Accepted Manuscript Title: Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse Author: Yaqing Shu, Rui Li, Yu Yang, Yongqiang Dai, Wei Qiu, Ying Chen, Zhihua Zhao, Zhengqi Lu, Xueqiang Hu PII: DOI: Reference:
S0197-0186(14)00255-1 http://dx.doi.org/doi: 10.1016/j.neuint.2014.12.008 NCI 3664
To appear in:
Neurochemistry International
Received date: Revised date: Accepted date:
26-7-2014 10-12-2014 22-12-2014
Please cite this article as: Yaqing Shu, Rui Li, Yu Yang, Yongqiang Dai, Wei Qiu, Ying Chen, Zhihua Zhao, Zhengqi Lu, Xueqiang Hu, Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse, Neurochemistry International (2015), http://dx.doi.org/doi: 10.1016/j.neuint.2014.12.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse Yaqing Shu, Rui Li, Yu Yang, Yongqiang Dai, Wei Qiu, Ying Chen, Zhihua Zhao, Zhengqi Lu, Xueqiang Hu#
Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510630, China
#
Corresponding author: Xueqiang Hu
Department of Neurology, The Third Affiliated Hospital, Sun Yat-Sen University 600 Tianhe Road, Guangzhou, Guangdong 510630, China Tel/Fax: +86-20-8751-6822; Email: [email protected]
Page 1 of 18
Abstract Background: Neutrophil-mediated inflammation plays an important role in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO). Concentrations of urinary trypsin inhibitor (UTI) have attracted attention as a useful index of the status of inflammatory response. Evidence suggests serum UTI levels are increased in some inflammatory diseases, but little attention has been paid to cerebrospinal fluid (CSF) levels of UTI. Objective: To measure CSF concentration of UTI and determine its relationship with disease activity in MS and NMO. Methods: CSF UTI was measured by an enzyme-linked immunosorbent assay in 18 MS patients, 28 NMO patients and eight controls. Results: CSF UTI concentrations in MS and NMO groups were both significantly lower than those in controls. Expanded disability status scale scores of MS and NMO revealed a trend of increased disease disability with decreased CSF UTI level. The CSF UTI concentrations were not significantly associated with CSF white blood cell counts, total protein, glucose and chlorine concentrations in MS and NMO subgroups. Conclusions: Our results indicate a reduced UTI level in CSF of MS and NMO patients. The levels were associated with the severity of the two demyelinating diseases during relapse.
Page 2 of 18
Highlights: 1. CSF UTI levels were reduced in MS and NMO patients during relapse. 2. CSF UTI levels were negatively associated with MS and NMO severity during relapse. 3. CSF UTI levels were not significantly associated with routine CSF in MS and NMO.
Page 3 of 18
Keywords: Urinary trypsin inhibitor; Cerebrospinal fluid; Multiple sclerosis; Neuromyelitis optica.
Abbreviations: Multiple sclerosis, MS; neuromyelitis optica, NMO; central nervous system, CNS; experimental autoimmune encephalomyelitis, EAE; urinary trypsin inhibitor, UTI; inter-α-trypsin inhibitor, ITI; cerebrospinal fluid, CSF; Expanded Disability Status Scale, EDSS; enzyme-linked immunosorbent assay, ELISA; oligoclonal bands, OCB; neutrophil elastase, NE; white blood cells, WBC; total protein, TP; glucose, Glu; chlorine, CL.
Page 4 of 18
1. Introduction Multiple sclerosis (MS) and neuromyelitis optic (NMO) are two typical inflammatory demyelinating diseases of the central nervous system (CNS). The pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, an animal model for MS) was characterized by the infiltration of CD4+T lymphocytes and other leukocytes into the CNS (Raine 1994). Recently, neutrophil-mediated inflammation was shown to play an important role in demyelinating diseases (Amadio and Volonte 2013; Ferrari et al. 2004; Herges et al. 2012; Liu et al. 2010; Naegele et al. 2012; Steinbach et al. 2013; Zehntner et al. 2005). In EAE, neutrophils that infiltrated the central nervous system could regulate T cell responses (Zehntner et al. 2005), and neutrophil granulocytes were important contributors to CNS inflammation (Steinbach et al. 2013). Urinary trypsin inhibitor (UTI) is one of the Kunitz-type trypsin inhibitors found in blood (Yasui et al. 2003), urine (Shikimi et al. 1994) and cerebrospinal fluid (CSF) (Shikimi et al. 1997). UTI can be synthesized by the inter-α-trypsin inhibitor (ITI) family. ITIs are degraded by neutrophil elastase (NE) to liberate UTI (Hirose et al. 1998). Several lines of evidence suggest that UTI has multiple effects including anti-inflammation (Chen et al. 2013; Fang et al. 2011; Huang et al. 2013; Kanai et al. 2011), anti-apoptosis (Hu et al. 2013; Wang et al. 2012), anti-proteases (Ohnishi et al. 1984), and neuroprotection (Cao et al. 2011; Li et al. 2013; Yano et al. 2003). Recently, our
previous
studies
showed
that
UTI
could
protect
against
EAE
by
anti-oligodendrocyte apoptosis (Shu et al. 2011) and anti-inflammation (Feng et al. 2014). In addition, UTI concentrations have already attracted attention as a useful index of the status of systemic inflammatory response syndrome (Mizon et al. 2002; Yamada et al. 2001). Yasui and colleagues found that serum concentration of UTI was
Page 5 of 18
increased in asthma exacerbation and proposed that monitoring UTI concentrations might be useful for evaluating the neutrophil-mediated inflammation in asthma attack (Yasui et al. 2003). Shikimi et al. reported lower CSF UTI levels in dementia compared with controls and that CSF UTI levels were associated with the severity of dementia (Shikimi et al. 1997). However, whether levels of CSF UTI change in MS and NMO patients is unknown. We performed a hospital-based study to measure CSF UTI concentration in MS and NMO patients to determine the correlation between CSF UTI level and clinical characteristics; these include routine CSF, disease duration and severity assessed by Expanded Disability Status Scale (EDSS) score (Kurtzke 1983).
2. Materials and Methods
2.1 Patients and controls Twenty-eight NMO patients based on the 2006 Wingerchuk diagnostic criteria (Wingerchuk et al. 2006) and 18 relapsing-remitting MS patients fulfilling 2010 McDonald’s diagnostic criteria (Polman et al. 2005) from the Demyelinating Disease Database of Neurology Department of the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China were enrolled. Eight controls with non-inflammatory and non-neurodegenerative diseases were recruited (cervical spondylosis n=5, migraine n=3). All patients had been hospitalized once or more during 2012 to 2013. Patients enrolled from the database were diagnosed by two neurologists specialized in CNS inflammatory demyelinating diseases. CSF samples were obtained during diagnostic lumbar punctures during relapses. All samples were taken during relapse phase before treatment. A clinical relapse was defined as a sudden appearance of new
Page 6 of 18
symptoms, lasting for at least 24 h, with an increase of EDSS over 1.0 before sampling. Patients were not receiving IFN-β or any other immunomodulatory therapies during remission. Demographic and clinical features of the patients are shown in Table 1. Indirect immunofluorescence test systems for human aquaporin 4 antibodies (AQP4-Ab, also called neuromyelitis optica immunoglobulin G (NMO-IgG)) detection from EUROIMMUN (Medizinische Labordiagnostika, Lübeck, Germany) were used. AQP4-Ab was assessed following the manufacturer’s instructions. All participants involved in this study provided written informed consent.
2.2 Study of CSF samples CSF samples were obtained during diagnostic lumbar punctures. CSF white blood cell (WBC) count, total protein concentration (TP), glucose (GLU) and chloride (CL), and oligoclonal bands (OCB) were determined by the hospital laboratories of the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
2.3 Enzyme-linked immunosorbent assay (ELISA) All CSF samples were processed within 30 min of withdrawal and stored at -80 °C until assay. UTI content was measured by a commercial ELISA kit (Cloud-Clone Corp, Houston, TX, USA). The detection limits of the assay were 0.47–30 ng/ml. The optimal dilution (1:1000) was determined and the ELISA was performed according to the manufacturer’s instructions.
2.4 Statistical analysis
Page 7 of 18
The data were presented as the mean ± standard deviation (CSF UTI, WBC, TP, Glu and CL levels) or median with range (onset age, disease duration, annualized relapse rate, and EDSS score). Differences in levels of CSF UTI between different subgroups were analyzed using the Mann-Whitney U test. Correlations between CSF UTI and CSF routine (CSF WBC, TP, GLU and CL) were analyzed using Spearman’s rank test. A P value
S0197-0186(14)00255-1 http://dx.doi.org/doi: 10.1016/j.neuint.2014.12.008 NCI 3664
To appear in:
Neurochemistry International
Received date: Revised date: Accepted date:
26-7-2014 10-12-2014 22-12-2014
Please cite this article as: Yaqing Shu, Rui Li, Yu Yang, Yongqiang Dai, Wei Qiu, Ying Chen, Zhihua Zhao, Zhengqi Lu, Xueqiang Hu, Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse, Neurochemistry International (2015), http://dx.doi.org/doi: 10.1016/j.neuint.2014.12.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse Yaqing Shu, Rui Li, Yu Yang, Yongqiang Dai, Wei Qiu, Ying Chen, Zhihua Zhao, Zhengqi Lu, Xueqiang Hu#
Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510630, China
#
Corresponding author: Xueqiang Hu
Department of Neurology, The Third Affiliated Hospital, Sun Yat-Sen University 600 Tianhe Road, Guangzhou, Guangdong 510630, China Tel/Fax: +86-20-8751-6822; Email: [email protected]
Page 1 of 18
Abstract Background: Neutrophil-mediated inflammation plays an important role in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO). Concentrations of urinary trypsin inhibitor (UTI) have attracted attention as a useful index of the status of inflammatory response. Evidence suggests serum UTI levels are increased in some inflammatory diseases, but little attention has been paid to cerebrospinal fluid (CSF) levels of UTI. Objective: To measure CSF concentration of UTI and determine its relationship with disease activity in MS and NMO. Methods: CSF UTI was measured by an enzyme-linked immunosorbent assay in 18 MS patients, 28 NMO patients and eight controls. Results: CSF UTI concentrations in MS and NMO groups were both significantly lower than those in controls. Expanded disability status scale scores of MS and NMO revealed a trend of increased disease disability with decreased CSF UTI level. The CSF UTI concentrations were not significantly associated with CSF white blood cell counts, total protein, glucose and chlorine concentrations in MS and NMO subgroups. Conclusions: Our results indicate a reduced UTI level in CSF of MS and NMO patients. The levels were associated with the severity of the two demyelinating diseases during relapse.
Page 2 of 18
Highlights: 1. CSF UTI levels were reduced in MS and NMO patients during relapse. 2. CSF UTI levels were negatively associated with MS and NMO severity during relapse. 3. CSF UTI levels were not significantly associated with routine CSF in MS and NMO.
Page 3 of 18
Keywords: Urinary trypsin inhibitor; Cerebrospinal fluid; Multiple sclerosis; Neuromyelitis optica.
Abbreviations: Multiple sclerosis, MS; neuromyelitis optica, NMO; central nervous system, CNS; experimental autoimmune encephalomyelitis, EAE; urinary trypsin inhibitor, UTI; inter-α-trypsin inhibitor, ITI; cerebrospinal fluid, CSF; Expanded Disability Status Scale, EDSS; enzyme-linked immunosorbent assay, ELISA; oligoclonal bands, OCB; neutrophil elastase, NE; white blood cells, WBC; total protein, TP; glucose, Glu; chlorine, CL.
Page 4 of 18
1. Introduction Multiple sclerosis (MS) and neuromyelitis optic (NMO) are two typical inflammatory demyelinating diseases of the central nervous system (CNS). The pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, an animal model for MS) was characterized by the infiltration of CD4+T lymphocytes and other leukocytes into the CNS (Raine 1994). Recently, neutrophil-mediated inflammation was shown to play an important role in demyelinating diseases (Amadio and Volonte 2013; Ferrari et al. 2004; Herges et al. 2012; Liu et al. 2010; Naegele et al. 2012; Steinbach et al. 2013; Zehntner et al. 2005). In EAE, neutrophils that infiltrated the central nervous system could regulate T cell responses (Zehntner et al. 2005), and neutrophil granulocytes were important contributors to CNS inflammation (Steinbach et al. 2013). Urinary trypsin inhibitor (UTI) is one of the Kunitz-type trypsin inhibitors found in blood (Yasui et al. 2003), urine (Shikimi et al. 1994) and cerebrospinal fluid (CSF) (Shikimi et al. 1997). UTI can be synthesized by the inter-α-trypsin inhibitor (ITI) family. ITIs are degraded by neutrophil elastase (NE) to liberate UTI (Hirose et al. 1998). Several lines of evidence suggest that UTI has multiple effects including anti-inflammation (Chen et al. 2013; Fang et al. 2011; Huang et al. 2013; Kanai et al. 2011), anti-apoptosis (Hu et al. 2013; Wang et al. 2012), anti-proteases (Ohnishi et al. 1984), and neuroprotection (Cao et al. 2011; Li et al. 2013; Yano et al. 2003). Recently, our
previous
studies
showed
that
UTI
could
protect
against
EAE
by
anti-oligodendrocyte apoptosis (Shu et al. 2011) and anti-inflammation (Feng et al. 2014). In addition, UTI concentrations have already attracted attention as a useful index of the status of systemic inflammatory response syndrome (Mizon et al. 2002; Yamada et al. 2001). Yasui and colleagues found that serum concentration of UTI was
Page 5 of 18
increased in asthma exacerbation and proposed that monitoring UTI concentrations might be useful for evaluating the neutrophil-mediated inflammation in asthma attack (Yasui et al. 2003). Shikimi et al. reported lower CSF UTI levels in dementia compared with controls and that CSF UTI levels were associated with the severity of dementia (Shikimi et al. 1997). However, whether levels of CSF UTI change in MS and NMO patients is unknown. We performed a hospital-based study to measure CSF UTI concentration in MS and NMO patients to determine the correlation between CSF UTI level and clinical characteristics; these include routine CSF, disease duration and severity assessed by Expanded Disability Status Scale (EDSS) score (Kurtzke 1983).
2. Materials and Methods
2.1 Patients and controls Twenty-eight NMO patients based on the 2006 Wingerchuk diagnostic criteria (Wingerchuk et al. 2006) and 18 relapsing-remitting MS patients fulfilling 2010 McDonald’s diagnostic criteria (Polman et al. 2005) from the Demyelinating Disease Database of Neurology Department of the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China were enrolled. Eight controls with non-inflammatory and non-neurodegenerative diseases were recruited (cervical spondylosis n=5, migraine n=3). All patients had been hospitalized once or more during 2012 to 2013. Patients enrolled from the database were diagnosed by two neurologists specialized in CNS inflammatory demyelinating diseases. CSF samples were obtained during diagnostic lumbar punctures during relapses. All samples were taken during relapse phase before treatment. A clinical relapse was defined as a sudden appearance of new
Page 6 of 18
symptoms, lasting for at least 24 h, with an increase of EDSS over 1.0 before sampling. Patients were not receiving IFN-β or any other immunomodulatory therapies during remission. Demographic and clinical features of the patients are shown in Table 1. Indirect immunofluorescence test systems for human aquaporin 4 antibodies (AQP4-Ab, also called neuromyelitis optica immunoglobulin G (NMO-IgG)) detection from EUROIMMUN (Medizinische Labordiagnostika, Lübeck, Germany) were used. AQP4-Ab was assessed following the manufacturer’s instructions. All participants involved in this study provided written informed consent.
2.2 Study of CSF samples CSF samples were obtained during diagnostic lumbar punctures. CSF white blood cell (WBC) count, total protein concentration (TP), glucose (GLU) and chloride (CL), and oligoclonal bands (OCB) were determined by the hospital laboratories of the Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
2.3 Enzyme-linked immunosorbent assay (ELISA) All CSF samples were processed within 30 min of withdrawal and stored at -80 °C until assay. UTI content was measured by a commercial ELISA kit (Cloud-Clone Corp, Houston, TX, USA). The detection limits of the assay were 0.47–30 ng/ml. The optimal dilution (1:1000) was determined and the ELISA was performed according to the manufacturer’s instructions.
2.4 Statistical analysis
Page 7 of 18
The data were presented as the mean ± standard deviation (CSF UTI, WBC, TP, Glu and CL levels) or median with range (onset age, disease duration, annualized relapse rate, and EDSS score). Differences in levels of CSF UTI between different subgroups were analyzed using the Mann-Whitney U test. Correlations between CSF UTI and CSF routine (CSF WBC, TP, GLU and CL) were analyzed using Spearman’s rank test. A P value
