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Enhanced expression of cytokine-induced neutrophil chemoattractant in rat hepatic allografts during acute rejection
Enhanced Expression of Cytokine-Induced Neutrophil Chemoattractant in Rat Hepatic Allografts During Acute Rejection YASUO YAMAGUCHI,1 OSAMU ICHIGUCHI,1 FUJIO MATSUMURA,1 EIJI AKIZUKI,1 TEISHI MATSUDA,1 KAZUTOSHI OKABE,1 SHINWA YAMADA,2 JIAN LIANG,1 KATSUTAKA MORI,1 AND MICHIO OGAWA1 The kinetics of messenger RNA (mRNA) and protein levels of cytokine-induced neutrophil chemoattractant (CINC) in rat hepatic allografts during acute rejection were investigated. Infiltrating cells were identified by double immunostaining with anti-CINC and anti-macrophage monoclonal antibodies, ED1 and ED2. The serum CINC concentration in untreated hepatic allograft recipients increased significantly at a constant rate over time after transplantation. No significant increases in serum CINC concentrations were observed in hepatic isografts or allografts treated with the immunosuppressant FK506. The number of neutrophils in untreated hepatic allografts increased significantly at a constant rate. Conversely, neutrophil accumulation in isografts or allografts treated with FK506 was much less than in untreated hepaticallografts. Immunostaining revealed that in the portal areas, mononuclear cells infiltrating untreated allograft liver were mainly positive for CINC and that CINC/ cells represented a subpopulation (Ç25%) of the ED1/ cells. On the other hand, in the sinusoidal areas CINC/ cells were scattered and mainly positive for ED2. Levels of CINC mRNA in liver tissues taken from untreated hepatic allografts increased after transplantation, peaked on day 5, and decreased thereafter. Hepatic allografts treated with FK506 or isografts showed much lower levels of CINC mRNA than untreated allografts. Allogeneic mixed lymphocyte reactions induced CINC production. The cellular source of CINC was mononuclear cells. CINC production in mixed lymphocyte reactions was inhibited in the presence of anti-tumor necrosis factor a (TNF-a) antibody. These results suggest that enhanced expression of CINC mRNA and prominent accumulation of neutrophils in the liver grafts are characteristic features of the immune response during acute rejection. (HEPATOLOGY 1997;26:15461552.) Neutrophil has long been regarded merely as a terminally differentiated cell capable of protein synthesis, and fulfilling only a passive effector role in inflammation via phagocytosis and the release of preformed enzymes and cytotoxic com-
Abbreviations: IL-1, interleukin-1; TNF-a, tumor necrosis factor a; IL-6, interleukin-6; IL-8, interleukin-8; CINC, cytokine-induced neutrophil chemoattractant; MLR, mixed lymphocyte reaction; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell. From the 1Department of Surgery II, Kumamoto University Medical School, Kumamoto; and the 2First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyusyu, Japan. Received March 19, 1997; accepted August 19, 1997. Address reprint requests to: Michio Ogawa, M.D., Department of Surgery II, Kumamoto University Medical School, Honjo 1-1-1, Kumamoto, 860, Japan. Fax: 81-96371-4378. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2606-0024$3.00/0
pounds.1 Thus, neutrophils were thought to be relevant only to the efferent limb of the immune response. However, it has been shown that neutrophils contribute significantly to the afferent or inductive limb of the immune response by modulating both cellular and humoral immunity, particularly via the synthesis and release of immunoregulatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor a (TNFa), and interleukin-6 (IL-6).2 Hence, neutrophils modulate both T- and B-cell activities in the evolution of an immune response. Neutrophils have been implicated as mediators of tissue damage in a variety of diseases.3,4 However, the role of neutrophils in organ allograft rejection is not well understood. Tissue injury may occur in the process of organ transplantation secondary to ischemia/reperfusion, rejection, and drug toxicity. Neutrophils may contribute to such injuries. In fact, ischemia/reperfusion generates mediators that include free radicals, and causes protease activation. In addition, many of the pathological changes noted after ischemia/reperfusion injury are thought to be induced by infiltrating neutrophils. Jaeschke et al.5 and Poggetti et al.6 have shown the importance of neutrophils in the development of schemia/reperfusion injury. Accumulation of neutrophils is mediated by local chemotactic agents. Increased production and release of these substances regulate neutrophil migration from the vascular compartment to the tissues. A tissue-derived neutrophil-activating peptide (NAP-1), which is identical to interleukin8 (IL-8), has been cloned.7 The effects of IL-8 on human neutrophil functions include enhanced chemotaxis, enzyme release, expression of surface adhesion molecules, and induction of the respiratory burst.7 No substance homologous to IL-8 has been identified in the rat. However, potent neutrophil chemotactic activity has been detected in rat inflammatory exudate induced by lipopolysaccharide, TNF-a, or IL-1.8-11 Cytokine-induced neutrophil chemoattractant (CINC) is an 8-kd polypeptide originally identified in conditioned media from IL-1 b-stimulated rat glomerular epithelial cells and subsequently purified from NRK52E rat epithelioid cells.8 CINC consists of 72 amino acids and has homology to human peptides with gro/melanoma growth stimulatory activities, indicating that rat CINC belongs to the IL-8 superfamily.9,10 Thus, the regulation of CINC and/or IL-8 production is a critical element in the control of inflammatory reactions associated with neutrophil infiltration. The mixed lymphocyte reaction (MLR) is an in vitro model of alloantigen-driven immunity and has provided significant insights into immune mechanisms. Therefore, the present study was undertaken to investigate the kinetics of CINC messenger RNA (mRNA) and protein levels in a rat model
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of hepatic transplantation in vivo and the allogeneic mixed lymphocyte reaction in vitro. MATERIALS AND METHODS Animals. Male LEW(RT1l) rats were used as recipients and ACI (RT1a) rats as donors. These rats were obtained from the Central Institute for Experimental Animals, Kawasaki, Japan. This strain combination is fully allogeneic and results in acute rejection of liver transplants.12 A LEW-to-LEW combination was used for control isografts. All animals were maintained under standard conditions and received water and rodent chow ad libitum. The animals weighed 225 to 250 g. Hepatic Transplantation. Orthotopic hepatic transplantation was performed using a cuff method. A simplified cuff was used for the portal and infrarenal caval anastomoses and a Teflon tube stent was used for the biliary anastomosis.13 Anesthesia during the operative procedure and postoperative care were consistent with the National Institutes of Health guidelines for the care and use of laboratory animals.14 Experimental Protocol. The experimental animals were divided into four groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with anti-CINC antibody; and group IV, allografts treated with FK506. Recipient animals were injected intraperitoneally with saline (0.5 mL), anti-CINC antibody (Peptide Institute, Osaka, Japan) (0.5 mg/kg body weight),15 or an immunoglobulin G control antibody (0.5 mg/kg body weight) on days 3, 4, 5, 6, and 7 after transplantation. FK506 was kindly provided by Fujisawa Pharmaceutical Inc., Osaka, Japan. FK506 was administered intramuscularly at a dose of 2 mg/kg/d immediately after transplantation during each experiment. Blood Chemistry. It is often difficult to obtain blood frequently from recipient rats following transplantation. Blood loss also may influence liver function and survival rate. Therefore, animals were killed to obtain blood 7 days after the transplantation, to determine serum concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP). The measurements were performed using the sequential multiple analyzer with a computer system (Technicon, Bad Vilbel, Austria). Measurement of cytokine-induced neutrophil chemoattractant activity A sensitive enzyme-linked immunosorbent assay (ELISA) for measuring rat CINC concentrations, using a biotin-conjugated polyclonal rabbit anti-CINC immunoglobulin G, has previously been described.16 The biotin-streptavidin sandwich enzyme-linked immunosorbent assay is linear for CINC concentrations ranging from 3 pg/mL to 30 ng/mL. Cytochemical Identification of Neutrophils Infiltrating Liver Grafts. A cytochemical method assaying activity of chloroacetate esterase has been reported to be a marker for neutrophils.17 Liver specimens were harvested at various time points following transplantation. Three different sections were stained with chloroacetate esterase from the liver specimens of each group and were photographed at a magnification of 1001 to enable visualization of the central and portal veins. The number of neutrophils that had infiltrated the liver graft were measured and represented as the number per square millimeter. Immunohistochemical Staining. ACI-to-LEW hepatic transplantation recipients that had or had not been pretreated with FK506 were killed at various times after transplantation. The LEW-to-LEW syngeneic hepatic transplants were used as controls. Four hepatic transplantation recipients were killed at each time point. The grafted liver was excised and frozen rapidly in liquid nitrogen. Cryosections were prepared and air-dried overnight. Following fixation in pure acetone for 10 minutes at room temperature, cryosections were stained with anti-CINC polyclonal antibody (Peptide Institute, Inc., Osaka, Japan) and were visualized by an indirect immuno-alkaline phosphatase method.18 Control experiments included 1) incubation of the sections with 10 mg/mL of nonimmune mouse immunoglobulin G (Sigma Chemical Co., St. Louis, MO) as the primary antibody;
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and 2) omission of the primary antibody, and 3) incubation of sections in Fast Red TR (Sigma, St. Louis, MO) medium only. Monoclonal Antibodies. The identity of the macrophage subpopulations in the liver grafts was determined using anti-rat macrophage (ED1 or ED2) monoclonal antibodies.19 ED1 reacts with macrophages, monocytes, and dendritic cells, but not granulocytes. Resident macrophages are positive for ED2, while monocytes, dendritic cells, and granulocytes are negative. Double Immunostaining. To identify CINC-positive cells of monocyte/macrophage lineage, double immunostaining was performed with anti-rat CINC polyclonal antibody and anti-macrophage (ED1 or ED2) monoclonal antibodies according to a method described previously.18 In brief, cryosections were first incubated with antiCINC polyclonal antibody and stained red with Fast Red TR as a substrate by indirect immuno-alkaline phosphatase staining. The specimen then was incubated with ED1 or ED2 and stained black with 4-chloro-1-naphthol as substrate by indirect immuno-peroxidase staining.18 The sections were lightly counterstained with hematoxylin. Immunostained specimens were examined by light microscopy (Microphot FX, Nikon, Tokyo, Japan) and photographed with a band pass filter (BPN45, Fuji, Tokyo, Japan) to reduce the intensity of the hematoxylin staining. RNA Isolation and Northern Blot Analysis. Liver specimens were obtained from transplantation recipients killed at various times after transplantation, quick frozen in liquid nitrogen, and stored at 0707C before RNA extraction. Total RNA was prepared according to Chomczynski et al.20 Twenty micrograms of RNA was subjected to electrophoresis on a 1% agarose-formaldehyde denaturing gel in 11 N-morpholino-propanesulfonic acid buffer containing 200 mmol/LM N-morpholinopropanesulfonic acid, 50 mmol/L sodium acetate, and 10 mmol/L ethylenediaminetetraacetic acid, pH 7.0. Following transfer of RNA to nylon filters in 201 SSC, blots were hybridized with 32P-labeled CINC DNA probes21 at 427C in 50% formamide, 0.2% polyvinyl-pyrrolidine, 0.2% bovine serum albumin, 0.2% Ficoll, 0.05 mol/L TRIS-HCl, 1.0 mol/L NaCl, 0.1% sodium pyrophosphate, 1.0% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and 100 mg/mL of salmon sperm DNA. After hybridization, filters were washed in the same buffer using deionized, distilled, and diethyl pyrocarbonate-treated water. The hybridization signal was assessed by radioisotope counting and autoradiography. PBMC Isolation. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described.22 Briefly, heparinized blood was obtained by aortic puncture from naive ACI and LEW rats. Blood samples were diluted 1:1 with pyrogen-free saline. PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque and washed three times. The mononuclear cells were a mixture of lymphocytes and monocytes, 74% { 5.5% and 26% { 7.2%, respectively, as determined by morphology. The viability of PBMC preparations was greater than 95% as determined by trypan blue exclusion. These cells were then resuspended in culture medium (RPMI-1640, HEPES, 100 mmol/L L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal calf serum). Mixed Lymphocyte Response. The mixed lymphocyte reaction was performed with a slight modification as previously described.23 In brief, PBMCs were divided and designated as stimulator (ACI) or responder (LEW) cells. Stimulator cells were incubated with Mitomycin C (MMC). In experiments assaying CINC production, stimulator (5 1 105) and responder (5 1 105) cells were mixed in each well of 24-well tissue culture plates (Costar, Cambridge, MA). In each experiment, MMC-treated PBMCs from LEW rats were used as autologous controls. Cell aliquots were also exposed to FK506 (50 ng/mL). All cultures were performed in triplicate and incubated in a humidified atmosphere containing 5% CO2 at 377C. The supernatant was sampled at each time point and CINC concentrations were determined by enzyme-linked immunosorbent assay. In neutralization experiments, mixed lymphocyte reactions were performed in the presence of varying dilutions of neutralizing rabbit anti-human TNF-a sera (100 ng/mL) (Genzyme Corporation, Cam-
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HEPATOLOGY December 1997 TABLE 1. Rat Hepatic Transplant Survival
Donor-to-Recipient
Treatment
— Anti-CINC AB* FK506† —
ACI-to-LEW LEW-to-LEW
Survival (d)
9, 9, 10, 10, 10, 10, 10, 11, 11 15, 15, 16, 17, 17, 17, 17, 18 ú60, ú60, ú60, ú60, ú60, ú60, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100
Mean { SD
P‡
10.1 { 0.7 16.6 { 0.3 ú60 ú100
— .001 õ.001 —
* Anti-CINC antibody was administered intraperitoneally at a dose of 0.5 mg/kg on days 3, 4, 5, 6, and 7 after transplantation. † FK506 was administered intramuscularly at a dose of 2 mg/kg/d. ‡ P values were obtained using the Student’s t test.
bridge, MA). For CINC mRNA determination and immunohistochemical localization of CINC, stimulator (3 1 106) and responder (3 1 106) cells were mixed in 35-mm culture plates (Costar) and cultured for 24 hours. RESULTS
Transplant survival is summarized in Table 1. Hepatic allografts from ACI to LEW rats survived 10.1 { 0.7 days. Treatment with peritoneal administration of anti-CINC antibody prolonged significantly the survival of hepatic allografts (16.6 { 0.3 days) compared with untreated allografts. In addition, treatment with FK506 significantly prolonged the survival of ACI livers in LEW recipients, to ú60 days. The LEW-toLEW isografts survived indefinitely (Table 1). Serum concentrations of aspartate transaminase, alanine transaminase, and alkaline phosphatase in untreated hepatic allograft recipients 7 days after transplantation were significantly increased compared with those in naive controls. In contrast, treatment with anti-CINC antibody attenuated the increment of liver enzymes 7 days after hepatic allografting (Table 2). The serum CINC concentrations in untreated hepatic allograft recipients increased significantly at a constant rate over time after transplantation. In contrast, no significant increases in serum CINC concentrations were observed with the isografts. The hepatic allograft recipients treated with FK506 maintained lower serum CINC concentrations throughout the experiment (Fig. 1A). The number of neutrophils was increased in the untreated hepatic allografts and rose significantly at a constant rate over time during acute rejection. In contrast, no significant increase in the number of neutrophils was observed in hepatic isografts. Treatment with FK506 resulted in lower numbers of neutrophils in the liver grafts than in untreated hepatic allografts (Fig. 1B). Immunostaining revealed that a number of infiltrating mononuclear cells in the portal areas of untreated liver allografts on day 5 were stained with an anti-CINC antibody. In addition, sinusoidal lining cells were positive for CINC (Fig.
2). On the other hand, a few cells in the portal and sinusoidal areas were positive for CINC in hepatic isografts and allografts treated with FK506 (data not shown). The localization of macrophage subpopulations infiltrating liver grafts was determined by immunostaining with anti-rat macrophage (ED1 or ED2) monoclonal antibodies. ED1/ cells gradually increased on days 5 and 7 in untreated hepatic allografts. The number of ED1/ cells in the hepatic isografts was sightly increased on day 3, but decreased thereafter. Treatment with FK506 greatly decreased the number of ED1/ cells infiltrating hepatic allografts, compared with the untreated hepatic allografts. These findings suggest that a progressive relative increase in the number of ED1/ cells was a characteristic feature of ongoing first-time rejection in the rat hepatic allografts (Fig. 3A). The number of ED2/ cells in untreated allografts increased after transplantation, peaked on day 5, and decreased thereafter. ED2/ cells in the hepatic isografts were slightly increased on days 5 and 7, but the number of these cells was much lower than in untreated allografts. There was no significant increase in the number of ED2/ cells in the hepatic allografts treated with FK506 (Fig. 3B). Double immunostaining revealed that in the untreated hepatic allografts CINC/ED1/ cells were distributed in the portal areas, whereas CINC/ED2/ cells were found in the sinusoidal areas. The number of CINC/ED1/ cells increased over time during rejection. CINC/ cells represented a subpopulation (Ç25%) of the ED1/ cells on day 7 in the untreated hepatic allografts (Fig. 4A). CINC/ED2/ cells were found in the sinusoidal areas (Fig. 4B). The level of CINC mRNA in the liver specimens peaked on day 5 in the untreated hepatic allografts. In contrast, hepatic allografts treated with FK506 showed lower levels of CINC mRNA compared with untreated allograft recipients (Fig. 5). The kinetics of CINC production during MLR are shown in Fig. 6. The CINC concentrations in the supernatant started to increase 48 hours after incubation and remained persistently elevated over time in the allogeneic MLR. No signifi-
TABLE 2. Serum Concentrations of AST, ALT, and ALP 7 Days After Transplantation Treatment
AST
ALT
54.6 { 13.8 G 427.8 { 56.7 * * 204.1 { 28.9 G†
Naive controls Untreated Anti-CINC Ab-treated
24.6 { 2.5 G 221.9 { 26.1 * * 153.6 { 31.8 G†
ALP
401.3 { 31.4 G 974.2 { 40.9 * * 598.1 { 44.3 G†
NOTE. Data are represented as the mean { SE. Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; anti-CINC Ab, anti-cytokine–induced neutrophil chemoattractant. * P õ .01. † P õ .05.
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FIG. 1. (A) Serum concentrations of CINC following rat hepatic transplantation. (B) Changes in the number of neutrophils infiltrating liver grafts after transplantation. * P õ .01; ** P õ .05; NS, not significant. Serum CINC concentrations and neutrophil accumulation in untreated hepatic allografts rose significantly at a constant rate over time during acute rejection.
cant increase in CINC production was observed in the MLR treated with FK506 or in autologous controls. Immunostaining revealed that the predominant cellular source of CINC expression in an allogeneic MLR was the mononuclear cells (data not shown). The level of CINC mRNA in an allogeneic MLR 48 hours after incubation was significantly increased compared with that observed in an allogeneic MLR treated with FK506 or in autologous controls (data not shown). To determine the role of TNF-a in the induction of CINC, the mixed lymphocyte reactions were performed in the presence of either rabbit anti-human TNF-a sera or control preimmune rabbit sera. Supernatants were sampled 96 hours after incubation and analyzed for CINC production. Anti– TNF-a antibody significantly attenuated the production of CINC in the MLR (Fig. 7). DISCUSSION
The present study has shown that the expression of CINC is significantly enhanced at the mRNA and protein levels
FIG. 3. (A) Changes in the number of ED1-positive cells accumulating in liver grafts following transplantation. * P õ .01; ** P õ .05; NS, not significant. (B) Changes in the number of ED2-positive cells infiltrating liver grafts after transplantation. * P õ .01; ** P õ .05; NS, not significant.
during acute rejection in rat hepatic transplantation. This elevated expression of CINC is associated with prominent neutrophil localization to untreated hepatic allografts during rejection. Recent studies have shown similar very early chemokine induction in human liver transplantation.24 In contrast, this induction of CINC was not observed in isografts or allografts treated with FK506. These results indicate that this inducible mediator contributes to acute rejection, suggesting the importance of neutrophil recruitment in organ transplantation. Double immunostaining revealed that CINC can be produced by cells of the monocyte/macrophage lineage. In addition, CINC/ cells represented a subpopulation of ED1/ cells. Some ED2/ resident macrophages, probably Kupffer cells, were positive for CINC. This suggests that at least some monocyte/macrophages localizing to the hepatic allografts were ‘‘activated’’ in the sense that they expressed CINC, an inducible gene product. A rejection episode begins with a prominent infiltration of cells into a liver graft during an early time period, before cellular immunity is induced. The infiltrating cells consist mainly of macrophages and lymphoid cells. Other infiltrating
FIG. 2. Immunohistochemical staining. Untreated allograft on day 7. (Magnification [A] 1100; and [B] 1200.) CINC/ cell: red. Most mononuclear cells in the portal and sinusoidal areas of untreated allografts were positive for CINC.
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FIG. 4. Changes in the number of (A) ED1/CINC/ and (B) ED2/CINC/ cells infiltrating liver grafts after transplantation. CINC/ cells represented a subpopulation (approximately 25%) of the ED1/ cells in untreated hepatic allografts.
cells, including neutrophils, basophils, and plasma cells, also increase at a constant rate.25 Histological examination of human liver graft biopsies taken during acute rejection reveals a mixed inflammatory infiltrate localized to small bile ducts and venous endothelium in portal tracts. In addition, the infiltrate consists of mononuclear cells and large numbers of neutrophils infiltrating the biliary epithelium.26,27 Furthermore, it has been reported that bile taken from patients with acute rejection contains chemoattractants that may be responsible for the recruitment of neutrophils.28 These observations suggest an important role for neutrophils in the immune response occurring during acute rejection. The present study also showed an increased number of neutrophils in the untreated hepatic allografts during acute rejection. Neutrophils have been implicated as mediators of tissue damage in a variety of diseases including emphysema and rheumatoid arthritis,3,4 but their role in allograft damage during rejection is unclear. Adherence of circulating leukocytes to endothelium may be a common early event in both ischemia/reperfusion injury and immune graft rejection. The vascular biology of early posttransplantation rejection may be complicated by the overlap between ischemia/reperfusion and immune phenomena. Adams et al.29 showed that neutrophil-mediated mechanisms may be important contributory factors in the tissue damage of acute rejection. After recruitment to the site of rejection by chemotactic factors, the activated neutrophils may spontaneously release proteolytic enzymes or be stimulated further by locally active cytokines.
FIG. 5. Northern blot analysis. A representative blot shows that expression of CINC mRNA in a liver specimen from an untreated allograft peaks on day 5 after transplantation and decreases thereafter.
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FIG. 6. CINC production during mixed lymphocyte reaction. * P õ .01; ** P õ .05 (vs. autologous or FK506-treated allogeneic MLR). CINC were produced in the allogeneic MLR but not in the FK506-treated allogeneic MLR. (l) Allogeneic MLR; (h) autologous MLR; and (s) allogeneic MLR treated with FK506.
Digestion of extracellular protein by neutrophil elastase may be important, not only in causing tissue damage, but also in allowing the movement of neutrophils from blood to inflamed tissues.30,31 Neutrophils are recruited to a graft by locally active chemotactic factors. A previous study showed that culture supernatants of lymphocytes isolated during episodes of acute rejection were both chemotactic for and capable of increasing superoxide production by neutrophils.29 Acute cellular rejection is associated with intragraft increases in the expression of several mRNAs. Kirk et al.32 reported that increased IL-8 mRNA in the human renal grafts by needle core biopsy was
FIG. 7. The effect of anti-TNF-a antibody on CINC production in an allogeneic mixed lymphocyte reaction 96 hours after incubation. Anti-TNFa antibody attenuated significantly the production of CINC in the MLR. *P õ .01. (j) Allogeneic MLR; and ( ) allogeneic MLR / anti–TNF-a antibody.
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correlated with irreversible loss of graft function. Similarly, the present study has shown that enhanced gene expression and production of CINC is associated with accelerated rejection reactions following rat hepatic allografting. The C-X-C chemokines mediate neutrophil chemotaxis and activation in vitro and are implicated in neutrophil influx to acute inflammatory sites in vivo.33,34 IL-8 stimulates upregulation of b2 integrins in vitro, enhancing adhesion to resting endothelial cells.35 In this regard, there are few in vivo data showing whether CINC may influence neutrophil functions involving adhesion and secretagogue activation in microcirculation. However, it has been reported that CINC is a novel stimulator that evokes not only locomotive but also secretagogue activation of neutrophils via a CD18-dependent mechanism in vivo.36 Subsequently, Frevert et al.37 reported that recombinant rat chemokine KC induces neutrophil chemotaxis and increases the expression of CD11b/CD18 on neutrophils. Recombinant KC also induces the respiratory burst in neutrophils. These biological activities suggest that elevated serum concentrations of CINC during acute rejection may modulate and further facilitate neutrophil activation and tissue injury. Neutrophils are also thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. Neutrophil adherence to endothelium can be significantly enhanced by the endothelial deposition of iC3b.38 Wieder et al.39 has reported that the immunosuppressive agent rapamycin prevents accelerated rejection of cardiac allografts in sensitized rats. This effect is associated with markedly reduced expression of a gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, operational rat homologues of the human intercrine-a cytokines that have proinflammatory IL-8–like neutrophil activation/chemotactic properties. The present study also showed that FK506 significantly decreased CINC expression and neutrophil accumulation in untreated hepatic allografts. Cell-mediated reactions are well known to be essential in the rejection of allogeneic transplanted organs. In the present study we have elucidated important aspects of CINC production in vitro, using a mixed lymphocyte reaction as well as in vivo studies. Delayed-type hypersensitivity and allograft rejection are dependent upon the generation of antigen-specific T-cell immune response. The mixed lymphocyte reaction is a model of alloantigen-driven immunity and has provided significant insight into mechanisms of T-cell proliferation. Larsen et al.40 showed that IL-8 plays an important pathophysiological role in the development of a delayed-type hypersensitivity reaction. In our study, allogeneic MLRs showed that CINC concentrations increased as early as day 2 and persisted over 4 days. Northern blot analysis confirmed early induction of CINC mRNA in the allogeneic response. Staining of cells recovered from allogeneic cultures showed that mononuclear cells are the predominant source for CINC production. This indicates that cells of the monocyte/macrophage lineage are capable of producing CINC under appropriate stimulation. It has been reported that serum TNF-a levels are increased during allograft rejection,41,42 and immunotherapy with anti– TNF-a antibody prolongs graft survival.43 These observations suggest that TNF-a may be involved in the pathogene-
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sis of acute rejection. Previous studies have shown the importance of TNF-a in the mixed lymphocyte reaction.44,45 Christensen et al.23 showed that MCP-1 and IL-8 are produced during the evolution of a mixed lymphocyte reaction and that their induction is TNF-a –dependent. CINC production can be induced by several inflammatory stimuli, including IL-1b, lipopolysaccharide, and TNF-a.8-11 NF-kB is a family of proteins that regulate the transcription of many genes. Blackwell et al.46 have shown that CINC production is transcriptionally regulated by NF-kB. The promoter region of the CINC gene contains an NF-kB binding site.46,47 TNFa contains NF-kB binding motifs in the proximal promoter regions which appear to be important in regulating transcription. These observations suggest that TNF-a stimulate CINC production through NF-kB activation. The present study also showed that the presence of neutralizing anti-human TNFa antibodies resulted in significant abrogation of CINC production in the MLR. In conclusion, enhanced CINC expression in association with neutrophil accumulation is an important mediator of acute rejection in rat hepatic transplantation. REFERENCES 1. Bainton DF, Ullyot JL, Farquhar MG. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med 1971;134:907-934. 2. Lloyd AR, Oppenheim JJ. Poly’s lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunol Today 1992;13:169-172. 3. Burnett D, Chamba A, Hill SL, Stockley RA. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet 1987;2:1043-1046. 4. Malech HL, Gallin JI. Current concepts: immunology. Neutrophils in human diseases. N Engl J Med 1987;317:687-694. 5. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/ reperfusion injury in rat liver in vivo. FASEB J 1990;4:3355-3359. 6. Poggetti RS, Moore FA, Moore EE, Bensard DD, Anderson BO, Benerjee A. Liver injury is a reversible neutrophil-mediated event following gut ischemia. Arch Surg 1992;127:175-179. 7. Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating peptide 1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 1989;84:1045-1049. 8. Watanabe K, Kinoshita S, Nakagawa H. Purification and characterization of cytokine-induced neutrophil chemoattractant produced by epithelial cell line of normal rat kidney (NRK-52E cell). Biochem Biophys Res Commun 1989;161:1093-1099. 9. Watanabe K, Suematsu M, Iida M, Takaishi K, Iizuka Y, Suzuki H, Suzuki M, et al. Effect of rat CINC/gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery. Exp Mol Pathol 1992;56:60-69. 10. Watanabe K, Konishi K, Fujioka M, Kinoshita S, Nakagawa H. The neutrophil chemoattractant produced by the rat kidney epithelial cell line NRK-52E is a protein related to the KC/gro protein. J Biol Chem 1989;264:19559-19563. 11. Watanabe K, Iida M, Takaishi K, Suzuki T, Hamada Y, Iizuka T, Turufuji S. Chemoattractants for neutrophils in lipopolysaccharide-induced inflammatory exudate from rats are not interleukin-8 counterparts but gro-gene-product/melanoma-growth-stimulating-activity-related factors. Eur J Biochem 1993;214:267-270. 12. Yamaguchi Y, Harland RC, Wyble C, Bollinger RR. The role of class I major histocompatibility complex antigens in prolonging the survival of hepatic allografts in the rat. Transplantation 1989;46:360-363. 13. Yamaguchi Y, Kikuchi N, Miyanari N, Ichiguchi O, Goto M, Mori K, Ogawa M. A technique for orthotopic reduced-size hepatic transplantation combined with ex vivo liver cut down in the rat. Dig Dis Sci 1996; 41:1713-1721. 14. National Institutes of Health. Guide for the care and use of laboratory animals. Public Health Service, NIH publication No. 86-23, Bethesda, MD, 1985. 15. Hisama N, Yamaguchi Y, Ishiko T, Miyanari N, Ichiguchi O, Goto M, Mori K, et al. Kupffer cell production of cytokine-induced neutrophil
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16.
17. 18.
19.
20. 21. 22.
23.
24.
25.
26. 27.
28. 29. 30. 31.
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chemoattractant (CINC) following ischemia/reperfusion injury in rats. HEPATOLOGY 1996;24:1193-1198. Nakagawa H, Ikesue A, Kato H, Debuchi H, Watanabe K, Tsurufuji S, Naganawa M, Mitamura M. Changes in the levels of rat interleukin 8/ CINC and gelatinase in the exudate of carrageenin-induced inflammation in rats. J Pharmacobio-Dynamics 1992;15:461-466. Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971;55:283-290. Matsuno K, Ezaki T, Kotani M. Splenic outer periarterial lymphoid sheath (PALS): an immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophage in the rat. Cell Tissue Res 1989;257:459-470. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2, and ED3. Immunology 1985;54:589-599. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;62:156-159. Konishi K, Takata Y, Yamamoto M, Yomogida K, Watanabe K, Tsurufuji S, Fujioka M. Structure of the gene encoding rat neutrophil chemoattractant Gro. Gene 1993;126:285-286. Strieter RM, Remick DG, Lynch JP, Genord M, Raiford C, Spengler R, Kunkel SL. Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis. Am J Respir Cell Mol Biol 1989;1:57-63. Christensen PJ, Rolfe MW, Standiford TJ, Burdick MD, Toews GB, Strieter RM. Characterization of the production of monocyte chemoattractant protein-1 and IL-8 in an allogeneic immune response. J Immunol 1993;151:1205-1213. Adams DH, Hubscher S, Fear J, Johnston J, Shaw S, Afford S. Hepatic expression of macrophage inflammatory protein-1 a and macrophage inflammatory protein-1 b after liver transplantation. Transplantation 1996;61:817-825. Yamamoto H, Kaneda K, Kamada N, Wake K. Morphometric analysis of infiltrating cells that adhere to the sinusoidal wall and migrate into the space of Disse in the orthotopically transplanted rat liver. Transplantation 1994;58:8-13. Snover DC, Freese DK, Sharp HL, Bloomer JR, Najarian JS, Ascher NL. Liver allograft rejection. An analysis of the use of biopsy in determining outcome of rejection. Am J Surg Pathol 1987;11:1-10. Demetris AJ, Lasky S, Van Thiel DH, Starzl TE, Dekker A. Pathology of hepatic transplantation: a review of 62 adult allograft recipients immunosuppressed with a cyclosporine/steroid regimen. Am J Pathol 1985;118:151-161. Adams DH, Burnett D, Stockley RA, Elias E. Patterns of leukocyte chemotaxis to bile after liver transplantation. Gastroenterology 1989;7:433438. Adams DH, Wang LF, Burnett D, Stockley RA, Neuberger JM. Neutrophil activation. An important cause of tissue damage during liver allograft rejection? Transplantation 1990;50:86-91. Campbell EJ, Senior RM, McDonald JA, Cox DL. Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro. J Clin Invest 1982;70:845-852. Smedly LA, Tonnesen MG, Sandhaus RA, Haslett C, Guthrie LA, Johnston RB Jr, Henson PM, et al. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 1986;77:1233-1243.
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32. Kirk AD, Bollinger RR, Finn OJ. Rapid, comprehensive analysis of human cytokine mRNA and its application to the study of acute renal allograft rejection. Hum Immunol 1995;43:113-128. 33. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines: CXC and CC chemokines. Adv Immunol 1994;55:97-179. 34. Miller MD, Krangel MS. Biology and biochemistry of the chemokines: A family of chemotactic and inflammatory cytokines. Crit Rev Immunol 1992;12:17-46. 35. Huber AR, Kunkel SL, Todd RF, Weiss SJ. Regulation of transendothelial neutrophil migration of endogenous interleukin-8. Science 1991;254: 99-102. 36. Suzuki H, Suematsu M, Miura S, Liu YY, Watanabe K, Miyasaka M, Tsurufuji S, Tsuchiya M. Rat CINC/gro: a novel mediator for locomotive and secretagogue activation of neutrophils in vivo. J Leukoc Biol 1994; 55:652-657. 37. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 1995;154:335-344. 38. Vercellotti GM, Platt JL, Bach FH, Dalmasso AP. Neutrophil adhesion to xenogeneic endothelium via iC3b. J Immunol 1991;146:730-734. 39. Wieder KJ, Hancock WW, Schmidbauer G, Corpier CL, Wieder I, Kobzik L, Strom TB, Kupiec-Weglinski JW. Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts. J Immunol 1993;151:1158-1166. 40. Larsen CG, Thomsen MK, Gesser B, Thomsen PD, Deleuran BW, Nowak J, Skodt V, Thomsen HK, Deleuran M, Thestrup-Pedersen K. The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody. J Immunol 1995;155:2151-2157. 41. Hoffmann MW, Wonigeit K, Steinhoff G, Herzbeck H, Flad HD, Pichlmayr R. Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection. Transplantation 1993; 55:329-335. 42. Pizarro TT, Malinowska K, Kovacs EJ, Clancy J Jr, Robinson JA, Piccinini LA. Induction of TNF alpha and TNF beta gene expression in rat cardiac transplants during allograft rejection. Transplantation 1993;56: 399-404. 43. Imanaga DK, Mills JM, Olthoff KM, Seu P, Dempsey RA, Hart J, Terasaki PI, et al. Anti-tumor necrosis factor antibody enhances allograft survival in rats. J Surg Res 1990;48:345-348. 44. Shalaby MR, Espevik T, Rice GC, Ammann AJ, Figari IS, Ranges GE, Palladino MJ. The involvement of human tumor necrosis factors-alpha and -beta in the mixed lymphocyte reaction. J Immunol 1988;141:499503. 45. Robinet E, Branellec D, Termijtelen AM, Blay JY, Gay F, Chouaib S. Evidence for tumor necrosis factor-alpha involvement in the optimal induction of class I allospecific cytotoxic T cells. J Immunol 1990;144: 4555-4561. 46. Blackwell TS, Holden EP, Blackwell TR, DeLarco JE, Christman JW. Cytokine-induced neutrophil chemoattractant mediates neutrophilic alveolitis in rats: association with nuclear factor kB activation. Am J Respir Cell Mol Biol 1994;11:464-472. 47. Ohtsuka T, Kubota A, Hirano T, Watanabe K, Yoshida H, Tsurufuji M, Iizuka Y, et al. Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kB activation. J Biol Chem 1996;271:1651-1659.
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Abbreviations: IL-1, interleukin-1; TNF-a, tumor necrosis factor a; IL-6, interleukin-6; IL-8, interleukin-8; CINC, cytokine-induced neutrophil chemoattractant; MLR, mixed lymphocyte reaction; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell. From the 1Department of Surgery II, Kumamoto University Medical School, Kumamoto; and the 2First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyusyu, Japan. Received March 19, 1997; accepted August 19, 1997. Address reprint requests to: Michio Ogawa, M.D., Department of Surgery II, Kumamoto University Medical School, Honjo 1-1-1, Kumamoto, 860, Japan. Fax: 81-96371-4378. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2606-0024$3.00/0
pounds.1 Thus, neutrophils were thought to be relevant only to the efferent limb of the immune response. However, it has been shown that neutrophils contribute significantly to the afferent or inductive limb of the immune response by modulating both cellular and humoral immunity, particularly via the synthesis and release of immunoregulatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor a (TNFa), and interleukin-6 (IL-6).2 Hence, neutrophils modulate both T- and B-cell activities in the evolution of an immune response. Neutrophils have been implicated as mediators of tissue damage in a variety of diseases.3,4 However, the role of neutrophils in organ allograft rejection is not well understood. Tissue injury may occur in the process of organ transplantation secondary to ischemia/reperfusion, rejection, and drug toxicity. Neutrophils may contribute to such injuries. In fact, ischemia/reperfusion generates mediators that include free radicals, and causes protease activation. In addition, many of the pathological changes noted after ischemia/reperfusion injury are thought to be induced by infiltrating neutrophils. Jaeschke et al.5 and Poggetti et al.6 have shown the importance of neutrophils in the development of schemia/reperfusion injury. Accumulation of neutrophils is mediated by local chemotactic agents. Increased production and release of these substances regulate neutrophil migration from the vascular compartment to the tissues. A tissue-derived neutrophil-activating peptide (NAP-1), which is identical to interleukin8 (IL-8), has been cloned.7 The effects of IL-8 on human neutrophil functions include enhanced chemotaxis, enzyme release, expression of surface adhesion molecules, and induction of the respiratory burst.7 No substance homologous to IL-8 has been identified in the rat. However, potent neutrophil chemotactic activity has been detected in rat inflammatory exudate induced by lipopolysaccharide, TNF-a, or IL-1.8-11 Cytokine-induced neutrophil chemoattractant (CINC) is an 8-kd polypeptide originally identified in conditioned media from IL-1 b-stimulated rat glomerular epithelial cells and subsequently purified from NRK52E rat epithelioid cells.8 CINC consists of 72 amino acids and has homology to human peptides with gro/melanoma growth stimulatory activities, indicating that rat CINC belongs to the IL-8 superfamily.9,10 Thus, the regulation of CINC and/or IL-8 production is a critical element in the control of inflammatory reactions associated with neutrophil infiltration. The mixed lymphocyte reaction (MLR) is an in vitro model of alloantigen-driven immunity and has provided significant insights into immune mechanisms. Therefore, the present study was undertaken to investigate the kinetics of CINC messenger RNA (mRNA) and protein levels in a rat model
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of hepatic transplantation in vivo and the allogeneic mixed lymphocyte reaction in vitro. MATERIALS AND METHODS Animals. Male LEW(RT1l) rats were used as recipients and ACI (RT1a) rats as donors. These rats were obtained from the Central Institute for Experimental Animals, Kawasaki, Japan. This strain combination is fully allogeneic and results in acute rejection of liver transplants.12 A LEW-to-LEW combination was used for control isografts. All animals were maintained under standard conditions and received water and rodent chow ad libitum. The animals weighed 225 to 250 g. Hepatic Transplantation. Orthotopic hepatic transplantation was performed using a cuff method. A simplified cuff was used for the portal and infrarenal caval anastomoses and a Teflon tube stent was used for the biliary anastomosis.13 Anesthesia during the operative procedure and postoperative care were consistent with the National Institutes of Health guidelines for the care and use of laboratory animals.14 Experimental Protocol. The experimental animals were divided into four groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with anti-CINC antibody; and group IV, allografts treated with FK506. Recipient animals were injected intraperitoneally with saline (0.5 mL), anti-CINC antibody (Peptide Institute, Osaka, Japan) (0.5 mg/kg body weight),15 or an immunoglobulin G control antibody (0.5 mg/kg body weight) on days 3, 4, 5, 6, and 7 after transplantation. FK506 was kindly provided by Fujisawa Pharmaceutical Inc., Osaka, Japan. FK506 was administered intramuscularly at a dose of 2 mg/kg/d immediately after transplantation during each experiment. Blood Chemistry. It is often difficult to obtain blood frequently from recipient rats following transplantation. Blood loss also may influence liver function and survival rate. Therefore, animals were killed to obtain blood 7 days after the transplantation, to determine serum concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP). The measurements were performed using the sequential multiple analyzer with a computer system (Technicon, Bad Vilbel, Austria). Measurement of cytokine-induced neutrophil chemoattractant activity A sensitive enzyme-linked immunosorbent assay (ELISA) for measuring rat CINC concentrations, using a biotin-conjugated polyclonal rabbit anti-CINC immunoglobulin G, has previously been described.16 The biotin-streptavidin sandwich enzyme-linked immunosorbent assay is linear for CINC concentrations ranging from 3 pg/mL to 30 ng/mL. Cytochemical Identification of Neutrophils Infiltrating Liver Grafts. A cytochemical method assaying activity of chloroacetate esterase has been reported to be a marker for neutrophils.17 Liver specimens were harvested at various time points following transplantation. Three different sections were stained with chloroacetate esterase from the liver specimens of each group and were photographed at a magnification of 1001 to enable visualization of the central and portal veins. The number of neutrophils that had infiltrated the liver graft were measured and represented as the number per square millimeter. Immunohistochemical Staining. ACI-to-LEW hepatic transplantation recipients that had or had not been pretreated with FK506 were killed at various times after transplantation. The LEW-to-LEW syngeneic hepatic transplants were used as controls. Four hepatic transplantation recipients were killed at each time point. The grafted liver was excised and frozen rapidly in liquid nitrogen. Cryosections were prepared and air-dried overnight. Following fixation in pure acetone for 10 minutes at room temperature, cryosections were stained with anti-CINC polyclonal antibody (Peptide Institute, Inc., Osaka, Japan) and were visualized by an indirect immuno-alkaline phosphatase method.18 Control experiments included 1) incubation of the sections with 10 mg/mL of nonimmune mouse immunoglobulin G (Sigma Chemical Co., St. Louis, MO) as the primary antibody;
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and 2) omission of the primary antibody, and 3) incubation of sections in Fast Red TR (Sigma, St. Louis, MO) medium only. Monoclonal Antibodies. The identity of the macrophage subpopulations in the liver grafts was determined using anti-rat macrophage (ED1 or ED2) monoclonal antibodies.19 ED1 reacts with macrophages, monocytes, and dendritic cells, but not granulocytes. Resident macrophages are positive for ED2, while monocytes, dendritic cells, and granulocytes are negative. Double Immunostaining. To identify CINC-positive cells of monocyte/macrophage lineage, double immunostaining was performed with anti-rat CINC polyclonal antibody and anti-macrophage (ED1 or ED2) monoclonal antibodies according to a method described previously.18 In brief, cryosections were first incubated with antiCINC polyclonal antibody and stained red with Fast Red TR as a substrate by indirect immuno-alkaline phosphatase staining. The specimen then was incubated with ED1 or ED2 and stained black with 4-chloro-1-naphthol as substrate by indirect immuno-peroxidase staining.18 The sections were lightly counterstained with hematoxylin. Immunostained specimens were examined by light microscopy (Microphot FX, Nikon, Tokyo, Japan) and photographed with a band pass filter (BPN45, Fuji, Tokyo, Japan) to reduce the intensity of the hematoxylin staining. RNA Isolation and Northern Blot Analysis. Liver specimens were obtained from transplantation recipients killed at various times after transplantation, quick frozen in liquid nitrogen, and stored at 0707C before RNA extraction. Total RNA was prepared according to Chomczynski et al.20 Twenty micrograms of RNA was subjected to electrophoresis on a 1% agarose-formaldehyde denaturing gel in 11 N-morpholino-propanesulfonic acid buffer containing 200 mmol/LM N-morpholinopropanesulfonic acid, 50 mmol/L sodium acetate, and 10 mmol/L ethylenediaminetetraacetic acid, pH 7.0. Following transfer of RNA to nylon filters in 201 SSC, blots were hybridized with 32P-labeled CINC DNA probes21 at 427C in 50% formamide, 0.2% polyvinyl-pyrrolidine, 0.2% bovine serum albumin, 0.2% Ficoll, 0.05 mol/L TRIS-HCl, 1.0 mol/L NaCl, 0.1% sodium pyrophosphate, 1.0% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and 100 mg/mL of salmon sperm DNA. After hybridization, filters were washed in the same buffer using deionized, distilled, and diethyl pyrocarbonate-treated water. The hybridization signal was assessed by radioisotope counting and autoradiography. PBMC Isolation. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described.22 Briefly, heparinized blood was obtained by aortic puncture from naive ACI and LEW rats. Blood samples were diluted 1:1 with pyrogen-free saline. PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque and washed three times. The mononuclear cells were a mixture of lymphocytes and monocytes, 74% { 5.5% and 26% { 7.2%, respectively, as determined by morphology. The viability of PBMC preparations was greater than 95% as determined by trypan blue exclusion. These cells were then resuspended in culture medium (RPMI-1640, HEPES, 100 mmol/L L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal calf serum). Mixed Lymphocyte Response. The mixed lymphocyte reaction was performed with a slight modification as previously described.23 In brief, PBMCs were divided and designated as stimulator (ACI) or responder (LEW) cells. Stimulator cells were incubated with Mitomycin C (MMC). In experiments assaying CINC production, stimulator (5 1 105) and responder (5 1 105) cells were mixed in each well of 24-well tissue culture plates (Costar, Cambridge, MA). In each experiment, MMC-treated PBMCs from LEW rats were used as autologous controls. Cell aliquots were also exposed to FK506 (50 ng/mL). All cultures were performed in triplicate and incubated in a humidified atmosphere containing 5% CO2 at 377C. The supernatant was sampled at each time point and CINC concentrations were determined by enzyme-linked immunosorbent assay. In neutralization experiments, mixed lymphocyte reactions were performed in the presence of varying dilutions of neutralizing rabbit anti-human TNF-a sera (100 ng/mL) (Genzyme Corporation, Cam-
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Donor-to-Recipient
Treatment
— Anti-CINC AB* FK506† —
ACI-to-LEW LEW-to-LEW
Survival (d)
9, 9, 10, 10, 10, 10, 10, 11, 11 15, 15, 16, 17, 17, 17, 17, 18 ú60, ú60, ú60, ú60, ú60, ú60, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100, ú100
Mean { SD
P‡
10.1 { 0.7 16.6 { 0.3 ú60 ú100
— .001 õ.001 —
* Anti-CINC antibody was administered intraperitoneally at a dose of 0.5 mg/kg on days 3, 4, 5, 6, and 7 after transplantation. † FK506 was administered intramuscularly at a dose of 2 mg/kg/d. ‡ P values were obtained using the Student’s t test.
bridge, MA). For CINC mRNA determination and immunohistochemical localization of CINC, stimulator (3 1 106) and responder (3 1 106) cells were mixed in 35-mm culture plates (Costar) and cultured for 24 hours. RESULTS
Transplant survival is summarized in Table 1. Hepatic allografts from ACI to LEW rats survived 10.1 { 0.7 days. Treatment with peritoneal administration of anti-CINC antibody prolonged significantly the survival of hepatic allografts (16.6 { 0.3 days) compared with untreated allografts. In addition, treatment with FK506 significantly prolonged the survival of ACI livers in LEW recipients, to ú60 days. The LEW-toLEW isografts survived indefinitely (Table 1). Serum concentrations of aspartate transaminase, alanine transaminase, and alkaline phosphatase in untreated hepatic allograft recipients 7 days after transplantation were significantly increased compared with those in naive controls. In contrast, treatment with anti-CINC antibody attenuated the increment of liver enzymes 7 days after hepatic allografting (Table 2). The serum CINC concentrations in untreated hepatic allograft recipients increased significantly at a constant rate over time after transplantation. In contrast, no significant increases in serum CINC concentrations were observed with the isografts. The hepatic allograft recipients treated with FK506 maintained lower serum CINC concentrations throughout the experiment (Fig. 1A). The number of neutrophils was increased in the untreated hepatic allografts and rose significantly at a constant rate over time during acute rejection. In contrast, no significant increase in the number of neutrophils was observed in hepatic isografts. Treatment with FK506 resulted in lower numbers of neutrophils in the liver grafts than in untreated hepatic allografts (Fig. 1B). Immunostaining revealed that a number of infiltrating mononuclear cells in the portal areas of untreated liver allografts on day 5 were stained with an anti-CINC antibody. In addition, sinusoidal lining cells were positive for CINC (Fig.
2). On the other hand, a few cells in the portal and sinusoidal areas were positive for CINC in hepatic isografts and allografts treated with FK506 (data not shown). The localization of macrophage subpopulations infiltrating liver grafts was determined by immunostaining with anti-rat macrophage (ED1 or ED2) monoclonal antibodies. ED1/ cells gradually increased on days 5 and 7 in untreated hepatic allografts. The number of ED1/ cells in the hepatic isografts was sightly increased on day 3, but decreased thereafter. Treatment with FK506 greatly decreased the number of ED1/ cells infiltrating hepatic allografts, compared with the untreated hepatic allografts. These findings suggest that a progressive relative increase in the number of ED1/ cells was a characteristic feature of ongoing first-time rejection in the rat hepatic allografts (Fig. 3A). The number of ED2/ cells in untreated allografts increased after transplantation, peaked on day 5, and decreased thereafter. ED2/ cells in the hepatic isografts were slightly increased on days 5 and 7, but the number of these cells was much lower than in untreated allografts. There was no significant increase in the number of ED2/ cells in the hepatic allografts treated with FK506 (Fig. 3B). Double immunostaining revealed that in the untreated hepatic allografts CINC/ED1/ cells were distributed in the portal areas, whereas CINC/ED2/ cells were found in the sinusoidal areas. The number of CINC/ED1/ cells increased over time during rejection. CINC/ cells represented a subpopulation (Ç25%) of the ED1/ cells on day 7 in the untreated hepatic allografts (Fig. 4A). CINC/ED2/ cells were found in the sinusoidal areas (Fig. 4B). The level of CINC mRNA in the liver specimens peaked on day 5 in the untreated hepatic allografts. In contrast, hepatic allografts treated with FK506 showed lower levels of CINC mRNA compared with untreated allograft recipients (Fig. 5). The kinetics of CINC production during MLR are shown in Fig. 6. The CINC concentrations in the supernatant started to increase 48 hours after incubation and remained persistently elevated over time in the allogeneic MLR. No signifi-
TABLE 2. Serum Concentrations of AST, ALT, and ALP 7 Days After Transplantation Treatment
AST
ALT
54.6 { 13.8 G 427.8 { 56.7 * * 204.1 { 28.9 G†
Naive controls Untreated Anti-CINC Ab-treated
24.6 { 2.5 G 221.9 { 26.1 * * 153.6 { 31.8 G†
ALP
401.3 { 31.4 G 974.2 { 40.9 * * 598.1 { 44.3 G†
NOTE. Data are represented as the mean { SE. Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; anti-CINC Ab, anti-cytokine–induced neutrophil chemoattractant. * P õ .01. † P õ .05.
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FIG. 1. (A) Serum concentrations of CINC following rat hepatic transplantation. (B) Changes in the number of neutrophils infiltrating liver grafts after transplantation. * P õ .01; ** P õ .05; NS, not significant. Serum CINC concentrations and neutrophil accumulation in untreated hepatic allografts rose significantly at a constant rate over time during acute rejection.
cant increase in CINC production was observed in the MLR treated with FK506 or in autologous controls. Immunostaining revealed that the predominant cellular source of CINC expression in an allogeneic MLR was the mononuclear cells (data not shown). The level of CINC mRNA in an allogeneic MLR 48 hours after incubation was significantly increased compared with that observed in an allogeneic MLR treated with FK506 or in autologous controls (data not shown). To determine the role of TNF-a in the induction of CINC, the mixed lymphocyte reactions were performed in the presence of either rabbit anti-human TNF-a sera or control preimmune rabbit sera. Supernatants were sampled 96 hours after incubation and analyzed for CINC production. Anti– TNF-a antibody significantly attenuated the production of CINC in the MLR (Fig. 7). DISCUSSION
The present study has shown that the expression of CINC is significantly enhanced at the mRNA and protein levels
FIG. 3. (A) Changes in the number of ED1-positive cells accumulating in liver grafts following transplantation. * P õ .01; ** P õ .05; NS, not significant. (B) Changes in the number of ED2-positive cells infiltrating liver grafts after transplantation. * P õ .01; ** P õ .05; NS, not significant.
during acute rejection in rat hepatic transplantation. This elevated expression of CINC is associated with prominent neutrophil localization to untreated hepatic allografts during rejection. Recent studies have shown similar very early chemokine induction in human liver transplantation.24 In contrast, this induction of CINC was not observed in isografts or allografts treated with FK506. These results indicate that this inducible mediator contributes to acute rejection, suggesting the importance of neutrophil recruitment in organ transplantation. Double immunostaining revealed that CINC can be produced by cells of the monocyte/macrophage lineage. In addition, CINC/ cells represented a subpopulation of ED1/ cells. Some ED2/ resident macrophages, probably Kupffer cells, were positive for CINC. This suggests that at least some monocyte/macrophages localizing to the hepatic allografts were ‘‘activated’’ in the sense that they expressed CINC, an inducible gene product. A rejection episode begins with a prominent infiltration of cells into a liver graft during an early time period, before cellular immunity is induced. The infiltrating cells consist mainly of macrophages and lymphoid cells. Other infiltrating
FIG. 2. Immunohistochemical staining. Untreated allograft on day 7. (Magnification [A] 1100; and [B] 1200.) CINC/ cell: red. Most mononuclear cells in the portal and sinusoidal areas of untreated allografts were positive for CINC.
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FIG. 4. Changes in the number of (A) ED1/CINC/ and (B) ED2/CINC/ cells infiltrating liver grafts after transplantation. CINC/ cells represented a subpopulation (approximately 25%) of the ED1/ cells in untreated hepatic allografts.
cells, including neutrophils, basophils, and plasma cells, also increase at a constant rate.25 Histological examination of human liver graft biopsies taken during acute rejection reveals a mixed inflammatory infiltrate localized to small bile ducts and venous endothelium in portal tracts. In addition, the infiltrate consists of mononuclear cells and large numbers of neutrophils infiltrating the biliary epithelium.26,27 Furthermore, it has been reported that bile taken from patients with acute rejection contains chemoattractants that may be responsible for the recruitment of neutrophils.28 These observations suggest an important role for neutrophils in the immune response occurring during acute rejection. The present study also showed an increased number of neutrophils in the untreated hepatic allografts during acute rejection. Neutrophils have been implicated as mediators of tissue damage in a variety of diseases including emphysema and rheumatoid arthritis,3,4 but their role in allograft damage during rejection is unclear. Adherence of circulating leukocytes to endothelium may be a common early event in both ischemia/reperfusion injury and immune graft rejection. The vascular biology of early posttransplantation rejection may be complicated by the overlap between ischemia/reperfusion and immune phenomena. Adams et al.29 showed that neutrophil-mediated mechanisms may be important contributory factors in the tissue damage of acute rejection. After recruitment to the site of rejection by chemotactic factors, the activated neutrophils may spontaneously release proteolytic enzymes or be stimulated further by locally active cytokines.
FIG. 5. Northern blot analysis. A representative blot shows that expression of CINC mRNA in a liver specimen from an untreated allograft peaks on day 5 after transplantation and decreases thereafter.
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FIG. 6. CINC production during mixed lymphocyte reaction. * P õ .01; ** P õ .05 (vs. autologous or FK506-treated allogeneic MLR). CINC were produced in the allogeneic MLR but not in the FK506-treated allogeneic MLR. (l) Allogeneic MLR; (h) autologous MLR; and (s) allogeneic MLR treated with FK506.
Digestion of extracellular protein by neutrophil elastase may be important, not only in causing tissue damage, but also in allowing the movement of neutrophils from blood to inflamed tissues.30,31 Neutrophils are recruited to a graft by locally active chemotactic factors. A previous study showed that culture supernatants of lymphocytes isolated during episodes of acute rejection were both chemotactic for and capable of increasing superoxide production by neutrophils.29 Acute cellular rejection is associated with intragraft increases in the expression of several mRNAs. Kirk et al.32 reported that increased IL-8 mRNA in the human renal grafts by needle core biopsy was
FIG. 7. The effect of anti-TNF-a antibody on CINC production in an allogeneic mixed lymphocyte reaction 96 hours after incubation. Anti-TNFa antibody attenuated significantly the production of CINC in the MLR. *P õ .01. (j) Allogeneic MLR; and ( ) allogeneic MLR / anti–TNF-a antibody.
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correlated with irreversible loss of graft function. Similarly, the present study has shown that enhanced gene expression and production of CINC is associated with accelerated rejection reactions following rat hepatic allografting. The C-X-C chemokines mediate neutrophil chemotaxis and activation in vitro and are implicated in neutrophil influx to acute inflammatory sites in vivo.33,34 IL-8 stimulates upregulation of b2 integrins in vitro, enhancing adhesion to resting endothelial cells.35 In this regard, there are few in vivo data showing whether CINC may influence neutrophil functions involving adhesion and secretagogue activation in microcirculation. However, it has been reported that CINC is a novel stimulator that evokes not only locomotive but also secretagogue activation of neutrophils via a CD18-dependent mechanism in vivo.36 Subsequently, Frevert et al.37 reported that recombinant rat chemokine KC induces neutrophil chemotaxis and increases the expression of CD11b/CD18 on neutrophils. Recombinant KC also induces the respiratory burst in neutrophils. These biological activities suggest that elevated serum concentrations of CINC during acute rejection may modulate and further facilitate neutrophil activation and tissue injury. Neutrophils are also thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. Neutrophil adherence to endothelium can be significantly enhanced by the endothelial deposition of iC3b.38 Wieder et al.39 has reported that the immunosuppressive agent rapamycin prevents accelerated rejection of cardiac allografts in sensitized rats. This effect is associated with markedly reduced expression of a gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, operational rat homologues of the human intercrine-a cytokines that have proinflammatory IL-8–like neutrophil activation/chemotactic properties. The present study also showed that FK506 significantly decreased CINC expression and neutrophil accumulation in untreated hepatic allografts. Cell-mediated reactions are well known to be essential in the rejection of allogeneic transplanted organs. In the present study we have elucidated important aspects of CINC production in vitro, using a mixed lymphocyte reaction as well as in vivo studies. Delayed-type hypersensitivity and allograft rejection are dependent upon the generation of antigen-specific T-cell immune response. The mixed lymphocyte reaction is a model of alloantigen-driven immunity and has provided significant insight into mechanisms of T-cell proliferation. Larsen et al.40 showed that IL-8 plays an important pathophysiological role in the development of a delayed-type hypersensitivity reaction. In our study, allogeneic MLRs showed that CINC concentrations increased as early as day 2 and persisted over 4 days. Northern blot analysis confirmed early induction of CINC mRNA in the allogeneic response. Staining of cells recovered from allogeneic cultures showed that mononuclear cells are the predominant source for CINC production. This indicates that cells of the monocyte/macrophage lineage are capable of producing CINC under appropriate stimulation. It has been reported that serum TNF-a levels are increased during allograft rejection,41,42 and immunotherapy with anti– TNF-a antibody prolongs graft survival.43 These observations suggest that TNF-a may be involved in the pathogene-
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sis of acute rejection. Previous studies have shown the importance of TNF-a in the mixed lymphocyte reaction.44,45 Christensen et al.23 showed that MCP-1 and IL-8 are produced during the evolution of a mixed lymphocyte reaction and that their induction is TNF-a –dependent. CINC production can be induced by several inflammatory stimuli, including IL-1b, lipopolysaccharide, and TNF-a.8-11 NF-kB is a family of proteins that regulate the transcription of many genes. Blackwell et al.46 have shown that CINC production is transcriptionally regulated by NF-kB. The promoter region of the CINC gene contains an NF-kB binding site.46,47 TNFa contains NF-kB binding motifs in the proximal promoter regions which appear to be important in regulating transcription. These observations suggest that TNF-a stimulate CINC production through NF-kB activation. The present study also showed that the presence of neutralizing anti-human TNFa antibodies resulted in significant abrogation of CINC production in the MLR. In conclusion, enhanced CINC expression in association with neutrophil accumulation is an important mediator of acute rejection in rat hepatic transplantation. REFERENCES 1. Bainton DF, Ullyot JL, Farquhar MG. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med 1971;134:907-934. 2. Lloyd AR, Oppenheim JJ. Poly’s lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunol Today 1992;13:169-172. 3. Burnett D, Chamba A, Hill SL, Stockley RA. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet 1987;2:1043-1046. 4. Malech HL, Gallin JI. Current concepts: immunology. Neutrophils in human diseases. N Engl J Med 1987;317:687-694. 5. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/ reperfusion injury in rat liver in vivo. FASEB J 1990;4:3355-3359. 6. Poggetti RS, Moore FA, Moore EE, Bensard DD, Anderson BO, Benerjee A. Liver injury is a reversible neutrophil-mediated event following gut ischemia. Arch Surg 1992;127:175-179. 7. Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating peptide 1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 1989;84:1045-1049. 8. Watanabe K, Kinoshita S, Nakagawa H. Purification and characterization of cytokine-induced neutrophil chemoattractant produced by epithelial cell line of normal rat kidney (NRK-52E cell). Biochem Biophys Res Commun 1989;161:1093-1099. 9. Watanabe K, Suematsu M, Iida M, Takaishi K, Iizuka Y, Suzuki H, Suzuki M, et al. Effect of rat CINC/gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery. Exp Mol Pathol 1992;56:60-69. 10. Watanabe K, Konishi K, Fujioka M, Kinoshita S, Nakagawa H. The neutrophil chemoattractant produced by the rat kidney epithelial cell line NRK-52E is a protein related to the KC/gro protein. J Biol Chem 1989;264:19559-19563. 11. Watanabe K, Iida M, Takaishi K, Suzuki T, Hamada Y, Iizuka T, Turufuji S. Chemoattractants for neutrophils in lipopolysaccharide-induced inflammatory exudate from rats are not interleukin-8 counterparts but gro-gene-product/melanoma-growth-stimulating-activity-related factors. Eur J Biochem 1993;214:267-270. 12. Yamaguchi Y, Harland RC, Wyble C, Bollinger RR. The role of class I major histocompatibility complex antigens in prolonging the survival of hepatic allografts in the rat. Transplantation 1989;46:360-363. 13. Yamaguchi Y, Kikuchi N, Miyanari N, Ichiguchi O, Goto M, Mori K, Ogawa M. A technique for orthotopic reduced-size hepatic transplantation combined with ex vivo liver cut down in the rat. Dig Dis Sci 1996; 41:1713-1721. 14. National Institutes of Health. Guide for the care and use of laboratory animals. Public Health Service, NIH publication No. 86-23, Bethesda, MD, 1985. 15. Hisama N, Yamaguchi Y, Ishiko T, Miyanari N, Ichiguchi O, Goto M, Mori K, et al. Kupffer cell production of cytokine-induced neutrophil
hepa
WBS: Hepatology
1552 YAMAGUCHI ET AL.
16.
17. 18.
19.
20. 21. 22.
23.
24.
25.
26. 27.
28. 29. 30. 31.
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chemoattractant (CINC) following ischemia/reperfusion injury in rats. HEPATOLOGY 1996;24:1193-1198. Nakagawa H, Ikesue A, Kato H, Debuchi H, Watanabe K, Tsurufuji S, Naganawa M, Mitamura M. Changes in the levels of rat interleukin 8/ CINC and gelatinase in the exudate of carrageenin-induced inflammation in rats. J Pharmacobio-Dynamics 1992;15:461-466. Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971;55:283-290. Matsuno K, Ezaki T, Kotani M. Splenic outer periarterial lymphoid sheath (PALS): an immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophage in the rat. Cell Tissue Res 1989;257:459-470. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2, and ED3. Immunology 1985;54:589-599. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;62:156-159. Konishi K, Takata Y, Yamamoto M, Yomogida K, Watanabe K, Tsurufuji S, Fujioka M. Structure of the gene encoding rat neutrophil chemoattractant Gro. Gene 1993;126:285-286. Strieter RM, Remick DG, Lynch JP, Genord M, Raiford C, Spengler R, Kunkel SL. Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis. Am J Respir Cell Mol Biol 1989;1:57-63. Christensen PJ, Rolfe MW, Standiford TJ, Burdick MD, Toews GB, Strieter RM. Characterization of the production of monocyte chemoattractant protein-1 and IL-8 in an allogeneic immune response. J Immunol 1993;151:1205-1213. Adams DH, Hubscher S, Fear J, Johnston J, Shaw S, Afford S. Hepatic expression of macrophage inflammatory protein-1 a and macrophage inflammatory protein-1 b after liver transplantation. Transplantation 1996;61:817-825. Yamamoto H, Kaneda K, Kamada N, Wake K. Morphometric analysis of infiltrating cells that adhere to the sinusoidal wall and migrate into the space of Disse in the orthotopically transplanted rat liver. Transplantation 1994;58:8-13. Snover DC, Freese DK, Sharp HL, Bloomer JR, Najarian JS, Ascher NL. Liver allograft rejection. An analysis of the use of biopsy in determining outcome of rejection. Am J Surg Pathol 1987;11:1-10. Demetris AJ, Lasky S, Van Thiel DH, Starzl TE, Dekker A. Pathology of hepatic transplantation: a review of 62 adult allograft recipients immunosuppressed with a cyclosporine/steroid regimen. Am J Pathol 1985;118:151-161. Adams DH, Burnett D, Stockley RA, Elias E. Patterns of leukocyte chemotaxis to bile after liver transplantation. Gastroenterology 1989;7:433438. Adams DH, Wang LF, Burnett D, Stockley RA, Neuberger JM. Neutrophil activation. An important cause of tissue damage during liver allograft rejection? Transplantation 1990;50:86-91. Campbell EJ, Senior RM, McDonald JA, Cox DL. Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro. J Clin Invest 1982;70:845-852. Smedly LA, Tonnesen MG, Sandhaus RA, Haslett C, Guthrie LA, Johnston RB Jr, Henson PM, et al. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 1986;77:1233-1243.
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32. Kirk AD, Bollinger RR, Finn OJ. Rapid, comprehensive analysis of human cytokine mRNA and its application to the study of acute renal allograft rejection. Hum Immunol 1995;43:113-128. 33. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines: CXC and CC chemokines. Adv Immunol 1994;55:97-179. 34. Miller MD, Krangel MS. Biology and biochemistry of the chemokines: A family of chemotactic and inflammatory cytokines. Crit Rev Immunol 1992;12:17-46. 35. Huber AR, Kunkel SL, Todd RF, Weiss SJ. Regulation of transendothelial neutrophil migration of endogenous interleukin-8. Science 1991;254: 99-102. 36. Suzuki H, Suematsu M, Miura S, Liu YY, Watanabe K, Miyasaka M, Tsurufuji S, Tsuchiya M. Rat CINC/gro: a novel mediator for locomotive and secretagogue activation of neutrophils in vivo. J Leukoc Biol 1994; 55:652-657. 37. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 1995;154:335-344. 38. Vercellotti GM, Platt JL, Bach FH, Dalmasso AP. Neutrophil adhesion to xenogeneic endothelium via iC3b. J Immunol 1991;146:730-734. 39. Wieder KJ, Hancock WW, Schmidbauer G, Corpier CL, Wieder I, Kobzik L, Strom TB, Kupiec-Weglinski JW. Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts. J Immunol 1993;151:1158-1166. 40. Larsen CG, Thomsen MK, Gesser B, Thomsen PD, Deleuran BW, Nowak J, Skodt V, Thomsen HK, Deleuran M, Thestrup-Pedersen K. The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody. J Immunol 1995;155:2151-2157. 41. Hoffmann MW, Wonigeit K, Steinhoff G, Herzbeck H, Flad HD, Pichlmayr R. Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection. Transplantation 1993; 55:329-335. 42. Pizarro TT, Malinowska K, Kovacs EJ, Clancy J Jr, Robinson JA, Piccinini LA. Induction of TNF alpha and TNF beta gene expression in rat cardiac transplants during allograft rejection. Transplantation 1993;56: 399-404. 43. Imanaga DK, Mills JM, Olthoff KM, Seu P, Dempsey RA, Hart J, Terasaki PI, et al. Anti-tumor necrosis factor antibody enhances allograft survival in rats. J Surg Res 1990;48:345-348. 44. Shalaby MR, Espevik T, Rice GC, Ammann AJ, Figari IS, Ranges GE, Palladino MJ. The involvement of human tumor necrosis factors-alpha and -beta in the mixed lymphocyte reaction. J Immunol 1988;141:499503. 45. Robinet E, Branellec D, Termijtelen AM, Blay JY, Gay F, Chouaib S. Evidence for tumor necrosis factor-alpha involvement in the optimal induction of class I allospecific cytotoxic T cells. J Immunol 1990;144: 4555-4561. 46. Blackwell TS, Holden EP, Blackwell TR, DeLarco JE, Christman JW. Cytokine-induced neutrophil chemoattractant mediates neutrophilic alveolitis in rats: association with nuclear factor kB activation. Am J Respir Cell Mol Biol 1994;11:464-472. 47. Ohtsuka T, Kubota A, Hirano T, Watanabe K, Yoshida H, Tsurufuji M, Iizuka Y, et al. Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kB activation. J Biol Chem 1996;271:1651-1659.
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