Interleukin-10 (IL-10) Augments Allograft Arterial Disease
1999; Elsevier BV; Volume: 155; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65512-5
ISSN1525-2191
AutoresYutaka Furukawa, Gerold Becker, Jennifer L. Stinn, Koïchi Shimizu, P Libby, Richard Mitchell,
Tópico(s)Chemokine receptors and signaling
ResumoInterleukin-10 (IL-10) is an anti-inflammatory helper T cell type 2 (Th2) cytokine that modulates Th1-type cytokine production. Graft arterial disease (GAD) is a vascular obliterative process mediated via the Th1 cytokine interferon-γ (IFN-γ); allografts in IFN-γ-deficient animals do not develop GAD. We investigated the effect of IL-10 and anti-IL-10 on GAD in murine heart transplants and whether anti-IL-10 reestablishes GAD in IFN-γ-deficient hosts. Major histocompatibility complex class II-mismatched hearts were transplanted for 8 weeks into wild-type or IFN-γ-deficient mice. In one set of experiments, wild-type hosts received daily administration of phosphate-buffered saline (PBS) or increasing IL-10; in a subsequent set of experiments, wild-type hosts received weekly PBS, rat IgG, or anti-IL-10 monoclonal antibody. IFN-γ-deficient recipients received weekly PBS or anti-IL-10 monoclonal antibody. Explanted allografts were assessed for parenchymal rejection and GAD, cytokine profiles, and adhesion/costimulatory-molecule expression. Exogenous IL-10 resulted in increased Th2-like cytokine production; nevertheless, it exacerbated parenchymal rejection and GAD and increased CD8+ infiltration. Anti-IL-10 did not significantly affect the extent of rejection or GAD, cytokine profiles, or immunohistology of the allografts in wild-type hosts. Adhesion molecule (CD54 and CD106) expression was not diminished by IL-10 treatment, and costimulatory-molecule (CD80 and CD86. expression was augmented by administration of exogenous IL-10. Allografts in IFN-γ-deficient recipients showed mild rejection and no GAD, regardless of anti-IL-10 treatment. IL-10 in vivo thus has markedly different effects than predicted from in vitro experience. Although allografts develop Th2-like cytokine profiles treatment with IL-10 causes exacerbated rejection and GAD. Interleukin-10 (IL-10) is an anti-inflammatory helper T cell type 2 (Th2) cytokine that modulates Th1-type cytokine production. Graft arterial disease (GAD) is a vascular obliterative process mediated via the Th1 cytokine interferon-γ (IFN-γ); allografts in IFN-γ-deficient animals do not develop GAD. We investigated the effect of IL-10 and anti-IL-10 on GAD in murine heart transplants and whether anti-IL-10 reestablishes GAD in IFN-γ-deficient hosts. Major histocompatibility complex class II-mismatched hearts were transplanted for 8 weeks into wild-type or IFN-γ-deficient mice. In one set of experiments, wild-type hosts received daily administration of phosphate-buffered saline (PBS) or increasing IL-10; in a subsequent set of experiments, wild-type hosts received weekly PBS, rat IgG, or anti-IL-10 monoclonal antibody. IFN-γ-deficient recipients received weekly PBS or anti-IL-10 monoclonal antibody. Explanted allografts were assessed for parenchymal rejection and GAD, cytokine profiles, and adhesion/costimulatory-molecule expression. Exogenous IL-10 resulted in increased Th2-like cytokine production; nevertheless, it exacerbated parenchymal rejection and GAD and increased CD8+ infiltration. Anti-IL-10 did not significantly affect the extent of rejection or GAD, cytokine profiles, or immunohistology of the allografts in wild-type hosts. Adhesion molecule (CD54 and CD106) expression was not diminished by IL-10 treatment, and costimulatory-molecule (CD80 and CD86. expression was augmented by administration of exogenous IL-10. Allografts in IFN-γ-deficient recipients showed mild rejection and no GAD, regardless of anti-IL-10 treatment. IL-10 in vivo thus has markedly different effects than predicted from in vitro experience. Although allografts develop Th2-like cytokine profiles treatment with IL-10 causes exacerbated rejection and GAD. Interleukin-10 (IL-10) is an 18-kD-homodimeric protein produced in mice by a helper T cell type 2 (Th2) subset of CD4+ T cells, by a subpopulation of activated B cells (CD5 and CD11 positive), and by activated macrophages. IL-10 blocks the synthesis of macrophage-derived cytokines including IL-1β, IL-12, and tumor necrosis factor-α, and it reduces macrophage cytotoxic activity and nitric oxide production.1Fiorentino DF Zlotnik A Mosmann TR Howard M O'Garra A IL-10 inhibits cytokine production by activated macrophages.J Immunol. 1991; 147: 3815-3822PubMed Google Scholar, 2Bogdan C Vodovotz Y Nathan C Macrophage deactivation by interleukin 10.J Exp Med. 1991; 174: 1549-1555Crossref PubMed Scopus (1153) Google Scholar IL-10 thus indirectly reduces helper T cell type 1 (Th1) differentiation by blocking macrophage IL-12 synthesis.3Fiorentino DF Bond MW Mosmann TR Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.J Exp Med. 1989; 170: 2081-2095Crossref PubMed Scopus (2652) Google Scholar, 4Abbas AK Murphy KM Sher A Functional diversity of helper T lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3917) Google Scholar In lymphocyte cultures, IL-10 can also directly inhibit T cell proliferation and the production of the Th1-type cytokines, IL-2, and interferon-γ (IFN-γ), mediators that initiate and/or regulate a delayed-type hypersensitivity response.5Danzer SG Kirchner H Rink L Cytokine interactions in human mixed lymphocyte culture.Transplantation. 1994; 57: 1638-1642Crossref PubMed Scopus (63) Google Scholar, 6Taga K Tosato G IL-10 inhibits human T cell proliferation and IL-2 production.J Immunol. 1992; 148: 1143-1148PubMed Google Scholar, 7Ding L Shevach EM IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function.J Immunol. 1992; 148: 3133-3139PubMed Google Scholar In addition to regulating macrophage cytokine production, IL-10 inhibits T cell activation by decreasing antigen presentation by macrophages via suppression of major histocompatibility complex (MHC) class II molecule (MHC II) expression and by limiting the expression of costimulatory molecules necessary for T cell activation.8de Waal Malefyt R Haanen J Spits H Roncarolo MG te Velde A Figdor C Johnson K Kastelein R Yssel H de Vries JE Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.J Exp Med. 1991; 174: 915-924Crossref PubMed Scopus (1794) Google Scholar Finally, IL-10 blocks the up-regulation of adhesion molecule expression, thereby theoretically reducing mononuclear cell emigration.9Ding L Linsley PS Huang LY Germain RN Shevach EM IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression.J Immunol. 1993; 151: 1224-1234PubMed Google Scholar, 10Chang CH Furue M Tamaki K Selective regulation of ICAM-1 and major histocompatibility complex class I and II molecule expression on epidermal Langerhans cells by some of the cytokines released by keratinocytes and T cells.Eur J Immunol. 1994; 24: 2889-2895Crossref PubMed Scopus (63) Google Scholar In organ transplantation, Th1 cells are purported to promote allograft rejection by inducing the development of alloantigen-specific cytotoxic T lymphocytes and delayed-type hypersensitivity responses.11Hall BM Cells mediating allograft rejection.Transplantation. 1991; 51: 1141-1151Crossref PubMed Scopus (344) Google Scholar In contrast, Th2 lymphocytes may theoretically promote long-term allograft acceptance. In a tolerogenic transplant model with anti-CD4 antibody, mouse hearts showed an increasing frequency of intragraft IL-10 and IL-4 expression, whereas untreated rejecting hearts expressed Th1 cytokines and IL-4, but not IL-10.12Mottram PL Han WR Purcell LJ McKenzie IF Hancock WW Increased expression of IL-4 and IL-10 and decreased expression of IL-2 and interferon-γ in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy.Transplantation. 1995; 59: 559-565Crossref PubMed Scopus (165) Google Scholar Similarly, in humans, a study of severe combined immunodeficient patients transplanted with allogeneic stem cells showed that only tolerized patients secreted a high level of IL-10.13Bacchetta R Bigler M Touraine JL Parkman R Tovo PA Abrams J de Waal Malefyt R de Vries JE Roncarolo MG High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.J Exp Med. 1994; 179: 493-502Crossref PubMed Scopus (373) Google Scholar In nonvascularized murine heart allografts, viral IL-10, homologous to murine and human IL-10 and sharing their inhibitory effect on cytokine synthesis, prolonged allograft survival.14Qin L Chavin KD Ding Y Tahara H Favaro JP Woodward JE Suzuki T Robbins PD Lotze MT Bromberg JS Retrovirus-mediated transfer of viral IL-10 gene prolongs murine cardiac allograft survival.J Immunol. 1996; 156: 2316-2323PubMed Google Scholar However, other groups have reported contradictory results. A long-acting IL-10 fusion protein in pancreatic islet allografts led to accelerated rejection, and, in vascularized heart transplants, grafts were rejected early after administration of a high dose of IL-10.15Qian S Li W Li Y Fu F Lu L Fung JJ Thomson AW Systemic administration of cellular interleukin-10 can exacerbate cardiac allograft rejection in mice.Transplantation. 1996; 62: 1709-1714Crossref PubMed Scopus (106) Google Scholar, 16Zheng XX Steele AW Nickerson PW Steurer W Steiger J Strom TB Administration of noncytolytic IL-10/Fc in murine models of lipopolysaccharide-induced septic shock and allogeneic islet transplantation.J Immunol. 1995; 154: 5590-5600PubMed Google Scholar IL-10 transgenic recipients also rejected their grafts earlier than wild-type recipients.17Lee MS Wogensen L Shizuru J Oldstone MB Sarvetnick N Pancreatic islet production of murine interleukin-10 does not inhibit immune-mediated tissue destruction.J Clin Invest. 1994; 93: 1332-1338Crossref PubMed Scopus (122) Google Scholar Another recent report demonstrated improved heart allograft survival by anti-IL-10 treatment and suggested potential immunostimulator effects of endogenous IL-10.18Li W Fu F Lu L Narula SK Fung JJ Thomson AW Qian S Systemic administration of anti-interleukin-10 antibody prolongs organ allograft survival in normal and presensitized recipients.Transplantation. 1998; 66: 1587-1596Crossref PubMed Scopus (30) Google Scholar Overall, the reported effects are contradictory; in some models, IL-10 promotes long-term survival; in others, IL-10 aggravates rejection. Moreover, no study has yet examined the role of IL-10 in graft arterial disease (GAD), the fibroproliferative intimal vascular lesion that is the major long-term limitation to solid-organ allograft survival.19Paul LC Fellstrom B Chronic vascular rejection of the heart and the kidney. Have rational treatment options emerged?.Transplantation. 1992; 53: 1169-1179Crossref PubMed Scopus (141) Google Scholar, 20Salomon RN Hughes CC Schoen FJ Payne DD Pober JS Libby P Human coronary transplantation-associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells.Am J Pathol. 1991; 138: 791-798PubMed Google Scholar Thus, although the in vivo data were equivocal regarding parenchymal rejection, the in vitro data with IL-10 suggested a number of expected beneficial effects of IL-10 administration potentially resulting in diminished GAD, including a shift to a predominant Th2-type response, and diminished macrophage activation. We and another group had previously demonstrated the critical role of IFN-γ in the pathogenesis of GAD, using IFN-γ-deficient (IFN-γKO) animals21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar or neutralizing antibody.22Russell PS Chase CM Winn HJ Colvin RB Coronary atherosclerosis in transplanted mouse hearts. III. Effects of recipient treatment with a monoclonal antibody to interferon-gamma.Transplantation. 1994; 57: 1367-1371Crossref PubMed Scopus (70) Google Scholar The corresponding secondary mixed lymphocyte reaction (MLR), using splenocytes from the IFN-γKO host animals, revealed increased IL-10 production in the supernatants (unpublished data). This result also suggested that exogenous IL-10 might prevent the development of GAD and that, conversely, neutralizing IL-10 activity could potentially exacerbate GAD. Therefore, to investigate the modulating effects of IL-10 on parenchymal rejection and/or GAD, we administered recombinant murine IL-10 or anti-IL-10 monoclonal antibody (mAb) in a well-characterized vascularized heart transplant model in which both pathologic lesions occur. Moreover, because of the reported effects on T cell cytokine profiles, we analyzed the induced immune response (Th1 versus Th2). Finally, because the absence of IFN-γ has been shown to prevent GAD,21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar potentially via augmented IL-10 production, we also investigated the effects of anti-IL-10 in transplants in IFN-γKO recipients. C57BL/6 (B6, H-2b) and B6 IFN-γ-deficient mice (IFN-γKO, H-2b), 25 to 30 g, were used as allograft recipients; C-H-2bm12KhEg (bm12, H-2bm12) mice were used as heart donors. The original IFN-γKOs were generated by homologous recombination and provided by Dr. Tim Stewart (Genentech, South San Francisco, CA).23Dalton DK Pitts-Meek S Keshav S Figari IS Bradley A Stewart TA Multiple defects of immune cell function in mice with disrupted interferon-γ genes.Science. 1993; 259: 1739-1742Crossref PubMed Scopus (1514) Google Scholar All IFN-γKOs were homozygotes and at least eighth-generation backcrossed into the B6 background.23Dalton DK Pitts-Meek S Keshav S Figari IS Bradley A Stewart TA Multiple defects of immune cell function in mice with disrupted interferon-γ genes.Science. 1993; 259: 1739-1742Crossref PubMed Scopus (1514) Google Scholar The backcrossed IFN-γKOs were confirmed as homozygotes by polymerase chain reaction amplification of tail DNA as reported previously.21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar B6 and bm12 mice aged 8 to 10 weeks were obtained from Taconic Farms (Germantown, NY) and the Jackson Laboratory (Bar Harbor, ME), respectively. The mice were maintained in the Harvard Medical School animal facilities on acidified water. sentinel animals in the same room that were surveyed serologically were consistently negative for all viral pathogens tested. All experiments conformed to approved animal care protocols. Heterotopic cardiac transplantation was performed using a 21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar of the method described by Corry et al.24Corry RJ Winn HJ Russell PS Primary vascularized allografts of hearts in mice: the role of H-2D, H-2K, and non-H-2 antigens in rejection.Transplantation. 1973; 16: 343-350Crossref PubMed Scopus (799) Google Scholar Ischemic time was routinely 30 to 35 minutes, with a success rate of approximately 90%. The viability of the cardiac allografts was assessed by daily abdominal palpation. Immunosuppression consisted of a pretransplant course of anti-CD4 (GK 1.5) and anti-CD8 (2.43) mAbs injected intraperitoneally 6, 3, and 1 days before transplantation.25Russell PS Chase CM Winn HJ Colvin RB Coronary atherosclerosis in transplanted mouse hearts. I. Time course and immunogenetic and immunopathological considerations.Am J Pathol. 1994; 144: 260-274PubMed Google Scholar Anti-CD4 and anti-CD8 antibodies were prepared from hybridoma clones (American Type Culture Collection, Manassas, VA) and used as ascites preparations or from comparable concentrations of antibody prepared from serum-free supernatants in an artificial capillary system (Cellmax, Celluco, Rockville, MD). In IL-10 treatment experiments, wild-type recipients were injected daily subcutaneously with recombinant murine IL-10 (rmIL-10, a generous gift of Schering-Plough, Kenilworth, NJ) or phosphate-buffered saline (PBS) (n = 6 per group). IL-10 doses used were 0.5, 1.0, and 2.5 μg/day, roughly corresponding to 17, 34, and 85 μg/kg per day in the 25- to 30-g-recipient animals. The rmIL-10 was a clinical-grade reagent containing no detectable lipopolysaccharide; this was confirmed by in vitro culture of adhesive splenocytes with 0 to 0.1 μg/ml rmIL-10 and measurement of IL-12 gene expression by RNase protection assay. With increasing concentration of IL-10, there was decreasing IL-12 mRNA synthesis; no IL-12 was detected at concentrations of rmIL-10 > 0.01 μg/ml (data not shown). For anti-IL-10 experiments, purified anti-IL-10 mAb, either SXC.1 (a generous gift of Schering-Plough) or JES5-2A5 (prepared from a hybridoma clone, American Type Culture Collection) was used. Animals were given intraperitoneal injections of 2 mg of purified anti-IL-10 mAb at the time of transplant and then 1 mg weekly, or an equivalent volume of PBS or rat IgG (Sigma Chemical Co., St. Louis, MO). All grafts were explanted 8 weeks after transplantation, a time shown previously to yield GAD lesions.25Russell PS Chase CM Winn HJ Colvin RB Coronary atherosclerosis in transplanted mouse hearts. I. Time course and immunogenetic and immunopathological considerations.Am J Pathol. 1994; 144: 260-274PubMed Google Scholar Harvested allografts were transversely sectioned in three roughly equal parts. The most basal section was fixed in 10% buffered formalin for morphological examination, the mid-portion was frozen in OCT compound (Ames Co., Elkhart, IN) and stored at −80°C for immunohistochemical staining, and the apical portion was used for intracellular cytokine analysis of mononuclear inflammatory cells by flow cytometry.26Prussin C Metcalfe DD Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.J Immunol Methods. 1995; 188: 117-128Crossref PubMed Scopus (422) Google Scholar, 27O'Garra A Murphy K Role of cytokines in determining T cell function.in: Weir DM Weirs Handbook of Experimental Immunology. Vol. 4. Blackwell Science, Cambridge, MA1996: 226.1-226.10Google Scholar, 28Stinn JL Taylor MK Becker G Nagano H Hasegawa S Furukawa Y Shimizu K Libby P Mitchell RN IFN-γ-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry.Am J Pathol. 1998; 153: 1383-1392Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The formalin-fixed sections were embedded in paraffin and stained with hematoxylin and eosin or the elastic fiber stain (Weigert's method). For immunohistochemistry, to 4- to 5-μm frozen heart sections were fixed in acetone for 10 minutes, then incubated with mAbs to Mac-3, CD4, CD8, CD40, CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), CD106 (VCAM-1), or MHC II I-Ab (PharMingen, San Diego, CA) for 90 minutes. Control, isotype-matched nonspecific antibodies were used to establish background staining. After appropriate secondary biotin-labeled antibodies against the primary mAbs, sections were stained with avidin-alkaline phosphatase (Vector Laboratories, Burlingame, CA) by a modified avidin-biotin complex method.29Hsu SM Raine L Fanger H The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics.Am J Clin Pathol. 1981; 75: 816-821Crossref PubMed Scopus (920) Google Scholar Sections were counterstained with hematoxylin. The basal third of each heart graft, where coronary arteries generally have largest caliber, was used for histological evaluation. Occasionally, some sections included coronary arteries at their take-off at the coronary sinus. Short axial sections were stained with hematoxylin and eosin (H&E) and elastin staining and were analyzed for severity of parenchymal rejection and GAD as previously reported.21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar Scores for parenchymal rejection and GAD were blindly graded by three independent observers (Y. F., G. B., R. N. M.). Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no rejection; 1, focal mononuclear cell infiltrates without necrosis; 2 focal mononuclear cell infiltrates with necrosis; 3, multifocal infiltrates with necrosis; 4, widespread infiltrate with hemorrhage and/or vasculitis),21Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar, 30Billingham ME Cary NR Hammond ME Kemnitz J Marboe C McCallister HA Snovar DC Winters GL Zerbe A A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group, The International Society for Heart Transplantation.J Heart Transplant. 1990; 9: 587-593PubMed Google Scholar and the GAD score was calculated from the number and severity of involved vessels (0, vascular occlusion 75% occlusion). Typically, 10 or more vessels were scored for each heart, and the degree of vascular occlusion for each was averaged. Scores for each specimen uniformly fell within a range of one grade for all observers and were averaged among observers. Immunohistochemical analyses (−, absent; +, weak, focal; ++, weak, diffuse; +++, strong, focal; ++++, strong, diffuse) were performed by three independent observers (Y. F., G. B., R. N. M.). One-way MLRs were performed using whole-splenocyte or CD4+ lymphocyte populations. Spleens were ground through a cytoscreen into RPMI 1640 (Gibco, Gaithersburg, MD). Cells and residue were pelleted at 300 g for 5 minutes and resuspended in 10 ml ammonium chloride buffer (0.83% NH4Cl, 5 mmol/L Tris, pH 7.2) at 37°C for 7 to 8 minutes to lyse erythrocytes, followed by washing in RPMI 1640. Cells were resuspended in RPMI 1640 supplemented with 1% nonessential amino acids, 1% l-glutamine, 1% 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid buffer, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.1% 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum (C/10). In primary MLR, immunobeads (Dynabeads, Dynal, Lake Success, NY) and polyclonal anti-Fab antibodies (Detachabeads, Dynal) were used for CD4+ purification after the protocol suggested by the manufacturer. Stimulator cells were radiated with 30 Gy. Responder cells and irradiated stimulator splenocytes (5 × 105 of each) were cultured in quadruplicate in 96-well plates in a 5. CO2 humidified atmosphere. In some primary MLR groups, 0.1 μg/ml IL-10 was also added. For proliferation assay, cells were exposed to [3H]thymidine (New England Nuclear, Boston, MA) for 6 hours on day 3, and incorporated radioactivity was measured in a Betaplate scintillation counter (Wallac, Gaithersburg, MD). Proliferation is reported as counts per minute, and results are expressed as the mean ± SD. IFN-γ was measured from primary MLR culture supernatants collected on day 3, using a two-site sandwich enzyme-linked immunosorbent assay, following the protocol recommended by the manufacturer. The explanted grafts were minced with a sterile razor blade and placed in 10 ml borate buffered saline with 2% bovine serum albumin and 20 mg collagenase (Sigma). These mixtures were rocked at 37°C for 2 hours and strained through a 70-μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Erythrocytes and dead lymphocytes were removed by centrifugation through Ficoll (Organon Teknika Corp., Durham, NY) for 20 minutes at 200 × g. Recovered interface cells were washed twice in RPMI 1640 and resuspended in C/10. Splenocytes were prepared as described above. Extracted cells were stimulated with 25 μmol/L ionomycin (Sigma) and 10 ng/ml phorbol myristate acetate (Sigma) for 4 hours at 37°C in a 5% CO2 humidified atmosphere, in the presence of 10 μg/ml brefeldin A (Sigma) to block cytokine secretion. Cells were then fixed at room temperature for 10 to 15 minutes with 4. paraformaldehyde in PBS and washed twice with PBS. The cells were permeabilized with a saponin buffer (0.5% saponin and 1% bovine serum albumin in PBS) and incubated with CD16/CD32 mAb (PharMingen) to block Fc receptors, thereby reducing background staining. For intracellular cytokine staining, biotin-labeled anti-IL-4, anti-IL-10, and anti-IFN-γ mAbs or isotype-matched control antibody was used in a final concentration of 10 μg/ml. After 30 minutes staining at room temperature and two washes with saponin buffer, the cells were incubated with allophycocyanin (APC)-conjugated streptavidin for a further 30 minutes. After two washes with saponin buffer, the cells were washed with PBS, allowing the membranes to reseal, and surface staining was performed using anti-CD11b fluorescein isothiocyanate (FITC) (macrophage marker), anti-CD8-phycoerythrin (PE) and anti-CD4. peridinin chlorophyll protein (PerCP).27O'Garra A Murphy K Role of cytokines in determining T cell function.in: Weir DM Weirs Handbook of Experimental Immunology. Vol. 4. Blackwell Science, Cambridge, MA1996: 226.1-226.10Google Scholar Flow cytometry was performed on a four-color FACScan flow cytometer (Becton-Dickinson, Mountain View, CA), using CellQuest software. For each sample, the threshold was adjusted to 5% for background staining in the isotype-matched control antibody staining of the same sample. The percentage of positively stained population for each cytokine was calculated by subtracting 5 from the percentage of the cells in the positive range.27O'Garra A Murphy K Role of cytokines in determining T cell function.in: Weir DM Weirs Handbook of Experimental Immunology. Vol. 4. Blackwell Science, Cambridge, MA1996: 226.1-226.10Google Scholar, 28Stinn JL Taylor MK Becker G Nagano H Hasegawa S Furukawa Y Shimizu K Libby P Mitchell RN IFN-γ-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry.Am J Pathol. 1998; 153: 1383-1392Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Hsu SM Raine L Fanger H The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics.Am J Clin Pathol. 1981; 75: 816-821Crossref PubMed Scopus (920) Google Scholar Values for parenchymal rejection and GAD scores, IFN-α concentration in the supernatants, and proliferative response in MLR are expressed as the mean ± SD. Statistical analyses of parenchymal rejection and GAD scores and proliferative response in MLR were performed by analysis of variance followed by Fisher's probable least-squares difference post hoc test. Values for IFN-α concentration in the supernatants were analyzed by Student's t test. P < 0.05 was considered statistically significant. Figure 1 summarizes parenchymal rejection and GAD scores; representative histology is shown in Figure 2. All allografts continued beating until harvest at 8 weeks. Compared with grafts in the control PBS-treated B6 recipients, there was a dose-dependent increase in parenchymal rejection scores in grafts in IL-10-treated hosts, achieving statistical significance at the highest concentration of IL-10 (Figure 1A). Grafts in the IFN-γKO recipients had significantly lower parenchymal rejection than those in the B6 recipients, and weekly injection of anti-10, using a protocol previously identified by Schering-Plough to block IL-10 activity, did not influence the scores. Similar results were found for GAD; allografts in the highest dose of IL-10-treated recipients showed a significant increase of GAD compared with the PBS-treated recipients. Anti-IL-10 treatment did not attenuate or accentuate GAD in wild-type hosts. As shown previously, grafts in IFN-γKO recipients had negligible GAD;20Salomon RN Hughes CC Schoen FJ Payne DD Pober JS Libby P Human coronary transplantation-associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells.Am J Pathol. 1991; 138: 791-798PubMed Google Scholar treatment with anti-IL-10 mAb did not promote GAD development. Although IL-10 treatment exacerbated parenchymal rejection and GAD in wild-type hosts, no significant difference was observed in transplanted hearts between recipients treated with anti-IL-10 or control rat IgG (Figure 1B).Figure 2Representative sections of bm12 allografts explanted from either B6 wild-type recipients or IFN-γKO recipients 8 weeks after transplantation. A–D: H&E staining; E–H: Elastic tissue staining. Note that bm12 allografts from B6 recipients show multifocal mononuclear infiltrates (A and B), whereas the graft from IFN-γKO recipients show reduced parenchymal rejection (C and D); coronary vessels of bm12 allografts from B6 recipients show well-developed GAD (E and F), and, in contrast, the vessels of grafts from IFN-γKO recipients show no sign of GAD (G and H). Scale bar = 50 μm.View Large Image Figure ViewerDow
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