Search

Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess alpha(4)beta(1) integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the alpha(4)beta(1) integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.

The integrin VLA-4 (alpha(4)beta(1)) mediates tethering and rolling events as well as firm adhesion of leukocytes to VCAM-1. Unlike selectins, VLA-4 integrin-mediated lymphocyte adhesiveness can be modulated by chemokines through intracellular signaling pathways. To investigate the effects of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) on VLA-4-mediated lymphocyte adhesion, human PBL were flowed over VCAM-1 substrates in a parallel plate flow chamber with surface-immobilized SDF-1alpha, a potent activator of firm adhesion. The initial tethering interactions had a median lifetime of 200 ms, consistent with the half-life of low-affinity VLA-4-VCAM-1 bonds. Immobilized SDF-1alpha acted within the lifetime of a primary tether to stabilize initial tethering interactions, increasing the likelihood a PBL would remain interacting with the surface. As expected, the immobilized SDF-1alpha also increased the ratio of PBL firm adhesion to rolling. An LDV peptide-based small molecule that preferentially binds high-affinity VLA-4 reduced PBL firm adhesion to VCAM-1 by 90%. The reduction in firm adhesion due to blockage of high-affinity VLA-4 was paralleled by a 4-fold increase in the fraction of rolling PBL. Chemokine activation of PBL firm adhesion on VCAM-1 depended on induction of high-affinity VLA-4 rather than recruitment of a pre-existing pool of high-affinity VLA-4 as previously thought.

Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn(-/-) mice (Lyn(-/-) BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn(-/-) BMMCs release more beta-hexosaminidase than wild-type BMMCs following FcepsilonRI cross-linking and show that multiple mast cell responses to FcepsilonRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cgamma isoforms, the mobilization of Ca(2+), the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the alpha(4)beta(1) integrin, VLA-4) are slow to initiate in Lyn(-/-) BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn(-/-) BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn(-/-) BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn(-/-) BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcepsilonRI signaling in Lyn(-/-) BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn(-/-) BMMCs may additionally contribute to their altered signaling properties.

The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below approximately 20 sites/microm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.

The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhydryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-(D,L-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl)-L-3-(tert-butyl)-alanyl-l -alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hypothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis.

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.

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.

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.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.

The activation of leukocytes by particulates is a critical event in certain inflammatory syndromes, including acute gout associated with microcrystals of monosodium urate monohydrate. In this study we have evaluated mechanisms of human neutrophil activation by urate crystals. Both N-formyl-nor-leu-leu-phe-nor-leu-tyr-lys and uncoated urate crystals (0.5 to 5 mg/ml) but not urate crystals coated with human low density lipoprotein (which suppresses crystal-induced neutrophil responsiveness), stimulated pertussis toxin (PT)-sensitive GTPase activity in purified preparations of human neutrophil membranes. Hydroxyapatite crystals (up to 5 mg/ml) were inactive. Pretreatment of neutrophil membranes with cholera toxin also inhibited crystal-induced and formylated peptide-induced GTPase activity. Pretreatment of whole neutrophils with PT resulted in nearly complete inhibition of formylated peptide-induced cytosolic calcium mobilization, release of superoxide and release of the azurophil granule constituent alpha-mannosidase. In contrast, identical pretreatment of whole neutrophils with PT only partially suppressed urate crystal-induced alpha-mannosidase and superoxide release and failed to inhibit crystal phagocytosis and increases in cytosolic free calcium. Mechanisms of neutrophil activation by monosodium urate crystals appear to be heterogeneous in comparison with activation by formylated peptides and there is no absolute requirement for PT-sensitive membrane G proteins in neutrophil responsiveness to urate crystals.

Leukotriene B4 (LTB4) was shown to be a potent stimulator of neutrophil actin polymerization as detected by right-angle light scatter and rhodamine-phalloidin staining of F-actin. When we compared the kinetics of this neutrophil cytoskeletal response to the chemoattractants formylpeptide and LTB4, we observed that the response to LTB4 was markedly shorter-lived. To understand the basis of this result, we re-examined the kinetics of superoxide generation, elastase release, intracellular calcium elevation, and phosphoinositide metabolism in neutrophils stimulated with LTB4 and N-formylhexapeptide. LTB4 was relatively inefficient in producing superoxide and in releasing elastase. Although both responses were initiated with similar rapidity, they turned off sooner with LTB4 as compared with N-formylhexapeptide stimulation. Intracellular calcium elevation, a signal that is necessary for superoxide generation and degranulation, was of similar magnitude but of shorter duration in response to LTB4 as compared with the N-formylhexapeptide. The LTB4-induced rise of phosphatidic acid also was not sustained as long as the N-formylhexapeptide-induced increase. Prior exposure of neutrophils to LTB4 did not inhibit subsequent stimulation of superoxide generation by N-formylhexapeptide. Thus, the inability of LTB4 to stimulate superoxide generation was not due to LTB4-induced global inhibitory signals. The deficiency in LTB4-induced superoxide and elastase responses may be related to short-lived LTB4-induced activation signals and/or the number of receptors contributing to these responses.

Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.

Murine monoclonal antibody (mAb) NMS-1 was generated which binds to the surface of living human neutrophils. The antigens on neutrophil plasma membranes recognized by mAb NMS-1 were solubilized in Nonidet P-40 and immunopurified on matrix-bound mAb NMS-1. mAb NMS-1 binds to four periodate-sensitive structures of 70,000, 95,000, 140,000, and 170,000 Da on the plasma membrane surface of human neutrophils as was shown by Western blot analysis. Binding of mAb NMS-1 to human neutrophils induced a rapid transient rise in cytosolic free calcium (Quin 2 fluorescence) but no detectable generation of reactive oxygen metabolites. The oxidative burst of N-formyl peptide-treated neutrophils, however, increased in the presence of mAb NMS-1. The kinetics of N-formyl peptide (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tryrosyl-lysine; FNLPNTL)-mediated hydrogen peroxide formation (p-hydroxy phenyl acetate oxidation) in the presence of mAb NMS-1 was analyzed quantitatively. 1) When neutrophils were incubated with mAb NMS-1 before FNLPNTL addition, an increase in rate, magnitude, and duration of hydrogen peroxide formation was observed compared with controls which received no mAb NMS-1 treatment. After termination of the initial linear phase of response, a second transient linear phase of hydrogen peroxide formation was induced. This second phase of activation was not observed in neutrophils which received no mAb NMS-1 treatment. The onset of the response and latency before attainment of the initial linear rate of hydrogen peroxide formation was not changed by mAb NMS-1 pretreatment. 2) When neutrophils were stimulated with FNLPNTL, the addition of mAb NMS-1--after termination of the FNLPNTL-induced response--without delay induced a second transient burst of hydrogen peroxide formation. Persistent activation of hydrogen peroxide formation by mAb NMS-1 in FNLPNTL-stimulated neutrophils was not observed.