Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.     

Bystander effect of normal fibroblasts for macrophages co-cultured with susceptible transformed target cells

Macrophages attack and kill pathologically changed, transformed and tumor cells. However, in some cases they may also support tumor growth, modulate the action of anticancer drugs, and even facilitate the development of drug resistance in tumor cells. Here we present data that bystander fibroblasts NIH3T3 were not only resistant to murine macrophages J774.2 but also blocked their killing action towards murine transformed fibroblasts L929. Macrophages were isolated from mixed cultures by means of CD11b specific immunomagnetic beads, and changes induced by their former co-culturing were studied using DNA microarray technology and other tests. An expression of candidate genes coding for cytokines and for signal transduction pathway proteins was estimated in macrophages in different variants of their co-culture with target cells. Changes in expression of mRNA for interleukin 1beta, NFkappaB, IkappaBalpha, gadd45, and CD5 were detected as the most prominent in the macrophages co-cultured with the transformed cells. Bystander NIH3T3 fibroblasts abolished these changes in the macrophages J774.2, and the level of expression of the above mentioned genes was close to the level seen in the macrophages which did not exert cytotoxicity towards the target fibroblasts. Potential implications and research perspectives of using the macrophage-target cell co-cultures with different bystander cellular partners are discussed.     

Enhanced release of oxygen metabolites by monocyte-derived macrophages exposed to proteolytic enzymes: activity of neutrophil elastase and cathepsin G

Macrophages at sites of inflammation are exposed to proteolytic enzymes derived from neutrophils, platelets, clotting factors, complement, and damaged tissues. To investigate the possible effect of proteases on the plasma membrane-mediated oxidative metabolic response of macrophages in inflammatory sites, cultured human monocyte-derived macrophages were treated in vitro with proteolytic enzymes and were then assayed for their ability to release superoxide anion (O2-) and hydrogen peroxide (H2O2) in response to stimuli. Macrophages pretreated for 1 to 20 min with trypsin, chymotrypsin, pronase, or papain, 0.1 to 200 micrograms/ml, released up to 3.5-times more O2- and H2O2 than did control (untreated) cells. This enhanced production of oxygen metabolites was observed by using either phorbol myristate acetate or opsonized zymosan as the stimulus. Macrophages were also "primed" for enhanced O2- release (2.3-fold) by pretreatment with a subfraction of granules extracted from human neutrophils. This subfraction contained primarily elastase and cathepsin G. Similar enhancement was observed with 60 ng/ml or purified human neutrophil cathepsin G (2.2-fold) and with 20 micrograms/ml of purified neutrophil elastase (3.3-fold). Priming by these neutrophil proteases could be blocked by specific inhibitors of their proteolytic activity. These results suggest that macrophages involved in an inflammatory response might be rapidly primed by proteases released from degranulating neutrophils. Primed macrophages could mount a more effective oxidative metabolic response to microorganisms or tumor cells, but might also cause greater tissue damage.     

Immortalized phenotype and the presence of active oncogenes correlate with the capacity of culture cells to induce reactivation of DNA synthesis in macrophage nuclei in heterokaryons

Several types of culture cells with limited life span (rat embryo fibroblasts, rat chondrocytes and mouse premacrophages) were found to be unable to induce the reactivation of DNA synthesis in the nuclei of non-dividing differentiated cells (mouse peritoneal resident macrophages) in heterokaryons. By contrast, malignant HeLa cells have this ability. In heterokaryons formed by fusion of mouse macrophages with HE239 cells (Syrian hamster fibroblasts transformed with a ts mutant of the SV40 virus), DNA synthesis in macrophage nuclei is reactivated only at the permissive temperature (33° C), at which viral T antigen is stable. Immortalization of rat chondrocytes by transfection with p53 gene enables to induce DNA synthesis in macrophage nuclei upon fusion. All the evidence indicates that the function of immortalizing oncogenes is necessary for the resumption of the DNA synthesis in macrophage nuclei in heterokaryons.     

Studies of the Numbers and Morphology of the Intracellular Form of Leishmania donovani Grown in Cell Culture*

SYNOPSIS. Macrophages were infected in vivo with the intracellular form of Leishmania donovani (LDs), harvested from the previously saline-stimulated peritoneal cavities of hamsters and explanted into Leighton tubes containing removable coverslips. Serum from either rabbit, chicken, human, calf, hamster or cotton rat blood was used as the 40% component of a Hanks' BSS₆₀ serum₄₀ medium used to maintain these Leighton tube cultures at 37 C. After varying lengths of time coverslips were removed from tubes, stained with Giemsa, and the parasites per infected macrophage, total number of hamster cells and total number of parasites on each coverslip were counted. Maerophages constituted more than 90% of the explanted cells on the coverslips. When cotton rat serum was used as a component of the medium, fibroblastic overgrowth of the coverslips followed. Some similarities and differences in the numbers of macrophages, fibroblasts and parasites were noted with regard to the serum used as part of the medium. Except for cotton rat serum, the serum component of the medium used apparently did not influence, to any great degree, the morphology of either the macrophages or parasites therein. Thus, vacuolarization and granularization of macrophages did not appear to be very distinctly correlated with the time of sampling or the type of serum in the medium used for maintenance nor could any morphologic variations of the LDs be ascribed to these factors. When cotton rat serum, but not any of the other sera, was the serum component of the medium, leptomonads were noted in the overlay fluid of the cultures after 6 days. Under these conditions of cell culture, fibroblasts could not be infected with LDs although macrophages on the same coverslip were heavily parasitized.     

Characterization of a rat liver epithelial cell line to detect inhibitors of metabolic cooperation

Summary A normal rat liver epithelial cell line, with phenotype characteristics of “oval” cells (WB-F344), was examined for its ability to perform gap-junctional intercellular communication as measured by metabolic cooperation. To test for gap-junctional intercellular communication, 6-thioguanine-sensitive cells were cocultivated with 6-thioguanine-resistant cells. It was found that the recovery of 6-thioguanine-resistant cells depended on the densities of the 6-thioguanine-sensitive cells. Higher densities of 6-thioguanine-sensitive cells reduced the recovery of 6-thioguanine-resistant cells. These observations demonstrate that rat liver epithelial cells could metabolically cooperate, implying they could perform gap-junctional intercellular communication. Two tumor-promoting organochlorine pesticides, aldrin and dieldrin, were potent inhibitors of metabolic cooperation for these cells, but 12-0-tetradecanoyl-phorbol-13-acetate and teleocidin, known mouse skin tumor promoters, were not significantly effective in inhibiting metabolic cooperation. The results suggest that these cells might provide the basis for an in vitro assay specifically to study liver tumor promoters. Research was sponsored by a grant from the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under grant AFOSR-86-0084. The U. S. Government is authorized to reproduce and distribute reprints for government purposes notwithstanding any copyright notation thereon.     

Fibroblasts acquire beta-glucuronidase by direct and indirect transfer during co-culture with macrophages

Macrophages can transfer beta-glucuronidase directly to co-cultured fibroblasts during cell-to-cell contact as well as indirectly via receptor-mediated endocytosis. The degree of enzyme activity acquired by the deficient fibroblasts was determined by the ratio of donor to recipient cells and by the length of time for which cells were allowed to interact. Both mechanisms of transfer were efficient so that 70% of normal enzyme activity was restored to deficient fibroblasts after 24 h of co-culture. These observations show that macrophages have great potential as donor cells in replacement therapy for the treatment of inherited lysosomal enzyme deficiency diseases.     

Role and function of macrophages in the metabolic syndrome

Macrophages are key innate immune effector cells best known for their role as professional phagocytes, which also include neutrophils and dendritic cells. Recent evidence indicates that macrophages are also key players in metabolic homoeostasis. Macrophages can be found in many tissues, where they respond to metabolic cues and produce pro- and/or anti-inflammatory mediators to modulate metabolite programmes. Certain metabolites, such as fatty acids, ceramides and cholesterol crystals, elicit inflammatory responses through pathogen-sensing signalling pathways, implicating a maladaptation of macrophages and the innate immune system to elevated metabolic stress associated with overnutrition in modern societies. The outcome of this maladaptation is a feedforward inflammatory response leading to a state of unresolved inflammation and a collection of metabolic pathologies, including insulin resistance, fatty liver, atherosclerosis and dyslipidaemia. The present review summarizes what is known about the contributions of macrophages to metabolic diseases and the signalling pathways that are involved in metabolic stress-induced macrophage activation. Understanding the role of macrophages in these processes will help us to develop therapies against detrimental effects of the metabolic syndrome.     

The non-selective junctional communication phenotype of normal and transformed human epidermal keratinocytes in vitro

The ability of cells to transfer tritium-labelled nucleotides to other cells is a measure of gap-junction-mediated communication based on metabolic cooperation. Here we report that human epidermal keratinocytes (HuK) transfer radiolabel to a variety of cell types including human skin fibroblasts (HuDF), human mammary fibroblasts (HuMF) and calf lens epithelial cells (CaLE). The metabolic cooperation pattern of HuK shows them to be non-selective communicators and in this respect they differ from another human epithelial cell type mammary epithelial cells (HuME), which communicates with CaLE but not HuDF or HuMF. SV40 transformation is known to modulate metabolic cooperation between some human cells in vitro. Here we further report that SV40 transformation of HuK has no obvious effect on their non-selective communication phenotype. Possible mechanisms involved in selective and non-selective junctional communication and their relevance to epithelial/stromal interactions in vivo are discussed.     

Macrophage requirements of CR- and CR+ B lymphocytes for antibody production in vitro.

A Sephadex G-10 column coated with antigen-antibody complexes and complement retains complement receptor-bearing (CR+) mouse spleen cells. The effluent is rich in thymus-derived cells (T cells), and contains bone marrow-derived cells (B cells) which carry surface immunoglobulin (Ig), Ir-associated antigen (Ia), and Fc receptors, but no complement receptors (CR-). Although both unfractionated and CR- B cell populations are capable of producing antibody to red cell antigens, they differ in their requirements for the initiation of the response. Unfractionated B cells cooperate with primed as well as unprimed helper T cells; macrophages are required for this cooperation but can be replaced by 2-mercaptoethanol. CR- B cells cooperate with primed but not with unprimed T cells provided macrophages are added to cultures. After addition of culture supernatant from BCG-activated macrophages CR- B cells cooperate with both unprimed and primed T helper cells.     

Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53

The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.     

Macrophage receptors]

Macrophages are involved in many immunological functions such as phagocytosis, cytotoxicity, antigen binding and cooperation with lymphocytes. The triggering of those functions involves membrane receptors. Several receptors species are well characterized, but some phenomena can be accounted for only if macrophages are endowed with nonspecific adhesive structures. A study of the effect of various physical or chemical factors on the binding of several particle species by rat peritoneal macrophages allowed us to classify nonspecific receptors and discuss the mechanisms involved in some types of cellular interaction.     

Relationship between connective tissue cells and fibronectin in a sequential model of experimental hepatic fibrosis

The cellular and non-cellular components of fibrous septa formed at early and late stages in a sequential model of experimental hepatic fibrosis have been investigated using ultrastructural and immunocytochemical techniques. In the early septa, cells with intermediate features between lobular Ito cells and active fibroblasts were formed. These cells frequently displayed subplasmalemmal microfilaments (myofibroblast-like cells). Macrophages were also present. Scanty typical fibroblasts were present in the late septa. This cellular recruitment might be related to an extracellular glycoprotein-fibronectin-which is at present under investigation as a chemotactic factor for fibroblasts. Strong positivity for fibronectin in early septa and its sharp decrease in late septa seems to support this view. Fibroblasts and/or macrophages are the likely source of fibronectin synthesis.     

Micro-environmental factors in the survival of human B-lymphoma cells

Burkitt lymphoma (BL) cells retain a high inherent propensity to undergo apoptosis indicating that net growth of the tumour population in vivo is likely to be influenced profoundly by its micro-environment. Here we investigate micro-environmental factors that affect BL-cell survival in vitro. We show that survival, and consequently net production, of tumour cells is enhanced by autocrine factors and, to a greater extent, by paracrine factors provided by relevant stromal elements of the tumour (fibroblasts and follicular dendritic cells) and by macrophages. Promotion of BL-cell survival by paracrine elements was mediated by cell/cell contact and by short-range soluble factor(s). IL-4, IL-10 and TNF-alpha promoted, whereas TGF-beta1 inhibited, tumour-cell production. Macrophages engaged in phagocytosis of apoptotic BL cells were less effective than untreated macrophages in supporting net expansion of BL populations. These results suggest that the net production of tumour cells in BL is supported by multiple micro-environmental factors that modulate apoptosis.     

Metabolic cooperation studied by a quantitative enzyme assay of single cells

A new method making use of a radiochemical enzyme assay at the single cell level is presented to investigate metabolic cooperation, a widely studied form of cellular communication. In this case metabolic cooperation between normal human fibroblasts and fibroblasts derived from a patient deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase has been studied.A mixture of an equal number of both cell types was cultured in close physical contact and after trypsinisation, replating and culturing the cells for several hours in a high dilution, quantitative enzyme measurements with individual cells isolated from the mixture were carried out. From the distribution curve of the enzyme activities of the individual cells the conclusion could be drawn that a macromolecule, either the enzyme itself or DNA or mRNA, coding for that enzyme, is transferred from normal to mutant cells.     

Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion, orientation, and movement of cells cultured on ultrathin fibronectin fibers

Summary This study examined the behavior of rat tendon fibroblasts, baby hamster kidney fibroblasts, macrophage-like P388D1 cells, and neurons from rat dorsal root ganglia, cultured on fibronectin strands 0.2–5 μm in diameter. We investigated cell spreading, orientation, formation of focal contacts, the speed of cell movement, and the speed of neurite outgrowth in cells cultured on fibronectin strands, glass covered with fibronectin, and plain, nontreated glass. Fibronectin strands significantly promoted cell spreading and caused a marked alignment of all kinds of cells to the direction of the fiber. The fibers caused the alignment of actin filaments in fibroblasts and focal contacts in fibroblasts and macrophages and increased polymerization of F-actin in cells. Fibronectin fibers also increased the speed and persistence of cell movement and the rate of neurite outgrowth. Macrophages grown on fibronectin fibers produced numerous actin-rich microspikes and adopted a polarized, migratory phenotype. These findings indicate that fibronectin strands, resembling natural components of the extracellular matrix, are more effective in activating various types of cells than two-dimensional, fibronectin-covered substrata. The results also confirm the suitability of the three-dimensionally oriented fibronectin form for use in clinical practice.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with Mycobacterium bovis (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE2. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE2. The major metabolites of resident cells were PGE2 and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts

Rat fibroblasts degraded human low density lipoprotein (LDL) very slowly, one-tenth to one-fortieth the rates observed in human fibroblasts. In rat cells, human LDL caused only very small increases in cell cholesterol content and acylCoA:cholesterol acyltransferase (ACAT) activity and caused only small decreases in beta-hydroxy-beta-methylglutaryl CoA (HMG CoA) reductase activity; in human cells, however, human LDL induced very large changes in all three of these parameters, as expected. The binding of human LDL to rat fibroblasts was not reduced by previous incubation with human LDL or with 25-hydroxycholesterol. Thus, in rat fibroblasts there appear to be few, if any, regulated high-affinity receptors that recognize human LDL. Rat LDL fractions (d 1.02-1.05 g/ml), in contrast, were degraded more rapidly than human LDL by rat fibroblasts, caused a significant increase in cell cholesterol content, an increase in ACAT activity, and a significant decrease in HMG CoA reductase activity. Moreover, the degradation of this rat LDL fraction by rat fibroblasts as a function of concentration was biphasic, i.e., there appeared to be a high-affinity component of degradation. Thus, it appears that rat fibroblasts do have a receptor for homologous lipoproteins. However, because both apoprotein B and apoprotein E are present in these rat lipoprotein fractions, the observed effects may relate to recognition of either or both of these apoproteins. The metabolism and metabolic effects of the conventionally defined high density lipoprotein (HDL) fraction of the rat by rat or human fibroblasts resembled those of human LDL in human fibroblasts. It is suggested that rat HDL may, because of its apo E content and higher concentration in rat plasma relative to that of LDL, play an important role in cholesterol homeostasis in vivo.     

Interferon-γ modulates lung macrophage production of PDGF-BB and fibroblast growth

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-γ (IFN-γ) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-γ would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-γ (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-γ or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-γ prior to the addition of iron spheres synergistically increased PDGF-BB production 2–10-fold after 24 h. In contrast, when IFN-γ was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-γ alone did not affect proliferation. [¹²⁵I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-γ affected PDGF-BB binding to its receptors.