We generated vascular cell adhesion molecule (VCAM)-1 “knock-in” mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1–deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a “TIE2Cre” transgene. Analysis of peripheral blood from conditional vcam-1–deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1–deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.

The IL receptor common gamma (gamma c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how gamma c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human gamma c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-gamma c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the gamma c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on gamma c chain required for its association with multiple ligands.

The anti-common gamma chain (gammac) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Ralpha and gammac receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Ralpha, and to PHA blasts expressing both IL-4Ralpha and gammac, were used to estimate the affinity of the key interaction between gammac and the binary IL-4Ralpha.IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Ralpha.IL-4.gammac]/[IL-4Ralpha.IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR approximately 9, implying that approximately 10% of the limiting IL-4Ralpha chain remains free of gammac even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.

DENDRITIC cells (DC) derived from bone marrow are critical in the function of the immune system, for they are the primary antigen-presenting cells in the activation of T-lymphocyte response. Their differentiation from precursor cells has not been defined at a molecular level, but recent studies have shown an association between expression of the relB subunit of the NF-kappa B complex(1-5) and the presence of DC in specific regions of normal unstimulated lymphoid tissues(4-6). Here we show that relB expression also correlates with differentiation of DC in autoimmune infiltrates in situ, and that a mutation disrupting the relB gene results in mice with impaired antigen-presenting cell function, and a syndrome of excess production of granulocytes and macrophages. Thymic UEA-1(+) medullary epithelial cells from normal mice show striking similarities to DC and, interestingly, these cells are also absent in relB mutant mice. Taken together, these results suggest that relB is critical in the coordinated activation of genes necessary for the differentiation of two unrelated but phenotypically similar cells (DC and thymic UEA-1(+) medullary epithelial cells) and is therefore a candidate for a gene determining lineage commitment in the immune system.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. VCAM1 cDNAs cloned from mouse, rat, rabbit, and human libraries contain six, seven, or eight extracellular Ig-like domains generated by alternate splicing, but to date shorter forms have not been found. We have cloned a novel cDNA encoding only the three N-terminal domains of murine VCAM1 followed by a unique C-terminal tail generated by alternate splicing of a previously undescribed exon. This truncated form of murine VCAM1 (3D-VCAM1) is expressed in COS cells as a functional adhesion molecule which is lost from the cell surface following treatment with phosphatidylinositol-specific phospholipase C. 3D-VCAM1 is found only in endotoxin-treated but not control murine and rat tissues. Thus in rodents alternate splicing of the VCAM1 gene generates a unique truncated inflammation-specific phosphatidylinositol-linked form of VCAM1.