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1.
Figure 4

Figure 4. CD11b–/loLy6ChiCX3CR1+ OCPs have both M1 and M2 characteristics and express many osteoclast-associated receptors. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(A) CX3CR1 expression assessed by GFP intensity in C57BL/6 CX3CR1-GFPhet BM demonstrates that CD11b–/loLy6ChiCX3CR1+ OCP (black line) have CX3CR1 expression levels similar to those of M1 monocytes (CD11b+Ly6ChiCD117, gray dashed line) and considerably lower than those of M2 monocytes (CD11b+Ly6CloCD117, dotted line). (B) CCR2 surface expression on OCP (black line) is positive compared with CD11b+Ly6Clo CCR2 M2 monocytes (dotted line), but lower than CD11b+Ly6Chi M1 monocytes (gray dashed line). α-CCR2 antibody staining of OCP from Ccr2–/– mice (gray area) is shown as a staining control. (C) OCPs express markers of M2 lineage, including Dectin1, CD36, and CD206. (D) OCPs express innate immune receptors previously reported to affect osteoclast function. Staining with antibodies to TREM-2, MDL-1, and PIRA/B (black line) are shown compared with isotype control (gray area).

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
2.
Figure 5

Figure 5. BM CD11b–/loLy6ChiCD117+ OCPs differentiate into osteoclasts in vivo. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(AC) Intramedullary injection of mT/mGOC OCPs into nfatc1Δ/Δ femurs demonstrates that CD11b–/loLy6ChiCD117+ are bona fide osteoclast precursors. (A) Donor-derived cathepsin K+ GFP+ osteoclasts are detected by anti-GFP immunohistochemistry (IHC) on femurs of nfatc1Δ/Δ mice 7 days after intramedullary injection of OCP; (B) Enlargement of boxed area in A, demonstrating multinucleation of donor-derived cells. (C) No GFP+ osteoclasts were present in control mice injected with C57BL/6 OCPs. (DG) Intravenous injection of mT/mGOC OCP into C57BL/6 mice 24 hours after calvarial LPS injection. (D) Anti-GFP IHC on calvarial sections 4 days after cell transfer demonstrates donor-derived cathepsin K+ GFP+ osteoclasts that also stain for TRAP (E). No GFP signal is detected in no–cell transfer control animals (F), although TRAP+ multinucleated cells can be detected (G). Counterstain, hematoxylin. Original magnification, ×40 (A, CG); ×300 (B).

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
3.
Figure 6

Figure 6. Adoptive transfer of CD11b-/loLy6Chi OCP does not increase erosive disease, but unexpectedly ameliorates inflammatory arthritis in the adoptive transfer SKG model. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(A) Schematic of adoptive transfer experiment. Adoptive transfer of conventional SKG CD4+CD25 T cells into Rag2–/– mice typically results in arthritis at 4 weeks. (B) One month after transfer, cross-sectional images from μCT of arthritic ankles were scored blindly for erosions. Coadoptive transfer of OCPs does not increase erosion score compared with mice receiving SKG CD4+ cells alone. Few erosions are seen in the group receiving Tregs. (C) Representative cross-sectional images used for erosion scoring; star denotes an erosion. (D) Blinded arthritis scores are significantly ameliorated by coadoptive transfer of CD11b–/loLy6Chi OCP compared with CD4+ group, although not as dramatically as with cotransfer of wild-type Tregs. *P = 0.03, Student’s t test. (E) Representative images from sagittal ankle sections shows inflammatory changes in the marrow of both OCP and CD4+ groups (stars), but more prominent synovial infiltrate (arrowheads) in the CD4+ group. H&E staining. Original magnification, ×10.

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
4.
Figure 8

Figure 8. Suppression of CD4+ T cell proliferation by CD11bloLy6Chi OCPs requires NOS2, caspase activity, and IFN-γ. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(A) Full suppression of T cell proliferation requires cell-cell contact. Maximal CD4+ T cell proliferation (black bars) is inhibited by CD11b–/loLy6Chi OCPs in the lower chamber (gray bars) more efficiently than OCPs in upper chamber (striped) in Transwell assays.**P = 0.02; *P = 0.035. (B) Suppression of T cell proliferation by OCP is not relieved by OPG. A ratio of 1:4 OCP/T cell inhibits proliferation (gray bars) compared with T cells alone (black bars). OPG at concentrations from 25–200 ng/ml had no effect. (C) CD4+ T cell proliferation (black bars) is suppressed by OCPs at a ratio of 1:4 (gray bars). The addition of the NOS2 inhibitor L-NIL or blocking antibody to IFN-γ relieves suppression compared with isotype/DMSO control. Inhibition of IL-10, arginase, and IDO have no effect. (D) OCPs from Nos2–/– mice (light gray bars) have decreased T cell–suppressive activity compared with C57BL/6 OCPs (dark gray bars). (E) OCPs express the IFN-γ receptor α-chain (black line) compared with isotype control (gray area). (FH) T cell–derived IFN-γ and intact IFN-γ signaling in OCPs is required for suppression. (F) Proliferation of wild-type CD4+ T cells (black bar) is suppressed by OCP derived from C57BL/6 (light gray bars) or Ifng–/– mice (striped bars) over a range of ratios from 1:2 to 1:8. In contrast, OCPs derived from Ifngr–/– mice (dark gray bars) do not suppress. (G) Proliferation of Ifngr–/– CD4+ T cells (black bar) is similarly inhibited by wild-type and Ifng–/– OCPs, but not Ifngr–/– OCP Ifng–/–. (H) Proliferation of Ifng–/– T cells (black bar) is not inhibited by OCPs of any genotype.

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
5.
Figure 3

Figure 3. CD11b–/loLy6ChiCX3CR1+ OCPs are distinct from other myeloid precursors. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(AC) FACS analysis of cell-surface markers on CD3B220Ter119 gated BM from CX3CR1-GFPhet distinguishes OCP from other myeloid populations. Staining of CD11bloLy6Chi CX3CR1 OCP population with the indicated antibody (lines) compared with isotype control (gray area) is shown. (A) OCPs are predominantly CD115+. (B) OCPs are CD135loCD117+/–, further distinguishing them from CD11bLy6CMDPs (CD135+CD117+, dashed line) and CD11b+Ly6Chi M1 monocytes (CD135CD117, dark gray line). (C) Both CD117+ and CD117 subsets are capable of differentiating into osteoclasts in vitro, although those in the CD117+ subset are more efficient. TRAP+ multinuclear osteoclast formed per 1 × 103 cells cultured in triplicate in M-CSF/RANKL, *P = 0.03. (D) OCPs are CD11c, in contrast with pre-DCs (CD11b+CX3CR1+CD11c+, dotted line). (E) GST-RANKL-biotin (black line) or GST-biotin control (gray area) demonstrate RANK expression on the surface of BM-derived day 2 osteoclasts (right panel) but not OCPs (left panel). Loss of osteoclast-binding RANKL-GST-bio binding in the presence of excess unlabeled RANKL (dashed line) demonstrates specificity. (F) Model of myeloid differentiation with the CD11b–/loLy6ChiCX3CR1+CD115+ OCP as distinct myeloid precursors derived from MDPs; Mo, monocyte; QOP, lineage committed QOP.

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
6.
Figure 7

Figure 7. CD11bloLy6Chi OCPs have cell-surface markers of MDSCs and suppress in vitro T cell proliferation. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(A) Wright-Giemsa stain of sorted OCPs cultured for 48 hours in M-CSF demonstrates monocytic nuclear morphology and characteristic gray blue cytoplasm. Original magnification, ×20. (B) Similar to MDSCs, BM CD11bloLy6Chi OCPs are CD49d and CD124 positive. Antibody-specific staining of cells gated on CD3B220Ter119CD11b–/loLy6Chi is shown in black line, isotype staining in gray area. (C) CD11b–/loLy6Chi OCPs purified from SKG BM suppress CD4+ T cell proliferation (light gray bars) in contrast with CD11b+Ly6C+ (white bar) or QN (dark gray bar) populations from the same BM. Data are representative of 3 independent experiments. (D) CD11bloLy6Chi OCPs sorted from C57BL/6 CX3CR1:GFPhet BM based on CX3CR1 expression demonstrate that, similar to osteoclast precursor potential, suppressor activity is seen in the CX3CR1+ (light gray bars) but not CX3CR1 (dark gray bar) population. (E) Inhibition of T cell proliferation by freshly isolated OCPs is maintained after differentiation of sorted OCPs in the presence of M-CSF and 100 ng/ml RANKL for 4 days. Addition of RANKL to proliferation medium does not significantly inhibit proliferation (white bar), whereas cultured OCPs retain suppressive activity (gray bars). (F) There is no significant difference in suppressor activity between OCPs derived from BALB/c mice (dark gray bars) and those from arthritic SKG mice (light gray bars).

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
7.
Figure 2

Figure 2. The CD11b–/loLy6ChiCX3CR1+ population contains the majority of osteoclast precursor activity. . From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(AC) Individual BM populations from SKG mice, sorted by CD11b and Ly6C staining as illustrated in A, were plated in triplicate at 2.5 × 103/well in M-CSF and RANKL. (B) Multinucleated TRAP+ cells were counted after 3 days, and (C) resorption pits on osteologic slides were quantitated after 14 days culture. The purity of each sorted population is stated in parenthesis in the x axis label. Results are representative of 4 independent experiments. (D) TRAP staining of CD11b–/loLy6Chi OCPs cocultured with BM stromal cell–derived osteoblasts demonstrates osteoclast formation that is absent in parallel osteoblast-only culture. (E) Original magnification, ×10. (F) CX3CR1 expression is variable in the CD11b–/loLy6Chi population. CX3CR1 expression by CD11b–/loLy6ChiCD3B220Ter119 gated BM from CX3CR1-GFPhet mice (black line) compared with C57BL/6 (gray area). (G) Triplicate cultures of 1 × 103 cells/well in M-CSF and RANKL demonstrates that only the CX3CR1+CD11b–/loLy6Chi population efficiently differentiates into multinucleated TRAP+ osteoclasts and (H) forms resorption pits on osteologic slides. Original magnification, ×4. Results are representative of 3 replicates. (I) CD11b–/loLy6ChiCX3CR1+ cells retain the ability to form osteoclast after long-term culture. The total number of colonies obtained from single cells sorted from the indicated populations after 60 days in culture is indicated by column height, with the number retaining osteoclast differentiation capacity denoted in gray.

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.
8.
Figure 1

Figure 1. Erosive arthritis, generalized bone loss, and increased BM OCPs in SKG arthritis.. From: Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

(AC) μCT analysis of female SKG mice with chronic arthritis (SKG-A) compared with healthy littermate controls (SKG-c) shows erosions and generalized bone loss. (A) Representative 3D reconstructions of rear paw, showing erosions (asterisks) and (B) representative images and quantitation of BV/TV of proximal tibia metaphysis and (C) cortical thickness, n = 4 per group. *P ≤ 0.03, Mann-Whitney test. (D) BM from SKG mice after 8 weeks arthritis shows increased osteoclast differentiation. BM cells derived from arthritic or control SKG mice, n = 4 each group, were cultured in M-CSF and RANKL and number of TRAP-stained multinuclear (MN) osteoclasts were quantified from triplicate wells of 2.5 × 103 cells plated from each individual, P = 0.03 Mann-Whitney test. A representative image of TRAP-stained cultures is shown. Original magnification, ×10. Data are representative of 3 experiments. (E) Representative dot plot showing an increased BM B220CD3CD11b–/loLy6Chi population in arthritic mice. Surface phenotype of CD11b–/loLy6Chi cells is Ly6G Gr1+. (F) CD11b–/loLy6Chi cells sorted from SKG BM are enriched for OCPs compared with the CD3B220Ter119 input; triplicate cultures of 12.5 × 103 cells were plated in M-CSF and RANKL for 3 days. Representative images are shown. Original magnification, ×10. (G) The CD11b–/loLy6Chi population increases in percentage (top) and total number (bottom) in SKG mice after 8 weeks arthritis compared with healthy littermate controls or age-matched BALB/c controls. P < 0.005, 1-way ANOVA. Representative data from 3 independent experiments are shown.

Julia F. Charles, et al. J Clin Invest. 2012 December 3;122(12):4592-4605.

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