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Mobilization with Cyclophosphamide Shifts Hematopoiesis From the Bone Marrow to the Spleen In Mouse.

Ludek Sefc, Ko-Tung Chang, Viktor Sykora and Emanuel Necas

Abstract

Abstract 1590

Introduction: Spleen is an active hematopoietic tissue in adult mice. Spleen niches supporting hematopoiesis were not extensively studied yet. Spleen does not contain osteoblastic niches which are considered to be essential for long-term hematopoiesis, and splenic hematopoiesis is assumed to originate mainly from progenitors with short-term repopulating ability. Normal spleen is primarily a lymphoid organ but it also contributes to myelopoiesis by containing approximately 5% of total body multipotent progenitors CFU-S. We have studied the effect of mobilization with cyclophosphamide on trafficking of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the spleen, and dependence of normal splenic hematopoiesis on continual supply of HSPC from the bone marrow.

Method: Murine congenic model C57Bl/6 Ly5.1/Ly5.2, and C57Bl/6 GFP, was used in the experiments. Kinetics of HSPC in the bone marrow and the spleen after a sublethal dose of cyclophosphamide (135 mg/kg) was measured by clonal assays (CFU-GM and CFU-S – progenitors) and by competitive repopulation assay (short- and long-term repopulating cells, STRC and LTRC). Parabiotic pairs were established from control and cyclophosphamide-treated partners in order to demonstrate HSPC exchange between hematopoietic organs. Selective irradiation of either spleen with bone marrow shielded, or irradiation of bone marrow with spleen shielded, was used to measure significance of HSPC migration from the spleen to the bone marrow during hematopoietic regeneration. Selected cytokine expression in the bone marrow and in the spleen was determined by real-time PCR.

Result: Bone marrow HSPC rapidly but transiently regenerated from cyclophosphamide damage. Secondary decrease in HSPC between 5 and 7 days after cyclophosphamide in the bone marrow was accompanied with significant reduction of SDF-1 mRNA expression, mobilization of HSPC into peripheral blood and their rapid accumulation in the spleen. Spleen thus transiently became the main hematopoietic organ in the mouse by containing 69% of total body CFU-S, and also contained significantly more LTRC compared to the femur (Fig).

Surprisingly, there was no increase in SDF-1 mRNA expression during homing of HSPC in the spleen. Spleens of parabiotic partners were rapidly colonized by partner cells including HSPC within two weeks. Bone marrow also contained small but significant amount of partner HSPC one and 2 weeks after parabiosis establishment. In pairs made from control and cyclophosphamide treated mice, mobilized HSPC preferentially engrafted in both own and partner spleens. Spleen cellularity and progenitor content was not affected by selective spleen irradiation and was restored from shielded bone marrow 6 days after irradiation. Expansion of extramedullary splenic hematopoiesis after cyclophosphamide occurred even when HSPC were destroyed in the spleen 1 day after cyclophosphamide, i.e. the spleen expansion was dependent on the bone marrow. Irradiation of bone marrow with spleen shielded led to abruption of splenic hematopoiesis in both control and cyclophosphamide treated mice.

Conclusion: There is a quantitatively significant trafficking of HSPC by means of the circulation in mice. The splenic hematopoiesis appears to be almost fully dependent on a continuous supply of HSPC from the bone marrow. This trafficking of HSPC can be enhanced by hematopoietic cell mobilization. In mice, damage caused by cyclophosphamide results in a transient, however quantitatively remarkable relocation of HSPC, including LTRC, from the bone marrow to the spleen. This accumulation of HSPC in spleen is not accompanied with increased expression of SDF-1 mRNA. HSPC circulating in peripheral blood also significantly colonize the bone marrow and actively participate in its hematopoiesis. Support: Projects LC06044 and MSM 0021620806 of the Ministry of Education, Youth and Sports of the Czech Republic.

Disclosures: No relevant conflicts of interest to declare.