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Rheumatology Advance Access originally published online on November 15, 2005
Rheumatology 2006 45(4):392-399; doi:10.1093/rheumatology/kei182
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Ex vivo CD4 + T-cell cytokine expression from patients with Sjögren's syndrome following in vitro stimulation to induce proliferation

S. Koarada, Y. Haruta, M. Mitamura, F. Morito, Y. Tada, A. Ohta and K. Nagasawa

Division of Rheumatology, Saga Medical School, 5–1–1 Nabeshima, Saga, 849-8501 Japan.

Correspondence to: S. Koarada, Division of Rheumatology, Saga Medical School, 5–1–1 Nabeshima, Saga, 849-8501 Japan. E-mail: koarada{at}med.saga-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To assess ex vivo CD4+ T-cell cytokine expression from patients with primary Sjögren's syndrome (SS) following in vitro stimulation to induce proliferation, as proliferation is closely related to differentiation of cytokine-producing cells.

Methods. Peripheral blood mononuclear cells (PBMCs) separated from primary SS patients (n = 28) and controls (n = 25) were analysed. PBMCs were stimulated with concanavalin A followed by phorbol 12-myristate 13-acetate and ionomycin. Intracellular interferon-{gamma} (IFN-{gamma}) and interleukin-4 (IL)-4 in proliferating CD4+ T cells were assessed by flow cytometry. The proportion of cytokine-producing cells and proliferating cells in each division cycle was assessed using [5(and 6)-carboxyfluorescein diacetate, succinimidyl ester]-labelled CD4± T cells.

Results. The proportion of IFN-{gamma}+ proliferating CD4+ T cells in each cell division cycle from extraglandular SS was increased in glandular SS patients compared glandular SS patients with controls (P<0.05~0.01). The percentage of IFN-{gamma} single positive proliferating CD4+ T cells was greater in extraglandular SS patients (26.7±14.1%) compared with glandular SS (9.9 ± 9.1%) (P<0.01) and controls (9.4 ± 5.8%) (P<0.001). There was no significant difference in the percentages of IL-4+ proliferating CD4+ T cells among the groups. However, the proliferating response of CD4+ T cells was significantly decreased in extraglandular SS patients (percentage of proliferating cells 38.4 ± 18.6%) compared with that in glandular SS patients (64.2 ± 17.2%) (P<0.05) and controls (63.1±10.6%) (P<0.01).

Conclusions. CD4+ T cells from extraglandular SS patients may have a predisposition for entry into the IFN-{gamma}-producing effector pathway as a result of the stimulations. These results are helpful for understanding the immunological difference between glandular and extraglandular SS and the mechanisms of disease progression.

KEY WORDS: Proliferation, CD4+ T cells, IFN-{gamma}, Sjögren's syndrome


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Sjögren's syndrome (SS) is a chronic autoimmune inflammatory disease characterized by diminished lacrimal and salivary gland secretions resulting in symptoms of dry eyes and dry mouth [1]. Although SS mainly affects the lacrimal and salivary glands, systemic immunological alterations occur as well. SS is subdivided into glandular SS and extraglandular SS [2]. Extraglandular SS patients have systemic organ manifestations including hepatic, musculoskeletal, pulmonary, gastric, haematological, vascular, renal and neurological involvements.

According to previous reports, in general, CD4+ T cells have been classified into two distinct subsets, Th1 and Th2, by their cytokine secretion patterns [3–5]; Th1 cells, producing interferon-{gamma} (IFN-{gamma}) and interleukin-2 (IL-2), are involved in cell-mediated immunity, and Th2 cells, secreting IL-4, -5, -6, -10 and -13, modulate humoral immunity. It has been believed that alteration of Th1/Th2 cytokine balance of CD4+ T cells may play a pivotal role in the pathophysiology of systemic and organ-specific autoimmune diseases. Previous studies on systemic immune diseases have suggested that different cytokine patterns are found at different stages in the same disease [6]. In studies of organ-specific chronic inflammatory diseases, it has been shown that memory CD4+ T cells accumulate in the target organs and secrete Th1 cytokines upon antigenic stimulation [7–9]. However, recent papers have suggested that the data do not fit with the model of the Th1/Th2 paradigm in its original form and it has been replaced by the idea that the pathogen itself (bacteria, virus or parasite) is driving the type of response [10, 11]. It was also reported that regulation of the T-cell cytokine repertoire links to proliferation following stimulation in murine models [12–16]. It was also found in human autoimmune diseases that proliferating CD4+ T cells play a role in determining the cytokine production profile [17]. In this study, we concentrated on the analysis of cell proliferation, one of the possible factors regulating cytokine production. In SS, various cytokines participate in immune dysregulation in pathophysiological process and contribute to the functional and structural tissue injury [18, 19]. However, cytokine production in proliferating cells from SS patients still remains to be elucidated. We analysed the proliferating response and cytokine production during proliferation of CD4+ T cells from patients with SS in vitro. We also assessed whether extraglandular SS differs immunologically from glandular SS.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
Twenty-eight patients with primary SS fulfilled a revised version of the European criteria proposed by the American–European Consensus Group [20] and 25 controls were studied (Table 1). All of the patients with SS showed positive tests for anti-Ro/La antibodies (anti-Ro/SSA and/or anti-La/SSB antibodies) and/or labial gland biopsy. Eighteen SS patients presented localized lacrimal gland and/or salivary gland manifestations (glandular SS) (mean age 42.7 ± 16.8 yr, all female) and 10 patients (mean age 54.9 ± 12.8 yr, nine female and one male) showed systemic organ involvement (extraglandular SS) consisting of: primary biliary cirrhosis (PBC) (two patients); autoimmune hepatitis (AIH; two patients); thyroiditis (two patients); interstitial pneumonia (one patient); pleurisies (one patient); vasculitis (one patient) and neuritis (one patient). None of the glandular or extraglandular primary SS patients were receiving immunosuppressive drugs at the time of examination.


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  		TABLE 1. Characteristics of patients with Sjögren's syndrome

 
Patients with SS consist of heterogeneous subgroups with a wide clinical spectrum, ‘primary SS versus secondary SS’. Patients are diagnosed as having primary SS when no associated connective tissue disease is found. Patients with secondary SS are those cases with an established systemic autoimmune disease. As secondary SS patients are not a homogeneous class for the purpose of interpretation of the results, we analysed only primary SS patients in this study.

Controls were volunteers in our hospital [mean (±S.D.) 36.7 ± 14.4 yr, 24 female and 1 male]. All subjects granted informed consent prior to sample acquisition. The Saga Medical School ethics committees approved the study.

Reagents
Concanavalin A (ConA) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A (BFA) and saponin were obtained from Sigma-Aldrich (St Louis, MO, USA).

Cell preparation and cell culture
Peripheral venous blood was drawn in a heparinized tube from patients with SS and controls. Peripheral blood mononuclear cells (PBMCs) were separated immediately by centrifugation over a Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. PBMCs were washed twice and resuspended at 1 x 106 cells/ml in complete medium consisting of RPMI 1640 supplemented with 10% FCS (fetal calf serum), 1 mM L-alanyl-glutamine (Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies) and 50 µM 2-ME (2-mercaptoethanol) (Sigma-Aldrich). A total of 1 ml of the cell suspension was placed in a 24-well plate (Falcon; BD Biosciences, Mountain View, CA, USA). PBMCs were activated with ConA (4 µg/ml) in vitro for 3 days and treated with PMA and ionomycin for 4 h [17, 21]. The purpose of this first treatment with ConA is to induce cell proliferation. The second treatment with PMA and ionomycin induced cytokine production during proliferation of CD4+ T cells. There was no rest interval between the two stimulations, as we intended to analyse cytokine secretion during proliferation. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere.

Flow cytometric intracellular cytokine analysis
For intracellular cytokine analysis of T cells, brefeldin A (final concentration at 10 µg/ml) was added to the culture 2 h before harvesting. The cells were harvested and stained for 20 min at 4°C with phycoerythrin-cyanine dye Cy5 (PE-Cy5)-conjugated anti-CD4 monoclonal antibodies (mAbs) (BD Biosciences) in staining buffer [2% FCS, 0.1% sodium azide in phosphate buffered saline (PBS)]. Then, the cells were fixed for 20 min at 4°C with 2% paraformaldehyde (Sigma-Aldrich) in PBS and permeabilized with 0.5% saponin in PBS (permeabilization buffer) for 10 min at room temperature. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IFN-{gamma} monoclonal antibodies (mAbs) (BD Biosciences) and phycoerythrin (PE)-conjugated anti-IL-4 mAbs (BD Biosciences) in permeabilization buffer for 20 min at 4°C. The cells were also stained with FITC-conjugated isotype control immunoglobulins (Igs) (BD Biosciences) and PE-conjugated isotype control Igs (BD Biosciences) in the same manner. In our previous study, it was demonstrated that the large-sized CD4+ T cells stimulated with ConA were proliferating T cells [17]. Large-sized CD4+ T cells were sorted by the cell size (FSC; forward scatter) and SSC (side scatter), and cytokine expression was analysed using a FACScan flow cytometer.

In a patient with primary extraglandular SS with liver involvement, a serial analysis of cytokine production with stimulation was also performed.

Analysis of cytokine production by proliferating CFSE-labelled CD4+ T cells
Thirty-three samples of [5 (and 6)-carboxyfluorescein diacetate, succinimidyl ester] (CFSE)-labelled (Molecular Probes, Lelden, OR, USA) CD4+ T cells were analysed (glandular SS, 10 samples; extraglandular SS, 8 samples; normal controls, 15 samples). CFSE is distributed equally through daughter cells and the fluorescence halves accordingly. Therefore, the cycles of cell division are clearly defined by the CFSE intensity. Single-cell suspensions of PBMCs (1 x 107/ml) were labelled with CFSE (10 µM) for 5 min at room temperature according to previous studies [17, 21]. The cells were washed three times with RPMI 1640 containing 10% FCS. The CFSE-labelled cells were cultured with ConA for 3 days. Then the cells were incubated with PMA and ionomycin for 4 h. Brefeldin A was added for the final 2 h and the cells were harvested. The cells were stained with PE-Cy5-conjugated anti-CD4 mAbs (BD Biosciences) for surface staining, and fixed and permeabilized. The cells were stained with PE-conjugated anti-IFN-{gamma} (BD Biosciences) or PE-conjugated anti-IL-4 mAbs. The cells were analysed using a FACScan and the percentage of events in each cell division cycle was calculated.

Statistical analyses
Analysis of variance with the Kruskal–Wallis test was used when three groups were compared. After a difference was detected among the groups, the groups were compared for statistical significance using the Mann–Whitney U-test. Analysis was performed using SPSS software (SPSS Japan Inc., Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Characteristics of patients with SS
The clinical and immunological features of patients with primary SS are summarized in Table 1. The mean age of patients with extraglandular SS was older than that of glandular SS patients and controls (P<0.05). The percentages of positive tests for antinuclear antibodies (ANA), anti-Ro/SS-A and anti-La/SS-B antibodies were similar in glandular SS and extraglandular SS.

Proliferating response and cytokine production of proliferating CD4+ T cells
The cytokine production during proliferation was analysed using CFSE-labelled CD4+ T cells stimulated with ConA followed by PMA and ionomycin. Figure 1A shows the production of IFN-{gamma} and IL-4 in representative cases. A larger population of IFN-{gamma} producing proliferating CD4+ T cells seemed to be found in an extraglandular SS patient compared with that in a glandular SS patient and a control.


Figure 1
Figure 1
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  		FIG. 1. (A) Representative FACS profiles of intracellular cytokine analysis of proliferating CFSE-labelled CD4+ T cells from patients with primary glandular SS and extraglandular SS, and a control. Expression of IFN-{gamma} and IL-4 in CFSE-labelled CD4+ T cells stimulated with ConA followed by PMA and ionomycin with BFA on day 3 were analysed by flow cytometry. (B) The percentages of IFN-{gamma}- and IL-4-producing CD4+ T cells in each cell division cycle. Bars show averages ± S.D. of the percentage of cytokine-producing CD4+ T cells in each cell division cycle. ({square}, glandular SS; {circ}, extraglandular SS; {triangleup}, controls) (*P<0.05, **P<0.01). (C) Numbers of proliferating cells in each cell division cycle. The percentages of events in each cell division cycle were calculated. (*P<0.05, **P<0.01).

 
Figure 1B shows the proportion of cytokine-producing CD4+ T cells at each subsequent cell division cycle in glandular SS (n = 10), extraglandular SS (n = 8) and controls (n = 15). The proportion of IFN-{gamma}-producing CD4+ T cells from extraglandular SS patients was remarkably increased at the first and the second cell division cycle, which contrasted with the results in glandular SS patients and controls (P<0.01 on the first and the second cell division cycle). Even in the non-dividing T cells and the third cell division cycle, the proportion of IFN-{gamma}-producing cells in extraglandular SS patients was increased compared with glandular SS patients and controls (P<0.05). There were no significant differences in the proportion of IFN-{gamma}-producing proliferating CD4+ T cells between glandular SS patients and controls. On the other hand, the proportion of IL-4-producing proliferating CD4+ T cells stayed at a low level and was not significantly different among the three groups.

Figure 1C shows the proliferating response of CD4+ T cells from SS patients and controls. The proportion of proliferating CD4± T cells was significantly decreased in extraglandular SS patients (38.4 ± 18.6%) compared with that in glandular SS patients (64.2 ± 17.2%) (P<0.05) and controls (63.1 ± 10.6%) (P<0.01) after 3 days of culture with ConA.

IFN-{gamma} and IL-4 single positive proliferating CD4+ T cells
In Fig. 1 we were not able to exclude double positive CD4+ T cells for IFN-{gamma} and IL-4 from the analysis. Therefore, to define IFN-{gamma} or IL-4 single positive proliferating CD4+ T cells, large-sized CD4+ T cells, consisting mainly of proliferating T cells [17], were stained with FITC-anti-IFN-{gamma} and PE-anti-IL-4 mAbs and were sorted by the cell size using a FACScan (Fig. 2A). Representative results of FACS analysis are shown in Fig. 2B. The proportion of IFN-{gamma} single positive proliferating CD4+ T cells from a patient with extraglandular SS was larger than that from a glandular SS patient and controls. The percentages of IFN-{gamma} and IL-4 single positive proliferating CD4+ T cells in SS patients and controls are shown in Fig. 2C. The percentages of IFN-{gamma} single positive proliferating CD4+ T cells from patients with extraglandular SS (26.7 ± 14.1%) were greater compared with glandular SS patients (9.9 ± 9.1%) (P<0.01) and controls (9.4 ± 5.8%) (P<0.001). There was no significant difference in the percentages of IFN-{gamma} single positive proliferating CD4+ T cells between glandular SS patients and normal subjects. On the other hand, the percentages of IL-4 single positive proliferating CD4+ T cells were low and were not different among the three groups. The ratio of IFN-{gamma}/IL-4 single positive proliferating CD4+ T cells was significantly increased in extraglandular SS patients (8.1 ± 8.0) compared with that in glandular SS patients (2.6 ± 2.6) and controls (3.1 ± 4.0) (P<0.01) (Fig. 2D).


Figure 2
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  		FIG. 2. (A) Setting of the FSC/SSC (forward scatter/side scatter). Peripheral CD4+ T cells were stimulated with ConA followed by PMA and ionomycin with BFA. Proliferating large-sized CD4+ T cells were sorted by FSC and SSC using flow cytometry on day 3. (B) Representative FACS profiles of intracellular cytokine analysis in proliferating CD4+ T cells from patient with glandular SS and extraglandular SS, and a control. The numbers indicate the percentages of IFN-{gamma} and IL-4 single positive cells in each quadrant. (C) The percentages of IFN-{gamma} and IL-4 single positive proliferating CD4+ T cells from SS patients and controls. Each dot represents the cytokine production of activated CD4+ T cells from the individual subject and means in the groups are shown. Data for the cytokine production of different groups were compared with each other. Only significant differences are shown (**P<0.01, ***P<0.001). (D) The ratio of IFN-{gamma}/IL-4 single positive proliferating CD4+ T cells (**P<0.01).

 
We performed a serial analysis of intracellular cytokine production by ConA-activated CD4+ T cells in a female patient with SS (Fig. 3). She was diagnosed as having glandular SS at 48 yr of age. At 60 yr she developed liver dysfunction and her liver biopsy showed autoimmune hepatitis. We observed cytokine production; both when she had only glandular SS at 59 yr, and when she had extraglandular organ involvement as well at 60 yr. As shown in Fig. 3, the proportion of IFN-{gamma} and IL-4 single positive proliferating CD4+ T cells were no different (12.6 vs 10.7%) when she still had only glandular SS. Interestingly, a year later when extraglandular SS developed, IFN-{gamma} single positive proliferating CD4+ T cells were markedly increased (from 12.6 to 53.7%).


Figure 3
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  		FIG. 3. FACS profiles in a serial analysis of cytokine production of proliferating CD4+ T cells in vitro from a primary SS patient with autoimmune hepatitis. The percentages of IFN-{gamma} and IL-4 single positive proliferating T cells were indicated before and after her liver disease.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
Patients with SS show hyperactivation of the immune system characterized by hypergammaglobulinaemia, production of autoantibodies and focal lymphocytic infiltration in exocrine glands and often even in extraglandular organs [22]. The profile of cytokine production of freshly isolated T cells from SS patients is still controversial. The decreased proportion of Th1 has been described in previous papers [23, 24]. Another study showed increased Th1 CD4+ T cells in the peripheral blood [25]. There has been only one report of cytokine production in SS patients with organ system involvement, in which no Th1/Th2 dominance was found [24]. In human SS, the difference in immunological events between extraglandular and glandular SS remains unclear.

Recently, it has been reported that regulation of the T-cell cytokine repertoire is closely linked to proliferation following stimulation [12–16]. Therefore, we studied the in vitro proliferating response and cytokine production of proliferating CD4+ T cells from SS patients. PBMCs were activated with ConA and then further treated with PMA and ionomycin. The purpose of the first treatment with ConA is to induce proliferation of T cells. To induce cytokine production during proliferation, the second stimulation was immediately introduced without any rest interval. The tissue injury in target organs may largely depend on the functional activity of memory T cells with the CD45RAlow CD45ROhigh phenotype in humans [7, 8]. Memory T cells, sensitized by foreign antigens, have an efficient homing capacity to the inflammatory tissues. The memory T cells consist of two subpopulations; central memory T cells and effector memory T cells [26]. The former have the capacity to readily proliferate and differentiate to effector cells in response to stimulation, and then home to T-cell areas of secondary lymphoid organs [26] and the latter selectively target to inflamed peripheral tissues, lung, liver, gut and so on, and display immediate effector function by producing cytokines efficiently [26–29].

Moreover, central and effector memory T cells show different abilities to produce cytokines. Central memory T cells were shown to produce IL-2 exclusively, whereas effector memory T cells secrete mainly IFN-{gamma} [30]. In previous studies of peripheral blood CD4+ T cells from patients with chronic beryllium diseases (CBD), the beryllium-specific CD4+ T cells are divided into two groups: CD4+ T cells capable of expressing both IFN-{gamma} and IL-2, and T cells expressing only IFN-{gamma} [30–32]. In addition, effector memory T cells have a decreased proliferating response compared with central memory cells. Beryllium-specific CD4+ T cells in the lung as the target organ proliferate poorly despite the increased ability to secrete IFN-{gamma} and tumour necrosis factor-{alpha} (TNF-{alpha}). These findings suggest that chronic antigen exposure may possibly induce IFN-{gamma}-producing poorly proliferating T cells. In our previous report, the phenotype of dividing CD4+ T cells stimulated with ConA was CD45RAlowCD45ROhigh memory type T cells [17]. Therefore, we analysed the proportion of proliferating memory type CD4+ T cells in each division cycle and non-proliferating CD4+ T cells in SS patients. While increased entry into the IFN-{gamma}-producing pathway was found in memory type CD4+ T cells from patients with extraglandular SS compared with glandular SS and controls, interestingly proliferating response of CD4+ T cells was significantly decreased. Our results in vitro are coincident with that in CBD. In extraglandular SS, CD4+ T cells might have a predisposition to enter into the IFN-{gamma}-producing effector pathway easily and differentiation into effector memory T cells during proliferation induced by the stimulations. However, this does not directly suggest the enlarged in vivo IFN-{gamma}-producing population in memory CD4+ T cells of extraglandular SS patients. In addition, these results suggest that IL-2 production may involve in this process. Therefore, further analysis of the cytokine response focusing on IL-2 production and effector memory CD4+ T cells in SS in vitro and in vivo is required.

In previous reports, immunological differences in peripheral blood T cells between glandular SS and extraglandular SS were not evident. In addition, the immunological mechanisms involved in the progression from glandular SS to extraglandular SS remain poorly defined. Investigation currently requires invasive tests such as liver biopsy, bronchoscopes and so on to confirm immunological difference between glandular SS and extraglandular SS. Therefore, a non-invasive assay using PBMCs to identify the immunological difference between glandular SS and extraglandular SS would advance our understanding of the immunophysiology of SS. In previous reports, a similar example has also been found in beryllium-sensitized (BeS) patients and CBD. In BeS individuals, who have no organ involvement, only small subsets progress to CBD, characterized by granulomatous inflammation in target organs. Patients with CBD show markedly increased beryllium-specific IFN-{gamma}-producing T cells in blood compared with BeS individuals. The results suggested that the analysis of antigen-specific CD4+ cells in blood could be useful as a biomarker for determining the stage of disease [31]. The results we obtained in extraglandular SS are also quite similar to previous data in Behçet's disease (BD) [17]. An increased IFN-{gamma}-producing response was found when the disease was active, and may reflect the different immunological background between active BD and inactive BD. In this study, increased IFN-{gamma} production in proliferating CD4+ T cells and decreased proliferation of T cells were found in extraglandular SS patients but not in glandular SS patients. The increased numbers of IFN-{gamma}-producing CD4+ T cells and/or their precursors detected by in vitro stimulation may be useful in monitoring an immunological change in SS. However, it is not clear whether enhanced IFN-{gamma} production of T cells during proliferation results in extraglandular diseases or whether increased IFN-{gamma}-producing cells just reflect the immunological hyperactivation of extraglandular SS.

A serial analysis of cytokine production in a case of primary SS shows increased IFN-{gamma} production during proliferation when the patient presented with extraglandular organ involvement. This result may be a suggestive observation for considering the relationship between expression of IFN-{gamma} in proliferating T cells and organ involvement. However, to confirm it, extensive serial analyses in SS patients are required.

In this study we could not exclude the possibility that our methodology using ConA is likely to select dividing IFN-{gamma}-producing cells rather than IL-4-producing cells. We have performed additional experiments to investigate this. Freshly isolated PBMCs, from SS patients and controls, were stimulated with PMA and ionomycin for 4 h without ConA. This methodology did not induce cell proliferation. The data demonstrate that IL-4 production was also lower than IFN-{gamma} (data not shown), in agreement with a previous study [25]. These results suggest that IL-4 production in CD4+ T cells may be originally lower than IFN-{gamma} production. We understand that an ideal system for analysis is to define the number of cytokine-producing autoantigen-specific T cells and the proliferating response of them in peripheral blood and infected organs of SS patients. However, in actuality, the analysis of pathogenic autoantigen-specific CD4+ T cells in human diseases, including SS, is a difficult task because of the low frequency of autoreactive CD4+ T cells circulating in peripheral blood, and the absence of definite stimulating and pathogenic autoantigens at present in SS. Therefore, we used ConA, a well-known T-cell-stimulating mitogen, in this study. However, to exclude the possibility of preferential IFN-{gamma} production by ConA, in future we have to confirm the precise cytokine production by specific antigenic stimulation and the proliferating response of autoreactive CD4+ T cells in SS.

Since the 1980s, the Th1/Th2 dichotomy has been believed to be important in cellular and humoral immune responses. However, recent papers have shown that the data did not fit with the model in its original form and the Th1/Th2 dichotomy has been replaced by the idea that the pathogen itself (bacteria, virus or parasite) drives the type of cytokine response [10, 11]. In particular, among pattern recognition receptors for the pathogen-associated molecular patterns (PAMPs), Toll-like receptors (TLRs) are crucial for the recognition of pathogen-derived products and initiation of the activation of immunological host defences [33]. TLRs play crucial roles in the regulation of the Th1 and Th2 response [11]. A recent study showed that flagellin (a TLR-5 ligand) and R-848 (a TLR7/8 ligand) up-regulate proliferation and IFN-{gamma} but not IL-4 production by human CD4+ T cells, and CD45RO+ memory CD4+ T cells are more sensitive to TLR ligands than CD45RA+-naive CD4+ T cells [34]. Memory T cells are more sensitive for stimulation than naive T cells [35]. To understand cytokine production and cell proliferation of CD4+ T cells in autoimmune diseases, we have to take the functions of TLRs and ligands into consideration in our next study.

In conclusion, increased IFN-{gamma}-producing proliferating CD4+ T cells and a decreased proliferating response of CD4+ T cells were found in the subtype of extraglandular SS. We present here the immunological difference between glandular SS and extraglandular SS patients. The analysis of cytokine and proliferation of CD4+ T cells with stimulation in vitro is helpful for considering the immunophysiology of development of disease from glandular SS to extraglandular SS.
Figure 4


   Acknowledgments
 
We thank M. Fujisaki for her assistance with the research. S.K. is supported by a grant-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan (No. 15591057).

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 14 July 2005; revised version accepted 30 September 2005.
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