Noninflammatory phagocytosis of monosodium urate monohydrate crystals by mouse macrophages: Implications for the control of joint inflammation in gout

Abstract

Objective. We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals.Methods. Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor ␣ (TNF␣) in response to MSU was measured by enzyme-linked immunosorbent assay.Results. The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNF␣ production, however, was not linked to phagocytic ability. Peak TNF␣ levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNF␣. Mature cell lines produced TNF␣ when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNF␣ (inhibited 80.6% by anti-TNF␣).Conclusion. We demonstrated that phagocytosis and TNF␣ production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.Acute gouty arthritis is a self-limiting inflammatory response to the intraarticular deposition of monosodium urate monohydrate (MSU) microcrystals. Previous studies investigating the effect of MSU crystals in mononuclear phagocytes have focused primarily on the ability of peripheral blood monocytes or monocytic cell lines to secrete proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), . Investigators in our group have further shown that TNF␣ and IL-1 released by peripheral blood monocytes can activate vascular endothelial cell expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, thereby stimulating both the recruitment of leukocytes to the site of crystal deposition and the amplification of the inflammatory response (6-8).Hyperuricemia and precipitation of inflammatory microcrystals in synovial fluid per se are not always predictive of acute synovitis, as the severity of disease Supported by grants from the British Heart Foundation and Glaxo Wellcome PLC.

Safe disposal of inflammatory monosodium urate monohydrate crystals by differentiated macrophages

“…In contrast, freshly isolated monocytes from the same donor challenged with MSU crystals secreted these proinflammatory cytokines, induced E-selectin expression, and promoted rolling and adhesion of neutrophils on HUVECs. These observations support and extend our previous observations made with a panel of mouse monocytic cell lines, which showed divergent responses to MSU crystals according to the state of differentiation (14). Our results suggest that the process of macrophage differentiation may break the cycle of inflammation triggered by urate crystals, preventing proinflammatory cytokine synthesis, endothelial cell activation, and secondary neutrophil recruitment.…”

“…In order to resolve these apparently disparate observations, we hypothesized that the state of differentiation of mononuclear phagocytes may determine how they respond to urate crystal uptake. Evidence in support of this idea has been demonstrated in a study of mouse monocyte/macrophage cell lines, which showed that incompletely differentiated monocytic cell lines synthesized TNF␣ and activated endothelial cells upon challenge with urate crystals, whereas well-differentiated macrophagic lines did not (14).…”

Rapid induction of peroxisome proliferator–activated receptor γ expression in human monocytes by monosodium urate monohydrate crystals

“…Although the mechanisms related to the initiation of acute attacks of gout have been well characterized, the mechanisms involved in the termination of these attacks are poorly understood. Removal of MSU crystals by phagocytes and dissolution of the crystals by increased solubility modulated by an increase of temperature, a decrease of pH, and a decrease of sodium and urate levels have been proposed (4)(5)(6)19). However, these explanations have not been widely accepted because crystals may remain in the inflamed joints for a long time after the acute attack has subsided (4).…”

“…Removal of MSU crystals by phagocytes, dissolution of the crystals, involvement of antiinflammatory factors, and alteration of the crystal surface by coating with serum factors have been suggested (1,(4)(5)(6). However, these explanations have not been generally accepted, and the precise mechanisms that actually cause spontaneous resolution of acute gouty arthritis remain unknown.…”

Innate immunity conferred by toll-like receptors 2 and 4 and myeloid differentiation factor 88 expression is pivotal to monosodium urate monohydrate crystal-induced inflammation

“…After 1 wash in the same solution, cells were seeded in tissue culture dishes in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 g/ml of streptomycin, 100 IU/ml of penicillin, and 40 ng/ml of recombinant granulocytemacrophage colony-stimulating factor (BioSource International, Camarillo, CA) (35) at 37°C for 7-9 days. Macrophages were then assessed by flow cytometry using a FACScan (Becton Dickinson Biosciences, San Jose, CA) by staining with allophycocyanin (APC)-conjugated anti-F4/80 (Caltag, Burlingame, CA), a marker preferentially expressed by mature macrophages (36). Double staining was performed using APCconjugated anti-F4/80 and phycoerythrin-conjugated anti-TLR-2 or anti-TLR-4 (eBioscience, San Diego, CA) for TLR-2 or TLR-4 expression, or APC-anti-F4/80 and MyD88 polyclonal primary antibodies (eBioscience, San Diego, CA) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) as secondary antibodies for MyD88 expression.…”