* Department of Cell and Molecular Biology, BMC, Lund University, Sweden; and
Division of Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada
Correspondence: Hans Tapper, Department of Cell and Molecular Biology, BMC, B14, Tornavägen 10, SE-22184 Lund, Sweden. E-mail: hans.tapper{at}medkem.lu.se
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and ß1 in adherent plasma membranes.
Key Words: secretion • exocytosis • inflammation • cellular activation
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RIIA and FcRIIIB [1
], and this interaction can trigger a degranulation response [2
]. Engagement of these receptors leads to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by src-related protein tyrosine kinases such as Hck and Lyn [3
, 4
]. Phosphorylation of two such YXXL motifs, typically located 10–12 amino acids apart in the intracytoplasmic tails, mediates association of proteins containing SH2 domains such as the protein tyrosine kinase syk. Activation of this kinase, which contains tyrosines that are phosphorylated upon binding to ITAMs, leads to propagation of the signaling cascade to several downstream pathways, which leads eventually to production of diacylglycerol [5
] and initiation of Ca2+-transients [6
], causing, in turn, the activation of protein kinase C (PKC). Neutrophils harbor at least four distinct types of granules that differ in regard to content, membrane proteins, and their propensity to be secreted [7
]. The secretion of neutrophil granules can be triggered by an increase in intracellular Ca2+, but additional signals are involved in the regulation of azurophilic degranulation [8
9
10
], and a role for G-proteins, tyrosine kinases, and PKC has been proposed [11
, 12
]. Neutrophil functions, such as phagocytosis and migration, are dependent on remodeling (and polymerization) of actin filaments [13
]. Fc receptor-triggered reorganization of actin filaments could possibly determine localization of a secretory response through their association with various signaling molecules. In this study, the spatial distribution of actin filaments during neutrophil adhesion was analyzed to evaluate a potential role in determining the localization of azurophilic degranulation. A highly localized azurophilic degranulation targeted toward forming phagosomes has been shown to occur recently in neutrophils phagocytosing IgG-opsonized zymosan [14 ]. It is not fully understood what determines the localization of this secretory response. The target membrane might be rendered "fusogenic" as a result of an enrichment of proteins that are part of a fusion complex, or alternatively, a spatially restricted production of second messengers could act to direct vesicle movement toward the target site where degranulation occurs. We devised a system to study the localization of tentative signaling molecules involved in the regulation of degranulation triggered by adhesion to surfaces coated with immune complexes.
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, 
, and 
were purchased from Transduction Laboratories (Lexington, KY). Antiphosphotyrosine mAb (G410) was bought from Upstate Biotechnology (Lake Placid, NY). Donkey serum and secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). GF109203X was purchased from Calbiochem (La Jolla, CA). FM1-43, ProLong, and rhodamine phalloidin were purchased from Moleular Probes (Eugene, OR). Paraformaldehyde was from J.B. EM Services Inc. (Dorval, Quebec). Dextran T500, Ficoll, Sepharose 4B, and cyanogen bromide-preactivated Sepharose 4B were purchased from Pharmacia Biotech (Uppsala, Sweden). All other chemicals including RPMI 1640 and 4-methyl umbelliferyl N-acetyl-ß-D-glucosaminide (NAG) were purchased from Sigma Chemical Co. (St. Louis, MO). Human IgG was a kind gift of the Department of Immunology at the Hospital for Sick Children (Toronto, Canada).
Buffers
All experiments were performed in medium containing 127 mM NaCl, 5.6 mM glucose, 5.4 mM KCl, 1.2 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, and 10 mM HEPES. The pH was adjusted to 7.4 using NaOH, and the medium was filtered before use. Coupling buffer contained 0.5 M NaCl and 50 mM NaHCO3, and pH was adjusted to 8.0 using NaOH. Acetate buffer contained 0.5 M NaCl and 5 mM Na acetate, and pH was adjusted to 4.0 using acetic acid. Borate buffer contained 0.5 M NaCl and 0.56 mM Na borate, and pH was adjusted to 8.0 by addition of boric acid.
Preparation of neutrophils
Dextran (9 ml; 4.5%) was added to 60 ml fresh blood from healthy donors. After 45 min, the top layer was centrifuged at 1500 rpm for 5 min. The pellet was resuspended in phosphate-buffered saline (PBS) and layered on top of 4 ml Ficoll. After centrifugation for 20 min at 3000 rpm, the pellet was resuspended in 0.5 ml HEPES-buffered RPMI. Any remaining erythrocytes were lysed by incubation with NH4Cl at 37°C for 10 min. Neutrophils were then washed twice in RPMI and counted in a Coulter counter. Neutrophils were kept on rotation at a concentration of 107 neutrophils/ml RPMI until experiments were performed.
Preparation of heat-aggregated IgG (HAIgG) and coating of beads
HAIgG was prepared by heating human IgG for 1 h at 63°C. CnBr-activated sepharose beads were washed three times in 1 mM HCl and three times in coupling buffer (pH 8.0). HAIgG (10 mg) was diluted in coupling buffer, pH 8.0, to a final volume of 8 ml. Diluted HAIgG was mixed with 800 mg washed sepharose, and coupling was performed at room temperature under constant rotation for 2 h. The coated sepharose was washed three times in coupling buffer and rotated for 2 h at room temperature in 10 ml coupling buffer containing 1 M ethanolamine to block remaining, noncoupled sites on the sepharose beads. A two-step washing procedure was then performed with acetate buffer, pH 4.0, followed by borate buffer, pH 8.0. This was repeated three times. The coated sepharose was rotated in borate buffer overnight, washed, and resuspended in borate buffer to a final concentration of 100 mg sepharose per ml borate buffer. The sepharose was stored at 4°C for not more than 1 week before use. The coated sepharose was always washed three times in experimental medium before use.
Immunofluorescence and confocal microscopy
Cells adherent to sepharose beads were fixed in 1.6% paraformaldehyde for 15 min on ice, followed by 45 min at room temperature. When imaging phosphorylated tyrosine and actin filaments, cells were permeabilized using ice-cold permeabilization buffer containing 0.1% Triton X-100, 100 mM PIPES (pH 6.8), 5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 100 mM KOH, and 2 mM MgCl2 for 5 min. When studying tyrosine phosphorylation or degranulation of specific or azurophilic granules, cells were washed three times in PBS and then blocked with 5% donkey serum for 1 h. After two washes in PBS, cells were incubated for 4 h with the primary antibody in PBS containing 1% bovine serum albumin (BSA). Cells were then washed three times and incubated for 1 h with a secondary Cy3-conjugated antibody in PBS containing 1% BSA. After final washing steps, all buffer was replaced by ProLong mounting medium, and the stained preparations were mounted on coverslips. Confocal microscopy was performed using a Leica TCS confocal microscope.
Quantification of localized versus nonlocalized degranulation
Microscopy slides were prepared as described above where exocytosis of azurophilic and specific granules was detected by antibodies against CD63 and CD66b, respectively, in nonpermeabilized cells. Light microscopy (Differential Interference Contrast) was used for selection of intact cells that were firmly attached to beads. Thereafter, the distribution of the markers for azurophilic and specific degranulation was analyzed further in these adherent cells. Degranulation was scored as localized to the adherent aspect of the cell when >75% of the staining was in the adherent membrane. Otherwise, the degranulation was scored as occurring in a nonlocalized way. Similar results were obtained by three different observers.
Enzyme assay for NAG
In these experiments, Na medium was supplemented with 10 µM aprotinin, 10 µM leupeptin, and 1 µM pepstatin A, and experiments were performed in culture dishes containing 24 wells. HAIgG-sepharose (40 mg) in 0.5 ml Na medium was sedimented in a culture-dish well on ice. Neutrophils (2x106) in 0.5 ml Na medium were added, and the cells were allowed to sediment for 10 min on ice. The culture dish was then placed in a 37°C water-bath for various times to allow cell adhesion. Adhesion was terminated by placing the dish on ice. Cells and beads were resuspended and placed in an Eppendorf tube and spun down, and the medium was collected. The pellet was then resuspended in Na medium containing 0.1% Triton-X. This solution was also used to wash off any remaining cells from the culture dish. Triton-X was added to the medium sample to a final concentration of 0.1%, and samples were kept on ice until enzyme measurements were performed. The substrate buffer contained 0.1 mg/ml 4-methyl umbelliferyl NAG and 0.3 M citric acid, pH 4.5. Substrate buffer (1.6 ml) was heated to 37°C and mixed with 0.4 ml sample, and analysis was performed at 37°C. Where pharmacological inhibitors were used, the cells were preincubated with the inhibitor at 37°C for 30 min (erbstatin or genistein) or 10 min (GF109203X). Enzyme release (%) was calculated as enzyme activity in the medium divided by the total activity in the medium and cell lysate.
Separation of adherent plasma membrane by sonication
Neutrophils (50x106) were suspended in 150 µl experimental medium and mixed with 60 mg HAIgG-sepharose on ice. After incubating cells and beads for 1 min at 37°C to allow adhesion, cross-linking was performed at 4°C for 15 min and at room temperature for 45 min in 1 ml PBS containing 2 mM 3,3'-dithio-bis(propionic acid H-hydroxysuccinimide ester) and 10% dimethyl sulfoxide. Nonadherent cells were washed away by centrifugating three times at 100 rpm for 10 min in PBS containing 5 mM ethylenediaminetetraacetate (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin A. Beads were then resuspended in 1 ml ice-cold PBS and sonicated repeatedly for 10 s in a Fisher sonic dismembrator model 300 until all adherent cells were broken, and only plasma membrane patches remained on the beads (verified by repeated inspection of FM1-43-labeled samples in a fluorescence microscope). The sepharose was then pelleted, and the supernatant containing the ripped-off cells was collected. This was repeated three times by washing the sepharose in 20 times diluted PBS containing 5 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin A. The ripped-off cells were sonicated further three times for 10 s in a Heat Systems Ultrasonic Processor XL sonicator, nuclei, and intact cells were spun down for 5 min at 3000 rpm, and membranes were retrieved by ultracentrifugation for 1 h at 200,000 g. The pellet was resuspended in 200 µl Laemlli sample buffer containing 50 mM dithiothreitol (DTT) and boiled for 15 min, and DNA was spun down. Supernatants were passed 10 times through a thin-gauged syringe. Boiling 2x concentrated Laemlli sample buffer (200 µl) containing 100 mM DTT was added to samples containing beads with adherent plasma membranes. These were then boiled for 15 min and passed through a syringe with a layer of glass fiber wool to remove the sepharose. The final volume was approximately 400 µl as a result of fluid from the beads. Samples were separated on 7.5% gels, transferred onto polyvinylidene difluoride membranes, and probed with antibodies. Protein bands were scanned, and densities were analyzed using NIH imaging software. We used two different approaches to determine the percent of total plasma membranes present in the adherent patch to estimate the relative enrichment of signaling molecules in fractions containing adherent membranes. In the first approach, cell-surface proteins were biotinylated prior to adhesion. Neutrophils were spun down and resuspended in 2 ml ice-cold PBS (pH 7.8) containing 200 µg/ml succinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin). The cells were then washed in two steps. In each step, the cells were incubated for 10 min on ice in PBS containing 1% BSA to block residual biotin. Such treatment has been shown not to activate neutrophils [15
, 16
]. Experiments were then performed as described, and membrane pools were collected and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotin was labeled using avidin-horseradish peroxidase (HRP) at a 1:5000 dilution for 1 h. A distinct band at approximately 150 kDa, visualized on a Western blot using avidin-HRP, was present in adherent and ripped-off samples. Protein densities were measured as described above, and the presence of biotinylated 150 kDa protein in adherent samples as percentage of total contents (adherent plus ripped-off samples) was calculated. Of the total amount of 150 kDa protein, 24.9 ± 2.9% (SEM, n=6) was present in the adherent samples. In the second approach, neutrophils were pretreated with tritiated 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) before adhesion. Aliquots from adherent plasma membranes and nonadherent plasma membranes were taken before running SDS-PAGE and quantified in a scintillation counter. Of the plasma membrane, 28.1 ± 2.4% (SEM, n=5) and was adhered to beads as estimated by amount of bead-associated, DIDS-labeled proteins. Values from the latter approach were used to normalize protein content in adherent and nonadherent pools for plasma membrane content.
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Figure 1 A shows neutrophils adherent to HAIgG-sepharose after 60 s incubation at 37°C. Neutrophils did not adhere nonspecifically to noncoated sepharose (Fig. 1C) . Further, adhesion to HAIgG-coated sepharose was almost eliminated totally in the presence of soluble IgG (10 µg/ml; Fig. 1B ).
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Figure 1. Adhesion of neutrophils to immune complex-coated sepharose beads. Neutrophils and beads were incubated for 60 s at 37°C in Na medium. (A and B) Beads were coated with HAIgG. (B) The medium contained soluble IgG (10 µg/ml). (C) Noncoated beads were used. Images were captured on Kodak slide film (ASA 400) using a Nikon F-601M camera mounted on a Nikon diaphot TMD-inverted microscope (40x objective, phase contrast). Slides were scanned in a Nikon slide-scanner.
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Figure 2. Release of azurophilic-granule content during neutrophil adhesion to immune complexes. Neutrophils were incubated with HAIgG-sepharose (•) or noncoated sepharose ( ) in Na medium at 37°C. NAG enzyme activity was measured in a Hitachi F-4000 fluorescence spectrophotometer with wavelengths for emission = 365 nm, and excitation = 460 nm. This experiment was repeated three times, and one representative experiment is shown.
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Figure 3. Time-course and spatial distribution of azurophilic- and specific granule exocytosis during adhesion to immune complexes. Confocal immunofluorescence cross-sections (1 µm) show CD63 staining of nonpermeabilized neutrophils (B, D, and F). The corresponding DIC images are shown (A, C, and E). (H) CD66b staining of nonpermeabilized cells; (G) the corresponding DIC image. Neutrophils were allowed to adhere to HAIgG-sepharose for 15 s (A and B), 30 s (C and D), or 60 s (E–H) at 37°C in Na medium.
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Table 1. Localization of Degranulation
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Figure 4. Organization of actin filaments and phosphorylation of tyrosine during neutrophil degranulation. Neutrophils were adhered to HAIgG-sepharose for 60 s at 37°C in Na medium. (B) CD63 staining of nonpermeabilized cells; (D) rhodamine-phalloidin staining of permeabilized cells; (F) phosphorylated-tyrosine staining of permeabilized cells. The corresponding DIC images are shown (A, C, and E). (G) Western blot of neutrophils adhered to HAIgG-sepharose for 0 or 5 min at 37°C in Na medium. Tyrosine-phosphorylated proteins were detected using a mAb (PY99). Cells in the two right-most lanes were pretreated with genistein for 10 min at 37°C. Neutrophils were presented to beads as in experiments where release of NAG was measured.
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Figure 5. Effect of cytochalasin B on localization of azurophilic degranulation and phosphorylation of tyrosine. Neutrophils were pretreated with 10 µM cytochalasin B for 20 min and then adhered to HAIgG-sepharose for 60 s at 37°C in Na medium. (B) CD63 staining of nonpermeabilized cells; (D) rhodamine-phalloidin staining of permeabilized cells; (F) phosphorylated-tyrosine staining of permeabilized cells. The corresponding DIC images are shown (A, C, and E).
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Table 2. Effect of Inhibitors on NAG Secretion
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and ß1 were enriched in the adherent plasma membrane after 60 s of adhesion (Fig. 8)
. The ß1 isoform was slightly more enriched in the adherent plasma membrane (2.38±0.63, SE, n=11) than the isoform (1.98±0.46, SE, n=8). Conversely, PKC was distributed evenly after 60 s of adhesion, with a relative enrichment in the adherent plasma membrane of 1.07 ± 0.12 (SE, n=7). Also the atypical isoform, , was found not to be enriched in the adherent plasma membrane (Fig. 8)
.
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Figure 6. Removal of nonadherent membrane by sonication. Neutrophils were adhered to HAIgG-sepharose for 60 s in Na medium at 37°C followed by cross-linkage and sonication as described in Materials and Methods. Cells were stained with 5 µM FM1-43. (A) Intact cell; (B) cell after sonication. Images were captured using a Leica DM IRB fluorescence microscope equipped with a 100x oil-objective and a Princeton-cooled CCD camera.
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Figure 7. Purity of sonicated, adherent plasma membranes. Neutrophils were adhered to HAIgG-sepharose for 60 s in Na medium at 37°C. EM images of an intact neutrophil adherent to HAIgG-coated sepharose (A; original magnification, 4000x) and adherent plasma membrane after sonication (B; original magnification, 12,000x). (B, Inset) Higher magnification of the plasma membrane lipid bilayer. Cells were fixed for 12 h with 4% glutaraldehyde at 4°C, and samples for EM were 90 nm thick.
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Figure 8. Syk and PKC in adherent and nonadherent plasma membrane. Western blots of adherent (P) and nonadherent (S) plasma membrane fractions from neutrophils adherent to HAIgG-sepharose in Na medium for 60 s at 37°C. Syk and PKC , ß1, , and were detected using primary antibodies and secondary HRP-labeled antibodies. The bar graph shows relative enrichment of syk and PKC , ß1, , and in the adherent plasma membrane compared with the rest of the plasma membrane (see Materials and Methods for calculations).
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For degranulation to occur, granules must translocate toward the target and then dock and fuse with the plasma membrane. In a model where a nonspecific, centrifugal movement of granules is triggered, localization of degranulation would depend on local accumulation of fusion proteins. In an alternative model, where the whole plasma membrane is accessible for docking and fusion, localization of degranulation would depend on guided movement of vesicles. We refined a technique to quantify the accumulation of signaling molecules in the adherent plasma membrane as compared with the nonadherent plasma membrane. An unequal distribution of a signaling molecule in the plasma membrane would indicate a possible role for such a protein in localization of degranulation.
Fc receptor-mediated stimulation of neutrophils with immune complexes results in a rapid tyrosine phosphorylation of several proteins [1
, 31
], and involvement of tyrosine phosphorylation in the triggering of degranulation has also been proposed in several other cell types [32
33
34
]. Signal propagation from cross-linked Fc receptors occurs by tyrosine phosphorylation of their ITAM motives [21
], and a role for members of the src family of tyrosine kinases in this event has been proposed [3
]. Phosphorylated ITAMs can associate with several different tyrosine kinases containing SH2 domains, and activation of FcRIIA in neutrophils and several other cell types induces the association, phosphorylation, and activation of the tyrosine kinase syk [24
]. Our finding that the release of azurophilic-granule content triggered by immune complexes was reduced by inhibitors of tyrosine kinases is in agreement with the central role of tyrosine phosphorylations in Fc receptor-mediated signaling and degranulation. Immunofluorescence data presented here show that tyrosine phosphorylation occurred mainly in the vicinity of the adherent plasma membrane, with a staining pattern similar to the azurophilic degranulation. Inhibition of actin-filament assembly diminished the accumulation of phosphorylated tyrosine but did not affect its localization. In our system, syk was enriched in the adherent plasma membrane after 60 s of adhesion. Our system did not exclude granules from the fraction containing a nonadherent plasma membrane. The enrichment of syk in the adherent plasma membrane might be an underestimation, because part of the syk content in the nonadherent plasma membrane could originate from granules. It is possible that altered distribution of syk or other proteins recruited to Fc receptor-associated multiprotein complexes could provide a localizing signal for azurophilic degranulation.
Fc-receptor activation by immune complexes results in elevated, cytosolic Ca2+ and production of diacylglycerol [35
36
37
], and conventional isoforms of PKC are activated by diacylglycerol in a Ca2+-sensitive manner. Release of azurophilic-granule content was inhibited by pretreatment with GF109203X, an inhibitor specific for PKC that does not discriminate between isoforms [38
]. At least 12 different isoforms of PKC have been described of which neutrophils express the , ßI, ßII, , and isoforms [39
]. Besides differential modes of activation, the isoforms of PKC translocate to specific, subcellular compartments upon activation [40
]. PKC has been detected in preparations of early phagosomes in monocytes [41
], and PKC ßII, , and have been shown to translocate to membranes in neutrophils stimulated with opsonized zymosan [42
]. The isoforms of PKC are primarily located in the cytosol in resting neutrophils, but the isoforms ßII, , and have also been detected in granular fractions [42
]. In neutrophils adhering to immune complexes, we found PKC , ßI, , and in the plasma membrane. The conventional isoforms and ßI accumulated in the adhering part of the plasma membrane, whereas isoforms and were not enriched at this site. It can be noted that a slight shift in electrophoretic mobility of some PKC isoforms upon activation has been described earlier [43
, 44
]. A possible granular location of PKC isoforms would lead to an undererstimation of the enrichment of PKC in the adherent aspect of the cells, because the enrichment was normalized using plasma membrane markers. Our data and several recent studies describe translocation of PKC to subcellular locations where azurophilic degranulation takes place [41
, 45
]. The inhibition of azurophilic degranulation by GF109203X and the enrichment of the conventional isoforms and ßI of PKC observed in the present study indicate a role for PKC in triggering and possibly also in localizing azurophilic degranulation.
Received May 6, 2001; revised December 5, 2001; accepted December 6, 2001.
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and C
upon mitogenic stimulation of quiescent rat 3Y1 fibroblasts J. Biol. Chem. 269,17495-17501This article has been cited by other articles:
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