Activated microglial cells acquire an immature dendritic cell phenotype and may terminate the immune response in an acute model of EAE

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Abstract

Antigen presentation, a key mechanism in immune responses, involves two main signals: the first is provided by the engagement of a major histocompatibility complex (MHC), class I or class II, with their TCR receptor in lymphocytes, whereas the second demands the participation of different co-stimulatory molecules, such as CD28, CTLA-4 and their receptors B7.1 and B7.2. Specific T-cell activation and deactivation are achieved through this signalling. The aim of our study is to characterise, in the acute experimental autoimmune encephalomyelitis (EAE) model in Lewis rat, the temporal expression pattern of these molecules as well as the cells responsible for their expression. To accomplish that, MBP-immunised female Lewis rats were daily examined for the presence of clinical symptoms and sacrificed, according to their clinical score, at different phases during EAE. Spinal cords were cut with a cryostat and processed for immunohistochemistry: MHC-class I and MHC-class II, co-stimulatory molecules (B7.1, B7.2, CD28, CTLA-4) and markers of dendritic cells (CD1 for immature cells and fascin for mature cells). Our results show that microglial cells are activated in the inductive phase and, during this phase and peak, they are able to express MHC-class I, MHC-class II and CD1, but not B7.1 and B7.2. This microglial phenotype may induce the apoptosis or anergy of infiltrated CD28+ lymphocytes observed around blood vessels and in the parenchyma. During the recovery phase, microglial cells express high MHC-class I and class II and, those located in the surroundings of blood vessels, displayed the B7.2 co-stimulatory molecule. These cells are competent to interact with CTLA-4+ cells, which indicate an active role of microglial cells in modulating the ending of the immune response by inducing lymphocyte activity inhibition and Treg activation. Once clinical symptomatology disappeared, some foci of activated microglial cells (MHC-class II+/B7.2+) were still present in concomitance with CTLA-4+ cells, suggesting a prolonged involvement of microglia in lymphocyte inhibition and tolerance promotion. In addition to microglia, during the inductive and recovery phases, we also found perivascular ED2+ cells and fascin+ cells which are able to migrate to the parenchyma and may play a role in lymphocytic regulation. Further studies to understand the specific function played by these cells are warranted.

Introduction

Antigen presentation is a crucial process in T-cell activation and modulation of immune responses. Two main signals are involved in this process. The first, provided by the engagement of either the MHC-class I or MHC-class II on antigen-presenting cells (APCs) with the T-cell receptor (TCR) on T lymphocytes, controls the specificity of the immune response, as MHC-class I is recognised by CD8+ T cells whereas MHC-class II interacts with CD4+ T cells (Janeway, 1992). The second signal, the co-stimulatory signal, is antigen non-specific, involves the interaction of different T-cell surface receptors with their respective ligands on APCs (Lanzavecchia, 1997, Lenschow et al., 1996) and is essential for the full T-cell activation, as TCR-MHC binding in the absence of co-stimulation can lead to T-cell apoptosis or anergy (Kishimoto and Sprent, 1999). Different combinations of co-stimulatory molecules and receptors providing stimulatory or inhibitory signals have been described (Nurieva et al., 2009), however the signal provided by the B7 molecules, B7.1 and B7.2 on APCs with their receptors CD28 and CTLA-4 in lymphocytes appear to be the predominant molecular interactions for T-cell activation (Salomon and Bluestone, 2001, Sharpe and Freeman, 2002). Binding of B7.1 or B7.2 with CD28 provides a potent stimulatory signal in T cells, whereas binding of the related but higher-affinity CTLA-4 receptor, delivers an inhibitory signal (Karandikar et al., 1996, Sansom, 2000). Therefore, the delicate balance established between these positive and negative signals may provide different outcomes of the immune response.

Evidence for the involvement of antigen presentation in experimental autoimmune encephalomyelitis (EAE) comes from the observations that microglial cells express MHC-class II in different EAE models in both rats (Craggs and Webster, 1985, Matsumoto and Fujiwara, 1986, Matsumoto et al., 1986, McCombe et al., 1994, McCombe et al., 1992) and mice (Juedes and Ruddle, 2001, Lindsey and Steinman, 1993, Ponomarev et al., 2005, Pope et al., 1998), and from studies that reported the expression of co-stimulatory molecules and the beneficial or detrimental effects derived from the lack of these molecules in EAE (Chang et al., 1999, Chang et al., 2003, Girvin et al., 2000, Hurwitz et al., 1997, Karandikar et al., 1998a, Miller et al., 1995, Perrin et al., 1999). It should be emphasised, however, that all the aforementioned studies have been focused only on the peak of the disease and did not address the co-expression of MHCs and B7.1/B7.2-CD28/CTLA-4 molecules in the same context. Thus, it remains to be determined, first, if MHCs expression matches with the presence of these co-stimulatory molecules and if this is the case, whether different combinations of B7.1/B7.2-CD28/CTLA-4 molecules differ during the different phases of EAE and may be playing a role in the initiation and resolution of the immune response. A second question that remains is which are the cells driving the antigen-presenting mechanism within the CNS in this acute EAE model, as in spite of the task of microglia as APCs, some recent studies have reported that a specific population of dendritic cells (DCs), the main APCs in the periphery, infiltrate the CNS in EAE rats (Matyszak and Perry, 1996, Serafini et al., 2000) and MS (Serafini et al., 2006) and play a central role in antigen presentation in murine EAE models (Bailey et al., 2007, Greter et al., 2005), opening the possibility that, in addition to microglia, DCs can be involved in the mechanism of antigen presentation also in this acute model.

Among the different EAE models in rodents, the acute EAE model induced in Lewis rat is of special interest, as it is characterised by a single peak of paralysis after which animals recover spontaneously (Swanborg, 2001, Tsunoda and Fujinami, 1996). Taking advantage of this model, which gave us the opportunity to investigate the mechanisms involved in the induction, peak and resolution of the inflammatory–immune response, the present study provides a detailed description of the temporal expression pattern of MHC/co-stimulatory molecules, and the phenotype of cells responsible for their expression.

Section snippets

EAE induction

A total of 77 female Lewis rats (180–200g) susceptible to develop experimental autoimmune encephalomyelitis (EAE) were used in this study. Animals were purchased from Charles River (IFFA Credo; Belgique) and maintained with food and water ad libitum in a 12 h light/dark cycle.

Rats were induced to develop EAE by a subcutaneous injection, in each hindfoot, of an emulsion containing 100 µg of MBP (M2295; Sigma; St Louis, USA), Complete Freud's Adjuvant (CFA) (Ref. 0638; Difco; USA) and 0.2 mg of

Results

MBP immunisation in Lewis rat produces an acute monophasic disease characterised by a progressive clinical motor impairment, starting around 9–11 days post-immunisation (dpi) reaching complete hindlimb paralysis (12–14 dpi) after which progressive–spontaneous recovery takes place (15–23 dpi). After 23 dpi, clinical symptoms were absent. In this work, in contrast to the major part of studies where experimental groups are determined on the basis of the days post-immunisation, the animals were

Discussion

In this study a detailed analysis of the temporal pattern of expression and cellular distribution of the different molecules (MHCs and co-stimulatory) related to the antigen-presenting mechanism along the different phases of EAE evolution was performed. Firstly, it is important to highlight the fact that no variability was observed in animals analysed in each specific clinical score, indicating that histopathological changes within the CNS are more associated with clinical symptomatology than

Conclusion

In conclusion, our study has shown that in this acute model of EAE, antigen-presenting mechanisms are not restricted to the inductive and peak phases of the disease, but rather they also play an important role during the recovery and post-recovery phases. Our results clearly indicate that during the inductive and the peak phases, antigen presentation to parenchymal CD28+ lymphocytes might take place in the context of MHC molecules without co-stimulatory B7.1/B7.2 signalling, thus may be

Acknowledgements

The authors would to thank Dr Iain L. Campbell for his comments about the manuscript. The authors wish to thank also Miguel A. Martil, Isabella Appiah and Maria González for their outstanding technical help and Mr Chuck Simmons, a native, English speaking Instructor of English in the Autonomous University of Barcelona for the proofreading of this manuscript. The authors also would thank the reviewers for their excellent comments that improve the quality of the manuscript. This work was

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