Absence of monocyte procoagulant activity during the immune response to influenza virus.

The ability of mononuclear phagocytes (MPh) to manifest procoagulant activity (PCA) resulting in the formation of fibrin is thought to be a key MPh effector function in tissue repair. The present study addresses the question of whether monocyte PCA is confined to tissue hypersensitivity reactions or is a general correlate of all immune responses. We show here that PCA is not the obligate outcome when the immune system is stimulated. In particular, under in vitro conditions in which a mitogen (phytohemagglutinin) or an antigen (purified protein derivative of tuberculin) elicits good PCA responses, incubation with influenza virus does not result in the generation of PCA, although other parameters of response to the virus appear to be intact. Moreover, influenza virus can cause suppression of PCA when cultures are stimulated with either phytohemagglutinin, purified protein derivative of tuberculin, or endotoxin, conditions which would otherwise result in good PCA responses. This lack of PCA persists throughout the culture period and is not caused by an effect of influenza virus on the viability of either MPh or leukocytes in general.

Changes in nucleolar morphology during human macrophage development: a morphometric study.

Morphometric methods were used to study the nucleolar ultrastructure during the development of human blood monocytes into macrophages in suspension culture. Nucleolar volume (Vn), surface area (Sn), volume fraction within the nucleus (VVn), surface-to-volume ratio [(S/V)n] and number of nucleolar profiles per section were measured in 20 healthy adults over a 6-day period, and the results examined using multivariate and univariate analyses of variance. Highly significant increases in Vn, Sn and VVn occurred, with no significant change in the number of nucleolar profiles per section; (S/V)n decreased during culture; no significant differences were found between male and female subjects. These nucleolar changes would be consistent with an increased protein synthesis during macrophage development. The results provide quantitative data against which changes in nucleolar morphology during macrophage development in disease states may be assessed.

Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells.

Treatment of human monocyte U937 and promyelocyte HL-60 cultures with agents known to induce differentiation (12-O-tetra-decanoylphorbol 13-acetate, calcitriol and dimethylsulfoxide) accelerates the maturation of cathepsin D and enhances the incorporation of [35S]methionine into cathepsin D. The most pronounced effects are obtained with calcitriol, which at a concentration of 10(-7) M increases the incorporation of [35S]methionine into cathepsin D from 0.08% to 0.4% of the detergent-soluble radioactivity. In addition, this treatment enhances the secretion of cathepsin D from about 8% to greater than or equal to 16% of the newly synthesized enzyme. In the presence of 10mM NH4Cl approximately half of the produced cathepsin D is secreted in both control and calcitriol-treated cells. It appears that in U937 cells two mechanisms are involved in sorting of cathepsin D. One of these is sensitive to NH4Cl and its efficiency is selectively decreased in cells pretreated with calcitriol.

Efecto del tratamiento sobre lo crecimiento de colonias gránulo-monocitarias en la médula ósea de pacientes con policitemia vera / Effect of treatment on the growth of monocyte-granulocytic colonies in bone marrow of patients with polycythemia vera

Se cultivó en agar semisólido la médula ósea de 15 pacientes con policitemia vera (PV) con el propósito de conocer la acción del tratamiento sobre el crecimiento de las colonias gránulo-monocitarias. Tres de los enfermos no habían recibido ningún tipo de tratamiento previo al estudio, 6 habían tenido como única terapéutica flebotomías seriadas y 6 solamente P a la 32. Se observó que en los enfermos sin tratamiento previo, el número de colonias fue similar al control. En los pacientes tratados con flebotomías el número de colonias fue menor que el control, pero sin diferencia significativa. Los cultivos de médula ósea en los casos tratados con P a la 32 mostraron una reducción marcada en el número de colonias que fue significativa con respecto al control (p < 0,005). Ninguno de los enfermos presentaban leucocitosis, ni hiperplasia granulopoyética. Se consideró que la disminución de colonias en los pacientes tratados con flebotomías refleja un tránsito de los progenitores granulopoyéticos de médula ósea hacia la sangre periférica, por el estrés que se produce en la hemopoyesis como consecuencia de las flebotomías. En cuanto a los enfermos tratados con P a la 32, todo parece indicar que el radionúclido inhibe la población de progenitores granulopoyéticos, sin que se produzca una disminución en el compartimiento de las células precursoras

Procoagulant cellular activity (PCA) in the classification of acute leukemia.

The procoagulant cellular activity (PCA) of leukemic cells was evaluated, before and after endotoxin stimulation, in 38 patients with acute leukemia at presentation subdivided according to the FAB classification. In the M4 and M5 subgroups the stimulated leukemic cells showed a significant increase in the production of PCA compared with freshly isolated cells. No evident PCA was documented in M1 and M2 AML as well as in the majority of acute lymphoid leukemias tested, both before and after endotoxin stimulation. The myeloid and lymphoid leukemic cells appear to behave similarly to normal leucocytes, within which only monocyte/macrophages are capable of producing PCA following endotoxin stimulation. These findings suggest that in human leukemic cells the endotoxin-induced production of PCA may be considered a indicator of monocyte/macrophage differentiation and thus represent a valuable diagnostic tool in the classification of acute leukemias.

Ferritin synthesis by monocyte-derived macrophages in thalassemic patients with intrinsic iron overload.

Macrophage ferritin content was determined following culture of peripheral blood monocytes for a period of 8 d in 40% autologous plasma to render them mature macrophages. Ferritin content was measured prior to and following culture using the radioimmunoassay. The normal range of values was established in a group of 22 healthy volunteer blood donors. A significant increase in the ratio of macrophage/monocyte ferritin was observed in every donor studied (range 1.2 - 1.8, p less than 0.001). Also, a further significant increase was observed when these macrophages were additionally incubated for 6 h with heterologous antibody-coated sheep red blood cells (range 1.2 - 1.57, p less than 0.001). Finally, the same studies were performed on a group of thalassemic patients with and without intrinsic iron overload. Again there were significant increases in monocyte ferritin content following culture as well as ingestion of heterologous sheep red cells, with magnitudes similar to those obtained with normal donor monocytes. Therefore we could not demonstrate the presence of a cellular ferritin synthesis defect in macrophages of thalassemic patients with intrinsic iron overload to explain the uncontrolled absorption of dietary iron from their gut.

In vitro induction of cytologic and functional differentiation of the immature human monocytelike cell line U-937 with phorbol myristate acetate.

The tumor-promoting ester 4 beta-phorbol 12-myristate 13-acetate (PMA) has been shown to induce the differentiation of the immature monocytelike cell line U-937 c in vitro into a heterogeneous population of cells, including small "dense" cells, large vacuolized or "foamy" cells, spindle-shaped cells, and cells with multiple filopodia ("stellate" cells). The effect of PMA was dose- and time-dependent, the optimal conditions being 40-162 nM PMA for 48 hours. The minimum time of exposure to PMA to ensure further differentiation of U-937 cells was about 5 hours. The PMA-stimulated cells acquired morphologic, ultrastructural, and functional characteristics typical of cells of the monocyte/macrophage lineage. The PMA-treated U-937 cells became adherent, ceased to proliferate, and exhibited increased expression of monocyte-specific antigens (Leu-M2, - M3, HLADr), surface receptors (FcR, C3bR), enzymes (nonspecific esterase, transglutaminase), and ability to mediate chemotaxis, phagocytosis, superoxide anion production, and antibody-dependent cytotoxicity reactions. The induced cells lost their morphologic differentiation and ability to attach to surfaces and regained proliferative capacity upon repeated subculture in PMA-free media.

Characterization of erythroid and granulocyte monocyte progenitors in human cord blood.

Some characteristics of both erythroid and granulocyte monocyte progenitors in human cord blood were compared to those in adult blood and bone marrow. The number of progenitors in cord blood was higher than that in adult blood and bone marrow. Most colonies in cord blood culture were monocyte-macrophage, whereas those from adult blood were largely eosinophilic. Cord blood progenitors had a slower sedimentation velocity than that reported for marrow, but sedimented faster than that for adult blood. A significant proportion of progenitors in cord blood as well as adult marrow was found to be in the DNA synthetic phase of the cell cycle whereas progenitors in adult blood were not. Cord blood BFU-E were more resistant than adult blood BFU-E but cord blood CFU-GM were not different from adult blood CFU-GM with regard to radiation sensitivity. Cord blood CFU-GM appeared to be more radio-resistant than adult marrow GFU-GM. From these results is seems clear that progenitors in cord blood differ in some aspects from those in adult blood and bone marrow.

Glucocorticoid therapy in the preleukemic syndrome (hemopoietic dysplasia): identification of responsive patients using in-vitro techniques.

Bone marrow cells from 54 patients with the preleukemic syndrome were cultured in agar (granulocyte colony forming units) in the presence and absence of cortisol. Twenty-four patients were given trials of prednisone therapy after the initial culture was performed. Cortisol (in vitro) failed to enhance colony growth in 29 of these 34 cases, and none of the 29 patients responded to prednisone therapy. Cortisol enhanced colony growth in five patients and three of these responded favorably to prednisone therapy. The correlation of in-vivo with in-vitro events is significant (P less than 0.005). Glucocorticoid therapy is of value in the management of a small number of patients with the preleukemic syndrome but is hazardous in those who fail to respond. These preliminary observations suggest that bone marrow cell culture techniques may aid in the identification of those patients who will and those who will not respond favorably to such therapy.

Human wound repair. II. Inflammatory cells, epithelial-mesenchymal interrelations, and fibrogenesis.

Connective tissue repair was studied in a series of skin wounds in young adult males. The tissues were examined at 3, 12, and 24 hr, and at 2, 3, 5, 7, 14, and 21 days after wounding. The neutrophilic leukocytes contain within membrane-bounded vacuoles some fibrin and serum protein from the wound; however, most of the granulocytes lyse and release their cytoplasmic contents into the extracellular space. The mononuclear cells undergo a series of morphologic alterations during which they develop a modest amount of relatively poorly developed rough endoplasmic reticulum and an extensive system of smooth-surfaced membranes prior to active phagocytosis. They could be clearly distinguished from immature fibroblasts by the differences in the development of their organelles, particularly the rough endoplasmic reticulum. The perivascular connective tissue adjacent to the wound contains cells which appear like poorly developed or immature fibroblasts. The development of these cells into mature fibroblasts can be followed during the different stages of wound repair. Intimate contact was observed between basal cells of the regenerated epidermis and monocytes in the wound below: cytoplasmic projections of the basal cells extended beneath the basement lamina to the surface of the monocytes. Such contacts were seen only on the 4th-7th day after wounding. Their possible significance is discussed.