DISCUSSION

In this study, we have demonstrated that the TLR2 and TLR4 are expressed in human melanocytes both at the mRNA and protein levels. We also showed that LPS induced TLR2, TLR4 expression in melanocytes, suggesting that the effect of LPS on melanocytes is mediated by TLR4. CD14, which is known to associate with TLR4, could also be involved in the mediation of LPS effects in melanocytes. We demonstrated that melanocytes constitutively express CD14. CD14 has a role as adapter molecule to increase the signaling sensitivity for LPS by the TLR4. CD14 is a 55-kDa glycosyl phosphatidylinositol (GPI)-anchored glycoprotein identified on the surface of monocytes, macrophages, and polymorphonuclear leukocytes (PMNs) (Song et al, 2001). TLR4, together with CD14, recognizes LPS of gram-negative bacteria. MyD88 is required for the intracellular signaling of TLRs. The signaling pathway of the TLR family leads to the activation of NF-κB and MAPK (p38 and JNK) through the adaptor proteins MyD88 and IRAK (Zhang and Ghosh, 2002; Akira and Takeda, 2004). We have shown here for the first time that MyD88 mRNA is also expressed and regulated by LPS treatment in melanocytes.

It is now well established that TLR4 mediated signaling events upon LPS stimulation (Molteni et al, 2006; Poltorak et al, 1998). LPS, a major component of the outer membrane of gram-negative bacteria. LPS and other bacterial or viral by-products bind to a family of specific receptors, the TLRs, which regulate both innate and adaptive immunity. Exposure to bacterial compounds such as LPS increased the expression of TLR4 in several cells including human keratinocytes and monocytes (Song et al, 2002, Guha and Mackman, 2001).

Some studies showed that LPS increased TLR2 expression and LPS stimulation of TLR2 initiated an interleukin 1 receptor-like NF-κB signaling cascade. These demonstrated that TLR2 also participated in sensing LPS (Yang et al, 1998; Kirschning et al, 1998). We

increased after stimulation with LPS. LPS also increased the expression of MyD88. We have also demonstrated that incubation of cultured melanocytes with LPS increased melanin, suggesting gram-negative microbial infection induces melanogenesis. It was suggested that human melanocytes produce nitric oxide in response to LPS (Tsatmali et al, 2000). Tsatmali et al. further found that melanin synthesis was increased in cultured human melanocytes in response to increased levels of nitric oxide, suggesting LPS up regulated both melanogenesis and nitric oxide production for antimicrobial role. These findings including ours are in keeping with an antimicrobial role for melanin. The expression of TLR4 mRNA in melanoma cells was shown recently by Molteni et al. The authors showed that LPS significantly up-regulated the production of IL-8 of the cells and suggested direct link between LPS and tumor cell metastasis. Because IL-8 is an important chemotactic factor, we presume that the induction of IL-8 in melanoma cells by pathogens could lead to the recruitment of inflammatory cells to the site of infection (Molteni et al, 2006). Indeed, melanocyte TLR4 may recognize pathogens leads to the production of melanin, which might be responsible for the defense mechanism. Our findings stress the importance of the melanocytes as a component of the innate immune response.

Melanocytes are not simply pigment-producing cells, but produce substances with a range of biological functions, including structural strengthening by cross-linking proteins, antimicrobial defense, photon shielding, and chemoprotection (Burkhart CG and Burkhart CN, 2005). There is evidence that a major function of melanocytes, melanosomes, and melanin in human skin is to inhibit the proliferation of bacterial, fungal, and parastitic infections in the epidermis and dermis (Mackintosh, 2001).

We are planning to study about role of TLRs during inflammatory stimulation, such as TNF-α or UVB irradiation. Because the inflammatory signaling including TNF-α may activate TLR, TLRs may play a role in inflammation induced pigmentary disorders. We observed expression of TLR2 was significantly decreased in vitiligo compared with in

melanocytes from the cutaneous epidermis. Although the exact etiology of vitiligo has not yet been established, the abnormal immune responses frequently observed in vitiligo patients have led to the suggestion that, in some cases, the condition has an autoimmune component (Kemp et al, 2001). Because vitiligo skin usually has no melanocytes, this decreased TLR2 expression may be on keratinocytes. It was reported that vitiliginous keratinocytes as well as melanocytes are more vulnerable to assaults from extracellular factors. Vitiligo skin having decreased TLR may not respond well against inflammatory insults compared with normal skin. Further study needs to delineate the role for TLR2 in vitiligo skin. Also, the role of TLRs in inflammation-induced pigmentary disorders required further investigation. Release of inflammatory mediators and cytokines from inflammatory cells, as well as epidermal cells and melanocytes play a role in inflammatory induced pigmentation. A study performed by Halder et al concerning acne in darker skin showed a high degree of histologic inflammation in lesions that did not show significant clinical inflammation.

Our finding suggested that microbial infection stimulates melanin production in human melanocytes. This increased melanogenesis may play a role in antimicrobial immunity. In summary, here we demonstrated that TLR2 and 4 are expressed in human melanocytes and TLR4 are involved in LPS-induced melanogeneis. TLRs would be important therapeutic target on microbial or inflammatory induced cutaneous pigmentation.

In this study, we investigated that TLR2, TLR4, and CD14 were expressed in cultured human melanocytes at mRNA and protein levels. mRNA expression of MyD88 which is TLRs adapter molecule, was also demonstrated in melanocytes. And we studied LPS increased the expression of TLR2, TLR4 and MyD88. This LPS induced pigmentation, suggesting that TLR4 may play a role in microbial-induced melanogenesis. TLR2 expression was decreased in vitiligo in vivo, suggesting that TLRs may play role in the inflammation-induced pigmentary disorders.

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인간 인간

인간 인간 멜라닌 멜라닌 멜라닌 멜라닌 세포에서 세포에서 세포에서 세포에서 Toll Toll Toll- Toll - - -Like Receptor 2 Like Receptor 2 Like Receptor 2 Like Receptor 2와 와 와 와 4 4 4의 4 의 의 의 발현과

발현과 발현과

발현과 기능 기능 기능 기능

아주대학교 대학원의학과 안 주 희

(지도교수: 강 희 영)

Toll-like receptors (TLRs)는 선천성 면역 체계에서 중요한 역할을 하는 세포막에 존재하는 수용체이다. 피부에서는 미생물과 같은 외부 병원체에 의한 세균성 감염질환 외에 건선이나 여드름 등의 만성 염증성 질환의 병인에 TLR 이 중요한 역할을 할 것으로 생각되고 있으며 외부 병원체에 대한 1 차 방어벽인 각질형성세포에서 TLR 의 역할이 밝혀지고 있다. 최근 멜라닌 세포가 멜라닌 형성뿐 아니라 자가면역에 관여한다는 사실이 보고되었고, 멜라닌 색소의 자외선 방어의 역할 뿐만 아니라 외부 미생물에 저항하는 기능이 보고되었다.

따라서 본 연구에서는 멜라닌 세포에서 세균감염방어에 있어서의 TLR 의 역할을 알아보고자 하였다.

먼저 인간 멜라닌 세포에서 TLR2, TLR4 와 adapter molecule 인 CD14 과

MyD88 이 발현됨을 확인하였다. 멜라닌 세포에 병원체 산물인

Lipopolysaccharide (LPS)를 처리한 결과 TLR2, TLR4 의 발현이 증가하였다.

결과는 멜라닌 세포에 존재하는 TLR2 또는 TLR4 가 병원체 감염에 의한 멜라닌 합성증가에 관여할 것을 시사하였다. 또한 TLR2 의 발현이 백반증 환자의 병변 표피 조직에서 인접 정상 표피보다 현저히 감소되어 있었는데, 이 결과는 염증성 색소질환의 병인에 TLR 의 역할을 시사하였다.

핵심어 : Toll-like receptor 2, 4, Lipopolysaccharide, 멜라닌 세포, 멜라닌

색소. 색소침착