Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL-1β

Abstract

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the pro-inflammatory cytokine IL-1β significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example, during bone healing or in inflammatory bone disorders.

Figure 1

Expression of complement regulatory proteins (A, B, C, F), complement receptors (D, E, F), and C3 and C5 (H) in bone cells: hMSC, o-hMSC, OB and OC. mRNA expression of the respective gene was related to the housekeeping gene GAPDH. (G) Increase in mRNA expression after OC formation from PBMNC. (H) Representative agarose gels, C3, and C5 expression in hMSC, o-hMSC, OB, PBMNC, and OC. Experiments were performed independently 3–7 times in triplicate or quadruplicate cultures; $ p<0.05 Abbreviations: hMSC: human mesenchymal stem cells; o-hMSC: hMSC cultivated under osteogenic conditions for 14, 21, and 28 days; OB: primary osteoblasts; OC: osteoclasts, multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells generated from peripheral blood mononuclear cells (PBMNC)

Figure 2

Fluorescence images of immunostained complement regulators proteins (CD46, CD55, CD59) and receptors (C3aR, C5aR) in bone cells.

Figure 3

Phase contrast and fluorescence images of immunostained C3aR and C5aR in osteoblasts without and after 45 min stimulation with 1µg/ml C3a or 0.1µg/ml C5a; first column: phase contrast images; second column: for the detection of membrane-bound receptors cells were fixed and the respective receptors were detected by a green fluorescent antibody as described in Materials and Methods; third column: for the detection of membrane-bound as well as internalized receptors cells were permeabilized before labelling with a red fluorescent antibody; fourth column: in the overlay the yellow colour indicates the membrane-bound receptors, whereas the internalized receptors are stained red.

Figure 4

C5a induction in supernatants of OB and OC after incubation with C5 for 4h. $ indicates a significant difference between osteoclasts treated with C5 alone and with C5 and phorbol 12-myristate 13-acetate (PMA); $ p>0.05; n=3 independent experiments

Figure 5

Stimulation of osteoblast with lipopolysaccharide (LPS; 0.1 µg/ml), C3a (1 µg/ml), C5a (0.1 µg/ml), IL-1β (0.1 ng/ml) or combinations of IL-1β/C3a and IL-1β/C5a for 24 h. The controls received medium without supplements. (A) IL-6 and IL-8 concentration in the supernatants of osteoblasts measured by ELISA (enzyme-linked immunosorbent assay); (B) IL-6 mRNA expression in stimulated osteoblasts relative to the untreated controls. Experiments were performed independently 3–7 times in triplicate or quadruplicate cultures; $ p<0.05

Figure 6

Osteogenic differentiation of hMSC after 28 days in differentiation medium containing 1 µg/ml C3a or 0.1 µg/ml C5a. First row: positive staining of alkaline phosphatase; second row: positive von Kossa staining indicating matrix mineralization.

Figure 7

(A) Formation of multinucleated TRAP-positive cells from PBMNC after stimulation with RANKL/M-CSF (20 ng/ml / 10 ng/ml), C3a (1 µg/ml) or C5a (0.1 µg/ml). The number of multinucleated TRAP-positive cells in stimulated cultures were related to control cultures without supplements (bar at 1). Experiments were performed independently 6 times in triplicate cultures; $ p<0.05 (B) Photomicrographs of TRAP-stained cells (left and middle column) after 21 days cultivation of PBMNC on plastic and von Kossa-stained 28 days cultures (right column) on calcium phosphate-coated culture vessels. TRAP-positive multinucleated cells containing at least 3 nuclei were generated in presence of medium with 10% FCS without supplementation with osteoclastogenic factors (FCS), in presence of 20 ng/ml RANKL and 10 ng/ml M-CSF (RANKL/M-CSF), in presence of 0.1 µg/ml C5a (C5a) or 0.1ng/ml C3a (C3a). Osteoclast resorption lacunae in calcium phosphate-coated cell culture surface (gray and white) were observed in presence of 40 ng/ml RANKL and 20 ng/ml M-CSF. After stimuation with 0.1 µg/ml C5a calcium phosphate coating was not resorbed, culture surface was black after von Kossa-staining. (C) RANKL and OPG mRNA expression in osteoblasts relative to the untreated controls after stimulation with LPS (0.1 µg/ml), C3a (1 µg/ml), C5a (0.1 µg/ml), IL-1β (0.1 ng/ml) or combinations of IL-1β/C3a and IL-1β/C5a for 24 h. The controls received medium without supplements. Experiments were performed independently 3–7 times in triplicate or quadruplicate cultures; $ p<0.05 Abbreviations: FCS: fetal calf serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; M-CSF: macrophage-colony stimulating factor; OPG: osteoprotegerin; RANKL: receptor activator of nuclear factor-kappaB ligand;