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Cell surface 4-1BBL mediates sequential signaling pathways 'downstream' of TLR and is required for sustained TNF production in macrophages

Nature Immunology volume 8, pages 601–609 (2007)Cite this article

Abstract

The stimulation of Toll-like receptors (TLRs) on macrophages triggers production of the cytokine tumor necrosis factor (TNF). TNF production occurs within 1 h of TLR stimulation and is sustained for 1 d. Here we document a function for the TNF family member 4-1BB ligand (4-1BBL) in sustaining TLR-induced TNF production. TLR signaling induced 4-1BBL, and 4-1BBL interacted with TLRs on the macrophage surface. The influence of 4-1BBL on TNF production was independent of its receptor (4-1BB) and did not require the adaptors MyD88 or TRIF. It did not influence TLR4-induced activation of transcription factor NF-κB (an early response) but was required for TLR4-induced activation of transcription factors CREB and C/EBP (a late event). Transient TLR4-MyD88 complexes appeared during the first hour after lipopolysaccharide stimulation, and TLR4–4-1BBL interactions were detected between 2 h and 8 h after lipopolysaccharide stimulation. Our results indicate that two different TLR4 complexes sequentially form and selectively control early and late TNF production.

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Figure 1: The ligand 4-1BBL is a TLR-interacting protein.
Figure 2: Sustained TNF production in LPS-stimulated macrophages requires 4-1BBL.
Figure 3: Induction and cell surface localization of 4-1BBL is required for sustained TNF production in LPS-treated macrophages.
Figure 4: Expression and crosslinking of 4-1BBL triggers TNF production.
Figure 5: In vivo effect of inhibition of 4-1BBL.
Figure 6: Signaling by 4-1BBL is sequential to MyD88 signaling in LPS-stimulated macrophages.

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Acknowledgements

We thank J. Peschon (Immunex), B. Beutler (The Scripps Research Institute) and S. Akira (Osaka University) for mice deficient in 4-1BBL, TRIF, MyD88, TLR4 and TLR2; and T. Wen (University of Toronto) for generating the mouse 4-1BBL adenovirus construct. Supported by the National Institutes of Health (GM67101, GM37696, AI41637 and AI54696 to J.H.; and EY13325, KRF-2005-084-E00001 and KRF-2005-201-E00008 to B.S.K.), the Canadian Institutes of Health (MT13220 to T.H.W., and MOP68841 to S.O.K.), the Science Research Center Fund from the Korea Science and Engineering Foundation (B.S.K.) and a Grant-in-Aid for the 21st Century Center of Excellence Program Topological Science and Technology from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (S.S.).

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Authors and Affiliations

  1. Department of Immunology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Young Jun Kang, Shigeki Shimada, Motoyuki Otsuka, Alim Seit-Nebi & Jiahuai Han

  2. Department of Microbiology & Immunology, University of Western Ontario, N6A 5C1, Ontario, Canada

    Sung Ouk Kim

  3. The Immunomodulation Research Center, University of Ulsan, Nam-ku, 680-749, Ulsan, Korea

    Byoung S Kwon

  4. Department of Immunology, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Tania H Watts

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Contributions

Y.J.K. designed the research, did most of the experiments, analyzed data and wrote the manuscript; S.O.K. and M.O. did experiments with macrophages from wild-type and 4-1BBL-deficient mice; S.S. did immunohistochemistry; A.S.-N. designed and generated '4-1BBL-knockdown' cell lines; T.H.W. and B.S.K. coordinated and organized the mouse experiments and contributed to the preparation of the manuscript; and J.H. designed the research, analyzed the data, supervised the experimental work and wrote the manuscript.

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Correspondence to Jiahuai Han.

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Supplementary information

Supplementary Fig. 1

4-1BBL is required for sustained TNF production in RAW264.7 macrophages. (PDF 160 kb)

Supplementary Fig. 2

4-1BB is not involved in LPS-induced TNF production in RAW264.7 macrophages. (PDF 139 kb)

Supplementary Fig. 3

TLR ligands, but not IL-1β, induce 4-1BBL expression in macrophages. (PDF 444 kb)

Supplementary Fig. 4

4-1BBL is involved in TLR-ligands-induced TNF production in macrophages. (PDF 204 kb)

Supplementary Fig. 5

A model of the sequential signaling in LPS-treated macrophages. (PDF 112 kb)

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Kang, Y., Kim, S., Shimada, S. et al. Cell surface 4-1BBL mediates sequential signaling pathways 'downstream' of TLR and is required for sustained TNF production in macrophages. Nat Immunol 8, 601–609 (2007). https://doi.org/10.1038/ni1471

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