Article |
Address correspondence to Barbara C. Furie, Center for Hemostasis, Thrombosis and Vascular Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Tel.: (617) 667-0620. Fax: (617) 975-5505. email: bfurie1{at}bidmc.harvard.edu
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Abstract |
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Key Words: secondary interactions; endocytosis; trafficking; lymphatics
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Introduction |
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Peritoneal macrophages are a population of resident mononuclear phagocytes of unknown function. In the first 2 h after induction of inflammation, the number of resident macrophages in the peritoneal cavity significantly decreases, suggesting their emigration out of the peritoneal cavity (Haskill and Becker, 1985; Barth et al., 1995). Activated resident peritoneal macrophages migrate into parathymic lymph nodes and to the gut-associated lymphoid tissues (Rosen and Gordon, 1990; Sminia et al., 1995; van Vugt et al., 1995; Bellingan et al., 1996). Adhesion molecules including L-selectin, VLA-4, Mac-1, LFA-1, and PSGL-1 that potentially mediate the interaction of monocytes under flow have been extensively studied (Luscinskas et al., 1994, 1996; Gerszten et al., 1998; Lim et al., 1998) in contrast to the interactions of macrophages with adhesive molecules under flow. Here, we demonstrate that mouse peritoneal macrophages adhere to VCAM-1 under conditions of laminar flow. We also demonstrate that homotypic interactions among macrophages occur in laminar flow and that these interactions are mediated by PSGL-1 and P-selectin on the surface of the peritoneal macrophages. P-Selectin expression has heretofore been thought to be restricted to platelets and endothelial cells. These studies are the first demonstration that a specific leukocyte subtype, resident peritoneal macrophages, can express functional P-selectin.
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Results |
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To demonstrate that nucleation is mediated by interaction of P-selectin and PSGL-1, we perfused a mixture of PSGL-1 null and P-selectin null leukocytes over a VCAM-1-coated surface. Combining the PSGL-1 null and P-selectin null macrophages restored cell string formation (Fig. 1, A and B, P-/- + PSGL1-/-). To observe the distribution of P-selectindeficient cells and PSGL-1deficient cells in strings, we labeled cells with two different fluorescent dyes. Strings were formed from cells of the two genotypes, P-selectin null macrophages (green) and PSGL-1 null macrophages (red) (Fig. 1 A, inset, P-/- + PSGL1-/-). Thus, PSGL-1 and P-selectin appear to be the major ligandreceptor pair mediating secondary interactions between resident peritoneal macrophages under these conditions.
Resident macrophages isolated from the mouse peritoneum have surface P-selectin
To date P-selectin has been observed only in platelets and endothelial cells (Hsu-Lin et al., 1984; Stenberg et al., 1985; Berman et al., 1986; Bonfanti et al., 1989; McEver et al., 1989). We used flow cytometry to confirm the presence of P-selectin on peritoneal leukocytes. Monoclonal rat antiP-selectin antibodies bound peritoneal leukocytes (Fig. 2 A) indicating that the mouse peritoneum contains a population of P-selectinpositive leukocytes in the absence of an inflammatory stimulus. In control experiments with peritoneal macrophages from P-selectindeficient mice, no cell labeling was observed.
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To confirm that the P-selectinpositive cells were macrophages, we observed their morphology by light microscopy. Peritoneal cells were isolated and the P-selectinpositive and negative fractions separated on a FACsorterTM with the aid of P-selectin antibodies. The total cell population (Fig. 2 C, left) and the fractionated cells were stained with Wright-Giemsa stain and analyzed by light microscopy. P-Selectinpositive cells had the morphological features of macrophages (Fig. 2 C, center), whereas P-selectinnegative cells appeared to be lymphocytic or plasmacytic in origin (Fig. 2 C, right). Thus, the P-selectinpositive cells isolated from the peritoneal cavity of mice appear to be macrophages.
We examined lysates of peritoneal leukocytes by Western blot analysis to confirm the presence of P-selectin and to determine its molecular weight. The P-selectin in peritoneal leukocytes was compared with P-selectin in lysates from mouse platelets and P-selectin purified from human platelets. The results of this analysis confirmed the presence of P-selectin on mouse peritoneal macrophages (Fig. 2 D). AntiP-selectin antibodies stained bands of 140,000 molecular weight in platelet lysates and in peritoneal leukocyte lysates from wild-type and PSGL-1deficient mice. P-Selectin m-RNA was also identified in peritoneal leukocytes from wild-type mice, but not P-selectin null mice, by Northern blot analysis (Fig. 2 E).
L-selectin is expressed on neutrophils, monocytes, and most lymphocytes, whereas PSGL-1 is expressed on essentially all blood leukocytes. We explored whether L-selectin and PSGL-1 were coexpressed with P-selectin on the surface of peritoneal macrophages. A subset of P-selectin bearing peritoneal macrophages also express L-selectin, whereas essentially all of the P-selectin bearing macrophages also express PSGL-1 (Fig. 2 F). P-Selectin bearing peritoneal macrophages express the integrins MAC-1 and VLA-1, important for monocyte trafficking (Fig. 2 F).
P-Selectin is synthesized in the peritoneal macrophage
P-Selectin is present on the surface of activated platelets and activated endothelial cells (Hsu-Lin et al., 1984; Stenberg et al., 1985; Sims et al., 1988; Bonfanti et al., 1989; McEver et al., 1989). We explored whether peritoneal macrophage P-selectin might arise from contamination with activated platelets or platelet-derived or endothelial cellderived microparticles bound to the PSGL-1 bearing leukocytes. Neither a platelet-specific marker, GPIIb, nor an endothelial marker, VE-cadherin, is observed on the surface of peritoneal leukocytes by flow cytometry (Fig. 3 A). Similarly, mRNA for GPIIb or VE-cadherin was not detectable in peritoneal leukocytes by RT-PCR analysis of peritoneal cells (Fig. 3 B). However, using RT-PCR we were able to amplify a portion of P-selectin cDNA including the coding sequence from the lectin domain to the transmembrane domain, indicating that macrophage-associated P-selectin is the membrane bound and not the soluble form of the protein (Fig. 3 C; Johnston et al., 1990). We did not detect any mRNA for von Willebrand factor, which resides within the same intracellular compartments as P-selectin in endothelial cells and platelets (Fig. 3 C; Bonfanti et al., 1989).
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P-Selectin undergoes internalization in macrophages
To address more precisely where P-selectin and PSGL-1 localize within peritoneal macrophages, we stained unfixed cells with antiP-selectin antibodies labeled with Texas red and antiPSGL-1 antibodies labeled with Alexa 488. Confocal microscopic analyses of stained macrophages show only small areas of colocalization of P-selectin and PSGL-1 (Fig. 4 A). Staining for PSGL-1 appears primarily on the periphery of the cell. P-Selectin is localized primarily in granule-like structures that appear to be beneath the cell surface although some P-selectin was observed on the cell surface. The granular appearance of the staining for P-selectin suggested that much of the P-selectin might be internalized.
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Incubation of peritoneal leukocytes with FITC-conjugated dextran results in colocalization of P-selectin with FITC-dextran particles, a fluid phase marker. Colocalization is likely within endocytic vesicles and lysosomes (Fig. 4 D).
In electron micrographs of cell sections from macrophages incubated at 0°C before fixation, P-selectin detected with nanogold-conjugated antibodies is localized to the external face of the plasma membrane and to the internal face of the membrane of intracellular vesicles (Fig. 5 A). Gold particles were not observed on the peritoneal macrophages of P-selectindeficient mice. In cell sections from macrophages incubated at 37°C, P-selectin is also detected in intracellular compartments (Fig. 5, B and C). To evaluate the nature of these compartments, cells stained with antiP-selectin and antiLAMP-1 antibodies were examined by confocal microscopy. Confocal images show that P-selectin is partially colocalized with the lysosomal marker, LAMP-1, in macrophages (Fig. 5 D).
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Macrophages are activated through a two-stage mechanism, a priming stage and a triggering stage (Adams and Hamilton, 1987). We determined the level of P-selectin surface expression on peritoneal macrophages after 24 h of stimulation with IFN, LPS or IFN
, and LPS. The extent of macrophage stimulation was determined by measuring secretion of TNF
into the cell supernatant. Activation of resident peritoneal macrophages with either agent or the combination of agents did not alter the level of P-selectin on the macrophage plasma membrane (Fig. 6). In contrast, the secretion of TNF
was significantly stimulated after activation of macrophages with LPS with or without IFN
.
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Discussion |
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Here, we demonstrate that peritoneal macrophages isolated from normal mice synthesize and express a functional form of P-selectin. This is the first example of a leukocyte that produces P-selectin. In contrast to platelets and endothelial cells, peritoneal macrophage P-selectin is constitutively expressed on the plasma membrane. We have demonstrated the presence of P-selectin on the cell surface by flow cytometry, by fluorescence and electron microscopy. However, distribution of P-selectin and PSGL-1 on the cell surface, as analyzed by fluorescence microscopy, reveals that, although there is some colocalization of the two proteins, PSGL-1 appears on the cell surface membrane, whereas most of the P-selectin is intracellular.
In cells with regulated secretory granules sequences within the cytoplasmic tail of P-selectin target this protein to the storage granules (Disdier et al., 1992; Koedam et al., 1992). Sequences within the C1 and C2 domains of the P-selectin cytoplasmic tail have been implicated in delivery of P-selectin to secretory granules during expression in heterologous cells (Modderman et al., 1998; Blagoveshchenskaya et al., 1999). After the stimulation of endothelial cells, P-selectin is redistributed to the plasma membrane from storage granules and then rapidly internalized (Subramanian et al., 1993; Steiadi et al., 1995; Hattori et al., 1989). When P-selectin is expressed in heterologous cells lacking regulated secretory granules, it is transported to the cell surface and then rapidly endocytosed for degradation (Green et al., 1994; Blagoveshchenskaya et al., 1998b). The cytoplasmic tail of P-selectin contains a lysosomal targeting signal in the C1 domain and lysosomal avoidance signals in the C2 domain (Blagoveshchenskaya et al., 1998a,b). Together these signals regulate P-selectin transport among early and late endosomes and lysosomes (Blagoveshchenskaya et al., 1998a). These same sequences within the P-selectin cytoplasmic tail likely also regulate distribution of the protein in peritoneal macrophages. In the absence of secretory granules, macrophages direct P-selectin to the plasma membrane. However, P-selectin is found primarily in vesicular compartments within peritoneal macrophages. Colocalization with dextran particles places P-selectin in endosomes, whereas colocalization with LAMP-1 places P-selectin in lysosomes. These results reflect the pathways of P-selectin expression and transport established in model systems of cells lacking regulated secretory organelles. Although de novo P-selectin synthesis is observed in endothelial cells after cytokine stimulation, macrophage activation does not appear to alter P-selectin surface expression.
Secondary leukocyteleukocyte interactions under flow conditions have been demonstrated previously for monocytes flowing over surfaces coated with P-selectin, E-selectin, or TNF-treated endothelial cells (Lim et al., 1998) and for neutrophils flowing over surfaces coated with E-selectin, P-selectin, L-selectin, PNAd, VCAM-1, cytokine-stimulated endothelial cells, and PSGL-1 or neutrophil monolayers (Alon et al., 1996; Walcheck et al., 1996). L-selectin has been demonstrated to mediate the secondary interactions resulting in string-like cell formations for both monocytes and neutrophils under hydrodynamic shear stress of 1.53.0 dynes/cm2 (Alon et al., 1996; Walcheck et al., 1996; Lim et al., 1998). PSGL-1 has been demonstrated to be the counter ligand for this interaction for both neutrophils and monocytes (Walcheck et al., 1996; Lim et al., 1998). However, macrophages are not found in blood but in the afferent lymph. The shear stress in the lymphatic system is much less than that in blood. Shear stress above a threshold level is critical for the optimal interaction of L-selectin with its ligands (Finger et al., 1996). At 0.7 dynes/cm2 wall shear stress, neutrophils flowing over a P-selectincoated surface do not form string-like formations; secondary accumulation of neutrophils does not occur at this wall shear stress as this is an L-selectinmediated interaction (Alon et al., 1996). Failure to observe a contribution of L-selectin to peritoneal macrophage string formation in our system may be a result of insufficient wall shear stress to induce L-selectinPSGL-1 binding. In contrast, P-selectin does not require shear above a critical threshold to promote and maintain interactions with its counterreceptor (Finger et al., 1996).
The functional significance of our observation that peritoneal macrophages express both the receptor and the counterreceptor, P-selectin and PSGL-1, remains unknown. We have demonstrated that at low temperature, when P-selectin internalization is slowed, homotypic peritoneal macrophage aggregates form (Fig. 7). It is possible that under certain physiologic conditions, P-selectin expression on the macrophage surface is stabilized. The presence of P-selectin and PSGL-1 on the same cell would potentially yield macrophage aggregates within the peritoneum. These peritoneal macrophages would not only adhere to each other but also to neutrophils, monocytes, and subsets of T lymphocytes. These cell aggregates may be part of a host defense system that clears the peritoneum of bacteria or other microbial particles. These aggregates may nucleate the formation of granulomas or possibly participate in the formation of giant cell granulomas.
Alternatively, P-selectin on the macrophage surface may be important in the immune response. Specifically, P-selectin and PSGL-1 may represent a ligand pair that contributes to the binding energy between macrophages and T lymphocytes. The interaction of antigen presenting cells with the lymphocyte T cell receptor complex is of low affinity, and several other ligand pairs are known to support both cellcell interaction as well as cell signaling in the immunologic synapse (Lee et al., 1998; Grakoui et al., 1999). It remains plausible that in the case of peritoneal macrophageT lymphocyte interaction, P-selectin on the macrophage participates in recognition and binding of T cell PSGL-1. LPS-induced up-regulation of P-selectin mRNA has been observed in both human dendritic cells and in Kupffer cells (Essani et al., 1995; Baltathakis et al., 2001) suggesting the potential for similar mechanisms in these cells.
In summary, these results suggest a functional role for the dual appearance of P-selectin and PSGL-1 on the peritoneal macrophage. These adhesion molecules may be important for microbial defense or for antigen presentation and immune response. In vivo experiments using genetically modified mice will offer an approach to testing these hypotheses.
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Materials and methods |
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Reagents
Rat antibodies specific to murine PSGL-1 (2PH1), P-selectin (RB40.34), L-selectin (MEL-14), CD41 (MWReg30), CD11b (M1/70), CD49D (MFR4.B), CD2 (RM25), and polyclonal rabbit antiP-selectin antibodies were obtained from BD Biosciences. F4/80 antibodies were obtained from Serotec Ltd. Goat antiVE-cadherin antibodies (C19) were obtained from Santa Cruz Biotechnology, Inc. Colloidal gold-conjugated goat antirat antibodies (10 or 18 nm particles) were obtained from Jackson ImmunoResearch Laboratories. Rabbit antiP-selectin cytoplasmic tail antibodies were prepared as described previously (Chong et al., 1994). The soluble form of mouse VCAM-1 was from R&D Systems. The soluble form of recombinant human PSGL-1, a gift from Genetics Institute (Andover, MA), was characterized in our laboratory (Croce et al., 1998). P-Selectin was purified from human platelets (Larsen et al., 1992). Texas red was obtained from Molecular Probes. Wright-Giemsa solution and low endotoxin BSA were obtained from Sigma-Aldrich.
Isolation of resident leukocytes from mouse peritoneum
Mice were killed and their peritoneal contents collected by flushing with 10 ml of ice-cold PBS containing 2 mM EDTA and 10 U/ml of heparin. Total leukocyte counts were determined using a counter (model T890; Beckman Coulter).
Macrophage binding studies
25 µl mouse recombinant VCAM-1 in 50 mM sodium bicarbonate containing 100 mM NaCl, pH 8.5, at a final concentration of 5 µg/ml was coated on a polystyrene dish and incubated overnight at 4°C. The dish was washed three times with PBS and blocked with BSA (20 mg/ml in PBS) for 2 h at 37°C. A VCAM-1coated plate was assembled in a parallel plate laminar flow chamber (Glycotech) and mounted on the stage of an inverted phase-contrast microscope (model Labovert FS; Leitz). To generate the desired shear stress, the outlet of the flow chamber was connected to an automated syringe pump (Harvard Apparatus).
Cells were prepared according to Chen et al. (1999). Resident leukocytes harvested from mouse peritoneum were washed twice with cation-free HBSS containing 2 mg/ml BSA, 2 mM EDTA, and 10 mM Hepes, pH 7.4. Cells were resuspended in the same buffer to a final concentration 107 cells/ml and kept at 4°C until use. For laminar flow assays, peritoneal cells were diluted in a binding buffer (HBSS containing BSA and where indicated, EDTA) to the desired concentration and perfused through the flow chamber at the indicated shear stress. Cell interactions with the surface-bound ligands were visualized with a 10x objective and videotaped using a digital color video camera (model SSC-S320; Sony) and a video recorder (model HS-U790; Mitsubishi).
String formation by peritoneal macrophages mediated by secondary macrophagemacrophage interactions was studied on VCAM-1coated substrate. Peritoneal cells (106 cells/ml) were perfused at 0.5 dyn/cm2 shear stress over a surface coated with VCAM-1. After 5 min of perfusion, free-floating cells were washed away and several fields with adherent cells were recorded for further analysis. A string was defined as three or more leukocytes aligned in the direction of flow and that were not separated by more than 10 µm. String formation was quantitated as the percentage of leukocytes in strings compared with the total number of adherent cells in the field of view.
Flow cytometric analysis and FACsortingTM
Peritoneal cells were isolated, washed, and resuspended in 200 µl of FACS® solution. After incubation for 5 min with 5 µl of anti-CD16/CD32 antibodies to minimize nonspecific binding, cells were stained for 30 min at 4°C with fluorescently labeled antibodies to the indicated cell surface antigens and analyzed by flow cytometry.
Purification of peritoneal leukocytes by FACsortingTM based on P-selectin expression was performed using a FACStar PlusTM cell sorter. Resident peritoneal cells, 107, were incubated for 30 min at 4°C with 20 µg/ml FITC-conjugated rat antiP-selectin antibodies in FACS® solution.
Fluorescent microscopy
For flow studies, peritoneal cells were labeled with Texas redconjugated F4/80 antibodies or with calcein. After washing, labeled cells were mixed at a 1:1 ratio to a final concentration of 106 cells/ml. Cells were perfused for 5 min over the VCAM-1coated surface. Adherent cells were washed with PBS, fixed for 15 min with 4% PFA solution, and examined by fluorescent microscopy. Excitation was at either 488 or 621 nm on a fluorescence microscope (model AX70; Olympus) equipped with a 40x water immersion objective.
For confocal microscopy, live or fixed Leucoperm permeabilized cells were stained with 20 µg/ml of antigen-specific mAbs directly conjugated to Texas red, Alexa 488, or FITC. After staining for 40 min at the indicated temperature, cells were washed twice with PBS at 4°C and examined microscopically using an 100x oil immersion objective.
Northern blot and RT-PCR analysis of gene expression
Peritoneal cells (8 x 106) in RPMI medium were plated on a 60 x 15-mm tissue culture dish. After a 2-h incubation, nonadherent cells were removed by washing with 2 mM EDTA in PBS. Total RNA from adherent cells (>98% macrophages) was isolated using TRIzol reagent (Life Technologies). Northern blots were performed using the NorthernMax-Gly system according to the manufacturer's protocol (Ambion). The P-selectin probe corresponding to exon 3 of P-selectin was radiolabeled by PCR using [32P]dCTP incorporation.
For RT-PCR analysis, RNA from total mouse peritoneal leukocytes, lung, and bone marrow was isolated and the mRNA reverse transcribed using oligo (dT)1218 primers and SuperScript Rnase H- reverse transcriptase (Life Technologies). The resulting cDNAs were used for amplification of mouse proteins. The gene-specific primers were: vWF, forward (26472673), 5'-CTTGGAGCTATTGCAGGCAGAGGAATG-3' and reverse (41634137), 5'-AGGCAGATCTCATACCTGAAAGGGTTC-3'; P-selectin, forward (311333), 5'-GGACCTGGGTGGGAACAAATAAG-3' and reverse (21702145), 5'-TTGTAGAAGCCACTGCACCACCCAAG-3'; GPIIb forward (720749), 5'-ATTGAGAACATCATCTCCACGTACCGC-3' and reverse (998972), 5'-CGAGTGCCCGAAATATGAAGCCATCTG-3'; VE-cadherin, forward (224249), 5'-CGCTGCCCCACTATGTGAAAGATCAG-3' and reverse (452427), 5'-TTGACAGTGAAGCTGGAAGGTTGTTC-3'.
Western blot analysis
500 µl of mouse blood was diluted twice with HBSS and 2 mM EDTA. Platelet-rich plasma was isolated by centrifugation at 400 g for 7 min. The platelets from platelet-rich plasma were washed twice with PBS and treated with lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM NaF, 10 mM sodium phosphate, and 10 mM sodium pyrophosphate) containing protease inhibitors (benzamidine, aprotinin, leupeptin, pepstatin A, and PMSF). Mouse peritoneal cells were treated with lysis buffer. The proteins from the cell lysates were separated under the reducing conditions on 7% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked for 2 h with 3% BSA in PBS and rabbit antibodies specific for P-selectin cytoplasmic tail were added to a final concentration of 10 µg/ml. After 2 h of incubation, the membrane was washed and bound antibodies were detected using goat antirabbit IgG conjugated to peroxidase.
Internalization assay
Freshly isolated peritoneal cells were first incubated for 40 min at 0°C in FACS® solution to inhibit endocytosis. After incubation, cells were stained with FITC-labeled monoclonal antiP-selectin antibodies or isotype-matched control IgG for 30 min at the indicated temperature. After washing with PBS at 4°C, half of the sample was incubated for 2 min in PBS, pH 7.0, at 4°C. The other half of the sample was treated with low pH buffer (500 mM NaCl, 0.2 N acetic acid, pH 2.3). The optimal pH for stripping antibody from the cell surface was determined using 10 µm polystyrene beads (Polysciences Inc.) with covalently bound mouse P-selectin. After incubation at low and neutral pH, cells were washed twice with PBS at 4°C and the mean fluorescence was determined by flow cytometry on a FACSCaliberTM after gating on the macrophage population.
EM
Peritoneal leukocytes were washed twice with FACS® solution and incubated for 30 min at 0°C with rat antiP-selectin antibodies. The cells were incubated for 30 min at either 0°C or 37°C with goat anti-IgG on 10- or 18-nm gold particles. Cells were washed twice with PBS and fixed for 15 min in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. Fixed cells were pelleted and resuspended in 5% low gelling temperature sea plaque agarose. Agarose blocks were fixed after in 1% osmium tetroxide in cacodylate buffer for 1 h and stained enbloc with 2% uranyl acetate in water overnight. Blocks were washed, dehydrated, infiltrated in Epon 812 propylene oxide, and polymerized for 48 h at 60°C. 80-nm sections were placed on formavir-coated nickel grids, counterstained with uranyl acetate and lead citrate, and viewed on an electron microscope (model JEM 100CX; JEOL).
Cell aggregation studies
Leukocytes (2.5 x 105) isolated from the peritoneum of wild-type mice, P-selectin null mice, or PSGL-1 null mice were resuspended in 50 µl of HBSS containing 2 mM CaCl2. Cells were incubated at the indicated temperature in the presence or absence of 10 µg/ml blocking antibodies or 5 mM EDTA for 1 h. Aggregates were observed and quantified by phase-contrast microscopy using a 10x objective. Aggregates were defined as containing two or more cells.
Measurement of P-selectin expression and TNF production in stimulated macrophages
Resident peritoneal cells were incubated on dishes for 4 h. Nonadherent cells were removed by washing and adherent macrophages were cultured with 20 ng/ml IFN and/or 100 ng/ml LPS for 24 h. Adherent cells were harvested by scraping dishes and stained with FITC-labeled rat antiP-selectin antibodies or FITC-labeled isotype matched control IgG. Stained cells were analyzed on a FACSCaliburTM flow cytometer. Concentrations of TNF
in the culture supernatants were determined by ELISA (R&D Systems).
Metabolic radiolabeling
CHO cells stably transfected with human P-selectin (2 x 107) or 5 x 107 resident peritoneal macrophages from wild-type or P-selectindeficient mice were grown in 150 µCi/ml [35S]methionine/cysteine containing DME supplemented with 10% FCS for 14 h. Cells were washed three times with cold PBS, and adherent macrophages and CHO cells harvested by scrapping. Cells were sedimented by centrifugation and lysed in buffer containing 1% Triton X-100, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.02% NaN3, and protease inhibitors. After a 1-h incubation at 4°C, cell lysates were clarified by centrifugation at 10,000 g for 15 min. Supernatants were precleared with protein GSepharose after a 4-h incubation with 10 µg/ml rabbit nonimmune IgG. Cell lysates were incubated overnight at 4°C with rabbit antibodies directed against the cytoplasmic tail of P-selectin (5 µg/ml). Immunoprecipitates were collected by incubation of cell lysates with protein GSepharose (Amersham Biosciences) for 4 h at 4°C. Supernatant was removed and the Sepharose beads washed three times with lysis buffer containing 500 mM NaCl. Bound protein was eluted from Sepharose beads by incubation with SDS sample buffer containing mercaptoethanol. Eluted proteins were separated by SDS-PAGE. Autoradiographs were analyzed using an imager (model Typhoon 9400; Amersham Biosciences).
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Acknowledgments |
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We thank Drs. Takako Hirata and Paul Gottlieb for helpful discussions about these studies.
Support for this work was provided by grants from the National Heart, Lung, and Blood Institute.
Submitted: 16 October 2003
Accepted: 4 November 2003
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References |
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Adams, D.O., and T.A. Hamilton. 1987. Molecular transductional mechanisms by which IFN gamma and other signals regulate macrophage development. Immunol. Rev. 97:527.[Medline]
Alon, R., R.C. Fuhlbrigge, E.B. Finger, and T.A. Springer. 1996. Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow. J. Cell Biol. 135:849865.[Abstract]
Baltathakis, I., O. Alcantara, and D.H. Boldt. 2001. Expression of different NF-kappaB pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling. J. Cell. Biochem. 83:281290.[CrossRef][Medline]
Barth, M.W., J.A. Hendrzak, M.J. Melnicoff, and P.S. Morahan. 1995. Review of the macrophage disappearance reaction. J. Leukoc. Biol. 57:361367.[Abstract]
Bellingan, G.H., H. Caldwell, S.E. Howie, I. Dransfield, and C. Haslett. 1996. In vivo fate of inflammatory macrophages during the resolution of inflammation: inflammatory macrophages do not die locally, but emigrate to the draining lymph nodes. J. Immunol. 157:25772585.[Abstract]
Berman, C.L., E. Yeo, J.D. Wencel-Drake, B.C. Furie, M.H. Ginsberg, and B. Furie. 1986. A platelet alpha granule membrane protein that is incorporated into the plasma membrane during activation. Characterization and subcellular localization of PADGEM protein. J. Clin. Invest. 78:130137.[Medline]
Blagoveshchenskaya, A.D., E.W. Hewitt, and D.F. Cutler. 1998a. A balance of opposing signals within the cytoplasmic tail controls the lysosomal targeting of P-selectin. J. Biol. Chem. 273:2789627903.
Blagoveshchenskaya, A.D., J.P. Norcott, and D.F. Cutler. 1998b. Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail. J. Biol. Chem. 273:27292737.
Blagoveshchenskaya, A.D., E.W. Hewitt, and D.F. Cutler. 1999. A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells. J. Cell Biol. 145:14191433.
Bonfanti, R., B.C. Furie, B. Furie, and D.D. Wagner. 1989. PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells. Blood. 73:11091112.[Abstract]
Chen, C., J.L. Mobley, O. Dwir, F. Shimron, V. Grabovsky, R.R. Lobb, Y. Shimizu, and R. Alon. 1999. High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow. J. Immunol. 162:10841085.
Chong, B.H., B. Murray, M.C. Berndt, L.C. Dunlop, T. Brighton, and C.N. Chesterman. 1994. Plasma P-selectin is increased in thrombotic consumptive platelet disorders. Blood. 83:15351541.
Croce, K., S.J. Freedman, B. Furie, and B. Furie. 1998. Interaction between soluble P-selectin and soluble P-selectin glycoprotein ligand 1: equilibrium binding analysis. Biochemistry. 37:1647216480.[CrossRef][Medline]
Disdier, M., J.H. Morrissey, R.D. Fugate, D.F. Bainton, and R.P. McEver. 1992. Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol. Biol. Cell. 3:309321.[Abstract]
Essani, N.A., G.M. McGuire, A.M. Manning, and H. Jaeschke. 1995. Differential induction of mRNA for ICAM-1 and selectins in hepatocytes, Kupffer cells and endothelial cells during endotoxemia. Biochem. Biophys. Res. Commun. 211:7482.[CrossRef][Medline]
Finger, E.B., K.D. Puri, R. Alon, M.B. Lawrence, U.H. von Andrian, and T.A. Springer. 1996. Adhesion through L-selectin requires a threshold hydrodynamic shear. Nature. 379:266269.[CrossRef][Medline]
Gerszten, R.E., Y.C. Lim, H.T. Ding, K. Snapp, G. Kansas, D.A. Dichek, C. Cabanas, F. Sanchez-Madrid, M.A. Gimbrone, Jr., A. Rosenzweig, and F.W. Luscinskas. 1998. Adhesion of monocytes to vascular cell adhesion molecule-1-transduced human endothelial cells: implications for atherogenesis. Circ. Res. 82:871878.
Grakoui, A., S.K. Bromley, C. Sumen, M.M. Davis, A.S. Shaw, P.M. Allen, and M.L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science. 285:221227.
Green, S.A., H. Setiadi, R.P. McEver, and R.B. Kelly. 1994. The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J. Cell Biol. 124:435448.[Abstract]
Hamburger, S.A., and R.P. McEver. 1990. GMP-140 mediates adhesion of stimulated platelets to neutrophils. Blood. 75:550554.[Abstract]
Haskill, S., and S. Becker. 1985. Disappearance and reappearance of resident macrophages: importance in C. parvum-induced tumoricidial activity. Cell. Immunol. 90:179189.[Medline]
Hattori, R., K.K. Hamilton, R.D. Fugate, R.P. McEver, and P.J. Sims. 1989. Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J. Biol. Chem. 264:77687771.
Hirata, T., G. Merrill-Skoloff, M. Aab, J. Yang, B.C. Furie, and B. Furie. 2000. P-Selectin glycoprotein ligand 1 (PSGL-1) is a physiological ligand for E-selectin in mediating T helper 1 lymphocyte migration. J. Exp. Med. 192:16691676.
Hsu-Lin, S.C., C. Berman, B.C. Furie, D. August, and B. Furie. 1984. A platelet membrane protein expressed during activation and secretion: studies using a monoclonal antibody specific for thrombin-activated platelets. J. Biol. Chem. 259:91219126.
Johnston, G.I., G.A. Bliss, P.J. Newman, and R. McEver. 1990. Structure of the human gene encoding granule membrane protein-140, a member of the selectin family of adhesion receptors for leukocytes. J. Biol. Chem. 265:2138121385.
Koedam, J.A., E.M. Cramer, E. Briend, B. Furie, B.C. Furie, and D.D. Wagner. 1992. P-Selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells. J. Cell Biol. 116:617625.[Abstract]
Larsen, E., A. Celi, G. Gilbert, B.C. Furie, J. Erban, R. Bonfanti, D.D. Wagner, and B. Furie. 1989. A receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell. 59:305312.[Medline]
Larsen, G.R., D. Sako, T.J. Ahern, M. Shaffer, J. Erban, S.A. Sajer, R.M. Gibson, D.D. Wagner, B.C. Furie, and B. Furie. 1992. P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities. J. Biol. Chem. 267:1110411110.
Lee, K.M., E. Chuang, M. Griffin, R. Khattri, D.K. Hong, W. Zhang, D. Stgraus, L.E. Samuelson, C.B. Thompson, and J.A. Bluestone. 1998. Molecular basis of T cell inactivation by CTLA-4. Science. 282:22632266.
Lim, Y.C., K. Snapp, G.S. Kansas, R. Camphausen, H. Ding, and F.W. Luscinskas. 1998. Important contributions of P-selectin glycoprotein ligand-1-mediated secondary capture to human monocyte adhesion to P-selectin, E-selectin and TNF--activated endothelium under flow in vitro. J. Immunol. 161:25012508.
Luscinskas, F.W., G.S. Kansas, H. Ding, P. Pizcueta, B.E. Schleiffenbaum, T.F. Tedder, and M.A. Gimbrone. 1994. Monocyte rolling, arrest and spreading on IL-4 activated vascular endothelium under flow is mediated via sequential action of L-selectin, ß1-integrins, and ß2-integrins. J. Cell Biol. 125:14171427.[Abstract]
Luscinskas, F.W., H. Ding, P. Tan, D. Cumming, T.F. Tedder, and M.E. Gerritsen. 1996. L- and P-selectins, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF-alpha-activated vascular endothelium under flow in vitro. J. Immunol. 157:326335.[Abstract]
McEver, R.P., J.H. Beckstead, K.L. Moore, L. Marshall-Carlson, and D.F. Bainton. 1989. GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J. Clin. Invest. 84:9299.[Medline]
Modderman, P.W., E.A. Beuling, L.A. Govers, J. Calafat, H. Janssen, A.E. Von dem Borne, and A. Sonnenberg. 1998. Determinants in the cytoplasmic domain of P-selectin required for sorting to secretory granules. Biochem. J. 336:153161.[Medline]
Moore, K.L., and L.F. Thompson. 1992. P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells. Biochem. Biophys. Res. Commun. 186:173181.[Medline]
Moore, K.L., N.L. Stults, S. Diaz, D.F. Smith, R.D. Cummings, A. Varki, and R.P. McEver. 1992. Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells. J. Cell Biol. 118:445456.[Abstract]
Randolph, G.J., S. Beaulieu, S. Lebecque, R.M. Steinman, and W. Muller. 1998. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science. 282:480483.
Randolph, G.J., K. Inaba, D.F. Robbiani, R.M. Steinman, and W.A. Muller. 1999. Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity. 11:753761.[Medline]
Rosen, H., and S. Gordon. 1990. Adoptive transfer of fluorescence-labeled cells shows that resident peritoneal macrophages are able to migrate into specialized lymphoid organs and inflammatory sites in the mouse. Eur. J. Immunol. 20:12511258.[Medline]
Sako, D., X.J. Chang, K.M. Barone, G. Vachino, H.M. White, G. Shaw, T. Veldman, K.M. Bean, T.J. Ahern, B. Furie, et al. 1993. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell. 75:11791186.[Medline]
Sato, K., Y. Imai, and T. Irimura. 1998. Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity. J. Immunol. 161:68356844.
Sims, P.J., E.M. Faioni, T. Wiedmer, and S.J. Shattil. 1988. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in membrane receptor for coagulation factor Va and express prothrombinase activity. J. Biol. Chem. 263:1820518212.
Sminia, T., M. Soesatyo, M. Ghufron, and T. Thepen. 1995. The migration of peritoneal cells towards the gut. Adv. Exp. Med. Biol. 371A:6165.
Steiadi, H., M. Disdier, S.A. Green, W.M. Canfield, and R.P. McEver. 1995. Residues throughout the cytoplasmic domain affect the internalization efficiency of P-selectin. J. Biol. Chem. 270:2681826826.
Stenberg, P.E., R.P. McEver, M.A. Shuman, Y.V. Jacques, and D.F. Bainton. 1985. A platelet -granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell Biol. 101:880886.[Abstract]
Subramanian, M., J.A. Koedam, and D.D. Wagner. 1993. Divergent fates of P- and E-selectins after their expression on the plasma membrane. Mol. Biol. Cell. 4:791801.[Abstract]
Thepen, T., E. Caassen, K. Hoeben, J. Breve, and G. Kraal. 1993. Migration of alveolar macrophages from alveolar space to paracortical T cell area of the draining lymph node. Adv. Exp. Med. Biol. 329:305310.[Medline]
van Furth, R., and Z.A. Cohn. 1968. The origin and kinetics of mononuclear phagocytes. J. Exp. Med. 128:415435.[Medline]
van Vugt, E., M. van Pelt, R.H. Beelen, and E.W. Kamperidjk. 1995. Migration of rat dendritic cells and macrophages from the peritoneal cavity to the parathymic lymph nodes. Adv. Exp. Med. Biol. 378:163167.[Medline]
Walcheck, B., K.L. Moore, R.P. McEver, and T.K. Kishimoto. 1996. Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. J. Clin. Invest. 98:10811087.
Weller, A., S. Isenmann, and D. Vestweber. 1992. Cloning of the mouse endothelial selectins. Expression of both E- and P- selectin is inducible by tumor necrosis factor alpha. J. Biol. Chem. 267:1517615183.
Yang, J., T. Hirata, K. Croce, G. Merrill-Skoloff, B. Tchernychev, E. Williams, R. Flaumenhaft, B.C. Furie, and B. Furie. 1999. Targeted gene disruption demonstrates that PSGL-1 is required for P-selectin mediated but not E-selectin mediated neutrophil rolling and migration. J. Exp. Med. 190:17691782.