Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

Immunologic stimulation of early murine hematopoiesis and its abrogation by cyclosporin A.

Mice injected chronically with antiplatelet serum develop an increase in the number of megakaryocytic progenitor cells compared to animals given normal rabbit serum. To examine the specificity of this response, progenitor cells giving rise to megakaryocyte, granulocyte-macrophage, erythroid, and mixed-cell colonies were assayed after injection of various heterosera or saline. All four colony types increased in the serum-treated groups. Since the in vitro proliferation of hematopoietic progenitor cells is promoted by supernatants of mitogen-stimulated spleen cells, we hypothesized that the immune response following antiserum administration resulted in the in vivo activation of T lymphocytes which produced or led to the production of colony stimulating activities. To test this hypothesis, cyclosporin A, a preferential inhibitor of T lymphocyte function, was given to mice concurrently with antiserum and also added to spleen cell cultures in the presence of pokeweed mitogen. Cyclosporin A abrogated the antiserum-related increases in progenitor cell numbers in vivo and the production of colony stimulating activity in vitro. The results suggest that the immune response related to antiserum administration results in the in vivo production of hematopoietic colony stimulating activities that may be identical to those produced in vitro by mitogen-stimulation of spleen cells.

Megakaryocytopoiesis in the mouse: response to varying platelet demand.

The response of murine megakaryocytopoiesis was studied under conditions of varying platelet demand. Twenty-four hours after mice were given a single injection of rabbit anti-platelet serum, megakaryocyte number and volume were increased, becoming maximal at 65 and 40 hr, respectively. Total body megakaryocytic colony-forming unit (CFU-M) numbers did not change until 90 hr, when a 35% increase in the experimental group was noted. The percentage of CFU-M in DNA synthesis in the experimental group was 38 +/-2% at 24 hr, 49 +/- 1% at 40 hr, and returned to normal (11 +/- 3%) at 90 hr. When mice were made thrombocytotic by platelet transfusions, both megakaryocyte number and volume were decreased compared to controls, while no difference was noted in the number and percentage of CFU-M in DNA synthesis. Finally, experiments were performed to examine the effect of platelet transfusions on regenerating marrow. Experimental mice were given platelet transfusions while control animals received platelet buffer solution. At sacrifice the number and volume of megakaryocytes in the experimental group (platelet count 2.568 X 10(6)/microliters) were less than controls (platelet count 0.363 X 10(6)/microliters), while the number and percentage of CFU-M in DNA synthesis were similar in both groups. These results demonstrate that CFU-M are not immediately responsive to acute changes in platelet demand. The data suggest that megakaryocytopoiesis is structured on at least two levels which are independently regulated.

Effect of red cell hypertransfusion on thrombocytopoietic regeneration following irradiation and bone marrow transplantation in the mouse.

To examine the influence of suppressed erythropoiesis on platelet recovery following marrow injury, C57B1/6 mice were given red cell transfusion or Ringer's citrate. Twenty-four hours later, the animals were given either lethal (9 Gy) irradiation followed by syngeneic marrow or sublethal (3.5 Gy) irradiation and allowed to recover. The adequacy of erythroid suppression was shown by a marked decline in erythroid colony-forming cells (CFU-E) in both marrow and spleen. In both irradiation groups, platelet counts were lower in the hypertransfused mice when compared to nontransfused controls. This decrease in platelet count was observed irrespective of the degree of polycythemia or the time of observation. To determine whether red cell hypertransfusion also affects megakaryocytopoiesis, megakaryocyte colony-forming cells (CFU-MK), megakaryocyte numbers in femoral marrow sections, and megakaryocyte volumes were measured. An increase in CFU-MK-derived colonies was observed in the marrows of hypertransfused animals in both irradiation groups, and in the spleen in the group which received 3.5 Gy. Megakaryocyte numbers and volumes in femoral marrow sections were increased in the hypertransfused mice given 3.5 Gy, but not in the group given 9 Gy. The data show that despite complex effects on megakaryocytes and their progenitors, red cell hypertransfusion delays rather than enhances platelet recovery.