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The Rs1143679 (R77H) Lupus Associated Variant of ITGAM (Cd11b) Impairs Complement Receptor 3 Mediated Functions in Human Monocyt

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The Rs1143679 (R77H) Lupus Associated Variant of ITGAM (Cd11b) Impairs Complement Receptor 3 Mediated Functions in Human Monocyt ARD Online First, published on August 8, 2012 as 10.1136/annrheumdis-2012-201390 Basic and translational research Ann Rheum Dis: first published as 10.1136/annrheumdis-2012-201390 on 14 May 2012. Downloaded from EXTENDED REPORT The rs1143679 (R77H) lupus associated variant of ITGAM (CD11b) impairs complement receptor 3 mediated functions in human monocytes Benjamin Rhodes,1 Barbara G Fürnrohr,2 Amy L Roberts,1 George Tzircotis,3 Georg Schett,4 Tim D Spector,5 Timothy J Vyse1 ▶ Additional supplementary ABSTRACT it is common (minor allele frequency ≈10%) in all data are published online only. except east Asian populations.3–5 It may be a mod- To view these fi les please visit Objectives The rs1143679 variant of ITGAM, the journal online (http://ard. erate risk variant for systemic sclerosis, but it is not encoding the R77H variant of CD11b (part of 6 7 bmj.com/content/early/recent) complement receptor 3; CR3), is among the strongest associated with other autoimmune diseases. 1Genetics and Molecular genetic susceptibility effects in human systemic lupus Interest has focussed on the rs1143679 variant, Medicine and Immunology, erythematosus (SLE). The authors aimed to demonstrate which encodes an arginine to histidine amino acid Infection and Infl ammatory R77H function in ex-vivo human cells. change at position 77 (R77H) in the beta-propeller Disease, King’s College London, domain of CD11b. This variant had already been London, UK Methods Monocytes/monocyte-derived macrophages 2 recognised as an antigen in neonatal alloimmune Molecular Immunology, from healthy volunteers homozygous for either wild 8 Department of Internal type (WT) or 77H CD11b were studied. The genotype- neutropenia. In European populations, linkage dis- Medicine-3, and Institute for specifi c expression of CD11b, and CD11b activation equilibrium between ITGAM variants leads to mul- Clinical Immunology, Nikolaus- using conformation-specifi c antibodies were measured. tiple genetic associations and diffi culty pinpointing Fiebiger Centre, University of functional effects to a single variant. Trans-ancestral Erlangen-Nuremberg, Germany Genotype-specifi c differences in iC3b-mediated 3Cancer Research Technology phagocytosis, adhesion to a range of ligands and the data support rs1143679, but without extensive rese- Ltd, London, UK secretion of cytokines following CR3 ligation were studied. quencing and replication, attributing function to a spe- 4Clinical Immunology and cifi c variant alone remains open to interpretation.5 Rheumatology, Department of The functionality of R77H was confi rmed by replicating fi ndings in COS7 cells expressing variant-specifi c CD11b. Direct experimental data have been confl icting. Internal Medicine-3, University A recent study reported differences in adhesion and of Erlangen-Nuremberg, Results No genotype-specifi c difference in CD11b Germany expression or in the expression of CD11b activation phagocytosis between cell lines transfected with 5 9 Department of Twin Research epitopes was observed. A 31% reduction was observed wild-type (WT) or 77H ITGAM. On the other and Genetic Epidemiology, in the phagocytosis of iC3b opsonised sheep erythrocytes hand, the 77H genotype was not reported to infl u- King’s College London, London, ence adhesion of ex-vivo neutrophils (although UK (sRBCiC3b) by 77H cells (p=0.003) and reduced adhesion 8 to a range of ligands: notably a 24% reduction in adhesion iC3b was not tested). http://ard.bmj.com/ Correspondence to to iC3b (p=0.014). In transfected COS7 cells, a 42% The strength of the ITGAM association in SLE Timothy J Vyse, Medical makes this a particularly important effect to under- and Molecular Genetics, reduction was observed in phagocytosis by CD11b (77H)- expressing cells (p=0.004). A signifi cant inhibition was stand. It may give us an insight to key pathogenic King’s College London, pathways that are potentially amenable to thera- London SE1 9RT, UK; seen in the release of Toll-like receptor 7/8-induced pro- [email protected] infl ammatory cytokines from WT monocytes when CR3 peutic manipulation. The relative lack of genetic data specifi cally supporting rs1143679, combined was pre-engaged using sRBCiC3b, but no inhibition in 77H BF and BR contributed equally with the confl icting early functional reports, means monocytes resulting in a signifi cant difference between on September 29, 2021 by guest. Protected copyright. to this study. genotypes (interleukin (IL)-1β p=0.030; IL-6 p=0.029; that existing studies give an inadequate picture. Accepted 9 April 2012 tumour necrosis factor alpha p=0.027). METHODS Conclusions The R77H variant impairs a broad range Reagents of CR3 effector functions in human monocytes. This study discusses how perturbation of this pathway may Culture media, sera and salt solutions and secondary predispose to SLE. antibodies were from Invitrogen (Life Technologies, Paisley, UK); rhM-CSF was from Peprotech (London, UK); anti-CD11b, anti-CD14 and control antibodies were from ebioscience (Hatfi eld, UK); Complement receptor 3 (CR3)/Mac1 is a heterodi- polyclonal antisheep erythrocyte IgM was from meric integrin receptor. The α-chain (CD11b), Cedarlane (Burlington, Ontario, Canada). Human encoded by the ITGAM gene, is unique to this CD11b and CD18 in the pRK5 vector was a gift of receptor, while the β-chain (CD18) is shared with Emmanuelle Caron, Imperial College, London. The other integrin receptors. CR3 is expressed widely R77H mutation was introduced using a Stratagene and at high levels on innate immune cells, including QuikChange site-directed mutagenesis kit (Agilent neutrophils, monocytes and macrophages. It binds Technologies, Stockport, UK). Protein ligands were a range of ligands including iC3b, ICAM-1 and from Calbiochem, Merck-Millipore, London, UK fi brinogen, and it participates in functions includ- (iC3b), R&D Systems, Abingdon, UK (ICAM-1) ing phagocytosis and adhesion.1 2 Variation at and Enzyme Research Laboratories Swansea, UK ITGAM is one of the strongest genetic risk factors (fi brinogen). Human DC-SIGN was a gift of Dan in human systemic lupus erythematosus (SLE), and Mitchell, University of Warwick. CopyrightRhodes B, FürnrohrArticle BG, author Roberts AL, (or et al their. Ann Rheum employer) Dis (2012). doi:10.1136/annrheumdis-2012-201390 2012. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.1 of 7 Basic and translational research Ann Rheum Dis: first published as 10.1136/annrheumdis-2012-201390 on 14 May 2012. Downloaded from Study participants Phagocytic assay Study volunteers were from the TwinsUK National Institute for Phagocytic assay was performed using monocyte-derived mac- Health Research (NIHR) bioresource. Individuals were selected rophages transferred to serum-free medium for 1 h. Sheep eryth- on the basis of imputed genotype but rs1143679 was checked rocytes (TCS Biosciences, Buckingham, UK) were opsonised with by TaqMan assay (Applied Biosystems, Life Technologies, rabbit antisheep erythrocyte IgM (sRBCIgM) with or without Paisley, UK). All volunteers were healthy with no history of human iC3b (sRBCiC3b) according to established protocols (see autoimmune disease, recent steroid or immunosuppressant use. supplementary text, available online only).10 Opsonised sRBC The study was approved by the South East London Research were added to the macrophages and incubated for 20 min before Ethics Committee and participants gave written informed con- halting phagocytosis on ice. External, engaged sRBC were distin- sent. Additional volunteers were recruited at the University of guished from internalised sRBC by staining as previously described Erlangen-Nuremberg, with approval from the ethics committee (see supplementary text, available online only) and the cover- of the Friedrich Alexander University of Erlangen-Nuremberg. slips were mounted for immunofl uorescent microscopy (Zeiss Axiophot, Welwyn Garden City, UK).11 12 Cells were scored blind Leucocyte preparation for the number of internalised sRBC (Alexa-555 labelled) and the number of bound but external sRBC (dual labelled with Alexa-488 Processing of heparinised blood was commenced within 1 h and Alexa-555). The association index was calculated as the mean of collection. For fl ow cytometry, a leucocyte-enriched frac- engaged (internal and external) sRBC per 100 phagocytes. The tion was obtained by sedimentation in 3% dextran-500, before phagocytic index was calculated as the mean internalised sRBC staining as outlined below. Untouched human monocytes were per 100 phagocytes. The percentage phagocytosis was calculated obtained by density gradient sedimentation (Histopaque; Sigma, as the mean internalised sRBC/engaged sRBC per phagocyte.12 Dorset, UK) and purifi cation by negative selection (Monocyte COS7 cell phagocytosis was quantifi ed 2 days after transfec- Isolation Kit II; Miltenyi Biotec, Bisley, UK). Monocyte-derived tion. The protocol was identical except the phagocytic incuba- macrophages were obtained by adherence of fresh monocytes, tion was 25 min and a modifi ed association index was calculated in serum-free medium, to glass coverslips for 1 h before being in which COS7 cells with no associated sRBC were not counted grown on in RPMI supplemented with 10% fetal bovine serum (even with CD11b staining it was hard to distinguish autofl uo- (FBS), Glutamax, pyruvate, penicillin/streptomycin, non-essen- rescent, but potentially untransfected, COS7 cells from those tial amino acids and 50 ng/ml M-CSF for 6 or 7 days at which expressing low level CD11b). point cells were spread and strongly
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