Multipotent-lineage nondominant growth factors, acting alone or in combination with lineage-dominant cytokines, are known to influence both myelopoiesis and thrombocytopoiesis. Interleukin (IL)-3 and IL-11 stimulate and expand multilineage progenitor cells and induce thrombocytopoiesis. These cytokines also act synergistically with various other lineage dominant and lineage-nondominant cytokines in vitro to expand primitive and committed hematopoietic stem cells. In this study we investigated the in vivo effects of IL-3 and IL-11 in combination with the c-mpl ligand, thrombopoietin (rhTPO), on neonatal rat hematopoiesis. Newborn Sprague-Dawley rats (24 36 hours old, weighing 6-8 g) were intraperitoneally injected with rhTPO (10 microg/kg) for 14 days, rmIL-3 (10 microg/kg) for 5 days followed by rhTPO (10 microg/kg) for 9 days, rmIL-3 (10 microg/kg) + rhTPO (10 microg/kg) for 14 days, rhIL-11 (250 microg/kg) + rhTPO (10 microg/kg) for 14 days, or PBS/human serum albumin (HSA) for 14 days. When compared with PBS/HSA, rhTPO at a dosage of 10 microg/kg significantly increased platelet count (10(-9) L) (day 6, 569 +/- 37.1 vs. 1446 +/- 43.8, p < 0.001; day 10, 796 +/- 68.3 vs. 1774 +/- 238.4, p < 0.01; day 14, 850 +/- 64.4 vs. 3441 +/- 98.1 /10(-9) l, p < 0.001) and absolute neutrophil count (ANC) (day 6, 335.2 +/- 59.6 vs. 752 +/- 335.2, p < 0.01; day 12, 664 +/- 54.1 vs. 1520 +/- 158.2, p < 0.01). However, rhTPO has no effect on the circulating hematocrit or red blood cell count. RhTPO-treated animals also displayed higher platelet counts (/10(-9) L) vs. rhIL-11 or rhIL-6 beginning on day 6 (day 6, 1597.6 +/- 134.7 vs. 930.7 +/- 67.3 vs. 863 +/- 19.6, p < 0.01; day 8, 1686 +/- 208.4 vs. 990 +/- 29.4, vs. 977 +/- 34.33, p < 0.05; day 10, 1774 +/- 238.4 vs. 1096 +/- 49.6, vs. 937 +/- 65, p < 0.01; day 14, 2187 +/- 127.5 vs. 1280 +/- 35.8 vs. 951 +/- 50.7 /10(-9) L, p < 0.01). Sequential administration of rmIL-3 followed by rhTPO resulted in no significant increase in platelet counts compared with PBS-HSA/rhTPO. RhTPO + rmIL-3 given simultaneously also had no additive effect on the circulating platelet count compared with rhTPO alone. Similarly, no additive effect on circulating platelet counts was observed with rhIL-11 + rhTPO vs. rhTPO alone. Bone marrow studies showed a significant increase in the number of megakaryocytes per high-power field in all the groups treated with rhTPO vs. control (p < 0.05), but no additive effect was seen in neonatal rats additionally receiving either rmIL-3 or rhIL-11. Colony forming unit (CFU)-Meg colony formation was also significantly increased in all the groups treated with rhTPO vs. control (p < 0.05), with no additive effect observed after the addition of either rmIL-3 or rhIL-11. These data suggest that rhTPO is more effective than rmIL-3 or rhIL-11 in inducing neonatal in vivo thrombocytopoiesis in rats, and that no additive effect is to be expected when rhTPO is combined sequentially with rhIL-3 or simultaneously with either rmIL-3 or rhIL-11. We hope that these preclinical data will provide insight into the design and future application of these thrombopoietic cytokines, alone or in combination, to prevent or treat thrombocytopenia.