Dendritic cells and dendritic-like microglia in focal cortical ischemia of the mouse brain

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Abstract

Intracerebral dendritic cells (DC) have recently been identified in neuroinflammation initiated peripherally by brain-targeted autoimmunity or infection. The present study detects DC in photochemically induced cortical ischemia of the mouse brain, a brain-intrinsic lesion model characterized by the lack of an overt T cell response. Concomitant to leukocyte infiltration of the infarcted area, cells expressing the pan-DC surface marker CD11c appeared at the lesion and persisted for weeks. These DC were located at the border zone of the infarct and remote from the lesion in degenerating corticothalamic fibre tracts and subcortical nuclei. All CD11c+ brain cells displayed a uniform CD11b+/CD8α/CD205 surface phenotype, indicating a myeloid origin, and were immature DC based on their MHC class II+/CD40/CD80+/CD86± profile. By expressing high levels of CD45, most DC from ischemic brain seemed to be blood-derived while a minority were CD45low, thus corresponding to resident microglia. Consistently, round-shaped CD11c+ cells were found at the lesion whereas CD11c+ cells at subcortical sites were ramified like parenchymal microglia. These findings evidence a recruitment of myeloid DC to ischemic brain lesions and suggest that reactive microglia in remote areas transform into dendritic-like cells. Brain-infiltrating DC and their microglial counterparts may play a role in the inflammatory response to cerebral ischemia independently of T cells.

Introduction

Inflammatory responses in the central nervous system (CNS) have primarily been studied in the context of T cell-mediated autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) which serves as a model for multiple sclerosis. However, recent evidence indicates that neurodegenerative conditions likewise initiate a profound activation of immune effector cells both resident within and recruited to the lesioned brain Perry et al., 1998, Stoll and Jander, 1999, Allan et al., 2001. Early activation of resident microglia and subsequent infiltration of the lesion by macrophages are hallmarks of focal brain ischemia which represents a brain-intrinsic lesion model Arvin et al., 1996, Stoll et al., 1998. In parallel to the rapid microglial reaction, proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1β are induced within hours after the insult, followed by the expression of inducible nitric oxide synthase Liu et al., 1993, Liu et al., 1994, Iadecola et al., 1995, Jander et al., 2000. Although small numbers of lymphocytes infiltrate the lesion (Jander et al., 1995), there is no evidence of a CNS antigen-specific T cell response. Postischemic brain inflammation is not restricted to the ischemic area but likewise involves remote nonischemic regions as evidenced by local microglia activation and cytokine induction Schroeter et al., 1999, Jander et al., 2000. Expression of class II major histocompatibility complex (MHC II) molecules is prominent and long-lasting in the subcortical fibre tracts undergoing secondary degeneration due to ischemic damage of cortical neurons.

Unlike other cells that express MHC II as result of an activation process, dendritic cells (DC) are per se MHC II+. DC comprise a heterogeneous family of bone marrow-derived cells with the unique ability to stimulate naı̈ve and resting T cell responses and to initiate antigen-directed immunity (Banchereau and Steinman, 1998). Circulating DC progenitors home to tissues where they reside as immature DC that can efficiently capture and process antigens but fail in stimulating T cells. Upon trigger by microbial products and CD40 ligation, they mature into antigen-presenting cells that exert the full repertoire of DC functions Banchereau et al., 2000, Schulz et al., 2000. Within the nervous system, DC have been detected in T cell-mediated inflammatory conditions, such as EAE, DTH reaction, and cerebral toxoplasmosis, by virtue of DC-associated surface markers Matyszak and Perry, 1996, Fischer et al., 2000, Serafini et al., 2000, Suter et al., 2000.

To address the question as to whether DC appear and mature at intracerebral sites of inflammation independently of T cells, photochemically induced focal ischemia of the cerebral cortex was chosen as a lesion model in which no overt brain-directed T cell response occurs. In comparison to the more frequently used models of middle cerebral artery occlusion, photothrombosis represents a noninvasive approach for the induction of circumscribed brain infarcts which are highly reproducible with respect to size and location, thereby allowing the precise topographical analysis of cellular responses at both ischemic and nonischemic sites. The present study provides evidence for a lesion-dependent appearance of DC within the brain and delineates the maturational state of these DC and their relation to microglia.

Section snippets

Induction of photothrombosis

Focal cerebral ischemia was induced in female C57BL/6 mice weighting 20–25 g (Tierversuchsanlage, Heinrich-Heine-University Düsseldorf, Germany) by photothrombosis of cortical microvessels as detailed elsewhere Jander et al., 1995, Schroeter et al., 2002. Briefly, the animals were anesthetized with enflurane in a 2:1 N2O/O2 atmosphere. A servo-controlled heating pad kept rectal temperature at 36.5–37.5 °C. The animal's head was fixed in a stereotactic holder and the skull was exposed via a

Composition of the ICL populations from ischemic versus nonischemic brain hemisphere

A comparative analysis of ICL from infarcted and contralateral nonischemic hemisphere was performed at day 6 after lesion induction, i.e. the peak stage of postischemic inflammation. Cells were enzymatically extracted from brain tissue, enriched for ICL by density gradient centrifugation, and double-stained for the common leukocyte marker CD45 in combination with the lineage markers CD11b or CD3 or the DC marker CD11c. By flow cytometric analysis, ICL were identified as a distinct population of

Discussion

This study provides evidence for the presence of DC in ischemic CNS lesions. Using a mouse photothrombosis model, CD11c+ cells were identified in the infarcted brain and characterized ex vivo. Upon focal cortical ischemia, infiltration of predominantly monocytes/macrophages into the lesion proved to coincide with a local emergence of phenotypically myeloid-related DC. These DC accumulate and persist at the lesion as well as in subcortical areas not directly affected by the ischemic insult, but

Acknowledgements

We thank Sabine Hamm, Helena Heinitz, and Anette Tries for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Ja 690/4-1, Re 1334/2-1, and SFB 194/B6 and B11).

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