RESUMO
Colonic and intestinal epithelial cells (EC) attach to a basement membrane of laminins, fibronectin, and collagen IV. Wounding of the epithelial layer can change the types of extracellular matrix (ECM) proteins to which the EC attach. In this study, we determined the effect of culturing Caco-2 cells on different ECM proteins on the capacity of EC to produce TNF-α- or IL-1-stimulated CXCL8. The effect of the ECM proteins was such that CXCL8 secretion by cells cultured on collagen I > collagen IV > fibronectin or laminin-111. However, suppression of ROCK activity resulted in a similar 75 to 85% suppression of CXCL8 secretion regardless of the ECM protein type. This suggests that EC can produce different levels of CXCL8 depending on the type of ECM proteins they attach to, but all cases result in a similar requirement for ROCK activity for optimal CXCL8 secretion. Furthermore, when confluent cells were compared to subconfluent cells, the level of TNF- or IL-1-stimulated CXCL8 secretion was greatly elevated with the subconfluent cells and inhibiting ROCK had no effect on CXCL8 secretion levels by the confluent cells. These experiments suggest that CXCL8 responses by confluent cells, which would model for intact, unwounded epithelial, do not involve ROCK activation. However, CXCL8 responses by subconfluent cells, which would model for cells attaching to and moving on ECM proteins in wounded epithelia, require ROCK activation for greatly elevated CXCL8 responses. These results provide a model to examine the important conditions which regulate chemokine production by EC in wounded epithelia.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas da Matriz Extracelular/metabolismo , Interleucina-8/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Células CACO-2 , Contagem de Células , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Humanos , Interleucina-1/farmacologia , Laminina/metabolismo , Piridinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Th17 cells of the intestine and colon can produce several important cytokines during mucosal inflammation. However, few studies have focused on the role of IL-26 in intestinal inflammations. Colonic epithelial cells express receptors for IL-26, and this cytokine has been shown to induce the HT-29 colonic epithelial cell line to produce the chemokine CXCL8. However, epithelial cells would function in a cytokine network environment during mucosal inflammation and any effect of IL-26 on colonic epithelial cell chemokine responses could be affected by the presence of other potent pro-inflammatory cytokines like TNF-α and IL-1. Therefore, we investigated the effect of IL-26 with TNF-α or IL-1 on colonic epithelial cell line secretion of CXCL8. IL-26 alone had no effect on HT-29 or DLD1 cell line CXCL8 secretion. Yet, IL-26 was found to significantly enhance TNF-α-induced, but not IL-1-induced, CXCL8 secretion, but only at high levels of TNF-α. Similar results were seen with DLD1 cells. IL-26 did not enhance TNF-α-induced CXCL8 mRNA levels and did not affect TNF-α-induced IκBα phosphorylation or degradation. However, signaling through ERK and p38 MAPK were determined to be involved in the enhancing effect of IL-26 on the TNF-α-induced CXCL8 secretion, perhaps through known post-translational effects. These results suggest that the role of IL-26 in intestinal inflammation may be limited to enhancing CXCL8 secretion in the presence high levels of TNF-α, such as may occur in inflammatory bowel disease. Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBD, inflammatory bowel disease; IL, interleukin; ITS, insulin, transferrin, selenium; TBS, Tris buffered saline; TNF, tumor necrosis factor.
Assuntos
Células Epiteliais/efeitos dos fármacos , Interleucina-8/metabolismo , Interleucinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Células Epiteliais/metabolismo , Células HT29 , Humanos , Interleucina-1/farmacologia , Interleucina-8/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
IL-22 is known to induce intestinal epithelial cells (IECs) to produce the chemokine CXCL8. However, IECs exist in a cytokine network during mucosal inflammation, such that IL-22 must act in concert with potent pro-inflammatory cytokines like TNF-α and IL-1. Our studies show that IL-22 alone increased CXCL8 secretion from HT-29 cells, but the levels were minimal compared to that of the cells treated with TNF-α or IL-1 only. More significantly, co-stimulation with IL-22 and TNF-α enhanced both CXCL8 secretion and mRNA levels well over that of TNF-α stimulation alone. A similar enhancing effect was seen with IL-22- and IL-1-stimulated CXCL8 secretion. The enhancing effect of IL-22 on TNF-α-induced CXCL8 secretion was then determined to require the p38 MAPK, but not STAT1/3, PI3K, Akt, c-Jun N-terminal kinase, ERK, or IκBα. These experiments indicate that more significant effect of IL-22 on IECs responses may not be in inducing CXCL8 by itself, but in enhancing TNF-α- and IL-1-induced CXCL8 secretion to augment the contribution of IECs to local inflammatory responses.
Assuntos
Células Epiteliais/metabolismo , Interleucina-1/farmacologia , Interleucina-8/metabolismo , Interleucinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células HT29 , Humanos , Intestinos/citologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Interleucina 22RESUMO
Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.
Assuntos
Corantes Fluorescentes/química , Ácidos Nucleicos Peptídicos/química , Rodaminas/química , Linhagem Celular Tumoral , Citometria de Fluxo , Fluoresceínas/química , Fluoresceínas/efeitos da radiação , Corantes Fluorescentes/efeitos da radiação , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/efeitos da radiação , Fotodegradação , RNA de Cadeia Dupla/química , Rodaminas/efeitos da radiaçãoRESUMO
Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.
Assuntos
Inflamação/genética , Interleucina-1/metabolismo , Interleucina-8/genética , MAP Quinase Quinase 4/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Quinases Associadas a rho/genética , Amidas/administração & dosagem , Células CACO-2 , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-8/biossíntese , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 7/biossíntese , MAP Quinase Quinase 7/genética , Fosforilação/efeitos dos fármacos , Piridinas/administração & dosagem , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/biossínteseRESUMO
Intestinal epithelial cells (IEC) play a role in mucosal inflammatory responses by producing important chemokines like CXCL8 when stimulated by TNF-α. Previously, we found that IEC cell lines required the Rho-associated kinase, ROCK, for CXCL8 responses after IL-1 stimulation. This study extends these findings by showing that inhibiting ROCK suppressed TNF-α-induced CXCL8 secretion by Caco-2 and DLD1 colonic epithelial cell lines and CXCL8 mRNA levels in Caco-2 cells. RNAi knockdown experiments indicated that the inhibitory effect was mediated by ROCK2, and not ROCK1. Inhibiting ROCK had no effect on TNF-stimulated IκBα phosphorylation and degradation or p38 MAPK phosphorylation indicating that ROCK plays no role in these signaling pathways. However, inhibiting ROCK suppressed TNF-induced phosphorylation of the p54 JNK isoform and phosphorylation of the upstream MKK4 kinase. These results suggest that ROCK is required for CXCL8 responses by TNF-stimulated IEC by affecting intracellular signaling through MKK4 and JNK.
Assuntos
Inflamação/imunologia , Interleucina-8/imunologia , Intestinos/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Quinases Associadas a rho/imunologia , Amidas/farmacologia , Western Blotting , Células CACO-2 , Inibidores Enzimáticos/farmacologia , Células Epiteliais/imunologia , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Intestinos/citologia , MAP Quinase Quinase 4/imunologia , Fosforilação/imunologia , Piridinas/farmacologia , Interferência de RNA/imunologia , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Intestinal epithelial cells (IEC) play a role in mucosal inflammation by producing pro-inflammatory chemokines that may initiate or amplify local responses. IL-1 is a potent activator of IEC and its receptor localizes to focal adhesions. Since the Rho-associated kinase, ROCK, also localizes to focal adhesions, we examined the role of ROCK in IL-1-induced chemokine responses in IEC cell lines. Suppressing ROCK with the Y27632 inhibitor suppressed IL-1-stimulated Caco-2 cell CXCL8/IL-8 and IEC-6 cell CCL2/MCP-1 secretion and mRNA levels. ROCK inhibition also suppressed IL-1-induced JNK phosphorylation in both cell lines, but high levels of the inhibitor had no significant effect on IL-1-stimulated Caco-2 IκBα phosphorylation and degradation or IKK phosphorylation and kinase activity. Therefore, ROCK may exert an effect on IL-1-stimulated JNK signaling to AP-1 activation, with little effect on IKK/IκBα signaling, defining a potentially important mechanism for regulating IL-1 signaling in IEC that may be essential for optimal cytokine responses.
Assuntos
Células Epiteliais/efeitos dos fármacos , Interleucina-1/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Quinases Associadas a rho/fisiologia , Células CACO-2 , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/fisiologia , Fosforilação , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Aurones, derivatives of 2-benylidenebenzofuran-3(2H)-one, are natural products that serve as plant pigments. There have been reports that some of these substances fluoresce, but little information about their optical properties is in the literature. In this report, series of aurone derivatives were synthesized as possible fluorescent probes that can be excited by visible light. We found that an amine substituent shifted the lowest energy absorption band from the near-UV to the visible region of the electromagnetic spectrum. Four amine-substituted aurone derivatives were synthesized to explore the effect of this substituent on the absorption and emission properties of the aurone chromophore. The emission maxima and intensities of the molecules are strongly dependent on the nature of the substituent and the solvent polarity. Overall, the emission intensity increases and the maximum wavelength decreases in less polar solvents; thus, the aurones may be useful probes for hydrophobic sites on biological molecules. A limited investigation with model protein, nucleic acid and fixed cells supports this idea. It is known that the sulfur analog of aurone can undergo photo-induced E/Z isomerization. This possibility was investigated for one of the aminoaurones, which was observed to reversible photoisomerize. The two isomers have similar absorption spectra, but the emission properties are distinct. We conclude that appropriately substituted aurones are potentially useful as biological probes and photoswitches.
Assuntos
Benzofuranos/química , DNA/química , Corantes Fluorescentes/química , Soroalbumina Bovina/química , Animais , Benzofuranos/síntese química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Humanos , Substâncias Macromoleculares/química , Masculino , Microscopia Confocal , Estrutura Molecular , Peso Molecular , Salmão , Espectrofotometria Ultravioleta , Espermatozoides/química , EstereoisomerismoRESUMO
A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.
Assuntos
Fator 7 de Crescimento de Fibroblastos/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Androstadienos/farmacologia , Células CACO-2 , Movimento Celular , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Interleucina-1/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/genética , Wortmanina , Cicatrização/fisiologiaRESUMO
Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Interleucina-1beta/farmacologia , Interleucina-8/metabolismo , Mucosa Intestinal/imunologia , Animais , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Mucosa Intestinal/citologia , RatosRESUMO
A series of synthetic (nonylphenol, diethylstilbestrol, and bisphenol A) and natural (quercetin, resveratrol, and genistein) phenolic estrogens were investigated for their ability to affect the viability and proliferation of A549 lung cancer cells. To assess and distinguish the cytotoxic effect of individual estrogens, we used both the MTT tetrazolium spectrophotometric method and the fluorescence assay, while the induction of the cell specific apoptotic process was examined by fluorescence microscopy after treatment of cells with SYTO 24 green fluorescent dye. A systematic study of interferences for both fluorescence and MTT methods is presented. The results showed that both natural and synthetic estrogens decreased the viability and proliferation of A549 lung cancer cells in a dose-dependent manner but at different sensitivities. Nonylphenol appeared very different as compared to the other estrogens, acting by inducing the higher inactivation rate of the cells within a very short time. The cytotoxic effect of the estrogens was directly related to their structural and conformational characteristics including chain length, number, and position of hydroxyl groups and degree of saturation.
Assuntos
Adenocarcinoma/tratamento farmacológico , Congêneres do Estradiol/toxicidade , Estrogênios não Esteroides/toxicidade , Neoplasias Pulmonares/tratamento farmacológico , Relação Estrutura-Atividade , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/análise , DNA de Neoplasias/biossíntese , Relação Dose-Resposta a Droga , Congêneres do Estradiol/química , Congêneres do Estradiol/classificação , Estrogênios não Esteroides/química , Estrogênios não Esteroides/classificação , Fluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fenóis/química , Fenóis/classificação , Fenóis/toxicidade , Reprodutibilidade dos Testes , Sais de Tetrazólio , TiazóisRESUMO
The cytotoxic effect of isoflavonoids in the development of different forms of cancer has been reported by epidemiological and dietary studies. Consequently, there is a search for an accurate and reliable method for monitoring the interactions of these chemicals with cancerous cells. We have developed and optimized a fully autonomous electrochemical biosensor for studying the role of isoflavonoids on A549 lung adenocarcinoma cell line. This advanced biosensor uses a prototype 96-electrode (DOX-96) well-type device that allows the measurement of cell respiratory activity via the consumption of dissolved oxygen. The system provides a continuous, real-time monitoring of cell activity upon exposure to naturally occurring polyphenols, specifically resveratrol, genistein, and quercetin. The system is equipped with a multipotentiostat, a 96-electrode well for measurements and cell culturing with 3 disposable electrodes fitted into each well. A comparison with classical "cell culture" techniques indicates that the biosensor provides real-time measurement with no added reagents. A detection limit of 1 x 10(4) was recorded versus 200 and 6 x 10(3) cells/well for MTT and fluorescence assays, respectively. This method was optimized with respect to cell stability, reproducibility, applied potential, cell density per well, volume/composition of cell culture medium per well, and incubation. Others include total measuring time, temperature, and sterilization procedure. This study represents a basic research tool that may allow researchers to study the type, level, and specific influence of isoflavonoids on cells.
Assuntos
Antineoplásicos/metabolismo , Isoflavonas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Eletrodos , Flavonoides/farmacologia , Humanos , Isoflavonas/química , Isoflavonas/farmacologia , Neoplasias Pulmonares/patologia , Fenóis/farmacologia , Polifenóis , Espectrometria de Fluorescência/métodos , Temperatura , Fatores de TempoRESUMO
Epithelial cells (EC) from various tissues can produce important cytokines and chemokines when stimulated by proinflammatory cytokines. These EC also receive signals from cell surface integrins, like the alpha3beta1 integrin, which is important in cell migration and wound healing of epithelial monolayers. However, little is known of the effect of integrin signals on cytokine responses by EC. Colonic Caco-2 cells treated with an anti-alpha3 integrin antibody prior to stimulation with the proinflammatory cytokine interleukin (IL)-1 yielded suppressed levels of mRNA and secreted IL-6, IL-8 and monocyte chemoattractant protein-1 as compared to cells treated with normal mouse immunoglobulin G. Lung A549 cells also showed a similar suppression of cytokine secretion. Likewise, treatment of the Caco-2 cells with the same antibody suppressed tumour necrosis factor-alpha-stimulated IL-6 secretion. Fab fragments of the anti-alpha3 integrin antibody did not induce the suppressive effect but did block the suppressive effect of the whole antibody suggesting that the effect of the antibody required cross-linking of the integrins. Finally, culture of the Caco-2 cells on laminin type 5 (the major ligand for this integrin) yielded depressed levels of IL-1-induced IL-6 secretion as compared to cells on laminin type 1. These data are the first indication that the alpha3beta1 integrin may cause a suppression of cytokine responses by EC which may be important in regulating the capacity of EC to respond during inflammation or wound healing.
Assuntos
Citocinas/biossíntese , Células Epiteliais/imunologia , Integrina alfa3beta1/imunologia , Anticorpos Monoclonais/imunologia , Células CACO-2 , Citocinas/genética , Humanos , Interleucina-1/imunologia , Interleucina-6/biossíntese , Laminina/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Intestinal inflammatory disease or infection often results in the loss of the epithelial layer as a result mainly of the action of proteases, including the leucocyte serine proteinases (neutrophil elastase), lysosomal cathepsins and the matrix metalloproteinases from recruited inflammatory cells. Previous studies have shown that bronchial or intestinal epithelial cells (IEC) can respond to proteolytic attack by producing cytokines. In this study, we have determined the effect of protease treatment on interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production by IEC lines. Both neutrophil elastase and trypsin treatment induced elevated levels of mRNA for IL-6 in rat IEC-6 cells. Non-proteolytic detachment of the IEC-6 cells also induced elevated levels of IL-6 mRNA, suggesting that the effect was not caused by a specific protease or degradation product, but probably by an effect on cell shape or cell detachment. Similar results were seen with the IEC-18 cell line. Trypsin treatment of the IEC-6 cells also enhanced unstimulated and IL-1 beta costimulated IL-6 secretion, but not MCP-1 secretion or mRNA levels. Finally, nuclear levels of the CCAAT/enhancer binding protein-beta (C/EBP-beta) were rapidly enhanced after proteolytic detachment of the IEC-6 cells, suggesting a mechanism for the enhancement of IL-6 mRNA responses. These data indicate that epithelial cells can respond to proteolytic attack or cell detachment by producing IL-6, a cytokine with several anti-inflammatory and antiprotease effects, which may be important in moderating the loss of the epithelial layer by its effects on nearby epithelial or inflammatory cells.