The Hemoglobin-Haptoglobin Receptor (CD163) Is Expressed by a Subpopulation of Erythroid and Granulo-Macrophagic Progenitors and Can Be Stimulated in Vitro and in Vivo by Physiological and Pharmacological Ligands to Stimulate Hematopoiesis

Abstract 1221

The receptor for the hemoglobin (Hb)-haptoglobin (Hp) complex, CD163, is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells (HPCs) (Matthews et al, 2006). The purpose of the studies presented here were two-fold – to demonstrate that the CD34⁺CD163⁺ double positive population could be isolated from normal adult bone marrow cells and these cells were functional as HPCs and, in addition, that these cells could be stimulated in vivo by ligands to CD163 to affect hematopoiesis. To investigate the clonogenic potential of CD34⁺/CD163⁺ HPCs, bone marrow CD34⁺ cells were examined for CD163 co-expression, sorted by fluorescence activated cell sorting (FACS) and plated into colony-forming assays (CFAs). 4.2% ± 1.4% (n=4) of CD34⁺ cells were found to co-express CD163 and this population consisted of two distinct sub-populations, CD34^{++ (hi)}CD163⁺ and CD34^+(lo)CD163⁺, each of which represented approximately half of the total CD34⁺CD163⁺ population. All three sorted populations (CD34^+(all)CD163⁻, CD34^++(hi) CD163⁺, CD34^+(lo)CD163⁺) were plated into CFAs (n=4) and were assessed for erythroid and myeloid colony formation. The clonogenic efficiency of CD34^++(hi)CD163⁺ had a 2.5-fold increase in the number CFU-E and CFU-GM when compared to both CD34^{+ (total)} CD163⁻ and CD34^+(lo) CD163⁺ cells. In contrast, CD34^{+(hi an low)}CD163⁺cells produced fewer BFU-E.

To determine how the expression of CD163 expression on progenitor cells may play a role in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb/Hp) as well as an agonistic antibody to CD163 (TBI 304) on HPCs in vivo. NOD-scid IL2R gamma^null (NSG) mice (HuMurine Technologies) were engrafted with human CD34⁺cells and animals with < 30% human CD45⁺ cells in the peripheral blood were administered either 2 mg Hb/mouse, or 100 or 500 μg/mouse TBI 304 every 4 days. At study termination (day 14), bone marrow cells (BMC) were examined by flow cytometry and enriched for CD34⁺ cells for enumeration in CFAs. Hb administration resulted in an increase of human CD34⁺cells ranging from 4% to 7% of BMC and a corresponding 57% increase in colony-forming cells (CFC) when compared to control (PBS-administered) animals. In contrast, TBI 304 produced a dose dependent decrease in CD34⁺ and CFC, possibly reflecting a depletion of CD34⁺/CD163⁺ cells from overstimulation due to the longer circulating antibody. To investigate this, human CD34⁺ cell engrafted animals were given a single dose of 10 or 100 μg/mouse of TBI 304 and bone marrow cells were examined on day 7. TBI 304 provided a 3.5-fold increase in human CD34⁺ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA⁺, huCD36⁺ and huCD71⁺) and a 2-fold increase in erythroid and myeloid colony-forming cells. No overall toxicities were observed with the administration of TBI 304 or Hb.

We have demonstrated that CD163 is expressed on a population of CD34⁺ hematopoietic progenitor cells, these cells have increased hematopoietic progenitor activity in vitro and that administration of physiological or pharmacological agonists of the CD163 receptor can measurably stimulate hematopoiesis in vivo.