Article Text

  1. D. A. Mills1,
  2. D. C. Brown1,
  3. S. Ramirez1,
  4. A. Chigaev1,
  5. L. A. Sklar1,
  6. M. Laurence1,
  7. R. S. Larson1
  1. 1University of New Mexico, Albuquerque, NM.


VLA-4 is critical for normal lymphocyte recirculation. Expressed on B lymphocytes, VLA-4 binds VCAM-1 on endothelium as a first 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 parameters and requirements for lymphocyte rolling and arrest mediated by VLA-4. In our first set of observations, we wished to define chemokines that stimulated VLA-4 on human B lymphocytes isolated from blood and determine the site densities over which VLA-4-mediated attachment could occur. We tested a panel of chemokines and show that four chemokines may stimulate VLA-4 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-lymphocyte accumulation does not occur below 50 sites/μm2 of VCAM-1 and shows increasing accumulation up to 200 sites/mm2. Interestingly, VLA-4 is activated to its high-affinity state using Mn2+ or SDF-1α stimulation, and increased accumulation is seen over the range of 50 to 200 sites/μm2 compared with no cellular stimuli, yet the site density over which attachment occurs remains unchanged. We then examined the effect of culturing B lymphocytes after isolation from blood on VLA-4-mediated binding. Using a VCAM-1 site density that would promote maximal binding (≈1,200 sites/μm2), we show that the proportion of VLA-4-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). Flow cytometry-based site density experiments indicate that the VLA-4 site density on B cells remains constant over the 4 days. To determine whether the change in VLA-4-mediated rolling and arrest on VCAM-1 was due to changes in the native receptor affinity or intracellular signaling, we performed a series of studies of pharmacologic studies. Exposure of day 1 or day 4 B lymphocytes to Mn2+ and DTT, activators of VLA-4, promoted firm attachment of greater than 90% of the cells, indicating that VLA-4 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 postisolation culture on the behavior of VLA-4-mediated adherence of B lymphocytes. Our observations also suggest that signaling both from and to the VLA-4 receptor occurs and changes during postisolation culture. These observations are important in resolving previous studies examining VLA-4-mediated lymphocyte rolling and arrest.

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