RESUMO
The 'flu, caused mostly by influenza A and B viruses, represents a major public health issue. Despite vaccines and antiviral drugs, the therapeutic arsenal is still suboptimal. Recently, several studies have reported the antiviral and anti-inflammatory properties of several host metabolites. Now, we show that a metabolite (called here "C2") has a potent anti-influenza activity by blocking the viral replication and by limiting the downstream pro-inflammatory signalling. These results pave the way for the development of innovative metabolic therapy against influenzal pneumonia.
Assuntos
Vacinas contra Influenza , Influenza Humana , Pneumonia , Antivirais/uso terapêutico , Humanos , Vacinas contra Influenza/uso terapêutico , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Pneumonia/tratamento farmacológico , Pneumonia/prevenção & controle , Replicação ViralRESUMO
Innate immunity is the host's first line of defence against infection. In this review, we present the innate immune response implicated in three examples of pulmonary infection of viral, fungal and bacterial origin. We show that this defence against infection can be a double-edged sword. Thus, the same cells, molecules and mechanisms involved in this protective process can also be involved in deleterious inflammation. A delicate balance between immunity and inflammation is therefore required, making it possible to fight pathogens effectively while limiting inflammation that might be damaging to the host.
Assuntos
Infecções/imunologia , Inflamação/imunologia , Animais , Infecções Bacterianas/imunologia , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/virologia , Pneumopatias/imunologia , Pneumopatias/microbiologia , Pneumopatias/virologia , Modelos Animais , Micoses/imunologia , Viroses/imunologiaRESUMO
Pneumonia is frequently complicated by occurrence of acute respiratory distress syndrome (ARDS), consequently to dysregulated inflammatory response. However, mechanisms driving this dysregulation are poorly understood. To address this, "unconventional T-cells (UTC)" -γδT, NKT and MAIT cells- appear to be relevant targets due to their key role in orchestrating anti-microbial immune response in the lung. Thus, using an experimental and translational approach, we test the hypothesis that a tight regulation of UTC is mandatory to fine-tune host response, and, subsequently to prevent emergence of an aberrant response leading to excessive tissue damages, and eventually, ARDS.
Assuntos
Pneumonia/imunologia , Síndrome do Desconforto Respiratório/imunologia , Linfócitos T/fisiologia , Doença Aguda , Dispneia/etiologia , Dispneia/patologia , Humanos , Pulmão/imunologia , Pulmão/patologia , Pneumonia/etiologia , Pneumonia/patologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Índice de Gravidade de DoençaRESUMO
Acute and chronic respiratory diseases account for major causes of illness and deaths worldwide. Recent developments of biotherapeutics opened a new era in the treatment and management of patients with respiratory diseases. When considering the delivery of therapeutics, the inhaled route offers great promises with a direct, non-invasive access to the diseased organ and has already proven efficient for several molecules. To assist in the future development of inhaled biotherapeutics, experimental models are crucial to assess lung deposition, pharmacokinetics, pharmacodynamics and safety. This review describes the animal models used in pulmonary research for aerosol drug delivery, highlighting their advantages and limitations for inhaled biologics. Overall, non-clinical species must be selected with relevant scientific arguments while taking into account their complexities and interspecies differences, to help in the development of inhaled medicines and ensure their successful transposition in the clinics.
Assuntos
Aerossóis/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Terapia Respiratória/métodos , Administração por Inalação , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Modelos AnimaisRESUMO
Human leukocyte elastase (HLE), a polymorphonuclear neutrophil (PMN) serine proteinase, is proteolytically active on some membrane receptors at the surface of immune cells. The present study focused on the effect of HLE on the expression of CD14, the main bacterial lipopolysaccharide (LPS) receptor at the surface of monocytes. HLE exhibited a time- and concentration-dependent downregulatory effect on CD14 surface expression. A 30-minute incubation of 3 microM HLE was required to display 95% disappearance of the receptor. This downregulation resulted from a direct proteolytic process, not from a shedding consecutive to monocyte activation as observed upon challenge with phorbol myristate acetate (PMA). To confirm that CD14 is a substrate for HLE, this enzyme was incubated with recombinant human CD14 (Mr approximately 57,000), and proteolysis was further analyzed by immunoblot analysis. Cleavage of the CD14 molecule was directly evidenced by the generation of short-lived fragments (Mr approximately 47,000 and 30,000). As a consequence of the CD14 proteolysis, a decrease in the responsiveness of monocytes to LPS was observed, as assessed by measuring tumor necrosis factor-alpha (TNF-alpha) formation. This inhibition was only observed with 1 ng/ml of LPS, i.e., when only the CD14-dependent pathway was involved. At a higher LPS concentration, such as 10 microgram/ml, when CD14-independent pathways were operative, this inhibition was overcome. The direct proteolysis by HLE of the membrane CD14 expressed on monocytes illustrates a potential anti-inflammatory effect of HLE through inhibition of LPS-mediated cell activation.
Assuntos
Elastase de Leucócito/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Baixo , Citometria de Fluxo , Formaldeído/farmacologia , Humanos , Immunoblotting , Cinética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP. Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation. Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment. In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment. We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB). We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular [Ca(++)] flux in neutrophils. We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion. This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.
Assuntos
Adenosina/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Neutrófilos/metabolismo , Transdução de Sinais/fisiologia , Fatores Ativadores da Transcrição , Adenosina/farmacologia , Animais , Proteínas Sanguíneas/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Colforsina/metabolismo , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Antagonistas de Receptores Purinérgicos P1 , Receptor A2B de Adenosina , Receptores Purinérgicos P1/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Intestinal epithelial cells express hPepT1, an apical transporter responsible for the uptake of a broad array of small peptides. As these could conceivably include n-formyl peptides, we examined whether hPepT1 could transport the model n-formylated peptide fMLP and, if so, whether such cellular uptake of fMLP influenced neutrophil-epithelial interactions. fMLP uptake into oocytes was enhanced by hPepT1 expression. In addition, fMLP competitively inhibited uptake of a known hPepT1 substrate (glycylsarcosine) in hPepT1 expressing oocytes. hPepT1 peptide uptake was further examined in a polarized human intestinal epithelial cell line (Caco2-BBE) known to express this transporter. Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1 recognized solutes, but not by related solutes that were not transported by hPepT1. Fluorescence analyses of intracellular pH revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide H+ cotransporter. Lumenal fMLP resulted in directed movement of neutrophils across epithelial monolayers. Solutes that inhibit hPepT1-mediated fMLP transport decreased neutrophil transmigration by approximately 50%. Conversely, conditions that enhanced the rate of hPepT1-mediated fMLP uptake (cytosolic acidification) enhanced neutrophil-transepithelial migration by approximately 70%. We conclude that hPepT1 transports fMLP and uptake of these peptide influences neutrophil-epithelial interactions. These data (a) emphasize the importance of hPepT1 in mediating intestinal inflammation, (b) raise the possibility that modulating hPepT1 activity could influence states of intestinal inflammation, and (c) provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/fisiologia , Fatores Quimiotáticos/metabolismo , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Simportadores , Animais , Cálcio/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Neoplasias do Colo/patologia , Dipeptídeos/metabolismo , Células Epiteliais/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Transporte de Íons/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Transportador 1 de Peptídeos , Células Tumorais Cultivadas , Xenopus laevisRESUMO
Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be up-regulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype.
Assuntos
Elastase de Leucócito/antagonistas & inibidores , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Proteínas/fisiologia , Inibidores de Serina Proteinase/sangue , Diferenciação Celular , Citoplasma/química , Citoplasma/enzimologia , Células HL-60 , Humanos , Elastase de Leucócito/sangue , Neutrófilos/efeitos dos fármacos , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , RNA Mensageiro/biossíntese , Inibidor Secretado de Peptidases Leucocitárias , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/isolamento & purificação , Escarro/química , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.
Assuntos
Plaquetas/fisiologia , Catepsinas/fisiologia , Inibidores Enzimáticos/farmacologia , Elastase de Leucócito/fisiologia , Fosfatidilinositol 3-Quinases/sangue , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Androstadienos/farmacologia , Plaquetas/efeitos dos fármacos , Catepsina G , Catepsinas/farmacologia , Cromonas/farmacologia , Fibrinogênio/metabolismo , Humanos , Técnicas In Vitro , Elastase de Leucócito/farmacologia , Morfolinas/farmacologia , Fosfatidilinositóis/sangue , Inibidores de Fosfoinositídeo-3 Quinase , Ativação Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Serina Endopeptidases , WortmaninaRESUMO
When polymorphonuclear neutrophil-platelet suspensions were stimulated by 0.5 microM N-formyl-Met-Leu-Phe in the presence of 40 U/mL of superoxide dismutase, a significant reduction of platelet secretion was observed (51.4 +/- 6.3% vs 62.4 +/- 4.6% for control; mean +/- SEM; N = 6; P < 0.01). This was due to the superoxide anion scavenging property of superoxide dismutase since neutrophil degranulation, cathepsin G and elastase enzymatic activities (the two main mediators of this cell-to-cell interaction) and platelet reactivity were not affected. Involvement of superoxide anions was confirmed using leukotriene B4, a neutrophil agonist which induces degranulation with minimal superoxide anion production. Indeed, serotonin release induced by this agonist was unchanged whether superoxide dismutase was added or not.
Assuntos
Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Superóxido Dismutase/farmacologia , Catepsina G , Catepsinas/análise , Células Cultivadas , Glucuronidase/análise , Humanos , Leucotrieno B4/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Elastase Pancreática/análise , Serina Endopeptidases , Serotonina/análise , Superóxidos/análiseRESUMO
While chemokines have received considerable attention for their role in leukocyte chemotaxis, their effects on platelets have not been well described. We found that two CC chemokine receptor 4 (CCR4) ligands, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) induce concentration-dependent platelet aggregation and calcium flux. Flow cytometric analysis revealed the expression of CCR4 on platelets and a monoclonal antibody (mAb) to CCR4 inhibited MDC- and TARC-induced platelet aggregation, confirming that this effect is mediated through their common receptor CCR4. MDC fully desensitized TARC-induced calcium mobilization in platelets, while TARC was unable to completely desensitize a subsequent MDC response, which is similar to observations made in Th2 CD4(+) lymphocytes and CCR4-transfected cells. Aspirin (ASA) treatment of platelets allowed reversible primary aggregation but inhibited irreversible complete aggregation, suggesting that MDC- and TARC-induced full platelet aggregation is dependent on cyclooxygenase metabolites of arachidonic acid. MDC and TARC were unable to induce platelet aggregation and platelet secretion in washed human platelets, even though they induced a calcium flux, suggesting that plasma components are required for MDC- and TARC-induced platelet aggregation. Since Th2-type cytokines induce the release of MDC and TARC from cells and the expression of these chemokines is increased in Th2-type inflammation, we hypothesize that MDC and TARC may play a role in platelet activation seen in Th2 diseases, such as asthma and atopic dermatitis.
Assuntos
Quimiocinas CC/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Receptores de Quimiocinas/efeitos dos fármacos , Receptores de Quimiocinas/fisiologia , Cálcio/sangue , Quimiocina CCL17 , Quimiocina CCL22 , Quimiocina CXCL12 , Quimiocinas CC/administração & dosagem , Quimiocinas CC/fisiologia , Quimiocinas CXC/administração & dosagem , Quimiocinas CXC/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Hipersensibilidade Imediata/sangue , Hipersensibilidade Imediata/etiologia , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Masculino , Receptores CCR4RESUMO
We previously showed that two polymorphonuclear neutrophil (PMN)-derived proteinases, namely cathepsin G (Cat. G) and elastase (HLE), acting in synergy activated nearby platelets in vitro. This cellular interaction could result in a pathology such as the adult respiratory distress syndrome (ARDS). Since elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were detected in these patients and therefore could be involved in this disease, we looked for their effects in the PMN-platelet cooperation system. Addition of IL-8 to mixed suspensions of PMN and platelets induced weak but significant platelet aggregations. Upon preincubation with TNF-alpha, aggregations triggered by IL-8 were significantly increased. The targets of these cytokines were not the platelets but the PMNs. This was shown by following beta-glucuronidase release and more interestingly by measuring the enzymatic activities of Cat. G and HLE in the supernatant. Inhibition of the platelet response upon addition of a serine proteinase inhibitor, eglin C, clearly demonstrated the involvement of these two enzymes. Taken together, these results constitute an additional argument for the role of the PMN-platelet interaction in ARDS.
Assuntos
Interleucina-8/administração & dosagem , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Fator de Necrose Tumoral alfa/administração & dosagem , Adulto , Catepsina G , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Interleucina-8/fisiologia , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Proteínas , Síndrome do Desconforto Respiratório/etiologia , Serina Endopeptidases , Serpinas/farmacologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca(2+), both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.
Assuntos
Receptores de Hialuronatos/fisiologia , Mucosa Intestinal/fisiologia , Neutrófilos/fisiologia , Adenilil Ciclases/metabolismo , Anticorpos Monoclonais/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Ativação Enzimática , Humanos , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 x 10(-7) M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 x 10(-8) and 5 x 10(-7) M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.
Assuntos
Neutrófilos/enzimologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Suramina/farmacologia , Catepsina G , Catepsinas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Cinética , Elastase de Leucócito/antagonistas & inibidores , Mieloblastina , Conformação Proteica , Cloreto de Sódio/farmacologia , Suramina/administração & dosagemRESUMO
BACKGROUND & AIMS: Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. METHODS: We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. RESULTS: As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. CONCLUSIONS: These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.
Assuntos
Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Proteínas , Adulto , Sequência de Bases , Transporte Biológico/fisiologia , Biópsia , Células CACO-2 , Polaridade Celular/fisiologia , Cloretos/metabolismo , Clonagem Molecular , Colo/enzimologia , Colo/microbiologia , Colo/patologia , DNA Complementar , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Células HT29 , Humanos , Técnicas In Vitro , Absorção Intestinal/fisiologia , Mucosa Intestinal/patologia , Jejuno/enzimologia , Jejuno/microbiologia , Jejuno/patologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Proteína Quinase C/metabolismo , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/análise , Infecções por Salmonella/metabolismo , Salmonella typhimurium , Inibidor Secretado de Peptidases Leucocitárias , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND & AIMS: The development of intestinal intraepithelial lymphocytes (IELs) requires the movement of lymphocytes into the epithelial compartment (i.e., IEL homing). The rules governing and the biologic consequences of IEL homing are poorly understood. The aims of this study were to examine the adhesion molecules involved in IEL homing and the phenotypic alteration of lymphocytes as a consequence of homing. METHODS: We previously developed an in vitro IEL homing model consisting of human IEL cell lines and a polarized monolayer of human intestinal epithelial T84 cells. Homing capacity of lymphocytes was assessed by measuring their migration into epithelial monolayers, and phenotypic analysis was performed by flow cytometry. RESULTS: In this model, approximately 30% of lymphocytes moved into the epithelial monolayer, regardless of the lymphocyte concentration. Flow cytometric screening of adhesion molecules revealed that homed lymphocytes expressed high levels of integrin alphaXbeta2 and alphaEbeta7 and low levels of alpha4beta7 compared with non-homed lymphocytes. In addition, subpopulations sorted as alphaXbeta2(high) or alphaEbeta7(high) independently showed greater homing capacities. After homing, alphaEbeta7 and intercellular adhesion molecule 1 (ICAM-1) on homed lymphocytes were significantly up-regulated, which was consistent with their high expression observed on freshly isolated human IELs. The up-regulation of alphaEbeta7 (but not ICAM-1) was completely dependent on epithelial-derived transforming growth factor beta1 (TGF-beta1). The expression of alphaXbeta2 was observed on a small population of freshly isolated human IELs, and was markedly induced by stimulation. Also, epithelial-derived TGF-beta1 down-regulated the alphaXbeta2 expression (an event likely to occur after homing). CONCLUSIONS: Our findings indicate a relationship between IEL alphaXbeta2 and alphaEbeta7 expression and homing into intestinal epithelia. We also show that phenotypic alteration of IELs is induced by close interaction with intestinal epithelia as a consequence of homing.
Assuntos
Integrinas , Mucosa Intestinal/imunologia , Linfócitos/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Linhagem Celular , Quimiotaxia , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Integrina alfaXbeta2 , Molécula 1 de Adesão Intercelular/genética , Ativação Linfocitária , Linfócitos/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
UNLABELLED: It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5'-(N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current (I(sc)) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I(sc) induced by apical and not basolateral adenosine. IN CONCLUSION: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.