Abstract
Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1–dependent expression of TNF-α in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4+ lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1–induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1–induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.
Highlights
Go6983, PP2, Mowiol 4-88, SB202190, and SP600125 were purchased from Merck (Nottingham, U.K.); cell-permeable C3 transferase was from Universal Biologicals (Cambridge, U.K.); PF573228 and focal adhesion kinase (FAK) inhibitor 14 were from Tocris (Bristol, U.K.); U0126 was from Promega (Southampton, U.K.); L-JNKi was from Alexis Biochemicals (Exeter, U.K.); actinomycin D, myelin basic protein (MBP), and anti-tubulin ascites were from Sigma (Poole, U.K.)
ICAM-1 engagement on brain microvascular EC (MVEC) leads to MAPK activation
Adhesion-dependent endothelial MAPK activation was studied in cocultures of brain MVECs (GPNT) and Con A–activated peripheral lymph node cell (PLNC) that adhere but do not undergo diapedesis [25]
Summary
Promoter analysis using transiently transfected luciferase reporters showed that ICAM-1 cross-linking enhanced the activity of the human VCAM-1 promoter by 50% and that this response was abolished by inhibition of ERK or p38, but not JNK (Fig. 3G). We investigated whether endothelial gene expression was critical for TEM of CD4+ Th1 cells across brain GPNT ECs. Pretreatment of ECs with the transcription inhibitor actinomycin D did not affect lymphocyte TEM, which occurred during the first 30 min of coculture (Fig. 5A).
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