Inflammation ( # 2014) DOI: 10.1007/s10753-014-9831-3
Elevated Serum IL-21 Levels in Hantavirus-Infected Patients Correlate with the Severity of the Disease Hongmei Chen,1 Hongli Liu,1 Yuefei Wang,1 Yucong Yang,1 and Yingren Zhao1,2
Abstract—Hemorrhagic fever with renal syndrome (HFRS) is an acute viral infection caused by Hantavirus (HTV). Capillary leakage is one of the hallmarks of HTV infection. The mechanisms underlying the pathogenesis of HFRS are not completely understood. However, it has been suggested that immune mechanisms, including cytokines, might have an important role in HFRS pathogenesis. In this study, we investigated the potential role of interleukin-21 (IL-21) which is a newly discovered cytokine that stimulates T-cell and B-cell responses in the pathogenesis of HFRS. Serum samples were collected from 58 patients hospitalized with laboratory-verified HTV infection and 20 healthy controls. Serum IL-21 concentration was measured using an enzyme-linked immunosorbent assay. Serum IL-21 levels began to increase in the fever phase when renal damage appeared. The highest serum IL-21 level was detected in oliguric phase along with the peak degree of urinary renal impairment. When entering the polyuric phase, with gradual increase in urine and recovered renal function, the serum IL-21 level was observed to fall, returning to normal level after the renal function recovered in the convalescent phase. The serum IL21 level was positively correlated with blood urea nitrogen (BUN) and creatinine (Cr), suggesting that the serum IL-21 level is associated with the disease severity of HFRS. This study indicated that IL-21 may act as an important inflammatory mediator in the immunopathogenesis of HFRS. The strategy to control IL-21 may hamper the immune response in patients with HFRS. KEY WORDS: IL-21; cytokine; HFRS.
INTRODUCTION Hemorrhagic fever with renal syndrome (HFRS) is an acute viral disease that is caused by infection with the Hantavirus (HTV) [1, 2]. There may be as many as 150,000 cases of HFRS reported annually worldwide, with more than half occurring in China [3]. Typically, there are five clinical phases in HFRS patients: fever, hypotensive shock, oliguric, polyuric and convalescent phase. The symptoms usually start with fever, then next is a drop in blood pressure, referred as fever and hypotensive-shock phase, respectively, which last for a couple of days, followed by the oliguric phase. In the oliguric phase, most patients develop acute renal failure, which lasts for days to 1
Department of Infectious Diseases, First Affiliated Hospital of Medical College, Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi 710061, China 2 To whom correspondence should be addressed at Department of Infectious Diseases, First Affiliated Hospital of Medical College, Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi 710061, China. E-mail: [email protected]
weeks before diuresis begins. The polyuric phase usually signals recovery and the patient become diuretic. The duration of the polyuric phase ranged from days to weeks, and even months in a few cases. Then enter convalescent phase [2, 4]. Several phases overlapping phenomenon often can be seen in severe cases, but some phases often been ignored in mild cases. HFRS is caused by HTV and immunopathogenesis has been suggested to be involved in HFRS. A common feature of HTV diseases is increased microvascular bed permeability, suggesting that vascular endothelium is a prime target of virus infection. Monocytes/macrophages also represent the important target cells, which may also have an important role in the spread of HTV from the primary site of infection. Both endothelial cells and monocytes/macrophages may act as a rich source of cytokines/ chemokines during the infection with HTV and additionally contribute to the HFRS immunopathogenesis. The efficient anti-hantaviral cell-immune response in patients is mainly due to the generation of cytotoxic T-lymphocytes (CTL). Specific IgM antibodies appear at the very
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Chen, Liu, Wang, Yang, and Zhao beginning of the diseases followed by an increase in specific IgG. In addition, IgE antibodies have been suggested to play a role in HFRS immunopathogenesis. Immune complexes have been detected in the sera, on the surface of red blood cells and platelets, in urine, glomerulus and dermal capillaries of HFRS patients. They may be responsible for increased capillary permeability, vascular injury, platelet lysis and kidney damage [5]. Interleukin-21 (IL-21) is a newly discovered cytokine, which is produced mainly by T-cells and acts on a variety of different immune and non-immune cells, controlling immune responses during innate and acquired immunity under physiologic conditions as well as in inflammatory and autoimmune diseases. T-cells, B-cells and NK-cells are the major target cells of IL-21 activity. IL-21 stimulates T-cell proliferation, supports B-cell differentiation into plasma cells, modulates IgE production and induces NK-cell differentiation [6, 7]. IL-21 was found to be associated with many inflammatory and autoimmune diseases, such as Crohn’s diseases, type 1 insulin-dependent diabetes, psoriasis, systemic sclerosis, rheumatoid arthritis and systematic lupus erythematosus [8–12]. Many studies demonstrated that IL21 is required to control chronic viral infection. In chronic lymphocytic choriomeningitis virus infection of mice, higher levels of IL-21 produced by virus-specific CD4 Tcells were shown to be essential for sustaining antiviral effector functions of CD8 T-cells and preventing them from immune exhaustion. [13–17] In chronic hepatitis B virus infected patients, increase in serum concentrations of IL-21 was associated with activation of the antiviral immune responses that could control hepatitis B virus replication [18]. In patients infected with the human immunodeficiency virus (HIV), existing data suggest that IL-21 could play an important role in modulating T- and B-cell function, thereby influencing the immunodeficiency associated with it [19, 20]. Up to date, few study reports can be found on the characters of IL-21 in acute viral infections. Based on the studies discussed above, we think that IL-21 may play a key role in the viral clearance and inflammatory reaction in the pathogenesis of HFRS. In China, the majority of causative strains of HFRS are known as Hantaan and Seoul viruses, which are the etiological agents for severe and mild types of HFRS, respectively [21, 22]. The epidemics involving the Hantaan and Seoul viruses have relatively stable geographical distributions [21, 22]. In view of the possible involvement of IL-21 in HFRS, changes of IL-21 levels in HFRS patients from a Hantaan virus epidemic area were determined at various stages of the disease and the relationships with disease progression and severity were analyzed in the present
study. These findings suggest that imbalance in production of proinflammatory and regulatory cytokines might be in part responsible for a more severe course of HFRS.
SUBJECTS AND METHODS Subjects The study protocol was approved by a joint institutional review board at First Affiliated Hospital and Xi’an Jiao Tong University (Xi’an China). The study subjects were composed of two populations: 58 patients (22 females and 36 males) who were prospectively identified as HFRS enrolled in this study between 2011 and 2012 at the Department of Infectious Diseases at the First Affiliated Hospital of Xi’an Jiao Tong University (Xi’an, China); and 20 healthy controls recruited from students and staff of the First Affiliated Hospital of Xi’an Jiao Tong University. All cases were clinically diagnosed by the detection of IgM (immunoglobulin M) specific antibodies to HTV in the patients’ serum specimens. According to the diagnosis criteria from the Prevention and Treatment Strategy of HFRS published by the Ministry of Health, People’s Republic of China [23], the disease severity in the patients is classified into four clinical types, namely, mild, moderate, severe, and gravis types: (1) mild patients with mild renal failure without an obvious oliguric stage; (2) moderate for those with obvious symptoms of uremia, effusion (bulbar conjunctiva), hemorrhage (skin and mucous membrane), and renal failure with a typical oliguric stage; (3) severe patients with severe uremia, effusion (bulbar conjunctiva and either peritoneum or pleura), hemorrhage (skin and mucous membrane), and renal failure with oliguria (urine output, 50–500 ml/day) for ≤5 days or anuria (urine output, 5 days, anuria (urine output, 2 days, or a blood urea nitrogen (BUN) level of >42.84 mmol/l. Moreover, according to the clinical observations, HFRS can be divided into five sequential stages: fever, hypotensive shock, oliguric, polyuric and convalescent. The patients who had other viral infection, kidney disease, diabetes, cardiovascular disease, hematological disease, autoimmune disease, viral hepatitis, and other liver diseases were excluded. From the first day of admission, blood samples were collected at different clinical phases.
IL-21 Levels in Patients with HFRS Ethical Considerations The study was conducted according to the Declaration of Helsinki guidelines, and was approved by the Ethical Committee of the First Affiliated Hospital of Xi’an Jiao Tong University. Written informed consent was obtained from all participants. Methods Detection of Serum IL-21 Serum IL-21 concentration was measured in duplicate using a commercial human IL-21 Platinum ELISA kit (eBioscience) according to the manufacturer’s instructions. The lowest detection limit of the kit was 20 pg/ml. Detection of BUN and Cr Two milliliters of fresh blood was collected aseptically from the vein into tubes containing sodium heparin in the morning. One milliliter of plasma was incubated in a 37°C water bath for 1 min. Plasma was isolated by centrifugation at 3,000 rpm for 4 min. BUN and creatinine (Cr) levels were measured by the CHEMIX-180 automated biochemistry analyzer (Sysmex, Kobe, Japan) following the manufacturer’s instructions. Statistical Analysis All statistical analyses were performed using SPSS version 17.0. The parametric data were analyzed using a one-way analysis of variance (ANOVA). For the homogeneity test of variance, Levene’s test was used. Dunnett’s T3 test was used in case of heterogeneity of variance among multiple groups. Student’s t-test was used to compare IL21 levels between two independent groups. Association between variables was evaluated with Spearman’s correlation coefficient. All statistical analyses were based on two-tailed hypothesis tests with a significance level of p
Elevated Serum IL-21 Levels in Hantavirus-Infected Patients Correlate with the Severity of the Disease Hongmei Chen,1 Hongli Liu,1 Yuefei Wang,1 Yucong Yang,1 and Yingren Zhao1,2
Abstract—Hemorrhagic fever with renal syndrome (HFRS) is an acute viral infection caused by Hantavirus (HTV). Capillary leakage is one of the hallmarks of HTV infection. The mechanisms underlying the pathogenesis of HFRS are not completely understood. However, it has been suggested that immune mechanisms, including cytokines, might have an important role in HFRS pathogenesis. In this study, we investigated the potential role of interleukin-21 (IL-21) which is a newly discovered cytokine that stimulates T-cell and B-cell responses in the pathogenesis of HFRS. Serum samples were collected from 58 patients hospitalized with laboratory-verified HTV infection and 20 healthy controls. Serum IL-21 concentration was measured using an enzyme-linked immunosorbent assay. Serum IL-21 levels began to increase in the fever phase when renal damage appeared. The highest serum IL-21 level was detected in oliguric phase along with the peak degree of urinary renal impairment. When entering the polyuric phase, with gradual increase in urine and recovered renal function, the serum IL-21 level was observed to fall, returning to normal level after the renal function recovered in the convalescent phase. The serum IL21 level was positively correlated with blood urea nitrogen (BUN) and creatinine (Cr), suggesting that the serum IL-21 level is associated with the disease severity of HFRS. This study indicated that IL-21 may act as an important inflammatory mediator in the immunopathogenesis of HFRS. The strategy to control IL-21 may hamper the immune response in patients with HFRS. KEY WORDS: IL-21; cytokine; HFRS.
INTRODUCTION Hemorrhagic fever with renal syndrome (HFRS) is an acute viral disease that is caused by infection with the Hantavirus (HTV) [1, 2]. There may be as many as 150,000 cases of HFRS reported annually worldwide, with more than half occurring in China [3]. Typically, there are five clinical phases in HFRS patients: fever, hypotensive shock, oliguric, polyuric and convalescent phase. The symptoms usually start with fever, then next is a drop in blood pressure, referred as fever and hypotensive-shock phase, respectively, which last for a couple of days, followed by the oliguric phase. In the oliguric phase, most patients develop acute renal failure, which lasts for days to 1
Department of Infectious Diseases, First Affiliated Hospital of Medical College, Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi 710061, China 2 To whom correspondence should be addressed at Department of Infectious Diseases, First Affiliated Hospital of Medical College, Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi 710061, China. E-mail: [email protected]
weeks before diuresis begins. The polyuric phase usually signals recovery and the patient become diuretic. The duration of the polyuric phase ranged from days to weeks, and even months in a few cases. Then enter convalescent phase [2, 4]. Several phases overlapping phenomenon often can be seen in severe cases, but some phases often been ignored in mild cases. HFRS is caused by HTV and immunopathogenesis has been suggested to be involved in HFRS. A common feature of HTV diseases is increased microvascular bed permeability, suggesting that vascular endothelium is a prime target of virus infection. Monocytes/macrophages also represent the important target cells, which may also have an important role in the spread of HTV from the primary site of infection. Both endothelial cells and monocytes/macrophages may act as a rich source of cytokines/ chemokines during the infection with HTV and additionally contribute to the HFRS immunopathogenesis. The efficient anti-hantaviral cell-immune response in patients is mainly due to the generation of cytotoxic T-lymphocytes (CTL). Specific IgM antibodies appear at the very
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Chen, Liu, Wang, Yang, and Zhao beginning of the diseases followed by an increase in specific IgG. In addition, IgE antibodies have been suggested to play a role in HFRS immunopathogenesis. Immune complexes have been detected in the sera, on the surface of red blood cells and platelets, in urine, glomerulus and dermal capillaries of HFRS patients. They may be responsible for increased capillary permeability, vascular injury, platelet lysis and kidney damage [5]. Interleukin-21 (IL-21) is a newly discovered cytokine, which is produced mainly by T-cells and acts on a variety of different immune and non-immune cells, controlling immune responses during innate and acquired immunity under physiologic conditions as well as in inflammatory and autoimmune diseases. T-cells, B-cells and NK-cells are the major target cells of IL-21 activity. IL-21 stimulates T-cell proliferation, supports B-cell differentiation into plasma cells, modulates IgE production and induces NK-cell differentiation [6, 7]. IL-21 was found to be associated with many inflammatory and autoimmune diseases, such as Crohn’s diseases, type 1 insulin-dependent diabetes, psoriasis, systemic sclerosis, rheumatoid arthritis and systematic lupus erythematosus [8–12]. Many studies demonstrated that IL21 is required to control chronic viral infection. In chronic lymphocytic choriomeningitis virus infection of mice, higher levels of IL-21 produced by virus-specific CD4 Tcells were shown to be essential for sustaining antiviral effector functions of CD8 T-cells and preventing them from immune exhaustion. [13–17] In chronic hepatitis B virus infected patients, increase in serum concentrations of IL-21 was associated with activation of the antiviral immune responses that could control hepatitis B virus replication [18]. In patients infected with the human immunodeficiency virus (HIV), existing data suggest that IL-21 could play an important role in modulating T- and B-cell function, thereby influencing the immunodeficiency associated with it [19, 20]. Up to date, few study reports can be found on the characters of IL-21 in acute viral infections. Based on the studies discussed above, we think that IL-21 may play a key role in the viral clearance and inflammatory reaction in the pathogenesis of HFRS. In China, the majority of causative strains of HFRS are known as Hantaan and Seoul viruses, which are the etiological agents for severe and mild types of HFRS, respectively [21, 22]. The epidemics involving the Hantaan and Seoul viruses have relatively stable geographical distributions [21, 22]. In view of the possible involvement of IL-21 in HFRS, changes of IL-21 levels in HFRS patients from a Hantaan virus epidemic area were determined at various stages of the disease and the relationships with disease progression and severity were analyzed in the present
study. These findings suggest that imbalance in production of proinflammatory and regulatory cytokines might be in part responsible for a more severe course of HFRS.
SUBJECTS AND METHODS Subjects The study protocol was approved by a joint institutional review board at First Affiliated Hospital and Xi’an Jiao Tong University (Xi’an China). The study subjects were composed of two populations: 58 patients (22 females and 36 males) who were prospectively identified as HFRS enrolled in this study between 2011 and 2012 at the Department of Infectious Diseases at the First Affiliated Hospital of Xi’an Jiao Tong University (Xi’an, China); and 20 healthy controls recruited from students and staff of the First Affiliated Hospital of Xi’an Jiao Tong University. All cases were clinically diagnosed by the detection of IgM (immunoglobulin M) specific antibodies to HTV in the patients’ serum specimens. According to the diagnosis criteria from the Prevention and Treatment Strategy of HFRS published by the Ministry of Health, People’s Republic of China [23], the disease severity in the patients is classified into four clinical types, namely, mild, moderate, severe, and gravis types: (1) mild patients with mild renal failure without an obvious oliguric stage; (2) moderate for those with obvious symptoms of uremia, effusion (bulbar conjunctiva), hemorrhage (skin and mucous membrane), and renal failure with a typical oliguric stage; (3) severe patients with severe uremia, effusion (bulbar conjunctiva and either peritoneum or pleura), hemorrhage (skin and mucous membrane), and renal failure with oliguria (urine output, 50–500 ml/day) for ≤5 days or anuria (urine output, 5 days, anuria (urine output, 2 days, or a blood urea nitrogen (BUN) level of >42.84 mmol/l. Moreover, according to the clinical observations, HFRS can be divided into five sequential stages: fever, hypotensive shock, oliguric, polyuric and convalescent. The patients who had other viral infection, kidney disease, diabetes, cardiovascular disease, hematological disease, autoimmune disease, viral hepatitis, and other liver diseases were excluded. From the first day of admission, blood samples were collected at different clinical phases.
IL-21 Levels in Patients with HFRS Ethical Considerations The study was conducted according to the Declaration of Helsinki guidelines, and was approved by the Ethical Committee of the First Affiliated Hospital of Xi’an Jiao Tong University. Written informed consent was obtained from all participants. Methods Detection of Serum IL-21 Serum IL-21 concentration was measured in duplicate using a commercial human IL-21 Platinum ELISA kit (eBioscience) according to the manufacturer’s instructions. The lowest detection limit of the kit was 20 pg/ml. Detection of BUN and Cr Two milliliters of fresh blood was collected aseptically from the vein into tubes containing sodium heparin in the morning. One milliliter of plasma was incubated in a 37°C water bath for 1 min. Plasma was isolated by centrifugation at 3,000 rpm for 4 min. BUN and creatinine (Cr) levels were measured by the CHEMIX-180 automated biochemistry analyzer (Sysmex, Kobe, Japan) following the manufacturer’s instructions. Statistical Analysis All statistical analyses were performed using SPSS version 17.0. The parametric data were analyzed using a one-way analysis of variance (ANOVA). For the homogeneity test of variance, Levene’s test was used. Dunnett’s T3 test was used in case of heterogeneity of variance among multiple groups. Student’s t-test was used to compare IL21 levels between two independent groups. Association between variables was evaluated with Spearman’s correlation coefficient. All statistical analyses were based on two-tailed hypothesis tests with a significance level of p
