Cytometry 11:272-282 (1990)
0 1990 Wiley-Liss, Inc.
Changes in Neutrophil Right-Angle Light Scatter Can Occur Independently of Alterations in Cytoskeletal Actin Eliyahu Kraus and Richard Niederman Department of Cell Biology, Forsyth Dental Research Center, Boston, Massachusetts 02115 Received for publication April 14,1989; accepted July 12, 1989
Forward-angle light scatter (FALS) and right-angle light scatter (RALS) are commonly employed to discriminate between leukocyte subclasses. Recently the application of RALS has expanded, and it is now also used as an indicator of neutrophil actin polymerization. In this communication we critically examine the relationship of RALS to changes in cytoskeletal actin. The data indicate that agonists which stimulate an increase, a decrease, or no change in F-actin content can all stimulate a biphasic change in RALS. We therefore conclude that chan-
Flow cytometric measurement of both forward- and right-angle light scatter is commonly employed to discriminate between leukocyte subclasses (10,19,25). Light scatter is also used to: sort cells (14,15,23,35), identify cell heterogeneity in similar cell types (30,31,34), and define cell morphology (2,321. Forward-angle light scatter (FALS) is the amount of light that is scattered off a n object and measured within 2" from the incident beam (25). FALS is attributed to the amount of light scattered from both the main contour of the cell and the nucleus (9,10,19,20). Thus FALS is primarily used in the identification of cell types according to their size (12). Similarly, rightangle light scatter (RALS) is the amount of light scattered off an object and measured a t 90" from the incident beam (25). However, RALS is attributed to the scattered light from subcellular structures having dimensions equal to or smaller than the wavelength of the exciting light. Because of this, RALS is thought to detect changes in membrane ruff ling and intracellular granules (40). This last application led to the observation that changes in RALS appear to parallel actin polymerization and depolymerization in chemotactic peptide stimulated neutrophils (27). This in t u r n led to the use of RALS as a n indicator of changes in F-actin content in neutrophils (6,13,16,17,26,27,39).
ges in RALS can occur independently of changes in F-actin content. This leads us to suggest that caution must be taken when interpreting RALS data in relation to changes in F-actin. Furthermore, the data also support the idea originally proposed by Yuli and Snyderman (J Clin Invest 73:1408-1417, 1984), that RALS may be an exceptionally sensitive indicator of cell activation. Key terms: Lactate, f-MLP, propionate, ammonium chloride
In this paper we critically examine the utility of RALS as a n indicator of cytoskeletal F-actin changes in neutrophils. The data indicate that agonists which stimulate a n increase, a decrease, or no change in cytoskeletal F-actin can all stimulate a biphasic change in RALS. We therefore conclude that changes in RALS can occur independently of actin polymerization. However, the data also suggests, as originally proposed by Yuli and Snyderman (40), that RALS may be a n exceptionally sensitive indicator of cell activation.
MATERIALS AND METHODS Preparation of Human Polymorphonuclear Leukocytes (PMN) PMNs were prepared essentially by the method of Boyum (4). Peripheral blood from healthy human volunteers was obtained by vein puncture and collected in vacutainer tubes containing 1,500 units/ml heparin (Sigma Chemical Co, St. Louis, MO). Heparinized blood was mixed with a n equal volume of 5% Dextran T-500 (Pharmacia, Piscataway, NJ) in 1x Hanks balanced salt solution (HBSS; Gibco Laboratories, Grand Island, NY) containing 10 mM HEPES (Sigma Chemical Co.) and allowed to sediment at lg for 30 min. The supernatant was layered over Ficoll-Hypaque (Pharmacia) and spun at 300g at room temperature for 30 min. The
CHANGES IN PMN RALS OCCUR INDEPENDENTLY O F F-ACTIN
red blood cells were removed by hypotonic lysis a s follows: the cell pellet was resuspended and diluted with 5 ml of a 0.2% NaCl solution; after 30 s a n equal volume of a 1.6% NaCl solution was added to reestablish iso-osmolarity; this was followed by the addition of 15 ml of a 1x calcium-magnesium-free-Hanks balanced salt solution (CMF-HBSS; Gibco Laboratories) containing 10 mM HEPES. The cells were then spun at 200g for 8 min, a t room temperature. Red blood cell lysis and sedimentation were then repeated and the cell pellet was resuspended to a final concentration of 1 ~ 1 cells/ml in a 1x HBSS + HEPES solution. The cell concentration was determined by using a hemocytometer and cell viability was determined by trypan blue exclusion (>95% neutrophils). All solutions were adjusted to pH 7.4 prior to cell isolation, and except for the vacutainer, all labware was plastic.
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for 30 min and then spun a t 20Og €or 10 min a t 4°C. The pellet was then resuspended and diluted with 0.25 ml PF + PHEM solution and analyzed on a flow cytometer. Once stained, the cells were kept covered to prevent photobleaching.
Analysis of Actin PMN fluorescence was measured on a Coulter EPICS C flow cytometer equipped with a Coulter MDADS data acquisition system. PMNs were identified by 0means ~ of forward- and right-angle light scatter. NBDstained PMN fluorescence was excited a t 488 nm and detected at 510 nm. Histograms of cell number vs. log fluorescence intensity were recorded for 5,000 cells for each time point. A standard series of calibrated fluorescent polystyrene beads (Flow Cytometry Standards Corporation, Research Triangle Park, NC) was used to create a standard curve relating the log fluorescence Stimulants channel to linear fluorescence. Interpolation of this Formyl-methionyl-leucyl-phenylalanine(f-MLP),Na- standard curve was then used to convert the mean log propionate, Na-lactate, NH,Cl, and butanol were all fluorescence channel number to mean linear fluoresobtained from Sigma Chemical Co. Solutions were pre- cence (22). Relative mean linear fluorescence was calpared in 1x HBSS+HEPES solution. The final pH of culated as follows: each solution was adjusted to 7.4. RMLF = (MLFt/MLFo) x 100. Actin Staining Here RMLF indicates relative mean linear fluoresCells (3 x lo6 celld3.0 ml at 22°C) were inoculated cence, MLFt indicates the mean linear fluorescence at with individual stimulants (f-MLP, propionate, lactate, a given time point, and MLFo indicates the mean linNH,Cl, or butanol). For f-MLP-, propionate-, and bu- ear fluorescence a t time zero. tanol-stimulated time courses, cell aliquots (0.2 ml) were taken at 15 s intervals for the first 2 min followRight-Angle Light Scatter ing stimulation and then at 1min intervals for the next RALS was measured by using a SLM-SOOC spectro3 min for a total of 5 min. For the lactate-stimulated time courses, time points were taken once every 30 s for fluorometer (SLM Instruments Inc, Urbana, IL) and the first 2 min and once every minute for the next 3 computer acquisition system (IBM PS/2) at a n excitamin. For the NH,Cl-stimulated time courses, aliquots tion and emission wavelength of 340 nm (27); 3 x lo6 were taken every 15 s for the first minute, once every cells in 3.0 ml of a Hanks + Hepes buffer at pH 7.4 were 30 s for the second minute, and once every minute for placed within a quartz cuvette and then monitored €or the remaining 3 min. Cell aliquots were placed in 50 p1 30 s to establish a baseline (data not shown). This was of ice-cold extraction-fixation buffer. The extraction- followed by injection of a stimulant through a n injecfixation buffer (stock solutions) consisted of 2.5% !hi- tion port. The cells were then monitored for 5 min with ton X-165, 2.5% paraformaldehyde (PF),and 300 mM readings taken every 0.5 s. Throughout the monitoring PIPES, 125 mM HEPES, 50 mM EGTA, and 10 mM process the cells were gently stirred to maintain them MgCl (PHEM) (21). Triton X-165 was chosen to facili- in suspension. The percentage of change in RALS was tate neutrophil identification after extraction. We determined by dividing the amount of RALS measured found that Triton X-100 decreased both right- and for- a t every time point of a single 5 min time course by the ward-angle light scatter to near baseline. Therefore amount of RALS measured a t time zero and then mulTriton X-165 which extracts less membrane (1) and tiplying by 100. still allows for specific binding of NBD-phallacidin to Statistics actin (unpublished observation) was used. This and all subsequent preparative procedures were carried out at All actin staining experiments consisted of duplicate 4°C. All chemicals in the extraction buffer were pur- or triplicate runs on a given day with a specified stimchased from Sigma Chemical Co. The cells were incu- ulant. Each experiment was repeated at least three bated in the extraction-fixation buffer for 30 min and times with cells from different donors. The data are then spun at 200g for 10 min. The pellet was resus- reported as a mean of the means t the standard error. pended in 0.2 ml of NBD-Phallacidin (Molecular Probe, All light-scatter experiments were performed at least Junction City, OR) in PF + PHEM buffer at a final con- three times with different donors and are reported as a centration of 2 pM of NBD-phallacidin in IX mean & standard error. The light-scatter data was PF+PHEM (7). The cells were incubated in the stain computationally smoothed prior to averaging. This was
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accomplished by plotting a running mean of every three points over a single 5 min time course (28).
RESULTS f-MLP Stimulation Figure 1A displays a 5 min time course of right-angle light scatter (RALS) following PMN stimulation with 1 pM f-MLP. The graph displays a 9 t 1%drop in RALS, 47 s after stimulation. This is followed by a rapid retur n to baseline, 79 s after the nadir. The RALS continued to increase past baseline, to a level 4 % 2% above the normal resting level, 300 s after stimulation. This response is similar to the f-MLP-stimulated response of neutrophils initially reported by Yuli and Snyderman (40). Figure 1B displays a 5 rnin time course of F-actin following PMN stimulation with 1 pM f-MLP. The graph indicates a biphasic response. F-actin reaches a zenith of 2.15 * .03 times the normal resting level of F-actin 30 s after stimulation with 1 pM f-MLP. This is followed by a slow return toward baseline, from the zenith, to a level 1.45 k .04 times the normal resting level of F-actin. This response is similar to the response of neutrophil stimulated with f-MLP, initially reported by Howard et al. (7,8). While both RALS and F-actin exhibit a n initial rapid change, the rate and magnitude of the return toward baseline differ. This suggests that the second phase of the RALS response can occur independently of changes in cytoskeletal actin. Short-Chain Carboxylic Acid (SCCA) Stimulation Because of the noted secondary phase differences in PMN RALS and F-actin responses to stimulation with f-MLP, we examined the responses of PMNs stimulated with propionate and lactate. These short-chain carboxylic acids were selected because propionate has been shown to stimulate a RALS response (39,401 as well as F-actin polymerization in PMNs (11,241 whereas work in this laboratory indicated that lactate does not stimulate F-actin polymerization. Figure 2A displays a 5 rnin RALS time course following PMN stimulation with 5 mM propionate. The data indicate that propionate stimulates a biphasic RALS response, with a 9 5 1% drop in RALS 36 s after stimulation. This is followed by a rapid return to the normal resting level of RALS 38 s after the nadir. Upon reaching baseline, 74 s after stimulation, there is a slow increase in RALS to 104 5 3% above baseline 5 min after stimulation. This response is similar to the RALS response observed after stimulation with f-MLP. Figure 2B displays a 5 rnin time course of F-actin following PMN stimulation with 5 mM propionate. The data indicate that propionate stimulates a biphasic change in F-actin, reaching a zenith 2.09 5 .08 times the normal resting level, 30 s after stimulation. After reaching the zenith there is a sharp decrease in the level of F-actin, reaching normal resting levels within 30 s. The F-actin level then remains a t baseline for the duration of the time course (4 rnin).
Figure 3A displays a RALS time course following PMN stimulation with 30 mM lactate. The data indicate that lactate stimulates a biphasic RALS response with a 16 0.6%decrease in RALS 46 s after stimulation. This is followed by a rapid return to baseline 130 s after the nadir. Furthermore, after reaching baseline there is a continued increase in RALS. Five minutes after stimulation the level of RALS is 6 5 1% above the normal resting level. Both the initial and secondary RALS responses to lactate are therefore much greater than that stimulated by f-MLP. Figure 3B displays a 5 min time course of F-actin following PMN stimulation with 30 mM lactate. The data indicate that lactate does not stimulate a change in F-actin content. Together, the RALS and F-actin data indicate th a t lactate stimulates a biphasic change in RALS without affecting a net actin polymerization or depolymerization in the PMN. This suggests that changes in RALS can occur without a net increase or decrease in cytoskeletal actin. Ammonium Chloride (NH,Cl) Stimulation NH,Cl was selected because of its alkaline properties. Figure 4A displays a RALS time course following PMN stimulation with 30 mM NH,Cl. The data indicate that NH&l stimulates a biphasic change in RALS with a 12 ? 3% drop 15 s after stimulation. This is followed by a slow increase in RALS to a level 6 % 5% below the normal resting level 5 min after stimulation. Figure 4B displays a 5 min time course of F-actin, following PMN stimulation with 30 mM NH,Cl. The data indicate that NH,Cl stimulates a rapid decrease in F-actin, with a drop to 0.62 2 0.02 times the normal resting level, 15 s after stimulation. This is followed by a further slow decrease in F-actin to a level 0.44 0.02 times the normal resting level 5 min after stimulation. These results indicate that NH,C1 can stimulate both a biphasic RALS response and a depolymerization of Factin. However, the RALS response is similar to the RALS response stimulated by f-MLP. This suggests that a biphasic RALS response can occur during a period of continued net F-actin depolymerization. Butanol Stimulation Butanol causes membrane fluidization (40) and can accelerate neutrophil responses (40). We therefore examined its effect on neutrophil F-actin and RALS. Figure 5A displays a RALS time course following PMN stimulation with 0.2% butanol. The data indicate that no change in RALS occurs following stimulation with butanol. Figure 5B displays a 5 rnin time course of F-actin, following PMN stimulation with 0.2% butanol. The data indicate that butanol stimulates no change in F-actin content. These results indicate that butanolstimulated membrane fluidization will not cause a change in PMN RALS or F-actin content. DISCUSSION Changes in right-angle light scatter are thought to reflect changes in subcellular structural elements
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which are smaller than the wavelength of the exciting light (9,10,19,20).This led Yuli and Snyderman (40) to propose that early changes in RALS are related to membrane ruffling. Sklar et al. (27) recognized the following two points: First, that actin polymerization was a n early, subcellular structural event following neutrophi1 stimulation (7,8,33); second, that actin polymerization is thought to be responsible for cell motility (18,29) and in particular neutrophil membrane ruffling (3,39-42). They therefore reasoned (27) and provided evidence to support the hypothesis that changes in RALS accurately reflected changes in actin polymerization. This method has now received some degree of acceptance (6,13,16,17,26,27,39). In this paper we critically examine this application of RALS. We reasoned that if RALS is indicative of the cytoskeletal F-actin, two conditions would reasonably be expected to be met. First, the levels of RALS and F-actin would be expected to move in concert. In other words during periods of actin polymerization, depolymerization, or no net change, RALS should change in a harmonious manner. Second, the magnitude of actin change should be reflected in a proportional magnitude of RALS change. As indicated below, RALS fulfills neither of these criteria. The data therefore suggest that changes in RALS can occur independently of changes in actin.
occur in parallel with stimulated neutrophil actin polymerization. The subsequent discussion examines more closely the relationship of RALS and actin polymerization by addressing two other questions. Can changes in RALS occur in the absence of net actin polymerization and depolymerization? Is net actin depolymerization always reflected in a n increase in RALS?
Lactate The stimulation of the PMNs with lactate causes a biphasic change in RALS. This change is greater than that stimulated by f-MLP, in terms of rate, extent, and duration. Lactate stimulates a 16% drop in RALS followed by a n increase to 6% above baseline 300 s after stimulation. In comparison, F-MLP stimulates a 9% drop in RALS followed by a increase to 4% above baseline, 300 s after stimulation. Based on this information, one would expect that lactate would stimulate a change in F-actin greater than or a t least similar to the change stimulated by f-MLP. However, lactate stimulates no net change in F-actin content. This response is similar to the response of PMN's when stimulated with 12(S)hydroxyeicosatetraenoic acid (13). These results suggest that changes in RALS can occur in the absence of both net actin polymerization and net actin depolymerization.
NH,CI EMLP As currently applied, decreases in RALS are thought The stimulation of PMNs with f-MLP results in a well-characterized biphasic RALS (6,13,16,39,40) and to reflect a n increase in cytoskeletal actin. The conactin (7,8,16,33,36-38) response. However, in compar- verse should also be true-namely, stimulation of net ing the data obtained by the two methods, similarities cytoskeletal F-actin depolymerization should effect a n and differences are apparent. Both responses exhibit a increase in RALS. This is not the case. PMNs stimusimilar rapid initial change. However, they exhibit dif- lated with NH4C1 display a rapid net F-actin depolyfering secondary responses. The RALS data indicate a merization, within 15 s. This is followed by a continquick return to baseline while the F-actin data indicate ued, slower decrease in F-actin content for the duration a slow return toward baseline. Therefore, a t the end of of the 5 min time course. In contrast to expectation, the time course (5 minutes after Stimulation), the level NH4C1 stimulates a rapid drop in RALS, followed by a of F-actin has not yet returned to the normal resting slow return toward baseline. The initial drop in RALS level. In contrast, the level of RALS has moved to, and is similar to that displayed by all the stimulants prethen beyond, the initial resting level. This suggests viously reported here. Thus PMN stimulation with that while the initial RALS changes may be indicative NH,Cl initiates a drop in RALS and a decrease in Fof actin polymerization, it is not necessarily indicative actin content. This suggests that RALS changes can, in some cases, reflect F-actin depolymerization. We conof actin depolymerization. clude that a decrease in RALS is therefore not a n acPropionate curate indicator of cytoskeletal actin polymerization in Like f-MLP, the stimulation of the PMNs with pro- PMNs. pionate causes a biphasic RALS and actin response. Membrane Fluidization Unlike the f-MLP data, the propionate-stimulated RALS and F-actin data resemble each other in rate, The final set of experiments used butanol to deterduration, and magnitude of change. However, 5 min mine whether simple membrane fluidization (40) could after stimulation the RALS is continuing to increase account for the observed RALS effects. The results indicate that membrane fluidization appears to have no while the F-actin remains at baseline. Taken together, the preceding data make three effect on either RALS or F-actin. points. First, changes in RALS can occur indepenConcluding Comment dently of changes in cytoskeletal actin depolymerizaThe data presented here lead one to conclude that tion. Second, changes in RALS can occur when there is no net change in F-actin. Third, changes in RALS can changes in RALS can occur independently of a n in-
CHANGES IN PMN RALS OCCUR INDEPENDENTLY OF F-ACTIN
crease, a decrease, or no change in PMN cytoskeletal F-actin. RALS changes do not therefore appear to be an accurate indicator of neutrophil actin changes. This then leads us to ask the question: What does RALS measure? Clearly RALS is sensitive to a change in internal cell structure. This light scatter can be attributed to diffraction, reflection, and refraction of light entering, moving through, and leaving the cell. Within the cell, subcellular constituents (e.g., cellular, nuclear, and organelle membranes) are in a constant state of flux, affecting the scattering of light. Also, the microfilament system (e.g., actin, microtubules, and intermediate filaments) can change its structure or position within the cell and affect the RALS. Thus great caution must be taken when interpreting RALS data. However, because RALS changes are so sensitive to the addition of all stimuli, RALS may be an accurate indicator for monitoring early events of cell activation (40).
ACKNOWLEDGMENTS This work was supported by NIH grants DE07675 and DE08415.
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Naccache PH, Feinstein MB, Sha’afi RI: Effects of chemotactic factors and other agents on the amounts of actin and a 65,000mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils. J Cell Biol 101:182-188, 1985. 38. Yuli I, Oplatka A: Cytosolic acidification as a n early transductory signal of human neutrophil chemotaxis. Science 235340-342, 1987. 39. Yuli I, Snyderman R: Rapid changes in light scattering from hu-
man polymorphonuclear leukocytes exposed to chemoattractants. J Clin Invest 73:1408-1417, 1984. 40. Zigmond S, Sullivan SJ: Sensory adaptation of leukocytes to chemotactic peptides. J Cell Biol 82:517-527, 1979. 41. Zigmond SH, Levitsky HI, BJ Kreel: Cell polarity: An examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis. J Cell Biol 89:585-592, 1981.
0 1990 Wiley-Liss, Inc.
Changes in Neutrophil Right-Angle Light Scatter Can Occur Independently of Alterations in Cytoskeletal Actin Eliyahu Kraus and Richard Niederman Department of Cell Biology, Forsyth Dental Research Center, Boston, Massachusetts 02115 Received for publication April 14,1989; accepted July 12, 1989
Forward-angle light scatter (FALS) and right-angle light scatter (RALS) are commonly employed to discriminate between leukocyte subclasses. Recently the application of RALS has expanded, and it is now also used as an indicator of neutrophil actin polymerization. In this communication we critically examine the relationship of RALS to changes in cytoskeletal actin. The data indicate that agonists which stimulate an increase, a decrease, or no change in F-actin content can all stimulate a biphasic change in RALS. We therefore conclude that chan-
Flow cytometric measurement of both forward- and right-angle light scatter is commonly employed to discriminate between leukocyte subclasses (10,19,25). Light scatter is also used to: sort cells (14,15,23,35), identify cell heterogeneity in similar cell types (30,31,34), and define cell morphology (2,321. Forward-angle light scatter (FALS) is the amount of light that is scattered off a n object and measured within 2" from the incident beam (25). FALS is attributed to the amount of light scattered from both the main contour of the cell and the nucleus (9,10,19,20). Thus FALS is primarily used in the identification of cell types according to their size (12). Similarly, rightangle light scatter (RALS) is the amount of light scattered off an object and measured a t 90" from the incident beam (25). However, RALS is attributed to the scattered light from subcellular structures having dimensions equal to or smaller than the wavelength of the exciting light. Because of this, RALS is thought to detect changes in membrane ruff ling and intracellular granules (40). This last application led to the observation that changes in RALS appear to parallel actin polymerization and depolymerization in chemotactic peptide stimulated neutrophils (27). This in t u r n led to the use of RALS as a n indicator of changes in F-actin content in neutrophils (6,13,16,17,26,27,39).
ges in RALS can occur independently of changes in F-actin content. This leads us to suggest that caution must be taken when interpreting RALS data in relation to changes in F-actin. Furthermore, the data also support the idea originally proposed by Yuli and Snyderman (J Clin Invest 73:1408-1417, 1984), that RALS may be an exceptionally sensitive indicator of cell activation. Key terms: Lactate, f-MLP, propionate, ammonium chloride
In this paper we critically examine the utility of RALS as a n indicator of cytoskeletal F-actin changes in neutrophils. The data indicate that agonists which stimulate a n increase, a decrease, or no change in cytoskeletal F-actin can all stimulate a biphasic change in RALS. We therefore conclude that changes in RALS can occur independently of actin polymerization. However, the data also suggests, as originally proposed by Yuli and Snyderman (40), that RALS may be a n exceptionally sensitive indicator of cell activation.
MATERIALS AND METHODS Preparation of Human Polymorphonuclear Leukocytes (PMN) PMNs were prepared essentially by the method of Boyum (4). Peripheral blood from healthy human volunteers was obtained by vein puncture and collected in vacutainer tubes containing 1,500 units/ml heparin (Sigma Chemical Co, St. Louis, MO). Heparinized blood was mixed with a n equal volume of 5% Dextran T-500 (Pharmacia, Piscataway, NJ) in 1x Hanks balanced salt solution (HBSS; Gibco Laboratories, Grand Island, NY) containing 10 mM HEPES (Sigma Chemical Co.) and allowed to sediment at lg for 30 min. The supernatant was layered over Ficoll-Hypaque (Pharmacia) and spun at 300g at room temperature for 30 min. The
CHANGES IN PMN RALS OCCUR INDEPENDENTLY O F F-ACTIN
red blood cells were removed by hypotonic lysis a s follows: the cell pellet was resuspended and diluted with 5 ml of a 0.2% NaCl solution; after 30 s a n equal volume of a 1.6% NaCl solution was added to reestablish iso-osmolarity; this was followed by the addition of 15 ml of a 1x calcium-magnesium-free-Hanks balanced salt solution (CMF-HBSS; Gibco Laboratories) containing 10 mM HEPES. The cells were then spun at 200g for 8 min, a t room temperature. Red blood cell lysis and sedimentation were then repeated and the cell pellet was resuspended to a final concentration of 1 ~ 1 cells/ml in a 1x HBSS + HEPES solution. The cell concentration was determined by using a hemocytometer and cell viability was determined by trypan blue exclusion (>95% neutrophils). All solutions were adjusted to pH 7.4 prior to cell isolation, and except for the vacutainer, all labware was plastic.
273
for 30 min and then spun a t 20Og €or 10 min a t 4°C. The pellet was then resuspended and diluted with 0.25 ml PF + PHEM solution and analyzed on a flow cytometer. Once stained, the cells were kept covered to prevent photobleaching.
Analysis of Actin PMN fluorescence was measured on a Coulter EPICS C flow cytometer equipped with a Coulter MDADS data acquisition system. PMNs were identified by 0means ~ of forward- and right-angle light scatter. NBDstained PMN fluorescence was excited a t 488 nm and detected at 510 nm. Histograms of cell number vs. log fluorescence intensity were recorded for 5,000 cells for each time point. A standard series of calibrated fluorescent polystyrene beads (Flow Cytometry Standards Corporation, Research Triangle Park, NC) was used to create a standard curve relating the log fluorescence Stimulants channel to linear fluorescence. Interpolation of this Formyl-methionyl-leucyl-phenylalanine(f-MLP),Na- standard curve was then used to convert the mean log propionate, Na-lactate, NH,Cl, and butanol were all fluorescence channel number to mean linear fluoresobtained from Sigma Chemical Co. Solutions were pre- cence (22). Relative mean linear fluorescence was calpared in 1x HBSS+HEPES solution. The final pH of culated as follows: each solution was adjusted to 7.4. RMLF = (MLFt/MLFo) x 100. Actin Staining Here RMLF indicates relative mean linear fluoresCells (3 x lo6 celld3.0 ml at 22°C) were inoculated cence, MLFt indicates the mean linear fluorescence at with individual stimulants (f-MLP, propionate, lactate, a given time point, and MLFo indicates the mean linNH,Cl, or butanol). For f-MLP-, propionate-, and bu- ear fluorescence a t time zero. tanol-stimulated time courses, cell aliquots (0.2 ml) were taken at 15 s intervals for the first 2 min followRight-Angle Light Scatter ing stimulation and then at 1min intervals for the next RALS was measured by using a SLM-SOOC spectro3 min for a total of 5 min. For the lactate-stimulated time courses, time points were taken once every 30 s for fluorometer (SLM Instruments Inc, Urbana, IL) and the first 2 min and once every minute for the next 3 computer acquisition system (IBM PS/2) at a n excitamin. For the NH,Cl-stimulated time courses, aliquots tion and emission wavelength of 340 nm (27); 3 x lo6 were taken every 15 s for the first minute, once every cells in 3.0 ml of a Hanks + Hepes buffer at pH 7.4 were 30 s for the second minute, and once every minute for placed within a quartz cuvette and then monitored €or the remaining 3 min. Cell aliquots were placed in 50 p1 30 s to establish a baseline (data not shown). This was of ice-cold extraction-fixation buffer. The extraction- followed by injection of a stimulant through a n injecfixation buffer (stock solutions) consisted of 2.5% !hi- tion port. The cells were then monitored for 5 min with ton X-165, 2.5% paraformaldehyde (PF),and 300 mM readings taken every 0.5 s. Throughout the monitoring PIPES, 125 mM HEPES, 50 mM EGTA, and 10 mM process the cells were gently stirred to maintain them MgCl (PHEM) (21). Triton X-165 was chosen to facili- in suspension. The percentage of change in RALS was tate neutrophil identification after extraction. We determined by dividing the amount of RALS measured found that Triton X-100 decreased both right- and for- a t every time point of a single 5 min time course by the ward-angle light scatter to near baseline. Therefore amount of RALS measured a t time zero and then mulTriton X-165 which extracts less membrane (1) and tiplying by 100. still allows for specific binding of NBD-phallacidin to Statistics actin (unpublished observation) was used. This and all subsequent preparative procedures were carried out at All actin staining experiments consisted of duplicate 4°C. All chemicals in the extraction buffer were pur- or triplicate runs on a given day with a specified stimchased from Sigma Chemical Co. The cells were incu- ulant. Each experiment was repeated at least three bated in the extraction-fixation buffer for 30 min and times with cells from different donors. The data are then spun at 200g for 10 min. The pellet was resus- reported as a mean of the means t the standard error. pended in 0.2 ml of NBD-Phallacidin (Molecular Probe, All light-scatter experiments were performed at least Junction City, OR) in PF + PHEM buffer at a final con- three times with different donors and are reported as a centration of 2 pM of NBD-phallacidin in IX mean & standard error. The light-scatter data was PF+PHEM (7). The cells were incubated in the stain computationally smoothed prior to averaging. This was
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accomplished by plotting a running mean of every three points over a single 5 min time course (28).
RESULTS f-MLP Stimulation Figure 1A displays a 5 min time course of right-angle light scatter (RALS) following PMN stimulation with 1 pM f-MLP. The graph displays a 9 t 1%drop in RALS, 47 s after stimulation. This is followed by a rapid retur n to baseline, 79 s after the nadir. The RALS continued to increase past baseline, to a level 4 % 2% above the normal resting level, 300 s after stimulation. This response is similar to the f-MLP-stimulated response of neutrophils initially reported by Yuli and Snyderman (40). Figure 1B displays a 5 rnin time course of F-actin following PMN stimulation with 1 pM f-MLP. The graph indicates a biphasic response. F-actin reaches a zenith of 2.15 * .03 times the normal resting level of F-actin 30 s after stimulation with 1 pM f-MLP. This is followed by a slow return toward baseline, from the zenith, to a level 1.45 k .04 times the normal resting level of F-actin. This response is similar to the response of neutrophil stimulated with f-MLP, initially reported by Howard et al. (7,8). While both RALS and F-actin exhibit a n initial rapid change, the rate and magnitude of the return toward baseline differ. This suggests that the second phase of the RALS response can occur independently of changes in cytoskeletal actin. Short-Chain Carboxylic Acid (SCCA) Stimulation Because of the noted secondary phase differences in PMN RALS and F-actin responses to stimulation with f-MLP, we examined the responses of PMNs stimulated with propionate and lactate. These short-chain carboxylic acids were selected because propionate has been shown to stimulate a RALS response (39,401 as well as F-actin polymerization in PMNs (11,241 whereas work in this laboratory indicated that lactate does not stimulate F-actin polymerization. Figure 2A displays a 5 rnin RALS time course following PMN stimulation with 5 mM propionate. The data indicate that propionate stimulates a biphasic RALS response, with a 9 5 1% drop in RALS 36 s after stimulation. This is followed by a rapid return to the normal resting level of RALS 38 s after the nadir. Upon reaching baseline, 74 s after stimulation, there is a slow increase in RALS to 104 5 3% above baseline 5 min after stimulation. This response is similar to the RALS response observed after stimulation with f-MLP. Figure 2B displays a 5 rnin time course of F-actin following PMN stimulation with 5 mM propionate. The data indicate that propionate stimulates a biphasic change in F-actin, reaching a zenith 2.09 5 .08 times the normal resting level, 30 s after stimulation. After reaching the zenith there is a sharp decrease in the level of F-actin, reaching normal resting levels within 30 s. The F-actin level then remains a t baseline for the duration of the time course (4 rnin).
Figure 3A displays a RALS time course following PMN stimulation with 30 mM lactate. The data indicate that lactate stimulates a biphasic RALS response with a 16 0.6%decrease in RALS 46 s after stimulation. This is followed by a rapid return to baseline 130 s after the nadir. Furthermore, after reaching baseline there is a continued increase in RALS. Five minutes after stimulation the level of RALS is 6 5 1% above the normal resting level. Both the initial and secondary RALS responses to lactate are therefore much greater than that stimulated by f-MLP. Figure 3B displays a 5 min time course of F-actin following PMN stimulation with 30 mM lactate. The data indicate that lactate does not stimulate a change in F-actin content. Together, the RALS and F-actin data indicate th a t lactate stimulates a biphasic change in RALS without affecting a net actin polymerization or depolymerization in the PMN. This suggests that changes in RALS can occur without a net increase or decrease in cytoskeletal actin. Ammonium Chloride (NH,Cl) Stimulation NH,Cl was selected because of its alkaline properties. Figure 4A displays a RALS time course following PMN stimulation with 30 mM NH,Cl. The data indicate that NH&l stimulates a biphasic change in RALS with a 12 ? 3% drop 15 s after stimulation. This is followed by a slow increase in RALS to a level 6 % 5% below the normal resting level 5 min after stimulation. Figure 4B displays a 5 min time course of F-actin, following PMN stimulation with 30 mM NH,Cl. The data indicate that NH,Cl stimulates a rapid decrease in F-actin, with a drop to 0.62 2 0.02 times the normal resting level, 15 s after stimulation. This is followed by a further slow decrease in F-actin to a level 0.44 0.02 times the normal resting level 5 min after stimulation. These results indicate that NH,C1 can stimulate both a biphasic RALS response and a depolymerization of Factin. However, the RALS response is similar to the RALS response stimulated by f-MLP. This suggests that a biphasic RALS response can occur during a period of continued net F-actin depolymerization. Butanol Stimulation Butanol causes membrane fluidization (40) and can accelerate neutrophil responses (40). We therefore examined its effect on neutrophil F-actin and RALS. Figure 5A displays a RALS time course following PMN stimulation with 0.2% butanol. The data indicate that no change in RALS occurs following stimulation with butanol. Figure 5B displays a 5 rnin time course of F-actin, following PMN stimulation with 0.2% butanol. The data indicate that butanol stimulates no change in F-actin content. These results indicate that butanolstimulated membrane fluidization will not cause a change in PMN RALS or F-actin content. DISCUSSION Changes in right-angle light scatter are thought to reflect changes in subcellular structural elements
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which are smaller than the wavelength of the exciting light (9,10,19,20).This led Yuli and Snyderman (40) to propose that early changes in RALS are related to membrane ruffling. Sklar et al. (27) recognized the following two points: First, that actin polymerization was a n early, subcellular structural event following neutrophi1 stimulation (7,8,33); second, that actin polymerization is thought to be responsible for cell motility (18,29) and in particular neutrophil membrane ruffling (3,39-42). They therefore reasoned (27) and provided evidence to support the hypothesis that changes in RALS accurately reflected changes in actin polymerization. This method has now received some degree of acceptance (6,13,16,17,26,27,39). In this paper we critically examine this application of RALS. We reasoned that if RALS is indicative of the cytoskeletal F-actin, two conditions would reasonably be expected to be met. First, the levels of RALS and F-actin would be expected to move in concert. In other words during periods of actin polymerization, depolymerization, or no net change, RALS should change in a harmonious manner. Second, the magnitude of actin change should be reflected in a proportional magnitude of RALS change. As indicated below, RALS fulfills neither of these criteria. The data therefore suggest that changes in RALS can occur independently of changes in actin.
occur in parallel with stimulated neutrophil actin polymerization. The subsequent discussion examines more closely the relationship of RALS and actin polymerization by addressing two other questions. Can changes in RALS occur in the absence of net actin polymerization and depolymerization? Is net actin depolymerization always reflected in a n increase in RALS?
Lactate The stimulation of the PMNs with lactate causes a biphasic change in RALS. This change is greater than that stimulated by f-MLP, in terms of rate, extent, and duration. Lactate stimulates a 16% drop in RALS followed by a n increase to 6% above baseline 300 s after stimulation. In comparison, F-MLP stimulates a 9% drop in RALS followed by a increase to 4% above baseline, 300 s after stimulation. Based on this information, one would expect that lactate would stimulate a change in F-actin greater than or a t least similar to the change stimulated by f-MLP. However, lactate stimulates no net change in F-actin content. This response is similar to the response of PMN's when stimulated with 12(S)hydroxyeicosatetraenoic acid (13). These results suggest that changes in RALS can occur in the absence of both net actin polymerization and net actin depolymerization.
NH,CI EMLP As currently applied, decreases in RALS are thought The stimulation of PMNs with f-MLP results in a well-characterized biphasic RALS (6,13,16,39,40) and to reflect a n increase in cytoskeletal actin. The conactin (7,8,16,33,36-38) response. However, in compar- verse should also be true-namely, stimulation of net ing the data obtained by the two methods, similarities cytoskeletal F-actin depolymerization should effect a n and differences are apparent. Both responses exhibit a increase in RALS. This is not the case. PMNs stimusimilar rapid initial change. However, they exhibit dif- lated with NH4C1 display a rapid net F-actin depolyfering secondary responses. The RALS data indicate a merization, within 15 s. This is followed by a continquick return to baseline while the F-actin data indicate ued, slower decrease in F-actin content for the duration a slow return toward baseline. Therefore, a t the end of of the 5 min time course. In contrast to expectation, the time course (5 minutes after Stimulation), the level NH4C1 stimulates a rapid drop in RALS, followed by a of F-actin has not yet returned to the normal resting slow return toward baseline. The initial drop in RALS level. In contrast, the level of RALS has moved to, and is similar to that displayed by all the stimulants prethen beyond, the initial resting level. This suggests viously reported here. Thus PMN stimulation with that while the initial RALS changes may be indicative NH,Cl initiates a drop in RALS and a decrease in Fof actin polymerization, it is not necessarily indicative actin content. This suggests that RALS changes can, in some cases, reflect F-actin depolymerization. We conof actin depolymerization. clude that a decrease in RALS is therefore not a n acPropionate curate indicator of cytoskeletal actin polymerization in Like f-MLP, the stimulation of the PMNs with pro- PMNs. pionate causes a biphasic RALS and actin response. Membrane Fluidization Unlike the f-MLP data, the propionate-stimulated RALS and F-actin data resemble each other in rate, The final set of experiments used butanol to deterduration, and magnitude of change. However, 5 min mine whether simple membrane fluidization (40) could after stimulation the RALS is continuing to increase account for the observed RALS effects. The results indicate that membrane fluidization appears to have no while the F-actin remains at baseline. Taken together, the preceding data make three effect on either RALS or F-actin. points. First, changes in RALS can occur indepenConcluding Comment dently of changes in cytoskeletal actin depolymerizaThe data presented here lead one to conclude that tion. Second, changes in RALS can occur when there is no net change in F-actin. Third, changes in RALS can changes in RALS can occur independently of a n in-
CHANGES IN PMN RALS OCCUR INDEPENDENTLY OF F-ACTIN
crease, a decrease, or no change in PMN cytoskeletal F-actin. RALS changes do not therefore appear to be an accurate indicator of neutrophil actin changes. This then leads us to ask the question: What does RALS measure? Clearly RALS is sensitive to a change in internal cell structure. This light scatter can be attributed to diffraction, reflection, and refraction of light entering, moving through, and leaving the cell. Within the cell, subcellular constituents (e.g., cellular, nuclear, and organelle membranes) are in a constant state of flux, affecting the scattering of light. Also, the microfilament system (e.g., actin, microtubules, and intermediate filaments) can change its structure or position within the cell and affect the RALS. Thus great caution must be taken when interpreting RALS data. However, because RALS changes are so sensitive to the addition of all stimuli, RALS may be an accurate indicator for monitoring early events of cell activation (40).
ACKNOWLEDGMENTS This work was supported by NIH grants DE07675 and DE08415.
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