The S100A8–serum amyloid A3–TLR4 paracrine cascade establishes a pre-metastatic phase

Abstract

A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.

Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis

“…To eliminate any contaminating submolar amounts of LPS in S100A8 and SAA3 preparation, we performed the experiments in the presence of an endotoxin inhibitor (see Materials and methods section). We also confirmed the results with those chemokines purified from mammalian cells (data not shown, Hiratsuka et al, 2008). The SAA3-induced upregulation was abrogated in Clara cells isolated from TLR4(À/À) mice (Figure 4a), indicating that TLR4 is a responsible receptor for SAA3 signaling in Clara cells.…”

“…WT/TLR4(À/À)-BMT mouse ( ¼ WT mouse that received TLR4(À/À) bone marrow) lungs had fewer numbers of CD11b þ cells and tumor cells ( Figure 3). As lung recruitment of CD11b þ cells was impaired in TLR4(À/À) mice as reported previously (Hiratsuka et al, 2008), it can safely be said that TLR4 in bone marrow cells makes an important contribution to the lung metastasis. However, TLR4(À/À)/WT-BMT mouse lungs did not show any significant difference from WT/WT-BMT control mouse lungs in terms of CD11b þ cell populations and metastasis ( Figure 3).…”

“…The difference between S100A8 and SAA3 stimulations to induce SAA3 and TNFa could be explained in several ways. First, the binding affinity with TLR4 is one magnitude different between the two (Hiratsuka et al, 2008). Second, the major receptor for S100A8 could be RAGE, abundantly expressed in the lungs (Ghavami et al, 2008).…”

“…Given that Clara cell ablation cancelled recruitment of CD11b þ TLR4 þ cells in the lungs and tumor metastasis and that tumor burden failed to maintain expression levels of SAA3 but not S100A8 in naphthalene-treated mice (Table 1; Supplementary Figure S6), it is highly likely that Clara cells are involved in SAA3 regulation. We have reported that SAA3 is found to be produced by S100A8 binding to TLR4 (Hiratsuka et al, 2008). SAA3 expression in Clara cells was confirmed by double immunostaining using antibodies against SAA3 and CC10 in the lungs of LPS-treated mice Table 1 Comparison of immunofluorescent, flow cytometric, quantitative PCR, western blot, ELISA and short-term metastasis assay data for control and tumor-bearing murine lungs with or without naphthalene treatment…”

“…We have previously shown that TNFa is necessary and sufficient to induce an endogenous Toll-like receptor (TLR) 4 agonist S100A8 that establishes the pre-metastatic soil in the lungs (Hiratsuka et al, 2006;Vogl et al, 2007). Although S100A8 induced another TLR4 agonist serum amyloid A3 (SAA3) in pulmonary macrophages and endothelial cells (Hiratsuka et al, 2008), it was also shown by other groups that TNFa can induce SAA3 expression in granulosa cells (Son et al, 2004) and nuclear factor-kB binds enhancer factor for SAA3 (Bing et al, 2000). S100A8 appears to have a homeostatic role in pulmonary defense against microbes because S100A8 promotes leukocyte chemotaxis in lungs (Ryckman et al, 2003) and transendothelial as well as transepithelial migration of leukocytes are impaired by inhibition of S100A8 (Raquil et al, 2008;Vogl et al, 2004).…”