Myocardial Pro-inflammatory Cytokine Expression and Cellular Rejection in Pediatric Heart Transplant Recipients

PEDIATRIC TRANSPLANTATION

Myocardial Pro-inflammatory Cytokine Expression and Cellular Rejection in Pediatric Heart Transplant Recipients John P. Breinholt, MD,a Jesus G. Vallejo, MD,a Corey M. Gates, RN,a Sarah K. Clunie, RN,a Debra L. Kearney, MD,b William J. Dreyer, MD,a Jeffrey A. Towbin, MD,a,c and Neil E. Bowles, PhDa Background: Accumulating evidence suggests that immune-mediated injury is important in the development of rejection after heart transplantation. We hypothesized that pro-inflammatory cytokine expression would increase in biopsy samples that manifest cellular rejection and that this would correlate with the development and progression of transplant cellular rejection. Methods: Children with heart transplants were prospectively enrolled from July 2004 to November 2005. Right ventricular endomyocardial biopsies were obtained during routine catheterization for rejection surveillance. Cellular rejection was graded using criteria established by the International Society for Heart and Lung Transplantation. RNA was extracted from biopsy samples and reverse transcription was used for complementary DNA synthesis. The cDNA product was evaluated by quantitative real-time polymerase chain reaction (PCR) to measure the following cytokines: interleukin (IL)-1␤, IL-6 and IL-18; tumor necrosis factor-alpha (TNF-␣); and interferon-gamma (IFN-␥). Normalized cytokine mRNA transcripts were correlated with cellular rejection scores and the presence of viral genome. Results: Seventy-four children (mean age 9.6 ⫾ 5.5 years, range 0.2 to 20.5 years) were enrolled and 95 biopsies were obtained. None of the cytokines demonstrated a correlation with the cellular rejection score, even within individual patients for whom multiple, serial biopsy samples were studied. Eighteen biopsy samples were found to have parvovirus B19 genome present, but there was no correlation between cytokine levels and the presence of parvovirus. Conclusions: Cytokine transcripts in heart transplants do not correlate with cellular rejection. In addition, there is no correlation between cytokine transcripts and the presence of viral genome. J Heart Lung Transplant 2008;27:317–24. Copyright © 2008 by the International Society for Heart and Lung Transplantation.

Cardiac transplantation in children is a life-saving procedure aimed at sustaining long-term, productive survival in recipients. A major risk preventing extended survival is allograft rejection and accumulating evidence

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From the Departments of Pediatrics (Cardiology), bPathology and Molecular and Human Genetics, Baylor College of Medicine and Texas Children’s Hospital, Houston, Texas. Submitted October 24, 2007; revised December 6, 2007; accepted December 13, 2007. Supported by the National Institutes of Child Health and Human Development, Pediatric Scientist Development Program (Grant Award K12-HD00850 to J.P.B., J.A.T.); the Abby Glaser Children’s Heart Fund (J.A.T.); and the Texas Children’s Foundation Chair in Pediatric Cardiac Research (J.A.T.). Present affiliation for Dr Bowles: Department of Pediatrics, University of Utah School of Medicine, Eccles Institute of Human Genetics, Salt Lake City, Utah. Reprint requests: John P. Breinholt, MD, Department of Pediatrics, Section of Pediatric Cardiology, Indiana University School of Medicine, 702 Barnhill Drive, Rm 127, Indianapolis, IN 46202. Telephone: 317-274-8906. Fax: 317-274-4022. E-mail: [email protected] Copyright © 2008 by the International Society for Heart and Lung Transplantation. 1053-2498/08/$–see front matter. doi:10.1016/ j.healun.2007.12.002 c

suggests that immune-mediated injury to the organ allograft plays an important role in the development of chronic rejection.1,2 Donor major histocompatibility complex (MHC) peptides presented by host antigenpresenting cells (APCs) have been implicated in the pathogenesis of chronic rejection.3 Graft injury also may expose organ-specific, non-MHC antigens, which can trigger a host immune response and contribute to the development of transplant coronary artery disease. A role for pro- and anti-inflammatory cytokines in promoting the rejection or acceptance of cardiac allografts remains controversial. A number of qualitative studies have assessed cytokine mRNA and protein expression during acute and chronic allograft rejection. Most studies have reported the detection of pro-inflammatory T helper type 1 cell (Th1)-associated cytokine mRNAs (interleukin [IL]-2 and interferon [IFN]-␥) along with macrophage-associated cytokine mRNAs (IL-1␤, IL-6 and tumor necrosis factor [TNF-␣]) within rejected allografts.4 –9 Furthermore, the accumulation of antiinflammatory Th2-associated cytokines (IL-4 and IL-10) has been suggested to play a role in the development of allograft acceptance.10 317

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One quantitative study, using real-time reverse transcription–polymerase chain reaction (RT-PCR) to detect cytokine mRNA expression in mouse cardiac allografts,11 reported that normal hearts had detectable baseline production of cytokine mRNAs. Interestingly, both accepted and rejecting cardiac allografts produced increased amounts of all cytokine mRNAs tested and displayed few quantitative differences in cytokine mRNA expression. Although prior studies in humans identified cytokine expression, they were limited by not quantitating the level of expression. This animal study suggests that cytokine expression is present regardless of rejection state, and that the level of expression may not be different in allograft acceptance and rejection. We hypothesized that pro-inflammatory cytokines would be detected in endomyocardial biopsy samples from pediatric heart transplant patients with histologic evidence of rejection and in samples prior to the development of the histologic changes. To test this hypothesis, we utilized real-time RT-PCR for surveillance of pro-inflammatory cytokine mRNA levels in an attempt to clarify the role of these molecules in the progression of cardiac transplant rejection. METHODS Study Design The institutional review board of Baylor College of Medicine approved the protocol for this study. From July 2004 to November 2005, all children with heart transplants, from whom right ventricular endomyocardial biopsy samples were obtained, were approached prospectively for enrollment. After informed consent was obtained, serial right ventricular endomyocardial biopsy samples were collected during routine cardiac catheterization for rejection surveillance in children with clinical evidence of rejection, or as follow-up after treatment for rejection. When possible, 3 ml of blood was obtained from the child during the catheterization procedure. Cardiac Catheterization Endomyocardial biopsy samples were obtained from the right ventricle by the standard percutaneous transvenous right femoral approach using a bioptome (Cordis, Haan, Germany), as modified by Olsen et al.12 Six biopsy samples are routinely collected for surveillance; five are sent to pathology for histology and the remaining biopsy sample is processed in the diagnostic laboratory. For the purposes of the study, a seventh sample was acquired to conduct the quantitative analysis. Biopsy samples for PCR analysis were immediately immersed in RNAlater (Qiagen, Valencia, CA) and stored at ⫺80°C. The research laboratory was blinded to patient identity and clinical status until all biopsies were analyzed and cytokine levels were determined. All of

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the samples were coded prior to nucleic acid extractions and quantitative PCR analysis, all of which was performed in a blinded fashion. Biopsy samples for histologic analysis were formalin-fixed and paraffinembedded while tissue for immunohistochemical analysis was frozen in OCT media. RNA Extraction and PCR Analysis RNA was extracted from frozen biopsy samples using the Qiagen EZ1 RNA Universal Tissue Kit on an EZ1 robot (Qiagen). For the synthesis of complementary DNA (cDNA), reverse transcription was employed. Ten microliters of extracted RNA was mixed with 1 ␮l (3 mg/ml) of random primers (Invitrogen, Carlsbad, CA) in the presence of 20 units (0.5 ␮l) of Prime RNase inhibitor (Eppendorf, Westbury, NY). Human heart total RNA (Ambion, Austin, TX) was used as a control. This mixture was heated to 94°C for 5 minutes, then snap-cooled on ice. To this we added 4 ␮l of 5⫻ reverse transcriptase buffer (Invitrogen), 2 ␮l of 100-mmol/liter dithiothreitol, 1 ␮l of 10-mmol/liter dNTPs, another 0.5 ␮l of RNase inhibitor, and 200 units (1 ␮l) of Superscript III reverse transcriptase (Invitrogen; 200 U/ml). The samples were incubated at 50°C for 1 hour, followed by 15 minutes at 70°C to inactivate the enzyme. This reaction was performed in triplicate, along with a water control and negative controls generated by treating the RNA as just described except replacing the reverse transcriptase with water. Quantitative PCR The cDNA products were then subjected to real-time PCR (TaqMan) to detect the pro-inflammatory cytokines IL-1␤, IL-6, IL-18, TNF-␣ and IFN-␥, using pre-developed assay reagent (PDAR) primer/probe combinations (Applied Biosystems, Foster City, CA). Total RNA was quantitated using a GAPDH PDAR (Applied Biosystems). Two microliters of the first-strand cDNA was added to 7 ␮l of distilled water, 10 ␮l of 2⫻ Mastermix (Applied Biosystems) and 1 ␮l of 20⫻ cytokine PDAR or GAPDH PDAR. The PCR reaction was performed by heating at 50°C for 2 minutes, then at 90°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, then 60°C for 1 minute. This was performed on a 7500 Real-Time PCR system (Applied Biosystems), using ABI Sequence Detection Software, version 1.2.3 (Applied Biosystems). Each quantitative PCR reaction set generated the cytokine expression profile of a selected cytokine for 12 to 15 samples, including negative controls. Using a control RNA sample and normalizing to the housekeeping gene, GAPDH, a relative quantity of the target cytokine was calculated. The relative quantities of mRNA encoding IL-1␤, IL-6, IL-18, TNF-␣ and IFN-␥ were calculated for each biopsy sample. Results were

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Table 1. ISHLT Histology Rejection Score Classification and Frequency in the Patient Cohort ISHLT grade Frequency

0 17

1A 26

1B 13

2 31

3A 6

3B 1

verified via two independent experiments on the extracted RNA. The relative quantity of cytokine was plotted against the transplant rejection score as determined by histologic examination. Serum IL-6 Analysis With numerous studies implicating IL-6 in myocarditis, heart failure and heart transplant dysfunction,1,13–17 available blood samples were evaluated for correlation with myocardial IL-6 levels. Patient blood was placed on ice immediately after drawing, and then centrifuged at 1,290g for 30 minutes at 4°C. Plasma was drawn off with a Pasteur pipette and frozen at ⫺80°C until processing. Serum IL-6 levels were quantitated by enzyme-linked immunosorbent assay (ELISA; Quantikine HS IL-6 Immunoassay Kit; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Viral Genome Analysis An association between viral genome in the myocardium and concomitant rejection has been reported previously.18 The diagnosis of allograft rejection relies on histopathologic criteria that are known to mimic myocarditis in non-transplanted patients.19,20 To determine whether increased inflammation secondary to viral genome in the myocardium would result in a concomitant increase in cytokine expression, all endomyocardial biopsies were evaluated for a panel of cardiotropic viruses that included adenovirus, enteroviruses, parvovirus B19, cytomegalovirus and Epstein– Barr virus (EBV). Viral genomes were detected by nested RT-PCR (RNA viruses) or PCR (DNA viruses), which were performed at the John Welsh Cardiovascular Diagnostic Laboratory, Baylor College of Medicine, as previously described.21,22 Histology For the evaluation of myocardial inflammation and determining a cellular rejection score, histologic assessments of sections of formalin-fixed, paraffin-embedded endomyocardial biopsies were performed according to the grading criteria of the International Society for Heart and Lung Transplantation (ISHLT) by a single pediatric cardiovascular pathologist (D.L.K.).23 Statistical Analysis To compare rejection score to cytokine relative quantities, a Q–Q plot was utilized to determine whether the data were normally distributed. As the data were highly

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skewed, a non-parametric approach was utilized and Spearman’s rho correlation was performed. To compare the presence or absence of viral genome in the myocardium to cytokine levels, a Mann–Whitney U-test was performed, comparing median cytokine levels. RESULTS Patient Cohort We prospectively enrolled 74 children (age 9.6 ⫾ 5.5 years, range 0.7 to 20.5 years) with a history of heart transplant, or transplanted during the study period. Forty-six of the children were male (62%). A total of 95 biopsies were obtained for RNA extraction. Each child provided 1.8 ⫾ 1.3 (maximum ⫽ 6) biopsy samples for analysis. These children were followed from their enrollment for 9.8 ⫾ 3.2 months. Biopsies and Rejection Scores Histopathologic rejection grading was performed in 93 of the 95 biopsies, of which 48 (51.1%) were obtained in the first year after transplantation, 123 (13.8%) in the second year, 7 (7.5%) in the third year, 10 (10.6%) in the fourth year, 7 (7.5%) in the fifth year and 9 (9.5%) at ⬎5 years post-transplantation. Histologic classification is summarized in Table 1. Acute cellular rejection, as defined by ISHLT Grade ⱖ3A, was detected in 7 (7.4%) biopsies, of which 4 were in the first year post-transplant, 2 in the second year post-transplant, and 1 after the fifth year post-transplant. Seventeen (18.3%) of the biopsies did not demonstrate any evidence of rejection (Grade 0) and 39 (42%) had evidence of mild cellular rejection (Grade 1). Thirty-seven (39.8%) had moderate cellular rejection, of which 31 (33.3%) had Grade 2 rejection and 6 (6.5%) had Grade 3A rejection. None of the endomyocardial biopsies demonstrated severe (Grade 4) rejection. PCR Analysis The correlation data in Table 2 demonstrate a small correlation for rejection score vs INF-␥ (0.225, p ⬍ 0.03) and TNF-␣ (0.242, p ⬍ 0.02). Both correlations indicate that the cytokine rank values accounted for about 5% to 6% of the variance in rejection score. This requires caution in interpreting significance as it reflects the testing of multiple correlations. This is sub-

Table 2. Spearman’s Rho Correlation Between Histologic Transplant Rejection Scores and Cytokine Level (Rank) For 94 Endomyocardial Biopsy Samplesa Correlation coefficient p-value a

IFN-␥ 0.225 0.03

TNF-␣ 0.242 0.02

IL-1␤ 0.119 0.26

IL-6 ⫺0.121 0.25

IL-18 0.14 0.18

Rank assigned to cytokine expression because of a highly skewed distribution. Correlation was determined between assigned rank and ISHLT score.

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Figure 1. Plot of cytokine quantity rank vs histologic transplant rejection score. The distribution of cytokine transcripts is highly skewed. Therefore, a non-parametric method was applied that assigned the transcript quantity a rank that was subsequently plotted against rejection score.

stantiated by the graphs representing the cytokine level rank vs rejection score (Figure 1). Because cytokine elevation may occur early relative to histologic manifestations of transplant rejection, we examined the results of 11 children who had Grade ⱖ3 biopsies during the study period to determine whether any longitudinal trend was evident. Table 3 exhibits the rejection scores and relative cytokine transcripts for each biopsy in a subset of these patients (those from whom more than four serial biopsies were obtained). There was both a wide range of values between patients and within patients when compared with the corresponding ISHLT rejection grade. Figure 2 depicts the values of 2 patients. No correlation was demonstrated.

With numerous studies implicating IL-6 in myocarditis, heart failure and heart transplant dysfunction,1,13–17 we evaluated serum IL-6 levels in 24 blood samples from 15 patients to ascertain any relationship between these levels, myocardial mRNA expression and rejection score. No correlation was found between serum levels of IL-6 and rejection score, nor between serum IL-6 and the myocardial IL-6 mRNA levels (Figure 3). Viral Influence on Cytokine Levels Among the study patients, 21 biopsies were found to have viral genome present. Fifteen PCR-positive specimens identified parvovirus B19 genome, 3 were EBVpositive, and 3 were positive for both parvovirus B19

Table 3. Transplant Patients With Serial Biopsies (ⱖ4) During the Study Period Patient 6 Bx 1 2 3 4 5 6

Time 0.05 0.12 0.24 0.29

Grade 2 2 2 2

IL-1 782 117 18 11,333

IL-6 4 4 0.22 58

Patient 24 IL-18 4,219 0 837 4,556

IFN-␥ 26 72,255 6,202 0

TNF-␣ 527 941 130 2,209

Time 0.09 0.13 0.18 0.25

Patient 34 Bx 1 2 3 4 5 6

Time 0.57 0.59 0.62 0.65

Grade 3a 3a 2 2

IL-1 58 553 289 38

IL-6 2 3 4 11

Grade 3a 1a 1a 1b

IL-1 3,661 47 29 1

Patient 37 IL-18 9,921 5,739 0 0

Each biopsy is reported from time of heart transplant. The relative transcript quantity is recorded for each cytokine evaluated. Bx, biopsy number; time, time from transplant (years);grade, ISHLT rejection grade.

IFN-␥ 259 100 139,069 114,412

TNF-␣ 368 153 943 710

Time 1.28 1.41 1.43 1.58

Grade 2 1b 3a 1b

IL-1 80 2,251 851 109

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and EBV. The relative quantity of each of the cytokines in children with viral genome in the myocardium was compared with levels in children without evidence of viral infection. No correlation was found between cytokine levels and presence of virus (Table 4). DISCUSSION The data presented herein demonstrate that, although pro-inflammatory cytokine transcripts in endomyocardial biopsy samples from heart transplant patients could be detected and quantitated, there was no correlation between the quantity of cytokine and the degree of histopathologic rejection. For each of the cytokines, low levels of expression could be detected in the majority of the samples tested, as well as the “normal” cardiac RNA used as a control. These data support the findings of Wu et al, who reported the detection of cytokines in cardiac transplant patients, both before and during rejection episodes. Moreover, the same cytokines were present in the pre-transplant donor heart, with the exception of IL-6 and IL-8.9 Baan et al detected IL-6 and IL-10 in most pre-transplant tissue samples,4 and the detection of IL-10 correlated with donor-specific cytotoxic hyporesponsiveness.24 These findings demonstrate that expression of cytokines is present in the absence of allograft rejection, and that detecting their presence is insufficient to describe their function in heart transplant rejection. We sought to determine that cytokine transcript quantity would elucidate the relationship between rejection and the detected cytokine. In a semi-quantitative study, de Groot-Kruseman showed an increase in TNF-␣

Figure 2. Longitudinal analyses of biopsy samples from 2 patients. Patient 30 underwent five serial biopsies. The bars represent the relative IL-6 copynumber and the line represents the corresponding ISHLT rejection score. The highest level of IL-6 corresponds with a peak biopsy score of Grade 3A; however, in the previous biopsy, with a score of Grade 2, the IL-6 level was low. Patient 51 underwent six serial biopsies. The peak IL-6 level corresponds to the lowest biopsy score in the series. Note the difference in y-axis scale (IL-6 expression) between the two graphs.

Table 3. Conrinued Patient 24 IL-6 17 3 0.147 0.097

Patient 30

IFN-␥ 38,296 0 0 0

IL-18 535 0 0 0

TNF-␣ 3,153 104 116 2

Time 0.04 0.07 0.15 0.19 0.3

Grade 2 3a 1b 1b 1b

IL-1 514 565 42 1,293 268

Patient 37 IL-6 2 297 0 0.201

IL-18 5,352 0 0 0

IFN-␥ 375 0 0 230

IL-6 2 17 0.417 2 4

IL-18 0 0 725 5,693 0

IFN-␥ 72,662 0 81,881 128,362 35

TNF-␣ 4,483 1,266 54 15,359 271

IFN-␥ 0 137 9,819 0 910 0

TNF-␣ 0 551 150 305 112 27

Patient 51 TNF-␣ 219 2,855 3,848 269

Time 0.03 0.06 0.13 0.18 0.2 0.5

Grade 1 1 2 2 1b 2

IL-1 0 181 55 4 22 80

IL-6 379 7 1 3 3 1

IL-18 0 3,282 0 0 2,812 0

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Figure 3. Comparison of serum IL-6 and myocardial biopsy IL-6 levels (left y-axis), and histologic rejection scores (right y-axis). Serum IL-6 level (pg/ml) and biopsy IL-6 level (relative quantity). Bars represent the serum or biopsy tissue expression of IL-6. Diamonds represent the corresponding ISHLT rejection score for the respective biopsy and serum sample. There is no correlation between IL-6 at any determined rejection score.

in acutely rejecting allografts from 16 patients.5 However, this study only showed increases in cytokines during the first 3 months post-operatively and it is difficult to know the influence of cytokine release at the time of organ donation.17 In our long-term follow-up study, we failed to demonstrate a consistent trend in cytokine expression in serial biopsies with any of the studied cytokines. There was a correlation between rejection score and the quantities of IFN-␥ and TNF-␣ mRNA; however, the strength of that correlation is diminutive due to the testing of multiple correlations. Both correlations indicate that the cytokine rank values accounted for only 5% to 6% of the variance in rejection score. Prior studies have implicated cytokines in animal models of heart transplant rejection and myocarditis.2,25–27 An explanation for the lack of correlation between heart transplant rejection and cytokine levels can be that most studies evaluating cytokines with respect to heart disease involve myocarditis, acute Table 4. Presence of Viral Genome in Biopsy sample compared with Median Cytokine Levela Median quantity p-value a

IFN-␥ 650.0 0.21

TNF-␣ 589.5 0.09

IL-1␤ 586.0 0.09

IL-6 721.0 0.61

IL-18 747.0 0.73

No correlation found between virus and cytokine level according to Mann– Whitney U-test.

myocardial infarction and heart failure, all diseases with significant inflammatory components. Historically, heart transplant rejection fits well with this family of diseases as inflammation is a key component of the ISHLT classification of organ rejection. Moreover, these histopathologic guidelines used to assess transplant rejection often mimic virus-induced myocarditis, particularly adenoviruses and enteroviruses.19 In the setting of a rejection episode related to infection with an enterovirus, significant inflammation would be expected. In our patient cohort, however, the predominant viral genome amplified from the myocardium was parvovirus B19. Recent evidence suggests that parvovirus B19 does not infect myocytes, but rather endothelial cells,28 and is implicated in endothelial cell dysfunction rather than in frank inflammation. Moreover, in a study of patients with parvovirus B19 –induced arthritis, a negative association was found between pro-inflammatory cytokines and arthritis.29 Further, the majority of biopsy samples (92.6%) in this study had ISHLT Grade ⱕ2 rejection scores. At our institution, significant rejection that results in medical therapy requires Grade ⱖ3A rejection. This implies that the degree of inflammation was low in most of the patient biopsy samples. If more samples with scores of Grade ⱖ3A were analyzed, it is possible that higher cytokine levels would be measured. However, those with Grade ⱖ3A rejection did

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not have notably higher cytokines levels than the samples with Grade ⬍3A.

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Limitations The population of patients studied was heterogeneous with potential confounding variables, including immunosuppression regimens, spectrum of Class I and Class II MHC mismatches, time from transplant, and individual variations in cytokine profiles. All patients transplanted at our institution are started on an immunosuppression protocol that includes mycophenolate mofetil, cyclosporine and a steroid regimen with tapering doses. In most cases, transplant patients remain on low-dose prednisone. In some cases, tacrolimus is substituted for cyclosporine due to side effects or restrictions in medication delivery (i.e., gastrostomy tubes). Potential environmental influences on cytokine profiles are minimized when children comprise the patient cohort. Sampling error is a limitation inherent to biopsy sampling as any one biopsy sample may not represent the status of the entire graft. Sampling error, however, cannot account for elevated cytokine transcript levels in patients without histologic evidence of rejection. In addition, extensive tissue damage from rejection could reduce local cytokine production; however, histologic evaluation should elucidate such tissue injury. In conclusion, we have demonstrated no correlation between the ISHLT rejection score and the levels of the cytokines IL-1␤, IL-6, IL-18, TNF-␣ and IFN-␥ in children with heart transplants. These data support prior studies in animal models that did not find a difference in quantitative cytokine expression between accepted and rejecting allografts.11 We speculate that cytokines may modulate aspects of the cellular rejection cascade, but they likely modulate other adaptive as well as maladaptive processes. In addition, cytokine expression did not increase in the presence of viral genome in the myocardium. Enteroviruses are known to cause significant inflammation and could possibly increase cytokine expression. It is unknown whether this degree of inflammation would affect cytokine expression, but these changes were not present in biopsies with the parvovirus B19 genome. With the prevalence of parvovirus in our patient cohort it would be important to further investigate the implications of this finding, particularly in the absence of increased cytokine expression and inflammation on histologic assessment. The authors thank Dorellyn B. Lee, MPA (Winters Center for Heart Failure Research), for analysis of serum IL-6 levels.

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