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.

Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

Antibiotic cyclic AMP signaling by "primed" leukocytes confers anti-inflammatory cytoprotection.

The mechanism underlying anti-inflammatory effects of macrolide antibiotics remains uncertain. In this study, we first show the evidences concerning the possible link between leukocytic cyclic adenosine monophosphate (cAMP) signaling and the mechanism of anti-inflammatory, cytoprotective actions of macrolides. The clinical range of macrolides (i.e., erythromycin, roxithromycin, and clarithromycin) preferentially inhibited nuclear factor-kappaB activation mediated by reactive oxygen intermediates, inducing cAMP-dependent signaling [i.e., cAMP and cAMP-responsive element-binding protein (CREB)] by "primed" but not "resting" leukocytes. In this context, cAMP/CREB inhibition with adenosine 3':5'-cyclic monophosphothioate, rp-isomer (rp-cAMPs) and CREB decoy oligonucleotides reduced the anti-inflammatory actions of macrolides. These results thus indicate that macrolide-induced cAMP/CREB signaling, selectively by primed leukocytes, plays a major role in the mechanism of anti-inflammatory actions of macrolides.