Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1β, IFN-γ, or TNF-α significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-κB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-α production and NF-κB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.

Toll-like receptor 2 (TLR2) is involved in the innate immunity by recognizing various bacterial components. We have previously reported that TLR2 gene expression is rapidly induced by LPS or inflammatory cytokines in macrophages, and by TCR engagement or IL-2/IL-15 stimulation in T cells. Here, to investigate the mechanisms governing TLR2 transcription, we cloned the 5′ upstream region of the mouse TLR2 (mTLR2) gene and mapped its transcriptional start site. The 5′ upstream region of the mTLR2 gene contains two NF-κB, two CCAAT/enhancer binding protein, one cAMP response element-binding protein, and one STAT consensus sequences. In mouse macrophage cell lines, deletion of both NF-κB sites caused the complete loss of mTLR2 promoter responsiveness to TNF-α. NF-κB sites were also important but not absolutely necessary for LPS-mediated mTLR2 promoter activation. In T cell lines, mTLR2 responsiveness to IL-15 was abrogated by the 3′ NF-κB mutation, whereas 5′ NF-κB showed no functional significance. The STAT binding site also seemed to contribute, as the deletion of this sequence significantly reduced the IL-15-mediated mTLR2 promoter activation. EMSAs confirmed nuclear protein binding to both NF-κB sites in macrophages following LPS and TNF-α stimulation and to the 3′ NF-κB site in T cells after IL-15 treatment. Thus, NF-κB activation is important but differently involved in the regulation of mTLR2 gene expression in macrophages and T cells following LPS or cytokine stimulation.

Mitogen-activated protein kinases (MAPKs) are activated by phosphorylation of threonine and tyrosine residues within a signature sequence of T-X-Y by dual-specificity MAPK kinases (MKKs). MKKs are in turn phosphorylated and activated by a family of serine/threonine MKK kinases (47). Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) (also called stress-activated protein kinase), and p38 (also called RK or CSBP) are distinct classes of MAPKs playing important roles in various cellular events. JNKs are activated by diverse stimuli, including DNA damage, heat shock, bacterial components, inflammatory cytokines, and Fas (31). Activated JNKs play an essential role in the activation of transcriptional factors, such as c-Jun (21), ATF-2 (16), Elk-1 (57), and ets-2 (50). In macrophages, activated JNKs mediate the expression of inducible nitric oxide synthase (6), cyclooxygenase-2 (56), chemokines such as RANTES (23), and cytokines such as tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), and IL-6 (39), all of which not only potently activate host defense mechanisms but also often lead to excessive inflammatory responses in microbial infection.MAPK activation is a reversible process, and an emerging family of dual-specificity protein phosphatases (DSPs) have been shown to inactivate MAPKs through dephosphorylation of both threonine and tyrosine residues that are essential for the enzymatic activity (4). DSPs share two common features: a catalytic domain with significant amino acid sequence homology to a vaccinia virus DSP, VH-1, and an N-terminal region homologous to the catalytic domain of the cdc25 phosphatase (rhodanase homology domain). Among DSP family members, some show highly selective substrate specificity while others efficiently inactivate all three classes of MAPKs. Interestingly, gene expression of many DSPs is significantly induced following stimulation with growth factors, cytokines, or cell stresses, and this induction of MAPK phosphatases (MKPs) may function as a negative feedback mechanism of MAPK activity.In this study, in order to elucidate the regulatory mechanisms of MAPK pathways in macrophages, we screened a cDNA library from a mouse macrophage cell line with a cDNA probe of MKP-1, a known MKP. We isolated a partial cDNA containing the extended active-site sequence motif, (V/L) X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), conserved in all DSPs (27,35,41). By rescreening the library and extending the cDNA by the method of 5Ј rapid amplification of cDNA ends (5Ј-RACE), we have isolated a full-length cDNA, which we named MKP-M (for MKP isolated from macrophages). It includes an N-terminal

Toll-like receptors (TLRs) are a family of mammalian proteins homologous to Drosophila Toll. Human TLR2 was shown to mediate the responsiveness to lipopolysaccharide (LPS). On the other hand, gene mutations of mouse TLR4 (mTLR4) in LPS-hyporesponsive strains have suggested that mTLR4 is essential for LPS-signaling in mice, but the role of mTLR2 has not been explored. This report describes molecular cloning of the mTLR2 cDNA. Overexpression of mTLR2 and mouse CD14 conferred LPS-inducibility of c-Jun N-terminal kinase phosphorylation and nuclear factor-κB activation to COS7 cells, suggesting that mTLR2 is a signaling receptor for LPS. BothmTLR2 and mTLR4 genes were expressed in T cells. Treatment with anti-CD3ɛ, PMA plus ionomycin, or interleukin-2 (IL-2)/IL-15 increased mTLR2 but not mTLR4 messenger RNA (mRNA) in some T cell lines. Specific inhibitors of mitogen-activated extracellular signal-regulated kinase and fusion protein 38 (p38) kinase inhibited mTLR2 mRNA up-regulation by PMA plus ionomycin. This suggests that extracellular signal-regulated kinase and p38 kinase pathways were involved. Additionally, LPS treatment of EL-4 cell line decreasedIL-4 gene expression. Our results indicate that both mTLR2 and mTLR4 are involved in LPS signaling, but their expressions are regulated differently in T cells, and that LPS may directly affect T-cell functions by binding to TLRs.