Talavera Dodanim, Castillo Aida M, Dominguez M C, Gutierrez Alejandro Escobar, Meza Isaura
Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México, DF 07360, Mexico.
Departamento de Investigaciones Inmunológicas, Instituto de Diagnóstico y Referencia Epidemiológicos, SSA, México, DF, Mexico.
J Gen Virol. 2004 Jul;85(Pt 7):1801-1813. doi: 10.1099/vir.0.19652-0.
Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [(3)H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml(-1). Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.
微血管内皮细胞的通透性改变可能是登革病毒感染导致血浆渗漏的一个因素。用人真皮微血管内皮细胞系HMEC-1的汇合单层细胞作为实验模型，研究病毒诱导的细胞反应。感染的单层细胞对[³H]甘露醇的通透性增加，但在感染后48小时（p.i.），对于4-70 kDa的葡聚糖未观察到变化，此时病毒滴度达到最大值，40%的细胞表达病毒蛋白。在72小时时通透性进一步增加，此时单层细胞仍无明显细胞病变效应。与此同时，感染细胞中的肌动蛋白发生重组，紧密连接蛋白闭合蛋白被转移至细胞质。在感染细胞邻近的未感染细胞中观察到应力纤维和黏着斑厚度增加。感染单层细胞的培养基在对照培养物中诱导通透性变化以及含肌动蛋白结构的增厚，类似于感染后48小时观察到的情况。在培养基中发现白细胞介素（IL）8的浓度范围为20至100 pg/ml⁻¹。抗IL8中和抗体部分抑制了培养基产生的变化以及添加IL8诱导的变化。染料木黄酮抑制了培养基的作用以及与黏着斑相关蛋白的磷酸化，并表明酪氨酸激酶参与其中。这些发现表明，感染单层细胞产生的IL8有助于病毒对细胞骨架和紧密连接的诱导作用，从而改变跨内皮通透性。
