A single dose of thrombopoietin shortly after myelosuppressive total body irradiation prevents pancytopenia in mice by promoting short-term multilineage spleen-repopulating cells at the transient expense of bone marrow-repopulating cells.
Thrombopoietin (TPO) has been used in preclinical myelosuppression models to evaluate the effect on hematopoietic reconstitution. Here we report the importance of dose and dose scheduling for multilineage reconstitution after myelosuppressive total body irradiation (TBI) in mice. After 6 Gy TBI, a dose of 0.3 microgram TPO/mouse (12 microgram/kg) intraperitoneally (IP), 0 to 4 hours after TBI, prevented the severe thrombopenia observed in control mice, and in addition stimulated red and white blood cell regeneration. Time course studies showed a gradual decline in efficacy after an optimum within the first hours after TBI, accompanied by a replacement of the multilineage effects by lineage dominant thrombopoietic stimulation. Pharmacokinetic data showed that IP injection resulted in maximum plasma levels 2 hours after administration. On the basis of the data, we inferred that a substantial level of TPO was required at a critical time interval after TBI to induce multilineage stimulation of residual bone marrow cells. A more precise estimate of the effect of dose and dose timing was provided by intravenous administration of TPO, which showed an optimum immediately after TBI and a sharp decline in efficacy between a dose of 0.1 microgram/mouse (4 microgram/kg; plasma level 60 ng/mL), which was fully effective, and a dose of 0.03 microgram/mouse (1.2 microgram/kg; plasma level 20 ng/mL), which was largely ineffective. This is consistent with a threshold level of TPO required to overcome initial c-mpl-mediated clearance and to reach sufficient plasma levels for a maximum hematopoietic response. In mice exposed to fractionated TBI (3 x 3 Gy, 24 hours apart), IP administration of 0. 3 microgram TPO 2 hours after each fraction completely prevented the severe thrombopenia and anemia that occurred in control mice. Using short-term transplantation assays, ie, colony-forming unit-spleen (CFU-S) day 13 (CFU-S-13) and the more immature cells with marrow repopulating ability (MRA), it could be shown that TPO promoted CFU-S-13 and transiently depleted MRA. The initial depletion of MRA in response to TPO was replenished during long-term reconstitution followed for a period of 3 months. Apart from demonstrating again that MRA cells and CFU-S-13 are separate functional entities, the data thus showed that TPO promotes short-term multilineage repopulating cells at the expense of more immature ancestral cells, thereby preventing pancytopenia. The short time interval available after TBI to exert these effects shows that TPO is able to intervene in mechanisms that result in functional depletion of its multilineage target cells shortly after TBI and emphasizes the requirement of dose scheduling of TPO in keeping with these mechanisms to obtain optimal clinical efficacy.
Institute of Hematology, Erasmus University Rotterdam, Rotterdam, The Netherlands.
K J
KJ
Neelis
T P
TP
Visser
W
W
Dimjati
G R
GR
Thomas
P J
PJ
Fielder
D
D
Bloedow
D L
DL
Eaton
G
G
Wagemaker
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002452:Cell Count; D003114:Colony-Forming Units Assay; D005260:Female; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010198:Pancytopenia / Q000209:etiology / Q000517:prevention & control*; D011994:Recombinant Proteins / Q000627:therapeutic use; D013154:Spleen / Q000166:cytology*; D013926:Thrombopoietin / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000627:therapeutic use*; D014916:Whole-Body Irradiation / Q000009:adverse effects*
D011994:Recombinant Proteins; D013926:Thrombopoietin