High CRMP2 expression in peripheral T lymphocytes is associated with recruitment to the brain during virus-induced neuroinflammation.
ABSTRACT Collapsin Response Mediator Protein (CRMP)-2 is involved in T-cell polarization and migration. To address the role of CRMP2 in neuroinflammation, we analyzed its involvement in lymphocyte recruitment to the central nervous system in mouse infected with neurotropic and non-neurotropic virus strains (RABV, CDV). A sub-population of early-activated CD69+CD3+ T lymphocytes highly expressing CRMP2 (CRMP2hi) peaked in the blood, lymph nodes and brain of mice infected with neurotropic viruses, and correlated with severity of disease. They displayed high migratory properties reduced by CRMP2 blocking antibody. These data point out the potential use of CRMP2 as a peripheral indicator of neuroinflammation.
High CRMP2 expression in peripheral T lymphocytes is associated with recruitment to the
brain during virus-induced neuroinflammation
Vuaillat C, Varrin-Doyer M, Bernard A, Sagardoy I, Cavagna S, N Chounlamountri, IA, Lafon M *,
Inserm, U842, Lyon, F-69372; Inserm, IFR19, Bron, F-69677 France; Université de Lyon, Lyon, F-
* Institut Pasteur, Viral Neuroimmunology, Paris, F-75015 France
Corresponding author: Pascale Giraudon, U842 Inserm, Rue Guillaume Paradin, Faculté de
Médecine Laënnec, F-69372 Lyon, Cedex, France
email@example.com; Fax 33 4 78 77 86 16
Funds: ARSEP, Internal grants from Institut Pasteur (ML) and INSERM
Collapsin Response Mediator Protein (CRMP)-2 is involved in T-cell polarization and migration. To
address the role of CRMP2 in neuroinflammation, we analyzed its involvement in lymphocyte
recruitment to the central nervous system in mouse infected with neurotropic and non-neurotropic
virus strains (RABV, CDV). A sub-population of early-activated CD69+CD3+ T lymphocytes highly
expressing CRMP2 (CRMP2hi) peaked in the blood, lymph nodes and brain of mice infected with
neurotropic viruses, and correlated with severity of disease. They displayed high migratory properties
reduced by CRMP2 blocking antibody. These data point out the potential use of CRMP2 as a
peripheral indicator of neuroinflammation.
Key words: neuroimmunology, neurovirology, neuroinflammatory disease, T-cell motility, rabies
virus, canine distemper virus
In acute viral encephalitis, the recruitment of immune cells to the central nervous system (CNS) plays
a crucial role in the outcome of this disease. Notably, T-lymphocytes are key players in the initiation of
specific intrathecal immune responses by directly destroying virus-infected cells, increasing the
phagocytotic activity of macrophages and stimulating the production of antibodies by B cells (Fujinami
et al., 2006; Salazar-Mather and Hokeness, 2006; Tishon et al., 2006). Thus, immune cells control the
spread of viruses and participate in viral clearance. However, T-lymphocytes can mediate
inappropriate inflammatory responses, producing multiple cytokines, chemokines, and reactive oxygen
species that contribute to severe neural tissue injury and CNS dysfunction (Carlson et al., 2006)
(Peterson and Fujinami, 2007; Galelli et al., 2000; Giraudon et al., 2004; Lane et al., 2000; Patterson
et al., 2003). Considering the importance of viral encephalitis in humans, the homing behavior and
functional status of immune cells that infiltrate the virus-infected brain need to be clarified.
Characterizing the mechanisms that favor the migration of T-lymphocytes to the CNS in response to
viral infection is of interest both for understanding the physiopathology of virus-induced
neuroinflammation and to propose new therapeutic approaches.
T-lymphocytes constantly move in and out of lymph nodes, circulating through the vasculature.
Probably following activation and as a result of chemotactic stimuli, T-cells acquire a polarized
morphology, leave the vasculature and start to crawl within neural tissue (Norman and Hickey, 2005;
Sanchez-Madrid and del Pozo, 1999; Westermann et al., 2005). Dynamic cytoskeletal remodeling is
fundamental to drive the rapid conversion of lymphocyte cytoarchitecture from a semi-rigid to a highly
deformable state needed for transendothelial migration and crawling within CNS parenchyma
(Vicente-Manzanares and Sanchez-Madrid, 2004). Thus, motile T-cells display a polarized
morphology, with two distinct T-cell compartments. At the advancing front, the leading edge is
enriched in chemokine receptors and acts as a sensor to the microenvironment. At the trailing edge,
the uropod is an adhesive and retractile structure required for T-cell migration (Barreiro et al., 2004).
Rapid reorganization of actin at the leading edge and the retraction of microtubule and vimentin
networks in the uropod during polarization and migration have been well described (Brown et al.,
2001; Mitchison and Cramer, 1996). Such a cytoskeletal reconfiguration requires a complex and still
not completely defined array of intracellular signaling pathways implicating second messengers,
several kinases and Rho family small GTPases (del Pozo et al., 1999; Vicente-Manzanares et al.,
2002). In search of molecules involved in cell motility and shared by the immune and nervous
systems, we recently identified Collapsin Response Mediator Protein-2 (CRMP2) in T-lymphocytes
(Vincent et al., 2005), a phosphoprotein described in neuron growth cone advance (Goshima et al,
1995) and neural cell migration via microtubule organization. We showed that CRMP2 redistributes in
the uropod in motile T-cells, binds cytoskeletal elements and is implicated in T-cell polarization and
migration (Vincent et al., 2005). A correlation between CRMP2 expression levels and cell migratory
rates was demonstrated by over-expression and knock-down experiments in primary T-lymphocytes.
The importance of CRMP2 in the immune response was emphasized by the observation that, in
humans suffering from virus-induced neuroinflammatory disease, peripheral activated immune cells
displayed high migratory rates associated with elevated CRMP2 expression (Vincent et al., 2005).
These observations led us to suspect a role for CRMP2 in immune cell trafficking and recruitment to
the inflamed CNS. In the present study, we evaluated the CRMP2 contribution in brain lymphocyte
recruitment by analyzing CRMP2 expression in peripheral and brain-infiltrating immune cells in mouse
models of infection using neurovirulent and non-neurovirulent strains of rabies virus (RABV) and
Canine distemper virus (CDV). After inoculation of the neurovirulent RABV strain Challenge virus
standard (CVS) in the muscle of the hind limb- to mimic the inoculation of this virus by an animal bite-
the virus enters nerve endings via the neuromuscular junction and then travels from one neuron to
another along the spinal cord before reaching the entire brain, causing severe neuronal dysfunctions
and fatal encephalitis (Lafon, 2004). Infection in the CNS triggers a strong innate immune response in
both neurons and astrocytes, resulting in inflammatory and chemo-attractive responses characterized
by IFN, IL-1, IL6, TNF CCL5, CXCL-10 expression capable of attracting T cells, B cells and
macrophages through the blood brain barrier (Camelo et al., 2001a; Baloul et al., 2004; Wang et al.,
2005; Roy et al., 2007). Inoculation of the Pasteur Virus (PV) RABV strain another neurovirulent RAV
strain causes only spinal cord and the bulb infection, resulting in abortive rabies: mice cleared the
CNS infection and survive despite paralytic sequel (6). By contrast, injection by the same route of the
non-neuronotropic vaccine RABV strain ERA does not cause infection of the CNS and animals remain
healthy. In the mouse model infected with CDV, intracerebral inoculation of a neurovirulent strain
results in acute encephalitis followed, in surviving animals, by the development of motor impairment
and metabolic disease (Bernard et al., 1999; Griffond et al., 2004; Verlaeten et al., 2001). CDV infects
the neurons of defined brain structures (Bernard et al., 1993) and provokes a brain inflammatory
reaction with the infiltration of T lymphocytes, the expression of the cytokines TNF, IL6, IFN and IL-4
associated with the disruption of the tissue inhibitor of the metalloprotease TIMP/ metalloprotease
MMP balance (Benscsik et al., 1996; Khuth et al., 2001) capable of modifying vascular permeability.
We show here that, in contrast of what was observed in mice infected with non-neurovirulent virus
strain, infection of the CNS with neuronotropic CDV and RABV enhanced both CRMP2 expression
and migratory rate of activated T lymphocytes. Interestingly, the presence in blood and lymph nodes of
lymphocytes expressing high CRMP2 level was associated with the intensity of recruitment of immune
cells into the brain of mice suffering from encephalitis and correlated with the severity of clinical score.