**2. The expression of CD69 and CD3+ T-lymphocytes in the diagnosis or therapy of AF**

Recently, the link between inflammation and AF appeals increasing attention. Many studies have demonstrated serum or plasma inflammation biomarkers have a link with the development of AF, which supported chronic inflammatory responses might partici‐ pate in the development of AF (Chung MK et al.,2006;Hemandez Madrid A et al.,2007;Li J et al.,2010;Psychari SN et al.,2005). Moreover, some other studies reported that activat‐ ed T-lymphocytes and macrophages infiltrated the endomyocardial of patients with AF, which supported the activation of local T-lymphocytes played a role in the pathogenesis of AF (Chen MC et al., 2008;Nakamura Y et al.,2003;Yamashita T et al.,2010). However, up to now, there are no evidences supporting the link between the activation of periph‐ eral blood T-lymphocytes and AF. As is well-known, CD69 and HLA-DR are markers of activated T-lymphocytes. In other words, they are both specifically expressed on the sur‐ face of activated T-lymphocytes.[11, 12](Caruso A et al.,1997;Reddy M et al.,2004) CD69 is known as an early activation marker of T-lymphocytes [13](Sancho D et al.,2005) and HLA-DR is known as a late activation marker of T-lymphocytes.[14] (Geraldes L et al., 2010)They both play a role in the specific immune response to inflammation. [15, 16, 14, 17-19, 13, 20] (Sancho D et al.,2005;Geraldes L et al.,2010;Afeltra A et al.,1993;Ferenczi K et al.,2000;McDonald GB et al.,1987;Miki-Hosokawa T et al.,2009;Oczenski W et al., 2003;Vance BA et al.,2005) To our knowledge, there is little information available about the expression of CD69 and HLA-DR on peripheral blood CD3+ T-lymphocytes in pa‐ tients with AF.Thus we designed an experiment to investigate the relationship between the activation of peripheral blood CD3+ T-lymphocytes and AF by flow cytometric analy‐ sis that we aimed to provide more evidences to support this phenomenon (Liu L, et al., 2012;53(4):221-4.)

**Figure 4.** Percentage of CD3+CD69+ T-lymphocytes (A) and CD3+HLA-DR+ T-lymphocytes (B) were shown, respectively.

Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial Fibrillation

http://dx.doi.org/10.5772/54167

51

During three-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group). The results demonstrated that the mean values of CD69 and HLA-DR in sinus group at follow-up (1.17%±0.38, 28.71%±8.70) were all signifi‐ cantly down-regulated compared with before cardioversion (1.45%±0.44, 34.71%±9.75; all *p*<0.05). However, there were no statistically significant differences between recurrence group at follow-up (1.57%±0.39, 36.40%±9.32) and before cardioversion (1.60%±0.35, 37.72%

**Figure 5.** The mean values of CD3+CD69+ T-lymphocytes (A) and CD3+HLA-DR+ T-lymphocytes (B) in the sinus group

±11.11; *p*=0.721, *p*=0.544. Figure.5)

and recurrence group at follow-up and before cardioversion.

Fifty paroxysmal AF patients and fifty-six persistent AF patients, underwent successful elec‐ trical cardioversion, were enrolled in this study. Percentage of CD69 and Human leukocyte antigen DR (HLA-DR) positive peripheral blood CD3+ T-lymphocyte, which indicates Tlymphocyte activation, were examined by flow cytometric analysis in the patients and fiftyone healthy controls. Patients groups had higher levels of CD69 and HLA-DR than healthy controls. During three-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group).

The results showed that Patients with AF groups had higher levels of CD69 and HLA-DR than healthy controls. The mean value of CD69 was significantly up-regulated in patients with paroxysmal (1.48%±0.42) and persistent AF (1.55%±0.38) compared with healthy indi‐ viduals (1.07%±0.37; all p<0.001). The mean value of HLA-DR was also significantly upregulated in patients with paroxysmal (35.16%±10.89) and persistent AF (37.73%±10.78) compared with healthy individuals (26.6%±8.41; all p<0.001. Figure 4)

Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial Fibrillation http://dx.doi.org/10.5772/54167 51

**2. The expression of CD69 and CD3+ T-lymphocytes in the diagnosis or**

Recently, the link between inflammation and AF appeals increasing attention. Many studies have demonstrated serum or plasma inflammation biomarkers have a link with the development of AF, which supported chronic inflammatory responses might partici‐ pate in the development of AF (Chung MK et al.,2006;Hemandez Madrid A et al.,2007;Li J et al.,2010;Psychari SN et al.,2005). Moreover, some other studies reported that activat‐ ed T-lymphocytes and macrophages infiltrated the endomyocardial of patients with AF, which supported the activation of local T-lymphocytes played a role in the pathogenesis of AF (Chen MC et al., 2008;Nakamura Y et al.,2003;Yamashita T et al.,2010). However, up to now, there are no evidences supporting the link between the activation of periph‐ eral blood T-lymphocytes and AF. As is well-known, CD69 and HLA-DR are markers of activated T-lymphocytes. In other words, they are both specifically expressed on the sur‐ face of activated T-lymphocytes.[11, 12](Caruso A et al.,1997;Reddy M et al.,2004) CD69 is known as an early activation marker of T-lymphocytes [13](Sancho D et al.,2005) and HLA-DR is known as a late activation marker of T-lymphocytes.[14] (Geraldes L et al., 2010)They both play a role in the specific immune response to inflammation. [15, 16,

17-19, 13, 20] (Sancho D et al.,2005;Geraldes L et al.,2010;Afeltra A et al.,1993;Ferenczi K et al.,2000;McDonald GB et al.,1987;Miki-Hosokawa T et al.,2009;Oczenski W et al., 2003;Vance BA et al.,2005) To our knowledge, there is little information available about the expression of CD69 and HLA-DR on peripheral blood CD3+ T-lymphocytes in pa‐ tients with AF.Thus we designed an experiment to investigate the relationship between the activation of peripheral blood CD3+ T-lymphocytes and AF by flow cytometric analy‐ sis that we aimed to provide more evidences to support this phenomenon (Liu L, et al.,

Fifty paroxysmal AF patients and fifty-six persistent AF patients, underwent successful elec‐ trical cardioversion, were enrolled in this study. Percentage of CD69 and Human leukocyte

lymphocyte activation, were examined by flow cytometric analysis in the patients and fiftyone healthy controls. Patients groups had higher levels of CD69 and HLA-DR than healthy controls. During three-month follow-up, 37 patients had recurrence of AF (recurrence

The results showed that Patients with AF groups had higher levels of CD69 and HLA-DR than healthy controls. The mean value of CD69 was significantly up-regulated in patients with paroxysmal (1.48%±0.42) and persistent AF (1.55%±0.38) compared with healthy indi‐ viduals (1.07%±0.37; all p<0.001). The mean value of HLA-DR was also significantly upregulated in patients with paroxysmal (35.16%±10.89) and persistent AF (37.73%±10.78)

antigen DR (HLA-DR) positive peripheral blood CD3+

group) and 50 patients remained in sinus (sinus group).

compared with healthy individuals (26.6%±8.41; all p<0.001. Figure 4)

14,

T-lymphocyte, which indicates T-

**therapy of AF**

50 Atrial Fibrillation - Mechanisms and Treatment

2012;53(4):221-4.)

**Figure 4.** Percentage of CD3+CD69+ T-lymphocytes (A) and CD3+HLA-DR+ T-lymphocytes (B) were shown, respectively.

During three-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group). The results demonstrated that the mean values of CD69 and HLA-DR in sinus group at follow-up (1.17%±0.38, 28.71%±8.70) were all signifi‐ cantly down-regulated compared with before cardioversion (1.45%±0.44, 34.71%±9.75; all *p*<0.05). However, there were no statistically significant differences between recurrence group at follow-up (1.57%±0.39, 36.40%±9.32) and before cardioversion (1.60%±0.35, 37.72% ±11.11; *p*=0.721, *p*=0.544. Figure.5)

**Figure 5.** The mean values of CD3+CD69+ T-lymphocytes (A) and CD3+HLA-DR+ T-lymphocytes (B) in the sinus group and recurrence group at follow-up and before cardioversion.

Before we received the result,we conduct the baseline clinical Caracteristics of the Studied Population to ensure facticity of the results (Table 2).

et al.,2002) HLA-DR belongs to the MHC class II system which is known as a late activation marker of lymphocytes. It is required for antigen presentation and activation of helper Tlymphocytes. (Geraldes L et al.,2010;Oczenski W et al.,2003;Bobryshev YV et al.,2011)They respectively expressed in some inflammatory infiltrates and played important roles in the pathogenesis of some inflammatory diseases such as allergic airway inflammation, (Miki-Hosokawa T et al.,2009;Wang HY et al.,2006) rheumatoid arthritis, (Afeltra A et al.,1993) psoriasis vulgaris lesional skin active inflammatory bowel disease (McDonald GB et al.,

Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial Fibrillation

T-lymphocytes in AF patients.

role in the pathogenesis of AF. A few studies reported that some inflammatory lympho‐

cardial in patients with AF, which supported the local activation of T-lymphocytes played a role in the pathogenesis of AF.(Chen MC et al.,2008;Nakamura Y et al.,2003;Yamashita T et al.,2010) On the other hand, a lot of studies reported that serum or plasma inflammation bio‐ markers, such as C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-10, tumor necrosis fac‐ tor (TNF)-α, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF), et al., increased in AF patients, which supported T-cell-associated chronic inflam‐ matory responses might involve in the pathogenesis of AF. (Chung MK et al.,2001;Heman‐ dez Madrid A et al.,2007;Li J et al.,2010;Psychari SN et al.,2005) In our study, we provided another evidence to support that there is an activation of peripheral blood CD3+ T-lympho‐ cytes in AF patients, demonstrated by the upregulation of CD69 and HLA-DR by flow cyto‐

Generally, our study here further emphasize that activation of T cells is involved in AF. As we all know, T-lymphocytes are the main cells participate in cell-mediated immunity, which is one of the primary ways of human immune. It can be suppressed by many immunosup‐ pressants such as Cyclosporine, Rapamycin, et al. If the activation of peripheral blood Tlymphocytes does participate in the pathogenesis of AF, maybe we can prevent recurrence

ipates in the progression of AF, we speculate the following three possibilities. Firstly, the ac‐

MCP-1, which could affect the contractility and electrical stability of myocytes inhomogene‐ ously and induce fibroblast activation leading to deposits of extracellular matrix fibrosis. (Ramos-Mondragon R et al.,2008) Secondly, activation of peripheral blood CD3+ T-lympho‐ cytes might promote the local immunologic inflammatory responses in the endomyocardial, and promote infiltrate of inflammatory lymphomononuclear in the endomyocardial in pa‐ tients with AF. Thirdly, the activation of peripheral blood CD3+ T-lymphocytes could acti‐ vate the calcineurin-nuclear factor, which involve in the T-lymphocytes signal transduction

It is important to note that there are several limitations that need to be addressed regarding this study. Firstly, The results cannot be taken as evidence to support that CD69 and HLA-

T-lymphocytes might cause the upregulation of IL-6 and

of AF through suppressing the activation of peripheral blood T-lymphocytes.

As for the underlying mechanisms of activated peripheral blood CD3+

T cells, and CD68+

T-lymphocytes implied there is an acti‐

http://dx.doi.org/10.5772/54167

53

T-lymphocytes maybe plays an important

macrophages, infiltrated in the endomyo‐

T-lymphocytes partic‐

1987). So the increase of CD69 and HLA-DR on CD3+

In fact, the activation of peripheral blood CD3+

cells, CD3+

vation of peripheral blood CD3+

cytes, such as CD45+

metric analysis.

tivation of peripheral blood CD3+

pathway. (Lin CC et al., 2004)


**Table 2.** Caracteristics of the Studied Population

ACEI indicates angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor block‐ ing agents; CCBs, calcium channel blockers; WBC, white blood cells; CRP, C-reactive pro‐ tein; *p*, probability of significance (difference among three groups); and \*, *p*<0.05 persistent AF vs paroxysmal AF.

In this study, we found that the respective expression of CD69 and HLA-DR on peripheral blood CD3+ T-lymphocytes in AF patients was significantly higher than control group, which might suggest that high expression of CD69 and HLA-DR was associated with AF. In the subsequent follow-up, we further found that the expression of CD69 and HLA-DR in si‐ nus group at follow-up was significantly down-regulated compared with before cardiover‐ sion. However, the expression of CD69 and HLA-DR in recurrence group at follow-up was not significantly down-regulated. It might further support that the CD69 and HLA-DR lev‐ els were related with the state of AF.

As demonstrated by the present study, there was a link between high expression of CD69 and HLA-DR and AF. CD69, known as an early activation marker of lymphocytes, is a type II transmembrane glucoprotein and may enhance activation and proliferation/differentiation of T-lymphocytes. (Sancho D et al. 2005;Vance BA et al.,2005;Beeler A et al.,2008;Creeners P et al.,2002) HLA-DR belongs to the MHC class II system which is known as a late activation marker of lymphocytes. It is required for antigen presentation and activation of helper Tlymphocytes. (Geraldes L et al.,2010;Oczenski W et al.,2003;Bobryshev YV et al.,2011)They respectively expressed in some inflammatory infiltrates and played important roles in the pathogenesis of some inflammatory diseases such as allergic airway inflammation, (Miki-Hosokawa T et al.,2009;Wang HY et al.,2006) rheumatoid arthritis, (Afeltra A et al.,1993) psoriasis vulgaris lesional skin active inflammatory bowel disease (McDonald GB et al., 1987). So the increase of CD69 and HLA-DR on CD3+ T-lymphocytes implied there is an acti‐ vation of peripheral blood CD3+ T-lymphocytes in AF patients.

Before we received the result,we conduct the baseline clinical Caracteristics of the Studied

Age(yrs) 64.4±8.5 64.3±9.5 67.2±9.7 0.176 Men 31 (61%) 32 (64%) 34 (61%) 0.927 Hypertension 13 (25%) 20 (40%) 25 (45%) 0.106 Hyperlipidemia 11 (22%) 8 (16%) 7 (13%) 0.448 Diabetes 2 (4%) 3 (6%) 5 (9%) 0.566

ACE-I/ARB 10 (20%) 11 (22%) 21 (38%) 0.074 Statins 8 (16%) 7 (14%) 6 (11%) 0.743 β-blockers 8 (16%) 26 (52%) 30 (54%) <0.001 CCBs 5 (10%) 12 (24%) 7 (13%) 0.108

WBC count(per uL) 6349±1891 6768±1859 6375±1663 0.819 Lymphocytes(%) 30.9±3.8 32.5±4.3 31.5±4.1 0.147 Monocytes(%) 5.5±1.2 5.8±1.1 5.4±1.1 0.123 CRP(mg/dl) 0.24±0.12 0.48±0.25 0.60±0.22\* <0.001

ACEI indicates angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor block‐ ing agents; CCBs, calcium channel blockers; WBC, white blood cells; CRP, C-reactive pro‐ tein; *p*, probability of significance (difference among three groups); and \*, *p*<0.05 persistent

In this study, we found that the respective expression of CD69 and HLA-DR on peripheral

which might suggest that high expression of CD69 and HLA-DR was associated with AF. In the subsequent follow-up, we further found that the expression of CD69 and HLA-DR in si‐ nus group at follow-up was significantly down-regulated compared with before cardiover‐ sion. However, the expression of CD69 and HLA-DR in recurrence group at follow-up was not significantly down-regulated. It might further support that the CD69 and HLA-DR lev‐

As demonstrated by the present study, there was a link between high expression of CD69 and HLA-DR and AF. CD69, known as an early activation marker of lymphocytes, is a type II transmembrane glucoprotein and may enhance activation and proliferation/differentiation of T-lymphocytes. (Sancho D et al. 2005;Vance BA et al.,2005;Beeler A et al.,2008;Creeners P

T-lymphocytes in AF patients was significantly higher than control group,

**Paroxysmal AF (n=50) Persistent AF (n=56)** *p*

Population to ensure facticity of the results (Table 2).

52 Atrial Fibrillation - Mechanisms and Treatment

Drugs:

**Table 2.** Caracteristics of the Studied Population

els were related with the state of AF.

AF vs paroxysmal AF.

blood CD3+

**Control (n=51)**

> In fact, the activation of peripheral blood CD3+ T-lymphocytes maybe plays an important role in the pathogenesis of AF. A few studies reported that some inflammatory lympho‐ cytes, such as CD45+ cells, CD3+ T cells, and CD68+ macrophages, infiltrated in the endomyo‐ cardial in patients with AF, which supported the local activation of T-lymphocytes played a role in the pathogenesis of AF.(Chen MC et al.,2008;Nakamura Y et al.,2003;Yamashita T et al.,2010) On the other hand, a lot of studies reported that serum or plasma inflammation bio‐ markers, such as C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-10, tumor necrosis fac‐ tor (TNF)-α, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF), et al., increased in AF patients, which supported T-cell-associated chronic inflam‐ matory responses might involve in the pathogenesis of AF. (Chung MK et al.,2001;Heman‐ dez Madrid A et al.,2007;Li J et al.,2010;Psychari SN et al.,2005) In our study, we provided another evidence to support that there is an activation of peripheral blood CD3+ T-lympho‐ cytes in AF patients, demonstrated by the upregulation of CD69 and HLA-DR by flow cyto‐ metric analysis.

> Generally, our study here further emphasize that activation of T cells is involved in AF. As we all know, T-lymphocytes are the main cells participate in cell-mediated immunity, which is one of the primary ways of human immune. It can be suppressed by many immunosup‐ pressants such as Cyclosporine, Rapamycin, et al. If the activation of peripheral blood Tlymphocytes does participate in the pathogenesis of AF, maybe we can prevent recurrence of AF through suppressing the activation of peripheral blood T-lymphocytes.

> As for the underlying mechanisms of activated peripheral blood CD3+ T-lymphocytes partic‐ ipates in the progression of AF, we speculate the following three possibilities. Firstly, the ac‐ tivation of peripheral blood CD3+ T-lymphocytes might cause the upregulation of IL-6 and MCP-1, which could affect the contractility and electrical stability of myocytes inhomogene‐ ously and induce fibroblast activation leading to deposits of extracellular matrix fibrosis. (Ramos-Mondragon R et al.,2008) Secondly, activation of peripheral blood CD3+ T-lympho‐ cytes might promote the local immunologic inflammatory responses in the endomyocardial, and promote infiltrate of inflammatory lymphomononuclear in the endomyocardial in pa‐ tients with AF. Thirdly, the activation of peripheral blood CD3+ T-lymphocytes could acti‐ vate the calcineurin-nuclear factor, which involve in the T-lymphocytes signal transduction pathway. (Lin CC et al., 2004)

> It is important to note that there are several limitations that need to be addressed regarding this study. Firstly, The results cannot be taken as evidence to support that CD69 and HLA-

DR play a role in the pathogenesis of AF, it only indicate the possible association of CD69 and HLA-DR with AF. Secondly, how CD69 and HLA-DR contribute to the pathogenesis of AF and what is the underlying mechanism need to be further investigated. Thirdly, we did not study the influence of other immune activation-associated molecules (CD25, CD71, and CD122, et al) and co-stimulatory molecules (CD28, CTLA-4, CD80, CD86, et al) during the progression of AF.

plement system during and after cardiopulmonary bypass surgery: postsurgery activation involves C-reactive protein and is associated with postoperative arrhyth‐

http://dx.doi.org/10.5772/54167

55

Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial Fibrillation

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[9] Chen MC, Chang JP, Liu WH, Yang CH, Chen YL, Tsai TH, Wang YH, Pan KL. In‐ creased inflammatory cell infiltration in the atrial myocardium of patients with atrial

[10] Nakamura Y, Nakamura K, Fukushima-Kusano K, Ohta K, Matsubara H, Hamuro T, Yutani C, Ohe T.Tissue factor expression in atrial endothelia associated with non‐ valvular atrial fibrillation: Possible involvement in intracardiac thrombogenesis.

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