have identified a new fibronectin receptor that is identical to the integrin receptor a4B1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the tx4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IrlCS for ot4B1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to or4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-glyasp) adhesion sequence, mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, ot5B1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both ot4/~l and ot5/31 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts.In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) ~x5~l is the receptor for the RGD containing cell adhesion domain, and (b) ot4~l is the receptor for a carboxyterminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that o~4~1 is the receptor for this adhesion site.

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors alpha4beta1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesis.

B-cell chronic lymphocytic leukemia (B-CLL IntroductionB-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal, slow-dividing CD5 ϩ B lymphocytes in the peripheral blood. [1][2][3] In most cases, these cells progressively infiltrate the bone marrow and secondary lymphoid tissue, resulting in poor prognosis. 1-3 Extravasation of B-CLL cells and migration through endothelium are mainly directed by 3 chemokines: CCL21, which is expressed in high endothelial venules (HEVs), and CCL19 and CXCL12, which are produced by stromal cells of lymph nodes and bone marrow, respectively. 4,5 The corresponding receptors for these chemokines, CCR7 (for CCL21 and CCL19) and CXCR4 (for CXCL12), are highly expressed in B-CLL with widespread involvement of lymph nodes. 5,6 Other molecules such as vascular endothelial growth factor (VEGF) and ␣L␤2/␣4␤1 integrins were also recently shown to be involved in B-CLL transendothelial migration in response to chemokines. 7 Moreover, high expression of ␣4␤1 (but not ␣L␤2) correlated with the presence of lymphadenopathy, 7 suggesting an important role for this integrin in B-CLL progression.Transendothelial migration and organ invasion of malignant cells also require proteolytic degradation of the vascular basement membrane and the extracellular matrix of lymphoid tissues. This can be accomplished by matrix metalloproteinases (MMPs), [8][9][10] in particular the gelatinases MMP-2 and MMP-9. MMPs also release matrix-bound growth factors that stimulate malignant cell expansion and angiogenesis. 11 Indeed angiogenesis is increased in the bone marrow of B-CLL patients, [12][13][14] and high levels of the angiogenic factors VEGF and basic fibroblast growth factor (bFGF) have been detected in the urine and serum of these patients. 12,15,16 Previous studies have shown that early-stage B-CLL cells produce and secrete MMP-9, which can be detected in the serum of these patients and in B-CLL cell culture supernatants. 17 It was later demonstrated that B-CLL cells constitutively produce MMP-9 in various molecular forms and that elevated levels of intracellular MMP-9 correlate with advanced stage and poor patient survival. 18 Moreover, MMP-9 was highly expressed by B-CLL cells present in the bone marrow (with a diffused pattern) and in lymph nodes, and contributed to B-CLL migration through artificial basement membranes or endothelial cells. 18 The presence of other MMPs in B-CLL cells has not been reported. performed research; R.S. performed the confocal microscopy analyses; M.J.T. contributed with patient samples and data; J.A.G.-M. contributed patient samples and data; and A.G.-P. designed and supervised research and wrote the paper.The online version of this article contains a data supplement.Reprints: Á ngeles García-Pardo, Departamento de Inmunología, Centro de Investigaciones Bioló gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; e-mail: agarciapardo@cib.csic.es.The publication costs of this article were defrayed in part by page charge payment. Therefore, an...

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As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti-MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and ␣4␤1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to ␣4␤1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of ␣4␤1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. ␣4␤1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-␣4, anti-␤1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that ␣4␤1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified ␣4␤1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cellassociated MMP-9 may regulate B-CLL cell migration and arrest. (Blood. 2008; 112:169-178) Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of CD5 ϩ B lymphocytes in the peripheral blood. 1,2 As the disease progresses, these cells infiltrate the bone marrow and secondary lymphoid tissue, resulting in poor prognosis. 1,2 Several molecules that participate in B-CLL cell migration and invasion have been identified. These include the CCL21, CCL19, and CXCL12 chemokines, 3,4 the vascular endothelial growth factor (VEGF), 5 and the ␣L␤2 and ␣4␤1 integrins. 5,6 Other molecules known to play a role in cell migration are matrix metalloproteinases (MMPs). 7-9 B-CLL cells produce and secrete progelatinase-B/proMMP-9 (92 kDa), 10 and elevated intracellular levels of this MMP correlate with advanced stage and poor patient survival. 11 We and others have shown that MMP-9 plays an important role in B-CLL transendothelial migration and invasion through basement membranes. 6,11 We also recently showed that the constitutive levels of proMMP-9 in B-CLL cells are significantly higher than in normal B cells, and that secretion of proMMP-9 by B-CLL cells is up-regulated by ␣4␤1 integrin, CXCR4, or CCR7 (the receptors for CXCL 12 and CCL19/CCL21, respectively) engagement. 6,12 Besides up-regulating proMMP-9 levels, ␣4␤1 integrin ligation also induces the formation of podosomes in B-CLL cells and the localization of proteolytically active MMP-9 to these invasive structures. 6 Although mainly present as a secreted soluble form, proMMP-9 has also been detected at the B-CLL cell surface by flow cytometry and cell biotinylation analyses. 11 Indeed, we recently showed that MMP-9 is present at the B-CLL ce...

SummaryHuman bone marrow (BM) is a relevant site for immunoglobulin (Ig) generation in vivo. The occurrence of BM cells capable of spontaneous and high rate Ig secretion for 14 d in vitro has been described previously. Accordingly, these cells provide a suitable model for studying terminal B cell maturation within the BM. We have reported recently that these BM cells are not totally differentiated when isolated from the body, as they require inductive signals from adherent stromal BM cells to complete their maturation. Interleukin (IL)-6 produced by these adherent BM cells was identified as one such signal. The present work shows that II.-6 was necessary, but not suf~cient, for the induction of BM Ig-secreting cells, since the cytokine was unable to restore missing IgG in nonadherent BM cell cultures. Supernatants (SN) obtained from cultures of stromal adherent BM cells, either freshly isolated or derived from long-term BM culture (LTBMC), restored Ig secretion by nonadherent BM cells, suggesting that additional soluble factors from BM stromal cells were required. Fibronectin (FN) was identified as that factor, as can be deduced from the following findings: (a) stromal, but not nonadherent, BM cells constitutively produced FN; (b) anti-FN antibodies markedly reduced the IgG secretion in cultures of BM mononuclear cells (BMMC), and blocked the inductive effect of stromal cell SN on nonadherent BM cells, and such a blockade could be reversed by exogenous FN; and (c) finally, although neither IL-6 nor FN alone exerted any effect, the combination of both factors induced optimal Ig secretion by nonadherent BM cells. Furthermore, VLA-4 molecules seemed to be the FN receptor that was active in this culture system, as indicated by: (a) BM Ig-secreting cells exhibited the phenotype VLA-4 + VLA-5-; (b) mAbs directed to VLA-4 (anti-CD29 and anti-CD49d), but not those directed to other adhesion molecules, inhibited Ig secretion by BMMC cultures, and this effect was reversed by FN; (c) the inductive role of the entire FN molecule could be replaced by a fragment containing the CS-1 region, but not by a fragment containing the RGDS sequence; and (d) only mAbs anti-CD49d capable of blocking VLA-4-FN interaction inhibited induction by either the FN or the CS-l-containing fragment of FN. These results suggest that, in addition to II~6, the interaction of FN produced by stromal BM cells with VLA-4 molecules present on the surface of BM producers is critical for the latter cells to differentiate into the prolonged and high rate Ig-secreting stage characteristic of these cells. Therefore, cells from the marrow microenvironment might contribute to the terminal maturation of Ig-secreting BM cells in vivo.

B cell chronic lymphocytic leukemia (B-CLL) consists of the accumulation of malignant cells that apparently escape normal apoptotic regulation. We have studied the role of ␣4␤1 integrin/fibronectin interaction in preventing apoptosis of these cells in vitro. B cells from 16 patients showed constant expression of ␣4␤1 and little or no ␣5␤1. B-CLL cells cultured on fibronectin or two previously described fibronectin recombinant fragments (H89 and H0) which contain the ligands for ␣4␤1, consistently showed higher viability than control cells cultured on poly-lysine. The H89 fragment, containing the high affinity ligand CS-1, was the most efficient substrate with mean cell viability values of 72, 60 and 35% at days 2, 5 and 8 of culture, respectively. For control cells these values were 40, 27 and 15%, respectively. Parallel cell cycle analysis confirmed these results. The anti-apoptotic effect required direct contact with immobilized substrata since it was not observed when using B-CLL conditioned media alone or when clustering ␣4␤1 with specific mAbs in suspension. Quantitation of the apoptosis regulatory proteins Bcl-2 and Bax revealed that cells cultured on the H89 fragment showed high/moderate levels of Bcl-2 (with some interpatient variation) and low levels of Bax resulting in an elevated Bcl-2/Bax ratio. These results indicate that adhesion of B-CLL cells to fibronectin upregulate the Bcl-2/Bax ratio and this may contribute to the anti-apoptotic effect induced via ␣4␤1 integrin.

B-cell chronic lymphocytic leukemia (B-CLL