Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.
OBJECTIVE
CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.


METHODS
Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits.


RESULTS
IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide.


CONCLUSIONS
These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.
Department of Periodontology, Showa University Dental School, Tokyo, Japan.
S
S
Mochizuki
M
M
Kobayashi
T
T
Suzuki
A
A
Oikawa
T
T
Koseki
T
T
Nishihara
K
K
Hasegawa
D048868:Adaptor Proteins, Signal Transducing; D016976:Aggregatibacter actinomycetemcomitans / Q000502:physiology; D000704:Analysis of Variance; D000943:Antigens, Differentiation / Q000096:biosynthesis*; D015153:Blotting, Western; D002478:Cells, Cultured; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism / Q000382:microbiology; D005786:Gene Expression Regulation / Q000187:drug effects; D005881:Gingiva / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D006801:Humans; D051550:I-kappa B Kinase; D007371:Interferon-gamma / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D011971:Receptors, Immunologic / Q000096:biosynthesis*; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D015850:Interleukin-6; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D008562:Membrane Glycoproteins; D053594:Myeloid Differentiation Factor 88; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D007371:Interferon-gamma; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D020928:Mitogen-Activated Protein Kinases