Antje Kroner

Repertoire of microglial and macrophage responses after spinal cord injury

Macrophages from the peripheral circulation and those derived from resident microglia are among the main effector cells of the inflammatory response that follows spinal cord trauma. There has been considerable debate in the field as to whether the inflammatory response is good or bad for tissue protection and repair. Recent studies on macrophage polarization in non-neural tissues have shed much light on their changing functional states. In the context of this literature, we discuss the activation of macrophages and microglia following spinal cord injury, and their effects on repair. Harnessing their anti-inflammatory properties could pave the way for new therapeutic strategies for spinal cord trauma.

TNF and Increased Intracellular Iron Alter Macrophage Polarization to a Detrimental M1 Phenotype in the Injured Spinal Cord

Macrophages and microglia can be polarized along a continuum toward a detrimental (M1) or a beneficial (M2) state in the injured CNS. Although phagocytosis of myelin in vitro promotes M2 polarization, macrophage/microglia in the injured spinal cord retain a predominantly M1 state that is detrimental to recovery. We have identified two factors that underlie this skewing toward M1 polarization in the injured CNS. We show that TNF prevents phagocytosis-mediated conversion from M1 to M2 cells in vitro and in vivo in spinal cord injury (SCI). Additionally, iron that accumulates in macrophages in SCI increases TNF expression and the appearance of a macrophage population with a proinflammatory mixed M1/M2 phenotype. In addition, transplantation experiments show that increased loading of M2 macrophages with iron induces a rapid switch from M2 to M1 phenotype. The combined effect of this favors predominant and prolonged M1 macrophage polarization that is detrimental to recovery after SCI.

A PD‐1 polymorphism is associated with disease progression in multiple sclerosis

T cells are considered to play a pivotal role in orchestrating the self‐reactive immune responses in multiple sclerosis (MS). Programmed death 1 (PD‐1) is a member of the B7/CD28 superfamily of costimulatory molecules exerting inhibitory functions on T cells. Recently, an intronic 7146G/A polymorphism within the PD‐1 gene was described and suggested to be associated with autoimmunity. We investigated whether this genetic polymorphism is a genetic modifier for risk and progression of MS. Blood samples from 939 German MS patients (mean age, 39 years; range, 13–71; 566 patients [60%] with relapsing‐remitting MS, 279 (30%) with secondary, and 94 (10%) with primary progressive MS) and 272 healthy white controls were tested. Genotyping was performed by polymerase chain reaction and restriction enzyme digestion; results were confirmed by automatic sequencing. A significant association of the mutated allele with a progressive disease course was detected (44% 7146G vs 56% 7146A, χ2 p = 0.002). Consequences of the PD‐1 mutation for T‐cell function were assessed ex vivo in some patients using microsphere‐stimulated peripheral blood lymphocytes and purified CD4 cells. Importantly, PD‐1–mediated inhibition of T‐cell cytokine secretion (interferon‐γ) is impaired in patients carrying the PD‐1 polymorphism. In conclusion, our data suggest that PD‐1 polymorphism is a genetic modifier of the progression of MS, possibly through inducing a partial defect in PD‐1–mediated inhibition of T‐cell activation. Ann Neurol 2005

Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

Refining genetic associations in multiple sclerosis

Genome-wide association studies involve several hundred thousand markers and, even when quality control is scrupulous, are invariably confounded by residual uncorrected errors that can falsely inflate the apparent difference between cases and controls (so-called genomic inflation). As a consequence such studies inevitably generate false positives alongside genuine associations. By use of Bayesian logic and empirical data, the Wellcome Trust Case Control Consortium suggested that association studies in complex disease should involve at least 2000 cases and 2000 controls, at which level they predicted that p values of less than 5×10 −7 would more commonly signify true positives than false positives.

Identification and molecular characterisation of a gene encoding a member of the insulin receptor family in Echinococcus multilocularis

Receptor kinases play a key role in the communication of cells with their environment and could be important mediators of the effects of host cytokines on endoparasitic organisms. In this paper we describe, for the first time, the characterisation of a receptor tyrosine kinase of the insulin receptor family from a parasitic helminth. Using a degenerative PCR approach, we identified and completely characterised the 5.5kb coding DNA for an Echinococcus multilocularis factor (EmIR) which displays significant homologies to insulin receptors of different phylogenetic origin. EmIR exhibited a domain structure which is typical for the protein family and contained all catalytically important residues at corresponding positions. One striking difference between EmIR and known insulin receptors was the presence of a 172 amino acid insert in the tyrosine kinase region of, as yet, unknown function. In yeast two-hybrid analyses, the ligand binding domains of the human insulin receptor and of EmIR showed comparable affinity to human insulin. The EmIR encoding chromosomal locus (emir) was characterised and comprised 16.5kb. Southern blot hybridisations demonstrated that emir is present as a single copy locus in E. multilocularis. Furthermore, structural comparisons indicated that emir and the insulin receptor genes from mammals and insects derive from a common ancestor. Based on reverse transcriptase-polymerase chain reaction analyses, emir was found to be expressed in the two larval stages metacestode and protoscolex. EmIR is, therefore, likely to play an important role in echinococcal development and possibly also in the interaction with the mammalian host.

Association of a common polymorphism in the promoter of UCP2 with susceptibility to multiple sclerosis

Uncoupling protein 2 (UCP2) is a member of the mitochondrial proton transport family that uncouples proton entry to the mitochondria from ATP synthesis. UCP2 expression levels have been linked to predisposition to diabetes and obesity. In addition, UCP2 prevents neuronal death and injury. Here we show that the common −866G/A promoter polymorphism is associated with susceptibility to multiple sclerosis (MS) in the German population. We analysed altogether 1,097 MS patients and 462 control subjects from two cohorts and found that the common G allele is associated with disease susceptibility (p=0.0015). The UCP2 −866G allele is correlated with lower levels of UCP2 expression as shown here in vitro and in vivo. Thus, UCP2 promoter polymorphism may contribute to MS susceptibility by regulating the level of UCP2 protein in the central nervous and/or the immune systems.

Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice

PLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants with RAG-1-deficient mice lacking mature lymphocytes, we could recently demonstrate a pathogenetic impact of the CD8+ cells. In the present study, we investigated the pathogenetic impact of CD11b+ macrophages by crossbreeding the myelin mutants with knockout mice deficient for the macrophage-restricted adhesion molecule sialoadhesin (Sn). In the wild-type mice, Sn is barely detectable on CD11b+ cells, whereas in the myelin mutants, almost all CD11b+ cells express Sn. In the double mutants, upregulation of CD8+ T-cells and CD11b+ macrophages is reduced and pathological alterations are ameliorated. These data indicate that in a primarily genetically caused myelin disorder of the CNS macrophages expressing Sn partially mediate pathogenesis. These findings may have substantial impact on treatment strategies for leukodystrophic disorders and some forms of multiple sclerosis.

B7-H1 restricts neuroantigen-specific T cell responses and confines inflammatory CNS damage : Implications for the lesion pathogenesis of multiple sclerosis

The co‐inhibitory B7‐homologue 1 (B7‐H1/PD‐L1) influences adaptive immune responses and has been proposed to contribute to the mechanisms maintaining peripheral tolerance and limiting inflammatory damage in parenchymal organs. To understand the B7‐H1/PD1 pathway in CNS inflammation, we analyzed adaptive immune responses in myelin oligodendrocyte glycoprotein (MOG)35–55‐induced EAE and assessed the expression of B7‐H1 in human CNS tissue. B7‐H1–/– mice exhibited an accelerated disease onset and significantly exacerbated EAE severity, although absence of B7‐H1 had no influence on MOG antibody production. Peripheral MOG‐specific IFN‐γ/IL‐17 T cell responses occurred earlier and enhanced in B7‐H1–/– mice, but ceased more rapidly. In the CNS, however, significantly higher numbers of activated neuroantigen‐specific T cells persisted during all stages of EAE. Experiments showing a direct inhibitory role of APC‐derived B7‐H1 on the activation of MOG‐specific effector cells support the assumption that parenchymal B7‐H1 is pivotal for delineating T cell fate in the target organ. Compatible with this concept, our data investigating human brain tissue specimens show a strong up‐regulation of B7‐H1 in lesions of multiple sclerosis. Our findings demonstrate the critical importance of B7‐H1 as an immune‐inhibitory molecule capable of down‐regulating T cell responses thus contributing to the confinement of immunopathological damage.

Evidence for VAV2 and ZNF433 as susceptibility genes for multiple sclerosis

In a genome wide association study consisting of 592 German multiple sclerosis (MS) patients and 825 controls we were able to replicate the association of the HLA region with MS independently of previous case control studies. No SNPs outside the HLA region reached a genome wide level of significance. Nevertheless, we found suggestive evidence for an association of MS with variants in two new genes, the VAV2 gene and the gene for ZNF433.