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* Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Turin, Italy, and
Department of Alimentary, Chemical, Pharmaceutical and Pharmacological Sciences, University of Piemonte Orientale, Novara, Italy
Correspondence: Roberto Fantozzi, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Università di Torino, Via Pietro Giuria 9, 10125 Torino, Italy. E-mail: roberto.fantozzi{at}unito.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cytosolic free-calcium concentration • neuropeptide • NK-1 receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments performed in vitro with human polymorphonuclear neutrophils (PMNs) have demonstrated that SP can transfer these cells from a resting to a primed state, thus allowing for an enhanced response to a given stimulus. SP works as a priming agent for PMNs triggered by various stimuli that are able to evoke different responses, such as the production of reactive oxygen species, the generation of nitric oxide (NO), and the formation of leukotriene B4 and 5-hydroxyeicosatetranoic acid [4 5 6 7 ]. Pretreatment of PMNs with SP has been shown to increase, in a dose- and time-dependent way, superoxide anion (O2-) production evoked by N-formyl-methionyl-leucyl-phenylalanine (fMLP) [8 ]. The same authors have reported that a single concentration of SP (3x10-5 M) stimulated a significant release of interleukin-8 (IL-8) fromPMNs [8 ].
IL-8 belongs to the subfamily of CXC chemokines, and it is involved in a wide variety of acute and chronic inflammatory diseases [9 ]. IL-8 induces a large array of PMN responses by interacting with specific cell surface receptors [9 ]. The activation of IL-8 receptors is followed by a rise in cytosolic free-calcium concentrations ([Ca2+]is) [10 ]. Two subtypes of IL-8 receptors, CXCR1 and CXCR2, have been described in PMNs. They appear to regulate different PMN responses, but they both stimulate Ca2+ movements [11 ]. The rise in [Ca2+]i is regarded as a key signaling event for PMN activation [12 ]. However, priming has been reported to have no effect on calcium signals triggered by subsequent stimulation [12 ].
SP and the other NKs, i.e., NKA and NKB, interact with three receptor types that recognize the common C-terminal sequence of the peptides [13 , 14 ]. The original criterion to distinguish the three receptor types (NK-1, NK-2, and NK-3) has been the rank order of potency of the NKs, SP being the preferred agonist for the NK-1 receptor, NKA being the preferred agonist for the NK-2 receptor, and NKB being the preferred agonist for the NK-3 receptor. Selective receptor agonists and antagonists have been successfully developed and used as suitable tools for receptor characterization of SP-evoked responses in different cells and tissues [13 ].
The SP receptor belongs to the family of guanosine triphosphate-binding protein-coupled receptors. However, a cationic amphiphilic peptide such as SP can insert itself into the cell membrane and interact with proteins on the inner side of the membrane [15 ]. This action may allow the positively charged domain of the molecule to interact with the carboxy terminus of G protein. Thus, SP might activate G protein directly, bypassing receptor structures. Such a mechanism has been proposed to explain the histamine-releasing effects of SP on rat peritoneal mast cells [16 ]. Conversely, evidence has been provided that rat basophilic leukemic cells can be activated by SP through NK-1 receptors [17 ]. Both receptor-dependent and -independent mechanisms might underlie the activating effects of SP on PMNs [3 ]. The stimulating effects of SP on PMN locomotion might depend on the N-terminal sequence and have been regarded as nonreceptorial effects [18 ]. SP-induced PMN accumulation in the murine air pouch can be prevented with selective NK-1 antagonists and reproduced with the selective NK-1 agonist [Sar-9, Met(O)2-11]SP, thus suggesting the involvement of NK-1 receptors [19 ]. To our knowledge, in studies in which SP has been evaluated as a priming agent on PMNs, no clear evidence of a receptor-mediated effect has been reported.
The experiments described here evaluated whether SP could prime PMNs challenged by IL-8. For this purpose, we measured calcium changes evoked by IL-8 in the absence and presence of SP, because this response is a convenient measure of receptor activation and is highly correlated with other functional responses to the chemokine. Additional experiments were performed to elucidate whether the priming effects of SP on IL-8-evoked calcium movements are mediated by a specific NK receptor type.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NKA, NKB, the selective NK-1 agonist [Sar-9, Met(O)2-11]SP [13 ], the selective NK-2 agonist [ß-Ala-8] NKA-(4–10) [13 ], the selective NK-3 agonist senktide (Suc-Asp-Phe-MePhe-Gly-Leu-Met-NH2) [13 ], the selective NK-1 antagonist L703,606, oxalate salt {[cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1-azabicyclo [2.2.2]octan-3 amine oxalate} [20 ] were purchased from RBI/Sigma (Natick, MA). SP fragments SP(1–7), SP(4–11), SP(6–11), and the selective NK1 antagonist CP96,345 {2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]octan-3 amine} [21 ] were kindly supplied by C. A. Maggi (A. Menarini Pharmaceuticals, Firenze, Italy). All the other reagents and solvents were from Merck (Darmstadt, Germany).
NKB and CP96,345 were dissolved in dimethyl sulfoxide (DMSO); the final
concentration of DMSO was 
0.1%. The same amount of DMSO was added to
the control samples, and it did not affect
[Ca2+]i in resting PMNs or the calcium
response in PMNs activated by rIL-8.
Cell preparation
PMNs were isolated from heparinized venous blood obtained from
healthy volunteers at the local hospital blood bank by using the
standard techniques of dextran sedimentation and Histopaque®1077
gradient centrifugation, as previously described [5
].
Residual erythrocytes were removed by hypotonic lysis. PMNs were
resuspended in a buffered salt solution (BSS) with the following
composition: 138 mM NaCl, 6 mM KCl, 1.0 mM
Na2HPO4, 5 mM NaHCO3, 1.1 mM
CaCl2, 5.5 mM glucose, and 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 7.4 at 37°C). The purity of the final cell suspension
averaged 98%. PMN viability, as assessed by the trypan blue exclusion
test, was >95% in all the experiments, and it was not affected by
drug treatment.
[Ca2+]i measurements
The effects of rIL-8 and SP on [Ca2+]i
were evaluated according to the procedure reported by Merritt et al.
[22
]. PMNs (5x106 cells/mL) were suspended
in BSS, as previously described, and loaded with 0.5 µM
fura-2/acetoxymethyl ester for 45 min at 37°C. After the cells were
loaded with the dye, they were washed, resuspended at 106
cells/mL in BSS, and maintained at room temperature until they were
assayed. PMNs were incubated with the NKs, SP fragments, and NK
receptor-selective agonists for the periods described in Results,
before rIL-8 was added. NK-1 antagonist CP96,345 or L703,606 was added
5 min before the NK receptor-selective agonists were added.
In some experiments, PMNs were exposed to a calcium-free medium that
had the same ionic composition as BSS but lacked CaCl2.
This medium was supplemented with the extracellular calcium chelator
EGTA (1 mM) [23
]. Fura-2 fluorescence was monitored in a
Jasco FP777 fluorimeter (Jasco International, Tokyo, Japan) at 37°C
with continuous stirring of the cells. Excitation and emission
wavelengths of 340 and 505 nm, respectively, were selected, and
[Ca2+]i values were calculated by the method
of Damaj et al. [24
]. The increase in
[Ca2+]i was measured as the difference
between the basal level and the peak reached after drug addition, and
it was expressed as 
[Ca2+]i. Each assay
was performed in triplicate.
Data analysis
The results were reported as means plus or minus standard errors
of the mean with n indicating the number of the experiments.
The percentage of inhibition of SP priming effects by the
NK1 antagonists was calculated as follows: percentage of
inhibition = [100 -
(z-x)/(y-x) x
100], where x is [Ca2+]i
evoked by rIL-8, y is [Ca2+]i
elicited by rIL-8 and SP, and z is
[Ca2+]i measured in the presence of rIL-8,
SP, and the NK-1 antagonist.
Origin version 4.10 (Microcal Software, Northampton, MA) was used as a nonlinear regression model for the analysis of dose-response data to obtain a 50% effective concentration, maximum effect, and 50% inhibitory concentration values. Statistical analysis was carried out by Student’s t-test for paired variants.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[Ca2+]i maximum plus or minus
SE=882±30 nM) (Fig. 1A
). The 50% effective concentration (EC50) was
2.4 ± 0.9 x 10-9 M. These results were in
agreement with published data and matched those recorded by measuring
calcium flux evoked by IL-8 in transfected cell lines expressing CXCR1
and CXCR2 [26
].
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[Ca2+]i max =
1,026±65 nM; the value is significantly different from that obtained
in the absence of SP: P<0.05). The EC50 was
5.4 ± 0.6 x 10-10 M, and it was significantly
different from that measured in the absence of SP (P<0.01). The contribution of extracellular calcium to the priming effects of SP was evaluated by incubating the cells in a calcium-free medium supplemented with EGTA (see Materials and Methods). Under these conditions, the changes in fluorescence of fura-2 that were recorded can be assumed to have been due to calcium release from internal stores. These experimental conditions caused a 20% decrease in the peak responses evoked by all the concentrations of rIL-8, thus confirming the observations made by Damaj et al. [24 ]. When PMNs were suspended in a calcium-free medium supplemented with EGTA, the enhancing effects of SP were abolished (Fig. 1B) . The dose-response curve of rIL-8 in the presence of SP was quite close to the one recorded in the absence of the neuropeptide.
In the experiments reported in Figure 1 , PMNs were treated with SP for 3 min before their exposure to rIL-8. This incubation time was selected according to the results of the time-course experiments shown in Figure 2 . The results demonstrated that the priming effects of 3 x 10-6 M SP reached the maximum after a 3-min incubation and were maintained up to 10 min. Thereafter, they declined and were lost at 30 min. Thus, an incubation time of 3 min proved to be optimal for eliciting the priming effects of SP, and it was used in all of the following experiments. When tested at the same concentration of 3 x 10-6 M for the same incubation times, NKA exerted weak effects that were statistically significant in comparison with those of rIL-8 alone only at 10 min, and NKB was inactive throughout (Fig. 2) .
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The priming effects of SP were inhibited by a potent nonpeptide antagonist, CP96,345, which has been described as selective for the human NK-1 receptor [27 ] (Fig. 5 ). When another potent nonpeptide-selective antagonist of the human NK-1 receptor, L703,606, was evaluated [20 ], the qualitative and quantitative effects of the two antagonists were so close that the respective dose-response curves could not be drawn separately (Fig. 5) . Both drugs acted in a dose-dependent way, with a 50% inhibitory concentration of 5.6 ± 0.35 x 10-8 M, and at 3 x 10-6 M they inhibited completely the priming effects of 3 x 10-6 M SP. The two antagonists by themselves did not affect [Ca2+]i in resting PMNs and did not modify the rise in [Ca2+]i induced by rIL-8 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the concentrations tested, SP did not affect [Ca2+]i in resting PMNs; but when the cells were exposed to the neuropeptide and then challenged with rIL-8, the calcium peak concentrations evoked by rIL-8 were significantly increased. The ability of SP to enhance, in a dose- and time-dependent way, the cell response to IL-8 can be regarded as a priming effect of the NK. SP acted at micromolar concentrations in the range from 10-6 to 3 x 10-6 M. These concentrations were close to those able to potentiate the oxidative response of PMNs to platelet-activating factor [5 ] and to the ones that prime the formation of hydrogen peroxide and NO in PMNs [7 ]. They were higher than the nanomolar concentrations of SP that were reported to exert no priming effect on calcium response of PMNs to the chemotactic peptide fMLP [6 , 28 ].
SP, by itself, was demonstrated to increase
[Ca2+]i in PMNs at two different
concentrations, 2 x 10-4 M [29
] and
3 x 10-5 M [30
]. The last one was
10-fold higher then the maximum concentration we assayed, and it was
the only concentration that stimulated a significant release of IL-8
from PMNs [8
]. SP, at 3 x 10-5 M,
primed fMLP-evoked O2- production from PMNs
[8
]. However, whereas a 5-min preincubation of the cells
with the neuropeptide was enough to prime O2-
production, significant amounts of IL-8 were detected only after a
60-min incubation [8
]. In our experiments, PMNs were
incubated with SP for 30 min, with the maximum priming effect being
achieved after a 3-min incubation. A similar time course for other
priming effects of SP on PMNs has been reported [5
,
12
].
The results described here add SP to the list of agents, such as
colony-stimulating factors, heparan sulfate, heparin, IL-1ß,
platelet-activating factor, and tumor necrosis factor-
, that were
shown to prime PMNs for IL-8 activation [31
32
33
]. fMLP
and lipopolysaccharide were shown to up-regulate IL-8 receptors in PMNs
[34
]. A similar effect was not exhibited by IL-1ß
[33
]. PMNs that had been incubated in the plasma from
patients with adult respiratory distress syndrome demonstrated an
increased [Ca2+]i response to IL-8
[35
]. IL-8 was more efficient in inducing a rise in
[Ca2+]i in IL-1ß-primed PMNs than in
untreated PMNs [33
]. The priming effects of IL-1ß were
related to its ability to increase calcium flux across the plasma
membrane [33
]. These results are similar to the ones
that were observed by exposing SP-primed PMNs to rIL-8. The priming
effects of SP were lost by removing extracellular calcium. Thus, the
response of IL-8-activated PMNs to SP may be attributed to calcium
entry from the medium. Calcium movements induced by IL-8 in PMNs have
been demonstrated to include at least two components
[10
]. One component was independent of extracellular
calcium, whereas the other one was shown to depend on calcium influx
through calcium-release-activated calcium channels. The effects of SP
might be added to the component of IL-8-induced calcium signaling that
was dependent on extracellular calcium.
In Chinese hamster ovary cells expressing the SP receptor, the NK elicited a prolonged phase of calcium entry that disappeared when extracellular calcium was depleted [36 ]. SP was shown to modulate [Ca2+]i in the cells by interacting with receptor-operated calcium channels [36 ]. Cultured rabbit osteoclasts showed a rise in [Ca2+]i after the addition of SP [37 ]. The [Ca2+]i elevation by SP was not observed in calcium-free medium and was blocked by the NK-1 receptor antagonist CP96,345 [36 ]. Another NK-1 receptor antagonist, GR 82334, at 10-5 M, completely inhibited the increase in [Ca2+]i that was recorded in PMNs exposed to 3 x 10-5 M SP [30 ].
In the experiments reported here, the priming effects of SP were mimicked by the C-terminal fragments SP(4–11) and SP(6–11) but not by the N-terminal peptide SP(1–7). The interaction between the NKs and their receptors has been reported to require the recognition of the C-terminal sequence of the neuropeptides [13 , 14 ]. Therefore, the ability of SP(4–11) and SP(6–11) to exhibit the same effects as SP suggests the involvement of an NK receptor. When selective agonists of the NK receptors [13 ] were compared with SP, the priming effects of the neuropeptide were mimicked by the selective NK-1 agonist [Sar-9, Met(O)2-11]SP but not by the selective NK-2 agonist [ßAla-8]-NKA(4–10) or the selective NK-3 agonist senktide. Furthermore, SP, the preferred natural agonist of the NK-1 receptor, was more active than NKA; NKB was inactive. The NK-1 antagonist CP96,345 inhibited, in a dose-dependent way, the priming effects of SP. Because this drug has been reported also to exert effects other than NK-1 antagonism (e.g., interaction with L-type calcium channels) [38 ], another NK-1 antagonist, L703,606, was tested. This other antagonist exerted the same inhibitory effects as CP96,345. CP96,345 and L703,606 both affected neither calcium levels in resting PMNs nor the changes in [Ca2+]i evoked by rIL-8, thus displaying full pharmacological antagonism against SP.
To determine whether the priming effects of SP were receptor mediated, an experimental procedure was followed that is widely used in pharmacological research, that is, evaluation of the ability of selective agonists to reproduce the effects of the endogenous ligand and of selective antagonists to counteract these effects. For this approach, the data pointed to an interaction between SP and a binding site with the pharmacological responsiveness of an NK-1 receptor. This positive conclusion must be considered with the fact that the putative receptor of SP on PMNs responds to micromolar concentrations of the neuropeptide, which indicates an affinity significantly lower than the nanomolar one reported for the NK-1 receptor in other cell types [13 , 15 ].
When the ability of SP to act on PMNs at concentrations higher than those effective in other cells or tissues was previously examined, the existence of isoforms of the NK-1 receptor was evaluated to explain the difference in SP efficacy [30 ]. The cloning and expression of two isoforms of the human NK-1 receptor, which differ only in the length of the encoded polypeptide, have been described elsewhere [39 ]. The short form binds SP with a lower affinity than does the long form, and it elicits a smaller electrophysiological response in Xenopus oocytes [39 ].Two isoforms of the rat NK-1 receptor, differing in the lengths of their cytoplasmic carboxy termini, have been transfected into Chinese hamster ovary cells [40 ]. The interaction of SP with either of these isoforms induces an increase in [Ca2+]i, but cells expressing the full-length receptor are activated by higher concentrations of the neuropeptide than cells expressing the truncated receptor [40 ].
According to these results, the presence of an isoform of the NK-1 receptor on PMNs that is distinct from those present in other cells, such as neurons, secretory cells, and smooth muscle cells, may allow us to explain why micromolar concentrations of SP are needed to prime PMNs. Although such a hypothesis must be taken into account, no direct experimental evidence is available, thus precluding a definite conclusion.
The fact that the binding site on PMNs, which has been labeled by the
selective NK-1 agonist [Sar-9, Met(O)2-11]SP and by the
selective NK-1 antagonists CP96,345 and L703,606, responds to high
concentrations of SP might depend on a low intrinsic binding affinity.
However, another attempt to explain the experimental results could be
made by evaluating the transmembrane signaling after the
agonist-receptor interaction. The NK-1 receptor has been demonstrated
to couple to G proteins of the Gq/11 subclass to
activate phospholipase Cß, resulting in formation of
inositol triphosphate and diacylglycerol [41
]. A weak
coupling to G proteins might account for the high concentrations of SP
that act on PMNs and underlie the apparent low affinity of the
interaction between the neuropeptide and its binding site on these
cells. It must be stressed that SP elicits a priming effect and not a
direct activating signal. Even this working hypothesis requires further
experimental evaluation before it can be accepted or rejected.
The micromolar concentrations of SP that are necessary to prime PMNs exposed to rIL-8 in vitro might suggest a limited pathophysiological role in vivo for the combined effects of the NK and the chemokine. Whether and when the working conditions described here can be transferred to an in vivo pathophysiological event is still difficult to ascertain. However, the stimulation of sensory nerves that occurs at inflammatory sites might induce high local concentrations of SP [3 , 4 , 8 ]. In such a case, PMNs might be challenged by SP concentrations of the same order as those ascribed to prime PMNs in vitro. Thus, our results indicated that the neuropeptide might contribute to the activation of PMNs in a dose- and time-dependent manner.
In summary, experimental evidence has been provided that SP primes IL-8-activated PMNs, thus amplifying the cell response to the chemokine. The effects of SP were reproduced by a selective NK-1 receptor agonist and were inhibited by selective NK-1 receptor antagonists. The data described here confirm and further extend existing information about the ability of SP to prime different PMN responses to various stimuli and add new information about the modulatory effects that the neuropeptide exerts on PMN activity. Our data support the role played by SP during inflammatory processes.
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ACKNOWLEDGEMENTS |
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Received August 9, 2000; revised January 23, 2001; accepted January 24, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits of the G proteins Gq and G11 Biochemistry 35,2909-2916[Medline]
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) or presence (•) of
3 x 10-6 M SP. Data are expressed as means ±
SE of five experiments. Asterisks indicate statistically
significant differences (**, P<0.01; *, P<0.05)
between the responses of PMNs exposed to rIL-8 and the responses of
PMNs exposed to rIL-8 plus 3 x 10-6 M SP.
), NKA (•) or NKB (
), all at 3 x
10-6 M, and then treated with 109 M rIL-8.
Data are means ± SE; n = 5. Asterisks
indicate statistically significant differences (**, P<0.01;
*, P<0.05) between the responses of PMNs exposed to rIL-8
and the responses of PMNs exposed to rIL-8 in the presence of each
neurokinin. Calcium response to 10-9 M rIL-8
(
, 
), and SP(6–11) (
). The
results are expressed as percentage increases over
