Analysis of functions of cutaneous mast cells using murine fetal skin-derived cultured mast cells

Toll样受体(TLR)通过与病毒和细菌产物结合，介导先天免疫反应。最近的研究表明，TLR3与病毒双链RNA结合，TLR9与CpG基序结合，CpG基序是细菌和病毒特有的共同寡核苷酸序列。本研究旨在确定皮肤肥大细胞是否表达TLR3和TLR9。首先，我们鉴定了TLR3和TLR9在小鼠胚胎皮肤来源的肥大细胞(FSMC)中的表达，这种细胞与皮肤肥大细胞非常相似。TLR3和TLR9在FSMC中的表达水平是代表未成熟(或粘膜)肥大细胞的骨髓源性肥大细胞(BMMC)的数倍。此外，与BMMC相比，FSMC对Poly(I：C)(TLR3的合成配体)和CpG寡脱氧核苷酸(但不对随机化的寡脱氧核苷酸)的反应更明显地使IkBA磷酸化，并分泌包括TNF-a和IL-6在内的细胞因子和包括MIP-1a和MIP-2在内的趋化因子。这些数据保证了功能性TLR3和TLR9在FSMC上的表达，并提示它们的表达可能取决于肥大细胞的分化。有趣的是，与通过FceRI刺激肥大细胞不同，通过TLR3和TLR9刺激不会导致脱颗粒，也不会诱导IL-13的产生。因此，皮肤相关的肥大细胞表达TLR3和TLR9，识别病原体特异性核苷酸，并释放促炎细胞因子。IL-13的缺失可能对以下Th1反应有利。我们的结果表明，皮肤肥大细胞在先天性免疫中的新作用不同于它们在IgE和Th2介导的过敏反应中的作用。

Toll-like receptors (TLR) mediate innate immune responses by their binding to viral and bacterial products. Recent studies revealed that TLR3 binds to viral double-stranded RNA and that TLR9 binds to CpG motifs, consensus oligodeoxynucleotide sequences specific to bacteria and viruses. This study aimed to determine whether mast cells in skin express TLR3 and TLR9. First we identified mRNA expression of TLR3 and TLR9 in murine fetal skin-derived cultured mast cells (FSMC) that closely resemble cutaneous mast cells. The expression levels of both TLR3 and TLR9 in FSMC were several-fold greater than those in bone marrow-derived cultured mast cells (BMMC) that represent immature (or mucosal) mast cells. Furthermore, FSMC phosphorylated IkBa and secreted cytokines including TNF-a and IL-6 and chemokines including MIP-1a and MIP-2 in response to poly (I:C) (synthetic ligand of TLR3) and CpG oligodeoxynucleotides (but not to randomized oligodeoxynucleotides) to much greater extents than BMMC did. These data assure expression of functional TLR3 and TLR9 on FSMC, and also suggest that their expression may be differentially regulated depending on differentiation of mast cells. Interestingly, unlike stimulation of mast cells through FceRI, stimulation through TLR3 and TLR9 did not cause degranulation and did not induce production of IL-13. Thus skin-associated mast cells express TLR3 and TLR9, recognize pathogen-specific nucleotides, and release proinflammatory cytokines. The absence of IL-13 may be advantageous for the following Th1 responses. Our results suggest novel roles of cutaneous mast cells in innate immunity distinct from their roles in IgE- and Th2-mediated allergic responses.