Keywords human neutrophils; formylpeptides; protein kinase C; MAP kinases; kinase inhibitors; chemotaxis Correspondence R.. Selvatici, Dipartimento di Medicina Sperimentale e Diagnostica
Trang 1a specific signalling pathway in human neutrophil
chemotaxis
Susanna Spisani1, Sofia Falzarano1, Serena Traniello1, Marianna Nalli2and Rita Selvatici3,4
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 PLA2, 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
Ca2+, known to be an important modulator of second messengers Our data demonstrate that specific PKC b1translocation and p38 MAPK phos-phorylation are strongly associated with the chemotactic response of the neutrophils triggered by both peptides, while Ca2+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.
Trang 2PKC is a multigene family of enzymes comprising
at least 11 isoforms These isoforms are characterized
by an NH2-terminal regulatory domain containing
binding sites for Ca2+, 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, b1, b2and 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,
b1, b2 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)9m fMLP-OMe or 10)9m [Thp1,Ain3] at 10¢¢, 30¢¢, 1¢, 2¢ and 5¢, both in normal KRPG and in Ca2+-free KRPG, as Ca2+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 Ca2+, the PKC
a, b1, b2 and f isoforms were all detected in the cyto-solic compartment and only PKC b1 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 Ca2+ Once again, PKC b1 translocation (Fig 4) and p38 MAPK phosphorylation
Fig 1 Peptide structures of fMLP-OMe and [Thp1,Ain3].
Trang 3(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 IC50, 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)9M fMLP-OMe (A) and 10)9M
[Thp1,Ain3] (B) in the presence of 1 m M
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, b1, b2and f The results are
repre-sentative of four separate experiments,
each performed with cells from different
donors.
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)9M fMLP-OMe (A) and 10)9M [Thp1,Ain3] (B) in the presence of 1 m M Ca2+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.
Trang 4MAPK 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 b1 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
Ca2+-free medium Neutrophils were stimu-lated with 10)9M fMLP-OMe (A) and
10)9M [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.
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)9M fMLP-OMe (A) and 10)9M [Thp1,Ain3] (B)
in Ca 2+ -free KRPG with 1 l M 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.
Trang 5P38 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 O2 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)9m (optimal concentration
to induce chemotaxis) and the ‘pure’ analogue [Thp1,Ain3], selectively trigger the translocation of PKC b1 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 b2integrins may not be coinciden-tal The genes encoding PKC b1 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 Ca2+ concentra-tion This was confirmed by our experiments, which showed PKC b1 translocation and p38 MAPK phos-phorylation in neutrophils triggered by fMLP-OMe and [Thp1,Ain3] in either the presence or absence of extracellular Ca2+ As the increase in intracellular
Ca2+is not important for chemotaxis, but is necessary for the activation of conventional PKC (a, b1, b2, 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.
Trang 6the PKC b1 translocation showed by both peptides
could be misleading However, it has been observed
that localized Ca2+ signalling spikes are present both
in the absence of extracellular Ca2+and in the presence
of transmembrane blocking Ni2+, thereby
demonstra-ting that the presence of localized Ca2+ signalling was
due to release from Ca2+ stores, and that there is no
requirement for transmembrane influx of extracellular
Ca2+ [30] Fluctuations of localized intracellular Ca2+
could explain the translocation of PKC b1 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 [DzLeu2], a peptide capable of eliciting superoxide anion (O2) 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 b1, anti-PKC b2 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)2m 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.
Trang 7buffer 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
CaCl2(normal KRPG) or Ca2+-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· 107
neutrophilsÆmL)1 were stimulated with fMLP-OMe or [Thp1,Ain3] 10)9m, 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 4C: 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· 107neutrophilsÆmL)1 were preincubated
at 4C 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)9m 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, b1, b2 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 mm2
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
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