Kent L W, Rahemtulla F, Michalek S M
Department of Microbiology, School of Dentistry, The University of Alabama at Birmingham, 35294-2170, USA.
J Periodontol. 1999 Mar;70(3):274-82. doi: 10.1902/jop.1999.70.3.274.
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.
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.
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.
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.
人牙龈成纤维细胞（HGF）在组织修复和破坏中起关键作用。这些细胞组成性地表达低水平的白细胞介素（IL）-6，在用脂多糖（LPS）或细胞因子IL-1和肿瘤坏死因子-α（TNF-α）刺激后表达水平升高。然而，关于患病组织来源的成纤维细胞产生IL-6的信息很少。本研究比较了健康和患病牙周组织来源的人牙龈成纤维细胞（HGF）组成性和诱导性IL-6的产生。我们还评估了IL-1是否与LPS协同作用诱导HGF产生IL-6，以及LPS是否通过CD14发挥作用。
将健康和患病组织来源的HGF以及包皮成纤维细胞培养至汇合，拍照并计数，通过酶联免疫吸附测定（ELISA）定量其组成性IL-6的产生。健康和患病的HGF也先用IL-1α预处理，然后与牙龈卟啉单胞菌或大肠杆菌LPS孵育。通过ELISA评估培养上清液中的IL-6蛋白，通过逆转录-聚合酶链反应（RT-PCR）评估细胞裂解物中的mRNA。为了确定LPS刺激是否通过LPS受体CD14介导，通过流式细胞术评估HGF上的表面受体，并评估总RNA中的CD14 mRNA。
患病的HGF在刺激前后产生的IL-6量高于健康的HGF。先用IL-1α预处理，然后用LPS刺激健康和患病的HGF细胞系，对IL-6的产生有累加效应。先用IL-1α预处理，然后再次与相同刺激物孵育产生的IL-6量高于单独用LPS孵育或IL-1α预处理后的培养物。未刺激的患病或健康组织的HGF以及仅用IL-1α孵育的HGF中存在相似量的IL-6 mRNA。在用IL-1α和LPS孵育后，患病的HGF产生的mRNA略多于健康的HGF。即使在用牙龈卟啉单胞菌或大肠杆菌LPS刺激后，健康或患病的HGF也不表达CD14。也未检测到CD14信息。
总之，这些结果表明牙龈成纤维细胞之间存在异质性，并且IL-1和牙龈卟啉单胞菌LPS对HGF产生IL-6有累加效应。此外，LPS对HGF的作用不是由CD14介导的。
