Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration - PubMed (original) (raw)

Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration

Mariam Al-Omari et al. Mol Med. 2011 Sep-Oct.

Abstract

A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (μ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.

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Figures

Figure 1

AAT inhibits neutrophil calpain I activity. Dose-dependent inhibitory effects of AAT on the enzymatic activity (expressed as relative fluorescence units) of calpain I on the specific substrate Ac-LLY-AFC in human neutrophils are shown. The fluorescence value of all samples is corrected by subtracting the value of the blank and then calculating the mean fluorescence value (mean ± SEM) for each sample from duplicate readings from six independent experiments.

Figure 2

AAT inhibits calpain I activity in vitro. Dose-dependent inhibitory effects of AAT on the enzymatic activity of purified human active calpain I on the specific substrate Suc-LLVY-AMC are shown. (A) The activity of purified calpain I was inhibited by AAT up to 90% in a concentration-dependent manner. Each point represents the mean ± SD of four independent experiments. (B) Active calpain I was incubated at a molar ratio of 1:1.3 with calpastatin or AAT for 5 min, and AMC release was measured upon Suc-LLVY-AMC cleavage with calpain I. The fluorescence value of all samples is corrected by subtracting the value of the blank and then calculating the mean fluorescence value (mean ± SD) for each sample from five independent experiments.

Figure 3

AAT effects on α-spectrin cleavage in neutrophils. Neutrophils were cultured alone or exposed to AAT for 18 h. Equal amounts of cell lysates were separated on 6.5% SDS-PAGE after Western blot analysis. Blots were immunostained with an anti–human α-spectrin monoclonal antibody (1:2,000 dilution) followed by incubation with horseradish peroxidase–conjugated rabbit anti–mouse secondary antibody diluted 1:10,000. The enhanced chemiluminescence kit was used to visualize immunolabeling. A 145-kDa calpain-mediated and 150-kDa (calpain- and caspase 3–mediated) fragments are clearly detectable in controls cells. The 145-kDa calpain-mediated fragment is not detectable in neutrophils treated with AAT. Weak staining for the 150-kDa (calpain- and caspase 3–mediated) fragment is present. A representative Western blot is shown (n = 3).

Figure 4

Effects of AAT on neutrophil adhesion to fibronectin. Calcein-AM–labeled neutrophils were added to fibronectin-coated 96-well plates and stimulated with AAT, fMLP (100 nmol/L), fMLP/AAT or medium alone, as described in Materials and Methods. Adherent cells were analyzed with a fluorescence spectrophotometer using an excitation λ of 485 nm and emission λ of 520 nm. The results are expressed as a percentage of neutrophil adhesion ([adherent cells/total cells] × 100%). Each bar represents the mean ± SD of three independent experiments, each performed in four repeats.

Figure 5

AAT-induced neutrophil polarization and reorganization of F-actin cytoskeleton. Controls and AAT-stimulated cells were labeled with Alexa Fluor 488-phalloidin for F-actin (green) and DAPI for DNA (blue). Intrinsic lipid fluorescence (red) was recorded as described in Materials and Methods. Spherical nontreated neutrophils with a subcortical F-actin arrangement showed no lipid fluorescence. After 5 min of exposure to AAT, cells spread out, acquiring wide F-actin–positive lamellas (small arrows) and accumulated lipid droplets in the cell body (small arrowheads). Gradually stimulated neutrophils obtained polarized elongated shape with uropods (large arrows) and F-actin redistribution from leading edges to F-actin foci in the central part of the cell. At the same time, larger lipid bodies (large arrowheads) accumulated at the rear of the cell. Representative images of three independent experiments are shown. Scale bar = 20 μm.

Figure 6

AAT increases intracellular cholesterol levels. Cells were incubated with AAT for the indicated time periods. The intra-cellular cholesterol was stained with filipin, as described in the Materials and Methods. Filipin fluorescence appears shortly after stimulation with AAT and enhances in a time-dependent manner. Images were acquired with constant exposure time and under the same magnification for the whole series. Representative images of three independent experiments are shown. Scale bar = 20 μm.

Figure 7

AAT induces the cytosolic lipid droplet formation. High amounts of cytosolic lipid droplets stained with Red Oil O (indicated by arrows) were found in neutrophils treated with AAT for 30 min (A) compared with nontreated control cells (B). The images were taken by digital camera (Sony, DKC-5000) at a magnification of 100×. Pictures are representative of five independent experiments. The arrow is pointing to lipid droplets stained with Red Oil O.

Figure 8

Neutrophil random migration induced by AAT. The videos were recorded with the temporal resolution of 5 s using a Hamamatsu ORCA-R2 CCD camera. Cell migration rates were determined using the Time Lapse Analyzer (TLA) software. Tracks of the cells that remained round after stimulation were manually excluded from the analysis. Values are expressed as mean ± SD, with track numbers n = 35 for control cells and n = 119 for AAT stimulated cells. In the presence of AAT, cell migration was significantly enhanced compared with nontreated controls.

Figure 9

Mobilization of intracellular calcium induced by AAT. Neutrophils loaded with the fluorescent Ca2+ indicator Fluo-3 were measured flow cytometrically in a buffer without Ca2+ containing 1 mmol/L EGTA. Fluo-3 fluorescence of neutrophils was measured for 20 s on unstimulated cells before the injection of AAT at various concentrations (arrows). After addition of AAT, measurement was continued until a total time of 100 s. (A) Fluo-3 fluorescence of single cells (gated on neutrophils on the basis of scatter properties) was plotted against time. The black curve indicates the median fluorescence of neutrophils. AAT (1 mg/mL) addition is marked by the arrow. (B) Comparison of Ca2+ kinetics after stimulation with various concentrations of AAT.

Figure 10

Comparison of Ca2+ mobilization by AAT in Ca2+-free versus Ca2+-containing buffer. Neutrophils loaded with the fluorescent Ca2+ indicator Fluo-3 were measured flow cytometrically in a buffer containing 1 mmol/L Ca2+ (A, C) or Ca2+-free buffer containing 1 mmol/L EGTA (B, D). Measurement was as described in Materials and Methods and in Figure 4. The black curves indicate the median fluorescence of neutrophils over time. The arrows indicate addition of 1 mg/mL (A, B) or 2 mg/mL (C, D) AAT.

Figure 11

AAT induces activation of Rac1/Cdc42 (A) and ERK1/2 (B). Neutrophils were treated with AAT (0.5 mg/mL) for the indicated periods at 37°C, 5% CO2. Cell lysates from 3 × 106 neutrophils were separated on 7.5% SDS-PAGE followed by immunoblotting, as described in Materials and Methods. Immunoblotting was performed using antibodies against Rac1 and Cdc42 and against the phosphorylated and unphosphorylated forms of ERK. Presumably because of the stress of the cell culture, the phosphorylation of ERK was also detected in unstimulated control cells to a lesser degree than in AAT-treated neutrophils incubated for 60 min. The results shown are representative of three or five independent experiments.

Figure 12

AAT inhibits LPS-induced IL-8 release from neutrophils. Neutrophils were re-suspended in RPMI 1640 medium at a concentration of 3 × 106 cells/mL and stimulated with LPS (10 ng/mL), AAT (0.5 mg/mL) alone and LPS/AAT together for 4 h at 37°C, 5% CO2. Supernatants of cell cultures were analyzed to determine the levels of IL-8. Each bar represents the mean ± SD from four independent experiments.

Figure 13

AAT does not decrease CD16 expression (A) and does not induce neutrophil apoptosis (B). Neutrophils were treated with AAT (0.5 mg/mL) for 4 h at 37°C, 5% CO2. (A) Cell lysates from 3 × 106 neutrophils were separated on 7.5% SDS-PAGE, followed by immunoblotting, as described in Materials and Methods. Immunoblotting was performed using a monoclonal antibody against human CD16. The results shown are representative of two independent experiments. (B) Neutrophil apoptosis was monitored using an Annexin V–FITC apoptosis detection kit (BD Biosciences PharmingenTM). Cells were analyzed by flow cytometry. Unstained cells, and cells stained with FITC Annexin V or PI, were used controls.

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Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration - PubMed (original) (raw)