Inductive effect of recombinant human interleukin-1 alpha and beta on differentiation of macrophage-like tumor cell line P388D1

We used the mouse monocyte/macrophage-like tumor cell line P388D1 to test whether or not interleukin-1 (IL-1) stimulates differentiation of monocyte/macrophage progenitors. Incubation of these cells with recombinant human interleukin-1 (rhIL-1) alpha and beta resulted in their increased adherence, stimulation of nonspecific esterase activity, and increased Fc rosette formation. rhIL-1s inhibited cell growth and stimulated Fc rosette formation in a dose-dependent fashion. The cell growth inhibition due to rhIL-1s depended on the concentration of serum in culture medium. Synergism between rhIL-1 and calcium ionophore A23187 was found for the cell growth inhibition and Fc rosette formation. The presence of ethylene glycol bis- (beta-aminoethyl ether) N,N,N,N,-tetraacetic acid(EGTA) in the medium abolished the stimulatory effect of rhIL-1 on Fc rosette formation of the cell line. These results demonstrate that rhIL-1s are a potent inducer of the differentiation of the macrophage-like tumor cell line P388D1.     

Iron Compounds after Erythrophagocytosis: Chemical Characterization and Immunomodulatory Effects

In humans, the lymphomyeloid system has a fundamental role on iron metabolism promoting its recycling due to a continuous removal of effete red blood cells. Additionally, one of the most intriguing aspects of metalloporphyrins in biology is their effect on the immune system. However, the process of erythrocyte catabolism is still poorly understood and needs further research. In the present study, we attempt to investigate the nature and the possible physiologic role of Fe compounds released after erythrophagocytosis during the removal of red blood cells. Monocyte erythrophagocytosisin vitroexperiments were done to characterize chemically the Fe compounds present inside the cells and in the culture supernatants. We tested the probable immunomodulatory functions of erythrophagocytosis products over lymphocyte cultures activatedin vitrowith T mitogens (α-CD3). Data obtained from atomic absorption spectroscopy confirmed the presence of Fe in the culture supernatants of monocyte cultures after erythrophagocytosis. Also, high-spin haem complexes derived from erythrocyte catabolism were detected by electron paramagnetic electronic resonance. Finally,in vitroactivated lymphocyte proliferation experiments indicate the co-mitogenic properties of monocyte culture supernatants after red blood cells phagocytosis. Thus, the results of the present work provide evidence that culture monocyte supernatants afterin vitroerythrophagocytosis contain Fe (III) high-spin haem complexes and show lymphocyte proliferation co-stimulatory properties.     

Redistribution of the Fc receptor on human blood monocytes and peritoneal macrophages induced by immunoglobulin G-sensitized erythrocytes.

The Fc receptor is a plasma membrane component exhibiting an affinity for the C gamma 3 domain of certain subclasses of immunoglobulin G. Using anti-Rho (D)-sensitized red cells (EA) in a slide rosette technique, we have demonstrated a translational mobility and polar redistribution of this receptor on the human blood monocyte and peritoneal macrophage. These cells, isolated from venous blood and malignant ascites and identified histochemically, showed a time- and temperature-dependent receptor capping defined by binding six or more EA confined to the cell half-perimeter. Caps formed in serum were mainly extreme caps in which bound EA appeared as a morula contiguous with the adherent cell. At 21 degrees C and 37 degrees C in serum 80% live rosettes formed caps; virtually none formed at 4 degrees C and about 25% were seen in PBS at 21 degrees C. Similarly, 10% extreme caps in PBS and 60 and 70% in serum were seen at room temperature and 37 degrees C, respectively, suggesting a serum factor(s) was important in promoting live rosette capping. Capping was reversibly inhibited by sodium azide although inhibition was incomplete even at 0.1 M, a concentration 10-fold higher than that giving complete inhibition of lymphocyte antigen-receptor capping. Azide also induced reversion of capped rosettes to diffuse, noncapped rosettes, and to levels comparable to those seen in inhibition studies. Re-exposure to EA after rosette capping failed to increase either the proportion of cells forming rosettes or the fullness of such rosettes indicating a critical number of receptors had been capped. Live rosettes induced a spherocytosis in bound EA irrespective of capping status; such changes appeared early in PBS where capping was minimal. Dead cells bore EA as normal biconcave discs. Some rosetting EA were ultimately hemolyzed upon prolonged incubation, and erythrophagocytosis was seen in about 15% of capped rosettes at 37 degrees C.     

Splenic monocyte-macrophage dependent suppression of autologous granulocyte-progenitors growth in Hodgkin's disease

The studies described compare the effect of spleen cell suspensions from 11 patients with Hodgkin's disease (HD) and 5 healthy subjects on the clonal growth of autologous marrow granulopoietic progenitors in diffusion chamber culture (CFU-G/D). Adherent monocyte/macrophage fraction of splenocytes from HD suppresses the proliferation of autologous CFU-G/D. This inhibition was mediated by an indomethacin-sensitive humoral factor(s). Non-adherent lymphoid cells stimulated myeloid colony formation. Dose response curves demonstrated a markedly increased inhibitory-activity production already by low numbers of splenic monocytes/macrophages from HD whereas a comparable counts of monocytes/macrophages from the spleens of healthy subjects stimulated the CFU-G/D growth. These results may suggest a possible activation of splenic monocytes/macrophages with an enhanced prostaglandin-mediated suppressor activity release for local granulocytopoiesis in the spleens of patients with HD.     

Disordered development of macrophages in non-Hodgkin's lymphoma

Macrophage development in 20 untreated patients with non-Hodgkin's lymphoma (NHL) has been studied and compared with that in 20 normal subjects. Morphometric measurements were carried out on ultrastructural features of cell, nucleus and mitochondria during 6 days suspension culture of blood monocytes in the presence of autologous serum and lymphocytes. The results were subjected to multivariate and univariate analysis of variance. Statistically significant differences were found between the subject groups with respect to the volumes and surface areas of cell, nucleus and mitochondria, to the excess surface membrane of cell and nucleus (as compared with equivalent spheres) and to the number of mitochondrial profiles per section. It would appear that the patients' cell grew less, showed less elaboration of surface features and had reduced nuclear and mitochondrial development, the latter affecting mitochondrial numbers rather than individual size. The findings provide further evidence that mononuclear phagocytes are deranged in NHL.     

Human monocyte-lymphocyte interaction: a new technique.

A rosette-type assay of the physical interaction between lymphocytes and monocytes after treatment with neuraminidase-galactose oxidase (NGAO) is reported. Monocyte-lymphocyte (ML) rosette formation and subsequent lymphocyte proliferation occurred when either lymphocytes or homologous monocytes were treated with NGAO and cultured together. Maximal ML rosette formation took place at 37 degrees C 4 hr after culture in media containing 10% serum at lymphocyte to monocyte ratios of 10:1 to 20:1. The percentage of rosette formation correlated with the extent of thymidine incorporation when increasing concentrations of NGAO were used. When NGAO-treated monocytes were added to untreated T and non-T lymphocytes, they bound preferentially to T lymphocytes and induced proliferation only in the T subpopulation. These results indicate that the ML rosette assay measures a highly specific monocyte-lymphocyte physical interaction after a mitogenic stimulus which is an early event in lymphocyte activation since it reflects the degree of subsequent lymphocyte proliferation.     

Continuous treatment with recombinant Mycobacterium tuberculosis CFP-10-ESAT-6 protein activated human monocyte while deactivated LPS-stimulated macrophage

Influence of the recombinant culture filtered protein 10 (CFP-10) and early-secreted antigenic target 6kDa protein (ESAT-6) (r-CFP-10-ESAT-6, rCE) of Mycobacterium tuberculosis (Mtb) on human monocyte and macrophage activation was investigated using human monocyte, monocyte like THP-1 cell line and monocyte derived macrophage (MDM). rCE solely enhanced TNF-alpha release from human monocytes and THP-1 cells in a dose- and time-dependent manner. rCE enhanced expression of CD80 and CD40, it also synergized with IFN-gamma in induction of TNF-alpha production and HLA-DR expression. Pharmacological agents that selectively inhibit mitogen activated protein kinase activation markedly suppressed rCE-induced TNF- alpha release. However, continuous presence of rCE (>72h) during monocyte to macrophage differentiation inhibited macrophage response to LPS stimulation. Collectively, these data suggest that rCE might have differential influence on monocyte and macrophage activation, which might be correlated with Mtb immune evasion.     

Cytokine and nitric oxide release by J774A.1 macrophages is not regulated by estradiol.

Estradiol is able to regulate the release of inflammatory mediators by macrophages; however, the presence, extent, and direction of this modulation varies with species, tissue of origin, and cell culture conditions. This study examines the effects of 17-beta-estradiol (E2) on the release of inflammatory mediators by the J774A.1 mouse macrophage cell line. For experiments, cells were plated in phenol red-free DMEM containing 5% charcoal-dextran stripped calf serum. Western analysis showed that J774A.1 cells contain the estrogen receptor alpha (ER alpha) protein. We found that physiological and pharmacological levels of E2 (10(-12) M-10(-6) M) have no effect on the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), or monocyte chemoattractant protein-1 (MCP-1). This suggests that J774A.1 cells grown under these culture conditions would be useful for the investigation of non-estrogen-dependent mechanisms by which certain endocrine disruptors may affect their targets in macrophages.     

In-vitro development and degeneration of stromal cells from the uterus of the rat at three stages of pregnancy

On Days 6, 11 or 16 of pregnancy, endometrial tissue (Day 6) or decidual tissue (Days 11 and 16) were removed from rat uteri, dissociated into single cells and grown in culture. At intervals during the culture period, the cells were examined and the cell density was calculated. The cells from Days 11 and 16 of pregnancy were similar in appearance, being very large and flattened. Initially, cells from Day 6 were much smaller, but showed an increase in size and came to resemble cells from the later stages of pregnancy. Growth curves for cells from each of the three stages of pregnancy over the first 16 days of culture differed. Cells from Day 6 of pregnancy increased exponentially in number. In cultures of cells from Day 11, cell numbers were stable, and in cultures of cells from Day 16, an exponential decline was seen. Cells from Days 6 and 11 of pregnancy were cultured for 42 days, by which time only a few cells remained viable. Cultures of cells from Day 16 degenerated within 3 weeks. These results indicate that, in vivo, decidual cells are not controlled solely by a 'programmed lifespan'. Changes occurring during pregnancy, however, limit the potential of the cells for division and survival in culture.     

Rosette formation of concanavalin A-treated erythrocytes around polymorphonuclear leucocytes and lymphocytes.

Concanavalin A (Con A) induces rosette formation of erythrocytes around polymorphonuclear leucocytes and lymphocytes in cell suspensions of autologous human blood cells. The effect which is most characteristic in a concentration between 25 and 50 microgram/ml is due to Con A bound on the erythrocyte membrane. A similar effect, although less pronounced, was observed with phytohaemagglutinin at concentrations of 10 and 25 microgram/ml. The treated erythrocytes showed a higher affinity to polymorphonuclears when compared with lymphocytes. At the contact area, the membrane of the erythrocyte became highly folded while its free surface was smooth and spherical. The effect of the local concentration and immunobilization of the lectin on the erythrocyte membrane and the similarity of the contact pattern to that of erythrophagocytosis are discussed.     

Disordered development of macrophages in non-Hodgkin’s lymphoma

Macrophage development in 20 untreated patients with non-Hodgkin’s lymphoma (NHL) has been studied and compared with that in 20 normal subjects. Morphometric measurements were carried out on ultrastructural features of cell, nucleus and mitochondria during 6 days suspension culture of blood monocytes in the presence of autologous serum and lymphocytes. The results were subjected to multivariate and univariate analysis of variance. Statistically significant differences were found between the subject groups with respect to the volumes and surface areas of cell, nucleus and mitochondria, to the excess surface membrane of cell and nucleus (as compared with equivalent spheres) and to the number of mitochondrial profiles per section. It would appear that the patients’ cell grew less, showed less elaboration of surface features and had reduced nuclear and mitochondrial development, the latter affecting mitochondrial numbers rather than individual size. The findings provide further evidence that mononuclear phagocytes are deranged in NHL.     

New method to quantify erythrophagocytosis by autologous monocytes.

BACKGROUND: Anemia is the net result of decreased red blood cell (RBC) production and increased removal of RBCs. Replication and maturation of erythroid precursors and RBC lysis can be measured by standardized in vitro methods and surrogate markers, respectively. In contrast, erythrophagocytosis by autologous phagocytes is more difficult to quantify. METHODS: We developed a method to assess erythrophagocytosis by autologous monocytes from 5 ml of whole blood. RBCs were labeled with carboxyfluorescein-diacetate-succinimidyl ester (CFDA-SE) and subsequently coincubated with autologous CD14(+) monocytes. Phagocytosis was quantified using flow cytometry. After standardization, the assay was validated in patients with severe malarial anemia (SMA), a condition that is associated with increased erythrophagocytosis. RESULTS: After labeling, CFDA-SE was stably incorporated into RBCs and no significant leakage leading to contamination of nonlabeled cells was observed. Monocytes ingested opsonized, labeled RBCs seven times more than nonopsonized controls. Erythrophagocytosis was significantly higher in SMA than in healthy controls. CONCLUSIONS: The established assay showed enhanced autoerythrophagocytosis associated with SMA and hence was able to detect clinically relevant erythrophagocytosis. This novel assay is well suited for rapid quantification of in vitro erythrophagocytosis by autologous monocytes.     

Alterations in thymocyte surface markers after in vivo treatment by serum thymic factor.

Three T cell markers (heterologous sheep rosette, autologous rosette, and theta antigen) have been studied in thymocytes after in vivo injection of a single dose of serum thymic factor (Facteur Thymique Sérique: FTS). Within 24 h after such treatment, sheep erythrocyte-binding cells increased 5 to 8 times, while autologous rosette-forming cells decreased by a factor of 3. Using cytotoxicity assays (trypan blue exclusion test and chromium release) it was observed that thymocytes from FTS-treated mice presented a higher sensitivity to antitheta serum and complement than control mice. These results are compatible with the hypothesis that exposure of immature T cells to thymic hormone may induce T cell maturation, as assessed by decreased number of immature autologous rosette forming cells (RFC) and increased number of mature T cells (sheep RFC and theta-positive cells). Facteur Thymique Sérique appears as an interesting probe to define the various intra-thymic cellular compartments of differentiation.     

Plasmodium berghei: correlation of in vitro erythrophagocytosis with the dynamics of early-onset anemia and reticulocytosis in mice.

Infection of BALB/c mice with Plasmodium berghei results in an anemia which is excessive to that which can be accounted for solely by direct destruction of infected erythrocytes by the mature schizonts at the time of merozoite release. Mice infected with 10⁴ infected erythrocytes exhibited a progressive anemia beginning on Day 7. Significant reticulocytosis was first observed on Day 9 and parasitemia tended to parallel reticulocytosis with a lag of about 1 day. In studies of erythrophagocytosis, washed erythrocytes from randomly selected mice infected with 10⁵ infected red blood cells were phagocytized by peritoneal macrophages in vitro to a significantly greater extent on Days 3–5 postinfection than were erythrocytes taken from normal controls. The degree of erythrophagocytosis reached a peak on Day 4 and returned to control levels on Days 6 and 7. Erythrocytes taken from infected animals on Day 7 and incubated in normal plasma were phagocytized to a significantly greater extent than were normal erythrocytes incubated in normal plasma or erythrocytes from infected mice incubated in plasma from infected animals. The enhanced in vitro erythrophagocytosis observed on Days 3–5, which preceded and coincided with the beginning of the early-onset anemia on Day 5, may correlate with in vivo phenomena which may contribute to the developing anemia. Furthermore, the restoration of enhanced erythrophagocytosis by normal plasma seems to indicate that some component(s) of normal plasma may be depleted during the early stages of P. berghei infection.     

Production of interleukin-1 and tumour necrosis factor in non-Hodgkin's lymphoma patients

Summary Peripheral blood monocytes from non-Hodgkin's lymphoma (NHL) patients were assessed for the monocyte functions with respect to their ability to secrete interleukin-1 and tumour necrosis factor (TNF) and their cytotoxic potential to tumour target WEHI 164 clone 13. Our results indicate comparable levels of interleukin-1 and TNF production by NHL patients. The cytotoxic potential by monocytes was also not depressed in these patients. The data obtained suggest normal monocyte functions in NHL patients.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Enhancement of colony-stimulating-factor--dependent clonal growth of murine macrophage progenitors and their phagocytic activity by retinoic acid

The effect of retinoic acid (RA) on the colony-stimulating-factor-dependent clonal growth of myeloid progenitors was assessed in semisolid agar cultures of mouse bone marrow cells using L-cell-conditioned medium that gave rise to macrophage colonies, granulocyte colonies, and mixed macrophage-granulocyte colonies and clusters. RA was found to enhance the overall formation of myeloid colonies (about 50%) and clusters in 7-day cultures. The increase was due to an enhanced formation of macrophage colonies (70-250%) and clusters which reached a maximal value at about 3 microM RA. In 4-day cultures, the effect of RA on macrophage colony formation was biphasic with a maximal enhancement at 10 nM. RA suppressed granulocyte-colony formation in 4-day cultures. RA increased the phagocytic activity of bone-marrow-derived macrophages at all stages of differentiation and/or maturation in culture. The Fc-receptor-mediated erythrophagocytosis as well as the phagocytosis of heat-killed yeast cells (HK-yeast) and starch particles increased by RA treatment in a dose-dependent manner, reaching an increase of 100-200% of the activity expressed in the absence of RA. Peritoneal exudate macrophages likewise exhibited an increased phagocytic response to a variety of particles, at both physiological and pharmacological concentrations of RA. Expression of an RA-mediated increase in phagocytic activity required a prolonged incubation with RA (greater than 19 hr). The data suggest that RA may be of physiological relevance in the regulation of proliferation and function of hemopoietic cells. Therapeutic doses of RA may potentiate macrophage proliferation and function, elements that are crucial at all phases of the various defense mechanisms that the organism possesses.     

Induction of differentiation of the human histiocytic lymphoma cell line U937 in the absence of vimentin expression

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.     

Activation of purified human thymus-derived (T) cells by mitogens. II. Monocyte- macrophage potentiation of mitogen-induced DNA synthesis.

Thymus-derived (T) cells from peripheral blood were purified by rosette formation with neuraminidase-treated sheep red blood cells (SRBC) and centifugation on Ficoll-Hypaque. T cells recovered from the pellet were freed of SRBC by treatment with Tris-NH4Cl. T cells purified by this method showed a diminished ability to take up 3H-thymidine (3H-TdR) after mitogen stimulation when compared to the mitogenic response of an equal number of autologous peripheral blood mononuclear lymphocytes (PBL). Autologous monocytes restored the capacity of purified T cells to take up 3H-TdR in the presence of phytohemagglutinin (PHA) or Concanavalin A (Con A). The effect was proportional to the number of monocytes added. Similar restorative effects could be obtained with allogeneic or xenogeneic monocytes. These data suggest that the mitogenic stimulation of human PBL and Con A may reflect the participation of more than one cell type: the T cells and monocyte and that the genetic origin of the monocyte is not critical for augmentation of the mitogenic activation of human T cells.     

Analysis of serum proteome profiles of non-Hodgkin lymphoma for biomarker identification

Serum samples from non-Hodgkin lymphoma (NHL) patients who had not undergone chemotherapy, lymphnoditis patients, and healthy adults were analyzed using surface-enhanced laser desorption–ionization time-of-flight mass spectrometry (SELDI-TOF MS) to detect the differentially expressed serum proteins. Models were developed to distinguish between the healthy adult group and the NHL group, with a sensitivity of 69% and specificity of 90%, and between the lymphnoditis group and the NHL group with a sensitivity of 74% and specificity of 84%. A protein with the m/z of M10 197.91 u was expressed at a significantly higher level in the NHL group, compared to the other groups. Furthermore, differences were also significant among different stages of NHL and among samples with different International Prognosis Index (IPI) scores or lactase dehydrogenase (LDH) levels. The three identified proteins may offer a new serological approach for early diagnosis, differential diagnosis, and pathogenic investigation of NHL. And the protein with the m/z of M10 197.91 u may be a new serological biomarker for monitoring treatment response and evaluating the prognosis of patients with NHL.