Signaling and Dynamics of Activation of LFA-1 and Mac-1 by Immobilized IL-8

Abstract

The dynamic response of neutrophils to interleukin-8 (IL-8) is of central interest in inflammation. Chemokine -induced β(2) integrin dependent adhesion can take several minutes after initial contact with IL-8 as evidenced by increased cell adhesion to intracellular adhesion molecule 1 (ICAM-1). The goal of this study is to identify signaling events that are critical for this response. We demonstrate that neither the PI3K inhibitor wortmannin, nor the PKC inhibitor bisindolymaleimide had any effect on IL-8 induced adhesion to ICAM-1. However, inhibition of PLC with U73122 or stopping the release of intracellular calcium by its downstream effector IP3 with caffeine or 2-aminoethoxydiphenyl borate completely blocked the adhesive response. Chelation of intracellular calcium with BAPTA or extracellular calcium with EGTA completely abrogated neutrophil adhesion to ICAM-1. This adhesion is mediated by LFA-1 (α(L)β(2)) within first 300 seconds after chemokine stimulation, followed by Mac-1 (α(M)β(2)) mediated adhesion, beginning 350 seconds after stimulus. Inhibition of p38MAP kinase results in a time course similar to Mac-1 inhibition, consistent with published evidence that Mac-1 mediated adhesion is p38MAP kinase dependent. These findings confirm a PLC dependent, PKC independent pathway from chemokine stimulus to integrin activation previously identified in other cell types, and demonstrate distinct dynamics and different requirements for LFA-1 vs. Mac-1 activation in primary human neutrophils.

Figure 1

Series of video microphotographs showing the experimental setup for the micropipette experiment. A. Initial setup. Left pipette is holding ICAM-1 coated bead (4.5µm in diameter) and right pipette is holding human neutrophil. During the experiment the adhesion probability between the neutrophil and ICAM-1 coated bead was measured. B–E. Time dependent IL-8 bead (2.8 µm in diameter) engulfment. Snapshots are taken at: B. 0 seconds (initial contact), C. 20 seconds, D. 70 seconds, E. 320 seconds – after IL-8 bead attachment to the neutrophil.

Figure 2

Effect of immobilized IL-8 (control), E-selectin or anti-CD45 antibody on neutrophil adhesion to ICAM-1. IL-8 was immobilized on the protein G coated beads at 1,800 sites/µm² and E-selectin was immobilized on tosyl-activated beads at 9,000 sites/µm². anti-CD45 antibody and E-selectin coated beads stayed attached to the surface of the cell, while IL-8 coated beads got engulfed. Experiments were performed at room temperature. 43 cells from 5 donors were analyzed for this experiment. Error bars represent standard error. The measured adhesion probability for time ≥ 325 s for E-selectin is significantly different from control (IL-8) assessed by Student’s t-test (p < 0.001). The probability of adhesion for CD 45beads for times ≥ 275 s is statistically different from control (p < 0.01).

Figure 3

Effect of signaling pathways inhibitors on IL-8 induced neutrophil adhesion to ICAM-1. A. PI3k inhibitor wortmannin (500 nM) and B. PKC inhibitor BIM (1µM) had no effect. C. PLC inhibitor U73122 (2 µM) completely blocked ICAM-1 dependent adhesion, while its inactive analog U73343 (2 µM), which was used as a control, had no effect. Experiments with BIM and PLC inhibitors were performed at 37°C, experiments with wortmannin were performed at room temperature. Ninety-five cells from six donors were analyzed for these experiments. Error bars represent standard error. Pair-wise t-tests at each time point show no statistically significant difference between wortmannin treated, BIM-treated or U73343-treated cells compared to control at the 95% confidence level, whereas the U73122-treated cells were statistically different at all time points (p < 0.01).

Figure 4

Effect of IP3 inhibitors caffeine and 2APB on IL-8 induced calcium release and neutrophil binding to ICAM-1. A. Inhibition of calcium spark by caffeine. While cells pre-incubation with 5mM caffeine was partially effective, 10mM caffeine completely inhibited IL-8 induced calcium release. B. Timing of calcium release inhibition by 2-APB. 20 µM 2-APB had a partial effect on calcium release induced by IL-8, while pre-incubation of cells with 60 µM 2-APB for 12 minutes completely inhibited IL-8 induced calcium release. C. Inhibition of IL-8 induced neutrophils adhesion to ICAM-1 by 2-ABP (60 µM) and caffeine (10 mM). 51 cells from 5 donors were analyzed for this experiment. Error bars represent standard error. Beginning at75 s and thereafter, adhesion probability in the presence of 2APBor caffeine is significantly different from control (Student’s t-test, p < 0.001). D. Video micrograph showing phagocytic cap in the control cells and in the cells pretreated with 2-APB or caffeine 90 seconds after IL-8 bead attachment.

Figure 5

Effect on intracellular and extracellular calcium chelation on neutrophils adhesion to ICAM-1. A. Inhibition of calcium release by 10 mM BAPTA. B. Inhibition of IL-8 induced neutrophils adhesion to ICAM-1 by 10 µM BAPTA or 0.5 mM EGTA. Experiments were performed in BSS supplemented with 1µM calcium and 1mM magnesium at room temperature. 15 cells from 3 donors were analyzed for this experiment. Error bars represent standard error. Adhesion probability in the presence of EGTAis significantly different from control (Student’s t-test, p < 0.001) beginning at seven minutes and thereafter. For BAPTA, statistical significance is reached at 8 minutes and thereafter.

Figure 6

Time dependent IL-8 induced neutrophil adhesion to ICAM-1. Experiments were performed in the presence of A. lovastatin (100 µM); B. Mac-1 blocking antibodies MEM174 and ICRF44 (each at 15 µl/ml); C. MAPK inhibitor SB203580 (15 µM). Eighty-eight cells from 6 donors were analyzed for these experiments. Error bars represent standard error. Adhesion probability is statistically different from control (Students t-test, p < 0.05) for lovastatin at 275 s and thereafter, and for SB203580 at 600 s and thereafter. The number of cells tested with Mac-1 blocking antibodies was small (n = 4 for MEM174 and n= 3 for ICRF44). Because of the small numbers, statistical significance was not reached for either antibody individually, but combining the seven cells tested, a significant difference (Student’s t-test, p < 0.05) is obtained at 375 s and thereafter.