Interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine CXCL11 expression

Abstract

In humans, type I interferon (IFN) is a family of 17 cytokines, among which the alpha subtypes and the beta subtype are differentially expressed. It has been suggested that IFN-beta activates a specific signaling cascade in addition to those activated by all type I IFNs. Nevertheless, no true biological relevance for a differential activity of alpha and beta IFN subtypes has been identified so far. Because type I IFNs are critical for the regulation of osteoclastogenesis in mice, we have compared the effect of IFN-alpha2 and IFN-beta on the differentiation of human monocytes into osteoclasts. Primary monocytes undergoing osteoclastic differentiation are highly and equally sensitive to both alpha2 and beta IFNs as determined by measuring the induction levels of several IFN-stimulated genes. However, IFN-beta was 100-fold more potent than the alpha2 subtype at inhibiting osteoclastogenesis. Expression profiling of the genes differentially regulated by IFN-alpha2 and IFN-beta in this cellular system revealed the chemokine CXCL11 as the only IFN-induced gene differentially up-regulated by IFN-beta. We show that recombinant CXCL11 by itself inhibits osteoclastic differentiation. These results indicate that autocrine-acting CXCL11 mediates, at least in part, the regulations of osteoclastogenesis by type I IFNs.

Fig. 1.

IFN-α2 and IFN-β exhibit a 100-fold difference in their specific activities toward the inhibition of the differentiation of monocytes in osteoclasts. Freshly purified monocytes from human blood donors were cultured in the presence of sRANKL and M-CSF for 2 days, and with different concentrations of IFN-α2 or IFN-β for an additional 4–6 days. Cells were then fixed and stained for TRAP and nuclei. (A) Photomicrographs of IFN-free culture (Left) or culture with 1 nM IFN (Right). (B) Quantification of the number of TRAP⁺ MNCs in IFN-α2-treated culture (○) or IFN-β treated culture (•). Each point represents the mean osteoclast number relative to IFN-free cultures ± SEM from 5 to 29 blood donors. The osteoclastic differentiation is inhibited by 30% with 1 pM IFN-α2, 80% with 1 pM IFN-β, 60% with 10 pM IFN-α2, and 95% with 10 pM IFN-β.

Fig. 2.

IFN-α2 and IFN-β exhibit the same specific activities for the induction of the expression of IFN-stimulated genes. Freshly purified monocytes from human blood donors were cultured in the presence of sRANKL and M-CSF for 2 days, and then left untreated (▵) or treated for 4 h with IFN-α2 (○) or IFN β (•). RNAs were then extracted and the levels of 6-16 (A) or 2′–5′ oligoadenylate synthetase, PKR, and MxA (B) transcripts were measured by quantitative RT-PCR. Each point in the vertical represents a different blood donor.

Fig. 3.

CXCL11 is the only ISG differentially up-regulated by IFN-β compared with IFN-α2 in monocytes purified from several blood donors. Candidate genes identified as preferentially up-regulated by 1 pM IFN-β in the gene array study (Table 1) were analyzed by quantitative RT-PCR. Freshly purified monocytes from human blood donors were cultured in the presence of sRANKL and M-CSF for 2 days, and then left untreated (▵) or treated for 4 h with IFN-α2 (○) or IFN-β (•). RNAs were then extracted and the levels of ISG20 (A), GBP5 (B), and CXCL11 transcripts (C) were quantified by quantitative RT-PCR. Each point in the vertical represents a different blood donor. Samples used for the gene array study are indicated by arrows. Statistical significance of the difference of the expression levels induced by IFN-α2 or IFN-β have been analyzed by the Mann–Whitney test. NS, nonsignificant. The median of the ratio of CXCL11 induced by IFN-β/CXCL11 induced by IFN-α2 is 4.2 for IFNs at 1 pM and 1.5 for IFNs at 10 pM.

Fig. 4.

CXCL11 but not CXCL9 or CXCL10 inhibits osteoclastic differentiation. Freshly purified monocytes from human blood donors were cultured in the presence of sRANKL and M-CSF for 2 days, and with different concentrations of the chemokine for an additional 4–6 days. Cells were then fixed and stained for TRAP and nuclei. (A) Photomicrographs of CXCL11-treated cultures. (B) Quantification of the number of TRAP⁺ MNCs in CXCL11-treated cultures; mean osteoclast number relative to untreated cultures ± SEM from eight blood donors. (C) Comparison of the effect of 100 nM CXCL11, CXCL9, and CXCL10 on the inhibition of osteoclastic differentiation.

Fig. 5.

The inhibitory effect of CXCL11 on osteoclastogenesis is independent of CCR5. (A) Identification of monocytes heterozygous (lane B) or homozygous (lane C) for the CCR5 Δ32 mutation. Lanes: A, wild type; D, molecular weight marker in base pairs. (B and C) Inhibitory activity of CXCL11 (B), IFN-α2(C, gray bars) and IFN-β (C, black bars) on osteoclastic differentiation of monocytes homozygous for the CCR5 Δ32 mutation. Cellular differentiation assay and analysis were performed as for Figs. 1 and 4.