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Published online before print January 14, 2004
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* Department of Bioengineering and The Whitaker Institute of Biomedical Engineering, University of California San Diego, La Jolla, California; and
Second Department of Surgery, Hamamatsu University School of Medicine, Japan
1Correspondence: Department of Bioengineering, The Whitaker Institute of Biomedical Engineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412. E-mail: gwss{at}bioeng.ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.5 dyn/cm2) by cytoplasmic reorganization. The cytoplasm is also influenced, however, by glucocorticoids. In this study, we explore how glucocorticoids may affect the leukocyte fluid shear response. Normal leukocytes, exposed to fluid shear in vitro during active migration, retract pseudopods accompanied by modestly decreasing intracellular calcium ions. In contrast, dexamethasone (DX)-treated leukocytes project pseudopods after shear exposure with a significant rise in intracellular calcium ions, an effect that can be blocked by voltage-dependent calcium channel blockers. Although a cyclic adenine monophosphate analog blocks calcium influx and pseudopod projection by DX, inhibition of A-kinase induces reversal of the shear response, as seen with DX treatment. DX also reverses the leukocyte shear response in vivo in the rat circulation. Leukocytes that adhere to the endothelium in postcapillary venules of control rats return into the circulation only after pseudopod retraction, and in DX-treated rats, adherent leukocytes return into the circulation still with projecting pseudopods. The fraction of circulating leukocytes with pseudopods in DX-treated rats is higher than in controls. Thus, the reversal of leukocyte shear response by glucocorticoids may contribute to an enhanced incidence of circulating leukocytes with pseudopods, a process that affects the kinetics of these cells in the microcirculation.
Key Words: mechanotransduction • fluid shear stress • pseudopod formation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.5 dyn/cm2) is applied to the membrane of fresh human leukocytes (neutrophils, monocytes, and lymphocytes) during migration on a surface, they respond within minutes by retraction of pseudopods as a result of actin depolymerization [1
2
3
]. Rat leukocytes suspended in whole blood also respond to fluid shear in a cone-and-plate shear device (5 dyn/cm2) by pseudopod retraction [2
, 3
]. Inflammatory mediators, such as platelet activating factor (PAF) and f-Met-Leu-Phe (fMLP), suppress the shear response of leukocytes, and nitric oxide derived from endothelial cells enhances the shear response via a cyclic guanosine monophosphate pathway [2
]. The shear stress response of leukocytes serves as a key mechanism to maintain circulatory leukocytes in a spherical shape without pseudopod formation. In the presence of pseudopods, leukocytes become trapped in capillaries [4
]. Thus, an elevated number of leukocytes with pseudopods as a result of an attenuated shear response may compromise normal passage of leukocytes through capillaries. Glucocorticoids have several anti-inflammatory activities [5 , 6 ]. The effect of glucocorticoids, however, on the fluid shear response is unexplored. We present a sequence of in vivo and in vitro studies designed to explore several mechanisms underlying the reversed fluid shear response in glucocorticoid-treated leukocytes. The evidence indicates that glucocorticoids reverse the normal leukocyte shear response; i.e., during shear exposure, they exhibit pseudopod projection instead of retraction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Shear response of adherent leukocytes
Erythrocytes in fresh blood from volunteers (30 U/ml ammonium heparin as anticoagulant) were separated by 1 g sedimentation. Aliquots of the supernatant with leukocytes were resuspended in buffer containing Plasma-Lyte (1:20, Baxter Health Care, Deerfield, IL; with 2.5 mM Ca2+), incubated with 1 µM Indo-1 AM (Molecular Probes, Eugene, OR) at 37°C for 30 min, and placed on a cover glass. After 15 min, the cover glass was gently rinsed with the buffer, and adherent leukocytes on the glass were observed with an inverted laser confocal microscope (MRC 1024, Bio-Rad Laboratories, Hercules, CA) with 60x objective (NA=1.4). The 410/490 nm emission ratio and transmission image were recorded. Using a calcium calibration buffer kit (Molecular Probes), the absolute values of intracellular-free calcium levels, [Ca2+]i, were computed using the Grynkiewicz equation. [7
].
Micropipettes with tip diameter between 4 and 6 µm were positioned adjacent to individual neutrophils so that a fluid flow could be applied over the cell membrane. The pipettes were filled with the same buffer in which the cells were suspended. The flow rate out of the pipette was adjusted by hydrostatic pressure inside the pipette, and in pilot studies, the peak (midstream) velocity at each pressure was measured by tracing the microsphere (0.2 µm diameter) positions [1
]. The magnitude of fluid stress was numerically computed for exactly the same setup (including size of cell, pipette, position of the pipette) by solution of the Stokes approximation of the equation of motion for a Newtonian fluid (plasma, 1.2 centipoise) and use of a finite element method [1
]. This computation shows that the flow field over the surface of the cell yields a spatially nonuniform shear stress. A peak shear stress of 1.5 dyn/cm2 is present on the upstream side of the cell close to the pipette; on the opposite, down-stream side, the shear stress is close to zero [1
].
As a measure for the degree of cell extension as a result of pseudopod formation, the maximum length across the cell (L) was measured and normalized by cell diameter without pseudopods (L0; Fig. 1 ).
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Shear response of leukocytes in suspension
Eighteen animals were used for the experiments. PAF, fMLP, DX, diltiazem, nifedipine, and 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP; all from Sigma Chemical Co., St Louis, MO), R-p-bromoadenosine 3',5'-cyclic monophosphorothioate (Rp-8-b-cAMPS; Life Science, San Diego, CA), or physiological saline were applied 30-min after arterial blood collection (30 U/ml ammonium heparin). Fifty minutes after blood collection, whole blood (0.3 ml) was sheared in the cone-and-plate device (5.0 dyn/cm2 for 10 min) [2
] and immediately fixed with 2% glutaraldehyde. Unsheared samples were fixed at the same time and analyzed in parallel with the sheared samples. After staining with 0.02% crystal violet, the fraction of leukocytes with pseudopods was counted in a blinded manner.
Fraction of circulating leukocytes with pseudopods in arterial blood
Femoral arterial blood (0.3 ml) was collected with 30 U/ml ammonium heparin from eight control rats and eight rats treated with 0.5 mg/kg/day (intramuscularly) for 7 days (DX-treated rats). The cells were immediately fixed with 2% glutaraldehyde, and the fraction of leukocytes with pseudopods was counted in the same way as explained above.
Shear response of leukocytes adhering to postcapillary venules in the microcirculation
The ileocecal portion of the mesentery of four control Wistar rats and four DX-treated rats (0.5 mg/kg/day for 7 days) was exteriorized. The tissue was carefully draped over a pedestal and superfused with a Krebs-Henseleit bicarbonate-buffered solution saturated with a 95% N2 and 5% CO2 mixture (36.5°C, pH 7.4). The mesenteric microcirculation was visualized with an intravital microscope (Leitz) through a 25x water immersion objective (Zeiss, NA=0.6) using a color CCD television camera (Optonics) and a Halogen light source with heat filter [1
2
3
]. fMLP (10 nM) was applied on the mesentery by superfusion 30 min before the experiment. To temporarily reduce fluid shear stress to near-zero values, one of the outflow postcapillary venules was occluded for 3 min with a blunt micropipette [1
, 2
]. Pseudopod formation on individual leukocytes downstream of the occlusion site was observed during stoppage and after flow restoration.
Statistics
Measurements are presented as mean ± SD. The data in Figure 2F
were analyzed using 
2 test. Differences between groups in other experimental data were analyzed by ANOVA and Fischer’s protected least significant difference test. The number of observations is indicated in the figure legends. P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, after DX treatment, pseudopod formation without shearing was less than in controls (Fig. 1E) , and after fluid shear exposure, leukocytes continued to project pseudopods and even spread (Fig . 1F 1G 1H and 2A) .
Pseudopod formation and [Ca2+]i
In control neutrophils, the [Ca2+]i insignificantly decreased during fluid shear in association with pseudopod retraction (Fig. 2A)
. However, after DX treatment, the [Ca2+]isignificantly increased in parallel with pseudopod projection during shear exposure (Fig. 2A)
. The calcium channel blocker, diltiazem, as well as the cAMP analog, 8-br-cAMP, almost completely inhibited the [Ca2+]i increase and pseudopod projection (Fig . 2B
and 2C) .
Treatment with 80 mM K+ had a similar influence on shear response as DX treatment (Fig. 2D) . The A-kinase inhibitor, Rp-8-b-cAMPS, also led to pseudopod projection and to an intracellular calcium increase (data not shown) after shear application, which diltiazem inhibited (Fig. 2E) . In the absence of shear stress, treatment with Rp-cAMPS caused leukocytes to remain in an almost spherical shape. In all groups, shear-induced pseudopod projection was consistently accompanied by a [Ca2+]i rise. The reversed shear response by DX was dose-dependent and observed regardless of the choice of the inflammatory stimulator (Fig. 2F) . Although in the absence of DX, 100% of leukocytes responded to shear application, in the presence of 0.1 µM DX, only 60% of leukocytes responded to shear exposure.
Shear response of circulating leukocytes after DX treatment
In the cone-and-plate device, the fraction of leukocytes with pseudopods in controls (n=5 animals) was lower after shear exposure, and with DX treatment (n=5 animals), the fraction was significantly higher after shear (Fig. 3A
). In the presence of inflammatory stimulators, the fraction was increased, and DX treatment served to impair the shear response.
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Shear response in DX-treated rats
The fraction of circulating leukocytes with pseudopods in arterial blood of controls was 5.1 ± 2.0% (n=8), and that in DX-treated rats (0.5 mg/kg/day for 7 days) was 41.0 ± 6.5% (n=8, Figs . 4
and 5A
).
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In contrast, in DX-treated rats (n=4, 0.5 mg/kg/day for 7 days) during topical application of fMLP, pseudopod formation was observed in 63% of leukocytes when blood flow was obstructed. During flow obstruction, fewer leukocytes (25%) adhered to the endothelium, and shorter pseudopods were projected (Fig. 5B) . Some leukocytes (25%) even retracted pseudopods. After flow restoration, all leukocytes continued to project pseudopods but were less adhesive and washed away in the blood stream.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In postcapillary venules of DX-treated rats, leukocytes continue to project pseudopods after flow restoration but are less adhesive and washed away in the blood stream (Fig. 5B) . Leukocyte transmigration, but not rolling or adhesion, is inhibited by DX in the inflamed hamster post-capillary venule [8 ]. With DX treatment, about two-thirds of adherent leukocytes become detached under normal flow conditions and returned to the blood stream, and transmigration requires a significantly longer period compared with controls [8 ]. These observations are consistent with the current in vivo data.
Mechanisms for reversed shear response by glucocorticoids
As the reversal of shear response was observed, even 20 min after DX treatment in vitro (Fig. 3A)
, the effect appears to require no new protein synthesis and instead, may be a direct, nongenomic effect on the plasma membrane. Glucocorticoids bind receptors on the cell surface and alter membrane ion permeability [9
]. Under the influence of shear stress, pseudopod projection on DX-treated leukocytes is accompanied by an increase in intracellular-free calcium level, and in untreated leukocytes, neither pseudopod projection nor a rise of calcium level is observed (Fig. 2A)
. The evidence suggests that the rise in intracellular calcium level during shear application as a result of glucocorticoids is closely associated with pseudopod projection.
Glucocorticoids modulate voltage-dependent Ca2+ channels [10 ]. Voltage-dependent Ca2+ channel blockers inhibit the ability of DX to induce pseudopod projection as well as an increase in cytoplasmic-free Ca2+ with shear application (Figs. 2B and 3B) . Moreover, after membrane depolarization by a high concentration of potassium ion, which serves to open voltage-dependent Ca2+ channels, pseudopod projection and a calcium rise were observed only during shear application, similar to the situation after DX treatment (Fig. 2D) . Shear-induced pseudopod projection in the presence of DX may be closely associated with calcium influx through voltage-dependent and shear-sensitive Ca2+ channels.
Shear-induced [Ca2+]i elevation and pseudopod projection as a result of DX were attenuated by a cAMP analog (Figs. 2C and 3C) . There are several mechanisms by which cAMP may act, such as activation of calcium pumps and inactivation of phospholipase C [11 ]. Another possibility is that cAMP may be associated with closure of a voltage-dependent and shear-sensitive Ca2+ channel via A-kinases. Several voltage-dependent Ca2+ channels are regulated by A-kinases, such that they have a stimulatory or an inhibitory effect on channel opening [12 ]. The A-kinase inhibitor, Rp-8-b-cAMPS, reverses the shear response and increases [Ca2+]i only during fluid shear application. This reversed shear response by Rp-8-b-cAMPS was also inhibited by diltiazem (Figs. 2E and 3C) . Therefore, A-kinase may have an inhibitory effect on opening the voltage-dependent, shear-sensitive Ca2+ channel so that glucocorticoid suppresses channel closure by A-kinase. It is interesting that in goldfish melanocytoma, DX induces cell spreading, which is inhibited by 8-br-cAMP or nifedipine [13 ].
Microvascular consequences of reversed leukocyte shear response by glucocorticoids
Glucocorticoids attenuate leukocyte activation and leukocyte-endothelial cell interaction by reduction of adhesion molecule expression [5
, 6
]. These actions may contribute to inhibition of leukocyte adhesion and emigration in postcapillary venules in inflammation (Fig. 5B)
. Generally, activated leukocytes increase their adhesion molecule expression on the membrane along with enhanced pseudopod formation. However, in the presence of fluid shear, glucocorticoids may cause dissociation between reduced expression of adhesion molecules and enhanced pseudopod formation. Reduced adhesion of leukocytes on venular endothelium and pseudopod projection under blood flow as a result of DX may contribute to the elevated fraction of leukocytes with pseudopods in the circulation (Figs. 4 and 5A)
. The evidence in turn clearly shows the importance of the leukocyte shear response to keep circulating leukocytes in a spherical state.
A high incidence of leukocytes with pseudopods as a result of a reversed shear response may change the transport and behavior of leukocytes in the microcirculation. As leukocytes in a spherical state are frequently larger than typical capillary lumen diameters, leukocytes need to change their shape to pass through most capillary networks. Therefore, their deformability is a key determinant for normal passage through the microcirculation [4 , 14 ]. Pseudopods are relatively rigid structures that exhibit reduced viscoelastic creep compared with the cytoplasm away from pseudopods [4 , 14 ]. Continued shear application leads to a passive spherical shape without pseudopods, which results in increased cell deformability. Thus, an elevated number of leukocytes with pseudopods as a result of attenuated shear response may compromise normal leukocyte passage through capillaries, leading to enhanced microvascular resistance and even capillary occlusion [4 , 14 15 16 17 ].
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ACKNOWLEDGEMENTS |
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Received October 8, 2003; revised November 16, 2003; accepted December 14, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  S. Fukuda, T. Yasu, N. Kobayashi, N. Ikeda, and G. W. Schmid-Schonbein Contribution of Fluid Shear Response in Leukocytes to Hemodynamic Resistance in the Spontaneously Hypertensive Rat Circ. Res., July 9, 2004; 95(1): 100 - 108. [Abstract] [Full Text] [PDF]
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, left Y axis) and pseudopod formation (L/L0; 
and 
, right Y axis) in human neutrophils. (A) Control (• and 
). n = 10 cells in each case. Responding leukocytes are defined as the cells in which more than a 20% reduction of L/L0 is observed during a 2-min shearing period. *, #, **, Versus values without DX treatment (0 µM) in DX, DX with PAF, and DX with fMLP, respectively (P<0.05, 
); DX and fMLP (
). (B) Control (
); 100 µM nifedipine (
