Division of Rheumatology and 1 Clinical Nursing, Saga Medical School, Saga, Japan.
Correspondence to: S. Koarada, Division of Rheumatology, Saga Medical School, 5-1-1 Nabeshima, Saga, 849-8501, Japan. E-mail: koarada{at}post.saga-med.ac.jp
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Abstract |
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Methods. Peripheral venous blood was drawn from patients with BD (n = 24; 10 patients with active and 14 patients with inactive BD) and normal subjects (n = 22). Peripheral blood mononuclear cells were separated immediately and were cultured with concanavalin A (Con A) followed by phorbol 12-myristate 13-acetate and ionomycin (PMA+Ion). Intracellular cytokine production of interferon- (IFN-
) (Th1) and IL-4 (Th2) in CD4+ T cells was determined by flow cytometry. Furthermore, CD4+ T cells labelled with CFSE [5 (and 6) carboxyfluorescein diacretate, succinimidyl ester] were stimulated and the cells were analysed for entry into the cytokine production effector pathway during cell division in active BD and normal subjects.
Results. In active BD, enhanced entry into the Th1 response effector pathway of CD4+ T cells was observed after stimulation with Con A followed by PMA+Ion. Analysis of CD4+ T cells at an identical cell division number in response to Con A followed by PMA+Ion revealed that IFN--producing cells were increased in active BD patients compared with normal subjects. These results suggest that the Th1 response of dividing CD4+ T cells is predominantly operating in active BD. Dividing CD4+ T cells stimulated with Con A followed by PMA+Ion showed a phenotype of activated effector memory T cells (CD45RAlow, CD45RO+, CD69high).
Conclusions. Cell kinetics play a crucial role in Th1 cell differentiation and pathophysiology in BD.
KEY WORDS: Behçet's disease, Cell division, Th1/Th2
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Introduction |
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Behçet's disease (BD) is a systemic rheumatic disease characterized by oral and genital ulcers and by cutaneous, ocular, arthritic, vascular and neurological lesions [8, 9]. Increasing numbers of reports show that various Th1 and/or Th2 cytokines may contribute to the pathophysiological process in BD [1016]. Recently, regulation of the T-cell cytokine repertoire linked to cell division following stimulation has been reported in murine models [1721]. These results prompted us to analyse the relationship between cell division and entry into the cytokine production effector pathway. In particular, dividing CD4+ T cells may play a key role in determining the Th1 and/or Th2 cytokine profile. In the present paper, levels of Th1 and Th2 cytokine production in dividing CD4+ T cells were investigated in BD. The results suggest that enhanced entry of CD4+ T cells into the Th1 cytokine effector pathway during cell division following stimulation may be associated with the pathogenesis of BD.
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Materials and methods |
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Agents
Concanavalin A (Con A) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin (Ion), brefeldin A and saponin were obtained from Sigma-Aldrich (St Louis, MO, USA).
Preparation and culture of cells
Peripheral venous blood was drawn in a heparinized tube from patients with BD and normal subjects. Peripheral blood mononuclear cells (PBMCs) were separated immediately by centrifugation on a FicollHypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. PBMCs were washed twice and were resuspended at 1 x 106 cells/ml in complete medium consisting of RPMI 1640 supplemented with 10% fetal calf serum (FCS), 1 mM L-alanyl-glutamine (Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin, 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 24-well plates (Falcon; BD Biosciences, Mountain View, CA, USA). PBMCs were stimulated with Con A for 3 days, and incubated with PMA plus ionomycin for 4 h (Con A followed by PMA+Ion). The cells were incubated at 37°C in a humidified 5% CO2 atmosphere.
Flow cytometric intracellular cytokine analysis
For intracellular cytokine analysis, brefeldin A (final concentration 10 µg/ml) was added to the culture 2 h before harvesting. At the end of the incubation, the cells were washed twice and stained for 20 min at 4°C with CyChrome [R-phycoerythrin (PE)-Cy5 tandem]-conjugated anti-CD4 monoclonal antibody (mAb) (BD Bioscience) in staining buffer [2% FCS, 0.1% sodium azide in phosphate buffered saline (PBS)]. The cells were then fixed for 20 min at 4°C with 2% paraformaldehyde (Sigma-Aldrich) in PBS and permeabilized for 10 min at room temperature with 0.5% saponin in PBS (permeabilization buffer). The cells were stained in permeabilization buffer for 20 min at 4°C with fluorescein isothiocyanate (FITC)-conjugated anti-IFN- mAb (BD Biosciences) and PE-conjugated anti-IL-4 mAb (BD Biosciences). The cells were also stained in the same manner with FITC-conjugated isotype control immunoglobulin (Ig) (BD Biosciences) and PE-conjugated isotype control Ig (BD Biosciences). Flow cytometric analyses were performed using a FACScan flow cytometer (BD Biosciences). PBMCs in culture with Con A followed by PMA+Ion were fractionated into large-sized cell populations by FACS (Fig. 4A) after being labelled with anti-CD4 mAbs, and the cytokine production of activated large-sized CD4+ T cells was analysed.
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Statistical analysis
Statistical analysis of the results was performed with Wilcoxon statistics using SPSS software (SPSS Japan, Tokyo, Japan).
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Results |
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Figure 1A and B shows the representative FACS profiles of IFN- and IL-4-producing CD4+ T cells in cultures of Con A followed by PMA+Ion stimulation from a BD patient and a normal subject. As shown in Fig. 1C, IFN-
-producing CD4+ T cells in active BD patients (mean ± S.E.D. 11.8 ± 5.5%) were significantly increased compared with normal subjects (6.3 ± 4.6%) (P = 0.005) and inactive BD patients (3.6 ± 2.7%) (P = 0.001). On the other hand, the numbers of IL-4-producing CD4+ T cells were not significantly different among the three groups in the same culture conditions.
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To determine whether cytokine production in CD4+ T cells was associated with cell division in BD, PBMCs labelled with CFSE were cultured with Con A followed by PMA+Ion. The cells were then analysed for the entry into the cytokine production pathway during cell division, using intracellular cytokine analysis. Figure 2A shows the production of IFN- and IL-4 in a representative case of active BD and in a normal subject (percentages of cytokine producing CD4+ T cells at each subsequent cell division are shown). It seems that proliferating cells from an active BD patient produce a greater amount of IFN-
than those from a normal subject.
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In the analysis of CFSE-labelled cells stimulated with Con A for 3 days followed by PMA+Ion stimulation, the size of dividing CD4+ T cells was measured by forward scatter (FSC) using a FACScan. The size of the dividing cells, shown for each division number gated by CFSE intensity, became larger as the number of cell divisions increased (Fig. 3A). Furthermore, dividing CD4+ T cells showed increased expression of CD69, an activated T-cell marker (Fig. 3B). Similarly, they developed CD45RO expression, a memory T-cell marker, and reduced expression of CD45RA, a naive T-cell marker. Thus, the phenotype of dividing CD4+ T cells was found to be that of activated, large, memory-type T cells.
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Figure 4C shows the numbers of cytokine-producing large CD4+ T cells stimulated with Con A followed by PMA+Ion from active and inactive BD patients and from normal subjects. As shown in Fig. 4C, IFN--producing cells from active BD patients were significantly increased (18.5 ± 7.1%) compared with other groups (inactive BD, 5.9 ± 3.9%, P = 0.001; normal subjects, 9.5 ± 6.4%, P<0.001). On the other hand, there was no significant difference in the percentage of IL-4-producing cells among three groups.
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Discussion |
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BD is characterized by immunopathological events, such as altered production of cytokines and/or chemokines, activation of neutrophils and the resulting vasculitis. In addition, lesions in BD have features similar to those of delayed type hypersensitivity [21]. These observations suggest that BD may be a Th1-dominant disease. In investigations on the roles of Th1 and Th2 cytokines, diverse findings have been described [10, 11, 13, 14, 16, 25, 26]. Several investigators have proposed association of the Th1 response, IL-12 and IFN- with BD [14, 15, 26]. These results reflect the cytokine balance in vivo. In the present ex vivo analysis using the culture protocol of Con A followed by PMA+Ion, we demonstrated that CD4+ T cells from active BD patients became capable of producing a significant amount of IFN-
during cell division. Labelling PBMC with CFSE proved to be a good technique to analyse the relationship between cell kinetics and cytokine production, although it has been used mainly in mice [2729]. The present study clearly showed that dividing cells stimulated by Con A followed by PMA+Ion were large in size and highly activated and that the cells responsible for the production of IFN-
increased further as cell division proceeded in active BD patients. Moreover, the phenotype analysis showed that these dividing CD4+ T cells corresponded to memory T cells that were CD45RO-positive and showed lower expression of CD45RA. On the other hand, IL-4 production in CD4+ T cells from both active BD patients and normal subjects was reduced as cell division advanced. As a result, the ratio of IFN-
to IL-4 (Th1/Th2) went on increasing as cell division advanced in active BD patients (Fig. 2C). These results suggest that activated memory CD4+ T cells may develop during cell divisions and that differentiation may occur, resulting in acceleration of entry into the cytokine (IFN-
) production pathway in BD patients.
In general, in vitro activation of antigen-specific memory T cells has been achieved by stimulating T cells with antigen-presenting cells pulsed with appropriate specific antigenic peptides. Tsotsiashvilli et al. described the use of the polyclonal activator Con A in the in vitro restimulation of memory cytotoxic T lymphocytes (CTLs) for 3 days [30]. CTLs stimulated with ConA effectively lysed virally infected targets. Moreover, Con A stimulation of allogenic CTLs induced a specific memory CTL response by bypassing the original priming antigen stimulation [30]. Therefore, CD4+ helper T cells stimulated with Con A may differentiate into long-term memory effector T cells in the same manner. In vitro activation of CD4+ T cells by Con A stimulation suggested the existence of memory-type T cell precursors. However, further research is required to clarify the precise mechanism of Con A stimulation in memory CD4+ T cells.
The results of cytokine production of ConA stimulated CD4+ T cells did not directly illustrate the in vivo Th1/Th2 cytokine balance in the patients with active BD. However, the cytokine data in this study of in vitro Con A stimulation agree with previous observations showing Th1 dominance in active BD [14, 15, 26] and suggest that CD4+ T cells from active BD patients may have a predisposition to a Th1 response during cell division on T-cell stimulation.
In mice, it has been shown that the number of divisions following activation is an important factor in T cell differentiation [27 29]. Entering the Th1/Th2 effector pathway requires induction of transcription factors, such as T-bet in the Th1 response and GATA-3 in the Th2 response [31]. Many factors control the cytokine genes, such as demethylation, enhanced ability to bind transcriptional factors and reduced histone acetylation. It is considered that these changes could occur during cell division and may contribute to the full differentiation of CD4+ T cells and the acquisition of effector functions.
The analysis of cytokine profile in proliferating and differentiated cells seems of great interest in human diseases as well as in mice. So far, however, there have been no reports discussing the association between the cytokine balance and the division of CD4+ T cells in human diseases.
In conclusion, dividing CD4+ T cells stimulated with Con A followed by PMA+Ion showed a phenotype of activated effector memory T cells, and activated dividing CD4+ T cells showed increased entry into the Th1 cytokine effector pathway during T-cell division in BD. It is suggested that cell division and differentiation play a crucial role in Th1 cell differentiation and pathophysiology in active BD.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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