Figure 1
Figure 1. Arthritic KSL cells have increased in vitro myeloid potential. (A) Experimental scheme for in vitro competitive culture of arthritic and control KSL cells. Arthritic and control mice have different CD45 (Ly5) alleles. Arthritic and control KSL cells (5000 each) were mixed in the same well of a 24-well plate with survival cytokines, KitL+, Flt3L, and media containing 10% serum. (B left panel) FACS plot depicting myeloid differentiation of KSL cells in vitro after 3.5 days of culture. Myeloid cells (red gate) are Mac1+ and/or Gr1+ and negative for B-cell (B220), T-cell (CD3, TCRβ), and erythrocyte (Ter119) markers. Approximately 40%-60% of cells were present in this gate. (Right panel) FACS plot depicting analysis to determine arthritic KSL-derived (Ly5.2+) and control KSL-derived (Ly5.1+) myeloid cells. (C) Summary of in vitro competitive culture results. Each bar represents outcome of KSL cells from 1 independent arthritic mouse competed with control KSL cells. In 5 of 6 trials, arthritic KSL cells (black) contributed greater to myeloid cell output. (D) Outcome of culture of arthritic and control KSL cells on irradiated OP9 cells and media containing 10% serum, KitL, Flt3L, IL-7 to promote lymphoid cell formation. To ensure that lymphoid cells were being analyzed, B220+ cells lacking Gr1 or lacking Mac1 and CD11c were analyzed. Control and arthritic KSL-derived cells were distinguished based on CD45 (Ly5) allele expression. In 6 of 6 trials, arthritic KSL cells were inferior to control KSL cells in lymphoid cell output. (E) Tartrate-resistant acid phosphatase (TRAP) stained wells at least 6 days after culturing sorted KSL and GMPs (Kit+Sca1−Lin−CD34+FcγRIII/IIbhi) with M-CSF and RANKL to promote osteoclastogenesis. Low-magnification camera images and high-magnification (40×; 4× objective, 10× eyepiece) microscopy images of wells are shown. TRAP+ cells are pink/purple. The few TRAP+ cells in wells seeded with control KSL cells are not multinucleate unlike arthritic KSL- and GMP-derived cells. The single high-magnification image labeled GMP is representative of osteoclastogenesis from arthritic or control GMPs which gave similar results. (F) Experimental scheme for arthritis induction in naive mice (B6xG7) by injection of serum. Control mice were injected with PBS. Serum injection was administered on day 0 (D0) and day 2 and KSL cells sorted for competitive in vitro stromal cell–free and OP9 culture with indicated cytokines on day 7. (G) Relative contribution of KSL cells from arthritic (serum-injected B6xG7 mice) and control (PBS-injected B6xG7 mice) to Mac1/Gr1+ cells generated in stromal cell–free culture (top panel) or to B220+CD11c−Mac1− cells generated in OP9 culture (bottom panel). See panel F for experimental scheme. Each bar represents outcome of KSL cells from 1 independent arthritic mouse competed with control KSL cells.

Arthritic KSL cells have increased in vitro myeloid potential. (A) Experimental scheme for in vitro competitive culture of arthritic and control KSL cells. Arthritic and control mice have different CD45 (Ly5) alleles. Arthritic and control KSL cells (5000 each) were mixed in the same well of a 24-well plate with survival cytokines, KitL+, Flt3L, and media containing 10% serum. (B left panel) FACS plot depicting myeloid differentiation of KSL cells in vitro after 3.5 days of culture. Myeloid cells (red gate) are Mac1+ and/or Gr1+ and negative for B-cell (B220), T-cell (CD3, TCRβ), and erythrocyte (Ter119) markers. Approximately 40%-60% of cells were present in this gate. (Right panel) FACS plot depicting analysis to determine arthritic KSL-derived (Ly5.2+) and control KSL-derived (Ly5.1+) myeloid cells. (C) Summary of in vitro competitive culture results. Each bar represents outcome of KSL cells from 1 independent arthritic mouse competed with control KSL cells. In 5 of 6 trials, arthritic KSL cells (black) contributed greater to myeloid cell output. (D) Outcome of culture of arthritic and control KSL cells on irradiated OP9 cells and media containing 10% serum, KitL, Flt3L, IL-7 to promote lymphoid cell formation. To ensure that lymphoid cells were being analyzed, B220+ cells lacking Gr1 or lacking Mac1 and CD11c were analyzed. Control and arthritic KSL-derived cells were distinguished based on CD45 (Ly5) allele expression. In 6 of 6 trials, arthritic KSL cells were inferior to control KSL cells in lymphoid cell output. (E) Tartrate-resistant acid phosphatase (TRAP) stained wells at least 6 days after culturing sorted KSL and GMPs (Kit+Sca1−Lin−CD34+FcγRIII/IIbhi) with M-CSF and RANKL to promote osteoclastogenesis. Low-magnification camera images and high-magnification (40×; 4× objective, 10× eyepiece) microscopy images of wells are shown. TRAP+ cells are pink/purple. The few TRAP+ cells in wells seeded with control KSL cells are not multinucleate unlike arthritic KSL- and GMP-derived cells. The single high-magnification image labeled GMP is representative of osteoclastogenesis from arthritic or control GMPs which gave similar results. (F) Experimental scheme for arthritis induction in naive mice (B6xG7) by injection of serum. Control mice were injected with PBS. Serum injection was administered on day 0 (D0) and day 2 and KSL cells sorted for competitive in vitro stromal cell–free and OP9 culture with indicated cytokines on day 7. (G) Relative contribution of KSL cells from arthritic (serum-injected B6xG7 mice) and control (PBS-injected B6xG7 mice) to Mac1/Gr1+ cells generated in stromal cell–free culture (top panel) or to B220+CD11c−Mac1− cells generated in OP9 culture (bottom panel). See panel F for experimental scheme. Each bar represents outcome of KSL cells from 1 independent arthritic mouse competed with control KSL cells.

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