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Neutrophils have been reported to acquire surface expression of MHC class II and co-stimulatory molecules as well as T-cell stimulatory activities when cultured with selected cytokines. However, cellular identity of those unusual neutrophils showing antigen presenting cell (APC)-like features still remains elusive. Here we show that both immature and mature neutrophils purified from mouse bone marrow differentiate into a previously unrecognized "hybrid" population showing dual properties of both neutrophils and dendritic cells (DCs) when cultured with granulocyte macrophage-colony-stimulating factor but not with other tested growth factors. The resulting hybrid cells express markers of both neutrophils (Ly6G, CXCR2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86). They also exhibit several properties typically reserved for DCs, including dendritic morphology, probing motion, podosome formation, production of interleukin-12 and other cytokines, and presentation of various forms of foreign protein antigens to naïve CD4 T cells. Importantly, they retain intrinsic abilities of neutrophils to capture exogenous material, extrude neutrophil extracellular traps, and kill bacteria via cathelicidin production. Not only do our results reinforce the notion that neutrophils can acquire APC-like properties, they also unveil a unique differentiation pathway of neutrophils into neutrophil-DC hybrids that can participate in both innate and adaptive immune responses.

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Bullous pemphigoid is an autoimmune subepidermal blistering disease associated with autoantibodies against BP180 and BP230. We report herein a rare case of bullous pemphigoid with newly formed annular erythematous lesions when bullous skin lesions were in remission. Various immunological studies revealed immunoglobulin (Ig)A antibodies against desmoglein 1, envoplakin, periplakin and BP230 in addition to IgG antibodies against BP180 and BP230. These clinical and immunological changes in a patient are a rare event, suggesting an epitope-spreading phenomenon.

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Short-term DC cultures generated with GM-CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c+ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL-33 was found to augment DC development time- and dose-dependently. Although the resulting CD11c+ cells generated in the presence of IL-33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL-12 p70, displayed PD-L1 and PD-L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL-33-induced expansion of CD11c+ cells was completely blocked by anti-GM-CSF mAb, and GM-CSF mRNA and protein expression in BM culture was markedly elevated by added IL-33, indicating that IL-33 promotes in vitro DC generation indirectly by a GM-CSF-dependent manner. With regard to the cellular source, IL-33-dependent GM-CSF production was observed exclusively within the CD45+/FcepsilonRI+ BM population. Not only do our results reinforce the notion that GM-CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.

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The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.

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Hailey-Hailey disease (HHD) is a blistering skin disease caused by malfunction of the Ca2+-dependent ATPase, ATP2C1. In this study, key regulatory regions necessary for the expression of the gene encoding human ATP2C1 were investigated. The transient reporter assay demonstrated that region +21/+57 was necessary for activation of the ATP2C1 promoter, and the electrophoretic mobility shift assay demonstrated that the region was recognized by the transcription factors, Sp1 and YY1. In accordance with this result, when Sp1 or YY1 was overexpressed in keratinocytes, an obvious increase in ATP2C1 promoter activity was observed, which was in contrast with the case where a mutant promoter lacking the binding sites for Sp1 and YY1 was used as the reporter. Ca2+-stimulation signal increased nuclear Sp1 proteins and ATP2C1 mRNA levels in normal keratinocytes. In contrast, both these increases were suppressed in keratinocytes from HHD patients. These results indicate that Sp1 and YY1 transactivate the human ATP2C1 promoter via cis-enhancing elements and that incomplete upregulation of ATP2C1 transcription contributes to the keratinocyte-specific pathogenesis of HHD. This is a report describing the regulation of the expression of ATP2C1.

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