F2Pal₁₀ induces pertussis toxin sensitive superoxide production and triggers mobilization of calcium from intracellular stores.

<p>A. Primary human neutrophils were treated with pertussis toxin (PTX, 500 ng/ml final concentration) and samples were withdrawn at indicated time points. These cells were activated by WKYMVM (50 nM, black bars) or F2Pal₁₀ (500 nM, white bars) and the release of superoxide anions was recorded continuously. The response induced by the peptides was gradually decreased (shown for 90 and 120 min incubation). As a control, the PMA response (5×10⁻⁸ M final concentration) from control cells (120 min incubation without PTX, solid line in the inset) or treated with PTX for 120 min (dashed line in the inset) is shown. Superoxide production (peak values) from pertussis toxin treated neutrophils was compared to that from non-treated control cells. A representative experiment out of more than five is shown. B and C. Fluo-3-AM/FuraRED labeled neutrophils were incubated without any additive (solid lines) or with EGTA (2 mM; dotted lines). The FPR2 specific peptide WKYMVM (100 nM final concentration; B) or F2Pal₁₀ (500 nM; C) was added and the concentration of free cytosolic calcium was monitored by the Fluo-3-AM/FuraRED fluorescence. Traces of representative calcium responses are shown and at least three experiments have been performed. Abscissa, time of study (sec); ordinate, fluorescence (arbitrary unit). D. Primary human neutrophils were incubated with different pharmacological kinase inhibitors (1 µM final concentration) for 15 min at 37°C. Control cells were incubated at the same condition but received no inhibitors. The cells were activated by WKYMVM (50 nM, black bars) or F2Pal₁₀ (500 nM, white bars) and the release of superoxide anions was recorded continuously. Data are presented as % inhibition compared to the control response (peak values of superoxide production were used, mean±SEM, n = 3).</p

The F2Pal₁₀ pepducin specifically activates FPR2.

<p><b>A.</b> Primary human neutrophils were activated by F2Pal₁₀ (500 nM) in the absence (solid line) or presence of the receptor specific inhibitor PBP10 (FPR2 specific inhibitor; 1 µM final concentration, dashed line) or the FPR1 antagonist cyclosporin H (CysH; 1 µM final concentration, dotted line) and the release of superoxide anions was recorded continuously. A representative experiment out of more than five is shown and the time point for F2Pal₁₀ addition is indicated by an arrow. Abscissa: Time of study (min); ordinate: Superoxide production (10⁶× counts per minute; Mcpm). <b>B.</b> F2Pal₁₀ desensitizes neutrophils in their response to the conventional FPR2 agonist WKYMVM. Primary human neutrophils were first activated by WKYMVM (40 nM; time point for addition is indicated by the first arrow, solid line) or F2Pal₁₀ (500 nM; time point for addition is indicated by the first arrow, dashed line) and five minutes later reactivated by F2Pal₁₀ (500 nM; time point for addition is indicated by the second arrow, solid line) or WKYMVM (40 nM; time point for addition is indicated by the second arrow, dashed line). The release of superoxide anions was recorded continuously. A representative experiment is shown. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). <b>C and D.</b> The F2Pal₁₀ pepducin triggers an increase in intracellular calcium when added to undifferentiated HL-60 cells overexpressing FPR2. The F2Pal₁₀ pepducin (broken lines; 10 nM final concentration) was added to Fura-2 labeled FPR2 overexpressing cells (<b>C</b>) or FPR1 overexpressing cells (<b>D</b>), and the concentration of free cytosolic calcium was monitored by the Fura-2 fluorescence. Representative calcium responses induced by control peptides (WKYMVM for FPR2 and WKYMVm for FPR1) are included (solid lines) for comparison. Abscissa, time of study (sec); ordinate, fluorescence (arbitrary units).</p

PAF- or ATP-stimulation induces reactivation of desensitized FPR2.

<p><b>A</b>. Naïve neutrophils desensitized with F2Pal₁₀ (500 nM final concentration) or WKYMVM (40 nM) were primed for subsequent PAF stimulation. A representative experiment is shown and the time point for addition of FPR2 agonists is indicated by the first arrow, the time point for PAF stimulation (100 nM final concentration) with the second arrow. The control PAF response induced in naïve (non-desensitized) neutrophils is shown for comparison (dotted line). Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). <b>B.</b> Naïve neutrophils were first desensitized with F2Pal₁₀ (F2Pal_10des., 500 nM final concentration) and subsequently stimulated with PAF (100 nM final concentration; time point for PAF addition is indicated by an arrow). The FPR2 specific inhibitor PBP10 (1 µM) was added 1 min prior to PAF stimulation (dashed line) and the release of superoxide anions was recorded continuously. The PAF responses induced in naïve cells (solid line), as well as in F2Pal₁₀-desensitized cells receiving no PBP10 (dotted line), are shown for comparison. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). <b>C.</b> Effect of calyculin A on receptor cross-talk induced FPR2 reactivation. Cells desensitized with F2Pal₁₀ (F2Pal_10des.) were incubated with the phosphatase inhibitor calyculin A (60 nM; solid line) or buffer as control (dashed line) for 10 min at 37°C prior to PAF stimulation (100 nM). The release of superoxide anions was recorded continuously. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). <b>D.</b> Naïve neutrophils were activated with WKYMVM (40 nM final concentration, solid line) or F2Pal₁₀ (500 nM final concentration, dashed line) and after the responses had declined the same cells were reactivated with ATPγS (50 µM final concentration; the addition is indicated by an arrow) and the release of superoxide anions was recorded continuously. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). Inset: The summary of three independent experiments in which superoxide production was determined using neutrophils desensitized with F2Pal₁₀ (500 nM) or WKYMVM (40 nM) when reactivated with ATPγS (50 µM final concentration). The results are given as peak values of superoxide production following addition of ATPγS (mean+SEM; n = 3).</p

The neutrophil response to the pepducin F2Pal₁₀ is primed by latrunculin A, TNFα and calyculin A.

<p>WKYMVM (50 nM final concentration) or F2Pal₁₀ (500 nM final concentration) were added to control (non-primed) or treated neutrophils and the release of superoxide anions was recorded continuously. <b>A.</b> A representative experiment of an F2Pal₁₀ induced response in control (solid line) and latrunculin A treated neutrophils (dashed line) is shown. The time point for addition of F2Pal₁₀ is indicated by an arrow. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). <b>B.</b> The priming effects of latrunculin A, TNFα and Calyculin A on the WKYMVM (50 nM final concentration, black bars) or F2Pal₁₀ (500 nM final concentration, white bars) are summarized as fold increase (peak values of the superoxide production) when compared to the response in non-treated cells (mean ± SEM; n = 3). A non-priming cut-off line (fold increase = 1) is included as a horizontal broken line in the figure.</p

The F2Pal₁₀ pepducin is a partial agonist in triggering superoxide release from human neutrophils.

<p>Human neutrophils were activated by different concentrations of WKYMVM (10–1000 nM; <b>A</b>) or F2Pal₁₀ (50–2000 nM; <b>B</b>) and the release of superoxide anions was recorded continuously. Representative experiments out of more than five are shown and an arrow indicates the time point for agonist addition. Abscissa: Time of study (min); ordinate: Superoxide production (Mcpm). The dose responses for WKYMVM (<b>A</b>, inset) and F2Pal₁₀ (<b>B</b>, inset) from three independent experiments are summarized in the insets, data are presented as normalized peak response with the fitted curves.</p

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

<div><p>Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPR_des) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPR_des back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPR_des as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPR_des also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPR_des initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.</p> </div

Characteristics of cytoskeleton interfering drugs used.

<p>Characteristics of cytoskeleton interfering drugs used.</p

More pronounced reactivation was induced by PAF in neutrophils desensitized by the pepducin F2Pal₁₀.

<p><b>A.</b> Naïve neutrophils were activated with different concentrations of WKYMVM (10–40 nM; black bars) or F2Pal₁₀ (100–500 nM; white bars) followed by a second stimulation with PAF (100 nM final concentration) and the release of superoxide anions was recorded continuously. The results are given as peak values of superoxide production from three separate experiments for the first stimulation with FPR2 agonists (mean+SEM, n = 3). <b>B.</b> Neutrophils desensitized with different concentrations of WKYMVM (10–100 nM; WKYMVM_des., black bars) or F2Pal₁₀ (10–500 nM; F2Pal_10des., white bars) were reactivated by a second stimulation with PAF (100 nM final concentration), after which the superoxide production (peak values) was recorded. The results are given as fold increase (peak values) of the PAF-induced response from FPR2-desensitized cells compared to the PAF response from naïve cells (mean+SEM; n = 3). The naïve PAF response (fold increase = 1) is indicated as a horizontal broken line.</p

PAF activates FPR1_des neutrophils also in the presence of latrunculinA.

<p>Human FPR1_des 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 FPR1_des 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 (FPR1_des neutrophils without latrunculin A pre-treatment, solid line) is shown.</p

Intracellular Ca²⁺ response triggered upon reactivation of FPR1_des by PAF is not cyclosporin H sensitive.

<p>FPR1_des 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).</p