Neurokinin-1 Receptor Agonists Are Involved in Mediating Neutrophil Accumulation in the Inflamed, But Not Normal, Cutaneous Microvasculature: An In Vivo Study Using Neurokinin-1 Receptor Knockout Mice

Thong Cao, Erika Pintér, Sabah Al-Rashed, Norma Gerard, J. Robin Hoult and Susan D. Brain
J Immunol May 15, 2000, 164 (10) 5424-5429; DOI: https://doi.org/10.4049/jimmunol.164.10.5424

Abstract

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1β (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1β-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 μg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at −5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.

Aneurogenic component has been identified in many types of inflammatory responses and the acute proinflammatory microvascular effects of neuropeptides such as substance P (SP)3 are well documented (1, 2). Neurogenic inflammation is defined as the edema formation, increased blood flow, and inflammatory cell involvement observed after stimulation of sensory nerves and release of neuropeptides. In particular, the activation of capsaicin-sensitive nerve fibers (usually C fibers) to release neuropeptides, of which the best known are members of the tachykinin family (e.g., SP) and calcitonin gene-related peptide has been studied. SP is a potent mediator of increased microvascular permeability acting via NK1 receptors most commonly situated on postcapillary endothelial cells or via non-receptor-dependent mast cell-dependent mechanisms, whereas calcitonin gene-related peptide is a potent arteriolar vasodilator. These microvascular effects are most commonly observed as edema formation or increased blood flow, and the importance of neurokinin-1 receptors (NK1 receptors) in neurogenic edema formation is demonstrated in a recent study with NK1 receptor (Sv129 + C57BL/6) knockout mice (3).

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-1β-induced neutrophil accumulation is significantly reduced in NK1 knockout mice in a murine air pouch model of inflammation. They suggested that either IL-1β 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-1β, carrageenin, and zymosan using wild-type and NK1 receptor knockout mice.

Materials and Methods

Animals

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-1β (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 λ (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 × 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 × 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 × 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 × 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 × 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 λ 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.

Results

Effect of SP and IL-1β on neutrophil accumulation in wild-type and NK1 receptor knockout mice

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. 1⇓B; see also Fig. 3⇓). The effect of the cytokine IL-1β on neutrophil accumulation is shown in Fig. 2⇓. IL-1β 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-1β-induced significant leukocyte recruitment (p < 0.001) in each case.

FIGURE 1.
FIGURE 1.

Effect of SP (300 pmol/site) on plasma extravasation during 1 h, n = 6–9 (A) and on neutrophil accumulation during 5 h, n = 4 (B) in the dorsal skin of wild-type and NK1 knockout mice. SP was injected i.d., and results with vehicle (Tyrode-treated sites) are also shown. Results are expressed as mean ± SEM. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. ∗∗, p < 0.01 vs. Tyrode-treated controls.

FIGURE 2.
FIGURE 2.

Effect of IL-1β on neutrophil accumulation in the dorsal skin of NK1 receptor knockout and wild-type mice. Mice received IL-1β (0.01–3 pmol/site i.d.), and cell accumulation was measured 5 h later. Results are expressed as the mean ± SEM, n = 8 per group. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. ∗∗∗ and ###, p < 0.001 vs vehicle-treated controls.

The effect of coadministration of SP and IL-1β is shown in Fig. 3⇓. As before, SP did not produce neutrophil accumulation when injected alone but significantly potentiated the effect of IL-1β (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 × 106/site, mean ± SEM, n = 6) but potentiated IL-1β-induced cell accumulation in wild-type animals after 5 h. The results were as follows: Tyrode (vehicle), 0.95 ± 0.13; IL-1β, 1.9 ± 0,19; IL-1β + septide, 2.52 ± 0.4,∗ (values given as number of neutrophils × 106/site, mean ± SEM, n = 11; ∗, p < 0.05 compared with IL-1β alone). These results show that either added SP or an alternative NK1 agonist can potentiate neutrophil accumulation induced by the cytokine IL-1β.

FIGURE 3.
FIGURE 3.

Potentiation of cytokine-induced neutrophil accumulation by SP. SP (300 pmol/site) was coinjected with IL-1β (0.3 pmol/site) into the mouse skin. Neutrophil accumulation was measured at 5 h. Results are expressed as mean ± SEM, n = 7–9. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 vs. Tyrode’s-treated control sites. #, p < 0.05 compared with corresponding treatment in knockout mice.

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. 4⇓A). 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. 4⇓B). 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⇓.

FIGURE 4.
FIGURE 4.

Effect of zymosan and carrageenin on neutrophil accumulation in the dorsal skin of NK1 receptor knockout and wild-type mice. Carrageenin (250, 500, and 1000 μg/site) (A) and zymosan (10, 20, and 50 μg/site) (B) was injected i.d., and neutrophil accumulation was measured during 5 h. Results are expressed as mean ± SEM, n = 6–10. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. ∗, p < 0.05; ∗∗∗; p < 0.001 vs vehicle-treated sites; ###, p < 0.001 vs wild-type animals.

View this table:
Table I.

Effect of carrageenin on edema formation in the dorsal skin, measured 4 h after intradermal injectiona

The hypothesis that NK1 receptors play a role in neutrophil accumulation induced by carrageenin is supported by additional experiments conducted in wild-type mice pretreated with the selective, long-lasting NK1 receptor antagonist, SR140333 (4, 14). SR140333 (480 nmol/kg i.v. 5 min before) significantly inhibited NK1 agonist-induced plasma extravasation, results not shown, in keeping with our previous studies in the rat (4). SR140333 attenuated carrageenin (500 μg/site)-induced neutrophil accumulation induced in wild-type mice in paired experiments to a level that was also observed in the same experimental series in knockout mice (Fig. 5⇓). These results further suggest that an NK1 agonist is released to play a pivotal role in carrageenin-induced neutrophil accumulation in normal mice.

FIGURE 5.
FIGURE 5.

Effect of NK1 receptor antagonist (SR140333) pretreatment (480 nmol/kg i.v., at −5 min) on carrageenin-induced neutrophil accumulation in the skin of wild-type mice. Carrageenin (500 μg/site) was injected i.d., and neutrophil accumulation was measured during 5 h. The effect of carrageenin on neutrophil accumulation in NK1 knockout mice is also shown. Results are expressed as mean ± SEM, n = 8–9. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. #, p < 0.05; ##, p < 0.01 vs wild-type solvent-pretreated animals.

Effects of bradykinin receptor antagonists on carrageenin-induced neutrophil accumulation.

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.

FIGURE 6.
FIGURE 6.

Effect of B1 and B2 receptor blockade on neutrophil accumulation induced by carrageenin (500 μg/site) or zymosan (20 μg/site) in the skin of wild-type mice and knockout mice. Neutrophil recruitment was measured during 5 h. B1 receptor antagonist desArg9[Leu8]bradykinin (3 μmol/kg, i.v., −5 min) or B2 antagonist HOE 140 (100 nmol/kg, i.v., −5 min) were injected. Controls received vehicle (solvent) alone. Results are expressed as mean ± SEM, n = 6. Statistical significance was evaluated by ANOVA followed by Bonferroni posttest. #, p < 0.05; ###, p < 0.001 vs wild-type animals.

Discussion

The results demonstrate that SP or related ligands cannot by themselves induce neutrophil accumulation into normal mouse skin, despite potent effects on microvascular permeability and edema formation. However, added SP, or an NK1 agonist (septide) potentiated IL-1β-induced neutrophil accumulation in vivo in wild-type mice, indicating an ability of SP to augment neutrophil accumulation, if produced at inflamed sites. The finding that carrageenin-induced neutrophil accumulation is strongly attenuated in knockout compared with wild-type mice provides evidence that tachykinins are released to play a proinflammatory role in neutrophil accumulation in a common model of dermal inflammation. This is supported by the finding that the NK1 antagonist SR140333 attenuated carrageenin-induced neutrophil accumulation in wild-type mice. Taken together, our results indicate that NK1 receptors play a pivotal role in neutrophil accumulation induced by carrageenin in the mouse and that kinins are involved in the response.

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-1β 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-α, up-regulate ICAM-1 and E-selectin expression (25). Our results, from experiments conducted in normal skin, indicate that IL-1β 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-1β is not primarily modulated by an NK1 receptor-dependent mechanism and suggests that IL-1β 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-1β. 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-1β 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-1β with either SP or the selective NK1 agonist septide leads to a potentiation of IL-1β 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-1β 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.

Acknowledgments

We thank Ms. E. Seeds for help with cell counts.

Footnotes

  • ↵1 T.C. holds a British Heart Foundation studentship. E.P. was supported by a North Atlantic Treaty Organization/Royal Society Postdoctoral Fellowship.

  • ↵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 December 20, 1999.
  • Accepted March 7, 2000.

References

  1. ↵
    Brain, S. D.. 1997. Sensory neuropeptides: their role in inflammation and wound healing. Immunopharmacology 37: 133
    OpenUrlCrossRefPubMed
  2. ↵
    Holzer, P.. 1998. Neurogenic vasodilatation and plasma leakage in the skin. Gen. Pharmacol. 30: 928
    OpenUrl
  3. ↵
    Cao, T., N. P. Gerard, S. D. Brain. 1999. Use of NK1 knockout mice to analyse substance P-induced edema formation. Am. J. Physiol. 277: R476
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Pinter, E., B. Brown, J. R. S. Hoult, S. D. Brain. 1999. Lack of evidence for tackykinin NK1 receptor-mediated neutrophil accumulation in the rat cutaneous microvasculature by thermal injury. Eur. J. Pharmacol. 369: 91
    OpenUrlCrossRefPubMed
  5. ↵
    Bozic, C. B., U. E. Lu, C. Gerard Hopken, N. P. Gerard. 1996. Neurogenic amplification of immune complex inflammation. Science 273: 171
    OpenUrlCrossRef
  6. ↵
    Ahluwalia, A, C. DeFelipe, J. O’Brien, S. P. Hunt, M. Perretti. 1998. Impaired IL-1β-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice. Br. J. Pharmacol. 124: 1013
    OpenUrlCrossRefPubMed
  7. ↵
    Perretti, M., A. Ahluwalia, R. J. Flower, S. Manzini. 1993. Endogenous tachykinins play a role in IL-1-induced neutrophil accumulation: involvement of NK1 receptors. Immunology 80: 73
    OpenUrlPubMed
  8. ↵
    Ahluwalia, A., M. Perretti. 1996. Involvement of bradykinin B1 receptor antagonist in the polymorphonuclear leukocyte accumulation induced by IL-1β in vivo in the mouse. J. Immunol. 156: 269
    OpenUrlAbstract
  9. ↵
    Schierwagen, C., A. C. Bylund-Fellenius, C. Lundberg. 1990. Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity. J. Pharmacol. Methods 23: 79
    OpenUrlCrossRefPubMed
  10. ↵
    Waller, J., L. Siney, J. R. S. Hoult, S. D. Brain. 1997. A study of neurokinins and other oedema-inducing mediators and mechanisms in thermal injury. Clin. Exp. Pharm. Physiol. 24: 861
    OpenUrlCrossRefPubMed
  11. ↵
    Lendrum, A. C.. 1944. The staining of eosinophil polymorphs adrenochromoffin cells in histological sections. J. Pathol. Bacteriol. 56: 441
    OpenUrlCrossRef
  12. ↵
    Moroney, M. A., M. J. Alcaraz, R. A. Forder, F. Carey, J. R. S. Hoult. 1988. Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids. J. Pharm. Pharmacol. 40: 787
    OpenUrlCrossRefPubMed
  13. ↵
    Pinter, E., J. Szolcsanyi. 1995. Plasma extravasation in the skin and pelvic organs. Neuroscience 68: 603
    OpenUrlCrossRefPubMed
  14. ↵
    Emonds-Alt, X., J. D. Doutremepuich, M. Heaulme, G. Neliat, V. Santucci, R. Steinberg, P. Vilain, D. Bichon, J. P. Ducoux, V. Proietto, et al 1993. In-vitro and in-vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur. J. Pharmacol. 250: 403
    OpenUrlCrossRefPubMed
  15. ↵
    Mantione, C. R., R. Rodriguez. 1990. A bradykinin (BK)1 receptor antagonist blocks capsaicin-induced ear inflammation in mice. Br. J. Pharmacol. 99: 516
    OpenUrlCrossRefPubMed
  16. ↵
    Hock, F. J., K. Wirth, U. Albus, W. Linz, H. J. Gerhards, G. Wiemer, G. St. Henke, W. Breipohl, J. Knolle Koning, et al 1991. Hoe 140, a new potent and long acting bradykinin-antagonist: in vitro studies. Br. J. Pharmacol. 102: 769
    OpenUrlCrossRefPubMed
  17. ↵
    Lembeck, F., T. Griesbacher, M. Eckhardt, S. Henke, G. Breipohl, J. Knolle. 1991. New, long-acting, potent bradykinin antagonists. Br. J. Pharmacol. 102: 297
    OpenUrlCrossRefPubMed
  18. ↵
    Wirth, K., F. J. Hock, U. Albus, W. Linz, H. G. Alpermann, H. Anagnostopoulos, St. Henke, G. Breipohl, W. Konig, J. Knolle, and B. A. Scholkens. Hoe 140, a new potent and long acting bradykinin-antagonists: in vivo studies. Br. J. Pharmacol. 102:774.
  19. ↵
    Baluk, P., G. Thurston, T. J. Murphy, N. W. Bunnett, D. M. McDonald. 1999. Neurogenic plasma leakage in mouse airways. Br. J. Pharmacol. 126: 522
    OpenUrlCrossRefPubMed
  20. ↵
    Iwamoto, I., S. Tomoe, H. Tomioka, S. Yoshida. 1993. Leukotriene-B4 mediates substance-P-induced granulocyte infiltration into mouse skin: comparison with antigen-induced granulocyte infiltration. J. Immunol. 15: 2116
    OpenUrl
  21. ↵
    Rampart, M., T. J. Williams. 1988. Evidence that neutrophil accumulation induced by interleukin-1 requires both local protein biosynthesis and neutrophil CD18 antigen expression in vivo. Br. J. Pharmacol. 94: 1143
    OpenUrlCrossRefPubMed
  22. ↵
    Sayers, T. J., T. A. Wiltrout, C. A. Bull, A. C. Denn, A. M. Pilaro, B. Lokesh. 1988. Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse: prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor. J. Immunol. 141: 1670
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Faccioli, L. H., G. E. Souza, F. Q. Cunha, S. Poole, S. H. Ferreira. 1990. Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms. Agents Actions 344: 9
    OpenUrl
  24. ↵
    Osborn, L., C. Hession, R. Tizard, C. Vassallo, S. Luhavskyj, G. Chirosso, R. Lobb. 1989. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59: 1203
    OpenUrlCrossRefPubMed
  25. ↵
    Panes, J., M. Perry, D. N. Granger. 1999. Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention. Br. J. Pharmacol. 126: 537
    OpenUrlCrossRefPubMed
  26. ↵
    Mason, M. J., D. E. Van Epps. 1989. In vivo neutrophil emigration in response to interleukin-1 and tumor necrosis factor-α. J. Leukocyte Biol. 45: 62
    OpenUrlAbstract
  27. ↵
    Appleton, I., A. Tomlinson, P. R. Colville-Nash, D. A. Willoughby. 1993. Temporal and spatial immunolocalization of cytokines in murine chronic granulomatous tissue: implications for their role in tissue development and repair processes. Lab. Invest. 69: 405
    OpenUrlPubMed
  28. ↵
    Dawson, J., A. D. Sedgwick, J. C. Edwards, P. Lees. 1991. A comparative study of the cellular, exudative and histological responses to carrageenan, dextran and zymosan in the mouse. Int. J. Tissue React. 13: 171
    OpenUrlPubMed
  29. ↵
    Damas, J., G. Remacle-Volon. 1986. Mast cell amines and the oedema induced by zymosan and carrageenan in rats. Eur. J. Pharmacol. 121: 367
    OpenUrlCrossRefPubMed
  30. ↵
    Ridger, V. C., E. R. Pettipher, C. E. Bryant, S. D. Brain. 1997. Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and l-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin. J. Immunol. 159: 383
    OpenUrlAbstract
  31. ↵
    Winter, C. A., E. A. Risley, G. W. Nuss. 1962. Carrageenin-induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111: 544
    OpenUrl
  32. ↵
    Vinegar, R., J. F. Truax, J. L. Selph. 1976. Quantitative studies of the pathway to acute carrageenan inflammation. Fed. Proc. 35: 2447
    OpenUrlPubMed
  33. ↵
    Damas, J., G. Remacle-Volon. 1992. Influence of a long-acting bradykinin antagonist, Hoe 140, on some acute inflammatory reactions in the rat. Eur. J. Pharmacol. 211: 81
    OpenUrlCrossRefPubMed
  34. ↵
    Lam, F. Y., W. R. Ferrell. 1989. Inhibition of carrageenan induced inflammation in the rat knee joint by substance P antagonist. Ann. Rheum. Dis. 48: 928
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Lam, F. Y., W. R. Ferrell. 1991. Neurogenic component of different models of acute inflammation in the rat knee joint. Ann. Rheum. Dis. 50: 747
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Raychaudhuri, A., C. Colombo, G. Pastor, M. Wong, A. Y. Jeng. 1991. Effect of capsaicin on carrageenan-induced inflammation in rat pleurisy and exudate substance P levels. Agents Actions 34: 251
    OpenUrlCrossRefPubMed
  37. ↵
    Vianna, R. M. J., J. B. Calixto. 1998. Characterization of the receptor and the mechanisms underlying the inflammatory response induced by des-Arg9-BK in mouse pleurisy. Br. J. Pharmacol. 123: 281
    OpenUrlCrossRefPubMed
  38. ↵
    Ahluwalia, A., M. Perretti. 1999. B1 receptors as a new inflammatory target. Could this B the 1?. Trends Pharmacol. Sci. 20: 100
    OpenUrlCrossRefPubMed
  39. ↵
    Bertrand, C., P. Geppetti. 1996. Tachykinin and kinin receptor antagonists: therapeutic perspectives in allergic airway disease. Trends Pharmacol. Sci. 17: 255
    OpenUrlCrossRefPubMed
Back to top

In this issue

The Journal of Immunology: 164 (10)
The Journal of Immunology
Vol. 164, Issue 10
15 May 2000
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section