Cerebrospinal fluid findings in multiple sclerosis patients before, during and after pregnancy

The mechanisms responsible for the lower relapse rate of multiple sclerosis during pregnancy and an increased relapse rate during the postpartum period are still undefined. Putative explanations include pregnancy‐related alterations in the function of the immune system, such as a shift towards T helper (Th)2‐type immune reactivity and expansion of regulatory T lymphocytes.¹,² Analysis of the cerebrospinal fluid (CSF) offers a unique opportunity to characterise immunological pathways in the pathogenesis of multiple sclerosis. The inflammatory activity in the central nervous system (CNS) of patients with multiple sclerosis is reflected in the composition of CSF cells and one hallmark of the disease is the production of intrathecal antibodies. The aim of our study was to investigate the alterations that take place in the CSF of patients with multiple sclerosis during pregnancy. To our knowledge, no previous attempts have been made to analyse the components of the CSF of pregnant patients with multiple sclerosis.

Methods

The study included six patients with relapsing remitting multiple sclerosis, who were enrolled at a time of known pregnancy status. Follow‐up with recording of relapses continued until 6 months after the delivery. Only one of the patients (case 5) was undergoing disease‐modifying treatment (subcutaneous interferon (IFN)β‐1a) before pregnancy, and the drug was discontinued 3 months before the onset of pregnancy. No immunomodulatory treatment was used during pregnancy or postpartum at the time of drawing the sample. All but one patient (case 4) chose to breast‐feed (duration range 6 weeks to 6 months). There were no signs of postpartum depression in any of the patients. One CSF sample was drawn at 26–28 weeks of pregnancy, at a time of putative low disease activity. The second CSF sample was drawn 4–5 weeks after the delivery, at a time of putative high disease activity. Lymphocyte number, IgG‐Index and number of oligoclonal bands were analysed from the CSF. At the same time points, venepuncture was performed for peripheral blood lymphocyte isolation and immunofluorescence staining for intracytoplasmic IFN‐γ and interleukin (IL)‐4 production, which was performed as described earlier.³ The study was approved by the Commission of Ethics of Turku University and the Turku University Central Hospital, and informed consent was obtained from all subjects after a full explanation of the procedure. A lumbar puncture is a standard procedure during diagnostic investigation in Finland and hence is performed often. All patients were fully aware of the scientific nature of the lumbar puncture, and were still willing to give the samples for analysis as accepted by the ethical committee.

Analysis of variance for repeated measurements and Newman–Keuls multiple comparison post‐test were used for comparisons of the relapse rate and the differences in the IgG indices. The Expanded Disability Status Scale values were compared by using a paired, two‐sided t test.

Results

Six women with definite relapsing remitting multiple sclerosis and a known pregnancy status were studied. The duration of the disease was 1–11 years (range) and the mean (SD) age of the patients was 26 (5) years. During the year before pregnancy, the relapse rate was 1 (0.9) relapses/patient/year (mean (SD)). During pregnancy, only one of the patients experienced a relapse; this was during the first trimester. In the 6‐month postpartum period, all but one patient experienced a relapse (relapse rate during pregnancy v postpartum: 0.2 (0.6) v 1.7 (0.9); p = 0.0336). The Expanded Disability Status Scale was 1.2 (0.6) during late pregnancy and 1.7 (0.9) at 6 months postpartum (mean (SD), p = 0.27). Table&#146>;1 shows the CSF parameters in samples taken before (diagnostic lumbar puncture), during and after pregnancy. Notably, the IgG‐Index was significantly higher during pregnancy than before pregnancy or after the delivery in all patients (p = 0.0390), but no difference was observed in the number of oligoclonal bands (table 1). Enhanced clinical disease activity was associated with a higher number of CSF cells, as expected (table 1), but no alteration was observed in the subtypes of CSF lymphocytes during versus after pregnancy (data not shown).

Table 1 CSF findings.

Patient	Before pregnancy		During pregnancy		OB	After pregnancy		OB
	IgG‐Ind	Cells	IgG‐Ind	Cells		IgG‐Ind	Cells
1	0.76	4	0.77	0	0	0.69	8	0
2	0.71	12	0.80	1	1–2	0.67	2	1–2
3	3.18	ND	4.77	0	3–4	4.06	4	3–4
4	2.28	26	3.19	1	1–2	2.08	9	1–2
5	1.22	12	2.19	2	1–2	1.14	2	1–2
6	0.70	0	0.74	0	0	0.72	8	0

Before pregnancy, diagnostic lumbar puncture; during pregnancy, at 26–28 gestational weeks; after pregnancy, 4–5 weeks postpartum; IgG‐Ind, IgG‐Index; cells, number of lymphocytes×10⁶/l. Cell number is in bold if sampling was performed during a relapse; OB, number of oligoclonal bands, was determined by using the Paragon Protein (SPE‐II) Electrophoresis Kit, Beckman Coulter, Fullerton, California, USA. ND, not detected.

To study whether the increased intrathecal IgG production was associated with a systemic Th2 shift, we measured intracytoplasmic IFN‐γ and IL‐4 production in peripheral blood monocyte cells after phorbol myristate acetate and ionomycin stimulation both during and after pregnancy. The percentage of IFN‐γ‐secreting cells within the peripheral blood lymphocyte pool was significantly lower during pregnancy than in the postpartum period (2.8 (0.7)% (mean (SEM)) IFN‐γ‐producing cells during pregnancy and 7.6 (0.8)% during postpartum, p = 0.003). The difference in IL‐4‐producing cells during and after pregnancy was less pronounced (1.0 (0.16)% (mean (SEM)) during pregnancy and 1.8 (0.15)% during postpartum, p = 0.028). This resulted in a Th2:Th1 ratio of 0.36 during pregnancy and 0.24 after delivery.

Discussion

This study describes, for the first time, that the production of intrathecal antibodies in patients with multiple sclerosis is higher during late pregnancy than in the postpartum period. A shift towards humoral (Th2)‐type immune responses was shown during pregnancy, and it is the most accepted theory to explain the amelioration of Th1‐type diseases such as multiple sclerosis and rheumatoid arthritis during pregnancy. Most of the data supporting the Th2 shift during pregnancy are derived, however, from mouse experiments, and the corresponding in vivo phenomena in pregnant women are poorly documented. Our finding of non‐altered oligoclonal bands is in line with previous studies, which have shown that oligoclonal banding in the CSF of patients with multiple sclerosis is persistent, and not affected by treatment, whereas the rate of IgG production is more susceptible to alterations.⁴

The CNS parenchyma is not a physiological site for B‐cell responses and antibody production, but, in multiple sclerosis, B cells are demonstrable in inflammatory infiltrates and in the CSF.⁵ Antibodies are indicative of chronic CNS inflammation and probably contribute to demyelination and axonal damage.⁵ Our data imply that factors other than intrathecal antibodies are crucial in the pathology leading to multiple sclerosis relapses in the postpartum period, as a reduction in the IgG‐Index was associated with an increase in the relapse rate. Our data suggest that the consequences of alterations in the immune system leading to a Th2 shift in the periphery reach beyond the blood–brain barrier and strengthen the humoral immune responses within the CNS.

Acknowledgements

The generous contribution of all patients with multiple sclerosis to the study is greatly acknowledged. We are grateful for the excellent technical help of Suvi Hyvönen and Terjo Huuhtanen, and we thank Dr David Smith for revising the language of the manuscript. The work was supported by the Finnish Medical Foundation and the Finnish Foundation of Neurology.