Mouse Protease-4 Deteriorates Renal Function by Contributing to and Fibrosis in Immune Complex-Mediated Glomerulonephritis This information is current as of October 1, 2021. Lisa Scandiuzzi, Walid Beghdadi, Eric Daugas, Magnus Åbrink, Neeraj Tiwari, Cristiana Brochetta, Julien Claver, Nassim Arouche, Xingxing Zang, Marina Pretolani, Renato C. Monteiro, Gunnar Pejler and Ulrich Blank

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 7, 2010, doi:10.4049/jimmunol.0902129 The Journal of Immunology

Mouse Mast Cell Protease-4 Deteriorates Renal Function by Contributing to Inflammation and Fibrosis in Immune Complex-Mediated Glomerulonephritis

Lisa Scandiuzzi,*,†,1 Walid Beghdadi,*,† Eric Daugas,*,†,‡ Magnus A˚ brink,x Neeraj Tiwari,*,† Cristiana Brochetta,*,† Julien Claver,*,† Nassim Arouche,†,{ Xingxing Zang,|| Marina Pretolani,†,{ Renato C. Monteiro,*,† Gunnar Pejler,# and Ulrich Blank*,†

Mast cells exert protective effects in experimental antiglomerular basement membrane-induced glomerulonephritis (GN), yet the responsible mediators have not been identified. In this study, we investigated the role of mouse mast cell protease (mMCP)-4, the

functional homolog of human , using mMCP-4–deficient mice. Compared with wild type animals, mMCP-4–deficient mice Downloaded from exhibited lower proteinuria, blood creatinine, and blood urea nitrogen levels, indicating an aggravating role of mMCP-4. Kidney histology confirmed less severe renal damage in mMCP-4–deficient mice with reduced deposits, glomerular and interstitial cellularity, and fibrosis scores. High amounts of mMCP-4 were detected in renal capsules, but not in the whole kidney, from wild type mice. Its expression in renal capsules was markedly decreased after GN induction, suggesting that locally released by degranulated mast cells could contribute to the functional and physiopathological hallmarks of GN. Supporting

a proinflammatory role, glomerular and interstitial macrophage and T cell infiltration, levels of proinflammatory TNF and http://www.jimmunol.org/ MCP-1 mRNA, and the expression of the profibrotic peptide angiotensin II together with type I collagen were markedly down- regulated in kidneys of mMCP-42deficient mice. We conclude that mMCP-4 chymase, contrary to the global anti-inflammatory action of mast cells, aggravates GN by promoting kidney inflammation. These results highlight the complexity of mast cell- mediated inflammatory actions and suggest that chymase inhibition may represent a novel therapeutic target in GN. The Journal of Immunology, 2010, 185: 000–000.

ast cells are key effectors of innate and adaptive im- granules, and most of them are present exclusively in mast cells. munity (1–3). Upon activation, they produce numerous These proteases can be divided into three classes: tryptases, car- by guest on October 1, 2021 M inflammatory mediators released from cytoplasmic boxypeptidase A, and , all of which are released in granules or after new synthesis, including arachidonic acid a complex with negatively charged serglycin proteoglycans (5–7). metabolites and a diverse set of cytokines and chemokines (3, 4). Chymases are -like serine proteases, which can be Neutral proteases are contained in high amounts in the cytoplasmic classified into a-chymases and b-chymases, according to their biochemical structure (5–7). Human mast cells express only one a-chymase, whereas rodents express one a-chymase, mouse mast † b *Institut National de la Sante´ et de la Recherche Me´dicale U699; Universite´ Paris 7, cell protease (mMCP)-5, and several -chymases, including Faculte´ de Me´decine Paris Diderot-Site Xavier Bichat; ‡Service de Ne´phrologie, Groupe mMCP-1, -2, and -4. While mMCP-4 and -5 are specifically Hospitalier Universitaire Bichat-Claude Bernard, Assistance Publique-Hoˆpitaux de Paris; { x expressed by mast cells in connective tissues, mMCP-1 and -2 Institut National de la Sante´ et de la Recherche Me´dicale U700, Paris, France; De- partment of Medical Biochemistry and Microbiology, Uppsala University; #Department are found in mucosal subtype mast cells. The former are stored of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, in tight complexes with serglycin proteoglycans and are released Uppsala, Sweden; and ||Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461 only upon degranulation, whereas the latter have low affinity for 1 serglycin proteoglycans and, accordingly, they may be constitu- Current address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY. tively secreted (8). Among the murine chymases, mMCP-4 is most Received for publication July 7, 2009. Accepted for publication April 21, 2010. likely the functional counterpart of the unique human chymase This work was supported by the French National Research Agency (R08171HS), the because it has similar substrate specificity, tissue distribution, ser- Fondation pour la Recherche Me´dicale, the Association pour l’Utilisation du Rein glycin proteoglycan-binding properties, and ability to convert an- Artificiel, and the Marie Curie Early Stage Research Training Fellowship of the giotensin (Ang) I into Ang II (5, 9, 10). European Community’s Sixth Framework Programme under contract no. 504926. In addition to their crucial role in allergy and asthma, mast cells Address correspondence and reprint requests to: Dr. Ulrich Blank, Institut National de la Sante´ et de la Recherche Me´dicale U699, Universite´ Paris 7, Faculte´ de Me´de- have recently been associated with renal diseases, because their cine Denis Diderot, Site Xavier Bichat, 16 Rue Henri Huchard, 75780 Paris Cedex numbers are strongly increased in nephropathies of different origins 18, France. E-mail address: [email protected] (11–13). Functional studies conducted using mast cell-deficient mice Abbreviations used in this paper: ACE, angiotensin-converting enzyme; Ang, angio- revealed protective properties of mast cells in an accelerated model tensin; anti-GBM, antiglomerular basement membrane; BMDM, bone marrow- derived macrophage; BMMC, bone marrow-derived mast cell; BUN, blood urea of antiglomerular basement membrane (anti-GBM)–induced glo- nitrogen; DTH, delayed-type hypersensitivity; GN, glomerulonephritis; mMCP, merulonephritis (GN), most likely through their capacity to initiate mouse mast cell protease; NRS, normal rabbit serum; PAS, periodic acid-Schiff; repair and remodeling and to favorably influence immune responses RT, room temperature; SA, streptavidin; WT, wild type. (14, 15). However, other results demonstrated a detrimental role in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 a nonaccelerated disease model of kidney disease (16). In this model,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902129 2 AGGRAVATING ROLE OF mMCP-4 CHYMASE IN GLOMERULONEPHRITIS mast cells promoted glomerular expression of adhesion molecules 8.7, 16.9 6 10.2, and 13.7 6 4.8; and for kidney capsules: 4.7 6 2.3, and enhanced Th1-dependent effector mechanisms. Overall, these 4.8 6 1.5, 1.8 6 1.3, and 1.2 6 0.7. observations indicate that mast cells can exert beneficial or detri- mental activities, depending on a given pathophysiological context. Induction of anti-GBM–induced GN Therefore, further studies are needed to clarify the mechanisms by Anti-GBM serum was produced in rabbits following immunization with pu- which mast cells affect renal disease parameters. rified mouse glomeruli obtained according to established procedures (22). The murine counterpart of human mast cell chymase, mMCP-4, An accelerated model of anti-GBM Ab-induced GN was used (23, 24). was shown to attract granulocytes and macrophages (17, 18), to Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g regulate homeostatic intestinal epithelial migration and barrier body weight) (Southern Biotechnology Associates) and CFA (Sigma- function (19), and to convert Ang I into Ang II, a vasoactive peptide Aldrich) 5 d before an i.v. administration of anti-GBM serum through the retro-orbital vein at a dose of 0.2 ml/20 g. For control purposes, with potent inflammatory and fibrogenic properties (20, 21). These NRS was also used. Mice were sacrificed at day 3 to evaluate early changes observations suggest that mMCP-4 may play an important role in of the heterologous phase following deposition of rabbit anti-GBM or at tissue inflammation and remodeling in the kidney. To explore this day 14 to evaluate late changes provoked by the developing immune re- issue, we examined the role of mMCP-4 in the accelerated model of sponse to the injected Abs. No sign of GN was observed when NRS was administered to WT and mMCP-4–deficient mice. Perfused kidneys were anti-GBM–induced GN using mMCP-4–deficient mice. We show fixed in 10% formalin and embedded in paraffin. Some portions of the that these mice develop less severe disease compared with their kidneys were also frozen in Tissue-Tek OCT compound (Polysciences) for wild type (WT) counterparts. This effect involved the ability of immunohistochemical and immunofluorescence analysis. Other portions mMCP-4 to mediate inflammatory and fibrotic processes in the were lysed in RA1 buffer (Nucleospin, Macherey-Nagel) for quantitative kidney. RT-PCR experiments or in buffer for protein analysis, as described below.

Experimental groups included male mMCP-4–deficient mice and their WT Downloaded from littermates between weeks 7 and 10 wk of age. All experiments were Materials and Methods approved by a local ethics committee. Induction of anti-GBM–induced GN Anti-GBM serum was produced in rabbits following immunization with pu- Evaluation of systolic blood pressure rified mouse glomeruli obtained according to established procedures (22). Systolic blood pressure of WT and mMCP-4–deficient mice (n = 6) before An accelerated model of anti-GBM Ab-induced GN was used (23, 24).

and after induction of anti-GBM GN was measured by the tail-cuff http://www.jimmunol.org/ Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g method, adapted to the mouse using Chart 4.1.1 software (ADInstruments, body weight) (Southern Biotechnology Associates, Birmingham, AL) and Chalgrove, U.K.) (26). To avoid variations in blood pressure that occur CFA (Sigma-Aldrich, St. Louis, MO) 5 d before an i.v. administration of during the day cycle, all measurements were carried out between 10 AM anti-GBM serum through the retro-orbital vein at a dose of 0.2 ml/20 g. For and 12 PM. Ten measurements from each mouse were taken on three control purposes, normal rabbit serum (NRS) was also used. Mice were consecutive days. sacrificed at day 3 to evaluate early changes of the heterologous phase following deposition of rabbit anti-GBM or at day 14 to evaluate late changes provoked by the developing immune response to the injected Immunoblotting Abs. No sign of GN was observed when NRS was administered into WT and mMCP-4–deficient mice. Perfused kidneys were fixed in 10% Tissue lysates were prepared in 50 mM Tris-HCl (pH 8) containing 1% SDS

formalin and embedded in paraffin. Some portions of the kidneys were and 5% glycerol. Lysates were migrated on a 12% SDS-PAGE, followed by by guest on October 1, 2021 also frozen in Tissue-Tek OCT (Polysciences, Warrington, PA) compound transfer onto nitrocellulose membrane (Schleicher & Schu¨ll Microscience, for immunohistochemical and immunofluorescence analysis. Other por- Dassel, Germany), as described (27). Membranes were blocked with 4% tions were lysed in RA1 buffer (Nucleospin, Macherey-Nagel, Hoerdt, BSA for 1 h, followed by incubation for 1 h at room temperature (RT) with France) for quantitative RT-PCR experiments or in buffer for protein anal- the following primary Abs: a rabbit anti-serum to mMCP-4 (a kind gift ysis, as described below. Experimental groups included male mMCP-4– from Lars Hellman, Uppsala University) (28), mouse mAb JRK directed to deficient mice and their WT littermates between 7 and 10 wk of age. All the b-chain of the high-affinity IgER (29), and a mouse mAb anti–b-actin experiments were approved by a local ethics committee. (Sigma-Aldrich). After several washes, blots were incubated with donkey anti-rabbit IgG HRP (1/30,000) or goat anti-mouse IgG HRP (1/20,000) Mouse strains and reconstitution experiments (Jackson ImmunoResearch Laboratories, Newmarket, U.K.) for 45 min Previously generated mMCP-4 knockout mice (25) were backcrossed into and were developed by ECL (GE, Paris, France). the C57BL/6J background, shipped from Uppsala, and a founder colony was independently established and housed under strictly controlled pathogen- Cell cultures free conditions at the mouse facilities of IFR02 at the Bichat Medical School. Homozygous mMCP-4–deficient and WT mice were screened by Bone marrow-derived macrophages (BMDMs) were obtained by isolating PCR using the following primers: forward primer 59- CAAGGTCCAAC- bone marrow cells from femurs and tibias of C57BL/6J mice, as described TAACTCCCTTTGTGCTCC-39, first reverse primer 59-GGGCCAGCTCA- (30). Cells were resuspended at a concentration of 1 3 106 cells/ml in TTCCTCCCACTCATGATCT-39, and second reverse primer 59-GGTGAT- BMDM complete medium (DMEM/Glutamax; BioWhittaker, Walkers- CTCCAGATGGGCCATGTAAGGGCG-39. They yielded a 380-bp and ville, MD), supplemented with 15% FCS, 10% L cell-conditioned medium a 900-bp amplified product for knockout and WT genes, respectively. Am- (as a source of M-CSF-1), 100 U/ml penicillin G, and 100 mg/ml strepto- plification was performed overnight with shuttle PCR using TaKaRa en- mycin (Life Technologies, Rockville, MD) and cultured at 37˚C in a hu- zyme (Takara Bio, Shiga, Japan), with annealing and melting temperatures midified 5% CO2 incubator in 75-ml flasks. After 24 h, the nonadherent of 68˚C and 98˚C. C57BL/6-Tg(OT-II)-RAG1tm1Mom mice were bred in cells were removed, counted, plated on bacteriologic (nontissue cultured- specific pathogen-free facilities at the Animal Care Facility of the Albert treated) plastic plates at a concentration of 1 3 106 cell/ml, and cultured in Einstein College of Medicine. BMDM complete medium that was replaced every 3 d. BMDMs were used For reconstitution experiments, bone marrow-derived mast cells between days 7 and 8. (BMMCs) grown from C57BL/6 WT or MCP-4–deficient mice were BMMCs were obtained by isolating bone marrow precursors from femurs transferred by retro-orbital i.v. injection (1 3 107 cells in 200 ml PBS) and tibias of WT and mMCP-4–deficient mice, as described (31). Cells W-sh/W-sh into mast cell-deficient Kit mice on the C57/BL/6 background were grown at 37˚C in a humidified 5% CO2 incubator in complete BMMC (The Jackson Laboratory, Bar Harbor, ME). After 12 wk, mice were medium (IMDM, 15% FCS, 25 mM HEPES [pH 7.4], 1 mM sodium subjected to anti-GBM GN. Successful reconstitution was monitored by pyruvate, 1% nonessential amino acid, 54 mM 2-ME with 100 U/ml pen- analysis of toluidine blue (see below)-stained sections of spleen, inguinal icillin, and 100 mg/ml streptomycin) (Invitrogen, Cergy Pontoise, France) lymph nodes, and kidney capsules after anti-GBM GN. Values (mast cell/ containing recombinant murine IL-3 and stem cell factor (10 ng/ml for mm2) for WT mice, mMCP-42/2 mice, KitW-sh/W-sh mice reconstituted with each cytokine) (PeproTech, Paris, France) for 4–8 wk to obtain differen- WT BMMCs, and KitW-sh/W-sh mice reconstituted with mMCP-42/2 tiated BMMCs. Medium was replaced every 5 d, and BMMCs were used BMMCs were, respectively, for spleen: 0.7 6 0.7, 0.5 6 0.7, 18.2 6 between weeks 4 and 8. Cell suspensions contained .96% mast cells, as 10.7, and 43.3 6 25.7; for inguinal lymph nodes: 54.7 6 15.7, 57.7 6 assessed by FACS analysis. The Journal of Immunology 3

Assessment of humoral and delayed-type hypersensitivity immune responses Sera from mice were collected at indicated days before and after the in- duction of anti-GBM GN. Sera from mice injected with NRS were used as a control. To detect normal rabbit IgG in serum, 96-well plates were coated overnight with F(ab9)2 goat anti-rabbit IgG (H+L) (Jackson ImmunoRe- search Laboratories, West Grove, PA) at 1 mg/ml in PBS (100 ml/well). As a standard, wells were incubated with defined concentrations of rabbit IgG (Southern Biotechnology Associates). After washing in 0.05% Tween-20 in PBS (pH 7.2), serum samples (100 ml/well) diluted in PBS/1% BSA were added for 1 h at RT. After washing, biotinylated donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) (1/100 in PBS/1% BSA) was added for 2 h at RT before the final addition of streptavidin (SA)-HRP diluted 1/200 (R&D Systems, Lille, France) for 20 min at RT. ELISA was developed by the addition of the substrate, and the reaction was stopped by adding 1 N HCl. The OD of each well was determined using a microplate reader set at 450 nm. To detect mouse anti-rabbit IgG, 96-well plates were incubated overnight with 100 ml/well rabbit IgG (Southern Biotechnology Associates) at a final concentration of 1 mg/ml in PBS. After washing, serum samples (100 ml/well) diluted in PBS/1% BSA were added for 1 h at RT. After washing, biotinylated donkey anti-mouse IgG biot (H+L) (Jackson ImmunoResearch Laboratories) (1/200) was added for 2 h at RT before the final addition of SA-HRP diluted 1/200 (R&D Sys- Downloaded from tems) for 20 min at RT. ELISAs were developed as described above. To evaluate dermal delayed-type hypersensitivity (DTH) to rabbit Ig, mice were challenged, 13 d after induction of GN, by injecting 250 mg normal rabbit or control mouse IgG into the right and left hind footpad, respectively. The swelling response was determined at 24 h using a mi- crometer. Hypersensitivity reaction was determined as the difference in

swelling response between the right and left footpads. http://www.jimmunol.org/ Evaluation of proteinuria, blood urea nitrogen, and creatinine levels FIGURE 2. Mice deficient in mMCP-4 have less decline in renal func- 2 2 Spot samples of urine were collected at indicated days. Urinary protein tion. WT mice (n =9),mMCP-4 / mice (n =13)(A), or in a separate concentrations (g/l) were normalized to urinary creatinine concentrations experiment, mast cell-deficient KitW-sh/W-sh mice reconstituted with WT (mmol/l) by calculating the ratio of urinary protein per urinary creatinine. BMMCs (n =6)ormMCP42/2 BMMCs (n =6)(D) were subjected to Proteinuria, blood urea nitrogen (BUN), and blood or urinary creatinine an accelerated model of anti-GBM–induced GN. The ratio of urinary levels were determined using commercially available kits (Olympus protein to urinary creatinine was determined. WT mice (n = 3 at day 3 Diagnostics, Hamburg, Germany) by means of an autoanalyzer (AU400; 2/2 and n = 9 at day 14), mMCP-4 mice (n = 3 at day 3 and n = 13 at day 14)

Olympus Diagnostics), as described (32). by guest on October 1, 2021 (B, C), or in a separate experiment, mast cell-deficient mice reconstituted Histological and immunohistochemical analysis with WT BMMCs (n = 6) or mMCP-4-deficient BMMCs (n =6)(E, F) were subjected to an accelerated model of anti-GBM–induced GN. Blood To assess histological changes by light microscopy, tissue sections were creatinine and BUN were measured after induction of anti-GBM–induced stained with periodic acid-Schiff (PAS) reagent and Masson’s trichrome. 6 Images were observed on a blinded basis with a Leica microscope (Leica GN on the days indicated. Data are mean SEM of urine or blood samples p , Microsystems, Rueil-Malmaisons, France) coupled to a MD2000 camera in each group. p 0.05. using 310, 320, 340, 363, or 3100 auxiliary lenses and a direct X1 C-mount. Deposits appear as pink- and green-stained areas for PAS and Germany) and L3T4 rat anti-mouse CD4 T cell Ab (Southern Biotechnol- Masson’s trichrome staining, respectively, which contrast with adjacent ogy Associates), as described (23). Rat anti-mouse IgG biotin (Vector glomerular structures. Glomerular deposits were scored in a minimum of Laboratories, Servion, Switzerland) was used for immunoperoxidase stain- 30 glomeruli per mouse as follows: 0 = no deposition of Masson’s tri- + ing. Staining with secondary Ab alone was negative. Scoring of positive chrome ; 1 = up to one third of the cross-sectional area of the glomerulus + cells was determined manually in a minimum of 30 high-power fields is Masson’s trichrome ; 2 = one third to two thirds of the cross-sectional + (340 or 363 objective) per experimental condition. Frozen kidney sec- area of the glomerulus is Masson’s trichrome ; and 3 = more than two thirds of the cross-sectional area of the glomerulus is Masson’s trichrome+. tions were also stained with Texas red goat anti-rabbit IgG or FITC goat anti-mouse IgG (Southern Biotechnology Associates) to evaluate heterol- Glomerular proliferation, interstitial infiltration, interstitial fibrosis, and ogous rabbit or autologous mouse anti-rabbit Abs deposited in the glomer- tubular necrosis were quantified using a semiquantitative score (0–3): uli, respectively, using immunofluorescence light microscopy. Fibrin 0 = absence of lesions, 1 = ,25%, 2 = 25–50%, and 3 = .50% of lesions. deposition was determined using direct staining with FITC-labeled anti- Mast cells in indicated tissues were evaluated after fixation in 10% fibrin Ab (Nordic Immunological Laboratories, Tilburg, The Netherlands). formalin and staining with toluidine blue. They were counted using a 340 Type I collagen and Ang II accumulation were evaluated, respectively, objective with a minimum of 30 high-power fields and are expressed as 2 using a goat anti-type I collagen plus FITC-labeled donkey anti-goat Ab number/mm . Macrophage and T cell infiltration was assessed by immu- (Southern Biotechnology Associates) and a guinea pig anti-Ang II Ab nohistochemistry using Mac-1 (anti-CD11b) Abs (AbD Serotec, Mu¨nster, (Peninsula Laboratories, Weil am Rhein, Germany), followed by staining with tetramethylrhodamine isothiocyanate-labeled donkey anti-guinea pig Ab (Jackson ImmunoResearch Laboratories) for 30 min. Quantitative anal- ysis of images was performed on a minimum of 30 high-power fields using NIH ImageJ software (National Institutes of Health, Bethesda, MD). Transwell cell-migration assay Chemotaxis of BMDMs and CD4+ T cells was measured by assessing migration through a polycarbonate filter with 8 mm and 5 mm pore size, FIGURE 1. Expression of mMCP-4 in tissues of WT and mMCP-4- respectively, in a 24-transwell microchemotaxis chamber (3422; Corning 2/2 deficient ( ) mice. Lysates (20 mg/lane) of indicated tissues were mi- Costar, Cambridge, MA). One hundred microliters BMDM cell suspension grated on SDS-PAGE and analyzed by immunoblotting using anti–mMCP-4 (1 3 106 cells/ml resuspended in IMDM medium, 5% FCS, 15% L cell- Ab. For loading controls, blots were probed with anti–b-actin Ab. conditioned medium, 100 U/ml penicillin, and 100 mg/ml streptomycin) or 4 AGGRAVATING ROLE OF mMCP-4 CHYMASE IN GLOMERULONEPHRITIS spleen cells freshly isolated (1 3 106 cells/ml in IMDM medium, 5% FCS, using reverse transcriptase and 1 mmol/l oligonucleotide-dT primer. Each 25 mM HEPES [pH 7.4], 1 mM sodium pyruvate, 1% nonessential amino cDNA sample was amplified by using specific primers to detect acid, 54 mM 2-ME with 100 U/ml penicillin, and 100 mg/ml streptomycin) MCP-1 (sense 59-GGCTCAGCCAGATGCAGTTAA-39 and antisense 59- from C57BL/6-Tg(OT-II)-RAG1tm1Mom mice were added to the upper CCTACTCATTGGGATCATCTTGCT-39), TNF (sense 59-CTGTCTAC- chamber. Anti-DNP IgE-sensitized BMMCs (1 3 106 cell/ml) derived TGAACTTCGGGGT-39 and antisense 59-GGTCTGGGCCATAGAACTG- from WT and mMCP-4–deficient mice were stimulated for 30 min with AT-39), IL-1b (sense 59-CTGTGTCTTTCCCGTGGACC-39 and antisense DNP-HSA (10 ng/ml), and 600 ml supernatant was added to the lower 59-CAGCTCATATGGGTCCGACA-39), TGF-b (sense 59-CCTGGGCAC- chamber (in duplicate). One hundred nanograms per milliliter of murine CATCCATGA-39 and antisense 59-CCGCACACAGCAGTTCTTCTC-39), MCP-1 (R&D Systems) in 5% serum complete BMMC medium and anti- and ribosomal protein L13a (RPL13a) (sense 59-GTGGTCCCTGCT- DNP IgE-sensitized BMMCs (1 3 106 cell/ml), stimulated for 2 h with GCTCTCAA-39 and antisense 59-CGATAGTGCATCTTGGCCTTTT-39). DNP-HSA (10 ng/ml), were used as positive control in separate lower Real-time PCR was performed in SYBR Green I dye (Sigma-Aldrich). chambers for the macrophage and for the T cell chemotaxis experiments, Measurements of the threshold cycles were made at the end of each ex- respectively. Chambers were incubated at 37˚C and 5% CO2 for indicated tension step by the second-derivative method, using LightCycler software, times. At the end of the incubation period, the total cell number in the version 4.0 (Stratagene, La Jolla, CA). Melting-curve analysis was per- lower compartment was determined microscopically by staining with try- formed to validate the identity of peaks of interest in all samples. Absolute pan blue (Thermo Scientific, Asheville, NC). For the CD4+ T cell chemo- quantification of the number of copies was calculated by determining half- taxis experiment, the percentages of CD4+ T cells migrated toward the maximal fluorescence values and comparing them to an internal standard lower chamber were determined by staining with specific Abs and sub- of amplification with a known number of copies. sequent FACS analysis. The chemotaxis index for BMDMs or CD4+ T cells was determined as the ratio of migrated cells per well/cells mi- Data analysis grated to wells containing medium from unstimulated BMMCs. Data are expressed as mean 6 SEM of the indicated number of experi- ments. Statistical analysis was performed using an unpaired Student t test; Flow cytometry values of p , 0.05 were considered significant. Downloaded from To evaluate the purity of BMMCs, cells were washed in FACS buffer (ice- cold 0.5% BSA in PBS) and stained with rat anti-mouse c-Kit–biotin and hamster anti-mouse FcεRI-biot Abs (both from eBioscience, San Diego, Results CA), followed by SA-PE (Southern Biotechnology Associates). For T cell mMCP-4 deficiency reduces renal disease in anti-GBM– chemotaxis experiments, migrated cells in the lower chamber were washed induced GN in FACS buffer and stained with anti-CD4 (L3T4) conjugated to allophy- cocyanin (eBioscience). To prevent unspecific binding, all samples were The expression of mMCP-4 was assessed by immunoblotting in preincubated with Fc-Block or unlabeled isotype-matched unspecific Abs kidney, ear, and renal capsule tissue lysates from WT and http://www.jimmunol.org/ (eBioscience). Data were acquired using a FACSCalibur flow cytometer mMCP-4–deficient mice (Fig. 1). Although mMCP-4 was prom- (BD Biosciences) according to standard protocols. inent in ear and renal capsule, no mMCP-4 was detected in total RT-PCR and real-time PCR kidney tissue, likely reflecting the relative paucity of mast cells in the kidney (12). As expected, mMCP-4 was absent in tissues from RNA was isolated from the cortex region of kidney of anti-GBM–treated mMCP-4–deficient and WT mice using Nucleospin purification system mMCP-4–deficient animals. (Macherey-Nagel). Kidneys from mice injected with NRS were used as The role of mMCP-4 in anti-GBM–induced GN was examined a control. The first-strand cDNA was synthesized from 2 mg total RNA in WT and mMCP-4–deficient mice using an accelerated model by guest on October 1, 2021

FIGURE 3. Mice deficient in mMCP-4 exhibit less deterioration in kidney morphology. A, Representative histological analysis of Masson’s trichrome- (1, 3, 4) and PAS- (2) stained sections of anti-GBM kidneys in the late autologous phase (day 14) of WT and mMCP-42/2 mice, as well as WT control mice that had received NRS (original magnifications: panel 1,103 objective; panel 2, 1003 objective; panels 3, 4,403 objective). Less extensive histological lesions are seen in all of the kidney compartments from mMCP-42/2 mice compared with WT mice. Progression of glomerular injury is characterized by subendothelial deposits (arrows in panel 2 and left arrow in panel 3), intraglomerular hypercellularity (lower right arrow in panel 2), tubular necrosis (arrows in panel 4) and mononuclear cell infiltrate in the interstitium (left pictures, right arrow on panel 3 and panel 4). B, Scoring of PAS+ and Masson’s trichrome+ material at day 14 in WT (n =9)ormMCP-42/2 mice (n = 13) after injection of anti-GBM Ab. pp , 0.05; ppp , 0.02. C, Scoring of Masson’s trichrome+ material at day 14 after injection of anti-GBM Ab of mast cell-deficient KitW-sh/W-sh mice reconstituted with WT BMMCs (n = 6) or mMCP-4– deficient BMMCs (n = 6) subjected to an accelerated model of anti-GBM–induced GN in separate experiments. Data are mean 6 SEM of urine or blood samples in each group. The Journal of Immunology 5

(23, 24). Both mouse strains developed marked proteinuria and after induction of GN (not shown). To determine whether mast showed high levels of blood creatinine and BUN following the cells present in kidney capsules became activated to release injection of anti-GBM serum (Fig. 2A–C). However, these param- mMCP-4 after the induction of anti-GBM GN, mMCP-4 expres- eters were significantly reduced in mMCP-4–deficient mice, in sion was assessed by immunoblotting of tissue lysates collected particular at the late (autologous) phase of the disease (days 10– from WT mice before and after the induction of GN. Fig. 5A 14), whereas no differences in proteinuria (Fig. 2A) were noted at shows that a marked decrease (90%) in mMCP-4 levels in the the time of the early (heterologous) phase (day 3). Both mouse renal capsules was observed at day 14 after the injection of anti- strains injected with NRS failed to develop functional features of GBM Abs. Under these conditions, mast cells were still present, as GN (data not shown). Together, these results demonstrated that demonstrated by the expression of FcεRI b-chain, which recog- mMCP-4 contributed to GN progression. Although mMCP-4 is nizes degranulated and nondegranulated mast cells. In agreement, a mast cell-specific enzyme, its absence may lead indirectly to histological analysis showed a decrease in toluidine blue-stained developmental defects or affect other cell types, which could con- mast cells in renal capsule tissue sections after anti-GBM treat- tribute to GN progression (33). To examine this possibility, mast ment, likely reflecting extensive mast cell degranulation (Fig. 5B). cell-deficient KitW-sh/W-sh mice were reconstituted with BMMCs By contrast, mast cell numbers did not significantly change in obtained from WT or mMCP-4–deficient mice for 12 wk before other tissues, including ear, skin, and tongue (Fig. 5C). Likewise, inducing anti-GBM GN. In agreement with the above studies, mMCP-4 levels in ear tissue were not significantly altered after mice reconstituted with mMCP-4–deficient mast cells showed a GN induction (Fig. 5D). Mast cell numbers were comparable tendency to lower proteinuria and lower blood creatinine levels between WT and mMCP-4–deficient mice (Fig. 5C). Taken and had significantly decreased BUN (Fig. 2D–F). Results in age- together, these results suggest that mast cells released mMCP-4 matched WT and mMCP-4–deficient mice were comparable (data locally in renal capsules upon anti-GBM–induced GN. Downloaded from not shown). This supports the conclusion that mast cell-secreted Inflammatory cytokines are decreased in kidneys of mMCP-4– mMCP-4 is directly responsible for the observed effects. deficient mice mMCP-4 aggravates histological features of anti-GBM– To establish whether mMCP-4 contributed to anti-GBM–induced induced GN kidney inflammation by sustaining the local production of

To delineate the involvement of mMCP-4 in kidney damage, we per- inflammatory cytokines and chemokines, we assessed mRNA http://www.jimmunol.org/ formed PAS and Masson’s trichrome staining in kidney tissue sec- steady-state levels of MCP-1, TGF-b, TNF, and IL-1b in kidney tions from WTand mMCP-4–deficient mice sacrificed at day 14 after anti-GBM. Both groups of mice showed significant renal damage compared with NRS-treated mice (Fig. 3A). However, semiquantita- tive evaluation showed lower deposits and cellularity in the glomer- ulus of mMCP-4–deficient mice compared with their WT counter- parts (Fig. 3B). This was accompanied by a decrease in cellular in- filtration, interstitial fibrosis, and tubular necrosis. Similar results by guest on October 1, 2021 were observed when mast cell-deficient KitW-sh/W-sh mice recon- stituted with WT or mMCP-4–deficient BMMCs were examined (Fig. 3C). These findings indicate that mMCP-4 directly modulates renal damage in this model. Evaluation of immune responses in WT and mMCP-4–deficient mice We next examined whether mMCP-4–deficient mice had altered humoral immune responses to injected rabbit IgG Abs compared with WT mice. Evaluation of frozen kidney sections at day 14 showed no significant differences in rabbit IgG or mouse anti- rabbit IgG glomerular deposits between WT and mMCP-4– deficient mice (Fig. 4A). Similarly, serum levels of rabbit IgG were comparable at day 3 and they had decreased to a similar extent by day 14 (Fig. 4B, left panel). Finally, the amounts of mouse anti-rabbit IgG at day 14 were indistinguishable between the two mouse strains (Fig. 4B, right panel). No significant levels FIGURE 4. Assessment of immune responses. A, Levels of heterologous of mouse anti-rabbit Abs were detected prior to the injection of anti-GBM rabbit Abs and autologous mouse anti-rabbit IgG in glomeruli. anti-GBM (data not shown). These results demonstrated that de- Cryostat kidney sections (day 14) from anti-GBM–treated WT mice (n = 9), ficiency in mMCP-4 fails to alter the deposition of heterologous mMCP-42/2 mice (n = 13), and healthy NRS-treated WT control mice were Abs and the onset of the humoral immune response to injected stained using Rhodamine goat anti-rabbit IgG (top panels) and FITC goat Abs. We also evaluated T cell responses by measuring dermal anti-mouse IgG (bottom panels) Abs and analyzed by immunofluorescence DTH activity to rabbit Ig. As shown in Fig. 4C, no differences microscopy. Objective magnification is indicated. The inset shows a single 3 in dermal DTH responses became apparent between WT and glomerulus at higher magnification (63 objective). B, Circulating rabbit and mouse anti-rabbit Abs in sera of mMCP-42/2 and WT mice were mMCP-4–deficient mice. measured by ELISA, as described in Materials and Methods. Sera were 2/2 mMCP-4 is released locally from mast cells in renal capsules collected at day 3 (n = 3) and at day 14 (WT mice, n = 9; mMCP4 after induction of GN mice, n = 13) after the induction of anti-GBM GN. C, Dermal DTH responses to rabbit IgG in WT mice (n = 5) and mMCP-42/2 mice (n = 5). The swelling Large amounts of mMCP-4 are expressed in renal capsule tissue, response was determined at day 14 after the induction of anti-GBM GN. All whereas we were unable to detect it in renal tissue before (Fig. 1) or data are mean 6 SEM. 6 AGGRAVATING ROLE OF mMCP-4 CHYMASE IN GLOMERULONEPHRITIS

FIGURE 5. mMCP-4 expression in renal capsules be- fore and after the induction of anti-GBM GN and mast cell quantification. A, Lysates (20 mg/lane) of renal capsule tissue from NRS- and anti-GBM–treated WT mice or an equivalent of 1 3 105 BMMC control lysates were migrated in parallel on 12% SDS-PAGE and analyzed by immunoblotting using anti–mMCP-4 or anti-FcεRI b-chain Abs. Numbers indicate relative expression levels compared with the first lane, which was set arbitrarily to 1. B, Several granulated mast cells stained with toluidine blue (arrows) can be observed in renal capsule tissue from control (NRS)-treated WT mice, whereas they are absent in anti-GBM–treated mice. Objective magnifications are indicated. Insets show high magnification (1003 objective) of intact granulated and degranulated mast cells (taken in a different field), the latter being observed often after anti-GBM treatment. Respective quantification of toluidine blue-stained mast cells in healthy NRS- treated mice (n = 6), anti-GBM–treated WT mice (n = 2 2 6), and mMCP-4 / mice (n = 6) is reported in the far right panel. pppp , 0.001. C, Quantification of toluidine Downloaded from blue-stained mast cells in indicated tissues. All data are mean 6 SEM. D, Lysates (20 mg/lane) of ear tissue from NRS- and anti-GBM–treated WT mice were migrated in parallel on SDS-PAGE and analyzed by immunoblotting using anti–mMCP-4 or anti–b-actin. Numbers indicate relative expression levels compared with the first lane, which was set arbitrarily to 1. http://www.jimmunol.org/ tissue extracts (34). All of the transcripts analyzed were elevated mMCP-4–deficient mice had slightly, but significantly, lower numbers at day 14 in GN-stimulated WT and mMCP-4–deficient mice of macrophages within the glomeruli, but not in the interstitial space, compared with NRS-injected mice (Fig. 6). mMCP-4–deficient compared with their WT counterparts (Fig. 7A). Similarly, intra- mice had significantly lower levels of MCP-1 and TNF mRNA glomerular and interstitial CD4+ T cells were markedly decreased than WT mice, a trend toward a decrease in the levels of IL-1b, in mMCP-4–deficient mice compared with WT mice (Fig. 7B). To but no significant changes in the expression of TGF-b. These examine whether mMCP-4 had chemotactic activity toward these results further support the view that mMCP-4 promotes a proin- cells, we determined the chemotactic potential of supernatants col- by guest on October 1, 2021 flammatory response that favors disease progression. lected from short-term IgE-activated WT or mMCP-4–deficient BMMCs toward macrophages or CD4+ T cells using an in vitro Role of mMCP-4 in mediating inflammatory cell infiltration transwell-migration assay. As shown in Fig. 7C, supernatants from We next examined the glomerular and tubulointerstitial inflammatory mMCP-4–deficient BMMCs had a significantly lower macrophage infiltrate in kidneys from anti-GBM–stimulated mice, in particular chemotactic activity than those collected from WT BMMCs. Su- macrophages and T cells, which are the main inflammatory cell pernatants from short-term IgE-activated BMMCs also induced sig- types that play a role in this disease model (35, 36). We found that nificant migration of CD4+ T cells; however, no differences were seen between WT and mMCP-4–deficient cells. Together, these results suggest that mMCP-4 contributes tokidneyinflammationbypromot- ing the infiltration and accumulation of macrophages and CD4+ T cells. This may be secondary to the increased inflammatory pro- cesses initiated, but it may also involve a direct recruitment of ma- crophages by mMCP-4 released locally from capsule tissue.

Renal fibrosis is attenuated in mMCP-4–deficient mice Next, a series of immunohistochemical studies was performed to analyze the possible consequences of mMCP-4 deficiency on known mMCP-4 target proteins, such as type I collagen, fibrin, and Ang II, which are associated with the renal-remodeling process (5, 9). Examination of kidney tissue sections from anti-GBM– treated mice showed a prominent increase of glomerular fibrin FIGURE 6. The expression of proinflammatory cytokine and chemokine and interstitial type I collagen deposits at day 14 compared with mRNA in kidneys is lower in mMCP-4–deficient mice. The cDNAs from 2 2 control Ab-treated kidneys (Fig. 8A,8B). The amounts of type I the cortex region of kidney of mMCP-4 / and WT mice, as well as from collagen were significantly decreased in mMCP-4–deficient mice, control (NRS)-treated WT mice, were used to evaluate the expression of MCP-1, TGF-b, TNF, and IL-1b mRNA by real-time PCR. The level of all whereas those of fibrin were unaffected. Finally, Ang II in the genes is expressed as the number of copies. Data are mean 6 SEM of interstitium was nearly absent in mMCP-4–deficient mice; its a minimum of six mice in each group. pp , 0.05. Similar results were levels were similar to those observed in NRS-injected mice (Fig. obtained when the number of copies was compared with the housekeeping 8C). By contrast, WT animals attained significant levels of Ang II, gene RPL13a. which appeared as a punctuate staining in localized areas through- The Journal of Immunology 7

FIGURE 7. Role of mMCP-4 in kidney macro- phage and CD4+ T cells infiltration in anti-GBM GN. Day 14 cryostat kidney sections from anti- GBM–treated WT mice (n = 9) and mMCP-42/2 mice (n = 13), as well as NRS-treated WT control mice (n = 6), were stained using anti-CD11b (A)or anti-CD4 (B) Abs (left panels). Cells infiltrating glo- meruli and interstitium were quantified (middle and right panels). Data are mean 6 SEM in each group. Objective magnifications for glomerulus and intersti- tium are indicated in the right panels. C, mMCP-4 increases the chemotaxis of macrophages but not of CD4+ T cells. BMDMs or spleen cells from OTII mice were added to the upper chamber in transwell plates, and migration was initiated by adding super- natants collected from short-term (30 min) IgE- stimulated BMMCs from mMCP-42/2 or WT mice

to the lower chamber. MCP-1–containing medium Downloaded from and supernatant of anti-DNP IgE-sensitized BMMCs stimulated for 2 h with DNP-HSA were used as posi- tive controls for BMDMs or CD4+ T cells, respec- tively. Relative migration was determined as described in Materials and Methods. Data are mean 6 SEM of three independent experiments. pp , 0.05;

pppp , 0.001. http://www.jimmunol.org/

out the interstitial space. Because chymase-mediated Ang II gen- Discussion eration has been implicated in blood pressure regulation with Mast cells produce a large variety of mediators with beneficial a potential impact on GN development (37), we also analyzed and deleterious roles in inflammatory diseases, depending on the systolic blood pressure in a separate experiment. However, no pathophysiological context (1–3, 39). Recently, we and other in- by guest on October 1, 2021 significant differences between the two types of mice were seen vestigators showed that mast cells protect against anti-GBM– before or after the induction of GN (Fig. 9). Together, these find- induced GN; however, relevant mast cell mediators involved ings indicated that mMCP-4 mediates type I collagen deposition have remained unclear (14, 15). Protection was seen in the in kidneys and local Ang II generation, both of which are associ- absence of any detectable mast cell infiltration into kidneys, ated with the fibrotic process (38). indicating that these cells could act systemically (14, 15). In the

FIGURE 8. mMCP-4–deficient mice have decreased glomerular and interstitial deposits of type I collagen and Ang II. At day 14, cryostat kidney sections from anti-GBM–stimulated mMCP-42/2 mice (n = 13) and WT mice (n = 9), as well as from NRS-treated WT control mice (n = 10) were stained with anti-fibrin Ab (A), anti-type I collagen Ab (B), or anti-Ang II Ab (C) and were analyzed by immunofluorescence light micros- copy (left panels). Fluorescence intensities were quan- tified (right panels). Objective magnifications for glomerulus and interstitium are indicated in the right panels. Data are mean 6 SEM per group. pp , 0.05. 8 AGGRAVATING ROLE OF mMCP-4 CHYMASE IN GLOMERULONEPHRITIS

function in the renal microenvironment to induce its biological effects. This is supported by the fact that no such mMCP-4 release and degranulation were noticed in distant ear tissues. Similarly, al- though the numbers of countable mast cells decreased in the cap- sule, likely as a result of their degranulated phenotype, these numbers were stable in other tissues (ear, skin, and tongue) exam- ined in WT and mMCP-4–deficient mice. Thus, our data indicate that mMCP-4 contributes locally to the aggravation of GN by mediating a variety of proinflammatory effects. In agreement, lower levels of TNF and MCP-1, and to a lesser extent, of FIGURE 9. No difference in systolic blood pressure between WT and IL-1b mRNA, were observed in mMCP-4–deficient mice com- mMCP-4–deficient mice. Systolic blood pressure of WT (n = 6) and pared with WT mice. All of these cytokines/chemokines were mMCP-42/2 mice (n = 6) before and after induction of anti-GBM GN shown to have a pathogenic role in anti-GBM–induced GN (35, was measured by the tail-cuff method. All measurements (10/mouse) were 41, 42). Although we cannot explain the mechanism behind the carried out between 10 AM and 12 PM on three consecutive days. Data are TNF and MCP-1 induction, possible scenarios include direct cell 6 mean SEM. No significant differences were seen. Urinary protein/cre- activation of kidney resident cells through G protein-coupled pro- atinine ratios after anti-GBM GN (day 10) were 10.9 6 3.7 g/mmol and tease-activated receptors (40, 43) or indirect effects through the 7.4 6 4.3 g/mmol for WT and mMCP-4–deficient mice, respectively. general activation of infiltrating cells.

Moreover,micelackingmMCP-4revealedanimpairedinfiltration Downloaded from current study, we examined the expression and role of mMCP-4 of inflammatory macrophages and CD4+ T cell upon anti-GBM chymase, the likely counterpart of human chymase, as a candidate treatment compared with their WT counterparts. In vitro experi- mediator accounting for this protective action of mast cells in GN. ments showed that supernatants of short-term IgE-activated WT This protease is one of the mast cell-specific proteases released BMMCs, which contained mediators stored in secretory granules, from the secretory granules by activated connective tissue type exhibited heightened macrophage chemotactic activity compared mast cells, and it plays an important role in tissue homeostasis with mMCP-4–deficient BMMCs. This is in agreement with pre- http://www.jimmunol.org/ (5–7). Interestingly, and contrary to our expectations, our results vious studies showing potent chemotactic properties of human chy- revealed that mice deficient in mMCP-4 developed attenuated mase and mMCP-4 for several types of leukocytes (17, 18). These glomerular disease, indicating that mMCP-4 has a detrimental role supernatants also promoted the migration of CD4+ T cells, although in GN. no specific mMCP-4–mediated enhancement was noticed. Altogether, Analysis of renal parameters following disease induction clearly these results suggested that the increased T cell infiltration in WT mice showed that the absence of mMCP-4 resulted in less severe kidney is likely the consequence of the generally aggravated inflammatory disease, with lower proteinuria and decreased serum parameters, response, whereas for macrophage infiltration, the possibility exists such as BUN and creatinine. This difference became apparent at that mMCP-4 diffused from capsules participates in the recruitment. the late stage (autologous phase) of GN development, whereas To establish whether mMCP-4 interferes with the remodeling com- by guest on October 1, 2021 the extent of the disease was similar in the two mouse strains during ponent of anti-GBM–induced GN, we examined the expression of the early stage (heterologous phase). Attenuation of renal disease Ang II, a peptide with multiple proinflammatory roles that promotes was confirmed by histological analysis showing less cellularity proliferation and fibrogenesis in the kidney (21, 44). The increase in and fewer deposits in the glomeruli and lower infiltration and tubular local expression of interstitial Ang II observed in WT mice upon anti- necrosis scores in the interstitium of mMCP-4–deficient mice. Although these findings contrast with the reported protective GBM stimulation was not apparent in mMCP-4–deficient animals. function of mast cells (15, 23), they are in agreement with recent Because human chymase and mMCP-4 can directly generate Ang II results showing that purified chymase preparations promote from Ang I as an alternative to the angiotensin-converting enzyme glomerular albumin permeability (40) and that mMCP-4 contrib- (ACE) pathway (10, 45), these data suggest that mMCP-4 is directly utes to the severity of autoimmune arthritis (28) and abdominal responsible for Ang II generation in vivo. This notion is also in agree- aortic aneurysm formation (33). ment with data showing that ACE-knockout mice do not display any Although we did not see any baseline effects on kidney de- major reduction in tissue Ang II, in contrast to its levels in the circu- velopment, kidney histology and renal function, in NRS-treated lation, indicating that ACE-independent Ang II-generation mecha- mMCP-4–deficient mice, mMCP-4 could have unknown develop- nisms, such as through chymase, are active (45). Based on the re- mental effects or affect the activity of other cells types. To analyze ported effects of chymase-mediated Ang II generation on blood this question directly, we reconstituted mast cell-deficient mice with pressure regulation that might contribute to the aggravation of GN WT or mMCP-4–deficient mast cells before the induction of anti- (37, 46), we measured systolic blood pressure in WT and mMCP-4– GBM–induced GN. Our data confirmed the less deteriorated renal deficient animals before and after the induction of GN. However, no phenotype in mice reconstituted with mMCP-4–deficient mast cells, measurable differences were found within the time frame examined, further supporting a direct effect of mast cell-derived mMCP-4 suggesting that blood pressure regulation does not contribute to the in renal disease progression. observed aggravation. Our findings extend previous observations Although the observed effects of mMCP-4 are compatible with suggesting that human chymase and mMCP-4 act as an alternate a role of mMCP-4 in kidney disease progression, we were unable to pathway to ACE for Ang II production in vivo, as shown in experi- detect mMCP-4 and mast cells in renal interstitium, even after GN mental and human nephropathies (46–49). Histological analysis of induction. Interestingly, however, large amounts of mMCP-4 were kidney tissue sections also revealed a partial inhibition of type I found in renal capsules, with levels decreasing markedly after collagen deposition in mMCP-4–deficient mice, an intriguing find- the induction of GN in conjunction with extensive mast cell degran- ing that differed from the known ability of mMCP-4 to trigger ulation. The agents responsible for this degranulation remain MMP-9 activity, which degrades collagen (9). However, during the unknown, but could be multiple, because of the induced inflamma- inflammatory phase, mMCP-4–induced Ang II may represent the tory response in anti-GBM kidneys. Once released, mMCP-4 may mechanism involved in this inhibition, because Ang II was shown The Journal of Immunology 9 to strongly contribute to the progression of renal disease and fibrosis 7. Stevens, R. L., and R. Adachi. 2007. Protease-proteoglycan complexes of mouse development (21, 24). and human mast cells and importance of their beta-tryptase-heparin complexes in inflammation and innate immunity. Immunol. Rev. 217: 155–167. Overall, our current results demonstrating that inflammatory and 8. Braga, T., M. Grujic, A. Lukinius, L. Hellman, M. Abrink, and G. Pejler. 2007. fibrotic responses in the kidney were markedly attenuated in the Serglycin proteoglycan is required for secretory granule integrity in mucosal absence of mMCP-4 contrast with previous findings showing that mast cells. Biochem. J. 403: 49–57. 9. Tchougounova, E., A. Lundequist, I. Fajardo, J. O. Winberg, M. Abrink, and mast cells exert global protective properties against renal diseases G. Pejler. 2005. A key role for mast cell chymase in the activation of pro-matrix (14, 15). One explanation for this discrepancy is that mast cells, in metalloprotease-9 and pro-matrix metalloprotease-2. J. Biol. Chem. 280: 9291– addition to mMCP-4, can release a set of mediators, including 9296. 10. Caughey, G. H., W. W. Raymond, and P. J. Wolters. 2000. Angiotensin II gen- tryptase, serglycin proteoglycans, and biogenic amines, with mul- eration by mast cell alpha- and beta-chymases. Biochim. Biophys. Acta 1480: tiple potential functions in relation to GN, as well as a plethora of 245–257. cytokines and growth factors that may counteract the pathological 11. Eddy, A. A. 2001. Mast cells find their way to the kidney. Kidney Int. 60: 375– 377. effects of this chymase. In addition, mast cells may be beneficial 12. Blank, U., M. Essig, L. Scandiuzzi, M. Benhamou, and Y. Kanamaru. 2007. Mast or detrimental, depending on the stage of disease. Indeed, follow- cells and inflammatory kidney disease. Immunol. Rev. 217: 79–95. ing the administration of anti-GBM Abs, WT mice showed a high 13. Holdsworth, S. R., and S. A. Summers. 2008. Role of mast cells in progressive renal diseases. J. Am. Soc. Nephrol. 19: 2254–2261. capacity to initiate repair functions and to activate the fibrinolytic 14. Kanamaru, Y., L. Scandiuzzi, M. Essig, C. Brochetta, C. Gue´rin-Marchand, system (14), suggesting that mast cells may decrease inflammation Y. Tomino, R. C. Monteiro, M. Peuchmaur, and U. Blank. 2006. Mast cell- at early time-points. Such early functions do not involve mediated remodeling and fibrinolytic activity protect against fatal glomerulone- phritis. J. Immunol. 176: 5607–5615. mMCP-4, because no protective effects were seen in mMCP-42 15. Hochegger, K., F. Siebenhaar, V. Vielhauer, D. Heininger, T. N. Mayadas, deficient mice at the initial stages of GN. Finally, mast cells G. Mayer, M. Maurer, and A. R. Rosenkranz. 2005. Role of mast cells in ex- may display immunoregulatory functions leading to downregula- perimental anti-glomerular basement membrane glomerulonephritis. Eur. J. Downloaded from Immunol. 35: 3074–3082. tion of inflammatory responses, as observed in other disease mod- 16. Timoshanko, J. R., R. Kitching, T. J. Semple, P. G. Tipping, and els (1–3, 39, 50, 51). Thus, the boundary between beneficial and S. R. Holdsworth. 2006. A pathogenetic role for mast cells in experimental detrimental functions can be tight, as also indicated by previous crescentic glomerulonephritis. J. Am. Soc. Nephrol. 17: 150–159. 17. Tani, K., F. Ogushi, H. Kido, T. Kawano, Y. Kunori, T. Kamimura, P. Cui, and data showing that mast cells can be protective or detrimental in S. Sone. 2000. Chymase is a potent chemoattractant for human monocytes and GN, depending on the experimental model (16). Similar contrast- neutrophils. J. Leukoc. Biol. 67: 585–589. ing examples were also reported in other pathophysiological con- 18. Terakawa, M., Y. Tomimori, M. Goto, and Y. Fukuda. 2006. Mast cell chymase http://www.jimmunol.org/ induces expression of chemokines for neutrophils in eosinophilic EoL-1 cells ditions, such as protein-induced fatal , in which and mouse peritonitis eosinophils. Eur. J. Pharmacol. 538: 175–181. the response is mediated by mast cells, but their proteases serve to 19. Groschwitz, K. R., R. Ahrens, H. Osterfeld, M. F. Gurish, X. Han, M. Abrink, detoxify these proteins (52). In addition, mast cell proteases may F. D. Finkelman, G. Pejler, and S. P. Hogan. 2009. Mast cells regulate homeo- static intestinal epithelial migration and barrier function by a chymase/Mcpt4- have protective effects during sepsis (53) or in allergic airway dependent mechanism. Proc. Natl. Acad. Sci. USA 106: 22381–22386. inflammation (54), but they can also aggravate disease by pro- 20. Urata, H., A. Kinoshita, K. S. Misono, F. M. Bumpus, and A. Husain. 1990. moting exacerbating inflammation (28, 33, 55), as also shown in Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart. J. Biol. Chem. 265: 22348–22357. this study. 21. Dussaule, J. C., and C. Chatziantoniou. 2007. Reversal of renal disease: is it In conclusion, we demonstrated that mice lacking mMCP-4 had enough to inhibit the action of angiotensin II? Cell Death Differ. 14: 1343–1349. by guest on October 1, 2021 fewer functional and histological signs of GN, particularly kidney 22. de Almeida, D. B., and M. F. de Franco. 1971. Isolated glomerular basement membrane as antigen in indirect immunofluorescent test for identification of inflammation and fibrosis, than WT mice, contrasting with the circulating rabbit anti-rat glomerular-basement membrane antibodies. Int. Arch. globally anti-inflammatory function of mast cells (14, 15). These Allergy Appl. Immunol. 41: 559–564. findings highlight the complexity of the inflammatory response, 23. Kanamaru, Y., M. Arcos-Fajardo, I. C. Moura, T. Tsuge, H. Cohen, M. Essig, F. Vrtovsnik, C. Loirat, M. Peuchmaur, L. Beaudoin, et al. 2007. Fc alpha receptor I which represents a double-edged sword, initially destined to re- activation induces leukocyte recruitment and promotes aggravation of glomerulone- store homeostasis but leading to pathology when dysregulated. phritis through the FcR gamma adaptor. Eur. J. Immunol. 37: 1116–1128. Our data suggest that strategies aimed at inhibiting chymase 24. Suzuki, Y., I. Shirato, K. Okumura, J. V. Ravetch, T. Takai, Y. Tomino, and C. Ra. 1998. Distinct contribution of Fc receptors and angiotensin II-dependent may represent a novel strategy for the treatment of GN. pathways in anti-GBM glomerulonephritis. Kidney Int. 54: 1166–1174. 25. Tchougounova, E., G. Pejler, and M. Abrink. 2003. 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