Abstract-In mammalian organs under normoxic conditions, O 2 concentration ranges from 12% to Ͻ0.5%, with O 2 Ϸ14%in arterial blood and Ͻ10% in the myocardium. During mild hypoxia, myocardial O 2 drops to Ϸ1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO 2 results in perceived hyperoxia. These effects were independent of NADPH oxidase function. CFs exposed to high O 2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-␤1, (2) lowered telomerase activity, and (3) Key Words: redox Ⅲ free radicals Ⅲ heart Ⅲ cell culture C ellular O 2 concentrations are maintained within a narrow range (normoxia) because of the risk of oxidative damage from excess O 2 (hyperoxia) and of metabolic demise from insufficient O 2 (hypoxia). 1 PO 2 ranges from 90 to Ͻ3 mm Hg in mammalian organs under normoxic conditions, with arterial PO 2 of Ϸ100 mm Hg or Ϸ14% O 2 . 2 Thus, "normoxia" for cells is a variable that is dependent on the specific localization of the cell in organs and functional status of the specific tissue. O 2 sensing is required to adjust to physiological or pathophysiological variations in PO 2 . Current work in this field is almost exclusively focused on the study of hypoxia. Reoxygenation, on the other hand, has been mostly investigated in the context of oxidative injury. Over 25 years ago, it was observed that PO 2 beyond the comfort of the "perceived normoxic range" is a significant stressor, leading to growth arrest. 3 The molecular bases of such observations remain to be characterized in light of current knowledge of signal transduction.During chronic hypoxia in the heart, cells adjust their normoxic set point such that the return to normoxic PO 2 after chronic hypoxia is perceived as relative hyperoxia. 4,5 We hypothesized that such challenge triggers changes in signal transduction processes. Although acute insult caused during reperfusion may be lethal to cells localized at the focus of insult, elevation of O 2 tension in the surrounding ischemic tissue triggers phenotypic changes in the surviving cells that may be associated with tissue remodeling.Ischemia in the heart results in a hypoxic area containing a central focus of near-zero O 2 pressure bordered by tissue with diminished but nonzero O 2 pressures. These border zones extend for several millimeters from the hypoxic core, with the O 2 pressures progressively increasing from the focus to the normoxic region. 6 Moderate hypoxia is associated with a 30% to 60% decrease (Ϸ1% to 3% O 2 ) in PO 2 . 7 Cardiac fibroblasts (CFs) are mainly responsible for the synthesis of major extracellular matrix (ECM) in the myocardium, including fibrillar collagen types I and III and fibronectin. More than 90% of the interstitial cells of the myocardium are fibroblasts, 8 which actively e...

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 lmol/L). All CCR5 -/-recipients rejected renal allografts within 21 days posttransplant (mean 13.3 ± 4 days) with elevated creatinine (90 ± 31 lmol/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, lMT -/-/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.

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-2d) kidneys into nephrectomized C57BL/6 (H-2b) 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-β production, but not by IL-10 production. These data suggest that a donor-reactive, cell-mediated immune mechanism involving TGF-β is associated with the spontaneous acceptance of renal allografts in mice.

Rejected MHC-mismatched cardiac allografts in CCR5−/− recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5−/− recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5−/− (H-2b) recipients of A/J (H-2a) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-γ, whereas CD4 T cells producing IL-4 were increased in CCR5−/− recipients. Numbers of donor-reactive CD8 T cells producing IFN-γ were reduced 60% in CCR5−/− recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5−/− allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1−/− recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5−/− recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12–60, mean 21 days) in μMT−/−/CCR5−/− recipients. These results indicate that the donor-specific Ab produced in CCR5−/− heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.

MHC-mismatched DBA/2 renal allografts are spontaneously accepted by C57BL/6 mice by poorly understood mechanisms, but both immune regulation and graft acceptance develop without exogenous immune modulation. Previous studies have shown that this model of spontaneous renal allograft acceptance is associated with TGF-β-dependent immune regulation, suggesting a role for T regulatory cells. The current study shows that TGF-β immune regulation develops 30 days posttransplant, but is lost by 150 days posttransplant. Despite loss of detectable TGF-β immune regulation, renal allografts continue to function normally for >200 days posttransplantation. Because of its recently described immunoregulatory capabilities, we studied IDO expression in this model, and found that intragraft IDO gene expression progressively increases over time, and that IDO in “regulatory” dendritic cells (RDC) may contribute to regulation associated with long-term maintenance of renal allografts. Immunohistochemistry evaluation confirms the presence of both Foxp3+ T cells and IDO+ DCs in accepted renal allografts, and localization of both cell types within accepted allografts suggests the possibility of synergistic involvement in allograft acceptance. Interestingly, at the time when RDCs become detectable in spleens of allograft acceptors, ∼30% of these mice challenged with donor-matched skin allografts accept these skin grafts, demonstrating progression to “true” tolerance. Together, these data suggest that spontaneous renal allograft acceptance evolves through a series of transient mechanisms, beginning with TGF-β and T regulatory cells, which together may stimulate development of more robust regulation associated with RDC and IDO.

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5−/− recipients was investigated. Heart grafts from A/J (H-2a) donors were rejected by wild-type C57BL/6 (H-2b) recipients on day 8–10 posttransplant vs day 8–11 by CCR5−/− recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4+ and CD8+ T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-γ-, but not IL-4-producing cells in the CCR5−/− recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4+ than CD8+ T cells in the spleens of allograft wild-type recipients and on CD4+ T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5−/− recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5−/− recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.

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.

Acute humoral rejection (AHR), which occurs in up to 8% of kidney transplant recipients, is a significant cause of renal allograft dysfunction and loss. More efficacious treatment modalities are needed to eliminate or curtail alloantibody production and its deleterious effects on the kidney. The availability of animal models mimicking human AHR is essential to understand its pathophysiology and develop new treatment strategies. Using a mouse kidney transplant model, we demonstrate that presensitization of recipients with donor skin grafts results in rejection of subsequent renal allografts. All presensitized mice developed renal failure 8.6 ؎ 4.3 days after engraftment, with serum creatinine values near 100 mol/dl. Graft histology revealed mild, diffuse, interstitial, mononuclear cell infiltrates; prominent peritubular capillary inflammatory cell margination; patchy interstitial hemorrhage; interstitial edema; and focal glomerular fibrin deposition. Complement (C3d) deposition was diffuse and prominent in peritubular capillaries. Serum analysis demonstrated high levels of circulating alloantibodies with broad cross-reactivity to many MHC haplotypes. The clinical setting and histological findings of our model strongly resemble AHR, which is frequently associated with cellular rejection, a situation commonly encountered in human renal allograft recipients. This animal model provides a valuable tool to study the pathogenesis of AHR, its relationship to cellular alloimmunity, its contribution to graft injury, and the effects of various potential therapeutic interventions.