Mechanical Properties of 1ll,60 Cells: Role of Stimulation and Differentiation in Retention in Capillary-sized Pores Serpil C. Erzurum, Michele L. Kus, Cynthia Bohse, Elliot L. Elson, and G. Scott Worthen Departments of Medicine, National Jewish Center for Immunology and Respiratory Medicine and University of Colorado School of Medicine, Denver, Colorado, and Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, S1. Louis, Missouri

Neutrophil sequestration in pulmonary capillaries occurs prior to the development of lung injury, but the mechanisms by which neutrophils are retained are unclear. We hypothesized that decreases in cell deformability, in the absence of an increase in cell surface adhesive properties, would be sufficient to cause cell retention in a filtration apparatus modeling the pulmonary microvasculature. The myelomonocytic cell line (HL60 cell line) was used to test the hypothesis since these cells were unable to increase adherence in response to n-formylmethionylleucylphenylalanine (FMLP) in either the undifferentiated state or when differentiated towards granulocytes. With differentiation, HL60 cell volume decreased, and f-actin organization changed from a thick cortical rim with focal areas of f-actin in undifferentiated cells to a thin rim in differentiated cells. Differentiated cells responded to FMLP by reorganizing f-actin and increasing stiffness. Undifferentiated cells did not exhibit changes in f-actin with stimulation, were stiffer than differentiated cells, and did not increase stiffness in response to FMLP. Cytochalasin D (CD), which disrupted the cytoarchitecture as assessed by confocal microscopy but did not affect cell volume or adherence, decreased the stiffness of undifferentiated and FMLP-stimulated differentiated cells, thus suggesting the importance of microfilament organization in the stiffness of these cells. Filtration of cells through 8-J.tm pores showed that undifferentiated cells were markedly retained and did not exhibit any further retention with FMLP. Differentiated cells exposed to FMLP exhibited a concentration-dependent increase in retention in 8-J.tm pores that was abolished by CD. In addition, CD reduced retention of undifferentiated cells, indicating that microfilament organization is an important factor in determining a cell's rheologic properties. In conclusion, FMLP-stimulated microfilament reorganization, which increased cell stiffness, was sufficient in the absence of adherence factors to cause cell retention in a filtration system. This lends support to the hypothesis that decreases in cell deformability contribute to neutrophil retention in the pulmonary microvasculature.

Neutrophils are retained in the lung as a critical early step in the genesis of inflammatory and infectious processes (1-4). The mechanisms by which neutrophils accumulate in lung capillaries are unclear, but the initial rapid response to chemotactic peptides results from an effect on neutrophils and not the endothelium (4). At least two mechanisms for initial retention have been proposed: adherence of neutrophils to the endothelium or retention by decreases in deformability of the neutrophil. In the past, we and others have

(Received in original form August 17, 1990 and in revised form March 7,

1991) Address correspondence to: G. S. Worthen, M.D., Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, CO 80206. Abbreviations: cytochalasin 0, CD; dimethyl sulfoxide, DMSO; n-formylmethionylleucylphenylalanine, FMLP; Hanks' balanced salt solution, HBSS; Kreb's Ringer phosphate with 0.2 % dextrose, KRPD; lipopolysaccharide, LPS; nitrobenzoxadiazole, NBD; relative fluorescence index, RFI. Am. J. Respir. Cell Mol. BioI. Vol. S. pp. 230-241, 1991

suggested that microfilament organization stimulated by chemotactic factors caused changes in the mechanical properties of the neutrophil which contributed to cell retention (5-9). We demonstrated, in a filtration apparatus, that retention of neutrophils induced by n-formylmethionylleucylphenylalanine (FMLP) was inhibited by disruption of microfilament organization with cytochalasin D (CD), but was not affected by a monoclonal antibody directed against specific adherence-promoting leukocyte glycoproteins (MoAb60.3) (5). Adherence, however, does seem to playa significant role in lipopolysaccharide (LPS)-induced retention of neutrophils in filters. Recently, we have shown that early LPSinduced retention (at 20 min) of neutrophils in filters is primarily due to microfilament organization, while later retention (at 40 min) depends upon both adherence (as assessed by MoAb60.3) and microfilament organization', In these I Erzurum, S. c., G. P. Downey, B. Schwab, E. L. Elson, and G. S. Worthen. Mechanisms oflipopolysaccharide-induced neutrophil retention: relative contributions of adhesive and cellular mechanical properties. Submitted.

Erzurum, Kus, Bohse et al.: Mechanical Properties of HL60 Cells

experiments, adherence to serum-coated plastic was inhibited by MoAb60.3. However, in the pulmonary microvasculature, other adherence molecules unaffected by MoAb60.3 may also playa role in retention of neutrophils (10). To begin to discriminate the contribution of adhesive forces from those due to resistance to cellular deformation without the use of this or other antibodies, we have used the human promyelocyte (HL60) cell line. We predicted that these cells would be capable of responding to stimulus by microfilament reorganization and changes in deformability but would not increase adherence. These studies further suggest a role for cellular deformability in controlling passage through 8-p.m pores and hence, perhaps, in the physiologic control of microvessel transit.

Materials and Methods Reagents Reagents used were LPS-free (containing < 0.01 ng/ml of LPS) as determined by the limulus amoebocyte assay kit from Associates of Cape Cod (Woods Hole, MA) (11). PercolI (colloidal silica coated with polyvinylpyrrolidone) was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Hanks' balanced salt solution (HBSS) was obtained from amco (Grand Island, NY). The assay buffer employed was KRPD (Krebs Ringer phosphate) (buffer, pH 7.2) with 0.2 % dextrose (5% dextrose in 0.2 % sodium chloride, injectable; Abbott Laboratories, North Chicago, IL). Salts for the buffer were obtained from Mallinckrodt (Paris, KY). KRPD in these experiments was made without the addition of calcium. All componentswere freshly diluted with LPS-free saline (0.9 % saline for irrigation; Abbott Laboratories) on each experimental day. Nitrobenzoxadiazole (NBD)-phallacidin and rhodamine-phalloidin were obtained from Molecular Probes (Eugene, OR). CD, glycerol, p-phenylenediamine dihydrochloride, paraformaldehyde, glutaraldehyde, poly-t-lysine hydrobromide (mol wt, > 300,000), dimethyl sulfoxide (DMSO), FMLP, and .polyoxyethylenesorbitan monolaurate syrup (Tween® 20) were obtained from Sigma Chemical Co. (St. Louis, MO). FMLP was stored in DMSO at 1 X 10-3 M. LPS from Escherichia coli Olll:B4 was purchased from List Biologicals (Campbell, CA). Lyophilized LPS was dissolved in LPS-free saline at 1 mg/ml. Aliquots of 100 p.l were sonicated using a Branson bath sonicator (50/60 Hz) for a period of 30 min before being diluted in KRPD for use. Cell Culture Methods The HL60 cell line was obtained from the American Type Culture Collection (Rockville, MD). Undifferentiated cells were maintained by passage 2 to 3 times per week with initial cell concentrations at 2 x 10s/ml . Cells were kept at 37° C with 5 % CO 2 in a humidified tissue culture incubator in 75-cm 2 surface area plastic culture flasks (Costar, Cambridge, MA) with RPMI 1640 (GIBCO), 20% heat-inactivated fetal bovine serum (Irvine Scientific, Santa Ana, CA), and glutamine (0.292 mg/ml), penicillin (100 U/ml), streptomycin sulfate (100 p.g/ml) (Irvine Scientific). Cells were used during passages 17 to 40. Cells were differentiated by the addition of 1.25% DMSO to the above culture media, and then incubated in the above conditions for 6 to 7 days (12). Cell viability was determined by trypan blue (Hazleton

231

Biologicals, Lenexa, KS) exclusion. Cell differentiation was determined by morphology on Diff-Quik" stain (American Scientific Products, McGaw Park, IL) and nitroblue tetrazolium reduction (13). Preparation of Plasma All plasma used in the following experiments was, human heat-inactivated, platelet-poor plasma to exclude any contribution of complement factors to the effects noted. Whole citrated blood was centrifuged, and the packed cells were separated from the platelet-rich plasma. The platelet-rich plasma was underlaid with 90% (wt/vol) Percoll and centrifuged at 2,500 x g for 20 min, then the supernatant was heat-inactivated at 56° C for 30 min. The heat-inactivated, platelet-poor plasma was then centrifuged at 2,500 x g for 15 min and stored at -20° C until use. Filtration Apparatus Filtration was performed as described previously (8). Briefly, polycarbonate filters (Nucleopore, Pleasanton, CA) with a pore size of 8 p.m were protein-coated with 20 % plasma at'37° C for 2 h. Filters with a pore diameter of 8 p.m were used in these experiments to maintain a similar ratio of cell diameter to filter pore diameter (rv1.2) as in previous experiments done with neutrophils (diameter, rv7.5 p.m) perfused through 6.5-p.m pores'. Diameter of differentiated HL60 cells ( rv 10 p.m) was determined by measurement of volume (see below) and calculation of diameter by D = (6Vhr)l/3, where D is diameter in p.m and V is volume in p.m3 (14). Polypropylene filter holder chambers (Millipore, Bedford, MA) and sterile plastic intravenous tubing (Venoset Microdrip; Abbott Hospitals, North Chicago, IL) were protein-coated prior to use by incubation with 20 % plasma at 37° C for 2 h. An infusion pump (Harvard Apparatus, South Natick, MA) was used to provide constant flow at I ml/min through the filters. Changes in pressure in this system were monitored by in-line pressure transducers (Bell and Howell, Pasadena, CA) connected to a strip chart recorder (Gilson Medical Electronics, Middleton, WI). During these experiments, pressure did not vary more than 5 mm Hg. Each individual experiment consisted of three filtrations run simultaneously. Filtration Experiments Differentiated or undifferentiated HL60 cells were labeled with "Tn (New England Nuclear, Boston, MA) by incubating 20 p.Ci of lIlInCl/I06 cells with 5 x 10-4 M tropolonate (Fluka AG, Buchs, Germany) for 5 min in KRPD, followed by a wash with KRPD. Labeled cells were suspended at 4 x 10s/ml in KRPD, and 0.25-ml aliquots were injected over 5 s into the ports of the filtration system. IdenticaI0.25-ml aliquots were also collected for determination of total counts. The cells were perfused with HBSS with 2 % heat-inactivated, platelet-poor plasma through the system at 1 ml/min for 5 min. The effluent was collected in polypropylene tubes. At the end of 5 min, filters and proximal and distal chambers were removed and placed in plastic scintillation vials and counted along with the effluent in a gamma counter (Gamma 7000; Beckman Instruments, Fullerton, CA). All filtrations were carried out at room temperature, and values expressed as % retention of cells in the filters determined by the following formula:

232

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

ti . filt - radioactivity in filter % o re ten Ion In er I d' .. . tota ra ioacnvity

Recovery of radiolabeled cells averaged 98 % for these filtration experiments. To study the effect of LPS on HL60 filtration, cells were incubated with LPS (10 to 1,000 ng/ml) prior to filtration in the presence of 1% heat-inactivated, platelet-poor plasma at 37° C for 40 min. The plasma effect on cell retention with LPS was further studied by incubating cells with LPS (1,000 ng/ml) for 40 min at 37° C in increasing plasma concentrations (1% to 50 %) prior to filtration. To examine the effect of FMLP on HL60 filtration, the cells were exposed to FMLP (10-7 to 10-9 M) in the presence of 1% plasma immediately prior to filtration. To determine the contribution of actin assembly on cell retention, HL60 cells were treated with CD (5 /-tg/ml) for 10 min prior to treatment with FMLP (10-7 M) before filtration using perfusate containing 2 /-tg/ml CD. Actin Assembly Actin polymerization in HL60 cells was determined by NBD-phallacidin staining. HL60 cells (2 X 106 cells/O.4 ml) were incubated in 1% heat-inactivated, platelet-poor plasma with FMLP (10-7 to 10-9 M) for 0.5 to 20 min. A concentration of 1% heat-inactivated, platelet-poor plasma was chosen as this did not interfere with fluorescence measurements in the cytofluorograph. Control cells were kept LPS-free and incubated for 0 to 20 min. At the end of incubation for the appropriate time and FMLP dose, cells were fixed by addition of 0.4 ml of 4 % paraformaldehyde at room temperature for 10 min. The fixed cells were then permeabilized by addition of 0.2 ml of 0.2 % Tween 20 at 37° C for 5 min. After this, 50 /-tl of NBD-phallacidin suspended in KRPD at 1.65 x 10-7 M was added to the cell suspension for 10 min. Stained cells were analyzed by a Coulter EPICs C cytofluorograph. The EPICs C utilizes an argon laser at 488 urn with a dichroic filter and a 514 urn long pass laser blocking filter. Emission is then read at wavelengths greater than 514 urn. For most experiments, 10,000 cells were analyzed. Results were initially obtained as log fluorescence then expressed as relative fluorescence index (RFI) (15). Photomicrographs of filamentous actin in the cells were performed after rhodamine-phalloidin staining since rhodamine is more resistent to bleaching than NBD. Cells (I X 1Q6 cells/0.2 ml) were exposed to FMLP 10-7 to 10-9 M) for increasing times in the presence of 1% heat-inactivated, platelet-poor plasma. At the end of the appropriate time period, the cells were fixed with 0.2 ml of 4 % paraformaldehyde at room temperature for 10 min. At the end of fixation, the cells were allowed to settle on coverslips previously coated with 0.03 % poly-r-lysine, After 20 min of incubation on the coverslips, the cells on the coverslips were gently washed with KRPD to remove nonadherent cells and then permeabilized by incubation with 0.2 % Tween 20 for 5 min. After this step, the cells were washed again, and the fixed and permeabilized cells then stained by addition of rhodamine-phalloidin (1.65 X 10-7 M) to the covers lips for 10 min at 37° C in the dark. The coverslips were mounted on slides in a 1:10 solution of saline:glycerol with p-phenyl-

enediamine (0.1%) as a quenching agent, and the edges sealed with nailpolish. Cells were then examined by confocal microscopy (Bio-Rad MC500) on a Nikon Diaphot inverted microscope using a Plan-Apo 60x objective with a numerical aperture of 1.4. The exciting light was passed through an excitation band pass filter of 514 urn and the emission filter was a 550 nm long pass. Images were acquired on a Compaq Deskpro 386 computer at 4 times normal magnification, resulting in a final magnification of 2,400 X using Kalman filtering and averaging of 30 images to reduce noise. Cells were imaged at 5 p,m above the substratum, and optical section thickness was set at the minimum value permitting ready delineation of edges in the dimmest cells in the experiment. This setting, which gave a thickness of approximately 1.0 /-tm, was then used for the rest of the experiment. Ultrastructural Studies The extension of pseudopodia by differentiated HL60 cells was determined by a semiquantitative ultrastructural analysis. After stimulation by FMLP (10-7 M) in the presence or absence of Cl) (5 /-tg/ml) for 2 or 5 min, the cells (including a buffer control) at a concentration of 5 x 106/ml were fixed overnight in 1.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3, postfixed in 1.0 % osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a graded series of ethanol and propylene oxide, and embedded in Embed/Araldite 502. Thin sections were cut from multiple blocks on an LKB Nova ultramicrotome, stained with 2.0% aqueous uranyl acetate and Reynolds' lead stain. Sections were examined on a Philips 40ar electron microscope at an accelerating voltage of 60 kV. Photographs were taken of HL60 cells, printed, and the number of pseudopodia graded in a blinded fashion and expressed as a mean over the population. Adherence Studies The assays of cell adherence to serum-coated plastic were performed as previously described (16), with minor modifications. In brief, 100 /-tl of medium 199 (GIBCO) containing 10% heat-inactivated fetal bovine serum was added to wells of 96-well microtiter tissue culture plates (Costar) and allowed to incubate for 2 h at 37° C. HL60 cells were labeled with II I indium as described above, then suspended in KRPD with 1% human serum at a concentration of 3 X 106 cells/mI. After aspirating the medium 199 from the microtiter tissue culture wells, 50 /-tl of cells (1.5 x 105 cells/ml) was placed in the wells. In these experiments, all tests were performed in triplicate. In addition, 50 /-tl of cells was placed in a polypropylene tube as a totals tube for each experimental condition. Stimulus in 50 /-tl ofKRPD with 1% serum was then added to the appropriate wells. Stimuli included LPS (10 ng/mI to 10 /-tg/ml) and FMLP (10-9 M). The plates were then incubated at 37° C, 5 % CO 2 in a humidified tissue culture incubator for 15 min for FMLP and for 40 min for LPS stimulus. At the end of the incubation period, 100 /-tl of 0.2 % glutaraldehyde in phosphate-buffered saline was added very slowly to each well, then incubated for 10 min. At the end of this time, the wells were aspirated and gently washed with saline, then 75 /-tl of KRPD was added to each well and the adherent cells harvested by thoroughly scrubbing each well with a cotton-tip swab. Each swab was placed into a polypropylene tube and counted in a gamma counter

Erzurum, Kus, Bohse et aI.: Mechanical Properties of HL60 Cells

along with totals tubes. The percentage of cells adherent to the serum-coated plastic was determined by: 01 /0

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To determine if CD had any effect on HL60 adherence, cells were exposed to CD 5 JLg/ml for 10 min at 37° C before exposure to FMLP in the adherence assay. Control for the CD experiments included cells exposed only to KRPD and 1% serum, cells exposed to CD without FMLP, and cells exposed to FMLP without CD. In addition, adherence of HL60 cells to serum-coated beads was assessed (17). Latex beads (1 JLm in diameter) were precoated with human serum albumin (10 mg/ml) for 10 min. The beads were then washed 3 times with saline to remove excess albumin. Beads were resuspended in KRPD. Differentiated and undifferentiated HL60 cells (1 x 107) were added to FMLP 1 x 10-7 M or KRPD alone and incubated at 37° C for 5 min. Albumin-coated latex beads were then added to give a final bead concentration of 1% (vol/vol), resulting in a bead to neutrophil ratio of 100:1. Tubes were then placed in an agitating water bath at 37° Cat 100 oscillations/minute for 10 min. The reaction was stopped by addition of an equal volume of 2 % glutaraldehyde, and the cells were allowed to incubate with the glutaraldehyde at room temperature for 30 min. The cells were then washed 3 times with saline to remove nonadherent beads and resuspended in 0.3 ml saline. Wet mounts were prepared, and adherence was examined by light microscopy. Adherence was scored by counting 150 cells. The percentage of cells that showed adherent albumin-coated latex beads was determined by scoring all cells as adherent that exhibited one or more beads on their surface. Cell Volume Determinations Determination of differentiated and undifferentiated HL60 cell volume was performed on a Coulter ZM (Coulter Counter Scientific Institutes, Hialeah, FL). Cells were suspended in KRPD at 4 x 1()4 cells/ml and studied in buffer alone, with FMLP (10-7 M) for 2 min, with CD (5 JLg/ml) for 10 min, or with CD for 10 min followed by exposure to FMLP for 2 min. To confirm these observations, water content was determined gravimetrically, using [I4C]polyethylene glycol ([I4C]PEG) '(4,000 mol wt; 100 JLCi/ml; Sigma) as the extracellular marker. Cells were suspended in preweighed microfuge tubes in 1 ml at a concentration of 5 x 107/ml with or without FMLP, 5 JLI [I4C]PEG was added, and the cells were centrifuged for 30 s in a microfuge. The supernatant was removed, and 0.5 ml added to scintillation vials containing 9.5 ml scintillant. The tube containing the pellet was weighed, dried overnight in a centrifugal vacuum evaporator, and reweighed repeatedly until dryness, and the pellet solubilized in 0.5 ml of 1 N NaOH and added to scintillant. Supernatant and pellet 14C counts were determined in a Beckman LS5000 TD, and the water content of the cells was obtained by calculation of the wet/dry weight ratio after correction for extracellular water. Cell Deformability HL60 cell stiffness was measured directly using the cell poker as previously described (18, 19). This instrument mea-

233

sured the deformability of the free surface of cells adherent to the bottom surface of a coverslip. The cell surface was indented by a glass microprobe (tip diameter, 2 JLm) attached to a flexible glass beam of known bending constant. The degree of bending of the beam was used to calculate the force (mdyne) with which a cell resisted indentation as a function of the depth (JLm) of indentation. As a convenient empirical parameter to characterize the cellular deformability, we define the "stiffness" of the cell as the initial linear slope [force (mdyne)/indentation (JLm)] of the ingoing limb of the deformation. Cells (500 JLI containing 4 x lQ6 cells) were pipetted onto a glass coverslip precoated with 15 JLg/ml of poly 2-hydroxyethyl methacrylate (polyHEMA) (Aldrich Chemical Co., Milwaukee, WI) to prevent cells from spreading when placed on the coverslip (20). The coverslips were then inverted and placed into the chamber ofthe cell poker that contained KRPD with 1% heat-inactivated, platelet-poor plasma. Unstimulated differentiated and undifferentiated cells were studied in KRPD with 1% plasma and during exposure to FMLP 1 x 10-6 and 1 x 10-8 M. The effect of CD on FMLP-induced cell stiffness was also examined. CD at 5 JLg/ml was incubated with neutrophils at 37° C for 5 m.in prior to placing the cells on the coverslips. The adherent cells on the coverslip were then inverted into the cell poker chamber that contained 1% heat-inactivated, platelet-poor plasma and CD 2 JLg/ml and FMLP 1 x 10-6 M. A distribution of cell stiffnesses was generated for each experimental condition. Statistical Analysis All data are reported as means ± SEM unless otherwise specified. Results were analyzed using proprietary statistical packages: Stat View 512+ and FASTAT (Systat Inc., Evanston, IL) running on a microcomputer (Macintosh SE, Apple, Cupertino, CA). For most analyses, t test or ANOVA was used for comparison of means. For the analysis of the data from the cell poker experiments, a nonparametric test (Kruskal-Wallis) was used because the data were not normally distributed.

Results Initial studies were performed to determine the magnitude of the adhesive responses to FMLP of HL60 cells.. Adherence of HL60 cells was assessed by two methods: adherence to serum-coated plastic and adherence of albumin-coated beads to cells. Figure 1 demonstrates that FMLP did not induce differentiated or undifferentiated HL60 cells to increase adherence to serum-coated plastic. CD at 2 or 5 JLg/ml did not affect HL60 cell adherence, and treatment of HL60 cells with CD 5 JLg/ml and FMLP 1 x 10-7 M did not affect adherence of HL60 cells (P > 0.05). Furthermore, there was no significant difference in adherence of albumin-coated beads to differentiated cells in the absence (21.0 ± 7.0% showing adherent beads) or presence (20.7 ± 6.6%) of FMLP. LPS in concentrations between 10 ng/ml to 10 JLg/ml was unable to induce any increase in adherence of undifferentiated or differentiated HL60 cells (28.8 ± 3.2 % differentiated cells adherent to serum-coated plastic in presence of LPS 10 JLg/ml versus 28 ± 5.3 % adherent in presence of KRPD).

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AMERICAN JOURNAL OF RESPIRATORY CELL AND.MOLECULAR BIOLOGY VOL. 5 1991

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Figure 1. Fraction of differentiated and undifferentiated HL60 cells adherent to serum-coated plastic. No significant increase in cell adherence occurred with n-formylmethionylleucyllphenylalanine (FMLP) stimulation (P > 0.05). There was no significant difference between adherence of differentiated and undifferentiated cells to serum-coated plastic (P > 0.05). Each value represents the mean ± SEM of three experiments, with each experiment done in triplicate.

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Having demonstrated no significant change in adherence of cells with stimulation or with CD, we then examined actin assembly and organization in HL60 cells. Figure 2 shows that with FMLP stimulation of differentiated HL60 cells there was an increase in fluorescence of cells, indicating a net increase of f-actin in these cells. Interestingly, there was a subpopulation of cells that returned to baseline unstimulated fluorescence more quickly than other differentiated cells. FMLP did not induce increases in f-actin in undifferentiated cells. Confocal microscopy of fluorescently labeled f-actin in differentiated HL60 cells revealed extensive redistribution of f-actin with FMLP stimulation. Unstimulated differentiated cells had a rim pattern of fluorescence of f-actin (Figure 3A). After FMLP stimulation, this rim pattern intensified at 30 s and 2 min (Figure 3B); by 5 min, polarization of f-actin in the cell occurred which corresponded to areas of shape change (Figure 3C). This redistribution off-actin persisted throughout 20 min (Figure 3D). Undifferentiated HL60 cells had a different pattern of f-actin distribution than differentiated cells (Figure 3E). The distribution of f-actin in the undifferentiated cells by confocal microscopy demonstrated a thick cortical pattern with aggregates of f-actin in foci within the cell (Figure 3E). Undifferentiated cells did not demonstrate redistribution of actin with exposure to FMLP (Figure 3F) or LPS. CD effectively disrupted actin organization in differentiated and undifferentiated HL60 cells (Figures 4A and 4C). After exposure to CD, the rim pattern of fluorescence in unstimulated cells was replaced by focal aggregates of fluorescence in the cells. CD also prevented the f-actin reorganization seen in differentiated cells with exposure to FMLP (Figure 4B). In addition, CD inhibited the increase in RFI induced by FMLP in differentiated cells (Figure 5A) and even decreased RFI in both differentiated and undifferentiated cells (Figures 5A and 5B) to below baseline unstimulated levels. With differentiation of HL60 cells, changes in volume oc-

Log Fluorescence Figure 2. Cytofluorograph tracings of changes in amount of f-actin (as determined by nitrobenzoxadiazole-phallacidin fluorescence) in HL60 cells stimulated with FMLP. Column A is fluorescence of differentiated cells with FMLP 1 x 10-7 M, and column B is undifferentiated cells with FMLP 1 x 10-7 M. The top panel in columns A and B is fluorescence of cells in buffer, while the four lower panels represent fluorescence of cells in the presence of FMLP for increasing periods oftime: 0.5, 2, 5, and 20 min, respectively. Fluorescence is shown on a log scale.

curred. Table 1 demonstrates that the average volume of undifferentiated cells was 883.8 ± 24.5 feintoliters and did not change significantly with exposure to FMLP, CD, or the two together. Differentiated HL60 cells were significantly smaller than undifferentiated cells (P < 0.001) and appeared to increase volume measured on the Coulter counter modestly but significantly (P = 0.02) with FMLP. In order to confirm estimates of volume by Coulter counter, we measured water content gravimetrically. Although not significantly different, the water content measurements demonstrate a trend toward increased cell water after FMLP (Table 1). However, there was no significant change in differentiated cell volume with exposure to CD or with exposure to FMLP in the presence of CD. Changes in deformability of HL60 cells were then examined by use of the cell poker (18, 19). Differentiated HL60 cells in KRPD had a medium stiffness of 0.080 mdyne/ ~m, which increased significantly (P < 0.001) to 0.152 mdyne/ urn in the presence of FMLP 1 x 10-8 M and to 0.194 mdyne/um in the presence of FMLP 1 x 10-6 (Figure 6A). Treatment of cells with CD prior to treatment with FMLP

Erzurum, Kus, Bohse et al.: Mechanical Properties of HL60 Cells

235

Figure 3. Confocal microscope images of f-actin in HL60 cells stained with rhodamine-phalloidin (original magnification: x600). f-actin in differentiated HL60 cells in buffer (A) was reorganized after FMLP 1 x 10-7 M for 2 min (B), 5 min (C), and 20 min (D). f-actin organization of undifferentiated cells in buffer (E) is not different from that in FMLP I x 10-7 M at 20 min (F).

prevented this increase in stiffness and narrowed the distribution of cell stiffnesses, so that the cells were more closely grouped about the median. Undifferentiated cells had a median stiffness of 0.206 mdyne/jtm with no significant change with exposure to FMLP I X 1O~ M, but CD treatment significantly decreased undifferentiated cell stiffness to 0.067 mdyne/um (P < 0.01) (Figure 6B).

Having shown that no adherence response was detectable but that changes in actin assembly and organization, deformability, and volume resulted from stimulation of differentiated cells by FMLP, we then examined retention of cells in a filtration system. In the absence of stimuli, retention in filters was markedly greater for undifferentiated than for differentiated cells (P = 0.004) (Figure 7). LPS had no effect

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 51991

cells, both differentiated (P = 0.006) and undifferentiated cells (P = 0.0004) (Figure 8). Moreover, CD treatment prior to exposure to FMLP prevented the FMLP-induced retention in differentiated cells (P = 0.0007) (Figure 8). Although these results implicated a role for decreased cell deformability in causing retention, the structural requirements for this mechanical resistance remain unclear. To determine whether extension of pseudopodia can itself account for retention in pores, a semiquantitative ultrastructural study was carried out and the results are depicted in Table 2, while examples of the appearance of these cells are shown in Figure 9. Differentiated HL60 cells demonstrate pseudopods even under control conditions and appear to demonstrate fewer within 2 min after stimulation after FMLP. This finding is consistent with the cortical shell of actin that appears immediately. By 5 min, similar numbers of pseudopods are seen either with or without CD, suggesting that pseupopods themselves cannot explain the altered retention of these stimulated cells.

Discussion

Figure 4. Confocal microscope images of f-actin in HL60 cells in the presence of cytochalasin D (CD) (5 p.g1ml) , cells stained with rhodamine-phalloidin (original magnification: X600). Panel A shows CD-disrupted f-actin organization in differentiated HL60 cells and in differentiated cells stimulated with FMLP I x 10-7 M (B), as well as in undifferentiated cells (C).

on HL60 cell filtration . However, FMLP induced retention in filters of differentiated HL60 cells in a concentrationdependent fashion (P = 0.03) (Figure 7). Pretreatment of cells with CD significantly reduced retention of unstimulated

Stimulation of circulating neutrophils leads to a coordinated series of events (neutrophil activation), an early manifestation of which is neutrophil sequestration in capillaries (1-4). A precise delineation of the mechanisms involved is complicated by the rapid and nearly simultaneous alterations in neutrophil adhesive and deformability properties that follow FMLP stimulation (8,10). Other studies have demonstrated that HL60 cells can express adherence-promoting glycoproteins on their surface (21), and that , with differentiation, cell adherence to surfaces increases nonspecifically. Nevertheless, stimulated adherence of these cells to surfaces other than to stimulated endothelial cells has not been demonstrated (22,23). Our findings demonstrated that HL60 cells differentiated towards granulocytes did not increase adherence to serum-coated plastic or to albumin-coated beads in response to FMLP. Thus, with this cell line the contribution of adherence in the response was minimal. As shown previously (24, 25), the differentiated HL60 cells increased and reorganized f-actin in response to FMLP. Cytofluorographic data demonstrated that the amount of f-actin in the differentiated cells increased with FMLP stimulation ; A subpopulation of cells returned to baseline levels of f-actin faster than the rest of the population, reflecting heterogeneity of the HL60 response to FMLP, perhaps related to degree of differentiation. Confocal microscope images showed that changes in f-actin organization occurred in the cells as they matured along the granulocytic cell line. It has been shown that the total amount of actin increases with differentiation of these cells (26), and demonstration of organizational changes has been suggested by electron microscope pictures (27). Whereas differentiated cells have a thin rim of f-actin by confocal microscopy, undifferentiated cells have a thick cortical rim with areas of focal fluorescence. The changes in organization of f-actin as these cells mature along the granulocytic cell line may playa large role in determining the time at which granulocytes leave the marrow as they mature (28,29). We have shown that dramatic changes in the organization of f-actin occurred not only upon

Erzurum, Kus, Bohse et al.: Mechanical Properties of HL60 Cells

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differentiation but also in differentiated cells in response to FMLP. Upon exposure to FMLP, the rim pattern seen in differentiated cells increased in intensity and thickness. Within 5 min after FMLP, the fluorescent pattern had polarized, with staining concentrated in regions corresponding to apparent lamellipodia. This polarized appearance persisted for at least 20 min. The changes observed in these differentiated HL60 cells are similar to those noted in neutrophils in response to FMLP (30). Undifferentiated cells did not demonstrate any organizational changes of f-actin in response to FMLP. It has been previously demonstrated that HL60 cells express very few chemotactic peptide receptors in the undifferentiated state and are unable to undergo actin polymerization upon stimulation with FMLP (25). We and others have shown that when these cells are induced to differentiate, they acquire the ability to undergo FMLPinduced actin polymerization (24, 25). Kinetic experiments, however, show that in the first 48 h after induction of

TABLE 1

Volume of HL60 cells with differentiation and stimulation * Differentiated Condition

Buffer FMLP CD CD + FMLP

Volume (jemtoliters)

Water Content (g/g dry cell)

± ± ± ±

2.32 ± 0.22 2.54 ± 0.35 ND ND

525.1 592.5 553.9 575.6

14.6 22.H 41.1 37.8

Undifferentiated Volume (jemtoliters)

883.8 886.7 924.8 886.3

± ± ± ±

24.5t 17.0 44.2 42.0

Definition ofabbreviations: FMLP = n-formylmethionylleucylphenylalanine; CD = cytochalasin D; ND = not determined. * Cells were treated with buffer (Kreb's Ringer phosphate with 0.2% dextrose), FMLP (1 x 10-7 M) for 2 min, CD (5 Jlg/ml) for 10 min, or CD (5 Jlg/ml) for 10 min prior to FMLP (1 x 10-7 M) for 2 min, then volume determined by Coulter counteror water contentby gravimetry. Each value represents the mean ± SEM of three experiments. t p < 0.001, undifferentiated cells are larger than differentiated cells. § p = 0.02, differentiated cells exposed to FMLP are larger than differentiated cells in buffer.

differentiation, no increase in FMLP receptors occurs, but the cells are able to polymerize actin in response to FMLP (25). This indicates that the few FMLP receptors on the undifferentiated cells are sufficient to respond to FMLP, if the intracellular mechanisms exist. Differentiation agents induce the maturation of intracellular mechanisms, such as actin-binding proteins (31), which allow the polymerization of actin in response to FMLP even in the presence of low numbers ofFMLP receptors. We also demonstrated that CD, an agent that disrupts f-actin assembly and organization (32-34) in cells, was able to prevent the increases and reorganization of f-actin seen in differentiated cells with FMLP. Likewise, f-actin organization in undifferentiated cells was disrupted by CD, and fluorescence intensity of NBDphallicidin measured by the cytofluorograph was decreased significantly, indicating .less actin in the filamentous form. Having shown that HL60 cell adherence is unaffected by stimuli, but that FMLP caused differentiated cells to increase and reorganize f-actin, we then examined the deformability of these cells with the cell poker (18, 19). Measurements of deformability with the cell poker yield stiffness values that depend upon both elastic and time-dependent (viscous) resistance to deformation. If the cell is modeled as a viscoelastic fluid interior enclosed by a tensed surface membrane cortex (35), the stiffness measured by the cell poker would be influenced by both the membrane cortex and cytoplasm. Transit through pores, and microvasculature, occurs at a finite rate and so should depend on both cellular viscosity and elasticity. Thus, stiffness values from the cell poker should predict the rheologic properties of cells, that is the ease with which cells can deform through pores of filters and through the microvasculature. The undifferentiated cells had a median stiffness of 0.206 mdyne/ p.m, while differentiated cells had a median stiffness of 0.080 mdyne/ p.m. With exposure to FMLP, differentiated cells increased cell stiffness, although not to levels seen in undifferentiated cells. CD was able to completely inhibit the development of increased stiffness induced by FMLP in differentiated cells and narrowed the distribution of cell stiffness seen, and re-

238

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

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duced undifferentiated cell stiffness to even below baseline levels as measured by the cell poker (Figures 6A and 6B). This supported the hypothesis that microfilament organization was of most importance in development of stiffness. Undifferentiated cell stiffness was dramatically affected by CD and may reflect the dynamic nature of f-actin organization and assembly in these immature cells and which may therefore be more vulnerable to the effects of CD. These findings and others support the postulate that organization of f-actin is important in determining the cell's stiffness (5, 8, 36), and we therefore examined the relationship between cell stiffness and retention by determining HL60 cell retention in a filtration system. Filtration was performed through 8-J-tm filters to maintain a similar cell diameter to pore diameter (f\J1.2) as used in previous neutrophil filtration experiments', Undifferentiated cells had high



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baseline retention and did not have any further increase in retention in response to stimuli. However, differentiated HL60 cells were able to respond to FMLP by significantly increasing retention in pores as FMLP concentration increased, just as stiffness increased with increasing FMLP concentration. CD was able to completely inhibit FMLPinduced cell retention and further was able to decrease differentiated and undifferentiated cell baseline retention. Others have demonstrated that CD is not toxic to HL60 cells (37), and, because CD did not affect adherence or size of these cells, its effect must be attributed to its demonstrated effect on microfilament organization and stiffness of cells. The effect of CD on retention of cells in pores of filters further suggests that transit through the pores is limited by cellular deformability, which is governed by the organization of microfilaments.

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0

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Figure 8. Effect of CD on HL60 cell retention in filters. CD reduced differentiated cell retention in buffer (P < 0.01) and prevented the increase in retention induced by FMLP 1 x 10-7 M (P < 0.001). CD also decreased undifferentiated HL60 cell retention (P < 0.001). Each value represents the mean ± SEM of three experiments, each experiment consisting of three simultaneously run filtrations.

Erzurum, Kus, Bohse et al.: Mechanical Properties of HL60 Cells

TABLE 2

Extension of pseudopodia by differentiated cells after stimulation (average number of pseudopodia/cell) * FMLP Buffer (n = 26)

1.2

FMLP

+

CD

2 min

5 min

2 min

5 min

(n = 31)

(n = 21)

(n = 17)

(n = 26)

0.55

1.3

0.88

1.35

Definition of abbreviations : see Table I. * Cells were treated with buffer (Kreb's Ringer phosphate with 0.2% dextrose), FMLP (l x 10-7 M) for 2 or 5 min, or CD (5 I'g/ml) for 10 min prior to FMLP (I x 10- 7 M) for 2 or 5 min, then processed for electron microscopyas indicated in MATERIALS AND METHODS, and pseudopodia counted. Each value represents the mean number/cell normalized over the population.

Figure 9. Ultrastructural appearance of differentiated HL60 cells. (A) Unstimulated cells. Arrow points to pseudopod (original magnification: x3,600). (B) FMLP stimulation for 2 min. Arrow points to pseudopod (original magnification: x3,600). (C) FMLP stimulation for 5 min. Arrow points to pseudopod (original magnification: x3,600) . (D) Cells were exposed to CD prior to FMLP stimulation for 5 min. Arrow points to a microvillus-like pseudopod (original magnification: x3,600).

239

Filterability of cells is also a function of cell diameter (38). As demonstrated previously (39) with differentiation of HL60 cells, average size of cells decreased. In this study, the undifferentiated cells (diameter, rv11.9 p,m) were more retained in filters than differentiated cells (diameter, rvlO p,m). However, the undifferentiated cells were also significantly stiffer than differentiated cells as determined by the cell poker. Cell deformability appeared to be more important than size in determining retention in filters. This was supported by the finding that while CD did not change cell volume, it was able to dramatically decrease retention of undifferentiated cells to levels of retention seen with

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240

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

differentiated cells. We did demonstrate that a small but significant increase in cell volume (as assessed by Coulter counter) occurred in differentiated cells with FMLP stimulation. A similar, although not significant, trend in cell water content was seen after FMLP stimulation. However, this increase in cell volume is small and would be unlikely to produce the degree of increased retention noted in the filtration experiments. Furthermore, FMLP-stimulated cells were not significantly different in volume from CD- and FMLPtreated cells, yet retention was dramatically different, suggesting that volume alone cannot explain retention. Nash and colleagues (40) demonstrated that volume changes induced osmotically are not independently capable of causing the same degree of retention in filters as stimulus-induced retention and therefore concluded that structural changes in cells are the major cause of rheologic defects inducing retention of cells in filters in response to stimuli. Stimulus-induced structural changes might include the extension of pseudopodia, but, if so, they would have to be stiff pseudopodia, as suggested by Schmid-Schonbein and associates (41). The semiquantitative ultrastructural study revealed no more pseudopodia in FMLP-stimulated cells than in control, and an equal number in cells pretreated with CD. These data argue that the mere presence or absence of pseudopodia cannot explain retention in this system. It remains possible, however, that if stiff pseudopodia are extended, that they exert a disproportionate effect on retention. The precise significance of the localization of actin and the geometry of the cell remain to be determined. In summary, HL60 cells stimulated with FMLP were retained in a filtration system under conditions in which they did not become adherent but did reorganize f-actin and increase stiffness. Furthermore, CD, which disrupted f-actin organization in these cells, was able to prevent FMLPinduced retention and stiffness, indicating that f-actin organization contributed to the development of stiffness and cell retention. In addition, stiffness and retention of the larger undifferentiated cells was markedly reduced by CD, despite no change of cell size, indicating that f-actin organization (and by implication, cell stiffness) was more important than cell size in determining the ease with which cells passed through pores of filters. These results support that resistance of cells to deformation is more important than cell size or adherence for retention in a filtration system. Likewise, an important factor in neutrophil retention in the pulmonary microvasculature may be alteration in cell deformability. Acknowledgments: The writers gratefully acknowledge Roberta Osborne for maintenance and culture ofHL60 cells, Scott Young for technical assistance, Bill McConnaghey for maintenance of the cell poker, Sheryl Campbell for assistance with ultrastructural studies, Bill Townsend for assistance with cytofluorometry and confocal microscopy, and Barry Silverstein and Leigh Landskroner for illustration services. This study was supported by a SCOR in ARDS HL-40784, and HL-07085 from the National Institutes of Health.

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Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillary-sized pores.

Neutrophil sequestration in pulmonary capillaries occurs prior to the development of lung injury, but the mechanisms by which neutrophils are retained...
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