BACKGROUND: We have recently shown that lipopolysaccharide (LPS), a major biologically active component of Gram-negative bacteria, mediate the activation of human keratinocytes by CD14 and Toll-like receptor (TLR 4). However, the mechanism of activation of keratinocytes by Gram-positive bacterial toxins remains unclear. OBJECTIVE: We investigated the mechanism of activation of human keratinocytes by lipoteichoic acid (LTA), a main stimulatory component of Gram-positive bacteria. METHODS: The effects of LTA on CD14, TLR2 and TLR4 mRNA expression were measured by quantitative RT-PCR in cultured human keratinocytes. To determine whether the effects of LTA on CD14, TLR2 and TLR4 expressions of the human keratinocytes were biologically functional, NF-kappaB nuclear translocation and IL-1alpha secretion were measured by immunofluorescence staining and ELISA, respectively. Furthermore, to determine whether these effects by LTA were specific for CD14, TLR2 and TLR4, some cells were pretreated with anti-CD14, anti-TLR2, or anti-TLR2 monoclonal antibodies prior to the addition of LTA. RESULTS: TLR4 mRNA expression on keratinocytes was augmented by exposure to LTA. LTA binding to keratinocytes resulted in NF-kappaB nuclear translocation and secretion of interleukin-1alpha. These responses by LTA were effectively abrogated by preincubating cells with anti-TLR4 monoclonal antibody, but not with anti-CD14 or anti- TLR2 monoclonal antibodies. CONCLUSION: These results indicate that, similar to LPS, LTA induces activation of human keratinocytes mainly through TLR4, however, in contrast to LPS signaling, LTA-induced keratinocyte activation is CD14-independent.
Antibodies, Monoclonal ; Bacterial Toxins ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Humans* ; Interleukin-1alpha ; Keratinocytes* ; NF-kappa B ; RNA, Messenger ; Toll-Like Receptor 4* ; Toll-Like Receptors*

Recent studies indicate that several Toll-like receptors (TLRs) are implicated in recognizing viral structures and instigating immune responses against viral infections. The aim of this study is to examine the expression of TLRs and proinflammatory cytokines in viral skin diseases such as verruca vulgaris (VV) and molluscum contagiosum (MC). Reverse transcription-polymerase chain reaction and immunostaining of skin samples were performed to determine the expression of specific antiviral and proinflammatory cytokines as well as 5 TLRs (TLR2, 3, 4, 7, and 9). In normal human skin, TLR2, 4, and 7 mRNA was constitutively expressed, whereas little TLR3 and 9 mRNA was detected. Compared to normal skin (NS), TLR3 and 9 mRNA was clearly expressed in VV and MC specimens. Likewise, immunohistochemistry indicated that keratinocytes in NS constitutively expressed TLR2, 4, and 7; however, TLR3 was rarely detected and TLR9 was only weakly expressed, whereas 5 TLRs were all strongly expressed on the epidermal keratinocytes of VV and MC lesions. In addition, the mRNA expression of IFN-beta and TNF-alpha was upregulated in the VV and MC samples. Immunohistochemistry indicated that IFN-beta and TNF-alpha were predominately localized in the granular layer in the VV lesions and adjacent to the MC bodies. Our results indicated that VV and MC skin lesions expressed TLR3 and 9 in addition to IFN-beta and TNF-alpha. These viral-induced proinflammatory cytokines may play a pivotal role in cutaneous innate immune responses.
Cytokines/metabolism ; *Gene Expression Regulation ; Humans ; Immunohistochemistry/methods ; Inflammation ; Interferon-beta/biosynthesis ; Keratinocytes/cytology ; Models, Biological ; Molluscum Contagiosum/*metabolism ; Toll-Like Receptor 3/biosynthesis ; Toll-Like Receptor 9/biosynthesis ; Toll-Like Receptors/*biosynthesis ; Tumor Necrosis Factor-alpha/biosynthesis ; Warts/*metabolism