Characterization of the interaction between the chlamydial adhesin OmcB and the human host cell.

In a previous study, we reported that the OmcB protein from Chlamydia pneumoniae mediates adhesion of the infectious elementary body to human HEp-2 cells by interacting with heparin/heparan sulfate-like glycosaminoglycans (GAGs) via basic amino acids located in the first of a pair of XBBXBX heparin-binding motifs (K. Moelleken and J. H. Hegemann, Mol. Microbiol. 67:403-419, 2008). In the present study, we show that the basic amino acid at position 57 (arginine) in the first XBBXBX motif, the basic amino acid at position 61 (arginine) in the second motif, and another amino acid (lysine 69) C terminal to it play key roles in the interaction. In addition, we show that discrimination between heparin-dependent and -independent adhesion by C. trachomatis OmcBs is entirely dependent on three variable amino acids in the so-called variable domain C terminal to the conserved XBBXBX motif. Here, the predicted conformational change in the secondary structure induced by the proline at position 66 seems to be crucial for heparin recognition. Finally, we performed neutralization experiments using different anti-heparan sulfate antibodies to gain insight into the nature of the GAGs recognized by OmcB. The results suggest that C. trachomatis serovar L2 OmcB interacts with 6-O-sulfated domains of heparan sulfate, while C. pneumoniae OmcB apparently interacts with domains of heparan sulfate harboring a diverse subset of O-sulfations.

Immunoregulatory natural killer cells suppress autoimmunity by down-regulating antigen-specific CD8+ T cells in mice.

Natural killer (NK) cells belong to the innate immune system. Besides their role in antitumor immunity, NK cells also regulate the activity of other cells of the immune system, including dendritic cells, macrophages, and T cells, and may, therefore, be involved in autoimmune processes. The aim of the present study was to clarify the role of NK cells within this context. Using two mouse models for type 1 diabetes mellitus, a new subset of NK cells with regulatory function was identified. These cells were generated from conventional NK cells by incubation with IL-18 and are characterized by the expression of the surface markers CD117 (also known as c-Kit, stem cell factor receptor) and programmed death (PD)-ligand 1. In vitro analyses demonstrated a direct lysis activity of IL-18-stimulated NK cells against activated insulin-specific CD8(+) T cells in a PD-1/PD-ligand 1-dependent manner. Flow cytometry analyses revealed a large increase of splenic and lymphatic NK1.1(+)/c-Kit(+) NK cells in nonobese diabetic mice at 8 wk of age, the time point of acceleration of adaptive cytotoxic immunity. Adoptive transfer of unstimulated and IL-18-stimulated NK cells into streptozotocin-treated mice led to a delayed diabetes development and partial disease prevention in the group treated with IL-18-stimulated NK cells. Consistent with these data, mild diabetes was associated with increased numbers of NK1.1(+)/c-Kit(+) NK cells within the islets. Our results demonstrate a direct link between innate and adaptive immunity in autoimmunity with newly identified immunoregulatory NK cells displaying a potential role as immunosuppressors.

Recruitment of blood-derived inflammatory cells mediated via tumor necrosis factor-α receptor 1b exacerbates choroidal neovascularization.

Purpose. Tumor necrosis factor (TNF)-α contributes to inflammation-associated angiogenesis, and TNF-α receptor 1b is selectively expressed on immuno-competent and endothelial cells. This study investigated the role of TNF-α receptor 1b in the recruitment of circulating inflammatory cells and the development of choroidal neovascularization (CNV). Methods. Lethally irradiated Tnfrsf1b(-/-) mice and their wild-type (WT) controls were transplanted with whole adult bone marrow (BM) cells, competent for both TNF-α receptors 1a and 1b (gfp(+) labeled), as well as with BM cells deficient for TNF-α receptor 1b. One month after transplantation CNV was induced by laser damage of Bruch's membrane. Pathologic angiogenesis was estimated qualitatively and quantitatively by histology on choroidal flatmounts and paraffin cross sections. Macrophage invasion was investigated by immunochemistry. Results. One month after transplantation the reconstitution rate measured by FACS analysis was >80% in gfp(+)-chimeric mice. Two weeks after laser injury reduced gfp(+)-cell invasion to the laser scars and decreased pathologic angiogenesis were observed in Tnfrsf1b(-/-) versus WT recipients. Approximately 70% of the invaded gfp(+) cells were labeled with macrophage marker F4/80. Transplantation of TNF-α receptor 1b-deficient BM cells in WT recipients reduced the CNV lesion compared with WT and Tnfrsf1b(-/-) recipients that received TNF-α receptor-competent BM cells. Transplantation of receptor 1b-deficient cells to Tnfrsf1b(-/-) recipients further reduced the degree of CNV formation. Conclusions. Signals through TNF-α receptor 1b expressed on BM -derived inflammatory cells mediate an increased inflammatory cell invasion and enhanced angiogenic response after laser-induced rupture of Bruch's membrane.

Conditions of retinal glial and inflammatory cell activation after irradiation in a GFP-chimeric mouse model.

PURPOSE: Microglia cells have been associated with immunologic defense and repair. The course of retinal disease after lethal irradiation for bone marrow depletion and substitution was evaluated with respect to macrophage and microglial involvement. METHODS: Lethal irradiation in C57BL/6 mice was conducted with a low-voltage radiation unit. The animals were randomized to shielded or unshielded radiation and subsequently received transplants of GFP+ bone marrow cells (beta-actin promoter). The GFP transformation rate was analyzed by flow cytometry. GFP+ cells in the retina were examined for co-localization with macrophage and dendritic cell markers at various time points between 1 and 7 months after irradiation. Clodronate liposomes were used to investigate the fate of migrated and residential microglia cells. Pathologic angiogenesis was investigated in laser-induced choroidal neovascularization (CNV) after unshielded and shielded irradiation. RESULTS: Flow cytometry revealed average transformation rates of 78.2% in unshielded and 64.1% in shielded group. Four weeks after transplantation, perfused flat mounts were virtually free of extravasal GFP+ cells in both groups, whereas 4 months after irradiation, cluster cell infiltrations, preferentially in the peripheral retina, became apparent exclusively in the unshielded group. Cell morphology ranged from oval, to a few extensions, to dendritiform with long-branched extensions. Clodronate treatment resulted in a reduction of GFP+ cells in the retinal tissue when applied 3 months after unshielded irradiation. Although GFP+ cells accumulated in the choroidal scar after laser treatment, in both the shielded and unshielded groups, GFP+ cells in the overlying retina were restricted to the unshielded group. CONCLUSIONS: Approximately 3 months after lethal full-body irradiation including the eye, bone marrow-derived leukocytes exhibit a wound-healing reaction, and unlike physiological turnover, infiltrate the retina and form microglial cells.

Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells.

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.