Dendritic cell activating factor produced by mouse macrophage hybridoma

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.     

Inhibition of erythroid cell growth in irradiated mice by allogeneic lymphoid cells: Specificity of the response

Allogeneic lymphocytes can inhibit proliferation of erythroid cells in the spleens of irradiated mice grafted with syngeneic bone marrow cells. Since there is a linear relationship between the number of injected lymphocytes and erythroid activity, the method is frequently used as a graft-vs-host assay. In this investigation we show that the reduction of erythroid activity is due to both a reaction of the lymphocytes against the tissues of the host and against the grafted bone marrow cells. Thus, reduced erythropoietic activity does not necessarily indicate that the bone marrow targets possess antigens against which the lymphocytes are reactive.     

Detection in a murine T lymphoma of a lipid-like factor that inhibits cell growth

In this paper we show that MCG3 cells, a murine T lymphoma, contain a factor(s) that inhibits the proliferation of cells of different histological origin. The lack of sensitivity of this cell proliferation-inhibiting factor (CPIF) to the treatment with proteolytic enzymes and its solubility in organic solvent demonstrated that it is a lipid-like substance. Separation by thin-layer chromatography showed it migrates before the prostaglandins with activity on cell proliferation. CPIF activity was reversible and more intense on bone marrow cells than on tumor cells, suggesting that it can play a role in cell growth regulation.     

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.     

Inhibition of adoptive secondary responses by lymphoid cell populations

The ability of normal and tolerant lymphoid cells to inhibit the adoptive secondary response was investigated in order to delineate the influence which the host can exert on a memory cell population as the host recovers from the effects of irradiation. LBN rats were irradiated with 500R whole body X-irradiation, reconstituted with either normal or tolerant lymphoid cells, then they were given immune spleen cells and challenged with soluble antigen (DNP-BGG). Cells capable of suppressing the adoptive secondary responses were shown to possess the following characteristics: 1) in nonimmune donors they were found in the greatest concentration in lymph nodes, followed by spleen and bone marrow and were practically absent in the thymus; 2) their numbers were not increased in donors rendered tolerant by long term treatment with deaggregated DNP-BGG plus cyclophosphamide nor in donors given large doses of DNP-BGG 48 to 72 hr before sacrifice; 3) in animals rendered tolerant by long term antigen treatment alone some enhancement of suppressor function was seen; 4) the suppressor cells could be shown among both glass wool adherent and nonadherent cells and 5) the nonantigen specific suppressor cells did not affect the kinetics of antibody formation nor the affinity of the antibody which was produced. These results are discussed in terms of the nature and source of the suppressor cell populations and their relevance to the control of secondary responses in intact animals.     

Inhibition of LPS-induced cytokines by Bcl-xL in a murine macrophage cell line

The antiapoptotic molecule Bcl-xL has been implicated in the differentiation and survival of activated macrophages in inflammatory conditions. In this report, the role of Bcl-xL in LPS-induced cytokine gene expression and secretion was studied. Bcl-xL-transfected RAW 264 macrophages were protected from gliotoxin-induced apoptosis, indicating the presence of functional Bcl-xL. Overexpression of Bcl-xL in this macrophage cell line was also associated with a marked inhibition of LPS-induced TNF-alpha, JE/monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 secretion. Inhibition of LPS-induced cytokine secretion was paralleled by a decrease in levels of steady-state mRNA for the above cytokines and for IL-1beta. Decreased production of TNF-alpha in Bcl-xL transfectants was not due to increased mRNA degradation, as the mRNA half-lives were the same in Bcl-xL transfectants and control macrophages. Although the composition of NF-kappaB complexes detected by EMSA and supershift analysis in nuclear lysates derived from Bcl-xL transfectants and control cells was indistinguishable, LPS-induced inhibitory kappaBalpha degradation, as well as NF-kappaB binding and AP-1 activation, were slightly decreased by ectopic expression of Bcl-xL. More strikingly, LPS-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase was strongly repressed by Bcl-xL overexpression, offering a possible mechanism for the inhibition of LPS-induced cytokine production. These data provide the first evidence for a novel role for Bcl-xL as an anti-inflammatory mediator in macrophages.     

Inhibition of influenza-immune T cell effector function by virus-specific hybridoma antibody.

The in vitro activity of influenza-specific cytotoxic T cells can be inhibited by incubation of the target cells with monoclonal anti-influenza antibodies. Hybridoma antibodies that bind to the virus HA inhibit the cytotoxic activity of TDL for the virus-infected target by as much as 80%, whereas these same antibodies never reduce splenic T cell function by more than 40%. This reflects the fact that TDL from anti-influenza strain A/WSN/33 (HON1) are highly subtype-specific, whereas splenic effector cells from the same mice are cross-reactive for target cells infected with heterologous influenza A viruses. These findings are discussed in the light of previous failures to block virus-immune T cell effector function with heterogeneous antisera produced in vivo, and are considered to favor the idea that at least some of the "virus-immune" T cells are indeed recognizing viral antigens.     

Purification and characterization of a cytotoxic factor produced by a mouse macrophage hybridoma

A mouse macrophage cytotoxic factor was purified to homogeneity from the serum-free culture supernatant of a mouse macrophage hybridoma clone, N/P-7-1, stimulated with lipopolysaccharide by gel filtration, affinity chromatography, anion-exchange chromatography, and polyacrylamide gel electrophoresis. The purified material was judged to be homogeneous as to the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and has a relative molecular mass of 17,500, as determined by SDS-PAGE, or 55,000, as determined by gel filtration on columns of both Sephacryl S-200 and TSK G3000SW. It has an isoelectric point of 5.0, and is trypsin sensitive, stable at 56 degrees C and labile at pH less than 6. The cytotoxic activity of the purified factor could not be inhibited by various sugars and lectins. The production of the factor from N/P-7-1 triggered by macrophage-activating factor for cytotoxicity, but not by mouse recombinant gamma-interferon. The factor should be synthesized after lipopolysaccharide stimulation because treatment of N/P-7-1 cells with a metabolic inhibitor, emetine or actinomycin D, prevents the production.     

Serum-free medium for murine and human lymphoid and hybridoma cell lines

We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS Fetal Bovine Serum - IMDM Iscove's Modification of Dulbecco's Medium - PBS Phosphate-Buffered Saline - TPA Tissue Plasminogen Activator     

Inhibition of macrophage activation and tumor cell cytolysis by cyclosporin-A

Cyclosporin (CsA)4, a fungal peptide used clinically for its immunosuppressive properties, was investigated for its ability to antagonize the activation of macrophages (PEM) to the tumoricidal state. The acquisition of tumoricidal properties by PEM challenged with macrophage activating factor (MAF) plus lipopolysaccharide (LPS) was inhibited in a dose-dependent fashion by CsA. Similarly, CsA antagonized activation of PEM exposed to the calcium ionophore, A23187. CsA also inhibited macrophage-mediated tumor cell cytolysis in a dose-dependent manner. These data indicate that in vitro, CsA can modulate directly the acquisition and expression of tumoricidal properties by PEM and suggests that the macrophage may be an important target cell for CsA in vivo.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Inhibition of lymphoid cell growth by adenine ribonucleotide accumulation. The role of phosphoribosylpyrophosphate-depletion induced pyrimidine starvation

The exact role of adenosine in the adenosine deaminase (EC 3.5.4.4) deficiency-related severe combined immunodeficiency disease has not been ascertained. We analysed the effects of adenosine, in the presence of the adenosine deaminase inhibitor, deoxycoformycin, on cell growth, cell phase distributions and intracellular nucleotide concentrations of cultured human lymphoblasts. Adenosine had a biphasic effect on cell growth and cell cycle distribution of a partial hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) deficient MOLT-HPRT cell line. After 24 h of incubation, 60 microM adenosine inhibited cell growth more extensively than did 100 and 200 microM adenosine. The distribution of the MOLT-HPRT cells in the various phases of the cell cycle showed a similar biphasic pattern. Adenosine concentrations in the medium below 10 microM caused accumulation of adenine ribonucleotides and depletion of phosphoribosylpyrophosphate, UTP and CTP in the cells. This was associated with inhibition of cell growth. Medium adenosine concentrations above 10 microM neither resulted in accumulation of adenine ribonucleotides nor in inhibition of cell growth.     

Effect of NH3 on the cell growth of a hybridoma cell line

Ammonia often has been reported to inhibit cell growth. The aqueous ammonia equilibrium between the un-ionized form (NH₃) and the ammonium ion (NH₄ ⁺) depends on the pH of the solution. Extensive studies in batch and continuous cultivation by varying pH and total ammonia concentration were carried out to investigate whether a kinetic model describing growth inhibition by ammonia has to be based on the total ammonia concentration, or the concentration of NH₃. A significant relationship between the specific growth rate and death rate, respectively, and the NH₃ concentration but not the total ammonia concentration, was detected. An adaptation of the cells to high ammonia levels was not observed. Based on these results a new kinetic model for ammonia mediated growth inhibition is suggested. For high density cultivation it is recommended to control the pH at the lower limit of the growth optimum to keep the NH₃ level low.     

Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells

Morphological studies of developing capillaries and observations of alterations in capillaries associated with pathologic neovascularization indicate that pericytes may act as suppressors of endothelial cell (EC) growth. We have developed systems that enable us to investigate this possibility in vitro. Two models were used: a co-culture system that allowed direct contact between pericytes and ECs and a co-culture system that prevented physical contact but allowed diffusion of soluble factors. For these studies, co-cultures were established between bovine capillary ECs and the following growth-arrested cells (hereafter referred to as modulating cells): pericytes, smooth muscle cells (SMCs), fibroblasts, epithelial cells, and 3T3 cells. The modulating cell type was growth arrested by treatment with mitomycin C before co-culture with ECs. In experiments where cells were co-cultured directly, the effect of co-culture on EC growth was determined by comparing the mean number of cells in the co-cultures to the mean for each cell type (EC and modulating cell) cultured separately. Since pericytes and other modulating cells were growth arrested, any cell number change in co-cultures was due to EC growth. In the co-cultures, pericytes inhibited all EC proliferation throughout the 14-d time course; similar levels of EC inhibition were observed in SMC-EC co-cultures. Co-culture of ECs with fibroblasts, epithelial cells, and 3T3 cells significantly stimulated EC growth over the same time course (30-192% as compared to EC cultured alone). To determine if cell contact was required for inhibition, cells were co-cultured using Millicell chambers (Millipore Corp., Bedford, MA), which separated the cell types by 1-2 mm but allowed the exchange of diffusible materials. There was no inhibition of EC proliferation by pericytes or SMCs in this co-culture system. The influence of the cell ratios on observed inhibition was assessed by co-culturing the cells at EC/pericyte ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. Comparable levels of EC inhibition were observed at ratios from 1:1 to 10:1. When the cells were co-cultured at a ratio of 20 ECs to 1 pericyte, inhibition of EC growth at 3 d was similar to that observed at other ratios. However, at higher ratios, the inhibition diminished so that by the end of the time course the co-cultured ECs were growing at the same rate as the controls. These results suggest that pericytes and SMCs can modulate EC growth by a mechanism that requires contact or proximity. We postulate that similar interactions may operate to modulate vascular growth in vivo.     

On-line monitoring of hybridoma cell growth using a laser turbidity sensor

A high-sensitivity turbidity probe was used for on-line monitoring of the cell concentration in batch hybridoma cultivation. Good correlation between off-line cell counts and the linearized sensor signal was found. The quality of the signal was sufficiently high to provide for on-line estimation of the specific growth rate using an efficient filtering procedure. These positive results suggest that such laser turbidity sensors will facilitate development of systems for on-line monitoring and control of animal cell cultivations. (c) 1992 John Wiley & Sons, Inc.     

Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen

Unlike other hamster phagocytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')2 fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')2 concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on 45Ca uptake was evaluated. It was observed that antigen-specific F(ab')2 fragments stimulated 45Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with our anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.     

Effect of mechanical agitation on hybridoma cell growth

Hybridoma cells (S3H5/2bA2) were grown in spinner flasks at different agitation speeds. It was found that cells in stationary and decline phases of growth were sensitive to shear force caused by agitation but cells in growth phase seemed less sensitive to the shear forces introduced. The death rate was found to be. 0.007 hr^–1 in T flasks but 0.018 hr^–1 and 0.028 hr^–1 at 100 and 200 rpm, respectively, while the growth rate was about 0.05 hr^–1 for all cases.