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Circadian changes in in vitro pharmacodynamic effects of recombinant mouse interleukin-3 (rmIL-3), rm granulocyte-macrophage colony-stimulating factor (rmGM-CSF), and recombinant human G-CSF (rhG-CSF) were investigated in 418 male B6D2F1 mice. Seven distinct experiments were staggered from July to December 1991. All mice were standardized for 3 weeks with a lighting schedule consisting of 12 hours of light and 12 hours of dark (LD12:12). In each experiment, bone marrow was sampled from separate groups of nine to 10 mice each every 4 hours for 24 hours. Data were analyzed with analysis of variance (ANOVA) and Cosinor. This latter method computes the probability of rhythm detection and its parameters. Femoral myeloid progenitors were quantified using the colony-forming units granulocyte/macrophage (CFU-GM) assay in the presence or absence of recombinant CSFs. For each CSF, the number of colonies is a function of circadian time of bone marrow exposure (ANOVA and Cosinor; p < 0.0001) with the values at peak time being double those found at the trough. Peak CSF efficacy occurred at 3 hours after light onset (HALO, early rest span) irrespective of CSF type or dose. Furthermore, in the absence of any added CSF, the number of clusters varied significantly according to sampling time, with a similar peak at 3 HALO (ANOVA and Cosinor; p < 0.001). Further in vivo chronopharmacologic experiments are needed to assess the relevance of these in vitro rhythms in bone marrow responsiveness to hematopoietic growth factors.

Cocultivation of erythroid leukemic cells (ELM-I-1) with hemopoietic supportive cells (MS-5) resulted in a specific adhesion of ELM-I-1 cells to MS-5 cells. This phenomenon was designated as rosette formation. After induction of differentiation of ELM-I-1 cells, rosette formation was reduced, and no rosette formation was observed between erythrocytes and MS-5 cells. Studies on anti-adhesion molecule antibody treatment have revealed that CD44 plays a key role in rosette formation. Expression of CD44 on (the membrane of) ELM-I-1 cells was reduced after differentiation, and no CD44 expression was detected on erythrocytes. CD44 was also expressed on MS-5. Hyaluronate is known as the ligand of CD44, but neither hyaluronidase treatment nor addition of excess hyaluronate to the assay system affected rosette formation. These data indicate that hyaluronate is not responsible for rosette formation. Anti-CD44 antibody (KM81), which recognized the hyaluronate binding site of CD44, inhibited rosette formation. But other monoclonal antibodies against different epitopes except for the hyaluronate binding site, even those against CD44's hyaluronate binding site, did not inhibit rosette formation. Thus, rosette formation between MS-5 cells and ELM-I-1 cells is mediated by CD44 but not by the hyaluronate binding site of CD44.

A subpopulation of granulocyte and macrophage precursors (GM-CFUc) differentiating in the agar colony technique of Bradley and Metcalf into mature granulocytes and macrophages, without the addition of granulocyte and macrophage colony stimulating factor (GM-CSF), can be detected in MPSV infected mice. These precursors were detected 5 days after virus infection, reaching a maximal concentration of 1/10,000 spleen or bone marrow cells, 25 days after viral infection. The number of the added GM-CSF independent GM-CFUc was linearly correlated with the number of seeded MPSV hematopoietic cells. No GM-CSF producing cells could be detected in the MPSV spleen using normal bone marrow GM-CFUc as responder cells. Study of the GM-CSF sensitivity of the GM precursors has demonstrated the existence of two GM-CFUc populations in the MPSV spleen: a) a GM-CSF dependent population with a GM-CSF sensitivity similar to that of normal GM-CFUc b) a GM-CFUc population which differentiated in the absence of detectable amount of GM-CSF and of which differentiation was not affected by the addition of progressive amounts of GM-CSF. A possible model explaining these results is proposed.

Cellular proliferation kinetics were investigated in the spleens of DBA/2 mice infected with a myeloproliferative sarcoma virus (MPSV) at three distinct phases of spleen growth: the rapid-growth phase, the slow-growth phase and the regression phase. Using injection of tritiated thymidine in vivo and autoradiographic techniques, we showed that most blast cells proliferate rapidly (cell cycle is equal to 10 h) even during the slow-growth phase. Very massive and rapid cell loss was found during the rapid-growth phase (cell loss factor phi = 80%). By comparing the decrease in specific splenic activity after in vivo injection of 125IUdR and 3HTdR, we were able to show a large reutilization of tritiated thymidine (R = 57%), visible less than 7 h after injection of isotopic DNA precursors. Thus, MPSV-infected spleen cells were shown, for the most part, to be short-lived cells: almost one-half mature into erythrocytes and the others rapidly die in situ.