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

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:
(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-

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-

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-

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

(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.

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

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+

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

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

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

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.
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