CCL3 Regulates Normal Hematopoiesis but Is Not Essential for the Maintenance of a Long-Term Engrafting Hematopoietic Stem Cell

Background/Rationale: Hematologic malignancies are known to remodel the bone marrow microenvironment, reducing support for normal hematopoiesis while increasing support for the malignant clone. The chemokine CCL3 has been demonstrated to play a role in microenvironmental dysfunction in multiple malignancies including myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome. In addition, CCL3 has been shown to be critical for the progression of chronic mylogenous leukemia in murine models. However, to consider anti-CCL3 therapy as an option for hematologic malignancies we must first understand its role in the regulation of normal hematopoiesis. To date the role of CCL3 in this process is poorly understood.

Methods/Results: In these experiments we utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. Peripheral blood counts revealed that monocytes, granulocytes, and red blood cells were all significantly decreased in the peripheral blood of CCL3KO mice as compared to WT controls at 12 weeks of age (9.78 ± 0.3 vs. 8.06 ± 0.2 RBCs*10⁶/μl, WT vs. CCL3KO p≤0.001 n=8 mice/group). CCL3KO mice also demonstrate a 2-fold increase in the frequency and number of phenotypic long-term hematopoietic stem cells (LT-HSCs: Lin-sca1+ckit+flt3-CD150+CD48-) at 12 weeks of age in the bone marrow by flow cytometric analysis (0.0053 ± 0.0005 vs. 0.0106 ± 0.0007 % of cells, WT vs. CCL3KO p≤0.0001 n=8 mice/group). A significant increase was also seen in short-term HSCs (ST-HSCs), but not in multipotent progenitor (MPP) populations (data not shown), suggesting that CCL3 regulates the most immature hematopoietic cells. To quantify functional hematopoietic stem cells in the marrow of CCL3KO mice competitive transplants were performed using whole bone marrow cells. In primary transplants CCL3KO mice demonstrated a small but significant decrease in engraftment over 22 weeks when compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Decreased engraftment was seen in B cells, T cells, and myeloid cells in the peripheral blood. Upon secondary transplantation the decrease in engraftment of HSCs from CCL3KO donor mice was much more profound. At 16 weeks post-transplant HSCs from CCL3KO donors contributed to hematopoiesis at a rate 5 times lower than WT littermate controls (64.67 ± 1.967 vs. 11.97 ± 5.322 % of cells, WT vs. CCL3KO p≤0.0001 n=10 mice/group). These results were seen in both male and female mice and suggest that, although phenotypic HSCs were increased in the bone marrow of CCL3KO mice, those HSCs were defective. To test this hypothesis we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for LT-HSCs in order to establish stem cell activity on a per cell basis through competitive transplantation. As with the whole bone marrow transplants, primary transplant of sorted Flt3-LSK cells resulted in reduced engraftment of CCL3KO cells as compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Surprisingly, upon secondary transplantation, CCL3KO Flt3-LSK donor cells performed better than the WT littermate controls (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). This result suggests that a transplantable population of cells excluded by the Flt3-LSK sorting parameters is responsible for repression of long-term engraftment capacity of marrow from CCL3KO mice. In establishing the mechanism by which CCL3 regulates hematopoiesis we investigated the rate of apoptosis by quantification of caspase 3 activation, as well as cell cycle status by quantification of Ki67 positivity and DNA content by flow cytometric analysis. We found no difference in the rate of apoptosis, however there was a significant decrease in the fraction of short term HSCs (ST-HSCs) (Flt3-CD48-CD150-LSK) that were actively cycling (2.06 ± 0.43 vs. 1.23±0.44 % of ST-HSCs WT vs. CCL3KO p<0.05 n=3 mice/group). This suggests that CCL3 regulates the proliferation of hematopoietic progenitor cells downstream of the LT-HSC.

Conclusions: These results highlight a role for the chemokine CCL3 in the maintenance of the hematopoietic system under benign, physiologic conditions. However, a long-term engrafting HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well tolerated by the hematopoietic system.