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

Receptor cross talk between neutrophil FPR1 and PAFR/CXCR1/2 determined as superoxide production.

<p>Human neutrophils desensitized with fMIFL were cross-desensitized to IL8 (<b>A</b>) but primed in their response to PAF (<b>B</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 <b>A</b> and <b>B</b>). A second stimulus (<b>A</b>; IL8, 100 ng/ml, <b>B</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 (<b>A</b>) and PAF (<b>B</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).</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

Phosphatase inhibition by CalyculinA has both inhibitory and priming effects on the neutrophil NADPH-oxidase response.

<p>(<b>A</b>) 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>B</b>) FPR1_des 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 FPR1_des neutrophils are expressed as percent of non-treated controls and is given as means ±SEM (n = 8).</p

Characteristics of the receptor antagonists used.

<p>Characteristics of the receptor antagonists used.</p

Receptor cross talk from the PAFR induces reactivation of FPR1_des.

<p>Human neutrophils (10⁵) were desensitized with the FPR1 agonist fMIFL (0.1 nM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060169#pone-0060169-g001" target="_blank">Figure 1</a>. (<b>A</b>) The FPR1_des 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>B</b>) Inhibition of the PAF-induced response in FPR1_des 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). (<b>C</b>) 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).</p

Model for FPR activation, desensitization and reactivation.

<p><b>A)</b> 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>B</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. <b>C</b>) 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.</p

The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue <i>In Vivo</i> and Give Rise to Distinct NETs <i>In Vitro</i>

<div><p>Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. <i>In vitro</i>, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites <i>in vivo</i>, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon <i>in vivo</i> transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue <i>in vivo</i>. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.</p> </div