Lipopolysaccharide induces expression of genes encoding pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human cervical smooth-muscle cells.
OBJECTIVE
Vaginal and amniotic infection with gram-negative bacteria is associated with preterm birth. We previously reported that human cervical smooth-muscle cells (CSMC) respond to pro-inflammatory cytokines by expressing enzymes that degrade the extracellular matrix. Our objective was to characterize the effects of lipopolysaccharide (LPS) from Escherichia coli (E coli), Bacteroides fragilis, (B frag)and Fusobacterium nucleatum (F nuc)on the expression of pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human CSMC.


METHODS
Human CSMC were exposed to LPS and the expression of mRNAs encoding pro-inflammatory cytokines and cathepsin S, and selected matrix metalloproteinases (MMPs) was analyzed by Northern blotting. The effect of cytokine-neutralizing antibodies on LPS-induced cathepsin S mRNA expression also was determined.


RESULTS
E coli LPS increased expression of cathepsin S 12.5-fold after 12 hours; MMP-1 and MMP-3 mRNAs also were increased 2.9- and 3.5-fold, respectively. Tumor necrosis factor (TNF)-alpha, interleukin (IL-1)alpha, and IL-1beta mRNAs were markedly up-regulated after 3 hours of LPS treatment. B frag and F nuc LPS also induced TNF-alpha and cathepsin S mRNAs. E coli LPS caused a sevenfold increase in TNF-alpha secretion after 5 to 8 hours. Antihuman TNF-alpha monoclonal antibody, but not a monoclonal antibody to the low-density lipoprotein receptor, reduced the LPS-induced increase in cathepsin S mRNA by 27%, whereas neutralizing antibodies against IL-1alpha and IL-1beta did not suppress the response. Human CSMC were shown to express the toll-like receptor (TLR-2) and TLR-4 genes, which mediate the action of LPS. TLR-2 mRNA was up-regulated by TNF-alpha.


CONCLUSIONS
CSMC respond to LPS with increased expression of pro-inflammatory cytokines and cathepsin S. Increases in cathepsin S mRNA result only in part from the rapid induction of TNF-alpha gene expression. TNF-alpha may also augment the CSMC response to LPS by increasing expression of the LPS signaling receptor, TLR-2, which probably directly mediates LPS action. These observations provide a mechanism by which gram-negative bacteria can precipitate cervical changes associated with preterm birth.
Watari
M
M
The Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA.
Watari
H
H
Nachamkin
I
I
Strauss
J F
JF
D002403:Cathepsins / Q000235:genetics*; D002478:Cells, Cultured; D002584:Cervix Uteri / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics*; D003609:Dactinomycin / Q000494:pharmacology; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D009130:Muscle, Smooth / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D002403:Cathepsins; C028217:cathepsin S; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1