Neuroscience 125 (2004) 449 – 459
MUSTARD OIL INDUCES A TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 RECEPTOR-INDEPENDENT NEUROGENIC INFLAMMATION AND A NON-NEUROGENIC CELLULAR INFLAMMATORY COMPONENT IN MICE ´ . BA ´ NVO ¨ LGYI,a G. POZSGAI,a S. D. BRAIN,c A ´ NYI,a M. GHOSH,b Z. S. HELYES,a J. SZOLCSA B. MELEGHb AND E. PINTE´Ra*
Key words: knockout mice, neurokinin 1 receptor, SR140333, capsaicin-desensitisation, oedema, neutrophil accumulation.
a Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pe´cs, Szigeti u. 12, H-7624 Pe´cs, Hungary
A neurogenic component has been suggested to play a pivotal role in pathomechanisms of numerous inflammatoryimmune diseases like bronchial asthma, allergic rhinitis, conjunctivitis, dermatitis, rheumatoid arthritis, inflammatory bowel diseases and migraine (Brain, 1997; Szolcsanyi, 1996). Mustard oil (allyl-isothiocyanate) as well as capsaicin, have been historically used as chemical algogens in studies of neurogenic inflammation, with topical administration resulting in increases in blood flow and vascular permeability in rodent skin (Jancso´ et al., 1967; Louis et al., 1989). Mustard oil was first identified as a pungent plant extract, in this case from several members of the genus Brassica (e.g. B. nigra, B. juncea), but the mode of action by which it activates sensory nerves remains unclear. The mechanism by which capsaicin, the active substance responsible for the irritating/pungent effects of hot peppers of the Capsicum family, acts has already been identified. It selectively excites and in high dose desensitises a major subpopulation of nociceptive sensory nerve fibres classified as “capsaicin-sensitive afferents” (Szolcsanyi, 1996) Recent studies have identified and cloned the receptor for capsaicin, so called transient receptor potential vanilloid 1 (TRPV1; Caterina et al., 1997). Peripheral endings of these sensory fibres release neuropeptide mediators, such as tachykinins and calcitonin gene-related peptide which mediate efferent responses leading to vasodilatation and plasma protein extravasation with subsequent oedema formation (Brain, 1997). These local vascular changes are major features of neurogenic inflammation. The TRPV1 receptor is associated with a cation channel, which can be activated by noxious heat, protons, and pungent agents as capsaicin (Caterina et al., 1997), resiniferatoxin (Szallasi and Blumberg, 1999), zingerone (Liu et al., 2000), or gingerol (Dedov et al., 2002). Trevisani et al. (2002) reported that a very small molecule, ethanol, activated primary sensory neurones and TRPV1-expressing human embryonic kidney (HEK293) cells in a concentration dependent manner. The TRPV1 antagonist, capsazepine, inhibited this response. Substantial efforts have been made to elucidate the nature and physiological roles of endogenous ligands at TRPV1 receptors. Recently, several potential candidates have been identified such as anandamide (arachidonyl ethanolamide; Di Marzo et al., 2002), N-arachinodyl-dopamine (Huang et al., 2002), Noleyldopamine (Chu et al., 2003) or lipoxygenase products
b Department of Medical Genetics and Child Development, Faculty of Medicine, University of Pe´cs, H-7624 Pe´cs, Hungary c
Centre of Cardiovascular Biology and Medicine KCL, Guy’s Campus London, London, UK
Abstract—A neurogenic component has been suggested to play a pivotal role in a range of inflammatory/immune diseases. Mustard oil (allyl-isothiocyanate) has been used in studies of inflammation to mediate neurogenic vasodilatation and oedema in rodent skin. The aim of the present study was to analyse mustard oil-induced oedema and neutrophil accumulation in the mouse ear focussing on the roles of neurokinin 1 (NK1) and vanilloid (TRPV1) receptors using normal (BALB/c, C57BL/6) as well as NK1 and TRPV1 receptor knockout mice. A single or double treatment of 1% mustard oil on the BALB/c mouse ear induced ear oedema with responses diminished by 6 h. However a 25–30% increase in ear thickness was maintained by the hourly reapplication of mustard oil. Desensitisation of sensory nerves with capsaicin, or the NK1 receptor antagonist SR140333, inhibited oedema but only in the first 3 h. Neutrophil accumulation in response to mustard oil was inhibited neither by SR140333 nor capsaicin pre-treatment. An activating dose of capsaicin (2.5%) induced a large oedema in C57BL/6 wild-type mice that was minimal in TRPV1 receptor knockout mice. By comparison, mustard oil generated ear swelling was inhibited by SR140333 in wildtype and TRPV1 knockout mice. Repeated administration of mustard oil maintained 35% oedema in TRPV1 knockout animals and the lack of TRPV1 receptors did not alter the leukocyte accumulation. In contrast repeated treatment caused about 20% ear oedema in Sv129ⴙC57BL/6 wild-type mice but the absence of NK1 receptors significantly decreased the response. Neutrophil accumulation showed similar values in both groups. This study has revealed that mustard oil can act via both neurogenic and non-neurogenic mechanisms to mediate inflammation in the mouse ear. Importantly, the activation of the sensory nerves was still observed in TRPV1 knockout mice indicating that the neurogenic inflammatory component occurs via a TRPV1 receptor independent process. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹36-72-536218; fax: ⫹36-72-536218. E-mail address:
[email protected] (E. Pinte´r). Abbreviations: NK1, neurokinin 1; OD, optical density; PCR, polymerase chain reaction; SP, substance P; TRPV1, transient receptor potential vanilloid 1.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.01.009
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(Hwang et al., 2000). It is hoped that the therapeutic use of TRPV1 antagonists will provide a new generation of analgesic and anti-inflammatory drugs. Substance P (SP), the best known member of the tachykinin family, is a potent mediator of increased microvascular permeability acting via neurokinin 1 (NK1) receptors, most commonly situated on post-capillary endothelial cells (McDonald et al., 1996). Several studies have shown that SP plays a pivotal role in mediating neutrophil accumulation, especially in the rat airways (Baluk et al., 1999). Evidence indicates that SP promotes leukocyte accumulation in vivo secondary to mast cell activation (Matsuda et al., 1989; Suzuki et al., 1995) and release of mediators which induce neutrophil accumulation (Walsh et al., 1995; Yano et al., 1989). Indeed, our previous studies indicate that exogenous SP itself does not induce significant cell recruitment when injected into naive rat and mouse skin at doses up to 1 nmol (Cao et al., 2000; Pinte´r et al., 1999). Furthermore, there is little clear evidence to show that released endogenous pro-inflammatory neuropeptides are able to induce cellular inflammation. Studies with mustard oil are in keeping with the concept that it acts to stimulate sensory nerves. Mustard oil solution (20%) caused a pure neurogenic inflammation, which was not observed in a denervated component of the rat hind paw skin (Bester et al., 1998). First Jancso´ et al. (1967) published the ineffectiveness of 5% mustard oil on denervated rat skin after transsection of the saphenous nerve. Ten years later Jancso´ et al. (1977) demonstrated that neonatal capsaicin pre-treatment inhibited cutaneus plasma protein extravasation induced by 5% mustard oil in the rat. Subsequently numerous papers presented evidence using selective NK1 receptor antagonists that mustard oil induced neurogenic oedema mediated by NK1 receptors (Pinte´r et al., 1999; Amann et al., 1995, 2000; Lembeck et al., 1992; Szolcsanyi et al., 1998). There are only a few studies regarding mustard oil-evoked inflammation in mice (Inoue et al., 1997; Laird et al., 2000) but importantly they provided evidence that mustard oil may act to stimulate inflammation via separate mechanisms from capsaicin. Laird et al. (2001) demonstrated that capsaicin (0.3%) evoked pure neurogenic extravasation, that was abolished by denervation of the extrinsic innervation of the mouse colon. In contrast, mustard oil (0.25%) elicited plasma extravasation in both the intact and denervated colon, indicating that it may provoke direct damage of the mucosa. They also found that mustard oil induced an equivalent degree of oedema in the colon of NK1 receptor knockout mice, whereas capsaicin evoked significantly less response (Laird et al., 2000). Inoue and colleagues (1997) examined the mechanism of mustard oil-induced (0.5–20%) plasma extravasation and oedema in the mouse ear. They demonstrated a major difference between capsaicin and mustard oil stimulation in that desensitisation of plasma extravasation was not shown by reapplication of mustard oil to ears unlike to capsaicin which shows desensitisation. In addition the inflammatory response was unaffected by pre-treatment with H1 and 5-HT2 receptor antagonists or the capsaicin functional in-
hibitor Ruthenium Red, which inhibited capsaicin-induced oedema. However, the tachykinin NK1 receptor antagonist SR140333 inhibited the response for 5% mustard oil, which suggests that mustard oil elicits plasma extravasation through the release of sensory neuropeptides, but its mechanism of action differs from that for capsaicin. The aim of the present work was to investigate mustard oil-induced inflammation in the mouse ear, focussing on the roles of NK1, and TRPV1 receptors through use of a pharmacogenetic approach involving use of antagonists and NK1 and TRPV1 receptor knockout mice. We have now developed a model where the contribution of endogenous sensory neuropeptides to leukocyte recruitment, in addition to oedema formation, can be determined in the mouse skin. This has allowed us to investigate the effect of single and repeated application of mustard oil on the acute as well as the more chronic (4 – 6 h) cellular inflammatory phase.
EXPERIMENTAL PROCEDURES Animals Experiments were performed in Hungarian and British laboratories and the design of this study was carried out according to the Animals (Scientific Procedures) Act 1986 (Great Britain) and Act for Animal Protection and Forbearance 1998 (Hungary). Efforts were made to minimize the number of animals used and their suffering. BALB/c (20 –25 g) and C57BL/6 (20 – 25 g) strains were obtained from Charles River Ltd., Hungary. TRPV1 receptor knockout transgenic mice were donated by Dr. J. B. Davis, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline, Research and Development Ltd., New Frontiers Science Park, Essex, Harlow, UK, and bred in the animal house of the Hungarian laboratory. Wild-type and NK1 receptor knockout Sv129⫹C57BL/6 mice were a gift from Dr. N. Gerard, Perlmutter Laboratory Children’s Hospital, Boston, MA, USA, then bred in the animal house of KCL in London, UK. Animals were given standard diet and water ad libitum in climatically controlled environment. Both knockout strains displayed normal growth and behavioural characteristics.
Generation of NK1 receptor knockout mice NK1 receptor knockout mice were generated by partial deletion of exon 1 of the NK1 receptor gene, and replacement with lacZ/ neomycin resistance cassette as described by Bozic and coworkers (1996). The NK1 receptor wild-type mice (Sv129⫹C57BL/6) were bred in parallel with the knockouts from pairs of homozygous wild-type mice. Functional tests of genotype were used, with an absence of capsaicin-induced plasma extravasation indicating the homozygous knockout genotype.
Generation of TRPV1 receptor knockout mice The generation of TRPV1 receptor knockout mice was by homologous recombination in embryonic stem cells to generate a mouse lacking transmembrane domains 2– 4 of the mTRPV1 gene. Germline chimaeras were crossed onto C57BL/6 females to generate heterozygotes, which were inter-crossed giving rise to healthy homozygous mutant offspring in the expected Mendelian ratio, as described by Davis and co-workers (2000). Successful targeting of the locus and germline transmission was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. After genotyping by PCR, they were bred from homozygous knockout breeding pairs, so all offspring were also homozygous knockouts (TRPV1⫺/⫺).
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Fig. 1. Effect of single and double application of mustard oil (mo) on ear inflammation in the BALB/c mice mouse over 6 h. (a) Data are expressed as percent increase of ear thickness compared with the initial control value. (* P⬍0.05 vs. single application of mustard oil; ** P⬍0.01 vs. single application of mustard oil.) (b) Numbers of accumulated neutrophils/mouse in response to mustard oil application. (* P⬍0.05 vs. paraffin oil.) Values are means⫾S.E.M.; n⫽4 – 6 per group.
Application of substances Anesthesia was induced by ketamine (Richter Gedeon, Budapest, Hungary) (100 mg/kg i.p., repeated as required) with xylazine (Lavet Ltd., Budapest, Hungary) (5 mg/kg i.m.). Both ears were smeared with 1% mustard oil dissolved in paraffin oil, with 10 l applied to each of the inner and outer surfaces. This procedure was repeated every hour for 6 h. In separate experimental groups animals received either single or double treatments at the beginning of the 6 h experiment. In the control group, paraffin oil was applied on both ears in the same volume at the required times. In
the relevant groups, SR140333 (Sanofi-Synthelabo, Montpellier, France) (240 nmol/kg s.c.) was applied 15 min before mustard oil to inhibit NK1 receptors (Cao et al., 2000). At the end of the incubation period animals were killed by cervical dislocation and ears were dissected for histological procedures or put on ⫺20 °C for the neutrophil accumulation assay.
Systemic capsaicin-pretreatment Depletion of sensory neuropeptides from the capsaicin-sensitive neurones was achieved by systemic capsaicin desensitisation.
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Capsaicin was dissolved in ethanol (10%), Tween 80 (10%) and isotonic saline (80%). BALB/c mice under anesthesia received 30 mg/kg s.c. capsaicin, in the neck region, on 3 consecutive days. The cumulative dose was 90 mg/kg (Girolomoni and Tigelaar , 1990). The animals were used in the experiment 14 days after pre-treatment with capsaicin. The success of the procedure was validated by the eye wiping test. The capsaicin pre-treatment was considered satisfactory, if one drop of 0.1% capsaicin solution did not evoke wiping movements in the eye.
Measurement of ear oedema Ear thickness was measured with an engineer’s micrometer (Moore and Wright, Sheffield, England) with 0.1 mm accuracy, before challenge with test agents and after the challenge at different time points. Data were expressed as percent increase of ear thickness compared with the initial values.
Measurement of neutrophil accumulation The number of accumulated neutrophil cells was determined from the frozen ear samples. The ears were thawed and chopped into small pieces then homogenised in phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide detergent (1 ml buffer/ ear). The homogenate was centrifuged at 10,000⫻g at 4 °C for 10 min and 0.5 aliquots of supernatant placed in Eppendorf tubes. Neutrophil accumulation was assessed by comparing the myeloperoxidase enzyme activity in sample extracts with mouse standard leukocyte preparation. Myeloperoxidase activity was assayed using H2O2-3,3⬘5,5⬘-tetramethyl-benzidine (Sigma, St. Louis, MO, USA). Reactions were performed in 96-well microtitre plates in room temperature. Known quantities of mouse neutrophils, purified from peritoneal lavage samples following injection of 6% oyster glycogen dissolved in physiologic saline, were used as standard (Cao et al., 2000). The optical density (OD) at 620 nm was measured at 5 min intervals for 30 min, using a microplate reader and plotted. The reaction rate (⌬ OD/time) was derived from an 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 ear samples homogenates.
Histological studies Dissected ears were fixed in 4% paraformaldehyde. Cross-sections (6 m) were cut at the base of the ears after paraffin embedding and stained with haematoxylin-eosin. Chloroacetateesterase staining as myeloid marker was used to analyse the cellular phase of inflammation.
Statistics Results are expressed as mean⫾S.E.M. Comparisons between treated and non-treated animals were made by ANOVA followed by Dunnett’s post-test for oedema data and by unpaired t-test for neutrophil accumulation studies. Probability values P⬍0.05 were regarded as significant.
RESULTS Mustard oil-induced inflammation in BALB/c mice A single application of 1% mustard oil to BALB/c mouse ear induced a 15% increase in ear thickness, whereas double treatment elicited more pronounced response, in both cases the response had diminished by 6 h (Fig. 1a). Whilst the single treatment provoked a non-significant in-
crease in the number of accumulated leukocytes by 6 h, the double application induced statistically significant recruitment of myeloperoxidase positive cells in the skin samples compared with the paraffin oil-treated control group, which was only slightly affected by systemic capsaicin pre-treatment (Fig. 1b). This was unaffected by a protocol designed to desensitise capsaicin-sensory nerves. A 25–30% increase in ear thickness was maintained by the hourly reapplication of mustard oil over 6 h (Fig. 2a). Systemic capsaicin pre-treatment significantly inhibited oedema formation in the first 3 h, but oedema in this group increased from 3 to 6 h. The NK1 receptor antagonist SR140333 also blocked the swelling in the first 3 h but it had only limited effect in the second half, despite a repeated administration during the third hour (Fig. 2a). This manner of mustard oil treatment elicited significant neutrophil cell accumulation in the ear samples, but neither NK1 receptor antagonist SR140333 nor systemic capsaicin pretreatment inhibited the cellular infiltration (Fig. 2b). Mustard oil-induced inflammation in TRPV1 receptor knockout mice In the present study we used three different strains of the mice; therefore, we cannot compare responses in different strains directly. However, it is important to point out that although there were minor differences in the absolute numerical values amongst the different strains, these variations were not of significance. Thus a single application of 2.5% capsaicin solution induced a large oedema in the wild-type mouse but it was only a minimal in the ear of TRPV1 receptor knockout mice (Fig. 3a). By comparison mustard oil generated the same degree of ear swelling in knockout animals as in wild-type C57BL/6 mice, moreover a longer-lasting effect was observed in knockout animals, but this response was completely abolished by the NK1 receptor antagonist SR140333 in both groups of animals (Fig. 3b). The repeated administration of mustard oil maintained 30 – 40% increase of ear thickness in C57BL/6 mice. The inhibitory effect of SR140333 pre-treatment followed the similar kinetics as observed in BALB/c mice. It almost completely abolished the ear oedema the first 3 h but was not effective in the second half of the 6 h experiment (Fig. 4a). The lack of TRPV1 receptors did not alter the amount of extravasated leucocytes in response to mustard oil (Fig. 4b). Mustard oil-induced inflammation in NK1 receptor knockout mice Repeated treatment with 1% mustard oil caused about 20 –25% ear oedema in Sv129⫹C57BL/6 wild-type mice compared with the vehicle treated control. The lack of NK1 receptors significantly decreased the ear swelling, confirming the important role of SP in mustard oil-evoked oedema (Fig. 5a). The activity of myeloperoxidase enzyme was almost three times higher after mustard oil treatment compared with the paraffin oil treated controls, but did not show significant differences between NK1 receptor knockout and wild-type mice (Fig. 5b). These results support our previ-
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Fig. 2. Effect of SR140333 pre-treatment (240 nmol/kg) and capsaicin desensitisation on ear inflammation induced by repeatedly applied 1% mustard oil in BALB/c mice. (a) Data are expressed as percent increase of ear thickness compared with the initial control values. (* P⬍0.05 vs. mustard oil, ** P⬍0.01 vs. mustard oil; ⫹ P⬍0.05 vs. mustard oil, ⫹⫹ P⬍0.01 vs. mustard oil.) (b) Numbers of accumulated neutrophils/ear in response to mustard oil application. (** P⬍0.01 vs. paraffin oil.) Values are means⫾S.E.M.; n⫽5–7 per group.
ous data (see Fig. 2b) where the NK1 receptor antagonist SR140333 did not inhibit the mustard oil-induced leucocyte accumulation in BALB/c mice. Histological findings Fig. 6 shows that the repeated application of 1% mustard oil induced about 20 –30% increase of ear thickness. Leukocyte accumulation can be also observed compared with the paraffin oil-treated control as observed in the chloroacetate esterase-stained slides. Disruption of TRPV1 or
NK1 receptors did not influence the number of accumulated myeloid cells after mustard oil.
DISCUSSION This study has revealed that mustard oil can act via both neurogenic and non-neurogenic mechanisms to mediate inflammation in the mouse ear. Mustard oil, whether given as a single or repeated administration evoked oedema formation, observed as ear swelling, that at the early time points (0 –3 h) was mediated by neurogenic mechanisms. However, surpris-
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mustard oil 1x Fig. 3. (a) Effect of single application of 2.5% capsaicin on ear oedema on C57BL/6 wild-type (TRPV1⫹/⫹) mice and receptor knockout (TRPV1⫺/⫺) animals. (b) Single mustard oil treatment-induced ear oedema in C57BL/6 wild-type (TRPV1⫹/⫹), knockout (TRPV1⫺/⫺), and SR140333 pre-treated (TRPV1⫹/⫹, TRPV1⫺/⫺) animals. Data are expressed as percent increase of ear thickness compared with the initial control values. Values are means⫾S.E.M.; n⫽5–7 per group.
ingly the activation of the sensory nerves was still observed in TRPV1 knockout mice and thus we propose that activation occurs via a mechanism that is independent of the TRPV1 receptor. Secondly we demonstrate that mustard oil, upon repeated administration induced a non-neurogenic inflammatory response over 3– 6 h that consisted of sustained oedema formation and neutrophil accumulation. Our present data revealed that single application of 1% mustard oil induces a transient swelling (15% increase) in
the ear of BALB/c, which gradually and completely disappears in 6 h. This stimulus is not able to provoke a detectable leukocyte accumulation in the ear tissue, which means that the transient vascular changes are not followed by cellular events. However when application of mustard oil is repeated 1 h later, a significant increase in neutrophil accumulation is observed that is unaffected by systemic capsaicin desensitisation. Thus we provide evidence that mustard oil, in repeated doses, can mediate chemotactic effects,
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Fig. 4. (a) Ear oedema to repeatedly administered 1% mustard oil in C57BL/6 wild-type (TRPV1⫹/⫹) and receptor knockout (TRPV1⫺/⫺) mice. Results are expressed as percent increase of the ear thickness. (** P⬍0.01 vs. mustard oil in TRPV1⫺/⫺ animals; ⫹ P⬍0.05 vs. mustard oil in TRPV1⫹/⫹ animals, ⫹⫹ P⬍0.01 vs. mustard oil in TRPV1⫹/⫹ animals.) (b) Effect of mustard oil treatment on neutrophil accumulation in the ears of C57BL/6 wild-type (TRPV1⫹/⫹) and receptor knockout (TRPV1⫺/⫺) mice over 6 h. Data are expressed as mean⫾S.E.M.; n⫽5– 8.
which seem to be independent from the neurogenic components. Hourly repeated mustard oil treatment maintained a 25–30% continuous enhancement of ear thickness. Although previous papers suggested that mustard-oil up to 20% induces an exclusive neurogenic inflammation in the mouse (Inoue et al., 1997) and in the rat (Bester et al., 1998), our present data are consistent with a neurogenic stage, in the first 3 h followed by a non-neurogenic stage in the second 3 h in oedema formation during the 6 h experiment. This hypothesis is supported by results from capsaicin-desensitisa-
tion and NK1 receptor antagonist studies. Capsaicin, which depletes the peptide-content of capsaicin-sensitive sensory neurones prevents the early swelling, but it is ineffective in the second phase, and the NK1 receptor antagonist shows a similar profile of activity. We conclude that a cumulative dose of mustard oil produces non-neurogenic inflammation by direct activation of inflammatory cells in the mouse ear. Mustard oil, given as a repeated application, induces a three-fold increase in myeloperoxidase enzyme activity in the ear samples. The rise of myeloperoxi-
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Fig. 5. (a) Ear oedema to 1% mustard oil in Sv129⫹C57BL/6 wild-type (NK1⫹/⫹) and receptor knockout (NK1⫺/⫺) mice. (* P⬍0.05 vs. wild-type animals, ** P⬍0.01 vs. wild-type animals.) Results are expressed as percent increase in the ear thickness, means⫾S.E.M.; n⫽6. (b) Effect of mustard oil treatment on neutrophil accumulation in the ears of Sv129⫹C57BL/6 wild-type (NK1⫹/⫹) and receptor knockout (NK1⫺/⫺) mice over 6 h. (* P⬍0.05 vs. paraffin oil, *** P⬍0.001 vs. paraffin oil.) Data are shown as mean⫾S.E.M.; n⫽5– 8.
dase activity indicates a significant accumulation of neutrophil granulocytes, as supported by histological analysis. Neutrophil accumulation is not affected by either systemic capsaicin-desensitisation, or pre-treatment with the NK1 receptor antagonist SR140333; therefore, participation of sensory neuropeptides can be excluded. The original aim of the present study was to develop a model for investigating the cellular phase of neurogenic inflammation. Instead, in agreement with of our previous
findings (Cao et al., 2000; Pinte´r et al., 2002), we did not find evidence to support the concept that stimulation of sensory nerves leads to neutrophil accumulation in the naive skin. The capsaicin-induced ear swelling does not occur in TRPV1 receptor animals, but ear oedema after mustard oil is unaffected by the absence of TRPV1 receptors. Our findings from TRPV1 receptor knockout mice suggest that mustard oil acts on the capsaicin sensitive neurones by a TRPV1 receptor independent process. This
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Fig. 6. Representative light micrographs of mustard oil-induced ear inflammation. (A) Paraffin oil-treated control ear in BALB/c mouse.(B) Mustard oil (1%)-treated ear in BALB/c mouse and (C) capsaicin pre-treated BALB/c mouse. (D) Mustard oil (1%)-treated ear in C57BL/6 wild-type (TRPV1⫹/⫹) and (E) knockout (TRPV1⫺/⫺) mouse (haematoxylin– eosin staining, 100⫻). (F) Mustard oil (1%)-treated ear in Sv129⫹C57BL/6 wild-type (NK1⫹/⫹) and (G) knockout (NK1⫺/⫺) mouse (haematoxylin– eosin staining, 200⫻). Chloroacetate-esterase positive accumulated myeloid cells in the ear of BALB/c mice. (H) Paraffin oil-treated control (200⫻). (I) Mustard oil-treated ear (200⫻). (J) Mustard oil-treated ear (600⫻).
is in agreement with a previous paper demonstrating that mustard oil induces mouse skin inflammation through a mechanism different from capsaicin (Inoue et al., 1997).
Furthermore they found that mustard oil-induced plasma extravasation is not inhibited by Ruthenium Red, a nonspecific TRPV1 receptor inhibitor. The fact that the NK1
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receptor antagonist, SR140333 inhibits the early oedema both in C57BL/6 wild-type and TRPV1 receptor knockout mice clearly demonstrates that the neurogenic phase of mustard oil-induced inflammation is mediated by NK1 receptors. The role of TRPV1 receptors remains unclear in mediation of neutrophil accumulation. Results obtained from NK1 receptor knockout mice demonstrate the pivotal role of NK1 receptors in ear swelling initiated by mustard oil, suggesting that SP mediates the oedema. These data are in agreement with previous results where specific NK1 receptor antagonists, such as SR140333 (Amann et al., 1995; Inoue et al., 1997), and CP-96,345 (Lembeck et al., 1992) inhibited the mustard oil-evoked oedema in the mouse or rat skin. Although, repeated administration of mustard oil is also able to induce neutrophil recruitment in these animals, there is no evidence for the role of NK1 receptors in the cellular events.
CONCLUSIONS In conclusion we have demonstrated that in contrast to capsaicin, mustard oil does not activate sensory nerves via the TRPV1 receptor and cannot be considered as a pure neurogenic stimulus in the mouse skin in that we describe mustard oil-induced neurogenic oedema formation and non-neurogenic neutrophil accumulation. The precise mechanisms involved in mustard oil-induced activation of sensory nerves are unknown. Whilst it is possible that mustard oil acts via a receptor-mediated site, a nonspecific neuronal activating effect cannot be ruled out at this stage. We provide evidence that mustard oil in small concentrations acts on the capsaicin-sensitive nerves via TRPV1 receptor independent mechanisms in TRPV1 knockout mice to release neuropeptides that mediate neurogenic inflammation. Its selectivity for sensory neurons is demonstrated by results that show that mustard oil does not induce neurogenic inflammation in the denervated skin or after capsaicin desensitisation protocols. The mechanisms involved in the separate non-neurogenic mechanism involved in the observed neutrophil accumulation are also unclear at this stage. Acknowledgements—This study was funded by The Wellcome Trust International Research Development Award and Hungarian Research Grant OTKA-T-032548. E.P. held Janos Bolyai Fellowship. We acknowledge Dr. J. B. Davis, GlaxoSmithKline, R&D Ltd. UK for the gift of TRPV1 knockout mice. Special thanks Dr. N. Gerard, Perlmutter Laboratory, Children’s Hospital, Boston, MA, USA, for the generously providing NK1 knockout animals. The authors wish to thank Dr. Xavier Emonds-Alt from the SanofiSynthelabo for the gift of compound SR140333, Dr. Robin Poston and Dr. Tere´zia La´szlo´ for valuable advice for histological work. We also acknowledge Ms Csilla Za´dor and Ms Aniko´ Perkecz for excellent technical assistance and Mr. Andy Grant for his critical comments.
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(Accepted 7 January 2004)
