Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. evidence that serum αl2‐hs glycoprotein is a potent and specific crystal‐bound inhibitor

Abstract

Tissue deposits of basic calcium phosphate (BCP) crystals are associated with various clinical manifestations of inflammation. We addressed the possibility that native proteins modify the ability of hydroxyapatite (HA) crystals to stimulate human inflammatory cells. Neutrophil superoxide release and chemiluminescence in response to HA crystals (0.3-4.0 mg/ml) were blunted by serum and plasma. Inhibitory activity was progressively removed from serum by sequential adsorption with HA crystals, suggesting that the inhibitors were crystal-bound proteins. Thus, we characterized HA crystal-bound plasma proteins by O'Farrell gels: Fibronectin, transferrin, albumin, a,-HS glycoprotein (AHSG), al-proteinase inhibitor, a,-acid glycoprotein, Gc globulin, haptoglobin, and high density lipoprotein apolipoproteins were major bound species. Of these, AHSG was the most active inhibitor of HA-induced neutrophil superoxide release, and this glycoprotein partially (60%) restored inhibitory activity to HAadsorbed serum. AHSG also bound in vitro to the related BCP crystal, octacalcium phosphate, but only minimally to calcium pyrophosphate dihydrate crystals and monosodium urate crystals. Suppressive effects on neutrophil stimulation exhibited by AHSG were also specific for BCP crystals. AHSG was present in noninflammatory synovial fluids bound to synthetic HA crystals in vitro, and AHSG could be detected on native synovial fluid HA crystals. We conclude that the binding of AHSG may modulate the inflammatory potential of BCP crystals.Synthetic hydroxyapatite (HA) crystals directly stimulate human neutrophils in vitro (1) and consistently provoke self-limited acute inflammation when injected into the articular and pleural cavities of animals, into the foot pad of rats, and into the dermis of human volunteers (2-4). Furthermore, acute periarticular inflammation, and less commonly, an acute synovitis that is similar to acute gout in presentation and in response to therapy, are associated with the presence of HA crystals and related basic calcium phosphates (BCP) (2,3,5-8).

Hierarchical Role of Fetuin-A and Acidic Serum Proteins in the Formation and Stabilization of Calcium Phosphate Particles

“…synthetic apatite, bone mineral, or calciprotein-like particles (4,17,18,20,21). These studies also suggested that the fetuin-A and albumin co-localized with mineral at ratios varying over a wide range.…”

“…For example, small molecules and ions like pyrophosphate, citrate, or magnesium (1) slow down crystal growth by interfering with crystal lattice assembly. On the protein level, in vitro binding studies of serum proteins to basic calcium phosphates helped to identify structurally diverse but generally acidic mineral-binding proteins like ␣ 2 -Heremans-Schmid glycoprotein/fetuin-A, ␥-carboxyglutamate proteins, albumin, fibronectin, or transferrin (2)(3)(4)(5). Acidic bone-derived proteins likewise have a high affinity for mineral and prevent their spontaneous precipitation from supersaturated salt solutions (6).…”

Fetuin-A Regulation of Calcified Matrix Metabolism

“…56 The antiinflammatory property of serum ␣ 2 -HS glycoprotein/human fetuin-A was further supported by the demonstration that fetuin-A is a potent and specific crystal-bound inhibitor of neutrophil stimulation by hydroxyapatite crystals. 57 Calcium phosphate crystals induce proinflammatory cytokine secretion through the NLRP3 inflammasome in monocytes/macrophages, 58 -60 cell death in human vascular smooth muscle cells, 61 and cell activation in chondrocytes. [62][63][64] Antiapoptotic activity of fetuin-A has been observed in smooth muscle cells 65 and dampening of the cell-specific responses would generally be expected to alleviate the detrimental consequences of local inflammation, cell death, and cartilage degradation.…”

“…[62][63][64] Antiapoptotic activity of fetuin-A has been observed in smooth muscle cells 65 and dampening of the cell-specific responses would generally be expected to alleviate the detrimental consequences of local inflammation, cell death, and cartilage degradation. The proven protective function of fetuin-A in many animal models of inflammation, 52-55 the inhibition of proinflammatory compounds, 50,51,66 -68 and the inhibition of crystalinduced neutrophil activation 57 collectively suggest that fetuin-A may generally protect during pathological mineralization as well. 69…”

Albumin and Its Role in Urolithiasis

“…HSA was one key component of the protein species that were adsorbed on the surface of hydroxyapatite (HAP) crystals when these crystals were incubated in whole human serum [23]. Bolstering the aforementioned findings, Atmani et al reports albumin to be the major component of the organic matrix of both CaOx as well as CaP crystals when the crystals were induced in the urine of stone formers, while Kaneko et al and Dussol et al reported albumin as a commonly occurring protein in all forms of kidney stone formations [8,24,25].…”