Extracellular Hmgb1 Functions as an Innate Immune-Mediator Implicated in Murine Cardiac Allograft Acute Rejection
2007; Elsevier BV; Volume: 7; Issue: 4 Linguagem: Inglês
10.1111/j.1600-6143.2007.01734.x
ISSN1600-6143
AutoresYun Huang, Hui Yin, Jun-Yan Han, Bo Huang, Juntao Xu, Fang Zheng, Zheng Tan, Min Fang, Li Rui, D. Chen, Shaogang Wang, Xiaodan Zheng, Cong‐Yi Wang, F Gonga,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoAmerican Journal of TransplantationVolume 7, Issue 4 p. 799-808 Free Access Extracellular Hmgb1 Functions as an Innate Immune-Mediator Implicated in Murine Cardiac Allograft Acute Rejection Y. Huang, Y. Huang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China These authors contributed equally to this work.Search for more papers by this authorH. Yin, H. Yin Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China These authors contributed equally to this work.Search for more papers by this authorJ. Han, J. Han Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GeorgiaSearch for more papers by this authorB. Huang, B. Huang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorJ. Xu, J. Xu Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorF. Zheng, F. Zheng Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorZ. Tan, Z. Tan Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorM. Fang, M. Fang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorL. Rui, L. Rui Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorD. Chen, D. Chen Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorS. Wang, S. Wang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorX. Zheng, X. Zheng Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorC.-Y. Wang, Corresponding Author C.-Y. Wang Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, Georgia Department of Pathology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia * Corresponding authors: Cong-Yi Wang, cwang@mcg.edu, and FeiliGong, flgong@163.comSearch for more papers by this authorF. Gong, F. Gong Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this author Y. Huang, Y. Huang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China These authors contributed equally to this work.Search for more papers by this authorH. Yin, H. Yin Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China These authors contributed equally to this work.Search for more papers by this authorJ. Han, J. Han Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GeorgiaSearch for more papers by this authorB. Huang, B. Huang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorJ. Xu, J. Xu Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorF. Zheng, F. Zheng Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorZ. Tan, Z. Tan Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorM. Fang, M. Fang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorL. Rui, L. Rui Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorD. Chen, D. Chen Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorS. Wang, S. Wang Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorX. Zheng, X. Zheng Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this authorC.-Y. Wang, Corresponding Author C.-Y. Wang Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, Georgia Department of Pathology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia * Corresponding authors: Cong-Yi Wang, cwang@mcg.edu, and FeiliGong, flgong@163.comSearch for more papers by this authorF. Gong, F. Gong Laboratory of Transplantation, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, ChinaSearch for more papers by this author First published: 28 February 2007 https://doi.org/10.1111/j.1600-6143.2007.01734.xCitations: 108 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Hmgb1, an evolutionarily conserved chromosomal protein, was recently re-discovered to be an innate immune-mediator contributing to both innate and adaptive immune responses. Here, we show a pivotal role for Hmgb1 in acute allograft rejection in a murine cardiac transplantation model. Extracellular Hmgb1 was found to be a potent stimulator for adaptive immune responses. Hmgb1 can be either passively released from damaged cells after organ harvest and ischemia/reperfusion insults, or actively secreted by allograft infiltrated immune cells. After transplantation, allografts show a significant temporal up-regulation of Hmgb1 expression accompanied by inflammatory infiltration, a consequence of graft destruction. These data suggest the involvement of Hmgb1 in acute allograft rejection. In line with these observations, treatment of recipients with rA-box, a specific blockade for endogenous Hmgb1, significantly prolonged cardiac allograft survival as compared to those recipients treated with either rGST or control vehicle. The enhanced graft survival is associated with reduced allograft expression of TNFα, IFNγ and Hmgb1 and impaired Th1 immune response. Abbreviation: DAMPs damage associated molecular patterns Hmgb1 high mobility group box 1 rA-box recombinant A-box rGST recombinant GST RAGE receptor for advanced glycation endproducts TLRs toll-like receptors Introduction The role of adaptive immunity in allograft rejection has been extensively studied for decades. It has been suggested that acute allograft rejection is principally a T cell-mediated adaptive immune response (1). However, the contribution of innate immunity to allograft rejection has generally been ignored until the discovery of the Toll-like receptor (TLR) system and the subsequent realization of innate immunity in driving and shaping adaptive immunity (2-4). Thus far, there is compelling evidence that in addition to responding to pathogen-associated molecular patterns (PAMPs) of microorganisms, TLRs also react to endogenous ligands such as heat shock proteins (HSPs), β-defensin, fibronectin, hyaluronan fragments and heparan sulfate (collectively known as damage-associated molecular patterns or DAMPs) (5-9). Particularly, a number of recent studies revealed that DAMPs not only participate in ischemia/reperfusion related early organ injury, but also play a pivotal role in perpetuating adaptive allograft rejection by signaling through TLRs or other innate receptors such as dectin-1 (10-13). Hmgb1 (high mobility group box 1), originally identified as a nuclear DNA-binding protein, is one of the most evolutionarily conserved proteins in the eukaryotic kingdom (14). It was recently re-discovered to be a potent innate 'danger signal' for the initiation of host defense or tissue repair when it is present extracellularly (15-18). It represents a long-searched-for DAMP adapted by the innate immune system after the split between the animal and plant kingdoms probably more than 500 million years ago. Hmgb1 can be either passively released from necrotic or damaged cells (19-21) or actively secreted by activated immune cells such as dendritic cells (DCs), macrophages and NK cells (22, 23). Extracellular Hmgb1 is a potent macrophage-activating factor and a proinflammatory mediator of inflammation (24). It is also potent in inducing DC maturation and Th1 polarization (23, 25). Hmgb1 contains two DNA-binding domains (A-box and B-box) and a highly acidic C-terminal tail. The stimulatory activity of Hmgb1 can be reproduced by the recombinant B-box, whereas the recombinant A-box (rA-box) acts as a specific antagonist attenuating the biologic function of the full length Hmgb1 (15). Previous studies implicated RAGE (receptor for advanced glycation endproducts) as an Hmgb1 receptor that mediates neurite outgrowth during brain development and migration of smooth muscle cells (26). Recently, studies have further implicated TLRs 2 and 4 in Hmgb1 signaling (18, 27, 28) and demonstrated that Hmgb1 not only enhances chemotactic and innate immune responses, but also acts as an adjuvant to initiate and enhance adaptive immune responses (19). Therefore, Hmgb1 has been found to play a pivotal role in the pathogenesis of inflammatory diseases such as sepsis (24) and autoimmune diseases such as systemic lupus erythematosus (SLE) (29), rheumatoid arthritis (RA) (30, 31), and type 1 diabetes (Han et al. unpublished data). Given the importance of Hmgb1 in both innate and adaptive immune responses, we postulate that Hmgb1 may act as a potent innate-immune mediator implicated in transplantation rejection. This hypothesis is based on (1) Hmgb1 passively released from damaged cells after organ harvest and ischemia/reperfusion insults contributes to the initiation of allograft rejection; and (2) Hmgb1 actively secreted by infiltrated immune cells (e.g. macrophages and DCs) contributes to the progression of acute allograft rejection. In this current study, we applied a murine cardiac transplantation model to test the above hypothesis. Our results demonstrate that blockade of extracellular Hmgb1 by a specific antagonist, rA-box, significantly delays cardiac allograft acute rejection. In addition, the prolonged graft survival is associated with reduced allograft proinflammatory cytokine expression and impaired Th1 immunity. Materials and Methods Animals BALB/c (H-2d) and C57BL/6 (H-2b) mice were maintained at the Animal Facility of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 8–12 week old male mice (weighed 20–25 g) were selected for the study. The experimental mice were housed in a specific pathogen-free facility (SPF) in microisolator cages supplied with autoclaved food and acidified water. All of the experimental mice were allowed to acclimatize in the local facility for one week before any experiments were started. All of the studies were performed under the guidelines of Tongji animal use regulations and approved by the institutional animal care and use committee (IACUC) at the Tongji Medical College. Expression and purification of recombinant full-length Hmgb1 and A-box Full-length mouse Hmgb1 was amplified from mouse spleen cDNA and cloned into a Pet28α vector (Novagen). The insert was confirmed by direct DNA sequencing. The DNA sequence encoding the A-box (261 bp) was then amplified from the above plasmid containing the full-length Hmgb1 using the following oligonucleotides; 5′-GAT GGG CAA AGG AGA TCC TAA G-3′ and 5′-TCA CTT TTT TGT CTC CCC TTT GGG-3′. The resulting amplified fragment was subsequently cloned into a pGEX5X1 vector with a glutathione S-transferase (GST) tag (Novagen). The recombinant plasmids were transformed into a protease-deficient Escherichia coli strain BL21 (DE3) and incubated in 2YT medium containing amplicilin (100 mg/mL) for 5–7 h at 37°C, with shaking, until the optical density at 600 nm reached 1–1.5. Recombinant protein expression was induced by addition of 1 mM IPTG (0.5 mM IPTG for A-box) and further incubated for 3–4 h at 30 or 37°C (A-box) with vigorous shaking. The cells were then collected and subjected to lysozyme treatment (1 mg/mL) for 30 min and subsequent sonication on ice in the presence of 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 1 mM PMSF. Recombinant Hmgb1 (rHmgb1) and A-box (rA-box) were then purified using the His-tagged affinity columns and the glutathione sepharose 4B resin columns (Amersham Biosciences, Piscataway, NJ) as instructed by the manufacturer. A GST vector protein was also expressed and purified to homogeneity. The affinity purified recombinant proteins were further subjected to gel filtration HPLC chromatography using the UPC 900 system (Pharmacia). The eluted protein was dialyzed extensively against PBS before passing over polymyxin B columns (PIERCE) to remove any contaminated LPS. The recombinant proteins were finally concentrated by stirred ultrafiltration cells (Millipore, Billerica, MA) and stored in aliquots at −80°C until use. The purity and integrity of each purified recombinant protein was verified by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Detection of LPS content in purified recombinant proteins The LPS content in purified recombinant proteins was measured by the Chromogenic Limulus Amoebocyte Lysate Assay (Sigma-Aldrich) according to the manufacturer's instruction. LPS content in purified rHmgb1, rA-box and rGST was 138 pg/μg, 18 pg/μg and 4 pg/μg, respectively. To prevent the possible effect of contaminating LPS, polymyxin B was added into cultures at six units of polymyxin B per pg of LPS. Separate controls confirmed that this amount of polymyxin B neutralized the maximal amount of contaminating endotoxin. Cell culture Murine macrophage-like RAW 264.7 cells (ATCC) were cultured in RPMI medium 1640 (Hyclone) supplemented with 10% FBS (Gibco, Gaithersburg, MD), 1 mM sodium pyruvate, 2 mM l-glutamine, 5 × 10−5 M 2-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were used at 90% confluence and treatment was carried out in serum-free Opti-MEM I medium (Life Technologies). Cardiac transplantation and rA-box administration For heterotopic cardiac transplantation, C57BL/6 (H-2b) mice were used as recipients, while Balb/c (H-2d) mice were used as donors. Abdominal heterotopic cardiac transplantation was performed according to the protocol described by Corry et al. (32). Briefly, the cardiac allograft was transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient's aorta and vena cava, respectively. rA-box (1.0 mg/day) was administered to each recipient intraperitoneally (i.p.) from the day of transplantation until day 4 posttransplantation. The same amount of rGST or PBS (control vehicle) was used as controls. A total of 18 mice were included in each study group. Six were used for monitoring allograft survival, while four mice were sacrificed at each of three time points: on days 1, 3 and 7 posttransplantation. Allograft and spleen from each sacrificed mouse were collected and subjected to real-time quantitative PCR, histological and flow cytometric analysis. Allograft survival was present as the mean survival time (MST) ± SD. Graft function was assessed by daily abdominal palpation. Rejection was defined as the absence of detectable heart beating and was verified in selected cases by direct visualization and histological examination. Real-time PCR and Western blot analysis Total RNA was extracted from grafts of recipient mice at the indicated time points using TRIzol® Reagent (Invitrogen, Carlsbad, CA) as instructed. The extracted RNA was further treated with DNase I to remove any contaminated genomic DNA. cDNA was synthesized from 1 μg total RNA using a first strand cDNA synthesis kit (MBI). Equal quantities of cDNA from each animal were used for real-time PCR (RT PCR) analysis of expression levels for Hmgb1, TNFα and IFNγ using an Icycler (BioRad) with a SYBR Green qPCR kit (Invitrogen) as previously reported (33). The relative expression levels for each target gene were normalized by GAPDH and present as fold increases versus heart tissues from normal mice. Oligonucleotides used in the PCR amplification were: Hmgb1 forward 5′-GCG GAC AAG GCC CGT TA-3′, reverse 5′-AGA GGA AGA AGG CCG AAG GA-3′; TNFα forward 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′, reverse 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; IFNγ forward 5′-TCA AGT GGC ATA GAT GTG GAA GAA-3′, reverse 5′- TGG CTC TGC AGG ATT TTC ATG-3′; and GAPDH forward 5′-AGG GCT GCT TTT AAC TCT GGT AAA-3′, reverse 5′-CAT ATT GGA ACA TGT AAA CCA TGT AGT TG-3′. For Western blot analysis, cytoplasmic proteins were prepared from allograft or normal tissues using an NE-PER cytoplasmic extraction kit (PIERCE). The relative amount of Hmgb1 in the cytoplasmic proteins was then determined using an ECL Plus™ Western blot kit (PIERCE) as previously reported (34). Intracellular IFNγ expression by flow cytometry Splenic single cell suspensions were prepared, followed by RBC lysis with ammonium chloride lysis buffer and washed with PBS. The cells (1 × 106 per sample) were first cultured for 4 h in the presence of 20 ng/mL PMA, 1 μg/mL of ionomycin (Sigma-Aldrich) and 2 μM of Monesin (Sigma-Aldrich), and then blocked with anti-mouse CD16/32 FcγIII/II receptor followed by staining with BD Cy-Chrome conjugated anti-mouse CD4 antibody and FITC-conjugated anti-mouse CD8 antibody. The cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) and stained with PE-conjugated anti-mouse IFNγ at 4°C in the dark for 30 min. After washes, the cells were resuspended in FACS buffer, and analyzed within 24 h on a FACSCalibur (BD Bioscience, San Jose, CA). At least 10,000 events were collected for the analysis of CD4+ or CD8+ T-cell intracellular IFNγ expression. Data were analyzed with the CellQuest v3.3 soft ware (BD Bioscience) as instructed. Appropriately conjugated isotype-matched control antibodies were used for negative controls. Histological and immunohistochemical analysis Cardiac grafts were fixed in 4% paraformaldehyde and embedded in paraffin. 5 μM tissue sections were prepared and stained with hematoxylin and eosin. For immunohistochemical staining, the slides were deparaffinized and boiled in 10 mM sodium citrate buffer (pH 6.0) for 25 min to unmask antigenic epitopes. After blocking with 10% goat serum for 1 h, the slides were incubated with an Hmgb1 monoclonal antibody (Abnova) in PBS with 1% bovine serum albumin (BSA, Fisher) overnight at 4°C. After washing, the slides were incubated with 3% H2O2 for 10 min to quench endogenous peroxidase activity and then stained with biotinylated anti-rabbit secondary antibody (1:200 in PBS, Santa Cruz). After incubating the slides in ABComplex (Santa Cruz) for 30 min in the dark, the color was developed using DAB (Santa Cruz). The slides were allowed to react for 5–10 min and were considered complete when a brown color was observed. The reaction was stopped by washing slides in ddH2O and counterstained with hematoxylin. The slides were then air dried and mounted using Faramount aqueous mounting medium. Normal rabbit IgG (instead of primary antibody) was used as a negative control. Statistical analysis Allograft survival curves were generated by the Kaplan and Meier method. Allograft survival differences between groups were determined using the log-rank (Mantel-Cox) test. Comparisons between groups for flow cytometry, immunohistochemistry, and RT-PCR data were accomplished by one-way ANOVA using SPS 11.5 for windows. Data were present as mean ± SD. P < 0.05 was considered statistically significant. Results rA-box effectively blocks rHmgb1-induced cytokine secretion in RAW264.7 cells We first performed in vitro studies to demonstrate the antagonistic effect of rA-box on the functionality of Hmgb1 in a mouse macrophage-like cell line (RAW264.7 cells). rHmgb1, rA-box and rGST were purified first by affinity columns and subsequently by gel filtration HPLC chromatography. The potential contaminated LPS in purified recombinant protein was further removed by passing through the polymyxin B columns. rHmgb1 was found to be a potent stimulator of RAW264.7 cells to secrete TNFα in a dose dependent manner. The highest stimulatory effect was observed when 20 μg/mL of rHmgb1 was added into the RAW264.7 cell cultures (data not shown). To demonstrate the antagonistic effect of rA-box, 10 μg/mL of rA-box was then added into the RAW264.7 cell cultures 1 h before the addition of 20 μg/mL rHmgb1. As shown in Figure 1, the addition of rA-box attenuated rHmgb1-induced TNFα release by 2.2-fold, while rGST failed to inhibit the stimulatory effect of rHmgb1. Of note, rA-box or rGST alone did not show any stimulatory effect on RAW264.7 cells. All of these observations indicate the possibility that rA-box can be used as a blockade for Hmgb1 for the studies in our murine cardiac transplantation model. Figure 1Open in figure viewerPowerPoint rA-box specifically attenuates Hmgb1-induced TNFα release. Murine RAW 264.7 cells were stimulated with 20 μg/mL rHmgb1 for 16 h in serum-free Opti-MEM medium in the absence or presence of purified rA-box (10 μg/mL) or rGST (as a control). Culture supernatants were collected and assayed for TNFα production using an ELISA kit. Data are present as mean ± SD of three independent experiments. Organ harvest and ischemia/reperfusion insults result in passive release of Hmgb1 It was suggested that ischemia/reperfusion is a major pathophysiological component of acute allograft malfunction after transplantation. This includes direct cellular damage resulting from ischemic/reperfusion insult and delayed dysfunction and damage caused by activation of adaptive immunity (acute rejection). In addition, allografts may also result in local tissue injury during the organ harvest process. As an innate 'danger signal', Hmgb1 can be passively released from necrotic or damaged cells. We, therefore, postulate that the initiation of acute allograft rejection involves the endogenous Hmgb1 passively released by damaged cells after organ harvest and ischemia/reperfusion insults. We first checked passive Hmgb1 release in allografts after organ harvest insult. To this end, cytoplasmic proteins were extracted from tissues of three cardiac allografts that underwent about 1 h of cold ischemic insult and were ready for transplantation. The isolated cytoplasmic proteins were then subjected to Western blot analysis for detection of Hmgb1 translocation. As can be seen from Figure 2A, the amount of Hmgb1 in the cytoplasmic fraction of allografts (lanes 2–4) was much higher than that of fresh heart tissues (without ischemic insult, lane 1). These data indicate that Hmgb1 is passively released from damaged cells after organ harvest insult. We next, checked Hmgb1 passive release from allografts after ischemia/reperfusion insult. We collected three cardiac allografts after 1 h of transplantation and performed Hmgb1 in situ immunohistochemical staining. In fresh heart tissues (without ischemia/reperfusion insult), Hmgb1 was only stained in the nucleus (stained in brown color, Figure 2B). In contrast, ischemia/reperfusion insult resulted in local allograft tissue injury. As expected, Hmgb1 was also present in the cytoplasm other than the nucleus in damaged cells (Figure 2C), which indicates that Hmgb1 is passively released from damaged cells after ischemia/reperfusion insult. Figure 2Open in figure viewerPowerPoint (A) Western blot analysis of cytoplasmic Hmgb1 in allografts after organ harvest insult. Lane 1: Cytosolic Hmgb1 from fresh control heart tissues; lane 2–4: cytosolic Hmgb1 originating from three allogarfts that were ready for transplantation (with approximately 1 h of cold ischemia). (B) Immunohistochemical staining of Hmgb1 in normal control heart tissues. Hmgb1 is stained brown, and can only be stained in the nucleus. The nuclei were also counterstained blue by hematoxylin. (C) Hmgb1 passive release after ischemia/reperfusion insult. Cardiac allografts after 1 h of transplantation were collected and subjected to immunohistochemical staining of Hmgb1. In contrast to the normal cells, Hmgb1 can be detected in the cytoplasm of damaged cells, indicating Hmgb1 passive release from damaged cells. (D) Allograft infiltrated immune cells actively secret Hmgb1 into intercellular milieu. After day 7 of transplantation, cardiac allograft underwent severe acute rejection characterized by inflammatory infiltration accompanied by allograft destruction. As indicated by the arrow, Hmgb1 in the infiltrated immune cells can be detected both in the cytoplasm and nucleus, indicating active Hmgb1 secretion by the infiltrated immune cells. (E) Hmgb1 staining in syngeneic cardiac grafts. Syngeneic grafts (BALB/c-BALB/c) after day 7 of transplantation were collected and subjected to Hmgb1 staining as above. In contrast to the allografts, no inflammatory infiltration was observed in the syngeneic grafts and Hmgb1 is predominantly localized in the nucleus. All data shown here are representatives of consistent results observed in three mice included in each group. Allograft infiltrated immune cells actively secret Hmgb1 into the intercellular milieu In addition to passive release by damaged cells, Hmgb1 can also be actively secreted by immune cells such as DCs and macrophages (15). We, therefore, assessed the active Hmgb1 secretion by infiltrated immune cells in cardiac allografts. We collected allografts on day 7 posttransplantation from three recipients without antirejection therapy. Histological analysis demonstrated severe acute rejection characterized by rigorous inflammatory infiltration accompanied by cardiac myocyte destruction (data not shown). Consistently, in situ immunohistochemical staining of all allografts revealed that Hmgb1 showed positive staining both in the nucleus and cytoplasmic vesicles (brown color) in the infiltrated immune cells (Figure 2D, indicated by the arrow), a characteristic proceeding for active secretion of Hmgb1. For controls we examined active Hmgb1 release in three syngeneic grafts (BALB/c- BALB/c) after day 7 of transplantation. As can be seen from Figure 2E, we failed to observe inflammatory infiltration and Hmgb1 active release in all of these grafts. These observations suggest that in the allografts the infiltrated immune cells (e.g. DCs and macrophages) secrete high levels of Hmgb1 into the intercellular milieu, which in turn, may function as an innate endokine to enhance allograft rejection. Hmgb1 expression is up-regulated in cardiac allografts after transplantation To further demonstrate the implication of Hmgb1 in the initiation and progression of acute rejection after transplantation, we analyzed the temporal expression of Hmgb1 in syngeneic and allogeneic grafts. Syngeneic (BALB/c- BALB/c) and allogeneic (BALB/c- C57BL/6) cardiac grafts were collected on days 1, 3 and 7 posttransplantation and subjected to RT PCR analysis. On day 1 posttransplantation, we observed >twofold increased Hmgb1 expression in both syngeneic and allogeneic grafts as compared to the control tissues. However, an increased temporal expression of Hmgb1 was observed in all allografts. On day 7 posttransplantation, the expression of Hmgb1 was 10.5-fold higher than that of the control tissues and sevenfold higher than the syngeneic grafts (p < 0.001). In contrast, a decreased temporal expression of Hmgb1 was observed in all the syngeneic grafts with the expression of Hmgb1 on day 7 posttransplantation decreased by 1.8-fold as compared to that of day 1 posttransplantation (Figure 3). More importantly, the increased temporal expression of Hmgb1 in allografts is accompanied by interstitial infiltration and active secretion
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