| Bacterial keratitis is a kind of suppurative keratitis caused by bacterial infection and can often lead to blindness. In many countries and areas,pseudomonas aeruginosa is the main pathogen which belongs to gram-negative opportunistic bacteria group.This strain can generate many pathogenic factors,among which, lipopolysaccharide(LPS)is the most important one.LPS plays a critical role in inflammation by producing TNF-α, IL-1, IL-6, IL-8 and IL-12 from corneal epithelial cells.The mammal's cells need some accessory proteins to response to LPS. It is currently known that LPS receptor complex is composed of 4 parts at least, which are LPS binding protein(LBP), CD 14, myeloid differentiation-2(MD-2)and toll-like receptor 4(TLR4). MD-2 is one of the most important progresses in this field until now and it acts as a necessary bridge in the signaling pathway associated with LPS and TLR4.But now it is still unclear whether the human corneal epithelial cells can response to LPS, if yes, are expression of MD-2 and location of TLR4 involved in the signaling pathway?Objects:To study the LPS responding mechanisms of human corneal epithelial cells in vitro.Methods:Spontaneously derived human corneal epithelial cell linel(SDHCEC1), donated by professor Wang Zhichong from Zhongshan Ophthalmic Center, were subcultured and specific protein CK3/CK12 were identified by immunohistochemistry. TLR4, CD 14, IL-8 and MD-2 expression in SDHCEC1 were analyzed by real-time PCR after incubating with different concentrations of LPS at different times. IL-8 in the supernatant was tested by ELISA. The activation of NF-κB was analyzed by western blot. The expression of TLR4 and CD 14 were tested by flow cytometry. MD-2 recombinant cloning and expression plasmids were constructed and identified. 293T cells were transfected with MD-2 recombinant expression plasmids.Western blot was used to test the MD-2 expression. SDHCEC1 was co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4, CD 14, IL-8, and NF-κB in SDHCEC1 were tested as the previous. Results:SDHCECl showed typical characters of corneal epithelial cells with the positive staining of CK3/CK12. SDHCEC1 cultured with different concentrations of LPS at different times only came up with weak expression of TLR4 and CD 14 (P＜0.05). IL-8 and MD-2 expression were negative. No IL-8 secretion was seen in the supernatant. Activation of NF-κB did not show up. TLR4 and CD 14 were not expressed at the cell surface with only weak expression within cytoplasm. MD-2 cloning plasmids and recombinant plasmids were successfully constructed and expressed in 293T cells. TLR4, CD 14 and IL-8 mRNA were up-regulated in the SDHCEC1 cells co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4 and CD 14 were also positive at the cell surface and within cell cytoplasm. IL-8 concentration was also up-regulated with the activation of NF-κB at the same time.Conclusions:MD-2 expression is absent in SDHCEC1, which may lead to the deficiency of TLR4 at the cell surface, inactivation of NF-κB and no secretion of IL-8 in LPS/TLR4/MD-2 signaling pathway. According to this, we speculate that under normal conditions, in order to maintain corneal immunosilence, corneal epithelial cells may use this way to inhibit the innate immune response induced by LPS and avoid corneal epithelial cells'destruction caused by invasive inflammatory cells. But with endogenous or exogenous MD-2, TLR4 may express at the cell surface with the activation of NF-κB and secretion of IL-8, which activates the whole signaling pathway and plays an important role in sequent inflammation.
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