A‘pure’chemoattractantformylpeptideanalogue triggers
a specificsignallingpathwayinhuman 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 specificsignalling requirements
for each physiological function, protein kinase C (PKC) isoforms and mito-
gen activated protein kinases (MAPKs) were studied inhuman 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 neutrophilchemotaxis (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 inhuman 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 inhuman 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 aspecific 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 inhuman 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 ina 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], neutrophilchemotaxis 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 signallingpathway 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 ina 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. We are grateful to Banca del
Sangue of Ferrara for providing fresh blood and
Dr Selena Harrison, from King’s College London, and
Anna Forster for the English revision of the text.
References
1 Wenzel-Seifert K & Seifert R (2001) Chemoattractant
receptor-G-protein coupling. In Physiology of
S. Spisani et al. Signal transduction pathway leading to chemotaxis
FEBS Journal 272 (2005) 883–891 ª 2005 FEBS 889
Inflammation. (Ley K, ed.), pp. 146–188. University
Press, Oxford.
2 Boulay F, Tardif M, Brouchon L & Vignais P (1990)
The human N-phormylpeptide receptor characterisation
of two cDNA isolated, and evidence for a new subfam-
ily of G-protein-coupled receptors. Biochemistry 29,
11123–11133.
3 Ye, RD & Boulay F (1997) Structure and function of
leukocyte chemoattractant receptors. Adv Pharmacol 39,
221–289.
4 Hallet MB (1997) Controlling the molecular motor of
neutrophils chemotaxis. Bioassay 19, 615–621.
5 Baggiolini M (1998) Chemokines and leukocyte traffic.
Nature 392, 565–568.
6 Prossnitz ER (1997) The N-formyl peptide receptor: a
model for the study of chemoattractant receptor struc-
ture and function. Pharmacol Ther 74, 3–102.
7 Hallet MB & Lloyds D (1997) Phospholipid signalling
of Ca
2+
in neutrophils. In The Molecular and Ionic Sig-
nalling of Neutrophils. (Hallet MB & Lloyds D, eds),
pp. 105–118. Landes Bioscience, Austin, TX.
8 Cockcroft S (1992) G-protein-regulated phospholipases
C, D and A2-mediated signallingin neutrophils. Bio-
chim Biophys Acta 1113, 135–160.
9 Cui DY, Inanami O, Yamamori T, Niwa K, Nagahata
H & Kuwabara M (2000) FMLP-induced formation of
F-actin in HL60 cells is dependent on PI3-K but not on
intracellular Ca2+, PKC, ERK or p38 MAPK. Inflamm
Res 49, 684–691.
10 Hofmann J (1997) The potential for isoenzyme-selective
modulation of protein kinase C. FASEB J 11, 649–669.
11 Faux MC & Scott JD (1996) Molecular glue: kinase
anchoring and scaffold proteins. Molecular glue: kinase
anchoring and scaffold proteins. Cell 85, 9–12.
12 L’Allemain G, Pouyssegur J & Webber MD (1991)
p42 ⁄ mitogen-activated protein kinase as a converging
target for different growth factor signalling pathways:
use of pertussis toxin as a discrimination factor. Cell
Regul 26, 75–84.
13 Kazlauskas A & Cooper JA (1988) Protein kinase C
mediates platelet-derived growth factor-induced tyrosine
phosphorylation of p42. J Cell Biol 106, 1395–1402.
14 Torrini I, Pagani Zecchini G, Paglialunga Paradisi M,
Lucente G, Gavuzzo E, Mazza F, Pochetti G, Spisani S
& Giuliani AL (1991) Synthesis and properties of che-
motactic peptide analogs. Int J Peptide Prot Res 38,
495–504.
15 Haines KA, Kolasinski SL, Cronstein BN, Reibman J,
Gold LI & Weissmann G (1993) Chemoattraction of
neutrophils by substance P and transforming growth
factor-beta 1 is inadequately explained by current mod-
els of lipid remodelling. J Immunol 151 , 1491–1499.
16 Fabbri E, Spisani S, Barbin L, Biondi C, Buzzi M,
Traniello S, Pagani Zecchini G & Ferretti ME (2000)
Studies on fMLP–receptor interaction and signal
transduction pathway by means of fMLP-OMe selective
analogues. Cell Signal 12, 391–398.
17 Brown R, Jarvis K & Hyland K (1989) Protein
measurement using bicinchoninic acid: elimination of
interfering substances. Anal Biochem 180, 136–139.
18 Zigmond SH & Hirsch JG (1973) Leukocyte locomotion
and chemotaxis. New methods for evaluation and
demonstration of a cell-derived chemotactic factor.
J Exp Med 137, 387–410.
19 Dang PM, Rais S, Hakim J & Perianin A (1995) Redis-
tribution of protein kinase C isoforms inhuman neutro-
phils stimulated by formyl peptides and phorbol
myristate acetate. Biochem Biophys Res Commun 212,
664–672.
20 Rane MJ, Carrithers SL, Arthur JM, Klein JB & McLe-
ish KR (1997) Formyl peptide receptors are coupled to
multiple mitogen-activated protein kinase cascades by
distinct signal transduction pathways: role in activation
of reduced nicotinamide adenine dinucleotide oxidase.
J Immunol 159, 5070–5078.
21 Rong Yu, Sandhya Mandlekar, WeiLei William E,
Fahl Tse-Hua Tan & A-N.Tony Kong (2000) p38
Mitogen-activated protein kinase negatively regulates
the induction of phase II drug-metabolizing enzymes
that detoxify carcinogens. J Biol Chem 275, 2322–2327.
22 Ferretti ME, Nalli M, Biondi C, Colamussi ML, Pavan
B, Traniello S & Spisani S (2001) Modulation of neutro-
phil phospholipase C activity and cyclic AMP-levels by
fMLP-OMe analogues. Cell Signal 13, 233–240.
23 Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV & Wu D
(2000) Roles of PLC-beta2 and -beta3 and PI3Kgamma
in chemoattractant-mediated signal transduction.
Science 287, 1046–1049.
24 Katanev VL (2001) Signal transduction in neutrophil
chemotaxis. Biochemistry (Moscow) 66, 351–368.
25 Marks PV & Maxfield FR (1990) Transient increases in
cytosolic free calcium appear to be required for the
migration of adherent human neutrophils. J Cell Biol
10, 43–52.
26 Juliano RL (2002) Signal transduction by cell adhesion
receptors and the cytoskeleton: functions of integrins,
cadherins, selectins, and immunoglobulin-superfamily
members. Annu Rev Pharmacol Toxicol 42, 283–323.
27 Keenan C & Kelleher D (1998) Protein kinase C and
the cytoskeleton. Cell Signal 10, 225–232.
28 Dekker LV & Parker PJ (1997) PKC isozymes and mye-
loid cell differentiation. In Protein Kinase C (Parker PJ,
Dekker LV, eds), pp. 121–129. Landes Bioscience,
Austin, TX.
29 Fabbri E, Spisani S, Biondi C, Barbin L, Colamussi
ML, Cariani A, Traniello S, Torrini I & Ferretti ME
(1997) Two for-Met-Leu-Phe-OMe analogues trigger
selective neutrophil responses: a differential effect on
cytosolic free Ca
2+
. Biochim Biophys Acta 1359, 233–
240.
Signal transduction pathway leading to chemotaxis S. Spisani et al.
890 FEBS Journal 272 (2005) 883–891 ª 2005 FEBS
30 Pettit EJ & Hallett MB (1996) Localised and global
cytosolic Ca
2+
changes in neutrophils during engage-
ment of Cd11b ⁄ CD18 integrin visualised using
confocal laser scanning reconstruction. J Cell Sci 109,
1689–1694.
31 Jongstra-Bilen J, Misener VL, Wang C, Ginzberg H,
Auerbach A, Joyner AL, Downey GP & Longstra J
(2000) LSP1 modulates leukocyte populations in resting
and inflamed peritoneum. Blood 96, 1827–1835.
32 Meng W, Swenson LL, Fitzgibbon MJ, Hayakawa K,
Ter Haar E, Behrens AE, Fulghum JR & Lippke JA
(2002) Structure of mitogen-activated protein kinase-
activated protein (MAPKAP) kinase 2 suggests a
bi-functional switch that couples kinase activation with
nuclear export. J Biol Chem 277, 37401–37405.
33 Huang CK, Zhan L, Ai Y & Jongstra J (1997) LSP1 is
the major substrate for mitogen activated protein
kinase-activated protein kinase 2 inhuman neutrophils.
J Biol Chem 272, 17–19.
34 Wang C, Hayashi H, Harrison R, Chiu B, Chan JR,
Ostergaard HL, Inman RD, Jongstra J, Cybulsky ML
& Jongstra-Bilen J (2002) Modulation of Mac-1
(CD11b ⁄ CD18)-mediated adhesion by the leukocyte-
specific protein 1 is key to its role inneutrophil polar-
ization and chemotaxis. J Immunol 169, 415–423.
35 Selvatici R, Falzarano S, Traniello S, Pagani Zecchini
G & Spisani S (2003) Formylpeptide trigger selective
molecular pathways that are required in the physiologi-
cal functions of human neutrophils. Cell Signal 15,
377–383.
S. Spisani et al. Signal transduction pathway leading to chemotaxis
FEBS Journal 272 (2005) 883–891 ª 2005 FEBS 891
. A ‘pure’ chemoattractant formylpeptide analogue triggers
a specific signalling pathway in human neutrophil
chemotaxis
Susanna Spisani
1
, So a Falzarano
1
,. 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