Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC50 = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation.

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1β, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1β may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1β. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1β release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1β, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1β release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1β from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1β is minimized.

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2′(3′)-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.