Leukocyte-Suppressing Influences of Interleukin (IL)-10 in Cardiac Allografts
1998; Elsevier BV; Volume: 153; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65737-9
ISSN1525-2191
AutoresAnne Räisänen‐Sokolowski, Troels Glysing-Jensen, Mary E. Russell,
Tópico(s)Viral Infections and Immunology Research
ResumoTo investigate the role of interleukin (IL)-10 in late graft outcomes, we compared BALB/c donor hearts transplanted into immunosuppressed wild-type or IL-10 gene-deficient (−/−) C57BL recipients (n = 49) at 50 ± 5 days. There was prominent leukocyte infiltration and parenchymal destruction with more severe vascular occlusion in grafts from IL-10 −/− recipients. An occlusive CD45+ arteritis with medial necrosis occurred with IL-10 deficiency instead of the α-smooth muscle actin-rich arteriosclerosis seen in wild-type recipients. Increased interferon (IFN)-γ as well as Mac-1, inducible nitric oxide synthase, and allograft inflammatory factor-1 (but not CD3 and IL-4) transcript levels were seen in allografts from IL-10 −/− recipients as assessed by 32P reverse transcription polymerase chain reaction. We then evaluated the contribution of IFN-γ-mediated responses by neutralizing IFN-γ. Anti-IFN-γ monoclonal antibody (MAb) treatment of IL-10 −/− recipients did not improve graft survival, parenchymal rejection, or occlusive arteritis, indicating that these processes are IFN-γ independent. However, medial smooth muscle cell loss in IL-10 −/− recipients was attenuated by anti-IFN-γ MAb. Hence, in this transplant model, IL-10 suppresses T cell and macrophage responses in the parenchyma and vasculature and confers a protective effect against late rejection. To investigate the role of interleukin (IL)-10 in late graft outcomes, we compared BALB/c donor hearts transplanted into immunosuppressed wild-type or IL-10 gene-deficient (−/−) C57BL recipients (n = 49) at 50 ± 5 days. There was prominent leukocyte infiltration and parenchymal destruction with more severe vascular occlusion in grafts from IL-10 −/− recipients. An occlusive CD45+ arteritis with medial necrosis occurred with IL-10 deficiency instead of the α-smooth muscle actin-rich arteriosclerosis seen in wild-type recipients. Increased interferon (IFN)-γ as well as Mac-1, inducible nitric oxide synthase, and allograft inflammatory factor-1 (but not CD3 and IL-4) transcript levels were seen in allografts from IL-10 −/− recipients as assessed by 32P reverse transcription polymerase chain reaction. We then evaluated the contribution of IFN-γ-mediated responses by neutralizing IFN-γ. Anti-IFN-γ monoclonal antibody (MAb) treatment of IL-10 −/− recipients did not improve graft survival, parenchymal rejection, or occlusive arteritis, indicating that these processes are IFN-γ independent. However, medial smooth muscle cell loss in IL-10 −/− recipients was attenuated by anti-IFN-γ MAb. Hence, in this transplant model, IL-10 suppresses T cell and macrophage responses in the parenchyma and vasculature and confers a protective effect against late rejection. Interleukin (IL)-10 is a pleiotrophic cytokine that can be produced by T cells, B cells, macrophages, mast cells, and keratinocytes.1Abbas A Lichtman A Pober J Cellular and Molecular Immunology. ed 2. WB Saunders, Philadelphia1994Google Scholar Reports of immunoregulatory properties of IL-10 include down-regulation of Th1-type cytokine production, suppression of macrophage and natural killer cell effector functions, and stimulation of B cell differentiation and immunoglobulin production.1Abbas A Lichtman A Pober J Cellular and Molecular Immunology. ed 2. WB Saunders, Philadelphia1994Google Scholar The role of IL-10 in transplantation has been widely debated with contrasting results among the different experimental systems studied.2Bromberg JS IL-10 immunosuppression in transplantation.Curr Opin Immunol. 1995; 7: 639-643Crossref PubMed Scopus (66) Google Scholar Elevated IL-10 expression in human and rodent allografts undergoing acute rejection suggested that IL-10 promotes alloimmune destruction.2Bromberg JS IL-10 immunosuppression in transplantation.Curr Opin Immunol. 1995; 7: 639-643Crossref PubMed Scopus (66) Google Scholar However, up-regulated expression of IL-10 in allografts from tolerant, long-surviving recipients had led some to speculate that IL-10 may promote allograft survival.2Bromberg JS IL-10 immunosuppression in transplantation.Curr Opin Immunol. 1995; 7: 639-643Crossref PubMed Scopus (66) Google Scholar Studies where IL-10 levels have been manipulated in transplantation models have not resolved the confusion. Systemic administration of IL-10-Fc fusion protein accelerated graft failure in islet cell allografts,3Zheng X Steele A Nickerson P Steurer W Steiger J Strom T 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 whereas pancreatic islet grafts overexpressing murine IL-10 had similar survival time to wild-type allografts.4Lee M-S Wogensen L Shizuru J Oldstone M 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 Retrovirus-mediated transfer of viral IL-10 gene into nonvascularized neonatal heart transplants prolonged graft survival.5Qin 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, in that study, murine IL-10 had no survival benefit.5Qin 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 The effect of systemic recombinant human IL-10 in a mouse heart transplant model appeared to depend on dosing and timing.6Lowry RP Konieczny B Alexander D Larsen C Pearson T Smith S Narula S Interleukin-10 eliminates anti-CD3 monoclonal antibody-induced mortality and prolongs heart allograft survival in inbred mice.Transplant Proc. 1995; 27: 392-394PubMed Google Scholar Daily injection of a high dose (100 μg/day) initiated on day 1 before the grafting shortened graft survival, whereas a lower dose (50 μg/day) did not alter it. If IL-10 was given only perioperatively (days −1, 0, +1; 50 μg/day), graft survival was improved. Hence, the efficacy of IL-10 manipulations (methods, doses, and timing) coupled with differences in transplant microenvironment may explain the inconsistent effects seen in graft survival to date. By using mice with targeted gene deletion as recipients, we have recently shown that the presence of IL-10 is protective in a heterotopic cardiac mouse transplant model of late or attenuated rejection.7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar After a 30-day course of T-cell-depleting immunosuppression, IL-10 −/− recipients rejected heterotopic mouse cardiac allografts twice as rapidly as wild-type controls.7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar Grafts from IL-10 −/− recipients had prominent mononuclear cell infiltration, myocyte loss, and fibrosis. Hence, this earlier study demonstrated that when present, IL-10 had a suppressive influence on the alloimmune response that culminates in graft failure. A number of mechanisms might be invoked to explain our findings. The original reports describing the phenotype of IL-10 −/− mice indicated augmented cell-mediated immune responses consistent with loss of suppressing influences.8Kühn R Löhler J Rennick D Rajewsky K Müller W Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3688) Google Scholar Although the IL-10 knockout mice appeared normal at birth, a chronic inflammatory bowel disease developed with age (especially if maintained in conventional animal facilities).9Rennick DM Fort MM Davidson NJ Studies with IL-10−/− mice: an overview.J Leukocyte Biol. 1997; 61: 389-396Crossref PubMed Scopus (260) Google Scholar The chronic enterocolitis involved large numbers of infiltrating mononuclear cells, including macrophages and Th1-type T cells in the bowel. This indicated that IL-10-deficient mice had an aggravated leukocyte response to gut flora present in the conventional facility.8Kühn R Löhler J Rennick D Rajewsky K Müller W Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3688) Google Scholar Since then, IL-10 −/− mice have been studied after other microbial challenges. Allergic bronchopulmonary aspergillosis, enterocolitis, andToxoplasma gondii and Trypanosoma cruziinfections showed increased mortality and morbidity in IL-10 −/− mice, whereas Listeria monocytogenes-infected mice were resistant to infection.10Berg DJ Leach MW Kuhn R Rajewsky K Muller W Davidson NJ Rennick D Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar, 11Grunig G Corry DB Leach MW Seymour BW Kurup VP Rennick DM Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.J Exp Med. 1997; 185: 1089-1099Crossref PubMed Scopus (267) Google Scholar, 12Berg DJ Davidson N Kuhn R Muller W Menon S Holland G Thompson-Snipes L Leach MW Rennick D Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses.J Clin Invest. 1996; 98: 1010-1020Crossref PubMed Scopus (971) Google Scholar, 13Dai W Köhler G Brombacher F Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10 deficient mice.J Immunol. 1997; 158: 2259-2267PubMed Google Scholar, 14Gazzinelli RT Wysocka M Hieny S Scharton-Kersten T Cheever A Kuhn R Muller W Trinchieri G Sher A In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-γ, and TNF-α.J Immunol. 1996; 157: 798-805PubMed Google Scholar, 15Hunter CA Ellis-Neyes LA Slifer T Kanaly S Grunig G Fort M Rennick D Araujo FG IL-10 is required to prevent immune hyperactivity during infection with Trypanosoma cruzi.J Immunol. 1997; 158: 3311-3316PubMed Google Scholar The altered immune response was typically associated with increased production of pro-inflammatory cytokines, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-12. Findings in these infection models suggested that aggravated Th1-type responses develop when leukocyte-suppressive effects of IL-10 are lost. This could convert protective immunity to a pathological response that ultimately leads to tissue destruction.9Rennick DM Fort MM Davidson NJ Studies with IL-10−/− mice: an overview.J Leukocyte Biol. 1997; 61: 389-396Crossref PubMed Scopus (260) Google Scholar Less is known about the molecular mechanism(s) through which IL-10 alters the alloimmune response. Intragraft cytokine analysis of hearts placed in IL-10 −/− recipients in our earlier study showed that early graft failure was associated with increased expression of IFN-γ.7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar We have also recently shown that IFN-γ has a role in promoting graft arteriosclerosis.16Räisänen-Sokolowski A Glysing-Jensen T Koglin J Russell M Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-γ knockout recipients.Am J Pathol. 1998; 152: 359-365PubMed Google Scholar In cardiac allografts placed in IFN-γ −/− recipients receiving T-cell-depleting immunosuppression, severity and frequency of vascular occlusion were significantly reduced as well as myointimal smooth muscle cell accumulation. Taken together, these findings suggest that augmented or accelerated Th1-type responses and interrelated macrophage activation might also promote graft loss in the setting of recipient IL-10 gene deletion. To gain more insight about how IL-10 protects the graft from rejection, we compared graft outcomes in IL-10 −/− recipients with wild-type recipients. Because graft failure was evident in IL-10 −/− recipients at 55 days in our earlier study,7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar we harvested grafts from wild-type recipients at this time point to have a time-matched control group and analyzed vascular as well as parenchymal features. Second, we addressed the contribution of IFN-γ-mediated Th1 responses to cardiac rejection in IL-10 −/− recipients. Our approach was to determine whether neutralization of IFN-γ with anti-IFN-γ monoclonal antibody (MAb) treatment in IL-10 −/− recipients would alter graft outcomes (survival, vascular and parenchymal histology, and inflammatory cell activation). BALB/cByJ (H-2d) donors were used for heterotopic cardiac transplantation17Corry RJ Winn HJ Russell PS Primarily 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 (785) Google Scholar into 6- to 8-week-old C57BL (H-2b) recipients that were either wild type or had targeted gene disruption in IL-10 (C57BL/10J).8Kühn R Löhler J Rennick D Rajewsky K Müller W Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3688) Google Scholar IL-10 −/− mice had been backcrossed seven times to C57BL background. All animals were maintained in a specific-pathogen-free (SPF) animal facility and appeared healthy at the time of the transplantation and harvest. The targeted gene disruption was confirmed using triple polymerase chain reaction (PCR) assays that amplify a portion of the neomycin cassette and a portion of the targeted exon as recommended by Jackson Laboratories (www.jax.org). All mice were purchased from Jackson Laboratories (Bar Harbor, ME). To attenuate acute rejection, we treated recipients with MAb against CD4 (clone GK1.5, rat IgG2b; American Type Culture Collection (ATCC), Rockville, MD) and CD8 (clone 2.43, rat IgG2b; ATCC) for days 1 to 4, 7, 14, 21, and 28 after transplantation at the dose of 500 μg/day/mouse of each MAb as previously described.7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar We have previously shown that a gradual repopulation of T cells occurs after the cessation of the a 30-day course of anti-CD4 and anti-CD8 treatment. At day 55 after transplantation, flow cytometry of splenocytes demonstrated that CD4+ T cells were 48% of normal level and CD8+ T cells were 15% of normal level.16Räisänen-Sokolowski A Glysing-Jensen T Koglin J Russell M Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-γ knockout recipients.Am J Pathol. 1998; 152: 359-365PubMed Google Scholar Hence, the animals were immunosuppressed at the time of harvest. Graft function was evaluated by regular palpation graded on a scale from 4 (functioning well) to 0 (no heart beat).17Corry RJ Winn HJ Russell PS Primarily 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 (785) Google Scholar Graft survival was defined as days after transplantation with palpation score of ≥1. Wild-type controls were harvested electively at 55 days after transplantation to serve as time-matched controls for grafts placed in IL-10 −/− recipients that began to fail by this time point. In our previous study,7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar we compared wild-type and knockout allografts at a comparable functional endpoint, ie, when graft function decreased. Two of seventeen allografts placed in IL-10 −/− recipients treated with anti-IFN-γ MAb rejected at 33 days after transplantation and were not included for further histological analysis. Hearts were collected and sections of grafts processed for the evaluation of histology and RNA extraction as previously described.18Räisänen-Sokolowski A Glysing-Jensen T Mottram P Russell M Sustained anti-CD4/CD8 blocks inflammatory activation and intimal thickening in mouse heart allografts.Arterioscler Thromb Vasc Biol. 1997; 17: 2115-2122Crossref PubMed Scopus (60) Google Scholar To determine whether IFN-γ-mediated Th1 forces contribute to the accelerated rejection in IL-10 −/− recipients, a subgroup of IL-10 −/− recipients received anti-IFN-γ MAb (clone R4–6A2, rat IgG1; ATCC). All ascites was purified in a protein G column as previously described18Räisänen-Sokolowski A Glysing-Jensen T Mottram P Russell M Sustained anti-CD4/CD8 blocks inflammatory activation and intimal thickening in mouse heart allografts.Arterioscler Thromb Vasc Biol. 1997; 17: 2115-2122Crossref PubMed Scopus (60) Google Scholar and administered days −1 and 1 and then biweekly until harvest (2 mg/mouse/week, intraperitoneally). Serum was collected from each mouse at the time of the harvest. An indirect enzyme-linked immunosorbent assay (ELISA) was used to measure anti-IFN-γ MAb (rat IgG1) levels in anti-IFN-γ MAb-treated animals. Briefly, a 96-well plate was coated with recombinant mouse IFN-γ (1.0 μg/ml; Genzyme, Cambridge, MA) in PBS (pH 9.0) overnight at room temperature. Additional binding sites were blocked with blocking buffer (0.017 mol/L Na2B4O7, 0.12 mol/L NaCl, 0.05% TWEEN 20, 1 mmol/L EDTA, 0.25% bovine serum albumin, 0.05% NaN3) for 30 minutes. Anti-IFN-γ MAb standards (range, 3 to 800 ng/ml) and mouse sera samples (dilution, 1:100 to 1:30,000) in triplicate were diluted in blocking buffer, applied to the wells, and incubated overnight at room temperature. Bound primary antibody was detected using a secondary antibody rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) followed by application of avidin-biotin complex (Vector Laboratories) according to the manufacturer's instructions. Horseradish peroxidase label was detected using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (ABTS substrate kit, Vector Laboratories). Negative controls included serum from naive IL-10 −/− mice and from transplanted wild-type mice that had received only anti-CD4/CD8 therapy. Final concentrations of anti-IFN-γ MAb in serum were derived from a standard curve. Anti-IFN-γ MAb concentrations in anti-IFN-γ MAb-treated IL-10 −/− recipients ranged from 59 to 425 μg/ml (mean, 198 ± 131 μg/ml; n = 13) in serum at the time of harvest. Activation of a murine macrophage cell line (RAW264.7, TIB 71; ATCC) by IFN-γ was used to test recipient sera at the time of harvest for anti-IFN-γ activity. RAW264.7 cells become activated after IFN-γ stimulation and produce nitric oxide products (nitrates and nitrites) that can be measured using the Griess reagent after treatment with nitrate reductase (colorimetric nitric oxide assay kit, Oxford Biomedical Research, Oxford, MI). All samples and control MAbs were assayed in triplicate, and the mean was derived. Figure 1 depicts how serum (1:10 dilution) from anti-IFN-γ MAb-treated IL-10 −/− recipients inhibited IFN-γ-mediated NO production in a dilution-dependent fashion to 15% of the control (recombinant IFN-γ-stimulated (100 U/ml) with no serum) level. Serum from nontransplanted IL-10 −/− animals and irrelevant MAb (anti-ICAM-1, clone YN1/1.7.4, rat IgG2a; ATCC) did not reduce nitric oxide products. These findings confirm the neutralizing capacity of anti-IFN-γ MAb in the recipient at time of harvest. The degree of acute rejection was evaluated and graded from paraffin sections using a modification of the International Society for Heart and Lung Transplantation (ISHLT) criteria.7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar, 19Billingham ME Nathaniel RB Cary NRB 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.J Heart Transplant. 1990; 9: 587-593PubMed Google Scholar Coded samples were evaluated by two observers. The scale was from 0 to 4: 0, no rejection; 1, mild focal (A) or diffuse (B) perivascular and interstitial infiltration with no parenchymal necrosis; 2, moderate, unifocal infiltration with/without focal myocyte injury; 3, moderate multifocal (A) or diffuse (B) infiltration with myocyte injury; 4, severe rejection with aggressive, diffuse infiltration, edema, myocyte necrosis, hemorrhage, and vasculitis. CD45 MAb (clone 30F11.1, leukocyte common antigen (LCA), Ly-5; PharMingen, San Diego, CA) was used to identify leukocytes within the grafts as previously described.20Shi C Russell ME Bianchi C Newell JB Haber E Murine model of accelerated transplant arteriosclerosis.Circ Res. 1994; 75: 199-207Crossref PubMed Scopus (79) Google Scholar Smooth muscle cell (SMC) accumulation, indicative of more advanced arteriosclerotic stages, was estimated by immunostaining for α-smooth muscle actin and desmin. Twenty-six grafts were stained using the previously described protocol with minor modifications.16Räisänen-Sokolowski A Glysing-Jensen T Koglin J Russell M Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-γ knockout recipients.Am J Pathol. 1998; 152: 359-365PubMed Google Scholar Briefly, Verhoeff's elastin-stained paraffin sections were blocked with 10% normal goat sera and then stained with α-smooth muscle actin antibody (clone 1A4, dilution 1:20,000; Sigma Chemical Co., St. Louis, MO) overnight at 4°C and detected using the avidin-biotin complex and 3-amino-9-ethylcarbazole substrate (Vectastain ABC kit, AEC-kit; Vector Laboratories) as described previously.16Räisänen-Sokolowski A Glysing-Jensen T Koglin J Russell M Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-γ knockout recipients.Am J Pathol. 1998; 152: 359-365PubMed Google Scholar Anti-desmin immunostaining was also performed on selected samples (n = 3) to confirm the presence or absence of neointimal and medial SMCs according to the manufacturer's instructions. Anti-human desmin MAb coupled with horseradish peroxidase (clone D33; Dako, Glostrup, Denmark) reacts with 53-kd intermediate filament protein in muscle cells. The reagent labels both striated (skeletal and cardiac) cells and SMCs and shows a broad interspecies cross-reactivity. The severity of disease (percentage of luminal occlusion) was analyzed in Verhoeff's elastin-stained sections from each graft.18Räisänen-Sokolowski A Glysing-Jensen T Mottram P Russell M Sustained anti-CD4/CD8 blocks inflammatory activation and intimal thickening in mouse heart allografts.Arterioscler Thromb Vasc Biol. 1997; 17: 2115-2122Crossref PubMed Scopus (60) Google Scholar, 21Russell ME Hancock WW Akalin E Wallace AF Glysing-Jensen T Willett T Saygeh MH Chronic cardiac rejection in the Lewis to F344 rat model. Blockade of CD28–B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis.J Clin Invest. 1996; 97: 833-838Crossref PubMed Scopus (161) Google Scholar Microscopic images of each elastin-stained vessel cross section (n = 692) were captured, and the percentage of luminal occlusion was tabulated by tracing the internal elastic lamina and the lumen with the ScionImage 1.60 software (National Institutes of Health, Bethesda, MD). The mean value for each individual graft was tabulated, and the mean ± SE for each group was reported. Image analysis using ScionImage 1.60 (National Institutes of Health) was performed to measure percent area of α-smooth muscle actin positivity within the neointima and media.16Räisänen-Sokolowski A Glysing-Jensen T Koglin J Russell M Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-γ knockout recipients.Am J Pathol. 1998; 152: 359-365PubMed Google Scholar Only larger vessels (area delineated by internal elastic lamina >350 μm2) with greater than 40% luminal occlusion were of sufficient resolution for measurement. The area staining for α-smooth muscle actin was determined by measuring the pixel area displaying the color intensity of immunopositive cells. Reverse transcriptase (RT)-PCR provides a global measurement of inflammatory markers in a more quantitative manner than immunostaining of individual microscopic sections. To estimate the contribution of T cells and macrophages, we elected to measure corrected CD3 and Mac-1 transcript levels because the low resolution of immunostaining in frozen sections often precludes quantitation. Hence, to measure relative differences in transcript levels between cardiac transplants we used a semiquantitative 32P-RT-PCR technique published in detail previously.18Räisänen-Sokolowski A Glysing-Jensen T Mottram P Russell M Sustained anti-CD4/CD8 blocks inflammatory activation and intimal thickening in mouse heart allografts.Arterioscler Thromb Vasc Biol. 1997; 17: 2115-2122Crossref PubMed Scopus (60) Google Scholar, 21Russell ME Hancock WW Akalin E Wallace AF Glysing-Jensen T Willett T Saygeh MH Chronic cardiac rejection in the Lewis to F344 rat model. Blockade of CD28–B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis.J Clin Invest. 1996; 97: 833-838Crossref PubMed Scopus (161) Google Scholar, 22Russell ME Wallace AF Hancock WW Sayegh MH Adams DH Sibinga NES Wyner LR Karnovsky MJ Upregulation of cytokines associated with macrophage activation in the Lewis to F344 rat chronic cardiac rejection model.Transplantation. 1995; 59: 572-578Crossref PubMed Scopus (120) Google Scholar Total RNA from 20 grafts was quantitated and reverse transcribed to cDNA simultaneously to generate a cDNA panel that allowed comparison among different groups. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a reference gene to normalize variations in cDNA or total RNA loading between samples. Transcript analysis was completed for CD3, Mac-1, IFN-γ, IL-4, TNF-α, inducible nitric oxide synthase (iNOS), and allograft inflammatory factor (AIF)-1. Primer sequences and PCR conditions were previously described7Räisänen-Sokolowski A Mottram P Glysing-Jensen T Satoskar A Russell M Heart transplants in interferon-γ, interleukin 4, and interleukin 10 knockout mice: recipient environment alters graft rejection.J Clin Invest. 1997; 100: 2449-2456Crossref PubMed Scopus (66) Google Scholar, 18Räisänen-Sokolowski A Glysing-Jensen T Mottram P Russell M Sustained anti-CD4/CD8 blocks inflammatory activation and intimal thickening in mouse heart allografts.Arterioscler Thromb Vasc Biol. 1997; 17: 2115-2122Crossref PubMed Scopus (60) Google Scholar except for the following: G3PDH, sense primer 5′-CAT CAA GAA GGT GGT GAA GCA GGC-3′ and antisense primer 5′-TTG TGA GGG AGA TGC TCA GTG TTG G-3′, with an annealing temperature of 58°C for 22 cycles; Mac-1, sense primer 5′-CAG AGG CTG TGA ATA TGT CCT TGG-3′ and antisense primer 5′-GTC ATT GAA GGT GAA GTG AAT CCG-3′, with an annealing temperature of 53°C for 28 cycles; AIF-1, sense primer 5′-GAC AGA CTG CCA GCC TAA GAC ACC-3′ and antisense primer 5′-CCA AGT TTC TCC TGC AGC ATT CGC-3′, with an annealing temperature of 60°C for 28 cycles; and macrophage lectin, sense primer 5′-TCA AGA ACA ACG GCT CGG AAG TG-3′ and antisense primer 5′-GAC ATC ATC ATT CCA GGG ACC ACC-3′, with an annealing temperature of 57°C for 30 cycles. The mean was obtained from triplicate analyses of the same cDNA. Corrected values were derived by dividing the measured incorporated 32P for the transcript of interest by the mean G3PDH value for the cDNA. Means ± SEM per group are reported. The product limit (Kaplan-Meier) estimate of the cumulative survival was assessed with the Breslow-Gehan-Wilcoxon test to evaluate significant differences in graft survival.23Brown M Engelman L Frane J Hill M Jennrich R Toporek J BMDP Statistical Software, 1983 printing with additions.in: Dixon W University of California Press, Berkeley1983Google Scholar The histological parameters and RT-PCR data were subjected to multiple analysis of variance without replication (StatView 4.5, Abacus Concepts, Berkeley, CA). If multiple analysis of variance was significant, individual comparisons were made by the Student'st-test, and the level of significance was corrected by the Bonferroni method.24Russell ME Adams DH Wyner LR Yamashita Y Halnon NJ Karnovsky MJ Early and persistent indu
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