Nucleotides Regulate Secretion of the Inflammatory Chemokine CCL2 from Human Macrophages and Monocytes

Abstract

CCL2 is an important inflammatory chemokine involved in monocyte recruitment to inflamed tissues. The extracellular nucleotide signalling molecules UTP and ATP acting via the P2Y2 receptor are known to induce CCL2 secretion in macrophages. We confirmed this in the human THP-1 monocytic cell line showing that UTP is as efficient as LPS at inducing CCL2 at early time points (2–6 hours). Expression and calcium mobilisation experiments confirmed the presence of functional P2Y2 receptors on THP-1 cells. UTP stimulation of human peripheral CD14+ monocytes showed low responses to LPS (4-hour stimulation) but a significant increase above background following 6 hours of treatment. The response to UTP in human monocytes was variable and required stimulation >6 hours. With such variability in response we looked for single nucleotide polymorphisms in P2RY2 that could affect the functional response. Sequencing of P2RY2 from THP-1 cells revealed the presence of a single nucleotide polymorphism altering amino acid 312 from arginine to serine (rs3741156). This polymorphism is relatively common at a frequency of 0.276 (n = 404 subjects). Finally, we investigated CCL2 secretion in response to LPS or UTP in human macrophages expressing 312Arg-P2Y2 or 312Ser-P2Y2 where only the latter exhibited significant UTP-induced CCL2 secretion (n = 5 donors per group).

Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration: a novel role for Pannexin1 in liver cells

“…In the presence of eATP alone, concentrations from 1 to 100 nM in the absence of chemokine increased primary monocyte migration by approximately 10 % (see supplementary data). This low level of activity is likely the result CCL2 secreted by monocytes in response to eATP [37]. In the presence of CCL2, Apyrase blocked migration of the control primary monocytes by 50 %, indicating endogenous production and release of eATP occurred in the Transwell assay, possibly by CCL2 activation of eATP release from migrating monocytes or as a result of eATP release from dying cells in the freshly isolated PBMC preparation.…”

“…For example, the P2Y2 receptor was identified as the nucleotide receptor for eATP and eUTP which induced CCL2 secretion from CD14+ monocytes and the THP-1 myeloid cell line. These researchers found that the selective antagonist, suramin, did not block receptor activity reproducibly at the higher concentrations of nucleotides often used for in vitro studies [37]. By contrast, Pam 3 CSK 4 -induced chemokine production by human monocytes was reported to be dependent on synergism between TLR2 and nucleotides, as the addition of Apyrase abrogated the response.…”

Identification of a Systemic Lupus Erythematosus Risk Locus Spanning ATG16L2, FCHSD2, and P2RY2 in Koreans

“…Mice null for P2RY2 showed a decreased ability to recruit monocytes and macrophages upon activation of nucleotides from apoptotic cells . P2RY2 is also known to induce CCL2 secretion in macrophages, and coding variants in the receptor have been shown to influence secretion of this proinflammatory chemokine .…”

The role of CD44H molecule in the interactions between human monocytes and pancreatic adenocarcinoma-derived microvesicles

“…Also, other molecules, such as TLR4, have to be taken under consideration as HA receptors [36]. Moreover, lipids, heat shock proteins [37] or nucleotides [38] carried by TMVs may be involved in the induction of chemokines. Other components of TMVs may address the question about the role of CD44H in the signalling pathway for the chemokines which secretion was unaffected by anti-CD44 mAb and TMVs (CXCL8, CCL2 and CCL3).…”