SummaryMacrophages are the major source of the chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), which play a major role in neutrophil migration to sites of inflammation. Although extracellular ATP from inflammatory tissues induces several immune responses in macrophages, it is unclear whether ATP-stimulated macrophages affect neutrophil migration. Therefore, the aim of the present study was to investigate the role of ATP-induced MIP-2 production by macrophages. When ATP was injected intraperitoneally into mice, the number of neutrophils within the peritoneal cavity markedly increased, along with the levels of MIP-2 and KC in the peritoneal lavage fluid. Consistent with this, ATP induced MIP-2 production, but not that of KC, by peritoneal exudate macrophages (PEMs) in vitro. This occurred via interactions with the P2X 7 receptor and P2Y 2 receptor. Furthermore, treatment of PEMs with ATP led to the production of reactive oxygen species. The ATP-induced MIP-2 production was inhibited by treatment with the antioxidant N-acetyl-L-cysteine. Also, MIP-2 production was inhibited by pre-incubating PEMs with inhibitors of extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase. The MIP-2 neutralization reduced the increase in neutrophil numbers observed in ATP-treated mice. Taken together, these results suggest that increased production of reactive oxygen species by ATP-stimulated macrophages activates the signalling pathways that promote MIP-2 production which, in turn, induces neutrophil migration.

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.Keywords: α-GalCer r IL-4 r Liver injury r NKT cell r Ras r RasGAP Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionRas proteins have been shown to play pivotal roles in proliferation, differentiation, and oncogenesis by functioning as a molecular switch for intracellular signaling pathways [1,2]. In order to act as a switch, Ras has two forms, an inactive GDP-bound form and an active GTP-bound form. GDP-bound Ras is dominant under the steady-state condition. Upon agonist binding to its receptor on the cell membrane, GDP-bound Ras is converted into active GTPbound Ras, which stimulates downstream signaling cascades, such as the Raf/Erk and phosphoinositide-3 kinase/Akt pathways [3]. Such switching of Ras forms is controlled by its intrinsic GTPase activity, and a balance between Ras guanine nucleotide exchange factors (RasGEFs) and Ras GTPase-activating proteins (RasGAPs) [4]. While RasGEFs act as a positive factor for Ras signaling by stimulating the conversion of Ras-GDP into Ras-GTP, RasGAPs act as negative factors by stimulating the rate of GTP hydrolysis from Ras-GTP into Ras-GDP [4]. Ras regulates the activation, differentiation, and proliferation of lymphoid cells. For instance, p21ras (K-Ras) regulates the function and growth of T cells and thymocytes during both immune activation and T-cell development [5][6][7]. In addition, TCR activation in T cells stimulates Ras, which initiates downstream signaling pathways leading to the production of cytokines such as IL-2, and subsequent IL-2-induced proliferation [8,9]. A deficiency of the RasGRP1 gene, a RasGEF, in mice inactivates the TCR activation-induced Ras signaling in thymocytes, and the mice have reduced single-positive (CD4 + CD8 − and CD4 − CD8 + ) thymocytes [10]....

Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin b1, enabling integrin b1 to initiate tumor invasion/metastasis. Integrin b1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin b1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin b1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

SummaryIn this study, normal adult mice carried B220 high conventional B cells in the spleen and liver, but carried both B220 high and B220 low in the bone marrow. However, at the neonatal stage, only B220 low unconventional B cells were found in all these organs. This pattern continued up to 2 weeks after birth, and at this stage autoantibodies were detected in the sera. This phenomenon was seen in all tested young mice (1-2 weeks), irrespective of their gender. Furthermore, at older stages (more than 20 weeks), B220 low cells reappeared in the spleen and liver, and these B220 low cells became dominant in the bone marrow. Autoantibodies also reappeared in the sera of these older mice. Cell-sorting experiments revealed that B220 low cells were able to produce autoantibodies upon lipopolysaccharide stimuli in vitro. These results suggest that B220 low cells appear at both neonatal and older stages as physiological responses and eventually produce autoantibodies.