Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Ectopic haematopoiesis in the mouse: roles of stroma and cytokines in granulocytopoiesis in in vivo diffusion chamber cultures.

We have examined a possible role of two different types of irradiated stromal cells, i.e. murine bone marrow (BM) stromal cells and stromal cell line MS-5R, when cocultured with murine blood-borne progenitors or sorted Lin- Sca-1+ bone marrow cells in vivo in peritoneal diffusion chambers (DC). Retrieval and quantification of the cultured cells were performed after 4, 7, and 14 d. Granulocyte and/or macrophage colony-forming cells (G/M-CFC) were enumerated in subcultures from the DC. G/M-CFC production was not enhanced in the stroma-contact cultures, in comparison with the standard stroma-non-contact cultures, but early granulocytopoiesis was stimulated. Perturbation of the humoral environment of DC was investigated in a number of ways, for example with continuous infusion of rhG-CSF from a subcutaneous implanted minipump to DC host mice, with DC host mice carrying a transplantable leukaemia, secreting interleukin 3 (IL-3), and with injections of various cytokines. None of these interventions sustained the expansion of the G/M-CFC population. In conclusion, for ectopic haematopoiesis to take place, several requirements must be met. Relevant stromal cells apparently affect haematopoiesis both via direct cell-cell interactions and via humoral mediators (viz. cytokines) which they secrete.

Maturation rate of mouse neutrophilic granulocytes: acceleration by retardation of proliferation, but no detectable influence from G-CSF or stromal cells.

Our purpose was to examine the possible influence of stromal and humoral mediators on granulocytic maturation rates. Sorted immature murine progenitor (Lin-Sca-1+) cells were cultured in peritoneal diffusion chambers (DCs) with or without a confluent layer of irradiated bone marrow stromal cells on one of the micropore membrane walls. In other experiments, 10 microg/kg/d recombinant G-CSF (rhG-CSF) was administered continuously into DC host mice through s.c. implanted osmotic minipumps. Operationally, maturation rate was assessed as the ratio between the number of polymorphonuclear cells (PMN) and proliferative granulocytes (PG) in short-term cultures, based on the differential cell counts, and supported by flow cytometric measurement of a granulocytic differentiation marker; and by the emergence time of PMN in the DCs, obtained by extrapolation. Also, increased maturation is associated with increased cell density, as reflected by the positioning of the granulocytes during centrifugation in a discontinuous Percoll gradient. This method, as well as the conversion rate of 3H-thymidine labeled PG into the heavier non-PG maturational stages, were also used as indicators of maturation rate. After five, six, and seven days of culture in the peritoneal cavity, DC cells were harvested. Their proliferative status, based on measurement of incorporated bromodeoxyuridine, was determined, and their maturation rates were evaluated. Proliferation of immature granulocytic progenitor cells was apparently inhibited by direct contact with bone marrow stromal cells, and stimulated by G-CSF during the early stage of culturing. However, the subsequent maturation rate, which could be accelerated by increasing culture cellularity, thus decreasing PG proliferation rate, was not detectably influenced by either stromal cells or G-CSF.

Effects of hTNFalpha expression in T cells on haematopoiesis in transgenic mice.

A transgenic line of mice carrying one copy of the hTNFalpha gene under the control of its own promoter and the CD2 locus control region has been analysed for the effects of TNFalpha on haematopoiesis. A low level constitutive expression of hTNFalpha in lymphoid tissue was observed. Human TNFalpha binds to and activates the murine p55 receptor, but not the p75 receptor. This implies that the observed effects of hTNFalpha in mice were mediated only through the p55 receptor. Various lymphoid tissues were depleted of lymphocytes, especially thymus, spleen and peripheral blood. Effects on thymus development were detected already at 3 wk of age, more general effects on haematopoiesis were evident by 5 wk: a drop in total blood leukocytes, mainly due to a 67% decline in lymphocytes. At 16 wk the mice had developed anaemia, whereas platelets, neutrophils and monocytes had increased. The fall in lymphocytes was due to lowered levels of T cells as well as B cells. The cause of the shortened lifespan of the transgenic mice was probably not the haematological effects of hTNFalpha directly. Absence of trophic factors supplied by the normal T cell population remains possible.

Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide.

We report herein, a new class of simple hematoregulatory semipeptides, formally derived from the cystine-dimerized peptide pGlu-Glu-Asp-Cys-Lys-OH, where the disulfide bond has been replaced by an isosteric dicarba bridge. The structure-activity relationship (SAR) of a series of analogues incorporating replacements at positions 1 and 2 of peptide 1 led to the design of active conformationally constrained cyclic peptides (12, 13). Ring closure was achieved by cyclization of the N-terminal amino groups at position 2 of peptide 2 using pyrazine-2,3-dicarboxylic acid. Subsequent excision of the putative C-terminal scaffold domain from the active cyclic peptides resulted in the discovery of a new class of low molecular weight hematoregulatory agents exemplified by compound 16. This semipeptide analogue, comprising two D-Ser residues connected via amide bonds to the acid groups of pyrazine-2,3-dicarboxylic acid, had comparable biological activity to the lead peptide 1. The stereochemical requirements for the observed biological activity of these novel compounds were examined. Furthermore, the hematopoietic synergistic activity induced by compound 16 in stromal cell cultures was blocked by an antibody known to neutralize the hematoregulatory effect of 1, indicating a common mechanistic end point. Compounds of the class typified by 16 may form the basis for the development of novel therapeutic agents within the area of immunoregulation.

Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

Indirect stimulation of hemopoiesis by hemoregulatory peptide (HP5b) dimer in murine long-term bone marrow cultures.

The hemoregulatory dipeptide (pEEDCK)2 was shown to stimulate production of a synergistic activity (SA) in 6- to 8-week-old primary stromal cell cultures. The SA increased colony formation by murine bone marrow cells (approximately 50% above control levels) in cultures stimulated by optimal concentrations of L-cell-derived macrophage colony-stimulating factor (M-CSF). An increased number of granulocyte-macrophage colony-forming cells (GM-CFC) was also observed in long-term bone marrow cell cultures following daily administration of dipeptide for 5 days. The increase in GM-CFC was approximately 90% above control as assessed by colony formation in soft agar and coincided with SA production. It appears that the dipeptide augments the production of myeloid progenitor cells through an indirect mechanism mediated by accessory cells.