Masako Unoshima

C-reactive protein induces high-mobility group box-1 protein release through activation of p38MAPK in macrophage RAW264.7 cells

BACKGROUND C-reactive protein (CRP) is widely used as a sensitive biomarker for inflammation. Increasing evidence suggests that CRP plays a role in inflammation. High-mobility group box-1 (HMGB1), a primarily nuclear protein, is passively released into the extracellular milieu by necrotic or damaged cells and is actively secreted by monocytes/macrophages. Extracellular HMGB1 as a potent inflammatory mediator has stimulated immense curiosity in the field of inflammation research. However, the molecular dialogue implicated between CRP and HMGB1 in delayed inflammatory processes remains to be explored. METHODS AND RESULTS The levels of HMGB1 in culture supernatants were determined by Western blot analysis and enzyme-linked immunosorbent assay in macrophage RAW264.7 cells. Purified CRP induced the release of HMGB1 in a dose- and time-dependent fashion. Immunofluorescence analysis revealed nuclear translocation of HMGB1 in response to CRP. The binding of CRP to the Fc gamma receptor in RAW264.7 cells was confirmed by fluorescence-activated cell sorter analysis. Pretreatment of cells with IgG-Fc fragment, but not IgG-Fab fragment, efficiently blocked this binding. CRP triggered the activation of p38MAPK and ERK1/2, but not Jun N-terminal kinase. Moreover, both p38MAPK inhibitor SB203580 and small interfering RNA significantly suppressed the release of HMGB1, but not the MEK1/2 inhibitor U-0126. CONCLUSION We demonstrated for the first time that CRP, a prominent risk marker for inflammation including atherosclerosis, could induce the active release of HMGB1 by RAW264.7 cells through Fc gamma receptor/p38MAPK signaling pathways, thus implying that CRP plays a crucial role in the induction, amplification, and prolongation of inflammatory processes, including atherosclerotic lesions.

Antiviral Effects of Geranylgeranylacetone: Enhancement of MxA Expression and Phosphorylation of PKR during Influenza Virus Infection

ABSTRACT A cyclic polyisoprenoid compound, geranylgeranylacetone (GGA), has been used as antiulcer drug. GGA is also a potent inducer of heat shock proteins (HSPs). HSPs are considered to induce an antiviral effect; however, the detailed mechanism is unknown. To determine whether GGA might show antiviral activity and what the mechanism is, the effect of GGA against influenza virus (strain PR8) infection in vivo and in vitro was investigated. The results demonstrated that GGA treatment strongly suppressed the deleterious consequences of PR8 replication and was accompanied by an increase in HSP70 gene expression in mice. Results from in vitro analyses demonstrated that GGA significantly inhibited the synthesis of PR8-associated proteins and prominently enhanced expression of human myxovirus resistance 1 (MxA) followed by increased HSP70 transcription. Moreover, GGA augmented the expression of an interferon-inducible double-strand RNA-activated protein kinase (PKR) gene and promoted PKR autophosphorylation and concomitantly α subunit of eukaryotic initiation factor 2 phosphorylation during PR8 infection. It is proposed that GGA-induced HSP70 has potent antiviral activity by enhancement of antiviral factors and can clinically achieve protection from influenza virus infection.

Neutrophilia and granulocyte colony-stimulating factor levels after cardiopulmonary bypass.

Purpose: The precise mechanism of neutrophilia after cardiac surgery is unknown. Granulocyte colony stimulating factor (G-CSF) can increase the number of leukocytes. The purpose of this study was to evaluate the relationship between serum G-CSF levels and peripheral blood leukocyte counts after cardiac surgery.Methods: We prospectively studied 10 patients undergoing cardiac surgery (coronary artery bypass grafting) using cardiopulmonary bypass (CPB). Plasma G-CSF levels and neutrophil count were measured before induction of anaesthesia, at the end of surgery, and on the first postoperative day. These changes were compared with those in patients undergoing non-cardiac major surgery (control group).Results: At the end of surgery, G-CSF levels increased (P<0.01) in both groups, but were higher in the control than in the cardiac group (3,250±690vs 194±29.5 pg·ml−1, respectively, mean±SEM,P<0.01). On the first postoperative day, G-CSF levels were still high in both groups, and were still higher in the control (710 ±179vs 122±19.9, respectively,P<0.01). However, neutrophilia was greater in the cardiac group than in the control. G-CSF response correlated positively with neutrophilia in the control group (r=0.656,P<0.05) but not in the cardiac group.Conclusions: Our results indicate that changes in leukocyte count following cardiac surgery are unique to patients undergoing CPB. G-CSF plays an important role as the mediator of neutrophilia after non-cardiac surgery, but not after cardiac surgery with CPB.RésuméObjectif: Le mécanisme précis de la polynucléose neutrophile qui survient après une intervention en cardiochirurgie est inconnu. Le facteur stimulant la formation de colonies de granulocytes (FSC-G) peut augmenter le nombre de leucocytes. L’objectif de la présente étude était d’évaluer la relation entre les niveaux sériques de FSC-G et la numération leucocytaire du sang périphérique à la suite d’une intervention cardiaque.Méthode: Nous avons mené une étude prospective auprès de 10 patients qui ont subi un pontage aorto-coronarien sous circulation extracorporelle (CEC). Les niveaux plasmatiques de FSC-G et la numération des neutrophiles ont été mesurés avant l’induction de l’anesthésie, à la fin de l’opération et au premier jour postopératoire. Les changements ont été comparés avec ceux qu’on a observés chez des patients qui ont subi une intervention importante, non cardiaque (groupe témoin).Résultats: À la fin de l’opération, les niveaux de FSC-G ont augmenté (P<0,01) dans les deux groupes, mais ont été plus élevés dans le groupe témoin que dans le groupe de cardiochirurgie (3,250±690vs 194±29,5 pg·ml−1, respectivement, moyenne±écart type,P<0,01). Au premier jour postopératoire, les niveaux de FSC-G étaient encore élevés dans les deux groupes et toujours plus hauts chez les témoins (710±179vs 122±19,9, respectivement,P<0,01). Toutefois, la polynucléose neutrophile était plus marquée dans le groupe de cardiochirurgie que dans le groupe témoin. La réponse du FSC-G était en corrélation avec la polynucléose neutrophile dans le groupe témoin (r=0,656,P<0,05) mais non dans l’autre groupe.Conclusion: Nos résultats indiquent que les changements de numération des leucocytes, à la suite d’une intervention cardiaque, sont uniques aux patients qui subissent une CEC. Le FSC-G joue un rôle important comme médiateur de la polynucléose neutrophile après une opération non cardiaque, mais pas après une intervention cardiaque sous CEC.