Shunichi Fukuda

Regulation of CD18 expression on neutrophils in response to fluid shear stress

When leukocytes adhere to endothelial cells and are exposed to fluid shear stresses, they often retract pseudopods and reduce their attachment. Leukocytes use CD18 for membrane adhesion, but the kinetics of such integrin adhesion molecules under fluid shear is unknown. We examine on neutrophils with confocal microscopy of single adherent cells and flow cytometry of cell suspensions the CD18 expression under fluid shear after labeling with fluorescent antibodies. Fluid shear causes reduction of CD18-associated immunofluorescence of extracellular epitopes, especially in areas of the membrane exposed to elevated levels of shear (1.5 dyne/cm2 maximum shear stress; 1 dyne = 10 mN). CD18 was also translocated over the leukocyte surface from regions of higher shear to lower shear and into the membrane contact areas with the substrate. We obtained no evidence for cytoplasmic internalization of CD18. Fluid shear (5 dyne/cm2) in a suspension of human leukocytes resulted in cleavage of the extracellular domain but not against a cytoplasmic domain of CD18. Chelation of extracellular Ca2+ abolished the down-regulation of CD18. Cysteine protease inhibitors and a selective inhibitor for cathepsin B, but no blockade of other cysteine proteases such as cathepsin L and calpain, aminopeptidases, elastase, or metalloproteinases, suppressed shear-induced CD18 down-regulation. The evidence suggests that physiological levels of fluid shear cause release of cysteine protease(s) including cathepsin B, leading to cleavage of the extracellular domain of CD18 molecules and possible membrane detachment.

Contribution of Fluid Shear Response in Leukocytes to Hemodynamic Resistance in the Spontaneously Hypertensive Rat

The mechanisms for elevation of peripheral vascular resistance in spontaneously hypertensive rats (SHR), a glucocorticoid-dependent form of hypertension, are unresolved. An increase in hemodynamic resistance caused by circulating blood may be a factor. Physiological fluid shear stress induces a variety of responses in circulating leukocytes, including pseudopod retraction. Due to high rigidity, leukocytes with pseudopods have greater difficulty to pass through capillaries. Because SHR have more circulating leukocytes with pseudopods, we hypothesize that inhibition of the leukocyte shear response by glucocorticoids in SHR impairs normal leukocyte passage through capillaries and causes enhanced resistance in capillary channels. Fluid shear leads to retraction of pseudopods in normal leukocytes, whereas shear induces pseudopod projection in SHR and dexamethasone-treated Wistar rats. The high incidence of circulating leukocytes with pseudopods results in slower cell passage through capillaries under normal blood flow and during reduced flow enhanced capillary plugging both in vivo and in vitro. SHR blood requires higher pressure (90.0±8.2 mm Hg) than Wistar Kyoto rat (WKY, 69.6±6.5 mm Hg; P <0.0001) or adrenalectomized SHR (73.5±2.1 mm Hg; P =0.0009) at the same flow rate in the resting hemodynamically isolated skeletal muscle microcirculation. Intravenous injection of blood from SHR, but not WKY, causes blood pressure increase in normal rats, which depends on pseudopod formation. We conclude that in addition to enhanced vascular tone, pseudopod formation with lack of normal fluid shear response may serve as mechanisms for an elevated hemodynamic resistance in SHR.

Centrifugation attenuates the fluid shear response of circulating leukocytes

Human leukocytes retract pseudopods in response to physiological fluid shear, a phenomenon that serves to keep circulating leukocytes in a spherical state. We show here that leukocyte fluid shear response is attenuated irreversibly by centrifugation. Inhibition of shear response depends on duration and magnitude of acceleration during centrifugation and time duration after centrifugation. Even after low‐speed centrifugation, leukocytes no longer retract pseudopods during shear application. After centrifugation at higher acceleration, leukocytes project pseudopods instead of retracting during shear application, which can be suppressed by a calcium channel blocker. We examined the role of fluid shear response in vivo by reintroduction of centrifuged cells into the rat circulation. Centrifuged leukocytes have a significantly enhanced tendency to migrate into tissue. These observations indicate that centrifugation may irreversibly damage the fluid shear response of leukocytes. It causes an impaired leukocyte behavior after reintroduction into the circulation, suggesting that shear response is a requirement for normal passage of leukocytes through the microcirculation.

Nicorandil and leukocyte activation.

Nicorandil, a hybrid compound of an ATP-sensitive potassium (KATP) channel opener and a nitric oxide donor, has been reported to preserve microvascular integrity in patients with reperfused myocardial infarction. The aim of the current study was to test the hypothesis that nicorandil suppresses activation of polymorphonuclear leukocytes (PMNLs), resulting in reduction of PMNL migration into tissue upon ischemia/reperfusion. Nicorandil, along with the mitochondrial KATP channel opener diazoxide and the nitric oxide donors nitroglycerin and isosorbide dinitrate, suppressed pseudopod projection in human PMNLs treated with 10−9M N-formyl-methionyl-leucyl-phenylalanine (FMLP) and subjected to shear stress (5 dyn/cm2) with a cone-and-plate shear device. Suppression by nicorandil and diazoxide was reversed by KATP channel blockers, 5 hydroxydecanoate and glibenclamide. FMLP-induced increase of [Ca2+]in in PMNLs was suppressed by nicorandil and diazoxide, and 5 hydroxy-decanoate and glibenclamide reversed this suppression. Results of reverse transcription polymerase chain reaction with rat PMNL mRNA indicated the presence of mRNAs of Kir6.2 and Kir6.1 but not mRNAs of sulfonylurea receptor 1 or 2. Isosorbide dinitrate, diazoxide, and nicorandil reduced leukocyte migration and microvascular obstruction in reperfused ischemic tissue of rat mesenteric microcirculation. In conclusion, nicorandil attenuates ischemia/reperfusion-induced PMNL activation via donation of nitric oxide and K channel–related cascade.

Leukocyte fluid shear response in the presence of glucocorticoid.

Leukocytes respond to physiological fluid shear stress (∼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.

Fluid shear-induced cathepsin B release in the control of Mac1-dependent neutrophil adhesion

There is compelling evidence that circulatory hemodynamics prevent neutrophil activation, including adhesion to microvessels, in the microcirculation. However, the underlying mechanism or mechanisms by which that mechanoregulation occurs remain unresolved. Here, we report evidence that exposure to fluid shear stress (FSS) promotes neutrophils to release cathepsin B (ctsB) and that this autocrine regulatory event is antiadhesive for neutrophils on endothelial surfaces through Mac1‐selective regulation. We used a combined cell‐engineering and immunocytochemistry approach to find that ctsB was capable of cleaving Mac1 integrins on neutrophils and demonstrated that this proteolysis alters their adhesive functions. Under no‐flow conditions, ctsB enhanced neutrophil migration though a putative effect on pseudopod retraction rates. We also established a flow‐based cell detachment assay to verify the role of ctsB in the control of neutrophil adhesion by fluid flow stimulation. Fluid flow promoted neutrophil detachment from platelet and endothelial layers that required ctsB, consistent with its fluid shear stress–induced release. Notably, compared with leukocytes from wild‐type mice, those from ctsB‐deficient (ctsB−/−) mice exhibited an impaired CD18 cleavage response to FSS, significantly elevated baseline levels of CD18 surface expression, and an enhanced adhesive capacity to mildly inflamed postcapillary venules. Taken together, the results of the present study support a role for ctsB in a hemodynamic control mechanism that is antiadhesive for leukocytes on endothelium. These results have implications in the pathogenesis of chronic inflammation, microvascular dysfunction, and cardiovascular diseases involving sustained neutrophil activation in the blood and microcirculation.

Fluid Shear Response in Leukocytes

The passage of leukocytes from the bone marrow into the active circulation requires cell migration with all its requirements, such as pseudopod projection by cytoplasmic actin polymerization in coordination with cyclic membrane adhesion and detachment. The cells have to be fully activated. But leukocytes that are collected in venous or arterial blood samples from a healthy individual have few of these indicators for cell activation. They are almost perfectly round cells, exhibit low levels of membrane adhesion, and pass readily through the microcirculation without entrapment in capillaries or adhesion to venular or arteriolar endothelium. In contrast, if an inflammatory stimulus is applied, circulating leukocytes are perfectly capable at any moment to adhere to and migrate on or across the endothelium. This evidence suggests that there exists a mechanism that serves to de-activate circulating leukocytes. Furthermore, in the presence of inflammatory mediators, this mechanism may be abolished and as a result activated leukocytes may appear in the circulation. We hypothesize here that this mechanism is due to mechanical fluid shear. This chapter is focused on the shear response of circulating leukocytes. The chapters by Dr. Frangos and Tarbel address the fluid shear response in endothelial cells and smooth muscle cells.

The Role of Biomechanics in Analysis of Cardiovascular Diseases: Regulation of the Fluid Shear Response by Inflammatory Mediators

Biomechanics at the level of individual cells, the microcirculation and the tissue is an effective tool to analyze important diseases such as stroke, myocardial infarction, or physiological shock. The biomechanics of inflammation needs to take center stage in the analysis of disease, organ rejection, or tissue engineering. There is a rich collection of problems that require analysis and finding solutions will be a rewarding exercise. The role of fluid shear stress in control of cells in the cardiovascular system is a major aspect of these problems.

A mechanisms for control of mechanotransduction in leukocytes

Human leukocytes retract pseudopods due to active actin polymerization and return into a spherical state in response to physiological fluid shear stresses (of the order of 1 dyn/cm/sup 2/). Fluid shear stress also serves to detach the leukocytes by a attenuation of the molecular membrane adhesion mechanism. All leukocytes derived from individuals without cardiovascular complications respond to fluid shear stress. In contrast, during inflammatory conditions pseudopod projection and firm adhesion on endothelium can be observed in a subgroup of leukocytes positioned on microvessels with normal blood flow and fluid shear stresses. Thus there exists a mechanisms that serves to regulate the shear stress response. The authors examine here several mechanisms that influence shear stress response of leukocytes and demonstrate that both inflammatory mediators as well as depletion of cGMP serves to attenuate the shear stress response in-vivo and in-vitro. These results indicate that there exist several mechanisms to attenuate mechanotransduction pathways in circulating leukocytes and thereby eliminate the anti-inflammatory action of normal physiological fluid shear stress.