The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇+/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on vascular endothelium in a variety of inflammatory conditions and mediates leukocyte recruitment from blood into tissues. In this study we report a novel role for VCAM-1 in the formation of the umbilical cord and placenta during development. The murine VCAM1 gene was disrupted by targeted homologous recombination, and a distinct phenotype was found in VCAM-1-deficient embryos. At 8.5 days of gestation, the allantois failed to fuse to the chorion, resulting in abnormal placental development and embryonic death within 1-3 days. In addition, a role for VCAM-1 in early placental formation after chorioallantoic fusion was observed. In a minority of VCAM-1-deficient embryos, the allantois was able to fuse with the chorion, but the allantoic mesoderm was abnormally distributed over the chorionic surface. A small number of VCAM-1-deficient embryos survived, presumably by circumventing the placentation defects. They became viable and fertile adult mice with lack of VCAM-1 expression, normal organs, and an elevated number of circulating blood mononuclear leukocytes.

Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow mesenchymal stromal cells (BM-MSCs). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSCs revealed that coculture with leukemia cells upregulated the transcription of genes associated with nuclear factor (NF)-κB signaling. Moreover, primary BM-MSCs from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of inhibitor κB-α (IκBα) in BM-MSCs markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemoprotection. Mechanistic in vitro studies revealed that the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSCs and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.

Lymphocyte homing, which contributes to inflammation, has been studied extensively in the small intestine, but there is little known about homing to the large intestine, the site most commonly affected in inflammatory bowel disease. GPR15, an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor, controlled the specific homing of T cells, particularly FOXP3(+) regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15 expression was modulated by gut microbiota and transforming growth factor-β1, but not by retinoic acid. GPR15-deficient mice were prone to develop more severe large intestine inflammation, which was rescued by the transfer of GPR15-sufficient Tregs. Our findings thus describe a T cell-homing receptor for LILP and indicate that GPR15 plays a role in mucosal immune tolerance largely by regulating the influx of Tregs.

Acute graft-versus-host disease (a-GVHD) is initiated primarily by immunologically competent cytotoxic T cells (CTLs) that express anti-host specificities. However, the host lymphoid compartment in which these precursor CTLs are initially stimulated remains unclear. Here we show that gut Peyer's patches (PPs) are required to activate anti-host CTL responses in a well characterized murine acute graft-versus-host reaction (a-GVHR) model, involving transfer of parent lymphocytes into F1 hybrid recipients. The a-GVHR was prevented when recruitment of donor T cells into PP was interrupted either by disrupting the gene encoding chemokine receptor CCR5 or by blocking integrin alpha(4)beta(7)-MAdCAM-1 (mucosal vascular addressin) interactions. Mice deficient for PPs failed to develop a-GVHD in two models of disease induction. Thus, blockade of CTL generation in PPs might offer new strategies for circumventing a-GVHD.

Antigen-dependent T cell activation drives the formation of signaling microclusters containing the adaptor SLP-76. Costimulatory integrins regulate SLP-76 phosphorylation and could influence SLP-76 microclusters in the integrin-rich periphery of the immune synapse. We report that costimulation by the integrin VLA-4 (alpha4beta1) required SLP-76 domains implicated in microcluster assembly. Pro-adhesive ligands enlarged the contact and increased the number of SLP-76 microclusters regardless of their costimulatory potential. Costimulatory VLA-4 ligands also prevented the centralization of SLP-76, promoted microcluster persistence, prolonged lateral interactions between SLP-76 and its upstream kinase, ZAP-70, and retained SLP-76 in tyrosine-phosphorylated peripheral structures. SLP-76 centralization was driven by dynamic actin polymerization and was correlated with inward actin flows. VLA-4 ligation retarded these flows, even in the absence of SLP-76. These data suggest a widely applicable model of costimulation, in which integrins promote sustained signaling by attenuating cytoskeletal movements that drive the centralization and inactivation of SLP-76 microclusters.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

The establishment of memory requires coordinated signaling between presynaptic and postsynaptic terminals in the CNS. The integrins make up a large family of cell adhesion receptors that are known to mediate bidirectional signaling between cells or between cells and their external environment. We show here that many different integrins, including alpha3 and alpha5, are expressed broadly in the adult mouse brain and are associated with synapses. Mice with genetically reduced expression of alpha3 integrin fail to maintain long-term potentiation (LTP) generated in hippocampal CA1 neurons. Mice with reduced expression of the alpha3 and alpha5 integrins exhibit a defect in paired-pulse facilitation. Mice with reduced expression of alpha3, alpha5, and alpha8 are defective in hippocampal LTP and spatial memory in the water maze but have normal fear conditioning. These results demonstrate that several different integrins are involved in physiological plasticity and provide the first evidence of their requirement for behavioral plasticity in vertebrates.

Mice chimeric for the expression of alpha4 integrins were used to dissect the roles of these receptors in development and traffic of lymphoid and myeloid cells. During fetal life, T cell development is alpha4 independent, but after birth further production of T cells becomes alpha4 dependent. Precursors for both T and B cells require alpha4 integrins for normal development within the bone marrow. In contrast, monocytes and natural killer cells can develop normally without alpha4 integrins. Thus, there are lymphocyte-specific, developmentally regulated requirements for alpha4 integrins in hematopoiesis in the bone marrow. We also show that alpha4 integrins are essential for T cell homing to Peyer's patches, but not to other secondary lymphoid organs, including spleen, lymph nodes, and intestinal epithelium.

The integrin α4β1 (VLA-4) is used by encephalitogenic T cells to enter the central nervous system (CNS). However, both Th1 and Th17 cells are capable of inducing experimental autoimmune encephalomyelitis (EAE), and the molecular cues mediating the infiltration of Th1 versus Th17 cells into the CNS have not yet been defined. We investigated how blocking of α4 integrins affected trafficking of Th1 and Th17 cells into the CNS during EAE. Although antibody-mediated inhibition of α4 integrins prevented EAE when MOG(35-55)-specific Th1 cells were adoptively transferred, Th17 cells entered the brain, but not the spinal cord parenchyma, irrespective of α4 blockade. Accordingly, T cell-conditional α4-deficient mice were not resistant to actively induced EAE but showed an ataxic syndrome with predominantly supraspinal infiltrates of IL-23R(+)CCR6(+)CD4(+) T cells. The entry of α4-deficient Th17 cells into the CNS was abolished by blockade of LFA-1 (αLβ2 integrin). Thus, Th1 cells preferentially infiltrate the spinal cord via an α4 integrin-mediated mechanism, whereas the entry of Th17 cells into the brain parenchyma occurs in the absence of α4 integrins but is dependent on the expression of αLβ2. These observations have implications for the understanding of lesion localization, immunosurveillance, and drug design in multiple sclerosis.

We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind directly to the chemokines that bind versican. Thus, versican appears to interact with chemokines via its GAGs. We next examined if versican or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced integrin activation and Ca(2+) mobilization in lymphoid cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both alpha(4)beta(7) integrin-dependent binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and Ca(2+) mobilization induced by SLC, versican or CS B inhibited these cellular responses, and the extent of inhibition was dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the function of SLC via its GAG chains.

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

Most mononuclear leukocytes and cell lines express the integrin alpha 4 beta 1 (VLA-4) heterodimer. In this study we have used Northern blotting and immunoprecipitation experiments to demonstrate that a B lymphoblastoid cell line (JY) expressed the integrin beta 7 subunit in association with alpha 4. These alpha 4 beta 7-positive JY cells bound poorly or not at all to VLA-4 ligands (soluble form of vascular cell adhesion molecule 1 (sVCAM-1) and the CS1 region of fibronectin). In contrast, a beta 1-positive variant of JY cells (selected to express a mixture of alpha 4 beta 1 and alpha 4 beta 7) bound avidly to VLA-4 ligands, and this binding was completely inhibitable by anti-alpha 4 and anti-beta 1 monoclonal antibodies. Thus, beta 1 expression appears to be a critically important component of VLA-4-mediated binding to its ligands. After either JY or JY-beta 1 cells were stimulated for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, the majority of adhesion to VCAM or fibronectin remained alpha 4- and beta 1-dependent, but a low amount of adhesion to sVCAM-1 or fibronectin became alpha 4-dependent, beta 1-independent, thus suggesting a role for alpha 4 beta 7. In summary, we have found (i) that alpha 4 beta 7 makes little or no contribution to fibronectin or VCAM-1 binding on unstimulated JY cells, (ii) that alpha 4 beta 7 perhaps makes a minor contribution to ligand binding on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated cells, and (iii) that alpha 4 beta 1 is the functionally dominant VCAM-1 and fibronectin receptor even when expressed in relatively low amounts compared to alpha 4 beta 7.

The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151.

Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observed when plerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSF-induced mobilization while decreasing both plerixafor- and BIO5192-induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells.

VLA-4 is a cell surface heterodimer in the integrin superfamily of adhesion receptors. Anti-VLA-4 antibodies inhibited cytolytic T cell activity, with inhibitory activity directed against the effector T cells rather than their targets. Thus, whereas other VLA receptors appear to mediate cell--matrix interactions, VLA-4 may have a cell--cell adhesion function. To facilitate comparative studies of VLA-4 and other integrins, cDNA clones for the human alpha 4 subunit of VLA-4 were selected and then sequenced. The 3805 bp sequence encoded for 999 amino acids, with an N-terminus identical to that previously obtained from direct sequencing of purified alpha 4 protein. The alpha 4 amino acid sequence was 17-24% similar to other integrin alpha chains with known sequences. Parts of the alpha 4 sequence most conserved in other alpha chains include (i) the positions of 19/24 cysteine residues, (ii) three potential divalent cation binding sites of the general structure DXDXDGXXD and (iii) the transmembrane region. However, alpha 4 stands apart from all other known integrin alpha subunit sequences because (i) alpha 4 has neither an inserted I-domain, nor a disulfide-linked C-terminal fragment, (ii) its sequence is the most unique and (iii) only alpha 4 has a potential protease cleavage site, near the middle of the coding region, which appears responsible for the characteristic 80,000 and 70,000 Mr fragments of alpha 4.

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.

Lymphocytes home to various lymphoid organs by adhering to and migrating through specialized high endothelial venules (HEV). The murine cell surface heterodimer LPAM-1 is involved in the homing of lymphocytes to mucosal sites (Peyer's patches). LPAM-1 has an alpha subunit (alpha 4m) analogous to the alpha chain of the human integrin molecule VLA-4. Here we show that the LPAM-1 beta subunit (beta p) is immunochemically and biochemically distinct from previously defined integrin beta subunits, suggesting that beta p represents a novel integrin beta subunit. Depending on the cellular source two alternative beta subunits, beta p and integrin beta 1, can be isolated in association with alpha 4m. Therefore, alpha 4m is the common subunit of the unique integrin LPAM-1 (alpha 4m beta p) and of the heterodimer LPAM-2 (alpha 4m beta 1), which is analogous to VLA-4. Antibody-blocking experiments suggest that, in addition to LPAM-1, LPAM-2 is also involved in the organ-specific adhesion of lymphocytes to Peyer's patch HEV.

The heterodimeric protein complex recognized by the human mucosal lymphocyte 1 (HML-1) monoclonal antibody is expressed on 95% of intraepithelial lymphocytes but on only 1-2% of peripheral blood lymphocytes [Cerf-Bensusson, N., Jarry, A., Brousse, N., Lisowska-Grospierre, B., Guy-Grand, D. & Griscelli, C. (1987) Eur. J. Immunol. 17, 1279-1285]. We purified the smaller HML-1 subunit (105 kDa under nonreducing conditions) from hairy-cell leukemia cells and determined the N-terminal amino acid sequence of this chain. The 17 residues determined were identical to the deduced amino acid sequence encoded by an integrin beta 7 cDNA clone [Yuan, Q., Jiang, W.-M., Krissansen, G.W. & Watson, J.D. (1990) Int. Immunol. 2, 1097-1108]. Biochemical analysis of the larger HML-1 subunit (175 kDa under nonreducing conditions) suggested that it was a distinct member of the cleaved group of integrin alpha chains, which we designated alpha E. The beta 7 chain also was associated with the integrin alpha 4 subunit, suggesting that the HML-1 antigen (alpha E beta 7) and alpha 4 beta 7 constitute a beta 7 integrin family on mucosal lymphocytes. Interestingly, regulation of the expression of the HML-1 antigen was reciprocal to that of lymphocyte function-associated molecule 1 in the presence of transforming growth factor beta 1. We suggest that these beta 7 integrins may play a specific role in mucosal localization or adhesion and that the expression of the HML-1 antigen might be regulated by transforming growth factor beta 1 produced at or near epithelial tissues.

The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrin-mediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian (beta)-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn(2+), which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its anti-adhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugar-dependent manner. Microsequencing and western immunoblotting revealed that (alpha)(3)(beta)(1 )integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 binding-protein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with (alpha)(3)(beta)(1) integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like (alpha)(6)(beta)(1 )integrins. In contrast, galectin-8 only minimally interacts with (alpha)(4 )or (beta)(3 )integrins. We propose that galectin-8 is an integrin binding-protein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.

CD49d/alpha4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, beta2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P<.001) and ZAP-70 (P<.001), or with IGHV mutations (P<.001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d=3.52, P=.02; HRIGHV=6.53, P<.001) or, if this parameter was omitted, with ZAP-70 (HRCD49d=3.72, P=.002; HRZAP-70=3.32, P=.009). CD49d was also a prognosticator for TTT (HR=1.74, P=.007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR=3.12; P=.023) or unmutated IGHV (HR=2.95; P=.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL.

Supporting roles of stromal cells in preferential colonization of myeloma cells in bone marrow and development of associated osteoclastic osteolysis through cell-cell interactions have been indicated. Here we examined the effects of a monoclonal antibody to alpha4 integrin (anti-alpha4 Ab) that disrupts myeloma cell-stromal cell interactions mediated via alpha4beta1 integrin and vascular cell adhesion molecule-1 (VCAM-1) on myeloma cell growth in bone marrow and accompanying osteolysis. The anti-alpha4 Ab decreased VCAM-1-stimulated 5TGM1/luc cell growth in culture. The 5TGM1 murine myeloma cells stably transfected with the firefly luciferase (5TGM1/luc) were inoculated from tail vein in bg/xid/nd mice. Preventative administration of the anti-alpha4 Ab suppressed the elevation of serum IgG2b levels, decreased 5TGM1/luc tumor burden with increased apoptosis in bone and spleen, reduced bone destruction with diminished number of osteoclasts, and prolonged survival of 5TGM1/luc-bearing mice. In contrast, therapeutic administration of the antibody failed to show these effects. However, therapeutic administration of the antibody combined with melphalan significantly suppressed serum IgG2b levels and tumor burden in bone. Our results suggest that the interactions with stromal cells via alpha4beta1/VCAM-1 are critical to the development of myeloma and associated osteolysis and that disruption of these interactions using anti-alpha4 Ab is a potential therapeutic approach for myeloma.