Injury to the donor organ prior to transplantation leads to the activation of the innate immune system which precedes and contributes to the activation of the adaptive alloimmune response (1). Brain death-associated hemodynamic, hormonal and molecular changes, the procurement procedure and ischemia-reperfusion injury promote cellular damage and activation of the innate immune response linked to the expression of toll-like receptors (2–7). Dendritic cells (DCs) connect the innate and the adaptive immune responses by providing the “second signal” to complete the alloresponse (1).
It has been proposed that graft survival may be substantially enhanced by minimizing initial cellular stress. Treatments that protect grafts and maintain its functionality prior to transplantation are receiving increased attention (3, 8–11).
Tissues exposed to stressful stimuli up-regulate protective genes (12). One of these genes encodes heme oxygenase-1 (HO-1) which has been associated with cytoprotection and a reduced alloresponse. Previous studies have shown that intragraft induction of the protective gene HO-1 is associated with increased graft survival (13). More recently, it has been shown that carbon monoxide (CO), a product of HO-1 metabolism, has anti-inflammatory properties and reproduces the protective effects of HO-1 induction (14). In the current experiment, using both low and high responder rat strain combinations, we tested whether treatment of kidney donors with CO may reduce graft immunogenicity and subsequent chronic graft deterioration.
MATERIALS AND METHODS
Effects of CO induction were tested both in a low (F-344/RT11v1→LEW; RT1 ) and high responder (DA/RTlavl→LEW) strain combination. Animals were bred at Harlan Winkelmann, Germany, aged 10–12 wks and weighted 220–250 g. Standard rodent chow and water were fed ad libitum and animals were cared for according to the National Institutes of Health guidelines for laboratory animal care. Permission for animal experiments was approved by the local authorities (G 0280/01).
Donor animals were either treated with methylene chloride (MC 100 mg/kg p.o.) four hr prior to organ harvesting or remained untreated. Grafts were perfused with UW solution and stored for six hr at 4°C. Following a unilateral nephrectomy in recipient animals, renal allografts were transplanted orthotopically under isoflurane anesthesia using standard microsurgical techniques (n=6). Time for vessel anastomosis averaged 25±5 min. The remaining contralateral native kidney in recipients was removed after a 10 days interval. Unilateral nephrectomized and age matched native animals were followed in parallel. Both, recipients and native controls received Cyclosporine A (CyA, Novartis, Switzerland; 1.5 mg/kg/day i.m. × 10 days) and were followed for 24 weeks. Early graft changes were tested in additional groups 24 hr following transplantation and compared to untreated controls for changes of gene expression by real time-PCR (n=4/group).
DC frequency, migration and alloreactivity were tested in the DA (RTlavl) → LEW (RTll) combination (n=4/group) as the low responder F-344→ LEW strain combination does not allow the detection of donor-derived DCs. Major histocompatibility complex (MHC)-class II + Donor-derived dendritic cells were determined by haplotype-specific monoclonal antibodies (mAb) and flow cytometry (RT1ab, OX62+). T-cell alloreactivity of recipient splenocytes was measured by ELISPOT. Analysis of grafts, spleens, perigraft lymph nodes, and blood of LEW recipients were performed by 24 hr in this model.
Carbon Monoxide Induction
Methylene chloride (Supelco, USA) was administered to deliver CO. MC was diluted in olive oil at a concentration of 62.5 mg/ml and given orally by gavage, at a dose of 100 mg/kg. MC is catabolized mainly by the cytochrome P-450 oxidative system in the liver producing (>98%) CO and CO2.
Carboxyhemoglobin (COHb) Determination
CO was measured spectrophotometrically by determination of COHb as a percentage of total hemoglobin in capillary blood, using a blood gas analyzer (Radiometer ABS 520, Copenhagen, Denmark). COHb levels were determined at baseline and 2, 4, 6, 12, and 24h after MC administration.
Urine was collected at two weeks, four weeks, and monthly intervals thereafter in metabolic cages over 24 hr. Urinary protein excretion which has been shown previously as the most sensitive marker of chronic allograft deterioration in this model was measured monthly by precipitation with 20% CCl3COOH. Turbidity was assessed at a wavelength of 415 nm using a Hitachi 911 analyzer.
Specimens were fixed in 4% buffered formalin and embedded in paraffin. Graft sections (4 μm) were stained with hematoxylin & eosin (H&E). Blinded sections were graded for overall severity of histopathological abnormalities using a semiquantitative scale. To determine the extent of glomerulosclerosis, >20 high power fields were evaluated at 400×; the ratio of sclerosed glomeruli/sections was examined and expressed as a percentage. The severity of chronic interstitial, tubular, and arteriosclerotic changes was graded using a semiquantitative scale from 0 to 4+ based on the percentage of parenchymal involvement, with zero representing a normal morphology, one representing less than 25%, two representing 25–50%, three representing 50–75%, and four representing 75–100% of the total parenchymal involved (more than 20 high-power fields were evaluated in each specimen and the mean per kidney section was calculated).
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
RT-PCR were performed using the 5′ nuclease activity of Taq polymerase to cleave a nonextendable hybridization probe during the extension phase of the PCR (n=5/experiment). The approach uses dual-labeled fluorogenic hybridization probes. One fluorescent dye serves as a reporter (FAM, 6-carboxy-fluorescein) and its emission spectrum is quenched by the second fluorescent dye (TAMRA, 6-carboxy- tetramethylrhodamine). During the extension phase of the PCR cycle, the probe is cleaved by the 5′-3′ nucleolytic activity of the DNA polymerase. The diminished energy transfer results in an increase of the reporter fluorescent emission intensity. During the early cycles of the PCR amplification, the reporter emission remains at baseline, increasing following cleaving by the Taq polymerase. The amplification plot is examined early in the reaction representing a log phase of product accumulation. Once the threshold is chosen, the point at which the amplification plot crosses the threshold is defined. For quantification, Ct is correlated to the constant expression of a housekeeping gene and then defined as ΔCt=Ct (gene of interest) – Ct (housekeeping gene); values are plotted as 2−ΔCt.
Flow Cytometry (Fluorescence-Activated Cell Sorter Analysis)
Heparinized blood, spleen, and graft sections from LEW recipients receiving DA kidney grafts were obtained by day one. Peripheral blood mononuclear cells (PBMC), splenocytes and graft infiltrating cells were isolated by a standard procedure using Pancoll density gradient centrifugation (density, 1.091 g/ml; Pan Biotech GmbH, Aidenbach, Germany). After isolation, PBMC were resuspended in RPMI 1640 containing 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin at a concentration of 5×106 cells/ml; 400-μL aliquots of the cell suspension were distributed for staining. MHC class II + donor-derived DCs were determined by haplotype-specific mAb (RTlab, OX62+). The following mouse anti-rat monoclonal antibodies were used: OX-62, detecting DCs, Fluorescein isothiocyanate (FITC)-conjugated anti-rat (DA) RT1a,b specific for the DA rat (both from BD PharMingen GmbH), and appropriate controls (corresponding immunoglobulin G-negative isotypes; Biosource Europe S.A., Nivelles, Belgium). Stained and fixed lymphocyte samples were analyzed with a FACScalibur (Becton Dickinson, Palo Alto, CA), and CELLQuest software was used for analysis. The cytometer was calibrated with CaliBRITE1 beads (Becton Dickinson) using FACSComp software and with QC-3 beads according to the manufacturer's recommendations (Flow Cytometry Standards Corp., San Juan, Puerto Rico). In all, 500,000 events were acquired.
Alloreactive ELISPOT Assay (Alloreactive Enzyme-Linked Immunospot)
Following the isolation and preparation of rat spleen cells, PVDF-bottomed, 96-well microtiter plates (Diaclone Besançon Cedex, France) were incubated with 70% ethanol for 10 min. This procedure was followed by the incubation with an anti-rat IFN-γ capture antibody for 12 hr, an additional washing step with phosphate-buffered saline (PBS) (Dulbecco's PBS; PAA Laboratories GmbH, Linz, Austria) and blocking of nonspecific binding by 2% skimmed dry milk in PBS for two hr. Subsequently, Roswell Park Memorial Institute medium (negative control) or phorbol myristate acetate (2 ng/ml) and ionomycin (2 μg/ml; both from Sigma Aldrich GmbH, Munich, Germany) in RPMI medium (positive control) or 5×105 stimulator cells (DA) were dispensed carefully into the wells. Thereafter, 5×105 responder cells (spleen cells of LEW rats) were added. Plates were incubated for 24 hr, washed three times with PBS containing 0.1% Tween 20 (Tween 20 pure; Serva Biochemica, Heidelberg, Germany), and then incubated with a biotinylated anti-rat IFN-γ detection antibody for 3 hr. After a final 45-min incubation period with a streptavidin-alkaline phosphatase conjugate and subsequent administration of the staining buffer (BCTP/NTB; Diaclone), the plates were dried and measured using the BIOREADER 3000 C/BIOCOUNTER system (BIOREADER 3000 C; BIO-SYS GmbH, Karben, Germany). The actual frequency of alloreactive T cells was determined as the ratio of the number of spots/number of responder cells. This ratio was then adapted to the actual percentage of T cells in the responder cell preparation assessed by flow cytometry (11, 12).
Data are shown as mean ± SD. Statistical significance was ascertained using the one-way ANOVA and Mann-Whitney U test. Differences were considered statistically significant at P value of <0.05. Statistical analysis was performed using SPSS 11.0, (SPSS Inc., Chicago, IL).
COHb levels in untreated DA were measured at 1.3±0.1%. Methylene chloride at 100 mg/kg p.o induced increased carboxyhemoglobin (COHb) levels. COHb levels peaked after two hr following oral application of MC (5.5%± 2.1 vs. controls, n=6) and were slightly maintained above basal levels at 24 hr (2.0±0.1 vs. 0.9±0.3; Figure 1).
Donor Treatment with Carbon Monoxide Improved Kidney Allograft Function Long-term
By 24 weeks, renal function was significantly better in the low responder strain combination (F-344 to Lewis model) in animals receiving an organ from MC treated donors (Proteinuria in recipients receiving untreated grafts: 60±20 mg/24 hr vs. MC treated donor grafts: 15±1 mg/24 hr; P<0.05). Proteinuria in rats receiving grafts from MC treated donors was comparable to that in nontransplanted, uninephrectomized controls (15±1 vs. 16±2, P=0.76; Figure 2).
Long-term Morphological Alterations in Grafts from Carbon Monoxide-treated Donors Were Comparable to Those in Uninephrectomized Nontransplanted Controls
Morphological alterations characteristic of chronic graft deterioration were significantly ameliorated in organs from CO treated F-344 donors and comparable to uninephrectomized controls (glomerulosclerosis by 24 weeks: 20±5% following donor treatment vs. 80±10% in untreated grafted controls (P<0.001) and 17.5±3.23 in native controls; P=0.59) Arteriosclerosis (P<0.01), tubular atrophy (P<0.0001), and fibrosis (P<0.0001) were significantly better in organs from treated donors (Figs. 3 and 4).
Treatment of Donor Animals with Carbon Monoxide Reduced CD3 mRNA Expression Intragraft and the Expression of Proinflammatory Cytokines
LEW recipients of F-344 grafts from MC treated donors demonstrated significantly reduced CD3 mRNA levels (P<0.05). A trend towards a Th1/Th2 shift based on cytokine levels (reduced IL-2 and IFN-γ, and increased IL-10 and IL-4 levels) was observed by 24 hr, although not reaching statistical significance. The expression of HO-1, bcl-2, TNF-α had increased, albeit not significantly (not shown).
Induction of CO Reduced the Frequency of DCs and Modulated Their Migration
As donor-derived DCs can not be detected in the F-344→LEW system, the migration and frequency of DCs were tested in the high responder DA→LEW combination. MC treatment of DA donors reduced the frequencies of donor-derived-DCs after transplantation in all compartments examined. By 24h, donor-derived DCs (RT1ab+/OX62+) were significantly reduced in the graft, as well as in blood and spleen of LEW recipients (P<0.05; Figure 5A). Reduction of donor derived DCs was also associated with reduced frequencies of intragraft CD4+ T-cells in this system (P=0.016; Figure 5B).
Induction of CO Reduced Graft Alloreactivity
Reduced numbers of donor-derived DCs were associated with reduced numbers of donor-reactive T cells by 24h (IFN-γ + cells/million T cells: 54.83±24.96 following donor treatment vs. 208.33±57.76 in controls, P<0.01; Figure 6).
Graft quality has a major impact on both short- and long-term graft outcome (15–18). Events occurring prior to organ transplantation such as brain death, organ procurement and ischemia-reperfusion lead to nonspecific inflammatory damages—“danger signals”—resulting in an increased expression of MHC antigens, proinflammatory cytokines, structural cellular damage (lipid peroxidation) and activation of proapoptotic pathways (19–21). Taken together, these changes may increase graft immunogenicity. Thus, blocking endogenous “alarm signals” may reduce the recipient immune response (1, 3, 10, 18, 20).
Intragraft dendritic cells initiate the immune response after transplantation. MHC class I is expressed on all parenchymal cells while MHC class II is expressed on dendritic cells, B-lymphocytes, monocytes, macrophages, thymic epithelial cells, and lymphocytes. MHC II expression can also be induced in nonprofessional APCs such as endothelial cells, fibroblasts, epithelial cells, keratinocytes, and by various cytokines. It has recently been shown that parenchymal cells are not only targets of the alloimmune response but are also playing an active role in the rejection process (3, 20–22). Tubular epithelial cells express Toll-like receptors potentially contributing to the activation of the immune response after ischemia-reperfusion, a process that mainly affects tubular cells (23). Solid and cellular grafts contain dendritic cells of donor origin in an immature stage (24). DCs are the most effective antigen presenting cells for the activation of an immune response and play an important role in eliciting rejection or inducing tolerance depending on the graft microenvironment (25). Inflammatory signals contribute to the maturation and migration of DCs (26).
Many reports have shown beneficial effects of heme oxygenase-1 (HO-1) induction in transplantation (27). Exogenous administration of CO a down stream product of HO-1 metabolisms may also play an important role in the inhibition or suppression of rejection processes (28). CO has been shown to reduce graft deterioration by preventing ischemia/reperfusion injury (29), inhibiting inflammation and apoptosis (30–34), optimizing microcirculation (35, 36) and reducing arteriosclerosis (37, 38). CO may induce immunomodulation through activation of the enzyme guanylate cyclase (36, 39); inhibition of NO synthase (40), and inhibition of protein kinases (e.g. MKK-3/p38 mitogen-activated protein kinase) (29, 30, 32–34, 41, 42). When CO was administered in recipient animals 1h before and 24h after kidney transplantation, graft survival and renal function improved. Those effects were associated with an inhibition of the initiating steps of immune activation and reduced levels of IL-6, IL-1β, TNF-α, ICAM-1, iNOS, apoptosis, cellular infiltration, tubular edema and necrosis (43). Otterbein et al. showed recently that preemptive use of CO shortly prior to carotid artery angioplasty injury reduced intimal hyperplasia (37).
To reduce graft immunogenicity we used a pro-drug methylene chloride (MC, CH2Cl2), also known as dichloromethane (44–46), for CO induction as a single treatment in donor animals. MC is a halogenated saturated hydrocarbon primarily metabolized in the liver. It is mainly catabolized by the cytochrome P-450 oxidative system and its main metabolites are CO and CO2 (>98% with a 100 mg/kg oral dose). A small amount of MC is eliminated unchanged by lungs and kidneys. The protective effects of MC have been associated with the production of CO. Toxicity associated with MC is dose, time, and vehicle dependent and primarily affects the liver and the central nervous system (44).
MC treatment has been previously reported as a recipient treatment. When MC was applied in a liver transplant model (500 mg/kg, at −2 hr and 500 mg/kg/day for 14 days posttransplant) median graft survival increased from 9 to 21 days with some animals living >120 days (31). In an aortic transplant model, intimal thickening had been reduced significantly after a 30-day treatment period (47).
One limitation of the clinical use of carbon monoxide is its narrow therapeutic window. Its affinity to the human hemoglobin molecule is 220× greater than that of O2. CO also binds to other heme proteins such as myoglobin and cytochrome oxidases. Furthermore, it shifts the hemoglobin curve, impairing the distribution of oxygen (48). Symptoms vary according to duration and levels of exposure and are in particular observed in organs with high metabolic rates, such as the heart and the brain (49, 50). Toxicity is also associated with the duration of exposure. In our experiment, the levels of COHb were low (below 6%). This level is considered safe as clinical symptoms such as exhaustion, dizziness, and angina are usually observed when COHb levels exceed 10% (49, 50).
The use of CO as donor treatment reduces concerns on toxicity in the clinical situation. A previous study by others demonstrated the beneficial effects of CO applied during a prolonged period prior to procurement (48 hr) in a syngeneic heart transplant model (51). In another study, using a completely mismatched islet transplant model, both donors and islet grafts were exposed to CO during 24h prior to transplantation and this was associated with long-term graft survival in untreated allogeneic recipients (52). In our experiment, we demonstrated for the first time the absence of chronic graft deterioration with a single treatment for the induction of CO in the donor shortly (4h) prior to procurement in an allogeneic vascularized organ transplant model (53). Renal function and the morphology of treated grafts were comparable to those of native, nontransplanted controls. In addition, CO donor treatment was associated with reduced frequencies of donor-derived DCs, intragraft CD4+ T cells and diminished alloreactivity.
We thank Dr. Fritz Bach and Dr. Nicholas L. Tilney for their most valuable comments and the critical reading of the manuscript.
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