In this study, we report the first demonstration of increased systemic and local expression of IL-18 in patients with SS. SS is an autoimmune disease characterized by the destruction of epithelial cells in salivary and lacrimal glands, leading to exocrine dysfunction. The histological hallmark of the disease is the presence of a periductal mononuclear-cell infiltrate that can become organized in follicle-like structures. Chronic inflammation within the salivary glands leads to the local production of autoantibodies and cell-mediated mechanisms of tissue damage. Although the presence of Th2 cytokines in salivary glands of patients with SS has been demonstrated [4
], several lines of evidence suggest that the immune cellular infiltrate in SS is mainly represented by CD4 lymphocytes expressing a Th1 profile. Accordingly, enhanced production of Th1-related cytokines such as IFN-γ, TNF-α, IL-2, and IL-1β has been demonstrated, both by mRNA and protein expression analysis [1
]. In particular, it has been observed that CD4+
T cells infiltrating salivary glands from patients with SS produce over 40-fold more IL-2 and IFN-γ mRNA than peripheral blood CD4 T cells isolated from the same patients as well as from salivary glands of normal controls [42
]. In addition, IFN-γ mRNA expression from cultured lymphocytes isolated from salivary glands of patients with SS correlates with the degree of lymphocytic infiltration in salivary gland, a finding that indirectly suggests that the increase in lymphocytic infiltration is accompanied by the up-regulation of Th1 cytokines [4
]. Despite these observations, factors regulating this Th1 response in SS have not been well characterized.
The crucial role for IL-18 in the development of Th1 immune responses has been established since its identification as a major IFN-γ-inducing factor [9
] in cooperation with IL-12 [44
]. Furthermore, IL-18 has been demonstrated to exert additional proinflammatory properties such as the ability to directly stimulate the production of TNF-α in macrophages, CD3+
cells, and natural killer cells, with subsequent release of IL-1β and IL-8 [13
]; to up-regulate the expression of both CC and CXC chemokines [12
]; and to stimulate adhesion molecule expression in different cell types [45
]. The broad range of proinflammatory activities and the demonstrated pathogenic role of IL-18 in other chronic Th1-mediated autoimmune diseases such as RA and Crohn's disease [13
] make this cytokine a prime candidate also in the pathogenesis of SS.
Increased serum levels and salivary gland expression of IL-18 in patients with SS is in keeping with an active role for this cytokine in the tissue pathogenesis. IL-18 serum concentrations in patients with primary SS were significantly higher than in normal controls and comparable with those observed in patients with RA, a disease in which elevated IL-18 serum levels have been reported by our group and others [28
]. Serum IL-18 levels were significantly increased in SS patients with anti-SSA/Ro+
antibodies. The strength of this association was further emphasized by the positive correlation of IL-18 serum levels with both anti-SSA/Ro and anti-SSB/La serum titers. The observation that anti-SSA/Ro and anti-SSB/La serum levels have been shown to correlate with the presence of anti-SSA/Ro- and anti-SSB/La-producing cells in the salivary glands [46
] induced us to investigate IL-18 expression at this site.
IL-18 protein was strongly expressed in periductal mononuclear cells infiltrating the salivary glands of all SS patients but not in patients with chronic sialoadenitis. Phenotypic analysis demonstrated that in SS samples, IL-18 production within the periductal inflammatory infiltrate was exclusively confined to CD68+
macrophages. Interestingly, the expression of IL-18 by CD68+
macrophages was observed only within periductal inflammatory foci and in periacinar macrophages adjacent to focal infiltrates, and not in isolated macrophages, an observation that suggests an activation state of macrophages. These observations reinforce an otherwise underestimated role of this cell type in the pathogenesis of SS, as also recently suggested [47
In addition to macrophages, ductal epithelial cells appeared to represent a major source of IL-18 in SS salivary glands. Although the range of ductal IL-18 expression was wide (18–82% positive ducts), expression at some level was detected in all SS patients studied, but interestingly only in a minority of patients with chronic sialoadenitis. No staining for IL-18 was found in acinar cells, indicating that IL-18 production is confined exclusively to ductal epithelial cells. The detection of IL-18 in this cell type is in accordance with the notion that, although classical antigen-presenting cells such as monocytes/macrophages and dendritic cells are regarded as the pivotal source of IL-18 in the regulation of Th1-mediated immune responses [7
], nonimmune cell types can also produce IL-18 [13
]. Notably, in salivary gland of SS, ductal IL-18 expression was observed both in the presence and in the absence of IL-18-producing cells in the periductal inflammatory foci and was also found in the absence of a focal infiltrate as well as in ducts surrounded by extensive fibrosis. The lack of association between ductal IL-18 expression and the presence of periductal inflammation in all cases raises the intriguing possibility that the dysregulation of ductal IL-18 expression may become uncoupled from or independent of the level of cellular infiltration. In addition, the demonstration of increased ductal expression of IL-18 in SS would be in keeping with other autoimmune conditions such as Crohn's disease and psoriasis, in which a dysregulation of IL-18 expression in intestinal epithelial cells and skin keratinocytes, respectively, is considered an important component in the development of local chronic inflammation [29
]. However, whether up-regulation of IL-18 expression in epithelial cells in SS, as well as in other autoimmune conditions, is an initiating event or is acquired later in the course of the inflammatory process is still unknown.
Because of the strong evidence that IL-18 expression was present only within the inflammatory infiltrates showing focal organization, we analyzed the anatomical relationship between the presence of IL-18 within the focus, the main lymphocytic subsets, and the degree of structural organization of the periductal inflammatory aggregates. While we found expression of IL-18 by infiltrating cells in the majority but not all of the nonsegregated foci, a large number of IL-18-producing cells was detected in 100% of the aggregates with well demarcated T–B-cell compartmentalization both in T- and B-cell areas. Thus, the increasing expression of IL-18 in larger and more structured infiltrates would suggest that IL-18 is involved in the amplification of the chronic inflammatory processes leading to the acquisition of a more complex organization of the periductal foci. This possibility is further supported by the observation of prominent IL-18 expression in all the ectopic GC-like structures in salivary glands of patients with SS. IL-18 production within these structures was exclusively co-localized with CD68+
tingible body macrophages. An identical pattern of IL-18 expression was also observed in secondary lymphoid organs of normal individuals. In this regard, a recent study demonstrated that IL-18R is expressed and functional on GC B cells isolated from human tonsil and is up-regulated by IL-12 [49
]. To our knowledge, this is the first report of IL-18 production within the GC by tingible body macrophages and suggests an active involvement of tingible body macrophages producing high levels of IL-18 in the regulation of GC reaction.
The relevance of IL-18 expression in B-cell-rich areas and GC-like structures in salivary glands of patients with SS relates to the demonstration that, as mentioned above, serum levels of IL-18 in our SS population were increased in patients positive for anti-SSA/Ro and anti-SSB/La in comparison with patients who were negative for these antibodies, and were closely correlated with the titers of these autoantibodies. Although our study did not address the direct relationship between IL-18 expression in GC-like structures, their functionality, and local production of autoantibodies, it has been reported that anti-SSA/Ro and anti-SSB/La are produced in SS salivary glands [46
], and their serum levels correlate with the presence of ectopic GC-like structures [41
]. Finally, although a conclusive demonstration of the functionality of ectopic GC-like structures in SS is required, Ig V gene rearrangement analysis has provided evidence of an antigen-driven B-cell response within microdissected GC-like structures in salivary glands of SS patients, suggesting their functionality in generating a local (auto) antibody response [38
]. In this regard, further studies will be required to assess the functional role of IL-18 in participating in physiologic and ectopic GC formation and function.