Calcium flux in neutrophils synchronizes beta2 integrin adhesive and signaling events that guide inflammatory recruitment - PubMed (original) (raw)
Calcium flux in neutrophils synchronizes beta2 integrin adhesive and signaling events that guide inflammatory recruitment
Ulrich Y Schaff et al. Ann Biomed Eng. 2008 Apr.
Abstract
Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity beta(2) integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner.
Figures
Figure 1
Dynamics of calcium flux in neutrophils rolling to arrest in presence of perfused chemokine. Neutrophils were loaded with Fluo-4 and perfused over monolayers expressing E-selectin at a shear stress of 2 dynes/cm2, then exposed to a dose range of IL-8 following 2 min of shear interaction. (a) Individual neutrophils that have rolled to arrest rapidly increase their intracellular calcium in response to IL-8 (0.1 nM) resulting in an increase in Fluo-4 emission. (b) On average, neutrophils exhibited a rapid increase in Fluo-4 emission indicative of calcium flux in response to IL-8 concentrations of 0.1 nM or higher, but did not significantly increase calcium in unstimulated or at low IL-8 of 0.01 nM. Plot is representative of 4 independent experiments with measurements from at least 60 neutrophils at each labeled concentration of IL-8
Figure 2
Dose dependence of calcium flux on stimulation with IL-8 in rolling or suspended neutrophils. Neutrophils were loaded with the ratiometric calcium indicator Fura-2 and perfused over a monolayer transfected with E-selectin. Calcium concentration was measured by ratiometric imaging in neutrophils sedimented onto the monolayer (Static) or rolling on the monolayer under shear stress (2 dynes/cm2) following exposure to a dose range of IL-8 from 0.001 to 5 nM. The average calcium concentration in all neutrophils in a field of view was measured over time, and the peak value was recorded. Data are the average of 4 or more independent experiments at each IL-8 concentration
Figure 3
Expression of high affinity β 2 -integrins is regulated by intracellular calcium. (a) Peak calcium concentration in suspended neutrophils loaded with Fura-2 was measured by fluorescent microscopy following stimulation by 5 nM IL-8 or a range of calcium ionophore concentrations (A2). Bars represent the average calcium concentration measured in the neutrophils, and are representative of 3 independent experiments. (b) Flow cytometry was conducted on neutrophils that were labeled with the monoclonal antibody 327C-Alexa488, which binds specifically to the high affinity conformation of β2 integrins. Labeled neutrophils were stimulated with 5 nM IL-8 or a range of ionophore concentration. (c) As in (b), neutrophils were labeled with 327C-Alexa488 and analyzed by flow cytometry. Cells were stimulated with 5 nM or 0.1 nM IL-8, and in certain experiments incubated with the calcium channel inhibitor 2-APB at 100 _μ_M. The upregulation of active CD18 induced by IL-8 or by each concentration of ionophore is displayed as the average fold-increase in 327C fluorescent signal compared to unstimulated controls from 3 independent experiments
Figure 4
Dependence of IL-8 mediated arrest and polarization on intracellular calcium. Neutrophils were pre-treated with inhibitors or agonists to the calcium signaling pathway, and were observed adhering to L-cell monolayers expressing E-selectin by phase contrast microscopy. 0.1 nM IL-8 was introduced into the flow chamber triggering neutrophil arrest and shape change. (a) The fraction of total neutrophils that were arrested on the monolayer was measured 1 min after IL-8 influx under conditions which stimulated or inhibited calcium flux. (b) The fraction of neutrophils undergoing polarization (as defined in the methods) was likewise measured 3 min following IL-8 infusion. Both polarization and arrest are significantly suppressed by 2-ABP, and rescued when ionophore A2 is introduced along with 2-APB. Bars represent the average of at least 3 independent experiments, and error bars represent the SEM
Figure 5
Neutrophils rolling on L–E cells decelerate to arrest before fluxing calcium and undergoing shape polarization. Neutrophils were pre-loaded with Fura-2 calcium indicator and their trajectories and shape change were simultaneously recorded at 1 s intervals in order to measure intracellular calcium, neutrophil deceleration, and polarization as a function of time. (a) The diagram depicts the stages of neutrophil response to infusion of IL-8. Neutrophils first decelerate from a steady state rolling velocity to a stable arrest in response to IL-8 stimulation, then exhibit a change in the ratiometric emission by Fura-2 indicating calcium flux. An increasing fraction of neutrophils become polarized (non-spherical) with time in preparation for migration as determined by the neutrophil aspect ratio. (b) A representative trace of instantaneous velocity and calculated deceleration of three individual neutrophils. Deviations in deceleration due to discontinuous rolling largely cancel each other outside of the two dotted lines, leaving a single spike in deceleration (downslope in velocity) marking arrest in response to IL-8. (c) Traces of intracellular calcium and deceleration in this panel are the average of at least 30 traces of individual neutrophils as in (b) from 3 separate experiments. The primary spike in average deceleration, intracellular calcium, and the fraction of neutrophils polarized are plotted for the minute following IL-8 infusion. Single asterisk indicates where calcium concentration significantly increases above background—3 s following significant deceleration
Figure 6
K_inetics of calcium flux, cell arrest and shape change in response to 2-APB and BAPTA._ Traces of intracellular calcium and deceleration in this panel are the average of at least 30 traces of individual neutrophils as in (Fig. 5c) from 3 separate experiments. (a) BAPTA abolishes intracellular calcium altogether, resulting in defective neutrophil arrest and migration. Traces of average deceleration, calcium concentration, and polarization as affected by administration of 0.1 nM IL-8 plus (b) the calcium channel inhibitor 2-APB at 100 _μ_M, (c) the ionophore A2 at 2 _μ_M, (d) a mixture of 2-APB and A2, and (e) A2 without IL-8 are shown as indicated. Asterisks indicate that neutrophils pretreated with the ionophore A2 take significantly less time to polarize than wildtype neutrophils (p < 0.05)
Figure 7
Shear stress activates calcium flux and cell polarization. Neutrophils were loaded with Fluo-4, perfused over an L–E monolayer, then stimulated with 0.1 nM IL-8 for 2 min at 0.2 dynes/cm2 (low) or 2 dynes/cm2 (normal) shear stress, and observed at high resolution with a 63× plan APO oil objective (Nikon). In some experiments, shear stress was increased to 2 dynes/cm2 after 2 min of incubation with 0.1 nM IL-8 at 0.2 dynes/cm2. (a) Changes in neutrophil shape and calcium flux following addition of IL-8 were monitored for 2 min by videomicrosopy in the presence and absence of 2-APB. (b) Intracellular calcium was quantified by the average fluorescent intensity of Fluo-4 emission (out of 255) at the centroid of the neutrophil and polarization was quantified by the fraction of visible cells that exhibited a radius ratio greater than 1.5. Both polarization and calcium flux were significantly lower in neutrophils under low shear than under normal shear conditions as indicated by * and ** respectively. Bars are representative of 3 or more independent experiments. (c) Neutrophils were pre-conditioned with 0.2 or 2 dynes/cm2 shear stress as indicated for 2 min, then observed 1 min at a shear stress of 2 dynes/cm2 (elicited by a step change in flow rate). Fraction of total neutrophils remaining adherent to an L–E monolayer in a microscope field were recorded. Bars are representative of <5 observations from 3 independent experiments and * indicates a significant difference (p < 0.05 by _t_-test)
Figure 8
Synchronization of selectin and chemokine signaling of neutrophil arrest via calcium flux. (a) β2 integrins in a resting neutrophil are expressed in a low affinity conformation clustered around a calcium channel and in close proximity to a chemokine receptor (CXCR 1). Upon recognition of IL-8, a G-protein coupled signal (represented here with PLCβ) is transmitted from the receptor to a nearby low affinity β2 integrin, via store release calcium triggering a shift to high affinity. (b) Under shear stress, high affinity β2 integrin is stabilized by binding to ICAM-1 and is put under tension, resulting in an outside-in signal which results in association of Src family kinases with the cytoplasmic domain of the integrin and PLCγ. (c) Depletion of a peripheral calcium store rapidly activates a local calcium channel, triggering calcium flux. A local hemispherical domain of high calcium concentration activates RhoA, causing surrounding β2 integrins to shift to high affinity, which is stabilized by ICAM-1 ligation, causing neutrophil deceleration and arrest
References
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