Effects of recombinant human granulocyte-macrophage colony-stimulating factor on platelet survival and activation using a nonhuman primate model.

In humans and nonhuman primates, the in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) consistently results in marked increase of megakaryocyte ploidy and size similar to that observed with interleukin-6 (IL-6). However, whereas the administration of IL-6 also results in an increase in circulating platelets, there is no predictable corresponding increase in peripheral blood platelets following treatment with rhGM-CSF. To determine whether the failure of rhGM-CSF to produce thrombocytosis is secondary to cytokine-related increased platelet activation and consumption in vivo, we quantified autologous platelet survival time and in vivo platelet activation before and during 5 days of administration of rhGM-CSF to two rhesus monkeys. Platelet survival was measured using autologous platelets labeled with 111Indium-oxine. Platelet activation was assessed by flow cytometric determination of the expression of the major platelet membrane glycoprotein (GP) IIb/IIIa complex, and an activation-dependent epitope on GPIIb/IIIa (recognized by monoclonal antibodies [MABs] LJ-P4 and PAC1, respectively). Platelet activation was also assessed by dose-response aggregometry using adenosine diphosphate (ADP). While megakaryocyte ploidy increased during rhGM-CSF administration, peripheral platelet counts were 418 x 10(9)/L and 525 x 10(9)/L before and 402 x 10(9)/L and 508 x 10(9)/L during cytokine treatment in animals 1 and 2, respectively. No changes were observed in the mean platelet volume. 111Indium-labeled platelet recovery in circulation was similar before (94.7%, 91.8%) and during (92.9%, 92.8%) rhGM-CSF administration, which indicates that cytokine-related in vivo sequestration of platelets does not occur. Autologous platelet survival was 5.6 and 6.2 days before and 5.0 and 5.4 days during the rhGM-CSF treatment (p = 0.07), without significant change in the corresponding platelet turnover rate (derived from the platelet count and survival time). The flow cytometric analysis showed no increase in the binding of either LJ-P4 or PAC1 MABs to the platelet membrane during rhGM-CSF administration. The aggregometry studies demonstrated similar concentrations of ADP inducing half-maximal aggregation (ED50). Overall, the above data indicate that treatment with rhGM-CSF is not associated with in vivo activation, sequestration, or increased consumption of platelets. The data suggest that the failure of rhGM-CSF-stimulated megakaryocytes to increase peripheral platelet count is a manifestation of ineffective megakaryocytopoiesis resulting from inability to increase platelet delivery to the circulation.

Protein S deficiency in men with long-term human immunodeficiency virus infection.

Decreases in protein S levels have recently been reported in some human immunodeficiency virus (HIV)-infected patients. To examine predisposing factors, 25 men randomly selected from a long-term study of HIV-infected patients were studied. The minimum mean duration of HIV seropositivity in this group was 106.6 months (range 15 to 143 months). No patients were anticoagulated at the time of the study. Three of the 25 randomly selected patients gave a history of thrombosis, in each instance occurring after the onset of HIV positivity. Two of the 3 patients with thrombosis had more than one episode. Coagulation studies showed that 3 of 3 (100%) of the patients with thrombosis and 16 of 22 (72.7%) of those without previous thrombosis had decreased free protein S. Mean-free and total protein S levels were statistically lower for HIV-infected patients with and without previous thrombosis compared with healthy male controls. C4b-binding protein was not increased in study patients with decreased protein S levels. Decreases in protein S levels did not correlate with CD4+ cell levels, CDC class, p24 antigen positivity, zidovudine (AZT) use, or Pneumocystis carinii prophylaxis. The duration of disease statistically correlated with decreases in protein S levels (r = .37, P < .05). A linear correlation existed between increasing IgG anticardiolipin antibody levels and decreasing free protein S antigen (r = .67, P < .005). This study shows that protein S deficiency is common in long-term HIV-infected patients and is caused by a decrease in the free protein, rather than by changes in the bound complex. The data suggest that protein S deficiency is not correlated with HIV disease severity but may predispose patients to thromboembolic complications.

Differential effects of sequential, simultaneous, and single agent interleukin-3 and granulocyte-macrophage colony-stimulating factor on megakaryocyte maturation and platelet response in primates.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM-CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.

Studies of an improved rhesus hematopoietic progenitor cell assay.

We have improved Rhesus monkey marrow cell growth in semisolid media by means of substituting supplemented calf serum for fetal bovine serum. The cloning efficiency of light-density marrow cells separated on 60% Percoll was 126 (+/- 54)/10(5) (n = 12, +/- SD), and for light-density peripheral blood cells 60 (+/- 46)/10(6) (n = 11). Thirty-five percent of the colonies were multilineage, whereas the remainder were unilineage colonies composed of erythrocytes, megakaryocytes, and neutrophilic or monocytic granulocytes. Unilineage megakaryocyte colonies comprised 12% of the total marrow progenitor cells. The [3H]TdR suicide index of marrow progenitor cells was 47% +/- 9% (n = 12). This progenitor cell assay should prove useful in preclinical studies of the effect of recombinant hematopoietic growth factors on the number and cycling status of Rhesus hematopoietic progenitor cells.

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates.

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.

Effects of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates.

Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in mice and primates. To elucidate the mechanisms underlying this phenomenon, serial analyses were performed on megakaryocytes obtained from rhesus monkeys treated for 8 days with 30 micrograms/kg/d of recombinant human IL-6. Platelet counts increased to a maximum of 7.8 x 10(5)/microL with biphasic peaks on days 7 and 12 without significant changes in platelet volumes. Large increases in DNA content were seen by two-color flow cytometry and digital image analysis. Ploidy distribution underwent a significant shift between study days 3 and 11 (P less than .0001) with large increases in the frequency of 64N and 128N megakaryocytes. The modal ploidy increased from the normal 16N to 64N. Megakaryocyte size, as measured by area, was increased 2- to 2.7-fold. On day 3, multiple megakaryocytes were seen in endomitosis, along with an abundance of young cells with wide, organelle-free peripheral zones. The giant megakaryocytes seen on days 5 to 7 exhibited marked membrane hyperplasia that occupied much of the cell. Emperipolesis occurred frequently, as did megakaryocyte cell death. No giant platelets were seen. We conclude that IL-6 significantly alters the process of megakaryocyte maturation and thrombocytopoiesis, and that these effects, at least in the doses of IL-6 administered, should not be equated with the physiologic mechanisms operative during accelerated platelet production.

Incomplete antigenic cross-reactivity between platelets and megakaryocytes: relevance to ITP.

Immune thrombocytopenias are usually associated with normal or increased numbers of megakaryocytes in the marrow. Therefore, the mechanism(s) responsible for the destruction of circulating platelets may not affect megakaryocytes in the same way. One of the possibilities which could account for the differential effect on the cells would be the development of antibodies to components of platelet membranes which are not exposed on the surface of all megakaryocytes. To investigate this possibility, a rabbit antiserum specific for mouse platelets was tested against fresh and cultured mouse megakaryocytes by indirect immunofluorescence. This antiserum cross-reacted with 46% of fresh murine megakaryocytes and 54% of cultured megakaryocytes. Phase-contrast microscopy revealed the reacting megakaryocytes to be fully granulated with irregular contours and in the process of releasing platelets. Nonreactive megakaryocytes demonstrated smooth contours and lacked morphological evidence of thrombocytopoiesis. Electron microscopy showed that only in megakaryocytes (MK) with an irregular contour had the demarcation membrane system (DMS) reached continuity with the plasma membrane. Ultrastructural analysis of megakaryocytes from patients with ITP showed approximately 25% to 50% of megakaryocytes without evidence of injury, whereas 50% to 75% had extensive damage. In undamaged cells, platelet territories had not yet reached the peripheral zone. The DMS of damaged megakaryocytes opened to the exterior elaborating platelets. The observations suggested that some platelet antibodies react only with megakaryocytes which have reached the stage of thrombocytopoiesis. Relevant target antigens may not be exposed on all megakaryocytes before cytoplasmic fragmentation occurs.