(723a) Evaluating the Use of HL60-Derived Neutrophilic Cells to Examine the Molecular Dynamics of the Regulation of CD18 Surface Expression By Shear Stress Exposure

Introduction

Chronic inflammation is a common denominator for a wide range of cardiovascular diseases and is associated with an impaired neutrophil response to fluid shear stress exposure [1]. In fact, fluid flow mechanotransduction by neutrophils has been shown to be part of a control mechanism to minimize inflammation in the blood under physiologic (i.e. non-pathogenic) conditions [1]. Specifically, shear stress exposure derived from blood flow deactivates neutrophils, even in the presence of low quantities of cell agonists. A key aspect of this mechanism is the mechanosensitive cleavage of CD18 cell-cell adhesion molecules off the surface of neutrophils, which prevents their heterotypic binding to other cells (e.g., endothelium, platelets) [2,3]. Recently, cathepsin B (catB) has been identified as a key protease involved in CD18 cleavage [2,4], but it is unclear how shear regulates the release of this protease into the extracellular milieu. To elucidate the dynamics of shear-induced catB release, we seek to overexpress a catB-GFP (i.e. green fluorescent protein) construct in neutrophils and track its release into the extracellular environment. Since transfection of human neutrophils with a catB-GFP construct is not feasible due to their short lifespan (<24 hours) outside the body, we tested the possibility of using HL60-derived neutrophilic cells as a transfectable cell culture model. For this study, we hypothesized that shear stress-induced CD18 cleavage is a characteristic of the neutrophilic phenotype.

Methods

HL60 cells were purchased from the American Type Culture collection (ATCC; CCL-240) and subcultured in RPMI 1640 media supplemented with 10% FSB plus 1% penicillin/streptomycin/L-glutamine solution. To induce a neutrophilic phenotype, HL60 cells were treated with 1.25% DMSO for 5 days (referred to as dHL60 cells). Immediately after neutrophil differentiation, dHL60 cells were resuspended in HBSS media. Some dHL60 cell populations were left untreated, while others were preincubated with 10 nM to 1 μM fMLP for 10 minutes. Following preincubation, the cells were exposed to 5 dynes/cm² steady laminar shear stress for 10 minutes using a custom cone-and-plate viscometer as described [3]. For some experiments, both unstimulated and fMLP-stimulated dHL60 cells were also pretreated with 30 μM E64, a known blocker of shear-induced CD18 cleavage [2], prior to shear exposure in the cone-and-plate viscometer. Controls were dHL60 cells maintained under static (no-flow, no-shear), but otherwise, similar experimental conditions. After shear and control experiments, dHL60s were fixed with 1% paraformaldehyde for 20 minutes. Immunofluorescence in conjunction with flow cytometric analyses was then conducted to assess dHL60 binding to fluorescent antibodies for CD18, CD11a, CD11b, and catB.

Results

Based on our analyses, sheared cells exhibited reduced expression of CD18 and CD11a, but not CD11b. E64, a cysteine protease inhibitor capable of blocking catB, attenuated the shear-induced CD18 and CD11a cleavage responses of cells. Furthermore, by increasing fMLP concentrations up to 1 μM, the shear-induced cleavage of CD18 and CD11a was dose-dependently attenuated. Notably, unlike human primary neutrophils, dHL60 cells did not appear to express appreciable quantities of catB.

Discussion

Based on our results, dHL60 neutrophilic cells exhibit preferential shear-induced cleavage of CD11a over CD11b isotypes, which differs from that of human neutrophils. Primary neutrophils have been shown to exhibit preferential cleavage of CD11b over CD11a isotypes [3]. This is consistent with the near absence of catB expression by dHL60 cells and, in conjunction with previous studies [2,4], further points to catB as responsible for the CD11b cleavage observed for sheared human neutrophils exhibiting abundant catB expression levels. Despite these differences, our results indicate that the CD18 cleavage response to shear is a distinct trait of the neutrophil phenotype. Additionally, this shear response appears to serve as an anti-inflammatory measure below 1 μM fMLP. It should be noted that this threshold concentration of fMLP is similar to that below which shear-induced pseudopod retraction, another neutrophil-deactivating effect of fluid flow, remains intact.

Conclusion

The results of the present study confirm the potential utility of the dHL60 neutrophilic cells in characterizing catB release dynamics using molecular cloning approaches. Moreover, our results further point to shear-induced CD18 cleavage as an important control mechanism that serves to restrict the neutrophil activation under physiological (i.e. non-inflamed, non-pathogenic) conditions.

References

[1] Makino A, Shin HY, Komai Y, Fukuda S, Coughlin M, Sugihara-Seki M, et al. Mechanotransduction in leukocyte activation: a review. Biorheology. 2007;44(4):221-49.

[2] Fukuda S, Schmid-Schonbein GW. Regulation of CD18 expression on neutrophils in response to fluid shear stress. Proc Natl Acad Sci U S A. 2003;100(23):13152-7.

[3] Zhang X, Zhan D, Shin HY. Integrin subtype-dependent CD18 cleavage under shear and its influence on leukocyte-platelet binding. J Leukoc Biol. 2013;93(2):251-8.

[4] Shin HY, Simon SI, Schmid-Schonbein GW. Fluid shear-induced activation and cleavage of CD18 during pseudopod retraction by human neutrophils. J Cell Physiol. 2008;214(2):528-36.