Intrathecal synthesis of IgM measured after a first demyelinating event suggestive of multiple sclerosis is associated with subsequent MRI brain lesion accrual

Abstract

Background: Previous studies have demonstrated that intrathecal synthesis of IgM is observed in multiple sclerosis (MS) and correlates with a worse disease course. These results suggest that IgM participates in the formation of MS lesions.

Objective: The aim of the present study was to assess the potential association between the level of intrathecal synthesis of IgM measured after a clinically isolated syndrome (CIS) and the subsequent formation of brain lesions.

Methods: Fifty seven patients with a CIS and a high risk developing MS were enrolled in a longitudinal study. Examination of cerebrospinal fluid was performed after the CIS and included measures of intrathecal IgM and IgG synthesis. Patients were assessed with the same 1.5 Tesla magnetic resonance imaging (MRI) system at baseline and after a mean follow-up period of 49 months (range 36–60). Spearman Rank correlation was used to assess the potential correlations between levels of intrathecal immunoglobulin synthesis and MRI data.

Results: The level of intrathecal IgM synthesis was correlated with the number of gadolinium-enhancing lesions at baseline (p = 0.01) and with accrual of brain lesions during the follow-up period (p = 0.02). By taking into account brain sub-regions, we demonstrated that the level of intrathecal IgM synthesis was only correlated with the increased number of lesions in the periventricular regions (p = 0.004). The level of intrathecal IgG synthesis was not correlated with any MRI data.

Conclusion: The present longitudinal study demonstrates that the level of intrathecal IgM synthesis measured after a CIS is associated with subsequent lesion accrual during the first years of MS. This result emphasizes the involvement of IgM in plaque formation.

Keywords

immunology MRI T2 lesions

Introduction

One of the major challenges in multiple sclerosis (MS) research is to define relevant surrogate markers that might predict future disease course. Over the last decade, numerous studies have demonstrated the accuracy of conventional magnetic resonance imaging (MRI) techniques in predicting future conversion to MS in patients suffering from a clinically isolated syndrome (CIS).^1-3 In addition, several studies have shown that detection of intrathecal synthesis of IgG in patients with CIS, evidenced by increased IgG index and/or IgG oligoclonal bands (OCB), represents an independent prognostic marker for future conversion to MS.^4-7 Although MRI techniques and cerebrospinal fluid (CSF) analysis are accurate in predicting conversion to MS, their ability to predict long-term disability remains limited.

Intrathecal IgM synthesis has recently been evaluated in patients with MS.^8-14 Intrathecal IgM synthesis has been associated with a shorter delay to convert to clinically definite MS and with a more severe long-term disability.^10,11 Moreover, IgM containing antibodies directed against some lipids constituting the myelin have been detected in patients with a worse disease course.^15,16 Although promising, these results, obtained by the same group, have been recently contradicted by two studies.^13,14 In a retrospective study, Schneider et al.¹⁴ demonstrated that intrathecal IgM synthesis (presence of IgM-OCB in the CSF) does not correlate with the risk of relapse in patients with CIS. In paediatric MS, Stauch et al.¹³ demonstrated that intrathecal IgM synthesis is not a negative prognostic marker of disease progression.

Only one study has assessed the potential relationship between intrathecal IgM synthesis and conventional MRI findings in patients with MS.¹⁷ This cross-sectional study demonstrated a significant association between IgM index and T2 lesion load in patients with MS.¹⁷ These results may reflect a pathogenic role of IgM in plaque formation. However, the cross-sectional design of this study prevents any inference concerning the potential causal relationship between IgM synthesis and lesion formation. To overcome this limitation, we conducted a longitudinal MRI study in patients included after a first clinical attack of MS in order to assess the potential association between baseline intrathecal IgM synthesis and subsequent plaque formation.

Methods

Subjects

In total, 57 patients were enrolled in a longitudinal study. Patients were recruited at the Department of Neurology (University Hospital of Marseille), based on the following criteria: 1) age between 18 and 50 years; 2) occurrence of a first presumed inflammatory demyelinating event in the central nervous system during the last 12 months, involving either the optic nerve, the spinal cord, the brainstem or a brain hemisphere; 3) no previous history of neurological symptoms; 4) no possible alternative diagnosis, excluded with clinically appropriate investigations; 5) a minimum delay since corticosteroid infusion of 1 month; and 6) ≥1 lesion in each of ≥2 characteristic locations of the central nervous system (periventricular, juxtacortical, posterior fossa, spinal cord) or one lesion in one characteristic location combined with OCB in the CSF. The last criterion meant that only CIS patients with a high risk developing MS were included.

Patients underwent a clinical examination during the first neurological episode. Other neurological examinations were performed the days of the first and second MRI to assess residual disability using the Expanded Disability Status Scale (EDSS).¹⁸

All the participants gave their informed consent to participating in this study, which was approved by the local Ethics Committee (Timone Hospital, Marseille, France).

MRI exploration

Patients were imaged with the same commercially available 1.5 Tesla MRI system (Magnetom Vision Plus; Siemens, Erlangen, Germany). The MRI protocol included transverse fast spin-echo (SE) proton density-weighted and T2-weighted sequences (echo time [TE]1/[TE]2/repetition time [TR] = 15 ms/85 ms/2600 ms, 44 contiguous slices, thickness = 3 mm, flip angle = 90°, field of view [FOV] = 240 mm, matrix = 256 × 256). A T1-weighted SE sequence (TE/TR = 10 ms/650 ms, 25 contiguous slices, thickness = 5 mm, flip angle = 90°, FOV = 240 mm, matrix = 256 × 256) was also carried out 5 min after injection of gadolinium (0.1 mM/kg).

In our study, the MRI data consisted of the lesion count at the two time points, allowing determination of the number of new T2 lesions occurring during the follow-up period. This parameter more appropriately reflected the formation of new plaques, compared with assessment of T2 lesion volume changes. The number of T2 lesions was determined at baseline and at follow-up by two readers (LD; BA) blinded to the clinical and CSF findings. In case of discrepancies, the final number of lesions was determined by a consensus re-reading. The number of lesions was determined in each brain sub-region, namely the periventricular, the subcortical and the infratentorial regions. The total number of enhanced lesions was determined at the two time points.

CSF examinations

All punctures were performed at the Department of Neurology (Timone Hospital, Marseille) and approximately 10 ml of colourless CSF was obtained. Albumin, IgG and IgM concentrations were quantified in serum and CSF by immunonephelemetry on a Siemens analyzer (BNII) using N Latex tests.

The intrathecal synthesis of IgG and IgM was quantified according to a non-linear, hyperbolic function for the relationship between the quotient of IgM and quotient of albumin.^13,19,20 The intrathecal, locally synthesized contribution of IgX (G, M) in CSF was calculated as intrathecal fraction IgX_LOC (mg/l).^13,19,20

Statistical analysis

Spearman Rank correlation was used to assess the potential correlations between immunological data, MRI data and EDSS.

Results

Clinical and MRI characteristics

Demographic and clinical characteristics of the patients are displayed in Table 1. The mean duration between the first clinical event and the first MRI was 4.2 months (range 1–12). According to the inclusion criteria no patients have suffered from a second relapse in this interval. The lumbar puncture was performed close to the first MRI (maximum 1 month). The mean interval between the first and the second MRI was 49 months (range 36–60). The mean number of relapses during the follow-up period (interval between the first and the second MRI) was 1.5 (SD = 2). At the point of inclusion, 26 out of 57 patients were diagnosed with MS according to the 2010 McDonald MRI Criteria for MS.²¹ At the end of the follow-up period, 46 out of 57 patients were diagnosed with MS according to the 2010 McDonald MRI Criteria.²¹ The median progression of the EDSS scores during the follow-up period was 0 (range 0–4.5). Patients were allowed to start immunomodulation during the follow-up period, and 30 out of the 57 patients started treatment.

Table 1.

Clinical and demographic characteristics of patients.

	Gender (F/M)	Age (years) (mean, range)	Residual EDSS (median, range)	Time to the clinical onset (month) (mean, range)	Type of symptom
	Gender (F/M)	Age (years) (mean, range)	Residual EDSS (median, range)	Time to the clinical onset (month) (mean, range)	ON	S	B	H
Patients n = 57	48/9	30 (19–48)	1 (0–1.5)	4.2 (1–12)	15	26	11	5

ON: Optic Nerve, S: Spinal Cord, B: Brainstem, H: Hemisphere

The numbers of T2 lesions at baseline and follow-up are displayed in Table 2. The mean number of lesions per patient was 18 (SD = 29) at baseline and 29 (SD = 21) at the follow-up. Statistical comparison (Wilcoxon test) between the number of lesions at the two time points demonstrated a significant (p < 0.005) increase during the follow-up period.

Table 2.

Number of brain lesions in the different sub-regions at baseline and at follow-up.

	Number of Lesions at Baseline (mean; median (SD))	Number of Lesions at the follow-up (mean; median (SD))
Total	18; 18.5 (21)	29; 26.5 (29)
Periventricular	9; 7.5 (8.5)	15; 14.5 (12)
Subcortical	8; 3.5 (12)	12; 8.5 (15)
Posterior fossa	1; 1 (2)	2; 1 (4)

The mean number of gadolinium-enhancing lesions was 2.4 (SD = 11.1) at baseline and 0.6 (SD = 1.7) at the follow-up.

Relationships between intrathecal immunoglobulin synthesis, MRI findings and clinical data

IgM_LOC was not correlated with the number of lesions at baseline, whatever the location of the lesions. IgM_LOC was correlated with the number of gadolinium-enhancing lesions at baseline (Rho = 0.32, p = 0.01), and with the accrual of T2 lesions in the whole brain during the follow-up period (Rho = 0.3, p = 0.02). IgM_LOC was correlated with lesion accrual in the periventricular regions (Rho = 0.37, p = 0.004) and not in the subcortical or the infratentorial regions.

No correlations were found between IgM_LOC and residual EDSS at baseline (Rho = 0.07, p = 0.6), progression of EDSS during the follow-up period (Rho = -0.01, p = 0.9) and residual EDSS at the follow-up (Rho = 0.009, p = 0.99). IgM_LOC was not correlated with the number of relapses during the follow-up period (Rho = 0.12, p = 0.34). Patients with positive 2010 McDonald MRI Criteria for MS²¹ at inclusion showed higher IgM_LOC compared with patients with negative criteria (p = 0.01). Patients with positive 2010 McDonald MRI Criteria for MS²¹ at the end of the follow-up period showed higher IgM_LOC compared with patients with negative criteria (p = 0.001).

No correlations have been demonstrated between IgG_LOC and any of the MRI and clinical parameters.

Discussion

The present longitudinal study demonstrates that intrathecal synthesis of IgM measured after the first clinical attack of MS is correlated with subsequent accrual of brain lesions.

The pathogenic role of IgM has been already suggested by animal studies demonstrating that injection of serum IgM in the central nervous system results in demyelination.²² In addition, Rosenbluth et al.²³ showed that implants of hybridoma cells that secrete an IgM anti-galactocerebroside cause focal MS-like plaques of demyelination. In the present study, we evidenced the existence of an association between the level of intrathecal IgM synthesis and the number of gadolinium-enhancing lesions. This finding emphasizes the potential involvement of IgM in the inflammatory processes occurring in plaques, but does not allow any inference concerning the causal relationship between IgM and lesion formation. However, the association found between intrathecal synthesis of IgM and subsequent lesion formation obtained from the follow-up data gives more relevant clues for the involvement of IgM in the pathogenesis of plaques formation. Furthermore, previous studies have provided other clues emphasizing that IgM may directly participate in lesion formation in patients with MS. Villar et al.¹⁶ demonstrated in MS patients that the antigenic specificity of IgM may influence the pathological process by demonstrating that patients with intrathecal synthesis of IgM directed against some lipids constituting the myelin present a worse disease course.

Although significant, the correlation between intrathecal IgM synthesis and lesion accrual during the follow-up period is limited. This finding may be explained by two points. First, the involvement of IgM in lesion formation may be different according to the brain regions involved. In accordance, we demonstrated that the IgM index was only correlated with increased number of lesions in the periventricular regions. Second, other pathological processes are probably involved in lesion formation.²⁴

Two previous studies performed by the same group have demonstrated that the level of intrathecally produced IgM is associated with long-term disability.^10,11 In addition, long-term disability has been associated with early brain lesion accrual during the first years following a CIS.^25,26 Therefore, the correlation demonstrated in the present study between intrathecal IgM synthesis and lesion accrual is a coherent clue to link IgM to long-term disability. As the population studied here consisted of patients followed during the first 4 years of MS, the progression of EDSS between baseline and follow-up was generally low (median 0, range 0–4.5), which may explain why no correlations between intrathecal synthesis of IgM and EDSS have been found.

The present longitudinal study demonstrates that the level of intrathecal IgM synthesis measured after a CIS is associated with subsequent lesion formation during the first 4 years of MS. This result emphasizes the involvement of IgM in the pathogenesis of MS.

Footnotes

Funding

This study was supported by grants from the Association pour la Recherche sur la Sclérose En Plaques (ARSEP) and CNRS.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

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