Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation

Abstract

β2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α-induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and αLβ2 or αMβ2 null mice suggest that extracellular PDI regulates αMβ2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for αMβ2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated αMβ2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with αMβ2 integrin in lipid rafts of stimulated neutrophils and regulates αMβ2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

Figure 1

Generation of myeloid-specific PDI CKO mice. (A) Targeting construct and homologous recombination. (B) Southern blotting analysis of genomic DNA isolated from mouse tails. (C) Polymerase chain reaction analysis of WT, heterozygous, and CKO mice with primers for floxed PDI and Lys-Cre. (D) Immunoblotting with lysates of cells isolated from WT (W), heterozygous (H), and homozygous PDI CKO (K) mice. The band density of PDI represents mean ± SD (n = 5-6 mice per group). (E-J) Flow cytometric analysis shows the expression of β2 integrins, L-selectin, PSGL-1, and CD44 on unstimulated (dot line) and fMLF-stimulated (black line) WT (WT) and PDI CKO (KO) neutrophils. The gray histogram represents the fluorescence intensity of control IgG on stimulated neutrophils. The mean fluorescence intensity of antibodies was normalized to that of control IgG, and data are shown as 100% (mean ± SD, n = 3-6 mice per group). PSGL-1, P-selectin glycoprotein ligand-1.

Figure 1

Generation of myeloid-specific PDI CKO mice. (A) Targeting construct and homologous recombination. (B) Southern blotting analysis of genomic DNA isolated from mouse tails. (C) Polymerase chain reaction analysis of WT, heterozygous, and CKO mice with primers for floxed PDI and Lys-Cre. (D) Immunoblotting with lysates of cells isolated from WT (W), heterozygous (H), and homozygous PDI CKO (K) mice. The band density of PDI represents mean ± SD (n = 5-6 mice per group). (E-J) Flow cytometric analysis shows the expression of β2 integrins, L-selectin, PSGL-1, and CD44 on unstimulated (dot line) and fMLF-stimulated (black line) WT (WT) and PDI CKO (KO) neutrophils. The gray histogram represents the fluorescence intensity of control IgG on stimulated neutrophils. The mean fluorescence intensity of antibodies was normalized to that of control IgG, and data are shown as 100% (mean ± SD, n = 3-6 mice per group). PSGL-1, P-selectin glycoprotein ligand-1.

Figure 2

PDI is required for neutrophil recruitment during TNF-α–induced vascular inflammation. Intravital microscopy was performed as described in “Materials and methods.” Neutrophils were visualized by infusion of an Alexa Fluor 647–conjugated anti–Gr-1 antibody. Six to 8 different inflamed venules were monitored in WT and PDI CKO mice. Then, wtPDI or dmPDI, 100 μg, was infused into PDI CKO mice and neutrophil recruitment was further monitored. (A) Representative images. Small (white and gray) and large arrows show rolling neutrophils and blood flow, respectively. (B-D) The rolling influx (rolling cells per minute) and velocity (micrometers per second) of neutrophils and the number of adherent neutrophils (number per field per 5 minutes) are shown. Data represent mean ± SEM (n = 17-24 venules in 3-4 mice per group). (E) The crawling population of adherent neutrophils was monitored over 5 minutes (n = 80-100 neutrophils in 3-4 mice per group). *P < .05, **P < .01, and ***P < .001 vs WT mice; #P < .05 and ###P < .001 vs PDI CKO mice with or without wtPDI or dmPDI after ANOVA and the Dunnett test. The diameter of microvenules observed was in the range of 33.3 to 45.5 μm and the wall-shear rate in the TNF-α–inflamed cremaster venules was approximately 400 to 650 s⁻¹ as described previously. (F-G) WT and PDI KO neutrophils were stimulated with fMLF in the presence of 50 μg/mL His-tagged wtPDI or dmPDI. Bound PDI was washed with RPMI media or carbonate buffer. Binding of recombinant PDI was analyzed by flow cytometry using a PE-conjugated anti-poly His antibody. The gray histogram represents the fluorescent signal of the anti-poly His antibody on PDI-untreated, stimulated neutrophils. (G) PDI binding is shown as a fold increase by the ratio of the geometric mean intensity value of a PE-conjugated anti-His antibody on PDI-treated vs untreated neutrophils (mean ± SD, n = 3). *P < .05 vs unstimulated neutrophils after the Student t test. (H-I) wtPDI or dmPDI, 100 μg, was infused into PDI CKO mice. Neutrophils, β2 integrin, and PDI were visualized by infusion of Alexa Fluor 647–conjugated anti-Gr-1 (red), Alexa Fluor 488–conjugated anti-β2 (green), and PE-conjugated anti-His antibodies (blue), respectively, into PDI CKO mice. Without recombinant PDI, no fluorescence signal was observed by the PE-conjugated anti-poly His antibody in PDI KO mice (data not shown). White arrows and arrowheads show blood flow and rolling neutrophils, respectively. Representative fluorescence images are shown at different time points following recording (n = 17-18 venules in 3 PDI CKO mice). Neutrophils and β2 (yellow); β2 and PDI (turquoise); neutrophils and PDI (magenta); neutrophils, β2, and PDI (white). (I) Fluorescence intensity of PE-conjugated anti-His antibodies was quantified over 2 minutes.

Figure 3

Extracellular PDI regulates αMβ2 integrin-mediated adhesion and crawling of neutrophils during vascular inflammation. Control IgG (mIgG2a) or anti-PDI antibody (RL90, 1 or 3 μg/g BW) was infused into WT mice. (A-E) Rolling, adherent, and crawling neutrophils were monitored and analyzed as described in the Figure 2 legend. (A) Representative images. Small (white, gray, and black) and large arrows show rolling neutrophils and blood flow, respectively. Data represent mean ± SEM (n = 24-28 venules in 3-5 mice per group). *P < .05 and **P < .01 vs control IgG after ANOVA and the Dunnett test. (F-I) Control IgG (rat IgG2a, rat IgG2b, or mouse IgG2a) or a blocking antibody against αM, αL, or PDI, 2 μg/g BW, was infused into αLβ2 or αMβ2 null mice. Adherent neutrophils and rapidly rolling (>10 μm/s) and embolized cells were counted. Data represent mean ± SEM (n = 18-22 venules in 3 mice per group). **P < .01 or ***P < .005 vs control IgG after the Student t test.

Figure 4

Surface PDI regulates αMβ2-mediated neutrophil adhesion under shear and static conditions. (A-B) Confluent HUVECs on FG-coated glass coverslips were stimulated with TNF-α (20 ng/mL) and placed into a flow chamber. Human neutrophils, 3 × 10⁶, were pretreated with blocking antibodies and stimulated with fMLF. (B) Anti-PDI (BD34, 30 μg/mL) and either anti-αM (15 μg/mL) or anti-αL (50 μg/mL) antibodies were coincubated. Neutrophils were perfused for 10 minutes over activated HUVECs under venous shear of 1 dyne/cm². Then, the medium was perfused for 5 minutes to wash out weakly bound cells. Adherent neutrophils were monitored in a field of 0.15 mm² and counted in 5 to 7 separate fields. (C) Human neutrophils were incubated with anti-PDI (30 μg/mL), anti-αM (10 μg/mL), and anti-αL antibodies (30 μg/mL). Cells were plated onto immobilized ICAM-1 in the presence of fMLF. (D) WT or PDI KO mouse neutrophils were treated with antibodies and plated onto immobilized mouse ICAM-1 in the presence of fMLF. (B-D) The number of adherent neutrophils treated with an inhibitor was normalized to that of adherent neutrophils treated with control IgG (100%). Data represent mean ± SD (n = 3-4 for human neutrophils and n = 6 WT and 6 PDI CKO mice). *P < .05, **P < .01, or ***P < .001 vs control IgG after ANOVA and the Dunnett test. #P < .05 and ##P < .01 vs either anti-PDI + anti-αL or anti-αL + anti-αM antibodies after ANOVA and the Dunnett test (for human neutrophils) or vs WT control after the Student t test (for mouse neutrophils). Round and spread neutrophils are shown as open and filled bars, respectively. Statistical significance was determined by comparison of the number of total adherent (round and spread) cells.

Figure 5

Surface PDI-αMβ2 interaction is enhanced on stimulated neutrophils. (A-B) Human neutrophils were incubated without or with fMLF. After SSB labeling, lysates were immunoprecipitated with anti-PDI antibodies and immunoblotted with indicated antibodies (total interaction). The blots were reprobed with peroxidase-conjugated avidin (surface interaction). (B) The band density was quantitated by densitometry (mean ± SD, n = 4-5). (C) Surface plasmon resonance assay was performed as described in “Materials and methods.” The extracellular domain of recombinant αMβ2 (25 μg/mL in 10 mM acetate buffer, pH 5.0) was immobilized on the surface of a CM5 chip. Recombinant PDI (0.03-21 μM) or purified FG (0.02-5 μM, data not shown) in running buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% P20 with 2 mM MnCl²) were infused over the reference and αMβ2-immobilized surfaces at a flow rate of 5 μL/min for 240 seconds, followed by a dissociation phase of 300 seconds. The representative graph is shown from triplicate. The dissociation constant, K_d, was calculated based on the K_on and K_off value. (D) WT and αMβ2 null neutrophils were stimulated with fMLF in the presence of 50 μg/mL His-tagged wtPDI or dmPDI. Binding of recombinant PDI was analyzed by flow cytometry as described in the panel G section of the Figure 2 legend. Data represent mean ± SD (n = 3-4). *P < .05 and **P < .01 vs unstimulated neutrophils, and #P < .05 vs PDI binding to stimulated WT neutrophils after the Student t test. (E) Using lysates of unstimulated and fMLF-stimulated neutrophils, fractions containing lipid rafts and nonrafts were collected and immunoblotted. Representative blots and densitometric analysis are shown (n = 3).

Figure 6

Surface PDI regulates ligand-binding activity and clustering of αMβ2 integrin without affecting its conformational change. (A-B) Human neutrophils were pretreated with blocking antibodies (10 μg/mL), followed by incubation with fMLF and Alexa Fluor 488–conjugated FG. The fluorescence intensity of FG on inhibitor-treated neutrophils was normalized to that on control cells (100%, white bar). Data represent mean ± SD (n = 3-6). *P < .05 or **P < .01 vs control IgG after ANOVA and the Dunnett test. #P < .05 vs each antibody alone after the Student t test. (C) Human neutrophils were pretreated with mouse IgG1 or IgG2a (IgG1 or IgG2a), or a blocking antibody against PDI (RL90 and BD34) or activated αM (CBRM1/5), 15 μg/mL, and then incubated with or without fMLF. Binding of the PE-conjugated CBRM1/5 was analyzed by flow cytometry. CBRM1/5 binding to neutrophils treated with control IgG was normalized as 100% (white bar). Data represent mean ± SD (n = 3-4). **P < .01 vs control IgG after the Student t test. (D-E) Confocal microscopy was performed as described in “Materials and methods.” Neutrophils were stimulated with fMLF in the presence of control IgG or a blocking anti-PDI antibody and plated onto ICAM-1 surfaces. Adherent cells were stained with PE-conjugated anti-activated αM (CBRM1/5) (red) and Alexa Fluor 488–conjugated anti-PDI antibodies (green). Representative images are shown as low (left panel) and high (right panel) magnifications (n = 4). Little signal was detected by isotype control IgG (data not shown). Colocalization histograms show the intensity of surface PDI and activated αMβ2 integrin along the white line. Bar = 10 μm. (E) The number of medium- (10-100 pixels) and large-sized (>100 pixels) punctates of activated αMβ2 integrin was quantified in confocal images (n = 25-30 cells in 4 independent experiments). P value was obtained from the Mann-Whitney test.

Figure 7

PDI regulates sulfhydryl exposure of αMβ2 integrin during neutrophil activation. (A-C) Human neutrophils pretreated without or with an anti-PDI antibody were stimulated with fMLF, followed by labeling with SSB or MPB. Lysates were pulled down with avidin agarose beads and immunoblotted. (D-F) WT and PDI KO neutrophils were stimulated with fMLF and used in the same pulldown assay. (B,E) The free thiol contents of surface αM, β2, and PDI (band density of MPB labeling) were normalized to the surface expression of each protein (band density of SSB labeling). Quantitative graphs are shown as mean ± SD (5-6 independent experiments for human neutrophils and 3 independent experiments for mouse neutrophils [5 WT and 5 KO mice per experiment in each group]). (C,F) The fold change of free thiol levels during cell activation was obtained by dividing the normalized value in stimulated cells by that in unstimulated cells in each group (mean ± SD). The number greater or lower than 1 implicates that sulfhydryl exposure per exposed surface protein increases or decreases following fMLF stimulation. *P < .05 vs unstimulated and IgG- or vehicle-treated control (MPB/SSB = 1).