Figure 1.
Receptor cross talk between neutrophil FPR1 and PAFR/CXCR1/2 determined as superoxide production.
Human neutrophils desensitized with fMIFL were cross-desensitized to IL8 (A) but primed in their response to PAF (B). Neutrophils (105 cells, 37°C) were first activated by the FPR1 specific agonist fMIFL (0.1 nM, added at time indicated by the arrows to the left) leading to receptor desensitization (solid lines in A and B). A second stimulus (A; IL8, 100 ng/ml, B; PAF, 100 nM) was added to the cells (solid lines) at the time point indicated by the arrows to the right. Activation of naïve (non-desensitized) neutrophils by IL8 (A) and PAF (B) was determined in parallel and is shown for comparison (broken lines). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×106; Mcpm).
Figure 2.
Receptor cross talk from the PAFR induces reactivation of FPR1des.
Human neutrophils (105) were desensitized with the FPR1 agonist fMIFL (0.1 nM) as described in Figure 1. (A) The FPR1des neutrophils were activated with PAF (100 nM, added at time indicated by arrow; solid line). The involvment of FPR1 and PAFR in the PAF-induced response was examined by addition of cyclosporin H (1 µM, FPR1 antagonist, broken line) or WEB2086 (1 µM, PAFR antagonist, dotted line) at 3 min prior to PAF addition. For comparison, the oxidative response to PAF in naïve neutrophils is shown (inset). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×106; Mcpm). (B) Inhibition of the PAF-induced response in FPR1des cells by cyclosporin H (1 µM, FPR1 specific antagonist) or WEB2086 (1 µM, PAFR antagonist) shown as mean peak values ±SEM of the responses (Mcpm, n = 5 for WEB2086, n = 19 for control, cyclosporine H). The PAF induced response in naïve neutrophils is shown for comparison (n = 19). (C) Human neutrophils (105) were activated/desensitized with different concentrations of the FPR1 agonist fMIFL (added at time indicated by arrow to the left). The neutrophils were then activated with PAF (100 nM final concentration, added at time indicated by arrow to the right). For comparison, a PAF-induced response in naïve neutrophils is shown (solid line). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×106; Mcpm).
Table 1.
Characteristics of the receptor antagonists used.
Figure 3.
Intracellular Ca2+ response triggered upon reactivation of FPR1des by PAF is not cyclosporin H sensitive.
FPR1des neutrophils (desensitized with 0.1 nM fMIFL) loaded with Fura-2 (2×106/ml) were activated by PAF (1 nM final concentration) in the absence (solid line) or presence (broken line) of the FPR1 specific antagonist cyclosporin H (1 µM added 30 sec before PAF). The changes in fluorescence were followed using dual excitation of Fura-2 at 340 and 380 nm, respectively, with an emission wavelength of 510 nm. For comparison, a PAF-induced intracellular Ca2+ response is shown for naïve neutrophils (inset). A representative experiment is shown, n = 3. Abscissa, time of study (sec); Ordinate, relative change in helloCa2+]i (arbitrary units, AU).
Figure 4.
Phosphatase inhibition by CalyculinA has both inhibitory and priming effects on the neutrophil NADPH-oxidase response.
(A) Human neutrophils were incubated without or with CalyculinA (CA; 60 nM) at 37°C for 10 min prior to stimulation with PAF (100 nM) or fMIFL (0.1 nM), and the release of superoxide anions was recorded. The graph shows ratios of superoxide production induced by PAF or fMLF between samples with and without calyculin A (fold increase, mean ±SEM; n = 5). (B) FPR1des neutrophils (desensitized with 0.1 nM fMIFL) were incubated at 37°C for 10 min without (control and inset, solid line) or with CalyculinA (CA, 50 nM; inset, broken line). The cells were then stimulated with PAF (100 nM) or latrunculin A (100 ng/ml final concentration) and the release of superoxide anions was recorded. A representative experiment for PAF stimulation is shown in the inset. The stimulus-induced responses in the CalyculinA treated FPR1des neutrophils are expressed as percent of non-treated controls and is given as means ±SEM (n = 8).
Table 2.
Characteristics of cytoskeleton interfering drugs used.
Figure 5.
The cytoskeleton disrupting agent latruculin A induces reactivation of FPR1des.
Latrunculin A (100 ng/ml) was added to FPR1des neutrophils (105 cells; desensitized with 0.1 nM fMIFL) in the absence (solid line) or presence (dotted line) of cyclosporin H (1 µM, FPR1 specific antagonist, added 1 min before latrunculin A) and the release of superoxide anions was determined. For comparison, a PAF-induced reactivation of FPR1des neutrophils is included (dashed line). A representative experiment is shown, n>5. Abscissa, time of study (min); Ordinate, superoxide production (counts per minute×106; Mcpm).
Figure 6.
Model for FPR activation, desensitization and reactivation.
A) The agonist-occupied FPR activates a G-protein and the second messengers generated activate the electron-transporting NADPH-oxidase that reduces oxygen to superopxide anion. The signaling state of the receptor is fairly short lived. B) The agonist-occupied receptor is desensitized and the functional response is terminated. This non-signaling state is hypothetically achieved through a physical separation of the receptor-ligand complex from the G-protein, made possible by binding of actin polymers and/or arrestin molecules to the receptor. C) The desensitized FPR is reactivated by signals generated when PAF binds to its neutrophil receptor (arrow, 1). Reactivation of the desensitized FPR is achieved also with cytoskeletal inhibitors, (shorter filaments, 2), suggesting a mechanism for reactivation that involves uncoupling of the receptor-ligand complex from the cytoskeleton. The described cross talk is hierarchial and unidirectional.
Figure 7.
PAF induces actin polymerization in both naïve and FPR1des neutrophils.
Human neutrophils (naïve or FPR1des) were activated with a receptor agonist or latrunculin A and the change in polymerized actin was determined att different time points (15 to 120 sec) after activation. Naïve neutrophils were activated by PAF (100 nM) or fMLF (0.1 nM) and FPR1des neutrophils were reactivated by PAF (100 nM) or latrunculin A (200 ng/ml). The stimulation at indicated time points was terminated by adding ice cold paraformyldehyde (final concentration 2%) to the cells. The amount of polymerized actin was determined by flow cytometry after phalloidin staining and compared to the amount of actin at time zero before activation. The values are shown as mean ratio ± SEM; n = 3.
Figure 8.
PAF activates FPR1des neutrophils also in the presence of latrunculinA.
Human FPR1des neutrophils were incubated in the absence or presence of latunculinA (LA, 50 ng/ml) and after return of the NADPH-oxidase activity to background levels (after around 20 min; not shown in the figure) the cells were activated with PAF (100 nM) and the measurement of oxidase activity was started. In some experiments, cyclosporinH (CA, 1 µM) was added to the cells just prior to PAF. The response induced was sensitive to this FPR1 specific antagonist. The results are expressed as peak response (Mcpm, open bars) and total production (area under curve; AUC, filled bars) in percent of control (PAF-induced peak response in FPRdes in the absence of LA and CA; mean±SEM, n = 3). The FPR1des neutrophils treated with latrunculin A (50 ng/ml) could not be reactivated by additional latrunculin A (100 ng/ml, inset, dotted line). For comparison, reactivation of control cells (FPR1des neutrophils without latrunculin A pre-treatment, solid line) is shown.