To determine whether the disease activity affected the senescence of the immune system, the correlation between the levels of CRP or ESR, and the frequencies of senescent immune cells were analyzed in BD group. The level of CRP did not correlate with the frequencies of senescent CD4+ T cells, CD8+ T cells, and B cells significantly, with the p value of .200, .236, and .359, respectively. Neither did the level of ESR with the frequencies of senescent CD4+ T cells, CD8+ T cells, and B cells (p value
= .458, .944, .476, respectively).
In BD group, 15 patients were receiving systemic steroid treatment and 14 were not.
The mean frequencies of senescent immune cells were compared. There was no difference in the mean frequencies of senescent CD4+ T cells, CD8+ T cells, and B cells (p value = .292, .414, .162, respectively).
HLA-B51 was positive in 11 among 26 BD patients. The mean frequencies of senescent immune cells between HLA-B51 postiive group and HLA-B51 negative group were compared. There was no difference in the mean frequencies of senescent CD4+ T cells, CD8+ T cells, and B cells (p value = .095, .447, .056, respectively).
In BD group, all the patients had oral ulcers. There were 69.2% of BD patients with genital ulcer, 69.2% with cutaneous involvement, 38.5% with ocular involvement, 41.0%
with joint involvement, 7.7% with gastrointestinal involvement, and 7.7% with cardiovascular involvement. The mean frequencies of senescent immune cells were compared between BD patients who had specific organ involvement and those who didn’t. None of the systems involved had effect on the frequencies of senescent immune cells with the p value > .05.
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E. SA-β-Gal activity of CD8+ T cells in active BD, inactive BD, DCs, and HCs
To confirm the senescence of CD8+ T cells, the activity of SA-β-Gal, a senescent marker, was measured. The percentage of SA-β-Gal+ CD8+ cells in CD8+ cells in each group was analyzed. The mean percentage and SD were 28.6 ± 11.0 %, 24.1 ± 10.3 %, 16.4 ± 5.1 %, and 14.1 ± 6.1 % in active BD (n=19), inactive BD (n=20), DCs (n=15), and HCs (n=15), respectively (Fig. 4). In inactive BD, the frequencies of CD8+ SA-β-Gal+ cells were increased compared to DCs and HCs with the p value < .05 each. In active BD, the frequencies of CD8+ SA-β-Gal+ cells were even more increased compared to DCs and HCs with the p value < .005.
Fig. 4. Dot plots of CD8+ SA-β-Gal+ cells in representative subjects in PBMCs of active BD, inactive BD, DCs, and HCs. Percentage of SA-β-Gal+ cells among CD8+
cells was analyzed in each group. Numbers shown are mean values of each group.
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Fig. 5. Frequency of CD8+ SA-β-Gal+ cells in PBMCs of active BD inactive BD, DCs, and HCs. The frequencies of CD8+ SA-β-Gal+ cells were increased in active BD and inactive BD compared to controls with statistical significance (*p < .05, **p < .005).
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DISCUSSION
The aim of the study was to investigate the frequencies of immunosenescent cells in peripheral blood mononulclear cells in BD patients and controls. The loss of expression of co-stimulatory molecules such as CD28 in CD4+ and CD8+ T cells is known to increase in an age-dependent manner in healthy subjects (Fagnoni et al., 1996; Vallejo et al., 1998). CD19+ CD27- IgD- cells, known as double-negative B cells, have also shown age-related increase in healthy subjects (Colonna-Romano et al., 2009). However, in the present study, only the frequency of senescent CD8+ cells revealed age-related increase with statistical significance in HC group. It could be partly explained that such phenotypic changes are much more frequently and dramatically found in the CD8+ T cells than in the CD4+ T cells (Fagnoni et al., 1996; Vallejo et al., 1998). Moreover, age-related loss of B cell function is a result of weakened interactions among immune cells, especially between B cells and senescent CD4+ T cells (Lazuardi et al., 2005). Due to present in proliferating and quiescent cells (Dimri et al.,1995). It is now widely used as a biomarker of cellular senescence in culture and in vivo (Dimri et al., 1995).
Cytochemical assay using the chromogenic substrate 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (X-gal) is the first used method of detection and is popularly used until now, but has limitations such as low sensitivity. In this study, using fluorescence-based assay coupled to flow cytometry, the SA-β-Gal activity could be detected more sensitively and quantitatively (Debacq-Chainiaux et al., 2009). The result confirmed the
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increased percentage of senescent CD8+ T cells in active and inactive BD compared to controls.
In the pathogenesis of BD, T cell response is accepted as more important than B cell response. For example, pathogenesis of BD is related to with increased Th1 cytokines such as IL-2 and IFNγ (Ben Ahmed et al., 2004). Recently, association with IL-17 has been also reported (Na et al., 2013; Ekinci et al., 2010). Moreover, the association of human leukocyte antigen (HLA)-B51 with BD implicate a role for class I molecules with the involvement of CD8+ T cells in disease development (Yu et al., 2004; Yasuoka et al., 2004). The results of this study were coinciding in that the frequency of senescent B cells did not show any trend or statistical significance. The frequency of senescent CD4+
T cells had a tendency to increase along the presence and activity of BD, but without significance. It suggests that the senescence of CD4+ T cell may be less related to BD in the pathogenesis of BD.
The study result shows that senescence of CD8+ T cells is associated with the immune dysfunction of BD. Considering the proportions of CD3+ CD8+ cells in the PBMCs were not significantly different between BD and controls (Bank et al., 2003), only the percentage of the senescent subpopulation has changed. The result does not explain whether the senescence is the cause or the consequence of the chronic inflammation in BD. Assuming the senescence of CD8+ T cells as the cause of the inflammation, tissue inflammation may result from proinflammatory cytokines and cytotoxic molecules released by senescent T cells. The loss of CD28 in T cells is associated with an increased production of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and IFNγ, and increased cytotoxicity, via expression of perforin and granzyme B and a propensity to migrate into tissues (Almanzar et al., 2004). However, there is a tendency for a higher incidence of BD in the third decade of life, which cannot be explained if the senescence is associated with the cause of inflammation. Therefore, it would be more natural to explain the senescence of CD8+ T cells as the result. There are several possible explanations. First, it could be the result of viral infection. Viruses such as
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herpes simplex I virus (HSV-I) and cytomegalovirus (CMV) are of the most explored viral etiology (Galeone et al., 2012). HSV-I DNA was detected more often in the saliva and the peripheral blood leukocytes of the patients with BD compared to those of HCs (Lee et al., 1996; Studd et al., 1991), suggesting HSV-I infection as one of the etiologic or triggering factors in BD. In addition, CMV infection is reported in relation with clinical flares of BD (Martin et al., 2010), and its DNA was detected more frequently in the blood of BD group compared to that of controls, but without statistical significance (Irschick et al., 2011). CMV infection is well known to induce the senescence of CD8+ T cells (Pawelec, 2014). Chronic antigenic load by HSV infected cells might as well result in the senescence of CD8+ T cells, as shown in HSV-infected mice (Lang et al., 2009).
Second, the immune activation by HSP may lead to the senescence of T cells. HSP is a immunoreactive protein produced in response to stressful conditions. Microbial 65-kDa HSP shows significant homology with the human 60-kDa mitochondrial HSP and shares antigenicity with an oral mucosal antigen (Lehner et al., 1991). T cells specific to HSP are known to mediate tissue damage by both the production of Th1 cytokines, such as IFN-r and TNF-a, and activation of cell-mediated cytotoxicity (Benagiano et al., 2005). The increased cell-mediated cytotoxicity may result in the senescence of CD8+ T cells.
Third, chronic inflammation caused by T cells in the involved tissues might bring about the immunosenescence. Infiltrates consisting of self-reactive lymphocytes and neutrophils are associated with skin lesion of BD (Mochizuki et al., 1997). Both the CD4+ and CD8+ T cells are known to regulate neutrophilc inflammation in autoinflammatory diseases (Keller et al., 2005). Chronic activation of T cells in the site of inflammation might be the cause of the senescence of T cells.
Meanwhile, accelerated immunosenescence of T cells is well known to occur in RA patients. Accumulation of DNA and impaired DNA repair mechanisms in naïve T cells facilitate their differentiation towards the senescent state and compensatory expansion of the senescent T cells in the peripheral blood, finally resulting in synovial inflammation by synovial senescent T cells (Chalan et al., 2015). This can explain why the incidence
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of RA increases with age. Other types of senescence cells related to BD needs to be further investigated.
The limitation of this study is that it only revealed phenotypic features of senescent immune cells. To further confirm the role of immunosenescence in pathogenesis of BD, functional studies are necessary. The effect of cytotoxic molecules and proinflammatory cytokines released by senescent T cells on inflammation of BD needs to be investigated.
Perforin, granzyme, TNFα, and IFNγ may be candidates, as they are known to be associated with inflammation in BD. (Accardo-Palumbo et al., 2010; Ahn et al., 2005). In addition, investigating the proneness to immunosenescence in patients with BD would help elucidate the role of immunosenescence. Oxidizing or DNA damaging agents can be used to induce senescence (Chrétien et al., 2008).
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CONCLUSION
In this study, CD3+ CD8+ CD27- CD28- cells, or senescent CD8+ T cells, were increased in the peripheral blood of patients with BD. It had a tendency to be more increased in active BD compared to inactive BD. Functional study or senescence-inducing study are necessary to further elucidate the role of immunosenescence in the pathogenesis of BD.
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높았다. 정상대조군에서 이미 알려진 바와 같이 CD3+ CD8+ CD27- CD28- 세포의 빈도가 연령에 따라 증가하는 경향을 보였다. 각 군에서 CD3+ CD4+
CD27- CD28- 세포 나 CD19+ CD27- IgD- 세포의 빈도는 통계적으로 유의 한 차이를 보이지 않았다. 베체트병 활성도와 관련한 C 반응성 단백(C reactive protein) 및 적혈구 침강 속도(erythrocyte sedimentation rate)와 면역 노화 세포의 빈 도 간 유의한 관련성은 보이지 않았다. 또, 스테로이드 치료 유무 및 다른 장기 침범 유무 역시 면역 노화 세포의 빈도에 영향을 미치지 않았다. 활동성 및 비활 동성 베체트변 환자군의 CD8+ 세포에서 노화 관련 베타갈락토시다아제의 활성 이 대조군과 비교했을 때 통계적으로 유의하게 증가되어 있는 것을 확인하였다.
결론: 본 연구 결과 대조군과 비교했을 때 베체트병 환자의 말초혈액에서 노화 CD8+ T 세포의 빈도가 증가되어 있음을 확인하였다. 더 나아가 베체트병 환자의 노화 CD8+ T 세포의 기능 및 노화 유도에 대한 반응에 관한 연구가 필요할 것이 다.
핵심어: 베체트병, 면역노화, CD8+ T 세포, 유동세포계수법, 노화 관련 베타갈 락토시다아제