Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

The data described here is related to the research article titled (Gabl et al., 2016) [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR) can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8) and CXCR4 (ATI-2341), respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe)6-NH2 (both are FPR2 specific inhibitors), but not to the FPR1 specific inhibitor cyclosporine H.


a b s t r a c t
The data described here is related to the research article titled   [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR) can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8) and CXCR4 (ATI-2341), respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal 10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP 10 and the peptidomimetic Pam-(Lys-betaNSpe) 6 The effects of receptor specific inhibitors and desensitization profiles were determined Data source location

Gothenburg, Sweden
Data accessibility Data are within this article

Value of the data
Receptor specific neutrophil responses can be determined by the sensitive assay to measure superoxide production.
The precise receptor involved can be identified by the desensitization profile and by the use of defined receptor specific antagonists.
The data provide insights into the highly variable effects of pepducins which includes receptor hijacking.
Direct neutrophil activation by ICL3-8 and its modulatory effect on FPR signaling are shown (Fig. 1). In addition, data on dose-dependent neutrophil activation induced by ATI-2341 and the effects on this response of FPR specific agonists as well as antagonists are provided (Fig. 2).

Neutrophil isolation
Human neutrophils were isolated from buffy coates as described [2], and diluted to 1 Â 10 6 /ml in Krebs-Ringer phosphate buffer (KRG) containing glucose (10 mM), Mg 2 þ (1.5 mM), and Ca 2 þ (1 mM). The cells were kept on ice until use. Ethics approval was not needed since the buffy coats were provided anonymously and could not be traced back to a specific individual. This is in line with Swedish legislation section code 4 § 3p SFS 2003:460 (Lag om etikprövning av forskning som avser människor).

The NADPH-oxidase assay and receptor desensitization protocol
The release of superoxide anions from activated neutrophils was measured using isoluminol/HPR amplified chemiluminescence systems [3]. Vials with reaction mixtures of 900 ml containing isoluminol (20 mM), HRP (4U), neutrophils (1 Â 10 5 /ml) with or without antagonist were incubated for Fig. 2. The CXCR4 pepducin ATI-2341 triggers a direct activation of the neutrophil NADPH-oxidase through FPR2. A) ATI-2341 dose-dependently triggers neutrophil superoxide production, as measured by isoluminol-amplified chemiluminescence. Data are presented as normalized peak response with the fitted curves. The EC 50 value and the 95% confidence interval were calculated from 8 independent experiments. Inset: A representative ATI-2341 (1 mM) induced neutrophil response is shown. Abscissa, Time of study; Ordinate, Superoxide production (arbitrary units). B) Desensitized neutrophils incubated for 5 min at 37°C without (solid lines) or with 1 mM ATI-2341 (dash lines) were activated with the FPR2 specific agonist F2Pal 10 (1 mM; main figure) or the FPR1 specific agonist fMLF (100 nM, inset). Arrows indicate the time point for agonist addition. C) Neutrophils were pre-incubated for 5 min at 37°C without any additive, with either of the two FPR2 specific inhibitors PBP10 (1 mM) or HF965A (1 mM), or with the FPR1 specific inhibitor cyclosporin H (CysH, 1 mM). The neutrophils were then activated with ATI-2341 (1 mM) and superoxide production was recorded by isoluminol-amplified chemiluminescence. The results are expressed as percent inhibition compared to the activity induced in cells incubated without any inhibitor (mean 7SD, n¼ 7). N.I. equals no inhibition. 5 min at 37°C. For receptor desensitization, cells were either activated or pre-incubated with receptor specific agonist for 5 min before stimulation. Stimuli (100 ml) were added and the light emission was recorded continuously.