High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mf) and define pathways activated by HMGB1 binding. Bone marrow Mf were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK-and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mf from interleukin (IL)-1 receptor type I -/-, Toll-like receptor 2 (TLR2 -/-) and RAGE -/-mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mf, phosphorylation of all investigated MAP kinase pathways and NF-kB translocation, and expression of MHC class II was increased. Mf from RAGE -/-mice produced significantly lower amounts of TNF, IL-1b and IL-6, while IL-1RI -/-and TLR2 -/-Mf produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mf, with RAGE as the major activation-inducing receptor.

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune disease and an animal model of multiple sclerosis. To investigate how upstream TLR signals influence autoimmune T cell responses, we studied the role of individual TLR and MyD88, the common TLR adaptor molecule, in the initiation of innate and adaptive immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in CFA. MyD88 -/-mice were completely EAE resistant. Purified splenic myeloid DC (mDC) from MyD88 -/-mice expressed much less IL-6 and IL-23, and serum and T cell IL-17 were absent. TLR4-/-and TLR9 -/-mice surprisingly exhibited more severe EAE symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were higher in TLR4 -/-mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6 expression by splenocytes was higher in TLR9 -/-mice. Our data suggest that MyD88 mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization, which in turn drives IL-17-producing encephalitogenic Th17 cell activation. Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOGinduced EAE.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. The underlying molecular mechanisms responsible for disease progression are still unclear. Herein, we demonstrate that high mobility group box chromosomal protein 1 (HMGB1), a DNA-binding protein with proinflammatory properties, is evident in active lesions of MS and experimental autoimmune encephalomyelitis (EAE) and that HMGB1 levels correlate with active inflammation. Furthermore, the expression of the innate HMGB1 receptors--receptor for advanced glycation end products, TLR2, and TLR4--was also highly increased in MS and rodent EAE. Additionally, in vitro activation of rodent CNS-derived microglia and bone marrow-derived macrophages demonstrated that microglia were equally as capable as macrophages of translocating HMGB1 following LPS/IFN-gamma stimulation. Significant expression of HMGB1 and its receptors on accumulating activated macrophages and resident microglia may thus provide a positive feedback loop that amplifies the inflammatory response during MS and EAE pathogenesis.