Charles G. Orosz

Oxygen Sensing by Primary Cardiac Fibroblasts: A Key Role of p21Waf1/Cip1/Sdi1

Abstract— In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 ≈14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to ≈1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of Po2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the Po2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the Po2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 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-&bgr;1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-&bgr;1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Transfusion of polarized TH2-like cell populations into SCID mouse cardiac allograft recipients results in acute allograft rejection

It has been hypothesized that TH1 cells mediate the archetypical cell-mediated immune response of acute allograft rejection, whereas TH2 cells promote allograft acceptance. To test this, we transfused SCID cardiac allograft recipients with polarized TH1-like or TH2-like graft-reactive T cells, and monitored graft function and graft-reactive immune responses in the graft recipients. Polarized THl-like cells, which were generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of anti-IL-4 mAb, produced IFNg but not IL-4 when restimulated with donor alloantigen. Polarized TH2-like populations, which are generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of IL-4, produced IL-4 but not IFNg when restimulated with donor alloantigen. Interestingly, bioassays of culture SN from restimulated TH1 but not TH2 cells revealed IL-2 production, although LDA analyses revealed that the TH1 and TH2 cells had identical frequencies of IL-2-producing cells. Transfusion of THl-like cells into SCID cardiac allograft recipients resulted in acute rejection within 7-10 days that was characterized by cellular infiltration, myocyte necrosis, and edema. Graft-infiltrating cells (GIC) recovered from TH1-transfused animals contained large numbers of graft-reactive IL-2-producing cells (68-269/10(6) GIC), but no LDA-detectable IL-4-producing cells. These data support the hypothesis that donor-reactive TH1 cells can promote acute allograft rejection. In contrast to the hypothesis, transfusion of the polarized TH2-like population into SCID cardiac allograft recipients also resulted in histologically similar acute rejection within 7-10 days. Infiltrating cells recovered from TH1-transfused allografts contained large numbers of graft-reactive (109-1458/10(6) GIC), LDA-detectable, IL-4-producing cells--indicating that the TH2 cells had arrived at the graft-but promoted acute allograft rejection rather than allograft acceptance.

The Basic Immune Simulator: An agent-based model to study the interactions between innate and adaptive immunity

BackgroundWe introduce the Basic Immune Simulator (BIS), an agent-based model created to study the interactions between the cells of the innate and adaptive immune system. Innate immunity, the initial host response to a pathogen, generally precedes adaptive immunity, which generates immune memory for an antigen. The BIS simulates basic cell types, mediators and antibodies, and consists of three virtual spaces representing parenchymal tissue, secondary lymphoid tissue and the lymphatic/humoral circulation. The BIS includes a Graphical User Interface (GUI) to facilitate its use as an educational and research tool.ResultsThe BIS was used to qualitatively examine the innate and adaptive interactions of the immune response to a viral infection. Calibration was accomplished via a parameter sweep of initial agent population size, and comparison of simulation patterns to those reported in the basic science literature. The BIS demonstrated that the degree of the initial innate response was a crucial determinant for an appropriate adaptive response. Deficiency or excess in innate immunity resulted in excessive proliferation of adaptive immune cells. Deficiency in any of the immune system components increased the probability of failure to clear the simulated viral infection.ConclusionThe behavior of the BIS matches both normal and pathological behavior patterns in a generic viral infection scenario. Thus, the BIS effectively translates mechanistic cellular and molecular knowledge regarding the innate and adaptive immune response and reproduces the immune systems complex behavioral patterns. The BIS can be used both as an educational tool to demonstrate the emergence of these patterns and as a research tool to systematically identify potential targets for more effective treatment strategies for diseases processes including hypersensitivity reactions (allergies, asthma), autoimmunity and cancer. We believe that the BIS can be a useful addition to the growing suite of in-silico platforms used as an adjunct to traditional research efforts.

Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.

Treatment with anti-vascular cell adhesion molecule 1 monoclonal antibody induces long-term murine cardiac allograft acceptance.

Daily in vivo treatment of murine H-2d --> H-2b cardiac allograft recipients with 400 micrograms/day i.p. of M/K-2, a mAb to the endothelial adhesion molecule VCAM-1, resulted in prolongation of graft survival. The surviving allografts showed little of the histologic changes observed in acutely rejecting control allografts. When antibody treatment was discontinued after 20 days, grafts continued to function for at least 40 more days. This was approximately 30 days after mAb was no longer detectable by ELISA in the circulation, or by immunoperoxidase staining at the graft site. The most notable feature of grafts that survived 60 days was the presence of mild interstitial fibrosis. Endothelial reactivity was minimal with the mAbs MECA-32 and M/K-2, which have been used in previous studies to visualize the extensive endothelial inflammation that develops during untreated acute rejection. There was a mild cellular infiltrate containing T cells, but few macrophages. However, infiltrating T cells appeared to be inactive in that IL-2R+ cells were immunohistologically undetectable and mRNA for IL-2, IL-4, or IFN-gamma was undetectable by polymerase chain reaction. In general, the immunologic conditions in these long-term grafts differed from those seen in normal cardiac tissue, cardiac isografts, or cardiac allografts. These data demonstrate that M/K-2 mAb can suppress cardiac allograft rejection and induce long-term graft acceptance. This graft survival appears to be associated with the development of a unique state of immunity at the graft site.

Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation.

Background. In vitro production of tumor necrosis factor-&agr; (TNF-&agr;), interferon-&ggr; (IFN-&ggr;), interleukin 10 (IL-10), and transforming growth factor-&bgr; (TGF-&bgr;) correlate with their respective genetic polymorphisms. We analyzed the relationship between these genetic polymorphisms and posttransplant outcome. Methods. Using DNA polymerase chain reaction (PCR) technology, polymorphisms for TNF- &agr;, IFN-&ggr;, IL-10, and TGF-&bgr; were determined for 82 kidney (K) and 19 simultaneous kidney-pancreas (SKP) recipients. These results were analyzed with regard to the incidence of acute rejection (AR), and the timing and severity of the first AR episode. Results. A high TNF-&agr; production phenotype correlated with recurrent acute rejection (AR) episodes (P <0.026). Compared with the low TNF-&agr; production phenotype, more patients with the high production phenotype had a post-AR serum creatinine >2.0 mg/dl, but this was not statistically significant (64 vs. 35%, P =0.12). There was no relationship between TNF-&agr; genotype and the time to first AR episode or incidence of graft loss. IFN-&ggr; production phenotype showed no correlation with any of these clinical outcome parameters. There was an increase in AR incidence as the IL-10 production phenotype increased (low, intermediate, high), but only in low TNF-&agr; producer phenotypes (P =0.023). Conclusions. Patients with a polymorphic cytokine genotype putatively encoding for high in vivo TNF-&agr; production, and to a lesser extent IL-10 cytokine genotypes putatively encoding for higher levels of in vivo IL-10 production, had a worse clinical outcome regarding AR episodes. These data support the hypothesis that the strength of alloimmune responsiveness after transplantation in part is genetically determined.

Clinical Significance of MHC‐Reactive Alloantibodies that Develop after Kidney or Kidney–pancreas Transplantation

The purpose of this study was to determine the relationships between acute rejection, anti‐major histocompatibility complex (MHC) class I and/or class II‐reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney‐pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post‐transplant (mean 2.6 years). The presence of anti‐MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC‐reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti‐MHC class II, but not anti‐MHC class I, alloantibodies detected post‐transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II‐reactive, but not MHC class I‐reactive, alloantibodies post‐transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post‐transplant detectable MHC class II‐reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post‐transplant alloantibody production, and avoidance of acute rejection episodes, may improve long‐term graft survival by reducing the incidence of chronic allograft rejection.

Immunobiology of Allograft Rejection in the Absence of IFN-γ: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions

Both wild-type (WT) and IFN-γ-deficient (IFN-γ−/−) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8+ cells, both WT and IFN-γ−/− mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4+ cells, WT mice accepted their allografts, while IFN-γ−/− recipients rapidly rejected them. Hence, IFN-γ−/−, but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-γ−/− mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-γ−/− mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-γ−/− mice, although grafts were eventually rejected when CD8+ T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-γ is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.

Morphometric analysis of neointimal formation in murine cardiac allografts.

BACKGROUND Transplant vascular sclerosis is expressed in transplanted human and murine hearts as a concentric intimal thickening. The purpose of this study was to characterize the location, distribution, and intensity of transplant vascular sclerosis in murine cardiac allografts using computerized morphometric analysis. METHODS Murine cardiac allograft recipients were treated with the immunosuppressant gallium nitrate to promote graft survival. The grafts were removed at 60 days after transplantation and histologically stained. The coronary arteries were analyzed for intimal thickening using a neointimal index (NI) derived with a computer imaging system. RESULTS A cross-section taken from the middle of a cardiac allograft showed four major coronary arteries, each with widely different NI values (65, 0, 92, and 0). The same four vessels in two other grafts also showed highly variable NI values, but different patterns of vessel involvement. Next, NI values were determined along the length of a single vessel from aorta to apex. This revealed variable, fluctuating intimal thickening along the length of the vessel. In general, arteries from the aortic versus apical regions of the grafted hearts expressed similar amounts of intimal thickening (analysis of variance, P=0.4826). Finally, a method was devised to quantitate intimal thickening from a sampling of three tissue cross-sections taken from the middle of each cardiac allograft. This value was statistically indistinguishable from values obtained by analysis of intimal thickening in multiple sections covering the entire heart (P=0.6734, 0.9021, and 0.1474). CONCLUSIONS Intimal thickening in the coronary arteries of murine cardiac allografts appears to be variable in terms of location, distribution, and intensity. This is true for different regions of the same vessel, different vessels in the same heart region, and the same vessels in different cardiac allografts.

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

Purpose: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4ds role as a prognostic adjunct in lung allografts. Material and Methods: Twenty‐three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. Results: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p < .0001) compared to other the immunoreactants C1q, C5b‐9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon‐BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. Conclusions: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.