (Figure 1B, right). All subsequent experiments were performed 2 weeks after TMPD injection as the absolute number of Ly6Chi monocytes in the peritoneal cavity was similar at 2 weeks and 2 months (data not shown). An early influx of granulocytes and Ly6Chi monocytes was also detected in the sterile peritonitis induced by thioglycollate (Figure 1C). However, the response was transient in comparison with TMPD as Ly6Chi monocytes elicited by thioglycollate were rapidly replaced by monocytes/macrophages with a mature Ly6Clo phenotype (Figure 1C). By 72 hours, few granulocytes were present and almost all monocytes/macrophages in the peritoneal cavity were Ly6Clo. Depletion of B lymphocytes was also not a feature of thioglycollate-induced inflammation (the reduced percentage of these cells was due to expansion of the myeloid populations). Proinflammatory cytokines including IL-6, TNF-α, IL-1β, IFN-γ, and IFN-I have been implicated in the immune response induced by TMPD.12Nacionales DC Kelly KM Lee PY Zhuang H Li Y Weinstein JS Sobel E Kuroda Y Akaogi J Satoh M Reeves WH Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane).Am J Pathol. 2006; 168: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 18Lee PY Weinstein JS Nacionales DC Scumpia PO Li Y Butfiloski E van Rooijen N Moldawer L Satoh M Reeves WH A novel type I IFN-producing cell subset in murine lupus.J Immunol. 2008; 180: 5101-5108PubMed Google Scholar, 23Kuroda Y Akaogi J Nacionales DC Wasdo SC Szabo NJ Reeves WH Satoh M Distinctive patterns of autoimmune response induced by different types of mineral oil.Toxicol Sci. 2004; 78: 222-228Crossref PubMed Scopus (52) Google Scholar, 24Patten C Bush K Rioja I Morgan R Wooley P Trill J Life P Characterization of pristane-induced arthritis, a murine model of chronic disease: response to antirheumatic agents, expression of joint cytokines, and immunopathology.Arthritis Rheum. 2004; 50: 3334-3345Crossref PubMed Scopus (53) Google Scholar We asked whether these cytokines play a role in the persistent inflammatory cell influx in response to TMPD. Compared with wild-type C57BL/6 controls, mice deficient in IL-6, TNF-α, or IL-1 receptor showed a similar influx of Ly6Chi monocytes to the peritoneal cavity 2 weeks after TMPD treatment (Figure 2A). The persistent inflammatory response also was not explained by the presence of endotoxin as a similar pattern was seen in C3H/HeJ mice (Figure 2A), which carry a mutation in Toll-like receptor (TLR)-4.25Poltorak A He X Smirnova I Liu MY Van Huffel C Du X Birdwell D Alejos E Silva M Galanos C Freudenberg M Ricciardi-Castagnoli P Layton B Beutler B Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6514) Google Scholar IFN-γ has been recently shown to mediate Ly6Chi monocyte recruitment in response to i.v. infection by Listeria monocytogenes.26Drevets DA Schawang JE Dillon MJ Lerner MR Bronze MS Brackett DJ Innate responses to systemic infection by intracellular bacteria trigger recruitment of Ly-6Chigh monocytes to the brain.J Immunol. 2008; 181: 529-536PubMed Google Scholar However, the absence of IFNγ did not affect recruitment of inflammatory cells (Figure 2B) induced by TMPD. Although granulocyte influx was higher in BALB/c than in B6 mice, there was no difference in the accumulation of granulocytes, Ly6Chi monocytes, or Ly6Clo monocytes/macrophages in IFNγ−/− mice versus wild-type controls (Figure 2B). Interestingly, although a sustained accumulation of granulocytes was found in TMPD-treated IFNAR−/−, the majority of monocytes/macrophages in the peritoneal cavity exhibited a mature, Ly6Clo phenotype (Figure 2C, left). A significant reduction in Ly6Chi monocytes paralleled the increase of Ly6Clo monocytes/macrophages in the absence of IFN-I signaling (Figure 2C, right). This was not due to genetic background differences because wild-type 129Sv and B6 mice demonstrated comparable monocyte responses to TMPD. The effects of IFNAR deficiency were also similar in both backgrounds (data not shown). Consistent with flow cytometry analysis, visual examination of PECs showed numerous polymorphonuclear cells in both wild-type and IFNAR−/− mice (Figure 2D). Whereas cells with monocytic morphology were found in the peritoneal cavity of wild-type mice, PECs from IFNAR−/− mice revealed the presence of larger, vacuolated cells consistent with a mature, macrophage-like morphology (Figure 2D). Supporting this view, monocytes/macrophages from IFNAR−/− mice displayed greater surface expression of several macrophages markers including F4/80, I-A, and CD115 (Figure 2E). Not surprisingly, expression of the interferon-stimulated gene Sca-1 on monocytes/macrophages was greatly reduced in the absence of IFN-I signaling. Under steady-state conditions, both residential (Ly6Clo) and inflammatory monocytes (Ly6Chi) are present in the circulation.3Geissmann F Jung S Littman DR Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2634) Google Scholar, 4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar Accumulation of Ly6Clo monocytes/macrophages in the peritoneum of TMPD-treated IFNAR−/− mice suggests that either this subset is preferentially recruited to the peritoneal cavity or that the infiltrating Ly6Chi monocytes rapidly acquire a mature phenotype. To distinguish between these possibilities, we monitored monocyte influx into the peritoneal cavity (2 weeks after TMPD injection) in vivo using fluorescently labeled liposomes. Neither Ly6Clo nor Ly6Chi circulating monocytes displayed fluorescence before labeling (CD11b+Ly6G− gate) (Figure 3A). As reported previously,4Sunderkötter C Nikolic T Dillon MJ Van Rooijen N Stehling M Drevets DA Leenen PJ Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417PubMed Google Scholar i.v. injection of liposomes containing the fluorescent dye DiD (DiD-liposomes) selectively labeled Ly6Clo monocytes in the peripheral blood of both wild-type and IFNAR−/− mice (Figure 3, B and C). Peripheral blood Ly6Chi monocytes were not labeled with the procedure, probably because of their lower phagocytic capacity compared with the Ly6Clo subset.27Peng Y Latchman Y Elkon KB Ly6Clow monocytes differentiate into dendritic cells and cross-tolerize T cells through PDL-1.J Immunol. 2009; 182: 2777-2785Crossref PubMed Scopus (41) Google Scholar After 24 hours, >40% of circulating Ly6Clo monocytes were DiD+ in TMPD-treated wild-type and IFNAR−/− mice, but few DiD+ cells had migrated into the peritoneal cavity in either strain. Thus, the absence of IFN-I signaling does not cause preferential recruitment of Ly6Clo monocytes from the peripheral blood. To track the migration of circulating Ly6Chi monocytes, clodronate-liposomes were first given to deplete the Ly6Clo subset. In the absence of Ly6Clo counterparts, liposome uptake was evident in the Ly6Chi subset as ∼50% of peripheral blood Ly6Chi monocytes were fluorescently labeled in TMPD-treated wild-type and IFNAR−/− mice 24 hours after administration of DiD-liposomes (Figure 3, D and E). Importantly, a similar percentage of Ly6Chi monocytes in the peritoneal cavity of wild-type mice were DiD+, indicating that most cells in this subset entered the peritoneal cavity within the past 24 hours. Although the absolute number of peritoneal Ly6Chi
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