Increased entry of CD4+ T cells into the Th1 cytokine effector pathway during T-cell division following stimulation in Behçet's disease

S. Koarada, Y. Haruta, Y. Tada, O. Ushiyama, F. Morito, A. Ohta1 and K. Nagasawa

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To investigate the relationship between the production of Th1/Th2 cytokines and cell kinetics, cell division and proliferation in patients with Behçet's disease (BD).

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-{gamma} (IFN-{gamma}) (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-{gamma}-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


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CD4+ T cells differentiate into two distinct functional subsets, Th1 and Th2, which differ in their cytokine production profiles and effector functions [13]. Th1 cells secrete interferon-{gamma} (IFN-{gamma}) and IL-2 and are associated with cell-mediated immunity. On the other hand, Th2 cells are responsible for humoral immunity and produce high levels of IL-4, IL-10 and IL-13. The Th1/Th2 balance plays an important regulatory role in the immune system [4]. It has been reported that imbalance of Th1/Th2 cytokines may account for the pathophysiology in murine autoimmune diseases [5]. Abnormalities of Th1/Th2 balance have also been demonstrated in human diseases [6, 7].

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.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Patients
Twenty-four patients with BD were enrolled in this study. BD patients (20 women and four men; mean ± S.E.D. age 37.7 ± 12.1 yr) were diagnosed using the diagnostic criteria of the International Study Group for Behçet's Disease [22]. Ten BD patients had active disease; they had at least one of the major clinical signs and symptoms (oral ulcer, genital ulcers, cutaneous, ocular and gastrointestinal lesions) and were positive for CRP (>1.0 mg/dl) or had an elevated ESR (>40 mm/h). None of the active BD patients were receiving immunosuppressive drugs at the time of examination. Fourteen BD patients were inactive. Normal subjects (n = 22) were volunteers in our hospital (16 women and six men; mean ± S.E.D. age 32.7 ± 4.4 yr). All subjects gave informed consent prior to sample acquisition. The study was approved by the Saga Medical School ethics committees.

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 Ficoll–Hypaque (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-{gamma} 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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FIG. 4. IFN-{gamma}/IL-4 production in activated CD4+ T cells stimulated with Con A followed by PMA+Ion (large cells sorted by FACScan). FACS analysis of cytokine production in large and activated CD4+ T cells was performed. Cells surrounded by a circle were sorted and used as large CD4+ T cells. (A) Setting of the FSC/SSC. (B) Representative FACS profiles from an active BD patient and a normal subject. (C) The proportions of IFN-{gamma} and IL-4-producing large and activated CD4+ T cells. Bars show the mean ± S.E.D. *P<0.05

 
Analysis of cell division
Single-cell suspensions of PBMCs (1 x 107/ml) were labelled with CFSE [5 (and 6) carboxyfluorescein diacretate, succinimidyl ester] (Molecular Probes, Lelden, OR, USA) (10 µM) for 5 min at room temperature, as described previously [23]. The cells were washed three times with RPMI 1640 containing 10% FCS. The labelled cells were cultured with or without Con A for 3 days at 37°C. The cells were then incubated with PMA+Ion for 4 h. Brefeldin A was added for the final 2 h and the cells were harvested. The cells were stained with CyChrome-conjugated anti-CD4 mAb (BD Bioscience) for surface staining, and fixed and permeabilized. The cells were stained with PE-conjugated anti-IFN-{gamma} (BD Bioscience) or PE-conjugated anti-IL-4 mAb. The CFSE-labelled cells were analysed using a FACScan. In the other experiments, the expression of CD69 (anti-CD69 mAb; BD Bioscience), CD45RA (anti-CD45RA mAb; BD Bioscience) and CD45RO (anti-CD45RO mAb; BD Bioscience) on CFSE-labelled PBMC stimulated with Con A followed by PMA+Ion was analysed.

Statistical analysis
Statistical analysis of the results was performed with Wilcoxon statistics using SPSS software (SPSS Japan, Tokyo, Japan).


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Cytokine profile in CD4+ T cells stimulated with Con A followed by PMA+Ion
It is known that the proliferation and cell division regulate cytokine production by CD4+ T cells [1721]. A total of 24 patients with BD and 22 normal subjects were analysed for cytokine production during cell division stimulated with Con A followed by PMA+Ion. Th1/Th2 profile was determined by measurement of intracellular IFN-{gamma} (Th1) and IL-4 (Th2) production in CD4+ T cells by flow cytometry.

Figure 1A and B shows the representative FACS profiles of IFN-{gamma} 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-{gamma}-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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FIG. 1. (A) Setting of the FSC/SSC. (B) Representative FACS profiles from an active BD and a normal subject. (C) Numbers of cytokine-producing CD4+ T cells in BD. The proportions of IFN-{gamma}-and IL-4-producing CD4+ T cells from active and inactive patients with BD and normal subjects are shown. Bars show the mean ± S.E.D. *P<0.05.

 
Increased CD4+ T cells entering the IFN-{gamma} production pathway during cell division in BD
As we had found that CD4+ T cells stimulated with Con A followed by PMA+Ion showed effective IFN-{gamma} cytokine production in active BD patients (Fig. 1), we hypothesized the association of cell kinetics of CD4+ T cells with cytokine production in BD. During cell division of CD4+ T cells, CFSE is distributed equally through daughter cells, and the amount of fluorescence is halved accordingly [17]. Therefore, the number of cell divisions is clearly defined by CFSE intensity.

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-{gamma} 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-{gamma} than those from a normal subject.




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FIG. 2. Differential IFN-{gamma} and IL-4 production in CD4+ T cells in relation to cell division. (A) Representative FACS profile of cytokines from a patient with BD and a normal subject. IFN-{gamma} and IL-4 expression in dividing CD4+ T cells stimulated with Con A followed by PMA+Ion was analysed by flow cytometry. Percentages of cytokine-producing CD4+ T cells at each subsequent cell division are shown. (B) Average percentages of cytokine-producing CD4+ T cells at each cell division from patients with BD and normal subjects. Bars show the mean ± S.E.D. (C) Ratio of IFN-{gamma}- to IL-4-producing CD4+ T cells at each cell division. Bars show the mean ± S.E.D. *P<0.05; **P<0.005.

 
Figure 2B shows the proportion of cytokine-producing CD4+ T cells at each subsequent cell division in active BD patients (n = 6) and normal subjects (n = 8). It is of interest that the proportion of IFN-{gamma}-producing CD4+ T cells from active BD patients increased markedly as the number of cell divisions increased, which was in contrast with the results in normal subjects (P = 0.039 at the second cell division, P = 0.003 at the third cell division). On the other hand, the percentages of IL-4-producing CD4+ T cells from active BD patients and normal subjects declined as the number of cell divisions increased. As a result, the ratio of IFN-{gamma}- to IL-4-producing cells increased markedly as the number of cell divisions increased in active BD patients compared with normal subjects (P = 0.038 at the third cell division) (Fig. 2C).

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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FIG. 3. Phenotype of dividing CD4+ T cells. (A) Sizes of dividing CD4+ T cells from an active BD patient were analysed by forward scatter (FSC) and side scatter (SSC) using a FACScan. The dividing cells at each division number were sorted by CFSE intensity. (B) CFSE-labelled dividing and non-dividing cells were stained with anti-CD69, anti-CD45RA or CD45RO and with anti-CD4 antibody. A representative FACS profile from a patient with active BD is shown.

 
Balance of IFN-{gamma}/IL-4 cytokine production in activated CD4+ T cells
As described above, entry into the cytokine production pathway seemed to depend on cell division and to differ between active BD patients and normal subjects. To distinguish the characteristics of activated CD4+ T cells, large-sized CD4+ cells stimulated with Con A followed by PMA+Ion were gated (Fig. 4A) and FACS analysis was performed to detect intracellular IFN-{gamma} and IL-4 production. Figure 4B shows the representative FACS profiles of IFN-{gamma} and IL-4-producing large CD4+ T cells from a BD patient and a normal subject. Predominant production of IFN-{gamma} was found in an active BD patient. In a normal subject, the cytokine balance was not shifted to either IFN-{gamma} or IL-4.

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-{gamma}-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.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Our understanding of certain immunological and infectious diseases on the basis of the function of helper T cells (Th1 and Th2) is growing. For instance, Th1 cells are thought to be predominantly involved in granulomatous diseases, intracellular-proliferating pathogen-associated infectious diseases and rheumatoid arthritis [24]. On the other hand, Th2 cells are suggested to play a role in the pathophysiology of certain allergic diseases and parasite infections [2, 7]. Despite numerous reports, the contribution of Th1 and Th2 cells to the pathophysiology of systemic autoimmune diseases is still controversial.

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-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} 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-{gamma}) 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 [2729]. 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.


    Acknowledgments
 
We thank M. Fujisaki for her assistance with the research. S.K. is supported by 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 21 January 2004; revised version accepted 16 March 2004.