Major histocompatibility complex (MHC2+) perivascular macrophages in the axotomized facial motor nucleus are regulated by receptors for interferon-gamma (IFNgamma) and tumor necrosis factor (TNF).

The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.

B7.2 on activated and phagocytic microglia in the facial axotomy model: regulation by interleukin-1 receptor type 1, tumor necrosis factor receptors 1 and 2 and endotoxin.

Co-stimulatory factors are involved in different forms of brain pathology and play an important role in the activation of T-cells. In the current study, we explored the regulation of B7.2, a prominent member of the B7 family of costimulatory factors, in the facial motor nucleus (FMN) following facial axotomy and systemic application of lipopolysaccharide (LPS, endotoxin) using light and electron immunohistochemistry and cytokine-receptor-deficient mice. Facial axotomy led to a gradual increase of B7.2 immunoreactivity (IR) on microglial cell surface; similar effects were also observed following application of LPS, but both effects were not additive, suggesting overlapping or saturated signaling pathways. Some B7.2-IR was already present on activated microglia surrounding injured neurons at days 1-4 after injury, but became particularly intense during neuronal cell death, peaking at day 14. Previous studies revealed that these late microglial changes are accompanied by a strong increase in the expression of proinflammatory cytokines such as interleukin-1 beta (IL1beta) tumor necrosis factor-alpha (TNFalpha) and interferon gamma (IFNgamma) [J. Neurosci. 18 (1998a) 5804]. Here, deletion of the receptors for these cytokines-IL1R1, TNFR1 or TNFR2, but not IFNgammaR1-caused a strong and significant reduction in B7.2-IR in reactive microglial cells, compared with their wild type (WT) controls on the same genetic strain background, with a 31% decrease in IL1R1-/- , 39% in TNFR1-/- and 49% in TNFR2-/- mice. These data underscore the significance of IL1beta, TNFalpha and LPS, and their receptors, as potent inflammatory signals that regulate the cellular response in the injured brain as well as the interaction with the rapidly recruited immune system. The broad susceptibility of B7.2 regulation to a wide range of different inflammatory signals also points to its role as a sensor of molecular pathology, and a factor that plays an important accessory role in allowing and shaping the microglia/T-cell interaction in the injured central nervous system.

Cytotoxic potential of proinflammatory cytokines: combined deletion of TNF receptors TNFR1 and TNFR2 prevents motoneuron cell death after facial axotomy in adult mouse.

Neural injury is known to trigger inflammatory changes, including the synthesis of proinflammatory cytokines such as interleukin-1-beta (IL1beta), tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) [G. Raivich, L. L. Jones, C. U. A. Kloss, A. Werner, H. Neumann, and G. W. Kreutzberg, 1998, J Neurosci, 18: 5804-5816] that may play a pivotal role in mediating the cellular response in the affected brain tissue. Here we examined the effects of transgenic deletion of receptors for these cytokines on neuronal cell loss in the adult mouse facial motor nucleus after a peripheral, facial nerve cut. Homozygous deletion of IL1 receptor 1 (IL1R1), TNF receptor 1 or 2 (TNFR1 or TNFR2), or IFNgamma receptor 1 (IFNgammaR1) alone had no effect but combined deletion of TNFR1 and TNFR2 caused a striking absence of alphaX beta2 integrin/IBA1-double-labeled, phagocytic microglial nodules in the axotomized facial motor nucleus 14 days after nerve cut. Moreover, this combined deletion also led to an almost complete prevention of cell loss by Day 29. Additional neuronal cell counts at Day 60 revealed a second phase of motoneuron cell disappearance, which did not depend on the presence of TNF receptors. However, there was still the same 22% difference in the total number of motoneurons between the wild-type and TNFR1 & 2-deficient mice, underlining the role of TNF ligands and both TNF receptors in mediating the early phase of neuronal cell loss after traumatic injury.

Thymus medulla consisting of epithelial islets each derived from a single progenitor.

The thymus is organized into medullary and cortical zones that support distinct stages of T-cell development. The formation of medulla and cortex compartments is thought to occur through invagination of an endodermal epithelial sheet into an ectodermal one at the third pharyngeal pouch and cleft, respectively. Epithelial stem/progenitor cells have been proposed to be involved in thymus development, but evidence for their existence has been elusive. We have constructed chimaeric mice by injecting embryonic stem (ES) cells into blastocysts using ES cells and blastocysts differing in their major histocompatibility complex (MHC) type. Here we show that the MHC class-II-positive medullary epithelium in these chimaeras is composed of cell clusters, most of which derive from either embryonic stem cell or blastocyst, but not mixed, origin. Thus, the medulla comprises individual epithelial 'islets' each arising from a single progenitor. One thymic lobe has about 300 medullary areas that originate from as few as 900 progenitors. Islet formation can be recapitulated after implantation of 'reaggregated fetal thymic organs' into mice, which shows that medullary 'stem' cells retain their potential until at least day 16.5 in fetal development. Thus, medulla-cortex compartmentalization is established by formation of medullary islets from single progenitors.

Interleukin-6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice.

Nerve injury triggers numerous changes in the injured neurons and surrounding non-neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin-6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6-deficient mice and wild-type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3-positive T-lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-kappaB activation.

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.

Aged mice exhibit in vivo defective peripheral clonal deletion of D(b)/H-Y reactive CD8(+) T cells.

We previously reported that T cells from aged mice were resistant to activation-induced cell death (AICD) in vitro. To determine whether the presence of AICD-resistant T cells is associated with defects in age-related peripheral clonal deletion in vivo, congenic male SCID mice were reconstituted with T cells from aged or young female D(b)/H-Y TCR (Tg71) transgenic mice. Compared with recipients of young cells, the recipients of T cells from aged mice exhibited a 3-fold increase in the percentage of autoreactive CD8(+) H-Y antigen-reactive T cells as defined by the clonotypic antibody, M33. There were significantly increased sera levels of interferon-gamma, a significantly decreased expression of FasL by M33(+)CD8(+) T cells, and significantly decreased apoptosis by DNA fragmentation staining of the spleen of mice reconstituted with T cells from aged mice compared to those from young mice. By day 21, the recipients of T cells from aged mice but not young mice, exhibited infiltration of CD3(+) cells into the non-lymphoid organs. These results indicate that there is defective peripheral deletion of the self-reactive T cells derived from aged female Tg71 mice, and that failure to delete these cells is associated with the defective T-cell clonal deletion in the recipient mice.

Synergism of Pseudomonas aeruginosa exotoxin A with endotoxin, superantigen, or TNF results in TNFR1- and TNFR2-dependent liver toxicity in mice.

Pseudomonas aeruginosa is a potentially dangerous Gram-negative nosocomial pathogen, causing bacteremia in debilitated patients, and a prominent cause of bacterial cholangitis. Opportunistic infections with other nosocomial pathogens, e.g. Staphylococcus aureus, are common. Hence, multi-intoxication with P. aeruginosa exotoxin A (PEA) and other bacterial toxins, including endotoxin (LPS) and the superantigen S. aureus enterotoxin B (SEB), is very likely. Here we show that PEA synergistically interacted with LPS, SEB, or recombinant murine tumor necrosis factor alpha (rmuTNF) in mice, resulting in severe liver injury. Enhanced and prolonged circulation of cytokines, including TNF, which depended on the presence of T cells, was a remarkable feature of synergistic PEA/LPS- or PEA/SEB-induced hepatotoxicity. PEA/LPS-, PEA/SEB- or PEA/rmuTNF-induced liver injury was mediated by both TNF receptors (TNFRs), i.e. TNFR1 and TNFR2. In view of the fact that TNFR1, but not TNFR2, signaling is unequivocally required for host defense, our results suggest that anti-TNFR2 strategies might be beneficial to protect the liver from inflammatory damage caused by synergistic interactions of PEA with other TNF-inducing bacterial toxins.

Enterotoxin adjuvants have direct effects on T cells and antigen-presenting cells that result in either interleukin-4-dependent or -independent immune responses.

In an in vitro study, Escherichia coli heat-labile toxin (LT) was shown to directly affect activated CD4(+) T cells and support interleukin (IL)-5 production in IL-4-deficient (IL-4(-/-)) mice, whereas cholera toxin (CT) did not. Both LT and CT enhanced B7-2 expression on B cells and macrophages. These effects were not influenced by CD40-CD40 ligand cosignaling. Addition of LT- or CT-treated antigen-presenting cells to anti-CD3-triggered CD4(+) T cells resulted in the induction of T cell proliferative responses. Further, these responses were inhibited by anti-B7-2 monoclonal antibody. Cocultivation of CD4(+) T cells with LT- or CT-treated antigen-presenting cells and anti-CD3 enhanced Th1- and IL-4-mediated Th2-type cytokine production. The results from in vitro studies were supported by in vivo studies in IL-4(-/-) mice, in which LT induced mucosal IgA responses but CT did not. Thus, although both LT and CT induce mucosal adjuvant responses via IL-4-dependent Th2-type responses, LT also elicits Th1- and IL-4-independent Th2-type responses.

Metallothionein induction by restraint stress: role of glucocorticoids and IL-6.

Restraint stress increased liver metallothionein-I (MT-I) mRNA and MT-I+II protein levels. The glucocorticoid receptor antagonist RU 486 decreased this response. In contrast, adrenalectomy only decreased MT-I+II protein levels. Moreover, corticosterone or progesterone did not reverse the effect of RU 486. These results suggest that glucocorticoids are important for MT-I+II protein synthesis but not for MT-I mRNA accumulation during restraint stress, and that other factors must be involved in this process. Interleukin-6 (IL-6) deficient mice showed a significant decrease of restraint stress-induced liver MT-I mRNA levels (approximately 30% of IL-6+/+ mice) up to approximately 4-5 hours after the onset of stress. Western blotting of hepatic nuclear proteins showed that the IL-6 responsive transcription factor Stat3, which has been shown to mediate MT induction by inflammation, was also activated by restraint stress. Results after extended periods of restraint stress indicate that IL-6 participates early and transiently in the process. The analysis of the expression of the acute phase plasma protein serum amyloid A suggests that restraint stress elicits an acute phase response similar to that caused by inflammation.

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract.

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.

Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature.

Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/-), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/- mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF-induced tumor necrosis.

Stress-induced enhancement of skin immune function: A role for gamma interferon.

Contrary to the widespread belief that stress is necessarily immunosuppressive, recent studies have shown that, under certain conditions, stress can induce a significant enhancement of a skin cell-mediated immune response [delayed-type hypersensitivity (DTH) or contact hypersensitivity]. Adrenal stress hormones and a stress-induced trafficking of leukocytes from the blood to the skin have been identified as systemic mediators of this immunoenhancement. Because gamma interferon (IFNgamma) is an important cytokine mediator of DTH, the studies described here were designed to examine its role as a local mediator of the stress-induced enhancement of skin DTH. The effect of acute stress on skin DTH was examined in wild-type and IFNgamma receptor-deficient (IFNgammaR-/-) mice that had previously been sensitized with 2,4-dinitro-1-fluorobenzene. Acutely stressed wild-type mice showed a significantly larger DTH response than nonstressed mice. In contrast, IFNgammaR-/- mice failed to show a stress-induced enhancement of skin DTH. Immunoneutralization of IFNgamma in wild-type mice significantly reduced the stress-induced enhancement of skin DTH. In addition, an inflammatory response induced by direct IFNgamma administration to the skin was significantly enhanced by acute stress. Our results suggest that IFNgamma is an important local mediator of a stress-induced enhancement of skin DTH. These studies are clinically relevant because, depending on the nature of the antigen, DTH reactions mediate numerous protective (e.g., resistance to viral, bacterial, parasitic, and fungal infections) or pathological (e.g., autoimmune reactions and contact sensitivity reactions such as that to poison ivy) immune responses.

Antiviral B cell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice.

TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.

TNF is essential for the cell-mediated protective immunity induced by the radiation-attenuated schistosome vaccine.

C57BL/6 mice exposed to the radiation-attenuated schistosome vaccine exhibit high levels of protective immunity. The cell-mediated pulmonary effector mechanism involves IFN-gamma-producing CD4+ T cells in a focal response around challenge larvae. IFN-gamma can promote production of TNF and can synergize with this cytokine in its actions on responder cells. We have examined whether TNF plays a role in lung phase immunity to schistosomes using mice with a disrupted gene for TNFRI (TNFRI-/-). The most dramatic finding was that the schistosome vaccine elicited no protection whatsoever in these mice. However, this could not be attributed to a lack of responder cells, because more lymphocytes were lavaged from the airways of TNFRI-/- than wild-type mice. Furthermore, CD4+ T cells were equally represented in airway populations from the two groups and produced IFN-gamma upon Ag stimulation in vitro. In contrast, pulmonary macrophage function was defective in TNFRI-/- mice, as indicated by a failure to up-regulate inducible NO synthase mRNA. Histopathological analysis revealed that focal infiltrates were of similar size and cell composition in the two groups but that more parasites were free of foci in the TNFRI-/- mice. These animals had a greatly impaired IgG response to schistosomes, which may explain their lack of residual protection due to Ab in a situation where cell-mediated immunity is disabled. We suggest that the absence of protective immunity could result from a retarded build-up of leukocytes around migrating lung worms and/or a deficit in accessory cell function within a focus, both of which would permit parasite escape.

The lectin-like domain of tumor necrosis factor-alpha increases membrane conductance in microvascular endothelial cells and peritoneal macrophages.

Herein, we show that TNF exerts a pH-dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor-independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin-like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17-amino acid peptide of TNF, which was previously shown to exert lectin-like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenous ion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology.

Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2-mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis.

The regulatory role of interferon-gamma receptor (IFN-gammaR)- and tumor necrosis factor receptor (TNFR)-mediated immune reactions for the activation of cerebral endothelial cells, microglia, and astrocytes was evaluated in a model of murine Toxoplasma encephalitis (TE). Brain endothelial cells of wild-type mice reacted in response to Toxoplasma infection with a strong up-regulation of the vascular cell adhesion molecule, the intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I and II antigens. A similar response was seen in mice genetically deficient for either TNFR1, TNFR2, or both TNFRs, whereas IFN-gammaR-deficient (IFN-gammaR0/0) mice were found to be defective in the up-regulation of these molecules. However, recruitment of leukocytes to the brain and their intracerebral movement were not impaired in IFN-gammaR0/0 mice. In addition, microglia of Toxoplasma gondii-infected IFN-gammaR0/0 mice failed to induce expression of ICAM-1, leukocyte function-associated antigen (LFA)-1, and MHC class I and II antigens, whereas wild-type and TNFR-deficient mice up-regulated these molecules. Moreover, TNF-alpha mRNA production of F4/80(+) microglia/macrophages was impaired in IFN-gammaR0/0 mice, but not in TNFR-deficient mutants. However, induction of interleukin (IL)-1beta, IL-10, IL-12p40, and IL-15 mRNA was independent of IFN-gammaR and TNFR signaling. In conclusion, IFN-gammaR, but not TNFR signaling, is the major pathway for the activation of endothelial cells and microglia in murine TE. These findings differ from observations in other inflammatory central nervous system disorders, indicating specific regulatory mechanisms in this parasitic cerebral infection.

Severity of symptoms and demyelination in MOG-induced EAE depends on TNFR1.

The individual role of tumor necrosis factor receptor 1 (TNFR1) and TNFR2 signaling in experimental autoimmune encephalomeylitis (EAE) was investigated using mice lacking TNFR1 (TNFR1-/-), TNFR2 (TNFR2-/-) as well as double receptor (TNFR1/2-/-) and double ligand (TNF/LT alpha-/-) knockout mice. In wild-type (wt) mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 the clinical course is characterized by an acute disease onset with peak disease scores and a consecutive chronic phase lasting up to 60 days. Compared to control mice, TNF/LT alpha-deficient mice showed a significant delay in disease onset and a remarkable reduction in demyelination which was, however, associated with increased inflammation. In TNFR1-/- and TNFR1/2-/- mice, the disease course was comparable to TNF/LT alpha-deficient mice but rather monophasic and less severe at late time points. Likewise only minimal spinal cord demyelination became apparent. In contrast, the course of EAE in TNFR2-/- mice was severe and associated with remarkable demyelination. Taken together these findings define TNFR1 as crucial mediator in MOG-induced EAE and suggest a protective role for TNFR2 signaling in the clinical course of EAE.

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells.

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.

Strongly compromised inflammatory response to brain injury in interleukin-6-deficient mice.

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin-6 (IL-6) during the inflammatory response in the brain following disruption of the blood-brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto-parietal cortex in interleukin-6-deficient (IL-6-/-) and normal (IL-6+/+) mice. In IL-6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and metallothionein-I+II (MT-I+II) were increased in these cells, while the brain-specific MT-III was only moderately upregulated. In IL-6-/- mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage-induced GM-CSF and MT-I+II expression were also markedly depressed compared to IL-6+/+ mice. In contrast, MT-III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT-I+II but not MT-III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL-6+/+ and IL-6-/- mice. The present results demonstrate that IL-6 is crucial for the recruitment of myelo-monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL-6 is important for neuroprotection and the induction of GM-CSF and MT expression. The opposing effect of IL-6 on MT-I+II and MT-III levels in the damaged brain suggests MT isoform-specific functions.