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1Department of Physiology, University of Bergen, N-5009 Bergan, Norway; and 2Department of Medical Biochemistry and Microbiology, University of Uppsala, S-751 23 Uppsala, Sweden
Submitted 26 September 2003 ; accepted in final form 2 November 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N-deacetylase/N-sulfotransferase-2; mast cell; capsaicin; peptides; micropuncture
(COPc – COPif)] = CFC x 
P, where c and if denote the capillary and the interstitial fluid, respectively, and is the capillary reflection coefficient for proteins. Pif has a dual role in the control of interstitial fluid balance. Under normal conditions, excess fluid filtered over the capillaries would increase Pif and reduce COPif and hence oppose further fluid filtration and prevent the formation of edema in a passive manner (1). Pif is also the filling pressure for the initial lymphatics and an increase in Pif will thus increase lymph flow (1). However, contrary to the situation in normal control of interstitial volume, Pif actively enhances edema formation rather than opposes further fluid filtration (18), because Pif is lowered from its slightly subatmospheric level to more negative values. This increases the net filtration pressure and hence, the driving force for transcapillary fluid filtration (18). Stimulation of sensory C fibers and the subsequent release of neuropeptides from capsaicin-sensitive nerve endings initiate neurogenic inflammation. Release of neuropeptides, including the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP), leads to an acute inflammatory response characterized by vasodilatation of the arterioles combined with increased vascular permeability, mainly in postcapillary venules, followed by edema formation. Recent studies in our group have demonstrated that the initial phase of neurogenic inflammation also involves lowering of Pif, which contributes to rapid swelling (8, 9, 28, 29).
It has been proposed that there is a functional relationship between mast cells and sensory nerves (13), although there are variations depending on both tissue and animal strain (22). This possible connection is supported by the fact that the nerve endings and mast cells have often been described to be spatially close. Mast cells can be activated by SP both by binding of the neurokinin-1 receptor known to be present on mast cells (4) and through a receptor-independent mechanism (6). Of special interest for our study is the finding that capsaicin-sensitive nerve endings are anatomically close to connective tissue mast cells (CTMCs) in mouse skin (3) and that activation of mast cells has been shown to induce lowering of Pif in skin of both rats and mice (12, 20).
NDST-2–/– mice lack the gene for N-deacetylase/N-sulfotransferase-2, an enzyme necessary for synthesizing sulfated heparin, a component found exclusively in CTMCs (7). Absence of sulfated heparin gives rise to mice with severe morphological and functional defects in their CTMCs (7). The number of CTMCs is reduced, and instead of normal dense core secretory granules, they contain large, empty vacuoles with a marked reduction of histamine and lack of proteases (7).
The present study was performed to examine the role of CTMCs in the process leading to lowering of Pif in the initial phase of neurogenic inflammation. First, the effect of mast cell activation induced by compound 48/80 (C48/80) on Pif and plasma protein extravasation (PPE) was compared in NDST-2–/– knockout mice and NDST-2+/+ wild-type mice. This was done to establish whether the NDST-2–/– mice could be used as an experimental model for studying the importance of CTMCs in acute inflammatory responses. Second, we established whether neurogenic inflammation, induced either by capsaicin to release endogenous neuropeptides or by exogenous SP or CGRP, leads to the same lowering of Pif in mouse skin as previously observed in the rat trachea (8). Thereafter, Pif in skin was compared in NDST-2+/+ and NDST-2–/– mice after neurogenic inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female NDST-2-deficient knockout mice (NDST-2–/–) and wild-type mice (NDST-2+/+) were obtained from a breeding stock developed at Uppsala University (Uppsala, Sweden) (7). Knockout mice were initially generated on a mixed genetic background (C57BL/6 x 129S4/SvJae), which were backcrossed with C57BL/6 mice. Mice used in this study were of the F6 generation. Female C57BL/6 mice were obtained from Möllegaard Breedings (Ry, Denmark). Mice weighed between 20 and 40 g and received water and food ad libitum before experiments. Experiments were performed on animals anesthetized with a 1:1 mixture (0.1 ml/10 g body wt) of midazolam (Dormicum; Roche) and fentanyl/fluanison (Hypnorm; Janssen) injected subcutaneously. The right carotid artery was catheterized for monitoring arterial blood pressure. Intravenous injections were performed through a PE-25 catheter in the external jugular vein. Cardiac arrest was induced under anesthesia with an intravenous injection of saturated KCl, either as a part of the experimental protocol or at the end of the experiment. All experiments were performed with the approval of and in the accordance with the Norwegian State Commission for Laboratory Animals.
Measurements
Pif. Pif was measured with sharpened glass capillaries (tip diameter, 4–7 µm), filled with 0.5 M NaCl colored with Evans blue dye to visualize the tip, connected to a servo-controlled counterpressure system (25, 27). The counterpressure generated by the servo-controlled pump (model 201; Ling Dynamic Systems; Royston, UK) was recorded with a pressure transducer (model 1280C; Hewlett-Packard) connected to an amplifier and a recorder (Gould Instruments; Ballainvilliers, France). Mice were kept in a supine position with the left hind paw immobilized by using surgical tape, with care taken not to cause visible compression or retraction of the skin. Pif measurements were obtained by puncturing with micropipettes through intact skin on the dorsal side of the left hind paw by using a micromanipulator (Leitz; Weitzlar, Germany) under the guidance of a stereomicroscope (Wild M5; Leitz). Measurements were performed after ensuring that there was no visible distortion of the punctured skin and accepted when the following three criteria were fulfilled. First, increased gain did not alter the measured pressure. Second, applying suction to the pipette by the servo-controlled pump resulted in increased electrical resistance in the capillary due to the influx of fluid with lower tonicity. This procedure was performed to ensure there was communication between the pipette and the surrounding tissue fluid. Third, recording of the zero pressure measured in a plastic cup filled with saline placed at the level of the puncture site did not change before and after the measurement.
To get an accurate measure of the lowering of Pif in the initial phase of the inflammatory process, it was necessary to induce circulatory arrest to minimize transcapillary fluid flux after giving either of the test substances. When fluid moves into the interstitium as part of an undisturbed inflammatory response this could cause an underestimation of the potentially lowered pressure, because the accumulated fluid will raise Pif.
Experimental Protocol for Pif
Pif was first measured with the circulation still intact in all animals and before the introduction of the test substance. Pressure was measured for 60 min after induction of circulatory arrest, and the pressure recordings were averaged for the following periods: 0–15, 16–30, 31–45, and 46–60 min. All test substances were provided by Sigma-Aldrich (Stockholm, Sweden). Doses of capsaicin, SP, and CGRP were chosen in accordance with the studies of Baluk et al. (2) and Gjerde et al. (8, 9).
Control. C57Black mice received 0.1 ml 0.9% NaCl iv, which circulated 2 min before cardiac arrest was induced by intravenous administration of saturated KCl (0.1 ml).
C48/80. The three groups of mice including C57Black, NDST-2+/+, and NDST-2–/– received 200 µg C48/80 iv (0.1 ml), which circulated 2 min before cardiac arrest was induced.
Capsaicin. C57Black, NDST-2+/+, and NDST-2–/– mice received 10 nmol capsaicin iv, which circulated 1 min before cardiac arrest was induced.
SP. C57Black, NDST-2+/+, and NDST-2–/– mice received 0.1 nmol SP iv, which circulated 1 min before cardiac arrest was induced.
CGRP. C57Black, NDST-2+/+, and NDST-2–/– mice received 0.1 nmol CGRP iv, which circulated 1 min before cardiac arrest was induced.
PPE
PPE was measured in NDST-2+/+ and NDST-2–/– mice by means of the microdialysis technique by using large pore size membranes as described by Schmelz et al. (21) and modified by Iversen et al. (14). Two hollow plasmapheresis fibers (0.4 mm in diameter, cutoff at 3,000 kDa; model PF200, Gambro) were placed subcutaneously at a length of 3 cm on the back and oriented longitudinally on both sides of the spine. For collection of dialysate, the outlet of the tubing was inserted in plastic vials (Eppendorf). Both microdialysis fibers were perfused with 0.9% NaCl by a microdialysis pump (model CMA 100) at a constant flow of 5 µl/min. After 45 min of postsurgical recovery, 0.2 ml of 125I-labeled human serum albumin (125I-labeled HSA; Institute for Energy Technique, Kjeller, Norway) at 10 Mbq was given intravenously and circulated for 10 min to fully distribute the 125I-labeled HSA. To establish a baseline control period, dialysate samples were collected every 10 min for a period of 30–50 min before stimulation with C48/80 (4 mg/ml) was given through the left fiber for the subsequent 70 min. The right fiber was perfused with 0.9% NaCl during the whole experiment for control measurements with each animal being its own control. Samples of the dialysate were collected for 10-min periods throughout the experimental period. Radioactivity in each sample was determined with a gamma counter (model 1282 LKB; Wallac, Turku, Finland). The mean of the measured radioactivity in the samples from the baseline control period was set as the basal extravasation of protein. The PPE after C48/80 injection was calculated relative to the baseline control period (%baseline).
Statistical Analysis
All values of Pif are given as means ± SD of the four experimental time periods from 0 to 60 min. Statistical analysis was performed on the mean of the four experimental time periods by using one-way ANOVA and subsequent Bonferroni and t-tests. The same analysis was performed for the comparison of PPE measurements. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In C57Black mice, Pif was stable during the entire 60-min period after intravenous injection of saline (Fig. 1). Pif averaged –0.41 ± 0.21 mmHg before circulatory arrest and –0.57 ± 0.17 mmHg after circulatory arrest was induced (Table 1).
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Pif C48/80
Measurements of Pif over time after intravenous C48/80 were compared in C57Black, NDST-2+/+, and NDST-2–/– mice (Fig. 1). C48/80 lowered Pif significantly (P < 0.05) in C57Black, NDST-2+/+, and NDST-2–/– mice compared with the vehicle-treated group (Table 1). However, Pif in NDST-2–/– mice was significantly (P < 0.05) less reduced compared with NDST-2+/+ and C57Black mice after treatment with C48/80.
PPE C48/80
The plasma protein level of 125I-labeled HSA in the dialysate from the control fiber in both NDST-2+/+ and NDST-2–/– mice was stable during the entire 70-min period (Fig. 2). In NDST-2+/+ mice, treatment with C48/80 gradually increased the plasma protein content in the dialysate 300% compared with control levels after 70 min (P < 0.05). C48/80 stimulation in NDST-2–/– mice did not increase protein content significantly in the dialysate at the end of the 70-min stimulation period compared with vehicle.
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Pif Neurogenic Inflammation
Capsaicin, SP, and CGRP lowered Pif over time in C57Black mice (Fig. 3). Pif was significantly (P < 0.05) lowered in all three groups compared with vehicle (Table 1). Capsaicin lowered Pif over time in both NDST-2+/+ and NDST-2–/– mice (Fig. 4A). Pif was significantly (P < 0.05) lowered compared with vehicle in both NDST-2+/+ and NDST-2–/– mice, whereas there was no significant difference in the response to capsaicin between the two groups. SP lowered Pif over time in both NDST-2+/+ and NDST-2–/– mice (Fig. 4B). Pif was lowered significantly (P < 0.05) compared with vehicle in both NDST-2+/+ and NDST-2–/– mice (Table 1). The two groups were not significantly different in their response to SP. CGRP lowered Pif over time in NDST-2+/+ and NDST-2–/– mice (Fig. 4C). Both groups were significantly (P < 0.05) different from vehicle, but there was no difference in the response when the two groups were compared (Table 1). Thus with C48/80, NDST-2–/– mice were different from NDST-2+/+ mice, whereas for capsaicin, SP, and CGRP, NDST-2–/– and NDST-2+/+ mice responded similarly.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neurogenic inflammation has been extensively studied in both rodents and humans, but the events downstream of neuropeptide release are not fully elucidated. Because of the mast cells' content of several inflammatory mediators and their presence in the vicinity of sensory nerve cells, the mast cells are likely to participate in neurogenic inflammation. A number of studies (5, 17, 24) have suggested a role for mast cells in the development of edema after neuropeptide challenge, but the ability to release histamine on neuropeptide stimulation seems to vary between species and different mast cell types. Nevertheless, activation of mast cells is associated with anaphylaxis in rats (23) and has been shown to lower Pif in both the trachea of rats (15) and skin of both rats and mice (12, 20). Histamine itself has also been shown to lower Pif in rat skin (19).
The aim of this study was to examine whether Pif is lowered in mouse skin during the initial phase of neurogenic inflammation and whether the presence of functional mast cells is a necessary condition for the eventual Pif changes. The NDST-2–/– mice used in this study have dysfunctional CTMCs due to the lack of the glucosaminyl N-deacetylase/N-sulphotransferase enzyme necessary to synthesize sulfated heparin, which is a part of the secretory granule content of CTMCs. Forsberg et al. (7) have demonstrated that the absence of this particular enzyme, and hence sulfated heparin, results in mice with a reduced number of CTMCs. The mast cells remaining contain less histamine and lack proteases (7). The phenotype of NDST-2–/– mice is restricted to the defect in their CTMCs, and the mice appear to be normal in other respects (11).
First, we examined the response of Pif and PPE to mast cell activation in skin of NDST-2–/– knockout mice and compared the response with that of NDST-2+/+ wild-type mice. Pif was measured in the intradermal layer of the skin, whereas PPE had to be measured subcutaneously due to the diameter of the microdialysis fiber (0.4 mm). Wiig et al. (26) demonstrated that there is no gradient in Pif either between superficial and deep skin layers or between skin and subcutis, thus indicating that the results are not affected by the localization of the measurements in these different layers. We found that C48/80 lowered Pif in paw skin of NDST-2+/+ mice from approximately –0.57 mmHg to an average of –2.31 mmHg. In C57Black mice, the strain that the NDST-2 mice have been developed from, Pif was lowered to the same extent. These results demonstrate that mast cell activation can induce lowering of Pif, an observation in accordance with previous findings in skin of mice after mast cell activation (12). In the NDST-2–/– mice, the response to C48/80 was significantly reduced compared with the NDST-2+/+ mice with a lowering of Pif to only –1.67 mmHg. Local administration of C48/80 through microdialysis fibers increased PPE threefold in the back skin of NDST-2+/+ mice, whereas C48/80 had no significant effect on PPE in the NDST-2–/– mice. We consider the reduced response to C48/80 in the NDST-2–/– mice to be an indirect evidence of the assumed dysfunctional CTMCs, with a lowered amount of inflammatory mediators released from a reduced number of mast cells. Although there is reduced histamine content and lack of proteases in the mast cell granules, it has yet not been examined whether TNF-
or other cytokines are still present and can be secreted on mast cell activation. TNF-, interleukin-1
, and interleukin-6 have previously been found to lower Pif in rat skin (16). Because the number of mast cells is markedly reduced, and the remaining mast cells contain large empty vacuoles instead of the normal secretory granules, it is also possible that the content of cytokines is severely altered. However, the presence of small amounts of inflammatory mediators could also explain that Pif is still lowered to some extent and not completely abolished in the NDST-2–/– mice after C48/80. Despite the presence of small amounts of inflammatory mediators, the NDST-2–/– mice were considered to be a suitable model for the following experiments, because a partial abolishment of the lowered Pif during neurogenic inflammation would also give information about mast cell involvement.
Pif was lowered in paw skin after SP, CGRP, and capsaicin in both C57Black and NDST-2+/+ mice, demonstrating the same changes in Pif in response to neurogenic inflammation as observed in the rat trachea (8). In the NDST-2–/– mice, Pif was lowered to the same extent as in the NDST-2+/+ mice after challenge with either of the three test substances. These experiments demonstrate that the response to neuropeptides is still intact in mice with dysfunctional mast cells. However, the involvement of mast cells in the lowering of Pif cannot be completely ruled out by this observation. The present experiments would seem to allow the conclusion that lowering of Pif in the initial phase of neurogenic inflammation is not exclusively dependent on intact CTMCs. Although the evidence is not conclusive, and the experiments preferentially should be repeated on mice completely lacking CTMCs, these results are a step forward in elucidating the events downstream of neuropeptide release culminating in lowered Pif and edema formation. Also, the use of genetically modified animals provides a valuable tool to further elucidate the physiological mechanisms involved in neurogenic inflammation.
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ACKNOWLEDGMENTS |
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This study received financial support from the Norwegian Research Council, the Norwegian Heart Association, the Swedish Research Council, Gustaf V:s 80-årsfond, and Polysackaridforskning AB.
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FOOTNOTES |
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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.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-trinositol. Am J Respir Crit Care Med 150: 924–928, 1994.[Abstract]This article has been cited by other articles:
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  O.S. Svendsen, M.M. Barczyk, S.N. Popova, A Liden, D. Gullberg, and H. Wiig The {alpha}11{beta}1 Integrin Has a Mechanistic Role in Control of Interstitial Fluid Pressure and Edema Formation in Inflammation Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1864 - 1870. [Abstract] [Full Text] [PDF]
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 O. S. Svendsen, A. Liden, T. Nedrebo, K. Rubin, and R. K. Reed Integrin {alpha}v{beta}3 acts downstream of insulin in normalization of interstitial fluid pressure in sepsis and in cell-mediated collagen gel contraction Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H555 - H560. [Abstract] [Full Text] [PDF]
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T. V. Karlsen, A. Bletsa, E.-A. B. Gjerde, and R. K. Reed Lowering of interstitial fluid pressure after neurogenic inflammation in mouse skin is partly dependent on mast cells Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1821 - H1827. [Abstract] [Full Text] [PDF]
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P < 0.05, significant difference from the response in NDST-2+/+ mice compared with the averages of the whole time period for both groups.
