Báo cáo khoa học: Human lactoferrin activates NF-jB through the Toll-like receptor 4 pathway while

Human lactoferrin activates NF-jB through the Toll-like

receptor 4 pathway while it interferes with the

lipopolysaccharide-stimulated TLR4 signaling

Ken Ando1

, Keiichi Hasegawa1

, Ken-ichi Shindo1

, Tomoyasu Furusawa1

, Tomofumi Fujino1

, Kiyomi

Kikugawa1

, Hiroyasu Nakano2

, Osamu Takeuchi3

, Shizuo Akira3

, Taishin Akiyama4

, Jin Gohda4

,

Jun-ichiro Inoue4 and Makio Hayakawa1

1 School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Japan

2 Department of Immunology, Juntendo University School of Medicine, Japan

3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan

4 Division of Cellular and Molecular Biology, Department of Cancer Biology, Institute of Medical Science, The University of Tokyo, Japan

Keywords

human lactoferrin; innate immunity;

lipopolysaccharide; nuclear factor-jB

(NF-jB); Toll-like receptor 4 (TLR4)

Correspondence

Makio Hayakawa, School of Pharmacy,

Tokyo University of Pharmacy and Life

Science, 1432-1 Horinouchi, Hachioji, Tokyo

192-0392, Japan

Fax: +81-42-676-4508

Tel: +81-42-676-4513

E-mail: [email protected]

(Received 26 August 2009, revised 15

February 2010, accepted 18 February 2010)

doi:10.1111/j.1742-4658.2010.07620.x

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal

the signal transduction pathway responsible for human LF (hLF)-triggered

nuclear factor-jB (NF-jB) activation. Endotoxin-depleted hLF induces

NF-jB activation at physiologically relevant concentrations in the human

monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts

(MEFs). In MEFs, in which both tumor necrosis factor receptor-associated

factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-jB activation

at a level comparable to that seen in wild-type MEFs, whereas TRAF6-

deficient MEFs show significantly impaired NF-jB activation in response

to hLF. TRAF6 is known to be indispensable in leading to NF-jB activa￾tion in myeloid differentiating factor 88 (MyD88)-dependent signaling

pathways, while the role of TRAF6 in the MyD88-independent signaling

pathway has not been clarified extensively. When we examined the

hLF-dependent NF-jB activation in MyD88-deficient MEFs, delayed, but

remarkable, NF-jB activation occurred as a result of the treatment of cells

with hLF, indicating that both MyD88-dependent and MyD88-independent

pathways are involved. Indeed, hLF fails to activate NF-jB in MEFs lack￾ing Toll-like receptor 4 (TLR4), a unique TLR group member that triggers

both MyD88-depependent and MyD88-independent signalings. Impor￾tantly, the carbohydrate chains from hLF are shown to be responsible for

TLR4 activation. Furthermore, we show that lipopolysaccharide-induced

cytokine and chemokine production is attenuated by intact hLF but not by

the carbohydrate chains from hLF. Thus, we present a novel model con￾cerning the biological function of hLF: hLF induces moderate activation of

TLR4-mediated innate immunity through its carbohydrate chains; however,

hLF suppresses endotoxemia by interfering with lipopolysaccharide-depen￾dent TLR4 activation, probably through its polypeptide moiety.

Abbreviations

ActE, actinase E; bLF, bovine lactoferrin; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;

hLF, human lactoferrin; IKK, IjB kinase; IL, interleukin; IP10, interferon-c-inducible protein-10; IRF, interferon regulatory factor; JNK,

c-Jun N-terminal kinase; LBP, LPS-binding protein; LF, lactoferrin; LPS, lipopolysaccharide; LRP, low-density lipoprotein receptor-related

protein; MD-2, myeloid differentiation-2; MEF, mouse embryonic fibroblast; MyD88, myeloid differentiating factor 88; NF-jB, nuclear

factor-jB; PMB, polymyxin B; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; TRIF,

Toll ⁄ interleukin-1 receptor-domain-containing adaptor inducing interferon-b.

FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2051