RESUMO
Whilst initial rates of insulin independence following islet transplantation are encouraging, long-term function using the Edmonton Protocol remains a concern. The aim of this single-arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end-point was an HbA1c of <7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥ 12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end-point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7-39 months) and 6 (35%) remain insulin independent. All recipients were C-peptide positive for at least 3 months. All subjects with unstimulated C-peptide >0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas/métodos , Adolescente , Adulto , Idoso , Austrália/epidemiologia , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/sangue , Feminino , Seguimentos , Rejeição de Enxerto/epidemiologia , Sobrevivência de Enxerto , Humanos , Incidência , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sirolimo/uso terapêutico , Tacrolimo/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
AIMS/HYPOTHESIS: For beta cells, contact with TNF-α triggers signalling cascades that converge on pathways important for cell survival and inflammation, specifically nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Here, we investigated the function of baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins in regulating TNF signalling cascades. METHODS: TNF regulation of Birc genes was studied by mRNA expression and promoter analysis. Birc gene control of cell signalling was studied in beta cell lines, and in islets from Birc2(-/-) and Birc3(-/-) mice, and from Birc3(-/-) Birc2Δ beta cell mice that selectively lack Birc2 and Birc3 (double knockout [DKO]). Islet function was tested by intraperitoneal glucose tolerance test and transplantation. RESULTS: TNF-α selectively induced Birc3 in beta cells, which in turn was sufficient to drive and potentiate NF-κB reporter activity. Conversely, Birc3(-/-) islets exhibited delayed TNF-α-induced IκBα degradation with reduced expression of Ccl2 and Cxcl10. DKO islets showed a further delay in IκBα degradation kinetics. Surprisingly, DKO islets exhibited stimulus-independent and TNF-dependent hyperexpression of TNF target genes A20 (also known as Tnfaip3), Icam1, Ccl2 and Cxcl10. DKO islets showed hyperphosphorylation of the JNK-substrate, c-Jun, while a JNK-antagonist prevented increases of Icam1, Ccl2 and Cxcl10 expression. Proteosome blockade of MIN6 cells phenocopied DKO islets. DKO islets showed more rapid loss of glucose homeostasis when challenged with the inflammatory insult of transplantation. CONCLUSIONS/INTERPRETATION: BIRC3 provides a feed-forward loop, which, with BIRC2, is required to moderate the normal speed of NF-κB activation. Paradoxically, BIRC2 and BIRC3 act as a molecular brake to rein in activation of the JNK signalling pathway. Thus BIRC2 and BIRC3 fine-tune NF-κB and JNK signalling to ensure transcriptional responses are appropriately matched to extracellular inputs. This control is critical for the beta cell's stress response.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular , Proteínas Inibidoras de Apoptose/genética , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína LigasesRESUMO
AIMS/HYPOTHESIS: This group of studies examines human genetic susceptibility conferred by the receptor for advanced glycation end-products (RAGE) in type 1 diabetes and investigates how this may interact with a western environment. METHODS: We analysed the AGER gene, using 13 tag SNPs, in 3,624 Finnish individuals from the FinnDiane study, followed by AGER associations with a high risk HLA genotype (DR3)-DQA1*05-DQB1*02/DRB1*0401-DQB1*0302 (n = 546; HLA-DR3/DR4), matched in healthy newborn infants from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study (n = 373) using allelic analysis. We also studied islets and circulating RAGE in NODLt mice. RESULTS: The rs2070600 and rs17493811 polymorphisms predicted increased risk of type 1 diabetes, whereas the rs9469089 SNP was related to decreased risk, on a high risk HLA background. Children from the DIPP study also showed a decline in circulating soluble RAGE levels, at seroconversion to positivity for type 1 diabetes-associated autoantibodies. Islet RAGE and circulating soluble RAGE levels in prediabetic NODLt mice decreased over time and were prevented by the AGE lowering therapy alagebrium chloride. Alagebrium chloride also decreased the incidence of autoimmune diabetes and restored islet RAGE levels. CONCLUSIONS/INTERPRETATION: These studies suggest that inherited AGER gene polymorphisms may confer susceptibility to environmental insults. Declining circulating levels of soluble RAGE, before the development of overt diabetes, may also be predictive of clinical disease in children with high to medium risk HLA II backgrounds and this possibility warrants further investigation in a larger cohort.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença/genética , Receptores Imunológicos/genética , Adulto , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Receptor para Produtos Finais de Glicação Avançada , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ß-cells sense glucose and secrete appropriate amounts of insulin by coupling glucose uptake and glycolysis with quantitative ATP production via mitochondrial oxidative pathways. Therefore, oxidative phosphorylation is essential for normal ß-cell function. Multiple cell types adapt to hypoxia by inducing a transcriptional programme coordinated by the transcription factor hypoxia-inducible factor (HIF). HIF activity is regulated by the von Hippel-Lindau (Vhl) protein, which targets the HIFα subunit for proteasomal degradation in the presence of oxygen. Several recent studies have shown that Vhl deletion in ß-cells results in Hif1α activation, impaired glucose-stimulated insulin secretion (GSIS) and glucose intolerance. This was found to be because of alterations in ß-cell gene expression inducing a switch from aerobic glucose metabolism to anaerobic glycolysis, thus disrupting the GSIS triggering pathway. Situations in which islets may become hypoxic are discussed, in particular islet transplantation which has been reported to cause islet hypoxia because of an inadequate blood supply post-transplant. Aside from this principal role for HIF in negatively regulating ß-cell glucose sensing, other aspects of hypoxia signalling are discussed including ß-cell differentiation, development and vascularization. In conclusion, recent studies clearly show that hypoxia response mechanisms can negatively impact on glucose sensing mechanisms in the ß-cell and this has the potential to impair ß-cell function in a number of physiological and clinical situations.
Assuntos
Hipóxia Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Células Secretoras de Insulina/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/fisiopatologia , Animais , Glicemia/fisiologia , Glicólise , Humanos , Insulina/metabolismo , Secreção de Insulina , Camundongos , Oxigênio/metabolismo , Fosforilação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genéticaRESUMO
AIMS/HYPOTHESIS: Islet transplantation is a potential therapeutic option for type 1 diabetes. However, the need for multiple donors per patient and heavy immunosuppression of the recipients limit its use. The goal of this study was to test whether the gene encoding activating transcription factor 3 (ATF3), a stress-inducible pro-apoptotic gene, plays a role in graft rejection in islet transplantation. METHODS: We compared wild-type (WT) and Atf3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation and hypoxia. We also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. RESULTS: ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets compared with KO islets. ATF3 upregulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (1) apoptosis (Noxa [also known as Pmaip1] and Bnip3), and (2) immunomodulation (Tnfalpha [also known as Tnf], Il-1beta [also known as Il1b], Il-6 [also known as Il6] and Ccl2 [also known as Mcp-1]). In vivo, Atf3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in the recipients and the reduction of the following variables in the KO grafts: caspase 3 activation, macrophage infiltration and expression of the above apoptotic and immunomodulatory genes. CONCLUSIONS/INTERPRETATION: ATF3 plays a role in islet graft rejection by contributing to islet cell death and inflammatory responses at the graft sites. Silencing the ATF3 gene may provide therapeutic benefits in islet transplantation.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Inflamação/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante Isogênico/imunologia , Fator 3 Ativador da Transcrição/genética , Animais , Apoptose/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Immunoblotting , Imuno-Histoquímica , Imunomodulação/genética , Imunomodulação/imunologia , Inflamação/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Apoptose/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas/genética , Transfecção/métodos , Receptor fas/metabolismo , Adenoviridae/genética , Animais , Apoptose/genética , Cisteína Endopeptidases , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Nucleares , Proteínas/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease resulting from apoptotic destruction of beta cells in the islets of Langerhans. Low expression of antioxidants and a predilection to produce nitric oxide (NO) have been shown to underscore beta cell apoptosis. With this perspective in mind, we questioned whether beta cells could mount an induced protective response to inflammation. Here we show that human and rat islets can be induced to rapidly express the antiapoptotic gene A20 after interleukin (IL)-1beta activation. Overexpression of A20 by means of adenovirus-mediated gene transfer protects islets from IL-1beta and interferon gamma-induced apoptosis. The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production. The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor kappaB at a level upstream of IkappaBalpha degradation. These data demonstrate a dual antiapoptotic and antiinflammatory function for A20 in beta cells. This qualifies A20 as part of the physiological cytoprotective response of islets. We propose that A20 may have therapeutic potential as a gene therapy candidate to achieve successful islet transplantation and the cure of IDDM.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , NF-kappa B/genética , Proteínas/farmacologia , Adenoviridae/genética , Animais , Linhagem Celular , Cisteína Endopeptidases , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Inflamação/imunologia , Interferon gama/farmacologia , Interleucina-1/metabolismo , Interleucina-1/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Proteínas Nucleares , Proteínas/genética , Ratos , Ratos Sprague-Dawley , Ativação Transcricional , Transfecção , Proteína 3 Induzida por Fator de Necrose Tumoral alfaAssuntos
Apoptose/genética , Interleucina-1/farmacologia , Ilhotas Pancreáticas/fisiologia , Óxido Nítrico/fisiologia , Proteínas/fisiologia , Adenoviridae , Animais , Células Cultivadas , Vetores Genéticos , Interleucina-1/fisiologia , Ilhotas Pancreáticas/citologia , Proteínas/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Transfecção/métodos , Fator de Necrose Tumoral alfa/fisiologiaAssuntos
Proteínas I-kappa B , Músculo Liso Vascular/fisiologia , Proteínas/fisiologia , Animais , Aorta , Células Cultivadas , Cricetinae , Citocinas/farmacologia , Proteínas de Ligação a DNA/fisiologia , Transplante de Coração/imunologia , Transplante de Coração/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Músculo Liso Vascular/citologia , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas Nucleares , Proteínas/genética , Ratos , Proteínas Recombinantes/biossíntese , Transfecção , Transplante Heterólogo/imunologia , Transplante Heterólogo/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.
Assuntos
Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Heme Oxigenase (Desciclizante)/fisiologia , Transplante Heterólogo/imunologia , Animais , Apoptose , Proteínas Inativadoras do Complemento/farmacologia , Ciclosporina/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Venenos Elapídicos/farmacologia , Rejeição de Enxerto/imunologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Imunossupressores/farmacologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miocárdio/citologia , RatosRESUMO
BACKGROUND: Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. METHODS AND RESULTS: Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses. CONCLUSIONS: Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.
Assuntos
Endotélio Vascular/metabolismo , Trombomodulina/biossíntese , Animais , Antioxidantes/farmacologia , Células Cultivadas , Clonagem Molecular , Humanos , Masculino , Retroviridae/genética , Superóxidos/metabolismo , Suínos , Trombomodulina/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-kappa B target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-kappa B in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-kappa B.
Assuntos
Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Selectina E/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , NF-kappa B/metabolismo , Fatores de Transcrição , Transcrição Gênica/fisiologia , Animais , Aorta , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Genes Reporter , Mutagênese Sítio-Dirigida , NF-kappa B/antagonistas & inibidores , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/biossíntese , Receptores Purinérgicos/biossíntese , Proteínas Recombinantes/metabolismo , Suínos , Fator de Transcrição RelB , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Human mononuclear phagocytes (MO) express a functional form of thrombomodulin (TM), the anticoagulant molecule typically considered purely in the context of regulation of conversion of protein C (PC) to activated PC (aPC) by thrombin-bound TM at the endothelial cell surface. We have been interested in the anti-inflammatory actions of aPC, including its ability to suppress MO production of multiple pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), leading us to consider whether MO surface expression of TM and resultant local aPC generation, might contribute to autoregulation of MO activation at sites of inflammation involving thrombin and fibrin formation. Since TNF-alpha and IL-1 are known to downregulate endothelial expression of TM, this study investigated the effects of TNF-alpha on production of TM by the monocytic leukemic cell line, THP-1. THP-1 cells display many monocyte-like properties, providing a convenient source for biochemical and molecular studies. Western blotting of lysates of THP-1 cells versus cultured human umbilical vein endothelial cells showed that after 24 h of stimulation, TNF-alpha decreased TM protein expression in endothelial but not THP-1 cells and comparable responses were noted by flow cytometry. Subsequent Northern blot analysis showed that at 24 h, TNF-alpha diminished TM steady state mRNA in endothelial but not THP-1 cells, although Northern analysis of the kinetics of TM steady state mRNA did show a rapid and transient modulation by TNF-alpha at 2 h of stimulation, which was confirmed by nuclear run-on analysis of the effect of TNF-alpha on TM gene transcription rates in THP-1 cells, analysis of protein expression by flow cytometry and Western blotting showed similar effects. In contrast to the divergent effects of TNF-alpha on THP-1 vs endothelial cells, agonists such as cyclic adenosine monophosphate (c-AMP) and phorbol ester (PMA) had comparable effects on THP-1 and endothelial cells, resulting in parallel increases or decreases in TM mRNA and protein expression, respectively. Hence, there is a 'split' in the nature of endothelial vs THP-1 cellular responses to TNF-alpha as compared to non-inflammatory stimuli, suggesting cell-specific differences in regulation of the TM promoter. We conclude that in contrast to its effects on TM expression by endothelial cells, exposure of THP-1 cells to TNF-alpha causes a rapid and transient decrease in TM mRNA production which is followed by sustained and high level expression, supporting the concept that MO expression of TM may contribute to regulation of MO activation and cytokine production at inflammatory sites.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Trombomodulina/genética , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Monócitos/metabolismoRESUMO
Expression of the NF-kappaB-dependent gene A20 in endothelial cells (EC) inhibits tumor necrosis factor (TNF)-mediated apoptosis in the presence of cycloheximide and acts upstream of IkappaBalpha degradation to block activation of NF-kappaB. Although inhibition of NF-kappaB by IkappaBalpha renders cells susceptible to TNF-induced apoptosis, we show that when A20 and IkappaBalpha are coexpressed, the effect of A20 predominates in that EC are rescued from TNF-mediated apoptosis. These findings place A20 in the category of "protective" genes that are induced in response to inflammatory stimuli to protect EC from unfettered activation and from undergoing apoptosis even when NF-kappaB is blocked. From a therapeutic perspective, genetic engineering of EC to express an NF-kappaB inhibitor such as A20 offers the mean of achieving an anti-inflammatory effect without sensitizing the cells to TNF-mediated apoptosis.
Assuntos
Apoptose/fisiologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiologia , NF-kappa B/fisiologia , Proteínas/genética , Fator de Necrose Tumoral alfa/farmacologia , Adenoviridae , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , SuínosRESUMO
BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.
Assuntos
Ativação do Complemento , Endotélio Vascular/fisiologia , Protrombina/metabolismo , Trombina/metabolismo , Transplante Heterólogo/fisiologia , Animais , Antitrombina III/análise , Aorta , Células Cultivadas , Rejeição de Enxerto , Humanos , Cinética , Masculino , Peptídeo Hidrolases/análise , Ativação Plaquetária , SuínosRESUMO
The endothelial molecule thrombomodulin (TM) regulates hemostasis by binding thrombin and promoting conversion of protein C to activated protein C (aPC). Apart from its anticoagulant actions, aPC modulates mononuclear phagocyte (M phi) activation, including TNF-alpha production, indicating interrelationships of the coagulation and immune systems. While the endothelium is considered to be the prime regulator of aPC generation, TM recently has been identified M phi and neutrophils. This study analyzes TM membrane expression by human blood monocytes, alveolar macrophages, and U937 cells cultured in the presence of various stimuli. All except U937 cell expressed high levels of surface TM. Surprisingly, stimulation with LPS or TNF-alpha further up-regulated TM expression by M phi, whereas cultured endothelial cells (EC) showed decreased TM expression. However, noninflammatory stimuli induced qualitatively similar changes in M phi and EC; all-trans retinoic acid and prostaglandin E up-regulated surface TM, and PMA decreased TM expression. Changes in M phi TM expression were accompanied by alteration in functional activity. Thus, LPS increased the TM cofactor activity of THP-1 cells by 27 +/- 6.9% (p < 0.05), and PMA decreased their cofactor activity by 53.2 +/- 11.5% (p < 0.05).In addition, in vivo relevance was demonstrated by the presence of TM on intragraft inflammatory M phi during cardiac rejection, whereas adjacent EC lacked TM expression. These studies demonstrate that expression of TM on human M phi is regulated differently to EC with respect to inflammatory stimuli, suggesting the potential for extravascular M phi to promote local production of aPC.
Assuntos
Anti-Inflamatórios , Macrófagos/metabolismo , Proteína C/metabolismo , Trombomodulina/metabolismo , Trombomodulina/fisiologia , Linhagem Celular , Relação Dose-Resposta Imunológica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Monócitos/enzimologia , Prostaglandinas E/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , Proteína C/metabolismo , Receptores de Superfície Celular/metabolismo , Divisão Celular , Humanos , Leucócitos Mononucleares/citologia , Monócitos/citologia , Fagocitose , Ensaio Radioligante , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Recent studies have shown that infusion of the anticoagulant protein, activated protein C (APC), can ameliorate many of the systemic effects of endotoxemia in experimental animals, although the mechanisms in this action are unknown. We investigated the effects of APC on the responses of blood monocytes, alveolar macrophages, and cells of the monocyte line, THP-1, to stimulation in vitro by LPS, IFN-gamma, or PMA. Mononuclear phagocyte (MO) activation was associated with rapid production of TNF-alpha, down-regulation of the glycophosphatidylinositol-linked protein CD14 (the key MO receptor for complexes of LPS and LPS-binding protein responsible for intracellular signaling), and down-regulation of the related LPS-binding proteins CD11b and CD18. Addition of APC, but not the zymogen, PC, or active site-blocked APC, inhibited selected MO responses involving the CD14-dependent LPS-induced pathway of MO activation, or activation induced by IFN-gamma or PMA. Thus, APC inhibited the production of TNF-alpha and prevented down-regulation of membrane CD11b, CD14, and CD18, but had no effect on up-regulation of MHC class II, ICAM-1, or IL-2R, down-regulation of MO expression of another glycophosphatidylinositol-linked protein, CD59, or production of reactive oxygen intermediates. These data show that APC inhibits host cytokine production but maintains MO responses associated with adhesion, phagocytosis, and killing of Gram-negative bacteria, such that use of APC may be a logical and potent adjunctive therapy in select inflammatory diseases involving MO activation and damaging host cytokine overproduction.
Assuntos
Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Monócitos/imunologia , Ésteres de Forbol/farmacologia , Proteína C/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos CD18/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática , Citometria de Fluxo , Humanos , Imunofenotipagem , Técnicas In Vitro , Receptores de Lipopolissacarídeos , Antígeno de Macrófago 1/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Cellular immune responses were observed in the skin of sheep after primary and secondary infection with sheep blowfly (Lucilia cuprina) larvae. Both primary and secondary infections resulted in a massive cellular infiltration within 48 h of wound initiation, with the majority of cells having the CD45 phenotype. Neutrophils comprised the major cell type at the skin surface. In the dermis, the number of CD4+ T helper, gamma delta-TCR+ cells and T19+ (CD4-, CD8-) T cells also increased significantly in skin during both primary and secondary infections compared with control sites. However, there was no significant difference in the numbers of these cells between primary and secondary infections. An increase in the expression of the CD1 antigen on Langerhans/dendritic cells was observed, along with an apparent increase in the number of these cells in secondary lesions, with the majority of the cells being concentrated in the upper dermis and epidermis. While there was no increase in mast cells, eosinophils increased significantly during infection compared with control sites. The cellular infiltration observed following primary and secondary infections suggests polyclonal activation of T cells and their selective recruitment to the lesion site.