Defective endochondral ossification-derived matrix and bone cells alter the lymphopoietic niche in collagen X mouse models

Abstract

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders.

FIG. 1.

Marrow stromal cell and osteoclast characterization. (A) Proliferation assay with marrow stromal cells from week-3 wild type (WT) and collagen X null and transgenic mice (Col X KO, Col X Tg; n=4, in 8 replicates). (B) Colony-forming unit alkaline phosphatase (CFU-ALP) colony counts and representative culture plate views depicting purple ALP staining of week-3 WT, Col X KO and Col X Tg mouse cells after 14 days of culture (n=4). (C) Colony-forming unit osteoblast (CFU-O) colony semi-quantification and representative culture plate views depicting Alizarin red S staining of week-3 WT, Col X KO, and Col X Tg mouse cells after 21 days in culture with ostogenic media (n=4). (D) Tibial longitudinal sections from week-3 WT, Col X KO and Col X Tg mice were reacted with tartrate-resistant acid phosphatase (TRAP; red color) for osteoclast activity. Growth plate (GP) and trabecular bone (TB) of the chondro-osseous junction are bracketed. While no discernable differences in TRAP staining or osteoclast distribution were noted, the average length of trabecular spicules was longer in WT mice (403.75 μM, n=4) than in Col X KO and Tg mice (340.75 and 291.75 μM, n=4, respectively). Bar=100 μM.

FIG. 2.

Characterization of cocultures with calvaria, trabecular and hypertrophic chondrocyte cells. Calvaria (A), trabecular (B) and hypertrophic chondrocyte (C) cocultures from WT and collagen X null (ColX-KO) mice were grown to confluency and cell-specific phenotypes were assessed by staining with antibodies for: collagen I, collagen II, collagen X, osteocalcin, and osteopontin. IgG controls are boxed on KO samples except for collagen X staining where the KO mice serve as controls. Magnification 10×, Bar=200 μM. (D) WT and Col X-KO mouse calveria and trabucular cultures were stained with Alizarin red S for mineralization, followed by quantitation of solubilized stain (OD 562). Chondrocyte cultures were stained with alcian blue, followed by quantitation of solubilized stain (OD 650) for identification of sulfated proteoglycans characteristically present in cartilage. (E) Expression of chondro-oseous markers (collagen I (Col I), osteocalcin (OC), osteopontin (Opn), collagen II (Col II), and collagen X (Col X) were assessed by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) in 3T3 fibroblasts, as well as in both WT and Col X-KO mouse calvaria (cal), trabecular (TB), and hypertrophic chondrocyte (HC) cultures. Genes with no to low expression fall in the red zone. Note, both cartilage and bone characteristics in all four cell types (B, E).

FIG. 3.

Coculture assays confirm a defect in the ability of collagen X mouse trabecular cocultures to support B lymphopoiesis. (A) The number of CD45.1+ (donor) CD19 (B cell marker) cells was determined from trabecular, calvarial, and hypertrophic chondrocyte cocultures from WT or collagen X null (Col X-KO) mice. (B) The number of CD45.1+ (donor) Gr-1 (granulocyte marker) cells was also determined from the same coculture experiments. Cells were analyzed 2 weeks post seeding of WT lineage negative cells. Standard error of the mean shown for >10 wells from multiple experiments per group.

FIG. 4.

Localization of SCF and SDF-1 in tibiae of WT and collagen X mice. Longitudinal sections from week-3 WT (A), collagen X null (Col X-KO) (B) and collagen X transgenic (Col X-Tg) (C) tibiae were stained with hematoxylin and eosin (H&E), or with either anti-SCF or anti-SDF-1 antibodies. Arrows indicate the growth plate (GP) and trabecular bone (TB) juncture. Q-dot rabbit controls are boxed. Note SCF localization to osteoblasts and bone lining cells along the trabecular bone and to osteocytes within trabecular bone. In WT, SDF-1 staining in the surrounding territorial matrix of terminal hypertrophic chondrocytes, as well as staining of osteoblasts and bone lining cells, but not osteocytes. In collagen X null and transgenic mice, note overall decrease in staining intensity. Bar=100 μM, Bar=50 μM in high magnification (high mag) views of SDF-1 staining.

FIG. 5.

Cytokine supplementation to trabecular cultures does not change chondro-osseous protein levels and rescues B lymphopoeisis in collagen X null cultures. (A) qRT-PCR was performed for the chondro-osseous markers collagen I (Col I), osteocalcin (OC), osteopontin (Opn), collagen II (Col II), and collagen X (Col X), following supplementation of WT trabecular cultures with IL-7, SCF, and SDF-1 (cytokines). (B) Immunostaining of WT trabecular cultures for collagen I (Col I) and osteopontin (OP) following supplementation with IL-7, SCF and SDF-1. Antibody controls are boxed in OP frames. Magnification 10×, bar=200 μM. (C, D) Quantitation of CD45.1+ (donor) CD19 (B cell marker) or Gr-1 (granulocyte cell marker) cells in WT and collagen X null (Col X-KO) mouse trabecular cultures following supplementation with IL-7, SCF, or SDF-1. Note the rescue of CD19+ cell outgrowth in Col X-KO trabecular cocultures after cytokine addition. Standard error of the mean shown for greater than five wells from multiple experiments per group.

FIG. 6.

In vivo IL-7 injections rescue B cell defect in collagen X mice. (A) Quantitation of Pro-B, and Pre-B marrow cells from WT, collagen X null and transgenic (KO and Tg) day 7 mice after 4 days of IL-7 injections. (B) Quantitation of total B220+ marrow cells in day 7 mice after IL-7 injections. (C) Early and late B marrow cells were quantified in day 21 mice after 4 days of IL-7 injections. *P<0.05, **P<0.001. n≥6 mice per group.