Complement dependent TNFα production in neutrophil-like HL60 cells

by Selleckchem | Aug 08 2023

Neutrophils develop in the bone marrow (BM) from hematopoietic stem cells (HSCs) through a series of progenitor cells and mature neutrophils play a critical role in the human immune system. Previous studies revealed that tumor necrosis factor α (TNFα) produced by immature neutrophils contributes to HSCs development and vascular regeneration in the BM niche. However, the precise mechanism of TNFα production in immature neutrophils remains unclear. This study aims to assess the relationship between complement C3 activation and TNFα production from immature neutrophils. We investigated the regulatory mechanism of TNFα production by complement components in neutrophil-like HL60 cells. Flow cytometric analysis showed that C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMβ2) are expressed on the surface of neutrophil-like HL60 cells. We found that zymosan-treated human serum leads to TNFα production in neutrophil-like HL60 cells, but not in human polymorphonuclear cells (PMNs). A C3-convertase inhibitor, compstatin suppresses TNFα production. These data suggest that the TNFα production is mediated by complement C3 activation. Furthermore, the TNFα production is enhanced by Ca2+ elevating agents, thapsigargin (TG), but is suppressed by treatment with Ca2+ chelators, EGTA, or BAPTA-AM. In addition, the soluble TNFα production is suppressed by treatment with immobilized-fibrinogen or -fibronectin. Thus, the TNFα production is enhanced by intracellular Ca2+ elevation and is negatively regulated by the interaction between the neutrophil-like HL60 cells and fibrinogen or fibronectin.

Comments:

The study aimed to investigate the relationship between complement C3 activation and TNFα production in immature neutrophils. The researchers used neutrophil-like HL60 cells as a model system to study the regulatory mechanism of TNFα production by complement components.

The flow cytometric analysis revealed that the C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMβ2) were expressed on the surface of neutrophil-like HL60 cells. This suggests that these cells have the potential to respond to complement activation.

The researchers observed that treating neutrophil-like HL60 cells with zymosan-treated human serum resulted in TNFα production. However, this effect was not observed in human polymorphonuclear cells (PMNs), indicating that the response may be specific to the neutrophil-like HL60 cells.

To further understand the role of complement C3 activation in TNFα production, the researchers used a C3-convertase inhibitor called compstatin. They found that compstatin suppressed TNFα production, suggesting that C3 activation is involved in mediating TNFα production.

The study also explored the involvement of calcium signaling in TNFα production. The researchers discovered that agents that elevate intracellular calcium levels, such as thapsigargin (TG), enhanced TNFα production. On the other hand, treatment with calcium chelators, such as EGTA or BAPTA-AM, which reduce intracellular calcium levels, suppressed TNFα production. This suggests that intracellular calcium elevation plays a role in enhancing TNFα production in neutrophil-like HL60 cells.

Furthermore, the researchers investigated the influence of the interaction between neutrophil-like HL60 cells and extracellular matrix proteins, specifically fibrinogen and fibronectin, on TNFα production. They found that soluble TNFα production was suppressed when the cells were treated with immobilized fibrinogen or fibronectin. This indicates that the interaction between neutrophil-like HL60 cells and these extracellular matrix proteins negatively regulates TNFα production.

In summary, this study provides evidence that complement C3 activation is involved in TNFα production in immature neutrophils. The findings suggest that C3 activation, calcium signaling, and interactions with extracellular matrix proteins play important roles in regulating TNFα production in neutrophil-like HL60 cells. Further research is needed to fully understand the precise mechanisms underlying TNFα production and its implications in the immune system.