Immunological Investigations, 2014; 43(5): 463–478 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.886260
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of peripheral blood mononuclear cells in patients with Behc¸et’s disease and association with HLA-B51 expression K. Sakly,1 R. Lahmar,1 F. Nefzi,2 S. Hammami,3 O. Harzallah,3 N. Sakly,1,4 W. Sakly,1 M. Hassine,5 S. Mahjoub,3 I. Ghedira,1 and S. Feki6 1
Research Unit 03/UR/07 ‘‘Autoimmunity and Allergy’’, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia, 2 Laboratory of Transmissible Diseases and Biological Active Substances, LR99ES27, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia, 3 Department of Internal Medicine, F.B. University Hospital, Monastir, Tunisia, 4 Laboratory of Microbiology, Unity of Immunology, F.B. University Hospital, Monastir, Tunisia, 5 Laboratory of Hematology, F.B. University Hospital, Monastir, Tunisia, 6 Department of Clinical Biology B, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behc¸et’s disease (BD) and to assess associations between the expression of HLA-B51 antigen and that of other cell markers. Thirty healthy volunteer blood donors and forty patients with BD were enrolled into this study. Phenotyping was performed using two color flow cytometry. HLA-B51 typing was performed using the complement dependent microlymphocytotoxicity assay. Unlike controls, patients with BD presented a modified immunophenotypic profile of lymphocytes. Compared to those in the remission phase, patients with active BD showed an increased mean of MFI ratio of CD56 on CD16+CD56+ cells (32.47 ± 14.26 versus 23.87 ± 10.3; p = 0.032), increased absolute numbers of CD4 CD8bright and CD4+CD8+ cells (657.1 ± 463.6 cells/mL versus 319.24 ± 116.4 cells/ mL; p = 0.017 and 40.77 ± 36.41 cells/mL versus 10.77 ± 9.78 cells/mL; p50.0001, respectively) and an elevated mean of MFI ratio of CD19 on B cells (252.3 ± 56.7 versus 205.67 ± 32.3; p = 0.021). However, expression of HLA-B51 was not associated with any specific immunophenotypic profile. In conclusion, abnormal immunophenotypic profile of peripheral lymphocytes was found in patients with BD, especially in active phase, reflecting an immune dysregulation. Moreover, HLA-B51 expression was not found to be related to the expression of other cell markers. Keywords Behc¸et’s syndrome, blood cells, CD antigens, histocompatibility antigens
INTRODUCTION Behc¸et’s disease (BD) is a multisystemic inflammatory disorder characterized by recurrent oral and genital ulcerations as well as ocular, arthritic, vascular, Correspondence: Nabil Sakly, Research Unit 03/UR/07 ‘‘Autoimmunity and Allergy’’, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia. Tel: +216 98 632 477. E-mail: [email protected]
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
464
K. Sakly et al.
cutaneous and neurological involvement, and generally presents with remissions and exacerbations (Mendes et al., 2009; Yurdakul & Yazici, 2008). The etiology and the pathogenesis of BD still remains poorly understood, although several clues have been proposed, including the interaction between environmental and genetic factors, leading to an inappropriate immunoinflammatory response (Pay et al., 2007; Sakane et al., 1999). Abnormalities of the immune system such as activated circulating T and B cells, NK and neutrophils responsible for endothelial damages have been recognized as important immunopathogenic cells in BD (Frassanito et al., 1999; Hegab & Al-Mutawa, 2000). The leukocytes immunophenotypic profile of patients with BD is characterized by multiple alterations mainly in those with active disease. A preferential skewing of T cells in patients with active BD towards an IFN-g and IL-2 producing Th1 phenotype has been previously demonstrated by intracellular cytokine staining (Pay et al., 2007). The levels of CD14+ monocytes, CD3+ TCRgd+, CD8+TCRgd+ cells, NK cells (CD56+CD16+) and activated regulatory T cells (CD4+CD25+, CD8+CD25+) were elevated in patients with BD, compared with healthy controls (Treusch et al., 2003). Dividing CD4+ T cells showed the phenotype of activated effector memory cells (Koarada et al., 2004). Interferon-gamma-producing CD8+ T cells were increased in patients with BD (Houman et al., 2004). An association between BD and HLA-B51 has been regarded as the strongest evidence of the involvement of genetic factors in its pathogenesis (Atalay et al., 2008; Direskeneli, 2001). Different hypotheses have been generated to explain the direct pathogenic role of HLA-B51 in Behc¸et’s disease, however, the exact mechanism of action of this molecule is still unknown (Gul, 2001). HLA-B51 molecules have multifunctional properties, including interaction with TCR and CD8 on T CD8+ cells and with KIRs expressed particularly by natural killer (NK) cells (Atalay et al., 2008). They may present endogenous peptides to CD8+ T cells, which could activate target cells and other cells of the immune system and may promote the expression of other surface molecules. In this regard, increased neutrophil function has been reported in both patients with BD and HLA-B51 positive healthy individuals (Takeno et al., 1995; Zierhut et al., 2003). On the other hand, Yasuoka et al. (2008) noted, exclusively in HLA-B51 positive patients with active BD, increased proportion of activated CD8+ and gd T cells. The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behc¸et’s disease and to assess possible associations between the expression of HLA-B51 antigen and that of other cell markers.
MATERIALS AND METHODS Samples Forty patients with BD, 24 males and 16 females (mean age: 41.3 ± 10.6 years; range: 11–60 years) were recruited from the Internal Medicine and Ophthalmology departments at Fattouma Bourguiba Hospital of Monastir (eastern coast of the Central region of Tunisia). The patients were diagnosed according to published BD criteria (ISGBD, 1990). Our patients were
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
classified on admission as patients with active BD (n = 15) and patients with inactive BD (n = 25). The mean duration of disease was 104 months (range: 12–252 months). All patients with active BD showed necessary active oral or genital ulcerations and one more sign of clinical activity: skin lesions (erythema nodosum, folliculitis), ocular lesions, joint symptoms, central nervous system involvement, or gastrointestinal inflammation. Inactive patients with BD were composed of asymptomatic patients (mean duration of inactive stage: 9 weeks). At the time of analysis, 36 patients were under treatments: Two patients were treated with colchicine and immunosuppressive drugs (prednisone or azathioprine), seven with colchicine and low doses of steroids and the remaining patients were treated with colchicine alone. One hundred and twenty-four healthy Tunisian subjects underwent HLA-B51 typing and constituted the control group for the HLA-B51 expression study (Sakly et al., 2009) and thirty healthy volunteer blood donors were recruited as controls for the flow cytometry tests. They were 23 males and 7 females, with a mean age of 30.6 ± 9.69 years (range: 18–62 years). At the time of blood sampling, none of the blood donors was receiving any medication or had an infection. The design of the study was approved by our Hospital Ethics Committee. Flow Cytometry Analysis Lymphocyte subsets were evaluated in whole fresh blood obtained from patients with BD and controls using different monoclonal antibodies: AntiCD3-PE, Anti-CD4-FITC, Anti-CD8-PE, Anti-CD16-FITC, Anti-CD56-PE, Anti-CD5-FITC, Anti-CD19-PE, Anti-CD14-FITC and Anti-CD45-FITC (Invitrogen, USA). One-color and two-color phenotypic characterizations of lymphocytes were performed. Venous blood samples were collected into EDTA containing vacuum tubes and processed on the same day. Briefly, blood samples (100 mL) were incubated for 20 min with the appropriate amounts of mAb. Red blood cells were then lysed (FACS lysing solution; Becton Dickinson, Stockholm, Sweden) and the white cells washed and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). The resulting data were analyzed with the commercial FlowJo software. Expression levels of different markers were investigated through the mean absolute number of positive cells and the mean MFI ratio of gated cells (the mean fluorescence intensity (MFI) of positive cells reported to the negative ones). HLA-B51 Typing Whole blood was collected into EDTA containing tubes. Peripheral blood mononuclear cells were isolated by density gradient centrifugation over FicollHypaque (lymphoprep, Axis-schield PoC AS, Oslo, Norway). The isolated cells were counted and adjusted to 106 cells/mL. Serological typing of HLA-B51 was performed immediately by the complement dependent microlymphocytotoxicity assay, using the HLA-B Locus Supplement Typing Tray (One Lambda, Canoga Park, CA, USA). The test was performed according to the manufacturer’s recommendations.
465
466
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Statistical Analysis All quantitative results are expressed as the mean ± standard deviation. Absolute numbers of cells were calculated by multiplying the cell percentages by the total lymphocyte numbers (per microliter). Comparisons between quantitative parameters were performed using the non parametric Mann– Whitney U-test. Comparison between frequencies was made by the 2 Pearson test. Odd ratios (OR) were calculated according to the Woolf ’s formula, and are given with 95% confidence interval [95% CI]. Statistical significance was considered when two-sided p values are under 0.05. Analyses were done with the aid of the SPSSÕ software.
RESULTS Clinical Features of Behc¸et’s Disease Patients Among the 40 patients with BD, 92.5% had oral ulcers, 82.5% had genital ulcers, 47.5% had skin lesions, 37.5% had arthritis, 22.5% had uveitis and 12.5% had thrombosis during the course of disease. Only a small proportion of patients had a history of neurological (15%) and/or gastrointestinal (15%) involvement. Only 17.5% of patients had a family history of BD. Clinical features of patients are reported in Table 1. Table 1. Characteristics of Behc¸et’s disease group.
Age (years) Sex (M:F) Disease duration (months) Organ involvement Oral ulcers Genital ulcers Arthritis Skin lesions Central nervous system disease Uveitis Gastrointestinal system lesions Thrombosis Joint symptoms Pathergy Test HLA-B51 Family history of BD
Active (n = 15)
Inactive (n = 25)
36.73 ± 8.9 9:6 92.06 ± 44.4 n (%) 14 (93.3) 12 (80) 4 (26.6) 7 (46.6) 2 (13.3) 6 (40) 1 (6.66) 4 (26.6) 10 (66.6) 6 (40) 5 (33.3) 3 (20)
44.04 ± 10.8 15:10 111.32 ± 77.9 n (%) 23 (92) 21 (84) 11 (44) 12 (48) 4 (16) 3 (12) 5 (20) 1 (4) 5 (20) 13 (52) 8 (61.5) 4 (16)
Flow Cytometry Analysis CD16 and CD56 Expression Absolute numbers of CD16+CD56+ and CD16 CD56+ cells were significantly increased in patients with BD compared to those of controls (313.67 ± 210.82 versus 245.69 ± 95.43 cells/mL [p = 0.006] and 525.44 ± 783.77 versus 256.50 ± 155.19 cells/mL [p = 0.019], respectively). similar results were found in patients with active BD compared to controls (421.55 ± 214.28 versus 245.69 ± 95.43 cells/mL [p = 0.002] and 925.92 ± 1255.9 cells/mL versus 256.50 ± 155.19 cells/mL [p = 0.005], respectively) (Table 2). No significant
245.69 ± 95.4 256.50 ± 155.1 54.43 ± 30.3 1406.18 ± 430.4 551.87 ± 248.4 779.83 ± 245.2 343.79 ± 141.8 14.85 ± 8.9 288.49 ± 212.2 249.04 ± 187.9 40.15 ± 32.9 371.9 ± 14.9
CD16+CD56+ CD16 CD56+ CD16+CD56 CD3+ CD4 CD8+ CD4+CD8 CD4 CD8bright CD4+CD8+ CD19+ CD19+CD5 CD19+CD5+ CD14+
421.55 ± 214.2c 925.92 ± 1255.9c 70.54 ± 51.7 1789.38 ± 1020.5d 904.98 ± 450d 1015.86 ± 476 657.1 ± 463.6d 40.77 ± 36.4c 206.074 ± 102.4 185.51 ± 118.4 20.99 ± 13.1d 456.69 ± 16.5
Active BD (n = 15) 283.46 ± 203.9 317.20 ± 162.3 61.14 ± 41.5 1426.77 ± 472.9 482.87 ± 216.2 642.36 ± 179.7 319.24 ± 116.4 10.77 ± 9.7 175.19 ± 79.4c 146.91 ± 65.8c 27.07 ± 20.2 473.29 ± 28.4
Inactive BD (n = 25) 313.67 ± 210.8 525.44 ± 783.7 64.36 ± 44.8 1537.57 ± 692.5 633.13 ± 339 674.9 ± 309.7 439.84 ± 283.4 19.72 ± 23.3 194.06 ± 111.5 167.46 ± 100.7 25.74 ± 18.6 459.4 ± 24.4
Total (n = 40)
0.006 0.019 NS NS NS NS NS 0.011 0.011 0.011 NSe NS
p Valuea
a
N.B. Results are shown as the mean absolute numbers of cells per mL ± SD, and Mann-Whitney U-test were used for the comparison of means. Comparison between all patients with BD and controls. b Comparison between patients with active BD and patients with inactive BD. c p 0.005 compared with controls d p50.05 compared with controls e Not significant.
Controls (n = 30)
Cell subsets
Patients with BD
NS NS NS NS 0.006 0.019 0.017 50.0001 NS NS NS NS
p Valueb
Table 2. Absolute numbers of lymphocytes subsets in peripheral blood from patients with BD, patients with active BD and patients with inactive BD and controls determined by flow cytometric analysis.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
467
468
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
difference was observed between patients with active disease and those with inactive disease for these cell subsets. With regards to expression levels of CD56 and CD16, the MFI ratio of CD56 of CD16+CD56+ cells was found to be significantly higher in patients with active BD (32.47 ± 14.26) than in those with inactive disease (23.87 ± 10.3; p = 0.032) and controls (22.29 ± 8.6; p = 0.018) (Figure 1). However, MFI ratio of CD16 of CD16+CD56 cells was higher in patients with inactive BD compared to that of controls (9.02 ± 2.7 versus 7.64 ± 2.2; p = 0.042) (Figure 2). In our study, we also noted that BD patients with oral ulcers showed increased absolute numbers of CD16+CD56+ cells compared to those without oral ulcers (297.73 ± 203.8 cells/mL versus 158.86 ± 15.5 cells/mL, p = 0.001) (Figure 3).
Figure 1. Comparison of the mean MFI ratios of CD56 on CD16+CD56+ and CD16 CD56+ cells from patients with active Behc¸et’s disease, those with inactive Behc¸et’s disease and controls. Comparison was performed using Mann-Whitney U-test. p50.05 was considered as significant. MFI: mean intensity fluorescence. ap = 0.018, compared with controls. bp = 0.032, compared with patients with active BD.
Figure 2. Comparison of mean MFI ratios of CD16 on CD16+CD56+ and CD16+CD56 cells in patients with active BD, patients with inactive BD and controls. Comparison of mean MFI ratios between different groups was performed using the Mann-Whitney U-test. p50.05 was considered as significant. MFI: mean intensity fluorescence. a p = 0.042, compared with controls.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
(A)
(B)
(C)
Figure 3. Associations of peripheral CD16+CD56+ NK cells and CD56 expression with clinical manifestations in Behcet’s disease. (A) Association of absolute numbers of CD16+CD56+ NK cells with oral ulcers (p = 0.001). (B) Association of CD56 expression on CD16+CD56+ with skin lesions (p = 0.05). (C) Association of CD56 expression on CD16+CD56+ with genital ulcers (p = 0.047).
469
470
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
In patients with BD, CD56 MFI ratio on CD16+CD56+ cells tended to be higher in patients with genital ulcers and with skin lesions than in those without genital ulcers (27.56 ± 12.78 versus 20.47 ± 4.53; p = 0.047) and without skin lesions (31.93 ± 15.95 versus 23.09 ± 7.21; p = 0.05) (Figure 3). CD3, CD4 and CD8 expression Absolute numbers of CD3+ cells were similar in patients with BD and controls. However, it is worthy to mention that CD3+ cells were significantly increased in patients with active BD compared to controls (1789.38 ± 1020.5 cells/mL versus 1406.18 ± 430.4 cells/mL; p = 0.032) (Table 2). Absolute numbers of CD4 CD8+ cells did not differ between patients with BD and controls. However, these absolute numbers were significantly increased in patients with active BD (904.98 ± 450.67 cells/mL) compared to those with inactive disease (482.87 cells/mL; p = 0.006) and controls (551.87 ± 248.49 cells/mL; p = 0.026). Similarly, absolute numbers of the CD4 CD8bright subset in patients with BD were comparable to those in controls. Yet, this cell subset was increased in patients with active BD (657.1 ± 463.61 cells/mL) compared to those with inactive BD (319.24 ± 116.49 cells/mL; p = 0.017) and controls (343.79 ± 141.85 cells/mL; p = 0.037) (Table 2). On the other hand, absolute numbers of CD4+CD8 cells were similar in patients with BD and controls. However, these cells were found to be significantly increased in patients with active BD compared to those with inactive disease (1015.86 ± 476.09 cells/mL versus 642.36 ± 179.77 cells/mL; p = 0.019) (Table 2). Among T cell subsets, interestingly, we detected a higher absolute number of double-positive CD4+CD8+ cells in patients with active BD (40.77 ± 36.41 cells/mL) compared to those with inactive disease (10.77 ± 9.78/mL; p50.0001) and controls (14.85 ± 8.91 cells/mL; p = 0.004) (Table 2). CD19 and CD5 expression Absolute numbers of CD19+ B cells were significantly decreased in patients with BD compared to controls (194.06 ± 111.5 cells/mL versus 288.49 ± 212.2 cells/mL; p = 0.011). This was also observed in patients with inactive BD compared to controls (175.19 ± 79.4 cells/mL versus 288.49 ± 212.2 cells/mL; p = 0.005) (Table 2). More precisely, this decrease interested the conventional CD19+CD5 B cells which were significantly lower in patients with BD compared to controls (167.46 ± 100.75 cells/mL versus 249.04 ± 187.98 cells/mL; p = 0.011) and especially in patients with inactive BD compared to controls (146.91 ± 65.8 cells/mL versus 249.04 ± 187.9 cells/mL; p = 0.005). In contrast, absolute numbers of CD19+CD5+ cells were significantly decreased in patients with active BD than in controls (20.99 ± 13.1 cells/ mL versus 40.15 ± 32.9 cells/mL; p = 0.045). No significant difference was observed between patients with active BD and those with inactive disease (Table 2). With regard to expression levels of CD19, MFI ratios of this marker for CD19+ and CD19+CD5 B cells were significantly lower in patients with BD than that found in controls (220.79 ± 46.6 versus 236.93 ± 52.6 [p = 0.043] and
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
216.2 ± 49.3 versus 235.45 ± 51.6 [p = 0.05], respectively) (Table 3) but they were significantly increased in patients with active BD compared to those with inactive disease (252.3 ± 56.7 versus 205.67 ± 32.3 [p = 0.021], and 238.78 ± 68.05 versus 204.46 ± 31.9 [p = 0.04], respectively). Similarly, expression levels of CD19 in CD19+CD5+ cells were higher in patients with active BD than in patients with inactive disease (262.036 ± 90.3 versus 220.07 ± 42.6 [p = 0.033]) (Table 3). Moreover, expression levels of CD5 on CD19+CD5+ B cells were significantly decreased in patients with BD compared to controls (7.46 ± 1.73 versus 8.38 ± 2.36; p = 0.032) and precisely in patients with inactive BD compared to controls (7.20 ± 1.34 versus 8.38 ± 2.36; p = 0.016) (Figure 4). CD14 expression The investigation of CD14+ cells showed no difference of absolute numbers between patients and controls or between patients with active or inactive disease (Table 2). HLA-B51 typing The HLA-B51 antigen was detected in 32.5% of patients with BD versus 16.1% in controls with OR = 2.50, 95% CI [1.10–5.66], p = 0.025. Table 3. Expression levels of CD19 on lymphocyte B subsets in patients with BD, patients with active BD, those with inactive disease and controls. Patients with BD CD19 expression (mean MFI ratio) CD19+ CD19+CD5+ CD19+CD5
Controls (n = 30) 236.93 ± 52.6 235.03 235.45 ± 51.6
Active (n = 15)
Inactive (n = 25)
252.3 ± 56.7 205.67 ± 32.3 262.03 ± 90.3 220.07 ± 42.6 238.78 ± 68.05 204.46 ± 31.9
Total (n = 40)
p Valuea
p Valueb
220.79 ± 46.6 237.52 ± 65.09 216.2 ± 49.3
0.043 NS 0.05
0.021 0.033 0.04
N.B. The Mann-Whitney U-test was used for the comparison of means. MFI: mean intensity fluorescence. a Comparison between all patients with BD and controls. b Comparison between patients with active BD and patients with inactive BD.
Figure 4. Mean MFI ratio of CD5 expression on CD19+CD5+ B cells according to Behc¸et’s disease activity. N.B. Mann-Whitney U-test was used for the comparison of means. MFI: mean intensity fluorescence ap = 0.016, compared with controls. bComparison between all patients with BD and controls (p = 0.032).
471
472
K. Sakly et al. Table 4. Absolute numbers and MFI ratios of lymphocyte subsets in peripheral blood from HLA-B51 negatif and HLA-B51 positive BD patients determined by flow cytometric analysis.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
HLA-B51 negative patients (n = 27) Cell subsets (Mean absolute numbers in cells/mL) 253.08 ± 213.56 CD16+CD56+ CD16+CD56 53.27 ± 33.08 408 ± 443.99 CD16 CD56+ 157.94 ± 76.91 CD3+ (103) 72.91 ± 35.29 CD4+CD8 CD4 CD8+ 70.41 ± 39.40 47.24 ± 32.27 CD4 CD8bright 23.16 ± 15.59 CD4 CD8dim 1.95 ± 1.90 CD4+CD8+ 189.90 ± 111.51 CD19+ CD19+CD5 164.06 ± 101.08 25.39 ± 19.38 CD19+CD5+ 46.21 ± 25.67 CD14+ CD56 expression (Mean MFI ratios) 27.71 ± 13.66 CD16+CD56+ 13.61 ± 3.27 CD16 CD56+ CD16 expression (Mean MFI ratios) 25.61 ± 16.24 CD16+CD56+ 8.34 ± 2.70 CD16+CD56 CD19 expression (Mean MFI ratios) 214.85 ± 53.81 CD19+ 211.99 ± 52.76 CD19+CD5 234.96 ± 72.92 CD19+CD5+ CD5 expression (Mean MFI ratios) 7.36 ± 1.66 CD19+CD5+
HLA-B51 positive patients (n = 13)
p Value
289.89 ± 177.48 61.88 ± 29.74 483.61 ± 509.38 142.86 ± 42.80 75.14 ± 22.17 66.61 ± 19.01 45.49 ± 17.15 21.11 ± 9.50 3.23 ± 3.98 205.70 ± 116.84 176.96 ± 104.60 26.69 ± 17.25 45.04 ± 21.51
NS NS NS NS NS NS NS NS NS NS NS NS NS
24.30 ± 7.48 13.35 ± 3.57
NS NS
31.56 ± 20.34 9.11 ± 3.62
NS NS
225.99 ± 36.17 227.98 ± 38.08 219.29 ± 35.56
NS NS NS
7.75 ± 1.99
NS
NS: not significant. Comparisons were made by the Mann-Whitney U-test.
Association Between the Expression of the HLA-B51 Molecule and the Immunophenotypic Profile of Leukocytes in Patients with BD For each studied marker, the mean absolute number of positive cells and the mean MFI ratio of gated cells are presented in Table 4 according to the status of the expression of the HLA-B51 molecule. The expression of the HLA-B51 molecule was not found to be associated with any specific immunophenotypic profile in patients with BD. The same observation was found between the status of the expression of the HLA-B51 molecule and disease activity (p40.05; data not shown).
DISCUSSION In the present study, we explored the immunophenotypic profile of peripheral mononuclear cell subclasses in Tunisian patients with Behc¸et’s disease and assessed the possible relation between the expression of HLA-B51 antigen and the expression of other cell markers. Our study showed increased absolute numbers of CD16+CD56+ and CD16 CD56+ cells in patients with BD compared to controls. Abnormalities
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
in the number and function of circulating NK cells have been shown in several autoimmune and inflammatory diseases, such as systemic lupus erythematosus (Cameron et al., 2003; Green et al., 2005; Park et al., 2009), rheumatoid arthritis (Aramaki et al., 2009), psoriasis (Cameron et al., 2003) and Hashimoto’s thyroiditis (Guo et al., 2013). Similarly, abnormalities in circulating NK cells have been reported in patients with BD. An elevated number of NK cells has been found in patients with inactive stage of BD compared to controls (Deniz et al., 2000; Onder et al., 1994). However, other studies reported no quantitative or functional significant differences in the NK cells between patients with BD and controls (Eksioglu-Demiralp et al., 1999a; Gul, 2005; Park et al., 2009). The discrepancies between different studies could be related to the series (number of patients, clinical status, disease activity, treatment modalities) and/or to the strategy used to identify the NK cell subset (number of monoclonals used in cytometry). With regard to disease activity, our study showed that patients with active BD had, in comparison to controls, increased absolute numbers of the above cited two cell subsets (CD16 CD56+ and CD16+CD56+). Similarly, increased percentages of CD16+CD56+ NK cells in patients with clinically active BD have been previously reported (Kaneko et al., 1985; Suzuki et al., 1992). Moreover, Yamaguchi et al. (2010) reported markedly increased proportion of peripheral blood CD69+ NK cells in patients with active BD. They also noted a shift to NK2 phenotype in patients with inactive disease, leading them to suggest a role of NK cells in modulating disease flare/remission. The investigation of CD56 and CD16 expression levels revealed an increase of CD56 expression in CD16+CD56+ NK cells in patients with active BD compared to those with inactive disease and controls, suggesting that there could be a shift to the CD16+CD56bright phenotype in active BD. Knowing that CD56bright NK cells are characterized by enhanced cytokine production, mainly IFNg, TNF-b, IL-10, IL-13 and GM-CSF, this could explain the findings previously reported by Hamzaoui et al. (2002), showing an increase in IL-10 and IFN-g in patients with active BD compared to those with inactive disease. It was also suggested that interferon-g may contribute to the local inflammatory response in BD (Mendoza-Pinto et al., 2010). On the other hand, our study showed that CD16+CD56+ cells expressing high levels of CD56 are associated with the presence of genital ulcers and skin lesions. The analysis of T cell subsets revealed an increased CD4+CD8 cells in patients with active BD compared to those with inactive disease. Similar findings were reported by Sugi-Ikai et al. (1998), who found higher absolute numbers of CD4+ cells in peripheral blood of patients with active BD compared to controls. Thus, CD4+ cells may be involved in the active phase of BD. This suggestion could be supported by the work of Frassanito et al., who analyzed intracytoplasmic cytokine expression using flow cytometry and revealed that IL-2 and IFN-g producing CD4+ cells was increased in patients with active BD (Frassanito et al., 1999; Sugi-Ikai et al., 1998) point to a strong polarized Th1 immune response in BD (Koarada et al., 2004). We found increased absolute numbers of CD4 CD8+ cells (mainly CD4 CD8bright cells) in patients with active BD compared to those with inactive disease and controls. These findings are consistent with those reported by Ahn et al. (2005), who demonstrated that activated effector
473
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
474
K. Sakly et al.
CD8bright T cells are expanded in patients with active Behc¸et’s uveitis compared to inactive disease and controls. Furthermore, the authors showed that the cytolytic activities of this subset against HUVECs were increased in active Behc¸et’s uveitis. One of the most striking findings, in our study, was the significant increase in the absolute number of peripheral double positive CD4+CD8+ cells in patients with active BD compared to those with inactive disease and controls. The origin of peripheral double positive T cells remains controversial, however, it is well documented that these cells are differentiated into functional effector/memory T cells (Nascimbeni et al., 2004; Wang et al., 2008) and being high producers of cytokines with immunomodulatory potential (Parel et al., 2007); It has been shown that these cells were expanded in several pathologic conditions such as systemic sclerosis (Parel et al., 2007), viral infections (Nascimbeni et al., 2011) and malignant tumors (Desfrancois et al., 2009). Our findings could suggest that the presence of such cells during active BD may play a role in the defence against a hypothetical etiological agent and in BD pathogenesis but they may also constitute a normal response to CD8+T-cell activation in inflammatory environment. In light of our results, CD4+CD8+ T cells should be tested for phenotypic and functional properties during BD. In our series, we observed a decrease in the absolute number of B cells and particularly the conventional CD19+CD5 B cells. Moreover, this decrease interested only patients with inactive BD. However, Eksioglu-Demiralp et al. (1999b), noted that the number of total B lymphocytes is normal during BD and these cells express both activation and memory markers suggesting a modified B cell function in BD. The investigation of CD19 expression levels of B cells showed that this marker was over-expressed in patients with active BD compared to those with inactive disease. Alterations in the expression of this marker have been previously reported in several autoimmune diseases (Korganow et al., 2010; Sato et al., 2004). Our results showed that expression levels of CD19 were higher in patients with active BD than in patients in remission. This increase could be related to various polymorphisms affecting CD19 gene and resulting in its overexpression as reported in other systemic autoimmune diseases (Kuroki et al., 2002; Tsuchiya et al., 2004). Alternatively, this increase may be the consequence of the augmented BAFF-derived signals during active BD, since BAFF, which enhances CD19 expression (Hase et al., 2004), was shown to be elevated in the sera of patients with active BD (Hamzaoui et al., 2008). Regarding peripheral CD19+CD5+ cells (B1 cells), they were found to be decreased in patients with active BD compared to controls. Our results are in agreement with those reported in previous studies (Eksioglu-Demiralp et al., 1999b; Evereklioglu, 2005) but in contrast with other observations showing that these cells are expanded in non organ-specific autoimmune diseases, (Bohm, 2004; Duan & Morel, 2006; Smith & Olson, 1990; Youinou et al., 1993; Zeher et al., 1990). The reduced absolute number of B1 cells during active BD may due to their suppression caused by some inhibitory stimuli. Moreover, the expression of the CD5 molecule in B1 cells was found to be decreased in patients with inactive disease. This could be due to the spontaneous downregulation of CD5 molecules (Dalloul, 2009) once the stimulatory signal
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
triggered by some unknown external antigen (Eksioglu-Demiralp et al., 1999b) decreases, notably, during disease remission. Besides the study of peripheral mononuclear cell populations, we investigated the expression of HLA-B51 in our series and we found that the HLA-B51 frequency was significantly increased in patients with BD compared to controls (32.5% versus 16.1%, respectively; OR = 2.5, 95% CI [1.10–5.66], p = 0.025). This supports our previously published data (Sakly et al., 2009) and corroborates the findings reported in the meta-analysis conducted by de Menthon et al. (2009) where 4800 patients with BD and 16.289 controls from 78 independent studies were included and where B5/B51 carriers had about six times more risk to develop BD (OR = 5.78; 95% CI [5.00–6.67]). Several cell surface molecular abnormalities have been found to be associated with HLA-B51 expression in patients with BD (Song et al., 2013). However, our study didn’t succeed to find any association between the expression of HLA-B51 antigen and a specific leukocyte immunophenotypic profile, even if, Yasuoka et al. had previously described that circulating CD8+ and gd T cells were activated in vivo in HLA-B51-positive patients with active BD, together with an increased ratio of activated Vd1+/Vd2+. They, therefore, suggested a direct involvement of CD8+ and gd T cells in the disease flare, potentially by exerting their capacity to directly damage the tissues (Yasuoka et al., 2008). Discrepancies could be attributed to the main limit of our study which is the reduced number of cell markers used to define different cell subsets.
CONCLUSION Behcet’s disease seems to be associated with an abnormal immunophenotypic profile of peripheral lymphocytes. Notably, active disease is associated with increased CD56+, CD4 CD8bright and CD4+CD8+ peripheral cells, together with an over-expression of the CD19 marker. Taken together these results may reflect an immune activation, probably related to the inflammatory status in patients with active behcet’s disease. On the other hand, HLA-B51 expression was not associated with any specific leukocyte immunophenotypic profile. Further investigations are needed to assess whether HLA-B51 molecules are involved in BD development.
ACKNOWLEDGMENTS We thank Mr. M. Rassas, English Department, Faculty of Medicine, Monastir, Tunisia) for his help.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES Ahn JK, Chung H, Lee DS, et al. (2005). CD8brightCD56+ T cells are cytotoxic effectors in patients with active Behcet’s uveitis. J Immunol, 175, 6133–42.
475
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
476
K. Sakly et al.
Aramaki T, Ida H, Izumi Y, et al. (2009). A significantly impaired natural killer cell activity due to a low activity on a per-cell basis in rheumatoid arthritis. Mod Rheumatol, 19, 245–52. Atalay A, Yildiz-Demirtepe S, Tatlipinar S, et al. (2008). HLA-B51 gene and its expression in association with Behcet’s Disease in Denizli Province of Turkey. Mol Biol Rep, 35, 345–9. Bohm I. (2004). Increased peripheral blood B-cells expressing the CD5 molecules in association to autoantibodies in patients with lupus erythematosus and evidence to selectively down-modulate them. Biomed Pharmacother, 58, 338–43. Cameron AL, Kirby B, Griffiths CE. (2003). Circulating natural killer cells in psoriasis. Br J Dermatol, 149, 160–4. Dalloul A. (2009). CD5: A safeguard against autoimmunity and a shield for cancer cells. Autoimmun Rev, 8, 349–53. de Menthon M, Lavalley MP, Maldini C, et al. (2009). HLA-B51/B5 and the risk of Behcet’s disease: A systematic review and meta-analysis of case-control genetic association studies. Arthritis Rheum, 61, 1287–96. Deniz G, Dum G, Aktas E, et al. (2000). Neutrophil functions and anti-candidal activity of CD3-NK cells in Behcet’s disease. In Abstracts of the 14th European Immunology Meeting: EFIS 2000, 23–27 September 2000: Elsevier; 2000. Desfrancois J, Derre L, Corvaisier M, et al. (2009). Increased frequency of nonconventional double positive CD4CD8 alphabeta T cells in human breast pleural effusions. Int J Cancer, 125, 374–80. Direskeneli H. (2001). Behcet’s disease: Infectious aetiology, new autoantigens, and HLA-B51. Ann Rheum Dis, 60, 996–1002. Duan B, Morel L. (2006). Role of B-1a cells in autoimmunity. Autoimmun Rev, 5, 403–8. Eksioglu-Demiralp E, Direskeneli H, et al. (1999a). Increased CD4+CD16+ and CD4+CD56+ T cell subsets in Behcet’s disease. Rheumatol Int, 19, 23–6. Eksioglu-Demiralp E, Kibaroglu A, Direskeneli H, et al. (1999b). Phenotypic characteristics of B cells in Behcet’s disease: Increased activity in B cell subsets. J Rheumatol, 26, 826–32. Evereklioglu C. (2005). Current concepts in the etiology and treatment of Behcet disease. Surv Ophthalmol, 50, 297–350. Frassanito MA, Dammacco R, Cafforio P, Dammacco F. (1999). Th1 polarization of the immune response in Behcet’s disease: A putative pathogenetic role of interleukin-12. Arthritis Rheum, 42, 1967–74. Green MR, Kennell AS, Larche MJ, et al. (2005). Natural killer cell activity in families of patients with systemic lupus erythematosus: Demonstration of a killing defect in patients. Clin Exp Immunol, 141, 165–73. Gul A. (2001). Behcet’s disease: An update on the pathogenesis. Clin Exp Rheumatol, 19, S6–12. Gul A. (2005). Behcet’s disease as an autoinflammatory disorder. Curr Drug Targets Inflamm Allergy, 4, 81–3. Guo H, Xu B, Yang X, et al. (2013). A high frequency of peripheral blood NKG2D+NK and NKT cells in euthyroid patients with new onset hashimoto’s thyroiditis-a pilot study. Immunol Invest [ePub ahead of print, December 11, 2013]. Hamzaoui K, Hamzaoui A, Guemira F, et al. (2002). Cytokine profile in Behcet’s disease patients. Relationship with disease activity. Scand J Rheumatol, 31, 205–10. Hamzaoui K, Houman H, Ben Dhifallah I, et al. (2008). Serum BAFF levels and skin mRNA expression in patients with Behcet’s disease. Clin Exp Rheumatol, 26, S64–71. Hase H, Kanno Y, Kojima M, et al. (2004). BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood, 103, 2257–65. Hegab S, Al-Mutawa S. (2000). Immunopathogenesis of Behcet’s disease. Clin Immunol, 96, 174–86. Houman H, Hamzaoui A, Ben Ghorbal I, et al. (2004). Tc1/Tc2 ratio in the inflammatory process in patients with Behcet’s disease. Mediators Inflamm, 13, 247–53. ISGBD. (1990). Criteria for diagnosis of Behcet’s disease. International Study Group for Behcet’s Disease. Lancet, 335, 1078–80.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Kaneko F, Takahashi Y, Muramatsu R, et al. (1985). Natural killer cell numbers and function in peripheral lymphoid cells in Behcet’s disease. Br J Dermatol, 113, 313–8. Koarada S, Haruta Y, Tada Y, et al. (2004). Increased entry of CD4+ T cells into the Th1 cytokine effector pathway during T-cell division following stimulation in Behcet’s disease. Rheumatology (Oxford), 43, 843–51. Korganow AS, Knapp AM, Nehme-Schuster H, et al. (2010). Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: Decreased memory B cells and membrane CD19 expression. J Autoimmun, 34, 426–34. Kuroki K, Tsuchiya N, Tsao BP, et al. (2002). Polymorphisms of human CD19 gene: Possible association with susceptibility to systemic lupus erythematosus in Japanese. Genes Immun, 3, S21–30. Mendes D, Correia M, Barbedo M, et al. (2009). Behcet’s disease–a contemporary review. J Autoimmun, 32, 178–88. Mendoza-Pinto C, Garcia-Carrasco M, Jimenez-Hernandez M, et al. (2010). Etiopathogenesis of Behcet’s disease. Autoimmun Rev, 9, 241–5. Nascimbeni M, Pol S, Saunier B. (2011). Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C. PLoS One, 6, e20145. Nascimbeni M, Shin EC, Chiriboga L, et al. (2004). Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood, 104, 478–86. Onder M, Bozkurt M, Gurer MA, et al. (1994). Natural cellular cytotoxicity in Behcet’s disease. J Dermatol, 21, 239–43. Parel Y, Aurrand-Lions M, Scheja A, et al. (2007). Presence of CD4+CD8+ doublepositive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosis. Arthritis Rheum, 56, 3459–67. Park YW, Kee SJ, Cho YN, et al. (2009). Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus erythematosus. Arthritis Rheum, 60, 1753–63. Pay S, Simsek I, Erdem H, Dinc A. (2007). Immunopathogenesis of Behcet’s disease with special emphasize on the possible role of antigen presenting cells. Rheumatol Int, 27, 417–24. Sakane T, Takeno M, Suzuki N, Inaba G. (1999). Behcet’s disease. N Engl J Med, 341, 1284–91. Sakly N, Boumiza R, Zrour-Hassen S, et al. (2009). HLA-B27 and HLA-B51 determination in Tunisian healthy subjects and patients with suspected ankylosing spondylitis and Behcet’s disease. Ann NY Acad Sci, 1173, 564–9. Sato S, Fujimoto M, Hasegawa M, Takehara K. (2004). Altered blood B lymphocyte homeostasis in systemic sclerosis: Expanded naive B cells and diminished but activated memory B cells. Arthritis Rheum, 50, 1918–27. Smith HR, Olson RR. (1990). CD5+ B lymphocytes in systemic lupus erythematosus and rheumatoid arthritis. J Rheumatol, 17, 833–5. Song, G.G., Kim, J.H., Lee, Y.H. (2013). The chemokine receptor 5 Delta32 polymorphism and Type 1 diabetes, Behcet’s Disease, and asthma: A meta-analysis. Immunol Invest 2014;43:123–36. Sugi-Ikai N, Nakazawa M, Nakamura S, et al. (1998). Increased frequencies of interleukin-2- and interferon-gamma-producing T cells in patients with active Behcet’s disease. Invest. Ophthalmol Vis Sci, 39, 996–1004. Suzuki Y, Hoshi K, Matsuda T, Mizushima Y. (1992). Increased peripheral blood gamma delta+ T cells and natural killer cells in Behcet’s disease. J Rheumatol, 19, 588–92. Takeno M, Kariyone A, Yamashita N, et al. (1995). Excessive function of peripheral blood neutrophils from patients with Behcet’s disease and from HLA-B51 transgenic mice. Arthritis Rheum, 38, 426–33. Treusch M, Stubiger N, Vonthein R, et al. (2003). Influence of interferon-alpha on lymphocyte subpopulations in Behcet’s disease. Adv Exp Med Biol, 528, 533–5. Tsuchiya N, Kuroki K, Fujimoto M, et al. (2004). Association of a functional CD19 polymorphism with susceptibility to systemic sclerosis. Arthritis Rheum, 50, 4002–7.
477
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
478
K. Sakly et al.
Wang X, Das A, Lackner AA, et al. (2008). Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection. Blood, 112, 4981–90. Yamaguchi Y, Takahashi H, Satoh T, et al. (2010). Natural killer cells control a T-helper 1 response in patients with Behcet’s disease. Arthritis Res Ther, 12, R80. Yasuoka H, Yamaguchi Y, Mizuki N, et al. (2008). Preferential activation of circulating CD8+ and gammadelta T cells in patients with active Behcet’s disease and HLA-B51. Clin Exp Rheumatol, 26, S59–63. Youinou P, Mackenzie LE, Lamour A, et al. (1993). Human CD5-positive B cells in lymphoid malignancy and connective tissue diseases. Eur J Clin Invest, 23, 139–50. Yurdakul S, Yazici H. (2008). Behcet’s syndrome. Best Pract Res Clin Rheumatol, 22, 793–809. Zeher M, Suranyi P, Nagy G, Szegedi G. (1990). B cells expressing CD5 in minor labial salivary glands of patients with primary Sjogren’s syndrome. Arthritis Rheum, 33, 453. Zierhut M, Mizuki N, Ohno S, et al. (2003). Immunology and functional genomics of Behcet’s disease. Cell Mol Life Sci, 60, 1903–22.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of peripheral blood mononuclear cells in patients with Behc¸et’s disease and association with HLA-B51 expression K. Sakly,1 R. Lahmar,1 F. Nefzi,2 S. Hammami,3 O. Harzallah,3 N. Sakly,1,4 W. Sakly,1 M. Hassine,5 S. Mahjoub,3 I. Ghedira,1 and S. Feki6 1
Research Unit 03/UR/07 ‘‘Autoimmunity and Allergy’’, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia, 2 Laboratory of Transmissible Diseases and Biological Active Substances, LR99ES27, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia, 3 Department of Internal Medicine, F.B. University Hospital, Monastir, Tunisia, 4 Laboratory of Microbiology, Unity of Immunology, F.B. University Hospital, Monastir, Tunisia, 5 Laboratory of Hematology, F.B. University Hospital, Monastir, Tunisia, 6 Department of Clinical Biology B, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behc¸et’s disease (BD) and to assess associations between the expression of HLA-B51 antigen and that of other cell markers. Thirty healthy volunteer blood donors and forty patients with BD were enrolled into this study. Phenotyping was performed using two color flow cytometry. HLA-B51 typing was performed using the complement dependent microlymphocytotoxicity assay. Unlike controls, patients with BD presented a modified immunophenotypic profile of lymphocytes. Compared to those in the remission phase, patients with active BD showed an increased mean of MFI ratio of CD56 on CD16+CD56+ cells (32.47 ± 14.26 versus 23.87 ± 10.3; p = 0.032), increased absolute numbers of CD4 CD8bright and CD4+CD8+ cells (657.1 ± 463.6 cells/mL versus 319.24 ± 116.4 cells/ mL; p = 0.017 and 40.77 ± 36.41 cells/mL versus 10.77 ± 9.78 cells/mL; p50.0001, respectively) and an elevated mean of MFI ratio of CD19 on B cells (252.3 ± 56.7 versus 205.67 ± 32.3; p = 0.021). However, expression of HLA-B51 was not associated with any specific immunophenotypic profile. In conclusion, abnormal immunophenotypic profile of peripheral lymphocytes was found in patients with BD, especially in active phase, reflecting an immune dysregulation. Moreover, HLA-B51 expression was not found to be related to the expression of other cell markers. Keywords Behc¸et’s syndrome, blood cells, CD antigens, histocompatibility antigens
INTRODUCTION Behc¸et’s disease (BD) is a multisystemic inflammatory disorder characterized by recurrent oral and genital ulcerations as well as ocular, arthritic, vascular, Correspondence: Nabil Sakly, Research Unit 03/UR/07 ‘‘Autoimmunity and Allergy’’, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia. Tel: +216 98 632 477. E-mail: [email protected]
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
464
K. Sakly et al.
cutaneous and neurological involvement, and generally presents with remissions and exacerbations (Mendes et al., 2009; Yurdakul & Yazici, 2008). The etiology and the pathogenesis of BD still remains poorly understood, although several clues have been proposed, including the interaction between environmental and genetic factors, leading to an inappropriate immunoinflammatory response (Pay et al., 2007; Sakane et al., 1999). Abnormalities of the immune system such as activated circulating T and B cells, NK and neutrophils responsible for endothelial damages have been recognized as important immunopathogenic cells in BD (Frassanito et al., 1999; Hegab & Al-Mutawa, 2000). The leukocytes immunophenotypic profile of patients with BD is characterized by multiple alterations mainly in those with active disease. A preferential skewing of T cells in patients with active BD towards an IFN-g and IL-2 producing Th1 phenotype has been previously demonstrated by intracellular cytokine staining (Pay et al., 2007). The levels of CD14+ monocytes, CD3+ TCRgd+, CD8+TCRgd+ cells, NK cells (CD56+CD16+) and activated regulatory T cells (CD4+CD25+, CD8+CD25+) were elevated in patients with BD, compared with healthy controls (Treusch et al., 2003). Dividing CD4+ T cells showed the phenotype of activated effector memory cells (Koarada et al., 2004). Interferon-gamma-producing CD8+ T cells were increased in patients with BD (Houman et al., 2004). An association between BD and HLA-B51 has been regarded as the strongest evidence of the involvement of genetic factors in its pathogenesis (Atalay et al., 2008; Direskeneli, 2001). Different hypotheses have been generated to explain the direct pathogenic role of HLA-B51 in Behc¸et’s disease, however, the exact mechanism of action of this molecule is still unknown (Gul, 2001). HLA-B51 molecules have multifunctional properties, including interaction with TCR and CD8 on T CD8+ cells and with KIRs expressed particularly by natural killer (NK) cells (Atalay et al., 2008). They may present endogenous peptides to CD8+ T cells, which could activate target cells and other cells of the immune system and may promote the expression of other surface molecules. In this regard, increased neutrophil function has been reported in both patients with BD and HLA-B51 positive healthy individuals (Takeno et al., 1995; Zierhut et al., 2003). On the other hand, Yasuoka et al. (2008) noted, exclusively in HLA-B51 positive patients with active BD, increased proportion of activated CD8+ and gd T cells. The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behc¸et’s disease and to assess possible associations between the expression of HLA-B51 antigen and that of other cell markers.
MATERIALS AND METHODS Samples Forty patients with BD, 24 males and 16 females (mean age: 41.3 ± 10.6 years; range: 11–60 years) were recruited from the Internal Medicine and Ophthalmology departments at Fattouma Bourguiba Hospital of Monastir (eastern coast of the Central region of Tunisia). The patients were diagnosed according to published BD criteria (ISGBD, 1990). Our patients were
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
classified on admission as patients with active BD (n = 15) and patients with inactive BD (n = 25). The mean duration of disease was 104 months (range: 12–252 months). All patients with active BD showed necessary active oral or genital ulcerations and one more sign of clinical activity: skin lesions (erythema nodosum, folliculitis), ocular lesions, joint symptoms, central nervous system involvement, or gastrointestinal inflammation. Inactive patients with BD were composed of asymptomatic patients (mean duration of inactive stage: 9 weeks). At the time of analysis, 36 patients were under treatments: Two patients were treated with colchicine and immunosuppressive drugs (prednisone or azathioprine), seven with colchicine and low doses of steroids and the remaining patients were treated with colchicine alone. One hundred and twenty-four healthy Tunisian subjects underwent HLA-B51 typing and constituted the control group for the HLA-B51 expression study (Sakly et al., 2009) and thirty healthy volunteer blood donors were recruited as controls for the flow cytometry tests. They were 23 males and 7 females, with a mean age of 30.6 ± 9.69 years (range: 18–62 years). At the time of blood sampling, none of the blood donors was receiving any medication or had an infection. The design of the study was approved by our Hospital Ethics Committee. Flow Cytometry Analysis Lymphocyte subsets were evaluated in whole fresh blood obtained from patients with BD and controls using different monoclonal antibodies: AntiCD3-PE, Anti-CD4-FITC, Anti-CD8-PE, Anti-CD16-FITC, Anti-CD56-PE, Anti-CD5-FITC, Anti-CD19-PE, Anti-CD14-FITC and Anti-CD45-FITC (Invitrogen, USA). One-color and two-color phenotypic characterizations of lymphocytes were performed. Venous blood samples were collected into EDTA containing vacuum tubes and processed on the same day. Briefly, blood samples (100 mL) were incubated for 20 min with the appropriate amounts of mAb. Red blood cells were then lysed (FACS lysing solution; Becton Dickinson, Stockholm, Sweden) and the white cells washed and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). The resulting data were analyzed with the commercial FlowJo software. Expression levels of different markers were investigated through the mean absolute number of positive cells and the mean MFI ratio of gated cells (the mean fluorescence intensity (MFI) of positive cells reported to the negative ones). HLA-B51 Typing Whole blood was collected into EDTA containing tubes. Peripheral blood mononuclear cells were isolated by density gradient centrifugation over FicollHypaque (lymphoprep, Axis-schield PoC AS, Oslo, Norway). The isolated cells were counted and adjusted to 106 cells/mL. Serological typing of HLA-B51 was performed immediately by the complement dependent microlymphocytotoxicity assay, using the HLA-B Locus Supplement Typing Tray (One Lambda, Canoga Park, CA, USA). The test was performed according to the manufacturer’s recommendations.
465
466
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Statistical Analysis All quantitative results are expressed as the mean ± standard deviation. Absolute numbers of cells were calculated by multiplying the cell percentages by the total lymphocyte numbers (per microliter). Comparisons between quantitative parameters were performed using the non parametric Mann– Whitney U-test. Comparison between frequencies was made by the 2 Pearson test. Odd ratios (OR) were calculated according to the Woolf ’s formula, and are given with 95% confidence interval [95% CI]. Statistical significance was considered when two-sided p values are under 0.05. Analyses were done with the aid of the SPSSÕ software.
RESULTS Clinical Features of Behc¸et’s Disease Patients Among the 40 patients with BD, 92.5% had oral ulcers, 82.5% had genital ulcers, 47.5% had skin lesions, 37.5% had arthritis, 22.5% had uveitis and 12.5% had thrombosis during the course of disease. Only a small proportion of patients had a history of neurological (15%) and/or gastrointestinal (15%) involvement. Only 17.5% of patients had a family history of BD. Clinical features of patients are reported in Table 1. Table 1. Characteristics of Behc¸et’s disease group.
Age (years) Sex (M:F) Disease duration (months) Organ involvement Oral ulcers Genital ulcers Arthritis Skin lesions Central nervous system disease Uveitis Gastrointestinal system lesions Thrombosis Joint symptoms Pathergy Test HLA-B51 Family history of BD
Active (n = 15)
Inactive (n = 25)
36.73 ± 8.9 9:6 92.06 ± 44.4 n (%) 14 (93.3) 12 (80) 4 (26.6) 7 (46.6) 2 (13.3) 6 (40) 1 (6.66) 4 (26.6) 10 (66.6) 6 (40) 5 (33.3) 3 (20)
44.04 ± 10.8 15:10 111.32 ± 77.9 n (%) 23 (92) 21 (84) 11 (44) 12 (48) 4 (16) 3 (12) 5 (20) 1 (4) 5 (20) 13 (52) 8 (61.5) 4 (16)
Flow Cytometry Analysis CD16 and CD56 Expression Absolute numbers of CD16+CD56+ and CD16 CD56+ cells were significantly increased in patients with BD compared to those of controls (313.67 ± 210.82 versus 245.69 ± 95.43 cells/mL [p = 0.006] and 525.44 ± 783.77 versus 256.50 ± 155.19 cells/mL [p = 0.019], respectively). similar results were found in patients with active BD compared to controls (421.55 ± 214.28 versus 245.69 ± 95.43 cells/mL [p = 0.002] and 925.92 ± 1255.9 cells/mL versus 256.50 ± 155.19 cells/mL [p = 0.005], respectively) (Table 2). No significant
245.69 ± 95.4 256.50 ± 155.1 54.43 ± 30.3 1406.18 ± 430.4 551.87 ± 248.4 779.83 ± 245.2 343.79 ± 141.8 14.85 ± 8.9 288.49 ± 212.2 249.04 ± 187.9 40.15 ± 32.9 371.9 ± 14.9
CD16+CD56+ CD16 CD56+ CD16+CD56 CD3+ CD4 CD8+ CD4+CD8 CD4 CD8bright CD4+CD8+ CD19+ CD19+CD5 CD19+CD5+ CD14+
421.55 ± 214.2c 925.92 ± 1255.9c 70.54 ± 51.7 1789.38 ± 1020.5d 904.98 ± 450d 1015.86 ± 476 657.1 ± 463.6d 40.77 ± 36.4c 206.074 ± 102.4 185.51 ± 118.4 20.99 ± 13.1d 456.69 ± 16.5
Active BD (n = 15) 283.46 ± 203.9 317.20 ± 162.3 61.14 ± 41.5 1426.77 ± 472.9 482.87 ± 216.2 642.36 ± 179.7 319.24 ± 116.4 10.77 ± 9.7 175.19 ± 79.4c 146.91 ± 65.8c 27.07 ± 20.2 473.29 ± 28.4
Inactive BD (n = 25) 313.67 ± 210.8 525.44 ± 783.7 64.36 ± 44.8 1537.57 ± 692.5 633.13 ± 339 674.9 ± 309.7 439.84 ± 283.4 19.72 ± 23.3 194.06 ± 111.5 167.46 ± 100.7 25.74 ± 18.6 459.4 ± 24.4
Total (n = 40)
0.006 0.019 NS NS NS NS NS 0.011 0.011 0.011 NSe NS
p Valuea
a
N.B. Results are shown as the mean absolute numbers of cells per mL ± SD, and Mann-Whitney U-test were used for the comparison of means. Comparison between all patients with BD and controls. b Comparison between patients with active BD and patients with inactive BD. c p 0.005 compared with controls d p50.05 compared with controls e Not significant.
Controls (n = 30)
Cell subsets
Patients with BD
NS NS NS NS 0.006 0.019 0.017 50.0001 NS NS NS NS
p Valueb
Table 2. Absolute numbers of lymphocytes subsets in peripheral blood from patients with BD, patients with active BD and patients with inactive BD and controls determined by flow cytometric analysis.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
467
468
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
difference was observed between patients with active disease and those with inactive disease for these cell subsets. With regards to expression levels of CD56 and CD16, the MFI ratio of CD56 of CD16+CD56+ cells was found to be significantly higher in patients with active BD (32.47 ± 14.26) than in those with inactive disease (23.87 ± 10.3; p = 0.032) and controls (22.29 ± 8.6; p = 0.018) (Figure 1). However, MFI ratio of CD16 of CD16+CD56 cells was higher in patients with inactive BD compared to that of controls (9.02 ± 2.7 versus 7.64 ± 2.2; p = 0.042) (Figure 2). In our study, we also noted that BD patients with oral ulcers showed increased absolute numbers of CD16+CD56+ cells compared to those without oral ulcers (297.73 ± 203.8 cells/mL versus 158.86 ± 15.5 cells/mL, p = 0.001) (Figure 3).
Figure 1. Comparison of the mean MFI ratios of CD56 on CD16+CD56+ and CD16 CD56+ cells from patients with active Behc¸et’s disease, those with inactive Behc¸et’s disease and controls. Comparison was performed using Mann-Whitney U-test. p50.05 was considered as significant. MFI: mean intensity fluorescence. ap = 0.018, compared with controls. bp = 0.032, compared with patients with active BD.
Figure 2. Comparison of mean MFI ratios of CD16 on CD16+CD56+ and CD16+CD56 cells in patients with active BD, patients with inactive BD and controls. Comparison of mean MFI ratios between different groups was performed using the Mann-Whitney U-test. p50.05 was considered as significant. MFI: mean intensity fluorescence. a p = 0.042, compared with controls.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
(A)
(B)
(C)
Figure 3. Associations of peripheral CD16+CD56+ NK cells and CD56 expression with clinical manifestations in Behcet’s disease. (A) Association of absolute numbers of CD16+CD56+ NK cells with oral ulcers (p = 0.001). (B) Association of CD56 expression on CD16+CD56+ with skin lesions (p = 0.05). (C) Association of CD56 expression on CD16+CD56+ with genital ulcers (p = 0.047).
469
470
K. Sakly et al.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
In patients with BD, CD56 MFI ratio on CD16+CD56+ cells tended to be higher in patients with genital ulcers and with skin lesions than in those without genital ulcers (27.56 ± 12.78 versus 20.47 ± 4.53; p = 0.047) and without skin lesions (31.93 ± 15.95 versus 23.09 ± 7.21; p = 0.05) (Figure 3). CD3, CD4 and CD8 expression Absolute numbers of CD3+ cells were similar in patients with BD and controls. However, it is worthy to mention that CD3+ cells were significantly increased in patients with active BD compared to controls (1789.38 ± 1020.5 cells/mL versus 1406.18 ± 430.4 cells/mL; p = 0.032) (Table 2). Absolute numbers of CD4 CD8+ cells did not differ between patients with BD and controls. However, these absolute numbers were significantly increased in patients with active BD (904.98 ± 450.67 cells/mL) compared to those with inactive disease (482.87 cells/mL; p = 0.006) and controls (551.87 ± 248.49 cells/mL; p = 0.026). Similarly, absolute numbers of the CD4 CD8bright subset in patients with BD were comparable to those in controls. Yet, this cell subset was increased in patients with active BD (657.1 ± 463.61 cells/mL) compared to those with inactive BD (319.24 ± 116.49 cells/mL; p = 0.017) and controls (343.79 ± 141.85 cells/mL; p = 0.037) (Table 2). On the other hand, absolute numbers of CD4+CD8 cells were similar in patients with BD and controls. However, these cells were found to be significantly increased in patients with active BD compared to those with inactive disease (1015.86 ± 476.09 cells/mL versus 642.36 ± 179.77 cells/mL; p = 0.019) (Table 2). Among T cell subsets, interestingly, we detected a higher absolute number of double-positive CD4+CD8+ cells in patients with active BD (40.77 ± 36.41 cells/mL) compared to those with inactive disease (10.77 ± 9.78/mL; p50.0001) and controls (14.85 ± 8.91 cells/mL; p = 0.004) (Table 2). CD19 and CD5 expression Absolute numbers of CD19+ B cells were significantly decreased in patients with BD compared to controls (194.06 ± 111.5 cells/mL versus 288.49 ± 212.2 cells/mL; p = 0.011). This was also observed in patients with inactive BD compared to controls (175.19 ± 79.4 cells/mL versus 288.49 ± 212.2 cells/mL; p = 0.005) (Table 2). More precisely, this decrease interested the conventional CD19+CD5 B cells which were significantly lower in patients with BD compared to controls (167.46 ± 100.75 cells/mL versus 249.04 ± 187.98 cells/mL; p = 0.011) and especially in patients with inactive BD compared to controls (146.91 ± 65.8 cells/mL versus 249.04 ± 187.9 cells/mL; p = 0.005). In contrast, absolute numbers of CD19+CD5+ cells were significantly decreased in patients with active BD than in controls (20.99 ± 13.1 cells/ mL versus 40.15 ± 32.9 cells/mL; p = 0.045). No significant difference was observed between patients with active BD and those with inactive disease (Table 2). With regard to expression levels of CD19, MFI ratios of this marker for CD19+ and CD19+CD5 B cells were significantly lower in patients with BD than that found in controls (220.79 ± 46.6 versus 236.93 ± 52.6 [p = 0.043] and
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
216.2 ± 49.3 versus 235.45 ± 51.6 [p = 0.05], respectively) (Table 3) but they were significantly increased in patients with active BD compared to those with inactive disease (252.3 ± 56.7 versus 205.67 ± 32.3 [p = 0.021], and 238.78 ± 68.05 versus 204.46 ± 31.9 [p = 0.04], respectively). Similarly, expression levels of CD19 in CD19+CD5+ cells were higher in patients with active BD than in patients with inactive disease (262.036 ± 90.3 versus 220.07 ± 42.6 [p = 0.033]) (Table 3). Moreover, expression levels of CD5 on CD19+CD5+ B cells were significantly decreased in patients with BD compared to controls (7.46 ± 1.73 versus 8.38 ± 2.36; p = 0.032) and precisely in patients with inactive BD compared to controls (7.20 ± 1.34 versus 8.38 ± 2.36; p = 0.016) (Figure 4). CD14 expression The investigation of CD14+ cells showed no difference of absolute numbers between patients and controls or between patients with active or inactive disease (Table 2). HLA-B51 typing The HLA-B51 antigen was detected in 32.5% of patients with BD versus 16.1% in controls with OR = 2.50, 95% CI [1.10–5.66], p = 0.025. Table 3. Expression levels of CD19 on lymphocyte B subsets in patients with BD, patients with active BD, those with inactive disease and controls. Patients with BD CD19 expression (mean MFI ratio) CD19+ CD19+CD5+ CD19+CD5
Controls (n = 30) 236.93 ± 52.6 235.03 235.45 ± 51.6
Active (n = 15)
Inactive (n = 25)
252.3 ± 56.7 205.67 ± 32.3 262.03 ± 90.3 220.07 ± 42.6 238.78 ± 68.05 204.46 ± 31.9
Total (n = 40)
p Valuea
p Valueb
220.79 ± 46.6 237.52 ± 65.09 216.2 ± 49.3
0.043 NS 0.05
0.021 0.033 0.04
N.B. The Mann-Whitney U-test was used for the comparison of means. MFI: mean intensity fluorescence. a Comparison between all patients with BD and controls. b Comparison between patients with active BD and patients with inactive BD.
Figure 4. Mean MFI ratio of CD5 expression on CD19+CD5+ B cells according to Behc¸et’s disease activity. N.B. Mann-Whitney U-test was used for the comparison of means. MFI: mean intensity fluorescence ap = 0.016, compared with controls. bComparison between all patients with BD and controls (p = 0.032).
471
472
K. Sakly et al. Table 4. Absolute numbers and MFI ratios of lymphocyte subsets in peripheral blood from HLA-B51 negatif and HLA-B51 positive BD patients determined by flow cytometric analysis.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
HLA-B51 negative patients (n = 27) Cell subsets (Mean absolute numbers in cells/mL) 253.08 ± 213.56 CD16+CD56+ CD16+CD56 53.27 ± 33.08 408 ± 443.99 CD16 CD56+ 157.94 ± 76.91 CD3+ (103) 72.91 ± 35.29 CD4+CD8 CD4 CD8+ 70.41 ± 39.40 47.24 ± 32.27 CD4 CD8bright 23.16 ± 15.59 CD4 CD8dim 1.95 ± 1.90 CD4+CD8+ 189.90 ± 111.51 CD19+ CD19+CD5 164.06 ± 101.08 25.39 ± 19.38 CD19+CD5+ 46.21 ± 25.67 CD14+ CD56 expression (Mean MFI ratios) 27.71 ± 13.66 CD16+CD56+ 13.61 ± 3.27 CD16 CD56+ CD16 expression (Mean MFI ratios) 25.61 ± 16.24 CD16+CD56+ 8.34 ± 2.70 CD16+CD56 CD19 expression (Mean MFI ratios) 214.85 ± 53.81 CD19+ 211.99 ± 52.76 CD19+CD5 234.96 ± 72.92 CD19+CD5+ CD5 expression (Mean MFI ratios) 7.36 ± 1.66 CD19+CD5+
HLA-B51 positive patients (n = 13)
p Value
289.89 ± 177.48 61.88 ± 29.74 483.61 ± 509.38 142.86 ± 42.80 75.14 ± 22.17 66.61 ± 19.01 45.49 ± 17.15 21.11 ± 9.50 3.23 ± 3.98 205.70 ± 116.84 176.96 ± 104.60 26.69 ± 17.25 45.04 ± 21.51
NS NS NS NS NS NS NS NS NS NS NS NS NS
24.30 ± 7.48 13.35 ± 3.57
NS NS
31.56 ± 20.34 9.11 ± 3.62
NS NS
225.99 ± 36.17 227.98 ± 38.08 219.29 ± 35.56
NS NS NS
7.75 ± 1.99
NS
NS: not significant. Comparisons were made by the Mann-Whitney U-test.
Association Between the Expression of the HLA-B51 Molecule and the Immunophenotypic Profile of Leukocytes in Patients with BD For each studied marker, the mean absolute number of positive cells and the mean MFI ratio of gated cells are presented in Table 4 according to the status of the expression of the HLA-B51 molecule. The expression of the HLA-B51 molecule was not found to be associated with any specific immunophenotypic profile in patients with BD. The same observation was found between the status of the expression of the HLA-B51 molecule and disease activity (p40.05; data not shown).
DISCUSSION In the present study, we explored the immunophenotypic profile of peripheral mononuclear cell subclasses in Tunisian patients with Behc¸et’s disease and assessed the possible relation between the expression of HLA-B51 antigen and the expression of other cell markers. Our study showed increased absolute numbers of CD16+CD56+ and CD16 CD56+ cells in patients with BD compared to controls. Abnormalities
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
in the number and function of circulating NK cells have been shown in several autoimmune and inflammatory diseases, such as systemic lupus erythematosus (Cameron et al., 2003; Green et al., 2005; Park et al., 2009), rheumatoid arthritis (Aramaki et al., 2009), psoriasis (Cameron et al., 2003) and Hashimoto’s thyroiditis (Guo et al., 2013). Similarly, abnormalities in circulating NK cells have been reported in patients with BD. An elevated number of NK cells has been found in patients with inactive stage of BD compared to controls (Deniz et al., 2000; Onder et al., 1994). However, other studies reported no quantitative or functional significant differences in the NK cells between patients with BD and controls (Eksioglu-Demiralp et al., 1999a; Gul, 2005; Park et al., 2009). The discrepancies between different studies could be related to the series (number of patients, clinical status, disease activity, treatment modalities) and/or to the strategy used to identify the NK cell subset (number of monoclonals used in cytometry). With regard to disease activity, our study showed that patients with active BD had, in comparison to controls, increased absolute numbers of the above cited two cell subsets (CD16 CD56+ and CD16+CD56+). Similarly, increased percentages of CD16+CD56+ NK cells in patients with clinically active BD have been previously reported (Kaneko et al., 1985; Suzuki et al., 1992). Moreover, Yamaguchi et al. (2010) reported markedly increased proportion of peripheral blood CD69+ NK cells in patients with active BD. They also noted a shift to NK2 phenotype in patients with inactive disease, leading them to suggest a role of NK cells in modulating disease flare/remission. The investigation of CD56 and CD16 expression levels revealed an increase of CD56 expression in CD16+CD56+ NK cells in patients with active BD compared to those with inactive disease and controls, suggesting that there could be a shift to the CD16+CD56bright phenotype in active BD. Knowing that CD56bright NK cells are characterized by enhanced cytokine production, mainly IFNg, TNF-b, IL-10, IL-13 and GM-CSF, this could explain the findings previously reported by Hamzaoui et al. (2002), showing an increase in IL-10 and IFN-g in patients with active BD compared to those with inactive disease. It was also suggested that interferon-g may contribute to the local inflammatory response in BD (Mendoza-Pinto et al., 2010). On the other hand, our study showed that CD16+CD56+ cells expressing high levels of CD56 are associated with the presence of genital ulcers and skin lesions. The analysis of T cell subsets revealed an increased CD4+CD8 cells in patients with active BD compared to those with inactive disease. Similar findings were reported by Sugi-Ikai et al. (1998), who found higher absolute numbers of CD4+ cells in peripheral blood of patients with active BD compared to controls. Thus, CD4+ cells may be involved in the active phase of BD. This suggestion could be supported by the work of Frassanito et al., who analyzed intracytoplasmic cytokine expression using flow cytometry and revealed that IL-2 and IFN-g producing CD4+ cells was increased in patients with active BD (Frassanito et al., 1999; Sugi-Ikai et al., 1998) point to a strong polarized Th1 immune response in BD (Koarada et al., 2004). We found increased absolute numbers of CD4 CD8+ cells (mainly CD4 CD8bright cells) in patients with active BD compared to those with inactive disease and controls. These findings are consistent with those reported by Ahn et al. (2005), who demonstrated that activated effector
473
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
474
K. Sakly et al.
CD8bright T cells are expanded in patients with active Behc¸et’s uveitis compared to inactive disease and controls. Furthermore, the authors showed that the cytolytic activities of this subset against HUVECs were increased in active Behc¸et’s uveitis. One of the most striking findings, in our study, was the significant increase in the absolute number of peripheral double positive CD4+CD8+ cells in patients with active BD compared to those with inactive disease and controls. The origin of peripheral double positive T cells remains controversial, however, it is well documented that these cells are differentiated into functional effector/memory T cells (Nascimbeni et al., 2004; Wang et al., 2008) and being high producers of cytokines with immunomodulatory potential (Parel et al., 2007); It has been shown that these cells were expanded in several pathologic conditions such as systemic sclerosis (Parel et al., 2007), viral infections (Nascimbeni et al., 2011) and malignant tumors (Desfrancois et al., 2009). Our findings could suggest that the presence of such cells during active BD may play a role in the defence against a hypothetical etiological agent and in BD pathogenesis but they may also constitute a normal response to CD8+T-cell activation in inflammatory environment. In light of our results, CD4+CD8+ T cells should be tested for phenotypic and functional properties during BD. In our series, we observed a decrease in the absolute number of B cells and particularly the conventional CD19+CD5 B cells. Moreover, this decrease interested only patients with inactive BD. However, Eksioglu-Demiralp et al. (1999b), noted that the number of total B lymphocytes is normal during BD and these cells express both activation and memory markers suggesting a modified B cell function in BD. The investigation of CD19 expression levels of B cells showed that this marker was over-expressed in patients with active BD compared to those with inactive disease. Alterations in the expression of this marker have been previously reported in several autoimmune diseases (Korganow et al., 2010; Sato et al., 2004). Our results showed that expression levels of CD19 were higher in patients with active BD than in patients in remission. This increase could be related to various polymorphisms affecting CD19 gene and resulting in its overexpression as reported in other systemic autoimmune diseases (Kuroki et al., 2002; Tsuchiya et al., 2004). Alternatively, this increase may be the consequence of the augmented BAFF-derived signals during active BD, since BAFF, which enhances CD19 expression (Hase et al., 2004), was shown to be elevated in the sera of patients with active BD (Hamzaoui et al., 2008). Regarding peripheral CD19+CD5+ cells (B1 cells), they were found to be decreased in patients with active BD compared to controls. Our results are in agreement with those reported in previous studies (Eksioglu-Demiralp et al., 1999b; Evereklioglu, 2005) but in contrast with other observations showing that these cells are expanded in non organ-specific autoimmune diseases, (Bohm, 2004; Duan & Morel, 2006; Smith & Olson, 1990; Youinou et al., 1993; Zeher et al., 1990). The reduced absolute number of B1 cells during active BD may due to their suppression caused by some inhibitory stimuli. Moreover, the expression of the CD5 molecule in B1 cells was found to be decreased in patients with inactive disease. This could be due to the spontaneous downregulation of CD5 molecules (Dalloul, 2009) once the stimulatory signal
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
triggered by some unknown external antigen (Eksioglu-Demiralp et al., 1999b) decreases, notably, during disease remission. Besides the study of peripheral mononuclear cell populations, we investigated the expression of HLA-B51 in our series and we found that the HLA-B51 frequency was significantly increased in patients with BD compared to controls (32.5% versus 16.1%, respectively; OR = 2.5, 95% CI [1.10–5.66], p = 0.025). This supports our previously published data (Sakly et al., 2009) and corroborates the findings reported in the meta-analysis conducted by de Menthon et al. (2009) where 4800 patients with BD and 16.289 controls from 78 independent studies were included and where B5/B51 carriers had about six times more risk to develop BD (OR = 5.78; 95% CI [5.00–6.67]). Several cell surface molecular abnormalities have been found to be associated with HLA-B51 expression in patients with BD (Song et al., 2013). However, our study didn’t succeed to find any association between the expression of HLA-B51 antigen and a specific leukocyte immunophenotypic profile, even if, Yasuoka et al. had previously described that circulating CD8+ and gd T cells were activated in vivo in HLA-B51-positive patients with active BD, together with an increased ratio of activated Vd1+/Vd2+. They, therefore, suggested a direct involvement of CD8+ and gd T cells in the disease flare, potentially by exerting their capacity to directly damage the tissues (Yasuoka et al., 2008). Discrepancies could be attributed to the main limit of our study which is the reduced number of cell markers used to define different cell subsets.
CONCLUSION Behcet’s disease seems to be associated with an abnormal immunophenotypic profile of peripheral lymphocytes. Notably, active disease is associated with increased CD56+, CD4 CD8bright and CD4+CD8+ peripheral cells, together with an over-expression of the CD19 marker. Taken together these results may reflect an immune activation, probably related to the inflammatory status in patients with active behcet’s disease. On the other hand, HLA-B51 expression was not associated with any specific leukocyte immunophenotypic profile. Further investigations are needed to assess whether HLA-B51 molecules are involved in BD development.
ACKNOWLEDGMENTS We thank Mr. M. Rassas, English Department, Faculty of Medicine, Monastir, Tunisia) for his help.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES Ahn JK, Chung H, Lee DS, et al. (2005). CD8brightCD56+ T cells are cytotoxic effectors in patients with active Behcet’s uveitis. J Immunol, 175, 6133–42.
475
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
476
K. Sakly et al.
Aramaki T, Ida H, Izumi Y, et al. (2009). A significantly impaired natural killer cell activity due to a low activity on a per-cell basis in rheumatoid arthritis. Mod Rheumatol, 19, 245–52. Atalay A, Yildiz-Demirtepe S, Tatlipinar S, et al. (2008). HLA-B51 gene and its expression in association with Behcet’s Disease in Denizli Province of Turkey. Mol Biol Rep, 35, 345–9. Bohm I. (2004). Increased peripheral blood B-cells expressing the CD5 molecules in association to autoantibodies in patients with lupus erythematosus and evidence to selectively down-modulate them. Biomed Pharmacother, 58, 338–43. Cameron AL, Kirby B, Griffiths CE. (2003). Circulating natural killer cells in psoriasis. Br J Dermatol, 149, 160–4. Dalloul A. (2009). CD5: A safeguard against autoimmunity and a shield for cancer cells. Autoimmun Rev, 8, 349–53. de Menthon M, Lavalley MP, Maldini C, et al. (2009). HLA-B51/B5 and the risk of Behcet’s disease: A systematic review and meta-analysis of case-control genetic association studies. Arthritis Rheum, 61, 1287–96. Deniz G, Dum G, Aktas E, et al. (2000). Neutrophil functions and anti-candidal activity of CD3-NK cells in Behcet’s disease. In Abstracts of the 14th European Immunology Meeting: EFIS 2000, 23–27 September 2000: Elsevier; 2000. Desfrancois J, Derre L, Corvaisier M, et al. (2009). Increased frequency of nonconventional double positive CD4CD8 alphabeta T cells in human breast pleural effusions. Int J Cancer, 125, 374–80. Direskeneli H. (2001). Behcet’s disease: Infectious aetiology, new autoantigens, and HLA-B51. Ann Rheum Dis, 60, 996–1002. Duan B, Morel L. (2006). Role of B-1a cells in autoimmunity. Autoimmun Rev, 5, 403–8. Eksioglu-Demiralp E, Direskeneli H, et al. (1999a). Increased CD4+CD16+ and CD4+CD56+ T cell subsets in Behcet’s disease. Rheumatol Int, 19, 23–6. Eksioglu-Demiralp E, Kibaroglu A, Direskeneli H, et al. (1999b). Phenotypic characteristics of B cells in Behcet’s disease: Increased activity in B cell subsets. J Rheumatol, 26, 826–32. Evereklioglu C. (2005). Current concepts in the etiology and treatment of Behcet disease. Surv Ophthalmol, 50, 297–350. Frassanito MA, Dammacco R, Cafforio P, Dammacco F. (1999). Th1 polarization of the immune response in Behcet’s disease: A putative pathogenetic role of interleukin-12. Arthritis Rheum, 42, 1967–74. Green MR, Kennell AS, Larche MJ, et al. (2005). Natural killer cell activity in families of patients with systemic lupus erythematosus: Demonstration of a killing defect in patients. Clin Exp Immunol, 141, 165–73. Gul A. (2001). Behcet’s disease: An update on the pathogenesis. Clin Exp Rheumatol, 19, S6–12. Gul A. (2005). Behcet’s disease as an autoinflammatory disorder. Curr Drug Targets Inflamm Allergy, 4, 81–3. Guo H, Xu B, Yang X, et al. (2013). A high frequency of peripheral blood NKG2D+NK and NKT cells in euthyroid patients with new onset hashimoto’s thyroiditis-a pilot study. Immunol Invest [ePub ahead of print, December 11, 2013]. Hamzaoui K, Hamzaoui A, Guemira F, et al. (2002). Cytokine profile in Behcet’s disease patients. Relationship with disease activity. Scand J Rheumatol, 31, 205–10. Hamzaoui K, Houman H, Ben Dhifallah I, et al. (2008). Serum BAFF levels and skin mRNA expression in patients with Behcet’s disease. Clin Exp Rheumatol, 26, S64–71. Hase H, Kanno Y, Kojima M, et al. (2004). BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood, 103, 2257–65. Hegab S, Al-Mutawa S. (2000). Immunopathogenesis of Behcet’s disease. Clin Immunol, 96, 174–86. Houman H, Hamzaoui A, Ben Ghorbal I, et al. (2004). Tc1/Tc2 ratio in the inflammatory process in patients with Behcet’s disease. Mediators Inflamm, 13, 247–53. ISGBD. (1990). Criteria for diagnosis of Behcet’s disease. International Study Group for Behcet’s Disease. Lancet, 335, 1078–80.
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
Phenotypic abnormalities of PBMC in Behc¸et’s disease
Kaneko F, Takahashi Y, Muramatsu R, et al. (1985). Natural killer cell numbers and function in peripheral lymphoid cells in Behcet’s disease. Br J Dermatol, 113, 313–8. Koarada S, Haruta Y, Tada Y, et al. (2004). Increased entry of CD4+ T cells into the Th1 cytokine effector pathway during T-cell division following stimulation in Behcet’s disease. Rheumatology (Oxford), 43, 843–51. Korganow AS, Knapp AM, Nehme-Schuster H, et al. (2010). Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: Decreased memory B cells and membrane CD19 expression. J Autoimmun, 34, 426–34. Kuroki K, Tsuchiya N, Tsao BP, et al. (2002). Polymorphisms of human CD19 gene: Possible association with susceptibility to systemic lupus erythematosus in Japanese. Genes Immun, 3, S21–30. Mendes D, Correia M, Barbedo M, et al. (2009). Behcet’s disease–a contemporary review. J Autoimmun, 32, 178–88. Mendoza-Pinto C, Garcia-Carrasco M, Jimenez-Hernandez M, et al. (2010). Etiopathogenesis of Behcet’s disease. Autoimmun Rev, 9, 241–5. Nascimbeni M, Pol S, Saunier B. (2011). Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C. PLoS One, 6, e20145. Nascimbeni M, Shin EC, Chiriboga L, et al. (2004). Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood, 104, 478–86. Onder M, Bozkurt M, Gurer MA, et al. (1994). Natural cellular cytotoxicity in Behcet’s disease. J Dermatol, 21, 239–43. Parel Y, Aurrand-Lions M, Scheja A, et al. (2007). Presence of CD4+CD8+ doublepositive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosis. Arthritis Rheum, 56, 3459–67. Park YW, Kee SJ, Cho YN, et al. (2009). Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus erythematosus. Arthritis Rheum, 60, 1753–63. Pay S, Simsek I, Erdem H, Dinc A. (2007). Immunopathogenesis of Behcet’s disease with special emphasize on the possible role of antigen presenting cells. Rheumatol Int, 27, 417–24. Sakane T, Takeno M, Suzuki N, Inaba G. (1999). Behcet’s disease. N Engl J Med, 341, 1284–91. Sakly N, Boumiza R, Zrour-Hassen S, et al. (2009). HLA-B27 and HLA-B51 determination in Tunisian healthy subjects and patients with suspected ankylosing spondylitis and Behcet’s disease. Ann NY Acad Sci, 1173, 564–9. Sato S, Fujimoto M, Hasegawa M, Takehara K. (2004). Altered blood B lymphocyte homeostasis in systemic sclerosis: Expanded naive B cells and diminished but activated memory B cells. Arthritis Rheum, 50, 1918–27. Smith HR, Olson RR. (1990). CD5+ B lymphocytes in systemic lupus erythematosus and rheumatoid arthritis. J Rheumatol, 17, 833–5. Song, G.G., Kim, J.H., Lee, Y.H. (2013). The chemokine receptor 5 Delta32 polymorphism and Type 1 diabetes, Behcet’s Disease, and asthma: A meta-analysis. Immunol Invest 2014;43:123–36. Sugi-Ikai N, Nakazawa M, Nakamura S, et al. (1998). Increased frequencies of interleukin-2- and interferon-gamma-producing T cells in patients with active Behcet’s disease. Invest. Ophthalmol Vis Sci, 39, 996–1004. Suzuki Y, Hoshi K, Matsuda T, Mizushima Y. (1992). Increased peripheral blood gamma delta+ T cells and natural killer cells in Behcet’s disease. J Rheumatol, 19, 588–92. Takeno M, Kariyone A, Yamashita N, et al. (1995). Excessive function of peripheral blood neutrophils from patients with Behcet’s disease and from HLA-B51 transgenic mice. Arthritis Rheum, 38, 426–33. Treusch M, Stubiger N, Vonthein R, et al. (2003). Influence of interferon-alpha on lymphocyte subpopulations in Behcet’s disease. Adv Exp Med Biol, 528, 533–5. Tsuchiya N, Kuroki K, Fujimoto M, et al. (2004). Association of a functional CD19 polymorphism with susceptibility to systemic sclerosis. Arthritis Rheum, 50, 4002–7.
477
Immunol Invest Downloaded from informahealthcare.com by University of Laval on 06/03/14 For personal use only.
478
K. Sakly et al.
Wang X, Das A, Lackner AA, et al. (2008). Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection. Blood, 112, 4981–90. Yamaguchi Y, Takahashi H, Satoh T, et al. (2010). Natural killer cells control a T-helper 1 response in patients with Behcet’s disease. Arthritis Res Ther, 12, R80. Yasuoka H, Yamaguchi Y, Mizuki N, et al. (2008). Preferential activation of circulating CD8+ and gammadelta T cells in patients with active Behcet’s disease and HLA-B51. Clin Exp Rheumatol, 26, S59–63. Youinou P, Mackenzie LE, Lamour A, et al. (1993). Human CD5-positive B cells in lymphoid malignancy and connective tissue diseases. Eur J Clin Invest, 23, 139–50. Yurdakul S, Yazici H. (2008). Behcet’s syndrome. Best Pract Res Clin Rheumatol, 22, 793–809. Zeher M, Suranyi P, Nagy G, Szegedi G. (1990). B cells expressing CD5 in minor labial salivary glands of patients with primary Sjogren’s syndrome. Arthritis Rheum, 33, 453. Zierhut M, Mizuki N, Ohno S, et al. (2003). Immunology and functional genomics of Behcet’s disease. Cell Mol Life Sci, 60, 1903–22.
