Introduction

The overall goal of the Alliance for Cellular Signaling (AfCS) is to understand, as completely as possible, how cells interpret signals in a context-dependent manner.  How do cells respond appropriately to individual signals while they are bombarded with many simultaneously?  How do various cellular signaling modules interact to form a robust control system?  The pathway to such understanding will involve identifying all the proteins that compose the various signaling systems, constructing a physical map of their relationships, assessing time-dependent information flow through the pathways, and, finally, reducing the mass of detailed data into a set of interacting theoretical models that describe cellular signaling.

One premise of the AfCS is that we must focus intense effort on a small number of cellular signaling systems in order to gather the requisite amount of data. An extensive list of criteria was evaluated to aid in these choices.  Particularly important among these were the capacity to observe interesting regulated phenotypes, to obtain relatively large numbers of homogeneous cells, and to manipulate gene expression in these cells based on reasonably complete knowledge of their genome.  We also wish to study relatively normal cells from a mammalian organism.  Based on these criteria, one of the cells chosen was the resting B lymphocyte (B cell) of the mouse.

By secreting antigen-specific immunoglobulin antibody, the B cell plays an essential role in host defense.  Resting B cells reside in the circulation and migrate to the spleen and lymph nodes.  Activation of the B-cell receptor by antigen in conjunction with appropriate co-stimulation causes the resting B cell to proliferate and differentiate into a plasma cell, which makes and secretes large amounts of immunoglobulin antibody.  In addition to the antigen receptor, B cells exhibit a large number of additional cell surface receptors, which are activated by T-cell surface molecules, cytokines, bacterial endotoxin, and other ligands.  Immunologists have begun to define key steps in signaling pathways downstream of these receptors that regulate steps in development of the mature B cell and modulate responses to antigen. (1, 2, 3)

The best-studied G protein-coupled receptors on B cells activate G_i and mediate directional motility in response to chemokines.  This chemotactic response, which is essential for homing of B cells to their proper location in the spleen and lymph nodes (4), offers a fitting challenge for the AfCS.  Chemotactic signals within migrating cells are just beginning to be identified but already include a vast array of different signaling protein families, including heterotrimeric G proteins; rho GTPases and their exchange factors, GTPase activating proteins, and effectors (e.g. kinases); tyrosine kinases; lipid kinases; and protein kinases A, B, and C (5, 6).  The challenge is not only that we know little about the wiring that connects these signaling elements, but even more importantly, we must identify the underlying compass mechanism-how the signaling machine interprets chemotactic gradients in space and time-to decide where the cell should go.

To approach this attractive and challenging cellular system, the AfCS must develop and apply standardized, reproducible procedures for isolating and purifying resting splenic B cells and for their short-term maintenance in culture under defined conditions.  These procedures must also be conveniently transferable between laboratories, not only within the AfCS, but to other interested laboratories as well.  This report describes the procedures that we have adopted to meet these needs and documents the utility of the isolated cells for short-term (hours) studies of signaling mechanisms.