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Am J Physiol Heart Circ Physiol 292: H1821-H1827, 2007. First published December 8, 2006; doi:10.1152/ajpheart.00365.2006
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Lowering of interstitial fluid pressure after neurogenic inflammation in mouse skin is partly dependent on mast cells

Tine V. Karlsen, Athanasia Bletsa, Eli-Anne B. Gjerde, and Rolf K. Reed

Department of Biomedicine, University of Bergen, Bergen, Norway

Submitted 6 April 2006 ; accepted in final form 27 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neurogenic inflammation is known to induce lowering of interstitial fluid pressure (Pif) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of Pif by using KitW/KitW-v mice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). Pif was measured in paw skin of anesthetized (fentanyl-fluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of Pif in KitW/KitW-v mice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant (P < 0.05) lowering of Pif in wild-type mice to –2.16 ± 0.28, –1.96 ± 0.11, and –2.22 ± 0.19 mmHg, respectively, compared with vehicle (–0.49 ± 0.11 mmHg). In KitW/KitW-v mice the Pif response to SP was completely abolished (–0.53 ± 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (–1.33 ± 0.13 and –1.42 ± 0.13 mmHg, respectively) although significantly (P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of KitW/KitW-v compared with wild-type mice. We conclude that lowering of Pif normally depends on mast cells. However, the sensory nerves can also elicit a lowering of Pif that is independent of mast cells.

KitW/KitW-v mice; neuropeptides; micropuncture

NEUROGENIC INFLAMMATION is initiated by the activation of sensory C fibers in response to heat, mechanical, or chemical stimuli. This activation results in release of neuropeptides, mainly substance P (SP), calcitonin gene-related peptide (CGRP), and neurokinin A, from the capsaicin-sensitive nerve endings. Capsaicin releases neuropeptides through binding to the vanilloid receptor subtype 1 (VR1) (9), which in turn induces an inflammatory response in various tissues of both rodents and humans (2, 21). This response is characterized by increased arteriolar vasodilatation and protein extravasation due to increased permeability of the postcapillary venules resulting in fluid accumulation in the tissue.

Previous reports from our research group have shown that the response in the initial phase of neurogenic inflammation involves lowering of interstitial fluid pressure (Pif) as observed both in rat trachea (16) and mouse skin (25). Pif is important in tissue fluid homeostasis both in the regulation of fluid filtration across the capillary and as the filling pressure for the lymphatics (1). In contrast to the classic view of Pif serving as an edema-preventing mechanism, Pif has been shown to play an active part in the initial phase of edema in a number of inflammatory models with a lowering of Pif to more negative values (16, 31, 34, 35). According to Starling's hypothesis, this lowering of Pif will increase net filtration pressure across the capillary and hence contribute to fluid accumulation in the interstitial space.

Mast cells have been thought of as possible participants in the neurogenic inflammatory response because they contain numerous inflammatory mediators and are localized in close proximity to the capsaicin-sensitive nerve endings (37, 39). Alterations in the number of SP-reactive fibers and mast cell-nerve contacts have been reported as parts of the pathology of several diseases (3, 19, 45). SP can activate mast cells both through binding of the neurokinin-1 receptor present on these cells (8) and through receptor-independent mechanisms (10). The significance of mast cell activation as a part of neurogenic inflammation has been questioned in a number of studies, and the results are yet not conclusive. Supporting the involvement of mast cells are the findings that stimulation of the rat saphenous nerve induces mast cell degranulation (27) and that SP in high concentrations (40) as well as topical application of capsaicin (7) induce cutaneous mast cell degranulation in human forearm. In contrast, Petersen et al. (32) detected no release of SP and histamine after capsaicin injection in the same area.

In light of the knowledge that capsaicin-sensitive nerve endings are localized close to connective tissue mast cells (CTMCs) in mouse skin (6) and that mast cell activation by compound 48/80 (C48/80) induces a lowering of Pif in skin of both rats and mice (20, 25, 36), the question arises as to whether mast cells are involved in the events leading to lowering of Pif in neurogenic inflammation. In a recent study (25) we examined this issue by measuring the response in Pif after challenge with capsaicin, SP, or CGRP in mice with deficient mast cells due to the lack of sulfated heparin (NDST-2–/– mice), a component found exclusively in CTMCs (14). In NDST-2–/– mice Pif was lowered to the same extent as in normal wild-type mice in response to neurogenic inflammation, and we concluded that lowering of Pif was at least not exclusively dependent on intact CTMCs. These mice have a reduced number of mast cells that are not functionally normal, but nevertheless they displayed a partial Pif lowering as well as a slight increase in protein extravasation in response to mast cell activation by C48/80; hence we were not able to conclusively exclude the involvement of CTMCs in our study. To further explore the potential involvement of mast cells in neurogenic inflammation, we have in the present study repeated the experiments in a mouse model appreciated for its almost complete absence of mast cells including CTMCs, namely, the KitW/KitW-v mouse (15, 26).

We investigated the effect on Pif in KitW/KitW-v mice in response to mast cell activation by C48/80 and explored whether capsaicin, SP, or CGRP was able to induce a lowering of Pif as seen in both control mice and NDST-2–/– mice. In agreement with the previous studies (25), the agents were administered systemically rather than topically in order to standardize the administration. Finally, we stained SP- and CGRP-containing fibers immunohistochemically in paw sections from wild-type and KitW/KitW-v mice. This immunostaining was performed to verify that the altered effect of capsaicin on Pif was not due to rearrangements of SP- and CGRP-containing fibers in the KitW/KitW-v mice in response to the absence of CTMCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Female mast cell-deficient (WBB6F1-KitW/KitW-v) mice and wild-type (WBB6F1+/+) mice were purchased from Jackson Laboratories (West Grove, PA). KitW/KitW-v mice have mutations affecting the c-kit tyrosine kinase receptor, which is necessary for normal mast cell development, and they have been shown to be virtually devoid of mast cells of all types including CTMCs (15, 26). The mice received water and food ad libitum and weighed between 20 and 30 g at the time of the experiment. During the experimental period the mice were anesthetized with a 1:1 mixture (0.1 ml/10 g body wt) of midazolam (Midazolam alpharma; Roche) and fentanyl-fluanison (Hypnorm; Janssen) injected subcutaneously. Intravenous injections were performed through a PE-25 catheter in the jugular vein, and blood pressure was monitored through a PE-25 catheter placed inside the carotid artery. As a part of the experimental protocol cardiac arrest was induced with an intravenous injection of saturated KCl while the mice were still anesthetized. All experiments were performed with the approval of and in accordance with the Norwegian State Commission for Laboratory Animals.

Interstitial Fluid Pressure

Pif was measured with sharpened glass capillaries (tip diameter 4–7 µm) connected to a servo-controlled counterpressure system (41, 42). The counterpressure generated by a servo-controlled pump (model 201, Ling Dynamic Systems, Royston, UK) was recorded with a pressure transducer (model 1280C, Hewlett-Packard). The mice were placed in the supine position, and the measurements were made through intact skin on the dorsal side of the left hind paw. The glass capillaries were filled with 0.5 M NaCl colored with Evans blue dye to visualize the tip when puncturing the skin. Zero measurements were made at the level of the puncture site in a cup filled with isotonic saline. Measurements were accepted after fulfillment of the following three criteria (42). 1) Increased feedback gain did not alter the measured pressure. 2) Suction applied to the pipette increased electrical resistance, confirming open communication with the tissue fluid. 3) Zero-pressure measurements before and after the Pif registration were unchanged.

Acute inflammation increases transcapillary fluid flux, which in turn could cause fluid to accumulate in the interstitium. Such fluid accumulation raises Pif and therefore cause an underestimation of an initial lowering of Pif. Thus circulatory arrest was induced shortly after the injection of test substances, i.e., when they had been distributed evenly by the circulatory system, but with minimum time for the inflammation to take place and therefore a response in Pif that has not been blunted by interstitial fluid accumulation.

Experimental Protocol

Pif was first measured with an intact circulation and before the introduction of test substance and subsequently measured for 60 min after cardiac arrest. To study the time course of Pif the pressure recordings were averaged for the following periods: 0–15, 16–30, 31–45, and 46–60 min. The entire period from 0 to 60 min was averaged for statistical comparison of the different experimental groups. All test substances were purchased from Sigma-Aldrich (Stockholm, Sweden). The doses and the circulation times of the different test substances were chosen in accordance with previous studies from our laboratory (16, 25). Both wild-type and KitW/KitW-v mice received the same treatments in the following five experimental groups.

Vehicle. NaCl (0.1 ml, 0.9%) was administrated intravenously and circulated 2 min before cardiac arrest was induced by intravenous injection of saturated KCl.

Compound 48/80. C48/80 (0.1 ml, 200 µg) was administrated intravenously and circulated 2 min before cardiac arrest was induced.

Capsaicin. Capsaicin (0.1 ml, 10 nmol) was administrated intravenously and circulated 1 min before cardiac arrest was induced.

Substance P. SP (0.1 ml, 0.1 nmol) was administrated intravenously and circulated 1 min before cardiac arrest was induced.

Calcitonin gene-related peptide. CGRP (0.1 ml, 0.1 nmol) was administrated intravenously and circulated 1 min before cardiac arrest was induced.

Immunohistochemistry

KitW/KitW-v mice (n = 3) and wild-type mice (n = 3) were anesthetized with a 1:1 mixture of midazolam and fentanyl-fluanison as previously described. Cardiac arrest was then induced by intracardiac injection of saturated KCl under anesthesia. The paws were cut with scissors and fixed overnight in Zamboni fixative (4% paraformaldehyde with 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). They were then rinsed in 0.1 M phosphate buffer and placed in 10% EDTA, pH 7.4. After decalcification, the paws were rinsed in phosphate buffer and soaked overnight in 30% sucrose solution for cryoprotection.

Serial 35-µm-thick sections were made in a freezing slide microtome. Immunoreactions were performed on precoated glass slides (SuperFrost Plus, MenzelGlaser, Braunschweig, Germany). After several rinses in phosphate-buffered saline (PBS), the sections were incubated for 30 min in methanol containing 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity. Preincubation was done in 2.5% normal goat serum (Vector Laboratories, Burlingame, CA) for 2 h. Afterwards, the sections were incubated in anti-rat SP (1:3,000 dilution, Peninsula Laboratories) or anti-human {alpha}-CGRP (1:6,000 dilution, Peninsula Laboratories) polyclonal antibodies raised in rabbit for 72 h at 4°C. After several rinses in PBS, secondary antibody incubation was performed in goat anti-rabbit biotinylated immunoglobulin G (1:1,000 dilution, Vector) for 1 h. Antigen-antibody complexes were detected by the avidin-biotin peroxidase (ABC) reaction with a commercially available kit (Vectastain ABC kit, Vector) and visualized with 3,3'-diaminobenzidine (Sigma) in the presence of 0.2% (NH4)2Ni(SO4)26H2O to enhance the immunostaining. Finally, the sections were counterstained with methylene blue/azure II in 1% sodium borate and distilled water. They were then dehydrated in graded alcohol series, cleared in xylene, and coverslipped with Eukitt (O. Kindler, Freiburg, Germany). Approximately 200 sections were stained for each of the primary antibodies and evaluated in a Nicon photomicroscope (Nikon Eclipse E600, Nikon Instruments).

Immunocontrols were routinely performed by replacement of the primary antibody with PBS. The controls did not show any immunolabeling.

Ten randomly selected microphotographs (x40) from each of the four groups were used for quantitative analysis of the immunostained nerves. A grid was placed on each photograph (Lucia 5.0 software, Laboratory Imaging, Prague, Czech Republic), and a visual field of 0.15 x 0.15 mm was evaluated. The immunostained nerves were quantified as nerve fibers crossing the grid lines on the visual field, and a mean score of fibers crossing an area of 0.05 x 0.05 mm was calculated for each visual field.

Statistical Analysis

All values of Pif are given as means (SD) for the entire experimental period from 0 to 60 min. Statistical analysis on Pif was performed on the mean of the 60-min period by using one-way ANOVA and subsequent Bonferroni and t-tests. To detect quantitative differences in the skin innervation between wild-type and KitW/KitW-v mice, a t-test was performed. Blood pressures were compared with paired t-tests within the experimental groups. A value of P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Interstitial Fluid Pressure

Pif was stable in vehicle-treated animals for the entire 60-min period in both wild-type and KitW/KitW-v mice, and there was no significant difference in Pif either between the two strains of mice or between measurements performed in vivo compared with measurements after circulatory arrest. Measurements of Pif over time were compared in wild-type and KitW/KitW-v mice in response to injection of C48/80, capsaicin, SP, or CGRP (Fig. 1). In wild-type mice, treatment with any of the different test substances lowered Pif significantly (P < 0.05) compared with the vehicle-treated mice (Table 1). KitW/KitW-v mice had no significant response in Pif after treatment with either C48/80 or SP compared with vehicle, while capsaicin and CGRP induced a significant (P < 0.05) lowering of Pif compared with vehicle (Table 1). However, the lowering of Pif induced by capsaicin and CGRP in KitW/KitW-v mice was also significantly (P < 0.05) less than the lowering in Pif observed in wild-type mice after the same treatment. Thus C48/80 and SP induced no response in KitW/KitW-v mice, while capsaicin and CGRP induced a significant, but attenuated, response compared with wild-type mice.


Figure 1
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  		Fig. 1. Effects of intravenous administration of 200 µg of compound 48/80 (C48/80; A), 10 nmol of capsaicin (B), 0.1 nmol of substance P (SP; C), and 0.1 nmol of calcitonin gene-related peptide (CGRP; D) on interstitial fluid pressure (Pif) in paw skin of wild-type mice and mast cell-deficient KitW/KitW-v mice. Vehicle (0.1 ml 0.9% NaCl)-treated wild-type and KitW/KitW-v mice are also shown. Values are means (SD). *P < 0.05, significant lowering of Pif comparing the average of the 60-min period with vehicle. §P < 0.05, significant difference from the response in wild-type mice comparing the average of the 60-min period in both groups.

 

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  		Table 1. Effects of C48/80, capsaicin, SP, and CGRP on interstitial fluid pressure in wild-type mice and mast cell-deficient KitW/KitW-v mice

 
Arterial Blood Pressure

Before the introduction of test substance the mean arterial blood pressure averaged 85.8 (10.8) and 86.3 (14.6) mmHg in wild-type and KitW/KitW-v mice, respectively. Challenge with C48/80 induced a significant (P < 0.05) lowering of blood pressure in wild-type mice [from 87.5 (6.9) to 57.5 (25.2) mmHg], while no lowering was observed in KitW/KitW-v mice (Table 2). Capsaicin had no significant effect on blood pressure in either of the groups. CGRP lowered blood pressure significantly (P < 0.05) in both wild-type [from 88.3 (9.3) to 72.5 (18.6) mmHg] and KitW/KitW-v [from 95.8 (7.4) to 68.3 (12.1) mmHg] mice. SP did not lower blood pressure significantly in either of the groups.


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  		Table 2. Mean arterial blood pressure in wild-type mice and mast cell-deficient KitW/KitW-v mice before and after injection of C48/80, capsaicin, SP, and CGRP

 
Immunohistochemistry

The immunohistochemical staining of paw sections revealed no qualitative differences in localization or number of CGRP- and SP-immunoreactive (IR) fibers between wild-type and KitW/KitW-v mice (Figs. 2 and 3). The mean score of CGRP- and SP-IR fibers crossing an area of 0.05 x 0.05 mm was 2.21 (0.99) and 1.57 (0.73), respectively, in wild-type mouse skin. No quantitative differences were found compared with KitW/KitW-v mice [2.57 (1.20) and 1.50 (0.54) for CGRP-IR and SP-IR fibers, respectively].


Figure 2
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  		Fig. 2. Immunohistochemical microphotographs of mouse paw skin stained for CGRP. Strongly positive CGRP-immunoreactive (IR) fibers are seen in the dermis of both wild-type (WT; A) and mast cell-deficient KitW/KitW-v (WW-v; B) mice. Note that the dermal CGRP-IR fibers are large and knoblike, whereas thinner fibers are associated with the hair follicles and even thinner free nerve endings are seen at the epidermis. Bars = 100 µm.

 

Figure 3
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  		Fig. 3. Immunohistochemical microphotographs of mouse paw skin stained for SP. No differences were observed in the distribution of SP-IR fibers between wild-type and KitW/KitW-v mice. A and B: thin SP-IR fibers localized in the dermis (A) and at the border of dermis-epidermis (B) in wild-type mice. C and D: sections from KitW/KitW-v mice show large SP-IR fibers associated with blood vessels in the dermis (C), whereas much thinner SP-IR fibers are seen around the hair follicles (D). Bars = 100 µm.

 
In both strains epidermal, dermal, and subcutaneous CGRP- and SP-IR fibers were identified and exhibited the same localization. Specifically, there were numerous strongly positive varicose dermal CGRP-IR nerve fibers associated with blood vessels and hair follicles. The follicular network was distributed around the hair shaft and the dermal papilla at the bottom of the hair follicle. In addition, thinner CGRP-IR fibers were associated with the erector pili muscle and the sebaceous glands next to the hair follicles. Freely terminating CGRP-IR fibers were seen in the epidermis, and they were thinner and less knoblike than those in the dermis.

In both strains SP-IR fibers were much thinner and less numerous than CGRP-IR fibers. The distribution was the same: they were associated with blood vessels and hair follicles, and they were rarely seen in clusterlike structures in the dermis. There were even fewer free nerve endings at the border of epidermis-dermis.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we report that lowering of Pif in response to the neuropeptide SP is completely dependent on the presence of CTMCs, while CGRP can induce a partial lowering of Pif in the absence of CTMCs. Capsaicin, which induces the release of both neuropeptides from capsaicin-sensitive nerve endings, is only capable of partly lowering Pif in the absence of CTMCs. Furthermore, as the distribution and density of SP- and CGRP-IR fibers is similar in wild-type and KitW/KitW-v mice, the difference in response in Pif is most likely not due to a difference in sensory neuropeptides and their nerves.

The present study is a continuation of our previous studies on the role of mast cells in neurogenic inflammation in mice (25). We extend our previous study on a knockout mouse with defective mast cells by studying lowering of Pif during neurogenic inflammation in mast cell-deficient mice. Lowering of Pif to more negative values contributes to edema in a number of inflammatory models in rodents, including mast cell activation (20, 36) and neurogenic inflammation (16, 25). Induction of neurogenic inflammation by exogenous SP and CGRP or endogenous release of neuropeptides by capsaicin lowers Pif in rat trachea and mice skin (16, 25) as well as in rat trachea after vagal nerve stimulation (43). Mast cells contain a range of inflammatory mediators and are located in the proximity of the sensory nerve fibers. Although mast cells express SP receptors, their involvement in neurogenic inflammation has been debated. Neuropeptides can induce histamine release in a number of tissues (12, 13, 23, 29), but it has been questioned whether endogenously released neuropeptides are present in sufficient amounts to induce mast cell activation and subsequent histamine release in human skin (22, 32, 40).

The agents used in the present study will have vascular effects such as vasodilatation, raising capillary pressure, and protein permeability, in turn lowering the capillary reflection coefficient for protein and the transcapillary oncotic gradient. Both phenomena will contribute in enhancing the capillary fluid filtration. Arterial blood pressure did not fall in response to C48/80 and capsaicin in KitW/KitW-v mice, while CGRP and SP induced a fall, although not significant for SP. Wild-type mice had similar responses, apart from an effect of C48/80 on blood pressure. According to a previous study by Gjerde et al. (17) Pif is not affected by a fall in arterial blood pressure induced by sodium nitroprusside. In the present study we focused on the agents’ role in lowering of Pif. Normally, Pif counteracts edema formation (18). However, in several studies, including those on neurogenic inflammation (16, 25, 43), Pif is initially lowered and will therefore enhance rather than limit the edema formation (33). The events involved in lowering of Pif involve the swelling properties of the hyaluronan and glycosaminoglycans of the tissues as well as the {alpha}2beta1-integrins attaching the connective tissue cells to the collagen scaffolding of the tissue (33). The connective tissue cells normally restrain the swelling of the hyaluronan-glycosaminoglycan gel by applying stress to the collagen fibers via the {alpha}2beta1-integrins (33). When this tension is released, the gel is allowed to expand and Pif is lowered until a new balance is achieved between the expanding gel, the fibers, and a lowered Pif. Although Pif is able to enhance capillary fluid filtration during the initial edema formation, Pif will rise when edema is formed and maintenance of the edema requires increased capillary pressure and/or capillary permeability. However, the phenomenon of lowering Pif is principally important in that it assigns an "active" role to the loose connective tissues with regard to transcapillary transport of fluid.

In partly CTMC-deficient mice (NDST-2–/– mice) Pif was lowered to the same extent in response to neuropeptides or capsaicin (25). Although the skin of NDST-2–/– mice contains a reduced number of severely altered mast cells (14), they revealed a partial response in Pif to C48/80, indicating that CTMC involvement could not be excluded (25), but nevertheless gave strong indications that lowering of Pif in response to neuropeptide challenge was not completely dependent on intact CTMCs.

In the present study we attempted to further elucidate CTMC involvement in neurogenic inflammation by using KitW/KitW-v mice, which are completely devoid of mast cells (15, 26). In wild-type mice Pif was lowered significantly from –0.49 mmHg in vehicle-treated mice to –2.03 mmHg after C48/80 challenge, comparable to the results obtained previously (25). In contrast, there was no significant response to C48/80 in KitW/KitW-v mice, and this was different than previously observed in the NDST-2–/– mice, in which C48/80 resulted in a partial Pif response (see Table 3). Thus we conclude from these findings that the presence of CTMCs in the skin of KitW/KitW-v mice is negligible and not able to induce an effect on Pif.


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  		Table 3. Overview of interstitial fluid pressure responses in mast cell-deficient mouse strains NDST-2–/– and KitW/KitW-v and their corresponding wild types after challenge with C48/80, capsaicin, SP, or CGRP

 
Challenge with SP, CGRP, or capsaicin induced a lowering of Pif in wild-type mice as observed previously (25). The effect observed by capsaicin could potentially be attributed to both endogenously released SP and CGRP from nerve fibers as well as direct mast cell activation through VR1 known to be present on mast cells (5, 38). While the response in Pif to either of these substances was unaffected in the partially CTMC-deficient NDST-2–/– mice (25), this was not repeated in the KitW/KitW-v mice (Table 3). The lowering of Pif observed in wild-type mice after SP was completely abolished in the KitW/KitW-v mice, while CGRP and capsaicin induced a lowering of Pif, which was, however, significantly attenuated compared with wild-type mice. Considering that the distribution of sensory nerve endings is dynamic and that mast cell-nerve interactions have been reported to be altered in certain diseases (3, 19, 45), we wanted to exclude the possibility that the absence of CTMCs could influence the presence of sensory nerve endings in KitW/KitW-v mice. Staining for SP-IR and CGRP-IR fibers in skin sections revealed no difference in distribution and density of such fibers between KitW/KitW-v mice and wild-type mice. Although not all the sensory axons immunoreactive for CGRP and SP are capsaicin sensitive (24), the majority of them in rodent skin are (11), and therefore this finding strongly suggests that the reduced Pif response after challenge with capsaicin in KitW/KitW-v mice is attributable to a direct effect of mast cell absence and not to a reduced release of endogenous SP or CGRP from a reduced number of capsaicin-sensitive nerve endings. Also, the effect of capsaicin without an effect of C48/80 strongly points to a direct effect of released neuropeptides from the sensory nerves on the connective tissue cells. The different response of SP and CGRP between KitW/KitW-v and wild-type mice could well be due to a dose-response effect but is nevertheless an effect due to presence and absence of mast cells. In an elegant study Yamamoto et al. (44) demonstrated that coculture of fibroblasts and mast cells significantly enhanced the contraction of a collagen fiber lattice. This effect was ascribed to stem cell factor/c-kit. This would not seem to contradict our conclusion that there is a direct effect of the neuropeptides on the connective tissue cells responsible for causing a lowering of Pif.

The attenuated lowering of Pif after challenge with neuropeptides and capsaicin in KitW/KitW-v mice, in contrast to the intact Pif lowering found in NDST-2–/– mice (Table 3), could be attributed to inflammatory mediators in the few mast cells present in NDST-2–/– mice. Although the histamine content of these mast cells is reduced (14), other inflammatory mediators like cytokines and prostaglandins could be present, although this has not been studied in NDST-2–/– mice. Such mediators were shown previously to lower Pif in rat skin (4, 30). Given our present findings in KitW/KitW-v mice, it seems plausible that there are enough mediators released on CTMC activation in NDST-2–/– mice to sustain a full response in Pif in response to neuropeptides.

To summarize, the present study clearly demonstrates the involvement of mast cells in lowering of Pif in neurogenic inflammation. Furthermore, the attenuated lowering of Pif after capsaicin also demonstrates an effect on Pif mediated directly via the sensory nerves to the connective tissue cells without the involvement of mast cells. Finally, the attenuated capsaicin and CGRP response could suggest that CGRP is responsible for this effect, although a contribution from SP at higher doses cannot be excluded. Nevertheless, since the present dose of SP otherwise elicits a full Pif-lowering response, it is likely that our observation demonstrates the lack of such an effect of SP.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study received financial support from the Norwegian Research Council and the Norwegian Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. V. Karlsen, Dept. of Biomedicine, Univ. of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway (e-mail:tine.karlsen{at}biomed.uib.no)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aukland K, Reed RK. Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol Rev 73: 1–78, 1993.[Abstract/Free Full Text]
  2. Baluk P, Thurston G, Murphy TJ, Bunnett NW, McDonald DM. Neurogenic plasma leakage in mouse airways. Br J Pharmacol 126: 522–528, 1999.[CrossRef][ISI][Medline]
  3. Batbayar B, Somogyi J, Zelles T, Feher E. Immunohistochemical analysis of substance P containing nerve fibres and their contacts with mast cells in the diabetic rat's tongue. Acta Biol Hung 54: 275–283, 2003.[CrossRef][ISI][Medline]
  4. Berg A, Ekwall AK, Rubin K, Stjernschantz J, Reed RK. Effect of PGE1, PGI2, and PGF2{alpha} analogs on collagen gel compaction in vitro and interstitial pressure in vivo. Am J Physiol Heart Circ Physiol 274: H663–H671, 1998.[Abstract/Free Full Text]
  5. Biro T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, Acs P, Paus R, Blumberg PM. Characterization of functional vanilloid receptors expressed by mast cells. Blood 91: 1332–1340, 1998.[Abstract/Free Full Text]
  6. Botchkarev VA, Eichmuller S, Peters EM, Pietsch P, Johansson O, Maurer M, Paus R. A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle-dependent changes in mast cell-nerve fiber contacts in murine skin. Arch Dermatol Res 289: 292–302, 1997.[CrossRef][ISI][Medline]
  7. Bunker CB, Cerio R, Bull HA, Evans J, Dowd PM, Foreman JC. The effect of capsaicin application on mast cells in normal human skin. Agents Actions 33: 195–196, 1991.[CrossRef][ISI][Medline]
  8. Cao T, Gerard NP, Brain SD. Use of NK1 knockout mice to analyze substance P-induced edema formation. Am J Physiol Regul Integr Comp Physiol 277: R476–R481, 1999.[Abstract/Free Full Text]
  9. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389: 816–824, 1997.[CrossRef][Medline]
  10. Devillier P, Drapeau G, Renoux M, Regoli D. Role of the N-terminal arginine in the histamine-releasing activity of substance P, bradykinin and related peptides. Eur J Pharmacol 168: 53–60, 1989.[CrossRef][ISI][Medline]
  11. Dux M, Sann H, Schemann M, Jancso G. Changes in fibre populations of the rat hairy skin following selective chemodenervation by capsaicin. Cell Tissue Res 296: 471–477, 1999.[CrossRef][ISI][Medline]
  12. Ebertz JM, Hirshman CA, Kettelkamp NS, Uno H, Hanifin JM. Substance P-induced histamine release in human cutaneous mast cells. J Invest Dermatol 88: 682–685, 1987.[CrossRef][ISI][Medline]
  13. Foreman JC, Jordan CC, Piotrowski W. Interaction of neurotensin with the substance P receptor mediating histamine release from rat mast cells and the flare in human skin. Br J Pharmacol 77: 531–539, 1982.[ISI][Medline]
  14. Forsberg E, Pejler G, Ringvall M, Lunderius C, Tomasini-Johansson B, Kusche-Gullberg M, Eriksson I, Ledin J, Hellman L, Kjellen L. Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme. Nature 400: 773–776, 1999.[CrossRef][Medline]
  15. Galli SJ, Tsai M, Gordon JR, Geissler EN, Wershil BK. Analyzing mast cell development and function using mice carrying mutations at W/c-kit or Sl/MGF (SCF) loci. Ann NY Acad Sci 664: 69–88, 1992.[Abstract]
  16. Gjerde EA, Karlsen TV, Reed RK. Lowering of interstitial fluid pressure in rat trachea after substance P alone and in combination with calcitonin gene-related peptide. Acta Physiol Scand 178: 123–127, 2003.[CrossRef][ISI][Medline]
  17. Gjerde EA, Woie K, Wei ET, Reed RK. Corticotropin-releasing hormone inhibits lowering of interstitial pressure in rat trachea after neurogenic inflammation. Eur J Pharmacol 352: 99–102, 1998.[CrossRef][ISI][Medline]
  18. Guyton AC, Granger HJ, Taylor AE. Interstitial fluid pressure. Physiol Rev 51: 527–563, 1971.[Free Full Text]
  19. Hagforsen E, Nordlind K, Michaelsson G. Skin nerve fibres and their contacts with mast cells in patients with palmoplantar pustulosis. Arch Dermatol Res 292: 269–274, 2000.[CrossRef][ISI][Medline]
  20. Heuchel R, Berg A, Tallquist M, Ahlen K, Reed RK, Rubin K, Claesson-Welsh L, Heldin CH, Soriano P. Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling. Proc Natl Acad Sci USA 96: 11410–11415, 1999.[Abstract/Free Full Text]
  21. Holzer P. Neurogenic vasodilatation and plasma leakage in the skin. Gen Pharmacol 30: 5–11, 1998.[ISI][Medline]
  22. Huttunen M, Harvima IT, Ackermann L, Harvima RJ, Naukkarinen A, Horsmanheimo M. Neuropeptide- and capsaicin-induced histamine release in skin monitored with the microdialysis technique. Acta Derm Venereol 76: 205–209, 1996.[ISI][Medline]
  23. Johnson AR, Erdos EG. Release of histamine from mast cells by vasoactive peptides. Proc Soc Exp Biol Med 142: 1252–1256, 1973.[Medline]
  24. Karanth SS, Springall DR, Kuhn DM, Levene MM, Polak JM. An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals. Am J Anat 191: 369–383, 1991.[CrossRef][ISI][Medline]
  25. Karlsen TV, Iversen VV, Forsberg E, Kjellen L, Reed RK, Gjerde EA. Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme. Am J Physiol Heart Circ Physiol 286: H884–H888, 2004.[Abstract/Free Full Text]
  26. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 52: 447–452, 1978.[Abstract/Free Full Text]
  27. Kowalski ML, Kaliner MA. Neurogenic inflammation, vascular permeability, and mast cells. J Immunol 140: 3905–3911, 1988.[Abstract]
  28. Lowman MA, Benyon RC, Church MK. Characterization of neuropeptide-induced histamine release from human dispersed skin mast cells. Br J Pharmacol 95: 121–130, 1988.[ISI][Medline]
  29. Lowman MA, Rees PH, Benyon RC, Church MK. Human mast cell heterogeneity: histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli. J Allergy Clin Immunol 81: 590–597, 1988.[CrossRef][ISI][Medline]
  30. Nedrebo T, Berg A, Reed RK. Effect of tumor necrosis factor-{alpha}, IL-1beta, and IL-6 on interstitial fluid pressure in rat skin. Am J Physiol Heart Circ Physiol 277: H1857–H1862, 1999.[Abstract/Free Full Text]
  31. Nedrebo T, Reed RK, Berg A. Effect of alpha-trinositol on interstitial fluid pressure, edema generation, and albumin extravasation after ischemia-reperfusion injury in rat hind limb. Shock 20: 149–153, 2003.[CrossRef][ISI][Medline]
  32. Petersen LJ, Winge K, Brodin E, Skov PS. No release of histamine and substance P in capsaicin-induced neurogenic inflammation in intact human skin in vivo: a microdialysis study. Clin Exp Allergy 27: 957–965, 1997.[CrossRef][ISI][Medline]
  33. Reed RK, Berg A, Gjerde EA, Rubin K. Control of interstitial fluid pressure: role of beta1-integrins. Semin Nephrol 21: 222–230, 2001.[CrossRef][ISI][Medline]
  34. Reed RK, Rodt SA. Increased negativity of interstitial fluid pressure during the onset stage of inflammatory edema in rat skin. Am J Physiol Heart Circ Physiol 260: H1985–H1991, 1991.[Abstract/Free Full Text]
  35. Rodt SA, Reed RK. Interstitial fluid pressure in rat skin becomes more negative in the initial phase of carrageenan-induced edema. Int J Microcirc Clin Exp 12: 299–312, 1993.[ISI][Medline]
  36. Rodt SA, Wiig H, Reed RK. Increased negativity of interstitial fluid pressure contributes to development of oedema in rat skin following application of xylene. Acta Physiol Scand 140: 581–586, 1990.[ISI][Medline]
  37. Scholzen T, Armstrong CA, Bunnett NW, Luger TA, Olerud JE, Ansel JC. Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems. Exp Dermatol 7: 81–96, 1998.[CrossRef][ISI][Medline]
  38. Stander S, Moormann C, Schumacher M, Buddenkotte J, Artuc M, Shpacovitch V, Brzoska T, Lippert U, Henz BM, Luger TA, Metze D, Steinhoff M. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures. Exp Dermatol 13: 129–139, 2004.[CrossRef][ISI][Medline]
  39. Wang L, Hilliges M, Jernberg T, Wiegleb-Edstrom D, Johansson O. Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin. Cell Tissue Res 261: 25–33, 1990.[CrossRef][ISI][Medline]
  40. Weidner C, Klede M, Rukwied R, Lischetzki G, Neisius U, Skov PS, Petersen LJ, Schmelz M. Acute effects of substance P and calcitonin gene-related peptide in human skin—a microdialysis study. J Invest Dermatol 115: 1015–1020, 2000.[CrossRef][ISI][Medline]
  41. Wiederhielm CA, Woodbury JW, Kirk S, Rushmer RF. Pulsatile pressures in the microcirculation of the frog's mesentery. Am J Physiol 207: 173–176, 1964.[Abstract/Free Full Text]
  42. Wiig H, Reed RK, Aukland K. Micropuncture measurement of interstitial fluid pressure in rat subcutis and skeletal muscle: comparison to wick-in-needle technique. Microvasc Res 21: 308–319, 1981.[CrossRef][ISI][Medline]
  43. Woie K, Koller ME, Heyeraas KJ, Reed RK. Neurogenic inflammation in rat trachea is accompanied by increased negativity of interstitial fluid pressure. Circ Res 73: 839–845, 1993.[Abstract/Free Full Text]
  44. Yamamoto T, Hartmann K, Eckes B, Krieg T. Mast cells enhance contraction of three-dimensional collagen lattices by fibroblasts by cell-cell interaction: role of stem cell factor/c-kit. Immunology 99: 435–439, 2000.[CrossRef][ISI][Medline]
  45. Zhao ZZ, Savage NW, Pujic Z, Walsh LJ. Immunohistochemical localization of mast cells and mast cell-nerve interactions in oral lichen planus. Oral Dis 3: 71–76, 1997.[Medline]



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