RESUMEN
Phospholipase D (PLD) activation is part of the complex signalling cascade induced during phagocyte activation. Two PLD isoforms have been cloned, but their role in phagocyte functions is still poorly defined. We report that resting fresh circulating human monocytes expressed PLD1. PLD1 protein expression was rapidly down-regulated during cell culture. Lipopolysaccharide and pathogen-derived agonists (Candida albicans, arabinoside-terminated lipoarabinomannan and Gram-positive bacteria, but not mannose-capped lipoarabinomannan or double-stranded RNA) strongly induced PLD1 expression at both the mRNA and protein levels. Pro-inflammatory cytokines [interleukin (IL)-1beta and tumour necrosis factor alpha] had only a weak effect, whereas immune cytokines (IL-6 and interferon gamma), anti-inflammatory cytokines (IL-13 and IL-10) and chemoattractants (fMet-Leu-Phe and macrophage chemoattractant protein 1) were inactive. None of the agonists tested induced significant changes in the basal expression of PLD2 mRNA. Consistent with PLD1 up-regulation was the observation that PLD enzymic activity was higher in monocytes treated with active-pathogen-derived agonists than in control cells, when stimulated with PMA or with chemotactic agonists (fMet-Leu-Phe and C5a). Thus PLD2 seems to be a constitutive enzyme in circulating monocytes. Conversely, PLD1 is an inducible protein, rapidly regulated during culture conditions and selectively induced during cell activation. Therefore PLD1 might have a relevant role in immune responses against pathogens and in chronic inflammation.
Asunto(s)
Monocitos/enzimología , Fosfolipasa D/metabolismo , Northern Blotting , Western Blotting , Movimiento Celular , Separación Celular , Quimiocina CCL2/metabolismo , Clonación Molecular , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fagocitosis , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
The use of tumor cells as vaccines in cancer immunotherapy is critically dependent on their capacity to initiate and amplify tumor-specific immunity. Optimal responses may require the modification of the tumor cells not only to increase their immunogenicity but also to improve their ability to recruit effector cells to the tumor sites or sites of tumor antigen exposure. It has been reported that CD40 cross-linking of acute lymphoblastic leukemia (ALL) cells significantly increases their immunogenicity and allows the generation and expansion of autologous antileukemia cytotoxic T lymphocytes. This study demonstrates that the CD40 ligation of these tumor cells also induces the secretion of the CC-chemokines MDC and TARC. Supernatants from malignant cells cultured in the presence of sCD40L promote the migration of activated T cells that express CCR4, the common specific receptor for MDC and TARC. More importantly, the supernatants from CD40-stimulated tumor cells also support the transendothelial migration of autologous CCR4(+) antileukemia T cells. Therefore, the results demonstrate that the delivery to leukemia cells of a single physiologic signal, that is, CD40 cross-linking, simultaneously improves tumor cell immunogenicity and induces potent chemoattraction for T cells. (Blood. 2001;98:533-540)
Asunto(s)
Linfocitos B/patología , Linfoma de Burkitt/patología , Antígenos CD40/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas CC/metabolismo , Células Madre Hematopoyéticas/patología , Linfocitos T Citotóxicos/inmunología , Presentación de Antígeno , Antígenos de Neoplasias , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/metabolismo , Ligando de CD40/metabolismo , Quimiocina CCL17 , Quimiocina CCL22 , Quimiocinas CC/farmacología , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Unión Proteica , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
Although it is commonly accepted that the anti-inflammatory effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is mainly associated to their ability to inhibit the cyclooxygenase (COX) enzyme system, several results indicate that non-COX mechanisms could be important in the therapeutical effect of these drugs. The aim of this study was to define if NSAIDs could exert, at least in part, their anti-inflammatory effect by inhibiting the activities of human polymorphonuclear leukocytes (PMNs) triggered by chemotactic stimuli and, if so, to understand the relationship of this effect with COX inhibition. A unique opportunity to dissociate the inhibition of prostaglandin (PG) synthesis from other therapeutical properties of NSAIDs is constituted by ketoprofen isomers being the S-isomer 100 time more potent than R-isomer on COX inhibition. Our results show that R- and S-ketoprofen, independently of their potency as PG inhibitors, proved very efficacious in selective inhibition of interleukin-8 (IL-8) chemotaxis. Inhibition of IL-8 chemotaxis was not restricted to ketoprofen isomer as it could be observed also with drugs belonging to different classes of NSAIDs and it was obtained at drug concentration superimposable to plasma levels after therapeutic administration in patients. Reduction of IL-8 migration by ketoprofen isomers was paralleled by selective inhibition of PMN response in terms of intracellular calcium concentration ([Ca(2+)]i) increase and extracellular signal regulated kinase(ERK)-2 activation, two intracellular mediators reported to be critical for PMN activities. It is concluded that inhibition of IL-8 chemotaxis could represent a new clinical target for ketoprofen isomers and, in fact, contribute to the anti-inflammatory activity of NSAIDs.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Interleucina-8/farmacología , Cetoprofeno/farmacología , Neutrófilos/efectos de los fármacos , Calcio/metabolismo , Quimiocina CCL2/farmacología , Complemento C5a/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Interacciones Farmacológicas , Activación Enzimática , Humanos , Técnicas In Vitro , Cetoprofeno/análogos & derivados , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/fisiología , EstereoisomerismoRESUMEN
Macrophage-derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL-1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E(2), which blocked the acquisition of IL-12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry-based techniques, DC supernatants were found to contain N-terminally truncated forms of MDC [MDC(3-69), MDC(5-69) and MD(C7-69)] as well as the full-length molecule. In vivo, CD1a(+), CD83(+), MDC(+) DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a(+) or CD1b(+) DC, and DC with a CD83(+) phenotype were responsible for MDC production in this Th2-oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3-69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.
Asunto(s)
Quimiocinas CC/genética , Células Dendríticas/inmunología , Células Cultivadas , Quimiocina CCL22 , Quimiocinas CC/biosíntesis , Colecalciferol/farmacología , Cromatografía Líquida de Alta Presión , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dermatitis/inmunología , Dermatitis/metabolismo , Dexametasona/farmacología , Dinoprostona/farmacología , Endocitosis , Histiocitosis de Células de Langerhans/inmunología , Histiocitosis de Células de Langerhans/metabolismo , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Enfermedades Linfáticas/inmunología , Enfermedades Linfáticas/metabolismo , Espectrometría de Masas , Monocitos/inmunología , Isoformas de Proteínas/biosíntesis , ARN Mensajero/biosíntesis , Activación Transcripcional/efectos de los fármacosRESUMEN
The production and action of primary proinflammatory cytokines are strictly controlled by a series of circuits to avoid damage that they can cause if produced in excess. Interleukin-10 and interleukin-1 receptor antagonist contribute to the control of the magnitude of the inflammatory responses in vivo. Benzydamine, a non-steroidal anti-inflammatory drug that has been shown to have suppressive activity for the proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, was investigated for its effects on interleukin-10 and interleukin-1ra production. The drug did not modify the production of interleukin-10 and interleukin-1ra by peripheral blood mononuclear cells stimulated with lipopolysaccharide, under conditions where tumor necrosis factor-alpha and interleukin-1beta were decreased. The antiinflammatory capacity of benzydamine might thus result from its ability to reduce the production of proinflammatory cytokines, without affecting antiinflammatory factors.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Bencidamina/farmacología , Quimiocina CCL2/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-10/biosíntesis , Interleucina-1/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Sialoglicoproteínas/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Células Cultivadas/efectos de los fármacos , Quimiocina CCL2/genética , Humanos , Inflamación , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-10/genética , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Sialoglicoproteínas/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Macrophage-derived chemokine (MDC) has been reported to inhibit different HIV-1 strains in activated peripheral blood mononuclear cells (T cell blasts), although other investigators have not confirmed these findings. Here we demonstrate that MDC inhibits the replication of CCR5-dependent (R5) HIV-1(BaL) in monocyte-derived macrophages (MDM), but not in T cell blasts, although with variable potency depending on donor variability. Analysis of HIV-1(BaL) proviral DNA synthesis in MDM indicated that the suppressive effect of MDC did not involve inhibition of early events such as entry or reverse transcription. Finally, an inverse correlation was observed between the levels of endogenous MDC secreted by uninfected MDM of different donors and the efficiency of different HIV strains, including two primary isolates with different coreceptor usage, to replicate in these cells. Thus, MDC represents an example of a chemokine inhibiting HIV replication in macrophages acting at one or more postentry levels in the virus life cycle.
Asunto(s)
Antivirales/fisiología , Quimiocinas/fisiología , VIH-1/fisiología , Macrófagos/virología , Linfocitos T/virología , Replicación Viral , Antivirales/metabolismo , Secuencia de Bases , Células Cultivadas , Quimiocinas/metabolismo , Cartilla de ADN , Humanos , Macrófagos/metabolismoRESUMEN
As originally demonstrated for the interleukin 1 (IL-1) type II receptor, some primary proinflammatory cytokines from the IL-1 and tumor necrosis factor families are regulated by decoy receptors that are structurally incapable of signaling. Here we report that concomitant exposure to proinflammatory signals and IL-10 generates functional decoy receptors in the chemokine system. Inflammatory signals, which cause dendritic cell (DC) maturation and migration to lymphoid organs, induce a chemokine receptor switch, with down-regulation of inflammatory receptors (such as CCR1, CCR2, CCR5) and induction of CCR7. Concomitant exposure to lipopolysaccharide (LPS) and IL-10 blocks the chemokine receptor switch associated with DC maturation. LPS + IL-10-treated DCs showed low expression of CCR7 and high expression of CCR1, CCR2 and CCR5. These receptors were unable to elicit migration. We provide evidence that uncoupled receptors, expressed on LPS + IL-10-treated cells, sequester and scavenge inflammatory chemokines. Similar results were obtained for monocytes exposed to activating signals and IL-10. Thus, in an inflammatory environment, IL-10 generates functional decoy receptors on DC and monocytes, which act as molecular sinks and scavengers for inflammatory chemokines.
Asunto(s)
Mediadores de Inflamación/metabolismo , Interleucina-10/farmacología , Receptores de Quimiocina/metabolismo , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Técnicas In Vitro , Ligandos , Lipopolisacáridos/farmacología , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunologíaRESUMEN
We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3'-azido-3'-deoxythimidine (AZT) or cell-exposure to heat-inactivated (triangle up degrees ) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain.
Asunto(s)
Quimiocina CCL2/biosíntesis , VIH-1/fisiología , Macrófagos/virología , Células U937/virología , Replicación Viral , Quimiocina CCL2/genética , Expresión Génica , Productos del Gen tat/genética , Productos del Gen tat/farmacología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Mutación , ARN Mensajero/metabolismo , Transfección , Células U937/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Three molecular forms of the IL-1 receptor antagonist (IL-1ra) have been identified and cloned. Secreted IL-1ra (sIL-1ra or IL-1ra1) contains a classical leader peptide giving a released mature protein. Two intracellular isoforms, icIL-1ra type I (IL-1ra2) and icIL-1ra type II (IL-1ra3), have no leader sequence, thus predicting that these proteins remain intracellular. In an effort to define its biological role, we structurally and functionally characterized IL-1ra3. Endogenous immunoreactive IL-1ra3 was detected in a variety of inflammatory cells and tissues. We used a gene transfer strategy to explore the possible intracellular functions of IL-1ra3 (and IL-1ra2) and the cell-associated agonist IL-1alpha. The intracellular IL-1ra3 isoform, as well as IL-1ra2, does not block the action of exogenous and endogenous IL-1 under these conditions. Intact IL-1ra3 was released from the cells killed by NK effectors. The intracellular isoforms may represent a reservoir of IL-1ra, released upon cell death, whose function is to limit the pro-inflammatory action of cell debris.
Asunto(s)
Sialoglicoproteínas/metabolismo , Animales , Exones , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/metabolismo , Interleucina-1/farmacología , Ratones , Isoformas de Proteínas , Sialoglicoproteínas/genética , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To investigate whether the CC-chemokine monocyte chemotactic protein (MCP)-1 could play a role in the pathogenesis of HIV infection of the central nervous system. This hypothesis was suggested by previous observations, including our finding of elevated cerebrospinal fluid (CSF) levels of this chemokine in patients with cytomegalovirus (CMV) encephalitis. DESIGN AND METHODS: CSF levels of MCP-1 were determined in 37 HIV-infected patients with neurological symptoms, and were compared with both the presence and severity of HIV-1 encephalitis at post-mortem examination and CSF HIV RNA levels. MCP-1 production by monocyte-derived macrophages was tested after in vitro infection of these cells by HIV. RESULTS: CSF MCP-1 levels were significantly higher in patients with (median, 4.99 ng/ml) than in those without (median, 1.72 ng/ml) HIV encephalitis. Elevated CSF MCP-1 concentrations were also found in patients with CMV encephalitis and with concomitant HIV and CMV encephalitis (median, 3.14 and 4.23 ng/ml, respectively). HIV encephalitis was strongly associated with high CSF MCP-1 levels (P = 0.002), which were also correlated to high HIV-1 RNA levels in the CSF (P = 0.007), but not to plasma viraemia. In vitro, productive HIV-1 infection of monocyte-derived macrophages upregulated the secretion of MCP-1. CONCLUSIONS: Taken together, these in vivo and in vitro findings support a model whereby HIV encephalitis is sustained by virus replication in microglial cells, a process amplified by recruitment of mononuclear cells via HIV-induced MCP-1.