Molecular Requirements and Cellular Effects on alpha4beta1 Mediated B Lymphocyte Adherence Under Flow Conditions.

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alpha4beta1 is critical for normal lymphocyte recirculation. Expressed on B lymphocytes, alpha4beta1 binds VCAM-1 on endothelium as an early step in lymphocyte extravasation. In this study, we examine the behavior of B lymphocytes on purified VCAM-1 as a first step toward quantifying the molecular and signaling requirements on B lymphocyte rolling and arrest mediated by alpha4beta1. In our initial set of observations, we wished to define chemokines that stimulated alpha4beta1 on human B lymphocytes isolated from blood and determine the site densities over which \uf0614Β1 mediated attachment could occur. We tested a panel of CXC and CC chemokines and show that 4 chemokines (MIP-3\uf061, MIP-3Β, SDF-1, BLC) may stimulate alpha4beta1 activation on human peripheral blood B lymphocytes. We next show that B lymphocytes isolated from peripheral blood will roll and attach to VCAM-1 in the absence of cellular stimulation; B lymphocytes accumulation does not occur below 50 sites/\uf06dm2 of VCAM-1 and shows increasing accumulation up to 200 sites/\uf06dm2. Interestingly, when alpha4beta1 was activated to its high affinity state using Mn2+ or SDF-1\uf061 stimulation, increased accumulation is seen over the range of 50-200 sites/\uf06dm2 compared with no cellular stimuli, yet the site density over which attachment and accumulation occurs remains unchanged. Since culturing B lymphocytes after isolation from blood may effect alpha4beta1 mediated binding but has not been studied, we examined B cell attachment to VCAM-1 (~1,200 sites/\uf06dm2) under different culture conditions. We show that the proportion of alpha4beta1 expressing B lymphocytes that roll on VCAM-1 progressively increases from 1 to 4 days of culture (90% firm arrest/10% rolling on Day 1 versus 45% firm arrest/55% rolling on Day 4). Although the \uf0614Β1 site density on B cells remains constant over the four days. In order to determine whether the change in alpha4beta1 mediated rolling and arrest on VCAM-1 was due to changes in the native receptor affinity or intracellular signaling, we performed a series of pharmacologic studies. Exposure of B lymphocytes cultured for 1 or 4 days to Mn2+ and DTT, activators of alpha4beta1, promoted firm attachment of greater than 90% of the cells, indicating alpha4beta1 could obtain a high affinity state regardless of culture duration. Inhibition of protein kinase C, farnesyl transferase, and G protein coupled receptor signaling increases the proportion of rolling cells with fewer overall attachments. This phenomenon is observed on Day 1 and is more pronounced on Day 4, suggesting that inside-out signaling remains intact despite culturing. In all, we quantify for the first time the effect of site density and post-isolation culture on the behavior of alpha4beta1-mediated adherence and signaling on B lymphocytes. These observations are important to understanding and interpreting studies on alpha4beta1 mediated lymphocyte rolling and arrest.

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