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We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

We have shown that macrophages in culture degrade the glycoproteins and amorphous elastin of insoluble extracellular matrices. Ultrastructural observation of the macrophage-matrix interaction revealed that connective tissue macromolecules were solubilized from the matrix extracellularly. At least part of the matrix breakdown was localized to the immediate vicinity of the cells, as shown by morphological and biochemical studies, although the rate of degradation correlated closely with the secretion of proteinases by various inflammatory stimuli in vivo, by glucocorticoids, prostaglandin E2 or colchicine, or by phagocytosis of latex, zymosan, or cholesterol-albumin complexes in culture was reflected in altered rates of glycoprotein and elastin degradation by the macrophages. Alteration of endocytosis and lysosomal digestion by cytochalasin B, NH4Cl, and proteinase inhibitors did not decrease the overall rate of matrix solubilization, but reduced the processing of the matrix fragments to peptides. Therefore, extracellular, pericellular, and lysosomal events each contribute to degradation of extracellular matrix macromolecules by inflammatory macrophages.

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.

Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.

Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.

During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, i.e., a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical "dense bodies" or mature lysosomes described in other cells are not present in resting neutrophils.

hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.

Binding of P-selectin on activated endothelium to P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils mediates the initial tethering and rolling of neutrophils on the vessel wall at inflammatory sites. Upon activation of rolling cells by locally expressed signaling molecules, integrin-dependent adhesion mechanisms are engaged and transendothelial migration proceeds. P-selectin binding sites are uniformly distributed on the surface of quiescent neutrophils, but are redistributed to the uropod of activated neutrophils. It is unclear whether this activation-induced change in the surface topography of P-selectin binding sites is due to surface redistribution of PSGL-1, shedding of PSGL-1 from the lamellapod, and/or movement of PSGL-1 from an intracellular compartment to the uropod of the polarized cell. With the use of immunogold electron microscopy we previously demonstrated that PSGL-1 was localized to the tips of microvilli on neutrophils. Here we document a similar localization for PSGL-1 on eosinophils, basophils, monocytes, and lymphocytes. On quiescent neutrophils, approximately 80% of the PSGL-1 label was on tips of microvilli, which are randomly distributed around the cell circumference. On activated, polarized neutrophils, the PSGL-1 label was restricted to a segment of approximately 42% of the cell circumference even though total labeling decreased by only approximately 26%. Latex microbeads coated with anti-PSGL-1 mAb bound preferentially to the uropod of activated neutrophils. Subcellular fractionation and immunogold analysis of frozen thin sections of neutrophils failed to detect PSGL-1 in any intracellular compartment. Taken together, these data indicate that the activation-induced change in the surface topography of PSGL-1 is due to surface redistribution of PSGL-1. This process may facilitate transendothelial migration by disrupting bonds between P-selectin and PSGL-1 at the leading edge of migrating cells.

Platelet responses to thrombin are at least partly mediated by a G-protein-coupled receptor whose NH2 terminus is a substrate for thrombin. In the present studies we have examined the location of thrombin receptors in resting platelets and followed their redistribution during platelet activation. The results reveal several new aspects of thrombin receptor biology. 1) On resting platelets, approximately two-thirds of the receptors were located in the plasma membrane. The remainder were present in the membranes of the surface connecting system. 2) When platelets were activated by ADP or a thromboxane analog, thrombin receptors that were initially in the surface connecting system were exposed on the platelet surface, increasing the number of detectable receptors by 40% and presumably making them available for subsequent activation by thrombin. 3) Platelet activation by thrombin rapidly abolished the binding of the antibodies whose epitopes are sensitive to receptor cleavage and left the platelets in a state refractory to both thrombin and the agonist peptide, SFLLRN. This was accompanied by a 60% decrease in the binding of receptor antibodies directed COOH-terminal to the cleavage site irrespective of whether the receptors were activated proteolytically by thrombin or nonproteolytically by SFLLRN. 4) The loss of antibody binding sites caused by thrombin was due in part to receptor internalization and in part to the shedding of thrombin receptors into membrane microparticles, especially under conditions in which aggregation was allowed to occur. However, at least 40% of the cleaved receptors remained on the platelet surface. 5) Lacking the ability to synthesize new receptors and lacking an intracellular reserve of preformed receptors comparable to that found in endothelial cells, platelets were unable to repopulate their surface with intact receptors following exposure to thrombin. This difference underlies the ability of endothelial cells to recover responsiveness to thrombin rapidly while platelets do not, despite the presence on both of the same receptor for thrombin.

We investigated the role of neutrophil microvilli in interactions with E-selectin and P-selectin in hydrodynamic shear flow by disruption with cytochalasin B, hypotonic swelling, and chilling. Cytochalasin B only marginally reduced microvilli numbers (from 30 +/- 6 to 16 +/- 6 per cell perimeter, p < 0.005) as shown by electron microscopy, completely disrupted tethering in shear flow to E-selectin and P-selectin, increased the strength of rolling adhesions on E-selectin and P-selectin, and increased cell deformability in shear flow with a likely increase in the area of cell:substrate contact. Hypoosmotic swelling markedly reduced microvilli number (to 6 +/- 5 per perimeter, p < 0.005), almost completely inhibited tethering on E- and P-selectin, and increased the strength of rolling adhesions on P-selectin but not on E-selectin. Chilling almost completely abolished microvilli (to 3 +/- 3 per perimeter, p < 0.005), but pseudopod-like structures were present, and had little effect on tethering in flow. Immunogold labeling of L-selectin, which is normally clustered on tips of microvilli, showed that in the absence of microvilli it remained in small clusters. Our studies show that alterations in cell morphology and viscoelasticity can have opposing effects on tethering and rolling, showing that they are independently regulatable. Furthermore, our results suggest that the association of molecules that mediate rolling with microvilli tips may be important not just to enhance presentation, but for other functions such as to promote resistance to extraction from the membrane or cooperative interactions among clustered receptors.

L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of inflammation and mediates lymphocyte attachment to high endothelial venules in peripheral lymph nodes. Localization of this receptor to the tips of PMN and lymphocyte microvilli has been demonstrated. However, its distribution on these cells has not been quantified, and its localization on other leukocytes and the morphometry of microvilli on different leukocyte subpopulations have not been previously examined. In this study, PMN and mononuclear leukocytes were isolated from anticoagulated blood by dextran sedimentation and density centrifugation, fixed in 2% paraformaldehyde and 0.05% glutaraldehyde, immunogold-labeled for L-selectin, and embedded in Epon resin. The distribution of L-selectin was determined by counting gold particles on the plasma membrane of sectioned cells, and the surface microstructure of these cells was surveyed on two-dimensional transmission electron micrographs. On average, 78% of PMN, 72% of monocyte, and 71% of lymphocyte L-selectin was observed on the microvilli, with more variance on lymphocytes than the other cell types. Typical PMN and monocyte sections had 26 microvilli, whereas typical lymphocyte sections had 23. Quantitation of the distribution of L-selectin and leukocyte surface topology offers a foundation from which to study the requirement of microvilli or microvillus-localized L-selectin for leukocyte tethering and rolling in model systems that mimic microvascular environments.

Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen.

Factor V stored in platelets is an important source of factor Va for the prothrombinase complex. Investigations of potential platelet factor Va-binding proteins, using factor Va light chain affinity chromatography, identified a disulfide-linked multimeric protein with a reduced mobility of 155 kDa in the column eluate. Immunodepletion and immunoblotting indicated that this protein was multimerin. Multimerin specifically bound factors V and Va and the isolated factor Va light chain, but not the heavy chain of factor Va. Factor V stored in platelets, but not plasma factor V, was found to be complexed with multimerin. Multimerin immunodepletion of resting platelet lysates was associated with the removal of factor V and the loss of factor V coagulant activity. Immunoelectron microscopic studies colocalized factor V with multimerin in the alpha-granules of resting platelets. With thrombin-induced platelet activation, we observed dissociation of factor Va-multimerin complexes, multimerin-independent membrane binding of factor Va, and prothrombinase activity that was not inhibitable by multimerin antibodies. This study indicates that platelet factor V is stored as a complex with multimerin and suggests a possible role for multimerin as a carrier protein for factor V stored in platelets.

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.

Activated platelets express P-selectin and release leukocyte chemoattractants; however, they have not been known to express integrin ligands important in the stabilization of leukocyte interactions with the vasculature. We now demonstrate the presence of intercellular adhesion molecular-2 (ICAM-2) (CD102), and lack of expression of other beta 2-integrin ligands, ICAM-1 (CD54) and ICAM-3 (CD50), on the surface of resting and stimulated platelets. ICAM-2 isolated from platelets migrates as a band of 59,000 M(r) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of bone marrow aspirates with anti-ICAM-2 mAb demonstrates strong reactivity to megakaryocytes. Using frozen thin sections and immunogold labeling, the antigen was shown to be present on the plasma membrane and surface-connected canalicular system of resting platelets. The average number of ICAM-2 molecules per platelet is 3,000 +/- 230 and does not change after activation. In adhesion assays, resting and stimulated platelets were capable of binding through ICAM-2 to purified leukocyte function-associated antigen-1. Activation of T lymphocytes with PMA stimulated binding to platelets that was Mg2+ dependent and could be specifically inhibited by mAbs to either ICAM-2 or leukocyte function-associated antigen-1. ICAM-2 is the only known beta 2-integrin ligand present on platelets, suggesting that it may play an important role in leukocyte-platelet interactions in inflammation and thrombosis.

The localization of the adhesion protein L-selectin in human neutrophils was determined by subcellular fractionation and immunoelectron microscopy and compared with the localization of Mac-1 (alpha m beta 2) and alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeløv, H., et al. J. Clin. Invest. (1993) 92, 1467-1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-1 and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-1 were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-1-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-1 and devoid of L-selectin, into the plasma membrane.

Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils.

Neutrophil tethering and rolling in shear flow are mediated by selectins and have been thought to be two indistinguishable manifestations of a single molecular interaction between selectin and ligand. However, we report that under physiologic flow conditions, tethering to E-selectin requires a ligand distinct from the one that supports neutrophil rolling. Tethering under shear to E-selectin requires a carbohydrate ligand that is closely associated with the lectin domain of L-selectin on the neutrophil surface, as enzymatic removal of L-selectin, chemotactic factor-induced shedding of L-selectin, and L-selectin MAbs effectively block tethering. In contrast, this ligand is dispensable for the ability to roll on E-selectin, since rolling adhesions formed after static incubations were not affected by the presence or absence of L-selectin. Thus, E-selectin interactions with ligands on neutrophils persist after L-selectin shedding. These findings add an additional step for regulation of leukocyte localization in inflammatory sites.

Apart from the diagnostic value of anti-neutrophil cytoplasmic antibodies (ANCA), their detailed characterization and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA are now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA-associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor-alpha (rhTNF-alpha) and two forms of IL-8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA-associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition, TNF-alpha and both forms of IL-8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.

The traditional classification of neutrophil granules as peroxidase-positive (azurophil, or primary) and peroxidase-negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase-positive and peroxidase-negative granules with regard to their content, mobilization and time of formation. Peroxidase-negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase-negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase-negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the secretory vesicle, has now been recognized as an important store of surface membrane-bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.

In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.

We studied the presence of proteinase 3 (PR3), myeloperoxidase (MPO) and elastase (HLE) on the plasma membrane of neutrophils in patients with biopsy-proven Wegener's disease (WG), pANCA-positive vasculitis, control patients (SLE, rheumatoid arthritis, ankylosing spondylitis), sepsis patients and healthy donors. We found an overexpression of PR3 on the cell surface of neutrophils in WG, ANCA-associated vasculitis and during infection (sepsis). Thus PR3 becomes accessible to ANCA. Furthermore we detected intracytoplasmic IgG antibodies in PMN from patients with WG by immunoelectron microscopy and direct immunofluorescence. Our findings support the pathophysiological role of ANCA.

Recent data indicate that megakaryocyte/platelet alpha-granule fibrinogen is endocytosed from plasma. Because fibrinogen is the major platelet protein present in high concentrations in alpha-granules, fibrinogen uptake into alpha-granules may occur via specific receptors. In that cells of the megakaryocyte/platelet lineage contain two integrins--alpha IIb beta 3 (GP IIb-IIIa) and the vitronectin receptor (alpha v beta 3)--that can bind fibrinogen, one or both of these receptors may mediate the endocytic uptake of fibrinogen. To test this hypothesis, we examined the effect of Kistrin, an RGD-containing protein purified from the venom of Agkistrodon rhodostoma that inhibits fibrinogen binding to human platelet receptors, on endocytosis of fibrinogen by megakaryocytes and platelets. Continuous intravenous infusion of kistrin into guinea pigs (200 micrograms/h) over a 24-h period inhibited collagen-induced platelet aggregation. When biotinylated fibrinogen was injected intravenously into animals receiving Kistrin, megakaryocytes failed to endocytose the labeled fibrinogen. Endocytosis of fibrinogen into platelets was also inhibited in these animals. In contrast, platelets and megakaryocytes obtained from sham-infused control animals contained the injected biotinylated fibrinogen. We conclude that, in addition to the well-known extracellular function of cell adhesion, integrins can also act as receptors that mediate endocytosis of exogenous proteins and incorporate them into regulated secretory granules.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.

To determine whether alpha-granule membranes are present in platelets of patients with storage pool deficiencies of both alpha and dense granules (alpha delta-SPD), we examined the content and surface expression of the alpha-granule membrane protein GMP-140 in one patient (J.C.) with a severe alpha-granule deficiency and in three members of a family (family C) with milder alpha-granule deficiencies. Surface expression of GMP-140 in stimulated platelets, assessed by flow cytometric measurements of the binding of two anti-GMP-140 monoclonal antibodies, was 24-38% of normal values in platelets from patient J.C., vs. 60-95% of normal values in family C. Total platelet content of GMP-140, determined in platelet lysates by antigen-capture ELISA, was 49% of normal in patient J.C., but normal in the members of family C. Platelets of patient J.C. were found to be heterogeneous with respect to GMP-140 content and surface expression by both flow cytometry and immunogold electron microscopy. Approximately 80% of her platelets expressed little or no GMP-140 after stimulation, whereas the remaining 20% expressed normal amounts of GMP-140 and showed extensive immunogold labeling of typical alpha-granules and clear vacuoles. No such heterogeneity was found in platelets from family C. These findings in the severe alpha delta-SPD patient are in clear contrast to the observations of normal GMP-140 content in the three other alpha delta-SPD patients, and in patients with the gray platelet syndrome, reported previously by others. These results illustrate the phenotypic heterogeneity of alpha-granule deficiencies in human platelets, and suggest that a defect in granule formation in the megakaryocytes may account for the alpha-granule defect in at least one form of alpha delta-SPD.

Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.

To investigate the distribution of proteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting normal human polymorphonuclear leukocytes and monocytes, the authors used immunocytochemical techniques on thin frozen sections. To obtain valuable tools for immunolabeling, two murine monoclonal antibodies (MAbs) directed against the ACPA antigen were produced. In neutrophils, the authors observed immunogold label for the ACPA antigen, predominantly in myeloperoxidase-positive azurophil granules, and in smaller amounts on the plasma membrane. In monocytes, the ACPA antigen could be detected in small granules, which occasionally also contained myeloperoxidase, and rare labeling was found on the monocyte membrane. The finding that the ACPA antigen is expressed on the plasma membrane of neutrophils and monocytes, thereby becoming accessible to circulating autoantibodies, supports the supposition that ACPA are not only markers of disease activity, but also are involved in the pathogenesis of Wegener's granulomatosis.