Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10^-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10^-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.     

Increased Larval Growth and Preference for Virus-Infected Leaves by the Mexican Bean Beetle, Epilachna varivestis Mulsant, a Plant Virus Vector

Phaseolus vulgaris L. cv. 'Black Valentine' is a systemic host for the plant viruses Southern bean mosaic virus (SBMV) and bean pod mottle virus (BPMV). The Mexican bean beetle, Epilachna varivestis Mulsant, is a vector of SBMV and BPMV. Our objective was to determine if the interaction of SBMV and BPMV with 'Black Valentine' bean plants would affect beetle behavior and growth. In adult feeding preference test assays, beetles preferred and ingested more of the virus-infected bean leaf tissue than the noninfected leaf tissue. Beetle larvae that fed on SBMV- or BPMV-infected plants weighed more than those that fed on healthy plants. Our experiments suggest that there might be a mutually beneficial relationship between the beetle and the viruses that it vectors. The virus benefits from being transmitted and the beetle benefits from better larval growth when feeding on virus-infected leaf tissue. This study further demonstrates the complexity of relationships between multiple organisms.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

The IS1167 Insertion Sequence Is a Phylogenetically Informative Marker Among Isolates of Serotype 6B Streptococcus pneumoniae

The phylogenetic utility of the IS1167 insertion sequence was examined with restriction fragment length polymorphism (RFLP) analyses of a sample of 50, predominantly invasive, capsular serotype 6B Streptococcus pneumoniae isolates previously characterized by multilocus enzyme electrophoresis (MLEE). The strains represented a genetically diverse assemblage of 34 distinct clonotypes composed of 26 restriction fragment types and 23 multilocus enzyme types. All isolates carried the IS1167 insertion sequence, with an average of 9.5 copies. The cross-classification of isolates based on RFLP and MLEE typing schemes was 81% concordant. Phylogenetic analyses demonstrated a significant (P < 0.0001) association between strains of a given RFLP lineage with those of a given MLEE lineage. A significant correlation (P < 0.00004) was also found between the proportion of restriction fragments shared by any given pair of isolates and their genetic distances estimated from the MLEE data. Parity between the two genetic markers implied that the sampled isolates were in linkage disequilibrium. The existence of nonrandom associations among genetic loci was confirmed by Monte Carlo analyses of the MLEE data. These studies, thus, demonstrated that invasive pneumococcal isolates of a single capsule type recovered on a regional scale can retain a largely clonal population structure over a period of 8 years. The ability to detect linkage disequilibrium and generate relatively congruent dendrograms based on distance and parsimony methods suggested that the restriction fragment data were robust to phylogenetic analysis. Received: 20 May 1997 / Accepted: 20 November 1997     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.