ME/CFS RESEARCH FORUM REPORT:
Adelaide Research Network 3 - 4 June 2005
UNIVERSITY OF ADELAIDE
Convenor: Alison Hunter Memorial Foundation
Donald R. Staines MBBS, MPH, FAFPHM, FAFOM
Public Health Physician
Gold Coast Public Health Unit
Southport, Queensland, Australia
An autoimmune theory of endogenous vasoactive neuropeptides in the aetiology of Chronic Fatigue Syndrome
Endogenous vasoactive neuropeptides (VNs) of the pituitary adenylate cyclase-activating polypeptide (PACAP)/ vasoactive intestinal peptide (VIP) family may be implicated in the aetiology of chronic fatigue syndrome (CFS) . These VNs are ubiquitous substances strongly preserved in evolutionary terms indicating their crucial roles for survival. They have extraordinary commonality between species. Substantial amino acid sequence homology exists between them, suggesting evolution from a common ancestral gene, and they demonstrate some degree of overlap of structure and function as well as potential for immunological cross-reactivity. They exert their effects at high level in controlling brain and hypothalamic-pituitary-adrenal axis functions as well as at important peripheral sites such as heart, gut, blood, lung, pancreas and reproductive tracts .
VN roles and functions
These VNs belong to the secretin-glucagon super-family , exerting significant control over carbohydrate and lipid metabolism. They have important roles in neurotrophic function, neuroregulation and neurotransmission, and immunological and hormonal modulation . They are susceptible to catalysis and antibody action . Compromise of their function is therefore likely to have serious consequences for homeostasis. Pain and fatigue in a number of vasoactive neuropeptide associated disorders are potentially explicable. Endogenous opioid activity is functionally related to cytokine and vasoactive neuropeptide activity. Thus pain mediation and perception will be altered in conditions where endogenous opioid function is impaired through these mechanisms . Nitric oxide metabolism is implicated in immuno-modulation as well as mediating chemical sensitivity in these conditions through co-location of nitric oxide synthases . These VNs influence receptor accumulation and activity for acetylcholine  and other neurotransmitters. Hence co-transmitter functions with acetylcholine may be the linkage to fatigue mediation in some syndromes. Vasoactive neuropeptides have complex roles and structures and are mediated by G protein-coupled receptors (GPCR). The secondary transmitter cyclic adenosine monophosphate (cAMP) is generated from ATP via adenylate cyclase which is known to exist in multiple isoforms and exhibits a range of processing functions .
Endogenous VNs exert a wide spectrum of immunological functions and have a critical role in homeostasis of the immune system through different receptors expressed in various immunocompetent cells . They operate via multiple signaling processes . Disturbances in their function are recognised as potential causes of autoimmune disease and they appear to have a role in protecting bystander lysis, a process in the pathogenesis of several autoimmune and inflammatory diseases .The development of autoantibodies to PACAP and VIP in mammalian tissues  is known but not extensively documented. However autoantibody responses to this group of neuropeptides indicate immunological tolerance may be readily broken. Hence it has been postulated that depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses .
Implications for neuroregulation
PACAP and VIP exert an extraordinary array of functions in the brain and peripheral tissues. VIP has been identified in all regions of the brain including cerebral vessels . PACAP is co-located with vesicular acetylcholine transporter in nerve terminals in all mouse adrenomedullary cholinergic synapses . PACAP has a potentiating additive effect with adrenal and noradrenaline on cAMP production in rat cerebral cortex indicating a crucial role in neuroregulation . Human PACAP and VIP have been shown to exert circadian oscillations in mice  and these substances have specific roles in setting rhythms at certain times of the circadian cycle . Disruption to these processes would therefore be expected to have significant impacts on physiological functions and homeostasis.
The role of VNs in protection from brain cell apoptosis is well documented. Cerebellar granule neurons cultured in the presence of KCl undergo spontaneous apoptosis which is reduced by exposure to PACAP . Interestingly apoptosis is known to be higher in sudden infant death syndrome (SIDS) cases than controls with hippocampus and brainstem, including dorsal nuclei being affected  and apoptotic neurodegeneration is postulated as the specific pathophysiological mechanism . Mechanisms associated with apoptosis would therefore indicate a suitable area for investigation, particularly those mechanisms usually protective against apoptosis. These roles are fulfilled by VNs, for example hippocampal ischaemia induced apoptosis in the rat is protected by PACAP through inhibition of the JNK/SAPK and p38 signalling pathways . PACAP is known to have a neuroprotective effect against a range of insults. Ethanol-induced apoptosis occurs via caspase pathways resulting in DNA fragmentation, mitochondrial permeability and cell death. PACAP, acting via its receptor PAC1, protects against ethanol-induced cell death and may have a therapeutic role in conditions such as fetal alcohol syndrome .
Other regulatory roles of VNs
PACAP is a powerful respiratory stimulant in dogs . A potentially important finding is that late-gestation blockade of VIP activity in pregnant mice produced distinct morphological abnormalities in the somatosensory cortex of offspring. Their response to hypoxia was subsequently impaired. A significant arousal deficit was seen in anti-VIP mice, which was not associated with deranged peripheral or brainstem-mediated responses to hypoxia during sleep . This finding may have significant implications for respiratory control mechanisms.
Cardiovascular function is in part regulated by VNs. Some variation in the cardiac roles of PACAP exists and is dose dependent, for example PACAP may promote both tachycardia and bradycardia . PACAP activates intracardiac postganglionic parasympathetic nerves by shortening the effective refractory period, and has a greater profibrillatory effect than vagal stimulation . Conceivably PACAP opposition, for example through autoimmune effects on the PAC1 receptor might result in decreased cardiac responsiveness. Adipose tissue metabolism and thermal stress have been linked to VN function. High levels of uncoupling protein are produced to regulate heat production. Neonatal adipose tissue is a primary site of cytokine and cytokine receptor action . While the precise mechanism of adipose tissue metabolism dysfunction is unclear, a combination of factors including metabolic stress, infection, necrosis and vascular hypoperfusion has been suggested .
Nitric oxide and chemical sensitivity
Chemical sensitivity to environmental substances is associated with CFS. This may be modulated via nitric oxide and its metabolites. PACAP and VIP coexist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues and have a role in NO modulation. PACAP and VIP also demonstrate neuroprotective effects against ischaemia and glutamate-induced toxicity . Hence symptoms attributable to nitric oxide and glutamate might therefore occur in association with VN disturbance. Peroxynitrites are associated with toxicity related to brain injury and infection. Loss of neurones is significantly higher in peroxynitrite exposed brains of rats [32,33] and neurotropic virus infection produces inflammatory responses dependent on the activity of peroxynitrite . NO may mediate both neuroprotective and neurodegenerative actions during ischaemic/reperfusion injuries as well as mediating neurotoxicity . Experimental autoimmune disease is known to cause an increase in the production of nitric oxide metabolites correlating with indices of autoimmune expression, morphological impairment and levels of cyclic nucleotides. Interestingly, inhibition of nitric oxide synthesis in rats causes rapid tachyphylaxis to the haemodynamic effects of PACAP-27 . This may prove to be an important modulating mechanism for diminishing vasoactive neuropeptide responsiveness in an NO generating environment with suppression of NO synthase expression.
Autoimmune dysfunction of VNs or their receptors may potentially arise via a number of pathways. Cytosine-guanosine dinucleotide DNA (CpG) elements in promoter regions of VN receptors [38,39] may be vulnerable to assault mechanisms such as hypomethylation, resulting in dysregulation of transcriptional/translational capacity. CpG elements are also known to be potent stimulators of immune responses  and antibody responses to these VN receptors might hypothetically occur, giving rise to IgM or IgG antibody types, resulting in long term autoimmunity to these vital substances. Such mechanisms might also result from mimicry with bacterial residues, giving rise to mistaken recognition of self VN-CpG for bacterial CpG and perverse VN autoimmunity. Heat shock proteins may also play a role. They are important chaperone molecules for proper intracellular functioning of VNs and are known to have a key role in immunoregulation [41,42,43,44].
Conclusion and future directions
The autoimmune hypothesis of VNs suggests that relatively minor infection or inflammation results in predictable pro-inflammatory cytokine and other responses. Other pro-inflammatory effects such as nitric oxide release and possible chemical sensitivities may also occur. Modulation and termination of these inflammatory responses is required by VNs. Autoimmune effects eg on PACAP/VIP or the PAC1/VPAC1/VPAC2 receptors will have a negating effect on neuropeptide function and also subsequent effects on intracellular mechanisms. While some inflammatory or infectious events may be trivial, compromise of the functions of PACAP/VIP is not. These vital functions in the brain include thermoregulation, olfaction, circadian rhythm, cardio-respiratory activation and sleep-wake cycles. Cardiac and other organs known to exhibit similar PACAP/VIP receptor function would also be expected to demonstrate dysfunction somewhat simultaneously. Further understanding of the roles of the autoimmune dysfunction of these VNs and their receptors may elucidate the mechanisms of CFS and open the way for routine laboratory investigations and prevention options.
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