Depletion of cellular cholesterol enhances macrophage MAPK activation by chitin microparticles but not by heat-killed Mycobacterium bovis BCG.
When macrophages phagocytose chitin (N-acetyl-d-glucosamine polymer) microparticles, mitogen-activated protein kinases (MAPK) are immediately activated, followed by the release of Th1 cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl-beta-cytodextrin (MBCD) and stimulated with chitin. These results were compared with the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guèrin (BCG) and an oligodeoxynucleotide (ODN) of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 min after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose-dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of cyclooxygenase-2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline-treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN induced TNF-alpha production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiological regulation of chitin microparticle-induced cellular activation.
Nishiyama
Akihito
A
Department of Biomedical Sciences, Florida Atlantic Univ., 777 Glades Rd., Boca Raton, FL 33431-0991, USA.
Shinohara
Tsutomu
T
Pantuso
Traci
T
Tsuji
Shoutaro
S
Yamashita
Makiko
M
Shinohara
Shizuka
S
Myrvik
Quentin N
QN
Henriksen
Ruth Ann
RA
Shibata
Yoshimi
Y
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002686:Chitin / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006358:Hot Temperature; D016753:Interleukin-10 / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009163:Mycobacterium bovis / Q000276:immunology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D010316:Particle Size; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology; D010588:Phagosomes / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000276:Adjuvants, Immunologic; D000954:Antigens, Surface; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D014409:Tumor Necrosis Factor-alpha; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D016753:Interleukin-10; D002686:Chitin; D002784:Cholesterol; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone