Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs.

In the phagocytosis field, ROS production by the phagocyte NOX has been associated with pathogen killing for the last 50 years. Since the discovery of nonphagocyte NOX, numerous other roles for ROS production have been identified. Oxidative stress and ROS-mediated signaling have received much attention in recent years. Much lower concentrations of ROS may be required for signaling compared with microbial killing. Based on the discoveries in nonphagocytic cells, it became logical to look for ROS functions distinct from pathogen killing, even in phagocytes. ROS are now linked to various forms of cell death, to chemotaxis, and to numerous modifications of cellular processes, including the NOX itself. ROS functions are clearly concentration-dependent over a wide range of concentrations. How much is required for which function? Which species are required for how much time? Is ROS signaling only a side effect of bactericidal ROS production? One major obstacle to answer these questions is the difficulty of reliable quantitative ROS detection. Signal transduction often takes place on a subcellular scale over periods of seconds or minutes, so the detection methods need to provide appropriate time and space resolution. We present examples of local ROS production, decreased degradation, signaling events, and potentially ROS-sensitive functions. We attempt to illustrate the current limitations for quantitative spatiotemporal ROS detection and point out directions for ongoing development. Probes for localized ROS detection and for combined detection of ROS, together with protein localization or other cellular parameters, are constantly improved.

Interleukin-33 (IL-33) is an IL-1-like ligand for the ST2 receptor that stimulates the production of Th2-associated cytokines. Recently, we showed that IL-33 is a chromatin-associated factor in the nucleus of endothelial cells in vivo. Here, we report the identification of a short IL-33 chromatin-binding peptide that shares striking similarities with a motif found in Kaposi sarcoma herpesvirus LANA (latency-associated nuclear antigen), which is responsible for the attachment of viral genomes to mitotic chromosomes. Similar to LANA, the IL-33 peptide docks into the acidic pocket formed by the H2A-H2B dimer at the nucleosomal surface and regulates chromatin compaction by promoting nucleosome-nucleosome interactions. Taken together, our data provide important new insights into the nuclear roles of IL-33, and show a unique example of molecular mimicry of a chromatinassociated cytokine by a DNA tumour virus. In addition, the data provide, to the best of our knowledge, the first demonstration of the existence of non-histone cellular factors that bind to the acidic pocket of the nucleosome.