Our results demonstrate that (i) micrococci and sPGN induce transcription of the gene for IL-8 in cells expressing TLR2 but not in cells expressing TLR1; (ii) micrococci and sPGN induce activation of NF-κB in cells expressing TLR2, and this activation requires the signal transduction molecules MyD88, IRAK1, NIK, IKKα, and IKKβ and, to a lesser extent, IRAK2 and TRAF6; and (iii) micrococcus- and sPGN-induced transcription of the gene for IL-8 requires MyD88, IRAK1, IRAK2, NIK, IKKα, IKKβ, and NF-κB and, to a lesser extent, TRAF6 and TRAF2. These data are the first to identify signal transduction molecules required for IL-8 induction in cells stimulated with bacteria and bacterial cell wall products.
These data also confirm our previous results showing that gram-positive bacteria and bacterial cell wall component PGN activate cells through TLR2, but not TLR1 or TLR4 (8
), and that this TLR2-mediated cell activation is enhanced by CD14 (27
). PGN binds CD14 (5
), and this binding may be the first step in PGN-induced cell activation. Although we have previously shown indirect activation of endothelial and epithelial cells by PGN-induced TNF-α and IL-1 secreted from monocytes (15
), in our current experiments, activation of 293 cells by PGN is a direct activation mediated through TLR2 and CD14 and not an indirect effect because 293 cells do not produce TNF-α and IL-1 (D. Gupta and R. Dziarski, unpublished data).
Of the six different TLRs that have been identified (published), only two have a known function, TLR2 and TLR4. TLR2 is a cell-activating receptor for gram-positive bacteria (8
), mycobacteria (2
), spirochetes (13
), and mycoplasmas (18
) and for the cell wall components PGN (8
), lipoteichoic acid (8
), lipopeptides, lipoproteins (2
), and ara-lipoarabinomannan (19
). Moreover, an accessory molecule, MD-2 (28
), enables TLR2 to respond to various nonactivating LPS structures (8
). TLR2 is recruited to macrophage phagosomes containing yeast, and dominant negative TLR2 abolishes TNF-α production in response to yeast and to gram-positive bacteria but not in response to gram-negative bacteria (31
). Thus, TLR2 appears to be a true pattern recognition receptor that recognizes a large variety of microbes and microbial products and mediates cell activation and host inflammatory responses. However, a structural feature common to these putative ligands that is needed for recognition by TLR2 has not been identified.
TLR4 is implicated in host immune responses to gram-negative bacteria and to their LPS cell wall component (22
) but not to gram-positive bacteria or PGN (8
). The Lps
gene in C3H/HeJ and C57BL/10ScCr mice, which is responsible for their defective responsiveness to LPS, was identified as Tlr4
). Moreover, TLR4-mediated cell activation by LPS requires coexpression of the accessory molecule MD-2 (8
Our data are the first to demonstrate that the signal transduction molecules MyD88, IRAK, NIK, and IKK are required for TLR2-mediated NF-κB activation in cells stimulated with micrococci and bacterial PGN (Fig. ). These data demonstrate that gram-positive bacteria and their cell wall components induce TLR2-mediated signal transduction pathways that are similar to the TLR-dependent LPS-induced signal transduction pathways and involve IRAK, MyD88, TRAF6, IKKα, IKKβ, NIK, and NF-κB (16
) and confirm the role of MyD88 as an essential component in this pathway (30
). This pathway is also similar to the IL-1-induced signal transduction pathway (4
), which is consistent with the homology of the cytoplasmic domains of TLR2 and IL-1R.
Our data also demonstrate TLR2-mediated transcriptional induction of IL-8 by micrococci and PGN and the requirement for NF-κB in induction of IL-8 by gram-positive bacterial stimulants. Although NF-κB is required for the induction of IL-8 transcription, it may not be sufficient, and other signal transduction pathways may also be required or may modulate IL-8 transcription. This study was done on a human embryonic kidney cell line, and this cell line, as many other cells in the body, such as endothelial cells, epithelial cells, and fibroblasts, can produce IL-8, in addition to the well-known production of IL-8 by bacterially activated monocytes (32
). However, the signal transduction pathways and transcription factors needed for activation of transcription of the gene for IL-8 are likely to be the same in all of these cells because the IL-8 promoter is the same in all of the cells. Therefore, our results should be applicable to all cells that are able to produce IL-8, and their responsiveness to bacterial products would depend on the expression of appropriate receptors, such as CD14 and TLR2.
The signal transduction molecules MyD88, IRAK, NIK, and IKK are required for TLR2-dependent induction of IL-8 expression in cells stimulated with micrococci and PGN. Both dominant negative TRAF6 and TRAF2 partially inhibit IL-8 induction in response to these bacterial products. Therefore, these results suggest that alternative pathways involving TRAF2 or TRAF6 may play an equal role in IL-8 induction, but not in NF-κB induction, in cells stimulated with bacteria and bacterial cell wall components (Fig. ). However, there may also be cross talk between TNF-R- and TLR2–IL-1R-induced pathways because of the partial inhibition of TNF-induced IL-8 activation by dominant negative MyD88 and IRAK.
Proposed signal transduction pathway activated by micrococci and sPGN.
In summary, our results demonstrate that gram-positive bacteria and PGN induce IL-8 transcription through the TLR2→MyD88→IRAK→TRAF6→NIK→IKK→NF-κB (i.e., IL-1R-like) signal transduction pathway, although additional signal transduction molecules may participate in this pathway, and other signal transduction pathways may also be involved in induction of IL-8 transcription.