These antibodies seem to block parasympathetic nervous signalling to exocrine glands, a signalling that is transmitted mainly via the parasympathetic M3R. Because the M3R is also found in other tissues, e.g., the bladder and the gastrointestinal system, these antibodies have also been proposed to cause symptoms such as irritable bladder and constipation [19]. However, anti-M3 antibodies do not account for all demonstrated disturbances of ANS function in pSS. Orthostatic hypotension, for example, often found in pSS patients, is usually considered a sign of sympathetic rather than parasympathetic dysfunction [25]. Orthostatic hypotension may be caused by inflammation of sympathetic ganglia or sympathetic nerves [22] or by cytokines that interfere with neurotransmission [24]. Thus, both subjective symptoms and objective signs of parasympathetic and sympathetic dysfunction have been described in pSS patients [1–5, 8, 9, 15].

The prevalence of AD in pSS is difficult to assess, for several reasons. First, the choice of methods, patients, and controls has varied between studies. Second, the standardization of autonomic test results and the definition of AD differ. Third, end-organ damage may obscure or mimic the effects of AD in some organ systems, e.g., in the exocrine glands. Fourth, cardiovascular autonomic nervous function, which is comparatively easy to assess, does not necessarily mirror ANS function in other parts of the body. Fifth, the proposed anti-M3R antibodies may mediate autonomic dysfunction in various organ systems but the effects of these antibodies are difficult to assess by the cardiovascular ANS tests that are used routinely. And finally, at this time no established, reproducible clinical test exists for the detection of these antibodies.

As a result of the limitations listed above, estimates of the prevalence of AD in pSS have varied widely. Taking all of the above limitations into account, objective signs of AD have been reported in 0–90% of pSS patients [3, 26] while approximately 50% have been reported to have an abnormal AD symptomatology using the Autonomic Symptom Profile questionnaire evaluating AD symptoms [26].

22.2Pathogenesis of Autonomic Dysfunction in pSS

In pSS, AD has been ascribed to various immunological mechanisms, namely anti-M3R antibodies, cytokines interfering with neurotransmission, and inflammation of autonomic nerves, nerve vessels, and ganglia [19–24]. Although many chronic AD symptoms may be explained by the anti-M3R antibodies that interfere with parasympathetic nervous transduction in organs containing the M3R (e.g., secretomotor dysfunction, bladder symptoms, and gastroparesis), a more subacute occurrence of AD symptoms, e.g., orthostatic intolerance [1], would fit better with an autoimmune ganglionitis or a vasculitic process affecting autonomic nerves.

Adie’s syndrome, a syndrome of parasympathetic nerve dysfunction involving the pupil, results in a dilated, tonic pupil that suffers from an impairment of the light reflex response. Adie’s pupils, which affect some pSS patients [27], can be explained by inflammation affecting the ciliary ganglion, with or without a role for anti- M3-receptor antibodies.

AD is probably due to different mechanisms in different organs as well as different patients. The clinical manifestations and time course for the development of AD symptoms in pSS vary substantially across patients, with some symptoms developing over months/years and some symptoms occasionally developing over days/weeks. In contrast to what is found in patients with AD associated with diabetes, AD in pSS does not seem to be associated with excessive cardiovascular mortality [28], which suggests that there may be different types of ANS involvement in patients with diabetes and pSS. Because it has been shown that parasympathetic nerve signalling may have an attenuating effect on macrophage-induced inflammation and production of various cytokines [29], parasympathetic dysfunction may also affect the inflammatory process in pSS and possibly result in an exaggerated inflammatory response. The parasympathetic nervous system appears to exert its anti-inflammatory effects on macrophages mainly through ACh and the nicotine receptor [29]. Thus, the reduced focus scores and reduced prevalence of anti-SS-A antibodies reported in pSS patients who smoke could fit with an immunomodulatory effect of nicotine in addition to that of the parasympathetic nervous system [30]. Antimuscarinic antibodies may also increase the inflammatory response in pSS by increasing cycloxyge- nase-2 expression and prostaglandin E2 production [31].

The fact that pSS patients often have large amounts of acinar tissue in the exocrine glands that is morphologically intact but which functions at a subnormal level in vivo [22, 23] has heightened the interest in the mechanisms governing exocrine secretion and in possible mechanisms interfering with the normal signal transduction to and within acinar cells. The main parasympathetic receptor in exocrine glands is the M3R, which is also found elsewhere, e.g., in the gastrointestinal system and the bladder. In contrast, other muscarinic receptor subtypes, namely the M1and M2-receptors, are more important in the brain and the heart, respectively. In healthy subjects, exocrine secretion starts when ACh stimulates the M3R. However, several factors could result in reduced stimulation of the M3R [18] with resulting exocrine dysfunction (Fig. 22.1), namely:

1.Reduced innervation of the exocrine glands. Although it is theoretically plausible that inflammation and neural degeneration could result in a reduced innervation of acinar, myoepithelial, and ductal cells in the exocrine glands, experimental data do not support this concept [18, 32].

2.Reduced ACh release. In vitro experiments using exocrine glands from MRL/lpr mice have indicated that certain cytokines (i.e., TNFa and IL-1b) may impair neuronal release of ACh [18, 33]. In addition, cytokines have also been proposed to affect the transcription and thus also the surface expression of neurotransmitter receptors, thereby interfering with signal transduction [34].

3.Increased degradation of ACh by cholinesterases. SS patients have been shown to have increased levels of cholinesterases, the enzymes that degrade ACh, in saliva. However, the implication of this finding is uncertain. Of note is that hydroxychloroquine, which is sometimes used as an immunomodulatory drug in pSS, is a cholinesterase inhibitor. This provides theoretical rationale for why treatment with this drug might affect ACh degradation in exocrine glands, thereby improving exocrine gland function [18, 35].

b)Various immunological techniques, e.g., a flow cytometric assay [37] and enzyme-linked immunosorbent assay (ELISA) [38]. However, neither technique has demonstrated sufficient reproducibility or specificity to date [18].

(c)Bioassays in mouse colon or bladder strips demonstrating reductions of car- bachol-induced contractility upon exposure to SS sera [19, 39].

(d)Bioassays using human salivary gland cells where carbachol-induced, fluorometrically assessed intracellular increase in Ca2+ is blunted by antibodies in the IgG fraction of SS sera. Using this technique, the M3R was found to be blocked by the M3R-antibodies in a reversible manner [40].

Additional studies have shown that after a period of blockade of cholinergic transmission, these antibodies may induce a cholinergic hyperresponsiveness with upregulation of the M3R. This may explain the bladder irritability that is commonly encountered in pSS patients [41, 42]. These antibodies have also been suggested to cause gastrointestinal symptoms, e.g., gastroparesis [41], and microvascular responses to cholinergic stimulation [12]. Reports on abnormal distribution of aquaporins (AQP), protein water channels, in acinar cells [43] also show a disturbed intracellular signalling downstream of the M3R, a finding that is compatible with the presence of anti-M3R antibodies in patients with pSS. Furthermore, the physiologically measurable effects of the anti-M3R antibodies and the symptoms probably associated with them have also been reported to be diminished by anti-idiotypic antibodies/intravenous immunoglobulins [44].

In conclusion, there is substantial evidence that a serological factor exists in the IgG fraction of sera from patients with primary and secondary SS. This factor appears to interact with the response of the M3R to cholinergic stimuli. The exact target of these antibodies is still a matter of debate, but the second and third extracellular loops of the M3R have been implicated [45, 46] as well as cross-reactivity with the muscarinic-1 receptor [47]. Probably due to their low concentration and our lack of knowledge about their exact specificity, they are difficult to detect using conventional immunological methods such as ELISA. The best methods at present for their detection seem to be various bioassays studying their effects on murine colon and bladder contractility [18] or on increase in intracellular Ca2+ in human salivary gland cells as a result of cholinergic stimulation [18].

22.3Diagnostic Tests

ANS functioning is assessed most commonly and easily in the cardiovascular system. Numerous cardiovascular tests can assess the effects of the parasympathetic and sympathetic nervous systems on the heart rate and blood pressure at rest and during various challenges. The cardiovascular tests are usually easy to perform, are reasonably standardized, and generally noninvasive. Autonomic reflex tests (ARTs) are used to measure various cardiovascular autonomic reflexes, which are modulated differently by the parasympathetic and sympathetic nervous systems. Examples of the ARTs are the deep-breathing test, which measures the degree of sinus arrhythmia to deep breathing (parasympathetic); the orthostatic test (Fig. 22.2),

reaction to the Valsalva manoeuvre (parasympathetic); and the sustained hand grip test, which measures the diastolic blood pressure reaction to sustained hand grip (sympathetic) [48].

More modern ways of assessing cardiovascular autonomic function include studies on heart rate variability (HRV), where fast HRV changes are considered to be mediated by the parasympathetic nervous system and slower changes by the sympathetic nervous system [48]. HRV can be studied in short-term electrocardiograms (ECGs) but it is better studied in 24-h ECGs. Baroreceptor sensitivity (BRS) involves the measurement of the sensitivity of the baroreflex by assessing the relationship between an increase in blood pressure and a decrease in heart rate, either by performing concomitant blood pressure and heart rate measurements during the day or during orthostatic challenge, but ideally during phenylephrine infusion [48].

ARTs are thought to measure ANS activity when the ANS is under stress while the HRV and BRS are thought to measure ANS function under basal conditions. The advantage of ARTs, in contrast to assessment of HRV and BRS, is their simplicity. However, ARTs depend upon the co-operation of the subject under study, a point that is less critical in the case of HRV. HRV and BRS investigations also have higher sensitivity and greater reproducibility compared to ARTs [49].

Because many factors affect the ART variables, it is of great importance that these be performed under standardized conditions, with uniform temperatures, during the same time of the day, without prior eating, drinking, or smoking and without medications that might affect ANS indices (e.g., various vasoactive drugs). Moreover, because autonomic function variables decline with advancing age and may demonstrate differences between the sexes, studies should include appropriate ageand sex-matched controls [50].

In addition to the cardiovascular ANS tests, additional assessments exist for the evaluation of other organs. Such tests include the quantitative sudomotor axon reflex test (QSART) and sympathetic skin response, which measure sudomotor function [51, 52]; pupillometry, which measures ophthalmologic ANS function [52]; urodynamic studies; gastric emptying scintigraphy [41]; and P-catecholamines, a surrogate marker of sympathetic nervous activity [52]. These tests are performed less frequently because of the need for specialized equipment, a lower degree of standardization of the methods, and greater invasiveness of the procedures. Because cardiovascular ANS tests do not necessarily mirror the function in other parts of the ANS and because AD is sometimes difficult to discriminate from the effects of endorgan damage (e.g., dryness can be both due to exocrine gland destruction and a parasympathetic dysfunction), it is ideal to combine different tests of autonomic function when evaluating the ANS [25].

In contrast to the numerous tests that exist to assess objective ANS function, few validated questionnaires exist for the documentation of subjective AD symptoms. One of the more commonly used is the Autonomic Symptom Profile (ASP) [53]. The ASP is the only questionnaire employed in pSS that addresses subjective AD symptoms. The questions in the ASP evaluate nine domains of autonomic symptoms: orthostatic intolerance, secretomotor dysfunction, male sexual dysfunction, urinary dysfunction, gastrointestinal dysfunction (divided into three subdomains, gastroparesis, diarrhoea, and constipation), pupillomotor dysfunction, vasomotor