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Early Chemokine Cascades in Murine Cardiac Grafts Regulate T Cell Recruitment and Progression of Acute Allograft Rejection This information is current as of August 9, 2017. Ken Morita, Masayoshi Miura, David R. Paolone, Tara M. Engeman, Anil Kapoor, Daniel G. Remick and Robert L. Fairchild J Immunol 2001; 167:2979-2984; ; doi: 10.4049/jimmunol.167.5.2979 http://www.jimmunol.org/content/167/5/2979 Subscription Permissions Email Alerts This article cites 33 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/167/5/2979.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 References Early Chemokine Cascades in Murine Cardiac Grafts Regulate T Cell Recruitment and Progression of Acute Allograft Rejection1 Ken Morita,* Masayoshi Miura,*† David R. Paolone,* Tara M. Engeman,† Anil Kapoor,* Daniel G. Remick,‡ and Robert L. Fairchild2*‡§ A llogeneic cardiac transplantation is an increasingly used approach for therapy of end-stage heart disease. The loss of cardiac allografts due to T cell-mediated acute rejection has been substantially decreased by current immunosuppressive strategies. Despite this improvement, acute rejection episodes remain a significant problem in allograft function and loss (1). Furthermore, recent clinical studies have clearly indicated that acute rejection episodes are a critical risk factor for the subsequent development of chronic rejection, the leading cause of cardiac allograft loss (2, 3). The key factors directing alloantigen-primed T cell infiltration into allografts during acute rejection remain largely unidentified. A critical factor facilitating leukocyte infiltration of tissue is the presence of inflammation. Inflammation in vascularized organ grafts induces up-regulated adhesion molecule expression on vascular endothelium and other cells (4 –7). Antagonism of adhesion molecules has inhibited acute rejection and prolonged graft survival in some clinical studies and animal models but not in Departments of *Urology and †Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195; ‡Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and §Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 Received for publication August 16, 2000. Accepted for publication June 20, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the National Institutes of Health (AI 40459) and the American Heart Association (0050538N) and a generous gift from the State of Qatar to the Renal Transplant Research Program at the Cleveland Clinic Foundation. 2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Department of Immunology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: [email protected] Copyright © 2001 by The American Association of Immunologists others (7–10), suggesting that other factors may also be important in directing T cell recruitment into allografts. Another important component of inflammation is the production of cytokines with chemoattractant properties for leukocyte populations, chemokines (11, 12). Chemokines are grouped into families (CXC, CC, C, and CX3C) based on cysteine motifs. The CXC family includes the neutrophil chemoattractants IL-8 and growthrelated oncogene ␣, of which KC is the murine homologue, and two potent chemoattractants for Ag-activated T cells, IFN-␥-inducible protein-10 (IP-10)3 and monokine induced by IFN-␥ (Mig). The CC family includes the monocyte/macrophage chemoattractant protein-1 (MCP-1), of which JE is the murine homologue, as well as macrophage inflammatory proteins (MIP)-1␣ and 1. Studies from this and several other laboratories have indicated the presence of chemokine mRNA and/or protein during acute rejection of allografts in experimental and clinical transplantation (13–17). IP-10 and Mig expression was observed in heterotopically transplanted cardiac allografts but not isografts at day 3 after transplant, whereas expression of MIP-1␣, MIP-1, and KC was absent in iso- and allografts (14, 18). The production of IP-10 and Mig may be an important step in the acute rejection process due to their strong chemoattractive properties for Ag-primed T cells (19). The induction and role of specific chemokines in the rejection of solid organ allografts remains unclear. In the current study, we have tested the expression of four chemokines 1.5– 48 h after transplant in murine cardiac iso- and allografts. The results indicate the induction of organized cascades of chemokine RNA expression initiated by KC and followed by JE, MIP-1, and MIP-1␣ at early 3 Abbreviations used in this paper: IP-10, IFN-␥-inducible protein-10; MCP-1, monocyte chemotactic protein-1; Mig, monokine induced by IFN-␥; MIP, macrophage inflammatory protein; NRS, normal rabbit serum. 0022-1767/01/$02.00 Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2a) and syngeneic heart grafts in C57BL/6 (H-2b) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene ␣ (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1 and 1␣. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN␥-inducible protein-10 and monokine induced by IFN-␥, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts. The Journal of Immunology, 2001, 167: 2979 –2984. 2980 times after transplant in both iso- and allografts. Administration of a single dose of rabbit antiserum to KC results in a substantial increase in allogeneic heart graft survival. The prolonged survival is associated with the decreased expression of Mig and IP-10, as well as decreased T cell infiltration into the allograft. These results indicate the importance of early inflammatory events on the subsequent production of T cell chemoattractants and T cell recruitment into the allograft during the acute rejection process. Materials and Methods Animals A/J (H-2a) and C57BL/6 (H-2b) mice were obtained through Dr. C. Reeder at the National Cancer Institute (Frederick, MD). Adult males of 8 –12 wk of age were used throughout this study. Abs and antiserum Immunohistology Heart grafts were retrieved at day 7 after transplant, embedded in OCT compound (Sakura Finetek, Torrence, CA), and frozen at ⫺80°C. Sections were cut at 8 m and mounted onto slides. For immunohistochemistry, sections were fixed in acetone for 10 min and air dried. Slides were immersed in PBS for 10 min and then in 0.03% H2O2 for 10 min to eliminate endogenous peroxidase activity. The slides were then stained for 1 h with 5 g/ml anti-CD4 mAb (GK1.5) or anti-CD8 mAb (53-6.7) in 0.05 TrisHCl with 1% BSA. Control slides were incubated with rat IgG as the primary Ab. After three washes in PBS for 5 min each, slides were incubated for 20 min with biotinylated goat anti-rat IgG diluted 1/300 in PBS. After three washes in PBS, slides were incubated with streptavidin-HRP (DAKO) for 20 min and washed another three times. To prepare the substrate-chromagen solution, a 10-mg tablet of 3,3⬘-diaminobenzidine (Sigma, St. Louis, MO) was dissolved in 15 ml of PBS plus 12 l of 30% H2O2. The solution was applied to the slides, which were incubated for 3–7 min and then rinsed in dH2O to stop the reaction. The slides were counterstained with hematoxylin for 3 min, rinsed with tap water, and immersed in 37 nM NH4OH for 10 s. Finally, the slides were dehydrated, viewed under light microscopy, and the images were captured using ImagePro Plus (Media Cybernetics, Silver Spring, MD). Numbers of cells staining positive were counted in eight random fields from three different tissue sections Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 Rabbit immune serum to a KC-specific peptide (sequence QTMAGIHLKNIQS) was made at BioSynthesis (Louisville, TX). This antiserum reacts with KC and not with other CXC chemokines including IP-10 and Mig in Western blot analyses and inhibits recombinant KC-mediated chemotaxis of thioglycolate-induced peritoneal neutrophils in in vitro chemotaxis assays (Ref. 20 and data not shown). Normal rabbit serum (NRS) was used as control Ig for in vivo Ab treatment experiments. Mice were depleted of neutrophils by giving 100-g aliquots of the rat anti-Ly6G mAb, RB6.8C5 (21), on two consecutive days. This treatment resulted in ⬍5% neutrophils in the peritoneal wash of mice 4 h after thioglycolate injection as assessed by staining the peritoneal cells with Wright’s stain. Previous studies have shown that treatment with either the KC antiserum or RB6.8C5 does not affect the viability of circulating or lymphoid T cell populations (20). For use in immunocytochemistry, GK1.5, rat anti-mouse CD4 mAb, was obtained from BD PharMingen (San Diego, CA); 53-6.7, rat anti-mouse CD8 mAb, and biotinylated goat anti-rat polyclonal Ab were purchased from DAKO (Carpinteria, CA); and control rat IgG was purchased from R&D Systems (Minneapolis, MN). EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS Heterotopic cardiac transplant Cardiac transplants were performed according to the method of Corry and coworkers (22). Briefly, donor and recipient mice were anesthetized with phenobarbital. Donor hearts were harvested and placed in chilled lactated Ringer’s solution while the recipient mice were prepared. The donor heart was anastomosed to the recipient abdominal aorta and vena cava using microsurgical techniques. Upon completion of the anastomosis and organ perfusion, the transplanted hearts resumed spontaneous contraction. The strength and quality of cardiac impulses were graded each day, as described previously (14, 18). Rejection of cardiac grafts was considered complete by the cessation of impulse and was confirmed visually for each graft by laparotomy. In C57BL/6 recipients, complete rejection of A/J cardiac grafts occurs between 8 and 10 days after transplantation. Cardiac isografts in the C57BL/6 recipients functioned for ⬎300 days. Significance in allograft survival between recipient treatment groups was analyzed by log rank test, and p ⬍ 0.01 was considered a significant difference between groups. Northern blot analysis Whole-cell RNA was isolated from transplanted and native heart tissue using TRIzol (Life Technologies, Grand Island, NY). Briefly, transplanted and naive hearts were excised from recipients, snap frozen, and homogenized in TRIzol. After extraction, precipitation, and resuspension in diethylpyrocarbonate-treated dH2O, 10-g aliquots of RNA were electrophoresed in 1% agarose formaldehyde-denaturing gels and analyzed by Northern blot analysis, as described previously (14). Blots were hybridized with 32P-labeled oligonucleotide probes specific for KC, MIP-1␣, MIP-1, JE, IP-10, and Mig. After hybridization and exposure with one cytokine oligonucleotide probe, the filter was stripped of the probe by washing the blot three times in 0.4% SDS at 90°C and then hybridized with the next test cytokine probe. After hybridization with the test cytokine probes was completed, the blot was stripped and probed with a rat GAPDH cDNA (23). Densitometry using Storage Phosphor Screen analyzer (Molecular Dynamics, Sunnyvale, CA) was used to comparatively measure the cytokine signal and the GAPDH signal for each sample of the blot. The cytokine signals for each sample of the blot were then normalized by expressing the density of the cytokine signal as a ratio to the signal of the GAPDH signal for each RNA sample. The mean ratio for each group (i.e., time point or treatment) was determined, and differences between means were tested using Welch’s t test. A p ⬍ 0.05 was considered a significant difference. FIGURE 1. Expression of chemokine genes following reperfusion of heart grafts. Groups of five cardiac isografts and A/J allografts were harvested from C57BL/6 recipients at 1.5, 3, 6, 9, 12, 18, 24, and 48 h after reperfusion, and total cellular RNA was prepared and analyzed by Northern blot for expression of the indicated genes. Intensity of the chemokine RNA signals were plotted as a ratio to the GAPDH signal, and the mean ratio ⫾ SD of the five grafts in each group is shown The Journal of Immunology 2981 from three different grafts, and significance between mean numbers of positive cells per field in different treatment groups was tested using MannWhitney U test. Results FIGURE 3. Histological analyses of A/J cardiac allografts from C57BL/6 recipients treated with NRS vs KC antiserum. C57BL/6 recipients were given 200 l KC antiserum (A and C) or NRS (B and D) within 30 min of cardiac allograft reperfusion. Allografts were harvested at day 7 after transplant and fixed with 10% buffered formalin. Sections were prepared and stained by H&E. Magnification: A and B, ⫻100; C and D, ⫻400. Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 FIGURE 2. Effect of KC antiserum on heart allograft rejection. Groups of C57BL/6 recipients were given 200 l KC antiserum (dotted line) or NRS (solid line) within 30 min of A/J cardiac allograft reperfusion. Survival of heart allografts in KC antiserum-treated C57BL/6 recipients (n ⫽ 6) was compared with survival of allografts in NRStreated recipients (n ⫽ 8). Allograft rejection in all recipient mice was visually confirmed by laparotomy. Isografts from recipients treated with KC antiserum or NRS were maintained ⬎300 days. Recipient treatment with KC antiserum significantly prolonged heart allograft survival (p ⬍ 0.01). In a previous study, expression of neutrophil and macrophage chemoattractant chemokines were absent from allogeneic and syngeneic vascularized cardiac grafts at day 3 after transplant (14). To begin to test if neutrophil and monocyte/macrophage chemoattractants were expressed at earlier time points in cardiac iso- and allografts, C57BL/6 (H-2b) mice received syngeneic or A/J (H-2a) heart grafts and at various times from 1.5 to 48 h after transplant groups of five grafts were harvested at each time point. RNA was then prepared and analyzed by Northern blot hybridization for expression of KC, JE, MIP-1␣, and MIP-1. KC and JE expression was first apparent at 3 h after transplant and peaked at 6 h after transplant in both iso- and allografts (Fig. 1). These levels dropped quickly by 9 h after ransplant, and by 48 h after transplant, levels of KC and JE expression in iso- and allografts had receded to background levels. Expression of MIP-1 in both syngeneic and allogeneic heart grafts also began to appear at low levels at 3 h after transplant. In contrast to KC and JE, expression levels of MIP-1 dipped slightly after 6 h after transplant and rose to a second peak at 24 h after transplant before beginning to fall to background levels 48 h after 2982 begins at day 2 after transplant and increases to the time of acute graft rejection (18). We tested the impact of KC on expression of these chemokines at day 2 after transplant by Northern blot hybridization. NRS or rabbit antiserum to KC was given i.v. to groups of allogeneic heart graft recipients 30 min after graft reperfusion. Two days later, the hearts were retrieved, and RNA was prepared and tested for expression of IP-10 and Mig. Recipient treatment with KC antiserum reduced intraallograft expression of both IP-10 and Mig ⬎50% (Fig. 6). The ability of Abs to the neutrophil chemoattractant KC to inhibit T cell infiltration into cardiac allografts and prolong survival suggested a role for neutrophils in the acute rejection process of these allografts. To examine this role more closely, we tested the effect of recipient neutrophil depletion on the survival of cardiac allografts. C57BL/6 mice were treated on two consecutive days with the neutrophil-depleting Ab RB6.8C5 or rat IgG as a control. On the day of the second treatment, groups of the mice received either syngeneic C57BL/6 or MHC-mismatched A/J cardiac allografts. Treatment with the control rat IgG did not prolong allograft survival when compared with nontreated allograft recipients (Fig. 7). However, depletion of recipient neutrophils at the time of transplantation extended the survival of the allografts up to day 21 after transplantation. In conjunction with the extended survival in recipients treated with the KC antiserum, these results indicate the importance of neutrophils in acute rejection of vascularized heart allografts. Discussion The introduction of wounds and ischemia/reperfusion injury initiates a tissue inflammatory response that includes production of TNF-␣, IL-1, and chemokines and the up-regulation of adhesion molecules (4 –7, 24, 25). Although chemokines play a critical role FIGURE 4. CD8⫹ T cell infiltration into heart allografts from KC antiserum- and NRS-treated recipients. C57BL/6 recipients were given 200 l KC antiserum (A) or NRS (B) within 30 min of cardiac allograft reperfusion. Allografts were harvested at day 7 after transplant, and frozen sections were prepared and stained with anti-CD8 mAb for immunohistochemistry. Magnification, ⫻200. Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 transplant. Expression of MIP-1␣ did not begin to become apparent until 12–18 h after transplant, reached peak levels at 24- 48 h after transplant, and then decreased. Because KC was the first chemokine we observed expressed in the cardiac grafts, we tested the effect of KC antagonism on the survival of the A/J cardiac allografts in C57BL/6 recipients. Groups of allograft recipients were given 200 l NRS or rabbit KC antiserum i.v. within 30 min after graft reperfusion. Heart graft beating in recipients was monitored daily by palpation and cessation of beating was confirmed by laparotomy. With one exception, heart allografts in recipients treated with NRS ceased beating between days 7 and 10 after transplant (Fig. 2). In contrast, significant prolongation of allograft survival up to day 23 after transplant was observed in recipients treated with the KC antiserum. This enhanced survival was accompanied by a visible decrease in cellular infiltration of the heart allografts from the KC antiserumtreated recipients when compared with allografts from NRStreated recipients at day 7 after transplant (Fig. 3, A vs B). The decrease in cellular infiltration was particularly evident around vessels and within the graft interstitium (Fig. 3, C vs D). Furthermore, vessels in allografts from NRS-treated recipients had signs of endothelial degeneration at day 7 after transplant that were absent in allografts from the KC antiserum-treated recipients. To compare T cell infiltration into allografts from control and KC antiserum-treated recipients, graft tissue sections were also prepared and stained with anti-CD8 or anti-CD4 mAb. KC antiserum treatment resulted in a clear decrease in CD8⫹ and CD4⫹ T cell infiltration into the allograft interstitium (Figs. 4, A vs B, and 5). There was little to no T cell infiltration evident in sections prepared from isografts at day 7 after transplant (data not shown). Recent results from this laboratory have shown that expression of the T cell chemoattractants IP-10 and Mig in cardiac allografts EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS The Journal of Immunology in leukocyte recruitment to peripheral tissues, few studies have tested the induction and role of chemokines during acute rejection of solid organ allografts. The results of the current report indicate coordinated cascades of chemokine gene expression in transplanted cardiac grafts within hours after graft revascularization. Northern blot analysis of RNA isolated from cardiac grafts 1.5– 48 h after revascularization indicated no difference between isografts and allografts with respect to patterns and levels of chemokine expression. The equivalent expression in allografts and isografts suggests that the early cascades occur without the influence of adaptive immune mechanisms. The chemokines expressed in cardiac grafts during the early time periods following transplant are those directing the recruitment of neutrophils and macrophages during wound healing. Whether the expression of these early chemokines is induced directly by the ischemia and trauma of the transplant surgery or induced indirectly by other cytokines produced during the initial inflammation is unclear at this time. Several investigators have reported the rapid after transplant induction of TNF-␣ in allo- and isografts (26, 27). Because TNF-␣ also induces expression and FIGURE 6. Expression of T cell chemoattractants in allografts at day 2 after transplant. KC antiserum or NRS was given, 200 l i.v., to groups of four C57BL/6 recipient mice within 30 min after A/J cardiac allograft reperfusion. At day 2 after transplant, allografts were retrieved, and RNA was prepared and analyzed for levels of IP-10 and Mig RNA by Northern blot hybridization. Intensity of intra-allograft IP-10 and Mig RNA signals was plotted as a ratio to the GAPDH signal, and the mean ratio ⫾ SD of the five grafts in each group is shown. ⴱ, p ⬍ 0.04. FIGURE 7. Depletion of recipient neutrophils extends cardiac allograft survival. C57BL/6 mice were given 100 mg RB6.8C or control rat IgG on two consecutive days. On the second day of treatment, the treated mice received A/J heart allografts. Survival of heart allografts in neutrophildepleted C57BL/6 recipients (n ⫽ 5) was compared with survival of allografts in control rat IgG-treated recipients (n ⫽ 8). Allograft rejection in all recipient mice was visually confirmed by laparotomy. Isografts from recipients treated with RB6.8C5 were maintained ⬎100 days. Recipient treatment with RB6.8C5 significantly prolonged heart allograft survival (p ⫽ 0.001). production of IL-8 and MIP-1␣ (28, 29), this cytokine may initiate the early chemokine cascades observed in the current studies. The results of the current report clearly indicate that induction of early inflammatory events in the cardiac grafts influences the progression of downstream events involving adaptive immune responses to the allograft. Recent results from this laboratory have demonstrated that Mig and IP-10 expression is observed as early as day 2 after transplant in cardiac allografts but not in isografts (18), ⬃1 day after intragraft expression of KC has subsided. Treatment of recipients with KC antiserum at the time of transplantation decreased intraallograft expression of the T cell chemoattractants IP-10 and Mig. Ab depletion studies and the use of recipients deficient in CD4⫹ vs CD8⫹ T cells have indicated that day 2 expression of IP-10 and Mig is mediated by IFN-␥-producing CD8⫹ T cells (18). On the basis of these results, we have proposed that circulating CD8⫹ T cells interact with inflammatory sites in the vascular endothelium and those T cells with allogeneic class I MHC specificity are stimulated to produce IFN-␥ that in turn stimulates endothelial cell production of Mig and IP-10. The importance of Mig production in the rejection of the heart allografts has been shown in our laboratory by the ability of Mig-specific Abs to inhibit T cell infiltration into the cardiac allograft and prolong survival from day 8 –9 to day 18 –20 after transplant.4 In light of these results, the effects of KC antagonism on T cell graft infiltration and survival observed in the current report may be indirectly mediated through decreases in Mig. The early chemokine cascades in cardiac grafts may be coordinated so that optimal induction of each cascade is dependent on induction of the preceding chemokine cascade. The mechanism by which treatment with the KC antiserum attenuates the expression of IP-10 and Mig and T cell infiltration into the graft remains unclear at this time. The chemoattractive properties of KC for neutrophils is suggestive of a potential role of these cells in initiating downstream events in the graft rejection process. Alternatively, recipient treatment with KC antiserum may inhibit a critical neutrophil-mediated activity without affecting M. Miura. Monokine induced by IFN-␥ is a dominant factor directing T cells into murine cardiac allografts during acute rejection. Submitted for publication. 4 Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 FIGURE 5. CD4⫹ and CD8⫹ T cell infiltration into allogeneic cardiac grafts from KC antiserum- and NRS-treated recipients. Cardiac allografts harvested from KC antiserum- and NRS-treated recipients were used to prepare frozen sections that were stained with anti-CD4 or anti-CD8 mAb for immunohistochemistry. The slides were viewed at ⫻200 magnification, and the number of positively staining cells in eight random fields from three different sections from three different grafts was counted. Each circle represents the number of positive cells per random field for the indicated stain, with the horizontal bar indicating the mean number of positive cells per field. Treatment with the KC antiserum significantly (ⴱ, p ⬍ 0.0001) inhibited infiltration of both CD4⫹ and CD8⫹ T cells into the allograft. 2983 2984 References 1. Hosenpud, J. D., L. E. Bennett, B. E. Keck, B. Fiol, M. M. Boucek, and R. J. Novick. 1998. The registry of the international society for heart and lung transplantation: fifteenth official report, 1998. J. Heart Lung Transplant. 17:656. 2. Billingham, M. 1995. 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Hechtman. 2000. Neutrophil mediated remote organ injury after lower torso ischemia and reperfusion is selectin and complement dependent. J. Trauma 48: 32. 33. Kasama, T., R. M. Strieter, T. J. Standiford, M. D. Burdick, and S. L. Kunkel. 1993. Expression and regulation of human neutrophil-derived macrophage inflammatory protein-1. J. Exp. Med. 178:63. 34. Taub, D. D., M. Anver, J. J. Oppenheim, D. L. Longo, and W. J. Murphy. 1996. T lymphocyte recruitment by interleukin-8 (IL-8): IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J. Clin. Invest. 97:1931. Downloaded from http://www.jimmunol.org/ by guest on August 9, 2017 neutrophil infiltration into the allograft. Further evidence for a neutrophil role in acute rejection of the heart allografts is indicated by the ability of recipient neutrophil depletion to extend heart allograft survival. Although Ab-mediated neutrophil depletion results in ⬃50% mortality of the heart allograft recipients from decreased resistance to infections, rejection of allografts in the surviving recipients is delayed until 18 –21 days after transplant, which is similar to that observed in the KC antiserum-treated recipients. Such observations support a role for neutrophils in the establishment of inflammatory foci in the allograft that facilitate T cell recruitment and graft infiltration. Several laboratories have documented the critical role of neutrophils in ischemia/reperufsion injury (30 –32). Antagonism of neutrophil adhesion to the endothelium effectively attenuates this injury in many models. Recent results from this laboratory have indicated marked inhibition of neutrophil infiltration into renal tissue and decreased tissue pathology by treating mice with KC-specific antiserum following reperfusion of kidneys subjected to 1 h of warm ischemia (M. Miura, manuscript in preparation). In addition to their role in ischemia/reperfusion injury, activation of neutrophils with solid-phase IL-8 stimulates neutrophils to produce or release many different chemoattractants for T cells (33, 34). Such events could certainly play a role in recruiting and/or focusing T cells to the sites of inflammation in the vascular endothelium of cardiac allografts during progression of acute allograft rejection. In summary, the results in the current report have indicated the induction of a programmed cascade of chemokines in both isografts and allografts at early times following the transplantation surgery. Treatment of recipients with Abs to one of the first components in these cascades attenuates downstream events including the expression of chemokines with chemoattractive properties for Ag-primed T cells and cellular infiltration into the graft. These data are the first to expose the induction of such cascades in transplanted organ allografts. Furthermore, these data indicate the profound effects of innate immune components on alloantigen-specific rejection responses and suggest that strategies aimed at antagonizing early inflammatory events in heart allografts will have substantial benefit in attenuating acute rejection of cardiac allografts. EARLY CHEMOKINES IN CARDIAC ALLOGRAFTS