Isolated from Iga Nephropathy Patients Cells Following Stimulation with Iga1 and Cell Proliferation in Human Mesangial Productio

Spleen Tyrosine Kinase Is Important in the Production of Proinflammatory Cytokines and Cell Proliferation in Human Mesangial Cells following Stimulation with IgA1 This information is current as Isolated from IgA Nephropathy Patients of September 24, 2021. Min Jeong Kim, John P. McDaid, Stephen P. McAdoo, Jonathan Barratt, Karen Molyneux, Esteban S. Masuda, Charles D. Pusey and Frederick W. K. Tam J Immunol 2012; 189:3751-3758; Prepublished online 5 Downloaded from September 2012; doi: 10.4049/jimmunol.1102603 http://www.jimmunol.org/content/189/7/3751 http://www.jimmunol.org/

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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. The Journal of Immunology

Spleen Tyrosine Kinase Is Important in the Production of Proinﬂammatory Cytokines and Cell Proliferation in Human Mesangial Cells following Stimulation with IgA1 Isolated from IgA Nephropathy Patients

Min Jeong Kim,*,†,‡ John P. McDaid,* Stephen P. McAdoo,* Jonathan Barratt,x Karen Molyneux,x Esteban S. Masuda,{ Charles D. Pusey,* and Frederick W. K. Tam*

IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the

progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell Downloaded from signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-g–inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC

proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA signiﬁcantly reduced the http://www.jimmunol.org/ synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the ﬁrst time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN. The Journal of Immunology, 2012, 189: 3751–3758.

eposition of polymeric IgA1 in the mesangium is the quently, mesangial cells release proinflammatory and profibrogenic hallmark of IgA nephropathy (IgAN). IgA1 is unusual in mediators, including IL-6, IL-8, IL-1b,IFN-g–inducible protein-10 D that it has a heavily O-glycosylated hinge region between (IP-10), TNF-a, MCP-1, macrophage migration inhibitory factor, by guest on September 24, 2021 the CH1 and CH2 domains of the a H chain (1, 2). The presence of and TGF-b (8–12). The IgA IC also modulates proliferation and poorly galactosylated IgA1 O-glycoforms in the serum of patients apoptosis of human mesangial cells (HMC) (13, 14). The subse- with IgAN favors formation of circulating immune complexes quent inflammation, cellular proliferation, and synthesis of extra- (IC), which are prone to deposition in the glomerular mesangium cellular matrix lead to the progression of IgAN (15). (3–6). Once deposited, these IgA IC trigger mesangial cell Spleen tyrosine kinase (SYK) was first discovered and cloned in activation. Binding of IgA IC induces an increase in intracellular porcine splenocytes in 1991 (16). SYK is a 72-kDa nonreceptor Ca2+, phospholipase C-g1 activation, production of inositol tri- tyrosine kinase that contains two SRC homology 2 domains and sphosphate, and protein tyrosine phosphorylation (7). Conse- a kinase domain. It was initially shown to be critical for the signaling of immunoreceptors, such as FcR and BCR, in association with ITAMs in hematopoietic cells (17). More recent studies *Imperial College Kidney and Transplant Institute, Hammersmith Hospital, Imperial College London, London W12 0NN, United Kingdom; †Clinic for Transplantation demonstrate the expression of SYK in nonhematopoietic cells, Immunology and Nephrology, University Hospital Basel, 4031 Basel, Switzerland; such as fibroblasts, mammary epithelial cells, hepatocytes, syno- ‡Department of Biomedicine, Molecular Nephrology, University Hospital Basel, x viocytes, and vascular endothelial cells. It is also involved in cell 4031 Basel, Switzerland; Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester LE5 4PW, United Kingdom; and {Rigel Pharma- signaling downstream of diverse cellular stimuli, including IL-1, ceuticals, South San Francisco, CA 94080 TNF-a, LPS, and b1-integrin in cells, that do not contain con- Received for publication September 9, 2011. Accepted for publication July 28, 2012. ventional ITAMs (18, 19). In a study using murine mesangial This work is supported by the Swiss National Science Foundation and the Novartis cells, reconstitution of the human FcaR and FcRg-chain, and Foundation (to M.J.K.), the Diamond Fund from Imperial College Healthcare Charity cross-linking of Fca receptor, led to the activation of SYK and (to F.W.K.T.), and the National Institute for Health Research Biomedical Research Centre funding scheme. subsequent MCP-1 production (20). This result confirms the ex- Address correspondence and reprint requests to Dr. Min Jeong Kim, Clinic for pression of SYK in murine mesangial cells. R406 (the active Transplantation Immunology and Nephrology, University Hospital Basel, Petersgra- component of fostamatinib) is a novel SYK inhibitor, and its ben 4, 4031 Basel, Switzerland. E-mail address: [email protected] therapeutic potential has been investigated in a range of animal Abbreviations used in this article: aIgA1, heat-aggregated IgA1 purified from an models of allergy, autoimmunity, and inflammation and also in IgAN patient; aIgG, heat-aggregated human IgG; cIgA1, IgA1 purified from a healthy subject; HMC, human mesangial cell; IC, immune complex; IgAN, IgA nephropathy; a limited number of clinical trials (21–24). Our group has recently IgAN–IgA1, IgA1 purified from an IgA nephropathy patient; IP-10, IFN-g–inducible shown that treatment with fostamatinib prevents the development protein-10; PDGF, platelet-derived growth factor; RA, rheumatoid arthritis; siRNA, of disease, and dramatically reduces glomerular injury and pro- small interfering RNA; SYK, spleen tyrosine kinase. teinuria in established disease, in a rat model of Ab-mediated Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 crescentic glomerulonephritis (25). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102603 3752 SYK AND IgA NEPHROPATHY

In this study, we investigated whether SYK is expressed in HMC medium after 48 h of transfection for 24 h before further stimulations. For and whether it is involved in downstream signaling of IgA1 the proliferation experiments, HMC were transfected in a 100 3 20-mm stimulation in HMC, leading to synthesis of proinﬂammatory dish and then seeded onto 96-well culture plate in 0.5% FCS containing RPMI 1640 medium after 48 h of transfection. Further stimulations were mediators by HMC and proliferation of HMC. performed after 72 h of transfection.

Materials and Methods Determination of MCP-1 gene expression by quantitative RT-PCR Sample collection Total cellular RNA was isolated using TRIzol reagents (Invitrogen). Two Serum samples were collected from healthy subjects and randomly selected micrograms of RNA was reverse transcribed with the First-Strand cDNA patients with IgAN attending the John Walls Renal Unit, Leicester General Synthesis kit for RT-PCR (avian myeloblastosis virus) (Roche Applied Hospital. All patients had biopsy-proven IgAN with microscopic hematuria Science, Burgess Hill, U.K.). Real-time PCR was performed on the and/or proteinuria. The study was approved by the local Research Ethics Mastercycler ep realplex (Eppendorf, Histon, U.K.) using the SYBR green Committee, and all subjects gave their written informed consent for serum MasterMix (ABgene, Epsom, U.K.). b-Actin served as the internal control. collection. The sequences of PCR primers were as follows: MCP-1, forward primer, Purification of serum IgA1 59-CCAAGCAGAAGTGGGTTCAG-39, and reverse primer, 59-CTTGG- GTTGTGGAGTGAGTG-39; and b-actin, forward primer, 59-TCACCC- Serum was precipitated with an equal volume of 45% ammonium sulfate ACACTGTGCCCATCTACGA-39, and reverse primer, 59-CAGCGGAA- solution and dissolved in 0.175 mol/l Tris-HCl (pH 7.5). The obtained fraction CCGCTCATTGCCAATGG-39. The expression of MCP-1 mRNA was 2 was incubated with Jacalin–Agarose (Vector Laboratories, Peterborough, analyzed by a relative quantification method, the 2 DDCt method. U.K.) at room temperature for 90 min. Unbound proteins were removed by Determination of cytokine concentrations in culture extensive washing of the Jacalin-Agarose with Tris-HCl, and IgA1 eluted Downloaded from from the Jacalin with 1 mol/l galactose in Tris-HCl. The IgA1 was then supernatants 3 dialyzed against 0.2 PBS, and the total volume reduced to 20% of the MCP-1 ELISA. MCP-1 was measured by specific ELISA using commer- 2 original volume and stored at 20˚C until use. The purity and concentration cially available paired Abs (R&D Systems, Abingdon, U.K.). The sensi- of IgA1 fractions were confirmed by SDS-PAGE, Western blot analysis, and tivity of the MCP-1 ELISA was 9.8 pg/ml. ELISA. Abnormal glycosylation of IgA1 purified from patients with IgAN was confirmed by lectin-binding assay using N-acetyl galactosamine- Multiplex cytokine assay specific lectin Helix aspersa as described previously (4). In all experiments, pooled IgA1 from randomly selected patients or healthy subjects was used. The expression of all other cytokines and chemokines was determined using http://www.jimmunol.org/ For each batch of IgA1, serum samples from eight subjects were used. the Bio-Plex Pro Human Cytokine 27-plex Assay (Bio-Rad, Hemel Hempstead, U.K.). Briefly, the principle of a sandwich immunoassay was SYK inhibitor combined with Luminex fluorescent bead-based technology (Luminex, Austin, TX), allowing measurement of large numbers of cytokines and The SYK inhibitor R406 was provided by Rigel Pharmaceuticals (South chemokines in small sample volumes. The assay was run on a Luminex 200 San Francisco, CA) and AstraZeneca (London, U.K.). The details of this system using low Photomultiplier Tube settings for broad range standard molecule have been reported previously (26). curve (system calibrated with CAL2 low RP1 target value) according to the Primary HMC culture manufacturer’s instructions. HMC were purchased at passage 3 (Lonza, Walkersville, MD). HMC were Western blotting by guest on September 24, 2021 grown in mesangial cell basal medium supplemented with 5% FCS, gen- Whole cell lysates were prepared using radioimmunoprecipitation lysis tamicin (30 mg/ml), and amphotericin B (15 ng/ml) at 37˚C in an atmo- buffer (Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with a sphere of 5% CO2/95% air. Cells grown at passage 5–8 were used in all protease inhibitor mixture and phosphatase inhibitor. The lysates were experiments. incubated at 4˚C for 20 min and then centrifuged at 14,000 rpm at 4˚C for Treatment of HMC with different IgA1 preparations, IgG and 20 min. The protein concentration of the supernatants was measured by the Bradford protein assay (Pierce, Thermo Fischer Scientific, Lough- SYK inhibitor R406 borough, U.K.). Equal amounts of protein extracts were separated by For measurement of cytokines, HMC were cultured in a 24-well culture 10% SDS-PAGE (NaDodSO4–PAGE) and transferred to Amersham plate (5 3 104 cells/well) to confluence, growth-arrested, and then incu- Hyperfilm ECL (GE Healthcare, Amersham, U.K.). After blocking with bated in RPMI 1640 medium containing 0.5% FCS with total IgA1 iso- 6% dry milk in TBS, membranes were incubated under agitation with lated from IgAN patients (IgAN–IgA1), IgA1 isolated from healthy control total SYK Ab (4D-10; Santa Cruz Biotechnology), phospho-SYK subjects (cIgA1) or heat-aggregated human IgG (aIgG) (Sigma-Aldrich, (Tyr525/526) Ab (2711; Cell Signaling Technology, Danvers, MA), or Gillingham, U.K., aggregated at 63˚C for 20 min) for 6 or 24 h. For anti-actin Ab (Sigma-Aldrich) diluted in 6% BSA containing TBS proliferation experiments, HMC were cultured in a 96-well culture plate overnight at 4˚C. Membranes were then washed with TBST (TBS with (1.5 3 104 cells/well), growth-arrested, and then stimulated with heat- 0.05% Tween 20) and incubated with peroxidase-conjugated secondary aggregated IgA1 isolated from IgAN patients (aIgA1, aggregated at Abs (Dakocytomation, Ely, U.K.) diluted in blocking buffer (1:3000) for 63˚C for 150 min (27)) for 24 h. For the experiments with R406, HMC 1 h at room temperature. Membranes were washed again in TBST, and were preincubated in medium containing either 0.04% DMSO (vehicle) or then, Western blots were developed using the Amersham ECL plus R406 in 0.04% DMSO for 1 h before further treatment with different IgA1 reagents (GE Healthcare). preparations. For RNA extraction, HMC were harvested after 6 h. For Cell proliferation ELISA using BrdU determination of cytokine concentrations, culture supernatants were collected after 24 h. For proliferation experiments, cell proliferation assay was After stimulation of HMC with aIgA1 for 24 h, cell proliferation ELISA performed after 24 h. The MTT assay (Sigma-Aldrich) was performed to using BrdU was performed using the cell proliferation ELISA kit (Roche assess cell viability. Applied Science, Rotkreuz, Switzerland), according to the manufacturer’s instructions. Transfection of HMC with small interfering RNAs Immunohistochemistry of kidney biopsies from patients with Lipofectamine RNAiMax (Invitrogen, Paisley, U.K.) was used for the IgAN transfection with small interfering RNAs (siRNA). siRNA directed against SYK and nonspecific, scrambled, control siRNAwere predesigned (Silencer Renal biopsies were performed according to the clinical need of the patients in select Predesigned siRNA; Ambion/Applied Biosystems, Paisley, U.K.). Hammersmith Hospital. The patients gave written consent for use of biopsied HMC were seeded onto 24-well culture plate (3 3 104 cells/ well) in tissues surplus to diagnostic analysis for research. The protocols were ap- antibiotic-free mesangial cell growth media and grown overnight. HMC proved by local research ethics committee. Immunhistochemistry was per- were then transfected with 50 nM of each siRNA mixed with Lipofect- formed on formalin-fixed paraffin-embedded tissues. Sections were subject to amine reagent in Opti-mem medium (Invitrogen). HMC were maintained heat-induced Ag retrieval in 0.1 M citrate buffer (pH 6) and then sequentially for 72 h and then subjected to Western blot analysis. For the cytokine blocked in 0.3% hydrogen peroxide and 20% goat serum (Sigma-Aldrich). experiments, medium was changed to 0.5% FCS containing RPMI 1640 Sections were then incubated with primary Abs for total SYK (N-19; Santa The Journal of Immunology 3753

Cruz Biotechnology) and for phospho-Syk (Tyr 525/526) (2711; Cell Effect of IgAN–IgA1 and R406 on other mediators produced by Signaling Technology) overnight at 4˚C, and developed using a secondary HMC polymer system (EnVision +; DakoCytomation, Cambridge, U.K.). The effect of stimulation with IgAN–IgA1 at 50 mg/ml and Statistical analysis treatment with R406 at two different concentrations on the syn- Differences between groups were analyzed by Mann–Whitney U test. All thesis of a panel of cytokines and chemokines by HMC was an- probabilities were two tailed. p values , 0.05 were considered signifi- alyzed using a multiplex cytokine assay. Among the 27 cytokines cant. Statistical analyses were performed using Prism 5.0 (GraphPad and chemokines tested, the synthesis of IL-6, IL-8, IP-10, Software, La Jolla, CA). The semiquantification of Western blot results was performed using ImageJ 1.45s program (National Institutes of Health, RANTES, and platelet-derived growth factor (PDGF)-BB was Bethesda, MD). significantly increased following stimulation with IgAN–IgA1 for 24 h. Treatment with R406 significantly downregulated the syn- Results thesis of these cytokines and chemokines (Fig. 2). Effect of SYK inhibitor R406 on the synthesis of MCP-1 by Effect of knockdown of SYK using siRNA on the synthesis of HMC following stimulation with IgAN–IgA1 MCP-1 and other mediators by HMC HMC were incubated with IgAN–IgA1, cIgA1, or aIgG at different To verify the inhibitory effect of R406 on SYK, HMC were concentrations for 24 h, and the concentration of MCP-1 was transfected with either siRNA directed against SYK (SYK siRNA) measured in culture supernatants by ELISA. The synthesis of MCP- or nonspecific, scrambled control siRNA. Cells were incubated for 1 following stimulation with IgAN–IgA1 was significantly upreg- 72 h in complete medium before Western blot analysis. Western ulated in a dose-dependent manner. At the same concentration of blots performed with total cell lysates showed clearly reduced Downloaded from each stimulus, the concentration of MCP-1 was significantly higher expression of SYK in HMC transfected with SYK siRNA compared following stimulation with IgAN–IgA1 than cIgA1 or aIgG (Fig. with HMC transfected with control siRNA or untreated HMC (Fig. 1A). There were no significant differences in cell viability, assessed 3A). For the stimulation experiments, cells were growth arrested by MTT assay, at different concentrations of IgAN–IgA1, cIgA1, or after 48 h of transfection and stimulated with IgAN–IgA1 at 50 aIgG (data not shown). In the next experiment, HMC were pre- mg/ml for 24 h. Knockdown of SYK in HMC by transfection with incubated with either vehicle or R406 at three different concen- SYK siRNA significantly reduced the IgAN–IgA1-induced pro- http://www.jimmunol.org/ trations for 1 h, and then, IgAN–IgA1 at 50 mg/ml was added to the duction of MCP-1, whereas transfection with control siRNA did media. Quantitative RT-PCR was performed using total RNA not affect the synthesis of MCP-1 (Fig. 3B). We then looked at the extracted from HMC after incubation with IgAN–IgA1 for 6 h. The effect of SYK siRNA on the synthesis of other mediators by HMC expression of MCP-1 mRNA was significantly upregulated in cells using a multiplex cytokine assay. Among the 27 cytokines and treated with either IgAN–IgA1 alone or vehicle and IgAN–IgA1. chemokines tested, the synthesis of IL-6, IL-8, IP-10, RANTES, In the cells treated with R406, the upregulation of MCP-1 and PDGF-BB was significantly reduced by the transfection with mRNA expression following stimulation with IgAN–IgA1 was SYK siRNA but not by the transfection with control siRNA (Fig. 4). significantly inhibited in a dose-dependent manner (Fig. 1B). Concentrations of MCP-1 in the culture supernatants from HMC Effect of SYK inhibition by R406 and knockdown of SYK by by guest on September 24, 2021 following stimulation with IgAN–IgA1, under treatment with SYK siRNA on the proliferation of HMC following stimulation vehicle or R406, were consistent with the MCP-1 mRNA expres- with aIgA1 sion in each condition (Fig. 1C). Treatment with R406 reduced the HMC were incubated with aIgA1 at 100 and 200 mg/ml for 24 h, and IgAN–IgA1-induced production of MCP-1 significantly in a dose- the cell proliferation was assessed by ELISA using BrdU. The dependent manner. There were no significant differences in cell proliferation of HMC was significantly increased following stimu- viability, assessed by MTT assay, in different treatment conditions lation with aIgA1 (data not shown). To examine the effect of R406 (data not shown). on the HMC proliferation, HMC were preincubated with either

FIGURE 1. Effect of SYK inhibitor R406 on the synthesis of MCP-1 by HMC following stimulation with IgAN–IgA1 is shown. (A) The concentrations of MCP-1 in the culture supernatant from HMC incubatedwithIgAN–IgA1increasedsignificantlyinadose-dependent manner. At the same concentration of each stimulus, the concentrations of MCP-1 were significantly higher in the culture supernatant from HMC incubated with IgAN–IgA1 than cIgA1 or aIgG. *p , 0.001 versus cIgA1 or aIgG. (B) The relative expression of MCP-1 mRNA was significantly upregulated in HMC incubated with IgAN–IgA1 at 50 mg/ml. Treatment with R406 at 0.2, 0.8, and 2 mM downregulated MCP-1 mRNA expression following stimulation with IgAN–IgA1. Treatment with vehicle did not affect MCP-1 mRNA expression significantly. *p , 0.01, +p , 0.05. (C) Treatment with R406 significantly reduced the synthesis of MCP-1 by HMC in a dose-dependent manner. Treatment with vehicle did not affect the synthesis of MCP-1. The results are representative of three experiments and reported as mean 6 SD. *p , 0.01, +p , 0.05. 3754 SYK AND IgA NEPHROPATHY Downloaded from http://www.jimmunol.org/

FIGURE 2. Effect of stimulation with IgAN–IgA1 and treatment with R406 on the synthesis of cytokines and chemokines by HMC was analyzed by guest on September 24, 2021 by multiplex cytokine assay. The synthesis of IL-6, IL-8, MCP-1, IP-10, RANTES, and PDGF-BB was increased significantly following stimulation with IgAN–IgA1 at 50 mg/ml. Treatment with R406 at 0.2 and 2 mM significantly reduced the synthesis of these cytokines following stimulation with IgAN–IgA1. The results are representative of three experiments and reported as mean 6 SD. *p , 0.01, +p , 0.05. vehicle or R406 at three different concentrations for 1 h before dependent manner (Fig. 5A). To verify the effect of R406, HMC stimulation with aIgA1 at 200 mg/ml for 24 h. The proliferation of were transfected with either SYK siRNA or control siRNA and then HMC was significantly inhibited by treatment with R406 in a dose- incubated with aIgA1 200 mg/ml for 24 h. The proliferation of

FIGURE 3. Concentrations of MCP-1 in the culture supernatants from HMC incubated with IgAN–IgA1 after transfection with siRNA against SYK or nonspecific scrambled control siRNA are shown. (A) Western blot analysis performedafter72hoftransfection shows clearly reduced expression of SYK in HMC transfected with SYK siRNA compared with HMC transfected with control siRNA or nontransfected HMC. Data are shown as the ratio of arbitrary intensity units for SYK to actin. (B) Concentrations of MCP-1 in the culture supernatant from nontransfected HMC increased significantly following stimulation with IgAN– IgA1 at 50 mg/ml. Transfection with SYK siRNA significantly reduced the synthesis of MCP-1 (p = 0.0006). Transfection with control siRNA did not affect the synthesis of MCP-1. The results are representative of three experiments and reported as mean 6 SD. The Journal of Immunology 3755 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Effect of knockdown of SYK using siRNA on the synthesis of other mediators by HMC was analyzed by multiplex cytokine assay. The synthesis of IL-6, IL-8, MCP-1, IP-10, RANTES, and PDGF-BB was increased signiﬁcantly following stimulation with IgAN–IgA1 at 50 mg/ml. The synthesis of these mediators by HMC transfected with SYK siRNA was signiﬁcantly downregulated compared with HMC transfected with control siRNA. The results are representative of three experiments and reported as mean 6 SD. *p , 0.05.

HMC was signiﬁcantly inhibited by knockdown of SYK by trans- SYK was performed with total cell lysates. The phosphorylation fection with SYK siRNA but not by control siRNA (Fig. 5B). of SYK was increased after stimulation with IgAN–IgA1 for up to 5 min and then decreased afterward when normalized with the Activation of SYK in HMC following stimulation with b-actin expression level (Fig. 6) IgAN–IgA1 To investigate whether SYK is activated following stimulation with Expression of SYK in kidney biopsies from patients with IgAN IgAN–IgA1, HMC were incubated with IgAN–IgA1 at 200 mg/ml We then investigated whether SYK is activated in the kidney bi- for various time periods, and Western blot analysis for phospho- opsies from patients with IgAN by immunohistochemistry for total

FIGURE 5. Effect of SYK inhibition by R406 and knockdown of SYK by SYK siRNA on the proliferation of HMC following stimulation with aIgA1 was shown. (A) The proliferation of HMC was significantly increased following stimulation with aIgA1 at 200 mg/ml. Treatment with R406 significantly reduced HMC proliferation in a dose- dependent manner. Treatment with vehicle did not significantly affect the HMC proliferation. *p , 0.0001, +p = 0.0016. (B) Transfection with SYK siRNA significantly inhibited the proliferation of HMC following stimulation with aIgA1 at 200 mg/ ml, whereas transfection with control siRNA did not affect the proliferation of HMC following stimulation with aIgA1. The results are representative of three experiments and reported as mean 6 SD. *p , 0.0001. 3756 SYK AND IgA NEPHROPATHY

experiments, we were able to see a clear difference in stimulatory effects on HMC between IgAN–IgA1 and cIgA1. It is unlikely that potential contaminants during the IgA1 purification process induced the observed stimulatory effects, because patient and healthy subject IgA1 were prepared in exactly the same manner. Exposure to aIgG did not result in any stimulatory effects in HMC, suggesting that the stimulatory effect of IgAN–IgA1 was not induced by binding of the IgG component of IgA IC to HMC. Using total serum IgA1 purified from patients with IgAN, we were not able to induce proliferation of HMC. Previous study showed circulating IC containing aberrantly glycosylated IgA1 isolated from patients’ sera stimulated mesangial cell proliferation (14). Because of technical issues, we were not able to isolate circulating IC from patients’ sera. However, using heat-aggregated patient IgA1 according to the previous study (27), we were able to induce the proliferation of HMC. Binding of IgA1 IC to mesangial cells with subsequent cell proliferation and release of proinflammatory and profibrotic mediators are widely accepted as key events in the pathogenesis of

IgAN (15, 28). Previous studies suggest that these mediators play Downloaded from FIGURE 6. Activation of SYK in HMC following stimulation with a signiﬁcant role in the pathogenesis and progression of IgAN. IgAN–IgA1 was examined. Western blot analysis for phospho-SYK with Mesangial cell-derived mediators induce proliferation of proximal total cell lysates of HMC incubated with IgAN–IgA1 at 200 mg/ml for tubular epithelial cells and synthesis of inﬂammatory mediators indicated time periods showed an increase in phosphorylation of SYK after (29). Moreover, mesangial cell-derived TNF-a and platelet- incubation for up to 5 min and then decreased afterward when normalized with the b-actin expression level. activating factor downregulate podocyte markers, resulting in

altered glomerular permeability and enhanced proteinuria in IgAN http://www.jimmunol.org/ (30, 31). These data indicate that such mediators play an important SYK and phospho-SYK. The levels of both total and phospho-Syk role in the cross-talk between the different cell types present in were increased in glomeruli of kidney biopsies from patients with glomeruli. In clinical studies of IgAN, patients with moderate to IgAN. In the crescentic IgAN, total and phospho-SYK were severe disease showed much higher renal MCP-1 mRNA and detected in both the glomerular tuft and the crescent. In the negative protein expression, and higher levels of urinary MCP-1, compared controls (kidney biopsy from patients with minimal change dis- with patients with mild and nonprogressive disease. Urinary levels ease), two images of the same glomerulus showed very slight of IL-6 and IL-8 also correlate with histological lesions and levels staining for total Syk but not for phospho-SYK (Fig. 7). of proteinuria (32–34). Furthermore, levels of renal gene expression of various cytokines and chemokines, including TNF-a, IL- by guest on September 24, 2021 Discussion 1b, IL-6, IL-8, and IFN-g, correlate with the pattern and extent In this study, we demonstrate that IgA1 from patients with IgAN of histological lesions and clinical findings (35). Another study activates HMC and induces the synthesis and release of a number of showed that serum PDGF-DD levels were elevated in patients with proinflammatory mediators and the proliferation of HMC. This is IgAN, although the levels of renal PDGF-DD mRNA expression consistent with previous data showing stimulatory effects of IgA1 did not differ between patients and control subjects (36). In this on HMC (9–12, 14, 27). Although we used total serum IgA1, study, stimulation of HMC with IgA1 purified from patients with which is a mixture of polymeric and monomeric IgA1, for these IgAN significantly increased synthesis of IL-6, IL-8, MCP-1,

FIGURE 7. Expression and activation of SYK in kidney biopsies from patients with IgAN was examined. Immunohistochemistry for both total- SYK (a, b) and phosphorylated-SYK (d, e) showed positive staining in glomeruli of the kidney biopsies from patients with IgAN. In the crescentic GN (b, e), total and phospho-SYK were detected in both the glomerular tuft and the crescent. In the negative controls [kidney biopsies from patients with minimal change disease (c, f)], there was very slight staining for total SYK but not for phospho-SYK. Orig- inal magniﬁcation 3200. The Journal of Immunology 3757

IP-10, RANTES, and PDGF-BB by HMC. These are all mediators and/or stock options for Rigel Pharmaceuticals. C.D.P. has received research involved in inflammation and cell proliferation in renal disease funding from Cyclacel, UCB Celltech, Proximagen, and GlaxoSmithKline. (37). Blocking the synthesis of these mediators and the prolifer- The other authors have no financial conflicts of interest. ation of HMC in response to binding of IgA1 to HMC may, therefore, have great relevance to the treatment of IgAN. Pharmacological inhibition of SYK by R406 effectively reduced References 1. Conley, M. E., M. D. Cooper, and A. F. Michael. 1980. Selective deposition of the synthesis of the above-mentioned mediators and also the immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura proliferation of HMC following stimulation with IgA1 purified nephritis, and systemic lupus erythematosus. J. Clin. Invest. 66: 1432–1436. from patients with IgAN. Although the effect of R406 is likely to be 2. Kerr, M. A. 1990. The structure and function of human IgA. Biochem. J. 271: 285–296. mediated through selective inhibition of SYK, it is possible that the 3. Tomana, M., J. Novak, B. A. Julian, K. Matousovic, K. Konecny, and compound also affects other kinases, such as Lck, c-Kit, and Flt3 J. Mestecky. 1999. Circulating immune complexes in IgA nephropathy consist of (18). We therefore verified the results by knockdown of the SYK IgA1 with galactose-deficient hinge region and antiglycan antibodies. J. Clin. Invest. 104: 73–81. gene using SYK siRNA. Both pharmacological inhibition of SYK 4. Allen, A. C., E. M. Bailey, P. E. C. Brenchley, K. S. Buck, J. Barratt, and and knockdown of the SYK gene showed consistent inhibitory J. Feehally. 2001. Mesangial IgA1 in IgA nephropathy exhibits aberrant O- glycosylation: observations in three patients. Kidney Int. 60: 969–973. effects on the synthesis of various mediators and the proliferation 5. Novak, J., H. L. Vu, L. Novak, B. A. Julian, J. Mestecky, and M. Tomana. 2002. of HMC. Interactions of human mesangial cells with IgA and IgA-containing immune Furthermore, we were able to demonstrate the activation of SYK complexes. Kidney Int. 62: 465–475. 6. Suzuki, H., R. Fan, Z. Zhang, R. Brown, S. Hall, B. A. Julian, W. W. Chatham, in the glomeruli of kidney biopsies from patients with IgAN using Y. Suzuki, R. J. Wyatt, Z. Moldoveanu, et al. 2009. Aberrantly glycosylated IgA1 immunohistochemistry for phospho-SYK, which strongly suggests in IgA nephropathy patients is recognized by IgG antibodies with restricted Downloaded from the involvement of SYK in the pathogenesis of IgAN. However, it is heterogeneity. J. Clin. Invest. 119: 1668–1677. 7. Go´mez-Guerrero, C., N. Duque, and J. Egido. 1996. Stimulation of Fc(a) unclear which cells expressed SYK in the glomeruli, because we receptors induces tyrosine phosphorylation of phospholipase C-g(1), phospha- did not perform double staining to identify cell types expressing tidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells. J. Immunol. 156: 4369–4376. SYK. Both migrating and intrinsic cells in glomeruli may have 8. Duque, N., C. Go´mez-Guerrero, and J. Egido. 1997. Interaction of IgA with Fca expressed SYK. Our results from the Western blot analysis for total receptors of human mesangial cells activates transcription factor nuclear factor-k B and induces expression and synthesis of monocyte chemoattractant protein-1, and phospho-SYK clearly demonstrate the expression of SYK in http://www.jimmunol.org/ IL-8, and IFN-inducible protein 10. J. Immunol. 159: 3474–3482. mesangial cells. 9. Leung, J. C. K., S. C. W. Tang, L. Y. Y. Chan, A. W. L. Tsang, H. Y. Lan, and To date, it is still controversial which receptors and mechanisms K. N. Lai. 2003. Polymeric IgA increases the synthesis of macrophage migration are involved in the binding of IgA1 to mesangial cells. The latest inhibitory factor by human mesangial cells in IgA nephropathy. Nephrol. Dial. Transplant. 18: 36–45. study suggest that complex interaction of IgA1, soluble CD89 10. Lai, K. N., S. C. W. Tang, J. Y. Guh, T. D. Chuang, M. F. Lam, L. Y. Y. Chan, receptor, transferrin receptor 1, and transglutaminase 2 is required A. W. L. Tsang, and J. C. K. Leung. 2003. Polymeric IgA1 from patients with IgA nephropathy upregulates transforming growth factor-b synthesis and signal for the binding of IgA1 leading to the activation of mesangial cells transduction in human mesangial cells via the renin-angiotensin system. J. Am. (38). Whether SYK is involved in the downstream signaling of this Soc. Nephrol. 14: 3127–3137. complex interaction remains to be further investigated. 11. Leung, J. C. K., S. C. W. Tang, L. Y. Y. Chan, W. L. Chan, and K. N. Lai. 2008. Synthesis of TNF-a by mesangial cells cultured with polymeric anionic IgA— by guest on September 24, 2021 The SYK inhibitor R788 (prodrug of R406) has been studied in role of MAPK and NF-kB. Nephrol. Dial. Transplant. 23: 72–81. patients with rheumatoid arthritis (RA) in phase II clinical trials 12. Tam, K. Y., J. C. K. Leung, L. Y. Y. Chan, M. F. Lam, S. C. W. Tang, and with overall favorable outcomes. The adverse events included K. N. Lai. 2009. Macromolecular IgA1 taken from patients with familial IgA nephropathy or their asymptomatic relatives have higher reactivity to mesangial diarrhea, increase in systolic blood pressure, and neutropenia (24, cells in vitro. Kidney Int. 75: 1330–1339. 39). RA is a disease with complex pathogenic mechanisms in- 13. Amore, A., P. Cirina, G. Conti, P. Brusa, L. Peruzzi, and R. Coppo. 2001. Glycosylation of circulating IgA in patients with IgA nephropathy modulates volving several inflammatory cascades and recruitment of in- proliferation and apoptosis of mesangial cells. J. Am. Soc. Nephrol. 12: 1862– flammatory cells. Inhibition of SYK by R788 may result in the 1871. inhibition of a wide range of downstream functional responses 14. Novak, J., M. Tomana, K. Matousovic, R. Brown, S. Hall, L. Novak, B. A. Julian, R. J. Wyatt, and J. Mestecky. 2005. IgA1-containing immune following immunoreceptor activation, including cell proliferation complexes in IgA nephropathy differentially affect proliferation of mesangial and differentiation, and effector functions such as cytokine re- cells. Kidney Int. 67: 504–513. lease, leading to favorable outcomes in RA (40). 15. Moura, I. C., M. Benhamou, P. Launay, F. Vrtovsnik, U. Blank, and R. C. Monteiro. 2008. The glomerular response to IgA deposition in IgA ne- To our knowledge, this is the first report showing the expression phropathy. Semin. Nephrol. 28: 88–95. of SYK in HMC and its involvement in the cellular responses to 16. Taniguchi, T., T. Kobayashi, J. Kondo, K. Takahashi, H. Nakamura, J. Suzuki, K. Nagai, T. Yamada, S. Nakamura, and H. Yamamura. 1991. Molecular cloning IgA1 stimulation. We demonstrate in our study that the synthesis of of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing proinflammatory and proproliferative mediators by HMC and the high susceptibility to proteolysis. J. Biol. Chem. 266: 15790–15796. proliferation of HMC can be effectively reduced by the inhibition 17. Wong, B. R., E. B. Grossbard, D. G. Payan, and E. S. Masuda. 2004. Targeting Syk as a treatment for allergic and autoimmune disorders. Expert Opin. Investig. of SYK. Use of a SYK inhibitor may, therefore, be a novel ther- Drugs 13: 743–762. apeutic strategy in IgA nephropathy, which is also well known for 18. Cha, H. S., D. L. Boyle, T. Inoue, R. Schoot, P. P. Tak, P. Pine, and its complex pathogenic mechanisms including inflammation, G. S. Firestein. 2006. A novel spleen tyrosine kinase inhibitor blocks c-Jun N- terminal kinase-mediated gene expression in synoviocytes. J. Pharmacol. Exp. proliferation, and fibrosis. Ther. 317: 571–578. 19. Moćsai, A., J. Ruland, and V. L. J. Tybulewicz. 2010. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat. Rev. Immunol. 10: 387–402. Acknowledgments 20. Tsuge, T., Y. Suzuki, T. Shimokawa, S. Horikoshi, K. Okumura, C. Ra, and We thank Dr. Simona Deplano, Dr. Maria Fragiadaki, and Kapil Kampe for Y. Tomino. 2003. Monocyte chemoattractant protein (MCP)-1 production via functionally reconstituted Fca receptor (CD89) on glomerular mesangial cells. technical advice and support, Prof. Terry Cook and Prof. John Feehally for Inflamm. Res. 52: 428–432. valuable advice on this project, Tricia Higgins for technical assistance, and 21. Tam, F. W. K., and S. P. McAdoo. 2011. Fostamatinib disodium: tyrosine-protein Dr. Andreas Jehle and Prof. Ju¨rg Steiger for generous support. kinase SYK/FLT3 inhibitor, treatment of rheumatoid arthritis, oncolytic. Drugs Future 36: 273–280. 22. Podolanczuk, A., A. H. Lazarus, A. R. Crow, E. Grossbard, and J. B. Bussel. Disclosures 2009. Of mice and men: an open-label pilot study for treatment of immune M.J.K. received research funding from Amgen. F.W.K.T. has received re- thrombocytopenic purpura by an inhibitor of Syk. Blood 113: 3154–3160. 23. Friedberg, J. W., J. Sharman, J. Sweetenham, P. B. Johnston, J. M. Vose, search project grants from Roche Palo Alto, AstraZeneca, and Baxter Bio- A. Lacasce, J. Schaefer-Cutillo, S. De Vos, R. Sinha, J. P. Leonard, et al. 2010. sciences. E.S.M. is employed by Rigel Pharmaceuticals and owns stock Inhibition of Syk with fostamatinib disodium has significant clinical activity in 3758 SYK AND IgA NEPHROPATHY

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