In vivo stimulation of murine granulopoiesis by human urinary extract from patients with aplastic anemia.

Significant in vivo stimulation of granulopoiesis was induced in mice by the administration of an extract from the urine of patients with aplastic anemia (AA). Sialic acid has been identified as an important molecular component for the in vivo biological activity of this granulopoietic factor, "granulopoietin," which is distinct and different from endotoxin. Urine from patients with AA was successively fractionated by Sephadex G-50 and DEAE-cellulose chromatography. The resultant extract, which we refer to as AA urinary extract, contained approximately equal to 44 international units of erythropoietin per A unit of protein and induced 15,000 colonies of granulocyte/macrophage precursor cells (granulocyte/macrophage colony-forming units, CFU-gm) per A unit of protein with mouse bone marrow. Eight daily intraperitoneal injections of this extract in mice induced a 6.2-fold increase in peripheral blood granulocytes and a 14.6-fold increase of splenic CFU-gm, with concomitant increases in the proliferation rates of CFU-gm in both bone marrow and spleen. Pretreatment of the AA urinary extract with sialidase significantly diminished these granulopoietic effects in vivo (P less than 0.001). In contrast, both extracts (i.e., native AA and sialidase-treated AA urinary extracts) revealed high granulocyte/macrophage colony-stimulating factor activity in vitro when clonal assays were performed with mouse bone marrow. Increased in vivo and in vitro granulopoietic activities were found in the concanavalin A "break-through" fraction, indicating that these activities were due to protein(s) that did not bind to the lectin. These results reveal that this urinary extract from patients with AA is capable of inducing significant granulopoiesis in mice and that sialic acid is an important component in the maintenance of this granulopoietic effect in vivo but not in vitro.     

Demonstration of granulopoietic factor(s) in the plasma of nude mice transplanted with a human lung cancer and in the tumor tissue.

A human lung cancer (OTUK-tumor) was transplanted serially to nude mice, which invariably developed a marked neutrophilia. In order to analyze this phenomenon, in vitro agar culture studies were carried out. A three- to fourfold increase of colony-forming units in culture was observed in the femurs of these nude mice. Moreover, the plasma of nude mice bearing this tumor, as well as the tumor extract, had a significantly higher colony-stimulating activity on mouse bone marrow cells than appropriate controls. This activity had the property to stimulate granulocyte and/or mixed cell type colonies rather than mononuclear cells. This activity was also demonstrated, in a dose-dependent way, on normal human bone marrow cells. These findings indicated that the OTUK-tumor produced human granulopoietic colony-stimulating activity, which may have stimulated granulopoiesis in vivo as well.     

In Vitro Colony and Cluster Growth in Haemopoietic Dysplasia (The Preleukaemic Syndrome).

The growth and differentiation of granulopoietic progenitor cells from 15 patients with haemopoietic dysplasia were studied by in vitro culture in agar-gel. After 14 d in culture whole colonies and clusters were transferred to glass slides and were stained with a modified Papanicolaou technique. The preparations were examined for cellular differentiation by counting the number of mature cells (band forms and polymorphonuclear cells) and a mitotic index was calculated from the number of mitotic cells. Patients with defective colony formation showed granulopoietic maturation defects and a reduced mitotic index was found in some colonies. Patients who had colony counts within the normal range, however, showed normal in vitro maturation. Defective colony growth in haemopoietic dysplasia generally indicates a malignant course and can occasionally be related to leukaemic transformation. The finding of in vitro maturation defects is an additional culture abnormality which may indicate a deteriorating clinical course. A defective maturation and a reduced mitotic index in vitro add support to the concept of clonal progression in malignant haemopoietic dysplasia.     

Adherent Cell Dependent Colony-Stimulating Activity in Human Serum: A Granulopoietic Regulator?

Bacterial infections and trauma which increase production of granulocytes and monocytes by the bone marrow, may do so through factors in serum capable of stimulating growth of granulocyte-macrophage cells in vitro. Human serum possesses two types of colony stimulating activity (CSA), one which stimulates granulopoietic progenitor cells directly, and another which results from the interaction of serum and bone marrow adherent cells (monocyte-macrophages) or peripheral blood leucocytes: adherent cell dependent CSA. These activities are due to different factors which may be separated by gel filtration. Sera of 7 patients undergoing hystectomy who developed post-operative infection showed post-operative elevation of the adherent cell dependent activity in all cases but no change in direct acting CSA. These results suggest that the direct acting CSA in human serum does not represent the principal humoral ‘message’ to the bone marrow from sites of trauma and infection in the tissues and that granulopoiesis may be controlled indirectly by the action of a different humoral factor which increases production of CSA by marrow monocyte-macrophages. Preliminary experiments suggest that lymphocytes stimulated by bacterial products may be one source of this factor.     

Granulopoietic progenitors in suspension culture: a comparison of stimulatory cells and conditioned media

Kinetic studies have been carried out to investigate the functional heterogeneity previously observed in populations of human marrow or peripheral blood cells separated by velocity sedimentation. The results obtained confirm the earlier results, in that slowly-sedimenting cells were found to stimulate both colony formation by granulopoietic progenitors and an increase in numbers of granulopoietic progenitors in suspension culture, while rapidly-sedimenting cells stimulated only colony formation and not increased progenitors in suspension cultures. Investigations of the properties of media conditioned by these two subpopulations of cells revealed no clear differences between them; both stimulated suspension cultures as well as colony formation, and both lost the former activity, but not the latter, after dialysis. The results contribute to the evidence that more than one process is regulated in cultures of granulopoietic progenitor cells.     

Myeloid membrane markers. I. Expression of marker antigens specific for murine granulocytes and their precursor cells.

Bone marrow cells were investigated by immunoelectromicroscopy and by quantitative photometric immunoradioautography with a rabbit antiserum against murine bone marrow cells. The serum was absorbed with murine spleen, liver and thymus cells until it no longer reacted with thymocytes and lymph node cells in a quantitative complement fixation test. The antiserum stained granulopoietic but not erythropoietic or lymphopoietic cells. The density of the myeloid antigen on single cells increased with the differentiation from immature to mature granulopoietic cells. While the increase of label was statistically not significant at the level of differentiation from promyelocyte to myelocyte and metamyelocyte to band neutrophil, there was a remarkable gain of label from myeloblast to promyelocyte and from band neutrophil to segmented neutrophil. This was evident under the electron microscopy using peroxidase-labeled antibodies and could be measured quantitatively with photometric immunoradioautography using 125I-labeled antibodies.     

Granulopoietic activity in Felty''s syndrome.

The levels of urinary and serum granulopoietic factor have been determined in nine patients with Felty's syndrome and compared with similar levels in patients with rheumatoid arthritis without Felty's syndrome and patients with neutropenic disorders of other causes. These studies have shown that urinary and serum levels of colony stimulating activity (CSA) in patients with Felty's syndrome are low (mean urine CSA 10.0 and serum CSA 4.0) when compared to patients with neutropenic disorders without rheumatoid arthritis (mean urinary CSA 26.8 and serum CSA 9.4). These findings suggest that Felty's syndrome may be, in part, the result of decreased production or activity of granulopoietic factors rather than due simply to increased granulocyte destruction.     

Specificity of Interacting Populations Affecting Granulopoiesis in Culture

Human marrow contains two interactingpopulations that affect granulopoiesis inculture. The first population consists ofcommitted granulopoietic progenitors,while the second contains cells that produce factors whose activity is essential forgranulopoiesis in culture. The two populations can be separated by an adherenceprocedure and assayed independently.Both populations proved to be heterogeneous: the granulopoietic progenitorpool contains a subpopulation which failsto respond to semipurified colony-stimulating activity derived from peripheralleukocytes but responds to the unpurifiedmaterial. The population of "factor-producing" cells contains two subpopulations that can be separated on the basis ofsize by velocity sedimentation. Both produce factors that stimulate granulopoieticcolony formation. However, the moreslowly sedimenting subpopulation is capable of interacting with granulopoieticprogenitors in suspension culture yieldingan increase in their number, while cellsfrom the more rapidly sedimenting population failed to support such an increase.The results are interpreted as indicatingspecificity in the interaction betweengranulopoietic progenitors and factor-producing cells.

Submitted on February 25, 1974 Accepted on May 13, 1974     

Different colony stimulating activity by tumoral ascitic fluid and conditioned media.

The effects of intraperitoneal Ehrlich ascites tumor (ET) growth on the kinetics of hemopoietic stem cells in the host bone marrow were studied using the spleen colony and the soft agar culture techniques. There is a decrease in spleen colony forming capacity of bone marrow of ET bearing mice, whereas in vitro assays of the committed macrophage granulocyte precursors, by the soft agar method, show that in the same circumstances a high yield of granulopoietic colonies can still be obtained. A shift of the CFU-c/CFU-s ratio from 15 to 36 thus occurs. Moreover, ascitic fluid from tumoral mice displays strong activity as CSF on normal mouse marrow, twice as strong as the standard mouse embryo CSF. When conditioned medium from cultures of ET cells (ET-CM) is tested, the pattern of agar colonies obtained is different from the previously obtained pattern of growth kinetics; furthermore many colonies are composed of undifferentiated cells. The hypothesis is suggested that among the variety of known CSF's, the ET-CM represents a unique factor, capable of inducing proliferation of marrow CFU-c, but only limited differentiation.     

Human placental conditioned medium contains monocyte-derived recruiting activity (MRA)

Monocyte-derived recruiting activity (MRA) stimulates the release of granulopoietic colony-stimulating factors (CSF) by endothelial cells. We carried out studies designed to test the hypotheses that human placental conditioned medium (HPCM), a widely utilized source of CSA for in vitro studies, contains both MRA and CSA and that these molecules could be separated on the basis of their isoelectric points. In five separate studies, concentrated samples of HPCM were chromatofocused and fractions tested in bioassays for MRA and CSA. We found that HPCM contains both MRA and CSA, that the majority of the MRA eluted at a pH of from 7.6 to 8.2 and was 2000-fold purified. CSA eluted at pH 5.6 or below. No CSA was detectable in the major MRA peak. We conclude that HPCM contains heterogeneous granulopoietic activities, that the isoelectric points of MRA and CSA are substantially different and that chromatofocusing provides a rapid single-step method for separating these two distinct granulopoietic factors from complex conditioned media.     

Enrichment of colony forming cells by methotrexate and isokinetic density gradient centrifugation.

Purification of target cells responsive to granulopoietic regulators would be of importance in understanding their mechanism of action. Previous studies have shown that mouse marrow could be enriched 3- to 7-fold with respect to agar colony forming cells (CFU-C) by appropriately scheduled doses of Methotrexate. To further increase this enrichment, such marrow has been subjected to centrifugation through an isokinetic gradient of Ficoll in culture medium. Under optimal conditions (17.5 min at 77.5 X g) the combination of chemotherapy and velocity sedimentation yielded a 17-fold enrichment of CFU-C's.     

Serum Myeloperoxidase and Lactoferrin in Neutropenia

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more mature granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Serum myeloperoxidase and lactoferrin in neutropenia.

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Virus-induced leukopenia: Colorado tick fever as a human model

Eight patients with Colorado tick fever were studied to determine whether alterations in the production of granulopoietic stimulatory or inhibitory factors (or both) could be found in association with the leukopenic state of the disease. The studies demonstrate that in the patients with Colorado tick fever the mononuclear cell production of colony-stimulating factor is decreased and that there is an increase in circulating inhibitory factors in the serum of such patients. The depressed mononuclear cell colony-stimulating activity does not appear to be reversible by addition of either endotoxin or normal human serum. Characterization of these serum inhibitory factors may facilitate understanding of leukopenia in human disease.     

Biological activity of synthetic human insulin

Summary The biological activity of human insulin, prepared by total chemical synthesis, was compared in various tests in vivo and in vitro with that of natural human or pork insulin. The potencies of the synthetic and natural hormone, as determined by the mouse convulsion assay, by hypoglycaemic effect and diaphragm glycogen increase in fasted rats in vivo, or by stimulation of¹⁴C-glucose metabolism in fat cells and binding to anti-insulin serum in vitro, did not differ significantly. It is concluded that this synthetic human insulin is biologically equivalent to the natural hormone.     

Rabbit anti-mouse brain serum (RAMBS): Lack of specific inhibition of late committed erythroid stem cells in culture (CFU-E)

Summary Heterologous antisera to mouse brain tissue have activity against mouse pluripotent hemopoietic stem cells (CFU-S), but not against granuloid/ macrophage committed precursor cells (CFU-C). In these studies we show that anti-mouse brain serum raised in a rabbit does not possess specific activity in vitro against late committed erythroid stem cells (CFU-E).From the Section of Hematology, Department of Medicine, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, U.S.A.     

Response of neutropenia and anaemia to immunosuppressive therapy: report and bone marrow culture studies.

Soft agar culture of bone marrow cells was used to investigate abnormalities of granulopoiesis in a patient with acquired anaemia and neutropenia which responded to immunosuppressive therapy. Results showed that the patient's peripheral blood leucocytes inhibited granulopoiesis in vitro. Variation in the response of different target marrows suggested that inhibition was not due to direct action on granulocyte-monocyte committed stem cells but was directed against or mediated through the cells in target marrow samples which produce granulopoietic stimulators. Cell separation showed that the inhibitory activity was associated with the patient's mononuclear cells rather than neutrophils. A tentative explanation of the mechanism of production of the anaemia is also offered.     

Relationship of the effects of interferon on chronic hepatitis B and the induction of 2',5'-oligoadenylate synthetase

Two years or more after 35 patients (29 men and six women) with chronic hepatitis B were treated by interferon, we studied relationships of age, ALT activity, activity of serum DNA polymerase associated with the hepatitis B virus, serum levels of hepatitis B e antigen and activity of 2',5'-oligoadenylate synthetase in peripheral blood mononuclear cells when treatment started in comparison with treatment results. Seventeen patients were given human lymphoblastoid interferon-alpha; the other 18 patients were given interferon-beta. We measured the activity of 2',5'-oligoadenylate synthetase in these mononuclear cells and found the rate of increase in vivo and in vitro; the correlation between the two was r = 0.68. This enzyme activity in the patients who became negative for DNA polymerase after interferon treatment increased more both in vivo and in vitro than in patients who did not became negative. Also, both the in vivo and in vitro activity increased more in patients who became negative for the e antigen after interferon therapy than in those who remained positive. In the first group, interferon was considered to be effective; in the second, ineffective. Of the patients who became negative, some developed e antibodies and some did not; the increase in this enzyme activity in the two groups was not significantly different. The increase in the activity of 2',5'-oligoadenylate synthetase activity could be used to predict the results of interferon treatment and is an index that can be used before treatment to predict the response.     

Relation between Life Expectancy and Composition of the Bone Marrow at Diagnosis of Chronic Myeloid Leukaemia

The proportions of myeloblasts (MB), promyelocytes (PMC) and myelocytes (MC) within the granulopoietic precursor pool of the bone marrow and a mitotic index of the precursor cells were determined in 43 patients with chronic myeloid leukaemia (CML) diagnosed in the chronic phase of the disease. There was a negative correlation between the percentages of MB + PMC and the mitotic indices, i.e. large proportions of MB + PMC were associated with low mitotic indices. With increasing proportions of MB + PMC at diagnosis, survival was significantly reduced. Furthermore, only 22 % of patients with MB + PMC above the median value (28 %) associated with a mitotic index below the mean value (1.02 %) survived for more than 2 years whereas 82 % of the patients with a percentage of MB + PMC below the median value combined with a mitotic index above the mean value survived for more than 2 years. It is concluded that the composition and mitotic activity of the granulopoietic precursor pool at diagnosis of CML has a prognostic significance. Successive accumulation of early granulocytic precursors with a low mitotic activity probably occurs in the bone marrow during the chronic phase of CML ending up in the final blast crisis characterized by a heavy predominance of such celis.