Modulation of endotoxin-induced neutrophil transendothelial migration by alveolar epithelium in a defined bilayer model.
Within the alveolus, epithelial cells, due to their close association with endothelial cells, can potentially influence endothelial cell responsiveness during inflammation and their interaction with leukocytes. To investigate this, three lung epithelial cell lines (A549, Calu-3, or NCI-H441) were grown with endothelium on opposing surfaces of Transwell filters and the formation and stability of bilayers was rigorously evaluated. All epithelial lines disrupted endothelial monolayer formation on filters with 3- or 5-microm pores by breaching the filter, and this occurred regardless of seeding density, matrix composition, or duration of culture. Endothelial disruption was not detectable by electrical resistance or permeability measurements but required cell-specific staining with immunofluorescence and microscopy. Distinct bilayers formed only on filters with 0.4-microm pores and only with A549 cells and human umbilical vein endothelial cells. Endotoxin (lipopolysaccharide [LPS]) stimulation of bilayers (4 hours) enhanced neutrophil transendothelial migration, but this was significantly decreased compared with the response of endothelium grown alone, irrespective of whether LPS exposure was via the epithelial or endothelial side of the bilayer. Down-modulation required epithelial-endothelial approximation and was not seen when these cells were separated by 0.5 to 1 mm. This study defines optimal conditions required for generation of intact bilayers of lung epithelial cells with endothelium for the study of leukocyte-transendothelial migration. Furthermore, it was demonstrated that lung epithelial cells can modulate endothelial cell responsiveness to an environmental inflammatory stimulus such as LPS and thus may have an important role in minimizing excessive and deleterious neutrophilic inflammation in the lung alveolus.
Weppler
Amy
A
Department of Microbiology-Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Rowter
Derek
D
Hermanns
Iris
I
Kirkpatrick
C James
CJ
Issekutz
Andrew C
AC
D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008851:Micropore Filters; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects* / Q000502:physiology
D008070:Lipopolysaccharides