Secreted phospholipase A2 activity in experimental autoimmune encephalomyelitis and multiple sclerosis

Abstract

Background: There is increased interest in the contribution of the innate immune system to multiple sclerosis (MS), including the activity of acute inflammatory mediators. The purpose of this study was to test the involvement of systemic secreted phospholipase A2 (sPLA2) enzymes in experimental autoimmune encephalomyelitis (EAE), an MS model, and to determine if enzyme activity is elevated in MS patients.

Methods: A non-invasive urinary assay was developed in order to monitor enzymatically active sPLA2 levels in Dark Agouti rats after induction of EAE. Some Rats were treated with nonapeptide CHEC-9, an uncompetitive sPLA2 enzyme inhibitor, during the initial rise in urinary enzyme levels. Body weight and clinical EAE score were measured for 18 days post immunization (PI), after which the rats were sacrificed for H&E and myelin staining, and for ED-1 immunocytochemistry, the latter to quantify macrophages and activated microglia. The urinary sPLA2 assay was also applied to un-timed samples collected from a cross section of 44 MS patients and 14 healthy controls.

Results: Mean levels of enzymatically active sPLA2 in the urine increased following immunization and peaked between days 8-10 PI which was just prior to the onset of EAE symptoms. At this time, a transient attenuation of activity was detected in the urine of CHEC-9 treated rats consistent with the activity-dependent properties of the inhibitor. The peptide also reduced or abolished EAE symptoms compared to vehicle-injected controls. Histopathological changes in the spinal cords of the EAE rats correlated generally with clinical score including a significant reduction in ED-1+ cells after peptide treatment. Multiple Sclerosis patients also showed elevations in sPLA2 enzyme activity. Mean levels of sPLA2 were increased 6-fold in the urine of patients with active disease and 4-fold for patients in remission, regardless of immunomodulating therapy.

Conclusion: The results suggest that sPLA2 enzymes, traditionally thought to be part the acute phase inflammatory response, are therapeutic targets for MS.

Figure 1

Secreted phospholipase A2 (sPLA2) activity and clinical disease in EAE rats treated with sPLA2 inhibitor CHEC-9. Top: Urinary sPLA2 enzymatic activity, normalized to average pre-immunization values, increased steadily to day 8 in both CHEC-9 and vehicle-treated rats. A significant reduction in activity was observed on days 10 and 12 post-immunization in the peptide treated group either by comparing values of peptide and vehicle directly (p = 0.049, 0.026 respectively, Mann Whitney), or by peak to trough comparison between days 8 and 12, where reduction in sPLA2 levels with peptide treatment was significant (p = 10^-3). Bottom: Mean clinical scores from day 10 onwards were also significantly lower in the peptide treated rats (see text).

Figure 2

Low power photomicrograph of hematoxylin & eosin (A, B) and cyanin R (C, D) stained sections through the lumbar spinal cord. Left panels (A, C): Spinal cord of a peptide-treated rat that was symptom-free showing limited small cell infusion and myelin degeneration. Right panels (B, D): Spinal cord of a vehicle treated rat that had an EAE score of 2.0. The tissue was characterized by EAE lesions consisting of small darkly staining cells in the ventral and lateral white matter (arrows), and myelin degeneration as shown in an adjacent section (D).

Figure 3

Macrophages and microglia were reduced by CHEC-9 treatment of EAE. Graph (left panel): The density of ED-1+ cells in the spinal cord was reduced over 60% following peptide treatment (p < 1 × 10^-3). Although the immunostained cells tended to accumulate at or near the surface of both groups, fewer cells, occupying a smaller area within the cord were found CHEC-9 treated rats (B) compared to vehicle treated rats (C). The majority of intraspinal ED-1 immunoreactive cells were activated microglia (arrows).

Figure 4

Level of sPLA2 enzyme activity in MS patients with active or stable disease compared to controls (see Table 1). All measurements were made using 600 μM substrate and normalized to the average control value. There was a significant 4 and 6-fold increase in PLA2 activity compared to controls in the stable and active MS patients respectively, (p = 0.049*; 0.0019**, for comparison with controls, Mann Whitney test). Treated and untreated stable patients were grouped since their average levels of active enzyme were almost identical (see text). Patients with active disease were mostly undergoing treatment with beta-interferon (9/12, Table 1).