Endothelial induction of fgl2 contributes to thrombosis during acute vascular xenograft rejection

Thrombosis is a prominent feature of acute vascular rejection (AVR), the current barrier to survival of pig-to-primate xenografts. Fibrinogen-like protein 2 (fgl2/fibroleukin) is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis and cytokine-induced fetal loss. We hypothesized that induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with AVR. We first examined fgl2 as a source of procoagulant activity in the pig-to-primate combination. The porcine fgl2 (pfgl2) was cloned and its chromosomal locus was identified. Recombinant pfgl2 protein expressed in vitro was detected on the cell surface and generated thrombin from human prothrombin. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells (ECs). Cultured porcine ECs activated by human TNF-alpha in vitro demonstrated induction of pfgl2 expression and enhanced activation of human prothrombin. The availability of gene-targeted fgl2-deficient mice allowed the contribution of fgl2 to the pathogenesis of AVR to be directly examined in vivo. Hearts heterotopically transplanted from fgl2(+/+) and fgl2(+/-) mice into Lewis rats developed AVR with intravascular thrombosis associated with induction of fgl2 in graft vascular ECs. In contrast, xenografts from fgl2(-/-) mice were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium plays a role in the pathogenesis of AVR-associated thrombosis. Manipulation of fgl2, in combination with other interventions, may yield novel strategies by which to overcome AVR and extend xenograft survival.     

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.     

The role of the CC chemokine, RANTES, in acute lung allograft rejection

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.     

Membrane permeability to macromolecules mediated by the membrane attack complex

A simple and well-defined system of purified phospholipids and human complement proteins was used to study membrane permeability to macromolecules mediated by the membrane attack complex (MAC) of complement. Large unilamellar vesicles (LUVs) of phosphatidylcholine (PC) or phosphatidylserine (PS) containing trapped macromolecules [bovine pancreatic trypsin inhibitor (BPTI), thrombin, glucose-6-phosphate dehydrogenase (G6PD), and larger molecules] were used to monitor permeability. Membrane permeability to macromolecules was measured by thrombin inhibition by an external inhibitor or by separation of released molecules by gel filtration. Membrane-bound intermediates (C5b-8 or C5b-93) were stable for hours, and macromolecular permeability occurred without fragmentation, fusion, or aggregation of the vesicles. Quantitative membrane binding by C5b-7 as well as essentially quantitative release of thrombin was obtained for PS vesicles. MAC binding to PS-LUVs approximated the theoretical Poisson distribution curve for full release of vesicle contents by one complex per vesicle. Reactions with PC-LUVs occurred with some fluid-phase MAC assembly. Therefore, results from experiments with these vesicles were interpreted in a relative manner. However, the values obtained closely corroborated those obtained with PS-LUVs. At low C9/C5b-8 ratios, the size of the lesion was proportional to the C9 content of the MAC. Half-maximum release of BPTI, thrombin, and G6PD, by a single MAC per vesicle, required approximately 3,5, and 7 C9/C5b-8 (mol/mol), respectively. Larger molecules (greater than or equal to 118-A diameter) were not released from the vesicles. Release of G6PD (95.4-A diameter) required 45% of saturating C9. Therefore, it appeared that the last half of the bound C9 molecules did not increase pore size and the pore which released G6PD approached the diameter of the closed circular lesion measured (by others) in electron micrographs (approximately 100 A). The results were consistent with the formation of a stable membrane pore by a single complex per vesicle in which C9 molecules line only one side of the pore at low C9/C5b-8 ratios and maximum pore size is attained by incomplete, noncircular polymers of C9.     

Prevention of neutrophil migration ameliorates rat lung allograft rejection

Chemokines activate and recruit specific leukocyte subpopulations. We sought to determine whether neutrophil migration, which can contribute to the development of ischemia-reperfusion injury, correlates with lung allograft rejection. Orthotopic left lung allotransplantation was performed from Brown Norway (donor) to Fisher 344 (recipient) rats. Because the role of activated neutrophils in the development of allograft rejection is believed to be biphasic, we used specific CXC receptor inhibition with antileukinate in 2 dosing regimens. Recipients were allocated into 4 groups; A (early administration) received 2 doses of antileukinate (10.0 mg/kg) intramuscularly 24 h before and immediately after transplantation; B (continuous administration) continuously received antileukinate intraperitoneally (10.0 mg/kg/day) for 7 days after surgery. Groups A or B were compared with individual controls that received PBS alone. The progression of rejection was assessed radiographically. Histologic evaluation of allograft rejection based on pathologic rejection grade, performed on day 7, demonstrated significantly lower histologic rejection in group B compared with the control group (2.1+/-1.0 vs. 3.3+/-0.5; P=0.018), whereas there was no significant difference in group A compared with the control group. There were no significant differences between the aeration scores of groups A or B compared with their control groups. Our data suggest that neutrophils may play a promoting role in the development of allograft rejection, and blockage of neutrophil migration may suppress acute lung allograft rejection.     

Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.     

Proteomic identification of human neutrophil alpha-defensins in chronic lung allograft rejection

Chronic allograft rejection remains a leading cause of morbidity and mortality in lung transplant recipients. Currently, diagnosis is based on lung biopsies or the presence of bronchiolitis obliterans syndrome (BOS). To identify a biomarker of rejection we performed a proteome survey of archived bronchoalveolar lavage fluid (BALF) acquired from lung transplant recipients between 1993 and 1996 using mass spectrometry (MS). A total of 126 BALF samples from 57 individuals were tested. Initial MS assessment revealed numerous differences in a majority of individuals who experienced BOS, but three unusually intense peaks at m/z = 3373, 3444, and 3488. These were identified as human neutrophil peptides 1-3 (HNP). Quantification by enzyme-linked immunoabsorbent assay showed an elevated HNP level (>0.3 ng/microg protein) in 89% of patients who developed BOS2-3 within 15 months, reaching as high as 6% of the total BALF protein. In control patients, 35% demonstrated a slightly elevated HNP level that declined in all who had subsequent BALF available for testing. HNP levels did not correlate with episodes of acute rejection, cytomegalovirus or fungal infection. In conclusion, elevated HNP levels are associated with the onset of BOS and can predate the clinical onset of disease up to 15 months.     

The role of TNF-related activation-induced cytokine-receptor activating NF-kappa B interaction in acute allograft rejection and CD40L-independent chronic allograft rejection

We analyzed the role of TNF-related activation-induced cytokine (TRANCE), a member of the TNF family expressed on activated T cells that shares functional properties with CD40L, and its receptor-activating NF-kappaB (RANK) which is mostly expressed on mature dendritic cells, during allogenic responses in vivo using a rodent heart allograft model. TRANCE mRNA was strongly up-regulated in acutely rejected allografts on days 4 and 5 posttransplantation whereas RANK was detected as early as day 1 but did not show further up-regulation during the first week. Immunofluoresence analyses of heart allografts showed that 80 and 100% of TRANCE and RANK-expressing cells were T cells and APCs, respectively. We show for the first time that short-term TRANCE blockade using a mouse RANKIg fusion molecule can significantly prolong heart allograft survival in both rat and mouse models. Similarly, rat heart allografts transduced with a RANKIg encoding recombinant adenovirus exhibited a significant prolongation of survival (14.3 vs 7.6 days, p < 0.0001). However, TRANCE blockade using RANKIg did not appear to inhibit allogeneic T and B cell priming humoral responses against RANKIg. Interestingly, TRANCE blockade induced strong up-regulation of CD40 ligand (CD40L) mRNA in allografts. Combined CD40L and TRANCE blockade resulted in significantly decreased chronic allograft rejection lesions as well as allogeneic humoral responses compared with CD40L blockade alone. We conclude that TRANCE-RANK interactions play an important role during acute allograft rejection and that CD40L-independent allogeneic immune responses can be, at least in part, dependent on the TRANCE pathway of costimulation.     

B lymphocytes differentially influence acute and chronic allograft rejection in mice

The relative contributions of B lymphocytes and plasma cells during allograft rejection remain unclear. Therefore, the effects of B cell depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, and CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Strikingly, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, whereas CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4(+) T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Moreover, CD19 mAb may represent a new approach for depleting both B cells and plasma cells to concomitantly impair T cell activation, inhibit the generation of new allograft-specific Abs, or reduce preexisting allograft-specific Ab levels in transplant patients.     

Evaluation of biomarker discovery approaches to detect protein biomarkers of acute renal allograft rejection

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.     

Renal allograft rejection: immunohistochemistry of inflammatory cellular subsets and vascular lesions

We have examined sixty-seven surgically removed allograft kidneys to identify the different leukocyte subsets of interstitial infiltration and the early vascular lesions which characterize renal allograft rejection. Histochemical and immunohistochemical results (mouse monoclonal antibodies anti: Leu 1, Leu 3a-3b, Leu 7, Leu 2a, OK Ia-Dr, OKB2, Leu M1, Leu M3; rabbit heteroclonal antibodies anti -: IgA, IgG, IgM, C3, fibrinogen, lysozyme; lectins-ABC: RCA, WGA, UEA) and routine histochemical staining have shown an increase of T-helper and T-activated lymphocyte subsets in acute rejection. Neutrophilic leucocytes were present in hyperacute rejection; macrophages were also noted. In chronic rejection, several lymphocyte subsets, in different ratios, were identified. Monocyte/macrophage leukocytes were the most abundant cell population. IgA deposits were noted on tubular epithelia in hyperacute and chronic rejections. IgM deposits were observed in vascular walls in chronic rejection. C3 and fibrinogen deposits were seen in glomerular capillaries and in arterial walls, although in different ratios, in all cases of renal allograft rejections. We have generally seen weak reactions to IgG deposits. Histochemical analysis of lectin receptors has given different results according to the type of rejection considered. In hyperacute rejection, receptors for WGA were found both on glomerular endothelial cells and on the tubular brush border. In the latter, receptors for RCA were also found. In acute rejection, receptors for UEA and WGA were found in a lower number of cases of acute vascular rejection. In acute cellular rejection, receptors for RCA, UEA and WGA were recognized in tubular epithelia. In acute vascular rejection, as well as in chronic rejection, only receptors for WGA were present on tubular epithelia and on capillary loop endothelium. The use of anti-human lysozyme antibodies has yielded the following results: in acute and hyperacute rejection, when renal failure occurred, we saw a high ratio of lysozyme, either coarsely granular or diffuse in the proximal tubular epithelia. Lysozyme was found in myelocyte/macrophage cells within capillary loops and arterial walls, when acute necrotizing vasculitis was present. In acute rejection, proximal tubular cells were lysozyme-negative or lysozyme-positive only segmentally, especially when obliterative vasculitis by fibrointimal proliferation was present and renal function progressively failed. In most of the chronic rejections, tubular cells were lysozyme-negative.     

Xanthine oxidase is increased and contributes to paraquat-induced acute lung injury

Two lines of investigation suggested that xanthine oxidase- (XO) derived O2 metabolites contribute to paraquat- (PQ) induced acute lung injury. First, PQ treatment increased lung XO activity and decreased lung xanthine dehydrogenase activity. Second, lung albumin uptake increased compared with control values in untreated XO-replete but not tungsten-treated XO-depleted lungs in rats treated with PQ.     

Inhibition of NF-kappaB-dependent T cell activation abrogates acute allograft rejection.

Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-kappaB(DeltaN)-transgenic mice that express a transdominant inhibitor of NF-kappaB in T cells. Our results show indefinite prolongation of graft survival in the I-kappaB(DeltaN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-kappaB(DeltaN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-kappaB(DeltaN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-kappaB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-kappaB activation in T cells is necessary for acute allograft rejection.     

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.     

Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection

Lung transplantation is the only definitive treatment modality for many forms of end-stage lung disease. However, the lung is rejected more often than any other type of solid organ allograft due to chronic rejection known as bronchiolitis obliterans (BO). Indeed, BO is the primary reason why the 5- and 7-yr survival rates are worse for the lung than for any other transplanted organ. Alloimmunity to donor antigens is established as the primary mechanism that mediates rejection responses. However, newer immunosuppressive regimens designed to abrogate alloimmune activation have not improved survival. Therefore, these data suggest that other antigens, unrelated to donor transplantation antigens, are involved in rejection. Utilizing human and rodent studies of lung transplantation, our laboratory has documented that a native collagen, type V collagen [col(V)], is a target of the rejection response. Col(V) is highly conserved; therefore, these data indicate that transplant rejection involves both alloimmune and autoimmune responses. The role of col(V) in lung transplant rejection is described in this review article. In addition, the potential role of regulatory T cells that are crucial to modulating autoimmunity and alloimmunity is also discussed.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.