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Clin Exp Immunol. Jun 2002; 128(3): 562–568.
PMCID: PMC1906267

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


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-γ 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-γ 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-γ 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-γ (17/19 cultures) and IL-13 (18/19) while IL-4 mRNA was also expressed at high frequency (14/19 cultures). Interestingly, the IFN-γ 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-γ 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.

Keywords: cytokines, IgE, Sjogren’s syndrome, Th1/Th2 balance, T lymphocytes


Sjogren’s syndrome (SS) is a chronic, slowly progressive autoimmune disease. The hallmark of SS is the mononuclear cell infiltration of the affected exocrine glands, which results in their functional impairment, evident clinically as xerostomia and keratoconjunctivitis sicca [1]. T lymphocytes form the majority of the infiltrate, while B cells constitute the 20–30% of the total cell population and macrophages are rarely observed. Approximately 70% of the T cells are CD4+ while 25% are CD8+. Most of the T cells are activated as they express HLA-DR and CD95 molecules [25].

T helper (Th) cells are divided into two main subsets. Th1 cells produce IFN-γ, IL-2 and lymphotoxin and are involved in macrophage activation and delayed type hypersensitivity reactions. Th2 cells produce IL-4, IL-5 and IL-13, play a major role in humoral responses and are involved in helminthic infections and atopy [6,7]. Studies on animal models of insulin-dependent diabetes mellitus or multiple sclerosis suggested that Th1 cytokines exhibit harmful properties, as opposed to Th2 cytokines that have a protective role [8,9]. Other studies, however, demonstrated that under different experimental conditions Th2 response may also be pathogenic [1012]. Studies on systemic autoimmune diseases such as systemic lupus erythematosus (SLE) have also reported contradictory results [1316].

In most studies concerning cytokine expression in SS, mRNA levels for IL-4 and IFN-γ were evaluated by RT-PCR of RNA extracted from the whole LSG. More often it has been reported that mRNA for IFN-γ was detectable and little or no IL-4 mRNA was found [1719]. However, it should be noted that later reports failed to find differential expression of IFN-γ between pathogenic and normal tissue [20,21]. In addition, other studies do not agree that Th1 cytokines are expressed preferentially in LSG [22]. In this regard, earlier work from our group, using in situ hybridization, failed to show significant IFN-γ production while IL-4 was present in early lesions [23]. Other studies have correlated IL-4 with strong B cell accumulation and progression to B cell lymphoma [24]. These contradictory results can be attributed in part to methodological or experimental differences as well as the diversity of LSG infiltration.

We reasoned that a more direct evaluation of the cytokines would divulge new information on this issue. To this end, LSG biopsies were cultured for 6 days in the presence of IL-2, at which time IFN-γ, IL-4 and IL-13 production in the culture supernatants (SN) was evaluated by ELISA. In parallel, the presence of mRNA for IFN-γ, IL-4 and IL-13 was evaluated by RT-PCR using RNA extracted either from lymphocytes recovered from LSG cultures or from whole LSG tissue. Our results show that LSG lymphocytes express a mixed cytokine profile at both protein and mRNA level. Morover, we observed that IFN-γ expression is associated with SS or high infiltration score while in low-grade infiltrates a type-2 response is more prominent as characterized by IgE production.


Patients and samples

Consecutive LSG were obtained from 58 patients that were referred for SS evaluation. The final diagnoses of the patients are shown in Table 1. Four LSG from each patient were used for the evaluation of focus lymphocytic score according to Chisholm’s classification [27] and one LSG from each patient was either placed immediately into culture (44 LSG) or stored at –80°C for whole tissue RT-PCR (14 LSG). The study had the approval of the ethical committee of the Laiko Hospital and informed consent was obtained from all patients.

Table 1
Diagnoses of the patients *

Biopsies and PBMC cultures

The first set (n = 44) of LSG was immediately depleted from the visible capillaries on the surface of the gland, cut into small pieces (<1 mm3), and washed three times for 1 h by shaking. LSG were cultured in RPMI-1640 supplemented with sodium bicarbonate (0·3%),l-glutamine (0·3 mg/ml), gentamycin (0·1 mg/ml), penicillin–streptomycin (50 U/ml and 50 μg/ml, respectively), fetal calf serum 10% (all purchased from Life Technologies) plus 40 U/ml recombinant human IL-2 (R&D systems, UK). Cultures were incubated at 37°C, 5% CO2. After 6 days of culture, usually 15 000–75 000 lymphocytes migrated out of the LSG tissue cultures.

Buffy-coats were obtained from healthy donors at the Laiko Hospital blood bank and PBMC were isolated after centrifugation on Ficoll-Paque (Pharmacia Biotech, UK). Cells were cultured in the presence of 40 U/ml of IL-2 or stimulated with plate-bound anti-CD3 (OKT3, at 2 μg/ml), and soluble anti-CD28 (9·3, at 1 μg/ml, kindly provided by Dr Carl June, University of Pennsylvania, USA). Culture conditions were same as for biopsies.

Fluorometric analysis

The following mouse monoclonal antibodies were used: anti-CD4-FITC/anti-CD8-PE, anti-Fas (CD95)-FITC, biotinylated anti-HLA-DR and γ 1-FITC/γ 1-PE as isotypic control (all from Pharmingen, CA, USA), anti-CD19-FITC (Diaclone, France) and streptavidin (SA)-conjugated PE (Serotec, UK). All analyses were performed with FACScalibur (Beckton Dickinson, CA, USA) using the CellQuest software.

Cytokine ELISA

Supernatants were collected on day 6 and day 12 of culture. To preserve the cytokines from proteolysis, a proteinase inhibitor cocktail consisting of AEBSF: 20 mm, EDTA: 100 mm, leupeptin: 1 mm and pepstatin: 1 mg/ml (all from Sigma-Aldrich, Germany) was added and the SN were stored at –80°C. Pairs of unlabelled and biotin labelled specific monoclonal antibodies against IL4, IL13 and IFN-γ were used according to the manufacturer’s instructions (Pharmingen, CA, USA). For detection, SA- conjugated horseradish peroxidase (HPO) at 1/10 000 (Jackson Immunoresearch, PA, USA) was added for 30 min at RT and TMB (Kirkegaard & Perry Laboratories, MD, USA) was finally used as substrate. The optical density of the reaction was measured at 450 nm. Standards for IL4, IL13 and IFN-γ were purchased from R&D systems (UK).


ELISA plates were coated with goat anti-human IgE (Cappel Laboratories) at 3 μg/ml by overnight incubation at 4°C in carbonate buffer pH 9·6. Blocking was performed with PBS, casein 2·5% (1 h at RT) and the SN were added in duplicates. Mouse monoclonal antihuman IgE antibody (clone 4·15 from ATCC) was added at 2 μg/ml, followed by biotinylated goat antimouse IgG (H + L) (Jackson Immunoresearch, PA, USA). Finally, SA-HPO and TMB were added as described above. An IgE myeloma protein was used as standard (kind gift of R. Wistar, Naval Medical Research Institute). To confirm our results further and eliminate the possibility of cross-reaction, the same ELISA was repeated with another monoclonal antihuman IgE antibody (clone He-2, kind gift of Alk-Albello, Spain). No cross-reactivity with other immunoglobulin isotypes was observed.

RNA extraction and RT-PCR

Extractions were performed using RNAwiz (Ambion, TX, USA) according to the manufacturer’s instructions. Transfer RNA (Type X-SA, Sigma-Aldrich, Germany) was added as carrier for RNA precipitation. DNAse treatment was performed for all samples with RQ1 RNAse-Free DNAse (Promega, WI, USA). The RNA was reverse transcribed after incubation for 50 min at 42°C with 0·5 μg oligo-dT (MWG Biotech), 0·5 mM dNTPs (Life Technologies, USA), 10× buffer and 5 mm MgCl2 (MBI Fermentas, Lithuania), 10 mm dTT (Life Technologies, USA), 40 U RNAse inhibitor (Ambion, WI, USA) and 200U M-MLV reverse transcriptase (Life Technologies, USA) at a total volume of 20 μl. The reaction was terminated at 70°C for 15 min.

Quantitative PCR

Polymerase chain reaction was performed on cDNA for IFN-γ, IL4, IL13 and the housekeeping genes GAPDH and actin-β. The primers used were: GAPDH: sense GCT CAG ACA CCA TGG GGA AGG T, antisense GTG GTG CAG GAG GCA TTG CTG A, actin-β: sense GGG TCA GAA GGA TTC CTA TG, antisense GGT CTC AAA CAT GAT CTG GG, IFN-γ: sense GCA GAG CCA AAT TGT CTC CT, antisense ATG CTC TTC GAC CTC GAA AC, IL4: sense TGC CTC CAA GAA CAC AAC TG, antisense AAC GTA CTC TGG TTG GCT TC and IL13: sense GAG TGT GTT TGT CAC CGT TG, antisense: TAC TCG TTG GCT GAG AGC TG. All primers were purchased from MWG Biotech. Conditions were as follows: 94°C for 40 s, 56°C/58°C/60°C (IFN-γ/IL4/IL13, respectively) for 1 min and 72°C for 1·5 min, for 40 cycles. The appropriate Mg+2 concentration for each PCR was: 2 mm for IFN-γ, 1 mm for IL4 and 1·5 mm for IL13. The sensitivity of the PCR and the quantification of the cDNA was performed using two plasmid constructs as internal standards (IS) (kind offer of Dr David Shire, Sanofi Recherche, France). These were pQA1 containing the IFN-γ and IL4 primers and pQB2 containing the IL13 and actin-β primers. The sensitivity of the IFN-γ and IL4 PCR was calculated at approximately 150 copies and that of IL13 at approximately 640 copies. PCR products were run in a 2% agarose gel; the intensity of the bands was measured by densitometry and the log of the ratio IS/sample density (y axis) was plotted against the log of the IS copies (x axis). The x intercept corresponds to the quantity of the sample. The quantification was performed using the Quantity One software (Bio-Rad, CA, USA) and the graphical calculations with the Prism 3·0 software (GraphPad Software Inc.).

Statistical methods

The association between INF-γ production and clinical status of the patients, the associations between Chisholm’s score, INF-γ and IgE, the association between Chisholm’s score and IL13 and the association between IL13 and IgE were investigated by fitting log-linear models [28]. The correlation between Chisholm’s score and IFN-γ mRNA copies was investigated using Spearman’s rank correlation test.


Phenotype of the lymphocytes in LSG cultures

The numbers of the lymphocytes that were generated in the cultures were estimated with an optical microscope on various time intervals and correlated proportionally to the infiltration of the gland according to Chisholm’s score (data not shown). Preliminary experiments demonstrated that the presence of recombinant IL-2 is necessary in order to have satisfactory cell number in the cultures.

To determine whether the cultured lymphocytes have a similar phenotype to that described by in situ studies, FACS analysis was performed on lymphocytes from 20 biopsies. In the forward scatter/side scatter plot, only cells of the lymphocytic lineage were detected (Fig. 1a). The mean CD4/CD8 ratio (Fig. 1b) was 3·2 (range 1·1–10·9) significantly higher than the average ratio that is found in our laboratory in peripheral blood from normal donors (ratio 1·7). The majority of the lymphocytes was positive for HLA-DR and the Fas antigen (Fig. 1c,d), indicating that they were not part of the naive repertoire. Together, the above data suggest strongly that the in vitro generated lymphocytes constitute part of the LSG infiltrating population and are not part of the circulating lymphocytes present in the capillaries of the gland.

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.

Cytokine production in the LSG cultures

Supernatants from 44 biopsies cultured in the presence of IL-2 were collected on day 6 and evaluated for the presence of IFN-γ, IL-4 and IL-13. IFN-γ was detected in 24/44 SN and IL-13 in 26/44 (Fig. 2). In 16/44 cases IFN-γ and IL-13 were present simultaneously, indicating the presence of both Th1 and Th2 lymphocytes. IL-4 was present in only 5/44 SN and in concentrations near the limit of detection. Similar results were obtained on day 12 SN (data not shown). Cultured LSG from patients with definite primary or secondary SS produce IFN-γ more frequently (79%) compared to patients not fulfilling the criteria for SS (33%,P < 0·01). No correlation was found between IL-13 production and the disease stage of the patients. To exclude the possibility that the cytokine production in the LSG cultures was due to the exogenously added IL-2, PBMC from three healthy donors were cultured at 105 cells/ml for 6 days in the presence of IL-2. No IL-4, IL-13 and IFN-γ were detected in the SN of these cultures (data not shown).

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.

IFN-γ, IL-4 and IL-13 mRNA expression in cultured lymphocytes

Total RNA was extracted successfully from lymphocytes of 19 cultured LSG at day 12. IFN-γ and IL-13 mRNA were present at detectable levels in 17/19 and 18/19 samples, respectively, and IL-4 mRNA was detected in 14/19 (Fig. 3a). Interestingly, in the majority of the cultured lymphocytes, all three mRNAs were simultaneously present, restating that the infiltrating lymphocytes are able to express both types of cytokines. Of the 14 LSG that expessed IL-4 mRNA only three secreted IL-4 protein. The discrepancy between IL-4 protein product and mRNA was also observed when PBMC from three normal donors were stimulated with anti-CD3 plus anti-CD28 monoclonal antibodies, i.e. mRNA was detected readily by PCR but no protein product was observed in the SN. It should be noted that the detection of IL-4 mRNA in the cultures was not due to excessive number of PCR cycles, because PCR product in positive samples could be detected after 36 or 32 cycles (data not shown).

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 ...

Next, the number of cytokines mRNA copies was evaluated by quantitative RT-PCR. Due to the limited quantity of cDNA, quantitative PCR was performed only for IFN-γ (n = 11 cultures) and IL-13 (n = 10). Quantitative PCR for actin-β was used for the normalization of the results and the copy numbers of IFN-γ and IL-13 was expressed per 100·000 copies of β-actin (Fig. 3b). Interestingly, the number of IFN-γ mRNA copies correlated strongly with the intensity of the lymphocytic infiltration (rS = 0·85,P < 0·01,Fig. 3c), indicating that increased lymphocytic infiltration is accompanied by up-regulated Th1 cytokine expression. No correlation was observed for IL-13 mRNA copies with either Chisholm’s score or the disease status of the patients.

IgE production in the SN of the cultured biopsies

Both IL-4 and IL-13 are known to act on B lymphocytes to induce switching to IgE isotype. The presence of IL-4 and IL-13, prompted us to investigate the production of IgE on day 6 SN from 44 cultures. Indeed, IgE was present in 21/44 SN. When the biopsies were categorized into two groups with Chisholm’s score < 0·5 or = 0·5, 17/25 SN from the first group were positive for IgE, in contrast to only 4/19 from the second group (P < 0·05,Fig. 4). The association between Chisholm’s score and IgE was affected by the presence of IFN-γ. Thus, the presence of IFN-γ inhibits the IgE production when Chisholm’s score was high (P < 0·05). Moreover, the presence of IgE in the culture SN was significantly associated with the simultaneous presence of IL-13 (P < 0·05), indicating the important role of this cytokine to the production of IgE.

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.

IFN-γ, IL-4 and IL-13 mRNA expression in frozen biopsies

The presence of IFN-γ, IL-4 and IL-13 mRNA was also evaluated in RNA extracted from 14 frozen biopsies. Consistent with the previous results, IL-13 was present in 8/14 samples, IL-4 in 9/14 and IFN-γ in 7/14 (Fig. 5). Again, IFN-γ mRNA was present mainly in those samples that had moderate to severe infiltration. Thus, 75% of patients with Chisholm’s score > 0·5,versus 18% of patients with Chisholm’s score < 0·5, expressed IFN-γ RNA (P < 0·05). Because IFN-γ inhibits IgE production, these results are in accordance with the low frequency of IgE in lesions with high-grade lymphocytic infiltration.

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.


The small volume of the LSG and consequently the relatively low number of the invading lymphocytes constitute a serious limitation in directly determining the cytokine profile produced by these cells. As a result, this question has been investigated previously by examining the presence of cytokine mRNA in whole LSG. Because mRNA expression may not always correlate with protein production [29], to obtain more accurate information both the mRNA and the protein product need to be examined. Previous studies on the production of cytokines at the protein level are limited to one study and for only IL-2, IL-6, IL-10 and TGF-α[30].

To investigate directly the potential of the infiltrating lymphocytes to produce type-1 and type-2 cytokines, LSG from consecutive patients that referred for xerostomia were cultured in the presence of IL-2. Under these culture conditions, infiltrating lymphocytes migrate out of the tissue, demonstrate a typical activated profile and proliferate slowly. The addition of recombinant IL-2 every 6 days was sufficient to keep the cultures viable for more than 25 days, suggesting that additional antigenic stimulation was provided by the LSG present in the cultures. Indeed, when the LSG was removed from the cultures lymphocytes died in the next 3–4 days (data not shown). Also, in preliminary cultures of seven LSG without lymphocytic infiltration (Chisholm’s score = 0) that are not included in this study, only minimal numbers of lymphocytes outgrew in the culture, most of which died after a few days. It is important that IL-2 has never been reported to possess polarizing activity, and therefore the addition of exogenous IL-2 in the cultures is highly unlikely to influence the cytokine profile [31]. Thus, this culture methodology allows the direct evaluation of the cytokine profile of LSG infiltrating lymphocytes.

Our results show that both types-1 and -2 cytokines are expressed by the lymphocytes infiltrating the LSG. IL-13 and IFN-γ are expressed both at the mRNA and protein level in the majority of LSG examined, while IL-4 was mainly detected at the mRNA level. The difference observed between IL-4 protein production and IL-4 mRNA expression (also found in stimulated PBMC) is probably attributed to the strong binding affinity of IL-4 to its receptors or to low half-life of the cytokine in the SN. However, we cannot exclude the possibility that IL-4 mRNA is unstable and rarely translated to protein product.

It is important that the proportion of SS patients that produced IFN-γ was significantly higher than that of non-SS patients. Moreover, IFN-γ mRNA expression in whole LSG, as well as the levels of IFN-γ mRNA expression in infiltrating cultured lymphocytes, correlated positively with high infiltration score. Together, these results show that increased IFN-γ expression in LSG is associated with definite SS and advanced lymphocytic infiltration. This observation is in agreement with previous studies in mice showing increased IFN-γ expression in late lesions [32]. Expression of IL-13 mRNA has been contradictory [18,33]. Our results agree with the work of Villareal et al.[33], who detected IL-13 mRNA in LSG of SS patients. IL-4 mRNA expression in SS LSG has been reported previously by our laboratory [23] and by Ogawa et al.[30]. De Vita et al. and Ohyama et al. have also found IL-4 expression in SS patients with B cell lymphoma or with strong B cell accumulation [24,25]. Others, however, have failed to detect IL-4 transcripts in SS patients [1719]. The difference between previous studies and our results may be due to differences in the sensitivity of the PCR that were employed or the intensity of the lymphocytic infiltration. Alternatively, it is possible that IL-4 expression is dependent on tissue microenviroment, as in some studies major salivary glands were used [17].

Our findings showing that type-1 and type-2 cytokines do not demonstrate a restricted profile in LSG of SS patients are not surprising. Numerous studies have provided evidence indicating that human T helper cells present a less polarized profile than their mouse counterparts and that Th1 and Th2 cells are still able to produce IL-4 and IFN-γ, respectively [3436]. Thus current understanding supports the idea that immune responses are influenced mainly by the balance between types-1 and -2 cytokines. The novel finding and of biological significance in our studies is that, depending on the grade of infiltration and the clinical status of the patients, the balance of Th1/Th2 cytokines changes in favour of Th1 cytokines in late lesions. The functional prevalence of Th2 cytokines in early lesions is characterized by IgE production in vitro. It is noteworthy that this IgE production probably reflects an in vivo phenomenon, as in vitro switching to IgE is unlikely to take place as early as 6 days of culture [37,38]. All the above support the hypothesis advanced by Ferracioli and De Vita [39].

The prevalence of type-2 cytokines in early lesions in SS, a systemic autoimmune disease, may be of more general significance and is consistent with recent studies showing that: (1) large numbers of low-affinity autoreactive T cells escape clonal deletion and are present in the periphery of normal animals [40] and (2) after antigenic stimulation, T cells bearing low affinity T cell receptor (TCR) preferentially express type-2 cytokines [41,42]. Based on these reports, a probable hypothesis explaining the prevalence of type-2 cytokines in early SS is that low affinity autoreactive T cells are recruited and activated (for unknown reasons) in the salivary glands at the initiation of the autoimmune reaction. Because these cells bear low-affinity TCR, they would express a type-2-biased cytokine profile, which is consistent with our results. The skewing of the cytokine profile during the progress of the disease may result from the accumulated changes in the microenvironment of the lesion, including fibrosis or other tissue components [43], and locally produced chemokines that may also contribute to the redirection from a typ-2 to a type-1 immune response.

In summary, in this study we provide evidence suggesting that in SS type-1 and type-2 cytokines are in dynamic balance. Type II microenvironment prevails in low-grade infiltration, while type I pattern increases in patients with definite SS and patients with advanced lymphocytic infiltration.


This work was supported by the Hellenic Secretariat for Research and Technology, programme EPET II. We are grateful to Dr S. Paikos for providing the LSG biopsies and to Dr S. Giannini for revising the manuscript.


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