Treatment of marrow stroma with interferon-alpha restores normal beta 1 integrin-dependent adhesion of chronic myelogenous leukemia hematopoietic progenitors. Role of MIP-1 alpha.

The mechanisms by which interferon-alpha (IFN-alpha) restores normal hematopoiesis in chronic myelogenous leukemia (CML) are not well understood. We have recently demonstrated that IFN-alpha acts directly on CML hematopoietic progenitors to restore their adhesion to marrow stroma by modulating beta 1 integrin receptor function. In the present study we examined the effect of IFN-alpha treatment of marrow stroma on subsequent adhesion of CML progenitors. Stromal layers were preincubated with IFN-alpha (10,000 microns/ml) for 48 h. Subsequent coincubation with CML progenitors for 2 h resulted in significantly increased adhesion of CML progenitors. We demonstrated that alpha 4 beta 1 and alpha 5 beta 1 integrin receptors were involved in the enhanced adhesion of CML progenitors, suggesting that IFN-alpha-treated stroma can upregulate CML integrin function. This effect is due, at least in part, to IFN-alpha-induced increased stromal production of the chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha), which upregulates beta 1 integrin-dependent adhesion of CML progenitors to stroma. Thus, IFN-alpha treatment of marrow stroma restores beta 1 integrin-dependent adhesion of CML progenitors, at least in part through induction of MIP-1 alpha production. These observations provide further insights into mechanisms by which IFN-alpha may restore normal hematopoiesis in CML.     

Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. Decreased adhesion to stroma and fibronectin but increased adhesion to the basement membrane components laminin and collagen type IV.

We studied the adhesion of primitive and committed progenitors from chronic myelogenous leukemia (CML) and normal bone marrow to stroma and to several extracellular matrix components. In contrast to benign primitive progenitors from CML or normal bone marrow, Ph1-positive primitive progenitors from CML bone marrow fail to adhere to normal stromal layers and to fibronectin and its proteolytic fragments, but do adhere to collagen type IV, an extracellular matrix component of basement membranes. Similarly, multilineage colony-forming unit (CFU-MIX) progenitors from CML bone marrow do not adhere to fibronectin or its adhesion promoting fragments but adhere to collagen type IV. Unlike committed progenitors from normal bone marrow, CML single-lineage burst-forming units-erythroid and granulocyte/macrophage colony-forming units fail to adhere to fibronectin or its components but do adhere to both collagen type IV and laminin. Evaluation of adhesion receptor expression demonstrates that fibronectin receptors (alpha 4, alpha 5, and beta 1) are equally present on progenitors from normal and CML bone marrow. However, a fraction of CML progenitors express alpha 2 and alpha 6 receptors, associated with laminin and collagens, whereas these receptors are absent from normal progenitors. These observations indicate that the premature release of malignant Ph1-positive progenitors into the circulation may be caused by loss of adhesive interactions with stroma and/or fibronectin and acquisition of adhesive interactions with basement membrane components. Further study of the altered function of cell-surface adhesion receptors characteristic of the malignant clone in CML may lead to a better understanding of the mechanisms underlying both abnormal expansion and abnormal circulation of malignant progenitors in CML.     

Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation.

In long-term bone marrow cultures, stroma-adherent progenitors proliferate significantly less than nonadherent progenitors. Thus, close progenitor-stroma interactions may serve to regulate or restrict rather than promote hematopoietic progenitor proliferation. We hypothesized that signaling through adhesion receptors on hematopoietic cells may contribute to the inhibition of proliferation observed when progenitors are in contact with stroma. We demonstrate that progenitors cultured physically separated from stroma in a transwell proliferate significantly more than progenitors adherent to stroma. Furthermore, proliferation of colony forming cells (CFC) is reduced after specific adhesion to stroma, metabolically inactivated glutaraldehyde-fixed stroma, stromal-extracellular matrix, or the COOH-terminal heparin-binding domain of fibronectin. Nonspecific adhesion to poly-L-lysine fails to inhibit CFC proliferation. That the VLA-4 integrin is one of the receptors that transfers proliferation inhibitory signals was shown using blocking anti-alpha 4 monomeric F(ab) fragments. Furthermore, when synthetic peptides representing specific cell attachment sites within the heparin-binding domain of fibronectin were added to Dexter-type marrow cultures, significantly increased recovery and proliferation of CFC was observed, suggesting that these peptides disrupt adhesion-mediated proliferation inhibitory events. Thus, negative regulation of hematopoiesis may not only depend on the action of growth inhibitory cytokines but also on growth inhibitory signals resulting from direct adhesive interactions between progenitors and marrow stroma.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function.

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine- dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine- stimulated adhesion to Fn. In contrast, genistein inhibited KL- stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.     

CD^＋34干／祖细胞与纤维连接蛋白的粘附在慢性粒细胞白血？…

探讨ＣＤ＾＋３４干／祖细胞与纤维连接蛋白（ＦＮ）的粘附在慢性粒细胞白血病（ＣＭＬ）发病中的作用。方法①采用细胞术双标记法检测初治ＣＭＬ慢性期３０例正常骨髓１０份ＣＤ＾＋３４细胞上整合素β１链（ＣＤ２９）和α４链（ＣＤ４９ｄ）的表达;②结晶紫染色法观察免疫磁珠分选的ＣＭＬ慢性期５例和５名正常人骨髓ＣＤ＾＋３４细胞ＦＮ的粘附功能;③极限稀释液体微培养观察ＦＮ对正常和ＣＭＬ慢性期骨髓粒细胞－巨噬细胞祖细     

Decreased stroma adhesion capacity of CD34+ progenitor cells from mobilized peripheral blood is not lineage- or stage-specific and is associated with low beta 1 and beta 2 integrin expression

Molecular mechanisms leading to mobilization of hematopoietic cells from bone marrow (BM) to peripheral blood (PB) involve modulation of adhesion molecule expression on these cells that probably result in changes in adhesion capacity to the microenvironment. However, it is not clear whether these changes involve different stages or lineages of progenitor cells. In this study, we compared the capacity of mature and immature clonogenic progenitor cells from granulocyte colony-stimulating factor (G-CSF)-mobilized PB and normal BM CD34+ cells to adhere to complete marrow stroma. This functional capacity was assessed concurrently with molecular expression on CD34+ cells of integrins VLA-4 (alpha 4/beta 1), VLA-5 (alpha 5/beta 1), and LFA-1 (alpha L/beta 2) by interindividual (between mobilized PB and normal BM) and intraindividual (between mobilized PB and steady-state BM and PB in the same patient) analysis. The proportion of adherent clonogenic progenitor cells was significantly lower in PB than in BM, not only for total progenitor cells but also for mature and immature progenitor cells, and the difference was found for granulocytic and particularly for erythroid lineages. The lower adhesion capacity of PB CD34+ cells to stroma was associated with decreased expression (signal/noise MFI ratio) of integrin alpha 4, beta 1, alpha L, and beta 2 chains whereas that of alpha 5 chain did not differ from BM cells with the lowest expression level. Similar differences in integrin expression levels were also found between mobilized PB and steady-state BM CD34+ cells in the same patient except for the alpha L chain. Moreover, we demonstrated for the first time a strong positive correlation between mobilizing capacity and expression levels on mobilized CD34+ cells for the LFA-1 alpha L chain but not for VLA-4 or VLA-5. In conclusion, the decreased adhesion capacity of mobilized PB progenitor cells to stroma involves different maturation stages and different lineages. This is associated with down-regulation of integrins VLA-4 and LFA-1, but mobilizing capacity appears positively correlated with LFA-1 levels.     

Interferon-alpha restores the deficient expression of the cytoadhesion molecule lymphocyte function antigen-3 by chronic myelogenous leukemia progenitor cells.

Hematopoietic cells from the malignant clone in chronic myelogenous leukemia (CML) maintain and expand a proliferative advantage over normal hematopoietic cells within the bone marrow. This advantage is often ameliorated or reversed in vivo by IFN alpha. Based upon earlier studies suggesting decreased adhesiveness of CML progenitor cells, we asked whether CML progenitor cells are deficient in their expression of the cytoadhesion molecule lymphocyte function antigen-3 (LFA-3, CD58) which is normally expressed on hematopoietic progenitors. Progenitor cells from untreated CML patients showed greatly reduced or absent LFA-3 expression, whereas progenitors from patients treated with IFN alpha in vivo or in vitro expressed surface LFA-3 at more normal levels. LFA-3-deficient CML progenitor cells were unable to stimulate normal regulatory proliferative responses in autologous T cells. We hypothesize that IFN alpha-sensitive LFA-3 deficiency reflects a cell surface cytoadhesion defect which may help explain adhesive abnormalities of CML progenitor cells in vitro and clonal proliferation in vivo.     

Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment.     

In vivo effects of imatinib mesylate on human haematopoietic progenitor cells

BACKGROUND: Imatinib mesylate has considerable antineoplastic activity in patients with chronic myeloid leukaemia (CML) and some solid tumours. Although originally regarded as nontoxic for normal haematopoiesis, mild to moderate myelosuppression is a commonly observed side-effect of this treatment. Recently, this molecule has been shown to suppress normal haematopoietic progenitor cells in vitro. This is the first study that has investigated the effect of imatinib on haematopoietic progenitor cells in vivo. MATERIALS AND METHODS: We investigated the number of circulating haematopoietic progenitor cells in 79 patients with CML and five patients with solid tumours who were treated with imatinib for at least 3 months. Bone marrow progenitor cells were assessed in a subgroup of 18 patients with CML after 12 months of imatinib treatment. Results were compared with haematopoietic progenitor cell numbers of normal controls. RESULTS: Circulating progenitors of all classes were significantly decreased in CML up to 24 months of imatinib therapy compared with healthy controls (median progenitor cells in CML after 12 months: CFU-GM 62, range 0-2543; BFU-E 216, range 0-3259; CFU-GEMM 0, range 0-139; versus controls: CFU-GM 208, range 50-936; BFU-E 690, range 120-1862; CFU-GEMM 20, range 4-77; P < 0.001). Similar reductions in the number of progenitor cells derived from bone marrow were found in a subgroup of 18 patients with CML. In patients with solid tumours the number of circulating progenitor cells was significantly lower under treatment with imatinib when compared with the controls. Withdrawal of imatinib in a patient with a malignant brain tumour resulted in a prompt normalization of circulating progenitors. CONCLUSIONS: This study suggests that imatinib exerts myelosuppressive effects through inhibition of haematopoietic progenitor cells.     

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.     

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins.Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.     

CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells

Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML. We proposed that tyrosine kinase inhibitors, imatinib or INNO-406, may restore CXCR4 expression and cause the migration of CML cells to bone marrow microenvironment niches, which in turn results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib, INNO-406, or IFN-alpha increased CXCR4 expression and migration. This increase in CXCR4 levels on CML progenitor cells was likewise found in samples from CML patients treated with imatinib or IFN-alpha. Imatinib induced G0-G1 cell cycle block in CML cells, which was further enhanced in a mesenchymal stem cell (MSC) coculture system. MSC coculture protected KBM-5 cells from imatinib-induced cell death. These antiapoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by inhibitor of integrin-linked kinase QLT0267. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to bone marrow stroma, causing the G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells.     

Design and characterization of orally active Arg-Gly-Asp peptidomimetic vitronectin receptor antagonist SB 265123 for prevention of bone loss in osteoporosis.

The Arg-Gly-Asp (RGD)-binding integrin alpha(V)beta(3) is highly expressed on osteoclasts and has been proposed to mediate cell-matrix adhesion required for osteoclast-mediated bone resorption. Antagonism of this receptor should prevent stable osteoclast adhesion and thereby inhibit bone resorption. We have generated an orally bioavailable, nonpeptide RGD mimetic alpha(v)beta(3) antagonist, SB 265123, which prevents bone loss in vivo when dosed by oral administration. SB 265123 binds alpha(v)beta(3) and the closely related integrin alpha(v)beta(5) with high affinity (K(i) = 3.5 and 1.3 nM, respectively), but binds only weakly to the related RGD-binding integrins alpha(IIb)beta(3) (K(i) >1 microM) and alpha(5)beta(1) (K(i) >1 microM). The compound inhibits alpha(v)beta(3)-mediated cell adhesion with an IC(50) = 60 nM and more importantly, inhibits human osteoclast-mediated bone resorption in vitro with an IC(50) = 48 nM. In vivo, SB 265123 completely blocks bone resorption in a thyroparathyroidectomized rat model of acute bone resorption when dosed at 2.5 mg/kg/h by continuous i.v. infusion. When dosed orally with 3 to 30 mg/kg b.i.d. , in the ovariectomy-induced rat model of osteoporosis, SB 265123 prevents bone resorption in a dose-dependent fashion. This is the first report of an orally active alpha(v)beta(3) antagonist that is effective at inhibiting bone resorption when dosed in a pharmaceutically acceptable fashion. Such a molecule may provide a novel therapeutic agent for the treatment of postmenopausal osteoporosis.     

Soluble factor(s) produced by adult bone marrow stroma inhibit in vitro proliferation and differentiation of fetal liver BFU-E by inducing apoptosis.

Hematopoiesis occurs in different organs during fetal development. Several studies suggest that the growth of hematopoietic progenitors at one stage of ontogenic maturation may not be supported by a microenvironment from a different ontogenic stage. To determine if human fetal liver (FL) clonogenic progenitors can develop in an adult bone marrow (ABM) microenvironment, we compared growth of BFU-E and CFU-GM from 7-14-wk-old FL, 11-20-wk-old fetal bone marrow (FBM), umbilical cord blood (UCB), or ABM in clonogenic medium with or without ABM stroma. In contrast to BFU-E from FBM, UCB, or ABM, soluble factor(s) produced by ABM stroma severely suppressed growth of 98% of FL BFU-E by inducing apoptosis of cells beyond early erythroblast stage. The nature of the soluble factor remains unknown, although we have evidence that it is heat labile with molecular mass < 10 kD. Antibody neutralization studies indicate that TGF-beta1, IL-1, TNF-alpha, macrophage inflammatory protein (MIP)-1alpha, or IFN-gamma are not responsible. The observation that FL progenitors may not be capable of differentiating when transferred to an ABM microenvironment may have important implications for FL transplantation into postnatal recipients. Further, this demonstrates that ontogenic stage-specific interactions between hematopoietic progenitors and their microenvironment are important for the normal development of hematopoiesis.     

Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors is accompanied by alterations in their interaction with fibronectin

We have previously demonstrated that primitive progenitors from human bone marrow termed long term bone marrow culture initiating cells (LTBMC-IC) adhere avidly to irradiated bone marrow stroma, while more mature clonogenic progenitors fail to do so. In this study we examine the interaction between these progenitors and components of the bone marrow stroma. (a) We demonstrate that both primitive LTBMC-IC and more mature clonogenic progenitors adhere to intact fibronectin. (b) Primitive LTBMC-IC and multi-lineage CFU-MIX progenitors adhere to the 33/66 kD COOH-terminal heparin-binding cell-adhesion promoting fragment of fibronectin, but adhere significantly less to its 75 kD RGDS-dependent cell-binding fragment. In contrast, more differentiated single-lineage progenitors adhere equally well to the 33/66 kD RGDS independent and the 75 kD RGDS-dependent cell-adhesion fragments of fibronectin. (c) Both primitive LTBMC-IC and clonogenic progenitors adhere to the three known cell-attachment sites in the 33/66 kD cell-adhesion promoting fragment, FN-C/H I, FN-C/H II and CS1. However, LTBMC-IC and CFU-MIX progenitors adhere significantly better to FN-C/H II than to the flanking FN-C/H I and CS1 cell-attachment sites. In contrast, single-lineage progenitors adhere equally well to all three cell attachment sites in the 33/66 kD cell-adhesion promoting fragment. (d) Finally, adhesion of primitive LTBMC-IC to intact irradiated stroma can be inhibited partially by peptide FN-C/H II and almost completely by a combination of FN-C/H II and peptide FN-C/H I and CS1. This study demonstrates that adhesive interactions between primitive hematopoietic progenitors and the extracellular matrix component fibronectin can occur. Specific changes in adhesion to the 33/66 kD cell-adhesion promoting fragment and the 75 kD RGDS-dependent cell-adhesion fragment of fibronectin are associated with differentiation of primitive multi-lineage progenitors into committed single-lineage progenitors. Such differences in adhesive interaction with fibronectin may allow hematopoietic progenitors at various stages of differentiation to interact with specific supportive loci of the bone marrow microenvironment. Finally, the ability to block adhesion of LTBMC-IC to intact irradiated stroma with peptides FN-C/H II, FN-C/H I and CS1 suggests that receptors responsible for this interaction may be important in the homing of primitive progenitors to the bone marrow.     

Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction

The integrin alpha L beta 2 (leukocyte function-associated molecule 1, CD11a/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of alpha L beta 2 have been demonstrated to modulate adhesiveness of alpha L beta 2. Affinity changes of alpha L beta 2 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the alpha L beta 2 cytoplasmic domains in postreceptor events we constructed cDNA encoding chimeric proteins with intracellular alpha L beta 2 domains, which are responsible for alpha L beta 2 specific intracellular interactions, and extracellular alpha IIIb beta 3 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The cDNA was stably transfected in Chinese hamster ovary cells and chimeric heterodimer formation proven by immunoprecipitations and flow cytometry. The chimeric receptors mediate adhesion to immobilized fibrinogen, and this adhesion is increased by phorbol myristate acetate and abolished by cytochalasin D. However, neither treatment affects the affinity state of the chimeric receptor, suggesting involvement of the cytoskeleton in the regulation of alpha L beta 2 mediated cell adhesion. To exclude the possibility of postoccupancy affinity changes of the chimeric receptors, we locked the receptors into a high affinity state by creating a deletion variant. The region deleted (VGFFK) is highly conserved in integrin alpha subunit cytoplasmic domains. Cotransfection of this deletion variant with a beta subunit truncation (beta 3 delta 724) and a triple mutation at 758-760 (TTT to AAA) of beta 2 abolishes adhesion without changing the affinity state. A single mutation (TTT to TAT) reduces adhesion by half without affinity change. Scanning electron microscopy reveals impaired spreading of these truncated/mutated chimeras. Immunofluorescence microscopy demonstrates a correlation between impaired adhesion and a decrease in the ability to form focal adhesions and to organize the cytoskeleton into stress fibers. These results describe the integrin/cytoskeleton interaction, the organization of the cytoskeleton, and cell spreading as postreceptor events modulating alpha L beta 2 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the beta 2 subunit, and within it the TTT region, are required for these postreceptor events. Additionally, we present a new approach, using deletion variants to lock integrins in a high affinity state without interfering with the investigated integrin/cytoskeleton interaction. This approach may be generally useful to investigate the role of postreceptor events in integrin- mediated cell adhesion and migration.     

Molecular basis for leukocyte integrin alpha(E)beta(7) adhesion to epithelial (E)-cadherin

Cadherins are expressed in tissue-restricted patterns and typically mediate homophilic adhesion. Cadherins also mediate lymphocyte adhesion, providing the opportunity for lymphocyte attachment to parenchymal cells. The best characterized example of lymphocyte adhesion to a tissue-specific cell adhesion molecule, as opposed to a vascular endothelial adhesion molecule, is the interaction between integrin alpha(E)beta(7) on intraepithelial lymphocytes and E-cadherin on epithelial cells. However, the molecular basis for an integrin-cadherin interaction is not well defined. Realization that the cadherin domain adopts a topology similar to the immunoglobulin (Ig) fold suggested that integrin recognition of E-cadherin might be similar to recognition of Ig superfamily ligands. Thus, we modeled domain 1 of human E-cadherin and studied the role of solvent-exposed loops that connect Ig-like core-forming beta strands. Mutational analyses localized the integrin alpha(E)beta(7) recognition site to the top of domain 1 at the face formed by the BC and FG loops, a site distinct from the region recognized in intercellular adhesion molecule (ICAM)-1, -2, and -3, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), vascular cell adhesion molecule 1 (VCAM-1), and fibronectin by their integrin ligands. Moreover, the integrin alpha(E)beta(7) binding site is distinct from the homophilic binding site on E-cadherin. These studies provide a conceptual basis for integrin-cadherin binding and extend the model that an Ig-like fold can serve as a scaffold for recognition.     

A homing mechanism for bone marrow-derived progenitor cell recruitment to the neovasculature

CD34+ bone marrow-derived progenitor cells contribute to tissue repair by differentiating into endothelial cells, vascular smooth muscle cells, hematopoietic cells, and possibly other cell types. However, the mechanisms by which circulating progenitor cells home to remodeling tissues remain unclear. Here we show that integrin alpha4beta1 (VLA-4) promotes the homing of circulating progenitor cells to the alpha4beta1 ligands VCAM and cellular fibronectin, which are expressed on actively remodeling neovasculature. Progenitor cells, which express integrin alpha4beta1, homed to sites of active tumor neovascularization but not to normal nonimmune tissues. Antagonists of integrin alpha4beta1, but not other integrins, blocked the adhesion of these cells to endothelia in vitro and in vivo as well as their homing to neovasculature and outgrowth into differentiated cell types. These studies describe an adhesion event that facilitates the homing of progenitor cells to the neovasculature.     

Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell-derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.