That Is Enabled by Mac-1 Deficiency Neutrophil-Mediated Organ

Human Lupus Serum Induces Neutrophil-Mediated Organ Damage in Mice That Is Enabled by Mac-1 Deficiency

This information is current as Florencia Rosetti, Naotake Tsuboi, Kan Chen, Hiroshi Nishi, of September 26, 2021. Thomas Ernandez, Sanjeev Sethi, Kevin Croce, George Stavrakis, Jorge Alcocer-Varela, Diana Gómez-Martin, Nico van Rooijen, Vasileios C. Kyttaris, Andrew H. Lichtman, George C. Tsokos and Tanya N. Mayadas

J Immunol published online 29 August 2012 Downloaded from http://www.jimmunol.org/content/early/2012/09/09/jimmun ol.1201594

Supplementary http://www.jimmunol.org/content/suppl/2012/08/29/jimmunol.120159 http://www.jimmunol.org/ Material 4.DC1

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 10, 2012, doi:10.4049/jimmunol.1201594 The Journal of Immunology

Human Lupus Serum Induces Neutrophil-Mediated Organ Damage in Mice That Is Enabled by Mac-1 Deﬁciency

Florencia Rosetti,*,† Naotake Tsuboi,*,1 Kan Chen,* Hiroshi Nishi,* Thomas Ernandez,*,2 Sanjeev Sethi,‡ Kevin Croce,x George Stavrakis,* Jorge Alcocer-Varela,{ Diana Go´mez-Martin,{ Nico van Rooijen,‖ Vasileios C. Kyttaris,# Andrew H. Lichtman,* George C. Tsokos,# and Tanya N. Mayadas*

Systemic lupus erythematosus (SLE) is a chronic, multiorgan inﬂammatory autoimmune disorder associated with high levels of circulating autoantibodies and immune complexes. We report that passive transfer of human SLE sera into mice expressing the uniquely human FcgRIIA and FcgRIIIB on neutrophils induces lupus nephritis and in some cases arthritis only when the mice

additionally lack the CD18 integrin, Mac-1. The prevailing view is that Mac-1 on macrophages is responsible for immune complex Downloaded from clearance. However, disease permitted by the absence of Mac-1 is not related to enhanced renal immune complex deposition or in situ C1q/C3 complement activation and proceeds even in the absence of macrophages. Instead, disease is associated with increased FcgRIIA-induced neutrophil accumulation that is enabled by Mac-1 deﬁciency. Intravital microscopy in the cremasteric vascu- lature reveals that Mac-1 mitigates FcgRIIA-dependent neutrophil recruitment in response to deposited immune complexes. Our results provide direct evidence that human SLE immune complexes are pathogenic, demonstrate that neutrophils are primary

mediators of end organ damage in a novel humanized lupus mouse model, and identify Mac-1 regulation of FcgRIIA-mediated http://www.jimmunol.org/ neutrophil recruitment as a key step in development of target organ damage. The Journal of Immunology, 2012, 189: 000–000.

ystemic lupus erythematosus (SLE) is a systemic autoim- Fcg-chain (g2/2) that results in the loss of activating FcgRs mune disease that affects multiple organs, including the protects lupus-prone New Zealand Black/New Zealand White S kidney, joints, and skin, with development of nephritis mice from spontaneously developing lupus nephritis (2, 3). Yet being one of the most serious complications of this disorder. SLE is there is little direct evidence that immune complexes (ICs) are characterized by a loss of tolerance to self-Ags and high titers of causative, as there are no reports that the passive transfer of SLE circulating autoantibodies (1). In mice, deletion of the common sera reproduces disease. Moreover, autoantibodies to classical lupus Ags such as dsDNA are not sufficient for development of by guest on September 26, 2021 nephritis in lupus-prone mouse strains, and renal biopsies of SLE *Department of Pathology, Center for Excellence in Vascular Biology, Brigham and patients without clinically evident nephritis can exhibit IC depo- Women’s Hospital and Harvard Medical School, Boston, MA 02115; †Immunology sition. Thus, factors in addition to ICs most likely dictate the Graduate Program, Division of Medical Sciences, Harvard Graduate School of Arts and Sciences at Harvard Medical School, Boston, MA 02115; ‡Division of Anatomic propensity to develop target organ damage, the identification of Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Roches- which remains a central question in the field (4–6). ter, MN 55905; xDepartment of Medicine, Brigham and Women’s Hospital and { Human genome-wide association studies have shown that small Harvard Medical School, Boston, MA 02115; Departamento de Inmunologıáy Reumatologıá, Instituto Nacional de Ciencias Me´dicas y Nutricioń Salvador Zubirań, variations in a large number of genes are associated with lupus. The Mexico City 1400, Mexico; ‖Department of Molecular Cell Biology, Vrije University current paradigm assumes that these traits modulate, through # Medical Center, 1081 Amsterdam, The Netherlands; and Division of Rheumatol- different pathways, the expression of disease in lupus. Variations ogy, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115 in one of these genes is the leukocyte integrin Mac-1 (ITGAM, 1Current address: Department of Nephrology, Internal Medicine, Nagoya University CD11b/CD18) (7), a member of the leukocyte CD18 integrin Graduate School of Medicine, Nagoya, Japan. family of adhesion molecules that are obligate heterodimers of 2Current address: Department of Medical Specialties, University Hospital Geneva, unique CD11 subunits paired with a common CD18 chain. Mac-1 Geneva, Switzerland. has multiple ligands, including complement fragment C3bi and Received for publication June 11, 2012. Accepted for publication August 4, 2012. ICAM-1 on the endothelium (8), and the Mac-1 R77H polymor- This work was supported by National Institutes of Health Grants RO1 HL065095 phism associated with lupus (9) leads to a reduction in its binding and AR050800 (to T.N.M.), AI42269 (to G.C.T.), and K23 AR055672 (to V.C.K.); to both these ligands (10). The question remains as to whether a Target Identification in Lupus Grant/Alliance for Lupus Research Foundation (to T.N.M.); Consejo Nacional de Ciencia y Tecnologia and FundaciońMe´xico en Har- Mac-1, which typically promotes inflammation and host defense vard (to F.R.); the Arthritis Foundation (to N.T.); Japan Society for the Promotion of (8), plays a role in the pathogenesis of lupus. The prevailing view Science Postdoctoral Fellowship for Research Abroad (to H.N.); and the Novartis Foundation (to T.E.). is that Mac-1 on macrophages clears circulating ICs (9), although there is no direct evidence of this. Likewise, polymorphisms in Address correspondence and reprint requests to Dr. Tanya N. Mayadas, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur NRB human FcgRIIA and FcgRIIIB are associated with lupus through 752O, Boston, MA 02115. E-mail address: [email protected] mechanisms that remain largely unclear (7). The physiological The online version of this article contains supplemental material. roles of the uniquely human neutrophil FcgRs, which structurally Abbreviations used in this article: GBM, glomerular basement membrane; hFcgR, differ from their mouse counterparts (11, 12), have been addressed human FcgR; IC, immune complex; RPA, reverse passive Arthus; SLE, systemic in genetically engineered mice. Expression of the single poly- lupus erythematosus; WT, wild-type. peptide FcgRIIA, containing an ITAM, and the GPI-anchored Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 FcgRIIIB selectively on neutrophils of Fcg-chain–deficient mice

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201594 2 Mac-1 DELETION ENABLES HUMAN LUPUS SERA-INDUCED NEPHRITIS

2 2 2 2 2 2 (IIA+g / , IIA+IIIB+g / ) restored susceptibility of g / animals 2, redness or swelling of .1 toe; 3, ankle or tarsal-metatarsal involvement; to progressive anti-glomerular basement membrane (GBM) ne- 4, redness or swelling of the entire paw. phritis and K/B3N serum-induced arthritis (13, 14). Although Histological assessment of tissue injury. Tissue sections were blindly both human FcgRs promoted neutrophil accumulation, only evaluated. Histological score included endocapillary proliferation, leukocyte infiltration, and crescents, as previously described (20). For joint FcgRIIA alone induced tissue injury most likely through its lesions, histological scores reflected leukocyte infiltration, synovial established ability to promote neutrophil cytotoxic functions thickening, and cartilage and bone erosion. (13). Histological studies Difficulty in understanding the underlying mechanisms of SLE arises from the multifactorial influences on disease course, in- Kidneys were fixed in formalin and paraffin embedded, or frozen in OCT cluding environmental, infectious, and hormonal factors, the as- medium, and 5-mm sections were prepared. Toe specimens were fixed in 4% paraformaldehyde and decalcified with modified Kristensen’s solution. sociation of lupus with abnormalities at all levels of the immune After dehydration, the tissues were embedded in paraffin and 5-mm sec- system, and the clinical heterogeneity of the disease (7). The tions were made (21). disparate disease courses in SLE most likely converge into a Immunohistochemistry. For kidneys, periodic acid-Schiff, H&E, and common route of end organ damage. In this study, in a new model dichloroacetate esterase (to identify neutrophils) on paraffin-embedded of lupus induced by the passive transfer of human SLE sera, we sections were performed, as described (22). Neutrophils in 100 glomeru- provide direct evidence that SLE ICs are pathogenic, show lar cross-sections were quantified and presented as neutrophils per glomerular cross-section. Immunohistochemistry on frozen sections was a central role for neutrophils in target organ damage, and dem- performed using a two-layer peroxidase method. Sections were immu- onstrate that two molecules, FcgRIIA and Mac-1, determine the nostained with anti-F4/80 for macrophages (BioLegend), anti-CD3 capacity of ICs to cause tissue damage. In addition, these results, (Serotech) for T cells, and anti-C1q (Hycult Biotech) for C1q deposi- Downloaded from coupled with the analysis of neutrophil behavior by intravital tion, and counterstained with Gill Hematoxylin No. 2 (NewComerSupply). Tissue area occupied by macrophages or glomerular C1q deposition in microscopy, revealed a new regulatory role for Mac-1 in FcgRIIA- four 320 consecutive fields was quantified using ImagePro. Sections of mediated neutrophil recruitment following deposition of circu- paraffin-embedded toes were stained with H&E, and immunohistochem- lating ICs. istry was performed with anti–NIMP-R14 (Abcam) for neutrophil quantification, anti-F4/80 (BioLegend) for macrophages, anti-CD3 (Serotech) for T cells, or anti-human IgG (Invitrogen). The total number of neu- Materials and Methods trophils in a toe cross-section was counted and divided by the total tissue http://www.jimmunol.org/ Mice area in each section, using ImagePro. Immunofluorescence. Human and mouse IgG deposition were evaluated on Fcg-chain–deficient mice, human FcgRIIA and FcgRIIIB-expressing frozen sections using anti-human IgG or anti-mouse IgG (Invitrogen) Alexa g-chain–deficient mice (IIA+g2/2 or IIA+IIIB+g2/2), Mac-1–deficient 2/2 2/2 fluor 488-conjugated Abs, respectively. Deposition of C3 was evaluated mice (Mac-1 ), and CD18-deficient mice (CD18 ) were as described using a FITC-conjugated anti-C3 Ab (ICN/CAPPEL). Images of at least 15 (13, 15–17). Mac-1–deficient mice were bred with IIA+g2/2 or IIA+IIIB+ 2/2 + 2/2 2/2 + + 2/2 2/2 glomeruli per mouse were captured, and glomerular fluorescence intensity g mice to generate IIA g Mac-1 and IIA IIIB g Mac-1 was measured using the acquisition and analysis software Metamorph animals or with wild-type (WT) mice to generate IIA+g+/+Mac-12/2 and + +/+ (Molecular Devices). To evaluate human IgG subclasses, immunofluores- IIA g mice. Mice were bred in a specific pathogen-free facility. Age- cence was performed using anti-human IgG1, IgG2, IgG3, and IgG4 Abs and gender-matched mice 8–12 wk old were used in all experiments. The (The Binding Site), followed by an anti-sheep DyLight-488–conjugated Ab by guest on September 26, 2021 Harvard Medical School Animal Care and Use Committee approved all (Jackson ImmunoResearch Laboratories). procedures used in this study. Electron microscopy. Kidney samples were taken 10 d after injection of SLE Passive transfer of human SLE sera or rabbit anti-GBM Ab sera and processed using standard techniques. For the human SLE sera transfer model, mice were injected s.c. in both Generation of a K562 stable cell line and analysis of binding to flanks with 2.5 mg human IgG (Jackson ImmunoResearch Laboratories) in ICAM-1 CFA (Thermo Scientific and Difco) on day 23. On days 0 and 2, 200 ml sterile serum was injected i.v. For induction of nephrotoxic nephritis, mice The R77H mutation was introduced into human CD11b DNA by standard were given rabbit IgG in CFA at day 23 in the footpad and rabbit aGBM PCR techniques. K562 cells were transfected with WT or mutant (R77H) antisera at day 0, as described (18), and urine and kidneys were collected at CD11b and CD18 plasmids by electroporation and confirmed by se- day 21. quencing. Single-cell sorting was used to select CD11b-positive cells. Cells were cultured in RPMI 1640 media supplemented with 10% heat- Human sera samples inactivated FBS, under constant selection using G418 (500 mg/ml; Human serum samples were obtained from the Lupus Clinic at the Beth Invivogen). CD11b expression was assessed by FACS analysis using Israel Deaconess Medical Center or Instituto Nacional de Ciencias Medicas anti-human CD11b-PE–labeled Ab (BD Pharmingen). To evaluate K562 y de la Nutricion Salvador Zubiran. Patients with SLE were diagnosed cell binding to ICAM-1-Fc, cells were preactivated with 1 mM MnCl2 for according to the classification criteria of the American College of Rheu- 10 min in HEPES buffer and drawn across a coverslip containing immo- m 2 matology. The inclusion criteria for SLE patients were patients who fulfilled bilized ICAM-1-Fc (15 g/ml; R&D Systems) under 0.1 dynes/cm shear $4 of 11 American College of Rheumatology criteria for SLE and at the flow. The number of cells bound in four different fields was quantified time of the first blood draw had active disease (i.e., SLE disease activity every 2 min over a 10-min period. index $6) and were diagnosed with nephritis. Normal serum was obtained IgG depletion and quantification of human IgG from healthy individuals matched for age, gender, and ethnicity. At the time of serum collection, all individuals showed no signs or symptoms of SLE serum was incubated with protein A/G-coated agarose beads (Thermo infection. Informed consent was obtained from all patients and healthy Scientific) or control agarose beads. Depletion was determined by mea- donors under a Beth Israel Deaconess Medical Center Institutional Review suring human IgG by ELISA (Bethyl Laboratories). Human ICs were Board-approved protocol. Unless indicated, all experiments were per- quantified in human and mouse serum using an ELISA kit with C1q as the formed using serum from SLE patient A. capture element (ALPCO Immunoassays). Due to cross-reactivity with mouse IgG, an anti-human HRP Ab (Bethyl Laboratories) was used as the Disease evaluation secondary Ab for the quantification of human ICs in murine serum samples. Functional assessment of renal damage. Spot urine samples were collected, and urine albumin and creatinine were evaluated by ELISA (Bethyl Lab- Macrophage depletion using clodronate liposomes oratories) and a chemical assay (Cayman Chemical), respectively (13), and Mice were given an i.p. injection of 500 ml liposomes containing clodronate expressed as a ratio of urine albumin to creatinine. or PBS (23) on day 25 before the first SLE injection, followed by 200 ml Clinical scoring of arthritis. Mice were evaluated every other day after every 5 d. Tissue was harvested at day 10 or 14 after SLE sera injection. induction of disease. Inflammation of each limb was scored, as reported Macrophage depletion was confirmed by immunohistochemistry of kid- previously (19): 0, no evident inflammation; 1, redness or swelling of 1 toe; neys and spleen using anti-F4/80 Ab. The Journal of Immunology 3

Intravital microscopy evaluation of reverse passive Arthus renal tissue were similar in disease-susceptible and nonsusceptible reaction in the cremaster muscle mice (Supplemental Fig. 1C, Fig. 1C). Although a role for mouse Rabbit IgG anti-BSA Ab (200 mg/300 ml; Sigma-Aldrich, St. Louis, MO) anti-human IgG and/or ICs formed from human IgG reacting with was injected intrascrotally, followed by an i.v. injection of BSA (300 mg/ Ags in the mouse serum cannot be formally ruled out, it is un- 100 ml; Sigma-Aldrich). After 3 h, leukocyte recruitment in the cremaster likely to be critical for disease development for the following two of anesthetized mice was evaluated by intravital microscopy, as previously reasons. First, SLE sera induced nephritis in the absence of described (13). Four venules per mouse were analyzed. Leukocyte rolling preimmunization with CFA/human IgG (data not shown), albeit velocities were measured by tracking single leukocytes (10/venule) over several frames and calculating the distance moved per unit time (mm/s). disease was more variable. Second, human FcgRs, and thus by Adherent leukocytes were defined as cells remaining stationary for 30 s inference human IgG, are required for development of nephritis. and were expressed as the number of cells/mm2 venule. In the anti-GBM murine model, rabbit IgG triggers a robust mu- MIP-2 was locally applied, as previously described (24). Briefly, rine anti-rabbit reaction; nonetheless, tissue injury is largely in- injections were done using a beveled glass pipette of 1 mm outer diameter mounted on a manual micromanipulator (MM-33; Warner Instruments, dependent of the humoral response (26). We propose that, as is Hamden, CT). The tip was filled with 100 nmol/L MIP-2 (U.S. Bio- known for other autoimmune models, activation of the innate logicals). The pipette was placed, and the number of rollers across a per- immune response by CFA in our SLE model may be important pendicular line and the number of adherent cells (Ab) in a 200-mm segment independent of the adaptive immune response (27). of the venule were recorded over a 1-min period. Next, air pressure was Histopathological and electron microscopy analyses of renal applied to the pipette, to trigger the injection of MIP-2 (,1 ml). Successful injection was verified by the presence of swelling of the interstitial tissue tissue from human SLE sera-treated mice were undertaken. surrounding the pipette tip. The process of injection and the following Congruent with proteinuria, glomerular damage and inflammation 2 2 2 2 minute were recorded. The number of cells that were adherent after MIP-2 were observed only in hFcgR+g / Mac-1 / animals (Fig. 1D, Downloaded from Δ injection (Aa) was evaluated. The adherent cells, which is the percentage 1E) and were associated with mesangial deposits of ICs (Fig. 1E). of rollers that adhered in response to MIP-2 injection, was calculated using Together these data suggest that Mac-1 deficiency enables SLE- the following equation: (Aa 2 Ab)/number of rollers 3 100. induced kidney damage. This may have relevance to human dis- Statistical analysis ease because the R77H polymorphism in the ITGAM gene (that Data are expressed as mean 6 SEM. For proteinuria data, the line repre- encodes the CD11b chain of Mac-1) associated with SLE abro- sents the median of the group. Differences were determined by Mann- gates the capacity of Mac-1 expressed in a cell line, to bind its http://www.jimmunol.org/ Whitney U test. For group analysis, one-way ANOVA was used for pro- ligand ICAM-1 (Fig. 2). teinuria data and two-way ANOVA was used for the cellular infiltration data. When significant differences were shown, data were subjected to Pathogenicity of SLE serum depends on the IgG fraction and Mann-Whitney U test or Bonferroni test, respectively, for comparison its capacity to deposit in glomeruli between two mouse strains. The p values ,0.05 were considered significant. Sera collected from different SLE patients were tested in susceptible IIA+IIIB+g2/2Mac-12/2 mice. Four of six SLE sera induced nephritis, whereas sera from five healthy controls (normal Results serum) or heat-aggregated human IgG (surrogate of ICs) failed to Human SLE serum induces nephritis in mice that express induce disease (Fig. 3A). Moreover, four of five sera from SLE by guest on September 26, 2021 neutrophil human FcgRs and lack Mac-1 patients at an independent clinical center induced nephritis com- Human SLE serum was obtained from patients that had active pared with normal controls (Supplemental Fig. 1D). Notably, disease (SLE disease activity index $6) and no clinical evidence nephritis was observed with multiple SLE serum samples from the of infection. Gender- and race-matched healthy individuals served same patient when treatment and disease activity varied, but cir- as controls. Mice preimmunized with human IgG/CFA received culating ICs remained elevated. IgG depletion of SLE serum A two i.v. injections of SLE sera at days 0 and 2. Nephritis was abrogated its nephritis-inducing capacity (Fig. 3B), which sug- monitored by analyzing albumin leakage in the urine over a 21-d gests that ICs in the sera are required for disease development period. SLE serum from patient A caused marked proteinuria in (Fig. 1A). mice that expressed human FcgRs (hFcgRs) only when these mice The sera that caused disease, henceforth referred to as patho- additionally lacked Mac-1 (IIA+g2/2Mac-12/2 and IIA+IIIB+g2/2 genic sera (e.g., SLE sera A and E), were associated with glo- Mac-12/2) (Fig. 1A). Proteinuria peaked at day 14 (Supplemental merular deposits of human IgG that exhibited a granular pattern Fig. 1A). Mac-1–deficient mice expressing FcgRIIA alone, characteristic of ICs (Fig. 3C). In contrast, no human IgG depo- without FcgRIIIB, exhibited significant proteinuria, suggesting sition was observed when nonpathogenic sera were injected; these that this hFcgR was sufficient for disease development (Fig. 1A). included SLE sera D and F and sera from healthy controls (Fig. No proteinuria was observed in Mac-1–sufficient mice expressing 3C). All four human IgG isotypes, IgG 1–4, were observed in the hFcgRs (IIA+g2/2 or IIA+IIIB+g2/2), Mac-1–deficient mice glomeruli, predominantly as deposits in the mesangium (Fig. 3D). without hFcgRs (g+/+Mac-12/2), or WT animals (Fig. 1A). Sur- Specificity of the Abs was confirmed by analyzing their reactivity face expression of human FcgRs and the sister CD18 integrin, against purified human IgG1–4 in Western blots (data not shown). LFA-1, was similar in Mac-1–deficient and sufficient groups In the case of serum A, focal tubular basement membrane depo- (Supplemental Fig. 1B). Any effect of g-chain deficiency super- sition of IgG was also detected (Fig. 3D), which is a pattern imposed on Mac-1 deficiency on disease susceptibility was ruled observed in human lupus nephritis. SLE sera pathogenicity out as SLE serum induced nephritis in mice that express (i.e., capacity of human ICs to deposit in glomeruli) did not cor- hFcgRIIA, lack Mac-1, and were g-chain sufficient (Fig. 1B). relate with the amount of ICs or IgG present in the sera (Sup- Mouse anti-human IgG was detected in the serum (Supplemental plemental Table 1A), nor did it correlate with the patient type or Fig. 1C) and in the kidney (Fig. 1C) after SLE sera transfer, but this lupus manifestations (Supplemental Table 1B). was much less compared with that in WT mice subjected to anti- GBM (nephrotoxic) nephritis (Fig. 1C), a widely used model of The susceptibility conferred by Mac-1 deficiency is not due to immune-mediated disease induced by preimmunization with rab- defects in IgG clearance bit IgG/CFA, followed by transfer of rabbit anti-GBM serum (25). Levels of circulating (Fig. 4A) and deposited human IgG-ICs (Fig. Importantly, mouse anti-human IgG levels in both the serum and 4B) as well as glomerular complement C1q and C3 deposition 4 Mac-1 DELETION ENABLES HUMAN LUPUS SERA-INDUCED NEPHRITIS Downloaded from http://www.jimmunol.org/

FIGURE 1. SLE sera induces nephritis in mice that express hFcgR and lack Mac-1. (A) Mice received two i.v. injections of SLE sera A on days 0 and 2, by guest on September 26, 2021 and urine albumin (normalized to creatinine) at day 14 was evaluated. The dashed line indicates the mean of proteinuria in mice not given SLE sera. Each data point represents one mouse, and the solid line is the median of the group. (B) Mice expressing the FcgRIIA in the presence of the g-chain (IIA+g+/+), and the same that are additionally Mac-1–deficient (IIA+g+/+Mac-12/2) were injected with SLE sera A and albuminuria was evaluated at days 7 and 14. Albuminuria in IIA+g2/2Mac-12/2 mice given SLE sera was analyzed in parallel for comparison. Each dot represents one animal, and the line indicates the median of the group. ns, not significant. (C) Murine anti-human IgG was quantified in renal tissue by immunofluorescence in indicated transgenic mice that were untreated (Unt) or 21 d following SLE sera A transfer (SLE). WT mice given rabbit anti-GBM injection (WT aGBM) served as a positive control. Mean 6 SEM are graphed. Dashed line is the average of pixel intensity in the Unt mice. (D) Representative images of renal sections (day 21) stained with H&E or periodic acid-Schiff (PAS). The arrow indicates a glomerular crescent that was observed in ∼5% of the glomeruli. A histological score was given, and mean 6 SEM was graphed (n $ 5 mice per group). (E) Electron microscopy of glomeruli of mice given SLE sera: (i) IIA+IIIB+g2/2 mice had normal glomerular architecture; (ii and iv) IIA+IIIB+g2/2Mac-12/2 mice exhibited mesangial expansion with increased cellularity (ii), neutrophils within capillary loops (iii)(34,730), mesangial electron-dense deposits (*), and lysosomal vacuoles containing protein reabsorption droplets (arrows) (iv) typically observed during proteinuria (311,100). *p , 0.05, **p , 0.01, ***p , 0.001. en, Endothelial; m, mesangial cell; n, neutrophils; p, podocyte.

(Fig. 4B) were comparable between hFcgR-expressing mice that joint inflammation with cellular infiltration associated with bone were Mac-1 sufficient (IIA+IIIB+g2/2) or deficient (IIA+IIIB+g2/2 and cartilage destruction (Fig. 5B). Significant deposition of hu- Mac-12/2), as were peripheral blood neutrophil and leukocyte man IgG-IC within synovial blood vessels was observed in both counts (Supplemental Table 1C). disease-susceptible and nonsusceptible mice (Fig. 5C). Neu- trophils were abundant in the lesions (Fig. 5D), whereas macro- Passive transfer of human SLE serum induces arthritis in mice phages and T cells were not detected (data not shown). Notably, two of the pathogenic SLE sera, A (Fig. 5) and E (data not shown), caused arthritis in mice that affected primarily small Marked glomerular neutrophil infiltration is observed in joints, namely the distal interphalangeal, and were evident at day susceptible mice, whereas macrophages do not contribute to 4. Patient A had clinical manifestations of arthritis, and patient E disease did not have arthritis, but presented with Raynaud syndrome. Susceptible mice (hFcgR+g2/2Mac-12/2) exhibited abundant There was no correlation between human symptoms and arthritis glomerular neutrophil infiltration at day 10 that significantly development, as some sera from patients who clinically presented exceeded that in nonsusceptible mice (Fig. 6A). In contrast, a mild with nephritis and arthritis did not induce arthritis in mice (data interstitial macrophage and T cell infiltration was observed in both not shown). Moreover, unlike nephritis, development of arthritis groups of mice (Fig. 6A). Thus, Mac-1 deficiency appears to se- was more variable with different sera samples. As in the case of lectively enable neutrophil accumulation. To distinguish whether nephritis, hFcgR-expressing mice lacking Mac-12/2 developed the Mac-1–expressing cell responsible for resistance to nephritis arthritis (Fig. 5A). Histopathological analyses revealed intense corresponded to circulating (i.e., monocytes or neutrophils) or The Journal of Immunology 5

FIGURE 2. The SLE-associated R77H mutation of Mac-1 abrogates its capacity to bind ICAM-1 under shear flow. (A) Flow cytometry analysis of K562 cells stably expressing Mac-1 WT or Mac-1 R77H. Similar levels of Mac-1 were observed on the surface of Mac-1 WT and Mac-1 R77H- transfected cells. (B) Binding of K562 cells expressing WT or the R77H Mac-1 variant to plate-immobilized ICAM-1-Fc was evaluated under shear flow. Mac-1 R77H mutation prevented interaction with ICAM-1 compared with WT Mac-1. Mean 6 SEM of four independent experiments is shown. Downloaded from **p , 0.01, ***p , 0.001. resident cells, we generated bone marrow chimeras. Bone marrow from IIAg2/2 or IIAg2/2Mac-12/2 mice was transferred into lethally irradiated g2/2Mac-12/2 or g2/2 mice, respectively 2/2 2/2 2/2 2/2 2/2 2/2 (i.e., IIAg →g Mac-1 and IIAg Mac-1 →g ), http://www.jimmunol.org/ and 3 mo later SLE serum was administered. IIAg2/2Mac-12/2 bone marrow provided susceptibility to nephritis when transferred into nonsusceptible g2/2 mice, suggesting that a lack of Mac-1 on hFcgRIIA-expressing circulating cells allows the development of disease (Supplemental Fig. 2A). Immunohistochemistry in tissue of g2/2Mac12/2 recipients of IIAg2/2 bone marrow revealed Mac-1–positive macrophages in the renal interstitium following human SLE serum transfer, suggesting that SLE serum induces renal infiltration of blood monocytes and their subsequent differ- by guest on September 26, 2021 entiation into macrophages (Supplemental Fig. 2A). To determine whether monocytes/macrophages contribute to renal injury, these FIGURE 3. The pathogenicity of SLE serum correlates with the ca- populations were depleted prior to SLE serum transfer by ad- 2 2 2 2 pacity of hIgG to deposit in the glomeruli. (A) IIA+IIIB+g / Mac-1 / ministering clodronate liposomes, which are internalized by these mice received sera from healthy donors (normal, data are pooled from five cells and leads to their apoptosis (Supplemental Fig. 2B). donors), six SLE patients (denoted A–F), or heat-aggregated human IgG 2/2 2/2 Macrophage-depleted IIAg Mac-1 mice remained highly (ICs). **p , 0.01, ***p , 0.001 compared with mice that received normal susceptible to nephritis (Fig. 6B), suggesting that macrophages do sera. Each data point represents one mouse, and the line indicates the not significantly contribute to renal injury. Moreover, macrophage median. (B) IIA+IIIB+g2/2Mac-12/2 mice were treated with SLE sera A depletion in disease-nonsusceptible (IIAg2/2) mice did not result subjected to sham or IgG depletion, and urine albumin was determined. (C) in proteinuria (Fig. 6C) or glomerular neutrophil infiltration (data Human IgG (hIgG) deposition in glomeruli was quantitated on day 21 in + + 2/2 2/2 not shown) at day 10, suggesting that Mac-1 deficiency in IIA IIIB g Mac-1 mice injected with normal human sera, non- monocytes/macrophages does not enable disease. pathogenic (D, F) or pathogenic (A, E) SLE sera, or aggregated hIgG (ICs) (n $ 3 mice per group). Mean 6 SEM is given, and representative pictures Mac-1 deficiency modifies the response of neutrophils toward of selected samples are shown. *p , 0.05. (D) Glomerular deposition of + tissue-deposited ICs in vivo human IgG isotypes (1–4) was evaluated by immunofluorescence in IIA IIIB+g2/2Mac-12/2 given normal or SLE sera A (SLE). Focal tubular To elucidate the mechanism(s) by which Mac-1 regulates FcgRIIA- basement membrane deposition (arrows) was detected in some cases. mediated neutrophil accumulation, we conducted intravital microscopy of the cremaster muscle that allows the real-time visu- alization of neutrophil behavior within the vessel wall. The reverse slow rolling required FcgRIIA, as it was not observed in g2/2 passive Arthus (RPA) reaction, induced by the i.v. administration Mac-12/2 and g2/2 mice (Fig. 7B). The downmodulatory effect of soluble BSA and an intrascrotal injection of anti-BSA, results of Mac-1 was selective for ICs, as the rolling velocity induced by in IC deposition within and outside the cremasteric vessels (13, an intrascrotal injection of TNF-a was actually increased in 28). The RPA induces neutrophil rolling that is P- and E-selectin IIAg2/2Mac-12/2 mice compared with IIAg2/2 mice (data not dependent (28), whereas the slowing of the rolling velocity and shown), as previously described (30). In the RPA, the observed adhesion is dependent on murine FcgRs (29) and FcgRIIA (13). slower neutrophil rolling in IIAg2/2Mac-12/2 mice was not as- Following induction of the RPA, neutrophils in the venules of sociated with an increase in the number of adherent cells com- IIAg2/2Mac-12/2 mice rolled significantly slower than in IIAg2/2 pared with IIAg2/2 mice (Fig. 7C). E-selectin–mediated mice (Fig. 7A). This was not a result of differences between the activation of LFA-1 has been shown to slow neutrophil rolling two groups in expression of LFA-1 on circulating neutrophils or velocity, and in turn lead to efficient firm adhesion only in re- neutrophils recruited to the cremaster (Supplemental Fig. 2C). The sponse to a local chemoattractant stimulus, MIP-2 (24). Accord- 6 Mac-1 DELETION ENABLES HUMAN LUPUS SERA-INDUCED NEPHRITIS

posited ICs and subsequent tissue injury, following human lupus sera transfer, is modulated by a functional association between an integrin and an IgG receptor. The role of Mac-1 in attenuating FcgRIIA-mediated neutrophil recruitment in the context of deposited ICs is unanticipated from previous work that has established Mac-1 largely as a positive regulator of neutrophil influx and inflammation (8, 31, 32). Our data predict that the R77H Mac- 1 variant, which leads to reduced binding to its ligands, may have functional consequences for SLE patients and offer a mechanism by which Mac-1 dysfunction may contribute to end organ injury in SLE. Disease susceptibility permitted by the absence of Mac-1 was not related to alterations in IC handling presumably by macrophages. Moreover, Mac-1 on macrophages and indeed macrophages themselves did not influence disease susceptibility. Thus, this cell type, abundant in SLE lesions (33), is not essential for target organ injury in our model at least in the time frame of our experiments. Instead, Mac-1 deficiency most likely predisposes to

disease development due to unrestrained FcgRIIA-mediated neu- Downloaded from trophil accumulation in the kidney. The reported physical interaction of Mac-1 with human FcgRs on the cell surface (34) and the sharing of intracellular ITAM-based signaling cascades by these two receptors (35) led us to the intriguing possibility that Mac-1 on neutrophils, in cis, modulates neutrophil FcgRIIA ac-

tivity and function. http://www.jimmunol.org/ FcgRIIA on neutrophils clearly has the capacity to induce tissue injury in vivo (13, 14). However, in the context of SLE-IC deposits in the kidney and joints, the regulation of FcgRIIA-mediated neutrophil recruitment appears to be a key step in conferring disease susceptibility. Recent work suggests that binding of FcgRIIA to ICs and FcgRIIA function may be regulated. In vitro, the G protein-coupled receptor agonists C5a and fmlp enhance (14, 36), whereas coengagement of FcgRIIB inhibits (37) FcgRIIA function. How is FcgRIIA regulated by Mac-1? Clues by guest on September 26, 2021 were provided by intravital microscopy. Slowing of the velocity of rolling cells improves their efficiency of firm adhesion (30), and, FIGURE 4. IC clearance and deposition are similar in susceptible and in the RPA, FcgRIIA plays an important role in mediating slow nonsusceptible mice. (A) Serum was collected from animals at intervals up rolling and leukocyte arrest (13). Additionally, CD18 integrins are to day 14 after SLE sera A transfer. Circulating human IgG (hIgG) and required as slow rolling, and MIP-2–mediated adhesion in the human IgG ICs (hICs) were evaluated, and the mean 6 SEM were RPA is abrogated in CD18-deficient mice. We postulate that graphed. (B) Kidneys were harvested at day 14 after SLE sera A transfer, FcgRIIA promotes the activation of LFA-1. A deficiency in Mac- and renal sections were stained for hIgG, murine C1q, and complement 1heightensFcgR activity toward ICs and subsequent LFA-1 6 C3. The mean fluorescence intensity (MFI; average pixel intensity activation that in turn leads to further deceleration of the roll- SEM) for glomerular hIgG and C3 and the percentage of glomerular area ing velocity and increased responsiveness to MIP-2. Mac-1 may positive for C1q (% area 6 SEM) were determined and found to be be responsible for mitigating FcgRIIA-mediated signaling or equivalent in both groups. limiting FcgRIIA mobility or clustering, the molecular details of which require further investigation. Human FcgRIIA appears to ingly, the local microinjection of MIP-2 favored adhesion of functionally interact with murine Mac-1 in cis, suggesting that 2 2 2 2 2 2 neutrophils in IIAg / Mac-1 / compared with IIAg / animals this interaction uses conserved features of human and mouse subjected to the RPA (Fig. 7D). IC induced slow rolling, and MIP- Mac-1. 2–induced neutrophil adhesion is most likely LFA-1 dependent, as The observed decrease in neutrophil accumulation in Mac-1– these steps were markedly diminished in CD18-deficient mice deficient mice in other models of IC-mediated disease, including (Fig. 7E, 7F). acute anti-GBM, thrombotic glomerulonephritis, and bullous In summary, FcgRIIA-dependent slow rolling, which correlates pemphigoid (22, 38, 39), suggests differential roles for Mac-1 in with efficient leukocyte adhesion in response to MIP-2, is regu- distinct models of IC-based diseases. Complex biological circuits lated by Mac-1, with a deficiency in this integrin, resulting in predict dual and sometimes opposing roles for the same receptor a further decrease in rolling velocity and subsequent enhanced that is cell type and context dependent. For example, incomplete responsiveness of neutrophils to a local chemotactic stimulus. phosphorylation of the ITAM following engagement of B or TCRs by low affinity/valency ligands favors inhibitory over activating Discussion signaling by these immune receptors (40). Moreover, unsustained In this study, we show in a novel lupus model that IC deposition and FcaRI clustering markedly inhibits, whereas sustained FcaRI organ damage are not inextricably linked, but that additional ge- aggregation promotes cell activation through differential recruit- netic factors dictate the propensity for target organ damage. In ment of the tyrosine kinase Syk versus an inhibitory protein particular, we demonstrate that the neutrophils’ response to de- phosphatase Src homology region 2 domain-containing phospha- The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Human SLE sera cause arthritis in mice that express hFcgR and lack Mac-1. Mice given SLE sera A were evaluated for the development of arthritis. (A) Inflammation of each limb was assessed, and a clinical score was given, as described in Materials and Methods. As shown in representative pictures, mostly small joint involvement (arrows) was observed. *p , 0.05 compared with IIA+IIIB+g2/2 and IIA+g2/2 given SLE sera. (B) Representative images of histological sections stained with H&E (upper panels) with magnification of boxed area (lower panels) are shown for indicated mice. Bone (b) erosion and inflammatory cell infiltration (*) were apparent only in IIA+IIIB+g2/2Mac-12/2 mice. The histological score in affected limbs of IIA+IIIB+g2/2 Mac-12/2 and similar digits in IIA+IIIB+g2/2 mice included leukocyte infiltration, synovial thickening, and cartilage and bone erosion. (C) Tissue hIgG staining by immunohistochemistry showed intravascular IC deposition (arrows) in nonsusceptible (upper panel) and susceptible (lower panel) mice treated with SLE sera. (D) Neutrophils in joint sections harvested at indicated days after SLE serum injection were quantitated. Robust neutrophil accumulation was observed only in IIA+IIIB+g2/2Mac-12/2. Representative pictures of indicated mice are shown. Only IIA+IIIB+g2/2 Mac12/2 have neutrophil infiltrates (arrow) adjacent to damaged articular surfaces as well as periarticular soft tissue. All data are mean 6 SEM. For (B) and (D), *p , 0.05. tase 1 (41, 42). We posit that Mac-1 relays similar context-dependent associated with SLE (44), high FcgRIIIB levels increase suscep- inhibitory and activating signals, a hypothesis that is supported by tibility to anti-neutrophil cytoplasmic Ab-associated systemic the finding that, although the R77H Mac-1 polymorphism is vasculitides, a disease associated with in situ rather than soluble a susceptibility factor for lupus (9), it does not predispose to ICs (45, 46). This concept is recapitulated in our mouse models. rheumatoid arthritis (43), another IC-mediated disease. Interest- Human SLE serum-induced nephritis was reduced in mice ingly, FcgRIIIB may also play context-dependent roles in IC- expressing both human FcgRIIA and FcgRIIIB versus FcgRIIA mediated inflammation. Whereas low expression of FcgRIIIB is alone, which contrasts with the greater proteinuria following anti- 8 Mac-1 DELETION ENABLES HUMAN LUPUS SERA-INDUCED NEPHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Mac-1 deficiency slows the rolling velocity and increases the chemoattractant-induced adhesion of neutrophils following the RPA reaction. The RPA was induced in the cremaster, and, 3 h later, the mice were prepared for intravital microscopy and neutrophil rolling velocity was measured in hemodynamically similar venules of (A) IIA+g2/2Mac-12/2 + 2/2 B 2/2 FIGURE 6. Disease in susceptible mice is associated with neutrophil and IIA g mice (n = 33 venules in 6 mice per group) and ( ) g Mac- 2/2 2/2 accumulation; macrophages do not participate in disease development. 1 and g mice (n = 13 venules in 3 mice per group). Cumulative C (A) Kidneys were harvested from mice at day 21 after injection of SLE histograms of rolling velocities are shown. ( ) The number of adherent A D sera A. Dotted line represents the mean value obtained in susceptible mice neutrophils in mice analyzed in ( ) was determined. ( ) Mice were sub- given normal human serum and harvested at day 10 (top panel)orday14 jected to the RPA, the cremaster was prepared for intravital microscopy, (middle and bottom panels). Neutrophil, macrophage, and T cell infiltra- and a local injection of MIP-2 was given as depicted in the diagram. The tion were quantified by immunohistochemistry. Mice treated with anti- increase in neutrophil adhesion after local MIP-2 injection (D of adherent + 2/2 + 2/2 2/2 GBM serum are included as a positive control for indicated leukocyte cells) in IIA g and IIA g Mac-1 mice was calculated (n = 15–16 6 , , venules in 6–7 mice per group). (E) Cumulative histogram of neutrophil infiltration. Mean SEM is shown. *p 0.05, **p 0.01 compared 2 2 2 2 / F with IIA+IIIB+g / injected with SLE sera. n = 3–7 mice per group. rolling velocities of WT and CD18 mice subjected to the RPA, and ( ) Animals were treated with liposomes that contained clodronate (+) or D of adherent cells upon MIP-2 injection are shown (n = 6 venules in 3 2 2 C D F 6 , PBS (2), and proteinuria was evaluated at indicated times in IIA+g / mice per group). For ( ), ( ), and ( ), mean SEM are graphed. **p 2 2 2 2 , Mac-1 / (B) and IIA+g / (C) mice. *p , 0.05. nd, Not determined; ns, 0.01, ***p 0.001. not significant. tentially autoantigenic, and drive IFN-a production by dendritic GBM nephritis in mice expressing both hFcgRs compared with cells (51, 52). SLE sera transfer results in the development of mice expressing FcgRIIA alone (13). lesions that recapitulate several features of lupus nephritis. The Our studies suggest that neutrophils are the primary cellular link mice obviously lack the imbalances in the adaptive immune re- between IgG and target organ damage in our model. Neutrophils sponse and therefore chronicity of the disease. However, like SLE are present in renal SLE lesions (47, 48), precede macrophage patients, in which development of lupus nephritis is not associated infiltration (47), and are appreciated as a sign of disease severity with autoantibodies of singular IgG specificity (53) a spectrum of (49). Importantly, recent studies identified a subset of neutrophils IgG1–4 subtypes was found deposited in the glomerulus with the in SLE patients that synthesize type I IFNs, induce endothelial cell capacity of SLE sera to induce proteinuria being directly corre- damage (50), release neutrophil extracellular traps that are po- lated with their ability to deposit in the glomerulus. 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