Differential effect of high-dose methotrexate on erythropoiesis and granulocytopoiesis in humans.

The kinetics of erythroid and granulocytopoietic cell production were investigated during Courses 1 and 4 of high-dose methotrexate therapy with citrovorum factor rescue in a patient suffering from metastatic osteogenic sarcoma. Using the technique of quantitative 14C autoradiography, relative production rates were determined before, as well as 2, 24, 48, and 72 hr after, methotrexate infusion. There was only a minor decrease of the relative granulopoietic cell production 2 hr after methotrexate infusion followed by an overshoot reaction after 48 hr with a maximum of 3 to 4 times the pretherapeutic value. The relative erythropoietic cell production dropped to less than one-third of the pretherapeutic level during both courses and remained low during the period of postinfusion observation. The results indicate a severe and long-lasting impairment of the erythropoietic cell series, which is likely to include the committed stem cell pool. The impairment of granulocytopoiesis was much smaller and was followed quite soon by a reaction of recovery. The rate of DNA synthesis of individual cells was subnormal in all cell types investigated prior to Course 4 and was hardly affected by the methotrexate. Intracellular accumulation of methotrexate polyglutamates and differences in this pattern of accumulation between the red and white cell series are discussed as one possible explanation in this context.

Drug testing in established cell lines by flow cytometric vitality measurements versus clonogenic assay.

The clonogenic assay is widely considered to be the most valid test for predicting tumor cell sensitivity to cytostatic drugs. In this study it was compared with early growth curves of human leukemic cell lines (HL-60, K562, Reh) after treatment with different types of cytostatic drugs (Adriamycin,cis-diamminedichloroplatinum(II):,1-beta-D-arabinofuranosyl- cytosine, and 5-fluorouracil) for 1 and 24 h. Following drug treatment two parallel cultures were started: a soft agar culture for the clonogenic assay; and a liquid suspension culture for vital cell counting by measuring esterase activity with fluorescein diacetate at different time points. The latter was recorded using flow cytometry during the following 3 days in 12-h intervals. For each drug concentration a survival factor was calculated from the growth curve between 24 and 72 h. This survival factor takes into account both the y intercept of the extrapolated growth curve and the slope of the growth curve. The dose-response curves resulting from either the survival factors or the clonogenic assay were always nearly identical. The results demonstrate that in established cell lines flow cytometric determination of vital cell increase rates provides a convenient alternative to the clonogenic assay for drug testing.

Cell kinetics in human malignancies studied with in vivo administration of bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BrdUrd) is a pyrimidine analogue which is incorporated into the DNA of proliferating cells. When in vivo BrdUrd infusion is coupled with bivariate flow cytometry to measure cell BrdUrd incorporation and DNA content, both the percentage of DNA-synthesizing cells [BrdUrd-labeling index (LI)] and the DNA synthesis time (TS) can be determined on the same tissue sample. From experimentally determined LI and TS, the potential doubling time of the population and its cell production rate are calculated. To ascertain whether the BrdUrd infusion method is clinically feasible and if data are reliable, we studied patients with leukemia, refractory anemia, multiple myeloma, and brain and gastric tumors. The BrdUrd incorporation data were compared with those determined on duplicate samples with the techniques conventionally used for LI and TS values, i.e., 3H- and 14C-labeled thymidine autoradiography, respectively. The complete BrdUrd procedure takes 6-9 h, and no immediate toxicity from BrdUrd administration has been observed. In an 8-month period, 154 patients were studied. Successful LI and TS determinations were obtained in 78.9 and 59.7% of cases, respectively, more often in hematological than in solid tumors. The values for LI and TS assessed with the BrdUrd technique were very close to those found with 3H- and 14C-labeled thymidine autoradiography (r = 0.88, P less than 0.005, and r = 0.89; P less than 0.005, respectively). The potential doubling time and production rate were accordingly similar. These data indicate that in vivo BrdUrd infusion coupled with flow cytometry measurements can be performed in clinical settings and that this method is reliable. It could be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and for planning radio- and/or chemotherapy.

Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia.

Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

Kinetics of myeloid progenitor cells in human micro long-term bone marrow cultures.

Hemopoiesis was analyzed in a miniaturized long-term culture of human bone marrow cells by quantifying the production of granulocyte-macrophage progenitor cells. As in the conventional long-term culture system, hemopoiesis was dependent on the presence of a marrow-derived adherent layer. Adipocytes proved to be essential for long-term proliferation. Optimal growth conditions were maintained by incubation in McCoy's medium supplemented with hydrocortisone, fetal calf serum, and horse serum. When calculated back to the volume of conventional cultures, the numbers and kinetics of nucleated cells and granulocyte-macrophage colony-forming cells were comparable in both culture systems. The microsystem is therefore suitable for performing multiple analyses on small samples of cells.

Antibodies to the beta 1-integrin chain, CD44, or ICAM-3 stimulate adhesion of blast colony-forming cells and may inhibit their growth.

Early hematopoietic progenitor cells adhere to bone marrow stromal cells (BMSCs) mainly through VLA-4/VCAM-1 interactions. However, many adhesion molecules are expressed by these cell types. Cell adhesion molecules not only mediate adhesion; some are also capable of triggering cellular signaling events. These signals can be induced by several anti-adhesion molecule antibodies. In this study, we investigated the effects of several of such stimulatory antibodies against alpha L (CD11a), alpha 4 (CD49d), and beta 1 (CD29) integrin chains, ICAM-3 (CD50), CD34, CD44, and CD45. All antibodies reacted strongly with CD34-positive bone marrow (BM) cells, but only those against beta 1 integrin (TS2/16, Lia1/2) or CD44 (NKI-P2) reacted with BMSCs. To test the ability of these antibodies to stimulate adhesive interactions, we analyzed their effect on stroma-adherent blast colony-forming cells (Bl-CFCs). We found that TS2/16 (anti-beta 1 integrin), NKI-P2 (anti-CD44), and 152-2D11 (anti-ICAM-3) enhanced adhesion of BM mononuclear cells to stroma (TS2/16:3.4-fold, NKI-P2: 3.8-fold, 152-2D11: 2.6-fold) when compared with isotype-control-treated cells. The increase in stroma-adherent cells was accompanied by an increase in Day 5-7 blast colonies of 3.8-, 2.6-, and 1.9-fold, respectively. One antibody against CD29:Lia1/2 strongly inhibited the formation of blast colonies, an effect that was at least partially caused by its growth-inhibitory activity. Of the other antibodies tested, none displayed growth-modulatory activity. We have found previously that Bl-CFCs depend strongly on VLA-4 and VCAM-1. However, in TS2/16- or 152-2D11-treated cultures, we observed not only these, but also VLA-5-dependent adhesive interactions. In contrast, VLA-5 did not appear to be involved in NKI-P2-treated cultures. Our data indicate that interactions mediated by beta 1-integrins are involved in the growth of Bl-CFCs. Furthermore, interactions mediated by beta 1-integrins, CD44, and ICAM-3 differentially modulate VLA-4- and VLA-5-dependent progenitor/BMSC interactions.

The effect of two different types of colony-stimulating factor on the expression of aminopeptidase on marrow-derived murine macrophages.

The expression of aminopeptidase, a surface-membrane-bound enzyme, on macrophages formed in liquid cultures of hemopoietic progenitor cells was studied over a period of 20 days. The cultures were stimulated by two biochemically distinct types of colony-stimulating factor (CSF) derived from mouse-lung-conditioned medium (MLCM) and L-cell-conditioned medium (LCCM), respectively. The enzyme content of single cells was determined microphotometrically after staining with Fast Blue B salt and leucine 4-methoxy-2-naphthylamide. In LCCM-stimulated cultures the number of cells expressing aminopeptidase, the enzyme content per cell and the enzyme concentration increased markedly from day 10 onward, while macrophages from MLCM-stimulated cultures only showed borderline yet significantly positive aminopeptidase levels. Maximum enzyme concentrations were found earlier than maximum enzyme content indicating an early local increase in the aminopeptidase concentration on the membrane and subsequently a more uniform distribution over the cell surface. The two types of CSF differ not only in their effect on macrophage production but also in their influence on the expression of the surface enzyme aminopeptidase on these cells.

The response of murine splenic granulocyte-macrophage colony-forming cells to lipid A in vivo.

The physical and biological properties of murine splenic granulocyte-macrophage colony-forming cells (GM-CFC) were analyzed after the injection of the splenic hemopoiesis stimulating agent lipid A. In continuous gradients of Percoll, the majority of the splenic GM-CFC of untreated mice peaked at a buoyant density of 1.090 g/cm3, while a small second GM-CFC peak could be detected at 1.065 g/cm3. One day after the injection of lipid A, the splenic GM-CFC were almost equally distributed among these two density peaks. This altered proportion was still detectable 72 to 96 h later, although to a smaller extent. No difference in the responsiveness to the colony-stimulating factor (GM-CSF) from mouse-lung conditioned medium (MLCM) was observed between these two density subpopulations. The differentiation pattern of splenic GM-CFC was altered after the injection of lipid A. However, this altered pattern was the same in both density subpopulations. The percentage of splenic GM-CFC as well as the percentage of multipotent hemopoietic stem cells (CFUs) in DNA synthesis were markedly elevated after the injection of lipid A. A striking difference in the proliferative activity was found between high- and low-density GM-CFC in post-lipid A spleens. 24 h after the injection of lipid A, 43% of the high-density GM-CFC subpopulation was found in S according to the suicide technique using tritiated thymidine, whereas in the low-density fraction only 10% of the population was killed. This finding allows alternative interpretations.

HLA-class II antigens on hemopoietic and stromal cells in human micro long-term bone marrow cultures.

Using a complement-dependent cytotoxicity assay (CDC), we analyzed the presence of HLA-class II antigens on both stromal and hemopoietic cells in a miniaturized human long-term bone marrow culture system. 4-Hydroperoxycyclophosphamide (4-HC)-resistant hemopoietic stem cells capable of restoring in vitro hemopoiesis on irradiated stromal cell layers were HLA-DR, -DP, and -DQ negative. In addition, these cells failed to bind the monoclonal antibody (mAb) Tü 39, previously proposed as a candidate for the recognition of a novel class II antigen, "-DY." On the other hand, the formation of confluent stromal cell layers was inhibited by HLA-DR- or -DP-specific mAbs, but not by the HLA-DQ-specific mAb Tü 22. This suggests the presence of HLA-DR- and/or HLA-DP-positive, but HLA-DQ-negative stromal precursor cells.

The role of the spleen in a lactate dehydrogenase mutant mouse (Ldh-1c/Ldh-1c) with hemolytic anemia.

The lactate dehydrogenase mouse mutant Ldh-1c/Ldh-1c is afflicted with a severe hemolytic anemia associated with extreme reticulocytosis (95%) and splenomegaly. Ninety-one percent of the total body colony-forming units--erythroid (CFU-E) have been quantified in the seven- to ten-times enlarged spleens of the mutant mice. Moreover, the splenic fraction of morphologically recognizable erythroid precursors was 134 times normal. From these data it was apparent that the spleen crucially contributes to the maintenance of steady state erythropoiesis in the mutants. On the other hand, an enhanced sequestration of red blood cells in the enlarged spleen may augment the anemia. Splenectomy experiments were performed with LDH mutant and wild type mice in order to investigate the role of the spleen in this particular hemolytic disease. Following splenectomy, the peripheral blood values and the frequency of femoral stem and progenitor cells were determined, and histological investigations were carried out. The life span of the splenectomized mutants was not shortened, in spite of a very low red blood cell count (25% of the untreated mutant value). Compared to the splenic loss only a moderate increase in bone marrow erythropoiesis was observed, such as a 250% increase of CFU-E. It is concluded that the reduction in red blood cell survival due to splenic sequestration in the mutants is of such a magnitude that it counterbalances a significant portion of splenic erythropoiesis.

Mechanisms of compensation of hemolytic anemia in a lactate dehydrogenase mouse mutant.

Hemopoiesis was studied in homozygous lactate dehydrogenase (LDH) mutant mice not showing noticeable impairment in viability and fertility but afflicted with a severe hemolytic anemia. In order to investigate the mechanisms of erythropoietic compensation, the numbers of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC), and early and late erythroid progenitors (BFU-E and CFU-E) in femur and spleen were determined, and the total body content of each cell type was computed. While the total CFU-S and GM-CFC numbers showed only slight deviations from normal, the total BFU-E pool was 1.4 and the CFU-E pool 18 times enlarged. No difference in cell cycle status could be detected in these compartments by means of tritiated thymidine (3H-TdR) suicide in vitro. However, splenic erythroblasts of homozygous LDH mutants had a shorter DNA synthesis time and a higher labeling index compared to the wild type mice. It is concluded that the hemolysis is compensated at a lower level of red blood cell count primarily by an increase in the total number of late erythroid progenitors resulting from roughly four extra divisions, and secondarily by an increase in the flux through the recognizable erythroblast compartments, predominantly a space-saving mechanism.

Recombinant murine interleukin 9 enhances the erythropoietin-dependent colony formation of human BFU-E.

Murine interleukin 9 (mIL-9) is a novel T-cell-derived lymphokine previously described as a T-cell growth factor (P40/TCGFIII) and as a mast cell growth-enhancing activity (MEA). In the present study we examined the potency of recombinant (r)mIL-9 to exhibit hemopoietic growth factor activity in the human system. In semisolid cultures of normal human bone marrow-derived mononuclear cells, rmIL-9 alone at a concentration range from 25 to 200 U/ml did not reveal any colony-stimulating activity on human granulocyte-macrophage colony-forming cells (GM-CFC), erythroid colony-forming units (CFU-E), and erythroid burst-forming units (BFU-E). Furthermore, we did not observe synergistic effects of rmIL-9 on the number, size, and morphological composition of human granulocyte-macrophage colonies in cultures stimulated with giant cell tumor-conditioned medium. However, a synergistic effect of rmIL-9 in the human erythropoietic culture system was clearly demonstrated in the presence of recombinant human erythropoietin (rhEpo). Recombinant murine IL-9 at a concentration of 200 U/ml enhanced the number of BFU-E-derived day-14 colonies about 3.6-fold as compared to control cultures stimulated with Epo alone. The formation of CFU-E-derived day-7 colonies was not significantly altered under the same conditions. Our results demonstrate that in the presence of rhEpo, rmIL-9 is synergistically active in human bone marrow cultures as an erythroid burst-promoting factor. The development of granulocyte-macrophage colonies obviously is not affected. This finding strongly suggests that mIL-9 can mediate signals via human IL-9 receptors and further extends the range of biological activities hitherto ascribed to mIL-9.

Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.

We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.

Quantitation of the Pap reaction on single cells by absorption cytophotometry.

The reaction product of the unlabeled antibody enzyme method was quantitated at the single cell level by absorption microphotometry. Attempts were made to demonstrate that the amount of dye deposited on individual cells can be taken as a measure of the number of antibody molecules bound. In an experiment with prefixed sheep erythrocytes loaded in excess with amboceptor a close relationship was observed between cell size and the amount of dye measured. Furthermore, the known differences in A1 antigen density on human A1 and A2 erythrocytes were satisfactorily reproduced with this quantitative approach. As regards T antigen density on human lymphocytes of different origin, the results presented tally with data in the literature. It is therefore concluded that the method is suitable for quantitating antibody amounts bound to individual cells.

Quantitative immunoautoradiography at the cellular level. I. Design of a microphotometric method to quantitate membrane antigens on single cells using 125I-labeled antibodies.

Iodine-125 has become a commonly-used radioisotope, especially for immunoautoradiographic investigations. Microphotometry of grain density, a well-established method in autoradiography with tritium and carbon-14, was applied to nucleated cells with 125I-labeled membranes. Geometric and absorption factors of radiation were investigated in order to find suitable conditions for quantitative evaluation. A preparatory device is given and a set-up of appropriate measuring conditions is presented. With these prerequisites the reflected-light bright-field photometry of immunoautoradiographs permits to determine automatically the content of surface antigens of single cells. Measurement examples were demonstrated.

Quantitative immunoautoradiography at the cellular level. II. Absolute measurements using labeled standard cells as a source of reference.

A quantitative autoradiographic method is presented for determining absolute amounts of 125I-labeled compounds on the surface of individual cells. Autoradiographic evaluation of single cell radioactivity is accomplished by comparing the silver grain densities over the specimen and a radioactive standard being exposed simultaneously. In order to obtain a reference source of comparable physical properties, surface-radioiodinated human erythrocytes are used, the radioactivity of which is determined in a crystal counter. A simple enzymatic method for preparing such standard erythrocytes of very uniform label density is described. Numerous experimental advantages derived from the use of the standard are discussed and demonstrated by examples employing various exposure times and different radioactive standards. Hereby, very similar results were obtained when the number of A-antigenic sites was quantified on single erythrocytes in different experiments. The quantification of membrane-bound IgM on single human lymphocytes is shown as another application of this scheme.

Discrimination in immunofluorescence between labeled and unlabeled cells by quantitative microfluorometry and computer analysis.

An automated procedure for discrimination in immunofluorescence between antibody-labeled and unlabeled cells has been developed on the basis of microfluorimetric determination of intensity distributions. After smoothing the raw data for irregularities caused by the scoring statistics optimum fit of the negative distribution to the corresponding positive one was achieved. The procedure was tested in a model system by mixing various known proportions of immunofluorescence-negative and -positive plastic beads. In addition, variable mixtures of T-negative CLL cells and normal mononuclear peripheral blood cells were labeled with FITC-conjugated anti-T-antiserum. The expected percentage of T-positive peripheral blood cells agreed satisfactorily with the data measured and computed. Finally, the measured percentage of Ig-positive mononuclear cells from normal peripheral blood was in agreement with the values obtained by other techniques.

Photometric methods in quantitation of light microscope radioautography.

Photometric evaluation of autoradiographic grain densities can be performed according to various optical principles. In all instances the amount of light recorded should be a measure of the radioactive substance amount in the specimens. It is shown that grain densities suitable for light microscopic autoradiography are indeed linearly related to radiation exposure. Dependent on the photometric system used, there is a larger or smaller section of linear correlation of the photometric response with grain density. The advantages of incident light bright-field illumination for silver grain counting are discussed. Quantitation of substance amounts in autoradiographs depends on the use of radioactive standard sources. Two different approaches of quantitation are discussed. In 14C-autoradiography which can be applied for cell-kinetic studies, standard plates are used consisting of 14C-polymethylmethacrylate. Allowance has to be made for the different condition of radiation geometry of the cells and the standards. In 125I-autoradiography, 125I-labeled red cells are used as standards. This technique allows for quantitating the number of antibodies bound to individual cell surfaces.

Tritium autoradiography of cell surfaces in smear preparations.

Experiments were performed in order to find out whether tritium-labeled cell surface markers can be quantified at the single cell level in autoradiographs of smear preparations. Mouse thymocytes were incubated with 3H-concanavalin A and subsequently spread on microscopic slides. The spreading techniques, either by cytocentrifugation or by the use of cover slips, were performed in such a way as to achieve preparations in which the mean flatness of the cells varied. By means of incident light microphotometry, the cellular areas and the grain counts of individual cells were determined. The results show a strong dependence of the mean grain yield per slide on the mean cellular area. Cytocentrifuge preparations resulted in larger mean cellular areas and higher mean grain counts than cover slip preparations. With the use of cytocentrifugation, however, the differences in the flatness of the cells were of such a magnitude that a reproducible quantification of 3H-labeled cell surface markers was not possible. Conventional techniques of spreading cells on slides failed to provide a degree of flatness that could approach the saturation grain count per cell without completely destroying the cellular morphology.

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.