Neurogenic inflammation, vascular permeability, and mast cells. II. Additional evidence indicating that mast cells are not involved in neurogenic inflammation.

Activation of cutaneous sensory nerves induces vasodilatation and vascular permeability, i.e., neurogenic inflammation. We examined the histology and possible mast cell involvement in cutaneous neurogenic inflammation induced by electrical nerve stimulation (ENS). Three lines of evidence indicated that mast cells were not involved in rodent cutaneous neurogenic inflammation induced by electrical stimulation of the saphenous nerve. 1) Most mast cells (86.5% of all mast cells in the dorsal skin of the paw) were found in the deep dermis, whereas vessels developing increased vascular permeability after nerve stimulation (visualized with the supravital dye Monastral blue B, a macro-molecular tracer) were localized predominantly in the superficial dermis. By contrast, i.v. substance P, which also causes increased cutaneous vascular permeability, predominantly caused deeper vessels to leak. As analyzed by electron microscopy, the vessels that developed permeability in response to nerve stimulation, and were thereby stained with Monastral blue B, were found to be exclusively postcapillary venules. 2) Disodium cromoglycate (DSCG), a mast cell stabilizing compound, inhibited the cutaneous vascular permeability induced by intradermal injections of anti-IgE in a dose-dependent manner. By contrast, vascular permeability induced by ENS was not influenced by disodium cromoglycate treatment. 3) ENS and i.v. substance P both induced cutaneous vascular permeability in mast cell-deficient W/Wv mice, despite the fact that their skin contained only 4.7% of the mast cells present in their normal +/+ litter mates. The magnitude of ENS-induced vascular permeability responses in W/Wv mice were similar to control +/+ and BALB/c mice. This study supports our earlier observations suggesting that mast cell activation is not essential for the initial, vascular permeability phase of neurogenic inflammation in rodent skin.     

Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs

Summary The association between mast cells (visualized by routine staining and immunohistochemistry for histamine) and capsaicin-sensitive nerves (containing calcitonin gene-related peptide (CGRP) and substance P (SP)) was studied in the pig. In the 1-ethyl-3(3-diethylaminopropyl)carbodiimide (EDCDI)-fixed skin tissue, histamine-containing mast cells and CGRP/SP-positive nerves were found in close association around blood vessels. In the EDCDI-fixed airway mucosa, only single histamine-containing mast cells were detected. However, many alcian blue-positive mast cells were found, sometimes close to the airway epithelium where CGRP/SP-containing nerve fibres were absent 2 days after systemic capsaicin pretreatment, but no changes in the number and distribution of tissue mast cells, granulocytes or lymphocytes, or the number of blood leukocytes were detected. Local injection of allergen, histamine and capsaicin into the skin of pigs actively sensitized with ascaris antigen caused a rapid light red-flare (vasodilation) reaction. Allergen and histamine, but not capsaicin, also produced plasma protein extravasation. In contrast to the absent flare, the protein extravasation response still occurred in capsaicin-treated pigs. The sensitivity to ascaris antigen was mediated by an IgE-like antibody. We conclude that a functional and morphological relationship exists between histamine-containing mast cells and capsaicin-sensitive sensory nerves in the pig skin. Mast cells and sensory nerves are also found in the airway mucosa and appear to be closely associated with the epithelium.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crossing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerves following traumatic injuries and surgical procedures.     

Neurogenic inflammation, vascular permeability, and mast cells

Electrical stimulation (ES) of sensory nerves causes increased vascular permeability and vasodilatation, a process known as neurogenic inflammation. The purpose of this study was to assess the role of mast cells in neurogenic inflammation induced by ES of sensory nerves. ES of the rat saphenous nerve for 1, 3, 5, 15, or 30 min induced a 166 to 436% increase in the amount of 125I-albumin deposited in the skin. Through the initial 15 min of ES, the histamine content of the skin remained unchanged. However, 30 min of ES caused a 22.1% decrease in skin histamine (p less than 0.05). ES for 5 min followed by measurement of vascular permeability from 0 to 30 min thereafter resulted in maximal increases in 125I-albumin in the skin immediately after cessation of the pulse of ES. When skin histamine was measured at various intervals after a 5-min pulse of ES, no change in the histamine content was observed through the subsequent 30 min. When mast cell degranulation was assessed histologically, 5 min of ES failed to stimulate mast cell degranulation. However, 30 min of ES caused a significant increase in the proportion of degranulating mast cells. When draining venous plasma histamine was monitored before, during and after ES, no change in plasma histamine was observed. In contrast, the intradermal injection of 5 micrograms of compound 48/80 produced a significant increase in plasma histamine. In order to examine the possibility that histamine might be released but remain in the skin after ES, skin "blisters" were developed by intradermal injections of saline. There was a significant increase in the amount of 125I-albumin extravasated into blister fluid measured after 3, 5, and 10 min of ES and a significant increase in histamine after 5 or 10 min. Therefore, prolonged ES of sensory nerves can cause mast cell degranulation. However, ES causes increased vascular permeability at times when no mast cell activation can be observed. These data suggest that the initial phases of neurogenic inflammation are independent of mast cell activation.     

Inhibition of neutral endopeptidase potentiates neurogenic inflammation in the rat trachea

The present study was performed to determine whether neurogenic inflammation in the rat trachea can be exaggerated by inhibiting neutral endopeptidase, an enzyme that degrades tachykinins that are believed to mediate neurogenic inflammation. Neurogenic inflammation was produced by antidromic electrical stimulation of one vagus nerve (2.5 Hz, 1 ms, 5 V for 5 min) in the presence of atropine or by an intravenous injection of capsaicin (100 micrograms/kg). Neutrophils that adhered to the endothelium of venules were visualized and counted in tracheal whole mounts that were stained by a histochemical reaction for myeloperoxidase. Neural inflammation increased the number of adherent neutrophils. Pretreatment with the neutral endopeptidase inhibitor phosphoramidon (1.0 or 2.5 mg/kg iv) increased neutrophil adhesion induced by neural inflammation. As assessed by the amount of extravasation of Monastral blue pigment, neural inflammation also increased vascular permeability, and this change was potentiated by phosphoramidon. These results are consistent with the concept that neuropeptides released from sensory nerves in the tracheal mucosa cause neutrophils to adhere to venules and increase vascular permeability and that these effects are modulated by neutral endopeptidase.     

Endoneurial mast cells in peripheral nerves of the armadillo dermis

Summary Electron microscopy of the dermis of the 9-banded armadillo, Dasypus novemcinctus, reveals an intimate relationship of mast cells to peripheral nerves. Mast cells are routinely found in the dermis in close proximity to nerves, and mast cells are also present within the perineurial cell sheath in the endoneurial space proper. These cells contain electron opaque granules and exhibit numerous surface folds as is characteristic of other species. The degranulation of the mast cells by injection of 0.5% aqueous trypan blue reveals sequential exocytosis of granules, similar to that described for the rat. Degranulation occurs in most dermal and endoneurial mast cells following a single intradermal injection of trypan blue. A small number of mast cells partially degranulate and a few exhibit no degranulation, suggesting a heterogeneous population. It is also noted that mitochondrial morphology differs in degranulating cells, in that they become swollen and vacuolated. The results of this study are discussed in light of previous results reported for other species, mainly at the light microscope level.Supported by institutional grant from the L.S.U. School of DentistryThe author thanks Ivis Insua and Angela Pepin for their technical and secretarial assistance and Garbis Kerimian for his photographic work     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

新生大鼠雌激素注射后睾丸肥大细胞的变化

新生大鼠注射雌二醇后,睾丸肥大细胞于第30天可见到,细胞数量随年龄增长而增多,生后4－6个月,睾丸网附近仍可见大量肥大细胞。睾丸内的肥大细胞比皮肤内的结缔组织肥大细胞（CTMC）小而与小肠粘膜的粘膜肥大细胞（MMC）相近,AB－S染色后基本着蓝色,硫酸小檗碱荧光染色后呈现中等强度黄色荧光,结果提示,新生大鼠雌激素注射后睾丸内肥大细胞的增多可能与免疫过程有关,睾丸内肥大细胞与CTMC和MMC皆有所不同。     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Summary Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crousing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerved following tramatic injuries and surgical procedures.     

牛蛙肥大细胞的组织化学与形态学

目的鉴定牛蛙组织中肥大细胞的存在。方法用于肥大细胞研究的一些常规组织化学技术与形态学方法。结果牛蛙的舌、肠、肠系膜和脾中肥大细胞数量较多,少量也见于神经、心、肾、肝和皮肤等多种组织中。肥大细胞有沿血管周和神经分布的倾向。脾脏中的肥大细胞形状比较一致,呈圆形或卵圆形,而在其它部位的肥大细胞则形态多样。Bouin氏液及Carnoy氏液是牛蛙肥大细胞优良的固定液。然而,与哺乳动物的黏膜肥大细胞相似的是,中性缓冲福尔马林(NBF)固定显著的阻断了牛蛙肠黏膜肥大细胞(MMC)的染色。有趣的是,甲苯胺蓝是牛蛙肥大细胞的最佳染料,它比阿尔新蓝能很好地显示牛蛙的肥大细胞。透射电镜下证实,牛蛙肥大细胞中含有大量特征性的胞浆颗粒。肥大细胞靠近雪旺氏细胞,并可见于神经束膜间,甚至以其突起与神经束膜相连。结论通过组织化学与形态学研究证实了牛蛙组织中肥大细胞的存在,再次证实肥大细胞与外周神经之间存在密切的解剖学关系。     

Role of capsaicin-sensitive nerve fibers in uterine contractility in the rat

The possible participation of capsaicin-sensitive sensory nerves in the modulation of neurogenic contractions was studied in nonpregnant and term pregnant rat uteri. Neurogenic contractions were elicited by electric field stimulation (40 V, 1-70 Hz, 0.6 msec) in intact uteri and uteri that were previously exposed to capsaicin in vitro. In capsaicin pretreated preparations obtained both from nonpregnant and term pregnant rats, a dose-dependent increase in the amplitude of uterine contractions was detected. Prior systemic treatment of the rats with capsaicin (130 mg/kg, s.c.) abolished the effect of in vitro capsaicin administration on the amplitude of neurogenic contractions. Use of a specific antagonist of calcitonin gene-related peptide revealed that depletion of this peptide, which normally elicits uterine smooth muscle relaxation, may be responsible for the increased responsiveness of the uterus to low-frequency stimulation. Experiments on the localization of calcitonin gene-related peptide in uterine tissue specimens exposed to capsaicin revealed dose-dependent depletion of calcitonin-gene related peptide-immunoreactive nerves innervating blood vessels and the myometrium. The findings indicate that capsaicin-sensitive afferent nerves, by the release of sensory neuropeptides, significantly contribute to the modulation of uterine contractility both in nonpregnant and term pregnant rats. It is suggested that uterine sensory nerve activation may be part of a trigger mechanism leading to preterm contractions evoked by, for example, inflammation.     

Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats

Respiratory syncytial virus (RSV) infection is associated with exaggerated neurogenic inflammation in the airways. This study sought to determine whether irritation of the mucosal sensory fibers affects the recruitment of lymphocytes and monocytes to RSV-infected airways. Pathogen-free rats were inoculated with RSV or with virus-free medium and were injected 5 days later with capsaicin to stimulate airway sensory nerves. Bronchoalveolar lavage was performed 1, 5, or 10 days after nerve stimulation, and samples were analyzed by differential cell count and flow cytometry. Without nerve stimulation, RSV caused a minimal increase in the number of lymphocytes and monocytes above pathogen-free control levels. After nerve stimulation, numerous lymphocytes, predominantly CD4+ T cells, and monocytes were recruited in the airways of infected rats, whereas no difference was found in pathogen-free controls. RSV induced overexpression of the neurokinin 1 (NK1) receptor for substance P on discrete lymphocyte subpopulations within the bronchial-associated lymphoid tissue (BALT), and treatment with a specific NK1 receptor antagonist abolished the recruitment of both lymphocytes and monocytes to infected airways. Our data suggest that airborne irritants stimulating mucosal sensory fibers during RSV infection exert important immunomodulatory effects by attracting to the infected airways selected lymphocyte subpopulations from the local BALT as well as monocytes.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Immunohistochemical analysis of substance P containing nerve fibres and their contacts with mast cells in the diabetic rat's tongue

Sensory neuropathy is common symptom of the diabetes mellitus and the prevalence of oral lesions is higher in diabetic patients. The distribution of substance P was studied immunohistochemically in streptozotocin induced diabetic rat's tongue. The morphological association of sensory nerves (substance P immunoreactive) with mast cells (nerve fibre-mast cell contact) was monitored. The substance P nerve fibre mast cell contacts were very scanty in control tongue. The number of substance P nerve terminals and mast cells was significantly increased (p < 0.05) in diabetes mellitus after 4 weeks of the treatment compared with the control tongue. The number of mast cell nerve contacts was even more significantly increased (p < 0.001) in diabetes. The distance between nerve fibres and mast cells was about 1 mm and very often less than 200 nm. In some instances, the mast cells were degranulated in the vicinity to nerve fibres. Increased number of mast cell nerve contacts in neurogenic inflammation might cause vasoconstriction and lesions of the oral mucosa, so some disorders such lichen planus, leukoplakia and cancer might frequently develop in diabetes mellitus.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

A study of chemically induced acute inflammation in the skin of the rat

Oedema due to application of benzene to the skin was reduced following prior sensory denervation and in animals systemically pre-treated with capsaicine (which is known to confer resistance to chemical irritants) or compound 48/80 (which depletes the body of mast cells). Increased degranulation of mast cells in the benzene-treated skin was unaffected by denervation but did not occur after treatment with capsaicine. Antidromic stimulation of a cutaneous nerve caused oedema and degranulation of mast cells, both of which were less severe than the corresponding effects of topically applied benzene. These effects were completely prevented by prior treatment with capsaicine and the oedema was less severe in 48/80-treated rats. Hence, the presence of mast cells was necessary for full development of the effects of antidromic stimulation. These observations indicate that axon reflexes in sensory fibres contribute to, but are not entirely responsible for, the development of oedema in chemically irritated skin. The prophylactic action of capsaicine may be due to the prevention of degranulation of mast cells rather than to a direct effect on cutaneous nerve endings. These conclusions are embodied in an hypothesis purporting to explain the involvement of axon reflexes, mast cells and various humoral mediators in chemically induced acute inflammation.     

Peripheral choline acetyltransferase in rat skin demonstrated by immunohistochemistry

Conventional choline acetyltransferase immunohistochemistry has been used widely for visualizing central cholinergic neurons and fibers but not often for labeling peripheral structures, probably because of their poor staining. The recent identification of the peripheral type of choline acetyltransferase (pChAT) has enabled the clear immunohistochemical detection of many known peripheral cholinergic elements. Here, we report the presence of pChAT-immunoreactive nerve fibers in rat skin. Intensely stained nerve fibers were distributed in association with eccrine sweat glands, blood vessels, hair follicles and portions just beneath the epidermis. These results suggest that pChAT-positive nerves participate in the sympathetic cholinergic innervation of eccrine sweat glands. Moreover, pChAT also appears to play a role in cutaneous sensory nerve endings. These findings are supported by the presence of many pChAT-positive neuronal cells in the sympathetic ganglion and dorsal root ganglion. Thus, pChAT immunohistochemistry should provide a novel and unique tool for studying cholinergic nerves in the skin.     

Peptidergic innervation of rat lymphoid tissue and lung: Relation to mast cells and sensitivity to capsaicin and immunization

Summary The peptidergic innervation of lymphoid tissue and the lung in relation to mast cells was studied in rat. The sensitivity of neuropeptide-containing nerves to capsaicin treatment and immunization was also examined. Measurements of the content of neurokinin A and calcitonin gene-related peptide revealed that the lung contained the highest content of both neuropeptides; lymph nodes had intermediate levels, whereas the spleen had the lowest content. Immuhohistochemistry showed that the calcitonin gene-related peptide- and neurokinin A-immunoreactive nerves in lymph nodes were mainly found around blood vessels, whereas in the lung the nerves were present within the lining respiratory epithelium, bronchial smooth muscle, around blood vessels and close to lymphoid aggregates. Combined immunohistochemistry for serotonin (5-hydroxytryptamine), as a marker for mast cells, and tachykinins or calcitonin gene-related peptide revealed that a close association was often present between the nerves and 5-hydroxytryptamine-positive cells in the bronchi of the lung, while 5-hydroxytryptamine-positive cells were not observed in lymph nodes. The neurokinin A and calcitonin gene-related peptide content in lymph nodes, spleen and lung, but not the content of neuropeptide Y, was markedly decreased by capsaicin treatment, suggesting a sensory origin for the two former peptides. Aerosol immunization increased the levels of calcitonin gene-related peptide in the lung, whereas the content in mediastinal lymph nodes was not affected. These data demonstrate a peptidergic innervation mainly of blood vessels in lymphoid tissue and a close relation between sensory nerves and mast cells as well as lymphoid aggregates in the bronchi of the lung. This further suggests that the sensory innervation of lymph nodes is mainly related to regulation of vascular tone and lymph flow. Furthermore, at the site of immunization, i.e., in the airway mucosa, sensory nerve mediators may interact both with mast cells and lymphoid cells.