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Intra-articularly localized bacterial DNA containing CpG motifs has been found to bring on arthritis. DNA from Escherichia coli and Staphylococcus aureus and oligonucleotides containing CpG was injected directly into knee joints of mice of various strains.

We demonstrate that humans have a phenotypically and functionally distinct subset of B lymphocytes that express the interleukin (IL)-2 receptor (IL-2R) α-chain, cluster of differentiation (CD) 25 . We found that one-third of the circulating CD20+ B cells expressed CD25 and, using fluorescence-activated cell sorter (FACS) analysis, that these cells were significantly larger and more granulated than B cells not expressing CD25 . The simultaneous expression of the other two subunits (CD122 and CD132) and the proliferative responses of cells expressing CD25 to IL-2 suggested that, in addition to CD25, functional IL-2 receptors were expressed on this cell population. CD25 expression on B cells was selectively up-regulated by Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands but not by a TLR3 ligand or Epstein–Barr virus (EBV) stimulation. Blockade of the nuclear factor (NF)-κB pathway completely abolished CD25 up-regulation by these B cells. Interestingly, CD25+ B cells expressed significantly higher levels of surface immunoglobulins but lacked the ability to secrete immunoglobulin (Ig), as compared with CD25− B cells. Furthermore, CD25+ B cells performed significantly better as antigen-presenting cells in allogeneic mixed lymphocyte reactions (MLR), which may be a result of their expression of high levels of the costimulatory molecules CD27 and CD80. Finally, blocking of CD25 on B cells led to an almost total abrogation of MLR. Our results indicate that CD25+ B cells have distinct phenotypic and functional properties, including the ability to contribute to antigen presentation, which is linked to their expression of CD25. Finally, the differential regulation of CD25 expression via selective TLR ligands suggests a role for CD25+ B cells in bridging innate and acquired immune responses. [ABSTRACT FROM AUTHOR]

The soybean-derived isoflavone genistein has been shown to exert beneficial effects on many disorders, including cancer and cardiovascular diseases. The effects of genistein on mammalian cells are mediated by its abilities to inhibit topoisomerase II and protein tyrosine kinase. In order to examine the potential antibacterial activities of genistein, we incubated the bacteria with various concentrations of this compound for different periods of time and assessed the viable counts. Exposure to genistein exhibited an inhibitory effect on all staphylococcal strains tested, including methicillin-resistant strains. Furthermore, the growth of Streptococcus pasteurianus, Bacillus cereus, and Helicobacter pylori was clearly inhibited by genistein, whereas Escherichia coli growth was not suppressed. Daidzein, which is structurally similar to genistein, but deficient in topoisomerase II inhibitory activity, also inhibited the growth of Staphylococcus aureus, albeit with lower potency than genistein. Our results indicate that genistein exerts potent antibacterial properties in vitro, which are possibly mediated by the stabilization of the covalent topoisomerase II-DNA cleavage complex. [Copyright &y& Elsevier]

Interactions between staphylococci and the joint tissues of the host lead typically to rapidly progressing and highly destructive processes. Staphylococci possess a vast arsenal of components and products that contribute to the pathogenesis of joint infection. Occasionally these compounds have overlapping activities and act either in concert or alone. Host responsiveness to staphylococcal infection displays an even more complex pattern. Most of the cells and molecules that participate in the innate immune system protect the host against bacteria. However, the staphylococci have developed systems that counteract endogenous protective mechanisms. Interestingly, certain cells and molecules of the acquired immune system potentiate the severity of infection by triggering exaggerated responses to the staphylococcal danger signals. This review deals with the intricate host–bacterium interactions that occur during experimental septic arthritis, and outlines potential preventive and treatment modalities. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) generated by the cellular NADPH-oxidase are crucial for phagocytic killing of ingested microbes and have been implicated as signaling molecules in various processes. For example, ROS are thought to be involved in activation of the transcription factor NF-kappaB, central for mediating production of proinflammatory cytokines in response to inflammatory stimuli. Several studies have demonstrated that inhibitors of the NADPH-oxidase interfere with NF-kappaB activation and production of proinflammatory cytokines. Curiously, patients with chronic granulomatous disease (CGD), an immunodeficiency characterized by an inability to produce ROS, are not only predisposed to severe infections, but also frequently develop various inflammatory complications indicative of exaggerated inflammatory responses. Here, we show that human CGD leukocytes display a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to stimulation with Toll-like receptor agonists. The hyperinflammatory phenotype was also evident in mononuclear cells from CGD mice (gp91(phox) -/-), but not in control cells in the presence of NADPH-oxidase inhibitor diphenyleneiodonium, probably reflecting NADPH-oxidase-independent effects of the inhibitor. Furthermore, we show that the major steps involved in NF-kappaB activation were intact in human CGD cells. These data indicate that ROS were nonessential for activation of NF-kappaB and their production may even attenuate inflammation.

CpG DNA functions via the toll-like receptor-9 (TLR-9) receptor, inducing B cell proliferation and promoting immunoglobulin production. B cell responses to CpG DNA-containing immune complexes could be important in chronic autoimmunity and immune responses to bacterial components. Therefore, we investigated the potential synergy of CpG DNA-stimulation with FcgammaR clustering (CFR) on splenic B cell activity. CFR-induced splenocyte proliferation was significantly increased compared to treatment with CpG DNA alone. While the levels of interleukin-10 (IL-10) were increased in CpG DNA-treated splenocyte cultures, particularly following FcgammaRII/III-clustering, CFR treatment reduced IL-6 levels. B-cell maturation in culture was enhanced by CFR. Indeed, the frequency of IgG expressing cells after stimulation with CpG DNA was increased and was even higher after CFR stimulation. Furthermore, the frequency of plasma cell precursors was markedly increased by stimulation with CFR. Late splenic B cell subsets, transitional type 2 (T2) and mature (M) B cells, responded strongly to CpG DNA with proliferation and the response was enhanced by FcgammaR-clustering. Immature transitional type 1 (T1) B cells showed distinctly lower proliferative response to CpG DNA and very small effects of FcgammaR-clustering, despite similar expression of Fcgamma-receptors by all B cell subsets. In conclusion, these data show synergistic impact of CpG DNA and simultaneous FcgammaR-clustering on B cell proliferation and differentiation.

The immunostimulatory effects of cytosine-phosphate-guanosine (CpG)-containing oligodeoxynucleotides (ODNs) have been extensively documented. In this paper, we describe the inhibitory effects of ODNs that contain natural phosphodiester backbones (O-ODNs) on the immunostimulation caused by CpG-containing phosphorothioated ODNs (CpG-S). CpG-S stimulation of mouse splenocyte proliferation was reduced by the addition of O-ODNs that contained or lacked the CpG-motif (CpG-containing phosphodiester oligodeoxynucleotide, CpG-O or GpC-O). The total number of cultured splenocytes was up-regulated by CpG-S, whereas repetitive addition of O-ODNs to the cell cultures inhibited this effect. The frequency of T2-like B cells was found to be increased by CpG-S. The culture supernatants of CpG-S-treated splenocytes contained elevated levels of IL-10 and IL-6. However, IL-10 and IL-6 production was down-regulated significantly by the combination of CpG-S and either CpG-O or GpC-O. The O-ODN mediated inhibition of proliferation was less pronounced in IL-10-/- mice. Thus, the O-ODNs, irrespective of CpG content, exerted inhibitory activities on the proliferation of B cells. These anti-proliferative effects appear to be mediated both by the down-regulation of IL-10 production and increased apoptosis.

Bacterial DNA and oligodeoxynucleotides containing cytosine-phosphate-guanosine sequences and thereby mimicking prokaryotic DNA, have recently been shown to exert potent immunostimulatory properties. As skin normally harbors bacteria, and as the bacterial content and the levels of bacterial degradation products increase during skin infection, we analyzed the potential inflammatogenic role of bacterial DNA and oligodeoxynucleotides in a mouse model of cutaneous inflammation. Bacterial DNA from Staphylococcus aureus was injected intradermally into mice and its inflammatogenic properties were compared with synthetic phosphodiester and phosphorothioate cytosine-phosphate-guanosine- or GpC-containing oligodeoxynucleotides. A peak inflammatory infiltrate in the skin was seen already 2 d after injection with either bacterial DNA or the phosphodiester cytosine-phosphate-guanosine-oligodeoxynucleotides. In contrast, nuclease-resistant phosphorothioate cytosine-phosphate-guanosine-induced dermatitis peaked 7 d after intradermal injection. The inflammatory infiltrates consisted mainly of macrophages, and depletion of this cell population resulted in a significant (p=0.0001) decrease in the severity of inflammation, which suggests that macrophages play a central part in inflammatory responses in the skin following exposure to cytosine-phosphate-guanosine-containing oligodeoxynucleotides. A significant decrease in local inflammatory infiltrate was also seen in mice with deficiencies in neutrophil or lymphocyte populations, which indicates that these cell populations may also be involved in mediating inflammatory signals after the injection of immunostimulatory DNA sequences. In summary, our results suggest that bacterial DNA is an important virulence determinant and inflammatory stimulus during skin infections.

Neutrophils play a key role at inflammatory sites where, in addition to destroying infecting microorganisms, they may also have deleterious effects on host tissues. Both activities involve activation of the NADPH-oxidase that produces bactericidal and tissue-destructive reactive oxygen species (ROS). We activated the murine NADPH-oxidase using different types of neutrophil activators and characterized the oxidative responses with respect to magnitude, localization, and kinetics. We show that agonist-induced activation of murine neutrophils results exclusively in extracellular release of ROS and no intracellular production could be detected. We also show that the formylated peptide, formyl-Met-Leu-Phe (fMLF), is a much less potent activator of the murine NADPH-oxidase than of the human analogue. Nevertheless, fMLF responses can be primed by pretreating the murine neutrophils with either cytochalasin B or bacterial lipopolysaccharide. Finally, we show that a synthetic hexapeptide, WKYMVM, is a more potent stimulus than fMLF for murine neutrophils and that these two agonists probably act via nonidentical high-affinity receptors.