Inflammatory Microcrystals Alter the Functional Phenotype of Human Osteoblast-Like Cells In Vitro: Synergism with IL-1 to Overexpress Cyclooxygenase-2

Abstract

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE2 in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D3-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.

Osteoblast retraction induced by adherent neutrophils promotes osteoclast bone resorption: implication for altered bone remodeling in chronic gout

“…However, the present data indicated that progressive MSU deposits have prolonged effects on OBs. While reducing bone formation by OBs, MSU stimulate these OBs to generate Bone remodeling in chronic gout I Allaeys et al inflammatory mediators like IL-6 and IL-8, 6 factors known to attract and activate neutrophils. Thus, these cells that are present locally in bone microvasculature and marrow space in non-pathological conditions, can easily migrate in different inflammatory bone tissues.…”

“…Normal human OBs were grown from trabecular bone explants until confluence before they were recovered and plated at starting densities of 0.5-1 Â 10 6 cells/well in aMEM þ 10% FBS with antibiotics. 6 All incubations were performed at the first passage and 80-90% cell confluence in six-well plates (371C, humidified atmosphere, 5% CO 2 ). Confluent OBs were preactivated with 1 mg/ml sterile pyrogen-free triclinic MSU (provided by Dr R De Médicis, University of Sherbrooke; mean size: 10 Â 1.25 mm, as determined by scanning electron microscopy) for 1 h at 371C, and washed twice to eliminate nonadherent MSU.…”

“…OBs coincubated with labeled neutrophils (short-term neutrophil adherence with an optimal neutrophil/OB ratio of 20/1) were vigorously washed two times to eliminate non-adherent cells. Since a 1-h contact of MSU with OBs was associated with partly engulfed crystals that could affect neutrophils, 6 amounts of MSU present in OBs and at their surface after washes were measured and averaged 0.52 ± 0.03 mg/ml (n ¼ 6). This latter concentration (half of MSU initially added to OBs) was used to stimulate neutrophils where appropriate.…”

“…Since MSU-stimulated OBs produce IL-8, 6 neutrophils present in bone space are likely attracted towards activated OBs, suggesting possible cell-to-cell interactions. Knowing that MSU can affect cellular viability, 24 the effect of MSU on neutrophil and OB viability was evaluated before studying interactions between neutrophils and OBs.…”

“…5 Moreover, osteoblasts (OBs), that originate from stromal cells, undergo functional deregulation by MSU with reduced parameters of bone formation and increased generation of inflammatory mediators like PGE 2 , interleukin (IL)-6, and IL-8. 6 Indeed, stromal cells and OBs are the major source of IL-6 and IL-8. 7 IL-8 is a chemokine that attracts and activates neutrophils, the major cell type involved in gouty arthritis.…”