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Local injection of nerve growth factor (NGF) triggers degranulation of mast cells in rat paw
Neuroscience Letters 221 (1997) 129–132
Local injection of nerve growth factor (NGF) triggers degranulation of mast cells in rat paw Michael Tal*, Ronit Liberman Department of Anatomy and Cell Biology, The Hebrew University-Hadassah, School of Dental Medicine, P.O. Box 12272, Jerusalem, 91120 Israel Received 30 October 1996; revised version received 30 November 1996; accepted 4 December 1996
Abstract We examined the relationship between hyperalgesia and degranulation of mast cells in rats following treatment with nerve growth factor (NGF) and inflammatory mediators. Intracutaneous injection to the rat’s paw of NGF (50–5000 ng, 50 ml) evoked dose-dependent thermal hyperalgesia as measured by paw withdrawal latency to noxious heat. Even more intense hyperalgesia was induced by an ‘inflammatory mediators soup’ containing BK, 5HT, PGE2 and histamine (10 mM). Counts of mast cells in the dermis were performed on coded slides under × 1000 magnification after fixation in Carnoy’s solution and staining with toluidine blue. After injection of three different concentrations of NGF (50, 500, 5000 ng in 50 ml), the proportion of mast cells that were degranulated was significantly higher (P , 0.001) than after saline injection. No significant differences were found among the three concentrations of NGF. The effect of inflammatory soup on the proportion of degranulated mast cells was similar to that of NGF. These results indicate that NGF activates cutaneous mast cells and stimulates the release of inflammatory tissue mediators in the acute phase of NGF-induced hyperalgesia in the rat’s paw. However, the finding that degranulation was not dose-dependent indicates that other mechanisms, independent of mast cells, are probably operating in response to NGF. 1997 Elsevier Science Ireland Ltd. All rights reserved Keywords: Nerve growth factor; Mast cells; Degranulation; Hyperalgesia
Several lines of evidence indicate that nerve growth factor (NGF) plays a role not only in development during cell growth and survival of sensory and sympathetic neurons, but also in hyperalgesia and inflammatory pain states in adulthood. Support for this notion came from experiments which showed that a single injection of NGF can lead to heat and mechanical hyperalgesia in adult rats [7]. NGF is upregulated in animal models of inflammation such as Freund’s adjuvant-induced arthritis, and carrageenan inflammation. It is also known that intracutaneous injection of NGF causes edema in the injected area and elevation of calcitonin gene-related peptide (CGRP) and substance P (SP) in the sciatic nerve after intracutaneous injection [1]. Although it is known that NGF is a potent releaser of peritoneal mast cell content [5], it has not yet been determined whether NGF has the potential to activate cutaneous mast cells. The mechanism by which NGF exerts its effects on * Tel.: +972 2 6758456; fax: +972 2 6757451.
nociceptive processing is still unclear. Lewin [8] suggested modes of action in the periphery and in the central nervous system (CNS). In this study we focus on the periphery. In peripheral tissues there are several possible processes on which NGF might act. These include sensitization of peripheral nociceptors to heat [2] and tonic activation of nociceptors [3]. We have shown previously that there may be both a direct sensitization and a small excitatory response of cutaneous nociceptors after treatment with either NGF or with other inflammatory agents [14]. Still to be addressed are the questions: (1) is NGF a potential mast cell activator in cutaneous tissue? and (2) does NGF directly activate nociceptors (many nociceptors have NGF receptors and hence could be directly sensitive to NGF [10]) or does it produce its effects via the release of inflammatory mediators from local sources in the tissue such as mast cells? We show here, using histological staining, that mast cells are degranulated following treatment with NGF as they are by
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (96 )1 3318-8
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M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
Fig. 1. Transverse sections of the rat cutaneous plantar paw injected with 500 ng in 50 ml rhNGF showing degranulated mast cells (A) and non-active cells in control (B). Magnification × 1000.
inflammatory mediators. This substantiates the hypothesis of NGF action through inflammatory intermediates. Part of these results have previously been reported in abstract form [Neuroscience Abstracts 711.2, 1996]. Under ether anesthesia, 50 ml of one of the following solutions was injected subcutaneously into the midplantar surface of the left hindpaw in rats using a fine 30-gauge
needle: 1, 10 and 100 mg/ml rhNGF (equivalent to the concentration of 50, 500 and 5000 ng, gift from Boehringer Mannheim), or a mixture that contained bradykinin 10 mM, prostaglandin E2 10 mM, histamine 10 mM and serotonin 10 mM (‘inflammatory soup’). Normal saline was injected in the same fashion into the right hindpaw as a control. Thirty minutes after NGF or ‘inflammatory soup’
M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
Fig. 2. Effect of intradermal injection of different NGF concentrations and ‘inflammatory mediator soup’ on the ratio of degranulated to nonactive mast cells. Values are mean ± SEM (**P , 0.001 relative to control).
injection, the rats were anesthetized (50 mg/kg pentobarbital i.p.) and a piece of the full thickness of the skin surrounding the injection site, measuring approximately 7 × 3 mm, was excised and placed into Carnoy’s fixative (60 ml absolute ethanol, 30 ml chloroform, 10 ml glacial acetic acid). After 48 h fixation, tissues were embedded in paraffin wax, sectioned (5–7 mm thick) serially and stained with toluidine blue (pH 0.5). This permits visualization of mast cells, as described by Strobel et al. [13]. Mast cell counting was performed under oil immersion (magnification × 1000). Mast cells with heavily stained granules scattered outside the cell membrane were defined as degranulated, whereas cells with a clearly outlined cell membrane, without stained granules, were defined as nonactive cells (Fig. 1). Both the injections and the mast cell counting were done blindly, i.e. the solutions were coded by numbers so that the individual doing the injections was not aware of which solution he was injected. Counting of mast cells was done using coded slides by an examiner who did not know the treatment. Statistical analysis was done using the non-parametric Kruskal-Wallis test. Significant level was P = 0.01. All means are given ± SEM. The average number of mast cells counted in each group of animals was 161 (range 124–301). The average ratio of degranulated mast cells over non-active cells after injection of the three concentrations of NGF was significantly higher 3.9 ± 0.38 (range 1.15–6.5) than after saline injection 1.33 ± 0.4 (range 0.75–2.8) (P , 0.001). The ratio of degranulated mast cells over non-active cells in the ‘inflammatory soup’ group was 5.06 ± 1.34 (range 1.8– 10.2) (P , 0.001 compared to saline control); see Fig. 2. No significant differences were found among the three concentrations of NGF. A significant difference at the level of P , 0.05 was found between the ‘inflammatory soup’ and the lower concentration of NGF treatment groups (10 and 100 ml/ml). The results demonstrate that NGF has the potential to
131
activate mast cells in cutaneous tissue. It is therefore tempting to speculate that degranulation, and subsequently release of inflammatory mediators, contributes to heat hyperalgesia induced by NGF injection. We and others [1,2,14] have previously shown that intracutaneous injection to the rat’s paw of NGF (50– 5000 ng) in a 50 ml bolus evoked a dose-dependent hyperalgesia to heat as measured by short latency paw withdrawal to noxious heat. An even more intense hyperalgesia was induced by ‘inflammatory soup’. The thermal hyperalgesia was short lived (,3 h) and limited to the injected side [14]. Intraplantar injection of NGF did not cause spontaneous pain behavior as judged by the absence of limping or guarding of the affected hindpaw. The rapid onset time and duration of NGF action suggests two possible modes of activity: (1) a direct local effect of NGF on cutaneous thermal nociceptors terminals results in sensitization of either afferent nerve endings to heat stimuli and/or actual excitation of action potentials, and (2) an indirect action of NGF by release of inflammatory mediators from mast cells which in turn trigger sensitization and/or excitation [4]. The notion that NGF can directly influence the threshold of nociceptive endings is supported by the observation that both NGF and inflammatory agents sensitize and caused small excitatory responses in cutaneous nociceptors in electrophysiological experiments [14]. Additional evidence comes from the report of Shen and Crain [12] who found that NGF could act directly and trigger action potentials in dorsal root ganglion (DRG) cells. On the other hand, there is also evidence for an indirect mode. Pretreatment with compound 48/80 abolished edema but failed to reduce thermal hyperalgesia 2 and 3 days after treatment while capsaicin treatment attenuated NGF-induced thermal hyperalgesia [1]. These data, therefore, suggest that NGF exerts its effect directly as well as indirectly; directly by releasing vasoactive compounds and by sensitization of afferent endings independently of mast cells, and indirectly by releasing histamine and serotonin from mast cells and by involvement of sympathetic postganglionic neurons. Our data show clearly that NGF induces mast cell degranulation and hence is involved in the degranulated products that may be NGF-induced hyperalgesia. Mast cell degranulation of course, may not be the sole indirect mechanism of NGF-induced thermal hyperalgesia. For example, NGF has the potential to release cytokines such as tumor necrosis factor, in skin, and these may contribute to the hyperalgesia [6]. The finding that three different concentrations of NGF yielded a marked increase in mast cell degranulation with no obvious difference among them, leads to two possible conclusions: (1) that even small amounts of NGF are sufficient to trigger maximal mast cell degranulation, and (2) that other mechanisms are operating in response to NGF treatment. The first conclusion is supported by the work of Andreev et al. [2] who reported that 50 ng NGF injected
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M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
intradermally is sufficient to cause plasma extravasation. We can safely assume that at the microscopic level, we see a saturation of degranulated mast cells that had been triggered by low levels of NGF. The suggestion that multiplicity of mechanisms is operating in response to NGF treatment is supported by the finding that the degranulation was not dose-dependent as found in the case of heat hyperalgesia. Lewin et al. [9] reported that following single systemic doses of NGF, the sensitivity of the animals to noxious heat and mechanical stimulation increased in two phases. The immediate phase of hyperalgesia began 30 min after the injection, and the delayed phase appeared several hours later and persisted for a few days. Thermal hyperalgesia was present in both the early and late phases, but mechanical hyperalgesia was found only in the late phase. It was suggested that the early phase of hyperalgesia was related to the peripheral action of NGF while the delayed phase involved changes in the central processing of nociceptive information. This suggestion was supported not only by the timing of the effects, but also by the finding that the early phase of heat hyperalgesia could be blocked by blocking mast cell degranulation using compound 48/ 80 [1], while the late phase was blocked by N-methyl-daspartate (NMDA) receptor antagonist [7]. In the periphery, a potential target for NGF is the mast cell. Mast cells express trkA receptors [11] and can release histamine and serotonin that sensitize primary afferent nociceptors [4,14]. There are other potential targets, for example, sympathetic terminals. These terminals may respond to circulating NGF by releasing nociceptor sensitizing substances such as prostaglandin [2]. Our observation of rapid mast cell degranulation and thermal hyperalgesia supports the notion that NGF initially induces mast cell degranulation and that mast cell products may contribute to the induction of nociceptor sensitization. However, NGF probably acts by a multiplicity of mechanisms, independently of mast cells. I thank Professor Marshall Devor for his comments on the manuscript. Supported by grant (3523) from the State
of Israel, Ministry of Health and by the Hebrew University Internal Fund. [1] Amann, R., Schuligoi, R., Herzeg, G. and Donnerer, J., Intraplantar injection of nerve growth factor into the rat hind paw: local edema and effects on thermal nociceptive threshold, Pain, 64 (1996) 323– 329. [2] Andreev, N.Y., Dimitrieva, N., Koltzenburg, M. and McMahon, S.B., Peripheral administration of NGF in the adult rat produces a thermal hyperalgesia that requires the presence of sympathetic post-ganglionic neurons, Pain, 63 (1995) 109–115. [3] Dimitrieva, N. and McMahon, S.B., NGF acutely sensitizes primary sensory neurons, Soc. Neurosci. Abstr., 20 (1994) 109.10. [4] Dimitrieva, N. and McMahon, S.B., Sensitization of visceral afferents by nerve growth factor in the adult rat, Pain, 66 (1996) 87– 97. [5] Horigome, K., Pryor, J.C., Bullock, E.D. and Johnson, E.M., Mediator release from mast cells by nerve growth factor, J. Biol. Chem., 268 (1993) 14881–14887. [6] Leon, A., Buriani, A., Dal Toso, R., Fabris, M., Romanello, S., Aloe, L. and Levi-Montalcini, R., Mast cells synthesize, store, and release nerve growth factor, Proc. Natl. Acad. Sci. USA, 91 (1994) 3739–3743. [7] Lewin, G.R., Rueff, A. and Mendell, L.M., Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci., 6 (1994) 1903–1912. [8] Lewin, G.R., Neurotrophic factors and pain, The Neurosci., 7 (1995) 227–232. [9] Lewin, G.R., Ritter, A.M. and Mendell, L.M., NGF induced hyperalgesia in the neonatal and adult rat, J. Neurosci., 13 (1993) 2136– 2148. [10] Lewin, G.R. and Mendell, L.M., NGF and nociception, Trends Neurosci., 16(1993) 353–359. [11] McMahon, S.B., Bennett, D.L.H., Priestly, J.V. and Shelton, D.L., The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule, Nature Med., 1 (1995) 774–780. [12] Shen, K.F. and Crain, S.M., NGF rapidly prolongs the action potential of mature sensory ganglion neurons in culture, and its effect requires activation of GS-coupled excitatory kappa-opioid receptors on these cells, J. Neurosci., 14 (1994) 5570–5579. [13] Strobel, S., Miller, H.R.P. and Ferguson, A., Human intestinal mucosal mast cells: evaluation of fixation and staining techniques, J. Clin. Pathol., 34 (1981) 851–858. [14] Tal, M., Schneider, M., Toyka, K.V. and Koltzenburg, M., Heat hyperalgesia and nociceptor excitation after application of NGF or inflammatory mediators, IASP Abstr., 33 (1996) 119.
Local injection of nerve growth factor (NGF) triggers degranulation of mast cells in rat paw Michael Tal*, Ronit Liberman Department of Anatomy and Cell Biology, The Hebrew University-Hadassah, School of Dental Medicine, P.O. Box 12272, Jerusalem, 91120 Israel Received 30 October 1996; revised version received 30 November 1996; accepted 4 December 1996
Abstract We examined the relationship between hyperalgesia and degranulation of mast cells in rats following treatment with nerve growth factor (NGF) and inflammatory mediators. Intracutaneous injection to the rat’s paw of NGF (50–5000 ng, 50 ml) evoked dose-dependent thermal hyperalgesia as measured by paw withdrawal latency to noxious heat. Even more intense hyperalgesia was induced by an ‘inflammatory mediators soup’ containing BK, 5HT, PGE2 and histamine (10 mM). Counts of mast cells in the dermis were performed on coded slides under × 1000 magnification after fixation in Carnoy’s solution and staining with toluidine blue. After injection of three different concentrations of NGF (50, 500, 5000 ng in 50 ml), the proportion of mast cells that were degranulated was significantly higher (P , 0.001) than after saline injection. No significant differences were found among the three concentrations of NGF. The effect of inflammatory soup on the proportion of degranulated mast cells was similar to that of NGF. These results indicate that NGF activates cutaneous mast cells and stimulates the release of inflammatory tissue mediators in the acute phase of NGF-induced hyperalgesia in the rat’s paw. However, the finding that degranulation was not dose-dependent indicates that other mechanisms, independent of mast cells, are probably operating in response to NGF. 1997 Elsevier Science Ireland Ltd. All rights reserved Keywords: Nerve growth factor; Mast cells; Degranulation; Hyperalgesia
Several lines of evidence indicate that nerve growth factor (NGF) plays a role not only in development during cell growth and survival of sensory and sympathetic neurons, but also in hyperalgesia and inflammatory pain states in adulthood. Support for this notion came from experiments which showed that a single injection of NGF can lead to heat and mechanical hyperalgesia in adult rats [7]. NGF is upregulated in animal models of inflammation such as Freund’s adjuvant-induced arthritis, and carrageenan inflammation. It is also known that intracutaneous injection of NGF causes edema in the injected area and elevation of calcitonin gene-related peptide (CGRP) and substance P (SP) in the sciatic nerve after intracutaneous injection [1]. Although it is known that NGF is a potent releaser of peritoneal mast cell content [5], it has not yet been determined whether NGF has the potential to activate cutaneous mast cells. The mechanism by which NGF exerts its effects on * Tel.: +972 2 6758456; fax: +972 2 6757451.
nociceptive processing is still unclear. Lewin [8] suggested modes of action in the periphery and in the central nervous system (CNS). In this study we focus on the periphery. In peripheral tissues there are several possible processes on which NGF might act. These include sensitization of peripheral nociceptors to heat [2] and tonic activation of nociceptors [3]. We have shown previously that there may be both a direct sensitization and a small excitatory response of cutaneous nociceptors after treatment with either NGF or with other inflammatory agents [14]. Still to be addressed are the questions: (1) is NGF a potential mast cell activator in cutaneous tissue? and (2) does NGF directly activate nociceptors (many nociceptors have NGF receptors and hence could be directly sensitive to NGF [10]) or does it produce its effects via the release of inflammatory mediators from local sources in the tissue such as mast cells? We show here, using histological staining, that mast cells are degranulated following treatment with NGF as they are by
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (96 )1 3318-8
130
M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
Fig. 1. Transverse sections of the rat cutaneous plantar paw injected with 500 ng in 50 ml rhNGF showing degranulated mast cells (A) and non-active cells in control (B). Magnification × 1000.
inflammatory mediators. This substantiates the hypothesis of NGF action through inflammatory intermediates. Part of these results have previously been reported in abstract form [Neuroscience Abstracts 711.2, 1996]. Under ether anesthesia, 50 ml of one of the following solutions was injected subcutaneously into the midplantar surface of the left hindpaw in rats using a fine 30-gauge
needle: 1, 10 and 100 mg/ml rhNGF (equivalent to the concentration of 50, 500 and 5000 ng, gift from Boehringer Mannheim), or a mixture that contained bradykinin 10 mM, prostaglandin E2 10 mM, histamine 10 mM and serotonin 10 mM (‘inflammatory soup’). Normal saline was injected in the same fashion into the right hindpaw as a control. Thirty minutes after NGF or ‘inflammatory soup’
M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
Fig. 2. Effect of intradermal injection of different NGF concentrations and ‘inflammatory mediator soup’ on the ratio of degranulated to nonactive mast cells. Values are mean ± SEM (**P , 0.001 relative to control).
injection, the rats were anesthetized (50 mg/kg pentobarbital i.p.) and a piece of the full thickness of the skin surrounding the injection site, measuring approximately 7 × 3 mm, was excised and placed into Carnoy’s fixative (60 ml absolute ethanol, 30 ml chloroform, 10 ml glacial acetic acid). After 48 h fixation, tissues were embedded in paraffin wax, sectioned (5–7 mm thick) serially and stained with toluidine blue (pH 0.5). This permits visualization of mast cells, as described by Strobel et al. [13]. Mast cell counting was performed under oil immersion (magnification × 1000). Mast cells with heavily stained granules scattered outside the cell membrane were defined as degranulated, whereas cells with a clearly outlined cell membrane, without stained granules, were defined as nonactive cells (Fig. 1). Both the injections and the mast cell counting were done blindly, i.e. the solutions were coded by numbers so that the individual doing the injections was not aware of which solution he was injected. Counting of mast cells was done using coded slides by an examiner who did not know the treatment. Statistical analysis was done using the non-parametric Kruskal-Wallis test. Significant level was P = 0.01. All means are given ± SEM. The average number of mast cells counted in each group of animals was 161 (range 124–301). The average ratio of degranulated mast cells over non-active cells after injection of the three concentrations of NGF was significantly higher 3.9 ± 0.38 (range 1.15–6.5) than after saline injection 1.33 ± 0.4 (range 0.75–2.8) (P , 0.001). The ratio of degranulated mast cells over non-active cells in the ‘inflammatory soup’ group was 5.06 ± 1.34 (range 1.8– 10.2) (P , 0.001 compared to saline control); see Fig. 2. No significant differences were found among the three concentrations of NGF. A significant difference at the level of P , 0.05 was found between the ‘inflammatory soup’ and the lower concentration of NGF treatment groups (10 and 100 ml/ml). The results demonstrate that NGF has the potential to
131
activate mast cells in cutaneous tissue. It is therefore tempting to speculate that degranulation, and subsequently release of inflammatory mediators, contributes to heat hyperalgesia induced by NGF injection. We and others [1,2,14] have previously shown that intracutaneous injection to the rat’s paw of NGF (50– 5000 ng) in a 50 ml bolus evoked a dose-dependent hyperalgesia to heat as measured by short latency paw withdrawal to noxious heat. An even more intense hyperalgesia was induced by ‘inflammatory soup’. The thermal hyperalgesia was short lived (,3 h) and limited to the injected side [14]. Intraplantar injection of NGF did not cause spontaneous pain behavior as judged by the absence of limping or guarding of the affected hindpaw. The rapid onset time and duration of NGF action suggests two possible modes of activity: (1) a direct local effect of NGF on cutaneous thermal nociceptors terminals results in sensitization of either afferent nerve endings to heat stimuli and/or actual excitation of action potentials, and (2) an indirect action of NGF by release of inflammatory mediators from mast cells which in turn trigger sensitization and/or excitation [4]. The notion that NGF can directly influence the threshold of nociceptive endings is supported by the observation that both NGF and inflammatory agents sensitize and caused small excitatory responses in cutaneous nociceptors in electrophysiological experiments [14]. Additional evidence comes from the report of Shen and Crain [12] who found that NGF could act directly and trigger action potentials in dorsal root ganglion (DRG) cells. On the other hand, there is also evidence for an indirect mode. Pretreatment with compound 48/80 abolished edema but failed to reduce thermal hyperalgesia 2 and 3 days after treatment while capsaicin treatment attenuated NGF-induced thermal hyperalgesia [1]. These data, therefore, suggest that NGF exerts its effect directly as well as indirectly; directly by releasing vasoactive compounds and by sensitization of afferent endings independently of mast cells, and indirectly by releasing histamine and serotonin from mast cells and by involvement of sympathetic postganglionic neurons. Our data show clearly that NGF induces mast cell degranulation and hence is involved in the degranulated products that may be NGF-induced hyperalgesia. Mast cell degranulation of course, may not be the sole indirect mechanism of NGF-induced thermal hyperalgesia. For example, NGF has the potential to release cytokines such as tumor necrosis factor, in skin, and these may contribute to the hyperalgesia [6]. The finding that three different concentrations of NGF yielded a marked increase in mast cell degranulation with no obvious difference among them, leads to two possible conclusions: (1) that even small amounts of NGF are sufficient to trigger maximal mast cell degranulation, and (2) that other mechanisms are operating in response to NGF treatment. The first conclusion is supported by the work of Andreev et al. [2] who reported that 50 ng NGF injected
132
M. Tal, R. Liberman / Neuroscience Letters 221 (1997) 129–132
intradermally is sufficient to cause plasma extravasation. We can safely assume that at the microscopic level, we see a saturation of degranulated mast cells that had been triggered by low levels of NGF. The suggestion that multiplicity of mechanisms is operating in response to NGF treatment is supported by the finding that the degranulation was not dose-dependent as found in the case of heat hyperalgesia. Lewin et al. [9] reported that following single systemic doses of NGF, the sensitivity of the animals to noxious heat and mechanical stimulation increased in two phases. The immediate phase of hyperalgesia began 30 min after the injection, and the delayed phase appeared several hours later and persisted for a few days. Thermal hyperalgesia was present in both the early and late phases, but mechanical hyperalgesia was found only in the late phase. It was suggested that the early phase of hyperalgesia was related to the peripheral action of NGF while the delayed phase involved changes in the central processing of nociceptive information. This suggestion was supported not only by the timing of the effects, but also by the finding that the early phase of heat hyperalgesia could be blocked by blocking mast cell degranulation using compound 48/ 80 [1], while the late phase was blocked by N-methyl-daspartate (NMDA) receptor antagonist [7]. In the periphery, a potential target for NGF is the mast cell. Mast cells express trkA receptors [11] and can release histamine and serotonin that sensitize primary afferent nociceptors [4,14]. There are other potential targets, for example, sympathetic terminals. These terminals may respond to circulating NGF by releasing nociceptor sensitizing substances such as prostaglandin [2]. Our observation of rapid mast cell degranulation and thermal hyperalgesia supports the notion that NGF initially induces mast cell degranulation and that mast cell products may contribute to the induction of nociceptor sensitization. However, NGF probably acts by a multiplicity of mechanisms, independently of mast cells. I thank Professor Marshall Devor for his comments on the manuscript. Supported by grant (3523) from the State
of Israel, Ministry of Health and by the Hebrew University Internal Fund. [1] Amann, R., Schuligoi, R., Herzeg, G. and Donnerer, J., Intraplantar injection of nerve growth factor into the rat hind paw: local edema and effects on thermal nociceptive threshold, Pain, 64 (1996) 323– 329. [2] Andreev, N.Y., Dimitrieva, N., Koltzenburg, M. and McMahon, S.B., Peripheral administration of NGF in the adult rat produces a thermal hyperalgesia that requires the presence of sympathetic post-ganglionic neurons, Pain, 63 (1995) 109–115. [3] Dimitrieva, N. and McMahon, S.B., NGF acutely sensitizes primary sensory neurons, Soc. Neurosci. Abstr., 20 (1994) 109.10. [4] Dimitrieva, N. and McMahon, S.B., Sensitization of visceral afferents by nerve growth factor in the adult rat, Pain, 66 (1996) 87– 97. [5] Horigome, K., Pryor, J.C., Bullock, E.D. and Johnson, E.M., Mediator release from mast cells by nerve growth factor, J. Biol. Chem., 268 (1993) 14881–14887. [6] Leon, A., Buriani, A., Dal Toso, R., Fabris, M., Romanello, S., Aloe, L. and Levi-Montalcini, R., Mast cells synthesize, store, and release nerve growth factor, Proc. Natl. Acad. Sci. USA, 91 (1994) 3739–3743. [7] Lewin, G.R., Rueff, A. and Mendell, L.M., Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci., 6 (1994) 1903–1912. [8] Lewin, G.R., Neurotrophic factors and pain, The Neurosci., 7 (1995) 227–232. [9] Lewin, G.R., Ritter, A.M. and Mendell, L.M., NGF induced hyperalgesia in the neonatal and adult rat, J. Neurosci., 13 (1993) 2136– 2148. [10] Lewin, G.R. and Mendell, L.M., NGF and nociception, Trends Neurosci., 16(1993) 353–359. [11] McMahon, S.B., Bennett, D.L.H., Priestly, J.V. and Shelton, D.L., The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule, Nature Med., 1 (1995) 774–780. [12] Shen, K.F. and Crain, S.M., NGF rapidly prolongs the action potential of mature sensory ganglion neurons in culture, and its effect requires activation of GS-coupled excitatory kappa-opioid receptors on these cells, J. Neurosci., 14 (1994) 5570–5579. [13] Strobel, S., Miller, H.R.P. and Ferguson, A., Human intestinal mucosal mast cells: evaluation of fixation and staining techniques, J. Clin. Pathol., 34 (1981) 851–858. [14] Tal, M., Schneider, M., Toyka, K.V. and Koltzenburg, M., Heat hyperalgesia and nociceptor excitation after application of NGF or inflammatory mediators, IASP Abstr., 33 (1996) 119.