Mononuclear Cell Suspensions Research Articles

Human bone marrow lymphocytes. Cytotoxic effector cells in the bone marrow of normal individuals.

This study was undertaken to determine the capability of lymphocytes in the bone marrow of normal individuals to mediate nonspecific killer cell functions in assays of phytohemagglutinin (PHA)-induced cellular cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) against 51Cr-labeled chicken erythrocyte target cells. Relatively pure mononuclear cell suspensions were obtained from bone marrow aspirates in 30 normal volunteers by sucrose gradient centrifugations and from the peripheral blood of the same individuals by Hypaque-Ficoll density centrifugations. At an effector: target ratio of 10:1, the PHA-induced cellular cytotoxicity of peripheral blood was 78.8 +/- 1.3%, while that of bone marrow was not significantly less at 66 +/- 9% (P greater than 0.1). At low effector:target ratios, the ADCC of bone marrow was negligible, while at higher effector:target ratios (20:1) bone marrow ADCC was 69 +/- 3.7%, which was comparable to that of peripheral blood. The lymphocytes themselves in the mononuclear cell suspensions of both peripheral blood and bone marrow were capable of cytotoxicity activity since depletion of monocytes from the suspensions by adherence to rayon wool and G-10 Sephadex columns did not remove the cytotoxic activity. Blocking of the Fc receptor on the effector cells by the addition of aggregated gamma globulin to the cultures suppressed the ADCC but not the PHA-induced cellular cytotoxicity of both peripheral blood and bone marrow, indicating that ADCC is dependent on an Fc receptor on the effector cell in both compartments. These studies demonstrate that the bone marrow of normal humans contains populations of lymphoid cells which have highly efficient killer cell capacities. It is uncertain what portion of these cells arise in the bone marrow and what portion enter the bone marrow parenchyma as part of the recirculating lymphocyte pool. These findings have relevance in the clearer understanding of the killer cell potential of grafted human marrow, as well as the bone marrow sequestration of functionally capable lymphocyte subpopulations in disease states and during chemotherapy.

Properties of Fcgamma receptors in normal and malignant human tissues.

Properties of the IgG receptors were studied by treating tissue sections or suspensions of peripheral mononuclear cells with various chemical reagents and determining changes in their ability to react with IgG-sensitized erythrocytes (EA). A heterogeneity of the Fc receptors was revealed. Iodoacetamide, 2-mercaptoethanol, sodium azide, EDTA, and a pH varying from 6.0 to 8.2 had no effect on Fc receptors in sections of normal lymphoreticular tissue and malignant tissue. Formaldehyde, low pH, and high salt concentrations affected receptor activity to various degrees. Receptors in liver sections and on monocytes were generally more resistant than receptors in spleen sections and on B lymphocytes. Receptors in malignant tissue behaved either like receptors in spleen or like receptors in liver. Although all tissues were sensitive to periodic acid, the Fc receptors in malignant tissue were always more resistant.

In Vitro Regulation of Granulopoiesis in Human Leukemia: Application of an Assay for Colony-inhibiting Cells

We describe two assays to detect the action of colony-inhibiting cells. In the first assay, we used a simple density separation technique to remove dense neutrophils (PMN) from suspensions of blood and of bone marrow cells prior to culture in semisolid agar. Conditions were arranged to ensure that control suspensions of unseparated cells and test suspensions of buoyant mononuclear cells differed only in their content of neutrophils. The control and test suspensions contained equal numbers of mononuclear cells (and granulocyte precursors). Colony (and cluster) formation was invariably enhanced in neutrophil-depleted cultures of normal cells. In the second assay, dense PMN, treated by an adherence separation procedure, were recovered, and the non-adherent dense PMN were added back to PMN-depleted cultures. A reproducible dose-related decrease in colony (and cluster) formation to basal levels resulted. The inhibitory effect was identical when the PMN were added directly to the culture (overlayer) or to the underlayer. In PMN-depleted cultures obtained from patients with leukemia and other hemopoietic disorders, neither colony nor cluster formation was enhanced, and sometimes it was reduced. When we compared the effect of adding patient and normal non-adherent PMN to target cultures of normal and patient PMN-depleted cells, some leukemic PMN were noninhibitory. Our results suggest that abnormalities of cellular interactions in vitro detected in the first assay may have more than one explanation, as shown when they are subjected to the closer scrutiny possible with the second assay.

In vitro regulation of granulopoiesis in human leukemia: application of an assay for colony-inhibiting cells

We describe two assays to detect the action of colony-inhibiting cells. In the first assay, we used a simple density separation technique to remove dense neutrophils (PMN) from suspensions of blood and of bone marrow cells prior to culture in semisolid agar. Conditions were arranged to ensure that control suspensions of unseparated cells and test suspensions of buoyant mononuclear cells differed only in their content of neutrophils. The control and test suspensions contained equal numbers of mononuclear cells (and granulocyte precursors). Colony (and cluster) formation was invariably enhanced in neutrophil-depleted cultures of normal cells. In the second assay, dense PMN, treated by an adherence separation procedure, were recovered, and the non-adherent dense PMN were added back to PMN-depleted cultures. A reproducible dose- related decrease in colony (and cluster) formation to basal levels resulted. The inhibitory effect was identical when the PMN were added directly to the culture (overlayer) or to the underlayer. In PMN- depleted cultures obtained from patients with leukemia and other hemopoietic disorders, neither colony nor cluster formation was enhanced, and sometimes it was reduced. When we compared the effect of adding patient and normal non-adherent PMN to target cultures of normal and patient PMN-depleted cells, some leukemic PMN were noninhibitory. Our results suggest that abnormalities of cellular interactions in vitro detected in the first assay may have more than one explanation, as shown when they are subjected to the closer scrutiny possible with the second assay.

Human bone marrow lymphocytes. I. Distribution of lymphocyte subpopulations in the bone marrow of normal individuals.

This study was undertaken to determine the proportions and in vitro immune capacities of lymphocyte populations in the bone marrows of normal humans. Relatively pure mononuclear cell suspensions were obtained from bone marrow aspirates by linear sucrose gradient centrifugations. Simultaneous peripheral blood and bone marrow specimens from each individual were assayed for lymphocyte surface markers and mitogen responsiveness. Maximal possible contamination of bone marrow aspirates by peripheral blood was determined by performing aspirates on individuals who had received 51chromium-labeled autologous erythrocytes. Rhymus-derived (T) lymphocytes, as determined by the sheep red blood cell (E) rosette assay, comprised 8.6-(plus or minus 1.6)% of the total bone marrow lymphocyte pool. Bone marrow-derived (B) lymphocytes, as determined by the presence of a complement receptor, made up 15.4-(plus or minus 1.9)% of the lymphocyte pool whereas 74.6 (plus or minus 2.4)% of mononuclear cells lacked easily detectable surface markers. These findings could not be explained by contamination with peripheral blood lymphocytes since contamination was corrected for in the calculations. Lymphocyte-enriched suspensions of bone marrow cells responded to stimulation with phytohemagglutinin, concanalin A, and particularly pokeweed mitogen. In vitro incubations of bone marrow and peripheral blood lymphocytes with tritiated thymidine followed by determinations of E and erythrocyte antibody complement (EAC) rosettes were performed. Simultaneous rosetteradioautographs demonstrated that the proliferative potential of bone marrow B lymphocytes was greater than peripheral blood B lymphocytes (P less than 0.01). On the other hand, the proliferative potential of bone marrow T lymphocytes was the same as that of peripheral blood T lymphocytes. These findings demonstrate that in addition to containing B lymphocytes the normal bone marrow contains a small fraction of T lymphocytes similar to the mature T lymphocyte pool found in the peripheral blood. These T cells most probably enter the bone marrow parenchyma as part of the normal recirculating lymphocyte pool.

Serial determinations of "blast cell count" during kidney allograft rejection in dogs.

Cellular and humoral responses were quantitated during acute and hyperacute kidney allograft rejection in dogs. Elevatednumbers of DNA synthesizing leukocytes appeared in the peripheral blood during primary rejection. These modifications were quantitated by serial determinations of the rate of tritiated thymidine incorporation in blood mononuclear cell suspensions. The rise of DNA synthesizing cells was paralleled by progressive mononuclear cell infiltrations in the kidney parenchyma. The decline in the level of tritiated thymidine incorporation coincided with the appearance of cytotoxic antibodies and with extensive kidney damage. Hyperacute rejections occurred in all these animals after secondary transplantation of a kidney from the original donor. All animals showed cytotoxic antibodies and displayed no increase in the rate of DNA synthesizing blood mononuclear cells.

Growth of human leukaemic peripheral blood cells in diffusion chambers

In these investigations, the diffusion chamber technique was used to characterise the growth of peripheral blood cells from 10 patients with acute leukaemia and, for comparison, from 4 normal persons. By means of the Isopaque-Ficoll method, erythrocytes and granulocytes were removed and the resulting mononuclear cell suspension cultured for various times up to 3 weeks in diffusion chambers implanted into preirradiated CBA mice. The growth pattern for cells from normal persons was uniform with an initial decrease in total cell number between 3 and 6 days after implantation and a maximum at day 13, due mainly to the appearance of blast cells, granulopoietic cells and macrophages, with few erythropoietic precursors and megakaryocytes. In the leukaemic patients, the growth patterns differed widely from normal and also between the patients themselves. Five patients had an almost continuous cell increase exceeding normal, largely due to leukaemic blast cells and granulopoietic cells, whereas other patients had growth curves similar to normal or a continuous decrease in cell number over the whole period. The extent of cell growth might be related to the diagnosis, since patients with marked increase in cell number had acute myeloid leukaemia or acute erythroleukaemia whereas patients with practically no growth at all had acute undifferentiated leukaemia or acute lymphoid leukaemia. In some patients the number of granulopoietic cells in diffusion chambers greatly exceeded the corresponding number from normal cells and it is assumed that some maturation of leukaemic blast cells took place. From the high number of leukaemic blast cells which developed in the diffusion chambers in spite of a low 3 H-thymidine labelling index in peripheral blood, it is concluded that either a small population of blast cells was capable of continued self-replication or that some non-proliferating “quiescent” leukaemic cells re-entered proliferation.

Activation of the Kinin-Forming System during Therapy with Cyclophosphamide

Abstract Venous blood kininogen is significantly decreased during therapy with cyclophosphamide in cases of advanced breast cancer. The maximum decrease in plasma kininogen coincided with onset of maximal leukopenia in the peripheral venous blood. In vitro studies showed that both human whole blood and mononuclear cell suspensions liberate free kinin when incubated with cyclophosphamide, but plateletfree human plasma does not. Evidently, the kinin-forming system is activated during therapy with cyclophosphamide, and this may account for some of its reported side effects. The in vitro studies suggest that this activation results from damage to peripheral blood leukocytes by the cyclophosphamide, with the release of intracellular proteases.

Characterization of Glass Adherent Human Mononuclear Cells

Abstract Although adherence to glass is commonly used to prepare monocyte monolayers from blood mononuclear cell suspensions (1–3), several recent reports suggest that lymphocytes are also able to adhere to either glass or glass substitutes like nylon or tissue culture dishes (4, 5). Using morphologic criteria, Levis (4) reported considerable lymphocyte binding to glass coverslips placed in mononuclear cell suspensions. Although many of the lymphocytes could be detached by rigorous mechanical washing, this resulted in a loss of monocytes as well. Rosenthal (5) has suggested that adherence to glass can be used to deplete selectively lymphocyte preparations of antibody-forming or “bursal-derived” (B)2 lymphocytes. Similarly, Boldt (6) has demonstrated that nylon columns can remove from a suspension of blood mononuclear cells a population of lymphocytes which are “highly responsive” to concanavalin A. These observations indicate that monocyte monolayers prepared by adherence of blood mononuclear cells to glass or tissue culture dishes may be contaminated by considerable numbers of lymphocytes.

Formation of granulocytes and macrophages in diffusion chamber cultures of mouse blood leucocytes.

Suspensions of mononuclear cells were prepared from mouse blood by a one stage centrifugation method. Iso‐ and homologous culturing of these cells were then carried out in cell‐impermeable diffusion chambers. These were implanted into the peritoneal cavities of host mice. The culture periods ranged from 1 to 13 days. The proliferation, differentiation and death of the chamber cells were quantitated by total and differential cell counts and by evaluation of 3H‐thymidine incorporation from liquid scintillation counting and radioautography.Proliferating and differentiating granulocytes and macrophages were consistently found in the cultures, whilst lymphocytes from the inoculum were gradually lost during the culture periods.