Brain, Vol. 122, No. 12, 2297-2307,
December 1999
© 1999 Oxford University Press
Review article |
Immunological profile of patients with primary progressive multiple sclerosis
Expression of adhesion molecules
Unitat de Neuroimmunologia Clínica, Servei de Neurologia, Hospital General Universitari Vall d'Hebron, Barcelona, Spain
Correspondence to:
Isabel Durán, Unitat de Neuroimmunologia Clínica, Escola d'Infermeria 5a planta, Hospital General Universitari Vall d'Hebron, Psg. Vall d'Hebron 119–129, 08035, Barcelona, Spain E-mail: iduran{at}hg.vhebron.es
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Abstract |
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Adhesion molecules are important in the trafficking of peripheral leucocytes into the central nervous system, a major event in the pathogenesis of multiple sclerosis, which is an inflammatory and demyelinating disease. The latest MRI evidence supports clinical divergence between forms of multiple sclerosis with relapses and the primary progressive form without relapses, which shows fewer and smaller inflammatory lesions. With the aim of elucidating whether different pathogenic mechanisms are involved in primary progressive multiple sclerosis, we compared membrane expression of the adhesion molecules ICAM-1 (CD54), LFA-1
(CD11a), VLA-4 [4/ß1 integrin (CD49d/CD29)], L-selectin (CD62L) and ICAM-3 (CD50) in peripheral blood and the serum-soluble forms ICAM-1, L-selectin, VCAM-1 and ICAM-3 in 89 patients (39 with the primary progressive form, 25 with the secondary progressive form and 25 with the relapsing–remitting form) and 38 healthy controls. We found a significant decrease in leucocyte surface expression of most of the adhesion molecules tested and an increase in soluble ICAM-1 and L-selectin levels in secondary progressive and relapsing–remitting multiple sclerosis compared with primary progressive multiple sclerosis, which gave results similar to those in controls. These results, which are supported by MRI evidence, show that trafficking of autoreactive leucocytes through the blood–brain barrier is crucial to the pathogenesis of secondary progressive and relapsing–remitting forms of multiple sclerosis, whereas other mechanisms leading to progressive axonal damage would account for primary progressive forms of the disease. multiple sclerosis; primary progressive; adhesion molecules; blood–brain barrier
CPMS = `chronic progressive' multiple sclerosis; ELISA = enzyme-linked immunosorbent assay; PBL = peripheral blood lymphocytes; PPMS = primary progressive multiple sclerosis; RRMS = relapsing–remitting multiple sclerosis; SPMS = secondary progressive multiple sclerosis. For abbreviations for monoclonal antibodies, see section headed Reagents.
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Introduction |
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Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS in which three major clinical courses have been identified (Lublin and Reingold, 1996
): relapsing–remitting multiple sclerosis (RRMS), characterized by exacerbations with subsequent total or partial remission of symptoms; secondary progressive multiple sclerosis (SPMS), in which progression follows an initial relapsing–remitting phase throughout the evolution of the condition; and primary progressive multiple sclerosis (PPMS), which is a progressive form with no evidence of relapses or remissions. In some cases the SPMS and PPMS multiple sclerosis forms have been grouped into `chronic progressive' multiple sclerosis (CPMS). However, in the first MRI study in which PPMS was specifically investigated it was shown that patients with PPMS, despite marked disability, had fewer and smaller gadolinium enhancing cerebral MRI lesions than patients with SPMS (Thompson et al., 1990, 1997). Furthermore, a recent study of a large cohort of PPMS cases from six European centres has shown clear MRI differences between PPMS and SPMS (Stevenson et al., 1999). The clinical and radiological differences between SPMS, RRMS and PPMS could imply that different mechanisms are involved, e.g. the trafficking of peripheral leucocytes into the CNS, which is a major element in the pathogenesis of relapsing forms of multiple sclerosis but not of PPMS. Adhesion molecules play an important role in this particular process (Springer, 1994). In the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), adhesion molecules have been shown extensively to play a central role in the pathogenesis of the disease (Kuchroo et al., 1993; Anderson et al., 1996; Previtali et al., 1997) and it has also been shown that antibodies against different adhesion molecules inhibit EAE (Yednock et al., 1992; Dopp et al., 1994; Steffen et al., 1994; Gordon et al., 1995; Kobayashi et al., 1995; Soilu-Hanninen et al., 1997). Based on their structure, adhesion molecules are classified as selectins, integrins and members of the Ig superfamily. Selectins are involved in the initial localization of leucocytes to inflammatory sites. Integrins and members of the Ig superfamily mediate strong adhesion and migration of leucocytes across the vascular endothelium (Hohlfeld, 1997).
Though a major role for adhesion molecules has been presumed in multiple sclerosis, in which the ability of leucocytes to cross the blood–brain barrier seems to be an early feature of most developing lesions, most authors have analysed individual adhesion molecules in specific subgroups of patients (Porrini et al., 1992; Svenningson et al., 1993; Salmaggi et al., 1996; Stüber et al., 1996; Lou et al., 1997) and a general overview of the process is still lacking. In relation to soluble forms of adhesion molecules in multiple sclerosis patients, several groups have reported elevated levels of soluble ICAM-1 (intercellular adhesion molecule-1, CD54), soluble L-selectin (CD62L), soluble VCAM-1 (vascular cell adhesion molecule, CD106) or soluble ICAM-3 (intercellular adhesion molecule-3, CD50) (Dore-Duffy et al., 1995; Hartung et al., 1995; Martin et al., 1995; Matsuda et al., 1995; Franciotta et al., 1997; Giovannoni et al., 1997). Most of these studies subdivided multiple sclerosis patients into RRMS and CPMS. Although lower frequencies of oligoclonal bands in CSF (Pirttila and Nurmikko, 1995) or increased levels of antiganglioside antibodies (Acarín et al., 1996; Sadatipour et al., 1998) and soluble E-selectin (Giovannoni et al., 1996; McDonnell et al., 1999) have been described in PPMS, we have not found any study showing definitely clear pathogenic differences between PPMS and relapsing forms of multiple sclerosis. It is thus important to attempt to define the matter clearly for a number of reasons (Thompson et al., 1997). In order to understand better the involvement of adhesion molecules in the pathogenesis of PPMS, we analysed a representative molecule of each family (leucocyte surface expression and soluble serum forms) in PPMS patients in comparison with SPMS and RRMS patients and healthy controls. We found that PPMS differs clearly from SPMS and RRMS in leucocyte surface expression and soluble serum levels of adhesion molecules, showing greater similarity to healthy controls. These findings support the idea that the trafficking of peripheral leucocytes into the CNS plays a major role in the pathogenesis of SPMS and RRMS but not in that of PPMS, and could explain the lack of attacks and the scarcity of gadolinium enhancing lesions observed in patients with PPMS.
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Methods |
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Patients
Eighty-nine patients (33 men and 56 women) with clinically definite multiple sclerosis were included in the study (Poser et al., 1983
). The mean age was 41 years [standard deviation (SD), 12 years; range, 20–67 years]. Thirty-nine had PPMS, 25 had SPMS and 25 had RRMS. None of the multiple sclerosis patients had received beta-interferon treatment or immunosuppressive medication, such as cyclophosphamide, azathioprine or methotrexate. No relapses were observed nor had the patients taken prednisone or methylprednisolone for at least 2 months before or after the samples were collected. No coincidental infections were reported by patients at the time of blood collection.
RRMS patients presented a clinical course of exacerbations followed by complete or partial remission. SPMS patients presented an initial course of RRMS followed by stepwise deterioration with or without superimposed relapses (11 out of 25 patients with SPMS had had superimposed relapses during the previous 2 years and the remaining 14 had not). PPMS patients presented progressive neurological deterioration without relapses. Baseline characteristics of the patients analysed in this work are shown in Table 1. Thirty-eight healthy controls (19 men and 19 women) were used as a control group [mean age ± SD, 39 ± 11 years; range, 25–60 years], and were age- and sex-matched with the multiple sclerosis group. The study was approved by the Ethics Committee of the Hospital General Universitari Vall d'Hebron, and all subjects gave informed consent.
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Reagents
The following conjugated monoclonal antibodies recognizing different surface molecules were used: anti-CD3-phycoerythrin (PE); anti-CD4-peridinium chlorophyll protein (PerCP); anti-CD14-allophycocyanin (APC); anti-CD49d (
4 integrin)-fluorescein isothiocyanate (FITC); anti-CD62L (L-selectin)-FITC (Becton Dickinson, Immunocytochemistry Systems, San Jose, Calif., USA); anti-CD29 (ß1 integrin)-FITC (Immunotech, Marseille, France); anti-CD54 (ICAM-1)– FITC; anti-CD11a (LFA-1)–FITC and anti-IgG control antibodies (Caltag Laboratories, Burlingame, Calif., USA); and streptavidin–FITC (Pharmingen, San Diego, Calif., USA). ICAM-3 was kindly donated by Dr R. Vilella (Hospital Clinic, Barcelona, Spain) and was biotinylated in our laboratory following standard protocols (Goding, 1996).
Serum collection and cell separation
Sera from 75 multiple sclerosis patients (25 PPMS, 25 SPMS and 25 RRMS) and 30 matched healthy controls were collected and were stored frozen at -80°C until use. Peripheral blood mononuclear cells (PBMC) were obtained from 59 multiple sclerosis patients (29 PPMS, 15 SRMS and 15 RRMS) and 16 healthy controls. Cells were isolated from heparinized blood by Ficoll–Isopaque density gradient centrifugation and kept frozen in liquid nitrogen until use. Samples were numbered and stored by a person blind to flow cytometry and enzyme-linked immunosorbent assays (ELISAs). Code was broken and added to the database only after analyses had been completed and introduced into the database, in order to ensure a fully blinded method.
Immunofluorescence and flow cytometry analysis
Four-colour immunofluorescence staining of surface markers was performed by the incubation of PBMC with saturating amounts of combinations of anti-CD3-PE, anti-CD4-PerCP, anti-CD14-APC and FITC-conjugated anti-CD54 (ICAM-1), anti-CD62L (L-selectin), anti-CD29 (ß1 integrin), anti-CD11a (LFA-1) or anti-CD49d (4 integrin) monoclonal antibodies for 30 min. Stained cells were washed with staining buffer (phosphate-buffered saline plus 1% foetal calf serum plus 0.1% sodium azide). After two washes of the cells, a second incubation with streptavidin–PE was performed in tubes containing biotinylated ICAM-3. All incubations were carried out at 4°C and the samples were protected from light. Anti-mouse matched-isotype IgG monoclonal antibodies were used as a negative control. After incubation, cells were resuspended in staining buffer and analysed by flow cytometry with a double laser FacsCalibur® (Becton Dickinson) flow cytometer. Cell analysis was performed with the CellQuest Program® (Becton Dickinson). The total population of peripheral blood lymphocytes (PBL), including T cells, B cells and natural killer cells, was gated according to forward and side light-scattering properties (Fig. 1A, gate R1). T cells were gated on CD3+ cells from the lymphocyte gate (Fig. 1B, gate R2). In the same way, CD4+ cells were gated by selecting cells positive for this marker in the R2 gate (Fig. 1C, gate R3). The monocyte gate was drawn according to forward and side light-scattering properties (Fig. 1D, gate R4) versus CD14+ cells (Fig. 1E, gate R5). The percentage of expression of adhesion molecules was calculated by subtracting the isotype control antibody signal from the specific anti-adhesion molecule antibody signal (Fig. 1F, G and H). The technique was performed on the same day on one sample from each group studied (1RRMS, 1SPMS, 1PPMS and healthy controls). Results are shown as the percentage of positive cells (percentage of the total events in a marker set compared with the number of events within the gate) and mean fluorescence intensity (intensity of expression of a given surface molecule per cell).
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Measurement of soluble adhesion molecules
Soluble serum concentrations of L-selectin, ICAM-1, VCAM-1 and ICAM-3 were measured by sandwich ELISA. Soluble L-selectin ELISA was provided by Dr P. Engel (University of Barcelona, Spain), soluble ICAM-1 and VCAM-1 were measured using commercial ELISA kits (Bender MedSystems, Vienna, Austria and Biosource International Calif., USA, respectively) and soluble ICAM-3 was measured by an ELISA protocol described by Pino-Otín and colleagues (Pino-Otín et al., 1995
) (antibodies required for this assay were provided by Dr A. Gayà, Hospital Clinic, Barcelona). In short, serum samples were applied in duplicate wells at appropriate dilutions on monoclonal antibody coated 96-well microtitre plates. Afterwards, a biotinylated monoclonal antibody was added. Finally, a horseradish-peroxidase-conjugated monoclonal antibody was used. Between incubation steps, unbound protein was removed by washing. Reaction products were measured spectrophotometrically at 450 nm for soluble L-selectin, ICAM-1 and VCAM-1 and at 620 nm for soluble ICAM-3 with an Anthos Labtec Reader 2001 (Salzburg, Austria). Quantitative results were obtained in relation to standard curves. The assays had a lower limit of detection of 3.3 ng/ml for soluble ICAM-1, of 0.8 ng/ml for soluble L-selectin, 0.5 ng/ml for soluble VCAM-1 and of 0.6 ng/ml for soluble ICAM-3. The interassay coefficient of variation was <7% for ICAM-1, 10% for L-selectin, 6% for VCAM-1 and 10% for ICAM-3.
Statistical analysis
Statistical analysis was performed with a microcomputer version of the Statistical Package for Social Sciences® (SPSS). In order to compare the differences in adhesion molecules between multiple sclerosis patients and healthy controls and between the different clinical forms of multiple sclerosis, we applied either the non-parametric test (Mann–Whitney U test) or the parametric test (Student's t test) for independent samples, depending on the size of the sample studied. We considered the level of statistical significance to be P < 0.05.
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Results |
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Down-regulation of surface expression of adhesion molecules in multiple sclerosis patients
To compare the levels of surface expression of adhesion molecules in the different clinical forms of multiple sclerosis, we analysed the percentage and mean fluorescence intensity of ICAM-1 (CD54), LFA-1
(CD11a), VLA-4 [4/ß1 integrin (CD49d/CD29)], L-selectin (CD62L) and ICAM-3 (CD50), in total PBL, in CD4+ T cells and monocytes (CD14+ cells) in 59 multiple sclerosis patients and 16 healthy controls by flow cytometry. A significant decrease in the percentage of ICAM-1-positive cells in all leucocyte populations tested was observed in the group of multiple sclerosis patients versus the healthy controls [mean ± standard error of the mean (SEM): in total PBL, 10.5 ± 0.7 in multiple sclerosis versus 14 ± 1.2 in healthy controls, P = 0.01; in monocytes, 89.7 ± 1.5 in multiple sclerosis versus 95.6 ± 0.8 in healthy controls, P = 0.01).
When analysing the mean fluorescence intensity we observed a significant decrease in LFA1- (in total PBL, P = 0.034), ß1 integrin (CD29) (in total PBL, P = 0.0007; in CD4+ cells, P = 0.0007; in monocytes, P < 0.0001), L-selectin (in total PBL, P = 0.0041; in CD4+ cells, P = 0.028) and ICAM-1 (in monocytes, P = 0.0016) in multiple sclerosis patients compared with healthy controls (Fig. 2).
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Differential expression of adhesion molecules between PPMS, SPMS and RRMS
In Figs 3 and 4
we show a separate analysis of the percentage of positive cells and mean fluorescence intensity in total PBL, CD4+ cells and monocytes (CD14+ cells) of 29 PPMS patients, 15 SPMS patients, 15 RRMS patients and 16 healthy controls.
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= P < 0.05 PPMS versus healthy controls.
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We did not find significant differences between PPMS and healthy controls in the percentage of expression of any of the adhesion molecules tested, except in the case of L-selectin, which was increased in total PBL (P = 0.03) (Fig. 3A
) as well as in CD4+ cells (P = 0.03) (Fig. 3B) in the PPMS group. In contrast, PBL of SPMS and RRMS patients showed significantly decreased levels in most of the adhesion molecules tested: percentage of LFA-1 (in total PBL, SPMS P = 0.015 and RRMS P = 0.047), 4 integrin (in total PBL, SPMS P = 0.0016 and RRMS P = 0.0062; in CD4+ cells, SPMS P = 0.023 and RRMS P = 0.0039), ß1 integrin (in total PBL, SPMS P = 0.0001 and RRMS P < 0.0001; in CD4+ cells, SPMS P = 0.0007 and RRMS P = 0.0001), ICAM-1 (in total PBL, SPMS P = 0.0001 and RRMS P = 0.0058; in CD4+ cells, SPMS P = 0.0002 and RRMS P = 0.0019) and L-selectin (in total PBL, SPMS P = 0.0058 and RRMS P = 0.027; in CD4+ cells, SPMS P = 0.018 and RRMS P = 0.017; ) compared with PPMS (Fig. 3A and B). In monocytes, we found a decrease in 4 integrin (SPMS P = 0.04 and RRMS P = 0.037) and in ICAM-1 (SPMS P = 0.037 and RRMS P < 0.0001) (Fig. 3C).
When analysing mean fluorescence intensity per cell, we observed in SPMS and RRMS a significant decrease in LFA-1 (in total PBL, SPMS P = 0.038 and RRMS P = 0.011; in CD4+ cells, SPMS P = 0.046 and RRMS P = 0.0024; in monocytes, RRMS P = 0.0047) and ß1 integrin (in total PBL, RRMS P = 0.019; in CD4+ cells, SPMS P = 0.032 and RRMS P = 0.0008; in monocytes, SPMS P = 0.0024 and RRMS P < 0.0001) in all cell subpopulations, and of L-selectin in PBL (in total PBL, SPMS P = 0.045 and RRMS P = 0.0041; in CD4+ cells, SPMS P = 0.0072 and RRMS P = 0.0002) and ICAM-1 in monocytes (RRMS P = 0.016) (Fig. 4). In the PBL we observed a slight increase in ICAM-1-positive cells (in total PBL, SPMS P = 0.04 and RRMS P = 0.034; in CD4+ cells, SPMS P = 0.032 and RRMS P = 0.024) (Fig. 4A and B).
There were no differences in ICAM-3 expression among groups (data not shown). We also analysed the percentage and mean fluorescence intensity of the SPMS patients in relation to the presence of previous superimposed relapses. In this respect, we did not find any differences in the percentage and mean fluorescence intensity between SPMS patients with and without relapses (data not shown).
Soluble ICAM-1 and L-selectin molecules are increased in serum of multiple sclerosis patients
Adhesion molecules in multiple sclerosis play an important role in the trafficking of peripheral leucocytes into the CNS. It has been postulated that soluble forms of membrane molecules have an important role as a feedback inhibitor to limit the extent of inflammatory cell infiltration and thus mediate tissue damage. Here we analysed the serum levels of soluble ICAM-1, L-selectin, VCAM-1 and ICAM-3 in 75 patients with multiple sclerosis and 30 healthy controls. A significant increase in soluble ICAM-1 (P = 0.018) (Fig. 5A) and soluble L-selectin (P = 0.035) (Fig. 5B) was observed in multiple sclerosis patients in comparison with healthy controls.
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No significant differences were found in VCAM-1 (mean ± SEM, 740.4 ± 49.6 ng/ml in multiple sclerosis and 721.4 ± 40.5 ng/ml in healthy controls) and ICAM-3 (18.3 ± 1.57 ng/ml in multiple sclerosis and 18.9 ± 3.2 ng/ml in healthy controls) concentrations between the groups.
Soluble adhesion molecules in PPMS, SPMS and RRMS
When results were analysed according to the clinical forms of multiple sclerosis, we found that serum concentrations of ICAM-1 were higher in patients with RRMS than in healthy controls (P = 0.007). Although not statistically significant, a trend was also observed in SPMS patients compared with controls (P = 0.052) (Fig. 6A). In the same way, we observed an increase in soluble L-selectin in RRMS and in SPMS patients compared with healthy controls (P = 0.022 and P = 0.04, respectively) (Fig. 6B). In contrast, similar levels of these soluble adhesion molecules were found between PPMS and healthy controls (Fig. 6).
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We did not find differences between groups, either in soluble VCAM-1 (621 ± 77 ng/ml in RRMS, 739.7 ± 89.9 ng/ml in SPMS, 855.2 ± 86.9 ng/ml in PPMS and 721.2 ± 40.5 in healthy controls) or in soluble ICAM-3 (16.8 ± 2.7 ng/ml in RRMS, 21.5 ± 2.9 ng/ml in SPMS, 16.5 ± 2.6 ng/ml in PPMS and 18.9 ± 3.2 ng/ml in healthy controls). No significant differences were observed in the soluble forms of SPMS patients in relation to the existence of previous superimposed relapses (data not shown).
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Discussion |
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We show here that the expression of adhesion molecules is clearly different between the primary progressive form (PPMS) and the relapsing forms (SPMS and RRMS) of multiple sclerosis. PPMS shows a pattern similar to that seen in healthy controls. In contrast, SPMS and RRMS show decreased leucocyte surface expression of most of the molecules tested and higher soluble serum levels of ICAM-1 and L-selectin. These results support the hypothesis that the pathogenic mechanisms that account for PPMS are different from those accounting for SPMS and RRMS.
In many studies analysing adhesion molecules—either the surface expression forms or the soluble forms—multiple sclerosis patients are divided into two groups, RRMS and CPMS (SPMS and PPMS, because of the progressive deterioration seen in both types). However, unlike SPMS and RRMS, PPMS exhibits deterioration without relapses, has fewer and smaller gadolinium enhancing lesions (Thompson et al., 1990, 1997; Stevenson et al., 1999) and is less responsive to anti-inflammatory drug therapy. This suggests that, besides inflammation, other mechanisms leading to progressive axonal injury may play a crucial role in PPMS (Lucchinetti et al., 1996; Trapp et al., 1998). In the relapsing disease, the importance of induction of adhesion molecule expression and an increase in the permeability of the blood–brain barrier in the context of the autoimmune response have been studied extensively (Butcher and Picker, 1996; Archelos and Hartung, 1997). On the basis of these hypothetical pathogenic differences we decided to analyse the three clinical forms separately.
The first difference found was the increased levels of soluble ICAM-1 and L-selectin in the serum of RRMS and SPMS patients (although in the case of the SPMS group it was not statistically significant for ICAM-1), while PPMS patients did not show statistically significant differences from healthy controls. Several authors have reported an increase in serum concentrations of ICAM-1 and L-selectin in different groups of multiple sclerosis patients (Rieckmann et al., 1994; Dore-Duffy et al., 1995; Trojano et al., 1996). Only a few authors have analysed the three clinical forms (RRMS, SPMS and PPMS) separately (Giovannoni et al., 1997; McDonnell et al., 1998, 1999). Giovannoni and colleagues found increased serum levels of soluble ICAM-1 in RRMS and SPMS, and non-significant levels in PPMS (Giovannoni et al., 1997). McDonnell and colleagues did not find differences in the ICAM-1 and L-selectin index among the different groups of patients (McDonnell et al., 1998). However, in a later study the same authors found increased levels of soluble ICAM-1 in PPMS in comparison with the other clinical forms of multiple sclerosis and increased levels of L-selectin in RRMS patients (McDonnell et al., 1999). Although Giovannoni and colleagues postulate that soluble ICAM-1 could be a marker of progression in multiple sclerosis (Giovannoni et al., 1997), we cannot confirm this result, given that we found this increase only in SPMS and in RRMS. Although some inflammation can be seen in PPMS lesions (Revesz et al., 1994), our results suggest that soluble adhesion molecules might indicate cell activation and might function as a feedback inhibitor to limit the extent of inflammatory cell infiltration in SPMS and RRMS, as has been postulated for other diseases (Lampeter et al., 1992; Gearing and Newman, 1993; Martin et al., 1995; Pino-Otín et al., 1995).
On the other hand, we did not find variation among the groups of patients in the serum levels of soluble VCAM-1, in contrast to the results of some authors (Hartung et al., 1995; Matsuda et al., 1995; McDonnell et al., 1999) and in agreement with those of others (Dore-Duffy et al., 1995; Giovannoni et al., 1997). A possible reason for this could be that IFN-
, which is associated with disease activity, increases the expression of ICAM-1 but not that of VCAM-1 (Cartwright et al., 1995; Giovannoni et al., 1997). In this context, we have observed increased production of IFN- by lymphocytes in relapsing forms of multiple sclerosis but not by those of PPMS patients (Durán et al., 1998).
Although we have not studied concentrations of other endothelial markers, such as soluble E-selectin, it is interesting to point out that Giovannoni and colleagues found a significant increase, measured by ELISA, in this molecule in five out of 10 patients with PPMS compared with nine SPMS and nine RRMS patients (Giovannoni et al., 1996). These authors found that patients with PPMS and increased levels of E-selectin had more rapid progression of disability than those patients with lower levels of E-selectin. More recently, McDonnell and colleagues also found increased serum levels of E-selectin in a large cohort of PPMS patients but not in SPMS and RRMS patients (McDonnell et al., 1999). The results support the immunological heterogeneity of the different clinical forms of multiple sclerosis and suggest, as pointed out by McDonnell and colleagues, that different leucocyte/endothelial cell interactions may be acting in the different clinical forms (McDonnell et al., 1999).
Regarding the surface expression of adhesion molecules, we observed a second important difference between the SPMS and RRMS forms compared with PPMS. In SPMS and RRMS we found a global decrease in leucocyte surface expression of most of the adhesion molecules tested, whereas PPMS showed a pattern similar to that seen in healthy controls.
We found decreases in the percentages of ICAM-1-positive cells (in PBL and monocytes) and L-selectin-positive cells (in PBL) compared with PPMS (Fig. 3). These decreases could be related to the increases in soluble ICAM-1 and L-selectin found in SPMS and RRMS that were mentioned above. Interestingly, a slight increase in surface mean fluorescence intensity of ICAM-1 was observed in PBL (Fig. 4) in contrast to the clear decrease found in monocytes, suggesting that the increase in soluble ICAM-1 was mainly due to the monocyte subpopulation.
Remarkably, the percentage of L-selectin-positive cells was higher in PPMS patients than in healthy controls and SPMS and RRMS patients (Fig. 3A and B). In fact, it has been demonstrated recently that L-selectin is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site (Kahn et al., 1998). The higher expression of membrane L-selectin and the low levels of soluble L-selectin in PPMS compared with SPMS and RRMS reinforces the hypothesis of a lower degree of lymphocyte activation in PPMS compared with SPMS and RRMS. However, further investigation is necessary in order to explain this difference.
In SPMS and RRMS patients we also observed a decrease in the percentage of 4 and ß1 integrins and a decrease in the mean fluorescence intensity of ß1 integrin (Figs 3 and 4). In this regard, Stüber and colleagues did not detect abnormalities in RRMS patients when analysing the mean fluorescence intensity of ß1 integrin in peripheral CD4+ cells (Stüber et al. 1996), but Svenningson and colleagues reported a decrease in the percentage of ß1 integrin in T cells in RRMS patients (Svenningson et al., 1993). As their study suggests, these results could be explained by a pronounced reduction in expression of ß1 integrin by the CD8+ population. However, we found similar levels of ß1 integrin both in total PBL and CD4+, which rules out a reduction in ß1 integrin levels caused by CD8+ cells.
Some investigators have reported a tendency towards a reduction in the percentage of LFA-1 and ICAM-1 expression in T cells in RRMS patients (Svenningsson et al., 1993) while others have reported an increase in mean fluorescence intensity of LFA-1 on circulating leucocytes in CPMS patients (Lou et al., 1997). However, we found an important decrease in LFA-1 in SPMS and RRMS in PBL and monocytes compared with PPMS (Fig. 4). This decrease in LFA-1 ( chain of LFA-1, ligand of ICAM-1) might be due to modulation by the high soluble levels of ICAM-1 seen in SPMS and RRMS.
Multiple sclerosis lesions are histopathologically characterized by multifocal perivenular infiltration of the white matter by lymphocytes and monocytes/macrophages within the CNS (Lassmann, 1997). At the molecular level, adhesion molecules are critically involved in adhesion and migration through the blood–brain barrier. Soluble adhesion molecules may function as a feedback inhibitor to limit the extent of inflammatory cell infiltration and thus mediate tissue damage. The increased levels of soluble ICAM-1 and L-selectin and the decreased levels of surface expression of adhesion molecules that we observed in SPMS and RRMS supports this hypothesis.
Although adhesion molecules levels have been found previously to vary considerably when measured longitudinally within individual patients and the interpretation of single measurements in patients is therefore controversial (Calabresi et al., 1997), the careful clinical selection, the absence of relapses in patients for at least 2 months before and after collecting the samples, the relatively high number of patients and the concordance between techniques (ELISA and flow cytometry) support the validity of our results.
We did not intend to find specific adhesion molecules as markers of the clinical course or activity of multiple sclerosis as more than one molecule is most probably involved in this process. Our results suggest that the PPMS form differs from the SPMS and RRMS forms in the expression of adhesion molecules. That PPMS shows levels of leucocyte surface expression that are similar to those seen in healthy controls and different from those in SPMS and RRMS patients suggests that crossing of the blood–brain barrier by activated T lymphocytes would be slight in PPMS and more active in SPMS and in RRMS patients, leading to sudden changes in the permeability of the blood–brain barrier that are characteristic of SPMS and RRMS, according to the MRI studies. Despite there being some inflammation in PPMS lesions, other mechanisms leading towards progressive axonal injury may play a crucial role in the pathogenesis of PPMS.
Taking all these results into account, we conclude that PPMS clearly differs from SPMS and RRMS and that PPMS should be considered a differentiated single group in further studies.
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Acknowledgments |
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We wish to thank Ms I. Saez-Torres for help in conducting blinded assays and Mr Josep Graells and Ms Julie Myers for language editing. We also wish to thank Dr R. Vilella and Dr A. Gayà for providing CD50 antibodies, Dr P. Engel for donating the CD62L ELISA and Dr H. P. Hartung for critical reading of the manuscript. I.D. was supported by a fellowship grant awarded by the Fundación Esclerosis Múltiple.
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References |
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Acarín N, Río J, Fernandez AL, Tintoré M, Durán I, Galán I, et al. Different antiganglioside antibody pattern between relapsing–remitting and progressive multiple sclerosis. Acta Neurol Scand 1996; 93: 99–103.[Medline]
Anderson JA, Lentsch AB, Hadjiminas DJ, Miller FN, Martin AW, Nakagawa K, et al. The role of cytokines, adhesion molecules, and chemokines in interleukin-2-induced lymphocytic infiltration in C57BL/6 mice. J Clin Invest 1996; 97: 1952–9.[Web of Science][Medline]
Archelos JJ, Hartung HP. The role of adhesion molecules in multiple sclerosis: biology, pathogenesis and therapeutic implications. [Review]. Mol Med Today 1997; 3: 310–21.[Web of Science][Medline]
Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. [Review]. Science 1996; 272: 60–6.[Abstract]
Calabresi PA, Tranquill LR, Dambrosia JM, Stone LA, Maloni H, Bash CN, et al. Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosis treated with interferon ß-1b. Ann Neurol 1997; 41: 669–74.[Web of Science][Medline]
Cartwright JE, Whitley GSJ, Johnstone AP. The expression and release of adhesion molecules by human endothelial cell lines and their consequent binding of lymphocytes. Exp Cell Res 1995; 217: 329–35.[Web of Science][Medline]
Dopp JM, Breneman SM, Olschowka JA. Expression of ICAM-1, VCAM-1, L-selectin, and leukosialin in the mouse central nervous system during the induction and remission stages of experimental allergic encephalomyelitis. J Neuroimmunol 1994; 54: 129–44.[Web of Science][Medline]
Dore-Duffy P, Newman W, Balabanov R, Lisak RP, Mainolfi E, Rothlein R, et al. Circulating, soluble adhesion proteins in cerebrospinal fluid and serum of patients with multiple sclerosis: correlation with clinical activity. Ann Neurol 1995; 37: 55–62.[Web of Science][Medline]
Durán I, Martínez-Cáceres E, Barberà N, Río J, Tintoré M, Montalban X. Immunological profile of primary progressive multiple sclerosis [abstract]. Multiple Sclerosis 1998; 4: 340.
Franciotta D, Piccolo G, Zardini E, Bergamaschi R, Cosi V. Soluble CD8 and ICAM-1 in serum and CSF of MS patients treated with 6-methylprednisolone. Acta Neurol Scand 1997; 95: 275–9.[Web of Science][Medline]
Gearing AJ, Newman W. Circulating adhesion molecules in disease [see comments]. [Review]. Immunol Today 1994; 14: 506–12. Comment in: Immunol Today 1993; 15: 140-1, Comment in: Immunol Today 1994; 15: 339, Comment in: Immunol Today 1995; 16: 251–2.
Giovannoni G, Thorpe JW, Kidd D, Kendall BE, Moseley IF, Thompson AJ, et al. Soluble E-selectin in multiple sclerosis: raised concentrations in patients with primary progressive disease. J Neurol Neurosurg Psychiatry 1996; 60: 20–6.
Giovannoni G, Lai M, Thorpe J, Kidd D, Chamoun V, Thompson AJ, et al. Longitudinal study of soluble adhesion molecules in multiple sclerosis: correlation with gadolinium enhanced magnetic resonance imaging. Neurology 1997; 48: 1557–65.
Goding JW. Monoclonal antibodies: principles and practice. 3rd ed. London: Academic Press; 1996.
Gordon EJ, Myers KJ, Dougherty JP, Rosen H, and Ron Y. Both anti-CD11a (LFA-1) and anti-CD11b (MAC-1) therapy delay the onset and diminish the severity of experimental autoimmune encephalomyelitis. J Neuroimmunol 1995; 62: 153–60.[Web of Science][Medline]
Hartung HP, Reiners K, Archelos JJ, Michels M, Seeldrayers P, Heidenreich F, et al. Circulating adhesion molecules and tumor necrosis factor receptor in multiple sclerosis: correlation with magnetic resonance imaging. Ann Neurol 1995; 38: 186–93.[Web of Science][Medline]
Hohlfeld R. Biotechnological agents for the immunotherapy of multiple sclerosis. Principles, problems and perspectives. [Review]. Brain 1997; 120: 865–916.
Kahn J, Walcheck B, Migaki GI, Jutila MA, Kishimoto TK. Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism. Cell 1998; 92: 809–18.[Web of Science][Medline]
Kobayashi Y, Kawai K, Honda H, Tomida S, Niimi N, Tamatani T, et al. Antibodies against leukocyte function-associated antigen-1 and against intercellular adhesion molecule-1 together suppress the progression of experimental allergic encephalomyelitis. Cell Immunol 1995; 164: 295–305.[Web of Science][Medline]
Kuchroo VK, Martin CA, Greer JM, Ju ST, Sobel RA, Dorf ME. Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T cell clones to mediate experimental allergic encephalomyelitis. J Immunol 1993; 151: 4371–82.[Abstract]
Lampeter ER, Kishimoto TK, Rothlein R, Mainolfi EA, Bertrams J, Kolb H, et al. Elevated levels of circulating adhesion molecules in IDDM patients and in subjects at risk for IDDM. Diabetes 1992; 41: 1668–71.[Abstract]
Lassmann H. Basic mechanisms of brain inflammation. [Review]. J Neural Transm 1997; Suppl 50: 183–90.
Lou J, Chofflon M, Juillard C, Donati Y, Mili N, Siegrist CA, et al. Brain microvascular endothelial cells and leukocytes derived from patients with multiple sclerosis exhibit increased adhesion capacity. Neuroreport 1997; 8: 629–33.[Web of Science][Medline]
Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 1996; 46: 907–11.
Lucchinetti CF, Bruck W, Rodriguez M, Lassmann H. Distinct patterns of multiple sclerosis pathology indicates heterogeneity on pathogenesis. [Review]. Brain Pathol 1996; 6: 259–74.[Web of Science][Medline]
Martin S, Rieckmann P, Melchers I, Wagner R, Bertrams J, Voskuyl AE, et al. Circulating forms of ICAM-3 (cICAM-3). Elevated levels in autoimmune diseases and lack of association with cICAM-1. J Immunol 1995; 154: 1951–5.[Abstract]
Matsuda M, Tsukada N, Miyagi K, Yanagisawa N. Increased levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in the cerebrospinal fluid and sera of patients with multiple sclerosis and human T lymphotropic virus type-1 associated myelopathy. J Neuroimmunol 1995; 59: 35–40.[Web of Science][Medline]
McDonnell GV, McMillan SA, Douglas JP, Droogan AG, Hawkins SA. Raised CSF levels of soluble adhesion molecules across the clinical spectrum of multiple sclerosis. J Neuroimmunol 1998; 85: 186–92.[Web of Science][Medline]
McDonnell GV, McMillan SA, Douglas JP, Droogan AG, Hawkins SA. Serum soluble adhesion molecules in multiple sclerosis: raised sVCAM-1, sICAM-1 and sE-selectin in primary progressive disease. J Neurol 1999; 246: 87–92.[Web of Science][Medline]
Moore CJ, Price CJ. Three distinct regions for word and picture naming in the ventral visual pathway. Neuroimage 1999; 10: 181–92.[Web of Science][Medline]
Mummery CJ, Patterson K, Hodges JR, Wise RJ. Generating `tiger' as an animal name or a word beginning with T: differences in brain activation. Proc R Soc Lond Biol Sci 1996; 263: 989–95.[Medline]
Pino-Otín MR, Viñas O, de la Fuente MA, Juan M, Font J, Torradeflot M, et al. Existence of soluble form of CD50 (intercellular adhesion molecule-3) produced upon human lymphocyte activation. Present in normal human serum and levels are increased in the serum of systemic lupus erythematosus patients. J Immunol 1995; 154: 3015–24.[Abstract]
Pirttila T, Nurmikko T. CSF oligoclonal bands, MRI, and the diagnosis of multiple sclerosis. Acta Neurol Scand 1995; 92: 468–71.[Web of Science][Medline]
Porrini AM, Gambi D, Malatesta G. Memory and naive CD4+ lymphocytes in multiple sclerosis. J Neurol 1992; 239: 437–40.[Web of Science][Medline]
Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983; 13: 227–31.[Web of Science][Medline]
Previtali SC, Archelos JJ, Hartung HP. Modulation of the expression of integrins on glial cells during experimental autoimmune encephalomyelitis. A central role for TNF-. Am J Pathol 1997; 151: 1425–35.[Abstract]
Price CJ. The functional anatomy of language processing. Trends Cognit Sci 1998; 2: 281–8.
Price CJ, Friston KJ. Cognitive conjunctions: a new approach to brain activation experiments. Neuroimage 1997; 5: 261–70.[Web of Science][Medline]
Price CJ. Functional anatomy of reading. In Frackowiak RSJ, Friston KJ, Frith CD, Dolan RJ, Mazziotta JC, editors. Human brain function. San Diego: Academic Press; 1997, p. 301–28.
Raichle ME, Ficz JA, Videen TO, MacLeod AM, Pardo JV, Fox PT, et al. Practice-related changes in human brain functional anatomy during nonmotor learning. Cereb Cortex 1994; 4: 8–26.
Revesz T, Kidd D, Thompson AJ, Barnard RO, McDonald WI. A comparison of the pathology of primary and secondary progressive multiple sclerosis. Brain 1994; 117: 759–65.
Rieckmann P, Martin S, Weichselbraun I, Albrecht M, Kitze B, Weber T, et al. Serial analysis of circulating adhesion molecules and TNF receptor in serum from patients with multiple sclerosis: cICAM-1 is an indicator for relapse. Neurology 1994; 44: 2367–72.
Sadatipour BT, Greer JM, Pender MP. Increased circulating antiganglioside antibodies in primary and secondary progressive multiple sclerosis. Ann Neurol 1998; 44: 980–3.[Web of Science][Medline]
Salmaggi A, Dufour A, Eoli M, Corsini E, La Mantia L, Massa G, et al. Low serum interleukin-10 levels in multiple sclerosis: further evidence for decreased systemic immunosuppression? J Neurol 1996; 243: 13–7.[Web of Science][Medline]
Shaywitz BA, Pugh KR, Constable RT, Shaywitz SE, Bronen RA, Fulbright RK, et al. Localization of semantic processing using functional magnetic resonance imaging. Hum Brain Mapp 1995; 2: 149–58.
Soilu-Hanninen M, Roytta M, Salmi A, Salonen R. Therapy with antibody against leukocyte integrin VLA-4 (CD49d) is effective and safe in virus-facilitated experimental allergic encephalomyelitis. J Neuroimmunol 1997; 72: 95–105.[Web of Science][Medline]
Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. [Review]. Cell 1994; 76: 301–14.[Web of Science][Medline]
Steffen BJ, Butcher EC, Engelhardt B. Evidence for involvement of ICAM-1 and VCAM-1 in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the central nervous system in the SJL/J mouse. Am J Pathol 1994; 145: 189–201.[Abstract]
Stevenson VL, Miller DH, Rovaris M, Barkhof F, Brochet B, Dousset V, et al. Primary and transitional progressive and transitional progressive MS: a clinical and MRI cross-sectional study. Neurology 1999; 52: 839–45.
Stüber A, Martin R, Stone LA, Maloni H, McFarland HF. Expression pattern of activation and adhesion molecules on peripheral blood CD4+ T-lymphocytes in relapsing–remitting multiple sclerosis patients: a serial analysis. J Neuroimmunol 1996; 66: 147–51.[Web of Science][Medline]
Svenningsson A, Hansson GK, Andersen O, Andersson R, Patarroyo M, Stemme S. Adhesion molecule expression on cerebrospinal fluid T lymphocytes: evidence for common recruitment mechanisms in multiple sclerosis, aseptic meningitis, and normal controls. Ann Neurol 1993; 34: 155–61.[Web of Science][Medline]
Thompson AJ, Kermode AG, MacManus DG, Kendall BE, Kingsley DP, Moseley IF, et al. Patterns of disease activity in multiple sclerosis: a clinical and magnetic resonance imaging study [see comments]. BMJ 1990; 300: 631–4. Comment in: BMJ 1990; 300: 1272.
Thompson AJ, Polman CH, Miller DH, McDonald WI, Brochet B, Filippi M, et al. Primary progressive multiple sclerosis. [Review]. Brain 1997; 120: 1085–96.
Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mörk S, Bö L. Axonal transection in the lesions of multiple sclerosis [see comments]. N Engl J Med 1998; 338: 278–85. Comment in: N Engl J Med 1998; 338: 323–5.
Trojano M, Avolio C, Simone IL, Defazio G, Manzari C, De Robertis F, et al. Soluble intercellular adhesion molecule-1 in serum and cerebrospinal fluid of clinically active relapsing–remitting multiple sclerosis: correlation with Gd-DTPA magnetic resonance imaging-enhancement and cerebrospinal fluid findings. Neurology 1996; 47: 1535–41.
Warburton E, Wise RJ, Price CJ, Hadar U, Ramsay S, et al. Noun and verb retrieval in normal subjects. Studies with PET. [Review]. Brain 1996; 119: 159–79.
Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against 4ß1 integrin. Nature 1992; 356: 63–6.[Medline]
Received May 24, 1999. Accepted June 30, 1999.
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