Disruption of murine cardiac allograft acceptance by latent cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well-described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had approximately 80% graft loss by 100 days after transplant, compared with approximately 8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN-alpha expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Acute humoral rejection of renal allografts in CCR5(-/-) recipients.

Increasing detection of acute humoral rejection (AHR) of renal allografts has generated the need for appropriate animal models to investigate underlying mechanisms. Murine recipients lacking the chemokine receptor CCR5 reject cardiac allografts with marked C3d deposition in the parenchymal capillaries and high serum donor-reactive antibody titers, features consistent with AHR. The rejection of MHC-mismatched renal allografts from A/J (H-2(a)) donors by B6.CCR5(-/-) (H-2(b)) recipients was investigated. A/J renal allografts survived longer than 100 days in wild-type C57BL/6 recipients with normal blood creatinine levels (28 +/- 7 micromol/L). All CCR5(-/-) recipients rejected renal allografts within 21 days posttransplant (mean 13.3 +/- 4 days) with elevated creatinine (90 +/- 31 micromol/L). The rejected allografts had neutrophil and macrophage margination and diffuse C3d deposition in peritubular capillaries, interstitial hemorrhage and edema, and glomerular fibrin deposition. Circulating donor-reactive antibody titers were 40-fold higher in B6.CCR5(-/-) versus wild-type recipients. Depletion of recipient CD8 T cells did not circumvent rejection of the renal allografts by CCR5-deficient recipients. In contrast, microMT(-/-)/CCR5(-/-) recipients, incapable of producing antibody, did not reject most renal allografts. Collectively, these results indicate the rapid rejection of renal allografts in CCR5(-/-) recipients with many histopathologic features observed during AHR of human renal allografts.

Transforming growth factor-beta and interleukin-10 subvert alloreactive delayed type hypersensitivity in cardiac allograft acceptor mice.

We have previously reported that temporary treatment of cardiac allograft recipients with gallium nitrate (GN) results in indefinite graft survival, and the inability to mount donor-reactive delayed type hypersensitivity (DTH) responses. We report that antibodies to either transforming growth factor-beta (TGFbeta) or interleukin-10 (IL10) can uncover DTH responses to donor alloantigens in cardiac allograft acceptor mice. The DTH responses uncovered with TGFbeta-reactive antibodies can be blocked by exogenous IL10, and those uncovered with IL10-reactive antibodies can be blocked by exogenous TGFbeta. These data demonstrate that allograft acceptor mice are fully allosensitized, and poised to make donor-reactive cell-mediated immune responses. However, such responses are subverted by a donor alloantigen-dependent mechanism that involves TGFbeta and IL10, which in turn interfere with local cell-mediated immune responses.

Real-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice.

BACKGROUND: The relative contribution of pro-inflammatory and anti-inflammatory cytokines in promoting the rejection or acceptance of experimental cardiac allografts remains controversial. We hypothesized that the posttransplantation induction of a new immune response to graft alloantigens at a distant delayed-type hypersensitivity (DTH) site would force the immune system to reveal its current disposition toward graft alloantigen as it initiates the new immune response. Thus, we should be able to monitor the evolution of the immunologic relationship between allograft recipients and their grafts at any time posttransplantation by challenging the recipient for DTH responses to donor alloantigen and evaluating the cytokine profiles displayed at the DTH site. METHODS: We have used the sensitive and quantitative technique of real-time polymerase chain reaction to evaluate the patterns of donor alloantigen-induced cytokine mRNA production for interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-10, and transforming growth factor (TGF)-beta. We evaluated cytokine mRNA expression in cardiac allografts and in donor alloantigen-challenged DTH sites in mice that have either accepted or rejected cardiac allografts. RESULTS: We observed the following. (1) Normal hearts and pinnae exhibited detectable baseline production of cytokine mRNAs: TGF-beta>IFN-gamma=IL-10>IL2->IL-4. (2) Both the accepted and rejecting cardiac allografts produced increased amounts of all cytokine mRNAs tested and displayed few quantitative differences in cytokine mRNA production. Notably, accepted allografts displayed enhanced IL-10 mRNA production on day 7 posttransplantation, but not on day 60 posttransplantation and reduced IFN-gamma mRNA production on day 60, but not day 7. (3) There was a high degree of variability in production levels among the various cytokine mRNAs, both for background levels and for allograft-stimulated levels. (4) Donor-reactive DTH sites of allograft rejector mice displayed a broad array of cytokine mRNAs, whereas the DTH sites of allograft acceptor mice displayed only IL-4 mRNA production. (5) Provision of exogenous TGF-beta or IL-10 at a DTH challenge site of allograft rejector mice caused a shift in the cytokine mRNA profile that minimized IFN-gamma and IL-2 mRNA production but spared IL-4, IL-10, and TGF-beta mRNA production. CONCLUSIONS: A broad array of cytokine mRNAs may be stockpiled for future use in cardiac allografts, regardless of whether the grafts will be accepted or rejected. This stockpile is continuously replenished for as long as the graft survives, thereby obscuring any changes in immune disposition of the graft recipient toward graft alloantigens. However, such changes can be revealed by challenge with donor alloantigens at a distant site (DTH challenge). In allograft acceptor mice, this disposition evolves from pro-inflammatory to anti-inflammatory.

Inhibition of angiogenesis-related endothelial activity by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective in the prevention/control of acute and chronic rejection in animal models and currently in phase I clinical trials in human transplant recipients. This agent is also effective in the control of graft-versus-host disease, autoimmune reactions, and the growth of certain tumors. The importance of the endothelium in these disease processes led us to hypothesize that leflunomide might act directly upon the endothelial cell (EC). METHODS AND RESULTS: Assay of human EC colony formation demonstrated dose-dependent, leflunomide-mediated inhibition of EC proliferation. In addition, the organization of EC into capillary-like networks, which occurs during 18 hr of incubation on Matrigel, was progressively disrupted with increasing concentrations of leflunomide. Finally, fibrin-embedded transverse sections of murine aorta, which sprout numerous microvessels during an 11-day incubation, were inhibited from doing so in the presence of this agent. All drug concentrations used in these experiments were nontoxic and pharmacologically relevant, and none of these effects were reversible by exogenous uridine, implying that inhibition of these processes was not due to intracellular pyrimidine depletion. Furthermore, neither cyclosporine nor tacrolimus exerted inhibitory activity in any of the experiments described above. CONCLUSIONS: Data generated by these studies distinguish leflunomide among immunosuppressants as uniquely capable of inhibiting angiogenesis-related endothelial functions and suggest additional mechanisms by which this agent might intervene in the diverse array of disease processes against which it has shown therapeutic potential.

Occult cytomegalovirus in vivarium-housed mice may influence transplant allograft acceptance.

We have recently shown that latent murine cytomegalovirus (MCMV) can influence murine transplant allograft acceptance. During these studies we became aware that vivarium-housed control mice can acquire occult MCMV infection. The purpose of this investigation was to confirm occult MCMV transmission and determine the timing, vehicle, and possible consequences of transmission. Mice arriving from a commercial vendor were negative for MCMV both by commercial serologic testing and by our nested PCR. Mice housed in our vivarium became positive for MCMV DNA 30-60 days after arrival, but remained negative for MCMV by commercial serologic testing. To confirm MCMV we sequenced PCR products for several genes and showed >99% homology to MCMV. Further sequence analyses show that the occult MCMV is similar to a laboratory strain of MCMV, but the vehicle of transmission remains unclear. Control tissues from historical experiments with unexplained graft losses were evaluated for occult MCMV, and mice with unexplained allograft losses showed significantly higher incidence of occult MCMV than did allograft acceptors. Deliberate infection with very low titer MCMV confirmed that viral transmission can occur without measurable virus specific antibody or T-cell responses. These data suggest that vivarium-housed mice can develop occult MCMV that is missed by currently available commercial serologic testing, and that these infections may influence transplant allograft acceptance.

Allogeneic stimulation causes transcriptional reactivation of latent murine cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well-described complication of transplantation that may be caused by allogeneic stimulation, immunosuppression, or both. These studies were performed to determine if allogeneic stimulation alone is sufficient to reactivate latent CMV. BALB/c mice latently infected with Smith strain murine CMV (MCMV) received allograft (n = 8), allograft plus cortisol (n = 5), or isograft (n = 4) skin. All allograft recipients rejected their grafts within 9 to 12 days of transplantation. Three weeks after grafting, recipients were evaluated for MCMV reactivation, and all allograft recipients (8/8) showed MCMV reactivation, while no isografts had reactivation (0/4). Surprisingly, cortisol therapy blocked MCMV reactivation (0/5). These data suggested that allogeneic stimulation alone can trigger systemic reactivation of latent CMV. Although immunosuppression is thought to contribute to reactivation, certain agents that impair NF-kappaB activation may actually reduce reactivation.

Mechanisms of graft acceptance: evidence that plasminogen activator controls donor-reactive delayed-type hypersensitivity responses in cardiac allograft acceptor mice.

We have used delayed-type hypersensitivity (DTH) responses to probe the mechanisms of drug-induced cardiac allograft acceptance in mice. DBA/2-->C57BL/6 cardiac allograft recipients treated transiently with gallium nitrate accept their grafts for >90 days and fail to display DBA/2-reactive DTH responses. These DTH responses are restored when anti-TGF-beta Abs are included at the challenge site, and cell depletion studies showed that this DTH inhibition is mediated by CD4+ cells. Real-time PCR analysis revealed that allograft acceptor mice produce no more than background levels of TGF-beta mRNA at DTH challenge sites. This suggests that DTH regulation in allograft acceptor mice may involve TGF-beta activation, rather than TGF-beta production. The protease, plasmin, can activate TGF-beta, and activated T cells can express a receptor for the plasmin-producing enzyme urokinase-type plasminogen activator (uPA), and can also produce both uPA and tissue-type plasminogen activator (tPA). We observed that Abs to tPA or uPA can replace anti-TGF-beta mAb for the restoration of donor-reactive DTH responses in allograft acceptor mice. Histologic analysis revealed that accepted cardiac allografts express uPA, tPA, and active TGF-beta, whereas accepted cardiac isografts express only tPA, but not uPA or activated TGF-beta. These data demonstrate that local tPA and uPA contribute to DTH regulation in allograft acceptor mice and suggest that these elements of the fibrinolytic pathway are used to control donor-reactive cell-mediated immunity in allograft acceptor mice.

Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity.

It was shown >20 yr ago that mice will spontaneously accept renal allografts in the absence of immunosuppression, but the mechanism responsible for this is not understood. We transplanted DBA/2 (H-2(d)) kidneys into nephrectomized C57BL/6 (H-2(b)) mice, and the allografts were spontaneously accepted for >60 days without immunosuppression. In contrast, nonimmunosuppressed cardiac and skin allografts in the same strain combination are rejected within approximately 10 days. The accepted renal allografts have a prominent leukocytic infiltrate, suggesting an ongoing, local immune response. At 60 days post-transplant, the recipients of accepted renal allografts display DBA/2-reactive alloantibodies. They also display DBA/2-reactive delayed-type hypersensitivity responses that are actively counter-regulated by DBA/2-induced TGF-beta production, but not by IL-10 production. These data suggest that a donor-reactive, cell-mediated immune mechanism involving TGF-beta is associated with the spontaneous acceptance of renal allografts in mice.