Three-dimensional architectures of P2X2-/P2X3-immunoreactive afferent nerve terminals in the rat carotid body as revealed by confocal laser scanning microscopy.

We investigated the three-dimensional architectures of P2X2-/P2X3-immunoreactive nerve terminals in the rat carotid body using immunohistochemistry with confocal laser microscopy. Nerve endings immunoreactive for P2X2 and P2X3 were associated with clusters of type I cells, whereas some nerve endings were sparsely distributed in a few clusters. Most nerve endings surrounding type I cells were hederiform in shape and extended several flattened axon terminals, which were polygonal or pleomorphic in shape and contained P2X2-/P2X3-immunoreactive products. Three-dimensional reconstruction views revealed that some flattened nerve endings with P2X3 immunoreactivity formed arborized, sac- or goblet-like terminal structures and were attached to type I cells immunoreactive for tyrosine hydroxylase (TH). However, P2X3-immunoreactive axon terminals were sparsely distributed in type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for P2X2, S100, and TH revealed that P2X2-immunoreactive axon terminals were attached to TH-immunoreactive type I cells on the inside of type II cells with S100 immunoreactivity. These results revealed the detailed morphology of P2X2-/P2X3-immunoreactive nerve terminals and suggest that sensory nerve endings may integrate chemosensory signals from clustered type I cells with their variform nerve terminals.

Expressions of angiotensin and cytokine receptors in the paracrine signaling of the carotid body in hypoxia and sleep apnea.

Arterial chemoreceptors in the carotid body are central to the chemical control of breathing in the chemotransduction of physiological stimuli in the arterial blood for eliciting the chemoreflex, which mediates the respiratory, cardiovascular and autonomic responses to hypoxia, hypercapnia and acidosis. Recent evidence suggests that signaling molecules locally produced in the carotid body, including angiotensin II and pro-inflammatory cytokines play an important role in the modulation of the activity of carotid chemoreceptors, via the angiotensin and cytokine receptors expressed in the chemosensitive cells in an autocrine-paracrine manner. The carotid chemoreceptor activity is augmented in subjects at high altitude and in patients with sleep-disordered breathing. Maladaptive responses of the paracrine signaling to hypoxia in the carotid body have been proposed to play a pathogenic role in sleep apnea. Specifically, recent findings show significant increases in expressions of angiotensin receptors and components of a local angiotensin-generating system in the carotid body in sustained or intermittent hypoxia, which augments the chemoreceptor activity and also mediates the inflammatory response of the carotid body to hypoxia. In addition, inflammation of the carotid body involves an increased local expression of cytokine receptors and pro-inflammatory cytokines in sustained or intermittent hypoxia. This review aims to summarize the evidence supporting that the upregulated expression of the angiotensin receptors and cytokine pathways in the carotid body leads to augmented activities of the carotid chemoreceptor in hypoxic conditions, which could play a role in the pathophysiology of sleep apnea.

Pathogenic roles of the carotid body inflammation in sleep apnea.

Breathing difficulties in sleep are a hallmark of sleep-disordered breathing commonly observed in patients with sleep disorders. The pathophysiology of sleep apnea is in part due to an augmented activity of the carotid body chemoreflex. Arterial chemoreceptors in the carotid body are sensitive to inflammatory cytokines and immunogenic molecules in the circulation, because cytokine receptors are expressed in the carotid body in experimental animals and human. Intriguingly, proinflammatory cytokines are also locally produced and released in the carotid body. Also, there are significant increases in the expression of proinflammatory cytokines, cytokine receptors, and inflammatory mediators in the carotid body under hypoxic conditions, suggesting an inflammatory response of the carotid body. These upregulated cytokine signaling pathways could enhance the carotid chemoreceptor activity, leading to an overactivity of the chemoreflex adversely effecting breathing instability and autonomic imbalance. This review aims to summarize findings of the literature relevant to inflammation in the carotid body, with highlights on the pathophysiological impact in sleep apnea. It is concluded that local inflammation in the carotid body plays a pathogenic role in sleep apnea, which could potentially be a therapeutic target for the treatment of the pathophysiological consequence of sleep apnea.

Inflammatory and immunomodulatory mechanisms in the carotid body.

Evidence is available about the role of inflammatory/immunological factors in the physiology and plasticity of the carotid body, with potential clinical implications in obstructive sleep apnea syndrome and sudden infant death syndrome. In humans, lymphomonocytic aggregations (chronic carotid glomitis) have been reported in aging and opiate addiction. Glomus cells produce prostaglandin E2 and the cytokines interleukin 1ß, interleukin 6 and TNF-α, with corresponding receptors. These factors modulate glomus cell excitability, catecholamine release and/or chemoreceptor discharge. The above cytokines are up-regulated in chronic sustained or intermittent hypoxia, and prevention of these changes, with ibuprofen or dexamethasone, may modulate hypoxia-induced changes in carotid body chemosensitivity. The main transcription factors considered to be involved are NF-kB and HIFs. Circulating immunogens (lipopolysaccharide) and cytokines may also affect peripheral arterial chemoreception, with the carotid body exerting an immunosensing function.

Effect of endothelin receptor antagonist bosentan on chronic hypoxia-induced inflammation and chemoafferent neuron adaptation in rat carotid body.

Chronic hypoxia (CH) induces an inflammatory response in rat carotid body that is characterized by immune cell invasion and the expression of pro-inflammatory cytokines. In the present study, we have investigated the role of type-A endothelin (ET-A) receptors in the development of CH-induced inflammation. After 7 days of CH (380 Torr), double-label immunofluorescence studies demonstrated elevated levels of ET-A receptor and tyrosine hydroxylase (TH) in O(2)-sensitive type I cells. Following CH, ET-A receptors were also expressed on resident and invasive CD45+ immune cells distributed in tissue surrounding chemosensory cell lobules. Immnofluorescence and quantitative PCR studies showed that concurrent treatment with the ET-A/B receptor antagonist, bosentan (200 mg/kg/day), blocked CH-induced ED-1+ macrophage invasion and the upregulation of cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and monocyte chemoattractant protein-1 (MCP-1). Moreover, bosentan treatment blocked the CH-induced increases in expression of acid-sensitive ion channels (ASICs) in chemoafferent neurons in the petrosal ganglion (PG). Our findings are consistent with the hypothesis that CH-induced inflammation involves the upregulation and release of ET-1 from type I cells. ET-1 may act in an autocrine/paracrine mechanism via ET-A receptors on chemosensory type I cells and immune cells to promote an inflammatory response.

Chronic intermittent hypoxia induces local inflammation of the rat carotid body via functional upregulation of proinflammatory cytokine pathways.

Maladaptive changes in the carotid body (CB) induced by chronic intermittent hypoxia (IH) account for the pathogenesis of cardiovascular morbidity in patients with sleep-disordered breathing. We postulated that the proinflammatory cytokines, namely interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α, and cytokine receptors (IL-1r1, gp130 and TNFr1) locally expressed in the rat CB play a pathophysiological role in IH-induced CB inflammation. Results showed increased levels of oxidative stress (serum 8-isoprostane and nitrotyrosine in the CB) in rats with 7-day IH treatment resembling recurrent apneic conditions when compared with the normoxic control. Local inflammation shown by the amount of ED1-containing cells (macrophage infiltration) and the gene transcripts of NADPH oxidase subunits (gp91(phox) and p22(phox)) and chemokines (MCP-1, CCR2, MIP-1α, MIP-1ß and ICAM-1) in the CB were significantly more in the hypoxic group than in the control. In addition, the cytokines and receptors were expressed in the lobules of chemosensitive glomus cells containing tyrosine hydroxylase and the levels of expressions were significantly increased in the hypoxic group. Exogenous cytokines elevated the intracellular calcium ([Ca(2+)](i)) response to acute hypoxia in the dissociated glomus cells. The effect of cytokines on the [Ca(2+)](i) response was significantly greater in the hypoxic than in the normoxic group. Moreover, daily treatment of IH rats with anti-inflammatory drugs (dexamethasone or ibuprofen) attenuated the levels of oxidative stress, gp91(phox) expression and macrophage infiltration in the CB. Collectively, these results suggest that the upregulated expression of proinflammatory cytokine pathways could mediate the local inflammation and functional alteration of the CB under chronic IH conditions.

Immunosensory signalling by carotid body chemoreceptors.

Injections of lipopolysaccharide (LPS) have been used to produce the signs of sepsis and study their underlying mechanisms. Intravenous (IV) injections of LPS in anesthetized cats induce tachypnea, tachycardia and hypotension, but ventilatory changes are suppressed after sectioning carotid and aortic nerves. Otherwise, LPS increases the basal frequency of carotid chemosensory discharges, but reduces ventilatory and chemosensory responses to hypoxia and nicotine injections. Increases in cytokines (IL-1ß, IL-6 and TNF-α) are observed in plasma and tissues after injecting LPS. In carotid bodies perfused in vitro, TNF-α reduces chemosensory discharges induced by hypoxia. The rat carotid body and its sensory ganglion constitutively express LPS canonical receptor, TLR4, as well as TNF-α and its receptors (TNF-R1 and TNF-R2). Increases of TNF-α and TNF-R2 expression occur after LPS administration. The activation of peripheral and central autonomic pathways induced by LPS or IL's is partly dependent on intact vagus nerves. Thus, the carotid and vagus nerves provide routes between the immune system and CNS structures involved in systemic inflammatory responses.

Chronic carotid glomitis in heroin addiction.

The aim of the present work was to investigate the occurrence and immunological characteristics of chronic carotid glomitis in opiate addicts. Carotid bodies were sampled at autopsy from 50 subjects who died of heroin intoxication (mean age 28 years), and from 16 young (24 years) and 10 older subjects (66 years) who died of trauma. Sections were stained with haematoxylin-eosin and azan-Mallory, and immunohistochemistry was carried out with anti-CD45, -CD3, -CD8, -CD4, -CD20, -CD68, -CD56. Inflammatory aggregates were not observed in young cases, but were found in 21/50 (42%) opiate cases and in 4/10 (40%) older cases. Infiltrates were mainly located in subcapsular and interlobular positions, and were also found around nerve fibres. Inflammatory aggregates were mainly composed of T suppressor/cytotoxic lymphocytes (50-80%). Monocytic/macrophagic cells and B lymphocytes comprised about 10% and 5-20% of inflammatory cells, respectively. T helper lymphocytes were fewer and only rare Natural Killer cells were found. Chronic carotid glomitis must be included among the autopsy findings of opiate addiction, and may be ascribed to inflammatory reactions to exogenous immunogens or to responses to drug-induced degenerative changes of carotid body components.

Morphological evidence for existence of IL-6 receptor alpha in the glomus cells of rat carotid body.

The carotid body (CB) senses changes in arterial blood PO2 and modulates respiratory movement. It is generally accepted that the dopaminergic type I cells in the CB are chemoreceptors. However, it has not been clarified whether the carotid body has the ability to perceive the stimulation of proinflammatory cytokines. Interleukin 6 (IL-6) as a multifunctional cytokine plays a pivotal role in host defense mechanism. In the present study, we observed the expression of IL-6Ralpha mRNA and protein in the carotid body using immunohistochemistry, Western blots, and in situ hybridization. The results confirmed the presence of IL-6Ralpha proteins and mRNAs in the glomus cells of rat carotid body. These results suggest that the function of the carotid body may be influenced by the proinflammatrory cytokines through their receptors.

Adrenomedullin immunoreactivity in the human carotid body.

We studied by immunocytochemistry the expression of AM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age+/-S.D.: 44.3+/-3.4 years) and from six fetuses (mean gestational age+/-S.D.: 167+/-11 days). No AM immunoreactivity was visible in the type II cells of both series. The percentage of immunoreactive type I cells was higher in the adult subjects (32.3+/-7.7%) with respect to the fetuses (11.8+/-2.7%, P < 0.001). Dark cells showed a higher percentage of positive immunoreaction with respect to light cells, both in adult subjects (61.7+/-13.4% versus 19.2+/-5.2%) and in fetuses (25.3+/-4.4% versus 6.2+/-2.0%). AM may play a role in the regulation of chemoreceptor discharge through paracrine releasing action and/or vasodilator effect. The low expression of AM in fetuses may be ascribed to the absence of pulmonary respiration with lack of regulatory role of the carotid body during the prenatal period.

Atrial natriuretic peptide increases cyclic guanosine monophosphate immunoreactivity in the carotid body.

The mammalian carotid body is a peripheral arterial chemoreceptor organ involved in the regulation of respiration, and in the modulation of blood pressure through reflex control of peripheral vascular resistance and cardiac output. In addition to its responsiveness to blood gases, the organ is also sensitive to hyperosmotic solutions, and we have recently shown that a systemic hormonal regulator of natriuresis and diuresis, atrial natriuretic peptide, is a potent inhibitor of chemoreceptor activity evoked by hypoxia in the cat carotid body. The present study demonstrates atrial natriuretic peptide immunoreactivity in type I cells of the carotid body, and shows further that a biologically active atrial natriuretic peptide fragment, atriopeptin III, increases cyclic guanosine monophosphate immunoreactivity in type I cells in a dose-dependent manner. Moreover, double-labeling techniques demonstrate co-existence of atrial natriuretic peptide immunoreactivity with the atriopeptin III-enhanced cyclic guanosine monophosphate reaction product. These findings indicate the probable existence of atrial natriuretic peptide receptors coupled to membrane-bound guanylate cyclase on the parenchymal type I cells. Our findings support the view that cyclic guanosine monophosphate functions as a second messenger in this organ, and may serve as a functional activity marker in identifying type I cells which respond to atrial natriuretic peptide.

Calcitonin gene-related peptide immunoreactivity in the nucleus of the tractus solitarius and the carotid receptors of the cat originates from peripheral afferents.

The presence and distribution of the calcitonin gene-related peptide was studied, using immunohistochemical techniques, in carotid receptors, in the nodose and glossopharyngeal ganglia and in the nucleus tractus solitarii of the cat. Seventy-seven per cent of the 42% of the nodose ganglion cells were labeled. Fine, sparsely branched immunoreactive terminal axonal arborizations were found in the carotid body; they disappeared after petrosal ganglionectomy. The intense immunoreactivity present in fibers in the commissural, medial, interstitial, gelatinosus, dorsal, intermediate and rostral gustatory subnuclei of the nucleus tractus solitarius was drastically reduced after removal of the ipsilateral nodose and petrosal ganglia. The central distribution of the immunoreactive axons, the morphology of the terminals in the carotid receptors and their dependence on an intact peripheral innervation are consistent with the idea that in the cat the calcitonin gene-related peptide is present in a high proportion of the primary visceral afferents, most of them unmyelinated.

Chemoreceptor A-fibres in the human carotid body contain tyrosine hydroxylase and neurofilament immunoreactivity.

Previous retrograde tracing studies on rat and guinea-pig showed a projection of sensory tyrosine hydroxylase-immunoreactive neurons to the region of the carotid bifurcation via the carotid sinus nerve. In the present study, focussing on the sensory innervation of the human carotid body, antisera to tyrosine hydroxylase and other catecholamine synthesizing enzymes were applied for an immunohistochemical investigation of carotid bodies obtained at autopsy. In addition, an array of antisera directed to non-enzyme antigens known to be present in viscero-afferent neurons were incorporated in the study. The glomic lobules consisting of glomus cells and sustentacular cells contained a variable number of enzyme-immunoreactive glomus cells. Arteries were supplied by nerve fibres displaying the full phenotype of sympathetic noradrenergic axons, i.e. immunoreactivity to tyrosine hydroxylase, aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. The glomic lobules, however, were densely innervated by tyrosine hydroxylase-immunoreactive axons lacking immunoreactivity to aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. These fibres reacted with neurofilament 160kD-antibody but were devoid of immunoreactivity to all neuropeptides tested (calcitonin gene-related peptide, somatostatin, substance P). Ultrastructurally, tyrosine hydroxylase/neurofilament 160kD-immunoreactive axons gave rise to large axonal swellings filled with mitochondria and vesicles, and established extensive contacts to glomus cells. Nerve bundles surrounded by a perineural sheath contained both myelinated (2.0-2.8 microns in diameter) and unmyelinated (0.14-3.0 microns) tyrosine hydroxylase-immunoreactive axons. Most of the unmyelinated immunoreactive axons were running singularly within a Schwann cell-sheath. Judged from the pattern of immunoreactivities as well as their preterminal and terminal ultrastructure, tyrosine hydroxylase-immunoreactive axons innervating glomus cells are of sensory origin. Although final proof by retrograde tracing cannot be presented in man, this conclusion is supported by experimental evidence in laboratory animals. The myelinated immunoreactive axons correspond to chemoreceptor A-fibres whereas the classification of the large unmyelinated immunoreactive axons has yet to be established. The lack of immunoreactivity to the dopamine-synthesizing enzyme, aromatic-L-amino-acid-decarboxylase, in this fibre type does not support the view of dopamine being the primary transmitter of chemoreceptor afferents.

Distribution of serotonin-immunoreactive cells around arteries arising from the common carotid artery in the chicken.

By using an immunoperoxidase method with antiserotonin antiserum, the distribution of serotonin-immunoreactive cells in the carotid body region was investigated in chickens. The thyroid gland, cranial and caudal parathyroid glands, carotid body, and ultimobranchial gland of chickens were located along the common carotid artery as a continuous series and were supplied with branches arising from the artery. Almost all chief cells of the chick carotid bodies were immunoreactive for serotonin. Furthermore, numerous serotonin-immunoreactive cells were widely distributed in the adventitial connective tissue around those arteries that issued from the common carotid artery to supply each endocrine organ, i.e., the carotid body artery, the esophagotracheobronchial artery, the ascending esophageal artery, and the inferior thyroid artery. These arteries usually arose by one trunk from the lateral aspect of the middle portion of the common carotid artery. The serotonin cells were most numerous around the carotid body artery and were dispersed along the whole length of the artery. In addition, they were detected around the common trunk of each artery and the roots of the ascending esophageal artery, the inferior thyroid artery, and the esophagotracheobronchial artery. The serotonin cells were also distributed in the tunica media of the common carotid artery. In that place, they were concentrated around the origin of the common trunk of each artery and were scattered below the origin along the longitudinal axis and on the opposite side of the origin. The serotonin-immunoreactive cells distributed around and in the arteries may be involved in the control of blood flow and may have chemoreceptive properties.

Chronic carotid glomitis.

Both carotid bodies were obtained at necropsy from each of 75 subjects, 38 male and 37 female, ranging in age from 14 to 90 years. In each case the histological features were classified as one of four age-related patterns. The presence and distribution of lymphocytes were noted. They presented either as a diffuse scattering throughout the stroma or as large focal aggregates. In subjects over the age of 50 years the diffuse infiltrates occurred in 56% of cases and were virtually all T-cells. In the same age group the aggregates of lymphocytes were found in 21% of cases. These findings indicate that there is a disease of human carotid bodies characterized by focal aggregates of lymphocytes that begins in middle age and becomes increasingly common with advancing years, affecting 29% of subjects aged 70 years or over. It is not related to any co-existing disease but seems to be a response to the degenerative changes in the glomic tissues that occur with age. By analogy with comparable changes in the thyroid and salivary glands, the disease may have a basis in auto-immunity.

Immunocytochemical demonstration of IGF-II-like immunoreactivity in human paraganglioma of the craniocervical region.

Insulin-like-growth factor (IGF)-II-like immunoreactivity was examined in two carotid bodies and six extra-adrenal paragangliomas with use of monoclonal antibody against rat IGF-II, which crossreacts with human IGF-II. Chief cells but not sustentacular cells of the carotid body were positive at about 10% in one case and less than 1% in another case. Among four carotid body tumours, a possible vagal body tumour and one glomus jugulare tumour, all but the glomus jugulare tumour exhibited positive tumour cells irrespective of histological variations. The frequency of positive cells ranged from 20 to 60%. IGF-II like immunoreactivity, therefore, might be widely distributed in human extra-adrenal paraganglionic tissues and tumours, although its biological role in these cells remains to be elucidated.

Retrograde neuronal labelling and double-staining immunohistochemistry of tachykinin- and calcitonin gene-related peptide-immunoreactive pathways in the carotid sinus nerve of the guinea pig.

The origin of tachykinin- and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibres in the guinea pig carotid body and carotid sinus was determined by retrograde labelling of the carotid sinus nerve with Fluoro-gold and immunohistochemical double staining with fluorescein- and rhodamine-conjugated second antisera. Fluoro-gold-labelled perikarya with characteristic features of primary sensory neurones were numerous in the glossopharyngeal (petrosal) ganglion and occurred rarely in the closely attached superior vagal (jugular) ganglion. An efferent pathway from the brainstem could not be detected. Co-existence of tachykinin- and CGRP-LI was observed in 25-47% of labelled sensory neurones; less than 1% of Fluoro-gold-containing perikarya were exclusively stained by CGRP antiserum. Co-existence of tachykinin- and CGRP-LI was also demonstrated in nerve fibres of the carotid body and carotid sinus. Somatostatin-, cholecystokinin- and dynorphin-LI did not co-exist with tachykinin-LI in these fibres. Thus, tachykinin/CGRP-LI fibres in the carotid presso- and chemoreceptive areas exhibit a peptide pattern being generally characteristic for sensory fibres supplying great vessels in the guinea pig. In view of the present findings doubt is raised as to a primary involvement of these fibres in presso- or chemoreception, although a modulatory influence on these specific functions appears to be likely.

Bombesin/gastrin-releasing peptide (GRP)- and Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivities in small intensely fluorescent (SIF) cells and nerve fibers of rat sympathetic ganglia.

Superior cervical and hypogastric ganglia were removed from rats that had been perfused with a mixture of 4% paraformaldehyde and 0.25% glutaraldehyde. Specific antisera against Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL) and bombesin/gastrin-releasing peptide (BN/GRP) were used in the immunofluorescence procedure. In hypogastric ganglia, a subpopulation of small intensely fluorescent (SIF) cells, as identified by their aqueous aldehyde (Faglu)-induced fluorescence, showed MEAGL- and BN/GRP-like immunolabeling. SIF cells in the superior cervical ganglia were unlabeled. In both ganglia, varicose nerve fiber networks and nerve terminals surrounding principal ganglion cells showed MEAGL- and BN/GRP-like immunoreactivities. Unlabeled SIF cells often were in close contact with nerve fibers that had MEAGL-like immunolabeling. Immunoreactivities against MEAGL- and BN/GRP-like neuropeptides in nerve fibers and terminals suggest a neurotransmitter or neuromodulator role for these peptides. In addition, labeling in SIF cells implies their possible endocrine function.

Localization of enkephalin-like immunoreactivity in the cat carotid and aortic body chemoreceptors.

The purpose of this study was to determine if enkephalin-like immunoreactivity was present in the glomus cells of the carotid and aortic body peripheral arterial chemoreceptors. Cat carotid and aortic bodies were reacted with antisera to met- and leu-enkephalin using the indirect peroxidase-antiperoxidase immunocytochemical method of Sternberger (1979). Both the carotid and aortic bodies demonstrated clusters of immunoreactive cells for both met- and leu-enkephalin. Additionally, met-enkephalin-like immunoreactivity was observed in many of the dense-core vesicles of the glomus cells of the carotid body. The glomus cells of these chemoreceptors are known to contain catecholamines which may modulate chemoreceptor activity. The presence of opioid peptide-like substances co-existing with the glomus cell catecholamines, perhaps in the same vesicles, may have important implications for a trophic influence of these peptides on glomus cell chemoreceptor modulation.