Sixty-seven consecutive female patients with pSS, classified according to the European–American criteria , with a mean age of 59 years (standard error of the mean (SEM) = 1.5) were enrolled. At the time of enrollment, serum samples were collected and stored at −20 °C until use and clinical and serological features were recorded. Concerning hematological abnormalities, leukopenia was defined as ≤3000 leukocytes/mL, hypergammaglobulinemia was defined as an IgG level over 16 g/L and hypocomplementemia was defined by a reduction of C4 or C3. One-hundred and eighty two healthy blood donors, 145 females and 37 males, with a mean age of 51 years (SEM = 1.2) acted as controls (healthy donors; HDs). Fifteen pSS MSGs, collected at the time of diagnosis, were also retrospectively evaluated. Ten MSGs obtained from subjects with sicca symptoms, but without any clinical and serological features of pSS, acted as controls (five normal MSGs and five showing different grades of nonspecific chronic sialadenitis (NSCS), without FLS). The whole study was approved by the local Ethics Committee (Comitato Etico delle Aziende Sanitarie dell’Umbria, CEAS) and written informed consent was obtained in accordance with the declaration of Helsinki.
Determination of extracellular IFI16 protein by capture ELISA
A capture enzyme-linked immunosorbent assay (ELISA) was employed for determination of circulating extracellular IFI16 protein following a procedure detailed elsewhere . Briefly, polystyrene micro-well plates (Nunc-Immuno MaxiSorp; Nunc, Roskilde, Denmark) were coated with a home-made polyclonal rabbit-anti-IFI16 antibody (aa 478–729). Subsequently, the plates were washed and free binding sites were saturated with phosphate-buffered saline (PBS)/0.05 % Tween/3 % bovine serum albumin (BSA). After blocking, sera were added in duplicate. Purified 6His-IFI16 protein was used as standard. BSA served as negative control. The samples were washed and in each case monoclonal mouse anti-IFI16 antibody (Santa Cruz, sc-8023) was added and incubated for 1 h at room temperature. After washing, horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare Europe GmbH, Milan, Italy) was added. Following the addition of the substrate (TMB; KPL, Gaithersburg, MD, USA), absorbance was measured at 450 nm using a microplate reader (SpectraCount, Packard, Packard BioScience Company). The determination of the concentration was carried out using a standard curve for which increasing concentrations of purified 6His-IFI16 were used.
Determination of antibody titers towards human recombinant IFI16 by ELISA
To detect anti-IFI16 antibodies, polystyrene micro-well plates (Nunc-Immuno MaxiSorp; Nunc) were coated with a solution of recombinant IFI16 in PBS and, after blocking, sera were added in duplicate. After washing, horseradish peroxidase-conjugated rabbit anti-human IgG (Dako Cytomation, Carpinteria, CA, USA) was added. Following the addition of the substrate (TMB; KPL), absorbance was measured at 450 nm using a microplate reader (SpectraCount, Packard). The background reactivity of the reference mixture was subtracted to calculate the results. A standard curve was constructed by serially diluting IgG from an anti-IFI16-positive patient serum [16, 17].
Histological analysis of MSG biopsies
Labial MSG specimens were obtained from 15 pSS patients and 10 HDs according to international guidelines. All sections were randomly analyzed by two expert observers, blinded to clinical and laboratory data. Each sample was independently evaluated and any discrepancies were resolved by consensus. The histological pattern (normal, NSCS, FLS) was assessed using hematoxylin-eosin stained sections [19, 20]. FLS pattern, which is diagnostic for pSS, is characterized by the presence of at least one focus, namely an aggregate of at least 50 lymphocytes, in the glandular tissue. NSCS pattern is characterized by scattered lymphocyte aggregates that do not reach the number of 50 cells and therefore cannot be classified as foci.
Both cellular infiltrate and lymphoid organization, including the presence of GC-like structures, were assessed by immunofluorescence staining of sequential sections with monoclonal antibodies recognizing CD3, CD20 and CD21 (all provided by DakoCytomation, Glostrup, Denmark), as previously described [2, 21].
IFI16 expression was evaluated using standard immunohistochemistry, as already described in a previous study . Images were acquired using an Olympus BX53 fluorescence microscope with CellSens software (Olympus America Inc., Center Valley, PA, USA).
Data analysis was performed using IBM-SPSS version 13.0 (IBM, Armonk, NY, USA). Mann Whitney U test or Chi square test were employed to compare variables among subgroups. Binary logistic regression was used to identify possible association between clinical and serological features and the presence of serum IFI16 protein and anti-IFI16 antibody. The significance level was two sided and set at p < 0.05.