Introduction
Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the human central nervous system (CNS) characterized clinically by a relapsing-remitting course. Neuropathologically MS manifests with development of demyelinating lesions which affect both gray and white matter [
1,
2] disruption of the blood brain barrier (BBB) and transendothelial migration of lymphocytes and macrophages across inflamed CNS endothelial monolayers appear to be among the earliest CNS and spinal cord abnormalities in MS [
3]. Activation of the cerebral endothelial cells and their binding with activated leukocytes are early crucial steps in creation of MS demyelinating lesions produced by immune cells [
4,
5].
Cerebral endothelial cells, the main components of the BBB, contain tight and adherens junctions which create a highly impenetrable and impermeable anatomic-physiologic barrier against circulating plasma neurotransmitters, cytokines, formed blood elements and soluble and insoluble molecular components of the circulating blood. Upon activation by pro-inflammatory cytokines such as IFN-γ or TNF-α, released by activated T-lymphocytes, endothelial cells shed small fragments of their membranes, known as endothelial microparticles (PMP) [
6]. These fragments contain some of the surface adhesion molecules and other endothelial markers of their parent cells [
7], including, but not limited to, platelet-endothelial cell adhesion molecule (PECAM-1/CD31), human endothelial marker CD146, and intercellular adhesion molecule (ICAM-1/CD54). Therefore while it is difficult to evaluate inflammatory endothelial markers in MS in situ, microparticles may provide a remote 'snapshot' of the surface of inflamed endothelium and provide information on the extent of platelet and leukocyte activity in MS.
We previously reported elevated plasma levels of PMP in MS patients and demonstrated that the plasma levels of PMP
CD31+ may reflect acute endothelial injury with positive association with presence of contrast enhancing lesions [
3]. Additionally, we reported that various sub-species of PMP form complex with different leukocytes and by formation of such complexes, they promote the inflammatory process by facilitating transendothelial migration of the leukocytes [
6]. In the current study, we hypothesized that prospective serial measurement of three sub-species of PMP CD31
+, PMP
CD146+, and PMP
CD54+ may reflect disease activity and serve as a surrogate marker of therapeutic response to high dose, high frequency interferon-β1a. We also assessed the correlation among these sub-species of PMP and MRI parameters.
Measurement of PMP using flow cytometry
Using a 21 gauge needle, venous blood was obtained in citrate loaded vacutainer tubes. Measurements of plasma PMP were performed within four hours from specimen collection. Briefly, blood specimen was centrifuged at 160 × g for 10 minutes to prepare platelet rich plasma (PRP). Next, the PRP specimen was centrifuged for 6 minutes at 1500 × g to generate platelet poor plasma (PPP). Then, each 25 μl aliquot of PPP was incubated with 2 μl of anti-CD31-PE, 2 μl of anti-CD146, and 2 μl of anti-CD54 at ambient temperature for 20 minutes with gentle shaking (80 rpm). The PMPs in the sample were measured using a Beckman Coulter FC500 MCL flow cytometer system equipped with CXP software (Beckman Coulter, Miami, FL). The sample flow rate and particle detection settings are identical to that described previously [
3]. Data collection is based on both multi-parameter and uni-parameter analyses of PMP
CD31+, PMPCD
146+, and PMP
CD54+. To evaluate the possibility that leukocyte adhesion of microparticles might be measured, the anti-leukocyte common antigen CD45 was used in the cocktails. With the exception of whole blood analysis (used as a positive control) negligible CD45
+ events were detected in the PPP preparations.
Discussion
High dose high frequency IFN-β1a is an FDA-approved treatment for MS which reduces annual relapse rate and improves brain MRI findings. The therapeutic benefits from IFN-β1a have been attributed to may involve effects on immune cells elevation of IL-10 and reduced synthesis of Th1 cytokines endothelial cell adhesion molecules MMPs resulting in lower leukocyte penetration of the CNS parenchyma, antigen presentation by microglia. We observed a decline in the plasma levels of CD31
+ microparticles following treatment of MS patients with IFN-β1a. The plasma levels of PMP
CD31+ were found to be statistically reduced by IFN-β1a therapy at 3, 6 and 12 months. Because these studies were performed with single staining protocols we cannot specifically designate these particles as 'endothelial', and these must be considered to be a mixture of platelet and endothelial derived microparticles. CD31/PECAM-1 is known to mediate the transendothelial migration of leukocytes [
10] in cytokine independent migration. Therefore, shedding of CD31 in microparticles, may represent an adaptive response aimed at limiting availability of CD31 on the endothelial, platelet or leukocyte membranes or alternatively may be another factor which facilitates transendothelial migration of the activated leukocytes. Therefore, a decrease in shedding of PMP
CD31+ into plasma following treatment with IFN-β1b may indicate a lessened interaction between activated leukocytes and inflamed underlying endothelium, which in turn translates into stabilization of the blood brain barrier and decrease in the number of contrast-enhancing T1-weighted lesions on brain MRI [
3]; [
11]. In addition, so-called 'insoluble' pools of CD31 found on microparticles may function as antagonists of leukocyte-bound CD31/PECAM-1 which functionally limits CD31 access, leading to reduced binding and transmigration.
Turning to another endothelial marker, PMP
CD146+, we observed the effect of treatment of IFN-β1a on the plasma levels of this biological marker. CD146 is abundantly expressed on the surface of brain endothelial cells where it may act as a specific functional ligand for Th17 cells [
12]. CD146 is also expressed on the surface of a subpopulation of activated T-cells [
13] and mediates lymphocyte-endothelial adhesion. CD146+ microparticles were nearly statistically significantly reduced over 12 months of therapy with IFN-β1a. This change in CD146
+ microparticle expression appeared at 3 months of therapy, and was not observed at 6 or 12 months. CD146, is also known as MUC18, which is considered to be a marker of endothelial cells, however it can also be found on a subset of T- and B-lymphocyte, NK cells [
14], pericytes [
15] and circulating endothelial cells [
16]. Plasma levels of endothelial derived 'soluble' CD146 were inversely correlated with congestive heart failure [
17], it is not clear whether how transient reductions in CD146 PMPs might be interpreted. Plasma levels of PMP
CD146+ cannot yet predict a long term stabilizing effect of IFN-β1a on cerebral endothelial cells.
During the course of this prospective study, we also assessed the plasma levels of PMP
CD54+ prior to treatment with IFN-β1a and at timed intervals (3, 6 and 12 months) after treatment. PMP
CD54+ appears to mainly represent endothelial microparticles-derived ICAM-1 (CD54), but ICAM-1 can also be expressed by macrophages and T-cells. CD54 is constitutively expressed on the surface of endothelial cells [
19], and its' expression is significantly upregulated by Th1 cytokines (TNF-α, IL-1b, IFN-γ) which are known to be elevated during MS exacerbations [
19]; [
20]. While some studies have shown elevation of soluble forms of ICAM-1 (CD54) during active MS disease, [
21] our study shows that levels of insoluble microparticles bearing CD54 in MS disease are significantly reduced by IFN-β1a therapy at 3, 6 and 12 months. In addition, previously Jy et al. [
6] demonstrated that PMP
CD54+ formed conjugates with monocytes and facilitated their migration through the endothelial cells monolayers and may represent an important mechanism supporting the penetration of immune cells into the CNS parenchyma in MS.
The correlation of plasma levels of PMP
CD31+ and volumes of contrast-enhancing T1-weighted lesions on brain MRI is presented as a bimodal graph in Figure
4. We found that at 12 months both markers (PMP
CD31+ and T1-weighted volumes) were significantly reduced by IFN-β1a therapy. Similarly, Figure
5 also depicts the bimodal graph relationship between PMP
CD54+ and volumes of contrast-enhancing T1-weighted lesion on brain MR images. Interestingly, when plotted in this manner, the magnitude of the reduction in these profiles appears to be similar for both PMP
CD31+ and PMP
CD54+ with volumes of the contrast-enhancing T1-weighted lesions. Whether and how these parameters are mechanistically linked to therapeutic benefit of IFN-b1α in MS is not clear but lower CD54 appears to reflect decreased endothelial activation. Because endothelial CD31/PECAM-1 is not as dramatically altered by exposure Th1 cytokines, as CD54 is for example [
22], diminished CD31 levels suggest stabilization of membrane integrity and reduced platelet activation both of which could contribute to therapeutic benefit in MS.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MLN - Helped to conceive study and directed MP flow analysis work, EE coordinated patient sample collection and interacted with AM, EGT carried out all patient MRI analyses, MKH interviewed, consented and collected patient specimens, KC collected clinical patient information and coordinated between AM, MKH and EGT, JMB collected patient clinical information data and coordinated between AM, MKH and EGT, CVG interpreted data, statistics and helped write manuscript, AM helped conceive the study and helped to write manuscript, DC worked on MP flow cytometry and helped perform statistics, JSA helped to conceive study, interpret data, statistics, figures and helped to write manuscript. All authors read and approved the final manuscript.