Formylpeptides trigger selective molecular pathways that are required in the physiological functions of human neutrophils

Cellular Signalling 15 (2003) 377 – 383 www.elsevier.com/locate/cellsig

Formylpeptides trigger selective molecular pathways that are required in the physiological functions of human neutrophils Rita Selvatici a, Sofia Falzarano b, Serena Traniello b, Giampiero Pagani Zecchini c, Susanna Spisani b,* a

Dipartimento di Medicina Sperimentale e Diagnostica, Sezione Genetica Medica, Via L. Borsari 46, Universita` degli Studi di Ferrara, 44100 Ferrara, Italy b Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari 46, Universita` degli Studi di Ferrara, 44100 Ferrara, Italy c Istituto di Chimica Biomolecolare CNR c/o Dipartimento di Studi Farmaceutici, P.zzale A.Moro 5, Universita` di Roma ‘‘La Sapienza’’, 00185 Rome, Italy Received 17 July 2002; accepted 10 September 2002

Abstract For-Met-DzLeu-Phe-OMe ([DzLeu2]) is a conformationally restricted for-Met-Leu-Phe-OMe (fMLP-OMe) analogue able to discriminate between different responses of human neutrophils. In contrast, [DzLeu2] significantly activates the transduction pathways—involving Ca2 +, inositol phosphate, and cyclic AMP (cAMP) enhancement, as is the case with the full agonist fMLP-OMe. Here, we have studied the specific involvement of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) in the presence or absence of extracellular Ca2 +, being the cation clearly involved in the activation of neutrophils by fMLP. A strong correlation has been found between PKC isoforms, MAPKs and the selective physiological functions by [DzLeu2]-activated neutrophils. In a calcium-free condition, our data suggest that the failure of PKC h1 translocation and of p38 MAPK phosphorylation by the analogue refers to its inability to induce chemotaxis, and that the failure by both fMLP-OMe and [DzLeu2] to evoke extracellular response kinase 1 and 2 (ERK1/2) phosphorylation would suggest a reduction in superoxide anion production. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Human neutrophils; Formylpeptides; Protein kinase C; MAP kinases; Chemotaxis; Superoxide anion release

1. Introduction Neutrophils are involved in host defence mechanisms against bacterial infections and their activation depends upon different extracellular signals, among which those mediated by chemotactic N-formylpeptides of bacterial origin [1] by binding to specific G-protein coupled receptors (FPR) [2]. FPR interaction generates multiple second messengers in neutrophils through the activation of phospholipase C (PLC), PLD, PLA2 and increase of cyclic AMP (cAMP) intracellular levels [3,4]. The involvement of protein kinase C (PKC), phosphatidilinositide 3 kinase (PI3-K) and mitogen activated protein kinases (MAPKs) [Jun Nterminal kinases (JNK), p38 and extracellular response kinase 1 and 2 (ERK1/2)] has also been demonstrated [5,6]. The activation of these transduction pathways is responsible for the modulation of neutrophil specific * Corresponding author. Tel.: +39-532-291424; fax: +39-532-202723. E-mail address: [email protected] (S. Spisani).

responses, i.e. adhesion, chemotaxis, exocytosis and activation of NADPH oxidase [7]. PKC is a multigene family of enzymes comprising at least 11 isoforms [8]. Upon activation, the kinase translocates from soluble to particulate compartment. The intracellular distribution of PKC isoforms suggests specific isoform-related biological functions, but this specialisation has only partly been explored. Interdependence between isozymes exists and may be important. PKC can be used by many receptor types to regulate the activation of MAP kinases, whose phosphorylations have an impact on processes in the cytoplasm, the nucleus, the cytoskeleton and the membrane. Upon activation, the phosphorylated proteins then move to the nucleus, where they catalyse phosphorylations and activation of specific target transcription factors. As a consequence of FPR interaction, an increase of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), as well as of Ca2 + cytosolic levels, is evoked. The release of Ca2 + from internal stores induces the opening of the store-

0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 2 ) 0 0 1 2 3 - 7

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operated Ca2 +-channels (SOCC) in the plasma membrane, and a sustained influx of Ca2 + takes place. Furthermore, PKC and MAPK pathways are both sensitive to changes in intracellular Ca2 + ([Ca2 +]i) levels [9]. We have previously reported studies on for-Met-LeuPhe-OMe (fMLP-OMe) and the conformationally constrained for-Met-DzLeu-Phe-OMe [DzLeu2] (Fig. 1). In [DzLeu2], the central Leu has been replaced by (Z)-2,3didehydroleucine (DzLeu) that reduces the ligand backbone flexibility. FMLP-OMe is able to induce a full transduction response by neutrophils, while the analogue is only able to elicit superoxide anion (O2 ) production and degranulation but not chemotaxis [10,11]. However, both peptides induce a considerable enhancement of PLC activity, as well as increasing Ca2 + and cAMP intracellular levels, and they are both effective in displacing the labelled peptide from their binding sites [12]. As a continuation of our studies, the present investigation was designed to determine the specific signalling cascade of PKC isoforms and MAPKs (JNK, p38 and ERK1/2) against fMLP-OMe and [DzLeu2] analogue on human neutrophils activation. Since the activation of neutrophils is dependent on store-operated Ca2 + influx, the effects of fMLP-OMe and [DzLeu2] have been studied in the presence or absence of extracellular Ca2 + to discover the pattern of second messengers that specifically align each peptide.

2. Materials and methods 2.1. Materials Dextran, Ficoll-Paque and ECL Western blotting detection reagents were purchased from Amersham-Pharmacia Biotech. FMLP-OMe, cytochalasin B (CB), dimethylsulfoxide (DMSO) were from Sigma. Polyvinilydene difluoride (PVDF) membranes were from Bio-Rad Laboratories. AntiPKC a, anti-PKC h1, anti-PKC h2 and anti-PKC ~ antibodies were from Santa Cruz Biotechnology. Polyclonal antibodies against p54/46 SAPK/c-JNK N-terminal kinase (JNK), p38 MAP kinase, p44/42 MAP kinase (ERK1/2) and the phospho-SAPK/JNK (pJNK), phospho-p38 MAP kinase (pp38) and phospho-p44/42 MAP kinase (pERK1/2) were from Cell Signalling Technology (Celbio). All the other reagents were of the highest grade commercially available. 2.2. Cell preparation Neutrophils were isolated from peripheral blood of healthy human volunteers and purified by the standard techniques [10]. Cells, 98 – 100% pure and z 99% viable, were resuspended in Krebs – Ringer-phosphate, pH 7.4, containing 0.1% w/v glucose supplemented with 1 mM CaCl2 (normal KRPG) or in Ca2 +-free KRPG added with 1 AM EGTA. All experiments were carried out according to the guidelines of the local and regional ethics committees. 2.3. Preparation of peptides For-Met-DzLeu-Phe-OMe was prepared as described previously [10,11]. 10 2 M in DMSO of fMLP-OMe and [DzLeu2] were diluted in buffer before use. At the concentrations used, DMSO did not interfere with any of the biological assays performed. 2.4. Stimulation and fractionation of neutrophils

Fig. 1. Structures of fMLP-OMe and [DzLeu2].

Suspensions of 1  107 neutrophils/ml were preincubated at 37 jC for 10 min with 5 Ag/ml cytochalasin B in normal KRPG or Ca2 +-free KRPG. Cells were stimulated with fMLP-OMe or [DzLeu2] 10 6 M, which is the optimal dose for the activation of O2 , or treated with 0.1% DMSO (vehicle) at the indicated times. Incubations were stopped by adding four volumes of ice-cold buffer and pelletted at 14,000 rpm for 5 min at 4 jC. Pellets were suspended in lysis buffer containing 20 mM Tris pH 7.5, 0.25 M saccharose, 2 mM EDTA, 10 mM EGTA, 2 mM phenylmethylsulfonyl fluoride and an anti-protease mixture consisting of 0.1% leupeptin, 10 Ag/ml aprotinin, 0.35 mM antipain, 0.35 mM pepstatin, 0.24 mg/ml chymostatin. Cells were disrupted by sonication (6  10 s) at 4 jC and the suspension was centrifuged at 5000 rpm for 10 min. The post nuclear supernatant was ultracentrifuged at 150,000  g for 1 h at 4 jC. The supernatant was the cytosolic fraction

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while the pellet, corresponding to particulate fraction, was resuspended in the same buffer, added with 0.2% Triton X100 and resonicated (2  10 s). Protein contents of cytosolic and particulate compartments were determined using the method of Bradford [13]. 2.5. Western blot analysis Equal amount of protein (50 Ag) was separated by 10% sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to PVDF membrane at 100 V for 1 h. Blots were incubated over night in Tris-buffered saline solution, pH 7.6, containing 5% non-fat dry milk added with 0.05% Tween 20. Western blots were performed using the following polyclonal antibodies against: (i) PKC a, h1, h2 and ~ (0.3 Ag/ ml); (ii) JNK, p38 and ERK1/2 (1:1000); (iii) pJNK, pp38 and pERK1/2 (1:1000). Signals were detected using enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). The molecular weight was calculated with prestained SDS-PAGE standards (New England BioLabs) that were applied to the same gel run samples. 2.6. Measurement of the densities of the immunoblot bands Densitometric analysis of specific autoradiographic bands was used for the statistical analysis. The densities were measured by the Bio-Rad densitometer GS700 and expressed as O.D. units/mm2. 2.7. Statistics Statistical analyses were performed with Student’s t-test for non-paired data. Differences were considered to be statistically significant if p values < 0.05.

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Table 2 Effect of Ca2 + chelator EGTA in the extracellular buffer on fMLP-OMe or [DzLeu2]-induced chemotaxis, O2 production and [Ca2 +]i levels in neutrophils Peptides

Chemotactic index

O2 (nmol)

D[Ca2 +]i levela (%)

fMet-LeuPhe-OMe fMet-DzLeuPhe-OMe

0.98 F 0.08 (10 9 M) 0.15 F 0.02 (10 7 M)

20 F 3 (10 7 M) 25 F 5 (10 6 M)

216 F 13b (10 6 M) 170 F 12b (10 6 M)

Data are means F standard errors of the mean (SEM) of at least six separate determinations. a Basal level was 92.4 F 4 nM. b p < 0.001 versus control conditions.

and particulate compartments to assay: (i) the rate of translocation of the PKC isoenzymes: a, h1, h2 and ~; (ii) the levels of the MAPKs: JNK, p38, and ERK1/2; and (iii) the active forms pJNK, pp38 and pERK1/2. For this purpose neutrophils were preincubated 10 min in normal KRPG or in Ca2 +-free KRPG, since Ca2 + is known to modulate the production of second messengers by regulating various transductional effectors. Preincubation was then followed by stimulation with 10 6 M fMLP-OMe or [DzLeu2] for 10 s, 30 s, 1 min, 2 min, and 5 min. In all the experiments, CB was added since it has been reported to increase the number of free fMLP binding sites [14] and [Ca2 +]i levels [15] to potentiate agonist-induced O2 production [16], but it can disrupt microfilaments decreasing chemotaxis [17]. We have previously reported [12,18] that in the presence of extracellular Ca2 +, [DzLeu2] similarly to fMLP and fMLP-OMe, induces a considerable enhancement of PLC activity, as well as Ca2 + and cAMP intracellular levels and shows the same fMLP receptor affinity, but it is unable to activate neutrophil locomotion (Table 1). In the absence of Ca2 + in the extracellular medium, neutrophil chemotaxis appears insensitive while superoxide anion release and [Ca2 +]i are present, but reduced (Table 2). 3.1. Preincubation of neutrophils in normal KRPG and stimulation with fMLP-OMe

3. Results In order to clarify further molecular mechanisms more tightly referred to the selection of the physiological functions by the two formylpeptides under investigation, Western blotting experiments were performed in cytosolic

When neutrophils were preincubated in KRPG supplemented with Ca2 + and then triggered with fMPL-OMe, a translocation of the PKC isoforms a, h1, h2 and ~ between

Table 1 Chemotactic activity, superoxide anion production, cAMP levels, intracellular Ca2 +, phospholipase C activity and binding activity (IC50) of fMLP-OMe derivatives Peptides

Chemotactic index

O2 (nmol)

9

M) 48 F 3 (10 M) 59 F 5 (10

fMet-Leu-Phe-OMe 1.15 F 0.08 (10 fMet-DzLeu-Phe-OMe 0.16 F 0.02 (10

7

cAMP (%)a 7 6

M) 250 F 3 (10 M) 350 F 4 (10

D[Ca+]i levela (%)b Phospholipase C (%)c IC50 (nM) 6 6

M) 450 (10 M) 200 (10

6 6

M) M)

245 F 18 (10 183 F 15 (10

6 6

M) M)

60 F 3 (1.4  10 55 F 2 (1.8  10

8 7

M) M)

Values indicate the efficacy (activity at the optimal peptide concentrations). Optimal concentration values in parentheses. Data are means F standard errors of the mean (SEM) of four separate determinations. a Basal cAMP level was 1.9 F 0.2 pmol/1  106 cells. b Basal Ca2 + level was 92.4 F 4 nM. c Basal level of inositol phosphate production was 1890 F 142 dpm/107 cells.

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Fig. 2. Western blot of cytosol and membrane fractions of human neutrophils treated with fMLP-OMe or with 0.1% DMSO (control) in normal KRPG and labelled with: (A) anti PKC-a, h1, h2 and ~ and (B) with anti MAP kinase-p38, pp38, ERK1/2, pERK1/2, JNK and pJNK specific antibodies, at the indicated times. The results are representative of three separate experiments, each performed with cells from different donors.

2 and 5 min was observed; the same isoforms were also present in the cytosolic compartment (Fig. 2A). The levels of JNK, p38, and ERK1/2 and of the phosphorylated forms pJNK, pp38 and pERK1/2 (Fig. 2B) were studied by Western blotting in cytosolic and particulate

extracts. The levels of p38 and pp38 were strongly detected both in cytosol and in membrane at all times; ERK1/2 and pERK1/2 were present only in the cytosol; and finally JNK was present in the cytosol only as p54, while its phosphorylated form was not observed either in cytosol or membrane.

Fig. 3. Western blot of cytosol and membrane fractions of human neutrophils treated with fMLP-OMe or with 0.1% DMSO (control) in calcium-free KRPG and labelled with: (A) anti PKC-a, h1, h2 and ~ and (B) with anti MAP kinase-p38, pp38, ERK1/2, pERK1/2, JNK and pJNK specific antibodies, at the indicated times. The results are representative of three separate experiments, each performed with cells from different donors.

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Fig. 4. Western blot of cytosol and membrane fractions of human neutrophils treated with [DzLeu2] or with 0.1% DMSO (control) in normal KRPG and labelled with: (A) anti PKC-a, h1, h2 and ~ and (B) with anti MAP kinase-p38, pp38, ERK1/2, pERK1/2, JNK and pJNK specific antibodies, at the indicated times. The results are representative of three separate experiments, each performed with cells from different donors.

3.2. Preincubation of neutrophils in Ca2+-free KRPG and stimulation with fMLP-OMe PKC isoform profile, shown in Fig. 3A, referred to neutrophils preincubated in Ca2 +-free medium and then triggered with fMLP-OMe. The cytosolic compartment

showed PKC a isoform only at 10 s and PKC h1 and h2 isoforms at all times studied. In the particulate compartment, only PKC h1 was barely detected from 10 s to 2 min and PKC ~ isoform was no longer observed. Cellular distribution of JNK, p38, ERK1/2 and the active forms were studied (Fig. 3B). In the cytosol, the rate of

Fig. 5. Western blot of cytosol and membrane fractions of human neutrophils treated with [DzLeu2] or with 0.1% DMSO (control) in calcium-free KRPG and labelled with: (A) anti PKC-a, h1, h2 and ~ and (B) with anti MAP kinase-p38, pp38, ERK1/2, pERK1/2, JNK and pJNK specific antibodies, at the indicated times. The results are representative of three separate experiments, each performed with cells from different donors.

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intensities of p38 and ERK1/2 were significantly decreased ( p < 0.05), as compared to the normal condition, and no signal was present in particulate compartment. JNK, as in Section 3.1, was present only as p54 and its level was not significantly modified by the absence of Ca2 +. It is to noteworthy that pp38 was the only MAPK active form observed in the cytosolic compartment, but its rate of intensity was significantly decreased ( p < 0.05), as compared to normal condition. 3.3. Preincubation of neutrophils in normal KRPG and stimulation with [DzLeu2] Fig. 4 shows the neutrophil signalling cascade triggered by [DzLeu2]. PKC a, h1 and h2, as well as ~ isoforms, were present both in the cytosol and the membrane (Fig. 4A). The rate of a and h2 isoforms translocation was much higher ( p < 0.05) than that of h1 and ~, and the effect was much greater ( p < 0.05) and earlier with respect to fMLP-OMe activation. The levels of p38 and pp38 once again were detected both in the cytosol and the membrane, while ERK1/2 and pERK1/2 appeared only in the cytosol. However, the intensities of the bands were decreased ( p < 0.001) when compared to fMLP-OMe stimulation. JNK or pJNK specific bands were not observed (Fig. 4B). 3.4. Preincubation of neutrophils in Ca2+-free KRPG and stimulation with [DzLeu2] As shown in Fig. 5A, the PKC a, h1, h2 and ~ isoforms were present in the cytosolic compartment, and a weak but specific translocation of PKC a and h2 isoforms were detectable in the particulate fraction when neutrophils were triggered with [DzLeu2]. The levels of MAPKs and MAPK phosphorylated forms were also assayed. As shown in Fig. 5B, p38 and ERK1/2 were evident in the cytosol and they appeared significantly decreased ( p < 0.05) when compared to those obtained with fMLP-OMe, whereas no signal by JNK was present; no MAPK phosphorylated forms were observed at any time.

4. Discussion The ability of the full agonist, fMLP-OMe, or the constrained analogue [DzLeu2] to distinguish the physiological responses renders them very useful tools for studies relating to specific neutrophil functions to specific second messengers pathways [12]. An increase in [Ca2 +]i has been postulated to be necessary for neutrophils to respond to fMLP, as it is one of the earliest detectable events induced by the exposure of the cells to the formylpeptide [19]. Following fMLP activation, PKC translocates to the particulate fraction only in the presence of Ca2 + [20]. In order to define the molecular pathways sensitive to the intracellular

increase of free Ca2 +, stimulation of neutrophils by fMLPOMe and [DzLeu2] were studied in normal conditions as compared to extracellular Ca 2 + -free conditions. The absence of extracellular Ca2 + decreases fMLP-induced neutrophil stimulation, but the cytosolic free Ca2 + levels appear to be sufficient for stimulus transduction [21]. In our experimental conditions, the presence of extracellular Ca2 +, both in fMLP-OMe or [Dz Leu2] stimulated neutrophils, contributes to the rise in [Ca2 +]i, leading to membrane-association of PKC a, h1, h2 and ~ isoforms and phosphorylation of p38 and ERK1/2 MAPKs. Unlike a number of previous studies, we have observed a small percentage of PKC ~ translocation [22,23] and the membrane localisation, besides the cytosol, of p38 MAPK. The PKC ~ discrepancy can be explained by the presence of CB during the experiments, that it is known to interfere with the association to the actin cytoskeleton regulating adhesion and cell motility and in the signalling cascade between fMLP receptor and NADPH oxidase activation [17]. The membrane localisation of p38 and its phosphorylated form could be referred to interactions with scaffold proteins (such as integrins) that take part in anchoring of signalling components at specialised subcellular sites, and relocalisation of active molecules during the signalling process [24]. Since activation of p38 MAPK has been reported to be indispensable for chemotaxis [25], the presence of the pp38 form by [DzLeu2] stimulation apparently contrasts with peptide inability to trigger chemotaxis. However, recent data reporting that p38 MAPK regulates exocytosis of intracellular granules [26] and phosphorylation of different NADPH oxidase components [22] or proteins inducing oxidase activation provides an explanation of our experimental data. In the absence of extracellular Ca2 +, the two formylpeptides assume different roles: fMLP-OMe stimulation selectively triggers the translocation of PKC h1 isoform and the phosphorylation of p38 MAPK, whereas [DzLeu2] induces the translocation of PKC a and h2 isoforms but failed to phosphorylate MAPKs. The rate of translocation in both the peptides was strongly reduced when compared with normal conditions. These latter findings suggest that the p38 activeform plays a central role in regulating neutrophil chemotaxis and also that PKC h1 translocation seems to contribute to the same physiological function. The selective translocation of different PKC isoforms by the two peptides can be explained by the presence in neutrophils of at least two fMLP-receptor isoforms [27] that may activate separate second messengers pathways, and hence specific biological responses. Studies with MAPK inhibitors [22] provided clear evidence that ERK1/2, but not p38 MAPK, was implicated in the phosphorylation cascade of p47phox in intact cells by the co-operation of PKC ~. Our data in Ca2 +-free conditions suggest that the decreased physiological function to produce O2 , by both peptides, could refer to the absence of ERK1/2 phosphorylation that is indeed consistent in normal conditions.

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In conclusion, our results obtained from this initial study reinforce the idea that selected PKC isoforms together with specific MAPK phosphorylations lead to transduction pathways in specific neutrophil functions. The knowledge gathered from this study, together with the use of new selective analogues, could facilitate future studies in this area.

Acknowledgements This work was supported by the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST). We are grateful to Banca del Sangue of Ferrara for providing fresh blood, and Dr. Selena Harrison, from King’s College London, for the English revision of the text.

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