RESUMO
A subset of Pts develops dysfunctional MO to inflammatory DC differentiation and immunosuppression. MDDC, a newly described DC subset, is pivotal in initiating antibacterial responses. Endogenous proteins are known to alter MO to MDDC differentiation. In particular, trauma-elevated TSP-1, a protein that is known to affect MO functions, could trigger MDDC differentiation defects. We hypothesized that TSP-1-deranged differentiation of inflammatory CD1a(+)MDDC would negatively alter activation of immune functions, thereby increasing the risk of postinjury infections. Post-trauma increased TSP-1 levels in patients' plasma and MO correlated with two distinct MDDC differentiation dysfunctions: the previously described decreased CD1a(+)DC yields but also, development of an immunoincompetent CD1a(+)MDDC. The Pts' development of Dysf DC correlated to increased infectious complications. TSP-1 triggered its inhibitory receptor, CD47, activating an inhibitory phosphatase, SHP-1. Increased pSHP-1, decreased antigen processing, and depressed T cell stimulation characterized Pt Dysf DC. TSP-1 mimics added during Cnt MDDC differentiation depressed CD1a(+)DC yields but more importantly, also induced defective CD1a(+)MDDC, reproducing Pts' MDDC differentiation dysfunctions. CD47 triggering during Cnt MDDC differentiation increased SHP-1 activation, inhibiting IL-4-induced STAT-6 activation (critical for CD1a(+)MDDC differentiation). SHP-1 inhibition during MDDC differentiation in the presence of TSP-1 mimics restored pSTAT-6 levels and CD1a(+)MDDC immunogenicity. Thus, postinjury-elevated TSP-1 can decrease CD1a(+)DC yields but more critically, also induces SHP-1 hyperactivity, deviating MDDC differentiation to defective CD1a(+) inflammatory MDDCs by inhibiting STAT-6.
Assuntos
Antígenos CD1/metabolismo , Antígeno CD47/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Trombospondina 1/metabolismo , Adulto , Diferenciação Celular , Núcleo Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Feminino , Humanos , Imunofenotipagem , Interleucina-4/farmacologia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Transporte Proteico , Fator de Transcrição STAT6/metabolismo , Índice de Gravidade de Doença , Trombospondina 1/sangue , Ferimentos e Lesões/diagnóstico , Ferimentos e Lesões/imunologia , Ferimentos e Lesões/metabolismo , Adulto JovemRESUMO
PURPOSE: Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. EXPERIMENTAL DESIGN: We applied 2D-LC-MS/MS-based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. RESULTS: A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs.
Assuntos
Neutrófilos/metabolismo , Proteômica , Ferimentos e Lesões/imunologia , Ferimentos e Lesões/metabolismo , Apoptose , Estudos de Casos e Controles , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Neutrófilos/imunologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Transporte Proteico , Transdução de Sinais , Transcriptoma , Ubiquitina/metabolismo , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologiaRESUMO
A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.
Assuntos
Genômica , Inflamação/genética , Doença Aguda , Adolescente , Adulto , Animais , Queimaduras/genética , Queimaduras/patologia , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Fatores de Tempo , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologia , Adulto JovemRESUMO
Immunosuppression resulting from excessive post-trauma apoptosis of hyperactivated T cells is controversial. TRAIL mediated T cell apoptosis decreases highly activated T cells' responses. Caspase-10, a particular TRAIL target, was increased in trauma patients' T cells with concomitantly elevated plasma TRAIL levels. These patients' T cells developed anergy, implicating increased TRAIL-mediated T cell apoptosis in post-trauma T cell anergy. Control T cells cultured with patients' sera containing high TRAIL levels increased their caspase-10 activity and apoptosis. Stimulated primary T cells are TRAIL apoptosis resistant. Increased plasma thrombospondin-1 and T cell expression of CD47, a thrombospondin-1 receptor, preceded patients' T cell anergy. CD47 triggering of T cells increased their sensitivity to TRAIL-induced apoptosis. Augmentation of T cell TRAIL-induced apoptosis was secondary to CD47 triggered activation of the Src homology-containing phosphatase-1 (SHP-1) and was partially blocked by a SHP-1 inhibitor. We suggest that combined post-trauma CD47 triggering, SHP-1 mediated NFκB suppression, and elevated TRAIL levels increase patients' CD47 expressing T cell apoptosis, thus contributing to subsequent T cell anergy.
Assuntos
Apoptose/imunologia , Anergia Clonal/imunologia , Soro/imunologia , Linfócitos T/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ferimentos e Lesões/imunologia , Adulto , Apoptose/genética , Antígeno CD47/genética , Antígeno CD47/imunologia , Estudos de Casos e Controles , Caspase 10/genética , Caspase 10/imunologia , Células Cultivadas , Anergia Clonal/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/patologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Trombospondina 1/genética , Trombospondina 1/imunologia , Ferimentos e Lesões/patologiaAssuntos
Choque , Animais , Queimaduras/fisiopatologia , Modelos Animais de Doenças , Enalaprilato/uso terapêutico , Humanos , Hipóxia/fisiopatologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Protoporfirinas/uso terapêutico , Sepse/terapiaRESUMO
Tumor cells release several factors that can help the progression of the tumor by directly supporting tumor growth and/or suppressing host antitumor immunity. Here, we report that human primary breast tumor cells not only express elevated levels of heat shock protein 27 (Hsp27) at the intracellular level but also release extremely high levels of Hsp27 compared with the same patients' serum Hsp27 levels, predicting an acutely increased concentration of soluble Hsp27 in the human breast tumor microenvironment (HBTM). We demonstrate that Hsp27 levels in the HBTM can be extremely elevated as evidenced by high soluble Hsp27 levels in patients' tumor interstitial fluid. Because increasing numbers of tumor-associated macrophages (TAM) in the HBTM negatively correlate to patients' clinical outcomes and we have previously reported the immunoregulatory activity of soluble Hsp27, here, we tested for any specific effects of soluble Hsp27 on human monocyte to macrophage differentiation. We demonstrate that soluble Hsp27 causes the differentiation of monocytes to macrophages with immuno-tolerizing phenotypes (HLA-DRlow, CD86low, PD-L1high, ILT2high, and ILT4high). We detected the presence of TAMs with similar phenotypes in breast cancer patients. Hsp27-differentiated macrophages induce severe unresponsiveness/anergy in T cells. Moreover, these macrophages lose tumoricidal activity but become extremely proangiogenic, inducing significant neovascularization, a process that is critically important for tumor growth. Thus, our data demonstrate a novel immune escape and tumor growth-supporting mechanism mediated by soluble Hsp27 that may be operative in human breast cancer.
Assuntos
Neoplasias da Mama/imunologia , Proteínas de Choque Térmico HSP27/imunologia , Macrófagos/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Proteínas de Choque Térmico HSP27/sangue , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Tolerância Imunológica , Macrófagos/patologia , Monócitos/imunologia , Monócitos/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
RATIONALE: The relationship between leukocyte gene expression and recovery of respiratory function after injury may provide information on the etiology of multiple organ dysfunction. OBJECTIVES: To find a list of genes for which expression after injury predicts respiratory recovery, and to identify which networks and pathways characterize these genes. METHODS: Blood was sampled at 12 hours and at 1, 4, 7, 21 and 28 days from 147 patients who had been admitted to the hospital after blunt trauma. Leukocyte gene expression was measured using Affymetrix oligonucleotide arrays. A linear model, fit to each probe-set expression value, was used to impute the gene expression trajectory over the entire follow-up period. The proportional hazards model score test was used to calculate the statistical significance of each probe-set trajectory in predicting respiratory recovery. A list of genes was determined such that the expected proportion of false positive results was less than 10%. These genes were compared to the Gene Ontology for 'response to stimulus' and, using Ingenuity software, were mapped into networks and pathways. MEASUREMENTS AND MAIN RESULTS: The median time to respiratory recovery was 6 days. There were 170 probe-sets representing 135 genes that were found to be related to respiratory recovery. These genes could be mapped to nine networks. Two known pathways that were activated were antigen processing and presentation and JAK-signaling. CONCLUSIONS: The examination of the relationship of gene expression over time with a patient's clinical course can provide information which may be useful in determining the mechanism of recovery or lack of recovery after severe injury.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Leucócitos/metabolismo , Ferimentos e Lesões/metabolismo , Adolescente , Adulto , Feminino , Expressão Gênica , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de TempoRESUMO
Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.
Assuntos
Queimaduras/fisiopatologia , Genômica/métodos , Microfluídica/métodos , Neutrófilos/fisiologia , Proteômica/métodos , Anticorpos Monoclonais , Antígenos CD/genética , Antígenos CD/imunologia , Biotinilação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , DNA/genética , DNA/isolamento & purificação , Proteínas Ligadas por GPI , Humanos , Neutrófilos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/isolamento & purificação , Ferimentos e Lesões/fisiopatologiaRESUMO
Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANOVA" and submitted to the Comprehensive R Archive Network at http://www.r-project.org/. It is also available for download at http://gluegrant1.stanford.edu/TANOVA/.
Assuntos
Queimaduras/genética , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Adulto , Fatores Etários , Análise de Variância , Queimaduras/imunologia , Criança , Pré-Escolar , Estudos Transversais , Interpretação Estatística de Dados , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Genes de Imunoglobulinas , Genes Mitocondriais , Humanos , Lactente , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Prognóstico , Software , Fatores de TempoRESUMO
Recent evidence indicates that human neutrophils can serve as non-professional antigen presenting cells (APC). Although expression of MHC class II and co-stimulatory molecules on human neutrophils is limited, these molecules can be significantly induced following in vitro exposure to the cytokines IFNgamma and GM-CSF. Since professional APCs such as dendritic cells express both co-stimulatory and co-inhibitory molecules for activation and regulation of adaptive immunity, we determined whether cytokines induce increased expression of specific co-signaling molecules on human neutrophils. We report here that circulating human neutrophils express co-inhibitory molecules such as immunoglobulin-like transcript (ILT) 4 and 5, and also comparatively low and highly variable levels of ILT2 and ILT3, but the expression of these ILTs was not significantly changed by cytokine treatment. In contrast, we demonstrate for the first time that human peripheral blood neutrophils, although do not express the co-inhibitory molecule, programmed death ligand (PD-L) 1 on their surface, can express this molecule at moderate levels following cytokine exposure. Although moderate PD-L1 levels on healthy volunteers' neutrophils were not inhibitory to T cells, our findings do not exclude a possible robust increase in neutrophil PD-L1 expression in pathological conditions with immunosuppressive functions. These results suggest a possible immunoregulatory role for human neutrophils in adaptive immunity.
Assuntos
Antígenos CD/imunologia , Apoptose , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ligantes , Neutrófilos/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Antígeno B7-H1 , Células Cultivadas , Citometria de Fluxo , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana/imunologia , Neutrófilos/imunologia , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/imunologiaRESUMO
OBJECTIVE: The aim of this study was to determine the effects of acetaldehyde on various steps of the monocyte recruitment cascade. METHODS: Human umbilical venous endothelial cells (HUVEC), primary blood monocytes (PBM) and THP-1 monocytes, were treated with acetaldehyde (0.1-0 microM) for 6h. Monocyte adherence experiments were performed using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethylester labeled PBM or (3)H-thymidine labeled THP-1 cells. HUVEC TNFalpha mRNA and protein levels were determined by quantitative real-time PCR and immunoassay, respectively, and HUVEC P-selectin and monocyte CCR2 expression were determined by FACS analysis. RESULTS: Acetaldehyde dose-dependently increased the number of CCR2 positive THP-1 monocytes, with a maximal increase of approximately 50% observed in the presence of 10 microM acetaldehyde. There was a significant increase in both the number of P-selectin positive cells and P-selectin receptor density when HUVEC were incubated with acetaldehyde. HUVEC TNFalpha mRNA expression and secretion were enhanced by acetaldehyde. Moreover, acetaldehyde increased THP-1 and PBM adhesion to HUVEC. Inhibition of P-selectin or TNFalpha, using antibodies or siRNA-directed gene knockdown, attenuated acetaldehyde-induced monocyte adhesion. In conclusion, acetaldehyde increased the number of CCR2 positive monocytes and stimulated endothelial cell P-selectin and TNFalpha expression. Moreover, acetaldehyde increased monocyte adhesion to endothelial cells, an effect that was both P-selectin- and TNFalpha-dependent. CONCLUSION: These effects of acetaldehyde may contribute, in part, to the increase in coronary heart disease that is associated with binge patterns of alcohol consumption.
Assuntos
Acetaldeído/metabolismo , Adesão Celular , Células Endoteliais/metabolismo , Monócitos/metabolismo , Selectina-P/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos , Células Cultivadas , Técnicas de Cocultura , Humanos , Selectina-P/genética , Selectina-P/imunologia , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores CCR2/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The effects of HSP27 on human monocytes (MO) are predominantly antiinflammatory through preferential interleukin (IL)10 induction and by alteration of MO to immature dendritic cells (iDCs) or MO to macrophage (Mac) differentiation. Initial HSP27 inclusion in IL4+GM-CSF MO to iDC induction cultures allows Mac differentiation (CD14++, CD16+), decreases iDC (CD1a+) differentiation, and depresses DC induction of allogeneic T lymphocyte proliferation (MLR). HSP27 increased MO IL10 and M-CSF production but subsequent increased Mac differentiation isn't responsible for depressed MO to iDC differentiation and function. Mac function after IL10 induced MO to Mac differentiation is also altered by HSP27 inclusion so that Mac phagocytic activity and scavenger receptor expression (CD163) are depressed. HSP27, in addition to immature DCs, doesn't increase Mac differentiation but instead generates inhibitory DCs, which depress rather than stimulate T cell proliferation even during anti CD3+CD28 induction. Upon maturation, these HSP27-altered inhibitory DCs have increased production of the T cell and DC suppressive mediator, thrombospondin 1. HSP27's anti-inflammatory and immunodepressive effects include deranging MO differentiation to both Mac and DCs, altering their receptor expression, and inducing production of inhibitory mediators such as thrombospondin-1 as well as IL10. These data suggest HSP27 belongs to a new group of 'anti-danger signals'.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Choque Térmico/farmacologia , Imunidade/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Fagocitose/efeitos dos fármacos , Fenótipo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Trombospondina 1/imunologiaRESUMO
Circulating heat shock protein (HSP)-27 is associated with tumor progression and increased post-injury infection. Extracellular HSP-27 might alter monocyte (MO)-derived DC and/or MPhi function to mediate immunosuppression. HSP-27 treatment inhibited expression of CD1a and CD1b/c, antigen uptake, and allogeneic T cell induction (MLR) by IL-4 + GM-CSF-differentiated human DC while increasing some MPhi characteristics ( upward arrowCD14, upward arrowCD16, upward arrowCD163). MO cytokine receptor profiles elicited by 24-h exogenous HSP-27 treatment remained supportive of immature DC (iDC) emergence ( upward arrowIL-4R, downward arrowIL-6R, downward arrowM-CSFR). IL-10, IL-6, and M-CSF (which promote MPhi differentiation) were significantly increased in IL-4 + GM-CSF + HSP-27 MO-->iDC differentiation cultures. However, HSP-27 treatment during MO differentiation to DC increased programmed cell death ligand 1 coinhibitor and depressed CD86 costimulator expression in parallel to decreased iDC MLR activity. This suggested that increased MPhi differentiation was not solely responsible for HSP-27 reduction of differentiating DC activity. HSP-27 treatment actually depressed the phagocytic capacity of MO differentiated to MPhi by IL-10 or M-CSF culture. CD163 (hemoglobin receptor) expression was depressed on M-CSF + HSP-27 MO-derived MPhi. HSP-27-mediated inhibition of MO-->iDC differentiation was reversed by p38alpha & beta inhibitor (SB202190) addition or TLR4 receptor modulation. HSP-27 impaired appropriate MO-->iDC and MO-->MPhi differentiation modulating expression of receptors necessary for their proper functions. This suggests that endogenous HSP-27 has immunoregulatory activities which could contribute to immunopathology.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/citologia , Inibidores do Crescimento/farmacologia , Proteínas de Choque Térmico/farmacologia , Monócitos/citologia , Proteínas de Neoplasias/farmacologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Proteínas de Choque Térmico HSP27 , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Chaperonas Moleculares , Monócitos/imunologia , Coelhos , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/biossíntese , Transdução de Sinais/imunologiaRESUMO
OBJECTIVE: Maintenance of postinjury T-lymphocyte immune paralysis or anergy could result from failure to activate costimulatory receptors during T-cell receptor activation and/or from chronic stimulation of a competing set of elevated corepressor receptors. Our objective was to assess whether elevated posttrauma T-lymphocyte surface expression of corepressor receptors was associated with immunodepressed lymphocyte responses and corresponded to increased inhibitory and decreased activating signal transduction molecules. DESIGN: Prospective observational study. SETTING: University trauma intensive care unit and research laboratory. PATIENTS: Sixty-one severe thermal and mechanical trauma patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Isolated trauma patients' and controls' peripheral blood T cells were assayed for negative and positive costimulation receptor expression. These receptor expression levels were compared (flow cytometry) between the two groups and correlated to T-cell levels of inhibitory and activating signal transduction molecules and proliferation capacity. Patients' proliferation hyporesponsive (anergic) T cells had increased expression of novel inhibitory receptors (corepressors) PD-1 (p < .05) and CD47 (p < .05) vs. patients' T-cell proliferation competent or controls' T cells. Patients' T-cell CD152 (CTLA-4) expression was also elevated vs. controls. Only patients' anergic T cells had simultaneously increased levels of the inhibitory signal transduction proteins, c-Cbl, a ubiquitin-ligase (p < .01) and SHP-1, a phosphatase (p < .01), concomitant to depressed phosphorylation of the activating signal kinases Erk, Zap70, and CD3Euro. T-cell receptor complex phosphorylation and activation of the interleukin-2 pivotal transcriptional complex protein CREB were also simultaneously depressed as c-Cbl and SHP-1 were elevated. CONCLUSIONS: Up-regulated corepressor receptor expression is novelly shown to characterize trauma patients' anergic T cells and correlate with predominance of inhibitory overactivating signal transduction molecules during T-cell stimulation. This could contribute to postinjury immunosuppression.
Assuntos
Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Queimaduras/imunologia , Antígeno CD47/metabolismo , Anergia Clonal/imunologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Ferimentos e Lesões/imunologia , Adulto , Antígenos CD/sangue , Antígenos de Diferenciação/sangue , Proteína de Ligação a CREB/sangue , Antígeno CTLA-4 , MAP Quinases Reguladas por Sinal Extracelular/sangue , Feminino , Citometria de Fluxo , Humanos , Tolerância Imunológica/imunologia , Unidades de Terapia Intensiva , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Receptor de Morte Celular Programada 1 , Estudos Prospectivos , Proteína Tirosina Fosfatase não Receptora Tipo 6/sangue , Proteínas Tirosina Quinases/sangue , Proteínas Proto-Oncogênicas c-cbl/sangue , RNA Polimerase I , Proteína-Tirosina Quinase ZAP-70/sangueRESUMO
Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.
Assuntos
Perfilação da Expressão Gênica , Monócitos/fisiologia , Linfócitos T/fisiologia , Ferimentos e Lesões/imunologia , Genoma Humano , Humanos , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos TestesRESUMO
Possible defective trauma patients' Mphi-T-cell feedback interactions between T cell IL-13 production and IL-1beta and IL-18 Mphi secretion were assessed. Mphi produced IL-1 and IL-18 augment T cell IL-13, which in turn limits excessive macrophage activation. Immunodepressed patients' T cells (depressed proliferation to alphaCD3 + alphaCD4) had decreased IL-13 production concomitant to aberrant Mphi activities ( upward arrow mTNFalpha, downward arrow IL-10) and consequent multiple organ failure (MOF). Decreased IL-13 levels in patients' T cell and diminished Mphi supernatant augmentation of healthy controls' T cell IL-13 production appeared concomitantly, suggesting patients' aberrant monokine levels might intensify in vivo T cell dysfunction severity. Patients' Mphi supernatants, which failed to augment controls' T cell IL-13 production, had depressed IL-1beta and lower induction of IL-18 than immunocompetent patients' Mphi, but combined addition of IL-1beta and IL-18 restored these Mphis' IL-13 enhancing activity. These data suggest that immunodepressed patients' aberrant monokine and depressed T cell IL-13 production are independent but synergistic contributors to emergence of MOF.
Assuntos
Interleucina-13/antagonistas & inibidores , Interleucina-13/fisiologia , Macrófagos/imunologia , Linfócitos T/imunologia , Ferimentos e Lesões/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Interleucina-13/biossíntese , Subunidade alfa1 de Receptor de Interleucina-13 , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Monocinas/metabolismo , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Receptores de Interleucina/metabolismo , Receptores de Interleucina-13 , Linfócitos T/metabolismo , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Although activation of human innate immunity after endotoxin administration is well established, in vivo endotoxin effects on human T cell responses are not well understood. Most naive human T cells do not express receptors for LPS, but can respond to endotoxin-induced mediators such as chemokines. In this study, we characterized the in vivo response of peripheral human T cell subsets to endotoxin infusion by assessing alterations in isolated T cells expressing different phenotypes, intracellular cytokines, and systemic chemokines concentration, which may influence these indirect T cell responses. Endotoxin administration to healthy subjects produced T cell activation as confirmed by a 20% increase in intracellular IL-2, as well as increased CD28 and IL-2R alpha-chain (CD25) expression. Endotoxin induced indirect activation of T cells was highly selective among the T cell subpopulations. Increased IL-2 production (36.0 +/- 3.7 to 53.2 +/- 4.1) vs decreased IFN-gamma production (33.8 +/- 4.2 to 19.1 +/- 3.2) indicated selective Th1 activation. Th2 produced IL-13 was minimally increased. Differentially altered chemokine receptor expression also indicated selective T cell subset activation and migration. CXCR3+ and CCR5+ expressing Th1 cells were decreased (CXCR3 44.6 +/- 3.2 to 33.3 +/- 4.6 and CCR5 24.8 +/- 2.3 to 12 +/- 1.4), whereas plasma levels of their chemokine ligands IFN-gamma-inducible protein 10 and MIP-1alpha were increased (61.4 +/- 13.9 to 1103.7 +/- 274.5 and 22.8 +/- 6.2 to 55.7 +/- 9.5, respectively). In contrast, CCR4+ and CCR3 (Th2) proportions increased or remained unchanged whereas their ligands, eotaxin and the thymus and activation-regulated chemokine TARC, were unchanged. The data indicate selective activation among Th1 subpopulations, as well as differential Th1/Th2 activation, which is consistent with a selective induction of Th1 and Th2 chemokine ligands.
Assuntos
Quimiocinas/fisiologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Adulto , Movimento Celular/efeitos dos fármacos , Quimiocinas/sangue , Citocinas/biossíntese , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Contagem de Linfócitos , Masculino , Receptores de Quimiocinas/análise , Subpopulações de Linfócitos T/imunologiaRESUMO
Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.
Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Genômica , Inflamação/genética , Leucócitos/metabolismo , Doença Aguda , Adolescente , Adulto , Endotoxinas/sangue , Endotoxinas/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamação/sangue , Inflamação/induzido quimicamente , Inflamação/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.
Assuntos
Expressão Gênica , Genoma Humano , Estudos Multicêntricos como Assunto/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise por Conglomerados , Genótipo , Humanos , Leucócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/normas , Seleção de Pacientes , Análise de Componente Principal , Reprodutibilidade dos Testes , Manejo de Espécimes/métodos , Ferimentos e Lesões/metabolismoRESUMO
Polymorphonuclear leukocytes (PMNs; commonly known as neutrophils) play essential roles in innate immunity and inflammation. Although there are standardized methods for the isolation of human neutrophils, they are time consuming and demand considerable technical expertise, making them unfeasible for many clinical applications. Here, we describe a simple and time-efficient technique for the isolation of human neutrophils, which adapts a readily available commercial cell preparation tube (CPT) currently in use for isolation of peripheral blood mononuclear cells (PBMC) and plasma and is now adapted to also yield neutrophils. The total time required for neutrophil isolation was less than 1 hr. Neutrophils isolated by this method were highly purified (> or =97%) as assessed by surface expression of the neutrophil specific marker, CD66b. Neutrophils isolated by this method were functional as demonstrated by their ability to secrete interleukin-1 receptor antagonist (IL-1RA). Neutrophils isolated using this new technique secreted significant amounts of soluble IL-1RA (929.3+/-197 pg/10(6)cells/mL) in response to lipopolysaccharide (LPS). Use of this adapted CPT method allows simultaneous isolation of functional human neutrophils as well as PBMC and plasma. Adoption of this new method will allow the conduct of different neutrophil assays at any clinical site without requiring trained laboratory personnel or a large staff time commitment.