RESUMO
Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up-regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis-associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid-derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue-specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC.-Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.
Assuntos
Ceramidase Ácida/biossíntese , Colite Ulcerativa/enzimologia , Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Mucosa Intestinal/enzimologia , Neutrófilos/enzimologia , Regulação para Cima , Ceramidase Ácida/genética , Animais , Quimiocinas/biossíntese , Quimiocinas/genética , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Humanos , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/enzimologia , Células Supressoras Mieloides/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neutrófilos/patologia , Microambiente Tumoral/genéticaRESUMO
The complement system is tightly regulated to safeguard against tissue damage that results from unwanted activation. The key step of C3 cleavage to C3b is regulated by multiple mechanisms that control the initiation and extent of activation. This study demonstrated that C3b:plasma protein complexes form in the fluid-phase during complement activation. Several different plasma proteins displayed a discrete high molecular SDS-resistant band when any of the three complement activating pathways were triggered in normal human serum or plasma. Serum depleted of individual complement proteins revealed that C3 and factors B and D were essential for complex formation. Inactivation of the thioester bond in C3 also prevented complex formation. In vitro, complexes could be generated using four purified proteins-C3, factor B, factor D, and target protein-and Mg(2+) to allow C3 convertase formation. These studies showed that the complexes consisted of a plasma protein covalently bound to C3b in a 1:1 molar ratio; the C3b portion was rapidly degraded by factors H and I. Analysis of plasma samples from patients with dense deposit disease and C3 glomerulonephritis demonstrated that C3b:protein complexes form spontaneously in the blood of patients with dense deposit disease and, to a lesser extent, in C3 glomerulonephritis patients, but not in healthy controls. This finding supports the underlying hypothesis that these C3 glomerulopathies are diseases of fluid-phase complement dysregulation. These complexes could normally function as a passive mechanism to intercept C3b from depositing on host cells. However, excessive generation and/or defective clearance of fluid-phase C3b:protein complexes may have pathological consequences.
Assuntos
Proteínas Sanguíneas/imunologia , Complemento C3/imunologia , Via Alternativa do Complemento , Glomerulonefrite/imunologia , Proteínas Sanguíneas/análise , Complemento C3/análise , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/análise , Fator H do Complemento/farmacologia , Fibrinogênio/farmacologia , Glomerulonefrite/sangue , Glomerulonefrite Membranoproliferativa/sangue , Glomerulonefrite Membranoproliferativa/imunologia , Humanos , Peso Molecular , Complexos Multiproteicos , Ligação Proteica , Desnaturação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Dodecilsulfato de Sódio/farmacologiaRESUMO
Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein-coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1ß. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174-180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37 °C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174-180 and 154-162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R.
Assuntos
Comunicação Autócrina , Proteínas de Transporte/metabolismo , Células Endoteliais/metabolismo , Proteínas Mitocondriais/metabolismo , Receptor B1 da Bradicinina/metabolismo , Transdução de Sinais , Comunicação Autócrina/efeitos dos fármacos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Células Endoteliais/efeitos dos fármacos , Fibrinogênio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptor B1 da Bradicinina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKß and IKKα-dependent NF-κB activation. IKKß-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKß and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKß, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Movimento Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/genética , Fator de Transcrição RelA/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Knowledge of how neutrophils respond to chemotactic signals in a complex inflammatory environment is not completely understood. Moreover, even less is known about factors in physiological fluids that regulate the activity of chemoattractants. The vitamin D-binding protein (DBP) has been shown to significantly enhance chemotaxis to complement activation peptide C5a using purified proteins in vitro, and by ex vivo depletion of DBP in physiological fluids, but this function has not been determined in vivo. DBP null ((-/-)) mice were used to investigate how a systemic absence of this plasma protein affects leukocyte recruitment in alveolitis models of lung inflammation. DBP(-/-) mice had significantly reduced (~50%) neutrophil recruitment to the lungs compared with their wild-type DBP(+/+) counterparts in three different alveolitis models, two acute and one chronic. The histology of DBP(-/-) mouse lungs also showed significantly less injury than wild-type animals. The chemotactic cofactor function of DBP appears to be selective for neutrophil recruitment, but, in contrast to previous in vitro results, in vivo DBP can enhance the activity of other chemoattractants, including CXCL1. The reduced neutrophil response in DBP(-/-) mice could be rescued to wild-type levels by administering exogenous DBP. Finally, in inflammatory fluids, DBP binds to G-actin released from damaged cells, and this complex may be the active chemotactic cofactor. To our knowledge, results show for the first time that DBP is a significant chemotactic cofactor in vivo and not specific for C5a, suggesting that this ubiquitous plasma protein may have a more significant role in neutrophil recruitment than previously recognized.
Assuntos
Quimiocina CXCL1/imunologia , Complemento C5a/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Pneumonia/imunologia , Proteína de Ligação a Vitamina D/metabolismo , Actinas/metabolismo , Animais , Movimento Celular/imunologia , Ativação do Complemento , Inflamação , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Proteína de Ligação a Vitamina D/deficiência , Proteína de Ligação a Vitamina D/genética , Proteína de Ligação a Vitamina D/farmacologiaRESUMO
HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)ß- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKß-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.
Assuntos
Comunicação Autócrina/imunologia , Movimento Celular/imunologia , Quimiocina CXCL12/biossíntese , Proteína HMGB1/fisiologia , Quinase I-kappa B/fisiologia , Subunidade p52 de NF-kappa B/fisiologia , Transdução de Sinais/imunologia , Fator de Transcrição RelB/fisiologia , Animais , Transformação Celular Neoplásica , Quimiocina CXCL12/antagonistas & inibidores , Quimiocina CXCL12/fisiologia , Quinase I-kappa B/biossíntese , Quinase I-kappa B/deficiência , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Subunidade p52 de NF-kappa B/biossíntese , Subunidade p52 de NF-kappa B/deficiência , Fator de Transcrição RelB/biossíntese , Células Tumorais CultivadasRESUMO
Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Proteína HMGB1/fisiologia , Imunidade Inata/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/prevenção & controle , Animais , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Proteína HMGB1/deficiência , Proteína HMGB1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Necrose , Neoplasias Experimentais/patologia , Distribuição AleatóriaRESUMO
Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.
Assuntos
Quimiotaxia/imunologia , Proteína HMGB1/fisiologia , Quinase I-kappa B/fisiologia , Animais , Células Cultivadas , Quimiotaxia/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/imunologia , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with beta(2)-microglobulin or BSA. The cell-DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1-191) and III (379-458), but not domain II (192-378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150-172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379-402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.
Assuntos
Fragmentos de Peptídeos/metabolismo , Receptores de Complemento/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Células U937/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Receptor da Anafilatoxina C5a , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ressonância de Plasmônio de SuperfícieRESUMO
There is an urgent need to identify the cellular and molecular mechanisms responsible for severe COVID-19 that results in death. We initially performed both untargeted and targeted lipidomics as well as focused biochemical analyses of 127 plasma samples and found elevated metabolites associated with secreted phospholipase A2 (sPLA2) activity and mitochondrial dysfunction in patients with severe COVID-19. Deceased COVID-19 patients had higher levels of circulating, catalytically active sPLA2 group IIA (sPLA2-IIA), with a median value that was 9.6-fold higher than that for patients with mild disease and 5.0-fold higher than the median value for survivors of severe COVID-19. Elevated sPLA2-IIA levels paralleled several indices of COVID-19 disease severity (e.g., kidney dysfunction, hypoxia, multiple organ dysfunction). A decision tree generated by machine learning identified sPLA2-IIA levels as a central node in the stratification of patients who died from COVID-19. Random forest analysis and least absolute shrinkage and selection operator-based (LASSO-based) regression analysis additionally identified sPLA2-IIA and blood urea nitrogen (BUN) as the key variables among 80 clinical indices in predicting COVID-19 mortality. The combined PLA-BUN index performed significantly better than did either one alone. An independent cohort (n = 154) confirmed higher plasma sPLA2-IIA levels in deceased patients compared with levels in plasma from patients with severe or mild COVID-19, with the PLA-BUN index-based decision tree satisfactorily stratifying patients with mild, severe, or fatal COVID-19. With clinically tested inhibitors available, this study identifies sPLA2-IIA as a therapeutic target to reduce COVID-19 mortality.
Assuntos
COVID-19/sangue , COVID-19/mortalidade , Fosfolipases A2 do Grupo II/sangue , SARS-CoV-2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A 2 Group IIA (sPLA 2 -IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA 2 -IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA 2 -IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA 2 -IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.
RESUMO
Neutrophils are the most abundant type of white blood cell in most mammals including humans. The primary role of these cells is host defense against microbes and clearance of tissue debris in order to facilitate wound healing and tissue regeneration. The recruitment of neutrophils from blood into tissues is a key step in this process and is mediated by numerous different chemoattractants. The neutrophil migratory response is essential for host defense and survival, but excessive tissue accumulation of neutrophils is observed in many inflammatory disorders and strongly correlates with disease pathology. The vitamin D binding protein (DBP) is a circulating multifunctional plasma protein that can significantly enhance the chemotactic activity of neutrophil chemoattractants both in vitro and in vivo. Recent in vivo studies using DBP deficient mice showed that DBP plays a larger and more central role during inflammation since it induces selective recruitment of neutrophils, and this cofactor function is not restricted to C5a, as prior in vitro studies indicated, but can enhance chemotaxis to many chemoattractants. DBP also is an extracellular scavenger for actin released from damaged/dead cells and formation of DBP-actin complexes is an immediate host response to tissue injury. Recent in vitro evidence indicates that DBP bound to G-actin, and not free DBP, functions as an indirect but essential cofactor for neutrophil migration. DBP-actin complexes always will be formed regardless of what initiated an inflammation, since release of actin from damaged cells is a common feature in all types of injury and DBP is abundant and ubiquitous in all extracellular fluids. Indeed, these complexes have been detected in blood and tissue fluids from both humans and experimental animals following various forms of injury. The published data strongly supports the premise that DBP-actin complexes are the functional neutrophil chemotactic cofactor that enhances neutrophil chemotaxis in vitro and augments neutrophilic inflammation in vivo. This review will assess the fundamental role of DBP in neutrophilic inflammation and injury.
RESUMO
Vitamin D binding protein (DBP) is a multifunctional plasma transport protein that is also found on the surface of many cell types. Cell surface DBP significantly enhances chemotactic activity of complement (C) peptides C5a and C5a des Arg. However, both DBP binding and C5a chemotaxis enhancement can vary among neutrophil donors. To test if activation during cell purification is responsible for this variability, neutrophils were isolated using both standard and lipopolysaccharide (LPS)-free protocols. Cells isolated by the LPS-free method had no DBP-enhanced chemotaxis to C5a or DBP binding to plasma membranes. Moreover, neutrophils treated with LPS bound more avidity to immobilized DBP than sham-treated cells. Subcellular fractionation of neutrophils (standard protocol) revealed a heavy plasma membrane (HM) band that contained components of light plasma membranes and all three granules. The HM band possessed most of the DBP binding activity (58%), and activation of cells with ionomycin greatly increased DBP binding to HM. Azurophil granules contained 33% of the total DBP binding sites and there was a highly significant positive correlation (r=0.988) between release of the granule marker myeloperoxidase and DBP binding. These results indicate that fusion of granules with the plasma membrane forms HM that contains DBP binding sites.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Ativação de Neutrófilo/imunologia , Regulação para Cima , Proteína de Ligação a Vitamina D/metabolismo , Sítios de Ligação/efeitos dos fármacos , Fracionamento Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Complemento C5a/imunologia , Complemento C5a/isolamento & purificação , Complemento C5a/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/imunologia , Humanos , Radioisótopos do Iodo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Peroxidase/metabolismo , Ligação Proteica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína de Ligação a Vitamina D/isolamento & purificação , Proteína de Ligação a Vitamina D/farmacologiaRESUMO
BACKGROUND: Severe acute muscle injury results in massive cell damage, causing the release of actin into extracellular fluids where it complexes with the vitamin D-binding protein (DBP). We hypothesized that a systemic DBP deficiency would result in a less proinflammatory phenotype. METHODS: C57BL/6 wild-type (WT) and DBP-deficient (DBP-/-) mice received intramuscular injections of either 50% glycerol or phosphate-buffered saline into thigh muscles. Muscle injury was assessed by histology. Cytokine levels were measured in plasma, muscle, kidney, and lung. RESULTS: All animals survived the procedure, but glycerol injection in both strains of mice showed lysis of skeletal myocytes and inflammatory cell infiltrate. The muscle inflammatory cell infiltrate in DBP-deficient mice had remarkably few neutrophils as compared with WT mice. The neutrophil chemoattractant CXCL1 was significantly reduced in muscle tissue from DBP-/- mice. However, there were no other significant differences in muscle cytokine levels. In contrast, plasma obtained 48 hours after glycerol injection revealed that DBP-deficient mice had significantly lower levels of systemic cytokines interleukin 6, CCL2, CXCL1, and granulocyte colony-stimulating factor. Lung tissue from DBP-/- mice showed significantly decreased amounts of CCL2 and CXCL1 as compared with glycerol-treated WT mice. Several chemokines in kidney homogenates following glycerol-induced injury were significantly reduced in DBP-/- mice: CCL2, CCL5, CXCL1, and CXCL2. CONCLUSIONS: Acute muscle injury triggered a systemic proinflammatory response as noted by elevated plasma cytokine levels. However, mice with a systemic DBP deficiency demonstrated a change in their cytokine profile 48 hours after muscle injury to a less proinflammatory phenotype.
Assuntos
Citocinas/metabolismo , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Proteína de Ligação a Vitamina D/deficiência , Doença Aguda , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Glicerol , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Coxa da PernaRESUMO
The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose-response curves demonstrated that as little as 1pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.
Assuntos
Calcitriol/farmacologia , Fatores Quimiotáticos/antagonistas & inibidores , Complemento C5a/antagonistas & inibidores , Proteína de Ligação a Vitamina D/antagonistas & inibidores , Actinas/farmacologia , Fatores Quimiotáticos/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C5a/metabolismo , Relação Dose-Resposta a Droga , Humanos , Radioisótopos do Iodo , Ligantes , Neutrófilos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Vanadatos/farmacologia , Proteína de Ligação a Vitamina D/metabolismoRESUMO
Dendritic cells (DCs) are recruited to inflammatory sites where they phagocytose and process antigens for subsequent presentation to the T lymphocytes in the lymphoid tissue. Several leukocyte chemoattractants and their specific receptors have been shown to induce the migration of DC. The complement protein C1q has multiple immune functions including acting as a chemoattractant for neutrophils, eosinophils and mast cells. Therefore, the objective of this study was to determine if soluble C1q can induce chemotaxis of DC. Culturing cells in GM-CSF and IL-4 for 5 to 7 days generated human monocyte-derived DCs. In addition, LPS was added from day 5 to 7 to induce DC maturation. Cells were classified as either immature or mature DC by assessing the cell surface markers by flow cytometry, phagocytosis of dextran-FITC and T cell proliferation in an allogenic MLR. Immature DCs express the C1q receptors (C1qR), gC1qR and cC1qR/CR and, accordingly, display a vigorous migratory response to soluble C1q with maximal cell movement observed at 10-50nM. In contrast, mature DCs neither express C1qR nor do move to a gradient of soluble C1q. Varying the concentration gradient of C1q (checkerboard assay) showed that the protein largely induces a chemotactic response. Finally, blocking gC1qR and cC1qR/CR by using specific antibodies abolished the chemotactic response to C1q but had no effect on a different chemoattractant C5a. These results clearly demonstrate that C1q functions as a chemotactic factor for immature DC, and migration is mediated through ligation of both gC1qR and cC1qR/CR.
Assuntos
Calreticulina/metabolismo , Proteínas de Transporte/metabolismo , Quimiotaxia/imunologia , Complemento C1q/metabolismo , Células Dendríticas/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Receptores de Complemento/metabolismo , Animais , Anticorpos/farmacologia , Calreticulina/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , Diferenciação Celular , Membrana Celular/imunologia , Fatores Quimiotáticos/metabolismo , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Complemento C1q/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Monócitos/citologia , Monócitos/imunologia , Fenótipo , Coelhos , Receptores de Complemento/antagonistas & inibidoresRESUMO
Bioactive sphingolipids are regulators of immune cell function and play critical roles in inflammatory conditions including ulcerative colitis. As one of the major forms of inflammatory bowel disease, ulcerative colitis pathophysiology is characterized by an aberrant intestinal inflammatory response that persists causing chronic inflammation and tissue injury. Innate immune cells play an integral role in normal intestinal homeostasis but their dysregulation is thought to contribute to the pathogenesis of ulcerative colitis. In particular, neutrophils are key effector cells and are first line defenders against invading pathogens. While the activity of neutrophils in the intestinal mucosa is required for homeostasis, regulatory mechanisms are equally important to prevent unnecessary activation. In ulcerative colitis, unregulated neutrophil inflammatory mechanisms promote tissue injury and loss of homeostasis. Aberrant neutrophil function represents an early checkpoint in the detrimental cycle of chronic intestinal inflammation; thus, dissecting the mechanisms by which these cells are regulated both before and during disease is essential for understanding the pathogenesis of ulcerative colitis. We present an analysis of the role of sphingolipids in the regulation of neutrophil function and the implication of this relationship in ulcerative colitis.
Assuntos
Colite Ulcerativa/imunologia , Imunidade Inata , Imunidade nas Mucosas , Neutrófilos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Esfingolipídeos/imunologia , Animais , Apoptose , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Expressão Gênica , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Infiltração de Neutrófilos , Neutrófilos/patologia , Fagocitose , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transdução de SinaisRESUMO
A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via surface expressed fibrinogen or other membrane molecules. The identification of the sites for these viral ligands should therefore provide additional targets for the design of peptide-based or antigen-based therapeutic strategies.
Assuntos
Proteínas de Transporte/química , Proteína gp41 do Envelope de HIV/imunologia , Proteínas Mitocondriais/química , Receptores de Reconhecimento de Padrão/química , Proteínas do Core Viral/imunologia , Sítios de Ligação/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/imunologia , Humanos , Proteínas Mitocondriais/imunologia , Monócitos/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Células U937RESUMO
Complement C3 is a key fluid-phase protein of the immune system that covalently tags pathogenic cells and molecules for subsequent clearance. Previously, we reported that complement activation results in the formation of multiple C3b:plasma protein complexes in serum. However, it is not known if C3b attaches to any plasma protein in close proximity or preferentially binds damaged proteins. The objective of this study was to determine if C3b couples to plasma proteins in a non-native state and if this could be a potential mechanism to detect and clear damaged proteins from the blood. Using a purified in vitro system with alternative pathway proteins C3, factors B and D it was observed that guanidinium-HCl denaturation of three purified plasma proteins (albumin, alpha-1 proteinase inhibitor, vitamin D binding protein) greatly increased their capacity to form covalent complexes with C3b. However, native vitamin D binding protein, covalently attached to C3b, still retained the ability to bind its natural ligand G-actin, indicating that C3b links to plasma proteins in their native configuration but denaturation substantially increases this interaction. Serum complement activation generated a large number of C3b:plasma protein complexes that bound red blood cell membranes, suggesting a CR1-mediated clearance mechanism. Thermally denatured (60°C) serum activated the alternative pathway when added to fresh serum as evidenced by factor B cleavage and iC3b generation, but this heat-treated serum could not generate the pro-inflammatory peptide C5a. These results show that C3 recognizes and tags damaged plasma proteins for subsequent removal from the blood without triggering proinflammatory functions.