Figure 2.
Stimulation of one neutrophil elicits NMDAR-mediated currents, Ca2+ rise, and glutamate release in neighboring neutrophils
(A) Illustration, dual whole-cell recordings from two human neutrophils (cell 1 and cell 2) semi-suspended in culture; patch-pipette tips can be seen; some neutrophils lie destroyed after unsuccessful dual-patch attempts.
(B) Example traces from experiments in (A). A 50 ms current pulse applied in cell 1 (upper trace; current clamp) triggers an inward current deflection in cell 2 (voltage clamp), which is blocked by 50 μM APV; bath medium contains 1 mM glycine.
(C) Summary of experiments shown in (A and B) for the five recorded neutrophil pairs (left), and theoretical estimates of the glutamate concentration time course at three different distances, as indicated, from a small source neutrophil (right); see supplemental methods for detail.
(D) Single-cell induced spread of Ca2+ signals among neutrophils. Time-lapse sequence (green Fluo-4 channel) in a semi-suspension of human neutrophils; dotted circle, neutrophil held in whole-cell mode (patch pipette fragment from DIC image shown) enabling one-cell electrical stimulation; electric stimulus (50 pA current) is applied at time zero to the patched cell; false color scale of Fluo-4 fluorescence F, as indicated; see Figure S1 for characteristic traces and Video S3 for the recorded image sequence.
(E) Statistical summary of experiments in (D): amplitude of Fluo-4 fluorescence response (peak response; mean ± SEM) at the stimulated cell (Stim), neighboring intact neutrophils (Intact), and in the presence of the NMDAR blocker 3-((+)-2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP, 10 μM; +CPP) or the wide-range metabotropic glutamate receptor blocker alpha-methyl-4-carboxyphenylglycine (MCPG, 400 μM, + MCPG); numbers inside bars, sample size; ∗∗∗, p < 0.001 (two-sample t test, with respect to the three other sample means); see Videos S4 and S5 for recoded examples in CPP and MCPG samples. Note that peak responses were detected in different cells at different times post-stimulus. The data represent 26 independent tests (coverslips) from 10 individual blood cell preparations.
(F) Left: Glutamate biosensor sensitivity test: a characteristic response to a 10 μM glutamate application, as indicated; dots, sensor record taken every 15 s. Right: A glutamate biosensor record during 10 s whole-cell current injection (within 1 min from the sensor recording onset; indicated by red cell-patch diagram) in an individual neutrophil held in whole-cell, as in (D), with sensor recording continuing for approximately 5 min (see Figures S2B–S2D for further detail).
(G) Top: Schematic of the glutamate imaging method; A fluorescent enzymatic-based assay involves conversion of β-nicotinamide adenine dinucleotide (NAD) by L-glutamic dehydrogenase (GDH) (in the presence of glutamate) to NADH that fluoresces upon UV light excitation. Bottom: Assay sensitivity text; a fluorescence response to a pressure pulse of 5 μM glutamate (∼1 μm pipette tip; fluorescence intensity average over a 10 μm × 10 μm area around the tip, to roughly represent the vicinity of an individual neutrophil); see Figure S2 for fluorescent time-lapse snapshots.
(H) Time-lapse series of fluorescence images (NADH channel) depicting a prominent rise in extracellular glutamate near the two neutrophils upon mechanical stimulation (light touch by a 1 μm micropipette tip) of one cell (dotted circle) at t = 0 s, as indicated; false color scale; note that the glutamate concentration signal drops sharply away from the cell surfaces reflecting rapid dilution in the bath medium.
(I) Statistical summary of experiments in (H): amplitude of glutamate-sensitive NADH fluorescence response (mean ± SEM) at the stimulated cell (Stim), neighboring intact neutrophils (Intact), and in the presence of the NMDAR blocker CPP (10 μM; +CPP); numbers inside bars, sample size; ∗∗∗, p < 0.001 (two-sample t test). The data represent 17 independent tests (coverslips) from 3 individual blood cell preparations; see Videos S6 and S7 for the illustration of recordings in Intact and CPP samples.