[Expressions of inflammatory cytokines in mouse peritoneal macrophages induced by MRP8/MRP14 in vitro].

OBJECTIVE To investigate MRP8/MRP14-induced expression of inflammatory cytokines in mouse peritoneal macrophages and explore the mechanism. METHODS Tumor necrosis factor-α (TNF-α), interferon-γ inducible protein 10 (IP-10), interleukin-2 (IL-2), IL-6, IL-5 and interferon-γ (IFN-γ) proteins in the culture supernatants of mouse peritoneal macrophages treated with recombinant MRP8/MRP14 were quantified using Luminex xMAP system. TNF-α, IP-10 and IL-6 levels were detected in the culture supernatants of the peritoneal macrophages after treatment with different domains of MRP14. Western blotting was used to detect the phosphorylation of p38 MAPK, JNK and ERK in the cells after MRP8/MRP14 treatment. The effects of p38 MAPK, JNK, ERK inhibitors, TLR4 or RAGE receptor antagonists on MRP8/MRP14-induced expressions of TNF-α, IP-10 and IL-6 were tested. RESULTS MRP8/MRP14 significantly increased TNF-α, IP-10 and IL-6 levels in mouse peritoneal macrophages by 98.2, 378.6 and 6.3 folds (P<0.01), respectively, but did not obviously affect IL-2, IL-5 and IFN-α levels. MRP14 protein and its calcium binding motifs such as EF hand-1, EF hand-2, EF hand-1+2, but not CT terminal domain, all induced TNF-α, IP-10 and IL-6 expressions (P<0.01). Phosphorylation of p38 MAPK, JNK and ERK were detected by Western blotting in the cells at 1 h after MRP8/MRP14 stimulation and sustained to 2 h. Compared with MRP8/MRP14, SB203580 (p38 MAPK inhibitor) significantly inhibited TNF-α, IP-10 and IL-6 expression (P<0.05), and SP600125 (JNK inhibitor) inhibited the expression of TNF-α and IP-10 (P<0.05) but not IL-6, PD98059 and U0126 (ERK and MEK1/2 inhibitor) reduced IL-6 expression (P<0.05). TNF-α, IP-10 and IL-6 levels were inhibited by TAK242 (P<0.05); IL-6 level in the cells was also partially inhibited by RAGE neutralizing antibody (P<0.05). CONCLUSION MRP8/MRP14 can induce the expression of TNF-α, IP-10 and IL-6 in mouse peritoneal macrophages. MRP14 protein, which contain calcium binding motifs, has the biological activity of inducing cytokine expression. TNF-α and IP-10 expressions are related with TLR4 and its downstream p38 MAPKs and JNK; IL-6 is regulated by both TLR4 and RAGE and their downstream p38 MAPKs and ERK.

[1]  T. Nagata,et al.  Calprotectin Induces IL‐6 and MCP‐1 Production via Toll‐Like Receptor 4 Signaling in Human Gingival Fibroblasts , 2017, Journal of cellular physiology.

[2]  A. Lourenço,et al.  A network perspective on antimicrobial peptide combination therapies: the potential of colistin, polymyxin B and nisin. , 2017, International journal of antimicrobial agents.

[3]  Peng Sun,et al.  Proinflammatory effects of S100A8/A9 via TLR4 and RAGE signaling pathways in BV-2 microglial cells , 2017, International journal of molecular medicine.

[4]  D. Viemann,et al.  S100-alarmin-induced innate immune programming protects newborn infants from sepsis , 2017, Nature Immunology.

[5]  D. Viemann,et al.  In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock , 2017, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  T. Vogl,et al.  Phagocyte-specific S100A8/A9 is upregulated in primary Sjögren's syndrome and triggers the secretion of pro-inflammatory cytokines in vitro. , 2017, Clinical and experimental rheumatology.

[7]  M. Aikawa,et al.  Macrophages in Vascular Inflammation: Origins and Functions , 2016, Current Atherosclerosis Reports.

[8]  Elizabeth M. Nolan,et al.  Calcium-induced tetramerization and zinc chelation shield human calprotectin from degradation by host and bacterial extracellular proteases , 2015, Chemical science.

[9]  M. Sperandio,et al.  Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion , 2015, Nature Communications.

[10]  Y. Vodovotz,et al.  Injury‐induced MRP8/MRP14 stimulates IP‐10/CXCL10 in monocytes/macrophages , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  A. Schmaier,et al.  Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis. , 2014, The Journal of clinical investigation.

[12]  M. Fieren.  The Local Inflammatory Responses to Infection of the Peritoneal Cavity in Humans: Their Regulation by Cytokines, Macrophages, and Other Leukocytes , 2012, Mediators of inflammation.

[13]  C. Geczy,et al.  S100 Calgranulins in inflammatory arthritis , 2010, Immunology and cell biology.

[14]  D. Foell,et al.  The endogenous Toll–like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer , 2009, Journal of leukocyte biology.

[15]  L. Juliano,et al.  Analgesic properties of S100A9 C‐terminal domain: a mechanism dependent on calcium channel inhibition , 2009, Fundamental & clinical pharmacology.

[16]  W. Nacken,et al.  Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock , 2007, Nature Medicine.

[17]  D. Foell,et al.  S100 proteins expressed in phagocytes: a novel group of damage‐associated molecular pattern molecules , 2007, Journal of leukocyte biology.

[18]  T. Vogl,et al.  Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity. , 2006, Journal of molecular biology.

[19]  D. Mayer,et al.  S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells. , 2006, Experimental cell research.

[20]  W. Nacken,et al.  The arachidonic acid‐binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac‐2 , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[21]  I. Thorey,et al.  MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. , 2004, Blood.

[22]  C. Kerkhoff,et al.  The Two Calcium-binding Proteins, S100A8 and S100A9, Are Involved in the Metabolism of Arachidonic acid in Human Neutrophils* , 1999, The Journal of Biological Chemistry.