Janeth C. Villanueva

Cytokine and T cell receptor gene expression at the site of allograft rejection

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1β, IL-6, and TNF-α was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-α was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-γ message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n=16) indicating the presence of T cells bearing the α/β TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x=7.47±2.4 families rearranged in samples obtained from nephrectomies, whereas x=5.33±0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.

Apoptosis as a mechanism of cell death in liver allograft rejection

It is generally recognized that there are two mechanisms of cell death, apoptosis and necrosis. Apoptosis--programmed cell death--is involved in numerous states of physiological cell deletion. Recent studies have demonstrated that hepatocytes, under certain conditions, undergo apoptosis. The purpose of this work was to determine if apoptotic cell death is involved in liver allograft rejection. Groups of Lewis (RT1l) rats underwent orthotopic liver transplantation (OLT) from disparate DA (RT1a) or syngeneic Lewis rats. Liver samples were harvested at 1, 2, 3, 4, and 7 days posttransplant and analyzed for apoptotic cell death. Since the characteristics of apoptosis are difficult to discern using routine hematoxylin and eosin staining, we utilized a novel method that detects the classic indicator of apoptosis, nonrandom DNA degradation. Paraffin-embedded tissue sections were end-labeled with nonradioactive dUTP and detection of apoptotic bodies accomplished by immunoassay. The incidence of apoptotic cells increased steadily over time in allografts, in contrast to syngeneic grafts. In this study apoptotic cell death paralleled standard indicators of liver allograft rejection including pathology, mononuclear cell infiltration, and increases in liver enzymes. Moreover, increased expression of TGF-beta 1 correlated with apoptosis in liver allografts, supporting the previously described role for this cytokine in hepatocyte apoptosis. Our results demonstrate, for the first time, that apoptosis may be a mechanism of cell death in liver allograft rejection.

Intragraft cytokine profile during human liver allograft rejection.

Forty-three human liver allograft biopsies and normal liver were directly analyzed for inflammatory and immunoregulatory cytokine gene expression by polymerase chain reaction (PCR). IL-5 gene expression was predominantly present in biopsies from liver allografts with histopathological evidence of acute rejection. IL-2 gene expression was rarely observed in rejecting allografts or allografts without evidence of rejection. In contrast, IL-4 message was readily detectable in the majority of liver allografts regardless of clinical status. The inflammatory mediators IL-1 beta, TNF-alpha, and IL-6 were detected with similar frequency in rejecting allografts and allografts without evidence of rejection. These findings suggest that inflammatory and immunoregulatory cytokines are produced within the allograft. Moreover, IL-5 may play a role in the local mechanisms of liver allograft rejection.

Evidence for a nonclassical pathway of graft rejection involving interleukin 5 and eosinophils

The role of IL-5 and eosinophils in allograft rejection was studied in human liver allograft recipients. Liver allograft biopsies were analyzed for intragraft IL-5 gene expression, and the percentages of eosinophils and plasma cells within the portal infiltrate as well as peripheral eosinophil levels were determined. The majority of allografts with evidence of rejection had concomitant IL-5 mRNA and eosinophilia, while no resolving or nonrejecting allografts had simultaneous IL-5 mRNA and eosinophilia. In fact, rejecting liver allografts that contain IL-5 mRNA and eosinophils also contain infiltrating cells that produce the cytotoxic mediator major basic protein. In contrast, intragraft plasma cell and peripheral eosinophil levels did not correlate with the histopathologic status of the allograft. Cyclosporine and FK506 had similar effects on the frequency of IL-5 gene expression in rejecting and nonrejecting allografts. However, OKT3 appeared to profoundly modulate IL-5 gene expression, since 0 of 11 biopsies obtained during OKT3 treatment for rejection contained IL-5 transcripts. These observations raise the possibility of a cellular pathway of liver allograft rejection mediated by IL-5-activated eosinophils.

CD30 expression identifies a functional alloreactive human T-lymphocyte subset

BACKGROUND CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and has been proposed as a marker of specific cytokine-producing subsets in humans. Previous studies have examined the expression of CD30 on established T helper type 1 and T helper type 2 cell clones and the function of CD30+ cells after mitogenic stimulation. In this study, we examined the development and function of CD30+ T cells generated in response to alloantigen. METHODS Primary one-way mixed lymphocyte reactions were established, and the expression of CD30 on T lymphocytes was determined by immunofluorescence and flow cytometry. Fluorescence-activated cell sorting was utilized to define the cytokine profile of alloactivated CD30+ cells after restimulation with anti-CD3 monoclonal antibodies or alloantigen. The effect of cyclosporine on the development of CD30+ cells, and on cytokines produced by CD30+ T lymphocytes, in response to alloantigen was determined. RESULTS CD30+ T lymphocytes could be detected on day 2 of mixed lymphocyte reactions and continued to increase in number and proportion through day 6. Both CD4 and CD8 T cells expressed CD30 after primary alloantigenic stimulation. CD30+ T cells are a subset of alloactivated T cells and are the major source of interferon-gamma and interleukin-5 produced in response to alloantigen. Cyclosporine partially, but not completely, inhibits the development of CD30+ cells, and has a greater effect on interferon-gamma production than on interleukin-5 production. CONCLUSIONS CD30+ T lymphocytes may constitute an important immunoregulatory subset in human allograft rejection.

Viral and immunologic aspects of Epstein-Barr virus infection in pediatric liver transplant recipients.

Pediatric allograft recipients in particular are at increased risk for Epstein-Barr virus (EBV)-associated disorders. Early identification and diagnosis of EBV-associated disorders is critical, since disease progression can often be halted by reduction of immunosuppression. In this study we examined viral and immunologic parameters of EBV infection in the circulation of pediatric liver recipients to identify factors associated with disease. Peripheral blood DNA from pediatric liver recipients was analyzed by PCR for the EBV genes coding for the nuclear antigen 1 (EBNA-1) and the viral capsid antigen gp220. Sequences for these viral genes could be readily detected in the circulation of 36.5% of patients. Moreover, identification of the EBV genome was associated with symptomatic infection, suggesting that circulating EBV may be a useful marker of disease. Since EBV-infected B cells release the low-affinity IgE receptor (sCD23), we measured sCD23 in the circulation of pediatric liver recipients and found it to be elevated in patients with detectable virus or symptoms of infection. However, sCD23 was also elevated in cases where no EBV was detectable, suggesting that factors other than viral infection could stimulate release of sCD23. To further characterize the immune response to EBV infection, the peripheral levels of IL-4, IL-5, IL-10, and IFN-gamma were determined in pediatric liver recipients. Each of these cytokines was elevated in patients with symptoms or circulating virus compared with stable, age-matched liver recipients. IL-4, in particular, was significantly increased, indicating an important role for this cytokine in EBV infection. Together, these findings suggest that (1) monitoring circulating levels of EBV may be useful in patients at high risk and (2) cytokines that promote B cell growth and differentiation contribute to EBV-associated disorders.

Expression of the cytotoxic T cell mediator granzyme B during liver allograft rejection.

Cytotoxic T lymphocytes (CTL) constitute a major component of the alloreactive response following organ transplantation. The molecular mechanisms of CTL killing remain to be determined but multiple candidate molecules involved in CTL-mediated cytotoxicity have been identified. Granzyme B, a serine protease, participates in perforin-dependent pathways of cytotoxicity and is necessary for induction of DNA fragmentation in target cells. In this study the expression of granzyme B in liver biopsies obtained from liver allograft recipients was determined by semiquantitative reverse transcriptase polymerase chain reaction. Biopsies were classified into four groups--no evidence of rejection, preservation injury, acute rejection, or resolving rejection--according to histopathological criteria. There was a significantly higher frequency of transcripts for granzyme B in the acute rejection group (82.8%) compared to the no rejection (20.0%), resolving rejection (12.5%) and preservation injury (0%) groups. Analysis of granzyme B gene expression in sequential samples from individual patients prior to, and after, treatment for rejection revealed an inverse correlation between granzyme B mRNA and response to treatment. These findings indicate that the cytopathic mediator granzyme B may participate in CTL-mediated cytotoxicity during liver allograft rejection.

IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro.

IL-2 and IL-5 gene expression in response to alloantigen was studied in liver allograft recipients and in an in vitro system. Seventy-seven sequential liver allograft biopsies from 22 patients were analyzed for IL-2 and IL-5 mRNA by polymerase chain reaction and Southern blot hybridization. Message for IL-5 was present in 74% of allografts with rejection, 46% of allografts with resolving rejection, and 33% of allografts with no evidence of rejection. The frequency of IL-5 transcripts in rejecting allografts was significantly different than the frequency of IL-5 transcripts in grafts without evidence of rejection (P = 0.003). Message for IL-2 was detected in 29% of rejecting allografts, 18% of allografts without evidence of rejection, and 43% of allografts with resolving rejection. There was no significant association between IL-2 gene expression and the histopathological status of the allograft. Interestingly, 9 of 15 biopsies that contained IL-2 message in the no rejection and resolving rejection categories went on to display rejection shortly thereafter. IL-2 and IL-5 gene expression rarely occurred simultaneously within allografts. An in vitro system consisting of irradiated, allogeneic stimulator cells and normal peripheral blood mononuclear cells as responders was established to further investigate alloantigen-driven IL-2 and IL-5 production. Both IL-2 and IL-5 were produced in response to alloantigen as determined by specific bioassays. Maximal levels of IL-5 activity in culture supernatants generally followed maximal IL-2 levels by 24 hr, but both IL-2 and IL-5 production were dramatically inhibited by CsA. Analysis of cytokine gene expression revealed that IL-2 transcription peaked within the initial 24 hr of culture, whereas IL-5 transcription was maximal at 120 hr of culture. The expression of a CTL-specific serine esterase gene was similar to IL-5 in that it was maximal during the latter phases of the culture period. Thus, both human IL-2 and IL-5 are produced in response to alloantigen and are inhibitable by CsA. These data suggest that IL-2 and IL-5 may participate in cellular pathways of tissue damage within the rejecting allograft.

Human hepatocytes produce an isoform of FAS that inhibits apoptosis.

BACKGROUND Fas (Apo-1/CD95), a member of the tumor necrosis factor receptor family, can mediate apoptosis when engaged by its ligand or by anti-Fas antibody. Fas is expressed by cells of the immune system and by some nonlymphoid tissues. Numerous studies have suggested that the Fas pathway may play a role in the rejection of allografts. Functional, soluble forms of the Fas receptor are produced by activated peripheral blood mononuclear cells and some transformed cell lines. The purpose of this study was to determine if soluble variants of Fas are produced in the liver and to determine if blockade of the Fas pathway, by liver-derived soluble Fas, inhibits Fas-mediated apoptosis. METHODS Liver and purified hepatocyte specimens were analyzed for Fas transcripts by reverse transcriptase-polymerase chain reaction with primers that span the transmembrane region of the molecule. Bile and cell lysates were analyzed for soluble Fas by specific enzyme-linked immunosorbent assay. Lysates were prepared from normal liver and hepatocytes and utilized to block Fas-mediated apoptosis of Jurkat cells as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometry. RESULTS A variant form of Fas is abundantly expressed in normal liver and purified hepatocytes. This variant form of Fas is expressed in all normal liver specimens but only in half of the liver specimens obtained during allograft rejection. The levels of soluble Fas diminish in patients undergoing liver allograft rejection in contrast to patients with stable grafts. Importantly, a soluble form of Fas is produced in the liver by hepatocytes and can specifically inhibit Fas-mediated apoptosis. CONCLUSION These data raise the possibility that soluble Fas, produced by hepatocytes, may influence the immune response by blocking Fas-mediated apoptosis and, thus, may have a role in liver transplantation.

Acute liver allograft rejection in the rat. An analysis of the immune response.

Liver allografts are vigorously rejected in 9-12 days in Lewis recipients of fully histoincompatible DA livers. The purpose of this study was to examine the initial events in this cascade, specifically the role of CD4+ T helper cells. Lewis recipients of DA or Lewis livers were killed at days 1, 2, 3, 4, and 7 days after transplant. Indicators of acute liver rejection, including a marked inflammatory infiltrate and decreased liver function, progressed in untreated recipients of allografts. Splenocytes taken from allogeneic recipients on days 1-4 and 7 proliferated in response to donor and third-party stimulators, whereas graft-infiltrating cells did not respond to donor and third-party antigens until day 3 after transplant, but thereafter maintained a good response. To further characterize the host T helper cell response to liver allografts, cytokine expression was analyzed in graft tissue and in the periphery. IL-4 mRNA was present in both syngeneic and allogeneic liver grafts, while message for IL-10 was present early in all liver grafts but persisted only in allografts. In contrast, IL-2 and IFN-gamma transcripts were specific to rejecting allografts. Similar patterns of cytokine expression were observed in the spleen, indicating the immune response to the graft involves the peripheral lymphoid organs. Thus, the cytokine profile detected during liver allograft rejection is extremely similar to that observed in other experimental models of transplantation.