Kensuke Miyake

Cloning of murine and rat vascular cell adhesion molecule-1

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.

CD44 and Other Cell Interaction Molecules Contributing to B Lymphopoiesis

The term “adhesion” seems much too narrow to describe the multifunctional molecules on which this book is based. In addition to mediating physical interactions, as the name suggests, they serve as receptors for cell-cell communication and in the binding of cells to the extracellular matrix. The distinction between cell adhesion molecules and growth factor receptors has become blurred by reports that the latter can immobilize cells. Moreover, the same, or closely related, molecules can serve different specialized functions in multiple tissues. Information is gradually accumulating about interaction molecules expressed in lymphohematopoietic tissues and to date none have been convincingly described which are restricted to those sites. Our experience with B lymphocyte precursors suggests that molecules critical for the formation and export of blood cells from bone marrow are closely related to those responsible for their recruitment from the bloodstream and migration within peripheral tissues. Like other cell adhesion molecules, they have the very interesting property of having multiple functional states, which are actively regulated by cells that express them. The emphasis of this review will be on studies which have implicated four cell adhesion/ interaction molecules in B lymphopoiesis.

Stromal-Cell and Cytokine-Dependent Lymphocyte Clones Which Span the Pre-B- to B-Cell Transition

Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation.They are useful for determining the molecular requirements for pre-B replication, for studying the stromal cells that supply those factors, and for delineating the final sequence of differentiation events as newly formed lymphocytes prepare to exit the bone marrow. The efficiency of lymphocyte growth at limiting dilution varied substantially on different stromal-cell clones and may reflect functional heterogeneity of stromal cells. Most lymphocyte clones were similar to uncloned lymphocytes from Whitlock-Witte cultures in that they responded only transiently to interleukin-7 (IL-7) and then died, unless maintained on a stromal-cell clone. One unusual lymphocyte clone (2E8) was propagated for more than 1 year in IL-7 alone and was selectively responsive to that cytokine. Most of the lymphocyte clones were not tumorigenic in immunodeficient mice. However, one pre-B clone (1A9)’grew autonomously in culture when held at high density, responded to conditioned medium from a number of cell lines, and was tumorigenic. Tumors derived from this clone were infiltrated by stromal cells and lymphocytes taken from the tumors retained characteristics of the original clone. Ly-6 antigens were inducible on 2E8 and 1A9 cells, but the lymphocytes were otherwise arrested in differentiation. The 2E8 cells had rearranged and expressed κ light-chain genes but displayed them on the surface along with surrogate light chains and μ heavy chains. Thus, expression of authentic Tight chain need not coincide with termination of surrogate light-chain utilization in newly formed B cells. Several glycoproteins have recently been demonstrated to be associated with surface immunoglobulin (Ig) on mature B-lineage cells and plasma-cell tumors. We now show that one member of this family (approximately 33 kD) was associated with the μ+surrogate light-chain complex on the 1A9 pre-B-cell clone. When compared to mature B lymphomas, fewer bands coprecipitated with the surface-labeled Ig isolated from pre-B- and early B-cell lines, suggesting that components of the antigen receptor are sequentially acquired during development. The normal replication and differentiation of pre-B cells is probably regulated by complex interactions with multiple cytokines and matrix components of the marrow microenvironment. Cloned lymphocyte lines that are dependent on stromal cells should continue to be important tools for molecular definition of those interactions.

Role of the endothelial adhesion molecule VCAM in murine cardiac allograft rejection.

Murine heterotopic cardiac isografts (C57B1/6----C57B1/6) undergo transient, non-destructive inflammation that is characterized by the acquisition of microvascular endothelial reactivity with the antibody MECA 32. Cardiac allografts (C57B1/6----DBA/2) undergo destructive inflammation that is characterized by the acquisition of reactivity with the antibody M/K-2, in addition to MECA 32. M/K-2 recognizes the murine endothelial adhesion molecule, VCAM-1. Hence, there appear to be antigen-dependent and antigen-independent forms of graft inflammation. Treatment of cardiac allograft recipients with 200 micrograms/day M/K-2 antibody retarded graft loss by only a few days, and did not interfere significantly with leukocytic infiltration, as detected by limiting dilution analysis of graft-reactive CTL, despite the fact that large amounts of M/K-2 could be detected on graft microvascular endothelia and in the peripheral blood as rejection progressed. These data indicate that VCAM is apparently not essential for the leukocytic infiltration and subsequent rejection of cardiac allografts, and is not involved in leukocytic infiltration of murine cardiac isografts.