Extracellular HMGB1 (High mobility group box 1 protein) functions as an inflammatory cytokine which stimulates monocytes to produce pro-inflammatory cytokines. HMGB1 is a ubiquitous non-histone nuclear protein and can be passively released by necrotic or late apoptotic cells and actively released by macrophages, monocytes, NK cells, and dendritic cells in response to exogenous and endogenous inflammatory stimuli. As a DNA chaperon, HMGB1 binding to pathogen DNA can be recognized by several pattern recognition receptors, such as Toll-Like Receptor (TLR) 2, TLR4, TLR9 and Receptor for Advanced Glycosylation End products (RAGE) resulting in activation of intracellular survival signaling pathways. It was recently reported that HMGB1-DNA complex binds to RAGE on B-cells and leads to the recruitment of TLR9 and enhances B-cell survival and proliferation. Overexpression of HMGB1 is associated with each of the hallmarks of cancer including unlimited replicative potential, angiogenesis, evading apoptosis, self-sufficiency in growth signals, inflammation, tissue invasion and metastasis. Elevation of serum HMGB1 has been detected in many cancer patients and the levels of serum HMGB1 increases after anti-cancer therapy. However, the potential roles of HMGB1 and the integrity of TLR9/RAGE pathway have not been studied in chronic lymphocytic leukemia (CLL).
CLL cells partially undergo spontaneous apoptosis when cells are cultured in vitro and this apoptosis can be decreased by addition of monocytes-derived nurse-like cells (NLCs). We aimed to investigate whether HMGB1 plays a role NLCs preventing apoptosis of CLL cells. The aims of this study were to determine whether HMGB1 is actively released by NLCs and whether CLL cells express pattern recognition receptors for HMGB1. After culture of CLL cells for one week, NLC formation was observed by MTT staining and phase contrast microscopy. HMGB1 and its receptors TLR9 and RAGE were constitutively expressed in all CLL samples studied. HMGB1 was initially expressed in the nucleus of both NLCs and CLL B-cells as detected by immuno-fluorescent microscopy. However, after one week of culture, NLCs started losing nuclear HMGB1 without changing their cellular structure. In NLC surrounding CLL cells, the HMGB1 receptors TLR9 and RAGE co-localized and formed large aggregates, indicating activation of the TLR9/RAGE signal pathway. This was followed by NF-kB (p65) nuclear translocation from its cytosolic localization in fresh CLL cells. To confirm whether the activation of TLR9/RAGE pathway is mediated by HMGB1, recombinant HMGB1 was used to stimulate CLL cells in vitro. Increased HMGB1/RAGE, HMGB1/TLR9 and TLR9/RAGE intracellular aggregations, TLR9 internalization, and NF-kB nuclear translocation, all essential for HMGB1-mediated activation of TLR9/RAGE pathway, were observed after 3 hours of incubation with HMGB1. HMGB1 also promoted CLL cell in vitro colony formation. Blocking HMGB1-induced TLR6/RAGE activation by anti-RAGE antibody reduced NLC-mediated CLL cell survival.
In summary, we report, for the first time, that NLCs release HMGB1, activating the TLR9/RAGE pathway in CLL cells. HMGB1 plays an important role in CLL cell survival by activation of TLR9/RAGE pathway and we propose that HMGB1 may be a potential target for treatment of CLL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.