TNFR1-dependent VCAM-1 expression by astrocytes exposes the CNS to destructive inflammation
Introduction
Multiple sclerosis (MS) is a debilitating autoimmune disease of the CNS. Neurological disabilities that are associated with this disease are a result of chronic CNS inflammation and subsequent demyelination (Lucchinetti et al., 1998). T cells and macrophages make up the majority of inflammatory cells found within MS lesions (Prineas, 1985). Based on studies with experimental autoimmune encephalomyelitis (EAE), an animal model for MS, it is believed that autoreactive myelin specific T cells are the initiators of this inflammatory and demyelinating process (Ando et al., 1989).
VCAM-1 is an adhesion molecule that facilitates extravasation of blood leukocytes into tissues Carlos et al., 1990, Osborn et al., 1989. VCAM-1 expression is not detected in the normal CNS (Brosnan et al., 1995). However, it has been detected on brain microvessel endothelial cells and microglia in MS lesions, and also in vitro on astrocytes upon stimulation with various inflammatory cytokines Brosnan et al., 1995, Cannella and Raine, 1995, Hurwitz et al., 1992, Rosenman et al., 1995. The ligands for VCAM-1 are the α4 integrins which are expressed on inflammatory leukocytes (Elices et al., 1990). EAE studies with a monoclonal antibody against VLA-4 (α4β1) demonstrated a suppression of clinical and histopathological signs of EAE Soilu-Hanninen et al., 1997, Yednock et al., 1992. More recently, a humanized monoclonal antibody against α4 integrins (Natalizumab) was shown to be effective in reducing the number of brain lesions in MS patients (Miller et al., 2003). In addition to facilitating leukocyte extravasation into the CNS, VCAM-1/α4 interaction is also important in retention of activated T cells within the CNS parenchyma. A study by Graesser et al. (2000) demonstrated that encephalitogenic T cells lacking α4 expression could enter the CNS but their numbers were greatly reduced in the brain parenchyma by day 3 post-transfer. In contrast, T cells expressing α4 integrin were continually maintained in the CNS.
Upregulation of VCAM-1 expression has been shown to be dependent on tumor necrosis factor receptor-1 (TNFR1) signaling. TNFR1, also known as the p55 kDa TNF receptor, binds to two ligands, TNF and LT-α (Hochman et al., 1995). The dominant signaling pathway for TNFR1 promotes inflammation by upregulating inflammatory cytokines, chemokines Butcher and Picker, 1996, Paul and Ruddle, 1988, Vassali, 1992 and adhesion molecules Barten and Ruddle, 1994, Bevilacqua, 1993, Lee and Benveniste, 1999, Neumann et al., 1996, and also suppresses apoptosis by the induction of IAP's (inhibitors of apoptosis) through NFκB-dependent pathways (Li and Li, 2000). Neumann et al. (1996) challenged wild type (WT) and TNFR1 deficient mice with TNF and showed that upregulation of VCAM-1 in the lung, liver, and kidney were dependent on TNFR1. In addition, Barten et al. showed that the administration of anti-TNF antibodies to mice with adoptively transferred EAE resulted in reduced upregulation of VCAM-1 on CNS endothelial cells (Barten and Ruddle, 1994).
In this report, we have utilized the adoptive transfer model in C57BL/6 WT and TNFR1 null mice to study the role of VCAM-1 expression in CNS inflammation. We demonstrate that encephalitogenic T cells traffic to the CNS of TNFR1 deficient mice, but are confined to the leptomeninges and perivascular spaces. As a result, the TNFR1 null mice did not develop clinical EAE and were resistant to demyelination and axonal damage. Currently, the majority of EAE studies have focused on the importance of VCAM-1 expression on CNS vascular endothelium Barten and Ruddle, 1994, Elices et al., 1990, Vajkoczky et al., 2001. Here, we show that VCAM-1 expression on spinal cord vascular endothelium occurs in the absence of TNFR1 expression and has no bearing on disease severity. Moreover, we reveal that parenchymal VCAM-1 expression on astrocytes is dependent on TNFR1 and coincides with T cell infiltration into the CNS parenchyma and disease initiation. In the absence of parenchymal VCAM-1 expression, we also found a lack of T cell retention within the CNS. Although others have demonstrated cytokine dependent VCAM-1 expression on astrocytes in culture Hurwitz et al., 1992, Rosenman et al., 1995 we demonstrate in vivo evidence of VCAM-1 expression on astrocytes in the CNS parenchyma during active disease.
Section snippets
Animals
C57BL/6 and congenic Thy1.1 mice (B6.PL-Thy1a/Cy) were purchased from Jackson Laboratories (Bar Harbor, ME). Breeders of the congenic TNFR1 null mutation (B6.129-Tnfrsf1atm1/Mak/J) were purchased from the same source and maintained by brother–sister matings in our colony. All experiments were approved by the Animal Care Committee and animals were housed in an AAALAC-approved facility.
Induction of EAE
For adoptive transfer, Thy1.1 congenic donor mice were immunized sc with 50 μg of MOG35–55 peptide (Sigma
TNFR1 null mice are resistant to adoptively transferred EAE
We performed a reciprocal adoptive transfer with B6 WT and TNFR1 null mice (Fig. 1, Table 1). In concert with earlier studies (Willenborg et al., 1998), our results indicated that TNFR1 expression in the host, and not on the encephalitogenic T cell, is important in EAE pathogenesis. The adoptive transfers also demonstrated that T cells from TNFR1 null mice were capable of mounting a normal immune response to MOG.
T cell trafficking to the CNS is independent of TNFR1 expression
Since the majority of TNFR1 null mice were nearly completely resistant to
Discussion
Early reports suggested that TNFR1 was important in inducing VCAM-1 expression on vascular endothelial cells, which allowed lymphocyte transmigration past that structure Barten and Ruddle, 1994, Neumann et al., 1996. However, our data demonstrates that TNFR1-dependent VCAM-1 expression is not at the level of the vascular endothelium, but rather at the level of the CNS parenchyma. Rosenman et al. (1995) previously demonstrated that TNF induces VCAM-1 on primary astrocytes in culture. We provide
Acknowledgements
This work was supported by grant # RG 2835 from the National Multiple Sclerosis Society.
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