RESUMEN

G protein-coupled receptors (GPCR) and cellular signaling elements are prime targets for drug discovery. Sensitive real-time methods that expand the analytical capabilities for these elements can play significant roles in basic research and drug discovery. Here, we describe novel approaches for the real-time fluorescence analysis of GPCRs. Using the G protein-coupled N-formyl peptide receptor (FPR) as a model system in concert with a fluorescent ligand, we showed the quantitative solubilization of his-tagged FPRs in 1% dodecyl maltoside. Solubilized receptors reconstitute in dodecyl maltoside with a mixture of bovine brain Gi/Go showing an apparent Kd of 100 nM. Solubilized receptors were also bound to Ni(2+)-silica particles and were detected in a flow cytometer by the binding of fluorescent ligand. The efficiency of receptor uptake by the particles was in excess of 80% with an apparent affinity for the bead in the nM range. The receptors had largely homogeneous dissociation characteristics, an appropriate Kd for the ligand in the low nM range and a high site number, with several million receptor molecules per particle. However, the G protein reconstitution was not detected on the beads, apparently for steric reasons. These approaches for displaying receptors could prove useful in drug discovery and in the analysis of the molecular assemblies in signal transduction.

Asunto(s)

Citometría de Flujo/métodos , Proteínas de Unión al GTP/análisis , Receptores Inmunológicos/análisis , Receptores de Péptidos/análisis , Factores Quimiotácticos , Sistemas de Computación , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Histidina , Humanos , Microesferas , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , Ácido Nitrilotriacético/análogos & derivados , Compuestos Organometálicos , Unión Proteica , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Transducción de Señal/genética , Dióxido de Silicio , Solubilidad , Espectrometría de Fluorescencia , Transfección , Células U937

RESUMEN

VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF165, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF165-specific aptamer NX1838 (2'-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCgamma, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF121-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.

Asunto(s)

Factores de Crecimiento Endotelial/antagonistas & inhibidores , Endotelio/metabolismo , Linfocinas/antagonistas & inhibidores , Oligonucleótidos/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Receptores de Factores de Crecimiento/química , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Factores de Crecimiento Endotelial/metabolismo , Endotelio/efectos de los fármacos , Humanos , Linfocinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.

Asunto(s)

Antígenos CD18/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Neutrófilos/citología , Animales , Adhesión Celular , Agregación Celular/fisiología , Comunicación Celular , Cloruros/farmacología , Citometría de Flujo , Humanos , Selectina L/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Antígeno de Macrófago-1/fisiología , Compuestos de Manganeso/farmacología , Melanoma Experimental/patología , Glicoproteínas de Membrana/fisiología , Ratones , Neutrófilos/metabolismo , Estrés Mecánico , Células Tumorales Cultivadas

RESUMEN

Anthracyclines, including daunorubicin (DnR) and doxorubicin (DoX), have shown clinical chemotherapeutic utility, albeit in association with cumulative dose-associated cardiotoxicities. Despite structural similarity, however, DnR and DoX treatments have been directed toward leukemias and solid tumor types, respectively. Due to a paucity of in vitro data regarding differential use of DnR or DoX, we assessed the cytotoxicity of these compounds against solid and hematological tumor cell types. In addition, we examined liposomal formulations of DnR (L-DnR) and DoX (PEG-DoX), which, in contrast to DnR or DoX, demonstrate antineoplastic activity with reduced cardiotoxicity in vivo. Accordingly, cytotoxicity testing (with [methyl/-(3)H]thymidine incorporation) of DnR, DoX, L-DnR, and PEG-DoX on a range of different human tumor cell lines (e.g., breast, lung, ovarian, prostate, melanoma, lymphoma, and leukemia tumor cell types) was performed. Our data indicate comparable activity for DnR, DoX, or L-DnR in all tumor cell types examined [e.g., SK-BR-3 (breast adenocarcinoma) cells: IC50 values = 5.9, 9.1, and 4.7 ng/mL for DnR, DoX, and L-DnR respectively]. In addition, several solid tumor cell types were more responsive to DnR than DoX [e.g., DU-145 (prostate carcinoma) cells: IC50 values = 10.4 and 41.2 ng/mL for DnR and DoX, respectively; p >. 001]. Interestingly, PEG-DoX was substantively less effective for all tumor cells (IC50 values were about 100-10,000 times greater for PEG-DoX than for DnR, DoX, or L-DnR; p >. 001, all cases). Reduced PEG-DoX activity in vitro may be related to polyethylene glycol (PEG) moieties present on the liposomal exterior of PEG-DoX, which are not present on L-DnR. Nonetheless, taken together, these data suggest that DnR and DoX demonstrate comparable efficacy in vitro and that specific liposomal encapsulation (L-DnR) does not mitigate DnR efficacy in vitro.

RESUMEN

In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L-selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL-1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.

Asunto(s)

Agregación Celular/fisiología , Selectina L/fisiología , Glicoproteínas de Membrana/fisiología , Neutrófilos/citología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD18/fisiología , Adhesión Celular , Citometría de Flujo , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Ligandos , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/inmunología , Metaloendopeptidasas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo

RESUMEN

MnCl2 and dithiothreitol (DTT) enhance the adhesive functions of beta 2 -integrins. We have used these agents and flow cytometry to distinguish the contributions of beta 2-integrins and L-selectin to neutrophil aggregation. Although neither compound induced aggregation, they prolonged N-formyl-methionyl-leucyl-phenylalanine-induced aggregation and produced larger aggregates. Because activated polymorphonuclear granulocytes (PMN) shed L-selectin in the presence of MnCl2, but not DTT, we could evaluate the role of L-selectin in the early and late stages of aggregation. Blocking L-selectin sites with DREG200 Fab and/or beta 2-integrin sites with IB4 Fab indicated that aggregation under all conditions remained beta 2-integrin- and L-selectin-dependent. Disaggregation was integrin-dependent whether L-selectin was present or shed. The disaggregation kinetics suggested that integrin bonds turned over at a slower rate in MnCl2-treated cells. Enhanced aggregation due to DTT and MnCl2 required sustained energy output, suggesting intracellular rather than strictly conformational control. These results provide evidence that PMN aggregation, like leukocyte-endothelial cell adhesion, utilizes L-selectin to form intercellular contacts that are maintained through activated integrins.

Asunto(s)

Antígenos CD18/fisiología , Cloruros/farmacología , Ditiotreitol/farmacología , Compuestos de Manganeso/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Reactivos de Sulfhidrilo/farmacología , Especificidad de Anticuerpos , Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Humanos , Neutrófilos/fisiología

RESUMEN

L-selectin is an adhesion molecule that mediates the recruitment of neutrophils to inflammatory sites and initiates the migration of lymphocytes into the peripheral lymph nodes. In response to cell activation, L-selectin is shed from the cell surface, and altered levels of functional soluble L-selectin are detected in the plasma of patients suffering from numerous inflammatory diseases as well as AIDS. The mechanism that regulates L-selectin shedding is poorly understood. Here we show that a hydroxamate-based metalloprotease inhibitor, N-(D,L-[2-(hydroxyaminocarbonyl)- methyl]-4-methylpentano)-L-3-(tert-butyl)-alanyl-L-alanine, 2-aminoethyl amide, which blocks leukocyte TNF, TNF receptor, and IL-6 receptor release, also inhibits L-selectin shedding from neutrophils, eosinophils, and lymphocytes. Moreover, we show that such inhibition of L-selectin shedding profoundly affects neutrophil aggregation and permits reaggregation in the presence of a heterologous stimulus.

Asunto(s)

Dipéptidos/farmacología , Ácidos Hidroxámicos/farmacología , Selectina L/efectos de los fármacos , Metaloendopeptidasas/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Agregación Celular/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Eosinófilos/fisiología , Humanos , Ácidos Hidroxámicos/química , Selectina L/fisiología , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Neutrófilos/fisiología

RESUMEN

The homotypic aggregation of neutrophils requires the participation of L-selectin and the beta 2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable beta 2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable beta 2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in beta 2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Selectina L/metabolismo , Neutrófilos/citología , Sialoglicoproteínas/metabolismo , Agregación Celular , Quimiotaxis de Leucocito , Humanos , Técnicas In Vitro , Yodoacetatos/farmacología , Ácido Yodoacético , Ligandos , Receptores de Superficie Celular/metabolismo , Reactivos de Sulfhidrilo/farmacología

RESUMEN

Emigration of leukocytes at sites of inflammation is initiated by the selectin family of carbohydrate-binding adhesion molecules. Molecular crossbridges initiate rolling of cells along the vascular endothelium where chemokines such as IL-8 and platelet activating factor (PAF) may be presented to their receptors on the leukocyte surface resulting in cell stimulation. Integrin activation appears to be a requirement for subsequent cell localization and diapedesis into the tissue. Several recent reports have demonstrated that ligation and cross-linking of neutrophil L-selectin results in neutrophil activation, including intracellular calcium release, superoxide production, and induction of mRNA for production of IL-8 and TNF-alpha. The purpose of this study was to examine whether ligation and cross-linking of L-selectin would specifically result in activation of beta 2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-microns diameter) were adsorbed with albumin or fibrinogen and added in excess to human neutrophils in a shear-stirred suspension. Following stimulation the kinetics of bead capture by neutrophils was continuously measured in real time on the flow cytometer. The onset of bead binding was detected in the presence of extremely low concentrations of PAF (10 pM) or formyl peptide (0.2 nM) stimulation. Ligation of L-selectin with whole IgG DREG200 or DREG56 Ab, but not controls (anti-CD44, -CD45, -CD11a), resulted in a significant potentiation of bead binding. Cross-linking F(ab')2 fragments of DREG200 with a goat anti-mouse F(ab')2 secondary Ab also stimulated beta 2-integrin-dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing gamma 4 or gamma 1 isotypes of human Fc domain also stimulated cell adhesion when cross-linked. Surface expression of CD18 and an activation-dependent epitope, as detected with mAb24, also increased in response to L-selectin cross-linking. Cross-linking L-selectin induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells. We propose that cross-linking of L-selectin results in a cell signal that directly stimulates beta 2-integrin adhesive responses.

Asunto(s)

Antígenos CD18/fisiología , Moléculas de Adhesión Celular/fisiología , Integrinas/fisiología , Antígeno de Macrófago-1/fisiología , Neutrófilos/fisiología , Agregación de Receptores , Transducción de Señal/fisiología , Adulto , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos CD18/química , Adhesión Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Colágeno/metabolismo , Endotelio Vascular/fisiología , Fibrinógeno/metabolismo , Citometría de Flujo , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Selectina L , Microesferas , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Conformación Proteica , Agregación de Receptores/efectos de los fármacos , Albúmina Sérica/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Human neutrophils are primed in the presence of complexes of lipopolysaccharide (LPS) with its serum binding protein (LBP) in a manner dependent on CD14. Cellular consequences of priming include increased responsiveness, the upregulation of surface proteins including the adhesive integrin CD11b/CD18 (Mac-1), the increased binding of certain ligands to CD11b/CD18, and the concurrent shedding of the L-selectin homing receptor. Because expression of both CD11b/CD18 and L-selectin is obligatory for formyl peptide-stimulated neutrophil aggregation in vitro (Simon et al, Blood 82:1097, 1993), we have examined the consequences of bacterial endotoxin on the expression of neutrophil adhesive molecules. We observed that the exposure of neutrophils to LPS/LBP, while enhancing the surface numbers and adhesive function of CD11b/CD18 for latex particles, did not induce aggregation. In contrast, as the LPS/LBP concentration increased (ED50 = 30 ng/mL LPS/LBP), the ability of neutrophils to aggregate decreased in parallel with the shedding of L-selectin. Moreover, when L-selectin adhesive activity was blocked by treatment with Fab fragments of Dreg-200, aggregation was inhibited to an extent roughly proportional to the available L-selection. Blocking of LPS/LBP with CD14-specific monoclonal antibodies suppressed L-selectin shedding and preserved formyl peptide-stimulated aggregation. Taken together, the data suggest that inhibition of neutrophil aggregation by LPS/LBP is related to the expression of L-selectin via CD14 rather than LPS inhibition of CD11b/CD18 function during cellular stimulation.

Asunto(s)

Proteínas de Fase Aguda , Lipopolisacáridos/farmacología , Antígeno de Macrófago-1/efectos de los fármacos , Glicoproteínas de Membrana , Neutrófilos/efectos de los fármacos , Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Proteínas Portadoras/farmacología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Agregación Celular/efectos de los fármacos , Humanos , Selectina L , Receptores de Lipopolisacáridos , Lipopolisacáridos/metabolismo , Antígeno de Macrófago-1/análisis , Antígeno de Macrófago-1/fisiología , Neutrófilos/fisiología

RESUMEN

We have recently reported that neutrophil aggregation is dependent on both L-selectin and the beta 2-integrin Mac-1, raising the possibility that carbohydrate interactions play a role in aggregation. We used mono- and polysaccharides known to inhibit L-selectin-dependent adhesion of lymphocytes to high endothelial venules to test whether these carbohydrates could inhibit neutrophil aggregation. Similar types and concentrations of carbohydrates found by others to inhibit lymphocyte adhesion were effective in blocking neutrophil aggregation. Thus, nanomolar concentrations of the polysaccharides dextran sulfate (m.w. 500,000) and fucoidan inhibited aggregation, whereas dermatan sulfate, alpha-carrageenan, and dextran sulfate (m.w. 5,000) showed no inhibition. All of the phosphorylated monosaccharides tested inhibited aggregation with ED50 values between 8 and 17 mM, the most potent being mannose-6-phosphate and fucose-1-phosphate. The nonphosphorylated monosaccharides glucose and fucose were noninhibitory. The inhibitory effects of fucoidan or dextran sulfate (m.w. 500,000) did not appear to be due to altered regulation of L-selectin after stimulation because fucoidan reduced the rate of L-selectin shedding, whereas dextran sulfate had no effect compared with control. Neither carbohydrate inhibited the binding of formyl peptide to its receptor. However, carbohydrates were able to compete with mAb binding to a number of known leukocyte adhesion proteins. We used endotoxin pretreatment to create L-selectin-deficient neutrophils to study the minimum adhesive requirements for aggregation using two-color fluorescence flow cytometry. Our results implicate a lectinlike contribution to neutrophil aggregation, and suggest that L-selectin is the molecule that mediates the carbohydrate-dependent adhesive event.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Lectinas/fisiología , Neutrófilos/citología , Anticuerpos Monoclonales , Unión Competitiva , Metabolismo de los Hidratos de Carbono , Agregación Celular , Humanos , Selectina L , Lipopolisacáridos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología

RESUMEN

An analysis of receptor modulation on the neutrophil cell surface is essential for gaining insight into the activation and function of neutrophils in host defense. Moreover, agents that regulate the expression of surface receptors may have profound implications in the management of host response to infection. With this study, we extend previous observations in isolated cells of the putative ability of methylprednisolone sodium succinate (MPSS) to inhibit ligand binding to the formyl peptide receptor. Because neutrophils are exquisitely sensitive to isolation conditions, we have analyzed the regulation of receptor expression using flow cytometry in whole blood. This technique allows discrimination of neutrophils from other formed elements without isolation. Quiescent cells in blood exhibit low levels of formyl peptide receptor, CD11b/CD18, and CD14. We show that MPSS blocks upregulation of each of these receptors in response to three different stimuli (formyl peptide, lipopolysaccharide, and granulocyte macrophage colony-stimulating factor). The inhibition is reversible with an ED50 of approximately 0.4 mg/ml. From these observations, we conclude that the action of MPSS on neutrophils blocks a common response of receptors. Since these receptors probably function in part through independent signaling pathways, MPSS may function at a common site related to vesicular trafficking. Further investigation is needed to determine the specific means by which corticosteroids interfere with neutrophil upregulation mechanisms.

Asunto(s)

Sangre/metabolismo , Metilprednisolona/farmacología , Neutrófilos/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Humanos , Técnicas In Vitro , Receptores de Superficie Celular/antagonistas & inhibidores

RESUMEN

We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2-integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates on the basis of size and color, as well as antibody binding to these two adhesive molecules. Formyl peptide-stimulated aggregate formation was measured for individual populations fluorescently labeled red (LDS-751) or green (CD44-FITC), and interpopulation red-green cell conjugates. Blocking either the beta 2-integrin or L-selectin adhesive epitope with monoclonal antibody on individual cell populations resulted in an approximately 50% reduction in two-color aggregation as compared with that in unblocked samples. Shedding the L-selectin on a cell population by preincubation with complexes of lipopolysaccharide and its plasma membrane binding protein also decreased aggregation to a control population by approximately 50%. We examined the aggregation of neutrophils from patients genetically deficient in beta 2-integrin and clinically leukocyte adhesion deficient (LAD). LAD adhesion to normal neutrophils was dependent on the expression of L-selectin on LAD cells and beta 2-integrin on normal cells. Thus, the minimum requirement for adhesion between two mixed populations of neutrophils was that one population expressed the beta 2-integrin and the other expressed the L-selectin adhesive epitope.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Integrinas/fisiología , Neutrófilos/fisiología , Animales , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Agregación Celular , Quimotripsina/farmacología , Humanos , Integrinas/análisis , Cinética , Selectina L , Lipopolisacáridos/farmacología , Ratones