RESUMO
Toll-like receptors (TLR) play an important role in pathogen recognition and innate immunity. We investigated the presence and function of TLRs in the BEAS-2B airway epithelial cell line and primary bronchial epithelial cells. Standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Taqman RT-PCR revealed that BEAS-2B cells express mRNA for TLR1-10. Several TLR ligands were tested for their ability to activate gene expression in BEAS-2B cells using limited microarray analyses focusing on genes of the chemokine and chemokine receptor family, cytokines, and signaling pathways. While the TLR3 ligand double-stranded RNA was the most effective epithelial activator, clear responses to flagellin, lipopolysaccharide, CpG, peptidoglycan, and zymosan were also observed. RT-PCR and/or enzyme-linked immunosorbent assay were used to confirm results obtained with microarrays for five of the induced genes: interleukin-8, serum amyloid A, TLR3, macrophage inflammatory protein-3alpha, and granulocyte-macrophage colony-stimulating factor. Stimulation of epithelial cells with double-stranded RNA induced levels of interleukin-8 exceeding 20 ng/ml and levels of serum amyloid A exceeding 80 ng/ml. Double-stranded RNA, lipopolysaccharide, zymosan A, and flagellin also induced expression of macrophage inflammatory protein-3alpha and granulocyte-macrophage colony-stimulating factor, which may facilitate immature dendritic cell migration and maturation. These results suggest that airway epithelial cells express several TLRs and that they are functionally active. Epithelial expression of TLRs may be of importance in inflammation and immunity in the airways in response to inhaled pathogens.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Glicoproteínas de Membrana/agonistas , RNA de Cadeia Dupla/farmacologia , Receptores de Superfície Celular/agonistas , Mucosa Respiratória/metabolismo , Alérgenos/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Flagelina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Receptor 1 Toll-Like , Receptor 3 Toll-Like , Receptores Toll-Like , Zimosan/farmacologiaRESUMO
Tissue structural cells are known in some situations to play a role in the presentation of antigen and in immunoregulation. We assessed the expression of B7 homologs, known to be involved in antigen presentation and lymphocyte costimulation, in human airway epithelial cells. Flow cytometry performed on the airway epithelial cell line BEAS-2B, as well as primary bronchial epithelial cells (PBEC), showed that B7-H2 was constitutively expressed on both BEAS-2B and PBEC, whereas B7-1 and B7-2 were undetectable on either epithelial cell type. B7-H2 expression was confirmed by Western blot using a specific antibody. Stimulation with various cytokines, including tumor necrosis factor-alpha, interferon-gamma, and interleukin-4, slightly downregulated B7-H2 expression detected by flow cytometry, but did not significantly alter the apparent level of protein as assessed by Western blotting. Northern blotting detected mRNA for B7-H2 and B7-1, but not B7-2. B7-H2 was cloned from BEAS-2B cells and the sequence verified. Expression of B7-H2 mRNA was detected by real-time reverse transcriptase-polymerase chain reaction in PBEC from three independent donors. Immunohistochemical analysis of airway derived from autopsies revealed expression of B7-H2 in human airway epithelial cells. These results demonstrate that airway epithelial cells express the costimulatory molecule B7-H2, and suggest the possibility that B7-H2 may participate in antigen presentation by epithelial cells.