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ABSTRACT: Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood.
In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation.
The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms.
Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.
Transplantation 10/2007; 84(6):771-7. · 4.00 Impact Factor
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ABSTRACT: Microarray technology can rapidly generate large databases of gene expression profiles. Our laboratory has applied these techniques to analyze differential gene expression in cardiac tissue and cells based on mouse heart transplantation. We have analyzed the different gene expression profiles such as stress or injury including ischemia following transplantation. We also have investigated the role of infiltrating inflammatory cells during graft rejection by purifying subsets of infiltrating cells using GFP transgenic mice and detailed all technical experiences and issues. The purpose of this study is to assist researchers to simplify the process of analyzing large database using microarray technology.
Methods in molecular biology (Clifton, N.J.) 02/2007; 366:3-12.
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ABSTRACT: The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains incompletely understood. The important role of foreign peptides in the alloimmune response was recently recognized.
We performed a comparative study of the functions of minor antigens Class I, Class II, and CD1 in murine cardiac allograft rejection by investigating the expression of a large panel of immune and inflammatory genes. To investigate the role of MHC Class II and I, our protocol analyzed allograft recipients deficient in MHC Class II and b2 microglobulin (b2-M), a critical component of the Class I heterodimer. We also included CD1 deficient recipients to differentiate effects in the beta2-M deficient strain due to CD1 deficiency versus the combined inactivation of CD1 and Class I. The serum cytokines tumor necrosis factor (TNF)-alpha interleukin (IL)-6, interferon (IFN)-gamma and IL-1beta were evaluated posttransplant by ELISA. The intragraft expression of 55 chemokines, chemokine receptors, and CD markers were measured by ribonuclease protection assay. The data were analyzed through hierarchical clustering dendrograms and self-organizing maps.
The analysis indicates that each gene deficiency induces both the upregulation and the downregulation of distinct subsets of genes and that similar kinetics of rejection can be attributed to different molecular mechanisms.
The study provides novel insights into the role of classical and non-classical MHC molecules in graft rejection.
Transplantation 10/2004; 78(6):788-98. · 4.00 Impact Factor
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Bianca Schaub,
Abdelouahab Bellou,
Fiona K Gibbons,
German Velasco,
Monica Campo,
Hongzhen He, Yurong Liang,
Matthew W Gillman,
Diane Gold,
Scott T Weiss,
David L Perkins,
Patricia W Finn
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ABSTRACT: Toll-like receptor 2 (TLR2) and TLR4 signaling may induce differential secretion of T helper 1 (Th1) and Th2 cytokines, potentially influencing the development of autoimmune or atopic diseases. To date, the influence of the type of stimulus, timing, and dose of TLR2 and TLR4 ligands on cytokine secretion has not been well established. We tested whether the innate stimuli peptidoglycan (Ppg, TLR2 agonist) and lipid A (LpA, TLR4 agonist) differentially affect the secretion of interleukin-13 (IL-13) (Th2) and interferon-gamma (IFN-gamma) (Th1). Further, we examined the influence of the maturity of the immune system, species, dose, and timing of stimuli in human cord and adult peripheral blood mononuclear cells (PBMC) and murine cells in vitro and in vivo. Stimulation with Ppg induced the secretion of both IL-13 and IFN-gamma, influenced by time and dose in neonates, adults, and mice. In contrast, stimulation with LpA induced primarily time-independent and dose-independent production of IFN-gamma. Pulmonary administration of Ppg in vivo in mice resulted in secretion of IL-13, whereas administration of LpA resulted in secretion of IFN-gamma in bronchoalveolar lavage (BAL). Therefore, TLR2 and TLR4 stimuli differentially influence IL-13 and IFN-gamma secretion in neonates, adults, and mice, supporting a critical role for innate stimuli in the modulation of cytokine responses.
Journal of Interferon & Cytokine Research 10/2004; 24(9):543-52. · 3.06 Impact Factor
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ABSTRACT: Lymphocyte antigen receptors are critical for allograft rejection, but their precise involvement is only partly understood. Some members of the immunoglobulin superfamily (e.g., alphabeta T-cell receptors [TCR]) are known to be crucial for acute allograft rejection, but the role of other members remains poorly defined.
In this study, the authors analyzed two poorly understood receptors, TCRgammadelta and B-cell receptors (BCR), in allograft rejection in a murine model of cardiac transplantation. Using ribonuclease protection assays, their approach was a comprehensive analysis of the expression of immune and inflammatory genes. The mice included those with deficiencies of gammadelta TCR or BCR and alymphoid (recombination activating gene [RAG] knockout) mice. Because the RAG mice lack all TCR and BCR, they provide a baseline with which to compare the effects of TCRgammadelta and BCR deficiency.
Graft survival was extended in the TCRgammadelta- and BCR-deficient mice. Furthermore, the authors were able to identify groups of genes modulated by specific receptors. Five and six genes were expressed at lower levels in the BCR- and TCRgammadelta-deficient groups, respectively. The authors also found eight genes that were at higher levels in the TCRgammadelta-deficient group, suggesting that these receptors have both pro- and antirejection effects.
Overall, the authors' study shows that the individual regulatory effects of these different lymphocyte antigen receptors are distinct. In addition, several genes are identified that are candidates as necessary for rejection. This has crucial implications for developing clinical therapies that target specific mechanisms for prolonging graft survival.
Transplantation 03/2004; 77(4):580-6. · 4.00 Impact Factor
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ABSTRACT: Background. Lymphocyte antigen receptors are critical for allograft rejection, but their precise involvement is only partly understood. Some members of the immunoglobulin superfamily (e.g., αβ T-cell receptors [TCR]) are known to be crucial for acute allograft rejection, but the role of other members remains poorly defined.
Methods. In this study, the authors analyzed two poorly understood receptors, TCRγδ and B-cell receptors (BCR), in allograft rejection in a murine model of cardiac transplantation. Using ribonuclease protection assays, their approach was a comprehensive analysis of the expression of immune and inflammatory genes. The mice included those with deficiencies of γδ TCR or BCR and alymphoid (recombination activating gene [RAG] knockout) mice. Because the RAG mice lack all TCR and BCR, they provide a baseline with which to compare the effects of TCRγδ and BCR deficiency.
Results. Graft survival was extended in the TCRγδ- and BCR-deficient mice. Furthermore, the authors were able to identify groups of genes modulated by specific receptors. Five and six genes were expressed at lower levels in the BCR- and TCRγδ-deficient groups, respectively. The authors also found eight genes that were at higher levels in the TCRγδ-deficient group, suggesting that these receptors have both pro- and antirejection effects.
Conclusions. Overall, the authors' study shows that the individual regulatory effects of these different lymphocyte antigen receptors are distinct. In addition, several genes are identified that are candidates as necessary for rejection. This has crucial implications for developing clinical therapies that target specific mechanisms for prolonging graft survival.
Transplantation 02/2004; 77(4):580-586. · 4.00 Impact Factor
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ABSTRACT: The function of interferon (IFN)gamma in the regulation of the immune response after allogeneic transplantation is still poorly understood. Previous studies have suggested that IFNgamma can promote rejection and be important in tolerance induction.
To analyze the various IFNgamma-dependent functions in terms of T helpers 1 and 2 responses during rejection, we investigated mice deficient in the transcription factors (signal transducer of activated T cells [STAT]4 and 6) and IFNgamma in fully major histocompatibility complex-mismatched vascularized cardiac transplants. Serum levels of the cytokines tumor necrosis factor-alpha, IFNgamma, and interleukin (IL)-1beta were evaluated by enzyme-linked immunosorbent assay, and the graft-infiltrating cells were examined by immunohistochemical staining. To analyze a large panel of immune parameters, we determined the expression of chemokines, chemokine receptors, and clusters of differentiation markers by RNAase protection assays. The data were analyzed with algorithms that generated hierarchic clustering dendrograms. Also, the expression profiles of individual genes were determined with self-organizing maps.
Our data show that both the STAT4- and STAT6-deficient groups have statistically prolonged graft survival (P<0.04 and P<0.01). Despite the absence of prolongation of graft survival in the IFNgamma-deficient group, our analysis of variance data show that more genes (18) were modulated in the IFNgamma-deficient group compared with the other two STAT4- and STAT6-deficient groups (five each).
Our results indicate that IFNgamma plays a distinct role in the modulation of gene expression that includes STAT4-independent mechanisms. Our study identifies eight genes (IL-1beta, IL-1RA, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, CC-chemokine receptor (CCR)-1, CCR2, CCR5, and F4/80) that are highly expressed in all of our experimental groups. Thus, these genes become candidates for essential functions during rejection.
Transplantation 01/2004; 76(12):1749-58. · 4.00 Impact Factor
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ABSTRACT: Background. The function of interferon (IFN)γ in the regulation of the immune response after allogeneic transplantation is still poorly understood. Previous studies have suggested that IFNγ can promote rejection and be important in tolerance induction.
Methods. To analyze the various IFNγ-dependent functions in terms of T helpers 1 and 2 responses during rejection, we investigated mice deficient in the transcription factors (signal transducer of activated T cells [STAT]4 and 6) and IFNγ in fully major histocompatibility complex-mismatched vascularized cardiac transplants. Serum levels of the cytokines tumor necrosis factor-α, IFNγ, and interleukin (IL)-1β were evaluated by enzyme-linked immunosorbent assay, and the graft-infiltrating cells were examined by immunohistochemical staining. To analyze a large panel of immune parameters, we determined the expression of chemokines, chemokine receptors, and clusters of differentiation markers by RNAase protection assays. The data were analyzed with algorithms that generated hierarchic clustering dendrograms. Also, the expression profiles of individual genes were determined with self-organizing maps.
Results. Our data show that both the STAT4- and STAT6-deficient groups have statistically prolonged graft survival (P <0.04 and P <0.01). Despite the absence of prolongation of graft survival in the IFNγ-deficient group, our analysis of variance data show that more genes (18) were modulated in the IFNγ-deficient group compared with the other two STAT4- and STAT6-deficient groups (five each).
Conclusions. Our results indicate that IFNγ plays a distinct role in the modulation of gene expression that includes STAT4-independent mechanisms. Our study identifies eight genes (IL-1β, IL-1RA, macrophage inflammatory protein-1β, monocyte chemoattractant protein-1, CC-chemokine receptor (CCR)-1, CCR2, CCR5, and F4/80) that are highly expressed in all of our experimental groups. Thus, these genes become candidates for essential functions during rejection.
The role of cytokines in allograft rejection remains poorly understood. It is commonly assumed that the inhibition of graft rejection by calcineurin inhibitors is mediated by blocking the production of a subset of T-cell-produced cytokines; however, in murine models the deficiency of single cytokines, or even groups of cytokines, has only modest effects on prolonging graft survival. For example, according to studies of antibody inhibitors or knockout mice, the inhibition of several cytokines, including interleukin (IL)-2, IL-4, and IL-12, have been shown to prolong graft survival (1-3). Conversely, in other models, some cytokines, including IL-4, IL-10, IL-13, and interferon (IFN)γ (4-8), have been shown to accelerate rejection. Thus, cytokines have complex interactions including both stimulatory and inhibitory effects on rejection; however, no cytokine has been shown to be required for acute rejection.
IFNγ, a potent proinflammatory cytokine, is induced by the signal transducer of activated T cell [STAT]4-dependent type 1 IFN. IFNγ-deficient mice do not demonstrate prolonged graft survival (9). In some transplant models, IFNγ-deficient recipients have accelerated rejection or are resistant to tolerance induction protocols (10). Thus, IFNγ may be important in tolerance induction. IFNγ is also the major cytokine involved in the stimulation of expression of major histocompatibility complex (MHC) class II molecules and increases the level of expression of MHC class I. Studies have shown that IFNγ produced by the recipient directly signals the graft to induce MHC expression, increase chemokines inducible protein (IP)-10 and MIG, and prevent immunologically mediated graft necrosis (11,12). To clarify the complex functions of IFNγ during rejection, we analyzed gene expression profiles in IFNγ-deficient allograft recipients.
Differential expression of cytokines can modulate the subsequent development of the immune response, for example, by promoting a T helper (Th)1- or Th2-type profile of cytokine production. During T-cell activation, the cytokine milieu regulates the Th1 and Th2 differentiation pathway. For example, the IL-12 receptor activates the STAT4, which in turn promotes production of Th1 cytokines, including IFNγ (13). Conversely, IL-4 receptor activates STAT6, which promotes production of the Th2 cytokines IL-4, IL-5, and IL-13 (14). Thus, STAT4 and STAT6 have profound effects on T-cell differentiation and cytokine production. Although some studies have reported differential graft survival because of skewed production of Th1 or Th2 cytokines, most studies have not detected a direct correlation between Th1 or Th2 cytokine profiles and graft survival. Nevertheless, studies have shown different mechanisms of rejection when cytokine production is skewed toward a Th1 or Th2 response (15,16). In this project, we investigated the Th1 versus Th2 functions of IFNγ with a comparative analysis of STAT4- and STAT6-deficient mice.
In this study, we performed a comparative evaluation of IFNγ-, STAT4-, and STAT6-dependent responses in allograft rejection by investigating a large panel of immune and inflammatory genes. The data were analyzed with algorithms that generated hierarchic clustering dendrograms. The distinct profile of gene expression of individual IFNγ-, STAT4-, and STAT6-dependent genes were determined with self-organizing maps (SOMs). We observed that knockout strains with similar kinetics of rejection have distinct profiles of gene expression. By analyzing large numbers of genes simultaneously during rejection in multiple transgenic strains, this study provides novel insights into the role of cytokines during rejection.
Transplantation 12/2003; 76(12):1749-1758. · 4.00 Impact Factor
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ABSTRACT: Little is known regarding the graft response to transplantation injury. This study investigates the posttransplantation response of genes that are constitutively expressed in the heart. Constitutive heart and lymph node tissue-restricted gene expression was first analyzed with DNA microarrays. To demonstrate changes following transplantation in genes constitutively expressed in the heart, we performed vascularized murine heart transplants in allogeneic (BALB/c to B6), syngeneic (B6 to B6), and alymphoid (BALB/c-RAG2-/- to B6-RAG1-/-) experimental groups. Temporal induction of genes posttransplant relative to constitutive expression was evaluated with DNA microarrays. Dendrograms and self-organizing maps were generated to determine the dissimilarity between the experimental groups and to identify subsets of differentially expressed genes within the groups, respectively. Expression patterns of selected genes were confirmed by real-time PCR. Biological processes were assigned to genes induced posttransplant using the AnnBuilder package via the Gene Ontology Database. Post-transplant, a shift was noted in genes classified as defense, communication, and metabolism. Our results identify novel components of the graft response to transplantation injury and rejection.
Physiological Genomics 10/2003; 15(1):52-64. · 2.73 Impact Factor
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ABSTRACT: Both clinical and experimental observations suggest that allograft rejection is a complex process with multiple components that are, at least partially, functionally redundant. Studies using graft recipients deficient in various genes including chemokines, cytokines, and other immune-associated genes frequently produce a phenotype of delayed, but not indefinitely prevented, rejection. Only a small subset of genetic deletions (for example, TCR(alpha) or beta, MHC I and II, B7-1 and B7-2, and recombinase-activating gene) permit permanent graft acceptance suggesting that rejection is orchestrated by a complex network of interrelated inflammatory and immune responses. To investigate this complex process, we have used oligonucleotide microarrays to generate quantitative mRNA expression profiles following transplantation. Patterns of gene expression were confirmed with real-time PCR data. Hierarchical clustering algorithms clearly differentiated the early and late phases of rejection. Self-organizing maps identified clusters of coordinately regulated genes. Genes up-regulated during the early phase included genes with prior biological functions associated with ischemia, injury, and Ag-independent innate immunity, whereas genes up-regulated in the late phase were enriched for genes associated with adaptive immunity.
The Journal of Immunology 08/2002; 169(1):522-30. · 5.79 Impact Factor
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ABSTRACT: Allograft rejection remains a major limitation to successful solid organ transplantation. Here, we investigated the biosynthesis and bioactions of the pro-resolving mediators lipoxin A(4) and resolvin E1 in host responses to organ transplantation. In samples obtained during screening bronchoscopy after human lung transplantation, bronchoalveolar lavage fluid levels of lipoxin A(4) were increased in association with the severity of allograft rejection that was graded independently by clinical pathology. Lipoxin A(4) significantly inhibited calcineurin activation in human neutrophils, and lipoxin A(4) stable analogs prevented acute rejection of vascularized cardiac and renal allografts. Transgenic animals expressing human lipoxin A(4) receptors revealed important sites of action in host tissues for lipoxin A(4)'s protective effects. Resolvin E1 displays counter-regulatory actions for leukocytes, in part, via increased lipoxin A(4) biosynthesis, yet RvE1 administered (1μg, iv) to donor (days -1 and 0) and recipient mice (days -1, 0 and +4) was even more potent than a lipoxin stable analog (1μg, iv) in prolonging renal allograft survival (median survival time=74.0 days with RvE1 and 37.5 days with a LXA(4) analog). Together, these results highlight the potential for pro-resolving mediators in prolonging survival of solid organ transplants.
Prostaglandins Leukotrienes and Essential Fatty Acids 84(1-2):43-50. · 3.37 Impact Factor
