RESUMO
Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.
Assuntos
Anticoagulantes/farmacocinética , Biomarcadores/sangue , Heparina/farmacologia , Fígado/efeitos dos fármacos , Adulto , Alanina Transaminase/sangue , Anticoagulantes/farmacologia , Aspartato Aminotransferases/sangue , Dalteparina/farmacologia , Enoxaparina/farmacologia , Glutamato Desidrogenase/sangue , Proteína HMGB1/sangue , Heparina/farmacocinética , Humanos , Queratina-18/sangue , L-Iditol 2-Desidrogenase/sangue , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset. MATERIALS AND METHODS: A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4. RESULTS: In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection. CONCLUSION: As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Interleucina-2/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Citometria de Fluxo/métodos , Humanos , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.
Assuntos
Imidazóis/farmacologia , Imidazolidinas , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Regulação Alostérica , Adesão Celular , Relação Dose-Resposta a Droga , Epitopos/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Conformação Proteica/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.
Assuntos
Citoplasma/fisiologia , Selectina L/metabolismo , Sequência de Aminoácidos , Calmodulina/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Endopeptidases/metabolismo , Humanos , Hidrólise , Interfase/fisiologia , Células K562 , Selectina L/genética , Selectina L/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neutrófilos/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , TransfecçãoRESUMO
The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.
Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Selectina L/metabolismo , Leucócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Humanos , Leucócitos/efeitos dos fármacos , Lipossomos , Metaloendopeptidases/metabolismo , Microinjeções , Oligonucleotídeos Antissenso/farmacologia , Fosfatidiletanolaminas , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
Neutrophil influx into the lung is an important event in the pathogenesis of acute lung injury in gram-negative sepsis. We hypothesized that administration of a monoclonal antibody to intercellular adhesion molecule 1 (ICAM-1, CD54), a molecule mediating neutrophil adhesion to endothelial cells, would decrease neutrophil sequestration and transmigration in the lung and attenuate lung injury in Escherichia coli sepsis. Sepsis was induced in 12 baboons primed with heat-killed E. coli (1 x 10(9) CFU/kg) 12 h before infusion of live bacteria (1 x 10(10) CFU/kg). Six animals received monoclonal antibody to CD54 (1 mg/kg) intravenously at the time of live E. coli infusion. After 48 h or when blood pressure could not be maintained, tissues were harvested and bronchoalveolar lavage (BAL) samples were obtained. Median survival time was decreased in anti-CD54-treated animals. This group also had decreased mean arterial pressure, increased metabolic acidosis, and decreased urine output. Measures of lung injury including gas exchange, lung lavage protein and lactate dehydrogenase (LDH), lung thiobarbituric acid-reactive species, and lung histology, including alveolar neutrophil volumes, were unaffected by treatment. The effect of anti-CD54 on neutrophil influx into tissues as measured by myeloperoxidase was organ specific. These data show that monoclonal antibody to CD54 does not ameliorate acute lung injury in E. coli sepsis, and septic primates given anti-CD54 have worsened metabolic parameters and decreased survival.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Molécula 1 de Adesão Intercelular/imunologia , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/prevenção & controle , Sepse/complicações , Animais , Hemodinâmica , Masculino , Papio , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/fisiopatologia , Sepse/fisiopatologia , Taxa de SobrevidaRESUMO
The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils.
Assuntos
Fatores Quimiotáticos/farmacologia , Selectina L/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Acetofenonas/antagonistas & inibidores , Acetofenonas/farmacologia , Antígenos CD/metabolismo , Benzopiranos/antagonistas & inibidores , Benzopiranos/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Selectina L/química , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Naftalenos/antagonistas & inibidores , Naftalenos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Neutrophil-endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil-endothelial cell interactions.
Assuntos
Comunicação Celular , Endotélio Vascular/metabolismo , Selectina L/metabolismo , Neutrófilos/metabolismo , Adulto , Tamanho Celular , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Microscopia Confocal , Microscopia de Fluorescência , Neutrófilos/citologia , Fatores de TempoRESUMO
Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.
Assuntos
Calmodulina/metabolismo , Endopeptidases/metabolismo , Selectina L/metabolismo , Sequência de Aminoácidos , Calmodulina/química , Calmodulina/isolamento & purificação , Antagonistas de Dopamina/farmacologia , Regulação para Baixo/fisiologia , Citometria de Fluxo , Humanos , Selectina L/química , Selectina L/isolamento & purificação , Linfonodos/química , Linfonodos/enzimologia , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Trifluoperazina/farmacologiaRESUMO
The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.
Assuntos
Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , DNA Complementar , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMO
Recruitment of polymorphonuclear leukocytes (PMN) through upregulation of cellular adhesion molecules is a proposed mechanism of injury in sepsis and acute respiratory distress syndrome (ARDS). We hypothesized that pretreatment of baboons with a monoclonal antibody to human E- and L-selectin (EL-246) during sepsis would decrease PMN influx into tissues and result in less organ injury during gram-negative sepsis. We studied 14 anesthetized, ventilated adult baboons; six animals received 1 mg/kg of EL-246 before infusion of an LD100 of live Escherichia coli and six received the E. coli infusion without antibody therapy. Two other animals received 1 mg/kg of EL-246 intravenously without an infusion of bacteria. Intermittent measurements were made of circulatory pressures, cardiac output, urine output, arterial blood gases, ventilation:perfusion ratio (VA/Q), and hematologic status. The experiments were ended at 48 h or at the time of death. Tissues were harvested for pathology and biochemical measurements. The E. coli infusions were associated with a hyperdynamic state, pulmonary hypertension, systemic hypotension, decreased urine output (UOP), and metabolic acidosis. The antibody partly blocked PMN migration, but there were few significant physiologic or biochemical differences between the EL-246-treated and untreated animals. In the antibody-treated animals, UOP was decreased, metabolic acidosis was worsened, and median survival time was decreased significantly. We conclude that treatment with an antibody to E- and L-selectin in gram-negative sepsis does not improve gas exchange or protect against lung injury, and is associated with decreased survival time in primates.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Selectina E/imunologia , Infecções por Escherichia coli/complicações , Selectina L/imunologia , Síndrome do Desconforto Respiratório/prevenção & controle , Sepse/complicações , Acidose/microbiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Pressão Sanguínea , Dióxido de Carbono/sangue , Débito Cardíaco , Moléculas de Adesão Celular/imunologia , Quimiotaxia de Leucócito/imunologia , Humanos , Hipertensão Pulmonar/microbiologia , Hipotensão/microbiologia , Injeções Intravenosas , Masculino , Ativação de Neutrófilo , Neutrófilos/imunologia , Oxigênio/sangue , Papio , Troca Gasosa Pulmonar , Respiração Artificial , Síndrome do Desconforto Respiratório/microbiologia , Taxa de Sobrevida , Regulação para Cima , Urina , Relação Ventilação-PerfusãoRESUMO
Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.
Assuntos
Adesão Celular/fisiologia , Selectina L/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Neutrófilos/fisiologia , Anticorpos Monoclonais/farmacologia , Fenômenos Biofísicos , Biofísica , Adesão Celular/efeitos dos fármacos , Humanos , Selectina L/imunologia , Ligantes , Movimento , FotomicrografiaRESUMO
Atherosclerosis is increasingly thought to be a chronic inflammatory disease. Inflammation requires transmigration of leukocytes from the circulation to the tissues. Adhesion of leukocytes to endothelial calls is the initial event in an inflammatory response and is mediated by expression of several adhesion molecules. In this study we characterize the contribution of intercellular adhesion molecules (ICAM-1) and L-selectin in patients with different coronary artery disease syndromes. Serum concentrations of cICAM-1 and sL-selectin were measured by enzyme-linked immunosorbent assay in 31 patients with stable angina, 30 patients with unstable angina, 18 patients with acute myocardial infarction and 20 healthy subjects in a control group. All patients underwent coronary angiography. Mean (+/-SE) cICAM-1 levels were higher (p < 0.05) in patients with stable angina (249 +/- 6 ng/ml), unstable angina (260 +/- 16 ng/ml), or acute myocardial infarction (261 +/- 24 ng/ml) compared with those in subjects in the control group (171 +/- 11 ng/ml). In contrast, levels of sL-selectin were lower (p < 0.01) in patients with stable angina (1.2 +/- 0.1 microg/ml), unstable angina (1.1 +/- 0.6 microg/ml), or acute myocardial infarction (1.1 +/- 0.1 microg/ml) compared with those in subjects in the control group (1.8 +/- 0.1 microg/ml). No difference was found in cICAM-1 or sL-selectin levels among patients with stable angina, unstable angina, or acute myocardial infarction. No correlation was seen between cICAM-1 or sL-selectin levels and extent (or severity) of coronary artery disease or leukocyte count. L-selectin expression was observed to be depressed in patients with severe angina compared with that in members of the control group. To examine the mechanism of reduction in sL-selectin levels and L-selectin expression on leukocytes, leukocytes from the control group were stimulated in vitro. Stimulation of leukocytes resulted in a rapid downregulation of surface L-selectin expression, measured by flowcytometry, similar to the suppressed expression of L-selectin found on leukocytes from patients with coronary artery disease. In conclusion, altered cICAM-1 and sL-selectin levels in patients with coronary artery disease reflect the presence of a chronic inflammatory process. This inflammatory process results in downregulation of leukocyte expression of L-selectin and thus lower circulating sL-selectin levels.
Assuntos
Molécula 1 de Adesão Intercelular/sangue , Selectina L/sangue , Isquemia Miocárdica/sangue , Angina Pectoris/sangue , Angina Instável/sangue , Adesão Celular , Movimento Celular , Doença Crônica , Angiografia Coronária , Doença da Artéria Coronariana/sangue , Doença das Coronárias/sangue , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Inflamação , Selectina L/genética , Contagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangueRESUMO
The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor alpha precursor (proTGF-alpha) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-alpha, cell adhesion receptor L-selectin, interleukin 6 receptor alpha subunit, beta-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.
Assuntos
Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Fenantrolinas/farmacologia , Inibidores de Proteases/farmacologia , Precursores de Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador alfa/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antígenos CD/metabolismo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Teste de Complementação Genética , Cinética , Selectina L/metabolismo , Mutagênese , Fenótipo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Transfecção , Fator de Crescimento Transformador alfa/biossínteseRESUMO
The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.
Assuntos
Selectina L/fisiologia , Neutrófilos/fisiologia , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Dimetil Sulfóxido/farmacologia , Dipeptídeos/farmacologia , Endopeptidases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Selectina L/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Selectina-P/fisiologiaRESUMO
Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-alpha.
Assuntos
Fosfatase Alcalina/genética , Ácidos Hidroxâmicos/farmacologia , Selectina L/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Sequência de Bases , Primers do DNA , Genes Reporter , Humanos , Hidrólise , Selectina L/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Dados de Sequência MolecularRESUMO
L-selectin expression is regulated in part by membrane-proximal cleavage from the cell surface of leukocytes and L-selectin-transfected cells. The downregulation of L-selectin from the surface of neutrophils is speculated to be a process involved in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. We previously reported that L-selectin is cleaved between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain. We demonstrate that replacing this cleavage domain of L-selectin with the corresponding region of E-selectin prevents L-selectin shedding, as judged by inhibiting the generation of the 68-kD soluble and 6-kD transmembrane cleavage products of L-selectin. Unexpectedly, we found that point mutations of the cleavage site, as well as mutations of multiple conserved amino acids within the cleavage domain, do not significantly affect L-selectin shedding. However, short deletions of four or five amino acids in the L-selectin cleavage domain inhibit L-selectin downregulation. Mutations that appeared to inhibit L-selectin shedding resulted in higher levels of cell surface expression, consistent with a lack of apparent proteolysis from the cell membrane. One deletion mutant, I327 delta N332, retains the native cleavage site yet inhibits L-selectin proteolysis as well. Restoring the amino acids deleted between I327 and N332 with five alanine residues restores L-selectin proteolysis. Thus, the proteolytic processing of L-selectin appears to have a relaxed sequence specificity at the cleavage site, and it may depend on the physical length or other secondary structural characteristics of the cleavage domain.
Assuntos
Moléculas de Adesão Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Endopeptidases/metabolismo , Humanos , Selectina L , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Receptores de Retorno de Linfócitos/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , TransfecçãoRESUMO
L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band comigrated with mitogen-activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab')2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Moléculas de Adesão Celular/fisiologia , Neutrófilos/fisiologia , Transdução de Sinais , Tirosina/análogos & derivados , Animais , Anticorpos/farmacologia , Moléculas de Adesão Celular/imunologia , Citosol/metabolismo , Ativação Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Técnicas In Vitro , Selectina L , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/fisiologia , Camundongos/imunologia , Fosfoproteínas/sangue , Fosfoproteínas/isolamento & purificação , Fosforilação , Fosfotirosina , Receptores Fc/fisiologia , Superóxidos/sangue , Tirosina/metabolismoRESUMO
L-selectin is a lectin cell adhesion molecule expressed on the cell surfaces of lymphocytes, monocytes and granulocytes. Upon leukocyte activation or L-selectin cross-linking the transmembrane-bound L-selectin is rapidly shed from the cell surface. Based on these observations, it has been proposed that L-selectin is proteolytically cleaved from the cell surface. However a panel of common protease inhibitors have no effect on L-selectin proteolysis. To further define the mechanism of L-selectin down-regulation we have produced reagents to study proteolytic fragments of L-selectin. We have developed a trapping ELISA for the detection of soluble L-selectin. In addition we have produced a high affinity polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled PHA lymphoblasts and peripheral blood neutrophils. We review here our progress in defining a 6 kD L-selectin transmembrane peptide (L-STMP) from PMA activated lymphoblasts and fMLP-activated neutrophils. Radiochemical sequencing data indicate that the cleavage site occurs between Lys321 and Ser322 in a short membrane-proximal region of the extracellular domain.
Assuntos
Moléculas de Adesão Celular/metabolismo , Membrana Celular/enzimologia , Endopeptidases/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/química , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas de Imunoadsorção , Selectina L , Ativação Linfocitária , Linfócitos/metabolismo , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Homologia de Sequência , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Production of reactive oxygen intermediates (ROI) by the NADPH oxidase of neutrophils is a major mechanism of bacterial killing and, in pathologic circumstances, tissue damage. Integrins and selectins participate in neutrophil adhesion but may also play a role in intracellular signaling. The role of L-selectin in ROI production and Ca2+ signaling in suspended neutrophils was examined using the DREG series of anti-L-selectin antibodies. NADPH oxidase activation was assessed in three ways: H2O2 production using either scopoletin or dihydrorhodamine and O2- production using cytochrome c. Alterations in [Ca2+]i were measured using Fura 2-AM and fluorescence spectrophotometry. Cross-linking of L-selectin with DREG and 2 degrees antibody did not trigger production of H2O2 by itself but significantly enhanced the subsequent response to two soluble activating agents; the formyl peptide formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor (TNF). Potentiation of the oxidative burst was observed using F(ab')2 fragments but not with irrelevant antibodies and was observed whether 2 degrees antibody was added before or after fMLP. Cross-linking of L-selectin also triggered a rise in [Ca2+]i, due, in part, to release from intracellular stores. The intracellular Ca2+ chelator BAPTA blocked both the rise in [Ca2+]i and the potentiation of the oxidative burst in response to fMLP or TNF. We conclude that cross-linking of L-selectin induces intracellular signals, including release of Ca2+, which may contribute to potentiation of the oxidative burst.