Per Anderson

Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis.

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.

Endothelial basement membrane laminin |[alpha]|5 selectively inhibits T lymphocyte extravasation into the brain

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 α4 and small amounts of laminin α5. In mice lacking laminin α4, laminin α5 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 α5 directly inhibited integrin α6β1–mediated migration of T lymphocytes through laminin α4. 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.

Ghrelin Protects against Experimental Sepsis by Inhibiting High-Mobility Group Box 1 Release and by Killing Bacteria

Sepsis, a life-threatening complication of infections and the most common cause of death in intensive care units, is characterized by a hyperactive and out-of-balance network of endogenous proinflammatory cytokines. None of the current therapies are entirely effective, illustrating the need for novel therapeutic approaches. Ghrelin (GHR) is an orexigenic peptide that has emerged as a potential endogenous anti-inflammatory factor. In this study, we show that the delayed administration of GHR protects against the mortality in various models of established endotoxemia and sepsis. The therapeutic effect of GHR is mainly mediated by decreasing the secretion of the high mobility box 1 (HMGB1), a DNA-binding factor that acts as a late inflammatory factor critical for sepsis progression. Macrophages seem to be the major cell targets in the inhibition of HMGB1 secretion, in which GHR blocked its cytoplasmic translocation. Interestingly, we also report that GHR shows a potent antibacterial activity in septic mice and in vitro. Remarkably, GHR also reduces the severity of experimental arthritis and the release of HMGB1 to serum. Therefore, by regulating crucial processes of sepsis, such as the production of early and late inflammatory mediators by macrophages and the microbial load, GHR represents a feasible therapeutic agent for this disease and other inflammatory disorders.

Inhibition of the transcription factors AP-1 and NF-κB in CD4 T cells by peroxisome proliferator-activated receptor γ ligands

The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, is essential for adipocyte differentiation and glucose homeostasis. PPARgamma has been found recently to regulate macrophage activation in response to mitogens and inflammation. Our study shows PPARgamma to be preferentially expressed in the nuclei of resting T cells and to increase upon activation of T cells by either anti-CD3 and anti-CD28 or phorbol myristyl acetate (PMA). We also found the PPARgamma ligand ciglitizone to attenuate the activation of T cells by inhibiting cytokine gene expression and anti-CD3 and anti-CD28 or PMA-induced proliferative responses. Inhibition of both the proliferative response and inflammatory cytokine expression in CD4 T cells was correlated with suppression of the activated transcription factors AP1 and NF-kappaB. PPARgamma ligands also strongly inhibited SEA-induced Vbeta3 T cell activation in vivo. These results, together with previous findings of the inhibitory effect of PPARgamma ligands on activated macrophages, provide clear evidence for PPARgamma as a negative regulator of the inflammatory activation of both macrophage and T cells. PPARgamma may thus be a potential therapeutic target for the treatment of autoimmunity.

Bcl-3 and NFκB p50-p50 Homodimers Act as Transcriptional Repressors in Tolerant CD4+ T Cells

The transcriptional events that control T cell tolerance are still poorly understood. To investigate why tolerant T cells fail to produce interleukin (IL)-2, we analyzed the regulation of NFκB-mediated transcription in CD4+ T cells after tolerance induction in vivo. We demonstrate that a predominance of p50-p50 homodimers binding to the IL-2 promoter κB site in tolerant T cells correlated with repression of NFκB-driven transcription. Impaired translocation of the p65 subunit in tolerant T cells was a result from reduced activation of IκB kinase and poor phosphorylation and degradation of cytosolic IκBs. Moreover, tolerant T cells expressed high amounts of the p50 protein. However, the increased expression of p50 could not be explained by activation-induced de novo synthesis of the precursor p105, which was constitutively expressed in tolerant T cells. We also demonstrate the exclusive induction of the IκB protein B cell lymphoma 3 (Bcl-3) in tolerant T cells as well as its specific binding to the NFκB site. These results suggest that the cellular ratio of NFκB dimers, and thus the repression of NFκB activity and IL-2 production, are regulated at several levels in tolerant CD4+ T cells in vivo.

CD1high B cells: a population of mixed origin

A specialized subpopulation of T lymphocytes is reactive to the MHC class I‐like molecule CD1d. It is not clear which cells are the major antigen‐presenting cells in vivo in the activation of CD1‐restricted immune responses. We have characterized a subset of B lymphocytes expressing six‐ to eightfold higher levels of CD1 than the bulk of B cells. The cells have a surface phenotype (CD21high, CD23low, IgMhigh, IgDlow) found previously to characterize B cells residing in the splenic marginal zones. CD1high B cells localize preferentially to the spleen, and appear late in ontogeny, at 3 – 4 weeks of age. The CD1high B cells were present in mice lacking conventional helper T cells, ruling out an exclusive origin from T cell‐dependent immune responses. Still, some CD1high B cells had been involved in T cell‐dependent immune responses as suggested by mutations in their rearranged immunoglobulin gene regions. The population could still be found in mice with severely reduced B cell reactivity to bacterial lipopplysaccharides (C3H / HeJ mice) and in mice unable to respond to thymus‐independent type 2 antigens (NFR.Xid mice), as well as in germ‐free mice, indicating that bacterial antigens are not major stimuli for the induction of CD1high B cells. In contrast, the CD1high B cell population was severely reduced in CD19‐deficient mice. Taken together, the results imply that the CD1high population is heterogenous and of mixed origin, dependent for its development or maintenance on signaling through the CD19 molecule.

The role of CD1d-restricted NK T lymphocytes in the immune response to oral infection with Salmonella typhimurium

CD1d‐restricted natural killer T (NKT) cells belong to the innate‐like lymphocytes which respond rapidly to stress and infectious challenge. We have studied murine CD1d‐restricted NKT cells in the early immune response to virulent Salmonella enterica serovar Typhimurium after oral infection. In the liver and spleen, neutrophil and macrophage numbers had increased several‐fold by day 5 post‐infection, while the frequency of B and T lymphocytes decreased. These cellular changes occurred independently of CD1d‐restricted NKT cells, and further, CD1d‐restricted T cells did not influence the bacterial load. However, in CD1d+ mice NK1.1+ T cells and invariant CD1d‐restricted T cells were activated by the infection, as demonstrated by an increase in size, up‐regulation of CD69 and production of IFN‐γ. The NK1.1 antigen was down‐modulated on these cells during the course of infection, while TCR levels were unaffected. While dendritic cells (DC) up‐regulated CD1d‐levels upon 24 h of in vitro exposure to the bacteria, increased CD1d expression was not evident on DC in vivo during infection. Furthermore, in vitro re‐stimulation of CD1d‐restricted T cells isolated from infected mice demonstrated a significant skewing of the cytokine profile, with suppressed IL‐4 and increased IFN‐γ production.

Porcine neural xenografts in rats and mice : donor tissue development and characteristics of rejection

Embryonic ventral mesencephalic tissue from the pig is a potential alternative donor tissue for neural transplantation to Parkinsons disease patients. For stable graft survival, the host immune response has to be prevented. This study was performed in order to analyze the mechanisms and dynamics of neural xenograft rejection, as well as neurobiological properties of the donor tissue. Adult normal mice and rats, and cyclosporin A-treated rats, received intrastriatal transplants of dissociated embryonic ventral mesencephalic pig tissue that was 27 or 29 embryonic days of age (E27 and E29). The animals were perfused at 2, 4, 6, and 12 weeks after grafting and the brains were processed for immunohistochemistry of dopaminergic (tyrosine hydroxylase positive) neurons, CD4(+) and CD8(+) lymphocytes, natural killer cells, macrophages, microglia, and astrocytes. Thirty-five rats received daily injections of BrdU for 5 consecutive days at different time points after transplantation and were perfused at 6 weeks. These animals were analyzed for proliferation of cells in the donor tissue, both in healthy and in rejecting grafts. No tyrosine hydroxylase-positive cells proliferated after grafting. Our results demonstrated that E27 was superior to E29 donor tissue for neurobiological reasons. Cyclosporin A immunosuppression was protective only during the first weeks and failed to protect the grafts in a long-term perspective. Grafts in mice were invariably rejected between 2 and 4 weeks after transplantation, while occasional grafts in untreated rats survived up to 12 weeks without signs of an ongoing rejection process. CD8(+) lymphocytes and microglia cells are most likely important effector cells in the late, cyclosporin A-resistant rejection process.

Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance

Repetitive antigen stimulation induces peripheral T cell tolerance in vivo. It is not known, however, whether multiple stimulations merely suppress T cell activation or, alternatively, change the transcriptional program to a distinct, tolerant state. In this study, we have discovered that STAT3 and STAT5 were activated in response to antigen stimulation in vivo, in marked contrast to the suppression of AP‐1, NF‐κB and NFAT. In addition, a number of transcription factors were induced in tolerant T cells following antigen challenge in vivo, including T‐bet, Irf‐1 and Egr‐2. The altered transcription program in tolerant cells associates closely with the suppression of cell cycle progression and IL‐2 production, as well as with the induction of IL‐10. Studies of T‐bet and Egr‐2 show that the function of T‐bet in peptide treatment‐induced regulatory T cells is not associated with Th1 differentiation, but correlates with the suppression of IL‐2, whereas expression of Egr‐2 led to an up‐regulation of the cell cycle inhibitors p21cip1 and p27kip. Our results demonstrate a balanced transcription program regulated by different transcription factors for T cell activation and/or tolerance during antigen‐induced T cell responses. Persistent antigen stimulation can induce T cell tolerance by changing the balance of transcription factors.

CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities

Administration of in vitro expanded mesenchymal stromal cells (MSCs) represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs) with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105) expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105+ and CD105- MSCs. CD105+ and CD105- mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105+ mMSCs was affected by passage number and cell confluency while CD105- mMSCs remained negative. The CD105+ and CD105- mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29+CD44+Sca1 + MHC-I+ and CD45-CD11b-CD31-) but varied in their differentiation and immunoregulatory properties. Interestingly, CD105- mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4+ T cells more efficiently compared to CD105+ mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.