Immunopathologic differences of Sjögren's syndrome versus sicca syndrome in HCV and HIV infection

A clinical picture of dry eye and dry mouth with the histological counterpart of focal lymphocytic sialoadenitis, usually detected in minor salivary glands, is considered the hallmark of Sjögren's syndrome. The association of sicca complaints and focal sialoadenitis can be also found in a number of other diseases, including some systemic viral infections. Among these conditions, chronic hepatitis C virus infection, associated with mixed cryoglobulinaemia and extra-hepatic manifestations, and HIV infection, particularly in the phase of diffuse interstitial lymphocytic infiltration, may mimic the clinical and histological aspects of Sjögren's syndrome. However, each disorder is characterised by specific, disease-related immunopathological aspects. Besides sicca complaints, the various disorders may also share a number of systemic extra-glandular features and the possible development of mucosa-associated lymphoid tissue lymphomas. This latter event represents in all of these diseases the final result of an antigen-driven chronic stimulation of B lymphocytes.

minimal score needed to defi ne a diagnosis of SS [5,8,9]. Moreover, some authors proposed that the presence of a given focal score in MSGs could be the mandatory criterion to classify a patient as having SS [5]. Th is point has been debated for a long time by the scientifi c community. Th e American-European Consensus Criteria for the classifi cation of SS fi nally established that, among the six items composing the criteria set, either focal sialoadenitis at MSG biopsy (with a focus score >1 for 4 mm 2 glandular tissue) or the presence of specifi c autoantibodies should be mandatory for the diagnosis of pSS [10].
Salivary glands (and also lachrymal glands) are not the exclusive infl ammatory targets for SS. Sicca features may be part of the clinical picture of other disorders, in which a focal sialoadenitis, with histological characteristics evocative of SS, can be found [11]. Besides sarcoidosis, where a focal granulomatosous sialoadenitis has been described, and the histological examination of MSGs can be useful in some cases to defi ne diagnosis [12], even hepatitis C virus (HCV) [13] and HIV [14] infections may exhibit, in diff erent phases of the disease, sicca complaints, with the histological counterpart of a focal sialoadenitis. Th is induced the American-European Consensus Group for the Classifi cation of SS to list HCV and HIV infections among the exclusion criteria for SS [10].
In clinical practice, however, the presence of these overlapping features in the various disorders may generate some diffi culties in the diff erential diagnostic defi nition. Th ese diffi culties can certainly be solved, alongside the viral investigations, by better defi ning the pathological diff erences between the focal sialoadenitis in the various conditions, using more sophisticated immunopathological techniques. In addition, the evidence that some viral infections may have salivary glands as one of the possible localisations, and that focal sialoadenitis may be the result of the local pathological process, again supports the theory -often proposed, but never completely proven -that some viral agent(s) could trigger the immunopathological cascade in pSS [15].

Immunopathological features of sialadenitis in pSS
Salivary epithelial cells certainly play a crucial role in the pathological process of pSS. In an adequate environment of stressor agents and hormonal stimulation, and in the presence of a specifi c genetic background, these cells probably become susceptible to viral aggression. Diff er ent viral agents such as Epstein-Barr virus, nonhuman immuno defi ciency retroviruses and, more recently, coxsackie viruses [15,16] have been postulated to be capable of triggering pSS. Th e expression of Toll-like receptor 3 on salivary glands and epithelial cell lines derived from patients aff ected by pSS may suggest that the engagement of epithelial cells by virus may happen via Toll-like receptor 3 [17]. Th e inadequate clearance of these viral agents and/or the consequent damage of the epithelial cells initiate a cascade of events that may lead to the selfmaintenance of infl ammatory mechanisms and induction of autoimmune manifestations. Th e insult may induce upregulation of type I interferon by epithelial cells and plasmacytoid dendritic cells, as demonstrated by IFNγ RNA overexpression in salivary gland tissue [18]. Secondly, viral injury may induce apoptosis or necrosis of glandular epithelial cells, with the consequent release of autoantigens, which become available for professional antigen-presenting cells [18]. Th e fact that epithelial cells may also work as nonprofessional antigen-presenting cells is still under debate [19]. Th is hypothesis has been suggested by the evidence that epithelial cells, when activated, may express class I and class II MHC molecules on their surface [20], together with co-stimulatory molecules [21], and are probably able to engage T cells for the presentation of autoantigens once relocated from the nucleus to the cellular surface [22]. Finally, epithelial cells are certainly important in T-cell recruitment since they produce both intracellular and vascular adhesion molecules [23] and diff erent types of chemotactic factors, including chemokines.
Once T cells are fi rst attracted into the glandular tissue, they assume the role of the main actors in the infl ammatory scene [24], by perpetuating the recruitment of other infl ammatory cells, mainly B cells, by expressing a restricted repertoire of T-cell receptors that can recognise autoantigens presented by antigen-presenting cells, and by activating macrophages (via the IFNγ signal). Th ese latter cells act as the main agents responsible for further salivary tissue injury, mainly by producing TNFα [25].
Th e great importance of T cells in the immmunopathological scenario of pSS is strongly confi rmed by the common observation that focal lymphocytic infi ltrates are mainly constituted of T cells with a large pre dominance of CD4 + with respect to the CD8 + T cells (CD4/ CD8 ratio ranging from 3:1 to 5:1) [24]. B cells represent approximately 20% of the infi ltrating cell population at least in some stages of disease [24]. In addition, macrophages, natural killer cells and dendritic cells are observed in more limited numbers [26]. More recent studies have also shown that two other diff erent T-cell subpopulations are represented in the salivary gland infi ltrates. Th 17 cells are a distinct CD4 + T-helper cell population expressing IL-17 that are strongly implicated in autoimmune diseases. Increased plasma levels of IL-17 have been observed in SS, and Th 17 cells seem to be signifi cantly represented in MSG infi ltrates [27]. Transforming growth factor beta (TGFβ), IL-23 and IL-6 are the main promoters of the Th 17 diff erentiation and proliferation [27]. TGFβ is also the pivotal diff eren tiation factor of Foxp3 + T-regulatory cells, which seems to have an immunosuppressive role in the chronic immunopathologic process [28]. T-regulatory cells have also been found in variable proportions in salivary biopsy specimens of patients with pSS [29].
Recent investigations have shown that the infl ammatory cell presence, proportion and organisation may greatly vary in the disease course, and in various subsets of patients, according to disease severity and evolution [30]. CD4 + T cells predominate in mild sialoadenitis and seem to be associated with the presence of a clinically signifi cant articular involvement. Th e B-cell presence pro gressively grows from intermediate to severe histological lesions. Th e predominance of B cells in infi ltrates and their organisation in the germinal-centre-like structure is strongly associated with hypergamma globu linaemia, hypocomplementaemia, rheumatoid factor and autoantibody production, and, from a clinical point of view, with salivary gland swelling, vasculitic manifestations and lymphoproliferation [30]. Foxp3 + T-regu latory cells are mainly represented in the intermediate grade of lymphocytic infi ltration and decline in severe histological lesions [29,30]. On the contrary, macrophages are more abundant in severe lesions, as well as dendritic cells. Th ese latter cells are largely represented in more advanced lesions [26,30], and particularly in the germinal-centre-like structure, where they appear to form a network that is essential for the organisation of the infi ltrates [30,31]. Finally, IL-17 protein expression progressively increases with a higher focus score, indicating an expansion of the Th 17 subpopulation in more severe phases of the disease [30].
Th e passage from a predominance of CD4 + cells to that of B lymphocytes in glandular infi ltrates is therefore characteristic of a more advanced severe disease pattern, which is correlated with systemic manifestations and higher risk of developing lymphoproliferative disorders [32].
B lymphocytes in salivary gland infi ltrates in SS are mainly represented by polyclonal CD27 + memory B cells [33]. Homing of this kind of B cell seems to predominate with respect to local proliferation of a few founder B cells [33]. A large number of chemokines may all contribute to B-lymphocyte homing and chronic maintenance. A particular profi le of chemokines, however, is predominantly active in SS. Th is profi le is represented by the CXCL13 and CXCL12 chemokines, which specifi cally attract CD27 + memory B cells expressing the corresponding receptors (CXCR4 and CXCR5) [33]. Recent data have shown a large expression of these receptors in the glandular infi ltrates of patients with SS. Th e interaction of B cells attracting chemokines with their specifi c receptors has been suggested to be essential for B-cell recruitment and organisation in a tertiary germinal-centre-like structure [34].
Behind memory B cells there is a convincing demonstration of the contemporary presence of marginal zone (MZ)-like B cells within the lymphoid infi ltrates in SS [35]. Th ese MZ-like B cells may work as reactive B cells against locally expressed autoantigens. Because of the permanent availability of autoantigens, the continuous stimulation of these autoreactive cells leads to the evolution from polyclonal to oligoclonal and then monoclonal selection, and fi nally to the development of mucosa-associated lymphoid tissue (MALT) B-cell lymphoma [35].
B-cell recruitment, survival, proliferation and organisation are strongly infl uenced in SS by the large production of B-cell activating factor (BAFF) and a proliferationinducing ligand (APRIL) [33,35]. In particular, enhanced levels of BAFF have been shown in most of the systemic autoimmune diseases, usually associated with B-cell hyperactivity. Th e highest plasma BAFF levels, however, have been found in patients with SS, and a strong local overexpression of this substance has been demonstrated in salivary gland infi ltrates [36], particularly in patients with a high focus score. BAFF is produced locally by diff erent cell types, including dendritic cells, T lymphocytes and macrophages, and certainly plays an important role in local B-cell proliferation, including that of MZ-like B-cell, germinal-centre-like formation, autoantibody produc tion and probably B-cell lymphomagenesis [37].

Immunopathological features of sialadenitis in chronic HCV infection
Besides hepatocytes and lymphocytes, HCV seems to have a special tropism for lachrymal and salivary epithelial cells. HCV RNA has been found both in saliva and salivary gland tissue [38], and in situ hybridisation studies have clearly demonstrated that HCV is exclusively localised in the salivary epithelial cell cytoplasma [39]. A chronic focal sialoadenitis, resembling that of SS, can be observed in approximately 50% of HCV-infected patients, while signs of mild infl ammatory infi ltration are present in a greater proportion of patients [40]. Sicca symptoms, however, seem to be less frequent and milder in HCVinfected patients. Th ese fi ndings have stimulated a long debate on the possibility that HCV could be considered at least one of the candidate viruses to work as trigger agents for pSS. Th is hypothesis has been supported by several papers in which HCV infection appeared to be more prevalent in patients with pSS than in healthy controls (reviewed in [41]). Other data, however, have shown that the prevalence of HCV infection in patients with defi ned pSS was similar to that observed in the normal population, and that the overestimation pre viously reported could by ascribed to diff erent classifi cation criteria used in diff erent studies, and to hyper gammaglobulinaemia frequently present in the sera of patients with SS [42]. Moreover, the co-existence of HCV infection and defi ned pSS (and lymphoma) in the same patients has been sporadically reported, and the demonstration that this association can be ascribed to chance alone is still under discussion [43]. According to the American-European Consensus Criteria, however, evidence of HCV infection is an exclusion criterion for the classifi cation of a patient as having SS [10].
Th e amount of information available on the charac teristics of the focal infi ltrates in salivary glands of HCVinfected patients is not as extensive as that accumulated in SS. Th is is undoubtedly due to the greater attention devoted to the predominance of liver involvement in HCV infection, and to the fact that features diff erent from sicca complaints are more clinically signifi cant in HCV infection with extra-hepatic manifestations. Histologic examination of salivary glands in HCVinfected patients shows diff erent aspects with respect to those found in SS patients. Lymphocytic infi ltrates often may be located in the pericapillary area rather than around the glandular ducts. In addition, a lack of damage (or mild damage) of the glandular tissue has also been reported [41,44]. Th e lymphocytic subpopulations present in glandular infi ltrates appear to be diff erent, sometimes being represented by a predominance of CD8 + T lymphocytes, at least in some areas. More commonly, CD4 + T cells constitute the most represented population in the infi ltrates. However, even in this latter case, the CD4/CD8 ratio is usually lower than that observed in SS patients with pSS [44]. Th ere is no demonstration of any translocation and expression of autoantigen peptides in salivary gland epithelial cells, and therefore the corresponding autoantibodies are not detectable in the sera of HCV-infected patients [45]. On the contrary, serum cryoglobulins and hypocomplement aemia are usually found in a large proportion of HCV-infected patients, and are considered markers or pre dic tors for the develop ment of extra-hepatic manifestations of the disease, such as cutaneous vasculitis (cryo globulin aemic purpura), peripheral neuropathy, glomerulo nephritis and sicca syndrome [45].
Th e pathogenetic mechanism underlying focal sialoadenitis in HCV-infected patients has not so far been clarifi ed. Transgenic mice carrying the HCV envelope genes for E1 and E2 proteins developed an exocrinopathy involving salivary and lachrymal glands [46]. Th is study clearly indicates a direct role for these viral proteins in the pathogenesis of HCV-related sialoadenitis. Molecular mimicry between HCV-E2 protein and an antigenic protein present in exocrine epithelial cells has been suggested as a potential autoimmune mechanism inducing lymphocyte homing and activation [47]. On the other hand, one could simply speculate that the HCV infection of the salivary epithelial cells may be the initial event that is suffi cient to induce the activation of innate immunity with the consequent production of proinfl ammatory cytokines, such as INFγ and IL-2, in a way similar to that demon strated to occur in HCV-infected hepatocytes [48].

Lymphomagenesis in Sjögren's syndrome and HCV chronic infection
Besides sharing similar clinical and serological features, such as cutaneous vasculitis, peripheral neuropathy and hypocomplementaemia, either patients with chronic HCV infection associated with mixed cryoglobulinaemia or patients with pSS may develop B-cell lymphomas with a higher prevalence than in the normal population [43]. Th e most common type of B-cell lymphoma in both disorders is a MZ low-grade lymphoma, where the pro liferating cells are rheumatoid factor-positive auto reactive B cells [43]. A signifi cant proportion of HCV-infected patients may also develop diff use large B-cell lymphoma, probably by completely diff erent patho genetic mechanisms [49]. In both diseases the low-grade B-cell lymphoma develop ment represents a pathological model of an antigen-driven expansion from polyclonal/oligoclonal to mono clonal proliferation of B cells [50]. Th e process is probably driven by autoantigen(s) in SS [43] and by viral antigen(s) in HCV infection [50]. Th e fact that the process of low-grade B-cell lymphoma develop ment is similar in both conditions is also supported by molecular studies demon strating a similar restricted usage of genes encoding for heavy and light variable regions and similar somatic mutations of the comple mentarydetermining region of the surface antigen receptor in monoclonal B cells from patients with SS and HCVrelated mixed cryoglobulinaemia [51].
Th e site of development of these B-cell lymphomas is typically extra-nodal, since the salivary gland is the preferential site for lymphomas that appear in the course of SS, and the liver and salivary gland are the preferential sites for lymphomas developing in HCV-infected patients with associated sicca complaints [52]. In both conditions, a possible evolution to diff use large-cell lymphoma has been reported [52].
Clonality studies revealing monoclonal B-cell expan sion in target tissues of both conditions are not strictly indicative of lymphoma [53]. Th ese clones may disappear after antiviral therapy in HCV-infected patients [54], or may persist in the same site for a long time without evolving to frank lymphoma. Clonality studies are therefore certainly useful at least to predict possible development of a frank lymphoma.

Sialoadenitis in HIV-infected patients
HIV-infected individuals are at an increased risk of developing associated rheumatic diseases [55]. Before the Vitali Arthritis Research & Therapy 2011, 13:233 http://arthritis-research.com/content/13/4/233 widespread use of highly active antiretroviral therapy (HAART), retrospective studies calculated the rates of rheumatic manifestations from 11 to 72% [56]. After the HAART implementation, rheumatic complications declined signifi cantly, with some change in the pattern of the incidental diseases [57].
A SS-like clinical picture may be present in HIVinfected patients who develop diff use infi ltrative lymphocytosis syndrome (DILS). DILS may be present in 3 to 50% of HIV-infected populations [58]. Th is great variability between the studies can be ascribed to ethnic diff erences, to the diff erent criteria used to defi ne the diagnosis (clinical vs. histological) and, fi nally, to the availability of eff ective antiviral therapy in the various populations described in the studies. Recent data strongly indicate that, similar to what has been reported for other rheumatic manifestations in HIV infection, the prevalence of DILS has also been remarkably reduced with the introduction of HAART therapy [59]. DILS may mimic pSS because DILS also presents with bilateral painless parotid gland enlargement, lachrymal gland enlargement and sicca symptoms [58]. DILS is characterised by circulating CD8 + T cells. Th e proliferation of CD8 + T cells is probably an antigen-driven process and leads to infi ltration of multiple organs, and probably refl ects an excessive host response to HIV [60]. Th e condition usually manifests several years after HIV seroconversion and, besides the lymphocitic infi ltration of salivary and lachrymal glands, is characterised by lymphocytic interstitial pneumonitis (31%), myositis (26%) and hepatitis (23%) [61]. DILS diff ers from pSS by the fact that the occurrence of extra-glandular involvement is more frequent, whilst the presence of autoantibodies and rheumatoid factor is observed in a lower number of patients In addition, the two conditions diff er in the nature of infi ltrating lympho cytes (CD4 + T cells in pSS, CD8 + T cells in DILS) and in the association with diff erent HLA haplotypes [58][59][60].
Interestingly, some cases of MZ indolent MALT lymphomas have rarely been reported in HIV-infected patients [62][63][64], among the large variety of incidental malignancies described in the course of the disease [65]. MALT lymphoma remission was obtained in these cases after HAART, in a way similar to that observed in HCVrelated and Helicobacter pylori-related MALT lymphoma after specifi c antiviral and antibiotic therapies [50]. Th is observa tion again suggests that infection-related lymphomas derived from MZ B cells may share common antigen-driven mechanisms [50].

Conclusion
Sicca symptoms and focal sialoadenitis are not exclusive features of pSS, but other diseases may present with involvement of salivary and lachrymal glands and a histological picture of focal infi ltration in the target tissues. Beyond viral serology, which may allow one to easily confi rm the presence of HCV or HIV infection as the primary causative agent of the syndrome, other clinical, genetic, immunological and histologic fi ndings can help to distinguish pSS from the other SS-like syndromes (Table 1).
Th e observation that MZ indolent MALT lymphomas may develop in all of these conditions has greatly contributed to a more precise understanding of the intriguing mechanisms of antigen-driven lympho pro liferation. Main characteristics of focal sialoadenitis in primary Sjögren's syndrome, hepatitis C virus-infected patients and HIV-infected patients developing diff use infi ltrative lymphocytosis syndrome (DILS). ANA: antinuclear antibodies; RF, rheumatoid factor. Modifi ed from Basu and colleagues [59].

Autoimmune Basis of Rheumatic Diseases
This article is part of a series on Sjögren' s syndrome, edited by Thomas Dörner, which can be found online at http://arthritis-research.com/series/Sjögrens This series forms part of a special collection of reviews covering major autoimmune rheumatic diseases, available at: http://arthritis-research.com/series/abrd