Migration of leukocytes across an endothelium-epithelium bilayer as a model of renal interstitial inflammation.
Interstitial inflammation has emerged as a key event in the development of acute renal failure. To gain better insight into the nature of these inflammatory processes, the interplay between tubular epithelial cells, endothelial cells, and neutrophils (PMN) was investigated. A coculture transmigration model was developed, composed of human dermal microvascular endothelial (HDMEC) and human renal proximal tubular cells (HK-2) cultured on opposite sides of Transwell growth supports. Correct formation of an endoepithelial bilayer was verified by light and electron microscopy. The model was used to study the effects of endotoxin (LPS), tumor necrosis factor (TNF)-alpha, and alpha-melanocyte-stimulating hormone (alpha-MSH) by measuring PMN migration and cytokine release. To distinguish between individual roles of microvascular endothelial and epithelial cells in transmigration processes, migration of PMN was investigated separately in HK-2 and HDMEC monolayers. Sequential migration of PMN through endothelium and epithelium could be observed and was significantly increased after proinflammatory stimulation with either TNF-alpha or LPS (3.5 +/- 0.58 and 2.76 +/- 0.64-fold vs. control, respectively). Coincubation with alpha-MSH inhibited the transmigration of PMN through the bilayer after proinflammatory stimulation with LPS but not after TNF-alpha. The bilayers produced significant amounts of IL-8 and IL-6 mostly released from the epithelial cells. Furthermore, alpha-MSH decreased LPS-induced IL-6 secretion by 30% but had no significant effect on IL-8 secretion. We established a transmigration model showing sequential migration of PMN across microvascular endothelial and renal tubular epithelial cells stimulated by TNF-alpha and LPS. Anti-inflammatory effects of alpha-MSH in this bilayer model are demonstrated by inhibition on PMN transmigration and IL-6 secretion.
Bijuklic
Klaudija
K
Clinical Dept. of Internal Medicine, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria.
Jennings
Paul
P
Kountchev
Jordan
J
Hasslacher
Julia
J
Aydin
Sonia
S
Sturn
Daniel
D
Pfaller
Walter
W
Patsch
Josef R
JR
Joannidis
Michael
M
D002460:Cell Line; D048430:Cell Shape; D002634:Chemotaxis, Leukocyte; D018920:Coculture Techniques; D017097:Electric Impedance; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D004847:Epithelial Cells / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology; D008833:Microcirculation / Q000166:cytology / Q000276:immunology; D009395:Nephritis, Interstitial / Q000276:immunology* / Q000503:physiopathology; D012867:Skin / Q000098:blood supply*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D000521:alpha-MSH / Q000276:immunology
C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000521:alpha-MSH