Expression of CXCR3 and its ligands CXCL9, -10 and -11 in paediatric opsoclonus-myoclonus syndrome

Abstract

Opsoclonus-myoclonus syndrome (OMS) is a neuroinflammatory disorder associated with remote cancer. To understand more clearly the role of inflammatory mediators, the concentration of CXCR3 ligands CXCL10, CXCL9 and CXCL11 was measured in 245 children with OMS and 81 paediatric controls using enzyme-linked immunosorbent assay (ELISA), and CXCR3 expression on CD4(+) T cells was measured by flow cytometry. Mean cerebrospinal fluid (CSF) CXCL10 was 2·7-fold higher in untreated OMS than controls. Intrathecal production was demonstrated by significantly different CXCL10 CSF : serum ratios. The dichotomized 'high' CSF CXCL10 group had higher CSF leucocyte count (P = 0·0007) and B cell activating factor (BAFF) and CXCL13 concentrations (P < 0·0001). CSF CXCL10 did not correlate with clinical severity or relapse using grouped data, although it did in some patients. Among seven types of immunotherapy, including rituximab or chemotherapy, only adrenocorticotrophic hormone (ACTH) monotherapy showed reduced CSF CXCL10, but prospective longitudinal studies of ACTH combination therapies indicated no reduction in CXCL10 despite clinical improvement (P < 0·0001). CXCL10 concentrations were 11-fold higher in CSF and twofold higher in serum by multiplexed fluorescent bead-based immunoassay than enzyme-linked immunosorbent assay, but the two correlated (r = 0·7 and 0·83). In serum, no group differences for CXCL9 or CXCL11 were found. CXCR3 expression on CD4(+) T cells was fivefold higher in those from CSF than blood, but was not increased in OMS or altered by conventional immunotherapy. These data suggest alternative roles for CXCL10 in OMS. Over-expression of CXCL10 was not reduced by clinical immunotherapies as a whole, indicating the need for better therapeutic approaches.

© 2013 British Society for Immunology.

Figures

Fig. 1

(a) Mean CXCL10 concentration measured by fluorescent bead-based immunoassay was increased in untreated opsoclonus–myoclonus syndrome (OMS) (uOMS) cerebrospinal fluid (CSF) but not in paired serum. Separate statistical comparisons were made between controls, untreated OMS (uOMS) and treated OMS (tOMS); and between controls, OMS without neuroblastoma (OMS − NB), and OMS with neuroblastoma (OMS + NB). Statistical brackets with P-values above them indicate analysis of variance (anova) results; those with P-values underneath indicate t-test results. The neuroblastoma without OMS group had only five patients, so it is presented only for visual comparison. (b) CSF CXCL10 concentration as measured by Luminex versus enzyme-linked immunosorbent assay (ELISA) in entire data set. (c) Serum CXCL10 measured by Luminex versus ELISA in entire data set.

Fig. 2

(a) Cross-sectional analysis of mean CXCL10 concentrations measured by enzyme-linked immunosorbent assay (ELISA) from controls, untreated opsoclonus–myoclonus syndrome (OMS) (group 1), various OMS treatment groups (group 2–8), previously but not currently treated OMS (group 9), and other inflammatory neurological disorders (OIND). Statistical brackets signify post-hoc tests. Among the seven OMS active treatment groups, adrenocorticotrophic hormone (ACTH) monotherapy was associated with the lowest cerebrospinal fluid (CSF) CXCL10 level. (b) The CSF: serum CXCL10 ratio showed similar results.

Fig. 3

Temporal relation of cerebrospinal fluid (CSF) CXCL10 concentration to relapse (a–d) and immunotherapy (e,f) in six children with opsoclonus–myoclonus syndrome (OMS). (a) A boy status/post (S/P) neuroblastoma with OMS onset at 13 months and our initial evaluation at 17 months. (b) A girl S/P neuroblastoma with OMS onset at 21 months and our first evaluation at 26 months. (c) A girl S/P neuroblastoma with OMS onset at 36 months and our first evaluation at 41 months. (d) A boy S/P neuroblastoma; OMS onset, 14 months; initial evaluation 31 months. (e) A boy without tumour; OMS onset, 31 months; initial evaluation, 32 months. (f) A girl S/P neuroblastoma; OMS onset, 11 months; initial evaluation, 14 months. Cellcept: mycophenolate mofetil; CPM: cyclophosphamide; Dex: pulse dose dexamethasone; 6MP: 6-mercaptopurine; Pred: prednisone; R: relapse of OMS; RTX: rituximab. Parentheses indicate continuing treatments.

Fig. 4

Serum concentrations of CXCL10 (a), CXCL9 (b), and CXCL11 (c). The other inflammatory neurological disorders (OIND)group, not used for statistical analysis, is shown for comparison. The asterisk denotes statistical significance on the Dunn's post-hoc test.

Fig. 5

Representative two-colour immunofluorescence analysis of CXCR3+CD4+ T cells in (a) cerebrospinal fluid (CSF) and (b) blood from a patient with untreated opsoclonus–myoclonus syndrome (OMS). (c) Correlation of CSF and blood CXCR3+CD4+ T cells.