Calcium Pyrophosphate Dihydrate Crystals Increase the Granulocyte/Monocyte Progenitor (GMP) and Enhance Granulocyte and Monocyte Differentiation In Vivo

Abstract

Calcium pyrophosphate dihydrate (CPPD) crystals are formed locally within the joints, leading to pseudogout. Although the mobilization of local granulocytes can be observed in joints where pseudogout has manifested, the mechanism of this activity remains poorly understood. In this study, CPPD crystals were administered to mice, and the dynamics of splenic and peripheral blood myeloid cells were analyzed. As a result, levels of both granulocytes and monocytes were found to increase following CPPD crystal administration in a concentration-dependent manner, with a concomitant decrease in lymphocytes in the peripheral blood. In contrast, the levels of other cells, such as dendritic cell subsets, T-cells, and B-cells, remained unchanged in the spleen, following CPPD crystal administration. Furthermore, an increase in granulocytes/monocyte progenitors (GMPs) and a decrease in megakaryocyte/erythrocyte progenitors (MEPs) were also observed in the bone marrow. In addition, CPPD administration induced production of IL-1β, which acts on hematopoietic stem cells and hematopoietic progenitors and promotes myeloid cell differentiation and expansion. These results suggest that CPPD crystals act as a "danger signal" to induce IL-1β production, resulting in changes in course of hematopoietic progenitor cell differentiation and in increased granulocyte/monocyte levels, and contributing to the development of gout.

Keywords: calcium pyrophosphate dihydrate crystals; granulocyte; monocyte.

Figure 1

Calcium pyrophosphate dihydrate (CPPD) injection induces reduction of white blood cells (WBCs) in the peripheral blood. (A) Experimental design for in vivo injection of CPPD. (B–D) The time course of cell number of WBCs (B), red blood cells (RBCs) (C), and platelets in the peripheral blood (PB) (D) were analyzed after vehicle (PBS) or CPPD (100 μg) injection. Data represent the mean ± s.d. of three independent experiments. * p < 0.01, ** p < 0.05.

Figure 2

CPPD injection induces myeloid cell expansion in vivo. (A,B) The percentage of granulocytes (Ly6G⁺CD11b⁺) and monocytes (Ly6G^intCD11b⁺) in the spleen (A) and peripheral blood (B) of the mice 3 days after the CPPD injection. (C,D) The total cell number of granulocyte and monocytes in the spleen (C) and PB (D) after CPPD injection of the indicated dose and at the indicated time course. (E,F) Total cell number of CD19⁺ B cells, and CD3⁺ T cell in the peripheral blood (E) and conventional dendritic cells (cDCs), plasmacytoid DC (pDC), and CD19⁺ B cells, and CD3⁺ T cells in the spleen (F). Data represent the mean ± s.d. of three independent experiments. * p < 0.01, ** p < 0.05.

Figure 3

CPPD injection induces granulocytes/monocyte progenitors (GMPs) expansion and megakaryocyte/erythrocyte progenitors (MEPs) reduction in vivo. (A–D) The percentage (A) and total cell number of lineage⁻ Sca-1⁺c-kit⁺ (LSK) cells (B) and megakaryocyte/erythrocyte progenitors (MEPs), common myeloid progenitors (CMPs), and granulocytes/monocyte progenitors (GMPs) in the bone marrow 3 days after vehicle (PBS) or CPPD (100μg) injection (C). (D) Time course of absolute cell number of GMP and MEP in the bone marrow after CPPD administration. Data represent the mean ± s.d. of three independent experiments. * p < 0.01.

Figure 4

CPPD injection induces IL-1β production in vivo. (A,B) Serum IL-1β production in WT mice 1 day after CPPD administration at the indicated concentration. (B) Kinetics of serum IL-1β production after CPPD administration. Data represent the mean ± s.d. of three independent experiments. * p < 0.01.