The Th1/Th2 cytokine balance changes with the progress of the immunopathological lesion of Sjogren's syndrome

Abstract

Expression of type-1 and type-2 cytokines at the mRNA level in labial salivary glands (LSG) of patients with Sjogren's syndrome (SS), as reported by several groups, have generated conflicting results. In the present study we have directly examined the production of IL-4, IL-13 and IFN-gamma by lymphocytes infiltrating the LSG of 44 consecutive patients referred for SS evaluation. Cytokines production was evaluated following in vitro culture of LSG in the presence of IL-2. IFN-gamma and IL-13 were detected in the majority of SN (24/44 and 26/44, respectively) while IL-4 was present in 5/44 SN. The presence of IFN-gamma was significantly higher in SS patients, as opposed to patients who did not fulfil the criteria for SS (P < 0.01). In addition, almost all cultured lymphocytes expressed mRNA for IFN-gamma (17/19 cultures) and IL-13 (18/19) while IL-4 mRNA was also expressed at high frequency (14/19 cultures). Interestingly, the IFN-gamma mRNA copies in cultured lymphocytes correlated significantly with the intensity of lymphocytic infiltration as evaluated by Chisholm's score (P < 0.01). Furthermore, RT-PCR of RNA extracted from whole LSG from 14 SS patients also demonstrated the presence of all cytokines in the majority of the cases and the prevalence of IFN-gamma in LSG with high-grade infiltration. Because IL-13 was produced by the majority of the cultured LSG, IgE production was also evaluated. Interestingly, IgE was detected in 21/44 LSG culture SN and mainly in those biopsies that had Chisholm's score less than 0.5 (P < 0.05). We conclude that lymphocytes infiltrating the LSG are capable of producing both Th1 and Th2 cytokines and that the balance between them shifts in favour of Th1 in LSG with high infiltration score and in patients with SS.

Fig. 1

Phenotypic analysis of infiltrating lymphocytes taken from a representative culture. (a) Forward scatter (FSC) versus side scatter (SSC) and staining for (b) CD4 and CD8, (c) HLA-DR and (d) CD95 (Fas) antigen.

Fig. 2

IFN-γ, IL-13 and IL-4 concentration in the supernatants (SN) of 44 labial salivary gland cultures. All SN were collected on day 6. The cut-off points of the ELISA were 8 pg/ml for IFN-γ, 6 pg/ml for IL-4 and 4 pg/ml for IL-13.

Fig. 3

IFN-γ, IL-13 and IL-4 mRNA expression in cultured lymphocytes. (a) RT-PCR analysis of mRNA extracted from LSG infiltrating lymphocytes. The samples (19) were divided into two sets (A + B) and the PCR products were electrophoresed in 2% agarose gel. GAPDH gene was used as internal control. (b) Quantitative-PCR and evaluation of IFN-γ and IL-13 mRNA copies. IFN-γ and IL-13 number of copies per 100 000 copies of actin-β. Quantitative-PCR was performed in 11 samples for IFN-γ and 10 samples for IL-13. (c) Correlation between IFN-γ copies per 100 000 copies of actin-β and infiltration of the gland (Chisholm’s score). Statistical analysis was performed with Spearman’s rank correlation test (rs).

Fig. 4

Total IgE concentration in the LSG culture on day 6. Dotted horizontal line represents the ELISA cut-off point (200 pg/ml). Statistical analysis was performed by log-linear modelling.

Fig. 5

RT-PCR analysis of total mRNA extracted from 14 frozen LSG biopsies. The samples were run in two sets (1 + 2). The GAPDH levels were not identical, due to different size of the glands. For technical reasons, sample 12 GAPDH band was absent.