Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα

Abstract

CCL23, also known as myeloid progenitor inhibitory factor (MPIF)-1, macrophage inflammatory protein (MIP)-3, or CKβ8, is a member of the CC chemokine subfamily exerting its effects via CCR1 binding. By doing so, CCL23 selectively recruits resting T lymphocytes and monocytes, inhibits proliferation of myeloid progenitor cells and promotes angiogenesis. Previously, we and other groups have reported that human neutrophils are able to produce chemokines upon appropriate activation, including CCR1-binding CCL2, CCL3, and CCL4. Herein, we demonstrate that human neutrophils display the capacity to also express and release CCL23 when stimulated by R848 and, to a lesser extent, by other pro-inflammatory agonists, including LPS, Pam3CSK4, and TNFα. Notably, we show that, on a per cell basis, R848-activated neutrophils produce higher levels of CCL23 than autologous CD14+-monocytes activated under similar experimental conditions. By contrast, we found that, unlike CD14+-monocytes, neutrophils do not produce CCL23 in response to IL-4, thus indicating that they express CCL23 in a stimulus-specific fashion. Finally, we show that the production of CCL23 by R848-stimulated neutrophils is negatively modulated by IFNα, which instead enhances that of CCL2. Together, data extend our knowledge on the chemokines potentially produced by neutrophils. The ability of human neutrophils to produce CCL23 further supports the notion on the neutrophil capacity of orchestrating the recruitment of different cell types to the inflamed sites, in turn contributing to the control of the immune response.

Cytokine production by human neutrophils: Revisiting the “dark side of the moon”

“…Another example of the differential cytokine expression between human neutrophils and monocytes concerns CCL23, which is a chemokine that binds to CCR1 and thus exerts chemotactic activities on monocytes, DCs and resting T lymphocytes. We found that while neutrophils and monocytes produce similar levels of CCL23 in response to R848, only monocytes, but not neutrophils, manufacture CCL23 upon incubation with IL‐4 . Under the latter experimental conditions, however, neutrophils promptly accumulate IL‐1ra mRNA, indicating that they do respond to IL‐4.…”

“…Human neutrophils have been shown to express and produce either in vitro or in vivo, particularly upon appropriate stimulation, many pro‐ and anti‐inflammatory cytokines (including TNFα, IL‐1β, IL‐1ra, IL‐6), chemokines (including CXCL1, CXCL8, CXCL10, CCL2, CCL3, CCL4 and, as more recently described, CCL23), colony‐stimulating and angiogenic factors (including G‐CSF and VEGF), TNF family members (including TRAIL, FasL and BAFF) and growth factors (HB‐EGF) (Figure ). These findings, in the face of multifold evidence are making it clear that neutrophils are functionally involved in a variety of physiological and/or pathological processes, such as hematopoiesis, angiogenesis, wound healing, autoimmune and neoplastic diseases, obviously in addition to acute inflammatory diseases.…”

“…It should be recalled that neutrophil‐derived cytokines are potentially detectable by a variety of methods, including enzyme‐linked immunosorbent assays (ELISA), cytokine secretion assays, immunoblotting and/or immunoprecipitation in cell‐free supernatants or cell lysates, immunohistochemistry (IHC) and/immunofluorescence (IF) in tissue samples, intracellular staining by flow cytometry or confocal microscopy in intact cells, and RT‐qPCR and RNA‐seq for mRNA expression . In our opinion, designating neutrophils as sources of a given cytokine, based on findings made only by IHC, or IF, or intracellular staining, does not guarantee a 100% certainty of being correct.…”

“…This function can be modulated either positively by IFNγ or negatively by IL‐10 . Doubts on the importance of such a neutrophil function in in vivo settings have no reason to exist at the light of multiple evidence . For example, a recent work has clearly shown that abrogation of CARD9 in murine neutrophils inhibits inflammatory reactions during autoantibody‐induced sterile inflammation, since it specifically suppresses the in vivo production and release of neutrophil‐derived chemokines and cytokines, but not LTB 4.…”

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

“…We recently identified R848 and CL075 as very powerful agonists able to trigger a remarkable extracellular production of cytokines, including TNF‐α, IL‐6, G‐CSF, and CCL23 . R848 and CL075 are synthetic compounds that in human neutrophils specifically act via TLR8, because TLR7, their other receptor, is absent and not inducible following cell activation .…”