Neutrophil elastase activates macrophage calpain as a mechanism for phagocytic failure

Abstract

Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease calcium ion-dependent papain-like cysteine protease-2 (Calpain-2) in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE-induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild-type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte-derived macrophages (hMDMs) from persons with CF (PwCF) and non-CF subjects were treated with NE or control vehicle, and cell lysates were prepared to evaluate Calpain-2 protein abundance by Western and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. hMDMs were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline, there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.NEW & NOTEWORTHY Neutrophil elastase, a cystic fibrosis airway inflammation biomarker, increases macrophage Calpain activity, and Calpain inhibition partially restores the decreased phagocytosis in neutrophil elastase-challenged macrophages. Neutrophil elastase increases Calpain-2 protein, degrades the Calpain inhibitor, Calpastatin, and increases intracellular calcium as potential mechanisms of Calpain activation. This presents a novel mechanism for macrophage dysfunction relevant to cystic fibrosis.

Keywords: calpain-2; calpastatin; cystic fibrosis; macrophage; phagocytosis.

Figure 1.. Calpain inhibition by PD150606 rescued the NE-mediated phagocytic defect in non-CF and CF murine alveolar macrophages.

Cftr null (A-D) or Cftr wild-type (F-I) murine alveolar macrophages were harvested and treated for 2 h with vehicle control (A, F), NE (500nM) (B, G), calpain inhibitor PD150606, 10μM (C, H), or NE and PD150606 (D, I), and then incubated with FITC-labeled E. coli K-12 bio-particles for 1h. After incubation, cells were washed, fixed with paraformaldehyde, stained with DAPI and imaged by confocal microscopy at 40x magnification. Fluorescence intensity is presented graphically (E, J); summarized as mean ±SD for n = 3 experiments. For CFKO alveolar macrophage (39 mice total, 23 female, 16 male) data, NE (0.400 ±0.132) significantly decreased phagocytosis vs control (0.930 ±0.07), p=0.005, which was significantly improved with Calpain inhibitor (0.589 ±0.222), p=0.036. For WT alveolar macrophage (39 mice total, 21 female, 18 male) data, NE (0.375 ±0.111) significantly decreased phagocytosis vs control (1.01 ±0.03), p=0.0004, which was significantly improved with Calpain inhibitor (0.596 ±0.212), p=0.004. Data summarize 4–6 replicates/experiment, 20–50 cells/group. Statistically significant differences are presented: *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 1.. Calpain inhibition by PD150606 rescued the NE-mediated phagocytic defect in non-CF and CF murine alveolar macrophages.

Cftr null (A-D) or Cftr wild-type (F-I) murine alveolar macrophages were harvested and treated for 2 h with vehicle control (A, F), NE (500nM) (B, G), calpain inhibitor PD150606, 10μM (C, H), or NE and PD150606 (D, I), and then incubated with FITC-labeled E. coli K-12 bio-particles for 1h. After incubation, cells were washed, fixed with paraformaldehyde, stained with DAPI and imaged by confocal microscopy at 40x magnification. Fluorescence intensity is presented graphically (E, J); summarized as mean ±SD for n = 3 experiments. For CFKO alveolar macrophage (39 mice total, 23 female, 16 male) data, NE (0.400 ±0.132) significantly decreased phagocytosis vs control (0.930 ±0.07), p=0.005, which was significantly improved with Calpain inhibitor (0.589 ±0.222), p=0.036. For WT alveolar macrophage (39 mice total, 21 female, 18 male) data, NE (0.375 ±0.111) significantly decreased phagocytosis vs control (1.01 ±0.03), p=0.0004, which was significantly improved with Calpain inhibitor (0.596 ±0.212), p=0.004. Data summarize 4–6 replicates/experiment, 20–50 cells/group. Statistically significant differences are presented: *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2.. NE increased cytosolic Calpain-2 protein levels and intracellular Calpain activity in non-CF and CF hMDM.

Non-CF hMDM (n=3 donors, 1 male, 2 female) (A) and CF hMDM (n=3 donors, 1 male, 2 female) (B) were treated with NE (500 nM, 2h) or control vehicle, and cytosolic proteins (20 μg) were separated on 4%–20% PAGE for western analysis for Calpain-2. Following incubation with secondary antibodies and Western blot development with ECL, band densities were quantified by ImageJ and then expressed as a Calpain-2:Actin densitometry ratios. Inserts show representative westerns. Results (mean ±SD) in non-CF hMDM cells (A) showed increased Calpain-2 with NE treatment (p=0.036), with 0.720 ±0.06 for control treated vs 1.10 ±0.20 for NE treated. Results (mean ±SD) in CF hMDM cells (B), showed increased Calpain-2 expression with NE treatment (p=0.047), with 0.929 ±0.472 for control treated vs 1.23 ±0.557 for NE treated. Calpain activity was measured in cytosolic proteins (40–50 μg) from non-CF hMDM (n=4 donors, 2 male, 2 female) (C) or CF hMDM (n= 4 donors, 1 male, 3 female) (D) using a Calpain activity kit. Results (mean ±SD) were reported as arbitrary relative fluoresce units (RFU) per μg total protein. In non-CF hMDM cells (C) NE treatment increased Calpain activity (p=0.048), with 173.9 ±61.78 for control treated vs 264.3 ±61.78 for NE treated. In CF hMDM cells NE also increase Calpain activity (p=0.046), with 198.5 ±71.82 for control treated vs 384.4 ±183.3 for NE treated control CF hMDM cells. Statistically significant differences were determined by unpaired t-test, *, p<0.05 and ○ denoting male; ● denoting female subjects.

Figure 3.. NE decreased cytosolic Calpastatin protein levels in non-CF and CF hMDM.

Non-CF hMDM (n=3 donors, 1 male, 2 female) (A) and CF hMDM (n=3 donors, 1 male, 2 female) (B) were treated with NE (500 nM, 2h) or control vehicle and cells were harvested for isolation of the cytosolic proteins. Proteins (20 μg) were separated on a 4%–20% PAGE and following transfer to nitrocellulose, filters were incubated with primary antibodies to Calpastatin. Following incubation with secondary antibodies and Western blot development with ECL, band densities were quantified by ImageJ and then expressed as a Calpastatin : Actin densitometry ratios. Inserts show representative westerns. Results (mean ±SD) in non-CF hMDM cells (A) showed decreased Calpastatin with NE treatment (p=0.046), with 0.967±0.415 for control treated vs 0.197±0.211for NE treated. Results (mean ±SD) in CF hMDM cells (B) showed decreased Calpastatin with NE treatment (p=0.01), 0.967±0.142 for control treated vs 0.443±0.204 for NE treated control CF hMDM cells. Statistically significant differences were determined by unpaired t-test and are noted by * p < 0.05, **, p<0.01 and ○ denoting male; ● denoting female subjects.

Figure 4.. NE increased intracellular calcium in non-CF and CF hMDM.

Intracellular calcium levels were measured in live non-CF hMDM (n=5 donors, 2 male, 3 female) and CF hMDM (n= 3 donors, 1 male, 2 female) from 0.2 x 106cells cultured in 96-well plate following treated with NE (500 nM) or control vehicle for 2 h. Calcium levels were quantified using a commercially available kit, Fluo-4 Direct Calcium assay. For non-CF hMDM (A) and CF hMDM (B) intracellular calcium levels were determined at 2h by Fluo-4 kit as arbitrary fluorescent units and normalized to the cell number loaded (200,000 cells) and then were expressed relative to the average control. Results (mean ±SD) in non-CF hMDM cells (A) showed increased intracellular calcium with NE treatment at 2 hour (p=0.047), with 0.96±0.07 for control treated and 1.53±0.54 for NE treated cells. In CF hMDM (B) NE also increased intracellular calcium at 2 hour (p=0.0003), with 1.06±0.08 for control treated cells and 1.75±0.06 for NE treated cells. Statistically significant differences were determined by unpaired t-test, where * denotes p<0.05 and *** denotes p<0.001 and ○ denoting male; ● denoting female subjects.

Figure 5.. NE increased extracellular Calpain activity in CF but not non-CF hMDM conditioned media and in CF BALF, Calpain activity was increased and correlated with NE activity.

Using 10x concentrated conditioned media (50 μl) from non-CF hMDM (n=3 donors, 3 male) CF hMDM cultures (n=4 donors, 2 male, 2 female) treated with control vehicle or NE (500 nM), 2 h, extracellular Calpain was evaluated using a Calpain activity kit and normalized to control-treated activity (A). Calpain activity was compared between BALF from subjects with CF (n=8 donors, 5 male, 3 female) and BALF from non-CF subjects (n=8 donors, 7 male, 1 female) (B). To determine whether there was a correlation between Calpain activity and NE activity in CF BALF samples (n=8 donors), NE activity was measured using a chromogenic 96-well microtiter plate assay with N-Methoxysuccinyl-Ala-Ala-pro-Val-p-nitroanilide as the substrate and was compared to Calpain activities by a scatter plot of log10-transformed Calpain activity vs log10-transformed NE activity (C). Results are presented as mean ±SD, show not statistically significant increase in Calpain activity in non-CF hMDM media (p=0.168), 0.960 ±0.176 (Control) versus 1.652+0.691 (NE) and show increased Calpain activity for CF hMDM media (p=0.024), 0.996 ±0.636 (Control) versus 1.59+0.449 (NE) (A). Calpain activity in BALF fluid, 899 ±81 (Non-CF) versus 16,804 ±17236 (CF) was significantly increased in CF individuals (p=0.0206) (B). Statistically significant differences were determined by unpaired t-test, *, p<0.01,***, p<0.001. Calpain activity correlated with NE activity in CF BALF by simple linear regression analysis (C). In all figures, ○ is denoting male and ● is denoting female subjects.

Figure 6.. There are no differences in baseline Calpain activity, Calpain-2 expression, Calpastatin expression, and intracellular calcium between CF and non-CF MDM.

Calpain activity (A) was measured in cytosolic proteins (50 μg) from non-CF hMDM (n = 6 donors, 3 male, 3 female) or CF hMDM (n= 5 donors, 1 male, 4 female) using a Calpain activity kit. Results (mean ±SD) reported as arbitrary relative fluoresce units (RFU) normalized to μg total protein, were no significant difference (p=0.981) between non-CF hMDM cells vs CF hMDM cells. Non-CF hMDM cells (n=4 donors, 3 male, 1 female) and CF hMDM (n=4 donors, 2 male, 2 female) were harvested for isolation of cytosolic proteins as described earlier for western blot analysis of either Calpain-2 (B) and Calpastatin (C). Following incubation with secondary antibodies and Western blot development with ECL, band densities were quantified by ImageJ and then expressed as a Calpain-2:Actin densitometry ratios (B) or Calpastatin:Actinratios (C). Inserts show representative westerns. Results (mean ±SD) were no significant difference in Calpain-2 expression (p=0.389) or Calpastatin expression (p= 0.593) between non-CF hMDM cells vs CF hMDM cells. Intracellular calcium (D) was measured in cytosolic lysates (30μg protein) using a commercially available kit using a red fluorescence probe. Results (mean ±SD) reported as μM intracellular calcium, were no significant difference (p=0.985) between non-CF hMDM cells vs CF hMDM cells. Statistically significant differences were determined by unpaired t-test, *, p<0.05 and ○ denoting male; ● denoting female subjects.