Increased circulating levels and salivary gland expression of interleukin-18 in patients with Sjögren's syndrome: relationship with autoantibody production and lymphoid organization of the periductal inflammatory infiltrate

Abstract

IL-18, an immunoregulatory and proinflammatory cytokine, has been shown to play an important pathogenic role in Th1-driven autoimmune disorders. In this study, we evaluated the circulating levels and salivary-gland expression of IL-18 in patients with Sjögren's syndrome (SS), a mainly Th1-mediated disease. IL-18 serum levels were measured by ELISA in 37 patients with primary SS, 42 with rheumatoid arthritis, and 21 normal controls. We demonstrated high IL-18 serum levels in SS, similar to those in rheumatoid arthritis patients and significantly higher than in controls (P < 0.01). In addition, IL-18 serum concentrations were significantly higher in anti-SSA/Ro+ and anti-SSB/La+ than in anti-SSA/Ro- and anti-SSB/La- SS patients (respectively, P = 0.01, P < 0.01). Serum IL-18 correlated strongly with anti-SSA/Ro (P = 0.004) and anti-SSB/La (P = 0.01) titers. Salivary gland IL-18 expression was investigated by single/double immunohistochemistry in 13 patients with primary SS and in 10 with chronic sialoadenitis, used as controls. The expression of IL-18 was also examined in periductal inflammatory foci in relation to the acquisition of features of secondary lymphoid organs such as T-B compartmentalization, formation of follicular dendritic cell networks, and presence of germinal-center-like structures. IL-18 expression in SS salivary glands was detected in 28 of 32 periductal foci of mononuclear cells (87.5%), while no IL-18 production by infiltrating cells was detected in patients with chronic sialoadenitis. Within the inflammatory foci, IL-18 immunoreactivity co-localized almost exclusively with CD68+ macrophages. In addition, IL-18 was found in 15 of 19 foci (78.9%) with no evidence of T-B cell compartmentalization (nonsegregated) but in 100% of the segregated aggregates, both in T- and B-cell-rich areas. Strikingly, IL-18 was strongly expressed by CD68+ tingible body macrophages in germinal-centre-like structures both in SS salivary glands and in normal lymph nodes. IL-18 expression was observed in the ducts of all SS biopsies but in only 4 of 10 patients with nonspecific chronic sialoadenitis (P < 0.01). This study provides the first evidence of increased circulating levels and salivary gland expression of IL-18 in SS, suggesting an important contribution of this cytokine to the modulation of immune inflammatory pathways in this condition.

Figure 1

Serum IL-18 concentrations in patients with Sjögren's syndrome (SS) and relationship with the presence of anti-SSA/Ro and anti-SSB/La antibodies. Box–whisker plots showing serum IL-18 concentration in patients with SS compared with patients with rheumatoid arthritis (RA) and normal healthy subjects (NHS) (a), and in patients with SS who are positive or negative for anti-SSA/Ro (b) or anti-SSB/La antibodies (c). See text for statistical analysis.

Figure 2

Immunohistochemical (IHC) detection of IL-18 in salivary glands of patients with Sjögren's syndrome (SS) (a–c,g–i) and in nonspecific chronic sialoadenitis (d–f). (a,b) Paraffin-embedded section of glands in SS, showing high amounts of IL-18-expressing cells distributed in a scattered fashion within the periductal mononuclear infiltrate. (c) IL-18-positive cells were also observed surrounding acini (arrows) in proximity with the inflammatory aggregate. (d–f) Paraffin-embedded sections of glands from patients with nonspecific chronic sialoadenitis, demonstrating the absence of IL-18 expression in mononuclear cells in nonfocal periductal infiltrates. (g) Paraffin-embedded sections of glands from patients with SS, double-stained for CD68 (brown) and IL-18 (purple), showed exclusive co-localization of IL-18 expression in most of the CD68⁺ macrophages (arrows) within the periductal inflammatory infiltrates. (h) Macrophages expressing a large amount of IL-18 (arrows) were also observed surrounding acini in contiguity with a focal lymphocytic aggregate. (i, same sample as g) Conversely, CD68⁺ macrophages adjacent to a nonfocal infiltrate remained single-stained. Original magnification (a,b,d) × 100, (c,e–i) × 200.

Figure 3

IL-18 expression in salivary gland ducts of patients with Sjögren's syndrome (SS) (a–d) and nonspecific chronic sialoadenitis (e,f). IL-18-positive ducts were detected in all the SS samples but in only a minority of those from chronic sialoadenitis. A considerable range of variability of IL-18 expression was observed in ducts among different samples. Within the same glandular lobule, positive and negative (arrowheads) adjacent ducts were observed (a). Ductal IL-18 expression was found in ducts surrounded (b) and not surrounded (c) by focal infiltrate, as well as in ducts characterized by periductal fibrosis (d). In contrast to ductal epithelial cells, no staining for IL-18 was found in acinar cells (a,d, stars). In a minority of patients with chronic sialoadenitis, we observed similar ductal staining patterns. Representative examples of positive (e) and negative (f) ductal IL-18 staining in different patients with chronic sialoadenitis are shown. Original magnification × 200.

Figure 4

Degree of lymphoid organization of the periductal lymphocytic infiltrates in salivary gland of patients with Sjögren's syndrome (SS). Paraffin-embedded sections were double-stained for CD3 (brown) and CD20 (purple) (a–c) and single-stained with CD21 (d). Inflammatory foci were classified as nonsegregated when T and B lymphocytes were not compartmentalized in distinct areas (a), as segregated in the presence of evident compartmentalization of T and B cells (b), and as segregated with germinal-centre-like structures (arrow) when a clear histological appearance (c) and networks of CD21⁺ follicular dendritic cells (d) were observed. Original magnification × 200.

Figure 5

Relationship between IL-18 expression and B-/T-cell compartmentalization (a,b) and germinal-center-like (GC-like) structures (c,d) in the salivary glands of patient with Sjögren's syndrome (SS) and, for comparison, in normal lymph nodes (e–h). Representative section of a large segregated aggregate double-stained for CD20 (brown) and IL-18 (purple) (a), and (b) sequential section with an irrelevant antibody replacing the anti-CD20, demonstrating the presence of IL-18-producing cells both in the T-cell (a, arrows) and B-cell (b, arrows) areas. (c) Single staining for IL-18, demonstrating a large number of IL-18-producing cells within ectopic GC-like structures in salivary gland from SS. (d) Double immunohistochemical staining for CD68 (brown) and IL-18 (purple), demonstrating the exclusive co-localization of IL-18 with CD68 macrophages. (e–h) An identical pattern of distribution in terms of IL-18 expression and co-localization with CD68 macrophages was observed in GCs of secondary lymphoid organs. Histomorphological analysis of the IL-18 positive cells within the GC showed evidence of engulfed apoptotic bodies in the cytoplasm (e) that identifies these cells as tingible body macrophages (TBMs). (f) Double immunohistochemical staining for CD68/IL-18 confirmed the exclusive co-localization of IL-18 with TBMs within the GC. Sequential sections in which the anti-CD68 (e), anti-IL-18 (g), or both the primary antibodies (h) were replaced with an isotype-matched irrelevant antibody confirmed the specificity of the double staining (h, negative control). Original magnification (a–d) × 200, (e-h) × 400