Renal -Type Plasminogen Activator (uPA) Receptor but not uPA Deficiency Strongly Attenuates Ischemia Reperfusion Injury and Acute Kidney Allograft Rejection This information is current as of September 24, 2021. Faikah Gueler, Song Rong, Michael Mengel, Joon-Keun Park, Julia Kiyan, Torsten Kirsch, Inna Dumler, Hermann Haller and Nelli Shushakova J Immunol 2008; 181:1179-1189; ; doi: 10.4049/jimmunol.181.2.1179 Downloaded from http://www.jimmunol.org/content/181/2/1179

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Renal Urokinase-Type Plasminogen Activator (uPA) Receptor but not uPA Deficiency Strongly Attenuates Ischemia Reperfusion Injury and Acute Kidney Allograft Rejection

Faikah Gueler,1* Song Rong,1* Michael Mengel,† Joon-Keun Park,* Julia Kiyan,* Torsten Kirsch,* Inna Dumler,* Hermann Haller,* and Nelli Shushakova2*‡

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not Downloaded from uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inade- quate ICAM-1 up-regulation in uPAR؊/؊ primary aortic endothelial cells after C5a and TNF-␣ stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory http://www.jimmunol.org/ responses mediating IR-injury and transplant rejection. The Journal of Immunology, 2008, 181: 1179–1189.

rokinase-type plasminogen activator (uPA)3 is a multi- lar signaling pathways (7), cytotoxicity (8), cell adhesion (9), and functional molecule that serves either as a proteolytic migration (10–12), all of which are critical steps in cell-mediated U enzyme or as a signal-inducing ligand. The urokinase immune responses. Furthermore, uPA potentiates neutrophil acti- receptor (uPAR; CD87) was originally identified as a proteinase vation (7) and superoxid production (13). Recently, we were also receptor for uPA, directing pericellular . However, ac- able to demonstrate an important link between the uPA/uPAR and cumulating data clearly demonstrate that uPAR can also activate a complement system in the regulation of immunological responses by guest on September 24, 2021 variety of intracellular signal pathways via its lateral interaction in kidney (14) and lung tissues (15). with different cell surface such as , growth factor Renal ischemia reperfusion (IR) injury after transplantation receptors, and G--coupled membrane proteins. These inter- leads to acute renal failure, which profoundly affects both early actions enable the uPA/uPAR system not only to control pericel- and long-term allograft function (16). Accumulating evidence lular fibrinolytic and proteolytic activities, but also to modulate demonstrates that prolonged cold ischemia time is a strong risk cell adhesion, migration, proliferation, and differentiation (1, 2). factor for unfavourable outcome after allogenic kidney transplan- Moreover, the important role of the uPA/uPAR system has re- tation (17, 18) and suggests that the severity of IR injury to the cently been demonstrated in both innate and adaptive immune- allograft determines its immunogenicity and subsequent graft fate mediated responses (3). The uPA/uPAR system modulates Ag pro- (19). The mechanisms underlying IR damage of kidney tissue cessing and presentation (4), lymphocyte activation (5), generation seem to be multifactorial and interdependent. The oxygen supply of pro- and anti-inflammatory signals (6), activation of intracellu- to the tissue by reperfusion leads to the generation of reactive oxygen species (ROS) exceeding the protective anti-oxidative ca- pacity of kidney cells. Oxidative stress is known to be a major *Department of Nephrology and †Department of Pathology, Medical School Han- nover, Hannover, Germany; and ‡Phenos GmbH, Hannover, Germany apoptotic stimulus in allograft nephropathy (20). Furthermore, IR Received for publication January 11, 2007. Accepted for publication May 13, 2008. injury stimulates the components of innate immune response, such The costs of publication of this article were defrayed in part by the payment of page as complement activation and up-regulation of multiple proinflam- charges. This article must therefore be hereby marked advertisement in accordance matory including chemokines, cytokines, cytokine receptors, with 18 U.S.C. Section 1734 solely to indicate this fact. and adhesion molecules. This inflammatory response induced by 1 F.G. and S.R. contributed equally to this work. innate mechanisms early after transplantation is markedly ampli- 2 Address correspondence and reprint requests to Dr. Nelli Shushakova, Nephrology fied by the subsequent adaptive response (16). Therefore, IR injury Department, Hannover Medical School, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany. E-mail address: [email protected] initiates and induces the alloimmune response leading to acute and 3 Abbreviations used in this paper: uPA, urokinase-type plasminogen activator; chronic allograft rejection (21, 22). uPAR, urokinase-type plasminogen activator receptor; IR, ischemia reperfusion; Recently, up-regulation of the uPA/uPAR system has been dem- ROS, reactive oxygen species; RT, room temperature; DHE, dihydroethidium; PFA, onstrated under hypoxia/reoxygenation conditions in vitro (23, paraformaldehyde; MAEC, mouse aortic endothelial cell; PBMC, peripheral blood mononuclear cell; MO, monocytes/macrophages; PMN, polymorphonuclear leuko- 24). Moreover, uPA/uPAR activation has been shown to correlate cyte; ATN, acute tubular necrosis; C5aR, receptor for C5a anaphylatoxin; MO, mac- with allograft rejection in a human biopsy study (25, 26) implying rophage/monocyte; WT, wild type. a probable involvement of this system in acute and chronic allo- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 graft rejection. However, the causal connection of uPA/uPAR in www.jimmunol.org 1180 uPAR AND KIDNEY TRANSPLANTATION mediating of IR injury and transplant rejection as well as the pos- monoclonal rat anti-mouse neutrophils (Gr-1, a gift from Prof. Hoffmann, sible molecular mechanisms for uPA/uPAR mediated initiation Hannover Medical School, Hannover, Germany), monoclonal rat anti- and perpetuation of inflammatory reaction after transplantation re- mouse T lymphocytes (CD4 and CD8; BD Pharmingen), rabbit polyclonal anti-mouse CD25 (Santa Cruz Biotechnology), rabbit polyclonal anti- main unexplored. mouse uPAR (Santa Cruz Biotechnology). For indirect immunofluores- The aim of our study was to assess the role of the renal uPA/ cence, nonspecific binding sites were blocked with 10% normal donkey uPAR system in renal IR injury and in allogenic kidney transplant serum (Jackson ImmunoResearch Laboratories) for 30 min. Thereafter, rejection. We demonstrate that uPAR but not uPA deficiency of the sections were incubated with the primary Ab for 1 h. All incubations were performed in a humid chamber at room temperature (RT). For fluorescent allograft protected from IR injury and acute allogenic transplant visualization of bound primary Abs, sections were further incubated with rejection. As underlying mechanisms we could elucidate impaired Cy3 conjugated secondary Abs (Jackson Immuno Research Laboratory) for susceptibility of the uPAR-deficient allografts to IR-induced ROS- 1 h. Specimens were analyzed using a Zeiss Axioplan-2 imaging micro- mediated apoptosis and reduced expression of adhesion molecules, scope with the computer program AxioVision 3.0 (Zeiss). Analysis of in- leading to impaired migration of host monocytes/macrophages and flammation was done by semiquantitative scoring of the infiltrating cells in Ϫ/Ϫ 10 randomly chosen, nonoverlapping fields of cortex and outer medulla neutrophils into uPAR allografts. (original magnification, ϫ200). Score: 0, no; 1, weak; 2, moderate; 3, high; and 4, very high numbers of infiltrating cells. For CD4 and CD25 expres- Materials and Methods sion, absolute cell numbers were counted in 20 nonoverlapping view fields Animals each specimen. Active caspase-3 expression in the outer strip of the outer medulla was scored as follows: 0 Ͻ 10%, 1 ϭ 10–30%, 30–50%, 4 Ͼ 50% Ϫ Ϫ Ϫ Ϫ The uPA- and uPAR-deficient mice (uPA / and uPAR / ), generated as of the tubuli affected. ICAM-1 expression in the cortex was scored as previously described (27), were a gift from Peter Carmeliet and Mieke follows: 0 Ͻ 10%, 1 ϭ 10–30%, 30–50%, 4 Ͼ 50% of the glomeruli

Dewerchin (Center for Transgene Technology and Therapy, Univer- affected. The analysis was done without knowledge of the animal Downloaded from sity of Leuven, Belgium). uPA/uPAR deficiency was verified by PCR assignment. genotyping. The uPAϪ/Ϫ mice on a C57BL/6J background and the corre- sponding WT controls (WT1), as well as uPARϪ/Ϫ mice on a mixed Mixed lymphocyte reactivity (MLR) C57BL/6J (75%) ϫ 129/Sv (25%) background and their WT littermate controls (WT2) served as kidney donors (H2b). BALB/c (H2d) mice served Priming of alloantigen-specific T cells from kidney graft recipients was as recipients in kidney transplantation experiments and were supplied by investigated by performing MLR assay based on the measurement of BrdU incorporation during DNA synthesis. The responder spleen cells obtained Charles River Laboratories. The animals were bred under pathogen-free Ϫ Ϫ

/ http://www.jimmunol.org/ conditions in the animal facility of Phenos GmbH (Hannover, Germany) from naive BALB/c mice or from WT2 or uPAR allograft recipients at and cared for in accordance with our institution’s guidelines for exper- day 6 after transplantation were treated with ammonium chloride solution imental animals. All experiments were approved by the animal protec- (Cell Systems) to lyse erythrocytes, washed three times, and resuspended at ϫ 6 tion committee of the local authorities. Mice weighing 25–30g were 3 10 cells/ml in complete RPMI 1640 medium (Life Technologies) sup- plemented with 10% FCS (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml used for all experiments. For IR injury 7–12 mice were used in each ␮ group (8 uPARϪ/Ϫ mice, 7 WT2 mice, 10 uPAϪ/Ϫ mice, and 12 WT1 penicillin, 100 mg/ml streptomycin, and 100- l aliquots were delivered in mice). For transplant experiments 6 mice for each group were used for triplicate to the wells of a 96-well, flat-bottom tissue culture plate. Stimulator the survival study and additional 6 mice for each group and each time cells were prepared from the spleens of syngeneic (i.e., BALB/c) and allograft donors (i.e.WT2, uPARϪ/Ϫ). After lysis of erythrocytes the stimulator cells point were sacrificed at 4 h, at day 1, and at day 6 after transplantation ␮ for histological and molecular analysis. were treated with 50 g/ml mitomycin C for 30 min at 37°C, washed and resuspended in culture medium at 3 ϫ 106 cells/ml, and 100-␮l aliquots per Renal ischemia reperfusion injury well were added to responder cells and cultured for 48 h. Afterward, BrdU was by guest on September 24, 2021 added and 18h later responder cell proliferation was quantified using colori- Ϫ/Ϫ Ϫ/Ϫ Renal IR-injury was induced in homozygous male uPA , uPAR , metric Cell Proliferation ELISA (Roche Diagnostics GmbH) in accordance WT1, and WT2 mice by bilateral clamping of both renal pedicals. The to the manufacturer’s instructions. Syngeneic stimulator cells were used as animals were anesthetized with isoflurane. After median laparatomy, renal background controls and were subtracted from alloresponses. pedicals were bluntly dissected and a nontraumatic vascular clamp was applied for 35 min. At 24 h, postischemia kidney function was estimated by ELISPOT assay serum creatinine measurement using an automated method (Beckman Analyzer). Priming of alloantigen-specific T cells from kidney allograft recipients was also tested by enumerating of IFN-␥-producing T cells using a mouse Kidney transplantation IFN-␥ ELISpot kit (BD Biosciences) in accordance to the manufacturer’s instructions. Spleen cell suspensions were prepared from naive BALB/c For transplant experiments, homozygous female uPAϪ/Ϫ, uPARϪ/Ϫ, WT1, d mice or from allograft recipients at day 6 after transplantation and used as and WT2 mice were used as kidney donors and female BALB/c (H2 ) mice responder cells. Spleen cells from WT2 or syngeneic BALB/c mice were served as recipients. Vascularized kidney transplantation was performed as prepared and treated with mitomycin C for use as stimulator cells in the described previously (28). In brief, the animals were anesthetized with assay as described above. Responder and stimulator cells were cultured isoflurane, and the left donor kidney attached to a cuff of the aorta and the together for 24 h at 37°C in 5% CO2. The resulting spots were counted renal vein with a small caval cuff and the ureter were removed en bloc. using the A.EL.VIS 4-Plate ELISPOT reader with the A.EL.VIS ELISPOT After left nephrectomy of the recipient, the vascular cuffs were anasto- analysis software. mosed to the recipient abdominal aorta and vena cava, respectively, below the level of the native renal vessels. The ureter was directly anastomosed ROS generation by oxidation of dihydroethidium to the bladder (29). The times of cold and warm ischemia of allografts were 60 and 30 min, respectively. The right native kidney was removed through The redox-sensitive fluorophore dihydroethidium (DHE) was used to eval- flank incision either on the day of transplantation or four days later. After uate O2-production in the kidney in situ (31). Frozen tissue Cryosections of transplantation, kidney function was estimated at designated time points 6 ␮M were incubated with 0.1 mM DHE dissolved in HEPES-Tyrode (ranging from 24 h to several weeks) by serum creatinine level measure- buffer solution (132 mM NaCl, 4 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, ment. The general physical condition of the animals was monitored for any 9.5 mM HEPES, and 5 mM glucose) for 12 min RT. After incubation, evidence of rejection. images were obtained using the Leica imaging system IM 500 (Ex, 520 nm; Em, 605 nm). Semiquantitative scoring was performed as follows: score: 0, Allograft pathology no; 1, weak; 2, moderate; 3, high; and 4, very high intensity. Kidney grafts were harvested 24 h and six days after transplantation and TUNEL-DAB assay one half of each allograft was immediately fixed in buffered formalin and embedded in paraffin. Sections of 3 ␮m were stained with hematoxylin- For TUNEL-assay (terminal deoxynucleotidyl transferase-mediated dUTP eosin and periodic acid-Schiff stain, and evaluated according to the updated nick-end labeling) 2 ␮m sections of 4% paraformaldehyde (PFA)-fixed Banff classification (30) by a nephropathologist, who was masked to the paraffin-embedded tissues were deparaffinized, treated with the terminal experimental groups. Immunohistochemistry was performed using the fol- deoxynucleotidyl transferase enzyme and incubated in a humidified cham- lowing primary Abs: rat anti-mouse monocytes/macrophages (F4/80; Se- ber at 37°C for 1 h. After washing, the tissue was treated with FITC-labeled rotec), polyclonal rabbit anti-mouse active caspase 3 (BD Pharmingen), anti-digoxygenin, incubated for 60 min, and washed. Negative controls The Journal of Immunology 1181 Downloaded from

FIGURE 2. uPA and uPAR expression is up-regulated in WT kidney FIGURE 1. Attenuation of IR-induced renal dysfunction and apoptosis allografts. uPAR (A) and uPA (B) mRNA expression in WT2 kidney al- Ϫ/Ϫ in uPAR mice. A, Renal function was investigated by measurement of lografts was investigated by qPCR before (control) and at 4 and 24 h after http://www.jimmunol.org/ serum creatinine level before (control) and at 24 h after IR. IR-injury allogenic kidney transplantation. Both uPAR and uPA mRNA expression ,ءء ;p Ͻ 0.05 ,ء) caused severe renal dysfunction with elevation of serum creatinine level in was time-dependently up-regulated after transplantation WT1, WT2, and uPA-deficient mice. In contrast, uPAR deficiency led to p Ͻ 0.01 vs control, six mice were investigated for each group). C, The -p Ͻ 0.001 uPAR protein expression in WT2 allografts was investigated by immuno ,ءءء) markedly attenuated loss of renal function after IR injury vs WT2, 7–12 mice were investigated for each group). B, Representative cytochemistry. The upper row, uPAR expression in the cortex area; the specimens after TUNEL assay 24-h postischemia are shown (original mag- lower row, uPAR expression in the outer strip of the outer medulla. Low nification, ϫ200). Because WT1 and WT2 mice demonstrated comparable baseline uPAR protein expression seen in normal WT2 kidney (control) results, representative data from WT2 kidney is shown. WT kidneys (a) was increased within 24 h and even more at 6 days after transplantation. and uPA-deficient kidneys (b) had increased numbers of apoptotic cells (Original magnification ϫ200; six mice were investigated for each group). by guest on September 24, 2021 mainly in the tubular epithelium. In contrast, postischemic uPAR-deficient kidneys (C) showed almost no TUNEL positive cells. The results of quan- p Ͻ 0.001 ,ءءء) tification are presented as mean Ϯ SEM in the lower panel vs WT2, six mice were investigated for each group). confluent monolayers, by Dil-Ac-LDL uptake and by surface expression of CD31 and CD106 analyzed by immunnocytochemistry (data not shown). MAEC immunocytochemistry WT and uPARϪ/Ϫ were prepared under the same conditions, with the omission of the terminal MAEC were seeded and cultured on glass coverslips. Serum-starved cells were fixed with 4% PFA in PBS for 20 min at RT. deoxynucleotidyl transferase enzyme. TUNEL pos. cell numbers were Nonspecific binding was blocked by 2 h incubation at RT with 3% BSA in counted in 20 nonoverlapping view fields each specimen without knowl- PBS; the preparations were washed three times with PBS. Incubations with edge of the animal assignment. primary Ab (rat anti-mouse receptor for C5a anaphylatoxin (C5aR) clone RNA extraction and real time quantitative PCR 20/70 from Hycult Biotechnology or polyclonal rabbit anti-TNFR1 from Santa Cruz Biotechnology) were performed for2hatRT.Incubations with Frozen kidneys were grinded in liquid nitrogen and total RNA was ex- Alexa 488-conjugated secondary Abs (Molecular Probes) were performed tracted using Trizol reagent (Invitrogen). For real-time quantitative PCR for 1 h. After staining, cells were embedded in Poly-Mount mounting me- (qPCR), 1 ␮g of DNase-treated total RNA was reverse transcribed using dium (Polysciences). The fluorescence cell images were captured using a Superscript II Reverse transcriptase (Invitrogen) and qPCR was performed Leica TCS-SP2 AOBS confocal microscope (Leica Microsystems). All the on an SDS 7700 system (Applied Biosystems) using Rox dye (Invitrogen), images were taken with oil-immersed ϫ 63 objective, NA ϭ 1.4. FastStart taq Polymerase (Roche Diagnostics) and gene specific primers and FAM-Tamra-labeled probes (BioTez). PCR amplification was con- ICAM-1 cell ELISA ducted at 10 min 96°C and 40 cycles at 10 s 95°C and 1 min at 60°C. ␤-actin served as the reference gene for normalization. Primer sequences Cell surface expression of ICAM-1 on MAEC was measured by cell are available on request. Quantification was conducted using qgene soft- ELISA as described previously (34). In brief, MAEC were grown in 96- ware (32). well plate until 80% confluent, starved for 4 h and then stimulated for 16 h with 100 ng/ml recombinant murine C5a (mrC5a; Sigma-Aldrich) or with Mouse aortic endothelial cell (MAEC) culture 5 ng/ml recombinant murine TNF-␣ (mrTNF␣; R&D Systems). These con- centrations were chosen in preliminary experiments ranging from 0.5 ng/ml Isolation of mouse aortic endothelial cells from 6- to 8-wk-old WT or to 200 ng/ml for each stimulus. For inhibition experiments MAEC were uPARϪ/Ϫ mice was performed as described previously (33). The cells were preincubated for 2 h with 100 ng/ml pertussis toxin (Sigma-Aldrich) or cultivated in medium consisting of endothelial cell growth medium 2 (Clo- with 20 ␮g/ml hamster anti-mouse-TNFR1 mAb (R&D Systems). MAEC netics/Cambrex) and DMEM (1:1) supplemented with 20% FCS, 100 incubated in medium without stimuli served as a control. After fixation ␮g/ml endothelial cell growth supplement (Sigma-Aldrich), 100 U/ml pen- with 3% PFA and blocking with 3% BSA to prevent nonspecific binding icillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 0.5% nonessential the cells were incubated for 2 h with polyclonal rabbit-anti-mouse ICAM-1 amino acids, and 0.1 mg/ml heparin. MAEC at passage 3 were used for Ab (Santa Cruz Biotechnology).The specific binding of Abs was then eval- endothelial cell characterization and for all experiments. The endothelial uated by incubation of cells for 1 h with secondary peroxidase conjugated of cells was confirmed by the typical cobblestone morphology of goat anti-rabbit IgG (Santa Cruz Biotechnology) followed by addition of 1182 uPAR AND KIDNEY TRANSPLANTATION Downloaded from http://www.jimmunol.org/

FIGURE 3. Effect of allograft uPA and uPAR deficiency on survival, renal function and renal morphology after allogenic kidney transplantation. A, Survival analysis of mice receiving uPA- and uPAR-deficient allografts as compared with the animals receiving corresponding WT allografts is shown. by guest on September 24, 2021 WT1 and WT2 allografts revealed identical results and are presented as WT. Survival of recipients of uPAϪ/Ϫ allografts was comparable to WT allograft controls. The majority of all these transplant recipients died within 4 wk after transplantation. In contrast, recipients of uPARϪ/Ϫ allografts showed prolonged allograft survival over 20 wk after transplantation. B, Renal function was estimated in recipient mice before (day 0) and at designated time points after transplantation by serum creatinine measurement. Recipients of uPAϪ/Ϫ and both WT1 and WT2 allografts had a similar steep rise of serum creatinine at 24 h and at 6 days after transplantation, correlating with acute rejection. In contrast, recipients of uPAR-deficient allografts had only a moderate initial rise in creatinine within 24 h after transplantation and remained stable at slightly increased serum creatinine levels thereafter. The results are presented as p Ͻ 0.001 vs WT2, six mice were investigated for each group). C, Morphological changes one day after kidney transplantation are ,ءءء) mean Ϯ SEM shown. All WT (a) and the uPA-deficient allografts (b) showed diffuse severe ATN. In contrast, the uPAR-deficient allograft recipients (c) had no signs of ATN. (PAS stain, original magnification ϫ200; six mice were investigated for each group).

tetramethylbenzidine substrate solution (R&D Systems), stopping the re- inhibitory experiments the pieces were preincubated for 2 h with 100 ng/ml ␮ action with 0.5 M H2SO4 and measuring the OD at 450 nm. The substrate pertussis toxin or with 20 g/ml neutralizing hamster anti-mouse-TNFR1 was then washed away with deionized water, the plate allowed to dry and mAb. Aorta pieces incubated in medium alone served as a control. After fluorescently labeled 103ءtrypan blue was added to stain for the number of cells/well. Excess 16 h, the aorta pieces were washed twice and 100 0.5% of trypan blue was washed away and 1% SDS was added to solubilize the WT2 PBMC in 200 ␮l medium were added. The cells were allowed to trypan blue stained cells. Each well was then read at 595 nm. The OD of adhere for 45 min at 37°C. Unbound cells were removed by washing three ICAM-1 staining was divided by the OD of trypan blue staining to yield times. Photographs of aorta pieces were then made using the Leica imaging ICAM-1 expression for each well. microscope with the digital image-processing program. The bound leuko- cytes were counted without knowledge of the group assignment in four Peripheral blood mononuclear cell (PBMC) adhesion assay different view-fields per aorta piece. ex vivo Statistical analysis The adhesion of WT2 PBMC isolated by Ficoll-Paque separation to the endothelial surface of aortas obtained from WT2 or uPARϪ/Ϫ mice was Data are shown as mean Ϯ SEM. Normal distribution was analyzed by determined by the counting of adherent cells fluorescently labeled with the Klomogorov-Smirnov-test and statistical significance was calculated by acetyloxymethyl ester of calcein (Calbiochem). A total of 1–2 mm pieces Student’s t test for independent groups. SPSS 12.01 software was used. of aortas cleaned carefully of periadventitial fat and connective tissue and opened longitudinally were placed adventitia-side down on collagen I coated 96-well plates containing 10 ␮l of endothelial cell basal medium 2 Results (Clonetics/Cambrex) supplemented with 5% FCS to allow adherence of the uPA/uPAR deficiency in IR injury aortic pieces to the substratum. When the pieces were well-attached (after 4 h), 200 ␮l of EBM-2 medium containing 50 ng/ml mrC5a, or 50 ng/ml The uPA/uPAR system is involved in a variety of signaling cas- mrTNF␣ was added. These concentrations were chosen in preliminary ex- cades which mediate IR injury. We first tested whether uPA or periments ranging from 0.5 ng/ml to 200 ng/ml for each stimulus. For uPAR are mediators or effectors of renal IR injury. We performed The Journal of Immunology 1183 Downloaded from

FIGURE 4. Allograft uPAR-deficiency strongly protected kidneys from transplantation-induced apoptosis. TUNEL assay was performed in WT2 FIGURE 5. uPAR-deficiency of the allograft strongly reduces ROS Ϫ/Ϫ and uPAR allografts at 4 (upper row) and 24 h (middle row) after generation after transplantation. Generation of ROS was investigated by http://www.jimmunol.org/ transplantation. WT2 allografts (a and c) had many TUNEL positive nuclei DHE stain. The upper row shows low basal level of DHE staining in WT2 Ϫ/Ϫ at4h(a) with further increase at 24 h (c) after transplantation. In contrast, (a) and uPAR (b) kidneys before transplantation. An increase in gen- uPAR-deficient allografts had almost no TUNEL positive cells at (b) and eration of ROS in the tubulo-interstitial compartment was detected in WT2 24h(d) after transplantation. (TUNEL assay, original magnification allografts as early as4h(c) after transplantation; this effect was still de- ϫ200). The quantification results are presented as mean Ϯ SEM in the tectable 24 h after transplantation (e). In contrast, uPAR-deficient allografts -p Ͻ 0.001 vs WT2, six mice were showed impaired generation of ROS at 4 (d) and 24 h (f) after transplan ,ءءء ;p Ͻ 0.01 ,ءء) right-hand panel investigated for each group). The immunohistochemistry for cleaved tation. The results of the semiquantitative analysis are presented as mean Ϯ p Ͻ 0.001 vs WT2, six mice were ,ءءء) caspase-3 showed in WT2 allografts an intense up-regulation of cleaved SEM in the right-hand panel caspase-3 level in the tubuli of the outer stripe of the outer medulla, the investigated for each group). area most sensitive to hypoxic damage (e). uPAR-deficient allografts in by guest on September 24, 2021 contrast showed only few cleaved caspase-3 positive tubuli (f). investigated in normal WT2 kidneys and in WT2 kidney allografts at 4 and 24 h after transplantation. We detected strong up-regula- bilateral renal pedical clamping in uPAϪ/Ϫ and uPARϪ/Ϫ mice tion of uPAR (Fig. 2A) mRNA 4 h after transplantation with fur- and their corresponding wild-type controls (WT1 and WT2, re- ther increase after 24 h. The up-regulation of uPA mRNA was less spectively). Both WT1 and WT2 controls and uPAϪ/Ϫ mice dem- pronounced but also could be observed at 24 h after transplantation onstrated severe loss of renal function within 24 h postischemia, as (Fig. 2B). At protein level (Fig. 2C), the weak expression of uPAR reflected in elevation of serum creatinine levels. In contrast, seen in normal WT2 kidney was strongly up-regulated within 24 h uPAR-deficient animals were protected from IR injury and dem- after transplantation and further increased at day 6. As expected, onstrated statistically significant attenuated loss of renal function no positive signal was detected in uPARϪ/Ϫ allografts (data not (Fig. 1A). Because apoptosis is a hallmark of IR injury, we per- shown). These data demonstrate that transplantation results in local formed a TUNEL assay in normal kidney tissue and in kidneys activation of the uPA/uPAR system in rejecting allografts from 24 h after ischemia (Fig. 1B). No TUNEL-positive cells were de- WT mice. tected in normal kidneys in any of the four groups of animals (data not shown). We detected increased numbers of TUNEL-positive uPAR but not uPA deficiency improved kidney allograft survival nuclei in WT1, WT2 and uPAϪ/Ϫ kidneys. Because WT1 and and attenuated loss of renal function WT2 mice showed almost identical results, only the data from Further, we analyzed an influence of uPA or uPAR deficiency of Ϫ Ϫ WT2 mice are shown. However, in uPAR / kidneys the amount the allograft on recipient survival (Fig. 3A). We performed allo- of TUNEL-positive cells was significantly reduced. These results genic kidney transplantation using uPA- and uPAR-deficient mice suggest that uPAR contributes to hypoxia-induced apoptosis inde- (H2b) and their corresponding WT controls as donors, and BALB/c pendently from uPA. mice (H2d) as recipients. All recipients of WT as well as uPAϪ/Ϫ allografts died shortly after allogenic kidney transplantation. In uPA/uPAR expression is up-regulated in rejecting kidney contrast, recipients of uPAR-deficient allografts showed superior allografts survival for more than 20 wk after transplantation. Because activation of uPA/uPAR system has been shown to cor- Next, we studied the effect of uPA/uPAR deficiency on renal relate with allograft rejection in a human biopsy study (25), we function after transplantation by measuring serum creatinine levels tested whether or not a similar phenomenon may be observed in (Fig. 3B). To monitor the effects of rejection on renal function we kidney allografts in mice. We performed allogenic kidney trans- removed both recipient kidneys and transplanted the donor kidney plantation using WT2 mice (H2b) as donors, and BALB/c mice in the same operation. An increase in serum creatinine was de- (H2d) as recipients. The expression of uPA and uPAR was then tected in all groups 24 h after transplantation; however this effect 1184 uPAR AND KIDNEY TRANSPLANTATION was strongly attenuated in mice which received uPARϪ/Ϫ allo- grafts. Six days after transplantation, recipients of uPA-deficient allografts and WT allografts developed deleterious loss of renal function as reflected in severe creatinine elevation. In contrast, the renal function of recipients of uPAR-deficient allografts remained stable without any significant increase in serum creatinine. Histo- logical analysis revealed severe diffuse acute tubular necrosis (ATN) in WT and uPAϪ/Ϫ but not in uPARϪ/Ϫ allografts at d1 after transplantation (Fig. 3C). These results indicate that the uPAR but not uPA of allograft origin contributes significantly to the loss of renal function and allograft rejection in this model of allogenic kidney transplantation. Because uPA deficiency had no protective effects in either the IR model or in kidney transplanta- tion, the following experiments were performed using only uPAR- deficient animals and their respective WT2 controls. Apoptosis was markedly reduced in uPAR-deficient allografts Because hypoxia-induced apoptosis is the pathophysiological cor- relate for ATN, we performed a TUNEL assay to investigate the Downloaded from cell death rate in uPARϪ/Ϫ and WT2 kidney allografts (Fig. 4). WT2 allografts ubiquitously developed TUNEL positive nuclei as early as4h(a) and more pronouncedly 24 h (c) after transplan- tation. In contrast, uPARϪ/Ϫ allografts demonstrated almost no signs of apoptosis at these time points (b and d). To elucidate Ϫ/Ϫ whether the differences in cell death between WT2 and uPAR http://www.jimmunol.org/ allografts were due to necrosis or to apoptosis we performed im- munohistochemistry for cleaved caspase-3. Especially in the outer stripe of the outer medulla, the area which is most sensitive to hypoxic damage, and in the medium of the vessels, cleaved caspase-3 level was increased in WT2 allografts at 24 h after trans- plantation (data not shown) and even more so at day 6 after kidney Ϫ/Ϫ transplantation (e). In comparison, uPAR allografts ( f) showed FIGURE 6. uPAR-deficiency of the allograft does not change host T markedly lower levels of cleaved caspase-3. These results were in cell infiltration and alloantigen-specific T cell priming after transplantation. by guest on September 24, 2021 line with our previous finding in the IR injury model and clearly A, At day 6 after transplantation, WT2 allografts (a) and uPAR-deficient demonstrate that local uPAR expression is also pivotal in ischemia allografts (b) showed similar CD8 positive T cell infiltration. Furthermore, triggered apoptosis in the allogenic kidney transplantation model. CD4 positive T cell infiltrates of similar density were seen in both WT2 and uPARϪ/Ϫ allografts (c and d). No differences were observed concern- Generation of ROS was reduced in uPAR-deficient allografts ing CD25 positive T cells between WT2 and uPARϪ/Ϫ allografts (e and f). Hypoxia leads to tissue damage via generation of ROS contribut- The results of the quantification analysis are presented as mean Ϯ SEM in ing significantly to the mitochondrial apoptotic pathway (35). the right-hand panel six mice were investigated for each group). B, In vitro MLR assay was performed with splenocytes isolated from naive BALB/c Scavengers for ROS, H O , and superoxide have been shown to Ϫ Ϫ 2 2 mice and from recipients of WT2 and uPAR / kidney allografts at day 6 inhibit apoptosis induced by ischemia-reperfusion (36). Therefore, after transplantation. No differences between WT2 and uPARϪ/Ϫ allograft we tested the hypothesis that strongly reduced apoptosis in recipients were observed. Experiments were performed in triplicate, n ϭ 5 Ϫ/Ϫ uPAR allografts might be due to decreased production of ROS. mice for each group, data are presented as mean Ϯ SEM. C, IFN-␥-pro- Ϫ/Ϫ DHE staining (Fig. 5) performed in normal WT2 (a) and uPAR ducing cells at day 6 after transplantation in spleen cells of naive BALB/c (b) kidneys revealed similar basal levels of ROS. However, in mice and recipients of WT2 and uPARϪ/Ϫ kidney allografts were deter- contrast to WT allografts demonstrating severe up-regulation of mined by ELISPOT analysis. Numbers of IFN-␥-producing cells were sim- Ϫ Ϫ ROS at 4 (c) and 24 h (e) after transplantation, ROS generation ilar in uPAR / and WT2 allograft recipients. Experiments were per- was markedly reduced in uPAR-deficient allografts (d and f). This formed in triplicate, n ϭ 5 mice for each group, data are presented as Ϯ result suggests that uPAR contributes to the initial apoptosis trig- mean SEM. gering step - the generation of ROS. uPAR deficiency of the allograft and inflammatory cell recipients showed a similar increase of proliferative response to infiltration WT2 stimulator cells compared with splenocytes from naive The inflammatory cell infiltration of the allograft is a hallmark of BALB/c mice. The frequency of alloantigen-specific IFN-␥-pro- allograft rejection (37, 38). Therefore, we performed immunohis- ducing T cells tested by ELISPOT assay was also comparable in tochemistry for different cell subsets to elucidate the composition WT2 and uPARϪ/Ϫ allograft recipients (Fig. 6C). This result of the cell infiltrates. No differences were observed between the shows that uPAR deficiency did not decrease the level of donor- groups in CD8 and CD4 infiltrates. Moreover, the CD25 expres- reactive T cell priming in this model. sion on CD4 T cells was comparable in WT2 and uPARϪ/Ϫ allo- Furthermore, we analyzed infiltration of neutrophils (Fig. 7A, grafts (Fig. 6A). In line with this observation the MLR assay did upper panel) and monocytes/macrophages (Fig. 7A, middle panel). not reveal any differences between splenocytes obtained from We found that uPAR deficiency of the allograft decreased the WT2 and uPARϪ/Ϫ allograft recipients at day 6 after transplanta- number of infiltrating neutrophils and monocytes/macrophages as tion (Fig. 6B). The splenocytes from WT2 and uPARϪ/Ϫ allograft compared with WT2 control allografts. These results underline the The Journal of Immunology 1185 Downloaded from

FIGURE 7. uPAR-deficiency of the allograft protects from host leukocyte infiltration after transplantation. A, At day six after transplantation, WT2 allografts (a) showed more tubulo-interstitial infiltrates of Gr-1 positive neutrophils as compared with uPAR-deficient allografts (b). Dense infiltration of F4/80 positive monocytes/macrophages seen in the tubulo-interstitium of WT2 allografts (c) was strongly reduced in uPAR-deficient allografts (d). ICAM-1 http://www.jimmunol.org/ expression was studied by immunohistochemistry. Twenty-four hours after transplantation ICAM-1 was heavily up-regulated in the glomeruli of WT2 allografts (e) whereas uPARϪ/Ϫ allografts ( f) showed almost no ICAM-1 up-regulation. The results of the semiquantification analysis are presented as .(p Ͻ 0.001 vs WT2, original magnification ϫ200, six mice were investigated for each group ,ءءء ;p Ͻ 0.01 ,ءء) mean Ϯ SEM in the right-hand panel B, The expression of MCP-1 (upper panel) and MIP-2 (low panel) mRNA was analyzed by TaqMan RT-PCR in normal kidneys (control) and in WT2 and p Ͻ 0.01 vs WT2, six mice were ,ءء) uPARϪ/Ϫ allografts obtained at 24 h and day 6 after transplantation. Data are expressed as the mean Ϯ SEM investigated for each group). by guest on September 24, 2021 importance of local renal uPAR for monocyte/macrophage and uPAR deficiency of blood vessel decreases TNF␣/C5a-induced neutrophil infiltration. ICAM-1 up-regulation and reduces WT leukocyte adhesion Adhesive interaction between up-regulated adhesion molecules in vitro on activated endothelium with blood leukocytes is a crucial step Recently, the interaction of complement activation product C5a for leukocyte infiltration into the site of inflammation. Therefore, with its receptor (C5aR) has been shown to induce a strong in- Ϫ/Ϫ we analyzed the expression of ICAM-1 in WT2 and uPAR crease in for cell adhesion molecules in endothe- allografts after kidney transplantation. Baseline ICAM-1 expres- lial cells similar to those induced by TNF-␣ (41). Because both Ϫ/Ϫ sion of uPAR and WT kidneys was comparable (data not TNF-␣ and activated complement are important mediators of IR shown). However, a strong ICAM-1 up-regulation seen in WT2 injury and transplant rejection (42, 43), we hypothesized that allografts after transplantation was significantly impaired in TNF-␣ and/or C5a-dependent up-regulation of endothelial adhe- Ϫ/Ϫ uPAR allografts (Fig. 7A, lower panel). sion molecules may be impaired in uPARϪ/Ϫ allografts and may Members of the chemokine family play a central role in in- be an explanation for the reduced monocyte/macrophage and gran- flammatory cell infiltration into extravascular sites by attracting ulocyte infiltration. To test this hypothesis, we performed addi- and stimulating specific subsets of leukocytes (39). MCP-1 is an tional in vitro experiments with the primary culture of MAEC from important mediator for monocyte recruitment (40) whereas WT2 and uPARϪ/Ϫ mice. After demonstration of similar cell sur- MIP-2 is necessary for an adequate neutrophil infiltration (39). face expression of both TNFR1 and C5aR on WT2 and uPARϪ/Ϫ Therefore, we compared the transplantation-induced changes of MAEC by immunocytochemistry (Fig. 8A) we investigated the Ϫ Ϫ MCP-1 and MIP-2 mRNA expression in WT2 and uPAR / expression of ICAM-1 after stimulation of cells with TNF-␣ and allografts. As expected, we observed a strong up-regulation of C5a for 16 h by cell ELISA. The treatment with 5 ng/ml mrTNF␣ these proinflammatory mediators in WT2 allografts. Surpris- resulted in a strong up-regulation of ICAM-1 expression in both Ϫ Ϫ ingly, uPAR / allografts demonstrated a practically identical WT2 and uPARϪ/Ϫ MAEC, however, this effect was significantly pattern of the transient overexpression of MIP-2 mRNA, with decreased in uPARϪ/Ϫ MAEC compared with WT2. The pretreat- a maximum at 24 h after transplantation. The kinetics of ment with TNFR1 blocking Ab strongly decreased the TNF-␣- MCP-1 mRNA up-regulation was also similar in WT and induced up-regulation of ICAM-1 suggesting the involvement of Ϫ Ϫ uPAR / allografts, and moreover, the grade of MCP-1 mRNA TNFR1 (Fig. 8B). The treatment of MAEC with 100 ng/ml mrC5a up-regulation was even significantly higher in uPARϪ/Ϫ kidney resulted in a moderate but significant up-regulation of ICAM-1 in (Fig. 7B). These results suggest that attenuated leukocyte infil- WT2 MAEC, however, this effect was completely abrogated in tration into uPAR-deficient allografts may be due to impaired uPARϪ/Ϫ MAEC. The pretreatment with the C5aR blocking per- up-regulation of ICAM-1 but not to altered chemokine tussis toxin prevented the C5a-induced up-regulation of ICAM-1 expression. in WT2 MAEC verifying the role of C5aR (Fig. 8C). 1186 uPAR AND KIDNEY TRANSPLANTATION Downloaded from http://www.jimmunol.org/

FIGURE 8. uPAR-deficiency strongly reduces C5a/TNF-␣-induced up-regulation of ICAM-1 in endothelial cells and completely abrogates C5a/TNF- ␣-induced increase of WT leukocyte adhesion in vitro. A, Nonstimulated serum-starved MAEC obtained from WT and uPARϪ/Ϫ mice were immunostained with monoclonal anti-C5aR (upper panel) or polyclonal anti-TNFR1 (lower panel) Abs and Alexa 488-conjugated secondary Abs. Basal expression of C5aR and TNFR1 was comparable in uPARϪ/Ϫ and WT MAEC. (The frame size of images is 240 ϫ 240 ␮m.) B, mrTNF␣ (left histogram) stimulation resulted in a strong up-regulation of ICAM-1 expression in WT2 and to a lesser extent also in uPARϪ/Ϫ MAEC. Pretreatment with TNFR1 blocking Ab strongly decreased the TNF-␣-induced up-regulation of ICAM-1. Nonstimulated cells served as controls. mrC5a stimulation (right histogram) resulted in a moderate but significant up-regulation of ICAM-1 in WT2 MAEC, however, this effect was completely abrogated in uPARϪ/Ϫ MAEC. Pertussis toxin prevented the C5a-induced up-regulation of ICAM-1 in WT MAEC. (Data are expressed as the mean Ϯ SEM from three independent experiments, by guest on September 24, 2021 performed in four parallels for each condition. Significant differences were determined by Student’s t test for C5a/TNF-␣-stimulated vs nonstimulated cells p Ͻ 0.001) and for WT2 vs uPARϪ/Ϫ cells (ϩϩ, p Ͻ 0.01; ϩϩϩ, p Ͻ 0.001). C, Adhesion of WT2 PBMC to the endothelial surface ,ءءء ;p Ͻ 0.01 ,ءء) of aortas was studied in vitro. Aortas from WT2 or uPARϪ/Ϫ mice were preincubated or not with 100 ng/ml pertussis toxin or with 20 ␮g/ml anti-TNFR1 Abs for 2 h and then stimulated for 16 h with 50 ng/ml C5a or TNF-␣. Unstimulated aortas served as a control. C5a- and TNF-␣-dependent increase of WT2 PBMC adhesion to uPARϪ/Ϫ aorta was completely abrogated as compared with WT2 aortas. The data are presented as mean Ϯ SEM for five p Ͻ 0.01 between C5a/TNF-␣-stimulated and nonstimulated aortas and ϩϩ, p Ͻ 0.01; ϩϩϩ, p Ͻ 0.001 ,ءء) individual experiments performed in triplicate between WT2 and uPARϪ/Ϫ aortas under the same stimulation conditions).

These results were confirmed by a PBMC adhesion assay ex allograft rejection. In the transplantation model, the resistance of vivo. In these experiments, adhesion of normal WT2 PBMC to the the allograft against hypoxia-induced apoptosis due to uPAR de- endothelial surface of aortas obtained from WT2 or uPARϪ/Ϫ ficiency was linked to better renal function and increased allograft mice and stimulated in vitro with mrC5a or mrTNF␣ was inves- survival. The inadequate up-regulation of ICAM-1 in the blood tigated. As demonstrated in Fig. 8D, 16-h stimulation of WT2 vessels of uPARϪ/Ϫ allografts resulted in attenuated monocyte/ aorta with TNF-␣ or C5a induced significant increase in adhesion macrophage and neutrophil infiltration. of WT2 PBMC. These effects could be strongly reduced or Allogenic transplant rejection is triggered by several stimuli completely abolished when aortas were pretreated with TNFR1 such as allograft hypoxia and subsequent apoptosis and inflam- blocking Ab and pertussis toxin, respectively. In contrast, the matory response. Tubular cell apoptosis is considered to be an TNF-␣ and C5a-dependent up-regulation of endothelial adhesion important pathway leading to tubular atrophy in progressive was completely abolished in uPARϪ/Ϫ aortas. These results renal disease. Recently, it has been shown that uPAR may mod- clearly demonstrate that uPAR expression on endothelial cells is ify the rate of apoptotic renal cell death (44). In vitro, human necessary for adequate TNF-␣ and C5a signaling leading to up- glioma cells exposed to uPAR antisense have been reported to regulation of adhesion molecules and mediating leukocyte undergo more apoptotic cell death (45). In contrast to these adhesion. observations, the major finding of this study was that uPAR deficiency strongly protected renal cells from apoptosis via re- Discussion duced ROS formation in both models, IR injury and kidney Our data provide the first evidence that local renal uPAR but not transplantation. One possible explanation for this discrepancy uPA expression plays a pivotal role in the pathogenesis of IR in- might be that uPAR exposes pro- or anti-apoptotic action and jury and allogenic transplant rejection. We ruled out that uPAR modulates the cell survival/apoptosis ratio depending on the deficiency protected kidney tissue from generation of ROS and cell type and/or the apoptosis-triggering pathways. This hypoth- consecutively from severe apoptosis in IR injury and acute kidney esis is supported, for example, by demonstration of the The Journal of Immunology 1187 anti-apoptotic and uPA-independent action of uPAR in endo- renal biopsies from patients with acute and chronic renal rejection thelial cells in which anti-uPAR Abs as well as soluble recom- (54). In line with this report, we observed a strong up-regulation of binant uPAR blocked the apoptotic effect of cleaved high mo- these proinflammatory mediators which was similar in WT2 and lecular mass kininogen by preventing of the binding of high uPARϪ/Ϫ allografts. Therefore, it could be concluded that the level molecular mass kininogen to these cells (46). of these chemoattractants was not responsible for the diminished Recently, it has been shown that uPA stimulates ROS produc- PMN/MO infiltration in uPARϪ/Ϫ allografts. tion in VSMC in vitro (47). Because hypoxia triggers ROS gen- Adhesion molecules are rapidly up-regulated early after trans- eration and mediates apoptosis, we investigated the possible role of plantation (55) and after induction of IR injury (56). Therefore, we uPA/uPAR interaction in ROS generation and subsequent ROS- analyzed expression of ICAM-1 in WT2 and uPARϪ/Ϫ allografts dependent apoptosis using both uPA- and uPAR-deficient mice. given the facts that, being the major counterreceptor for leukocyte Our results clearly demonstrate that in both models uPAR defi- ␤2-integrins, this adhesion molecule is rapidly up-regulated within ciency protected renal tissue from ROS generation independently the first 2–3 h after transplantation (57) and the blockade of from uPA. The molecular mechanisms underlying the attenuated ICAM-1 strongly attenuated PMN infiltration in some experimen- Ϫ/Ϫ ROS formation in uPAR allografts remain to be investigated. tal models of renal ischemia (58–60). Indeed, we could demon- uPAR can interact laterally with a wide variety of membrane pro- strate that the transplantation-induced up-regulation of ICAM-1 teins including integrins, endocytic receptors, caveolin, the gp130 observed in WT2 allografts was strongly attenuated in uPARϪ/Ϫ cytokine receptor, the epidermal growth factor receptor, chemoat- allografts. However, the reduced expression of ICAM-1 in tractant receptors (1) and platelet-derived growth factor receptor uPARϪ/Ϫ allografts was not sufficient to prevent T cell infiltration. (2). This interaction might activate both pro- and anti-apoptotic This result coincides with observation of Zhang and coworkers Downloaded from downstream signaling pathways. For example, recently ROS-trig- (61) demonstrating that primed alloreative T cells do not require gered apoptosis in polymorphonuclear leukocytes (PMNs) cells allograft expression of ICAM-1 to infiltrate heart allografts. The ␤ has been shown to be 2 -dependent (48). Therefore, interaction of integrin leukocyte function Ag-1 with its alternative uPAR may stimulate ROS production via its lateral interaction ligand ICAM-2, or interaction of other T cell homing receptors with other molecules. with their ligands on endothelium, such as CD44/E- inter- The early inflammatory response during reperfusion of allo- action, may be involved in lymphocyte adhesion to the vascular http://www.jimmunol.org/ grafts is initiated by the infiltration of PMNs into the graft (38) endothelium of the graft and its subsequent infiltration bypassing followed by infiltration of monocytes/macrophages (MO). Mac- the requirement for ICAM-1 (62). rophages constitute 38 to 60% of infiltrating cells during acute By performing in vitro experiments with primary aortic endo- allograft rejection and increased influx of MO has been strongly thelial cells we could elucidate impaired TNF-␣ and C5a signaling correlated to complement activation and acute rejection in a Ϫ Ϫ in uPAR / cells leading to strongly decreased TNF-␣-induced human protocol biopsy study (49). Furthermore, it has been and completely abrogated C5a-induced up-regulation of ICAM-1. shown that MO infiltration 3 mo after transplantation correlated In line with a prior report that only the TNFR1 receptor is involved inversely with graft survival (50). It is well known that uPAR ␣ in TNF- -induced ICAM-1 up-regulation (63) we could demon- by guest on September 24, 2021 deficiency on the surface of leukocytes strongly reduces their strate that the up-regulation of ICAM-1 in MAEC after TNF-␣ migratory capacity both in vivo and in vitro (15, 51, 52). How- stimulation was mediated predominantly via TNFR1 because the ever, it should be noted that in our transplant experiments, the neutralizing anti-mouse TNFR1 Ab almost completely blocked host leukocytes expressed uPAR normally. Despite the normal this effect. These results coincide with strongly reduced adhesion expression of uPAR on host leukocytes, we found strongly re- Ϫ/Ϫ duced PMN/MO infiltration in kidney allografts from uPARϪ/Ϫ of WT2 PBMC to isolated aortas from uPAR mice compared ␣ as compared with those from WT2 mice. This finding stresses with WT2 aortas after stimulation with TNF- and C5a. This result the importance of the local uPAR expression on resident renal is in line with a prior report that platelet uPAR is critical for the ␣ cells in the allograft for adequate cell adhesion and subsequent response to TNF- (64) and coincides also with our previous ob- PMN/MO accumulation after kidney transplantation, indepen- servation that uPAR is necessary for C5a/C5aR-mediated re- dently from the uPAR expression on infiltrating cells. Recently sponses in mouse alveolar macrophages (15) and in human mes- it has been shown that inhibition of PMN infiltration into car- angial cells (14). Collectively, these data suggest that uPAR plays diac allografts may have a significant downstream impact on the a major role in mediating IR injury and subsequently influences efficacy of recipient T cell responses to the allograft (38). In early inflammation and allograft survival in renal transplantation contrast, in our model of acute renal allograft rejection we did rather than directly affecting T cell mediated alloimmune not find any differences in CD4 and CD8 positive cell infiltra- responses. tion between WT2 and uPARϪ/Ϫ allografts after transplanta- In summary, this study shows that the local expression of uPAR tion. Moreover, the expression of T cell activation Ag CD25 on resident renal cells of the allograft contributes to the develop- used as a marker for T cell activation (53) was also similar in ment of acute allograft rejection via at least two different path- WT2 and uPARϪ/Ϫ allografts. Furthermore, by investigating of ways, ischemia-induced apoptosis and the infiltration of host leu- priming of alloreactive T cells by MLR and IFN-␥ ELISPOT kocytes. These results suggest that uPAR, whose role was assays we did not found differences of T cell function of WT2 presumed to be involved in the migratory behavior of infiltrating and uPARϪ/Ϫ allografts recipients. These results suggest that cells, has a broader critical function as an early regulator of isch- superior survival of uPARϪ/Ϫ deficient allografts is not medi- emia-triggered initial generation of ROS, which, in turn, induces ated by T cell-dependent alloimmune response. the apoptosis in intrinsic renal cells. uPAR is also necessary for The migration of inflammatory cells into an extravascular site adequate TNF-␣ and C5a signaling, leading to up-regulation of requires a series of coordinated signals including the generation of ICAM-1 on endothelial cells of the allograft which is a crucial step a chemotactic gradient by the cells of the extravascular compart- for adequate leukocyte recruitment to the inflamed tissue. These ment and up-regulation of adhesion molecules on activated endo- observations underscore a new role of uPAR in acute allograft thelium. The strong up-regulation of MCP-1 and MIP-2 has been rejection and highlights uPAR as a target for prevention of organ demonstrated in animal models during renal ischemia as well as in dysfunction and damage in IR injury. 1188 uPAR AND KIDNEY TRANSPLANTATION

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