A ‘pure’ chemoattractant formylpeptide analogue triggers a specific signalling pathway in human neutrophil chemotaxis Susanna Spisani 1 , Sofia Falzarano 1 , Serena Traniello 1 , Marianna Nalli 2 and Rita Selvatici 3,4 1 Dipartimento di Biochimica e Biologia Molecolare, Universita ` degli Studi di Ferrara, Italy 2 Istituto di Chimica Biomolecolare CNR c ⁄ o Dipartimento di Studi Farmaceutici, Universita ` di Roma ‘La Sapienza’, Italy 3 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione Genetica Medica, Universita ` degli Studi di Ferrara, Italy 4 Centro di Neuroscienze, Universita ` di Ferrara, Italy for-Met-Leu-Phe (fMLP), an N-formylpeptide that represents a series of prototypic peptide chemoattract- ants, plays a key role in the defence against bacterial infections by binding with specific G-protein coupled receptors (FPR), expressed on neutrophils and mono- cytes [1–3]. Upon stimulation, neutrophils develop a polarized shape with front lamella and a contracted tail and start to migrate. It has been demonstrated [4,5] that this reaction is accompanied by a reorganiza- tion of actin filaments, but how these events are regu- lated is not fully understood. The interaction of fMLP with its receptor expressed on neutrophils, triggers multiple second messengers, through the activation of phospholipase (PL) C, PLD and PLA 2 , and rapidly stimulates phosphatidylinositol-3-kinase, as well as activating tyrosine phosphorylation. An increase in intracellular levels of cAMP [6,7] and the involvement of kinases, such as protein kinase C (PKC) and mito- gen activated protein kinases (MAPKs) [Jun N-ter- minal kinases (JNK), p38 and extracellular response kinase 1 and 2 (ERK1 ⁄ 2)], has also been demonstrated [8]. The activation of these transduction pathways is known to be responsible for different biochemical responses, which contribute to the physiological defence against bacterial infections and cell disruption [6], but it has not yet been demonstrated whether sig- nalling requirements are identical or specific for each physiological function. Keywords human neutrophils; formylpeptides; protein kinase C; MAP kinases; kinase inhibitors; chemotaxis Correspondence R. Selvatici, Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Universita ` degli Studi di Ferrara, Via Fossato di Mortara 74, 44100 Ferrara, Italy Fax: +39 0532 236157 Tel: +39 0532 424474 E-mail: svr@unife.it (Received 12 October 2004, accepted 22 November 2004) doi:10.1111/j.1742-4658.2004.04497.x As it has not yet been established whether the second messengers involved in the neutrophil response have identical or specific signalling requirements for each physiological function, protein kinase C (PKC) isoforms and mito- gen activated protein kinases (MAPKs) were studied in human chemotaxis triggered by the full agonist for-Met-Leu-Phe-OMe (fMLP-OMe) and the ‘pure’ chemoattractant for-Thp-Leu-Ain-OMe [Thp1,Ain3] analogue. Experiments were performed in the presence or absence of extracellular Ca 2+ , known to be an important modulator of second messengers. Our data demonstrate that specific PKC b 1 translocation and p38 MAPK phos- phorylation are strongly associated with the chemotactic response of the neutrophils triggered by both peptides, while Ca 2+ is not necessary for che- motaxis to occur. PKC and MAPK inhibitors were used in Western blot- ting assays and in cell locomotion experiments to investigate if the MAPK signalling pathway was controlled by PKC activation. The most important finding emerging from this study is that PKC and MAPK activate the chemotactic function of human neutrophils by two independent pathways. Abbreviations Ain, 2-aminoindane-2-carboxylic acid; ERK1 ⁄ 2, extracellular response kinase 1 and 2; fMLP-OMe, for-Met-Leu-Phe-OMe; JNK, Jun N-terminal kinases; KRPG, Krebs–Ringer phosphate containing 0.1% (w ⁄ v) glucose; LSP1, leukocyte-specific gene 1; MAPK, mitogen activated protein kinases; PKC, protein kinase C; Thp, 4-amino-tetrahydrothiopyran-4-carboxylic acid. FEBS Journal 272 (2005) 883–891 ª 2005 FEBS 883 PKC is a multigene family of enzymes comprising at least 11 isoforms. These isoforms are characterized by an NH 2 -terminal regulatory domain containing binding sites for Ca 2+ , phosphatidylserine and diacyl- glycerol, a small central hinge region and a COOH- terminal catalytic domain [9–11]. Upon activation, the kinase translocates from soluble to particulate compartments (plasma membrane, nucleus, cytoskele- ton) inducing a variegated pattern of regulatory func- tions. The cellular and intracellular distribution of PKC isoforms suggests isoform-related biological functions, but this specialization has only partly been explored. PKC is used by many receptor types to regulate the MAPK pathway, either alone or in con- junction with other mechanisms [12,13], and may act at several steps in the cascade. MAPK phosphoryla- tions have an impact on cascade processes in the cyto- plasm, the nucleus, the cytoskeleton and the cell membrane. We have described the structures of the formylpep- tide for-Met-Leu-Phe-OMe (fMLP-OMe) and the con- strained analogue for-Thp-Leu-Ain-OMe [Thp1,Ain3], depicted in Fig. 1, in previous studies. FMLP-OMe is characterized by a pronounced backbone conforma- tional flexibility, which seems to be an important fea- ture for establishing efficient interactions with FPR, and it is able to induce not only chemotaxis, but also adhesion, exocytosis and activation of NADPH oxidase in neutrophils. In the synthetic analogue [Thp1,Ain3], the native Met and Phe external residues have been replaced by 4-amino-tetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain), respectively [14], thus reducing the backbone flexibility of the peptide and inducing the adoption of a pre- ferred conformation. As this structure only allows [Thp1,Ain3] to elicit chemotaxis, it can therefore be considered a ‘pure’ chemoattractant [15,16]. The present study was designed to investigate the role of PKC isoforms (a, b1, b2, f) and MAPKs (p38, ERK1 ⁄ 2 and JNK) in the signal transduction pathway leading to chemotaxis triggered by the classi- cal peptide fMLP-OMe, and the ‘pure’ chemoattractant [Thp1,Ain3] using PKC and MAPK inhibitors. Results Western blotting of fMLP-OMe- or [Thp1,Ain3]- stimulated human neutrophils When human neutrophils are stimulated with formyl- peptides, they show MAPK activation and predomin- antly express the PKC isozymes a, b 1 , b 2 and f [19,20]. In order to clarify molecular mechanisms closely rela- ted to the chemotactic function, we examined by West- ern blotting: (a) the rate of translocation of PKC a, b 1 , b 2 and f isoforms; (b) the levels of MAPKs p38, ERK1 ⁄ 2 (p44 ⁄ 42) and JNK; and (c) the active forms pp38, pERK1 ⁄ 2 and pJNK. To this end, neutrophils were stimulated with 10 )9 m fMLP-OMe or 10 )9 m [Thp1,Ain3] at 10¢¢,30¢¢,1¢,2¢ and 5¢, both in normal KRPG and in Ca 2+ -free KRPG, as Ca 2+ is known to regulate various transductional effectors. The cellular distribution (cytosolic and membrane compartments) of PKC isoforms is shown in Fig. 2. When neutrophils were triggered by fMLP-OMe (Fig. 2A) or [Thp1,Ain3] (Fig. 2B) in KRPG supplemented with Ca 2+ , the PKC a, b 1 , b 2 and f isoforms were all detected in the cyto- solic compartment and only PKC b 1 translocated to the membrane fraction. Total lysates, obtained from neutrophils stimulated with fMLP-OMe (Fig. 3A) or [Thp1,Ain3] (Fig. 3B), were analysed by Western blotting in order to investi- gate MAPK activation. Both formylpeptides showed p38 and pp38 MAPK at all times and ERK 1 ⁄ 2, but not pERK1 ⁄ 2, while JNK and pJNK were not detected. The same experiments were carried out in the absence of extracellular Ca 2+ . Once again, PKC b 1 translocation (Fig. 4) and p38 MAPK phosphorylation Fig. 1. Peptide structures of fMLP-OMe and [Thp1,Ain3]. Signal transduction pathway leading to chemotaxis S. Spisani et al. 884 FEBS Journal 272 (2005) 883–891 ª 2005 FEBS (Fig. 5) were the only processes activated by fMLP- OMe or [Thp1,Ain3]. Chemotactic assays with PKC and MAPK inhibitors in fMLP-OMe- or [Thp1,Ain3]- stimulated human neutrophils In order to investigate the role of various intracellular signalling pathways on the neutrophil chemotactic response, we evaluated the effect of different pharma- cologic agents on fMLP-OMe- or [Thp1,Ain3]-medi- ated neutrophil chemotaxis (Fig. 6). To distinguish the effects of each inhibitor we used the IC 50 , the concentration required to reduce to 50% the maximum effect; the concentration–effect curve was performed for each inhibitor assaying the chemo- tactic activity (data not shown). As indicated in Experimental procedures, neutrophils were pretreated with the agents at the indicated concentrations for 40 min prior to the initiation of chemotaxis. Pre-treat- ment with GF109203X (0.8 lm), a PKC inhibitor, reduced fMLP-OMe- or [Thp1,Ain3]-induced chemo- taxis by 67% and 52%, respectively (P<0.01). Simi- larly, pretreatment with SB203580 (3 lm), a p38 A B Fig. 2. PKC distribution in human neutroph- ils stimulated with formylpeptides under normal conditions. Neutrophils were stimula- ted with 10 )9 M fMLP-OMe (A) and 10 )9 M [Thp1,Ain3] (B) in the presence of 1 mM Ca 2+ for the indicated times, or treated with 0.1% (v ⁄ v) dimethylsulfoxide as control (c). Cytosolic and membrane fractions were pre- pared, subjected to SDS ⁄ PAGE and electro- blotted as described in Experimental procedures. Blots were probed with anti- PKC a, b 1 , b 2 and f. The results are repre- sentative of four separate experiments, each performed with cells from different donors. AB Fig. 3. MAPK activation in human neutrophils stimulated with formylpeptides in normal conditions. Western blots of p38, ERK1 ⁄ 2 and JNK MAPKs and their phosphorylated forms pp38, pERK1 ⁄ 2 and pJNK in neutrophils stimulated with 10 )9 M fMLP-OMe (A) and 10 )9 M [Thp1,Ain3] (B) in the presence of 1 mM Ca 2+ for the indicated times, or treated with 0.1% (v ⁄ v) dimethylsulfoxide as control (c). Lysates prepared from freshly purified neutrophils were subjected to SDS ⁄ PAGE and Western blotting as described in Experimental procedures. Results are representative of four independent experiments, each performed with cells from different donors. S. Spisani et al. Signal transduction pathway leading to chemotaxis FEBS Journal 272 (2005) 883–891 ª 2005 FEBS 885 MAPK inhibitor, attenuated chemotaxis by 38% (P<0.01) in fMLP-OMe-stimulated neutrophils and by 54% (P<0.05) in [Thp1,Ain3]-stimulated neu- trophils. This appears to be a specific effect of the p38 kinase inhibition as the inactive analogue, SB202474 [21], had no effect on chemotaxis. When the neutro- phils were pretreated with SB20358 + GF109203X and then stimulated with formylpeptides, the chemo- taxis was further reduced: by 84% for fMLP-OMe (P<0.01) and by 85% for [Thp1,Ain3] (P<0.01). In contrast, PD98059 (25 lm), an ERK1 ⁄ 2 MAPK inhibitor, had no effect on the chemotaxis induced by either peptide. Western blotting analysis with PKC and MAPK inhibitors in fMLP-OMe- or [Thp1,Ain3]- stimulated human neutrophils Human neutrophils preincubated with or without GF109203X or SB203580, as described in Experimen- tal procedures, and then exposed to fMLP-OMe or [Thp1,Ain3], were also evaluated by Western blotting experiments. PKC b 1 membrane translocation (Fig. 7) was significantly decreased by GF109203X (lane 2); it was not modified by SB203580 (lane 3), as compared with neutrophils preincubated without inhibitors (lane 1). A B Fig. 4. PKC distribution in human neutro- phils stimulated with formylpeptides in Ca 2+ -free medium. Neutrophils were stimu- lated with 10 )9 M fMLP-OMe (A) and 10 )9 M [Thp1,Ain3] (B) in Ca 2+ -free KRPG supplemented with 1 l M EGTA for the indicated times or treated with 0.1% (v ⁄ v) dimethylsulfoxide as control (c). Cytosolic and membrane fractions were prepared, subjected to SDS ⁄ PAGE and electroblotted as described in Experimental procedures. Blots were probed with anti-PKC a, b 1 , b 2 and f. The results are representative of four separate experiments, each performed with cells from different donors. AB Fig. 5. MAPK activation in neutrophils stimulated with formylpeptides in Ca 2+ -free medium. Western blots of p38, ERK1 ⁄ 2 and JNK MAPKs and their phosphorylated forms, pp38, pERK1 ⁄ 2 and pJNK, in neutrophils stimulated with 10 )9 M fMLP-OMe (A) and 10 )9 M [Thp1,Ain3] (B) in Ca 2+ -free KRPG with 1 lM EGTA for the indicated times, or treated with 0.1% (v ⁄ v) dimethylsulfoxide as control (c). Lysates prepared from freshly purified neutrophils were subjected to SDS ⁄ PAGE and Western blotting as described in Experimental procedures. Results are representative of four independent experiments, each performed with cells from different donors. Signal transduction pathway leading to chemotaxis S. Spisani et al. 886 FEBS Journal 272 (2005) 883–891 ª 2005 FEBS P38 MAPK phosphorylation (Fig. 8) was not modi- fied by GF109203X (lane 2), while pretreatment of neutrophils with SB203580, down-regulated the kinase (lane 3), as compared with neutrophils preincubated without inhibitors (lane 1). Discussion It has long been known that the transduction pathway underlying the chemotactic response is different from those responsible for O 2 – production or lysozyme release [16,22,23] and several previous experiments, car- ried out utilizing pharmacological manipulation of the signal transduction pathway, have highlighted the fact that distinct mechanisms are involved in each of these neutrophil responses. Neutrophil motility is a complex process which requires integrated pathways including actin polymerization, cytoskeletal reorganization, morphological polarization, specific adhesiveness and cell-substratum detachment [24–26]. This report demonstrates that the formylpeptide fMLP-OMe, at a concentration of 10 )9 m (optimal concentration to induce chemotaxis) and the ‘pure’ analogue [Thp1,Ain3], selectively trigger the translocation of PKC b 1 isoform. Cellular functional assays using the specific PKC inhibitor, GF109203X indicated that the activation of PKC was indispensable for both fMLP- OMe- and [Thp1,Ain3]-induced chemotaxis of human neutrophils. PKC is considered an important regulator of cytoskeletal functions, and it has previously been associated with intermediate filament proteins, mem- brane-cytoskeletal cross-linking proteins, components of the actin filaments and microtubules, as well as with b-integrin vesicle trafficking [27] and therefore the link between PKC b and b 2 integrins may not be coinciden- tal. The genes encoding PKC b 1 isoform, leukocyte adhesion receptor (CD43) and CD11a, CD11b and CD11c, occur in a cluster on human chromosome 16, suggesting that they could be functionally linked [28]. PKC has also been shown to phosphorylate proteins localized in specialized regions including talin, vinculin and integrins (focal adhesions). Within these regions, several components of the cytoskeleton are concentra- ted, together with a number of signalling proteins. As previously demonstrated [29], neutrophil chemotaxis is almost insensitive to any variation of Ca 2+ concentra- tion. This was confirmed by our experiments, which showed PKC b 1 translocation and p38 MAPK phos- phorylation in neutrophils triggered by fMLP-OMe and [Thp1,Ain3] in either the presence or absence of extracellular Ca 2+ . As the increase in intracellular Ca 2+ is not important for chemotaxis, but is necessary for the activation of conventional PKC (a, b 1 , b 2 , c), Fig. 6. Chemotactic assays with PKC and MAPK inhibitors in fMLP- OMe- or [Thp1,Ain3]-stimulated human neutrophils. Effect of phar- macologic inhibitors on chemotaxis induced by fMLP-OMe (A) or [Thp1,Ain3] (B). Neutrophils were pretreated with the inhibitors for 40 min, stimulated with peptides and then the chemotactic response was evaluated. FMLP-OMe or [Thp1,Ain3] indicate the chemotactic index without the inhibitors. *P<0.05, **P < 0.01 compared to fMLP or [Thp1,Ain3] alone. Fig. 7. Western blotting analysis with a PKC inhibitor in neutrophils stimulated with formylpeptides. Western blotting of cytosolic and membrane PKC b 1 distribution in neutrophils stimulated with fMLP- OMe or [Thp1,Ain3] for 5 min as control (lane 1), or preincubated with PKC inhibitor GF109203X (lane 2) or p38 MAPK inhibitor, SB203580 (lane 3) for 40 min before stimulation. Fig. 8. Western blotting analysis with p38 MAPK inhibitor in neu- trophils stimulated with formylpeptides, Western blotting of p38 MAPK phosphorylation in neutrophils stimulated with fMLP-OMe or [Thp1,Ain3] for 5 min as control (lane 1) or preincubated with PKC inhibitor GF109203X (lane 2) or p38 MAPK inhibitor SB203580 (lane 3) for 40 min before stimulation. S. Spisani et al. Signal transduction pathway leading to chemotaxis FEBS Journal 272 (2005) 883–891 ª 2005 FEBS 887 the PKC b 1 translocation showed by both peptides could be misleading. However, it has been observed that localized Ca 2+ signalling spikes are present both in the absence of extracellular Ca 2+ and in the presence of transmembrane blocking Ni 2+ , thereby demonstra- ting that the presence of localized Ca 2+ signalling was due to release from Ca 2+ stores, and that there is no requirement for transmembrane influx of extracellular Ca 2+ [30]. Fluctuations of localized intracellular Ca 2+ could explain the translocation of PKC b 1 by both formyl peptides observed in our study. Functional chemotactic experiments using MAPK inhibitors revealed that fMLP-OMe- and [Thp1,Ain3]- induced chemotaxis of human neutrophils was reduced by the p38 MAPK inhibitor SB20358, but not by the p44 ⁄ 42 MAPK inhibitor PD98059. In addition, West- ern blotting analysis confirmed that exposure of neu- trophils to formylpeptides induced phosphorylation and activation of p38 MAPK, but not of p44 ⁄ 42 MAPK. These observations strongly suggest that the p38 MAPK-mediated signalling pathway plays a cen- tral role in regulating neutrophil chemotaxis. Sche- matic signalling pathways of chemotaxis are proposed in Fig. 9, in response to stimulation of human neutro- phil with formyl-peptides. Although we have not yet studied downstream pro- teins, as they are potential candidates for phosphoryla- tion by p38 MAPK and may be involved in cytoskeletal rearrangement of neutrophils, a number of these molecules should be considered. Molecules asso- ciated with neutrophil motility, downstream from p38 MAPK include leukocyte-specific gene 1 (LSP1) protein, which is an F-actin binding protein [31] and a major substrate of MAPK-activated protein kinase 2 [32]. LSP1 negatively regulates fMLP-induced polariza- tion and chemotaxis of neutrophils through its func- tion on adhesion via specific integrins, such as CD11b ⁄ CD18 [33] and may be phosphorylated by MAPK-activated protein kinase 2 of pathway p38 and so dissociate from F-actin to allow cytoskeletal rear- rangement [34]. Therefore, elucidation of the mechanism of inhibi- tion of neutrophil movement is of great importance in models of inflammation. The data here presented com- pared with the results obtained by [D z Leu 2 ], a peptide capable of eliciting superoxide anion (O 2 – ) production alone [35], support the idea that fine tuning of neutro- phil activation occurs through differences in activation of a spectrum of signalling pathways. Moreover our data not yet published, utilizing PKC and MAPK inhibitors, demonstrate that PKC, p38 and ERK1 ⁄ 2 are associated with superoxide generation and are acti- vated independently from each other, but converge in regulation of this function. For each stimulus capable of a unique set of cellular responses, a distinctive imprint of signal protein activation may exist. Through more complete understanding of intracellular signal- ling, new drugs could be developed for the selective inflammatory blockade. Experimental procedures Materials Dextran, Ficoll-Paque and enhanced chemiluminescence Western blotting detection reagents were from Amersham- Pharmacia Biotech (Milan, Italy) FMLP-OMe and dimeth- ylsulfoxide were from Sigma; SB203580 and inactive analogue SB202474, PD98059 and GF109203X were from Calbiochem (Milan, Italy). Poly(vinylidene difluoride) mem- branes were from Bio-Rad Laboratories (Milan, Italy) and anti-PKC a, anti-PKC b 1 , anti-PKC b 2 and anti-PKC f were from Santa Cruz Biotechnology (Milan, Italy). Poly- clonal antibodies against p54 ⁄ 46 SAPK ⁄ c-JNK N-terminal kinase (JNK), p38 MAPK, p44 ⁄ 42 MAPK (ERK1 ⁄ 2) and the phospho-SAPK ⁄ JNK (pJNK), phospho-p38 MAPK (pp38) and phospho-p44 ⁄ 42 MAPK (pERK1 ⁄ 2) were from Cell Signalling Technology, Inc. (Celbio, Milan, Italy) and all other reagents used were of the highest grade commer- cially available. Preparation of peptides For-Met-Leu-Phe-OMe and for-Thp-Leu-Ain-OMe were prepared at 10 )2 m in dimethyl sulfoxide and diluted in Fig. 9. Schematic signalling pathways of chemotaxis. Upon formyl- peptide binding, trimeric G-proteins are uncoupled from FPR and a series of signal transduction events ensue that results in chemotac- tic activation. Signal transduction pathway leading to chemotaxis S. Spisani et al. 888 FEBS Journal 272 (2005) 883–891 ª 2005 FEBS buffer before use. At the concentrations used, dimethyl sulfoxide did not interfere with any of the biological assays performed. Cell preparation Neutrophils were isolated from the peripheral blood of healthy human volunteers and purified using standard tech- niques [14]. Cells, 98–100% pure and ¼ 99% viable, were resuspended in Krebs–Ringer phosphate pH 7.4, containing 0.1% (w ⁄ v) glucose (KRPG), and supplemented with 1 mm CaCl 2 (normal KRPG) or Ca 2+ -free KRPG supplemented with 1 lm EGTA. All experiments were carried out accord- ing to the guidelines of local and regional ethics committees. Neutrophil stimulation Suspensions of 1 · 10 7 neutrophilsÆmL )1 were stimulated with fMLP-OMe or [Thp1,Ain3] 10 )9 m, the optimal dose for chemotactic activity, lysed using ice-cold lysis buffer containing: 20 mm Tris pH 7.5, 0.25 m saccharose, 2 mm EDTA, 10 mm EGTA, 2 mm phenylmethylsulfonyl fluor- ide, 1% (w ⁄ v) NP-40, 0.25% (w ⁄ v) sodium deoxycholate, an antiprotease mixture consisting of 0.1% (w ⁄ v) leupeptin, 10 lgÆmL )1 aprotinin, 0.35 mm antipain, 0.35 mm pepsta- tin, 0.24 mgÆmL )1 chymostatin and then centrifuged at 17 500 g for 5 min to pellet nuclei and unbroken cells. Con- trol samples were resuspended with 0.1% (v ⁄ v) dimethyl- sulfoxide (vehicle). The supernatant, corresponding to the total lysate, was recovered in a separate tube, sonicated six times with 10-s bursts and then used to analyse the levels and the rate of phosphorylation of MAPKs by Western blotting. In order to study the PKC activation, the total lysate was ultracentrifuged at 150 000 g for 1 h at 4 °C: the super- natant, corresponding to the cytosolic fraction and the pel- let, resuspended in the same buffer supplemented by 0.2% (v ⁄ v) Triton X-100, corresponding to the membrane frac- tion, were analysed by Western blotting. Protein content was determined by the bicinchoninic acid (BCA) method [17]. Pre-treatment of neutrophils with inhibitors Suspensions of 1 · 10 7 neutrophilsÆmL )1 were preincubated at 4 °C for 40 min with SB203580 (3 lm) to modify cellular p38 MAPK activity or inactive analogue SB202474 (3 lm) and with PD98059 (25 lm) to block the activation of p42 ⁄ 44 MAPK indirectly. In addition, GF109203X (0.8 lm) was used as a PKC inhibitor. Cells were then sti- mulated with fMLP-OMe or [Thp1,Ain3] 10 )9 m and used for chemotaxis experiments or Western blotting assays. Control samples were re-suspended with 0.1% (v ⁄ v) dimethylsulfoxide (vehicle) without peptides. Western blot analysis Equal amounts of proteins (50 lg) were separated by SDS ⁄ PAGE on 10% gels and then electrophoretically transferred to poly(vinilydene difluoride) membrane at 100 V for 1 h. Blots were incubated in Tris-buffered saline pH 7.6 containing 5% dry nonfat milk and 0.1% (v ⁄ v) Tween 20. Western blots were performed using polyclonal antibodies a, b 1 , b 2 and f (0.3 lgÆmL )1 ) against PKC and 1 : 1000 dilutions of p38, pp38, ERK1 ⁄ 2, pERK1 ⁄ 2, JNK and pJNK against MAPK. Signals were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The molecular weight was calculated with prestained SDS ⁄ PAGE standards (New England Bio- Labs, Inc.) Densitometric analysis of specific autoradio- graphic bands was used for the statistical analysis. The densities were measured by the Bio-Rad densitometer GS700 and expressed as absorbance units per mm 2 . Chemotaxis Random locomotion and chemotaxis were evaluated using a 48-well micro chemotaxis chamber (BioProbe, Milan, Italy). Cell migration in the presence or absence of the chemotactic factor was evaluated by estimating the distance (in lm) migrated by the leading-front of the cell, after the method of Zigmond and Hirsch [18]. All data are expressed as the mean ± SEM of six separate experiments performed in duplicate. Data are expressed in terms of chemotactic index using the following ratio: migration towards test attractant minus migration towards the buffer ⁄ migration towards the buffer. Statistics Statistical analyses were performed by Student’s t-test for unpaired data. Differences between treatment groups were judged statistically significant at P £ 0.05. Acknowledgements This work was supported by the Ministero dell’Univer- sita ` e della Ricerca Scientifica e Tecnologica (ex 40%, 60%) and Associazione E and E. Rulfo of Medical Genetics, Parma, Italy. 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