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

Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1

<div><p>Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits.</p></div

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

NADPH-oxidase activity induced in mouse (A) and human (B) neutrophils by formyl peptides differing in their M3 binding properties, fMWYYLF (a representative of the low M3 binding group), fMFLIDV (a representative of the middle M3 binding group) and, fMILLV (a representative of the low M3 binding group).

<p><b>(A)</b> Neutrophils (5x10⁴) from wild type (WT; solid lines) and Fpr2^-/- (broken lines) were activated by by fMWYYLF (5 nM), fMFLIDV (5 nM) or fMILLV (5 nM) and the oxidase activity was measured. The time point for addition of the agonist is indicated by an arrow. <b>B)</b> Human neutrophils incubated 5 min without (solid lines) or with an FPR antagonist, either the FPR2 antagonist PBP₁₀ (1 μM, dashed lines) or the FPR1 antagonist CysH (1 μM, dotted lines) were activated with fMWYYLF (100 nM), fMFLIDV (500 nM) or fMILLV (5 nM) and the oxidase activity was measured. The time point for addition of the agonist is indicated by an arrow. Arrows indicate the time points for addition of the peptide agonists. One representive experiment out of three is shown. Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6).</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

NADPH-oxidase activity induced in bone marrow neutrophils derived from wild-type and Fpr2^-/- mice by fMIFL and PSMα2.

<p>The oxidase activity was measured by an isoluminol/HPR chemiluminescence system. <b>(A)</b> Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 10 nM fMIFL. <b>(B)</b> Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 50 nM PSMα2. <b>(C)</b> Neutrophils (5x10⁴) from wild type (WT; solid line) and Fpr2^-/- (broken line) were activated by 50 nM fMYFINILTL. Arrows indicate the time points for agonist addition and a representative experiment out of four is shown. Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6).</p

fMYFINILTL induced superoxide production from bone marrow derived mouse neutrophils.

<p>Neutrophil NADPH-oxidase activity was measured by an isoluminol/HPR chemiluminescence system. (<b>A)</b> Neutrophils (5x10⁴) were activated by either fMYFINILTL (50 nM) or fMIFL (10 nM). The time point for addition of agonists is indicated by an arrow and the curves are from one representative response (n > 3). Abscissa, Time of study (min); ordinate, superoxide production (cpmx10^-6). <b>(B)</b> Mouse neutrophil NADPH-oxidase activity induced by different concentrations of fMYFINILTL. The peak values of the responses in relation to the concentration of fMYFINILTL was determined and is expressed as a percent of the maximal response. Data are expressed as mean ± SD from three independent experiments. Abscissa, agonist concentration (M); ordinate, superoxide production (percent of max).</p

fMYFINILTL and MYFINILTL induced superoxide production from human neutrophils.

<p>Neutrophil NADPH-oxidase activity was measured by an isoluminol/HPR chemiluminescence system. (<b>A)</b> Neutrophils were pre-incubated at 37°C for 5 min in the absence (solid line) or presence of FPR1 antagonist Cyclosporin H (CysH, 1 μM, dashed line) or FPR2 antagonist PBP₁₀ (1 μM, dotted line) before stimulation with fMYFINILTL (10 nM; time for addition indicated by an arrow). The curves are from one representative response (n > 10). <b>Inset:</b> The superoxide production induced by the non-formylated peptide variant MYFINILTL (1 μM) in the presence or absence (control) of FPR specific inhibitors. Abscissa, Time of study (min); ordinate, superoxide production (in arbitrary light units, cpmx10^-6). <b>(B)</b> Neutrophil NADPH-oxidase activity induced by different concentrations of fMYFINILTL. The peak values of the responses in relation to the concentration of fMYFINILTL was determined and is expressed as a percent of the maximal response. Data are expressed as mean ± SD from four independent experiments. Abscissa, agonist concentration (M); ordinate, superoxide production (percent of max).</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

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