Interleukin (IL)-1 and Porphyromonas gingivalis lipopolysaccharide stimulation of IL-6 production by fibroblasts derived from healthy or periodontally diseased human gingival tissue.
BACKGROUND
Human gingival fibroblasts (HGF) play a key role in tissue repair and destruction. These cells express low levels of interleukin (IL)-6 constitutively and increased levels after stimulation with lipopolysaccharide (LPS) or the cytokines IL-1 and tumor necrosis factor-alpha (TNF-alpha). However, little information is available on IL-6 production by fibroblasts derived from diseased tissue. The present study compared constitutive and induced IL-6 production by human gingival fibroblasts (HGF) derived from healthy and diseased periodontal tissue. We also evaluated whether IL-1 acted in a synergistic manner with LPS in inducing IL-6 production by HGF and whether LPS acted via CD14.


METHODS
HGF derived from healthy and diseased tissue and foreskin fibroblasts were grown to confluency, photographed, and counted, and their constitutive IL-6 production was quantitated by ELISA. Healthy and diseased HGF were also pretreated with IL-1alpha, followed by incubation with Porphyromonas gingivalis or Escherichia coli LPS. Culture supernatants were assessed for IL-6 protein by ELISA and cell lysates for mRNA by reverse transcription-polymerase chain reaction (RT-PCR). In order to determine if LPS stimulation was mediated through the LPS receptor, CD14, surface receptors on HGF were assessed by flow cytometry and the total RNA CD14 mRNA was assessed.


RESULTS
Higher quantities of IL-6 were produced by the diseased HGF before and after stimulation than by the healthy HGF. Pretreatment with IL-1alpha followed by LPS stimulation of the healthy and diseased HGF cell lines resulted in an additive effect on IL-6 production. Pretreatment with IL-1alpha followed by a second incubation with the same stimulant produced higher amounts of IL-6 than cultures incubated with LPS alone or following IL-1alpha pretreatment. Similar amounts of IL-6 mRNA were present in unstimulated HGF from either diseased or healthy tissue and in those incubated with IL-1alpha only. After incubation with IL-1alpha and LPS, diseased HGF produced slightly more mRNA than healthy HGF. CD14 was not expressed by healthy or diseased HGF even after stimulation with either P. gingivalis or E. coli LPS. CD14 message was also undetectable.


CONCLUSIONS
Taken together, these results indicate heterogeneity among gingival fibroblasts and an additive effect of IL-1 and P. gingivalis LPS on IL-6 production by HGF. Furthermore, the LPS effect on HGF was not mediated by CD14.
Kent
L W
LW
Department of Microbiology, School of Dentistry, The University of Alabama at Birmingham, 35294-2170, USA.
Rahemtulla
F
F
Michalek
S M
SM
D002452:Cell Count; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D005881:Gingiva / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D018950:Lipopolysaccharide Receptors / Q000032:analysis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D010510:Periodontal Diseases / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha