Background
Chemokines attract various types of leukocytes to sites of infection and inflammation and act as immunoregulatory molecules in driving T-helper (Th)1/Th2 responses [
1]. The role of different chemokines and chemokine receptors in the pathogenesis of multiple sclerosis (MS) has been extensively investigated [
2‐
5]; however, little is known about the role of chemokine receptors in the pathogenesis of neuromyelitis optica (NMO).
NMO is an immune-mediated inflammatory disorder of the central nervous system and predominantly affects the optic nerves and spinal cord [
6]. Recent evidences such as the discovery of serum anti-aquaporin-4 (AQP4) antibody in NMO patients [
7] (now recognised as a specific biomarker for NMO), differences in pathology [
8], neuroimaging [
9], cytokine profiles [
10,
11] and responses to some immunotherapies between MS and NMO [
12], supports the hypothesis that NMO is distinct from MS.
In the present study, we report the expression of chemokine receptors on peripheral blood lymphocytes (PBL) in NMO and MS during relapse compared with healthy controls (HC) to determine differences in the expression of chemokine receptors between these disorders.
Methods
Patients
This study included 12 Japanese patients with relapsing NMO fulfilling Wingerchuk's revised criteria [
6] that uses seropositivity of the anti-AQP4 antibody [
13] instead of NMO-IgG. In addition, the study included 24 patients with relapsing-remitting MS fulfilling McDonald's criteria [
14]. Their whole blood samples were obtained during relapse, which was defined as the period within one month of clinical relapse and before beginning relapse treatment. Blood samples from 25 healthy participants (9 women and 16 men; mean age, 32.7 years) served as controls. In addition, blood samples during remission were obtained from only 3 of the 12 NMO patients and 5 of the 24 MS patients.
We also reviewed the clinical characteristics of NMO and MS patients such as gender, age, disease duration, expanded disability status scale (EDSS) and the proportion of patients receiving immunomodulating treatment.
Ethics approval was granted by the Ethics Committee of Chiba University School of Medicine, Chiba, Japan. All study subjects gave informed consent for their participation.
Flow cytometry
Blood samples collected in heparinised tubes were washed with phosphate buffered saline (PBS), supplemented with 0.5% foetal calf serum and resuspended in PBS with 0.5% foetal calf serum. Subsequently, the aliquots of the cell suspensions were double-stained with fluorescein isothiocyanate- and phycoerythrin-labelled monoclonal antibodies for 30 min at 4°C in the dark. Table
1 summarises the combinations of monoclonal antibodies used to identify chemokine receptor-positive cells as well as for other lymphocyte subsets. After lysing the red blood cells with FACS Lysing Solution (BD Biosciences, San Jose, CA, USA.), the cells were washed with PBS and resuspended in 0.5 ml PBS. IgG1 coupled to fluorescein isothiocyanate and IgG2a coupled to phycoerythrin were used as negative controls. The percentages of chemokine receptor-positive cells or lymphocyte subsets were obtained using a FACScan (BD Biosciences).
Table 1
Combinations of monoclonal antibodies used.
CCR3c × CD4b
| CD3a × CD19a
|
CD4b × CCR4a
| CD3a × CD4a
|
CCR5c ×CD4b
| CD3a × CD8a
|
CXCR3c × CD4b
| CD4b × CD25a
|
CD8b × CCR4a
| CD45RAa × CD4b
|
CXCR3c × CD8b
| CD4b × CD45ROa
|
Statistical analysis
For baseline variables, the groups were compared using Fisher's exact test for categorical outcomes, the Mann-Whitney U test for continuous variables and paired t-test for paired continuous measures. All comparisons were planned, tests were two-sided and P values < 0.05 were considered to be statistically significant. In addition, multiple testing problems were resolved by applying the Bonferroni correction on the computed P values to reduce type I errors. All statistical analyses were performed using SPSS 16.0J (SPSS Japan Inc., Tokyo, Japan).
Discussion
In this study, we analysed the expression of chemokine receptors on circulating PBL during relapse in NMO and MS patients and compare it with that of HC. We confirmed the dominance of the Th1- and Tc1-related chemokine receptors in MS patients; however, no remarkable differences were observed between NMO and HC patients.
Th1 and Th2 subsets of lymphocytes can be characterised by their expression of chemokine receptors. CXCR3 and CCR5 are associated with a Th1 phenotype, whereas CCR3 and CCR4 are expressed preferentially on activated Th2 cells [
15]. Several studies have reported Th1 dominance over the Th2 response associated with chemokine receptors in MS, increased percentages of T cells expressing CXCR3 and CCR5 chemokine receptors on PBL and cerebrospinal fluid (CSF) in the active phase of MS and the identification of CXCR3
+ and CCR5
+ T cells in active demyelinating MS brain lesions [
2,
3]. Th1 dominance over the Th2 response is also indicated by decreased expression of the chemokine receptor CCR4 on blood T cells and brain tissue [
4,
5]. The results of our study indicates higher expression of Th1-associated chemokine receptors CD4
+CXCR3
+ on peripheral T cells and lower expression of Th2-associated chemokine receptors such as CD4
+CCR3
+ in MS patients than in HC, which is in accordance with the results of the above-mentioned reports. Ratios of CD4
+CXCR3
+/CD4
+CCR4
+ and CD4
+CXCR3
+/CD4
+CCR3
+ were higher in MS patients than HC, which also indicates Th1 dominance in balancing Th1/Th2 responses. Furthermore, we confirmed higher percentages of peripheral CD4
+CD25
+ (regulatory or activated T cells) and CD4
+CD45RO
+T cells (memory T cells) in MS patients than in HC, which is in accordance with previous studies [
16,
17].
Our results estimated that the ratios of CD8
+CXCR3
+/CD8
+CCR4
+, indicating Tc1/Tc2, were significantly higher in MS patients than in HC. In addition, the percentage of CD4
+CXCR3
+ and CD8
+CXCR3
+ T cells was correlated with disease activity in MS. The roles and functions of chemokine receptors on CD8
+ T cells remain to be elucidated. CD8
+CXCR3
+ T cells are associated with migration and differentiation of memory T cells [
18] and also have a similar function as regulatory T cells (Treg) [
19]. Our results indicate Tc1 dominance over Tc2 in MS, which may reflect the abnormality of Treg in MS.
To the best of our knowledge, no studies have reported the expression of chemokine receptors on PBL in NMO patients. A few studies have investigated chemokine levels in NMO, in which CXCL10/IP-10 and CCL17/TARC levels were significantly elevated in NMO patients [
20] and CXCL-8/IL-8, CCL4/MIP-1β and CCL2/MCP-1 levels showed significant elevation in opticospinal MS patients (part of them are considered NMO) compared with normal subjects [
10].
Recent studies have shown that anti-AQP4 antibody and complements play a critical role of in the pathogenesis of NMO [
21]. Thus, we expected the up-regulated expression of Th2-related chemokine receptors in NMO; however, no remarkable deviation in the Th1/Th2 balance was observed in this study. Recent studies have revealed that Th17-related cytokines and chemokines play a dominant role in the pathogenesis of NMO from elevated protein levels of IL-17 and IL-8 in the CSF of opticospinal MS patients [
10] and IL-6 in the CSF of NMO patients [
11]. Th17 cells belong to a distinct lineage of Th1 and Th2 cells, which are responsible for organ-specific autoimmune diseases. Although the association between Th17 cells and NMO remains unclear, Th17 related-responses may be more important than Th1-and Th2-related responses in NMO pathogenesis. Hence, no remarkable changes might be seen in the Th1/Th2-related chemokine receptors on peripheral T cells. However, our study has a limitation because the subject was chemokine receptors on only PBL, and not on CSF, and thus we were unable elucidate the complete Th1/Th2 balance in the pathogenesis of MS and NMO.
In this study, some patients received oral prednisolone treatment. It has been reported that high-dose intravenous methylprednisolone reduced CD4
+CXCR3
+ Th1 cells [
17] and CCR5
+ Th1 cells in MS patients [
22]. Hence, it can be suggested that steroid treatment affected the expression of chemokine receptors in this study. However, only one (4%) patient with MS and three (25%) with NMO received oral prednisolone at sampling PBL, and no statistical difference was found between the percentage of patients treated with steroid in NMO and MS. We hypothesise that steroid treatment had a relatively small effect on the results. Meanwhile, higher WBC counts and lower lymphocyte percentages in NMO and MS patients were possibly affected by immunomodulating therapy.
Conclusions
In conclusion, we have identified several differences, such as Th1 and Tc1 dominance and the association between CXCR3+ (Th1 and Tc1) and disease activity in T cell subsets on PBL, in MS patients compared with HC, but these differences did not appear in NMO patients. We cannot determine characteristic lymphocyte subsets on PBL involved in the pathophysiology of NMO. Although further studies will be required to determine the immunological features of NMO, these differences in PBL could be based on immunological differences between NMO and MS.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AU drafted the manuscript and carried out the statistical analysis. MM diagnosed MS and NMO patients, collected blood samples and provided other information of the patients. SH and FN participated in the design of the study. MM and SK critically reviewed the manuscript. All authors have read and approved the final manuscript.