Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes.

BACKGROUND: Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE. METHODS: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35₋55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR. RESULTS: We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35₋55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site. CONCLUSIONS: These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases. However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

Deficiency of gelatinase B/MMP-9 aggravates lpr-induced lymphoproliferation and lupus-like systemic autoimmune disease.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a key enzyme involved in inflammatory, hematological, vascular and neoplastic diseases. In previous studies, we explored the intracellular substrate set or 'degradome' of MMP-9 and found many systemic autoantigens as novel intracellular gelatinase B substrates. Little is known, however, about the functional role of MMP-9 in the development of systemic autoimmunity in vivo. B6(lpr/lpr) mice with defective Fas-mediated apoptosis were used to investigate the functions of MMP-9 in lymphocyte proliferation and in the development of systemic autoimmunity. Combined Fas and gelatinase B deficiency resulted in extreme lymphoproliferative disease with enhanced lymphadenopathy and splenomegaly, and significantly reduced survival compared with single Fas deficiency. At the cellular level, this was corroborated by increased lymph node accumulation of 'double negative' T cells, B cells and myeloid cells. In addition, higher autoantibody titers and more pronounced autoimmune tissue injury were found in the absence of MMP-9, culminating in chronically enhanced systemic lupus erythematosus (SLE)-like autoimmunity. After cleavage by MMP-9 the SLE autoantigens U1snRNP A and ribosomal protein P0 were hardly recognized by plasma samples of both B6(lpr/lpr).MMP-9⁻/⁻ and B6(lpr/lpr).MMP-9+/+ mice, pointing to a destruction of B cell epitopes by MMP-9-mediated proteolysis. In addition, the same loss of immunodominant epitopes was observed with plasma samples from SLE patients, suggesting that MMP-9 suppresses systemic antibody-mediated autoimmunity by clearance of autoepitopes in immunogenic substrates. Thus, new protective functions for MMP-9 were revealed in the suppression of lymphoproliferation and dampening of systemic autoimmunity, cautioning against the long-term use of MMP inhibitors in autoimmune lymphoproliferative syndrome (ALPS) and SLE.

Interferon-γ orchestrates the number and function of Th17 cells in experimental autoimmune encephalomyelitis.

Th17 cells are suggested to be pathogenic in mediating experimental autoimmune encephalomyelitis (EAE), but their relation to interferon (IFN)-γ-producing Th1 cells in vivo is not well understood. We studied the numbers and functions of Th17 cells in CREAE in Biozzi ABH mice, both in peripheral lymphoid organs and in the central nervous system. Th1 and Th17 cells alternated in secondary lymphoid organs and infiltrated into the central nervous system during chronic relapsing experimental autoimmune encephalomyelitis (CREAE). In the absence of IFN-γ the numbers and secretion of Th17 cells was enhanced, whereas exogenous administration of IFN-γ decreased the Th17 cells. In mice with intact IFN-γ genes, in vivo neutralization of interleukin (IL)-17 protected against EAE development by enhancing the number of IFN-γ-producing cells. IFN-γ knockout mice were partially protected by anti-IL-17 antibodies by decreasing cell numbers and production of IL-17. Our findings suggest that, whereas IFN-γ as such is not necessary for EAE development in the mouse, the lack of suppression of Th17 cells by IFN-γ enhances the susceptibility to develop EAE. IFN-γ thus orchestrates the number and function of Th17 cells.

Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection.

Insight into molecular and cellular mechanisms of innate immunity is critical to understand viral pathogenesis and immunopathology and might be exploited for therapy. Whereas the molecular mechanisms of the IFN defense are well established, cellular mechanisms of antiviral immunity are only emerging, and their pharmacological triggering remains unknown. COAM is a polysaccharide derivative with antiviral activity but without comprehension about its mechanism of action. The COAM mixture was fractionated, and prophylactic treatment of mice with COAM polymers of high MW resulted in a conversion from 100% lethal mengovirus infection to an overall survival rate of 93% without obvious clinical sequelae. Differential and quantitative analysis of peritoneal leukocytes demonstrated that COAM induced a profound influx of neutrophils. Selective cell depletion experiments pointed toward neutrophils and macrophages as key effector cells in the rescue of mice from lethal mengovirus. COAM was able to induce mRNA and protein expression of the mouse neutrophil chemokine GCP-2. Binding of GCP-2 to COAM was demonstrated in solution and confirmed by SPR technology. Although COAM was not chemotactic for neutrophils, COAM-anchored muGCP-2 retained chemotactic activity for human and mouse neutrophils. In conclusion, this study established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, as proposed through the induction, binding, and concentration of endogenous chemokines. These findings reinforce the role of neutrophils and macrophages as critical cells that can be manipulated toward antiviral defense.

Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome.

RATIONALE: Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. OBJECTIVES: Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. METHODS: C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. MEASUREMENTS AND MAIN RESULTS: In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. CONCLUSIONS: We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

IL-12 contributes to allergen-induced airway inflammation in experimental asthma.

Lack of sufficient IL-12 production has been suggested to be one of the basic underlying mechanisms in atopy, but a potential role of IL-12 in established allergic airway disease remains unclear. We took advantage of a mouse model of experimental asthma to study the role of IL-12 during the development of bronchial inflammation. Administration of anti-IL-12p35 or anti-IL-12p40 mAb to previously OVA-sensitized BALB/c mice concomitantly with exposure to nebulized OVA, abolished both the development of bronchial hyperresponsiveness to metacholine as well as the eosinophilia in bronchoalveolar lavage fluid and peripheral blood. Anti-IL-12 treatment reduced CD4(+) T cell numbers and IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid and the mRNA expression of IL-10, eotaxin, RANTES, MCP-1, and VCAM-1 in the lung. Anti-IL-12p35 treatment failed to show these effects in IFN-gamma knockout mice pointing to the essential role of IFN-gamma in IL-12-induced effects. Neutralization of IL-12 during the sensitization process aggravated the subsequent development of allergic airway inflammation. These data together with recent information on the role of dendritic cells in both the sensitization and effector phase of allergic respiratory diseases demonstrate a dual role of IL-12. Whereas IL-12 counteracts Th2 sensitization, it contributes to full-blown allergic airway disease upon airway allergen exposure in the postsensitization phase, with enhanced recruitment of CD4(+) T cells and eosinophils and with up-regulation of Th2 cytokines, chemokines, and VCAM-1. IFN-gamma-producing cells or cells dependent on IFN-gamma activity, play a major role in this unexpected proinflammatory effect of IL-12 in allergic airway disease.

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling.

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.

Exogenous IL-12 suppresses experimental autoimmune encephalomyelitis (EAE) by tuning IL-10 and IL-5 levels in an IFN-gamma-dependent way.

Endogenous IL-12 is considered to be required for the generation and function of pathogenic Th1 effector cells in experimental autoimmune encephalomyelitis (EAE). We show here that IL-12 administration together with the immunization suppressed actively induced CREAE in SJL/J and in Biozzi/ABH mice and even subsequent spontaneous relapse incidence and severity in Biozzi ABH mice. IL-12 given during remission of primary disease inhibited re-induced relapses in SJL/J, but not spontaneous relapses in Biozzi mice. The protective effect of IL-12 is time- and dose-dependent. Protection is accompanied by subsequent increased production of IL-10 and IL-5 by lymph node and spleen cells and an inhibition of cell proliferation. Mice depleted of IFN-gamma by administration of neutralizing antibodies were poorly protected by exogenous IL-12, indicating that the inhibitory effect of IL-12 is partially IFN-gamma dependent.

The homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1).

1alpha,25-Dihydroxyvitamin D3 modulates the murine antibody response to pneumococcal capsular polysaccharide serotype 3 through IL-12.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a steroid hormone that regulates calcium metabolism. Besides, 1alpha,25(OH)(2)D(3 )also has pronounced immunomodulatory effects: it strongly inhibits dendritic cell (DC) maturation and impairs IL-12 production. We studied the effect of 1alpha,25(OH)(2)D(3 )on the antibody response to pneumococcal capsular polysaccharide (caps-PS) serotype 3. 1alpha,25(OH)(2)D(3) inhibited the IgG2a antibody response to caps-PS serotype 3. Besides, 1alpha,25(OH)(2)D(3) also inhibited IL-12 production and maturation of DC. Anti-IL-12 and exogenous IL-12, respectively, inhibited and stimulated the IgG2a antibody response to caps-PS serotype 3. Exogenous IL-12 abrogated the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3, indicating that the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3 was mediated through IL-12. In conclusion, we demonstrate that 1alpha,25(OH)(2)D(3) has an inhibitory effect on the IgG2a antibody response to caps-PS serotype 3, and that this effect was mediated trough IL-12.

Dependence on interferon-gamma for the spontaneous occurrence of arthritis in DBA/1 mice.

OBJECTIVE: Male DBA/1 mice are known to spontaneously develop arthritis in the hind legs. The present study was undertaken to investigate the role of endogenous interferon-gamma (IFNgamma) in the pathogenesis of this ankylosing enthesopathy. METHODS: The role of IFNgamma was studied by examining the development of arthritis in IFNgamma receptor-knockout (IFNgammaR-KO) DBA/1 mice as compared with wild-type mice, and by treatment of wild-type mice with monoclonal anti-IFNgamma antibody. IFNgamma-disrupted and wild-type mice were mixed and housed in the same cage, and clinical symptoms of arthritis were assessed weekly for at least 9 weeks. Histologic examination was performed at the end of the experiment. RESULTS: In DBA/1 wild-type mice, 70% of the animals developed clinical symptoms of spontaneous arthritis, such as redness and swelling of the proximal interphalangeal joints, toe stiffness, and ankylosis. As evident from microscopic evaluation, the arthritis was mainly characterized by formation of new cartilage and bone, originating at the entheses and leading to ankylosis. The incidence and severity of arthritis, both clinically and histologically, were significantly reduced in IFNgammaR-KO mice. In wild-type mice, neutralizing anti-IFNgamma antibody inhibited the occurrence of the disease for the duration of treatment. CONCLUSION: The results suggest that endogenous IFNgamma plays an important role in the initial stages of spontaneous arthritis, and that the inflammatory components in its pathogenesis are more prominent than has been believed. In view of the similarity between this disease and spondylarthropathies in humans, the data suggest that endogenous IFNgamma may also play a disease-promoting role in the human condition and thus may serve as a target for therapy.

Oncoviral bovine leukemia virus G4 and human T-cell leukemia virus type 1 p13(II) accessory proteins interact with farnesyl pyrophosphate synthetase.

G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic alpha-helix rich in arginine residues. Subtle mutation of this alpha-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13(II) was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13(II) and G4 accessory proteins opens new prospects for treatment of retrovirus-induced leukemia.

Immunomodulatory properties of interferon-gamma. An update.

During the early aspecific phase of host defense, production of interferon (IFN)-gamma by natural killer cells plays an important role in bringing about acute inflammation, mainly because of the activating effects of IFN-gamma on adhesive properties of endothelial cells and on mediator production by mononuclear phagocytes (MPCs). In the subsequent antigen-specific phase of the immune response, IFN-gamma acts as a regulator of antigen presentation and of proliferation and differentiation of lymphocyte populations. Immunosuppressive as well as immunostimulatory effects may result from these actions. High-level production of IFN-gamma during this phase of host defense is now classically seen as a hallmark of a T-helper 1 (TH1)-type reaction, characterized by activation of antimicrobial activity of macrophages and by inflammatory reactions with a DTH character. Development of TH1-type lymphocyte populations producing IFN-gamma is regulated by other cytokines including interleukin (IL)-12. In many systems IL-12 and IFN-gamma act in a similar fashion, and a current subject of debate is the question of whether all activities of IL-12 are mediated by IFN-gamma. Another question is whether IFN-gamma, by its ability to potentiate MPCs' ability to produce IL-12, plays a role in bringing about or stabilizing TH1 type responses. In two model systems of autoimmune disease, experimental autoimmune encephalomyelitis and collagen-induced arthritis, IL-12 and IFN-gamma were found to act independently.