G-CSF and Neutrophils Are Nonredundant Mediators of Murine Experimental Autoimmune Uveoretinitis
2015; Elsevier BV; Volume: 186; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2015.09.008
ISSN1525-2191
AutoresGabrielle L. Goldberg, Ann L. Cornish, Jane Murphy, Ee Shan Pang, Lyndell L. Lim, Ian K. Campbell, Karen Scalzo‐Inguanti, Xiangting Chen, Paul G. McMenamin, Eugene Maraskovsky, Brent S. McKenzie, Ian P. Wicks,
Tópico(s)Immune Response and Inflammation
ResumoGranulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis–experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF–deficient mice and in anti–G-CSF monoclonal antibody–treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF–deficient and anti–G-CSF monoclonal antibody–treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF–driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis. Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis–experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF–deficient mice and in anti–G-CSF monoclonal antibody–treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF–deficient and anti–G-CSF monoclonal antibody–treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF–driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis. Uveitis is a leading cause of blindness in developed countries. Intermediate/posterior uveoretinitis damages the retina and accounts for most visual morbidity. Infections cause approximately 50% of uveitis cases,1Forrester J.V. Intermediate and posterior uveitis.Chem Immunol Allergy. 2007; 92: 228-243Crossref PubMed Scopus (39) Google Scholar but the remainder are believed to be autoimmune.2Lee R.W. Nicholson L.B. Sen H.N. Chan C.C. Wei L. Nussenblatt R.B. Dick A.D. Autoimmune and autoinflammatory mechanisms in uveitis.Semin Immunopathol. 2014; 36: 581-594Crossref PubMed Scopus (101) Google Scholar Although current therapies control autoimmune uveitis in many patients, some fail to respond, or treatment is limited by adverse effects. There is consequently a need for alternative therapeutic approaches. Experimental autoimmune uveoretinitis (EAU), a widely used murine model of noninfectious posterior uveoretinitis, is CD4+ T-cell–mediated and mimics the clinical and histopathological features of human disease.3Chen J. Qian H. Horai R. Chan C.C. Caspi R.R. Mouse models of experimental autoimmune uveitis: comparative analysis of adjuvant-induced vs spontaneous models of uveitis.Curr Mol Med. 2015; 15: 550-557Crossref PubMed Scopus (25) Google Scholar Type 1 helper T (Th1) cells contribute to EAU, but recent studies suggest that Th17 cells (but not IL-17 itself) and IL-23, a potent stimulator of Th17 cell differentiation, are nonredundant in this disease model.4Luger D. Silver P.B. Tang J. Cua D. Chen Z. Iwakura Y. Bowman E.P. Sgambellone N.M. Chan C.C. Caspi R.R. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.J Exp Med. 2008; 205: 799-810Crossref PubMed Scopus (595) Google Scholar In contrast, although innate immune cells, including neutrophils and macrophages, feature prominently in the ocular infiltrate in EAU,5Forrester J.V. McMenamin P.G. Immunopathogenic mechanisms in intraocular inflammation.Chem Immunol. 1999; 73: 159-185Crossref PubMed Scopus (50) Google Scholar, 6Kerr E.C. Raveney B.J. Copland D.A. Dick A.D. Nicholson L.B. Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations.J Autoimmun. 2008; 31: 354-361Crossref PubMed Scopus (126) Google Scholar the pathogenic role of myeloid cells, or regulators of myeloid cell production and function, has been far less studied. Granulocyte colony-stimulating factor (G-CSF) is a major regulator of steady-state neutrophil production and survival,7Lieschke G.J. Grail D. Hodgson G. Metcalf D. Stanley E. Cheers C. Fowler K.J. Basu S. Zhan Y.F. Dunn A.R. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.Blood. 1994; 84: 1737-1746Crossref PubMed Google Scholar and granulopoiesis is reduced in G-CSF– and G-CSF receptor (G-CSF-R)–deficient mice. During infection or inflammation, neutrophil production and export increases, under the influence of G-CSF, in a response known as emergency granulopoiesis.8Nakamura H. Ueki Y. Sakito S. Matsumoto K. Yano M. Miyake S. Tominaga T. Tominaga M. Eguchi K. 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Recurrent idiopathic iridocyclitis after autologous peripheral blood stem-cell transplantation followed by G-CSF administration for acute lymphoblastic leukemia.Ann Hematol. 2000; 79: 269-271Crossref PubMed Scopus (5) Google Scholar We have previously reported that G-CSF–deficient mice are resistant to collagen-induced arthritis.31Lawlor K.E. Campbell I.K. Metcalf D. O'Donnell K. van Nieuwenhuijze A. Roberts A.W. Wicks I.P. Critical role for granulocyte colony-stimulating factor in inflammatory arthritis.Proc Natl Acad Sci U S A. 2004; 101: 11398-11403Crossref PubMed Scopus (97) Google Scholar Wild-type (WT) mice treated with anti–G-CSF also had reduced collagen-induced arthritis, even when anti–G-CSF was administered therapeutically.31Lawlor K.E. Campbell I.K. Metcalf D. O'Donnell K. van Nieuwenhuijze A. Roberts A.W. Wicks I.P. Critical role for granulocyte colony-stimulating factor in inflammatory arthritis.Proc Natl Acad Sci U S A. 2004; 101: 11398-11403Crossref PubMed Scopus (97) Google Scholar In this study, we investigated the role of endogenous G-CSF and neutrophils in EAU. Our data show a strong relationship between neutrophilia and elevated G-CSF in PB, and in the eye, in WT mice. In contrast, G-CSF–deficient mice did not develop EAU-associated neutrophilia and were markedly protected from disease, as were WT mice treated with a neutralizing anti–G-CSF monoclonal antibody (mAb; anti-G-CSF). Analysis of the ocular infiltrate revealed a neutrophil-rich infiltrate in WT mice with EAU, which was almost absent in G-CSF–deficient mice and in WT mice treated with anti–G-CSF. Furthermore, expression of CXCR2 on circulating neutrophils was markedly decreased in G-CSF–deficient mice and anti–G-CSF–treated mice with EAU. Lower CXCR2 expression was associated with reduced neutrophil actin polymerization and reduced in vitro and in vivo neutrophil migration. Intriguingly, decreased activation of the neutrophil compartment impaired the differentiation of pathogenic Th17 cells, linking innate and adaptive immunity. These results reveal a fundamental contribution of endogenous G-CSF and neutrophils to the pathogenesis of EAU. Experiments were performed in accordance with National Health and Medical Research Council of Australia guidelines and approved by the Animal Ethics Committee of the Walter and Eliza Hall Institute (WEHI; Parkville, VIC, Australia). C57BL/6 mice were obtained from WEHI. G-CSF–deficient (B6;129P2-Csf3tm1Ard/J) mice were kindly provided by Ashley Dunn (Ludwig Institute for Cancer Research, Parkville, VIC, Australia),32Liu F. Wu H.Y. Wesselschmidt R. Kornaga T. Link D.C. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice.Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar and G-CSF-R–deficient [B6.129X1(Cg)-Csf3rtm1Link/J] mice were kindly provided by Daniel Link (Washington University Medical School, St. Louis, MO).33Betsuyaku T. Liu F. Senior R.M. Haug J.S. Brown E.J. Jones S.L. Matsushima K. Link D.C. A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation.J Clin Invest. 1999; 103: 825-832Crossref PubMed Scopus (61) Google Scholar C57BL/6 RAR-related orphan receptor (ROR) γT–enhanced green fluorescence protein (eGFP) mice were kindly provided by Gabrielle Belz, and the G-CSF–deficient/RORγT-eGFP mice were bred at WEHI by crossing the G-CSF–deficient mouse (B6;129P2-Csf3tm1Ard/J) with the C57BL/6 RORγT-eGFP reporter mice. G-CSF–deficient and G-CSF-R–deficient mice were backcrossed onto the C57BL/6 background for >20 and >8 generations, respectively. LysM-cre-eGFP mice were kindly provided by Michael Hickey (Monash University, Clayton, VIC, Australia) and did not carry the rd8 mutation in the Crb1 gene.34Mattapallil M.J. Wawrousek E.F. Chan C.C. Zhao H. Roychoudhury J. Ferguson T.A. Caspi R.R. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.Invest Ophthalmol Vis Sci. 2012; 53: 2921-2927Crossref PubMed Scopus (485) Google Scholar Mice were housed under standard conditions in the WEHI animal facility. Unless otherwise stated, EAU was induced as described previously.35Agarwal R.K. Silver P.B. Caspi R.R. Rodent models of experimental autoimmune uveitis.Methods Mol Biol. 2012; 900: 443-469Crossref PubMed Scopus (138) Google Scholar Each mouse received 150 μg interphotoreceptor retinoid-binding protein (IRBP), 250 μg Mycobacterium tuberculosis in complete Freund's adjuvant, given by s.c. injection, and 0.5 μg pertussis toxin i.p. In some experiments, complete Freund's adjuvant was replaced by incomplete Freund's adjuvant (IFA). Rat anti–G-CSF mAb (clone 67604) and biotinylated rabbit anti–G-CSF polyclonal Ab (R&D Systems, Minneapolis, MN) were used in a sandwich enzyme-linked immunosorbent assay. Recombinant murine G-CSF (Peprotech, Rocky Hill, NJ) was used as a standard. Vitreous humor samples and serum were evaluated for cytokine and chemokine levels with the Bio-Plex mouse cytokine 23-plex panel, used according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). The assay was analyzed using a Bio-Plex 200 instrument and Bio-Plex Manager software version 5.0 (Bio-Rad Laboratories). IL-17 concentrations were measured using this kit. Rat anti-murine G-CSF mAb (clone 67604, as above) was used for G-CSF blockade in EAU, on the basis of our previous G-CSF neutralization experiments.31Lawlor K.E. Campbell I.K. Metcalf D. O'Donnell K. van Nieuwenhuijze A. Roberts A.W. Wicks I.P. Critical role for granulocyte colony-stimulating factor in inflammatory arthritis.Proc Natl Acad Sci U S A. 2004; 101: 11398-11403Crossref PubMed Scopus (97) Google Scholar mAb (0.25 mg) (or a rat IgG1 control mAb) was injected i.p. on day 8 and then every second day up to day 20 of EAU. For chemokine receptor expression and migration experiments, 2 μg G-CSF per mouse (Neupogen; Amgen, Thousand Oaks, CA) was administered s.c. twice, 12 hours apart, and organs were harvested the next day. For the IFA and G-CSF experiment, 2 μg G-CSF per mouse was administered s.c. for seven doses, 12 hours apart, over 3.5 days. MPO was quantified in enucleated, homogenized whole eyes with a mouse ELISA test kit (Hycult Biotech, Uden, the Netherlands), used according to the manufacturer's instructions. Eyes were harvested from mice 21 days after immunization. Hematoxylin and eosin sections were scored by two separate observers (blinded to experimental group; X.C., P.G.M.) and averaged. Infiltrative and structural scores were combined to give the total histological score. The scoring system used was on the basis of the system described by Dick et al.36Dick A.D. Cheng Y.F. Liversidge J. Forrester J.V. Immunomodulation of experimental autoimmune uveoretinitis: a model of tolerance induction with retinal antigens.Eye (Lond). 1994; 8: 52-59Crossref PubMed Scopus (66) Google Scholar Eye homogenates were clarified by centrifugation, and sample proteins (20 μg) were run on 4% to 12% Bis-Tris NOVEX gels (Invitrogen, Carlsbad, CA), then transferred to a polyvinylidene difluoride membrane. IRBP was detected with rabbit–anti-IRBP (Santa Cruz Biotechnology, Santa Cruz, CA), followed by sheep–anti-rabbit Ig–horseradish peroxidase (Silenus, Melbourne, VIC, Australia). Immunoblots were developed with Amersham ECL (GE Healthcare, Little Chalfont, UK). Single-cell suspensions of PB, BM, and spleen were made by passage through a 70-μm cell strainer. Single-cell suspensions were made by crushing enucleated eyes between two frosted slides. Antibodies used to stain leukocyte subsets are listed in Table 1.Table 1Flow Cytometry ReagentsAntigen/fluorophoreCloneSupplierProducts CD45.2-PercP-Cy5.5104BD Biosciences (San Jose, CA) CD11b-FITCM1/70BD Biosciences Gr-1–APCRB6.8C5BD Biosciences F480-FITCF4/80WEHI monoclonal antibody facility CXCR2-PE242216R&D Systems CD62L-PE-TX-REDMEL-14Invitrogen CXCR4-APC2B11eBioscience (San Diego, CA) γδ-TCR–PEB1BD Biosciences IL-17–APCeBio17B7eBioscience GM-CSF-PEMP1-22E9BD Biosciences IFN-γ–FITCXMG1.2eBioscienceReagent AnnexinV-FITCBD Biosciences FluoroGoldSigma-Aldrich (St. Louis, MO)APC, allophycocyanin; Cy5.5, cyanine5.5; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PercP, peridinin-chlorophyll proteins; TX-RED, Texas Red. Open table in a new tab APC, allophycocyanin; Cy5.5, cyanine5.5; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PercP, peridinin-chlorophyll proteins; TX-RED, Texas Red. Single-cell suspensions of the eye and blood were stimulated for 4 hours with 50 ng/mL phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO) and 500 ng/mL ionomycin, 1 mg/mL brefeldin A, and 2 mg/mL monensin. Cells were stained with surface antibodies (Table 1). Cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA), used according to the manufacturer's instructions, and stained with anti-cytokine antibodies (Table 1), followed by resuspension in 1× BD Perm/Wash buffer (BD Biosciences). Unstimulated cells were used as a control. Whole splenocytes from WT and G-CSF–deficient mice, harvested 21 days after the induction of EAU, were stained with 5 μmol/L CellTrace Violet (Invitrogen). Cells (2 × 105 per well) were cultured for 5 days in the presence or absence of 5 μg/mL IRBP 1-20. On day 5, cells were stained for CD3, CD4, and CD8 and propidium iodide to exclude dead cells. Stained cells were analyzed on a BD Fortessa flow cytometer. The severity of retinal disease was assessed on the basis of examination of bright-field and fluorescent fundal images obtained from in vivo examination (Micron III; Phoenix Laboratories, Palmdale, CA). A modified version of a previously described grading scheme was used to score fundal images37Xu H. Koch P. Chen M. Lau A. Reid D.M. Forrester J.V. A clinical grading system for retinal inflammation in the chronic model of experimental autoimmune uveoretinitis using digital fundus images.Exp Eye Res. 2008; 87: 319-326Crossref PubMed Scopus (108) Google Scholar (Supplemental Table S1) by two observers (blinded to experimental group; X.C., P.G.M.), and the scores were averaged. Splenocytes (2 × 106) were stimulated with 5 ng/mL human G-CSF (Neupogen) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech), or incubated in media alone for 16 hours. Live neutrophils were defined as AnnexinV−FluoroGold−Gr–1hiCD11b+F4/80− (BD Biosciences). A total of 1 × 106 cells (1 × 107 cells/mL) were placed in the top chamber of a 3-μm pore, 24-well transwell plate (Corning, Tewksbury, MA). Media (600 μL) containing 500 ng/mL recombinant murine CXCL2/macrophage inflammatory protein (MIP)-2 (R&D Systems), or media alone, were placed in the bottom chamber, and the plate was incubated for 30 minutes at 37°C/5% CO2. The ViaLight plus kit (Lonza, Basel, Switzerland) was used, according to the manufacturer's directions, to calculate migrating cells. The remaining cells were stained and analyzed by flow cytometry, with neutrophils defined as Gr-1hiCD11b+F4/80−. A previously reported actin polymerization assay was adapted.38Carulli G. Mattii L. Azzara A. Brizzi S. Galimberti S. Zucca A. Benedetti E. Petrini M. Actin polymerization in neutrophils from donors of peripheral blood stem cells: divergent effects of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor.Am J Hematol. 2006; 81: 318-323Crossref PubMed Scopus (18) Google Scholar White blood cells were stained (neutrophils defined as Gr-1hiCD11b+F4/80−) and resuspended at 2 × 107/mL, and 50 μL was transferred to 5-mL polystyrene tubes on ice. Samples were incubated at 37°C for 2 minutes, then murine MIP-2 (R&D Systems) was added (final concentration of 500 ng/mL). Cells were incubated for 15, 30, 60, or 120 seconds before BD Cytofix/Cytoperm was added. Samples were stained with phalloidin–fluorescein isothiocyanate and analyzed by flow cytometry. B6/G-CSF–deficient and B6/G-CSF-R–deficient mice were injected s.c. with either 2 μg hG-CSF or phosphate-buffered saline. Sixteen hours later, mice were injected i.p. with 500 ng murine CXCL2/MIP-2 (Peprotech). After 4 hours, mice were bled and peritoneal lavages were performed with cold phosphate-buffered saline. Cell counts and flow cytometry were performed after lysis of red blood cells (RBCs). Cells were analyzed on an ADVIA blood analyzer (Siemens, Munich, Germany) and BD Fortessa. Neutrophils were defined as CD45+Gr-1hiCD11b+F4/80−. Prism software version 6 (GraphPad, La Jolla, CA) was used for statistical analysis. The U-test was used to compare sample groups, unless otherwise stated. Analysis of variance was used for measurements taken at multiple time points. Data shown are means ± SEM. EAU was induced in age- and sex-matched C57BL/6 mice, and PB leukocytes were analyzed by automated cell counting (Figure 1A). Neutrophils increased approximately 35-fold at day (d) 7, 20-fold at d11, and 10-fold at d15 and d21. In contrast, the increase in lymphocyte numbers was modest (2.5-fold) on d7 and normalized by d15. G-CSF levels increased markedly by d7 and remained high throughout disease, correlating closely with increased circulating neutrophils (Figure 1B). In mice, MPO generation in tissues arises mainly from neutrophils, because monocytes/macrophages down-regulate MPO production outside of the BM.39McMillen T.S. Heinecke J.W. LeBoeuf R.C. Expression of human myeloperoxidase by macrophages promotes atherosclerosis in mice.Circulation. 2005; 111: 2798-2804Crossref PubMed Scopus (147) Google Scholar MPO was, therefore, used as a surrogate marker for neutrophil infiltration in the eye. Ocular MPO40Graff G. Gamache D.A. Brady M.T. Spellman J.M. Yanni J.M. Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis.J Pharmacol Toxicol Methods. 1998; 39: 169-178Crossref PubMed Scopus (68) Google Scholar increased throughout EAU and correlated with increased G-CSF levels within the eye (Figure 1C). Anti–Gr-1 mAb41Eyles J.L. Hickey M.J. Norman M.U. Croker B.A. Roberts A.W. Drake S.F. James W.G. Metcalf D. Campbell I.K. Wicks I.P. A key role for G-CSF-induced neutrophil production and trafficking during inflammatory arthritis.Blood. 2008; 112: 5193-5201Crossref PubMed Scopus (129) Google Scholar was administered to WT mice to examine the effect of neutrophil deplet
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