Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Cortisol modulates the induction of inflammatory gene expression in a rainbow trout macrophage cell line

Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1β, IFN-γ), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1β, rIFN-γ and poly I:C, of γIP by rIFN-γ or poly I:C, and of Cox-2 by rIL-1β was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment.     

A continuous alveolar macrophage cell line: Comparisons with freshly derived alveolar macrophages

Summary Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonizedPseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H₂O₂ and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations. This work was supported in part by grant A119811 and SCOR HL23578, from the National Institutes of Health, Bethesda, MD. Portions of these studies appeared as a poster presentation at the American Society for Microbiology, Atlanta, GA, 1987.     

Expression of the erythropoietin receptor on a human myeloma cell line

We demonstrated the expression of the erythropoietin (EPO) receptor by a human myeloma cell line (MM-S1) which was established in our laboratory. EPO dose-dependently stimulated the proliferation of MM-S1 cells. Binding of radioiodinated EPO (125I-Epo) to MM-S1 cells was competitively inhibited by unlabeled EPO, but not by other recombinant cytokines. Specific binding of 125I-Epo to MM-S1 cells was saturable, and the Scatchard analysis revealed 330 EPO binding sites per cell with a Kd of 0.56 nmol/L. Bound EPO was internalized by MM-S1 cells during incubation at 37 degrees C. This is the first report describing the expression of the EPO receptor by human cells other than those of the erythroid lineage.     

Gene expression changes induced by bismuth in a macrophage cell line

We have investigated the effect of bismuth by autometallography, cell viability, TUNEL assay and microarray analysis of a macrophage cell line. The cells accumulate bismuth in their lysosomes in a time- and dose-dependent manner. Cell viability assays show a significant decrease in the number of viable cells related to both bismuth concentrations and exposure time. TUNEL assays after 12 h and 24 h at a bismuth-citrate concentration of 50 M revealed the presence of 30% and 70% TUNEL-positive cells, respectively, compared with 8% in the controls. We have analysed gene expression profiles for cells exposed to 50 M bismuth-citrate and for untreated controls at 12 h and 24 h by microarray analysis, which confirmed that bismuth is a powerful metallothionein inducer. A number of glycolytic enzymes are induced by bismuth, suggesting that bismuth is able to induce hypoxia-like stress. BCL2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) has been suggested as a regulator of hypoxia-induced cell death independent of caspase-3 activation and cytochrome c release. Bnip3 is up-regulated indicating the involvement of Bnip3 as a possible mechanism for bismuth-induced cell death. Differences have been noticed in cell viability and in the modification of the mRNA expression levels at 12 and 24 h. Only 13 genes are modified at both these times, suggesting a time-dependent molecular cascade in which bismuth-exposed cells enter a dormant stage with mRNA down-regulation being followed by cell death of susceptible cells. This study was supported by the Aarhus University Research Foundation, Aase og Ejnar Danielsens Fond and Direktør Jacob Madsen og Hustrus Fond.     

Characteristics of macrophage activation by gamma interferon for tumor cytotoxicity in peritoneal macrophages and macrophage cell line J774.1

We investigated the characteristics of macrophage-mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN-gamma). In PMP, MTC was demonstrated when they were stimulated with IFN-gamma for 12 hr (short-term stimulation) and was abrogated when they were stimulated for 48 hr (long-term stimulation). Enhanced H2O2 generation was observed in PMP activated by long-term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non-treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short- and long-term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN-gamma stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN-gamma for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN-gamma. The results suggest that TNF-like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.     

Expression of differentiated functions in hepatoma cell hybrids. II. Aldolase

A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro–in vivo passage results in a reversible modulation in aldolase A activity. Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.     

Uptake of histamine by mouse peritoneal macrophages and a macrophage cell line,RAW264.7

We have previously demonstrated that dietary histamine is accumulated in the spleens of L-histidine decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis. To characterize the clearance system for dietary histamine in mice, we investigated the cell type and mechanism responsible for histamine uptake in the spleens of HDC-deficient mice. Immunohistochemical analyses using an antihistamine antibody indicated that a portion of the CD14⁺ cells in the spleen is involved in histamine storage. Peritoneal macrophages obtained from Balb/c mice and a mouse macrophage cell line, RAW264.7, had potential for histamine uptake, which was characterized by a low affinity and high capacity for histamine. The histamine uptake by RAW264.7 cells was observed at physiological temperature and was potently inhibited by pyrilamine, chlorpromazine, quinidine, and chloroquine, moderately inhibited by N-methylhistamine, dopamine, and serotonin, and not affected by tetraethylammonium and 1-methyl-4-phenylpyridinium. Intracellular histamine was not metabolized in RAW264.7 cells and was released at physiological temperature in the absence of extracellular histamine. These results suggest that histamine uptake by macrophages may be involved in the clearance of histamine in the local histamine-enriched environment. cation transporter; chlorpromazine; pyrilamine; quinidine     

Modulation of cyclic AMP-dependent protein kinase isozyme expression associated with activation of a macrophage cell line

We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the Mr of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 micrograms/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was not sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO.     

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.     

Glucose-6-phosphate dehydrogenase activity in a hepatoma cell line: preliminary evidence for negative genetic control.

A line of cells, positive for glucose-6-phosphate dehydrogenase activity, has been separated from a negative parental stock derived from the H4-II-E-3C rat hepatoma line. Cytogenetic analyses showed specific marker chromsomes of the parent line were present in the variant. On the other hand, cells of the variant line contained three chromosomes less, on the average, than the total present in parent cells. Karyotypic analyses indicated the diminution in chromosome numbers to be in the subacrocentric group. The appearance of glucose-6-phosphate dehydrogenase associated with loss of chromosomes suggests this enzyme is suppressed or under negative control in the parental stock. The consistent loss of chromosomes of the subacrocentric group suggests that the locus bearing regulatory activity is in this specific group.     

Cell hybrids between SV40-transformed macrophage cell lines and a Chinese hamster cell line: growth responsiveness and induction of colony-stimulating factor

Three cell lines from resident macrophages of BALB/c mice and four from activated macrophages of the same strain were isolated by infection with simian virus 40 (SV40). A majority of these cells showed dependency on L cell-conditioned medium (LCM), which is necessary for proliferation of normal macrophages in vitro. Somatic cell hybridization was applied in the study of macrophage growth responsiveness. A macrophage cell line (BR15) with strict dependency on LCM for growth was fused to a Chinese hamster cell line (hs222-16); it was found that dependency on LCM was a dominant trait in the hybrids. Following fusion of a macrophage cell line (BAM3) which grew without LCM to hs222-16, a large number of colonies appeared in the selection medium containing LCM. Four hybrids not requiring LCM for growth were selected in an LCM-free culture, and their hybrid properties were examined. Three out of the four hybrids secreted colony-stimulating factor (CSF) constitutively, whereas the fourth secreted no CSF. The level of acid phosphatase activity in the hybrids was higher than in the parent cells. Two peaks of CSF activity were observed after gel filtration chromatography of conditioned medium: One was eluted at molecular weight of 36,000 and the other at 17,000.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.