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PubMed1996

Interaction of monocytoid cells with the mucosal addressin MAdCAM-1 via the integrins VLA-4 and LPAM-1.

The differentiation of myeloid cells into macrophages and granulocytes is accompanied by marked changes in adhesive phenotype. Here we seek to understand the regulation of expression and functionality of the VLA-4 (alpha 4 beta 1), LPAM-1 (alpha 4 beta 7) and HML-1 (alpha E beta 7) integrins on monocytes/macrophages and granulocytes, given that these integrins including LFA-1 (alpha L beta 2) mediate the entry, retention and signalling events of pathogenic leucocytes within chronically inflamed tissues. Phorbol ester-induced monocytic differentiation of the promyelocyte cell line HL60 led to increases in the steady-state levels of beta 2 and beta 7 mRNA transcripts, requiring a period of 10 and 24 h, respectively, of de novo protein synthesis. There was a parallel de novo expression of LPAM-1 on the cell surface, despite the fact that alpha 4 mRNA transcripts were rapidly down-regulated. At 72 h, HML-1 was not coexpressed with LPAM-1 on HL60 cells, although it was weakly expressed on peripheral blood monocytes/macrophages after a prolonged period of in vitro culture. Retinoic acid-induced granulocytic differentiation of HL60 cells led to the appearance of low levels of LPAM-1 at the cell surface. LPAM-1 was not found expressed on peripheral blood neutrophils, raising the possibility that it is transiently expressed during granulocyte differentiation. In accord with the above findings, differentiated monocytes and HL60 cells bound to recombinant MAdCAM-1 in an alpha 4- and beta 7-integrin-dependent fashion, whereas a population of undifferentiated HL60 cells and Mn(+2)-activated monocytes bound in an alpha 4-integrin-dependent beta 7-integrin-independent manner via VLA-4 expressed abundantly at all stages of differentiation. Four h after attachment, some of these VLA-4+ LPAM-1- HL60 cells could be seen to start spreading. These finding suggest that MAdCAM-1 can bind to VLA-4 when LPAM-1 is absent, and thus has the potential to recruit both VLA-4-bearing monocytes and VLA-4+ LPAM-1+ macrophages into chronically inflamed tissues.

Antigens, CD↗
PubMed1996

Differing roles of CD18 and VLA-4 in leukocyte migration/activation during anti-GBM nephritis.

The mechanisms underlying leukocyte migration into inflamed glomeruli and their in situ activation are incompletely understood. We addressed this issue by investigating the effects of monoclonal antibodies (mAbs) to CD18 and VLA-4 on these process in the heterologous phase of anti-glomerular basement membrane (GBM) nephritis in rat. Anti-CD18 mAb substantially attenuated neutrophil (PMN) migration into glomeruli and the accompanying proteinuria which is a function of in situ leukocyte activation (ca. 60%). Anti-VLA-4 mAb modestly inhibited PMN migration (ca. 20%) and had no significant effect on proteinuria. Combination of both mAbs was no more effective than anti-CD18 mAb alone. Despite continued mAb blockade of CD18 or VLA-4 (or both), macrophage (M phi) migration following PMN influx was unaltered. However, combined CD18/VLA-4 mAbs diminished the proteinuria associated with M phi influx (ca. 50%). Abrogation of the acute influx of PMNs in this model (via complement depletion or anti-PMN antibody) did not diminish the following influx of M phi s, although the associated proteinuria was abolished. In this context, M phi migration was substantially decreased by anti-VLA-4 mAb (ca. 50%), but not anti-CD18 mAb (either alone or with anti-VLA-4 mAb). In sum, leukocyte migration and activation in the heterologous phase of anti-GBM nephritis are dependent on CD18 and VLA-4, although to varying degrees depending on the leukocyte subtype and the presence of prior inflammation. Nonetheless, a significant component of both PMN and M phi migration/activation is CD18/VLA-4 independent.

Animals↗
PubMed2001

Potential role of the chemokine receptors CXCR3, CCR4, and the integrin alphaEbeta7 in the pathogenesis of psoriasis vulgaris.

Various adhesion molecules have been implicated in T lymphocyte binding to dermal vascular endothelium in psoriasis vulgaris, but the chemotactic signals that promote subsequent homing into the adjacent dermis and overlying epidermis are poorly defined. We studied chemokine receptor (CCR1-CCR5, CXCR1-CXCR3), chemokine (interferon-gamma inducible protein 10 [IP-10]), monokine induced by interferon-gamma (MIG), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and adhesion molecule (cutaneous lymphocyte antigen [CLA], E-selectin, lymphocyte function-associated antigen-1 [LFA-1], intercellular adhesion molecule-1 [ICAM-1], very late antigen 4 [VLA-4], vascular cell adhesion molecule-1 [VCAM-1], alphaEbeta7, and E-cadherin) expression in psoriasis by immunohistology, flow cytometry, and molecular techniques. CXCR3 and CCR4 were expressed by dermal CD3+ lymphocytes, and their chemokine ligands, IP-10, MIG, TARC, and MDC, were up-regulated in psoriatic lesions. Keratinocytes stimulated with tumor necrosis factor-alpha and interferon-gamma up-regulated expression of IP-10, MIG, and MDC mRNA, whereas dermal endothelial cells, similarly stimulated, up-regulated expression of IP-10, MDC, and TARC mRNA, suggesting that these cell types were sources of the chemokines detected in biopsies. There was enhanced expression of E-selectin, CLA, LFA-1, ICAM-1, VLA-4, VCAM-1, and alphaEbeta7 in psoriatic lesions versus nonlesional skin. Finally, intra-epidermal CLA+ and alphaEbeta7+ T lymphocytes selectively expressed the chemokine receptor CXCR3. Collectively, these data suggest that CXCR3 and CCR4 may be involved in T lymphocyte trafficking to the psoriatic dermis and that CXCR3 is selectively involved in subsequent T cell homing to the overlying epidermis.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2001

Loss of expression of alpha4beta7 integrin and L-selectin is associated with high-grade progression of low-grade MALT lymphoma.

Expression of adhesion molecule in low-grade B-cell mucosa-associated lymphoid tissue (MALT) lymphoma of the gastrointestinal tract has been reported in recent years, but these reports have primarily focused on low-grade gastrointestinal MALT lymphoma. In this study, we examined the lymphocytic homing receptor alpha4beta7 integrin, L-selectin, and VLA-4 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in low-grade lymphoma of the gastrointestinal tract and other organs such as the ocular adnexa and thyroid. We also observed changes in the expression pattern associated with high-grade transformation. Neoplastic cells in the gastrointestinal low-grade lymphoma and the low-grade component of high-grade MALT lymphoma were found to be alpha4beta7 integrin(+), L-selectin(+), whereas the gastrointestinal high-grade component and diffuse large B-cell lymphoma were found to be alpha4beta7 integrin(-), L-selectin(-). High endothelial venules in the gastric MALT lymphomas expressed MAdCAM-1. In the ocular adnexa low-grade MALT lymphoma, most cases were alpha4beta7 integrin(-), L-selectin(+); and in the thyroid, most cases of both low- and high-grade MALT lymphoma were alpha4beta7 integrin(-), L-selectin(-). These findings show that alpha4beta7 integrin and L-selectin may play an important role in the lymphocyte homing of gastrointestinal low-grade MALT lymphoma and in the loss of alpha4beta7 integrin expression throughout the course of high-grade progression.

Cell Adhesion Molecules↗
PubMed2003

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia.

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Animals↗
PubMed1997

The effect of the selectin binding polysaccharide fucoidin on eosinophil recruitment in vivo.

1. In order to accumulate at sites of inflammation, leukocytes initially roll on endothelial cells of postcapillary venules before becoming firmly attached. This process of rolling is mediated by selectins which bind to carbohydrate counter-ligands present on the surface of both leukocytes and endothelial cells. The polysaccharide fucoidin has been previously shown to inhibit leukocyte rolling in the mesenteric circulation and to reduce neutrophil accumulation in the skin and meninges in experimental inflammation. 2. In the present study we have assessed the effects of fucoidin on eosinophil function in vitro and eosinophil accumulation at sites of inflammation in guinea-pig skin. 3. At concentrations of up to 1200 micrograms ml-1, fucoidin inhibited phorbol myristate acetate (PMA)-induced eosinophil homotypic aggregation by up to 60% but had no inhibitory effect on PMA-induced eosinophil adhesion to serum-coated plates. 4. Fucoidin effectively reduced the binding of the anti-L-selectin mAb MEL-14 to guinea-pig eosinophils. Binding of a P-selectin-IgG chimera to eosinophils was also partially inhibited by fucoidin, but binding of an anti-CD18 or an anti-VLA-4 mAb were unaffected. 5. When given systemically to guinea-pigs, fucoidin suppressed 111In-labelled eosinophil recruitment to sites of allergic inflammation. 111In-labelled eosinophil accumulation induced by platelet-activating factor (PAF) and zymosan-activated plasma (as a source of C5a des Arg) was also inhibited. 6. These results demonstrate a role for fucoidin-sensitive selectins in mediating eosinophil recruitment in vivo.

Animals↗
PubMed1997

A novel murine model of allergic inflammation to study the effect of dexamethasone on eosinophil recruitment.

1. We have developed a novel model of allergen-induced eosinophil into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex). 2. Injection of 10 micrograms Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08 +/- 0.03 and 4.0 +/- 1.0 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02 +/- 0.01 and 6.0 +/- 0.8 x 10(5) leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression. 3. Subcutaneous (s.c.) administration of Dex (50 or 100 micrograms per mouse, -1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P < 0.05) and 75% (P < 0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (-75%) by Dex administration at the 24 h time-point (n = 12, P < 0.05). 4. Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7 +/- 0.3 x 10(5); sensitized mice: 8.7 +/- 1.7 x 10(5), P < 0.05) and blood (naive mice: 0.5 +/- 0.2 x 10(5); sensitized mice: 1.5 +/- 0.3 x 10(5), P < 0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7 +/- 0.8 x 10(5) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached. 5. Dex administration provoked a profound eosinopaenia in the blood of naive (5.2 +/- 1.5 to 0.9 +/- 0.6 x 10(4)) and sensitized mice (1.5 +/- 0.3 to 0.08 +/- 0.02 x 10(5)) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge. 6. We show for the first time that express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373 x 10(3)). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 micrograms) did not alter LC1 levels in blood eosinophils, such that 235 +/- 21 x 10(3) LC1-like molecules per cell were measured after vehicle treatment (n = 5), and 224 +/- 8 x 10(3) LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n = 5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P < 0.05). 7. Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 micrograms s.c., -1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) the LCS3-treated mice (-56% and 59%, respectively, n = 15-21, not significant). 8. In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and

Animals↗
PubMed1998

Priming and induction of eosinophil trafficking in guinea-pig cutaneous inflammation by tumour necrosis factor alpha.

Tissue eosinophilia is a hallmark of allergic and parasitic diseases. Priming mechanisms may play an important role in mediating the process of eosinophil accumulation in these conditions. We have previously shown that blockade of tumour necrosis factor alpha (TNFalpha) inhibited the capacity of lipopolysaccharide to prime skin sites for chemoattractant-induced eosinophil recruitment. The present study was carried out to investigate the capacity of TNFalpha to prime an inflammatory site for enhanced eosinophil accumulation. Initial experiments investigated the capacity of TNFalpha itself to induce eosinophil accumulation. Intradermal injection of murine TNFalpha (10-300 ng per site) in the guinea-pig induced significant accumulation of 111In-eosinophils. Kinetic studies showed the response to be delayed in onset and inhibited by cycloheximide, consistent with a dependency on protein synthesis. Trafficking of 111In-eosinophils to sites treated for 2 h with TNFalpha (10-100 ng per site) was inhibited by monoclonal antibodies (mAbs) against beta2 or alpha4 integrins. Intradermal injection of a low dose (3 ng) of TNFalpha (which by itself had no significant effect on eosinophil trafficking) prior to chemoattractants or antigen in sensitized skin sites, induced significant priming of eosinophil accumulation. Recruitment of both 111In-eosinophils and endogenous eosinophils was enhanced. Trafficking to TNFalpha-primed responses was dependent on protein synthesis and beta2 integrins. In contrast, the alpha4 integrin mAb failed to inhibit the TNFalpha primed response. Thus, TNFalpha can induce and also prime eosinophil recruitment in guinea-pig skin. Our results provide further evidence that this cytokine may be an important mediator of allergic- or parasite-induced eosinophilic inflammation.

Animals↗
PubMed2006

A small molecule, orally active, alpha4beta1/alpha4beta7 dual antagonist reduces leukocyte infiltration and airway hyper-responsiveness in an experimental model of allergic asthma in Brown Norway rats.

alpha(4)beta(1) and alpha(4)beta(7) integrins are preferentially expressed on eosinophils and mononuclear leukocytes and play critical roles in their recruitment to inflammatory sites. We investigated the effects of TR14035, a small molecule, alpha(4)beta(1)/alpha(4)beta(7) dual antagonist, in a rat model of allergic asthma. Actively sensitized rats were challenged with aerosol antigen or saline on day 21, and the responses evaluated 24 and 48-h later. TR14035 (3 mg kg(-1), p.o.) was given 1-h before and 4-h after antigen or saline challenge. Airway hyper-responsiveness to intravenous 5-hydroxytryptamine was suppressed in TR14035-treated rats. Eosinophil, mononuclear cell and neutrophil counts, and eosinophil peroxidase and protein content in the bronchoalveolar lavage fluid (BALF) were decreased in TR14035-treated rats. Histological study showed a marked reduction of lung inflammatory lesions by TR14035. At 24-h postchallenge, antigen-induced lung interleukin (IL)-5 mRNA upregulation was suppressed in TR14035-treated rats. By contrast, IL-4 levels in BALF were not significantly affected by TR14035 treatment. IL-4 selectively upregulates vascular cell adhesion molecule-1 (VCAM-1), which is the main endothelial ligand of alpha(4) integrins. Intravital microscopy within the rat mesenteric microcirculation showed that 24-h exposure to 1 microg per rat of IL-4 induced a significant increase in leukocyte rolling flux, adhesion and emigration. These responses were decreased by 48, 100 and 99%, respectively in animals treated with TR14035. In conclusion, TR14035, by acting on alpha(4)beta(1) and alpha(4)beta(7) integrins, is an orally active inhibitor of airway leukocyte recruitment and hyper-responsiveness in animal models with potential interest for the treatment of asthma.

Allergens↗
PubMed1997

GM-CSF-mobilized peripheral blood CD34+ cells differ from steady-state bone marrow CD34+ cells in adhesion molecule expression.

To determine the effect of growth factor mobilization on the expression of adhesion molecules, we compared CD34+ progenitor cell (PC) populations from steady-state bone marrow (BM) with granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized apheresis products (peripheral blood stem cell (PSC)) using flow cytometry. To increase the accuracy of this analysis, CD34+ cells were enriched (MiniMACS) before cytometric analysis. A significantly lower expression of very late antigen-4 (VLA-4), leukocyte function antigen-1 (LFA-1) and LFA-3 were observed on PSC compared to BM CD34+ cells. In addition, significantly lower mean fluorescence intensity (MFI) of VLA-4, VLA-5, intercellular adhesion molecule-1 (ICAM-1), and sialyl Lewisx were observed on PSC as compared to BM CD34+ cells. Significantly higher levels of L-selectin and CD44 expression were observed on PSC as compared to BM CD34+ cells based on frequency and MFI (P < or = 0.05). In addition, the duration of GM-CSF administration or number of prior aphereses had no effect on adhesion molecule expression. These data suggest that decreased expression of adhesion molecules including VLA-4, LFA-1, ICAM-1 and LFA-3 play a role in PC mobilization. Based on these studies, we suggest that PC mobilization occurs as a stochastic process and is associated with the selection of CD34+ cells with low adhesion molecule expression.

Adult↗
PubMed1999

Modulation of VLA-4 and L-selectin expression on normal CD34+ cells during mobilization with G-CSF.

We have evaluated the immunophenotype, functional activity and clonogenic potential of CD34+ cells from peripheral blood (PB) of normal donors before and after 4 and 6 days of G-CSF administration. The percentage and absolute number of CD34+ cells significantly increased at days 4 and 6 of G-CSF administration, compared to the steady-state level (P < 0.0001). Two-colour fluorescence analysis showed, at days 4 and 6, a lower proportion of CD34+/c-kit+ compared to the steady-state level (P < 0.0001), but a similar expression of CD13, CD33, CD38, HLA-DR and Thy-1 antigens on CD34+ cells. The expression of adhesion molecules on CD34+ cells revealed a significant reduction of CD11a (P = 0.009), CD18, CD49d and CD62L (P < 0.0001) at days 4 and 6, compared to the baseline level. Three-colour staining showed a reduction of the more immature compartment (34+/DR-/13-) and an increase of the more differentiated compartment (34+/DR+/13+). Downregulation of VLA-4 during mobilisation was seen almost exclusively on more committed cells (34+/13+); downregulation of CD62L, on the contrary, was observed on both early progenitors (34+/13-) and more committed cells (34+/13+). The expression of 34+/VLA-4+ decreased on both c-kit+ and c-kit- cells, while the expression of 34+/62L+ decreased on the c-kit+ cells only. In vivo administration of G-CSF reduced the adherence capacity of CD34+ cells to normal BM stroma; in vitro incubation with SCF or IL-3 enhanced the expression of CD49d on CD34+ cells, while GM-CSF reduced the expression of CD62L. SCF was the only cytokine able to induce a significant increase of CD34+ cell adherence to preformed stroma. Pre-incubation with the blocking beta2 integrin monoclonal antibody caused a reduction of CD34+ cell adherence. In conclusion, the decrease of CD49d expression on mobilized CD34+ cells correlates with a poor adhesion to BM stroma; CD34+ cells incubated in vitro with SCF showed, conversely, a higher expression of CD49d and a greater adherence capacity on normal preformed stroma.

Antigens, CD↗
PubMed1998

Stem cell factor enhances the adhesion of AML cells to fibronectin and augments fibronectin-mediated anti-apoptotic and proliferative signals.

Acute myeloid leukaemia (AML) cells express the SCF receptor c-kit (CD117) on their cell surface and demonstrate enhanced adhesion to fibronectin (FN) following exposure to stem cell factor (SCF). Increased adhesion occurs within 5 min, is dose dependent, and persists beyond 2 h. Baseline and enhanced adhesion occur through the surface FN receptor very late antigen-5 (VLA-5, CD49e/CD29) which is expressed by AML cells. Unstimulated AML cells exposed to FN undergo less apoptosis than controls (inhibition 22.5 +/- 7.0%, P = 0.02, n = 8). Exposure to SCF alone without FN also inhibits AML cell apoptosis (by 19.0 +/- 7.7% compared to controls, P = 0.06, n = 8). Simultaneous exposure to SCF and FN increases the inhibition of AML cell apoptosis to 37.8 +/- 7.9% (P = 0.005 compared to control, P = 0.04 compared to FN alone, P = 0.06 compared to SCF alone) demonstrating that SCF not only enhances the propensity of AML cells to adhere to FN, but also results in an additive survival benefit following FN contact. Some but not all the reduction in apoptosis is mediated through VLA-5. The combination of SCF and FN also affects proliferation, resulting in a synergistic enhancement of AML cell proliferation in half the cases studied. When normal CD34+ human haemopoietic progenitors were studied, FN had little effect on their apoptosis and failed to enhance the anti-apoptotic effect of SCF. It did, however, synergise with SCF in promoting CD34+ cell proliferation. Exposure of AML cells to SCF and FN, both of which can be found in high concentration in the bone marrow stroma, inhibits apoptosis. Cytokines and extracellular matrix proteins augment each others' effects since SCF enhances adhesion to fibronectin, which in turn augments the survival signal delivered by the cytokine alone. Cytokine and adhesion receptors can combine to affect cell characteristics including proliferation and survival.

Acute Disease↗
PubMed2000

Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers.

Acute lymphoblastic leukemia (ALL) blasts undergo migration into layers of bone marrow fibroblasts (BMF) in vitro, utilizing the beta1 integrins VLA-4 and VL-5 as adhesion molecules. However, it has been unclear as to whether this is a selective process mediated by specific chemoattractant molecules, or simply a reflection of the highly motile nature of early B cell precursors. We further characterized this process using a transwell culture system, in which the two chambers were separated by an 8 microm diameter microporous membrane, through which leukemic cells could move. When a BMF layer was grown on the upper surface of the membrane there was an 84.1% reduction in transmigration of the human pre-B ALL cell line NALM-6 into the lower chamber, compared to control membrane with no BMF layer. Localization of leukemic cells under the BMF layer was confirmed ultrastructurally, suggesting the possibility that the migration of leukemic cells was directed by a chemotactic agent secreted by BMF. The involvement of the chemokine stromal cell-derived factor-1 (SDF-1) in this process was next investigated. BMF were shown to express m-RNA for SDF-1. Addition of SDF-1 at 100 ng/ml into the lower chamber increased transmigration of NALM-6 across the membrane by 2.2-fold, and also induced a 1.4- to 6.1-fold increase in movement of NALM-6 through a BMF layer into the lower chamber. The receptor for SDF-1, CXCR4, was demonstrated by flow cytometry on all 10 cases of precursor-B ALL analyzed, as well as on NALM-6, KM-3 and REH lines. An inhibitory antibody to CXCR4 was able to block the migration of NALM-6 cells into BMF monolayers grown on plastic by 51%, and in nine cases of ALL by 8-40%, as well as partially inhibit transmigration of leukemic cells through BMF layers along an SDF-1 concentration gradient. These results confirm that precursor-B ALL cells selectively localize within bone marrow stroma in vitro, and that this process is partially due to the stromal chemokine SDF-1 binding to its receptor CXCR4 on leukemic cells. SDF-1 may be important in influencing the localization of precursor-B ALL cells in marrow microenvironmental inches which regulate their survival and proliferation.

Bone Marrow↗
PubMed2001

Endothelial expression of vascular cell adhesion molecule-1 correlates with metastatic pattern in spontaneous melanoma.

OBJECTIVE: Adhesive interactions between tumor cell surface receptors and endothelial cell adhesion molecules are thought to contribute to tumor cell arrest and extravasation during hematogenous metastasis. Recent reports suggest that melanoma cell integrin alpha4beta1 (very late antigen-4, VLA-4) interaction with the inducible cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), is critical for tumor cell arrest. However, no information is available regarding microvascular VCAM-1 expression during spontaneous melanoma metastasis. The objectives of this study were to evaluate VCAM-1 expression in pulmonary and extrapulmonary vascular beds during melanoma progression, and to determine whether there is an organ-specific profile for VCAM-1 expression which corresponds with the clinical pattern of melanoma metastasis. METHODS: The dual-radiolabeled monoclonal antibody (mAb) technique for quantification of VCAM-1 in different vascular beds was applied to a physiological model of melanoma (B16-BL6) metastasis. Measurements of VCAM-1 were obtained when primary tumors reached 5 mm in size, and every 7 days following removal of the primary lesion. Histological examinations were performed, and mice were placed into 2 groups, based on the presence (+colonies) or absence (-colonies) of pulmonary metastases. VCAM-1 measurements obtained from several organ systems were then compared between these 2 groups of mice. Localization of VCAM-1 was achieved through immunohistochemical staining of tissues. Plasma collected from each experimental animal, as well as melanoma-conditioned media, was assayed to determine levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). RESULTS: Data collected from the dual-radiolabeled mAb technique indicate that 3 weeks following removal of the primary lesion, there is a tendency for VCAM-1 expression to increase in cardiac, hepatic, and cerebral vascular beds. Four weeks following primary resection, when pulmonary metastatic burden was maximal, VCAM-1 was significantly upregulated in each of these vascular beds. Results obtained from the lung indicate that VCAM-1 remains unchanged in pulmonary vessels at all time points examined. Immunohistochemical staining of heart and brain support the radiolabeled mAb measurements, and reveals that these organs exhibit an inflammatory phenotype in mice with heavy pulmonary tumor burden. Furthermore, 25% of these mice had histological evidence of melanoma metastases in heart and brain. Transplantation of liver fragments from mice with advanced pulmonary metastases into subcutaneous tissue of donor mice resulted in the formation of melanotic outgrowths. Plasma levels of the cytokines TNF-alpha and IL-1alpha were negligible in both groups of mice. CONCLUSIONS: Our results indicate that upregulation of VCAM-1 is not a prerequisite for the formation of pulmonary metastases during spontaneous melanoma metastases. However, once lung metastases become well established, organ-specific increases in VCAM-1 expression become apparent. Furthermore, these organ-specific increments in VCAM-1 expression correspond with documented clinical patterns of melanoma metastasis. The enhanced expression of VCAM-1 is independent of systemic levels of TNF-alpha and IL-1alpha, but may be the result of melanoma-induced alterations at the local level, as we found evidence of melanoma cell occupation in heart, brain, and liver in pulmonary metastases-bearing mice.

Animals↗
PubMed1995

Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

Animals↗
PubMed1996

Integrin alpha 4 cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to alpha 4/180 formation.

Here we describe experiments in which we mutated four of the six integrin alpha 4 subunit cysteine residues that are not present in most other integrin alpha subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin alpha 4 beta 1 and/or to CS1 peptide required the presence of both alpha 4 Cys 278 and Cys 717. In addition, optimal ligand binding required divalent cations and reduced cysteines, as evidenced by EDTA and N-ethylmaleimide inhibition results. In a control experiment, an alpha 4 mutation that completely eliminated the alpha 4 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys 278 an Cys 717 mutations markedly altered ligand binding, they had no adverse effect on cell adhesion. Thus, compared with cell adhesion, ligand binding is a distinct and apparently more stringent test of VLA-4 integrin-ligand interactions. In addition, we have established that the formation of the previously described alpha 4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028-2035] also requires Cys 278 and Cys 717, divalent cations and reduced cysteines. thus alpha 4/180 appears to be more functionally relevant than alpha 4/150.

Animals↗