Samuel A. Burstein

Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the diameters of these cells. From a Bayesian analysis of the likelihood that some megakaryocytes increased in DNA content during the culture period, we estimate that 61% of the cells increased in ploidy. These data indicate that the action of erythropoietin is not restricted to the erythroid lineage.

Elevated serum interleukin-6 levels in patients with reactive thrombocytosis

Summary Interleukin‐6 (IL‐6) is known to promote mega‐karyocytopoiesis in vitro and raise platelet counts in vivo. To determine if there is a relationship between circulating IL‐6 and thrombocytosis in man, we measured bioactive IL‐6 in the serum of 13 patients with myeloproliferative disorders and 143 patients with reactive thrombocytosis having platelet counts > 600 × 109/I. IL‐6 activity was assayed using the IL‐6‐responsive B9 cell line. Seventy‐one controls with normal platelet counts had a mean IL‐6 level of 2.19 U/ml × 1.08 (SD). None of the 13 patients with myeloproliferative disorders had elevated IL‐6 levels (1.56 U/ml × 1.2). In contrast, serum IL‐6 levels of 143 patients (158 samples) with reactive thrombocytosis were significantly greater than controls (38.3 U/ml × 94.6; P < 0.001), with 83% of the samples showing elevated serum IL‐6. No significant correlation was observed between serum IL‐6 levels and platelet counts in the reactive thrombocytosis group. We conclude that elevated IL‐6 is associated with reactive thrombocytosis, and hypothesize that the increased platelet count in many cases is causally related to elevated IL‐6.

Biotinylated platelets: a new approach to the measurement of platelet life span

Summary The in vivo life span of dog platelets was determined by derivatizing whole blood with N‐hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 ± 1.1 d as determined by curve‐fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either 111Indium‐oxine or 51Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.

Serum Interleukin-6 Levels in Patients with Thrombocytosis

Interleukin-6 (IL-6) has been shown to increase platelet counts in several animal models and to enhance megakaryocytopoiesis in vitro. In order to investigate the possible relationship between IL-6 and thrombocytosis, serum IL-6 levels in patients with platelet counts > or = 6 x 10(5)/microliters were measured using an IL-6-responsive bioassay. A cohort of healthy volunteers with normal platelet counts was used to establish a control mean serum IL-6 level [2.19 U/ml +/- 1.08 SD (range 0-5.5)]. Patients with primary thrombocytosis had a mean serum IL-6 level not significantly different from controls. In comparison, serum IL-6 levels of patients with reactive thrombocytosis were significantly greater than controls (38.3 U/ml +/- 94.6; range 0-933; P < 0.001). Although no significant correlation was observed between the degree of serum IL-6 elevation and the height of the platelet count in any individual, elevated serum IL-6 was highly correlated with reactive thrombocytosis.

Recombinant human macrophage colony‐stimulating factor‐induced thrombocytopenia in dogs

Summary. To characterize recombinant human macrophage‐colony stimulating factor (rhM‐CSF)‐associated thrombocytopenia (TCP), in vivo studies were performed in dogs, including the biodistributions and recoveries of radiolabelled autologous and allogeneic platelets. rhM‐CSF induced a reversible, dose‐dependent decrease in platelet counts. The number of megakaryocytes in spleen and marrow of rhM‐CSF‐treated dogs was increased two to threefold. Recoveries of allogeneic platelets transfused from rhM‐CSF‐treated donors into tolerized recipients (n = 3) were not significantly different from allogeneic baseline studies (93 ± 10% of baseline values at 24 h and 90 ± 1% at 40 h), whereas autologous platelets infused back into rhM‐CSF‐treated donors had decreased recoveries (45 ± 2% of baseline values at 24 h, P = 0·03 and 20 ± 4% at 40 h, P = 0·001). Platelet biodistribution studies showed increased accumulation of radiolabelled platelets over the spleens and livers of rhM‐CSF‐treated dogs. Histochemistry showed increased levels of platelet‐specific antigen (CD41; glycoprotein IIb) associated with Kupffer cells. The sensitivity of platelets from rhM‐CSF‐treated dogs to activation from thrombin, as measured by expression of P‐selectin (CD62P), was not significantly different when compared with baseline studies (P = 0·18; n = 4). These results support the concept that rhM‐CSF induces an activation of the monocyte–macrophage system (MMS), which causes a reversible TCP in a dog model.

Inhibition of the acute‐phase response in vivo by anti‐gp130 monoclonal antibodies

The acute‐phase response is believed to be an important systemic defence reaction to inflammation during infection, trauma, injury or neoplasia. Although the interleukin‐6 (IL‐6) family of cytokines appear to be the major regulators of the acute‐phase reaction, the exact biological significance of this process remains unknown. In this study, a panel of monoclonal antibodies (Mabs) was raised against the extracellular domain of human gp130 (the common signal transducing chain of the IL‐6 cytokine family) in order to inhibit the biological activity of IL‐6‐like cytokines in vivo. Mabs designated 4B11 and 2H4 were most effective in the inhibition of the in vitro acute‐phase response on hepatoma cells and prevented the IL‐6‐induced growth inhibition of A375 cells. Administration of the antibodies to dogs at a dosage of 8 mg/kg/d showed that 2H4 was a potent inhibitor of the IL‐6‐induced (40 μg/kg/d) acute‐phase response, abrogating IL‐6‐mediated increments in fibrinogen, C‐reactive protein and the platelet count. This antibody, the first described to abrogate the acute‐phase response in vivo, may not only permit development of a new anti‐inflammatory strategy, but provides an excellent tool for defining the function of acute‐phase proteins in inflammation and infection.