Correlation between disease severity and in vitro cytokine production mediated by MSRV (Multiple Sclerosis associated RetroViral element) envelope protein in patients with multiple sclerosis

Journal of Neuroimmunology 160 (2005) 195 – 203 www.elsevier.com/locate/jneuroim

Correlation between disease severity and in vitro cytokine production mediated by MSRV (Multiple Sclerosis associated RetroViral element) envelope protein in patients with multiple sclerosis Alexandre Rollanda, Evelyne Jouvin-Marchea, Marina Saresellab, Pasquale Ferranteb, Rosella Cavarettac, Alain Cre´anged, Patrice Marchea,*, Herve´ Perrone,* a Laboratoire d’Immunochimie, CEA/ INSERM-U548, 17 rue des Martyrs 38054 Grenoble, France Laboratorio di Biologia, Don C. Gnocchi Foundation, IRCCS, Via Capecelatro, 66, 20148 Milan, Italy c Multiple Sclerosis dpt, Don C. Gnocchi Foundation, IRCCS, Via Capecelatro, 66, 20148 Milan, Italy d Service de Neurologie, Hoˆpital Henri Mondor, 51, Av Mar de Lattre de Tassigny 94000 Cre´teil, France e De´partement des Immunoessais, bioMe´rieux, Chemin de l’Orme 69280 Marcy l’Etoile, France b

Received 3 September 2004; accepted 19 October 2004

Abstract MSRV is a retroviral element previously isolated in cell cultures from patients with multiple sclerosis. It is part of a new multi-copy endogenous retrovirus family named HERV-W and displays pro-inflammatory properties both in vitro in human PBMC cultures and in vivo in a humanized SCID mice model. In the present study, we have evaluated potential links between the pro-inflammatory properties of MSRV envelope protein and MS disease. Thus, cytokine productions mediated by the surface unit of MSRV envelope protein were evaluated in PBMC of MS patients and compared with healthy controls. Divergent reactivity to ENV-SU between MS and control PBMC was observed and was reflected by a significant increase of IFN-g, IL-6 and IL-12p40 production by the tested MS population. Interestingly, the overproduction of IL-6 and IL-12p40 was found to correlate with disease severity (EDSS) in most patients. Altogether our data suggest that MSRV envelope protein may induce an abnormal cytokine secretion, thus contributing to the inflammatory process in MS. D 2004 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Endogenous retrovirus; Inflammation; Cytokines; Th1/Th2; HERV-W; MSRV

1. Introduction Multiple sclerosis (MS) is a common inflammatory demyelinating disease of the central nervous system (CNS) characterized by multifocal plaques of demyelination associated with activated macrophage/microglia and massive lymphocyte infiltration through disrupted blood/brain barrier (Bar-Or et al., 1999). The aetiology of MS remains unknown, * Corresponding authors. P.N. Marche is to be contacted at Tel.: +33 4 38 35 75; fax: +33 4 38 78 98 03. H. Perron, Tel.: +33 4 78 87 75 28; fax: +33 4 78 87 73 65. E-mail addresses: [email protected] (P. Marche)8 [email protected] (H. Perron). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.10.019

although evidences indicate the involvement of autoimmune, genetic and environmental factors (Compston and Sadovnick, 1992). Numerous immunological events are also involved in the development/persistence of MS and cytokines are thought to play a major role in the inflammatory demyelinating process. Despite recent critical re-evaluation, MS is related to a dysregulation of the balance between proinflammatory Th1 cytokines and anti-inflammatory Th2 cytokines (Hintzen and Polman, 1997). Thus, several studies have shown that pro-inflammatory cytokines such as TNF-a are up-regulated in MS serum and/or cerebrospinal fluids and that levels observed are often correlated with disease progression (Chofflon et al., 1992; van Oosten et al., 1998). In parallel, production of inhibitory or anti-inflammatory

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cytokines such as IL-10 is often associated with less disability (Petereit et al., 2003) and observed in patients treated with hinterferon (Rep et al., 1999). If immunological dysfunctions are intimately associated with MS pathogenesis, the role of environmental factors such as viruses has also been argued in the epidemiology of MS. As for immunological aspects, their role may not be univocal. Contrary to the opinion that one virus or bacteria would alone be a causative agent, as in classical infectious disease, new concepts are now arising in diseases resulting from specific interactions of several infectious and/or latent pathogens (e.g. HIV-1 associated diseases grouped in a unifying bAcquired Immune Deficiency SyndromeQ). Similar concepts are now suggested in MS, which may be caused by an immunological pathogenic process, activated and possibly maintained by a retroviral agent, itself triggered by certain viral infections (Haahr and Munch, 2000; Perron, 2001). Such a retroviral agent, named MSRV (for multiple sclerosis associated retroviral element) was characterized in MS but, contrary to known human infectious retroviruses (HIV, HTLV), it belongs to a family of multicopy human endogenous retrovirus (HERV-W) (Komurian-Pradel et al., 1999). HERV comprise between 5% and 8% of the human genome and represent the proviral phase of exogenous retroviruses that have integrated into the germline of their host (Belshaw et al., 2004). The complete significance of such HERV expression in humans is far from being understood and is only beginning to be addressed in genetic and functional studies. Nonetheless, such retroviral agents are well known in mice with the prototypic Mouse Mammary Tumor Virus (MMTV) and, from a functional point of view, were found to encode the first viral superantigen ever characterized thus linking their presence in mice genome with an immunological function or, rather, dysfunction (Choi et al., 1991; Marrack et al., 1991). MSRV was previously isolated from leptomeningeal, choroid plexus and Monocyte/Macrophage cultures of patients with MS (Perron et al., 1989, 1991). Independent studies have now confirmed detection of virion-associated MSRV RNA in sera and/or cerebrospinal fluids (CSF) of MS patients (Dolei et al., 2002; Garson et al., 1998; Serra et al., 2001) and have shown that CSF detection correlates with disease activity when detected in early cases (Sotgiu et al., 2002). In recent reports, we have demonstrated that MSRV particles cause T lymphocyte-dependent death with brain haemorrhage in humanized SCID mice (Firouzi et al., 2003). We have also observed that MSRV appears to possess superantigen (SAg) like properties (Perron et al., 2001) mediated by its envelope protein. Interestingly, the stimulation of human PBMC by MSRV envelope protein leads to the production of pro-inflammatory cytokines such as TNF-a, IL-6 and IFN-g. Since MSRV particles are often found in MS patient’s body fluids, and that MSRV envelope protein has the ability to induce the production of cytokines, one should expect that recurrent and/or chronic exposure to this

retroviral immunotoxin would at least create an increasing dysfunction or bias in the immune system of MS patients. Moreover, a recent report has evidenced a positive feed back loop on MSRV expression in carrier cells generated by pro-inflammatory cytokines such as those triggered by MSRV envelope itself (Serra et al., 2003). These data have incited us to search for eventual bias in MS peripheral immune system compared to appropriate controls after exposure to the surface soluble unit (ENV-SU) of MSRV envelope protein, naturally present at the virion surface.

2. Patients and methods 2.1. Patients Thirty-three patients (22 females and 11 males) with clinical definite MS were included in the study (Table 1). Both treated and untreated patients were included. The Unita Sclerosi Multipla of the Don Gnocchi Foundation, Table 1 Patients characteristics Patient number

Sex

Age

Disease duration

Course

Therapy

EDSS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

M F F F F M F M F F F M M M F F F F F M M M F M F F F M F F F F F

36 28 30 50 37 25 36 44 39 35 42 41 28 34 28 48 28 39 48 26 52 63 39 30 32 31 29 53 55 46 63 51 54

8 3 10 20 16 3 21 23 4 15 15 21 5 8 2 18 1 18 28 18 23 38 3 7 6 5 3 6 0 0 3 16 5

RR RR RR SP/R RR RR RR RR RR RR RR RR RR RR RR RR RR RR RR SP SP SP SP/R RR RR RR RR PP RR RR SP RR PP

No No No No No No Azathioprine No No No No No No No No No No No No IFN-h No No No Photopheresis No No No No No No No IFN-h No

1.5 2 2.5 3 3 2 4 3 3 1 1.5 1 2.5 4 1 4 1 3.5 6 7 8 8 7 5 3.5 2 2 1 1 1 4.5 1 4.5

RR=relapsing–remitting form; SP=secondary progressive form; PP=primary progressive form.

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Milan, Italy and the Mondor Hospital, Cre´teil, France, respectively, followed patients 1–26 and 27–33. The mean age was 40F9 years (range 25–63 years). Assessment of disability using Expanded Disability Status Scale (EDSS) was made (Kurtzke, 1983) for each patient. Twenty healthy volunteers obtained from the e´tablissement francais du sang, Grenoble, France were also included in the study. 2.2. ENV-SU protein and mock control ENV-SU is a 293 amino acids fraction of the full length MSRV envelope protein (ENV pV14; genbank AF331500). Recombinant ENV-SU protein was synthesized in Escherichia coli and purified on FPLC column by Protein Expert (Grenoble, France). Quality and purity of the protein were assessed by mass spectrometry and western blot. A mock protein (Mock) with no immunological properties was synthesized and purified under the same conditions as ENV-SU and used as a negative control. Both proteins were tested for the presence of endotoxins by a Lymulus Amebocyte Lysate (LAL) test performed by CleanCells (Bouffere, France) and all fractions were below the detection level of 5 UI/ml. In order to further rule out the possibility of an endotoxin contamination in our protein preparations, several specific anti-ENV-SU monoclonal antibodies were tested for the inhibition of ENV-SU mediated cytokine productions. Two of these antibodies gave rise to significant inhibitions (more than 50%) whereas several anti-GAG antibodies did not inhibit ENV-SU properties (data not shown). 2.3. Isolation of peripheral blood mononuclear cells (PBMC) Blood was obtained by venous puncture and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation on Ficoll Paque. Cells at the interphase were collected and washed three times in PBS supplemented with 2% fetal calf serum (FCS) (BioWest, Nouaille, France). Cells were then frozen at 80 8C in FCS+10% DMSO, prior to use. 2.4. PBMC culture Cells were thawed and washed three times in RPMI 1640 (Gibco, Rockville, MD) supplemented with 1% lglutamine (Sigma, St Louis, MO), 1% penicillin/streptomycin (Sigma), 1% sodium pyruvate (Sigma), 1% non essential amino acids (Sigma), and 10% heat inactivated human AB serum (Sigma). Cell viability was then assessed by Trypan blue exclusion and viable cells were resuspended in complete medium at 1106 cells/ml. One milliliter of cell suspension was then plated in wells of a 24-well plate and incubated for 6 h at 37 8C in 5% CO2 in humidified atmosphere with either mock control or ENV-

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SU, both at 1 Ag/ml. IL-2 at 10 UI/ml (R&D Systems, Minneapolis, MN) was then added to each well and cells were further incubated for 18 hours in order to obtain the optimal cytokine production. 2.5. Cytokine production analysis Cell supernatants were collected and IL-1h, IL-6, TNFa, IFN-g, IL-12p40 and IL-10 productions were determined using commercial ELISA kits from BD-Pharmingen (San Diego, CA). In all cases, the manufacturer’s instructions were followed without modifications. All samples were tested in duplicates. The sensitivities of the assays, expressed by the manufacturer as the minimum detectable level of cytokine production were as followed: IFN-g, b4.7 pg/ml; TNF-a, b7.8 pg/ml; IL-1h, b15.4 pg/ml; IL-6, b4.7 pg/ml; IL-10, b7.8 pg/ml and IL-12p40, b7.8 pg/ml. 2.6. Statistical analysis All statistical analysis were conducted with the software Statview. Cytokine productions induced by mock and ENVSU in healthy volunteers and MS patients were compared using a Student’s t-test. Rank correlation with the Spearman correlation coefficient was used to examine the relationships between clinical data and cytokine productions. For both tests, p values b0.05 are referred to as statistically significant in the text.

3. Results 3.1. Analysis of cytokine production induced by ENV-SU and mock control treatment in healthy individuals and MS patients We first evaluated the ability of ENV-SU to stimulate cytokine production in PBMC cultures from 33 patients with MS and 20 healthy individuals. ENV-SU mediated cytokine production was compared with Mock treatment, the latter representing the spontaneous cytokine release. Cells were stimulated for 24 h and productions of TNF-a, IFN-g, IL-1h, IL-6, IL-10 and IL-12p40 were measured by ELISA. Both ENV-SU and Mock were used at a concentration of 1 Ag/ml, found to be the optimal concentration for cytokine release. Mean cytokine productions obtained with both healthy controls and MS patients are presented in Table 2. Cytokine productions obtained by incubation with ENVSU were significantly increased in both groups when compared with Mock, which gave rise to only marginal amounts of cytokine. It thus appears that ENV-SU has the ability to induce the production of cytokines in both healthy controls and MS patients. In order to evaluate the specificity of ENV-SU mediated cytokine productions and to compare responses obtained between healthy controls and MS patients, we

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Table 2 Cytokine production following Mock and ENV-SU treatment in PBMC cultures from healthy controls and MS patients IFN-g

TNF-a

IL-1h

IL-6

IL-12p40

IL-10

(A) MS patients (n=33) Mock 14.9F10.5 ENV-SU 72.3F51.6

31F41 501F178

92F107 873F401

2467F3524 22,698F16,462

22F21 265F165

29.3F16 210F130

(B) Healthy controls (n=20) Mock 7.7F6.2 ENV-SU 30.5F17.7

26F28 411F192

86F88 1016F437

1329F1461 8286F4417

12.1F12.4 111F60

22.2F14.4 191F124

Mean valuesFstandard errors are presented. ENV-SU mediated cytokine production was always considered to be significantly increased when compared with Mock stimulation ( pN0.05 as assessed by a Student’s t-test).

first studied in detail the spontaneous cytokine release (mock control treatment) obtained in both groups (Fig. 1). When we looked at the mean cytokine releases, only IFNg was significantly ( p=0.02) increased in the patient’s group. However, it appears that the significant difference observed with IFN-g between patients and healthy

controls is only due to one single patient who shows high spontaneous IFN-g release. No significant differences were observed for the mean TNF-a, IL-1h, IL-6, IL-10 and IL-12p40 productions. Although some individuals with MS displayed increased spontaneous cytokine productions in every cytokines tested, no highly signifi-

Fig. 1. Spontaneous cytokine production following Mock treatment (1 Ag/ml) on PBMCs from healthy controls (HC) and MS patients (MS). Mean values (–) and statistical significance are shown (significant when pb0.05; NS: not significant).

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cant differences were observed for spontaneous productions between MS and controls. We then studied in detail the cytokine productions induced by ENV-SU in PBMC from both groups. Several main points emerged from this study. First, as shown in Fig. 2 and already presented in Table 2, the mean production of all cytokines was highly increased when compared to the mock treatment. Secondly, the mean production of TNF-a, IL-1h, and IL-10 remained similar between patients and controls. Then, IFN-g production that was already significantly increased with mock treatment displayed a significant increase in MS following ENV-SU treatment when compared with healthy donors. Finally, two additional cytokines, IL-12p40 and IL-6, which were not spontaneously elevated in most MS patients were shown to be significantly increased following ENV-SU treatment. Taken together, our findings suggest that ENV-SU specifically induces the

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over production of pro-inflammatory cytokines such as IFNg, IL-12p40 and IL-6 in MS patients. 3.2. Analysis of the IL-12p40/IL-10 ratio IL-12p40 is a subunit of IL-12, a cytokine involved in the development of Th1 responses, and IL-10 an immunoregulatory cytokine known to inhibit IFN-g secretion by Th1 lymphocytes. We have here analyzed and compared the ratio of IL-12p40/IL-10 productions in both patients and controls. This ratio was significantly increased in MS patients treated with ENV-SU when compared with any other condition (Fig. 3). Healthy controls displayed a similar ratio between both treated and untreated PBMCs. It thus appears that the effect of ENV-SU treatment on PBMCs from MS patients is capable to promote a cytokine microenvironment known to facilitate Th1 like responses.

Fig. 2. Cytokine production following ENV-SU treatment (1 Ag/ml) on PBMCs from healthy controls (HC) and MS patients (MS). Mean values (–) and statistical significance are shown (significant when pb0.05; NS: not significant).

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Fig. 3. Evaluation of the IL-12p40/IL-10 ratio in both stimulated and unstimulated PBMCs from MS patients and healthy controls. Statistical significance between MS patients treated with ENV-SU and all other conditions is shown (*pb0.05).

3.3. Clinical and immunological correlations We have shown that the mean productions of IL-6, IL12p40 and IFN-g were increased in MS patients following ENV-SU treatment when compared with healthy donors. However, it appears that within the MS population tested, approximately one third to one half of the patients tested

with MS gave rise to normal amounts of cytokine when compared to the control group. Thus, in order to determine the significance of the immunological bias observed in MS patients in response to ENV-SU, correlations between clinical and immunological factors were then evaluated. All clinical data such as EDSS, treatment and disease duration were thus assessed for correlation with the corresponding cytokine production. No correlations were obtained in any condition with the spontaneous cytokine production (data not shown). Significant correlations were nonetheless obtained with ENV-SU induced cytokine production and are presented in Fig. 4. IL-6 production that was significantly increased in the tested MS population following ENV-SU treatment, was positively correlated with EDSS ( p=0.003). Similarly, IL-12p40 following EN-SU treatment was also positively correlated with EDSS, though with a lower significance ( p=0.026). Disease duration was not correlated with any parameter including IL-6. Finally, INF-g production, which was also increased in the tested MS group, was not correlated with EDSS ( p=0.26). 3.4. Comparison of the mean cytokine production induced by ENV-SU in MS subgroups EDSS is an imprecise measure that mainly relies on the physical severity of the disease but not on the inflamma-

Fig. 4. Correlation between EDSS and IL-6 (A), EDSS and IL-12p40 (B), IL-6 and disease duration (C) and between IFN-g and EDSS (D). The solid line represents the regression line and the r value shown is the Spearman rank correlation coefficient. Significant correlations between two groups when pb0.05.

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Table 3 Comparison of the mean cytokine production induced by ENV-SU in patients with Relapsing–Remitting (RR), Primary Progressive (PP) and Secondary Progressive (SP) MS RR (n=25) PP (n=2) SP (n=6)

Mean EDSS

IFN-g

TNF-a

IL-1

IL-6

IL-10

IL-12p40

2.48F1.14 2.75F1.75 6.25F1.67

58F51 14F14 71F55

426F99 520F32 634F367

768F345 295F132 1033F517

15,000F9163 23,077F6047 41,143F19,147

154F98 92F58 309F200

222F128 75F48 478F305

NB: Given low numbers of patients with primary progressive and secondary progressive MS, no statistical analysis could be performed.

tory process itself. We thus decided to compare the cytokine productions induced by ENV-SU in PBMC cultures from patients with different MS subgroups. The correlation between EDSS and ENV-SU induced proinflammatory cytokines is statistically significant with the overall MS population (33 patients) tested. However, numbers are becoming too low in sub-categories of MS (RR/PP/SP) for reaching any statistical significance, but they nonetheless show interesting tendencies as well, which indicate that patients with SP show higher cytokine response than RR forms (Table 3). This was expected from the general correlation with EDSS, since SP patients are more evolved than RR patients by definition. Interestingly, PP forms appear rather discordant in their tendency, which should be confirmed in further studies with larger numbers of PP cases.

4. Discussion Multiple sclerosis is an inflammatory demyelinating disease of the CNS that is believed to be immune-mediated. In particular, pro-inflammatory cytokines seem to play a central role in the development and the biological progression of MS pathogenesis. MSRV is a retroviral element often found in MS body fluids such as serum and/or CSF, with the ability to induce the production of pro-inflammatory cytokines in human PBMC. In order to further evaluate potential effects of MSRV expression in MS pathogenesis, we have in the present study compared the inflammatory response (cytokine secretion) induced by the surface unit (ENV-SU) of the envelope protein of MSRV in PBMCs from MS patients and healthy controls. We have first demonstrated the ability of ENV-SU to induce the production of cytokines in both groups, as expected from previously described properties of this retroviral protein (Perron et al., 2001). Following treatment with ENV-SU, all cytokines tested (IFN-g, IL-6, IL-12p40, TNF-a, IL-1h and IL-10) were significantly ( pb0.05) increased in both groups when compared with Mock control. These results confirm that MSRV envelope protein has the ability to stimulate the immune system of both healthy controls and MS patients, independently from a specific immune recognition pathway (Lafon et al., 2002; Perron et al., 2001). Contrary to these ubiquitous immunotoxic effects, we observed divergent reactivity to ENV-SU between MS and

control PBMCs. Interestingly, this was reflected by a significant increase of IFN-g, IL-6 and IL-12p40 production by the overall MS population when compared with the control group. TNF-a, IL-1h and IL-10 were similarly stimulated by ENV-SU in MS and healthy controls. When PBMCs were stimulated with Mock, only IFN-g production was found significantly increased in MS patients. However, this IFN-g over production was mainly due to one single patient who showed high spontaneous IFN-g production when compared with the others. The spontaneous cytokine release observed being similar between patients and healthy controls, we can therefore exclude a non-specific general over-reactivity of MS PBMCs in the context of an autoimmune hyper activation revealed by any antigen. This suggests that the divergent reactivity of MS PBMCs probably reflects a relatively specific bias in the MS immune system through IFN-g, IL-6 and IL-12p40 towards ENV-SU. Moreover, an interesting correlation with expanded disease status scale (EDSS) was observed with ENV-SU induced IL-6 and IL-12p40. However, it could appear difficult to claim a correlation with EDSS since the clinical scale is not absolutely the result of a precise measure. But errors and fluctuations in EDSS scoring that are likely to occur are indeed randomly distributed throughout the studied population. Therefore, these scores can be taken into account, since statistical analysis deals with such measure variation. It thus suggests that the immune bias induced by ENV-SU parallels the disease evolution and severity. Given that MSRV virions expressing the envelope protein can be detected in cell cultures, blood and CSF from MS patients, this bias may be induced in vivo by this pathogenic expression. Very interestingly, this is supported by the recent report from Sotgiu et al. (2002) who observed a predictive value of MSRV detection in CSF and a retrospective disease outcome in Sardinian patients series. Several reports have also highlighted the potential role of both IL-6 and IL-12p40 in MS pathogenesis. Thus, in some studies elevated levels of IL-6 have been found in MS serum and/or CSF (Frei et al., 1991; Maimone et al., 1991; Stelmasiak et al., 2000). IL-6 has also been detected in acute and chronic active plaques from brain of MS patients and was concentrated at the sites of ongoing demyelination and immune activation (Maimone et al., 1997). Moreover, IL-6 deficient mice were found to be resistant to Experimental Autoimmune Encephalomyeltis (EAE) (Samoilova et al., 1998), an animal model for MS and treatment of these mice

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with recombinant IL-6 during the preclinical phase caused typical EAE (Okuda et al., 1999; Samoilova et al., 1998). In a more recent study, it has also been reported that IL-6 could promote the development of autoimmunity as seen in MS by suppression of the regulatory response naturally induced by regulatory T cells (Treg) (Pasare and Medzhitov, 2003), which can suppress in vivo the inflammatory and autoreactive responses in EAE (Kohm et al., 2002). Although its precise role in MS pathogenesis is unknown, IL-6 could thus be involved in the activation and differentiation of autoreactive T cells and thus in the induction phase of MS. Interestingly, IL-12p40 deficient mice have also been found to be resistant to the induction of EAE (Cua et al., 2003) whereas IL-12p35 deficient mice were susceptible to EAE (Gran et al., 2002). Moreover, similar to IL-6, several studies have demonstrated that active MS is associated with increased percentages of monocytes positive for IL-12 (Comabella et al., 1998; Makhlouf et al., 2001). Thus, both IL-6 and IL-12p40 are associated with active MS and could play a major role in MS pathogenesis. IL-12 is involved in Th1 mediated responses by promoting the development of Th cells secreting IFN-g. The general consequence of these individual cytokine effects mediated by ENV-SU is also illustrated by IL12p40/IL-10 ratio, which is increased in MS as compared to healthy individuals, IL-10 being an immunoregulatory cytokine. This observation together with the over production of IFN-g observed in MS patients following ENV-SU stimulation can thus reinforce the possibility of a Th1 bias induced by ENV-SU in MS patients. In conclusion, we can now consider that we have significant evidence that the surface unit of MSRV envelope protein can affect the immune system of patients with MS. The importance of the immune bias thus created appears to increase with disease severity (correlation with EDSS). The pro-inflammatory properties of ENV-SU, and independent reports on MSRV detection in MS, are thus compatible with a relationship between MSRV expression, MS immunopathology and MS disease evolution.

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