Shape F-actin and surface morphology changes during chemotactic peptide-induced polarity in human neutrophils.код для вставкиСкачать
THE ANATOMICAL RECORD 241519-528 (1995) Shape, F-Actin, and Surface Morphology Changes During Chemotactic Peptide-Induced Polarity in Human Neutrophi Is EDUARDO FERNANDEZ-SEGURA, JOSE M. GARCfA, JUAN L. SANTOS, AND ANTONIO CAMPOS Departamento de Biologia Celular e Histologia, Facultad de Medicina, Uniuersidad de Granada (E.F.S., J.M.G., A.C.), Seruicio de Inmunologia, Hospital Clinic0 Universitario (J.L.S.), Granada, Spain ABSTRACT Background: The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cellular polarization induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in human neutrophils in suspension. Methods: Neutrophil shape changes induced by lo-' M fMLP were observed with DIC microscopy. Size and cellular granularity were analyzed by flow cytometry measuring their forward and side scattered light. To visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and confocal laser scanning microscopy. Cell surface morphology was assessed with scanning electron microscopy (SEM). Results: The stimulation of round-smooth neutrophils with nanomolar concentrations (lo-' M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized by 1)increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cytoplasmic localization to the cell periphery, and 3) rapid reorganization of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identified with SEM after stimulation of roundsmooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell. Conclusions: These findings show the relation between reorganization of the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils. o 1995 Wiley-Liss, Inc. Key words: Neutrophils, N-formyl peptide, Cell polarity, Flow cytometry, F-actin, Confocal microscopy, Scanning E M Polymorphonuclear neutrophils are recruited to inflammatory sites by a variety of soluble mediators (chemoattractants) that stimulate neutrophil migration (chemotaxis). Two types of chemoattractants can be distinguished: exogenous (i.e., N-formyl peptides), and endogenous (C5a, LTB,, PAF, IL-8) (Harvath, 1991). The N-formyl peptides have been extensively studied as probes of neutrophil activation because these peptides stimulate a variety of biochemical and physiological responses in addition to chemotaxis. The cellular response begins when this signal molecule interacts with its specific receptor, formyl peptide receptor (FPR), on the plasma membrane of neutrophils. This induces a series of responses coordinated a t the 0 1995 WILEY-LISS, INC biochemical and cellular levels: activation of pertussis toxin-sensitive G protein and increased activity of phospholipase C, which induces the formation of inosito1 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate increases intracellular free Ca2+, and diacylglycerol activates protein kinase C (PKC) (for a review, see Omann et al., 1987). These signaling Received February 17, 1994; accepted September 16, 1994. Address reprint requests to Prof. Antonio Campos, Departamento de Biologia Celular e Histologia, Facultad de Medicina, Universidad de Granada, E-18071 Granada, Spain. 520 E. FERNANDEZ-SEGURA pathways interact with each other to initiate or enhance a wide range of neutrophil activities, including initiation of the respiratory burst leading to superoxide production, degranulation, up-regulation of receptors, and cell shape changes with projections of membrane ruffles and pseudopodia. These cell shape changes are correlated with dynamic changes in the organization of the cytoskeleton, mainly in relation with microfilamentous actin (F-actin), the major cytoskeletal element in neutrophils. Exposure of neutrophils to N-formyl peptides generates transient actin nucleation activity, rapid polymerization of F-actin followed by slower depolymerization (Howard and Meyer, 1984; Howard and Oresajo, 1985; Howard and Wang, 1987; Watts et al., 1991), F-actin cross-linking activity, and incorporation of actin-binding proteins (Niggli and Jenni, 1989; Fujimoto and Ogawa, 1990). These processes give rise to morphological and functional polarization of the cytoplasm and plasma membrane of neutrophils. Previous reports have indicated the spatial reorientation of microtubules (Anderson et al., 1982), centrosomes (Schliwa et al., 1982), and membrane proteins such as FPR (Sullivan e t al., 1984), concanavalin A-binding sites (Weinbaumn et al., 1980), and Fc and C3b receptors (Davis et al., 1982; Petty et al., 1989; Pytowski et al., 1990; Walter et al., 1980). However, conflicting results were reported when cells adherent to a substrate were studied. Previous studies have shown that the same morphological cell polarization process occurs in suspension, after the exposure of human neutrophils to a n isotropic concentration of N-formyl peptides in the absence of a gradient or attachment to a substrate (Davis et al., 1982; Shields and Haston, 1985). Although most studies have examined the kinetics of chemotactic peptideinduced changes in the content and distribution of F-actin (Coates et al., 1992; Howard and Meyer, 1984; Howard and Oresajo, 1985; Howard and Wang, 1987; Lepedi et al., 1992; Wallace et al., 1984; Watts et al., 1991), little is known about the relationship between temporal changes in the cell shape, F-actin and cell surface morphology. Moreover, detailed morphologic analyses were not reported in any of these investigations. Consequently, the purpose of the present study was to analyze the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during the cellular polarization induced by fMLP in human polymorphonuclear leukocytes. To examine the relation between F-actin reorganization and changes in cell shape and surface morphology, we used spherical-smooth neutrophils stimulated in suspension. DMSO, and stored frozen at -20°C in aliquots until use. FITC-phalloidin stock solution was prepared as 3.3 x lop6 M in methanol and was stored at -20°C. The HBSS used in polarization assays was supplemented with 25 mM Hepes, 1.2 mM calcium chloride, and 1.0 mM magnesium chloride. Cell Preparation Human peripheral blood was obtained from healthy volunteer donors by venipuncture and collected into heparin (20 U/ml). Heparinized blood was mixed with HBSS without Ca2+ and Mg2+,pH 7.2, and was sedimented in 6% Dextran 70 in 0.9% sodium chloride for 1 h. The leukocyte-rich plasma was layered on a FicollPaque density gradient and centrifuged at 400g for 30 min. Cells were washed (3 times, 400g, 5 min) and resuspended in HBSS without Ca2+ and Mg2+ at a concentration of 1 x lo6 cell/ml. The contaminating red blood cells were lysed with distilled water for 20-30 sec. Isolated neutrophils were washed and resuspended in HBSS/Hepes buffer with Ca2+ and Mg2+. All the above procedures were carried out under sterile conditions. Cell preparations contained >95% neutrophils as detected by Giemsa staining and immunofluorescence flow cytometry. Neutrophil viability was >95% by trypan blue exclusion. Cells were used for polarization assays within 1h after isolation. To avoid the effects of cell purification on neutrophil response to fMLP stimulation, we used total leukocyte suspensions for polarization assays. Briefly, the blood was immediately treated twice for 30 sec with distilled water to lyse the erythrocytes. The leukocytes were washed twice in HBSS without Ca2+ and Mg2+, and resuspended in HBSS/Hepes buffer with Ca and Mg2+ a t a final concentration of 1 x lo6 cell/mI. + Neutrophil Polarization Assays Neutrophils (5 x lo5 cell/ml) were incubated in suspension with M fMLP in HBSS/Hepes buffer with Ca2+ and Mg2+ in a water bath at 37°C. After fMLP had been added, 250 pl of cell suspension was removed and fixed with 2.5% glutaraldehyde in 0.1 M buffer phosphate, pH 7.2, a t room temperature, at the times indicated. Cells were fixed overnight at 4"C, then washed (3 times) with PBS, pH 7.2. Control cells were incubated with 0.01% DMSO in HBSS/Hepes buffer with Ca2+ and Mg2+. Shape Analysis Glutaraldehyde-fixed neutrophils were mounted on slides and coverslips and cell shape changes were deMATERIAL AND METHODS termined with differential interference contrast miChemicals croscopy (DIC, Nomarski optics, Zeiss Axiphot). Also, Hanks' balanced salt solution (HBSS), Hepes, Dex- cell shape was defined by morphological parameters, tran T-70, N-formylmethionyl-leucyl-phenylalanine such as perimeter, area, and shape factor (SF). The SF (fMLP), poly-L-lysine (PLL), Triton X-100, and fluores- is a measure of the deviation from a circle of the shape cein (F1TC)-phalloidin were obtained from Sigma of the cell, with a prefect circle resulting in a SF of 1.0, Chemicals Company (St. Louis, MO). Ficoll-Paque was whereas elongation results in a decrease in SF. The obtained from Pharmacia Fine Chemicals (Uppsala, value of SF was determined according to the equation Sweden). Phosphate buffered saline (PBS) was pur- SF = 4 x 7~ x area/(perimeter)2.Cell outlines were chased from Flow Laboratories (Irvine, Scotland). Glu- traced using a camera lucida drawing tube fitted to a taraldehyde EM grade and dimethylsulfoxide (DMSO) Leitz microscope with a x 100 oil immersion objective were obtained from Merck (Darmstadt, Germany). A on a digitizing tablet. The tracings were analyzed on a stock solution of fMLP was prepared as M in personal computer with a n image analysis program. fMLP INDUCES POLARITY IN NEUTROPHILS Each morphological parameter was measured in a t least 50 cells a t the times indicated. Flow Cytometry Single cell suspensions of fixed neutrophils, stimulated with chemotactic peptide, were analyzed according to their size and cellular granularity by measuring their forward (narrow angle) and side (right angle) scattered light, respectively. Steady state analysis was performed on a n Ortho Cytoron Absolute flow cytometer (Ortho Diagnostics Systems, Raritan, NJ) with excitation at 488 nm using a 5 W argon ion laser. In total leukocyte suspensions, neutrophils were easily discriminated from lymphocytes and monocytes by their characteristics forward and side scattered light. Frequency histograms were obtained by analyzing 2,000 cells at the times indicated. Data are expressed as the mean channel number of the histograms. Fluorescence and Confocal Microscopy FITC-phalloidin was used to stain F-actin in neutrophils. After stimulation with lo-' M fMLP, cell suspensions were fixed by adding equal volumes of 0.2% glutaraldehyde EM grade (final concentration 0.1%) in PBS, pH 7.2, for 10 min at room temperature. After fixation, neutrophils were washed with 0.05 M glycine in PBS, pH 7.2, for 15 min at 4°C in order to quench aldehyde groups. The cells were washed, and then permeabilized by incubation with 0.1% buffered Triton X-100 for 5 min at room temperature, followed by washing with PBS, pH 7.2, prior to incubation with 1.65 x M FITC-phalloidin for 20 min at room temperature in the dark. Neutrophils were then washed with PBS, pH 7.2, dropped onto PLL-precoated glass coverslips, and mounted on slides with PBS/glycerol. For conventional fluorescence microscopy, the samples were visualized in a Leitz Laborlux 12 epifluorescence microscope. For confocal microscopy, the samples were observed in a Zeiss 310 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). Photographs were taken using Kodak Tri-X 400 film. Scanning Electron Microscopy Glutaraldehyde-fixed cells were washed, and then dropped onto PLL-precoated coverslips overnight a t 4°C. The cells were dehydrated in a graded series of alcohols, critical-point dried (CPD30, Balzers Union, Liechtenstein) with COs, and gold-sputter coated (E5000, Polaron, UK). The samples were visualized in a Philips SEM 505 scanning electron microscope (Philips, Eindhoven, Holland) at 20-30 kV. Micrographs were made using Polaroid type 52 film. RESULTS Shape of fMLP Stimulated Neutrophils Human isolated neutrophils incubated with lo-' M fMLP showed rapid, time-dependent shape changes (Fig. 1A). Cellular perimeter and area increased, and SF decreased significantly as a result of polarization (Fig. lB,C). Examination with DIC microscopy showed that >90% of the unstimulated (time 0) neutrophils were round-smooth (Fig. 2A). Polar neutrophils were not observed. After 30 sec to 2 min of stimulation, the cells were rounded with small surface projections (Fig. 2B). After 5 min of chemotactic peptide stimulation, 521 the neutrophils were irregularly shaped, with large surface projections. Some cells were elongated, with clear signs of polarization. After incubation with fMLP for 10 min, most neutrophils displayed a polar shape with front-tail polarity (Fig. 2C). Control cells showed no morphological changes. We also analyzed the size and cellular granularity of chemotactic peptide-stimulated neutrophils by cytometric measurements of forward (size) and side scattered light (cellular granularity). Stimulation with lo-' M fMLP increased the size of neutrophils in a time-dependent manner, in comparison with control cells (Fig. 3). The isotropic concentration of chemoattractants induced little change in the cellular granularity of stimulated neutrophils as compared with control cells. Figure 4 illustrates the response of neutrophils in total leukocyte suspensions after stimulation with lo-' M fMLP. The changes were similar to those seen with purified neutrophils. fMLP Induces F-actin Reorganization in Neutrophils F-actin was stained with FITC-phalloidin in glutaraldehyde-fixed cells. Fixation with paraformaldehyde induced the formation of blebs on the plasma membrane, and cell lysis of chemotactic peptide-stimulated neutrophils (not shown). Exposure of neutrophils in suspension to lo-' M fMLP induced a dramatic, timedependent rearrangement of F-actin, a s seen with conventional fluorescence and confocal microscopy. The use of confocal microscopy made it possible to observe optical sections of individual cells and thus improved the resolution of fluorescence imaging. In unstimulated neutrophils, F-actin was distributed diffusely and homogeneously throughout the cytoplasm (Fig. 5A). Within 30 sec after the addition of fMLP, F-actin was redistributed to form a peripheral submembranous ring (Fig. 5B). After 2 min of exposure to chemotactic peptide, actin microfilaments were redistributed as focal aggregates at the cell periphery (Fig. 5C). Five minutes after the addition of chemotactic peptide, F-actin was asymmetrically redistributed near the pseudopods (Fig. 5D). After 10 min of stimulation, FITC-phalloidin stained neutrophils displayed a strongly fluorescent ruffled lamellipodium and a weakly fluorescent tail (Fig. 5E). fMLP Induces Changes in Cell Surface Morphology of Neutrophils In an attempt to identify and correlate the time-dependent reorganization of cell surface morphology of neutrophils after fMLP stimulation with changes in cell shape and F-actin reorganization observed by flow cytometry and confocal microscopy, we used SEM. The morphological polarization of neutrophils in suspension was accompanied by a time-dependent redistribution of the cell surface. Neutrophils of five different shapes were identified by SEM: round-smooth, roundridged, round-ruff led, nonpolar ruff led, and polarized cells. Round-smooth cells displayed a spherical smooth surface with few short microvilli and microridges (Fig. 6A). Round-ridged cells were spherical with ridges and short microvilli on cell surface (Fig. 6B). Round-ruffled cells were characterized by a rough surface with welldeveloped ridges and folds (Fig. 6C). Nonpolar ruffled cells displayed a n asymmetric rough surface with ruf- 522 E. FERNANDEZ-SEGURA 100 90 . 80 1BI 70 70 % 3 60 60 50 m f 50 40 40 30 0.90 . ’ 30’ 20 t 0.80 0.70 0.60 0 2 4 6 8 1 0 1 2 Time (min) Fig. 1. A: Time-course of shape changes of neutrophils in suspension after stimulation with lo-’ M fMLP. Cell shape was determined in cells fixed with 2% glutaraldehyde and observed using Nomarsky optics. Non-spherical, irregular shaped cells with surface projections plus front-tail polarized cells, 0 ; front-tail polarized cells, a. Results are given as percentages of cells that changed in shape (means 2 SD of ten separate experiments). B Time-course of changes in the area (m, 0 ) and perimeter ( 0 , 0 ) of neutrophils stimuIated with lo-’ M fMLP (closed symbols) and control cells incubated with 0.01% DMSO (open symbols). C: Time-course of changes in shape factor (SF),determined according to the equation SF = 4 X TI x areaiperimeter’. Neutrophils .; stimulated with [email protected] fMLP, control cells incubated with 0.01% DMSO, 0. Results are means i SD of three separate experiments with cells isolated from different donors. Fig. 2. Differential interference contrast (DIC) micrographs of the effect of stimulation with lo-’ M fMLP on neutrophil shape. After stimulation, neutrophils were fixed with 2% glutaraldehyde in suspension and then dropped onto PLL-coated coverslips. A, unstimuM fMLP for 2 lated neutrophils; B, Neutrophils stimulated with min; and C, for 10 min. Scale bar: 10 pm. 523 fMLP INDUCES POLARITY IN NEUTROPHILS 250 . 160 . .' 4P 120 200 80 40 0 150 v) I 3 100 L L"" 160 120 50 80 40 I . 50 . .. . 100 I 150 R T-S FW-SC 200 250 C RT-SC Fig. 3. Flow cytometry frequency histograms of changes in forward (FW-SC) and side (RT-SC) scatter parameters of chemotactic peptidestimulated neutrophils in suspension. Frequency histograms were obtained by analyzing 2,000 cells at the time indicated. Cells after the addition of lo-' M fMLP for 30 see (A,E), 2 rnin (B,F), 5 rnin (C,G), and 10 min (D,H). Unstimulated neutrophils (-); neutrophils stimulated with lo-' M fMLP (--). Histograms are representative of five separate experiments. Fig. 4. Dot mapping of changes in forward (narrow angle) and side (right angle) scatter parameters in neutrophils after 10 min of stimulation with lo-* M M L P in total leukocyte suspensions. Neutrophils were gated by their characteristic forward (FW-SC) and side (RT-SC) scattered light. Dot maps were obtained by analyzing 2,000 cells. A, unstimulated cells; B, cells stimulated with lo-' M fMLP. Note the increase in forward scatter in neutrophils (N). Lymphocytes (L) and monocytes (MI show no changes in scatter parameters. Plots are representative of three separate experiments. fles and folds (Fig. 6D). These cellular projections arose from two or three sites, giving the cell a multipolar appearance. Polar cells were elongated with front-tail polarity, i.e., ruffles and folds at the frontal pole, and a contracted rear end with a distinct tail knob at the posterior pole (Fig. 6E). The exposure of total leukocyte M fMLP during 10 min induced a suspensions to polar morphology in neutrophils, similar to that seen in purified neutrophils. Lymphocytes and monocytes showed no changes in cell shape (not show). Figure 7 illustrates the sequential modifications in the cell surface morphology of lo-' M fMLP stimulated neutrophils. Before stimulation, neutrophils were round-smooth (83%).Round-ridged neutrophils developed after 30 sec of chemotactic peptide stimulation (64.2%). Cells with ridges and folds were observed after 2 min (47.8%). The formation of nonpolar ruffled cells reached its peak at 5 min (56.8%), decreasing after 10 min of fMLP stimulation, whereas the proportion of polar cells increased after 2 min, and reached its peak after 10 rnin of exposure to fMLP (57.6%). Control cells, incubated with 0.01% DMSO, showed no changes in cell surface morphology. DISCUSSION In this report we analyzed the changes in cell shape, F-actin, and surface morphology that occur during cell polarization induced by nanomolar concentration (lo-' M) of fMLP in human neutrophils in suspension. Our results not only confirm earlier findings, but also provide new evidence of the relationship between the pattern of F-actin distribution and the different cell 524 E. FERNANDEZ-SEGURA Fig. 5. Confocal laser scanning microscopic images of the reorganization of F-actin in neutrophils stimulated with lo-' M fMLP in suspension. Neutrophils were fixed with 0.1%glutaraldehyde, permeabilized, stained with FITC-phalloidin, and dropped onto PLL-coated coverslips. F-actin distribution in unstimulated neutrophils (A) and in cells after the addition of lo-' M fMLP for 30 sec (B), 2 min ( C ) , 5 min (D), and 10 min (E). Each micrograph is representative of five separate experiments with cells isolated from different donors. Scale bar: 10 pm. shapes and surface features observed during the phases of polarization. These modifications are discussed below with reference to the signal transduction pathways in neutrophils t h a t may come into play as a result of chemotactic peptide stimulation. This model should provide a n appropriate morphological substrate to establish, with immunogold-labeling techniques and SEM, the topological distribution of antigenic receptor sites on cell surface domains throughout the polarization process (Fernandez-Segura et al., 1994) Purified neutrophils are able to reversibly bind chemoattractants, and are capable of responding to their continued presence after warming to physiological temperatures (English and Graves, 1992). Nevertheless, the different isolation procedures used to purify neutrophils may have caused cellular activation (Kuijpers e t al., 1991). Although the reasons for activation are unknown, possible factors are the exposure of cells to endotoxin and/or centrifugation on density gradients during cell preparation (Howard et al., 1990). Activation may influence cell surface morphology (Watts et al., 1991) and neutrophil response in vitro (Haslett et al., 1985), and hence may modulate the expression of surface molecules, such as Fc receptors, LFA-1, Mac-1, p150, CR1, or FPR (Kuipjers et al., 1991; Norgauer et al., 1991). However, our purification procedure did not affect cellular response, as shown by the findings with neutrophils in total leukocyte suspensions: after stimulation with lo-' M fMLP, the changes in cell shape were similar to those found with purified neutrophils. Our cell preparations of purified neutrophils contained no polarized cells, and more than 90% of the neutrophils were round as determined by morphometric analysis (e.g., shape factor) and flow cytometry; F-actin was diffusely distributed as determined by confocal microscopy. Previous studies with purified neutrophils under endotoxin-free conditions reported similar results (Howard et al., 1990). The neutrophil response to chemotactic peptide is sensitive to different isotropic concentrations (Shields and Haston, 1985). At low concentrations of fMLP, neutrophils rapidly acquire a polarized appearance upon activation. However, at high concentrations, neutrophils display a multipolar morphology (McKay et al., 1991). Isotropic concentrations of chemotactic peptide (lo-' M fMLP) induced the sequential development of polar cell shape, as we demonstrated by light microscopy and flow cytometry. We also found that stimulation with fMLP of neutrophils in suspension induced a sequence of cell shape changes, cytoskeletal F-actin reorganization, and topographic redistribution of cell surface features, which was different from the changes observed in neutrophils stimulated on substrate (Davis et al., 1982). Neutrophil adhesion to a substrate may alter the cellular response, leading to cellular activation, characterized by a rise in free intracellular Ca2' (Jaconi et al., 1991; Southwick et al., 1989), sustained burst of superoxide production (Ginis and Tauber, 19901, increased cytoskeleton-associated actin (F-actin) (Ginis et al., 1992), F-actin reorganization, and membrane ruffling (Boyles and Bainton, 1979). These adhesiondependent events are mediated by heterodimer membrane proteins-integrins-which link the cytoskeleton to the extracellular environment (Hynes, 1987). It therefore seems logical to assume that all modifications that occur in cell shape, F-actin, and cell surface morphology in adherent neutrophils after fMLP stimulation may be caused by a two-pronged effect: 1)activation in response to stimulation by the chemotactic peptide itself, and/or 2) activation a s a result of a transitory, cyclic process of adhesion-detachment on the substrate during cell polarization. However, the stimulation of neutrophils in suspension makes i t possible to observe changes in cell shape and cell surface morphology in the absence of other stimuli. These changes reflect only the effects of cytoskeletal F-actin reorganization during morphological polarization (Coates et al., 1992). SEM observations of neutrophils in suspension allowed us identify different time-dependent transitional cell shapes. Our results show that cell shape changes from round-smooth to polar after chemotacticpeptide stimulation in suspension progresses through three different intermediate types: round-ridged, round-ruffled, and non-polar ruffled cells. Each cell fMLP INDUCES POLARITY IN NEUTROPHILS 525 Fig. 6. Scanning electron micrographs of changes in cell surface morphology of neutrophils in suspension stimulated with lo-' M fMLP. A Cell surface morphology of unstimulated neutrophils. B: 30 sec after fMLP stimulation, round-ridged cell. C: 2 min after chemoattractant stimulation, round-ruffled cell. D 5 min after the addition of chemotactic peptide, nonpolar cell with ruffles and folds. E: Polarized front-tail cell. Each micrograph is representative of neutrophils from five separate experiments with cells isolated from different donors. Scale Bar: 1 km. shape showed a distinctive subcellular localization of F-actin. Under our isolation conditions, unstimulated neutrophils displayed a regular round-smooth appearance. Watts et al. (1991) reported basal, endotoxin-free neutrophils with a similar cell surface appearance in scan- ning EM observations. These results differ from earlier studies that showed typical unstimulated neutrophils after Ficoll-dextran sedimentation purification, bearing short ruffles and membrane folds (Davis et al., 1982; Hoffstein et al., 1982). This morphological phenotype corresponds to cells that were preactivated dur- E. FERNANDEZ-SEGURA 526 100 ~~ 90 = u) 70 h - T 60 -ln 0 s 50 40 30 20 10 0 0 0.5 2 5 Smooth cell Ridged cell Ruffled cell Nonpolar c e l l Polar c e l l 10 Time (min) Fig. 7. Kinetic analysis of neutrophil surface changes observed with scanning EM. Neutrophils were stimulated with M fMLP in suspension during the indicated times, fixed with 2% glutaraldehyde, and then dropped onto PLL-coated coverslips. Data are given as percentages of cells that changed in shape (means ? SD of five separate experiments). ing isolation procedures (Haslett et al., 1985; Howard et al., 1990; Watts et al., 1991). Two different F-actin pools have been described in unstimulated neutrophils: 1)a stable Triton-insoluble, gelsolin-poor F-actin pool representing 35-40% of total cellular F-actin, and 2) a labile, Triton-soluble, gelsolin-rich F-actin pool representing the 60-65% of total cellular F-actin (Watts and Howard, 1992). The spatial distribution of the two F-actin pools in the cytoplasm is different (Zaffran et al., 1993). Watts and Howard (1992) reported t h a t in formaldehyde-prefixed neutrophils, which retain both stable and labile actin pools, F-actin was diffusely distributed throughout the cytoplasm. Our results showed a similar subcellular distribution of F-actin labeled with FITC-phalloidin after glutaraldehyde fixation. These findings suggest that our basal cell preparations retained both F-actin pools. The first phase (<30 sec) of neutrophil stimulation by fMLP is characterized by a rapid, transient rise in free intracellular Ca2+, increased F-actin content (Howard and Meyer, 1984; Howard and Oresajo, 1985; Howard and Wang, 1987; Wallace et al., 1984), and spatial redistribution of F-actin (Coates et al., 1992; Haston, 1987; Lepedi et al., 1992). At this time of stimulation, we observed a subcortical localization of F-actin with both fluorescence and confocal microscopy. This reorganization of F-actin coincided with a rapid redistribution of actin-associated proteins to the cell surface upon cell activation. In ultrastructural studies with immunogold staining, Hartwig et al. (1989) reported a submembranous localization of gelsolin, and suggested that this actin-binding protein translocated short actin oligomers to the membrane surface. Furthermore, other authors have noted the rapid incorporation into the cytoskeleton of specific actin cross-linking proteins, such as a-actinin (Niggli and Jenni, 1989), which are implicated in the extension of surface projections. During the early phase of stimulation, we found that neutrophils displayed a rough-ridged cell surface under SEM. The development of ridges on the cell surface may be related with the rearrangement of actin cytoskeleton, i.e., F-actin and actin-associated proteins move to the cell periphery after exposure to chemoattractants. The transductional pathway involved in actin polymerization, F-actin distribution, and subsequent cell shape changes remains unclear, specifically with regard to the role of intracellular calcium (Zaffran et al., 1993). Recent studies showed that depletion and chelation of cytosolic free calcium have no effect on actin assembly (Howard and Wang, 1987), F-actin distribution, or membrane ruff ling in neutrophils stimulated with fMLP (Zaffran et al., 1993).However, free intracellular Ca2 may modulate the physical organization of F-actin (Omann et al., 1987). Round-ruff led and non-polar ruff led neutro hils, cell shapes that appear within 2-5 min after 10- M fMLP stimulation, showed a reorganization of F-actin in peripheral focal aggregates. Kinetic studies with flow cytometry and video image analysis reported that this asymmetric redistribution of F-actin coincides with the depolymerization phase of F-actin, and probably precedes the development of polar shape in neutrophils stimulated with fMLP in suspension (Coates et al., 1992; Lepedi et al., 1992; Watts e t al., 1991). The signal transduction pathways responsible for these various shapes are unknown. Previous studies showed that exposure of neutrophils to PKC activators (e.g., diacylglycerols or phorbol esters) induced reorganization of F-actin and membrane ruffling (Roos et al., 1987; Zimmermann et al., 1988; Downey et al., 1992) similar to the changes described in the present study. However, these chemotactic peptide-induced effects cannot be prevented by PKC inhibitors, such as H7, staurosporine, or calphostine C; thus neither the reorganization of F-actin nor pseudopod extension induced by fMLP appears to depend on PKC activation. Protein kinase C has been implicated in the maintenance of cell polarity, i.e., pseudopod extension and tail contraction (Niggli and Keller, 1993). In our study, after 10 min of stimulation with chemotactic peptide, most neutrophils displayed the typical polarized shape, with a broad, ruffled lamellipodium and a contracted, granular tail. Like other workers (Coates et al., 1992; Lepedi et al., 1992), we found that F-actin was distributed in two foci: a main focus in the anterior pole, and a less intense, secondary focus in the posterior end. Keller and Niggli (1993)reported the accumulation of a-actinin in the anterior pole. In contrast, fodrin (Fujimoto and Ogawa, 1990) and myosin (Keller and Niggli, 1993) were preferentially concentrated at the rear end of polarized neutrophils. The co-localization of F-actin and myosin may be related with the development of the contracted, granular tail. The reorganization of actin cytoskeleton and the cell surface morphological features after fMLP stimulation induced a n asymmetrical distribution of molecules on the plasma membrane. The causes of this differential reorganization of surface molecules are under debate. Some authors have suggested that the differences are caused by a n increase in the density of the molecules a t the anterior pole (Haston and Maggs, 1990; McKay et al., 1991; Sullivan et al., 1984; Walter et al., 1980; Weinbaum et al., 1980). Alternatively, they may result from the accumulation of plasma membrane in the front of polar cells (Petty et al., 1989; Pytowski et al., 1990). We hope that our findings will make it possible to analyze the expression, spatial distribution, and mobilization of different cell surface molecules linked to + r fMLP INDUCES POLARITY IN NEUTROPHILS the F-actin based cytoskeleton (e.g., integrins and selectins) during polarization and cellular motility, and will shed light on the role of these molecules during neutrophil-mediated cell functions. 527 tide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils. J . Cell Biol., 101t1078-1085. Howard, T.H. and D. Wang 1987 Calcium ionophore, phorbol ester, and chemotactic peptide-induced cytoskeleton reorganization in human neutrophils. J. Clin. Invest. 79t1359-1364. Howard, T.H., D. Wang, and R.L. Berkow (1990) Lipopolysaccharide ACKNOWLEDGMENTS modulates chemotactic peptide-induced actin polymerization in neutrophils. J . Leukoc. Biol., 47t13-24. This work was supported in part by the Direccion Hynes, R.O. 1987 Integrins: a family of cell surface receptors. Cell, General de Investigacion Cientifica y Tecnica (DGI48,549-554. CYT) through grant PM91-0114. The authors thank Jaconi, M.E.E., J.M. Theler, W. Schlegel, R.D. Appel, S.D. Wright, and P.D. Lew 1991 Multiple elevations of cytosolic-free Ca2+ in Dr. Navascues (Dept. of Cell Biology, Univ. of Granhuman neutrophils: initiation by adherence receptors of the inada) for providing DIC microscope, Dr. Garcia del tegrin family. J . Cell Biol., 112r1249-1257. Moral (Dept. of Pathology, Univ. of Granada) for use of Keller, H.U. and V. Niggli 1993 Colchicine-induced stimulation of PMN motility related to cytoskeletal changes in actin, a-actinin, the Ortho Cytoron Absolute flow cytometer, Dr. Casero and myosin. Cell Motil. Cytoskeleton, 25:lO-18. and Dra. Casimiro (Dept of Cell Biology, Univ. of BadaT.W., A.J.T. Tool, C.E. van der Schoot, L.A. 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