The endothelial cell monolayer of cerebral vessels and its basement membrane (BM) are ensheathed by the astrocyte endfeet, the leptomeningeal cells, and their associated parenchymal BM, all of which contribute to establishment of the blood–brain barrier (BBB). As a consequence of this unique structure, leukocyte penetration of cerebral vessels is a multistep event. In mouse experimental autoimmune encephalomyelitis (EAE), a widely used central nervous system inflammatory model, leukocytes first penetrate the endothelial cell monolayer and underlying BM using integrin β1-mediated processes, but mechanisms used to penetrate the second barrier defined by the parenchymal BM and glia limitans remain uninvestigated. We show here that macrophage-derived gelatinase (matrix metalloproteinase [MMP]-2 and MMP-9) activity is crucial for leukocyte penetration of the parenchymal BM. Dystroglycan, a transmembrane receptor that anchors astrocyte endfeet to the parenchymal BM via high affinity interactions with laminins 1 and 2, perlecan and agrin, is identified as a specific substrate of MMP-2 and MMP-9. Ablation of both MMP-2 and MMP-9 in double knockout mice confers resistance to EAE by inhibiting dystroglycan cleavage and preventing leukocyte infiltration. This is the first description of selective in situ proteolytic damage of a BBB-specific molecule at sites of leukocyte infiltration.

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFα release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide–binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.

CD4+CD25+ T cells have been shown to inhibit experimentally induced organ‐specific autoimmune disease and depletion of these regulatory T cells from normal mice results in development of such conditions. Furthermore, CD4+CD25+ T cells suppress the IL‐2 production and thereby the proliferation of polyclonally activated CD4+CD25– T cells in vitro. The suppression in vitro is independent of secreted factors but requires interactions between CD4+CD25– and CD4+CD25+ T cells and antigen‐presenting cells (APC). We have now further investigated the function of CD4+CD25+ T cells in vitro and have focused on their interactions with APC. We found that CD4+CD25+ T cells down‐regulated the expression of the co‐stimulatory molecules CD80 and CD86 on dendritic cells. The steady‐state level of CD80 mRNA was also decreased, while the steady‐state level of CD86 mRNA was not, suggesting that distinct mechanisms regulate the expression of these molecules. The down‐regulation occurred even in the presence of stimuli that would normally increase the expression of CD80 and CD86 molecules. Thus, down‐regulation of co‐stimulatory molecules may be an additional effector function of these regulatory T cells.

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin alpha4 and small amounts of laminin alpha5. In mice lacking laminin alpha4, laminin alpha5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin alpha5 directly inhibited integrin alpha(6)beta(1)-mediated migration of T lymphocytes through laminin alpha4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population.

We have previously demonstrated that CD4+ CD25+ natural regulatory T cells (Treg cells) induce down-modulation of CD80 and CD86 (B7) molecules on dendritic cells (DCs) in vitro. In this report we show that the extent of down-modulation is functionally significant because Treg-cell conditioned DCs induced poor T-cell proliferation responses. Further, we report that down-modulation was induced rapidly and was inhibited by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4), which is constitutively expressed by the Treg cells. Even though Treg cells have previously been reported to kill antigen-presenting cells, the down-modulation was not due to selective killing of DCs expressing high level of the costimulatory molecules. We propose that Treg cells down-modulate B7-molecules on DCs in a CTLA-4-dependent way, thereby enhancing suppression of T-cell activity.