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Human leukocyte antigen–G is upregulated in heart failure patients: A potential novel biomarker
Human Immunology 72 (2011) 1064-1067
Contents lists available at SciVerse ScienceDirect
Human leukocyte antigen–G is upregulated in heart failure patients: A potential novel biomarker Ali Almasood a,*, Rohit Sheshgiri b, Jemy M. Joseph b, Vivek Rao b, Mahsa Kamali b, Laura Tumiati b, Heather J. Ross b, Diego H. Delgado b a b
Prince Sultan Cardiac Center, Riyadh, Saudi Arabia University of Toronto, Toronto, Ontario, Canada
A R T I C L E
I N F O
Article history: Received 24 January 2011 Accepted 25 August 2011 Available online 1 September 2011
Keywords: Human leukocyte antigen-G Heart failure Biomarker
A B S T R A C T
Immune activation and inflammation play critical roles in the development of heart failure (HF). Human leukocyte antigen–G (HLA-G) is a nonclassical, major histocompatibility complex class I (MHC–I) protein, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. We sought to evaluate the utility of plasma HLA-G in identifying patients with HF. We conducted a single-center, cross-sectional pilot study involving 82 patients diagnosed with HF and 10 healthy controls. Concentrations of circulating HLA-G and inflammatory markers were detected with specific enzyme-linked immunosorbent assay kits and quantified according to purified protein standards. The mean age of the patients was 49.1 ⫾ 12.0 years and 62.2% were male. The median and interquartile range of HLA-G levels (U/ml) were significantly higher (p ⬍ 0.001) in HF patients (63, 36 –98) compared with controls (28, 22– 40). Moreover, HLA-G levels that were similarly (p ⫽ 0.766) upregulated across all New York Heart Association functional classes. There was no significant correlation between serum HLA-G and other biomarkers. In conclusion, HLA-G is upregulated in patients diagnosed with HF. Its marked elevation even in New York Heart Association class I patients might indicate that serum HLA-G is a more sensitive marker than other classical HF biomarkers. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Despite extensive research and the development of novel treatment options, heart failure (HF) remains a major and growing public health concern, with poor prognosis. It remains the only cardiovascular diagnosis to show an increasing incidence [1,2]. Several studies reported detection and infiltration of circulating immune and inflammatory mediators in the myocardium [3,4]. Mounting evidence has suggested that immune activation and inflammation play critical roles in the pathogenesis of HF [5,6]. The mechanisms by which immune and inflammatory responses are activated in HF and their affects on the myocardium are exceptionally complex and difficult to investigate [7]. Trials suggested that these proinflammatory and anti-inflammatory mediators might be used as a predictor for the development of HF, its progression, and the associated increase in mortality [8,9]. HLA-G is a nonclassical, major histocompatibility complex class I (MHC-I) molecule, described as a tolerogenic antigen primarily expressed by cytotrophoblast cells of the placenta [10,11]. To date, there are seven isoforms of HLA-G produced from alternate splicing of the mRNA transcript. These include four membrane-bound pro-
* Corresponding author. E-mail address: [email protected] (A.S. Almasood).
teins (HLA-G 1– 4) and three soluble proteins (HLA-G 5–7) [12]. Soluble HLA-G (sHLA-G) has immunosuppressive effects similar to those of the the cell-surface isoforms, which include the inhibition of cytotoxic T-lymphocytes and natural killer cells. HLA-G has been reported to play a role in mediating maternal tolerance of the fetal graft [11,13]. It is upregulated in response to transplantation [14,15], malignancies [16,17] and inflammation [18,19], and has also been correlated with various clinical outcomes. This molecule has potent immunoregulatory effects that are directed at different elements of the immune system. Because HF is in part characterized by activation of inflammatory markers, we sought to determine whether HLA-G might be expressed in HF patients and to compare serum concentrations with other clinical and laboratory markers of HF. 2. Subjects and methods 2.1. Study group This single-center, cross-sectional pilot study included patients followed in specialized HF clinics at our institution. We included 82 consecutive patients with a diagnosis of HF who were clinically stable at the time of enrollment in the study. HF patients were identified based on clinical history, physical examination and echocardiographic parameters. Demographic, clinical, and laboratory
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.016
A. Almasood et al. / Human Immunology 72 (2011) 1064-1067
variables were collected from the HF clinic database and hospital charts. The severity of HF was reported according to the NYHA classification. Patients with significant co-morbidities, including active infection, autoimmune disease or renal failure requiring renal replacement therapy were excluded. The study was approved by the ethical review board of the University Health Network. All patients provided written informed consent. We included 10 healthy individuals (six males and four females) as a control group for HLA-G measurement. 2.2. Biomarker measurement Blood samples for HLA-G and other biomarkers were collected into ethylenediaminetetraacetic acid (EDTA)– containing tubes and centrifuged for 20 minutes at 4⬚C. Plasma was isolated, flash frozen and stored at ⫺80⬚C within 4 hours of blood collection. Concentrations of circulating HLA-G and other cytokines, including interleukins (IL)–1b, IL-2, IL-6, IL-10, and tumor necrosis factor (TNF)–␣ were detected with specific antibodies by enzyme-linked immunosorbent assay (ELISA) kits and quantified according to purified protein standards (R&D Systems, Minneapolis, MN). The HLA-G kit detected plasma HLA-G1 and HLA-G5 isoforms only (BioVendor, Candler, NC.) All samples were run in duplicate. Measurements that fell at or below the minimum detectable dose specific to each kit were considered not detectable. 2.3. Cardiopulmonary exercise test A cardiopulmonary exercise test (CPET) was done at the time of a clinical visit unless contraindicated. Following a standard ramp protocol, patients performed upright bicycle exercise to maximum tolerance using a progressively increasing workload at rate of 10 to 20 W/min. Maximum oxygen consumption (VO2 Max), carbon dioxide production (VCO2), Minute ventilation (VE), respiratory exchange ratio (RER), VE/VCO2 slope (minute ventilation divided by carbon dioxide output), and anaerobic threshold (AT) were calculated. 2.4. Statistical analysis Data were tested for normality. Data were presented as frequencies and percentages for categorical variables, with mean ⫾ standard deviations for continuous variables with normal distribution, and median and interquartile range (IQR) for continuous variables with nonnormal distribution. Significant differences between HF patients and control as well as between NYHA classes and control were tested using the 2 for categorical variables (such as gender), Student t test for continuous parametric variables (such as age), and Mann–Whitney test for continuous nonparametric variables (as HLA-G). Because we have multiple (four) NYHA comparisons to control for, individual comparisons were made at the 0.0125 alpha level to keep overall risk of type I error equal to 0.05. Differences in HLA-G between NYHA classes were tested using the Kruskal– Wallis test. Correlations between HLA-G and other HF parameters were determined with Spearman’s rho statistic. A p value ⬍0.05 was considered significant. SPSS software (release 17.0, SPSS, Inc., Chicago, IL) was used for all statistical analyses. 3. Results 3.1. Patient characteristics The clinical characteristics of all 82 HF patients are summarized in Tables 1 and 2. Patients had mean age of 49.1 ⫾ 12.0 years (range, 24.7– 73.7 years), and males accounted for 62.2% of the HF population. The etiology of HF was ischemic cardiomyopathy in 27 patients (32.9%), idiopathic dilated cardiomyopathy in 23 (28.1%), congenital heart disease in 10 (12.2%) and others diagnoses in 22 (26.8%). The distribution of patients according to NYHA class was as follows: 12.2%, class I; 56.1%, class II; 20.7%, class III; and
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Table 1 Baseline demographic and clinical characteristics of heart failure (HF) patients Variable Age (y) Gender Male Female Body mass index Blood pressure (mm Hg) Systolic Diastolic Hypertension Diabetes Paroxysmal nocturnal dyspnea HF diagnosis ischemic Dilated cardiomyopahy Congenital Others NYHA class I II III IV Medications -Blockers Angiotensin-converting enzyme inhibitor Furosemide Hemoglobin (g/dl) Sodium (mEq/l) Potassium (mEq/l) Creatinine (mg/dl) B-type natriuretic peptide (pg/ml) HLA-G (U/ml)
Valuea 49.1 ⫾ 12.0 51 (62.2%) 31 (37.8%) 28.8 ⫾ 6.1 101.7 ⫾ 14.0 68.1 ⫾ 8.9 21 (25.9%) 12 (14.6%) 10 (12.2%) 27 (32.9%) 23 (28.1%) 10 (12.2%) 22 (26.8%) 10 (12.2%) 46 (56.1%) 17 (20.7%) 9 (11.0%) 77 (93.9%) 59 (72.0%) 52 (63.4%) 14.0 ⫾ 1.4 138.7 ⫾ 2.8 4.26 ⫾ 0.53 1.05 ⫾ 0.34 236 (85–536) 63 (36–98)
NYHA, New York Heart Association. Mean ⫾ standard deviation for normal continuous data, median (interquartile range) for non-normal continuous data, and number (%) for categorical data. a
11.0%, class IV. Mean of maximal oxygen consumption (VO2 Max) was 16.11 ⫾ 4.54 ml/kg/min. All patients underwent echocardiography. The average (%) of left ventricular ejection fraction (LVEF) was 31.5 ⫾ 11.4, with 21.4% (n ⫽ 15) having preserved ejection fraction (⬎40%); left ventricular end-diastolic diameter (LVDD) was 62.0 ⫾ 8.9 mm, and right ventricular systolic pressure (RVSP) was 36 (30 – 45) mm Hg. The majority of patients were on -blockers (93.9%), angiotensin-converting enzyme inhibitors (72.0%), and furosemide (63.4%). The median (interquartile range [IQR]) value of B-type natriuretic peptide (BNP) levels was 236 (85–536) pg/ml in the HF population. Levels of other biomarkers are shown in Table 2. The age of the control cohort (n ⫽ 10) was 33.4 ⫾ 5.7 years, with 60% being male. 3.2. HLA-G concentrations and association with HF markers HLA-G was measured in all HF patients and controls. HLA-G distribution was clearly skewed to the right. HLA-G levels were significantly higher in patients diagnosed with HF compared with the control cohort (p ⬍ 0.001). The mean HLA-G levels in the HF patients were 84 ⫾ 63 U/ml, with a median (IQR) of 63 (36 to 98) U/ml. The mean HLA-G levels in the healthy control group were 30 ⫾ 9 U/ml, with a median (IQR) of 28 (22 to 40) U/ml. HLA-G had a weak but significant negative correlation with age (r ⫽ ⫺0.22, p ⫽ 0.048) and was similar in both sexes (p ⫽ 0.616). There were no significant correlations between HLA-G and LVEF (R ⫽ 0.04, p ⫽ 0.716), VO2 max (r ⫽ ⫺0.02, p ⫽ 0.855), or BNP levels (r ⫽ 0.08, p ⫽ 0.501). In addition, there were no signification correlations between HLA-G level and other cytokines, including IL-1 (r ⫽ 0.12, p ⫽ 0.294), IL-6 (r ⫽ 0.03, p ⫽ 0.805), IL-10 (r ⫽ 0.10, p ⫽ 0.362), and TNF-␣ (r ⫽ 0.17, p ⫽ 0.129).
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A. Almasood et al. / Human Immunology 72 (2011) 1064-1067
Table 2 Correlations between clinical and biochemical markers of heart failure (HF) and New York Heart Association (NYHA) classes n
LVEF (%) LVDD (mm) RVSP (mm Hg) Vo2 max (ml/kg/min) BNP (pg/ml) HLA-G (U/ml) IL-1 (pg/ml) IL-6 (pg/ml) IL-10 (pg/ml) TNF-␣ (pg/ml)
70 70 65 82 82 82 81 81 82 82
Overall
31.5 ⫾ 11.4 62.0 ⫾ 8.9 36 (30–45) 16.11 ⫾ 4.54 236 (85–536) 63 (36–98) 0.0 (0.0–0.0) 1.7 (1.0–4.0) 0.0 (0.0–4.5) 0.0 (0.0–0.0)
NYHA classes I
II
III
IV
40.50 ⫾ 9.25 5.72 ⫾ 0.76 32 (26–36) 23.69 ⫾ 2.26 58 (31–123) 61 (35–76) 0.0 (0.0–0.0) 0.9 (0.0–1.6) 0.0 (0.0–6.1) 0.0 (0.0–0.0)
31.15 ⫾ 11.71 6.12 ⫾ 0.83 36 (31–43) 15.94 ⫾ 3.87 210 (95–493) 74 (43–104) 0.0 (0.0–0.0) 1.4 (0.8–2.2) 0.0 (0.0–0.9) 0.0 (0.0–0.0)
29.29 ⫾ 9.38 6.40 ⫾ 0.95 36 (29–56) 13.32 ⫾ 2.48 366 (84–1243) 70 (40–115) 0.0 (0.0–0.8) 4.9 (2.8–8.5) 0.0 (0.0–5.4) 0.0 (0.0–0.0)
25.14 ⫾ 11.04 6.91 ⫾ 0.94 43 (37–65) 13.84 ⫾ 3.45 519 (387–958) 64 (38–101) 0.0 (0.0–0.0) 3.7 (2.5–6.3) 0.0 (0.0–7.1) 0.0 (0.0–2.8)
Rhoa
p Value
⫺0.33 0.33 0.28 ⫺0.54 0.44 0.05 0.02 0.56 0.06 0.23
0.006 0.006 0.025 ⬍0.001 ⬍0.001 0.638 0.842 ⬍0.001 0.602 0.037
LVDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; RVSP, right ventricular systolic pressure. a Spearman correlation.
3.3. HLA-G and other biomarkers levels according to NYHA class Patients in all NYHA classes had HLA-G plasma levels that were significantly higher than those in the control group, even after adjusting for multiple comparisons (p ⫽ 0.003, p ⬍ 0.001, p ⫽ 0.001, and p ⫽ 0.007 for class I, II, III, and IV, respectively; Fig. 1). However, HLA-G levels were not significantly different among classes. Median levels for class I, II, III, and IV were 61 (35– 76), 74 (43–104), 70 (40 –115), and 64 (38 – 101), respectively (p ⫽ 0.766). LVEF and VO2 max were negatively correlated, whereas LVDD, RVSP, BNP, IL-6, and TNF-␣ were positively correlated, with increasing NYHA classes (Table 2). By contrast, HLA-G levels were not significantly correlated with NYHA class (r ⫽ 0.05, p ⫽ 0.638). 4. Discussion In this study, we demonstrated higher levels of serum HLA-G among HF patients compared with healthy controls. To our knowledge, this is the first report to suggest an association of HLA-G with HF. Serum HLA-G levels among the healthy controls in the current study (28, 22– 40 U/ml) was similar to those reported by previous studies (⬍20 U/ml) among in healthy individuals [20,21]. The increase on HLA-G levels among HF patients in the current study was independent of NYHA class, LVEF, or other HF biomarkers. Unlike the elevation of other HF markers (such as BNP and IL-6), which significantly correlated with higher NYHA classes, the higher levels of HLA-G were evident across all NYHA classes including the lower
ones. The finding may suggest that serum HLA-G could be a more sensitive HF biomarker than these markers but less useful as a measure for HF severity. However, the cross-sectional design used in the current study does not allow for testing the temporal relation between HLA-G level and HF course. Moreover, the fact that our patients are controlled HF patients who most likely have reduced their NYHA class with treatment may complicate the interpretation of the association between HLA-G level and HF course. HLA-G exerts anti-inflammatory and strong inhibitory effects that are directed at different levels of the immune system. The immunosuppressive role of HLA-G is apparent by its ability to inhibit CD8-T cell toxicity [22], CD4-T cell alloreactivity [23], and natural killer cell–mediated cytolysis [13], and activate regulatory CD4⫺ T cells [24]. Numerous studies have shown the relevance of HLA-G in several inflammatory and autoimmune diseases, such as systemic lupus erythematosis (SLE) [19]. HLA-G was found to be markedly elevated during septic shock, with a possible role in negative feedback signals that limit the process of inflammation [20]. It is now recognized that HF may perturb immune homeostasis by inducing a systemic inflammatory response that is followed by an anti-inflammatory process, acting as negative feedback [25,26]. This compensatory inhibitory response may become deleterious, as nearly all immune functions are compromised. Although there is no clear demonstration that these alterations are directly responsible for worsening outcome, it is speculated that they play a major role in the decreased resistance to infections in these patients. However, the mechanistic and molecular bases for HF induced immunomodulation have not been clearly established. The role that HLA-G may play in the immune response to HF is beyond the scope of the current study. Moreover, any suggested association between HLA-G an HF is further complicated by other confounding sources of HLA-G up-regulation as viral, inflammatory, neoplastic, and autoimmune diseases [16 –21]. In addition, nitric oxide (NO), which has been shown to mediate and to enhance spironolactone effect in HF patients [27,28], was recently linked to HLA-G up-regulation [29]. Unfortunately, these potential confounding cannot be tested using current data. 4.1. Study limitations and future perspectives
Fig. 1. Median and inter-quartile range (boxplot) of plasma HLA-G levels (U/ml) in different New York Heart Association (NYHA) classes of heart failure (HF) patients compared with healthy controls. p Values represent individual NYHA class– control comparisons.
Although the current study suggested a novel association between an immune marker and HF, we acknowledge several limitations. First, it was performed in a single tertiary academic cardiac center. Patients managed in our center might differ from those treated in primary care or community hospitals. Second, the study had limited data and a relatively small number of controls, which precludes detecting independent associations. Finally, although the cross-sectional design is important as a preliminary hypothesis testing, it cannot detect predictability or temporal relation. It will therefore be important to replicate the current research question in
A. Almasood et al. / Human Immunology 72 (2011) 1064-1067
a prospective cohort design with detailed clinical data among individuals with and without HF to better characterize any role that HLA-G may play in the immune response to HF. 4.2. Conclusion In conclusion, we have observed high levels of serum HLA-G in patients diagnosed with HF. Unlike the elevation of other HF markers (such as BNP and IL-6), which significantly correlated with higher NYHA classes, the higher levels of HLA-G were evident across all NYHA classes, including the lower ones. The finding may suggest that serum HLA-G could be a more sensitive HF biomarker than these markers. The role that HLA-G may play in the immune response to HF is beyond the scope of the current study and remains to be investigated. Acknowledgments The authors thank Susan Carson for assistance in data collection. References [1] Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, et al. Heart disease and stroke statistics—2007 update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2007o;115:e69 –171. [2] Liu P, Arnold M, Belenkie I, Howlett J, Huckell V, Ignazewski A, et al. The 2001 Canadian Cardiovascular Society consensus guideline update for the management and prevention of heart failure. Can J Cardiol 2001;17(Suppl E):5E–25E. [3] Gong KZ, Song G, Spiers JP, Kelso EJ, Zhang ZG. Activation of immune and inflammatory systems in chronic heart failure: Novel therapeutic approaches. Int J Clin Pract 2007;61:611–21. [4] Fedak PW, Verma S, Weisel RD, Li RK. Cardiac remodeling and failure: From molecules to man (part I). Cardiovasc Pathol 2005;14:1–11. [5] Braunwald E. Biomarkers in heart failure. N Engl J Med 2008;358:2148 –59. [6] Anker SD, von Haehling S. Inflammatory mediators in chronic heart failure: An overview. Heart 2004;90:464 –70. [7] Mehra VC, Ramgolam VS, Bender JR. Cytokines and cardiovascular disease. J Leukoc Biol 2005;78:805–18. [8] Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, Mann DL. Cytokines and cytokine receptors in advanced heart failure: An analysis of the cytokine database from the Vesnarinone Trial (VEST). Circulation 2001;103:2055–9. [9] Torre-Amione G, Kapadia S, Benedict C, Oral H, Young JB, Mann DL. Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: A report from the Studies of Left Ventricular Dysfunction (SOLVED). J Am Coll Cardiol 1999;27:1201– 6. [10] Carosella ED, Rouas-Freiss N, Paul P, Dausset J, HLA G. A tolerance molecule from the major histocompatibility complex. Immunol Today 1999;20:60 –2. [11] Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990;248:220 –3. [12] McMaster MT, Librach CL, Zhou Y, Lim KH, Janatpour MJ, DeMars R, et al. Human placental HLA-G expression is restricted to differentiated cytotrophoblasts. J Immunol 1995;154:3771– 8.
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[13] Rouas-Freiss N, GonÈalves RM, Menier C, Dausset J, Carosella ED. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci U S A 1997;94:11520 –5. [14] Lila N, Amrein C, Guillemain R, Chevalier P, Latremouille C, Fabiani JN, et al. Human leukocyte antigen-G expression after heart transplantation is associated with a reduced incidence of rejection. Circulation 2002; 105:1949 –54. [15] Creput C, Le Friec G, Bahri R, Amiot L, Charpentier B, Carosella E, et al. Detection of HLA-G in serum and graft biopsy associated with fewer acute rejections following combined liver-kidney transplantation: Possible implications for monitoring patients. Hum Immunol 2003;64:1033– 8. [16] Urosevic M, Kurrer MO, Kamarashev J, Mueller B, Weder W, Burg G, et al. Human leukocyte antigen G up-regulation in lung cancer associates with high-grade histology, human leukocyte antigen class I loss and interleukin-10 production. Am J Pathol 2001;159:817–24. [17] Urosevic M, Willers J, Mueller B, Kempf W, Burg G, Dummer R. HLA-G protein up-regulation in primary cutaneous lymphomas is associated with interleukin-10 expression in large cell T-cell lymphomas and indolent B-cell lymphomas. Blood 2002;99:609 –17. [18] Chen CH, Liao HT, Chen HA, Liu CH, Liang TH, Wang CT, et al. Human leukocyte antigen-G in ankylosing spondylitis and the response after tumour necrosis factor-alpha blocker therapy. Rheumatology (Oxford) 2010;49:264 –70. [19] Rosado S, Perez-Chacon G, Mellor-Pita S, Sanchez-Vegazo I, Bellas-Menendez C, Citores MJ, Losada-Fernandez I, Martin-Donaire T, Rebolleda N, PerezAciego P. Expression of human leukocyte antigen-G in systemic lupus erythematosus. Hum Immunol 2008;69:9 –15. [20] Morandi F, Levreri I, Bocca P, Galleni B, Raffaghello L, Ferrone S, et al. Human neuroblastoma cells trigger an immunosuppressive program in monocytes by stimulating soluble HLA-G release. Cancer Res 2007;67:6433– 41. [21] Torres MI, LÔpez-Casado MA, Luque J, PeÒa J, RÎos A. New advances in celiac disease: Serum and intestinal expression of HLA-G. Int Immunol 2006;18: 713– 8. [22] Fournel S, Aguerre-Girr M, Huc X, Lenfant F, Alam A, Toubert A, et al. Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand mediated apoptosis in activated CD8⫹ cells by interacting with CD8. J Immunol 2000;164:6100 – 4. [23] Lila N, Rouas-Freiss N, Dausset J, Carpentier A, Carosella ED. Soluble HLA-G protein secreted by allo-specific CD4⫹ T cells suppresses the allo-proliferative response: A CD4⫹ T cell regulatory mechanism. Proc Natl Acad Sci U S A 2001;98:12150 –5. [24] Paul P, Cabestre FA, Ibrahim EC, Lefebvre S, Khalil-Daher I, Vazeux G, et al. Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells. Hum Immunol 2000;61:1138 – 49. [25] Torre-Amione G. Immune activation in chronic heart failure. Am J Cardiol 2005;95:3C– 8C; discussion 38C– 40C. [26] Celis R, Torre-Martinez G, Torre-Amione G. Evidence for activation of immune system in heart failure: Is there a role for anti-inflammatory therapy? Curr Opin Cardiol 2008;23:254 – 60. [27] Ghali JK, Tam SW, Sabolinski ML, Taylor AL, Lindenfeld J, Cohn JN, et al. Exploring the potential synergistic action of spironolactone on nitric oxideenhancing therapy: Insights from the African-American Heart Failure Trial. J Card Fail 2008;14:718 –23. [28] Thai HM, Do BQ, Tran TD, Gaballa MA, Goldman S. Aldosterone antagonism improves endothelial-dependent vasorelaxation in heart failure via upregulation of endothelial nitric oxide synthase production. J Card Fail 2006;12: 240 –5. [29] DÎaz-Lagares A, Alegre E, LeMaoult J, Carosella ED, GonzÂlez A. Nitric oxide produces HLA-G nitration and induces metalloprotease-dependent shedding creating a tolerogenic milieu. Immunology 2009;126:436 – 45.
Contents lists available at SciVerse ScienceDirect
Human leukocyte antigen–G is upregulated in heart failure patients: A potential novel biomarker Ali Almasood a,*, Rohit Sheshgiri b, Jemy M. Joseph b, Vivek Rao b, Mahsa Kamali b, Laura Tumiati b, Heather J. Ross b, Diego H. Delgado b a b
Prince Sultan Cardiac Center, Riyadh, Saudi Arabia University of Toronto, Toronto, Ontario, Canada
A R T I C L E
I N F O
Article history: Received 24 January 2011 Accepted 25 August 2011 Available online 1 September 2011
Keywords: Human leukocyte antigen-G Heart failure Biomarker
A B S T R A C T
Immune activation and inflammation play critical roles in the development of heart failure (HF). Human leukocyte antigen–G (HLA-G) is a nonclassical, major histocompatibility complex class I (MHC–I) protein, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. We sought to evaluate the utility of plasma HLA-G in identifying patients with HF. We conducted a single-center, cross-sectional pilot study involving 82 patients diagnosed with HF and 10 healthy controls. Concentrations of circulating HLA-G and inflammatory markers were detected with specific enzyme-linked immunosorbent assay kits and quantified according to purified protein standards. The mean age of the patients was 49.1 ⫾ 12.0 years and 62.2% were male. The median and interquartile range of HLA-G levels (U/ml) were significantly higher (p ⬍ 0.001) in HF patients (63, 36 –98) compared with controls (28, 22– 40). Moreover, HLA-G levels that were similarly (p ⫽ 0.766) upregulated across all New York Heart Association functional classes. There was no significant correlation between serum HLA-G and other biomarkers. In conclusion, HLA-G is upregulated in patients diagnosed with HF. Its marked elevation even in New York Heart Association class I patients might indicate that serum HLA-G is a more sensitive marker than other classical HF biomarkers. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Despite extensive research and the development of novel treatment options, heart failure (HF) remains a major and growing public health concern, with poor prognosis. It remains the only cardiovascular diagnosis to show an increasing incidence [1,2]. Several studies reported detection and infiltration of circulating immune and inflammatory mediators in the myocardium [3,4]. Mounting evidence has suggested that immune activation and inflammation play critical roles in the pathogenesis of HF [5,6]. The mechanisms by which immune and inflammatory responses are activated in HF and their affects on the myocardium are exceptionally complex and difficult to investigate [7]. Trials suggested that these proinflammatory and anti-inflammatory mediators might be used as a predictor for the development of HF, its progression, and the associated increase in mortality [8,9]. HLA-G is a nonclassical, major histocompatibility complex class I (MHC-I) molecule, described as a tolerogenic antigen primarily expressed by cytotrophoblast cells of the placenta [10,11]. To date, there are seven isoforms of HLA-G produced from alternate splicing of the mRNA transcript. These include four membrane-bound pro-
* Corresponding author. E-mail address: [email protected] (A.S. Almasood).
teins (HLA-G 1– 4) and three soluble proteins (HLA-G 5–7) [12]. Soluble HLA-G (sHLA-G) has immunosuppressive effects similar to those of the the cell-surface isoforms, which include the inhibition of cytotoxic T-lymphocytes and natural killer cells. HLA-G has been reported to play a role in mediating maternal tolerance of the fetal graft [11,13]. It is upregulated in response to transplantation [14,15], malignancies [16,17] and inflammation [18,19], and has also been correlated with various clinical outcomes. This molecule has potent immunoregulatory effects that are directed at different elements of the immune system. Because HF is in part characterized by activation of inflammatory markers, we sought to determine whether HLA-G might be expressed in HF patients and to compare serum concentrations with other clinical and laboratory markers of HF. 2. Subjects and methods 2.1. Study group This single-center, cross-sectional pilot study included patients followed in specialized HF clinics at our institution. We included 82 consecutive patients with a diagnosis of HF who were clinically stable at the time of enrollment in the study. HF patients were identified based on clinical history, physical examination and echocardiographic parameters. Demographic, clinical, and laboratory
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.016
A. Almasood et al. / Human Immunology 72 (2011) 1064-1067
variables were collected from the HF clinic database and hospital charts. The severity of HF was reported according to the NYHA classification. Patients with significant co-morbidities, including active infection, autoimmune disease or renal failure requiring renal replacement therapy were excluded. The study was approved by the ethical review board of the University Health Network. All patients provided written informed consent. We included 10 healthy individuals (six males and four females) as a control group for HLA-G measurement. 2.2. Biomarker measurement Blood samples for HLA-G and other biomarkers were collected into ethylenediaminetetraacetic acid (EDTA)– containing tubes and centrifuged for 20 minutes at 4⬚C. Plasma was isolated, flash frozen and stored at ⫺80⬚C within 4 hours of blood collection. Concentrations of circulating HLA-G and other cytokines, including interleukins (IL)–1b, IL-2, IL-6, IL-10, and tumor necrosis factor (TNF)–␣ were detected with specific antibodies by enzyme-linked immunosorbent assay (ELISA) kits and quantified according to purified protein standards (R&D Systems, Minneapolis, MN). The HLA-G kit detected plasma HLA-G1 and HLA-G5 isoforms only (BioVendor, Candler, NC.) All samples were run in duplicate. Measurements that fell at or below the minimum detectable dose specific to each kit were considered not detectable. 2.3. Cardiopulmonary exercise test A cardiopulmonary exercise test (CPET) was done at the time of a clinical visit unless contraindicated. Following a standard ramp protocol, patients performed upright bicycle exercise to maximum tolerance using a progressively increasing workload at rate of 10 to 20 W/min. Maximum oxygen consumption (VO2 Max), carbon dioxide production (VCO2), Minute ventilation (VE), respiratory exchange ratio (RER), VE/VCO2 slope (minute ventilation divided by carbon dioxide output), and anaerobic threshold (AT) were calculated. 2.4. Statistical analysis Data were tested for normality. Data were presented as frequencies and percentages for categorical variables, with mean ⫾ standard deviations for continuous variables with normal distribution, and median and interquartile range (IQR) for continuous variables with nonnormal distribution. Significant differences between HF patients and control as well as between NYHA classes and control were tested using the 2 for categorical variables (such as gender), Student t test for continuous parametric variables (such as age), and Mann–Whitney test for continuous nonparametric variables (as HLA-G). Because we have multiple (four) NYHA comparisons to control for, individual comparisons were made at the 0.0125 alpha level to keep overall risk of type I error equal to 0.05. Differences in HLA-G between NYHA classes were tested using the Kruskal– Wallis test. Correlations between HLA-G and other HF parameters were determined with Spearman’s rho statistic. A p value ⬍0.05 was considered significant. SPSS software (release 17.0, SPSS, Inc., Chicago, IL) was used for all statistical analyses. 3. Results 3.1. Patient characteristics The clinical characteristics of all 82 HF patients are summarized in Tables 1 and 2. Patients had mean age of 49.1 ⫾ 12.0 years (range, 24.7– 73.7 years), and males accounted for 62.2% of the HF population. The etiology of HF was ischemic cardiomyopathy in 27 patients (32.9%), idiopathic dilated cardiomyopathy in 23 (28.1%), congenital heart disease in 10 (12.2%) and others diagnoses in 22 (26.8%). The distribution of patients according to NYHA class was as follows: 12.2%, class I; 56.1%, class II; 20.7%, class III; and
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Table 1 Baseline demographic and clinical characteristics of heart failure (HF) patients Variable Age (y) Gender Male Female Body mass index Blood pressure (mm Hg) Systolic Diastolic Hypertension Diabetes Paroxysmal nocturnal dyspnea HF diagnosis ischemic Dilated cardiomyopahy Congenital Others NYHA class I II III IV Medications -Blockers Angiotensin-converting enzyme inhibitor Furosemide Hemoglobin (g/dl) Sodium (mEq/l) Potassium (mEq/l) Creatinine (mg/dl) B-type natriuretic peptide (pg/ml) HLA-G (U/ml)
Valuea 49.1 ⫾ 12.0 51 (62.2%) 31 (37.8%) 28.8 ⫾ 6.1 101.7 ⫾ 14.0 68.1 ⫾ 8.9 21 (25.9%) 12 (14.6%) 10 (12.2%) 27 (32.9%) 23 (28.1%) 10 (12.2%) 22 (26.8%) 10 (12.2%) 46 (56.1%) 17 (20.7%) 9 (11.0%) 77 (93.9%) 59 (72.0%) 52 (63.4%) 14.0 ⫾ 1.4 138.7 ⫾ 2.8 4.26 ⫾ 0.53 1.05 ⫾ 0.34 236 (85–536) 63 (36–98)
NYHA, New York Heart Association. Mean ⫾ standard deviation for normal continuous data, median (interquartile range) for non-normal continuous data, and number (%) for categorical data. a
11.0%, class IV. Mean of maximal oxygen consumption (VO2 Max) was 16.11 ⫾ 4.54 ml/kg/min. All patients underwent echocardiography. The average (%) of left ventricular ejection fraction (LVEF) was 31.5 ⫾ 11.4, with 21.4% (n ⫽ 15) having preserved ejection fraction (⬎40%); left ventricular end-diastolic diameter (LVDD) was 62.0 ⫾ 8.9 mm, and right ventricular systolic pressure (RVSP) was 36 (30 – 45) mm Hg. The majority of patients were on -blockers (93.9%), angiotensin-converting enzyme inhibitors (72.0%), and furosemide (63.4%). The median (interquartile range [IQR]) value of B-type natriuretic peptide (BNP) levels was 236 (85–536) pg/ml in the HF population. Levels of other biomarkers are shown in Table 2. The age of the control cohort (n ⫽ 10) was 33.4 ⫾ 5.7 years, with 60% being male. 3.2. HLA-G concentrations and association with HF markers HLA-G was measured in all HF patients and controls. HLA-G distribution was clearly skewed to the right. HLA-G levels were significantly higher in patients diagnosed with HF compared with the control cohort (p ⬍ 0.001). The mean HLA-G levels in the HF patients were 84 ⫾ 63 U/ml, with a median (IQR) of 63 (36 to 98) U/ml. The mean HLA-G levels in the healthy control group were 30 ⫾ 9 U/ml, with a median (IQR) of 28 (22 to 40) U/ml. HLA-G had a weak but significant negative correlation with age (r ⫽ ⫺0.22, p ⫽ 0.048) and was similar in both sexes (p ⫽ 0.616). There were no significant correlations between HLA-G and LVEF (R ⫽ 0.04, p ⫽ 0.716), VO2 max (r ⫽ ⫺0.02, p ⫽ 0.855), or BNP levels (r ⫽ 0.08, p ⫽ 0.501). In addition, there were no signification correlations between HLA-G level and other cytokines, including IL-1 (r ⫽ 0.12, p ⫽ 0.294), IL-6 (r ⫽ 0.03, p ⫽ 0.805), IL-10 (r ⫽ 0.10, p ⫽ 0.362), and TNF-␣ (r ⫽ 0.17, p ⫽ 0.129).
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Table 2 Correlations between clinical and biochemical markers of heart failure (HF) and New York Heart Association (NYHA) classes n
LVEF (%) LVDD (mm) RVSP (mm Hg) Vo2 max (ml/kg/min) BNP (pg/ml) HLA-G (U/ml) IL-1 (pg/ml) IL-6 (pg/ml) IL-10 (pg/ml) TNF-␣ (pg/ml)
70 70 65 82 82 82 81 81 82 82
Overall
31.5 ⫾ 11.4 62.0 ⫾ 8.9 36 (30–45) 16.11 ⫾ 4.54 236 (85–536) 63 (36–98) 0.0 (0.0–0.0) 1.7 (1.0–4.0) 0.0 (0.0–4.5) 0.0 (0.0–0.0)
NYHA classes I
II
III
IV
40.50 ⫾ 9.25 5.72 ⫾ 0.76 32 (26–36) 23.69 ⫾ 2.26 58 (31–123) 61 (35–76) 0.0 (0.0–0.0) 0.9 (0.0–1.6) 0.0 (0.0–6.1) 0.0 (0.0–0.0)
31.15 ⫾ 11.71 6.12 ⫾ 0.83 36 (31–43) 15.94 ⫾ 3.87 210 (95–493) 74 (43–104) 0.0 (0.0–0.0) 1.4 (0.8–2.2) 0.0 (0.0–0.9) 0.0 (0.0–0.0)
29.29 ⫾ 9.38 6.40 ⫾ 0.95 36 (29–56) 13.32 ⫾ 2.48 366 (84–1243) 70 (40–115) 0.0 (0.0–0.8) 4.9 (2.8–8.5) 0.0 (0.0–5.4) 0.0 (0.0–0.0)
25.14 ⫾ 11.04 6.91 ⫾ 0.94 43 (37–65) 13.84 ⫾ 3.45 519 (387–958) 64 (38–101) 0.0 (0.0–0.0) 3.7 (2.5–6.3) 0.0 (0.0–7.1) 0.0 (0.0–2.8)
Rhoa
p Value
⫺0.33 0.33 0.28 ⫺0.54 0.44 0.05 0.02 0.56 0.06 0.23
0.006 0.006 0.025 ⬍0.001 ⬍0.001 0.638 0.842 ⬍0.001 0.602 0.037
LVDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; RVSP, right ventricular systolic pressure. a Spearman correlation.
3.3. HLA-G and other biomarkers levels according to NYHA class Patients in all NYHA classes had HLA-G plasma levels that were significantly higher than those in the control group, even after adjusting for multiple comparisons (p ⫽ 0.003, p ⬍ 0.001, p ⫽ 0.001, and p ⫽ 0.007 for class I, II, III, and IV, respectively; Fig. 1). However, HLA-G levels were not significantly different among classes. Median levels for class I, II, III, and IV were 61 (35– 76), 74 (43–104), 70 (40 –115), and 64 (38 – 101), respectively (p ⫽ 0.766). LVEF and VO2 max were negatively correlated, whereas LVDD, RVSP, BNP, IL-6, and TNF-␣ were positively correlated, with increasing NYHA classes (Table 2). By contrast, HLA-G levels were not significantly correlated with NYHA class (r ⫽ 0.05, p ⫽ 0.638). 4. Discussion In this study, we demonstrated higher levels of serum HLA-G among HF patients compared with healthy controls. To our knowledge, this is the first report to suggest an association of HLA-G with HF. Serum HLA-G levels among the healthy controls in the current study (28, 22– 40 U/ml) was similar to those reported by previous studies (⬍20 U/ml) among in healthy individuals [20,21]. The increase on HLA-G levels among HF patients in the current study was independent of NYHA class, LVEF, or other HF biomarkers. Unlike the elevation of other HF markers (such as BNP and IL-6), which significantly correlated with higher NYHA classes, the higher levels of HLA-G were evident across all NYHA classes including the lower
ones. The finding may suggest that serum HLA-G could be a more sensitive HF biomarker than these markers but less useful as a measure for HF severity. However, the cross-sectional design used in the current study does not allow for testing the temporal relation between HLA-G level and HF course. Moreover, the fact that our patients are controlled HF patients who most likely have reduced their NYHA class with treatment may complicate the interpretation of the association between HLA-G level and HF course. HLA-G exerts anti-inflammatory and strong inhibitory effects that are directed at different levels of the immune system. The immunosuppressive role of HLA-G is apparent by its ability to inhibit CD8-T cell toxicity [22], CD4-T cell alloreactivity [23], and natural killer cell–mediated cytolysis [13], and activate regulatory CD4⫺ T cells [24]. Numerous studies have shown the relevance of HLA-G in several inflammatory and autoimmune diseases, such as systemic lupus erythematosis (SLE) [19]. HLA-G was found to be markedly elevated during septic shock, with a possible role in negative feedback signals that limit the process of inflammation [20]. It is now recognized that HF may perturb immune homeostasis by inducing a systemic inflammatory response that is followed by an anti-inflammatory process, acting as negative feedback [25,26]. This compensatory inhibitory response may become deleterious, as nearly all immune functions are compromised. Although there is no clear demonstration that these alterations are directly responsible for worsening outcome, it is speculated that they play a major role in the decreased resistance to infections in these patients. However, the mechanistic and molecular bases for HF induced immunomodulation have not been clearly established. The role that HLA-G may play in the immune response to HF is beyond the scope of the current study. Moreover, any suggested association between HLA-G an HF is further complicated by other confounding sources of HLA-G up-regulation as viral, inflammatory, neoplastic, and autoimmune diseases [16 –21]. In addition, nitric oxide (NO), which has been shown to mediate and to enhance spironolactone effect in HF patients [27,28], was recently linked to HLA-G up-regulation [29]. Unfortunately, these potential confounding cannot be tested using current data. 4.1. Study limitations and future perspectives
Fig. 1. Median and inter-quartile range (boxplot) of plasma HLA-G levels (U/ml) in different New York Heart Association (NYHA) classes of heart failure (HF) patients compared with healthy controls. p Values represent individual NYHA class– control comparisons.
Although the current study suggested a novel association between an immune marker and HF, we acknowledge several limitations. First, it was performed in a single tertiary academic cardiac center. Patients managed in our center might differ from those treated in primary care or community hospitals. Second, the study had limited data and a relatively small number of controls, which precludes detecting independent associations. Finally, although the cross-sectional design is important as a preliminary hypothesis testing, it cannot detect predictability or temporal relation. It will therefore be important to replicate the current research question in
A. Almasood et al. / Human Immunology 72 (2011) 1064-1067
a prospective cohort design with detailed clinical data among individuals with and without HF to better characterize any role that HLA-G may play in the immune response to HF. 4.2. Conclusion In conclusion, we have observed high levels of serum HLA-G in patients diagnosed with HF. Unlike the elevation of other HF markers (such as BNP and IL-6), which significantly correlated with higher NYHA classes, the higher levels of HLA-G were evident across all NYHA classes, including the lower ones. The finding may suggest that serum HLA-G could be a more sensitive HF biomarker than these markers. The role that HLA-G may play in the immune response to HF is beyond the scope of the current study and remains to be investigated. Acknowledgments The authors thank Susan Carson for assistance in data collection. References [1] Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, et al. Heart disease and stroke statistics—2007 update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2007o;115:e69 –171. [2] Liu P, Arnold M, Belenkie I, Howlett J, Huckell V, Ignazewski A, et al. The 2001 Canadian Cardiovascular Society consensus guideline update for the management and prevention of heart failure. Can J Cardiol 2001;17(Suppl E):5E–25E. [3] Gong KZ, Song G, Spiers JP, Kelso EJ, Zhang ZG. Activation of immune and inflammatory systems in chronic heart failure: Novel therapeutic approaches. Int J Clin Pract 2007;61:611–21. [4] Fedak PW, Verma S, Weisel RD, Li RK. Cardiac remodeling and failure: From molecules to man (part I). Cardiovasc Pathol 2005;14:1–11. [5] Braunwald E. Biomarkers in heart failure. N Engl J Med 2008;358:2148 –59. [6] Anker SD, von Haehling S. Inflammatory mediators in chronic heart failure: An overview. Heart 2004;90:464 –70. [7] Mehra VC, Ramgolam VS, Bender JR. Cytokines and cardiovascular disease. J Leukoc Biol 2005;78:805–18. [8] Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, Mann DL. Cytokines and cytokine receptors in advanced heart failure: An analysis of the cytokine database from the Vesnarinone Trial (VEST). Circulation 2001;103:2055–9. [9] Torre-Amione G, Kapadia S, Benedict C, Oral H, Young JB, Mann DL. Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: A report from the Studies of Left Ventricular Dysfunction (SOLVED). J Am Coll Cardiol 1999;27:1201– 6. [10] Carosella ED, Rouas-Freiss N, Paul P, Dausset J, HLA G. A tolerance molecule from the major histocompatibility complex. Immunol Today 1999;20:60 –2. [11] Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990;248:220 –3. [12] McMaster MT, Librach CL, Zhou Y, Lim KH, Janatpour MJ, DeMars R, et al. Human placental HLA-G expression is restricted to differentiated cytotrophoblasts. J Immunol 1995;154:3771– 8.
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