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*
Centre for Cardiovascular Biology and Medicine and
Messengers and Signaling Group, King’s College London, London, United Kingdom; and
Perlmutter Laboratory, Children’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is also evidence that SP has effects on the cellular component in inflammation, in particular on neutrophil accumulation, the subject of this study (1). However, our recent experiments in rat skin in vivo, using a thermal injury model, has revealed that although the early edema formation is tachykinin NK1 receptor dependent, the later neutrophil infiltration occurs independently of NK1 receptor activation (4). Furthermore, neither administration of SP nor stimulation of sensory nerves had any direct effect on neutrophil accumulation during a 5-h experimental period (4). By comparison, there is evidence from inflammatory models, in NK1 knockout mice, that NK1 receptor activation can contribute to neutrophil accumulation. Bozic et al. (5) demonstrated in an immune complex-mediated model in the lung that neutrophil accumulation was significantly inhibited in NK1 knockout mice. They suggested that the early NK1-mediated edema provided plasma constituents for Ag-mediated responses (e.g., complement components), thus amplifying the response independently of an effect on neutrophils. More recently, Ahluwalia et al. (6) have shown that IL-1ß-induced neutrophil accumulation is significantly reduced in NK1 knockout mice in a murine air pouch model of inflammation. They suggested that either IL-1ß can act to directly release an NK1 agonist from sensory nerves or perhaps an intermediate such as a bradykinin B1 agonist may be involved. The proposals are supported by earlier results obtained through the use of selective NK1 receptor antagonists (7) and through the use of kinin bradykinin 1 receptor (B1 receptor) antagonists (8) in the same air pouch model in normal mice. Thus, although it is generally described in the literature that activation of NK1 receptors contribute to neutrophil accumulation, it remains contentious, and little is known about the mechanisms involved. In this study, we have investigated the role of NK1 receptors in mediating neutrophil accumulation in skin induced by IL-1ß, carrageenin, and zymosan using wild-type and NK1 receptor knockout mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice (Sv129 + C57BL/6) strain, either genetically unaltered wild-type (+/+) or with the gene for the NK1 receptor removed, knockout (-/-) were developed at Perlmutter Laboratory, Children’s Hospital (Boston, MA) (5) and bred at King’s College London (London, U.K.). The mice were raised in a climatically controlled environment and allowed food and water ad libitum. Both colonies of mice displayed normal growth and behavioral characteristics, and both male and female mice (25–30 g) were used in experiments. Experiments were conducted according to the U.K. Animals (Scientific Procedures Act) 1986.
Effect of agents on neutrophil accumulation
Studies were conducted on anesthetized animals, using 2%
halothane (Rhone-Merieux, Harlow, U.K.) in gas (95%
O2, 5% CO2). Body
temperature was maintained at 37–37.5°C using a heating pad
controlled by a rectal thermistor probe (Harvard Apparatus, Edenbridge,
U.K.). The dorsal skin of the animals was shaved and prepared for
intradermal (i.d.) injection (up to 4 sites per mouse, each in a
randomly allocated balanced site pattern). The
NK1 receptor antagonist SR140333 (gift from
Sanofi, Toulouse, France, 480 nmol/kg); the B1
receptor antagonist
desArg9[Leu8]bradykinin
(Bachem, Saffron Walden, U.K., 3 µmol/kg); the
B2 receptor antagonist HOE 140
([D-Arg,Hyp3,Thi5,D-Tic7,Oic8]bradykinin,
100 nmol/kg, Peninsula Laboratories, Merseyside, U.K.) or their
vehicles were given as pretreatments (i.v. 5 min before i.d.
injections) when required. Agents were injected i.d. (50 µl/site) at
the following doses: murine-recombinant IL-1ß (R&D Systems, Abingdon,
U.K., 0.03–3.0 pmol/site); SP (Sigma, Poole, U.K., 300 pmol/site);
septide
([pGlu6,Pro9]SP(6, 7, 8, 9, 10, 11),
Peninsula (30 pmol/site). The agents were dissolved in Tyrode’s
solution (composition as follows (in mmol/l): NaCl 137; KCl 2.7;
MgCl2 0.5;
NaH2PO4 0.4;
NaHCO3 11.9; glucose 5.6.). Carrageenin 
(Sigma, 250-1000 µg/site) and zymosan (Sigma, 10–50 µg/site) were
dissolved in saline (0.9% sodium chloride, pyrogen free, Baxter
Healthcare, Thetford, Norfolk, U.K.). Experiments were terminated at 3
or 5 h after the i.d. treatments as required. In the case of 5-h
experiments, injection of SP (300 pmol/site) and septide (30 pmol/site)
was repeated at the 3-h time point. At the end of the accumulation
period, the mice were killed by cervical dislocation. The skin was
removed and treated, and control dorsal skin sites were punched out
(16-mm-diameter samples). The skin samples were frozen and kept at
-18°C until required.
Measurement of neutrophil accumulation
The samples were thawed, chopped, and homogenized in a phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide (Sigma) detergent. The homogenate was centrifuged at 13,000 x g for 5 min (Microcentaur MSE, Scotlab, Strathclyde, Lanarkshire, U.K.). Supernatants (1 ml) were then frozen at -18°C. A previously validated method for measurement of neutrophils in the rat skin (4, 9, 10) was adapted for mouse skin. Neutrophil accumulation was measured by comparing myeloperoxidase activity in extracts from known amounts of mouse neutrophils and compared with that at skin sites. Myeloperoxidase activity was determined using the H2O2 oxidation of 3,3',5,5'-tetramethylbenzidine (Bionostics, Skybio, Wyboston, Beds, U.K.). Reactions were performed in a 96-well microtiter plate at room temperature. OD readings at 620 nm were taken at 5-min intervals for 30 min with a microplate reader (Anthos HTIII, Labtech International, Saltzburg, Austria). These values were plotted, and a reaction rate (OD/time) was measured from the initial slope of the curve. A calibration curve was then produced, with the rate of reaction plotted against the number of neutrophils in the standard samples. This was used to convert reaction rates to number of neutrophils for the skin sample homogenates.
Preparation of leukocytes from mouse peritoneal cavity for determining myeloperoxidase activity per neutrophil
Mixed leukocytes were elicited from the mouse peritoneal cavity
by inducing leukocyte-rich acute peritonitis. For this, 6% oyster
glycogen (Sigma, Poole, U.K.) dissolved in 1 ml isotonic saline was
injected i.p. Then 16–20 h later, the mice were sacrificed by cervical
dislocation, and 5 ml ice-cold modified HBSS (Sigma) free of
Ca2+ and Mg2+ was injected
into the peritoneal cavity. After 
90 s of massage, the peritoneal
fluid was collected with a syringe and centrifuged at 4°C for 10 min
at 400 x g. Supernatant was removed, and any
contaminating erythrocytes were lysed by brief exposure to hypotonic
saline (0.2% sodium chloride). After tonicity was restored, the cells
were centrifuged again (4°C, 10 min, 400 x g).
Supernatant was discarded, and the pellet was taken up in 2 ml HBSS
containing 1.26 mM Ca2+ and 0.9 mM
Mg2+. Solution (0.1 ml) was mixed with 0.9 ml
complete HBSS again. A sample (0.1 ml) was stained with 0.1 ml trypan
blue (0.4% solution), and the number of white blood cells was
determined using hemocytometry. For determination of the number of
neutrophils, cell smears were prepared by cytospin. Thus, 2–3 drops of
cells, from a sample count of 5 x 104
cells/ml, were placed in a cytospin cup attached to a microscope slide
and spun for 1 min at 1300 rpm in a cytospin centrifuge
(Shandon-Cytospin 2, Shandon Scientific, Runcorn, Cheshire, U.K.).
Slides were fixed in acetone (BDH, Merck, Poole, Dorset, U.K.) for 10
min and air dried, followed by 10 min in Mayer’s hematoxylin (BDH).
Slides were washed with tap water and placed in 1% chromotrope 2R, 1%
phenol, (11). Microscopic examination showed that 70–75%
of the leukocytes are polymorphonuclear neutrophil granulocytes.
Aliquots of standard preparations were made up (2.5 x
106 cells/ml) for daily use
(12).
Effect of agents on edema formation
Animals were prepared for i.d. injection (up to 4 sites per
mouse) of drugs as described above. Plasma extravasation was determined
by Evans blue leakage method (13). Evans blue (Sigma;
2.5%) was injected i.v. using 50 µl/10 g body weight, 5 min before
i.d. treatments. After 5 min, NK1 receptor
agonists or carrageenin or vehicle controls were injected i.d. into
the dorsal skin. Animals were left for 30 min
(NK1 agonists) or 4 h (carrageenin)
to allow plasma extravasation to occur. The tachykinin
NK1 receptor antagonist, SR140333
((S)-1-{2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl}-4-phenyl-1-azoniabicyclo[2.2.2]octane
chloride, 480 nmol/kg) or vehicle was injected i.v. when required.
SR140333 was dissolved in a minimal volume of 100% ethanol
and made up to the final volume with 0.9% sodium chloride.
At the end of the accumulation period, the mice were killed by cervical
dislocation, and 16-mm-diameter skin pieces were punched out and placed
in 1 ml formamide solution (Sigma). The extravasated dye was extracted
at room temperature after 72 h. Evans blue content of the samples
was determined by a 96-well microplate reader at 620 nm (100 µl
sample/well). Plasma extravasation was expressed as µg Evans
blue/skin site, by comparing the experimental values with a known
standard.
Statistical analysis
All results are expressed as the mean ± SEM. Statistical differences between groups were evaluated by ANOVA followed by Bonferroni’s modified t test. A threshold of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first experiments, the effect of intradermal SP on edema
formation and neutrophil accumulation is shown. SP induced significant
edema formation in the dorsal skin of wild-type mice, but not knockout
mice, as expected (Fig. 1
A)
(3). In comparison, in separate experiments, no effect of
SP on neutrophil accumulation was observed in either wild-type or
knockout mice (Fig. 1B; see also Fig. 3). The effect of the
cytokine IL-1ß on neutrophil accumulation is shown in Fig. 2. IL-1ß was injected i.d. (0.03–3.0
pmol/site). The dose-response curve was bell shaped. The results were
similar in wild-type and knockout mice, and 0.3 pmol/site
IL-1ß-induced significant leukocyte recruitment
(p < 0.001) in each case.
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. As before, SP did not produce
neutrophil accumulation when injected alone but significantly
potentiated the effect of IL-1ß (0.3 pmol/site) over 5 h in
wild-type, but not in knockout mice (Fig. 3). The effect of the
selective NK1 receptor agonist, septide
([pGlu6,Pro9]SP(6, 7, 8, 9, 10, 11),
30 pmol/site) was also investigated. In a manner similar to that of SP,
septide had no effect on neutrophil accumulation when given alone:
Tyrode (vehicle), 1.93 ± 0.27; septide, 1.97 ± 0.18 (values
given as number of neutrophils x 106/site,
mean ± SEM, n = 6) but potentiated
IL-1ß-induced cell accumulation in wild-type animals after 5 h.
The results were as follows: Tyrode (vehicle), 0.95 ± 0.13;
IL-1ß, 1.9 ± 0,19; IL-1ß + septide, 2.52 ± 0.4,*
(values given as number of neutrophils x
106/site, mean ± SEM, n =
11; *, p < 0.05 compared with IL-1ß alone). These
results show that either added SP or an alternative
NK1 agonist can potentiate neutrophil
accumulation induced by the cytokine IL-1ß. Neutrophil accumulation in wild-type and NK1 receptor knockout mice in response to zymosan and carrageenin
The effect of neutrophil accumulation induced by either
carrageenin or zymosan was investigated in wild-type and
NK1 receptor knockout mice. Increasing doses of
i.d. zymosan induced neutrophil accumulation in a dose-related manner
in both types of mice. The number of accumulated neutrophil cells did
not differ significantly between wild-type and knockout animals (Fig. 4A). This indicates that the
activated NK1 receptor does not play a major role
in the neutrophil accumulation observed in response to zymosan. By
comparison, carrageenin, induced substantial neutrophil accumulation in
normal mice with a bell-shaped dose-response curve, and this was
markedly attenuated in NK1 knockout mice (Fig. 4B). This indicates that the NK1
receptors must have a role in neutrophil accumulation induced by
carrageenin, but not that induced by zymosan. In separate experiments,
during 4 h, edema formation induced by carrageenin (500 µg) was
partially, but significantly, attenuated in NK1
knockout mice, as shown in Table I.
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). These results further suggest that an
NK1 agonist is released to play a pivotal role in
carrageenin-induced neutrophil accumulation in normal mice.
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The possible mechanisms by which the NK1
agonist was involved in carrageenin-induced inflammation was further
investigated by determining the effect of selective bradykinin
B1 and B2 antagonists.
Pretreatment with either B1,
(desArg9[Leu8]bradykinin,
3 µmol/kg i.v.) (15) or B2 (HOE
140, 100 nmol/kg i.v.) (16, 17, 18) receptor antagonists
inhibited the carrageenin induced neutrophil accumulation in wild-type
mice after 5 h (Fig. 6, third set of
columns). However, these antagonists did not affect the suppressed
response of knockout mice (Fig. 6, fourth set of columns). Furthermore,
neutrophil accumulation induced by zymosan (20 µg/site i.d.) was not
affected by the bradykinin receptor antagonists in wild-type mice (Fig. 6, fifth set of columns), thus demonstrating the different kinin
receptor involvement in neutrophil recruitment between the zymosan and
carrageenin models.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lack of effect of SP, NK1 agonists and endogenous tachykinins to induce neutrophil accumulation in normal skin has been previously demonstrated by this group in the rat (4). The present results show that in the naive skin of the mice under study, SP, at doses up to 300 pmol, that induce substantial edema formation, do not alone induce neutrophil accumulation. These results differ from those observed in the normal mouse lung because SP or an NK1 agonist both induced edema formation and neutrophil accumulation (19). This may indicate a tissue difference in the ability of NK1 agonists to induce neutrophil accumulation in vivo. Alternatively, there are studies that indicate that SP can induce neutrophil accumulation into mouse skin as a consequence of its ability to activate mast cells, via an NK1 receptor-independent mechanism. It has been shown that SP can cause neutrophil accumulation in mice, secondary to mast cell degranulation and leukotriene B4 release (20). Our recent study in wild-type and knockout mice, suggests that SP cannot activate mast cells via an NK1-independent mechanism to mediate edema formation in skin (3). Thus, this mechanism appears to be lacking in these mice, possibly due to differences in specific technical protocols or due to the strain of mice used.
The proinflammatory cytokine IL-1ß is a potent mediator of neutrophil
accumulation in vivo, inducing local neutrophil accumulation after
injection into a range of specific tissue sites (21, 22, 23).
In 1989, Osborn et al. (24) reported that IL-1 was able to
augment the adhesiveness of the vascular endothelium. This cytokine,
along with others such as TNF-
, up-regulate ICAM-1 and E-selectin
expression (25). Our results, from experiments conducted
in normal skin, indicate that IL-1ß induced significant neutrophil
accumulation at 0.3 pmol/site, but this was not observed with higher
doses, with similar results in wild-type and knockout mice. This
bell-shaped dose-response curve is in keeping with results from other
in vivo studies of IL-1-induced neutrophil accumulation, e.g., into a
s.c. sponge in the mouse (26). The results clearly
indicate that neutrophil accumulation in response to IL-1ß is not
primarily modulated by an NK1 receptor-dependent
mechanism and suggests that IL-1ß does not directly release an
NK1 agonist in normal mouse skin. By comparison,
Ahluwalia et al. (6) showed, using a 6-day murine
air-pouch model with NK1 receptor knockout mice,
that NK1 receptors play a significant role in the
cellular response to IL-1ß. It is interesting that the murine air
pouch model involves skin tissues, but the cells alter during its
development such that the lining cells are substantially changed to
include macrophage and fibroblast-like cells (27). It is
possible that in this chronic inflammatory model IL-1ß can influence
neuropeptide release via mediator systems that are not present in
normal naive skin. This suggestion is supported by our results, which
show that the combined administration of IL-1ß with either SP or the
selective NK1 agonist septide leads to a
potentiation of IL-1ß neutrophil accumulation in wild-type mice. This
is clear evidence that an NK1 agonist, if
released at an inflammatory site can potentiate neutrophil
accumulation.
We then turned our attention to learning more about the release and role of NK1 agonists in cutaneous inflammation. Our aim was to study mechanisms in a system where endogenous tachykinins may be released and contribute to neutrophil accumulation. Zymosan (yeast cell walls) induces well-defined neutrophil accumulation in many species, including the mouse (28). The chemotactic complement factor C5a plays a major role in this model, but other mediators including mast cell amines, but not bradykinin, have been suggested to contribute to these inflammatory responses in vivo (29, 30). Zymosan induced dose-dependent neutrophil accumulation, but results were similar in wild-type and knockout mice, indicating a lack of functional involvement of NK1 agonists. These results were in contrast to those obtained using carrageenin. Carrageenin (sulfated polysaccharide from seaweed) induces inflammation that includes substantial edema formation (31). The complement factors play a minor role (28, 29, 32), but the involvement of neutrophils (32) and the kinin system (28, 33) has been demonstrated. In addition, there is evidence for a tachykinin neurogenic component (34, 35, 36). Our results demonstrated a small but significant decrease in edema formation in knockout compared with wild-type mice, but a substantial and highly significant decrease in neutrophil accumulation. The selective NK1 antagonist SR140333 inhibited neutrophil accumulation induced by carrageenin in wild-type mice, which is in keeping with the suggestion that sufficient tachykinins are released to substantially modulate carrageenin-induced neutrophil accumulation. Thus, the results provide direct evidence that endogenous tachykinins can influence neutrophil accumulation in inflamed skin.
The release of endogenous tachykinins in inflamed skin is of interest. Our results discussed above rule out the possibility that IL-1ß can directly mediate neuropeptide release. However, there is evidence from studies in the mouse air pouch (6) and the mouse pleural cavity (37), that kinins are involved in mediating release of tachykinins from sensory nerves in inflamed tissues. The released tachykinins, probably SP, then act as NK1 agonists. The kinin B2 receptor, for which bradykinin has high affinity, is constitutively and widely expressed in peripheral tissues. By comparison, the kinin B1 receptor is not normally expressed in basal conditions but is induced in situations of stress, shock, and tissue inflammation (38). The endogenous agonist of the B1 receptor is desArg9 bradykinin, which is formed from bradykinin and elevated in inflammatory exudates (38). We investigated the effect of pretreatment with B1 and B2 receptor antagonists in carrageenin-induced cell accumulation. The inhibitory effect of the B1 and B2 receptor antagonists in wild-type animals but not in knockout mice indicates that kinins are key intermediates of the NK1 receptor-dependent component of carrageenin-induced neutrophil accumulation, acting via both the constitutive B2 receptor and the inducible B1 receptor. These combined results obtained from wild-type and NK1 knockout mice adds further evidence to the suggestion that kinins may act via B1 or B2 receptors to release SP from sensory nerves in skin. The lack of effect when inflammation is induced by zymosan indicates some selectivity in this response. Thus, the present results are indicative that these mediator pathways only become operational in skin under certain circumstances. This selectivity may be related to the presence and activity of other mediators at inflammatory sites. The results extend findings from other laboratories where a similar phenomenon has been observed in inflammation studies in the airways (39), in a murine pleurisy model (37) as well as in the murine air pouch (6, 7).
In conclusion, the results in this study demonstrates that NK1 agonists such as SP cannot act on their own to mediate neutrophil accumulation in naive skin. By comparison, the results provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in the potentiation of neutrophil accumulation during the ongoing inflammatory process. Plasma extravasation and neutrophil accumulation were both attenuated in carrageenin-induced (but not zymosan-induced) inflammation in NK1 knockout mice. The carrageenin-induced cellular response was inhibited by a NK1 receptor antagonist, SR140333, in the wild-type group. Furthermore, B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) also attenuated neutrophil accumulation to carrageenin in wild-type mice but not in knockout mice. The results provide further evidence that B1 and B2 receptors participate in the NK1 receptor-dependent component of the neutrophil cell recruitment induced by carrageenin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. S. D. Brain, Centre for Cardiovascular Biology and Medicine, New Hunt’s House, King’s College London, Guy’s Campus, London, SE1 1UL, U.K.
3 Abbreviations used in this paper: SP, substance P; NK1 receptor, neurokinin-1 receptor; B1 and B2 receptors, bradykinin-1 and bradykinin-2 receptors; i.d., intradermal.
Received for publication December 20, 1999. Accepted for publication March 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Leukocyte Biol. 45:62.[Abstract]
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