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 cellengineering 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 ctsBdeficient (ctsB 2/2 ) 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.
Shear stress-induced cleavage of cell surface CD18 integrins is reported to be part of an anti-inflammatory control mechanism that minimizes neutrophil activity in the blood under physiologic conditions. The cysteine protease, cathepsin B (catB), has been implicated in this mechanoregulatory mechanism, but its molecular dynamics remain to be elucidated. Moreover, attempts to do so using molecular approaches are hindered by the limited ex vivo life span of primary neutrophils. As an alternative, we explored the potential use of HL60-derived neutrophilic cells as a transfectable culture model that exhibits a shear-induced CD18 cleavage response comparable to primary neutrophils. HL60 cells were differentiated into neutrophil-like cells (dHL60-NCs) and exposed to laminar shear stress ([Formula: see text] for 10 min). Based on cytometric analyses, sheared cells cleaved CD18 and CD11a, but not CD11b, integrins. Treatment of cells with E64 or doxycycline prior to and during shear exposure inhibited CD18, but only attenuated CD11a, cleavage. Neither aprotinin nor pepstatin affected shear-induced CD18 or CD11a cleavage. Notably, dHL60-NCs expressed minimal catB. Thus, multiple cysteine proteases in addition to catB may cleave CD18 on sheared leukocytes. In fact, our findings indicate that multiple non-cysteine proteases also participate in the shear-related cleavage of CD11/CD18 heterodimers. Finally, shear-induced cleavage of CD18 and CD11a by dHL60-NCs was inhibited by fMLP concentrations of at least [Formula: see text]. Collectively, our findings indicate that shear-induced CD11/CD18 cleavage is phenotypic of neutrophilic cells, including those derived from HL60 cells. Moreover, our results verify shear stress as a key anti-inflammatory stimulus for neutrophils under physiologic conditions.
Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s−1 shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).
In this chapter, we discuss evidence that potentially implicates impaired neutrophil responses to shear stress as a putative factor in obesity-related cardiovascular disease. We do so by presenting evidence connecting obesity with microvascular disorders due to chronic inflammation and highlighting this link in the setting of hypertension and hypercholesterolemia. Furthermore, the potential impact of neutrophils on these obesity-related disease processes is discussed. Notably, both hypertension and hypercholesterolemia are associated with a deficiency in the neutrophil responsiveness to fluid shear stress. In this regard, we define the neutrophil responses to shear stress exposure and how these responses may play a role in microvascular function under physiological conditions as well as how their impairment may result in microcirculatory dysfunction. Finally, we point to data consistent with an impaired neutrophil shear sensitivity being a manifestation of obesity, particularly as it relates to hypertension and hypercholesterolemia. In this regard, we aim to suggest a novel perspective; specifically, that impaired neutrophil shear sensitivity precedes a chronic inflammatory state and serves as a putative source of obesity-related pathobiology.
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