Published online before print October 22, 2008
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* Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Centre National de la Recherche Scientifique UMR 6184, IFR Jean Roche, and Pôle de Biologie, Laboratoire d’Immunologie, Hôpital de la Conception, Assistance Publique–Hôpitaux de Marseille, Marseille, France;
Pôle de Neurosciences Cliniques, Service de Neurologie, Hôpital de la Timone, Marseille, France; and
Autoimmunity and Drug Discovery, Biogen Idec, Cambridge, Massachusetts, USA
1 Correspondence: NICN, CNRS UMR 6184, IFR Jean Roche, 51 Bd Pierre Dramard, 13 916 Marseille Cedex 20, France. E-mail: sophie.jego-desplat{at}ap-hm.fr
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Key Words: inflammation • cytokine • central nervous system
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS involving an infiltration of CNS tissue by immune cells, a reactive astrogliosis, an activation of microglial cells, and neuronal damage. We reported previously that the TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14-kDa protein (Fn14) pathway is involved in an experimental model of CNS inflammation [1 , 2 ]. TWEAK is a type II transmembrane protein belonging to the TNF family, which can be cleaved to function as a soluble cytokine [3 ]. TWEAK transcripts have been found in many tissues including the brain [3 ]. TWEAK exerts pleiotropic effects on a variety of cell types including proangiogenic activities on endothelial cells [4 , 5 ] and proinflammatory activities on astrocytes [6 ], dermal fibroblasts, and synoviocytes [7 ]. TWEAK induces cellular responses ranging from proliferation to cell death depending on the cell type and the microenvironmental context (for review, see ref. [8 ]). It is now understood that biological effects of TWEAK are mediated by its receptor, Fn14 [9 ]. In the CNS, astrocytes and neurons have been shown to express Fn14 as described for endothelial cells [2 , 10 ]. A proinflammatory role of the soluble form of TWEAK was demonstrated by an increased severity of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in transgenic mice overexpressing soluble TWEAK as compared with wild-type [2 ]. Moreover, we have shown that treatment of MOG-EAE mice after the priming phase with neutralizing anti-TWEAK antibodies results in a reduction in the severity of the disease and leukocyte infiltration [1 ]. Mueller et al. [11 ] also reported that inhibitory antibodies to TWEAK or Fn14 elicited by vaccination with their respective extracellular domains reduced EAE severity. Moreover, TWEAK has been shown to increase the blood brain barrier (BBB) permeability and thereby, to lead to edema in animal models of cerebral ischemia [12 13 14 ].
TWEAK is thought to be expressed primarily as a soluble cytokine as a result of efficient furin-mediated cleavage along the secretory pathway [7
]. However, cell-surface expression of TWEAK has been reported on human monocytes after IFN-
exposure in culture [15
] and on T cells from patients with systemic lupus erythematosus [16
]. Maecker et al. [17
] showed that human NK cells, macrophages, and dendritic cells expressed intracellular TWEAK and membrane Fn14 and that other lymphoid cells including T and B cells did not express significant levels of TWEAK or Fn14. To further characterize TWEAK/Fn14 involvement in MS, we evaluated the expression of TWEAK and Fn14 after short-term culture of PBMC, stimulated or not with IFN- and IFN-β and the levels of soluble TWEAK in serum and cerebrospinal fluid (CSF) in MS patients and non-MS disease controls.
Thirty-six consecutive patients from the same Department of Neurology were enrolled in this study. Neurological examination, cerebral magnetic resonance imaging (T1, with and without gadolinium injection; T2, fluid attenuated inversion recovery), and CSF analysis were performed in all patients. MS diagnosis was assessed according to McDonald’s criteria, which required dissemination in time and dissemination in space to be satisfied. Eleven patients fulfilled both criteria and suffered from a relapsing-remitting form of MS [RRMS; in relapse (n=5) or stable (n=6) at the time of sampling]. When the dissemination in time criterion was not satisfied, patients have been classified as having clinical isolated syndrome suggestive of MS (CISSMS; n=11). At the time of sampling, these patients suffered from their first CNS inflammatory episode, and they have now all converted to definite MS. Six patients suffering from inflammatory non-MS diseases, such as lupus disease and infectious disorders affecting the CNS (meningoencephalitis or meningitis), were also included. At least eight patients having other neurological disorders such as psychiatric or neurovascular disorders or peripheral nerve involvement were enrolled and served as control subjects. None of the patients received immunomodulatory therapy during the last 6 months before blood and CSF sampling. All patients gave written, informed consent after approval by the local ethics committee.
PBMC were isolated from heparinized venous blood by gradient centrifugation on separation media and managed blind with respect to the clinical status of the patient. PBMC at the interface were washed three times with PBS and seeded in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 µg/ml penicillin and streptomycin, 2 mM L-glutamine, and nonessential amino acid solution. PBMC were cultured unstimulated or stimulated with IFN- (R&D Systems, Abingdon, UK) or IFN-β (Avonex®, Biogen Idec, Cambridge, MA, USA) for 24 h. Flow cytometry analyses were performed on live cells (surface staining) and on fixed and permeabilized cells (intracellular staining). AB.G11 anti-TWEAK mAb was generated in Armenian hamsters using immunization with soluble human TWEAK protein and standard hybridoma generation procedures as described previously [4
]. The hamster control isotype-matched Ig (clone Ha4/8-3.1) was obtained from the American Type Culture Collection (Manassas, VA, USA). Purified anti-human TWEAK receptor (Fn14) mAb (clone ITEM-4) was obtained from eBioscience (San Diego, CA, USA) and other control and secondary antibodies (allophycocyanin-conjugated anti-hamster IgG and anti-mouse IgG), from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Samples were analyzed on a FACSCalibur flow cytometer using the CellQuest software (Becton Dickinson, San Jose, CA, USA) after gating on monocytes (R1) and second, on lymphocytes (R2), according to forward and side scatter properties and immunostaining with anti-CD3, -CD19, and -CD14 antibodies. Results are expressed as the difference between mean fluorescence index (MFI) obtained with anti-TWEAK or anti-Fn14 antibody and MFI obtained with isotype-matched control antibody.
Results for TWEAK and Fn14 expression are illustrated in Figure 1A
and summarized in Figure 1 B and C
. Intracellular detection of TWEAK could be observed without stimulation in lymphocytes and/or in monocytes in any group of patients and controls. None of the 36 patients displayed TWEAK expression at the cell surface of lymphocytes. On the contrary, interestingly, 12 out of the 36 included patients were positive for membrane TWEAK expression on their monocytes. Among these patients, eight were from the CISSMS group (eight of 11), and four suffered from RRMS (two in relapse and two stable). The median of MFI differences for TWEAK membrane expression were statistically higher in the CISSMS group (median: 36) than in the non-MS CNS inflammatory diseases control group (median: 7.5) or in the other neurological diseases control group (median: 4.5; Fig. 1B
), neither of which exhibited monocyte membrane TWEAK (n=14). Moreover, we observed a positive quantitative correlation between intracellular and membrane TWEAK expression in CISSMS patients (Fig. 1C)
. The patients displaying the highest TWEAK expression (n=5) fulfilled the Barkhoff’s criteria in less than 2 years and presented a highest number of gadolinium-enhanced cerebral lesions (up to 24). Concerning Fn14 studies, we failed to detect the TWEAK receptor in PBMC by flow cytometry. All of the patients were negative for Fn14 expression for lymphocytes and monocytes, independent of the clinical group to which they belong. Exposure of PBMC to IFN-β or IFN- had no notable effect on TWEAK or Fn14 expression.
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Figure 1. TWEAK expression on monocytes and lymphocytes by flow cytometry. (A) The flow cytometry profile of a representative patient suffering from a CISSMS. Cells were stained with anti-TWEAK mAb ABG11 (open gray curves) or with isotypic control hamster mAb HA4/8 (filled black curves). (B and C) Data are expressed by the differences of MFI obtained with anti-TWEAK antibody and MFI obtained with isotypic control antibody. The cut-off (B) is represented by the horizontal black line and corresponds with the median + two SEM of the values obtained for the 14 control patients. P values are calculated according to the Mann-Whitney t-test. Mb, membrane; RRMSR, RRMS in relapse; RRMSS, RRMS stable; Non MS CNSInfl Disease and Non MS CNSID, non-MS CNS inflammatory diseases; OND, other neurological diseases.
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Figure 2. TWEAK quantification in serum and CSF. (A) Results of the ELISA test are expressed by medians and ranges (in brackets). Sensitivity of the test is 9 pg/ml. F, Female; M, male. *Ages are expressed in years and by medians and ranges (in brackets). (B) TWEAK indices of the five patient groups (A–E; see A). Horizontal bars represent medians of TWEAK indices, which have been calculated as following: (CSF TWEAK/serum TWEAK)/(CSF albumin/serum albumin).
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. It could also represent a predictive marker of quick MS conversion. A longitudinal study with a larger number of CISSMS patients, analyzing TWEAK and other monocyte activation markers, is required. TWEAK expression on monocytes potentially favors their interaction with Fn14 expressed on endothelial cells and could facilitate the migration of monocytes through the BBB and their interaction with astrocytes and neurons. In vivo, the presence of soluble TWEAK and monocyte cell-surface TWEAK in MS patients also supports the contention that TWEAK plays a role in the physiopathology of CNS inflammation in MS. Our results support the proposal that circulating monocytes expressing surface TWEAK and/or secreting TWEAK could be a target for anti-TWEAK therapy in MS patients.
Received June 10, 2008; revised September 21, 2008; accepted September 23, 2008.
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B pathway activation J. Neurosci. 25,10094-10100-stimulated monocyte cytotoxicity J. Exp. Med. 192,1373-1380 production as a possible predictor of relapse in patients with multiple sclerosis Eur. Cytokine Netw. 3,523-531[Medline]
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