Intragraft mRNA Detection of Cell Surface, Messenger, and Effector Molecules of the Immune System in Human Heart Transplantation

Natalia Shulzhenko, MD,^a Andriy Morgun, MD,^a , Marcello Franco, MD, PhD,^b Márcia M. Souza, MD, PhD,^b Dirceu R. Almeida, MD, PhD,^c Rosiane V.Z. Diniz, MD,^c Antonio C.C. Carvalho, MD, PhD,^c Álvaro Pacheco-Silva, MD, PhD,^d Maria Gerbase-DeLima, MD, PhD^a

From the Division of Allergy, Clinical Immunology and Rheumatology, Department of Pediatrics,^a Department of Pathology, ^b Division of Cardiology, ^c and Division of Nefrology, ^d Department of Medicine, Universidade Federal de São Paulo - Escola Paulista de Medicina (UNIFESP-EPM); São Paulo, Brazil.

Corresponding author

Natalia Shulzhenko, MD
Rua dos Otonis, 725
04025-002 São Paulo, SP, Brazil;
business telephone number: 55 11 5764426;
home telephone number: 55 11 5390679;
fax number: 55 11 5701590;
e-mail: morgun@sti.com.br

This investigation was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

ABSTRACT; INTRODUCTION; METHODS; RESULTS; DISCUSSION; REFERENCES

ABSTRACT

Background: The purpose of the present was to investigate the intragraft expression of immunologic markers in relation to the occurrence and severity of acute cardiac allograft rejection as well as its possible value in predicting acute rejection.

Methods: A series of 46 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months post-transplantation was evaluated for the presence of mRNA for CD3, CD40L, IFN-g, IL-8, granzyme B and FasL, using the reverse transcriptase -polymerase chain reaction method.

Results: CD3 gene transcripts were found in all biopsy samples, indicating the presence of T cells, regardless of rejection. The presence of mRNA of the other molecules was significantly associated with acute rejection. Moreover, the detection of CD40L and IFN-g gene transcripts was more frequently observed in cases of severe than non-severe rejection. The data also suggested that the detection of IFN-g and IL-8 may be of predictive value for the occurrence of rejection.

Conclusions: Overall, our results clearly indicate that intragraft mRNA detection of cell surface (CD40L), messenger (IFN-g, IL-8), and effector molecules (granzyme B, FasL) of the immune system represents a valuable tool, in addition to histology, in the monitoring of cardiac allograft rejection.

INTRODUCTION

Cardiac transplantation is a well established and effective therapy for end-stage cardiac disease. Despite immunosuppression, the majority of heart transplant recipients experience at least one rejection episode during the first year post-transplant and acute and chronic rejection continue to represent a major menace to the success of the transplant, being responsible for approximately one third of the mortality in heart transplant recipients.^1,2 Although histological evaluation of endomyocardial biopsy (EMB) is currently the gold standard for the diagnosis of rejection,³ its sensitivity is not absolute and it may not always discriminate between mild episodes of rejection which might be self-limiting from those that may progress.^4-8 Furthermore, it has no predictive value for rejection episodes. Therefore, it is of interest to search for other markers that could be of additive value to histological grading and that could predict rejection. Although a number of studies on cytokine and immune activation molecules expression in human cardiac allografts have been reported, a consistent pattern of expression preceding or occurring during rejection episodes has not yet emerged. ^9-24

The purpose of the present study was to investigate, by reverse transcriptase -polymerase chain reaction (RT-PCR) method, transcripts of CD3, CD40L, IFN-g, IL-8, granzyme B, and FasL genes in sequential EMB collected within the first 6 months post-transplantation and to evaluate the findings in terms of relation to rejection.

CD3 is constitutively expressed on CD4⁺ and CD8⁺ T lymphocytes and was included in the study to assess the presence of T cells. The other genes encode for inducible molecules that are involved in different steps of the cell mediated immune response.

CD40L (CD154), mainly present on activated T cells, is the ligand for CD40 which is expressed on various antigen presenting cells (APC), including dendritic cells, macrophages, B cells, and endothelial cells. Following the stimulation of the TCR/CD3 complex, CD40L is rapidly induced on T cells and binds to CD40 on APC. This interaction itself constitutes a costimulatory signal and, in addition, enhances and prolongs the expression of B7-1 (CD80) and B7-2 (CD86) which further stimulate T cells through the CD28 receptor. The CD40-CD40L interaction also contributes to the inflammatory response, since it induces or up-regulates the expression of other accessory/costimulatory molecules (e.g., ICAM-1) and cytokine/chemokine production (e.g. IL-6, IL-8, IL-12, GM-CSF and TNF). ^25-32

Interferon-g (IFN-g), an immunoregulatory cytokine secreted by activated T lymphocytes and NK cells, has been shown to play an essential role in acute allograft rejection. It enhances antigen presentation by up-regulating MHC expression and promotes cell-mediated immunity by activating macrophages, NK cells and Th1 lymphocytes. ^33,34

Major inflammatory cytokines, like TNF and IL-1, induce the production of IL-8 by endothelial cells and by other cell populations. IL-8 is a member of the chemokine superfamily and exerts its effect on leukocyte-endothelial cell adhesion. In addition, IL-8 plays a role in the activation, degranulation and production of superoxide anions in leukocytes. ^35-37

Granzyme B and FasL are involved in the effector phase of cytotoxic T cell (CTL) response. Differentiation from pre-cytotoxic T cells to CTLs involves the activation of the machinery to perform lysis. The "lethal hit" by the activated CTLs on their targets can be mediated by granule exocytosis-dependent (i.e., perforin/granzime B-mediated) or by granule exocytosis-independent mechanisms (i.e., Fas ligand-mediated). ^38-40

METHODS

Patient Population, Collection and Classification of Biopsies

The study was conducted on 46 endomyocardial biopsy samples from 10 adult recipients of cardiac allografts, transplanted between October 1996 and October 1997. Ethical committee approval and informed consent were obtained.

All patients were maintained on standard triple therapy immunosuppression consisting of cyclosporine (4-6 mg/kg/day), azathioprine (2 mg/kg/day), and prednisone (0.2 mg/kg/day). Treatment for rejection consisted of pulse therapy with methylprednisolone (1 g daily for 3 days) and/or augmentation of the oral doses of prednisone and cyclosporine.

EMB were routinely collected weekly for the first 4 weeks, every 15 days during the second month, and once a month from the third to the sixth month. In addition, biopsies were performed because of a clinical hypothesis of rejection or for the follow-up of a rejection episode.

Pathologic assessment was performed by two of us (MF; MMS), who were unaware of the mRNA study results. Rejection was graded according to the criteria of the International Society for Heart and Lung Transplantation (ISHLT) criteria,³ after the examination of three or four EMB fragments of each biopsy. Twenty-two biopsies presented grade 0; 2, grade 1B; 1, grade 2; 16, grade 3A; and 3, grade 3B. One fragment of the biopsy was snap-frozen and stored in liquid nitrogen until the time of mRNA extraction.

For mRNA data analysis, all the biopsies with grade 1B or more were grouped (biopsies with rejection). Biopsies with grade 3B and/or those that were followed by anti-rejection treatment were considered as severe rejection. Zero grade biopsies were sub-divided into two groups: those that were preceded or followed by a biopsy with rejection within 15 days were considered as borderline biopsies (post- or pre-rejection, respectively); otherwise, zero grade biopsies were considered without rejection. Two biopsies from a borderline group that were collected between two rejection episodes were not included in the analysis. There was no difference between all the groups of biopsies regarding the day after transplantation in which they were performed.

Detection of mRNA by the RT-PCR

Experiments to test each primer pair and to optimize the amplification conditions for each set of primers were performed with the mRNA extracted from phytohemagglutinin (PHA-P, Difco, Detroit, MI) stimulated peripheral blood mononuclear cells (PBMC) from a normal donor. Briefly, PBMCs were isolated from fresh blood by ficoll-hypaque density gradient centrifugation, resuspended at a concentration of 1 x 10⁶ cells/ml in complete RPMI 1640 medium (Sigma, St Louis, MO), containing 20% fetal calf serum and 10 µg of PHA/ml, and distributed in tissue culture microplates (Corning, New York, NY) at a volume of 100 µl per well. After incubation in a humidified chamber with 5% CO₂ at 37ºC, for 48 h, the cells from 10 wells were pooled, pelleted, and resuspended in 0.4 ml of extraction buffer (QuickPrep Micro mRNA Purification Kit, Pharmacia Biotech, UPPSALA, Sweden). The other steps of mRNA extraction were performed according to the manufacturer's instructions.

The biopsy fragment was placed in 0.4 ml of the extraction buffer and homogenized with a homogenizer (Handishear AC, The VirTis Company, Gardiner, NY). The other steps were the same as for cells.

The extracted mRNA was resuspended in 60 µl of diethylpolycarbonate-treated water (0.1 g%).

Reverse transcription was performed on 19 µl of the isolated mRNA with 2 µl of Moloney murine leukemia virus (MMLV) reverse transcriptase (200 U/µl; Gibco-BRL, Gaithersburg, MD), 2 µl of oligo(dT)_12-18 primer at 100 µg/ml (Pharmacia Biotech, Sweden), and 10 µl of 10 mM deoxyribonucleoside triphosphates (dNTPs), dATP, dCTP, dGTP, and dTTP (Pharmacia Biotech). The remainder of the mixture comprised 10 µl of reverse transcription buffer, 5 µl of 0.1 M dithiothreitol solution, 1 µl of bovine serum albumin at 1 mg/ml (all from Gibco BRL), and 2 µl (20 U) of placental ribonuclease inhibitor (Pharmacia Biotech). The mixture with the mRNA was first incubated at 37^oC for 60 min, and then at 65^oC for 10 min.

First-strand cDNA was amplified using Taq polymerase (Cenbiot, Porto Alegre, Brazil) and primers designed for the following molecules: b-actin, CD3, CD40L, IFN-g, IL-8, granzime B and FasL. The primers were synthesized by Gibco BRL and the sequences of the oligonucleotides and the predicted size of the PCR products generated with each primer pair are listed in Table 1. Sterile injection-grade water was used, instead of cDNA, as a negative control. The reaction mixture for PCR amplification consisted of 5 µl of cDNA,10 mM of each dNTP, 250 ng of each primer, 5 µl of 10 x concentrated PCR buffer, 2.5 mM MgCl2, and 2.5 U of Taq polymerase in a total volume of 50 µl. Samples were denatured at 94^oC for 60 sec, annealed at 55^oC for 45 sec, and extended at 72^oC for 45 sec. This cycle was carried out 40 times in a thermocycler (GeneAmp PCR System 9600, Perkin Elmer Cetus, Norwalk, CT). All samples were initially tested with the b-actin primers (internal control to confirm successful RNA extraction and cDNA amplification). Thereafter, each sample was tested in two independent experiments with the other primer pairs. In the rare cases of discrepancy between the two experiments, the test was repeated. A 14 µl aliquot of the PCR product was analyzed by electrophoresis on ethidium-bromide-stained 2% agarose gels (w/v). Products were identified by comparison with a molecular weight marker (f-174-RF DNA/Hinc II Digest, Pharmacia Biotech).

Statistical analysis

The data were analyzed by the Kappa (k) test and the level of significance was set at p<0.05.⁵⁴

RESULTS

A representative experiment on mRNA extracted from PHA-stimulated PBMN cells, where the bands corresponding to the amplification of the cDNA of all the markers are present, is shown in Figure 1.

CD3 mRNA was detected in all biopsy samples.

The results concerning the mRNA of the other molecules are shown in Table 2.

The positivity for all the markers, except granzyme B, was statistically higher in grade 0 biopsies collected within a 15-day interval from a biopsy with rejection (borderline group) than in grade 0 biopsies collected outside this period (no rejection group). The difference was extremely important for IFN-g (82 vs. 15%) and IL-8 (82 vs. 8%). The higher positivity of these two transcripts was seen both in pre-and in post-rejection biopsies as compared to no rejection biopsy group. mRNA expression of all the molecules did not differ between borderline biopsies and biopsies with rejection (Table 2).

The comparison between biopsies with rejection and grade zero biopsies collected more than 15 days before or after rejection (no rejection group) revealed a higher percentage of positive biopsies in cases with rejection, concerning all the markers (Table 2).

The detection of CD40L and IFN-g transcripts was more frequent in biopsies with severe rejection than in biopsies with non-severe rejection (Table 2). Higher difference was observed when simultaneous expression of CD40L and IFN-g genes was considered (severe rejection vs. non-severe rejection: 100% vs. 46%, p<0.005, k =0.49).

DISCUSSION

The purpose of the present was to investigate the intragraft expression of immunologic markers in relation to the occurrence and severity of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. We evaluated the expression of mRNA of CD3 and of a series of inducible molecules that participate in various steps of the immune response to the allograft using the RT-PCR method in 46 EMB collected from 10 heart transplant recipients, during the first 6 months post- transplantation.

The mRNA for CD3 was present in all biopsy specimens, indicating the presence of T lymphocytes in heart allografts, regardless of rejection. This result is in agreement with other studies.^13,14,17 Since the intensity of the lymphocyte infiltrate is definitely higher during rejection,³ it is conceivable that with the utilization of a quantitative RT-PCR method, higher CD3 mRNA levels would be detected in biopsies with rejection. In fact, it has been demonstrated, by a semi-quantitative immunohistochemical staining method that the intensity of CD3 detection could discriminate between non-rejection and rejection.²⁶

The percentage of biopsies in which mRNA for CD40L, IFN-g, IL-8, granzyme B, and FasL was detected was significantly higher during rejection episodes than during quiescent periods.

The association herein reported of CD40L mRNA and rejection represents the first report in the literature concerning human cardiac allografts. Two previous studies on CD40L in human heart transplantation, both utilizing immunohistochemistry for protein detection, showed discrepant results. In one of them, CD40L could not be demonstrated in any biopsy.²¹ In the other one, CD40L expression was shown to be prominent in CD3-positive cell infiltrates and in microvascular endothelial cells.²⁶ In human renal transplantation, a recent work showed a correlation between heightened CD40L gene expression and acute allograft rejection.⁵¹

Our results concerning association of IFN-g and IL-8 transcripts and rejection agree with the concept of the important role of the DTH response in allograft rejection.^34,37,45,53 The association between IFN-g, or its mRNA, and kidney or heart allograft rejection has been found by some^11,12,43 but not by other^13,19,24 authors. Our data on the association of IL-8 mRNA and rejection are in agreement with studies performed on human heart²⁰ and renal grafts.^43,46

We have also shown that the expression of mRNA of the cytotoxic effector molecules granzyme B and FasL was significantly associated with rejection. While the association of granzime B transcripts and acute rejection has been previously described,^9,10,16,18 intragraft FasL mRNA expression has not been investigated in human heart transplantation. In kidney transplantation, there are a few recent studies demonstrating, by semi-quantitative RT-PCR, higher intragraft FasL mRNA expression in acute rejection episodes.^{43, 48, 49}

In order to investigate the relationship between the presence of mRNA of the different markers and the severity of the rejection episodes, we compared mRNA expression between biopsies with severe rejection (i.e., with histological grade 3B and/or followed by treatment for rejection) and biopsies with non-severe rejection. In general, the percentage of biopsies positive for any of the markers was higher in cases of severe rejection, but a statistically significant difference was detected only for CD40L and IFN-g. The greatest difference between these groups was observed when the simultaneously expression of CD40L and IFN-g transcripts was considered (100% in severe rejection vs. 46% in non-severe rejection; p<0.05).

Increased expression of IFN-g in cases of severe rejection has been also observed by other authors¹¹, whereas the association of CD40L mRNA detection with severe rejection is an unexpected result, considering that the expression of CD40L is an early event in the T cell activation process. However, this finding is in agreement with a recent work that showed that the treatment with anti-CD40L antibody was successful in reversing ongoing acute renal allograft rejection in primates.⁵⁰ Therefore, a continuous co-stimulation of newly recruited lymphocytes appears important for the development and maintenance of the rejection process. Moreover, the CD40L-CD40 interaction leads to macrophage activation, IL-12 release, and consequently, IFN-g production by T lymphocytes.⁵² We believe that the essential role of macrophage-dependent pathway in the pathogenesis of acute rejection and its dependence on CD40L-CD40 interaction could explain our finding of the association of CD40L and IFN-g co-expression with severe rejection.

Another interesting finding of our study was the higher detection of immune activation markers in borderline (pre- and post-rejection) biopsies as compared to biopsies without rejection, while no difference in the expression of any of the markers was detected between borderline biopsies and biopsies with rejection (Table 2).

Since our data have clearly demonstrated that the pattern of immunological reactivity may differ among zero grade biopsies depending on their proximity to a histologically proven rejection, the subdivision of the zero grade biopsies that we have utilized in the present work seems to be an important approach to the study of the association between immunological markers and rejection.

Despite the limited number of samples in pre-rejection group, the significantly higher detection of IFN-g and IL-8 mRNA in pre-rejection biopsies than in biopsies taken in quiescent periods was observed suggesting that these markers could have a predictive value for the occurrence of rejection. In fact, it has been shown in primates that the intragraft detection of IFN-g mRNA preceded by 4 days the rejection episode¹². In another study, an elevation of serum IL-8 level was shown to predict the onset of cardiac rejection in humans⁴⁷.

In biopsies that were collected within a 15-day interval after the rejection episode (post-rejection group), we have also observed a higher expression of mRNA of all the markers, as compared to biopsies from quiescent periods. As in the pre-rejection biopsies, the main difference was in the expression of IFN-g and IL-8. We have not found in the literature any work that has focused the attention on the intragraft expression of markers after rejection. In a study on cytokine mRNA in PBMC from human heart transplant recipients, a significant decrease in the amount of IFN-g mRNA was observed after the initiation of antirejection treatment²⁴. It would be interesting to investigate, with the use of a semi-quantitative RT-PCR assay, whether the amount of mRNA of immune activation molecules in post-rejection biopsies could be of value for the assessment of the efficacy of anti-rejection therapy.

It is interesting that both at the beginning (pre-rejection), and at the final stages (post-rejection) of a rejection episode we found increased expression of mRNA of the two pro-inflammatory cytokines.

In conclusion, our results clearly indicate that intragraft mRNA detection of cell surface (CD40L), messenger (IFN-g, IL-8), and effector (granzime B, FasL) molecules of the immune system allows a deeper insight into the ongoing receptor-allograft interactions than morphology, and could represent a valuable tool, in addition to histological evaluation, in the monitoring of cardiac allograft rejection.

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