Distinct role of hydrodynamic shear in leukocyte-facilitated tumor cell extravasation

Previously, we found polymorphonuclear neutrophils (PMNs) increased melanoma cell extravasation under flow conditions (Intl J Cancer 106: 713–722, 2003). In this study, we characterized the effect of hydrodynamic shear on PMN-facilitated melanoma extravasation using a novel flow-migration assay. The effect of shear stress and shear rate on PMN-facilitated melanoma extravasation was studied by increasing the medium viscosity with dextran to increase shear stress independently of shear rate. Under fixed shear rate conditions, melanoma cell extravasation did not change significantly. In contrast, the extravasation level increased at a fixed shear stress but with a decreasing shear rate. PMN-melanoma aggregation and adhesion to the endothelium via ₂-integrin/intracellular adhesion molecule-1 (ICAM-1) interactions were also studied. Lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, whereas Mac-1 (CD11b/CD18) affected prolonged PMN-melanoma aggregation. Blockage of E-selectin or ICAM-1 on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. We have found PMN-melanoma adhesion is correlated with the inverse of shear rate, whereas the PMN-endothelial adhesion correlated with shear stress. Interleukin-8 (IL-8) also influenced PMN-melanoma cell adhesion. Functional blocking of the PMN IL-8 receptors, CXCR1 and CXCR2, decreased the level of Mac-1 upregulation on PMNs while in contact with melanoma cells and reduced melanoma extravasation. We have found PMN-facilitated melanoma adhesion to be a complex multistep process that is regulated by both microfluid mechanics and biology. neutrophil; melanoma; shear stress; shear rate; ₂-integrins; intracellular adhesion molcule-1; CXCR1/2; adhesion; migration     

Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.     

Fibrin serves as a divalent ligand that regulates neutrophil-mediated melanoma cells adhesion to endothelium under shear conditions

Elevated soluble fibrin (sFn) levels are characteristic of melanoma hematogeneous dissemination, where tumor cells interact intimately with host cells. Melanoma adhesion to the blood vessel wall is promoted by immune cell arrests and tumor-derived thrombin, a serine protease that converts soluble fibrinogen (sFg) into sFn. However, the molecular requirement for sFn-mediated melanoma-polymorphonuclear neutrophils (PMNs) and melanoma-endothelial interactions under physiological flow conditions remain elusive. To understand this process, we studied the relative binding capacities of sFg and sFn receptors e.g., α(v)β(3) integrin and intercellular adhesion molecule-1 (ICAM-1) expressed on melanoma cells, ICAM-1 on endothelial cells (EC), and CD11b/CD18 (Mac-1) on PMNs. Using a parallel-plate flow chamber, highly metastatic melanoma cells (1205Lu and A375M) and human PMNs were perfused over an EC monolayer expressing ICAM-1 in the presence of sFg or sFn. It was found that both the frequency and lifetime of direct melanoma adhesion or PMN-facilitated melanoma adhesion to the EC in a shear flow were increased by the presence of sFn in a concentration-dependent manner. In addition, sFn fragment D and plasmin-treated sFn failed to increase melanoma adhesion, implying that sFn-bridged cell adhesion requires dimer-mediated receptor-receptor cross-linking. Finally, analysis of the respective kinetics of sFn binding to Mac-1, ICAM-1, and α(v)β(3) by single bond cell tethering assays suggested that ICAM-1 and α(v)β(3) are responsible for initial capture and firm adhesion of melanoma cells. These results provide evidence that sFn enhances melanoma adhesion directly to ICAM-1 on the EC, while prolonged shear-resistant melanoma adhesion requires interactions with PMNs.     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.     

Tumor Cell Extravasation Mediated by Leukocyte Adhesion is Shear Rate Dependent on IL-8 Signaling^*

To complete the metastatic journey, cancer cells have to disseminate through the circulation and extravasate to distal organs. However, the extravasation process, by which tumor cells leave a blood vessel and invade the surrounding tissue from the microcirculation, remains poorly understood at the molecular level. In this study, tumor cell adhesion to the endothelium (EC) and subsequent extravasation were investigated under various flow conditions. Results have shown polymorphonuclear neutrophils (PMNs) facilitate melanoma cell adhesion to the EC and subsequent extravasation by a shear-rate dependent mechanism. Melanoma cell-PMN interactions are mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells andb2integrins on PMNs. In addition, the fluid convection affects the extent of activation ofb2integrins on PMNs by endogenously secreted interleukin 8 (IL-8) within the tumor microenvironment. Results also indicate that shear rate affects the binding kinetics between PMNs and melanoma cells, which may contribute to the shear-rate dependence of melanoma extravasation in a shear flow when mediated by PMNs.     

Kinetics analysis of binding between melanoma cells and neutrophils

It has been determined previously that polymorphonuclear leukocytes, or PMNs, can facilitate melanoma cell extravasation through the endothelium under shear conditions. The interactions between melanoma cells and PMNs are mediated by the beta2-integrins expressed by PMNs and intercellular adhesion molecules (ICAM-1) expressed on melanoma cells. In this study, the kinetics of these interactions was studied using a parallel plate flow chamber. The dissociation rates were calculated under low force conditions for ICAM-1 interactions with both beta2-integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), together and separately by using functional blocking antibodies on PMNs. The kinetics of PMNs stimulated with IL-8 was also determined. It was concluded that the small number of constitutively expressed active beta2-integrins on PMNs are sufficient to bind to ICAM-1 expressed on melanoma cells and that the intrinsic dissociation rate for these adhesion molecules appear to be more dependent on what method is used to determine them than on what cells express them.     

Hydrodynamic shear regulates the kinetics and receptor specificity of polymorphonuclear leukocyte-colon carcinoma cell adhesive interactions.

The ability of tumor cells to metastasize hematogenously is regulated by their interactions with polymorphonuclear leukocytes (PMNs). However, the mechanisms mediating PMN binding to tumor cells under physiological shear forces remain largely unknown. This study was designed to characterize the molecular interactions between PMNs and tumor cells as a function of the dynamic shear environment, using two human colon adenocarcinoma cell lines (LS174T and HCT-8) as models. PMN and colon carcinoma cell suspensions, labeled with distinct fluorophores, were sheared in a cone-and-plate rheometer in the presence of the PMN activator fMLP. The size distribution and cellular composition of formed aggregates were determined by flow cytometry. PMN binding to LS174T cells was maximal at 100 s(-1) and decreased with increasing shear. At low shear (100 s(-1)) PMN CD11b alone mediates PMN-LS174T heteroaggregation. However, L-selectin, CD11a, and CD11b are all required for PMN binding to sialyl Lewis(x)-bearing LS174T cells at high shear (800 s(-1)). In contrast, sialyl Lewis(x)-low HCT-8 cells fail to aggregate with PMNs at high shear conditions, despite extensive adhesive interactions at low shear. Taken together, our data suggest that PMN L-selectin initiates LS174T cell tethering at high shear by binding to sialylated moieties on the carcinoma cell surface, whereas the subsequent involvement of CD11a and CD11b converts these transient tethers into stable adhesion. This study demonstrates that the shear environment of the vasculature modulates the dynamics and molecular constituents mediating PMN-tumor cell adhesion.     

Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion

This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.     

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.     

Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.     

Sequential binding of αVβ3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.     

Influence of beta 1 integrin intracytoplasmic domains in the regulation of VLA-4-mediated adhesion of human T cells to VCAM-1 under flow conditions

The VLA-4 integrin supports static cell-cell, cell-matrix adhesion, and dynamic interactions with VCAM-1. Although functions for well-conserved beta(1) integrin cytoplasmic domains in regulating static cell adhesion has been established, the molecular basis for beta(1) integrin-dependent arrest on VCAM-1 under flow conditions remains poorly understood. We have transfected the beta(1) integrin-deficient A1 Jurkat T cell line with beta(1) cDNA constructs with deletions of the NPXY motifs and specific mutations of tyrosine residues. Deletion of either NPXY motif impaired static adhesion induced by CD2 or CD47 triggering or direct beta(1) integrin stimulation. In contrast, PMA-induced adhesion to VCAM-1 was unaffected by deletion of the NPIY motif and only slightly impaired by deletion of NPKY. Moreover, deletion of the NPIY motif resulted in enhanced rolling and reduced arrest on VCAM-1 under shear flow conditions. In contrast, deletion of the NPKY motif did not alter arrest under flow. Although tyrosine to phenylalanine substitutions within two NPXY motifs did not alter static adhesion to VCAM-1, these mutations enhanced arrest on VCAM-1 under flow conditions. Furthermore, although deletion of the C'-terminal 5 AA of the beta(1) cytoplasmic domain dramatically impaired activation-dependent static adhesion, it did not impair arrest on VCAM-1 under flow conditions. Thus, our results demonstrate distinct structural requirements for VLA-4 function under static and shear flow conditions. This may be relevant for VLA-4 activity regulation in different anatomic compartments, such as when circulating cells arrest on inflamed endothelium under shear flow and when resident cells in bone marrow interact with VCAM-1- positive stromal cells.     

Regulation of interleukin-8 expression in melanoma-stimulated neutrophil inflammatory response

Inflammation facilitates tumor progression including metastasis. Interleukin-8 (IL-8) is a chemokine that regulates polymorphonuclear neutrophil (PMN) mobilization and activity and we hypothesize that this cytokine influences tumor behavior. We have demonstrated that IL-8 is crucial for PMN-mediated melanoma extravasation under flow conditions. In addition, IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells. Melanoma cells induce IkappaB-alpha degradation in PMNs indicating that NF-kappaB signaling is active in PMNs. Furthermore, the production of IL-8 in PMNs is NF-kappaB dependent. We have further identified that interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) from PMN-melanoma co-cultures synergistically contribute to IkappaB-alpha degradation and IL-8 synthesis in PMNs. Taken together, these findings show that melanoma cells induce PMNs to secrete IL-8 through activation of NF-kappaB and suggest a model in which this interaction promotes a microenvironment that is favorable for metastasis.     

Unique ability of integrin alpha(v)beta 3 to support tumor cell arrest under dynamic flow conditions

Shear-resistant arrest of circulating tumor cells is required for metastasis from the blood stream. Arrest during blood flow can be supported by tumor cell interaction with attached, activated platelets. This is mediated by tumor cell integrin alpha(v)beta3 and cross-linking plasma protein ligands. To analyze the mechanism of tumor cell ligand interactions under dynamic flow conditions, we used real-time video microscopy and tested human melanoma cell binding to fibrinogen, von Willebrand Factor, or fibronectin matrices in a buffer perfusion system. When perfused at venous flow, melanoma cells arrested abruptly and began to spread immediately. This was uniquely mediated by integrin alpha(v)beta3 on all tested ligands, and required alpha(v)beta3 activation and actin polymerization. Under static conditions, alpha(v)beta3 cooperated with alpha(v)beta1 and alpha5beta1 in supporting melanoma cell adhesion to fibronectin. But even when activated, beta1 integrins did not contribute to melanoma cell arrest during flow. Soluble ligand served as a cross-linker between attached and circulating tumor cells and enhanced melanoma cell arrest. Cohesion of activated melanoma cells was restricted to the matrix surface and did not occur in suspension. We conclude that the presence of alpha(v)beta3 in a functionally activated state provides a unique advantage for circulating tumor cells by promoting tumor cell arrest in the presence of flow-dependent shear forces.     

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.     

Endothelial cell prostacyclin production induced by activated neutrophils

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.     

Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils

The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC-associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins.     

Determining beta2-integrin and intercellular adhesion molecule 1 binding kinetics in tumor cell adhesion to leukocytes and endothelial cells by a gas-driven micropipette assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.