(401) Neutrophil Elastase Increases Intracellular Calpain Activity and Calcium in CF and non-CF Macrophages

Background: Patients with cystic fibrosis have defective macrophage phagocytosis, resulting in inadequate innate immune response and airway inflammation. Several etiologies have been postulated, including CFTR dysfunction and proteolysis of cell-surface receptors [1]. Neutrophil elastase (NE) is a major inflammatory protease in the CF airway. We and others have shown that NE independently contributes to impaired macrophage phagocytic function [2]. During pilot proteomic studies, we found that NE increased secretion of calcium ion-dependent papain-like Cysteine protease-2 (Calpain-2), a protease associated with leukocyte migration, adhesion, and phagocytosis [2]. Increased Calpain activity has been exploited by Group B Streptococcus to evade macrophage phagocytosis [3]. We thus sought to investigate the effect of NE-challenge on intracellular calpain activity and its effect on the Calpain-Calpastatin axis in CF and non-CF macrophages.

Methods: Human monocyte derived macrophages (hMDM) from CF and non-CF individuals differentiated by GMCSF were exposed to either 500nM NE or vehicle control buffer, followed by NE inhibitor treatment and cytoplasmic lysates were collected. The relative abundances of Calpain-2 and Calpastatin were determined by Western analyses. The relative Calpain activity of NE vs control treated cytosolic lysates reflecting intracellular calpain activity was assayed using a fluorometric commercial kit. Intracellular calcium was assayed in NE (200nM, 500nM) or control treated CF and non-CF hMDM cells using a Fluo-4 based commercial kit for dose and time dependence of the NE challenge. Intracellular calcium was then assessed in hMDM cells in a similar fashion with 500nM NE vs control in the context of extracellular calcium chelators (EDTA, 10μM) and inhibitors of extracellular calcium entry (Gadolinium, 10μM).

Results: In human CF and non-CF MDM cells, NE increased intracellular calpain activity. NE increased Calpain-2 protein expression and decreased the abundance of the endogenous Calpain inhibitor Calpastatin. 500nM NE but not 200nM NE increased intracellular calcium in hBMDM cells and in a time dependent manner. Neither EDTA nor Gadolinium decreased the NE-mediated increase in intracellular calcium in hMDM cells.

Conclusions: NE-mediated calcium increase was not decreased in the presence of the extracellular calcium chelator EDTA or Gadolinium, which blocks receptors and channels for calcium entry, suggesting that NE increased intracellular calcium in macrophages from release of intracellular stores. The mechanism of NE mediated increase in calpain activity could be attributed to NE upregulation of Calpain-2 protein, degradation of the endogenous calpain inhibitor calpastatin, or increased intracellular calcium, which were all observed.

Acknowledgements: This work was supported by The Cystic Fibrosis Foundation (Harry Shwachman Clinical Investigator Award Grant MA20Q0).

References: [1] Lévêque M LTS, Del Porto P, Martin-Chouly C., The impact of impaired macrophage functions in cystic fibrosis disease progression. J CystFibros. 2017;16(4):443-453.

[2] Ma, J., Kummarapurugu, A.B., Hawkridge, A., Ghosh, S., Voynow, J., Neutrophil Elastase-Regulated Macrophage Sheddome/Secretome and Phagocytic Failure. AJP-Lung Cellular and Molecular Physiology. 2021 Sep 1;321(3):L555-L565.

[3] Fettucciari K, Quotadamo F, Noce R, Palumbo C, Modesti A, Rosati E, et al., Group B Streptococcus (GBS) disrupts by calpain activation the actin and microtubule cytoskeleton of macrophages. Cell Microbiol 2011;13(6):859-884.