- Email: [email protected]
Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion
Journal of Infection (2015) xx, 1e4
www.elsevierhealth.com/journals/jinf
LETTER TO THE EDITOR Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion
Dear Editor, We read with interest the article of Delacour et al. about the potential impact of the analytical systems on adenosine deaminase (ADA) determination.1 ADA has been widely used as a complementary diagnostic test for tuberculous pleural effusion (TPE) in tuberculosis (TB)-prevalent areas.2 The levels of ADA vary among TPE cases. Also, the diagnostic threshold values for TPE differ among studies. However, the factors associated with pleural fluid ADA levels in terms of study population remains still unclear. Thus, we examined the factors affecting pleural fluid ADA levels in TPE patients. Data from 353 consecutive patients who were diagnosed with TPE between 2009 and 2013 at Kyungpook National University Hospital, a South Korean tertiary referral hospital, were retrospectively reviewed. Of these, 202 patients with a confirmed TPE (positive Mycobacterium tuberculosis (MTB) culture [n Z 192] and histologic [n Z 10] results) were analyzed with regard to clinical, laboratory, radiological, and pleural fluid parameters. Linear regression analyses were performed to identify independent factors predictive of pleural fluid ADA levels. Univariate linear regression analysis identified a significant negative association between ADA levels and age (b Z 0.158, p Z 0.024), current smoking (b Z 0.170, p Z 0.016), pleural fluid glucose (b Z 0.396, p < 0.001), and pleural fluid pH levels (b Z 0.450, p < 0.001), and a significant positive association between ADA levels and loculated pleural effusion (b Z 0.257, p < 0.001), pleural fluid protein (b Z 0.431, p < 0.001), pleural fluid LDH levels (b Z 0.482, p < 0.001), and positive pleural fluid MTB PCR (b Z 0.211, p Z 0.004) and MTB culture results (b Z 0.295, p < 0.001) (Table 1). Stepwise multiple linear regression analysis identified current smoking (b Z 0.213, p < 0.001), pleural fluid protein (b Z 0.381, p < 0.001), LDH (b Z 0.312, p < 0.001), and glucose levels (b Z 0.139, p Z 0.040), and a positive MTB culture result (b Z 0.128, p Z 0.036) as independent factors associated with ADA levels.
To find further evidence to support a new finding of a positive association between the pleural fluid MTB load and ADA levels obtained from this single-center descriptive study, we performed in-vitro experiments using monocyte-derived macrophages (MDMs). Monocytes were purified from peripheral blood mononuclear cells of healthy volunteer donors and then were differentiated into macrophages using granulocyte-macrophage colonystimulating factor. MDMs were infected with MTB H37Rv at different multiplicity of infection (MOI) (1:1 and 10:1). The total ADA and ADA2 activities were assayed using a commercial Adenosine Deaminase Assay Kit (BQKITS Diagnostics, CA, USA) in unstimulated and MTB-infected MDM culture supernatants. In addition, ADA2 coding CECR1 (cat eye syndrome chromosome region, candidate 1) mRNA expression was measured in these MDM cells. Total RNA was extracted from the unstimulated and MTBinfected MDM cells by using an RNeasy mini kit (Qiagen, UK). Synthesis of the first-strand complementary DNA was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, CA, USA). To evaluate the level of expression of CECR1 mRNA, real-time quantitative reverse transcriptase polymerase chain reaction was performed by using a 7500 Fast Real-Time PCR system (Applied Biosystems, CA, USA). Fig. 1A and B show the total ADA and ADA2 levels, respectively, from the unstimulated and MTB H37Rv-infected MDM culture supernatants. The median levels of total ADA and ADA2 from the culture supernatants of MDM infected with MTB H37Rv at a lower MOI (1:1) were significantly higher than those of unstimulated MDM culture supernatants (total ADA: 16.5 [interquartile range (IQR), 16.4e19.3] vs. 14.1 [11.9e16.1], p Z 0.003; ADA2: 15.3 [14.9e17.0] vs. 13.2 [12.1e16.1], p Z 0.035). The total ADA levels from MTBinfected MDM culture supernatants were significantly greater at a higher MOI (10:1) than at a lower MOI (1:1) (median [IQR]: 19.3 [17.5e21.6] vs. 16.5 [16.4e19.3] U/ L, p Z 0.003) (Fig. 1A). Similarly, ADA2 levels were significantly higher in the MDMs infected with MTB H37Rv at a higher MOI than at a lower MOI (median [IQR]: 17.8 [14.1e21.1] vs. 15.3 [14.9e17.0] U/L, p Z 0.014) (Fig. 1B). Relative levels of CECR1 mRNA expression were significantly greater in MTB H37Rv-infected MDM cells compared to unstimulated MDM cells (p Z 0.008 for MOI 10:1; p Z 0.008 for MOI 1:1) (Fig. 1C). CECR1
http://dx.doi.org/10.1016/j.jinf.2015.05.015 0163-4453/ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
2
Letter to the Editor Table 1 Factors affecting pleural fluid adenosine deaminase levels in 202 patients with a confirmed tuberculous pleural effusion. Variable
Median (IQR) or No (%) Univariate analysis B
Age, y Male Current smoker Immunosuppressive conditiona Symptoms lasting > 2 wks Fever Chest pain Laboratory findings WBC, cells/mL CRP, mg/dL Albumin, g/dL Radiographic findings Parenchymal lesionb Loculated effusion Pleural fluid analyses Total cell count, cells/mL Protein, g/dL LDH, IU/L Glucose, mg/dL pH Positive AFB smear Positive MTB PCR Positive MTB culture
SE
p Value Multivariate analysis
b
B
64 (39e74) 143 (71) 54 (27) 23 (11) 113 (56) 107 (53) 98 (49)
0.212 0.028 0.099 0.109 0.026 0.015 0.059
0.093 0.158 0.040 0.049 0.041 0.170 0.057 0.134 0.037 0.051 0.037 0.029 0.036 0.114
0.024 0.489 0.016 0.057 0.474 0.677 0.105
7275 (5713e8998) 6.4 (3.0e10.3) 3.3 (2.9e3.7)
0.063 0.113 0.040 0.054 0.036 0.104 0.013 0.193 0.005
0.576 0.142 0.945
155 (77) 63 (31)
0.003 0.043 0.006 0.938 0.144 0.038 0.257 <0.001
1500 (750e2800) 5.0 (4.5e5.6) 904 (570e1635) 97 (68e127) 7.42 (7.37e7.46) 3/200 (2)c 22/182 (12)c 71/199 (36)c
0.008 1.106 0.371 0.432 14.042 0.015 0.177 0.160
0.033 0.164 0.048 0.072 1.998 0.151 0.061 0.037
0.016 0.431 0.482 0.396 0.450 0.007 0.211 0.295
0.818 <0.001 <0.001 <0.001 <0.001 0.921 0.004 <0.001
SE
p Value
b
0.122 0.033 0.213 <0.001
1.011 0.156 0.381 <0.001 0.246 0.054 0.312 <0.001 0.157 0.076 0.139 0.040
0.069 0.032
0.128
0.036
IQR Z interquartile range; B Z unstandardized regression coefficient; SE Z standard error; b Z standardized regression coefficient; WBC Z white blood cell; CRP Z C-reactive protein; LDH Z lactate dehydrogenase; AFB Z acid-fast bacilli; MTB Z Mycobacterium tuberculosis; PCR Z polymerase chain reaction. All continuous variables were log-transformed. All categorical variables were classified as “0”, absent (or a negative result) or as “1”, present (or a positive result). There was no evidence of multicollinearity because the variance inflation factor for independent variables was less than 1.2 in all models. R2 (adjusted R2) Z 0.455 (0.439) for the goodness of fit (using multivariate analysis). a Includes patients with malignancy (n Z 9), immunosuppressive drug therapy (n Z 2), transplantation (n Z 1), end-stage renal disease (n Z 4), advanced chronic liver disease (n Z 4), and human immunodeficiency virus infection (n Z 3). b Includes consolidation, nodules, masses, cavities, and calcified lesions. c Expressed as positive number/available number (%).
mRNA expression was also significantly more abundant in MDMs infected with MTB at MOI 10:1 than at MOI 1:1 (p Z 0.027). ADA2, accounting for most of the total ADA activity in TPE, is elevated when monocyte-macrophages are infected with intracellular microorganisms such as human immunodeficiency virus (HIV) or MTB.2,3 The macrophage is the first line cell to encounter MTB at the site of infection and plays an essential role in the initiation and maintenance of immune response to MTB infection. ADA2 production by MTB-infected macrophages might therefore be involved in the innate immune response network to MTB infection.4 Our finding that ADA2 mRNA expression levels of MTBinfected MDM correlated with MTB load supports clinical observation that a positive MTB culture result affected pleural fluid ADA levels. This is similar to the previous finding that serum ADA concentrations are positively associated with viral load in patients with HIV infection.5 These increased ADA2 activities may play a role in counteracting
immune suppression mediated by adenosine which is increased at the time of infection.6 A correlation between ADA levels and the extent of pleural inflammation in this study is in accordance with previous studies.7 A recent study8 has also shown that smoking negatively affected ADA levels of TPE patients. Cigarette smoke attenuated pro-inflammatory cytokine rin-infected MDM.9 In production by Bacillus Calmette-Gue addition, macrophage polarization toward the M2 phenotype, exhibiting immunosuppressive or anti-inflammatory characteristics, was induced by cigarette smoke.10 Thus, a suppressive effect of smoking on the pro-inflammatory responses of macrophage or macrophage phenotype alteration may be associated with decreased ADA levels in TPE patients. In conclusion, the present study showed that pleural fluid ADA levels in TPE patients were associated with smoking status, the extent of pleural inflammation, and pleural MTB burden. And, ADA2 mRNA expression by MTB-
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
Letter to the Editor
3
Figure 1 Comparison of total adenosine deaminase (ADA) and ADA2 concentrations, and cat eye syndrome chromosome region, candidate 1 (CECR1) mRNA expression levels from the culture supernatants and cells of unstimulated and Mycobacterium tuberculosis (MTB) H37Rv-infected monocyte derived macrophages (MDMs). (A) and (B). Total ADA and ADA2 concentrations from the culture supernatants of unstimulated and MTB H37Rv-infected MDMs (multiplicity of infection [MOI] 1 and 10), respectively, were measured using ADA assay kit (n Z 18). Each box outline represents the 25th and 75th percentiles, the central line the median value, and the whiskers minimum and maximum values. (C). Relative levels of CECR1 mRNA expression were determined using real-time reverse transcription-polymerase chain reaction (n Z 8). Human acidic ribosomal protein (HuPO) served as an internal control. Each column and bar represent the mean and standard error values. NC Z unstimulated; MTB1 Z infected with MTB at MOI 1:1; MTB10 Z infected with MTB at MOI 10:1. Differences were analyzed by Wilcoxon-paired signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001.
infected MDMs was positively affected by MTB load. Taken together, these findings provide evidence to suggest that pleural fluid ADA levels of TPE patients are affected by pleural fluid MTB load. The pleural fluid mycobacterial burden needs to be taken into account when pleural fluid ADA levels are interpreted in patients suspected of having TPE.
6.
7.
Conflict of interest None of the authors have any conflicts of interest to declare.
8.
Acknowledgments
9.
JL, JC and SS receive salary support from the EU grants IDEA, TANDEM (Grant No. 305279), and NORAD.
References 1. Delacour H, Bousquet A, Fontan E, Ceppa F. Pleural adenosine deaminase determination: an inter-laboratory comparison is required. J Infect 2014;68:103. 2. Piras MA, Gakis C, Budroni M, Andreoni G. Adenosine deaminase activity in pleural effusions: an aid to differential diagnosis. Br Med J 1978;2:1751e2. 3. Gakis C, Calia G, Naitana A, Pirino D, Serru G. Serum adenosine deaminase activity in HIV positive subjects. A hypothesis on the significance of ADA2. Panminerva Med 1989;31: 107e13. 4. Zavialov AV, Gracia E, Glaichenhaus N, Franco R, Zavialov AV, Lauvau G. Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. J Leukoc Biol 2010; 88:279e90. 5. Ipp H, Zemlin AE, Glashoff RH, van Wyk J, Vanker N, Reid T, et al. Serum adenosine deaminase and total immunoglobulin G correlate with markers of immune activation
10.
and inversely with CD4 counts in asymptomatic, treatment-naive HIV infection. J Clin Immunol 2013;33: 605e12. Pacheco R, Martinez-Navio JM, Lejeune M, Climent N, Oliva H, Gatell JM, et al. CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci USA 2005;102: 9583e8. Tay TR, Tee A. Factors affecting pleural fluid adenosine deaminase level and the implication on the diagnosis of tuberculous pleural effusion: a retrospective cohort study. BMC Infect Dis 2013;13:546. Lee SJ, Kim HS, Lee SH, Lee TW, Lee HR, Cho YJ, et al. Factors influencing pleural adenosine deaminase level in patients with tuberculous pleurisy. Am J Med Sci 2014;348:362e5. van Zyl-Smit RN, Binder A, Meldau R, Semple PL, Evans A, Smith P, et al. Cigarette smoke impairs cytokine responses and BCG containment in alveolar macrophages. Thorax 2014; 69:363e70. Yuan F, Fu X, Shi H, Chen G, Dong P, Zhang W. Induction of murine macrophage M2 polarization by cigarette smoke extract via the JAK2/STAT3 pathway. PLoS One 2014;9:e107063.
Chang Ho Kim Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea Jaehee Lee* Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom Jisook Lee Jacqueline M. Cliff Frederic Toulza Steven Smith Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
4
Letter to the Editor Seung Soo Yoo Shin Yup Lee Seung Ick Cha Jae Yong Park Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea
Hazel M. Dockrell Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom E-mail address: [email protected] Accepted 26 May 2015
* Corresponding author. Department of Internal Medicine, Kyungpook National University School of Medicine, 680 Gukchaebosangro, Jung-gu, Daegu, 700-842, Republic of Korea. Tel.: þ82 53 420 5536; fax: þ82 53 426 2046. Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
www.elsevierhealth.com/journals/jinf
LETTER TO THE EDITOR Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion
Dear Editor, We read with interest the article of Delacour et al. about the potential impact of the analytical systems on adenosine deaminase (ADA) determination.1 ADA has been widely used as a complementary diagnostic test for tuberculous pleural effusion (TPE) in tuberculosis (TB)-prevalent areas.2 The levels of ADA vary among TPE cases. Also, the diagnostic threshold values for TPE differ among studies. However, the factors associated with pleural fluid ADA levels in terms of study population remains still unclear. Thus, we examined the factors affecting pleural fluid ADA levels in TPE patients. Data from 353 consecutive patients who were diagnosed with TPE between 2009 and 2013 at Kyungpook National University Hospital, a South Korean tertiary referral hospital, were retrospectively reviewed. Of these, 202 patients with a confirmed TPE (positive Mycobacterium tuberculosis (MTB) culture [n Z 192] and histologic [n Z 10] results) were analyzed with regard to clinical, laboratory, radiological, and pleural fluid parameters. Linear regression analyses were performed to identify independent factors predictive of pleural fluid ADA levels. Univariate linear regression analysis identified a significant negative association between ADA levels and age (b Z 0.158, p Z 0.024), current smoking (b Z 0.170, p Z 0.016), pleural fluid glucose (b Z 0.396, p < 0.001), and pleural fluid pH levels (b Z 0.450, p < 0.001), and a significant positive association between ADA levels and loculated pleural effusion (b Z 0.257, p < 0.001), pleural fluid protein (b Z 0.431, p < 0.001), pleural fluid LDH levels (b Z 0.482, p < 0.001), and positive pleural fluid MTB PCR (b Z 0.211, p Z 0.004) and MTB culture results (b Z 0.295, p < 0.001) (Table 1). Stepwise multiple linear regression analysis identified current smoking (b Z 0.213, p < 0.001), pleural fluid protein (b Z 0.381, p < 0.001), LDH (b Z 0.312, p < 0.001), and glucose levels (b Z 0.139, p Z 0.040), and a positive MTB culture result (b Z 0.128, p Z 0.036) as independent factors associated with ADA levels.
To find further evidence to support a new finding of a positive association between the pleural fluid MTB load and ADA levels obtained from this single-center descriptive study, we performed in-vitro experiments using monocyte-derived macrophages (MDMs). Monocytes were purified from peripheral blood mononuclear cells of healthy volunteer donors and then were differentiated into macrophages using granulocyte-macrophage colonystimulating factor. MDMs were infected with MTB H37Rv at different multiplicity of infection (MOI) (1:1 and 10:1). The total ADA and ADA2 activities were assayed using a commercial Adenosine Deaminase Assay Kit (BQKITS Diagnostics, CA, USA) in unstimulated and MTB-infected MDM culture supernatants. In addition, ADA2 coding CECR1 (cat eye syndrome chromosome region, candidate 1) mRNA expression was measured in these MDM cells. Total RNA was extracted from the unstimulated and MTBinfected MDM cells by using an RNeasy mini kit (Qiagen, UK). Synthesis of the first-strand complementary DNA was performed using the SuperScript III First-Strand Synthesis System (Invitrogen, CA, USA). To evaluate the level of expression of CECR1 mRNA, real-time quantitative reverse transcriptase polymerase chain reaction was performed by using a 7500 Fast Real-Time PCR system (Applied Biosystems, CA, USA). Fig. 1A and B show the total ADA and ADA2 levels, respectively, from the unstimulated and MTB H37Rv-infected MDM culture supernatants. The median levels of total ADA and ADA2 from the culture supernatants of MDM infected with MTB H37Rv at a lower MOI (1:1) were significantly higher than those of unstimulated MDM culture supernatants (total ADA: 16.5 [interquartile range (IQR), 16.4e19.3] vs. 14.1 [11.9e16.1], p Z 0.003; ADA2: 15.3 [14.9e17.0] vs. 13.2 [12.1e16.1], p Z 0.035). The total ADA levels from MTBinfected MDM culture supernatants were significantly greater at a higher MOI (10:1) than at a lower MOI (1:1) (median [IQR]: 19.3 [17.5e21.6] vs. 16.5 [16.4e19.3] U/ L, p Z 0.003) (Fig. 1A). Similarly, ADA2 levels were significantly higher in the MDMs infected with MTB H37Rv at a higher MOI than at a lower MOI (median [IQR]: 17.8 [14.1e21.1] vs. 15.3 [14.9e17.0] U/L, p Z 0.014) (Fig. 1B). Relative levels of CECR1 mRNA expression were significantly greater in MTB H37Rv-infected MDM cells compared to unstimulated MDM cells (p Z 0.008 for MOI 10:1; p Z 0.008 for MOI 1:1) (Fig. 1C). CECR1
http://dx.doi.org/10.1016/j.jinf.2015.05.015 0163-4453/ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
2
Letter to the Editor Table 1 Factors affecting pleural fluid adenosine deaminase levels in 202 patients with a confirmed tuberculous pleural effusion. Variable
Median (IQR) or No (%) Univariate analysis B
Age, y Male Current smoker Immunosuppressive conditiona Symptoms lasting > 2 wks Fever Chest pain Laboratory findings WBC, cells/mL CRP, mg/dL Albumin, g/dL Radiographic findings Parenchymal lesionb Loculated effusion Pleural fluid analyses Total cell count, cells/mL Protein, g/dL LDH, IU/L Glucose, mg/dL pH Positive AFB smear Positive MTB PCR Positive MTB culture
SE
p Value Multivariate analysis
b
B
64 (39e74) 143 (71) 54 (27) 23 (11) 113 (56) 107 (53) 98 (49)
0.212 0.028 0.099 0.109 0.026 0.015 0.059
0.093 0.158 0.040 0.049 0.041 0.170 0.057 0.134 0.037 0.051 0.037 0.029 0.036 0.114
0.024 0.489 0.016 0.057 0.474 0.677 0.105
7275 (5713e8998) 6.4 (3.0e10.3) 3.3 (2.9e3.7)
0.063 0.113 0.040 0.054 0.036 0.104 0.013 0.193 0.005
0.576 0.142 0.945
155 (77) 63 (31)
0.003 0.043 0.006 0.938 0.144 0.038 0.257 <0.001
1500 (750e2800) 5.0 (4.5e5.6) 904 (570e1635) 97 (68e127) 7.42 (7.37e7.46) 3/200 (2)c 22/182 (12)c 71/199 (36)c
0.008 1.106 0.371 0.432 14.042 0.015 0.177 0.160
0.033 0.164 0.048 0.072 1.998 0.151 0.061 0.037
0.016 0.431 0.482 0.396 0.450 0.007 0.211 0.295
0.818 <0.001 <0.001 <0.001 <0.001 0.921 0.004 <0.001
SE
p Value
b
0.122 0.033 0.213 <0.001
1.011 0.156 0.381 <0.001 0.246 0.054 0.312 <0.001 0.157 0.076 0.139 0.040
0.069 0.032
0.128
0.036
IQR Z interquartile range; B Z unstandardized regression coefficient; SE Z standard error; b Z standardized regression coefficient; WBC Z white blood cell; CRP Z C-reactive protein; LDH Z lactate dehydrogenase; AFB Z acid-fast bacilli; MTB Z Mycobacterium tuberculosis; PCR Z polymerase chain reaction. All continuous variables were log-transformed. All categorical variables were classified as “0”, absent (or a negative result) or as “1”, present (or a positive result). There was no evidence of multicollinearity because the variance inflation factor for independent variables was less than 1.2 in all models. R2 (adjusted R2) Z 0.455 (0.439) for the goodness of fit (using multivariate analysis). a Includes patients with malignancy (n Z 9), immunosuppressive drug therapy (n Z 2), transplantation (n Z 1), end-stage renal disease (n Z 4), advanced chronic liver disease (n Z 4), and human immunodeficiency virus infection (n Z 3). b Includes consolidation, nodules, masses, cavities, and calcified lesions. c Expressed as positive number/available number (%).
mRNA expression was also significantly more abundant in MDMs infected with MTB at MOI 10:1 than at MOI 1:1 (p Z 0.027). ADA2, accounting for most of the total ADA activity in TPE, is elevated when monocyte-macrophages are infected with intracellular microorganisms such as human immunodeficiency virus (HIV) or MTB.2,3 The macrophage is the first line cell to encounter MTB at the site of infection and plays an essential role in the initiation and maintenance of immune response to MTB infection. ADA2 production by MTB-infected macrophages might therefore be involved in the innate immune response network to MTB infection.4 Our finding that ADA2 mRNA expression levels of MTBinfected MDM correlated with MTB load supports clinical observation that a positive MTB culture result affected pleural fluid ADA levels. This is similar to the previous finding that serum ADA concentrations are positively associated with viral load in patients with HIV infection.5 These increased ADA2 activities may play a role in counteracting
immune suppression mediated by adenosine which is increased at the time of infection.6 A correlation between ADA levels and the extent of pleural inflammation in this study is in accordance with previous studies.7 A recent study8 has also shown that smoking negatively affected ADA levels of TPE patients. Cigarette smoke attenuated pro-inflammatory cytokine rin-infected MDM.9 In production by Bacillus Calmette-Gue addition, macrophage polarization toward the M2 phenotype, exhibiting immunosuppressive or anti-inflammatory characteristics, was induced by cigarette smoke.10 Thus, a suppressive effect of smoking on the pro-inflammatory responses of macrophage or macrophage phenotype alteration may be associated with decreased ADA levels in TPE patients. In conclusion, the present study showed that pleural fluid ADA levels in TPE patients were associated with smoking status, the extent of pleural inflammation, and pleural MTB burden. And, ADA2 mRNA expression by MTB-
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
Letter to the Editor
3
Figure 1 Comparison of total adenosine deaminase (ADA) and ADA2 concentrations, and cat eye syndrome chromosome region, candidate 1 (CECR1) mRNA expression levels from the culture supernatants and cells of unstimulated and Mycobacterium tuberculosis (MTB) H37Rv-infected monocyte derived macrophages (MDMs). (A) and (B). Total ADA and ADA2 concentrations from the culture supernatants of unstimulated and MTB H37Rv-infected MDMs (multiplicity of infection [MOI] 1 and 10), respectively, were measured using ADA assay kit (n Z 18). Each box outline represents the 25th and 75th percentiles, the central line the median value, and the whiskers minimum and maximum values. (C). Relative levels of CECR1 mRNA expression were determined using real-time reverse transcription-polymerase chain reaction (n Z 8). Human acidic ribosomal protein (HuPO) served as an internal control. Each column and bar represent the mean and standard error values. NC Z unstimulated; MTB1 Z infected with MTB at MOI 1:1; MTB10 Z infected with MTB at MOI 10:1. Differences were analyzed by Wilcoxon-paired signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001.
infected MDMs was positively affected by MTB load. Taken together, these findings provide evidence to suggest that pleural fluid ADA levels of TPE patients are affected by pleural fluid MTB load. The pleural fluid mycobacterial burden needs to be taken into account when pleural fluid ADA levels are interpreted in patients suspected of having TPE.
6.
7.
Conflict of interest None of the authors have any conflicts of interest to declare.
8.
Acknowledgments
9.
JL, JC and SS receive salary support from the EU grants IDEA, TANDEM (Grant No. 305279), and NORAD.
References 1. Delacour H, Bousquet A, Fontan E, Ceppa F. Pleural adenosine deaminase determination: an inter-laboratory comparison is required. J Infect 2014;68:103. 2. Piras MA, Gakis C, Budroni M, Andreoni G. Adenosine deaminase activity in pleural effusions: an aid to differential diagnosis. Br Med J 1978;2:1751e2. 3. Gakis C, Calia G, Naitana A, Pirino D, Serru G. Serum adenosine deaminase activity in HIV positive subjects. A hypothesis on the significance of ADA2. Panminerva Med 1989;31: 107e13. 4. Zavialov AV, Gracia E, Glaichenhaus N, Franco R, Zavialov AV, Lauvau G. Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. J Leukoc Biol 2010; 88:279e90. 5. Ipp H, Zemlin AE, Glashoff RH, van Wyk J, Vanker N, Reid T, et al. Serum adenosine deaminase and total immunoglobulin G correlate with markers of immune activation
10.
and inversely with CD4 counts in asymptomatic, treatment-naive HIV infection. J Clin Immunol 2013;33: 605e12. Pacheco R, Martinez-Navio JM, Lejeune M, Climent N, Oliva H, Gatell JM, et al. CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci USA 2005;102: 9583e8. Tay TR, Tee A. Factors affecting pleural fluid adenosine deaminase level and the implication on the diagnosis of tuberculous pleural effusion: a retrospective cohort study. BMC Infect Dis 2013;13:546. Lee SJ, Kim HS, Lee SH, Lee TW, Lee HR, Cho YJ, et al. Factors influencing pleural adenosine deaminase level in patients with tuberculous pleurisy. Am J Med Sci 2014;348:362e5. van Zyl-Smit RN, Binder A, Meldau R, Semple PL, Evans A, Smith P, et al. Cigarette smoke impairs cytokine responses and BCG containment in alveolar macrophages. Thorax 2014; 69:363e70. Yuan F, Fu X, Shi H, Chen G, Dong P, Zhang W. Induction of murine macrophage M2 polarization by cigarette smoke extract via the JAK2/STAT3 pathway. PLoS One 2014;9:e107063.
Chang Ho Kim Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea Jaehee Lee* Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom Jisook Lee Jacqueline M. Cliff Frederic Toulza Steven Smith Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom
Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015
4
Letter to the Editor Seung Soo Yoo Shin Yup Lee Seung Ick Cha Jae Yong Park Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Republic of Korea
Hazel M. Dockrell Department of Immunology and Infection, London School of Hygiene & Tropical Medicine, London, United Kingdom E-mail address: [email protected] Accepted 26 May 2015
* Corresponding author. Department of Internal Medicine, Kyungpook National University School of Medicine, 680 Gukchaebosangro, Jung-gu, Daegu, 700-842, Republic of Korea. Tel.: þ82 53 420 5536; fax: þ82 53 426 2046. Please cite this article in press as: Kim CH, et al., Mycobacterial load affects adenosine deaminase 2 levels of tuberculous pleural effusion, J Infect (2015), http://dx.doi.org/10.1016/j.jinf.2015.05.015