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S W Russell

Publications and source records attributed to S W Russell.

At least 55 records · Page 3Linked to original sources
PubMed1989

A monoclonal antibody against the ligand binding site of the receptor for mouse interferon-gamma.

A rat monoclonal IgG2a antibody, GR-20, has been produced against the receptor for mouse interferon-gamma (MuIFN-gamma). Comparison of competitive binding studies performed with either 125I-labeled GR-20 or recombinant (r) MuIFN-gamma, as well as a variety of other studies, suggested that the epitope recognized by the monoclonal antibody (mAb) is in the domain of the receptor that binds ligand. The binding of GR-20 to cells of the monomyelocytic line WEHI-3 was of high affinity (1-2 nM). Approximately 20,000 binding sites were found per cell, a value that is in close agreement with the number of MuIFN-gamma receptors quantified on cells of the same type by ligand binding studies. The mAb also bound to a variety of other mouse cells, suggesting that the same epitope is shared by receptors for MuIFN-gamma, regardless of cell type. The epitope was not detected on two human cell types that were tested, while cells of a rat cell line shown to be minimally responsive to rMuIFN-gamma gave equivocal binding results when they were interacted with GR-20. Binding of the mAb to the receptor did not mimic the effects of ligand. In fact, the opposite was true: binding blocked the induction of three biological effects of MuIFN-gamma, including priming of macrophages for tumor cell killing, upregulation of the expression of class II major histocompatibility antigens (Ia) on the same cell type, and induction of antiviral activity in L cells. Following intravenous injection, initial removal of GR-20 was precipitous, followed after 1 h by a phase which was more gradual, resulting in 5-10% of biologically active mAb remaining in the circulation after 24 h. Such retention should make this mAb useful in a variety of studies in vivo.

Animals↗
PubMed1988

Enrichment and initial characterization of the solubilized receptor for mouse gamma interferon.

The work reported here constitutes a first step in characterizing the receptor for mouse gamma interferon at the biochemical level. The myelomonocytic cell line, WEHI-3, was the source of starting material. Iodinated recombinant mouse gamma interferon incubated with WEHI-3 cells, as well as membranes prepared from them, bound specifically to a single class of sites with a Kd of 7 x 10(-9)M. Membranes were solubilized with the non-ionic detergent octyl-beta-D-glucopyranoside. As solubilization proceeded, binding activity could be assayed by precipitating the receptor with acetone in the presence of egg phosphatidylcholine liposomes. The Kd of the receptor in association with liposomes was 13 nM. Again here, only a single class of binding activity was found, and specificity for gamma, compared to other interferons, was maintained. This is the first time that the receptor for mouse gamma interferon has been solubilized and recovered in functional form. Further characterization included at least a 200-fold enrichment of binding activity by ligand affinity chromatography, resulting in the identification of a 95 kDa protein as the most likely candidate for either the receptor or a binding subunit thereof.

Animals↗
PubMed1988

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.

Animals↗
PubMed1987

Protein phenotypes of mouse macrophages activated in vivo for tumor cell killing.

We have shown previously that the cellular proteins 47b and 71/73 could be used to construct phenotypes that distinguish among bone marrow culture-derived (BMCD) macrophages that were either unstimulated or primed by gamma interferon or fully activated for tumor cell killing by gamma interferon in combination with lipopolysaccharide (LPS). In the present study we examined in vivo-derived correlates for each of these stages of macrophage activation and found that the same protein phenotypes held true: both p47b and p71/73 were expressed by cytolytic peritoneal macrophages, including macrophages from a tumoral effusion, whereas macrophages primed in vivo by the intraperitoneal injection of either concanavalin A or methyl vinyl ether copolymer II expressed p47b but lacked p71/73. Neither resident nor inflammatory macrophage populations expressed p47b, and acute inflammatory macrophages, like unstimulated BMCD macrophages, expressed little or no p71/73. By contrast, resident and thioglycollate-elicited macrophages synthesized moderate levels of p71/73. When p71/73 also was expressed, there was a quantitative relationship between p47b expression and cytolytic activity in five different in vivo-activated macrophage populations. The results suggest that, regardless of the macrophage source or stimulus, it may be possible to assess macrophage activation status by reference to protein phenotypes utilizing p47b and p71/73.

Animals↗
PubMed1986

Protein changes associated with stages of activation of mouse macrophages for tumor cell killing.

Bone marrow-derived mouse macrophages become activated for tumor cell killing by traversing a series of stages. The stages studied here were as follows: unstimulated (exposed to nothing but medium), primed (prepared to become cytolytic), fully activated (primed macrophages exposed to a triggering agent), and postcytolytic (previously activated macrophages that had gradually lost cytolytic activity after the removal of stimuli). Macrophages were labeled with [35S]methionine, lysed, and subjected to 2-D gel electrophoresis and fluorography. The priming agent used was recombinant mouse IFN-gamma, 10 to 20 U/ml. Bacterial lipopolysaccharide (LPS), 0.4 to 1 ng/ml, was used as the triggering agent. A total of 40 major changes was identified in macrophages treated with both agents. Twenty-six of these were seen in macrophages treated with IFN-gamma, and 35 were found in LPS-treated macrophages. Twenty-two of the 40 changes were found in both IFN-gamma- and LPS-treated macrophages. The major reason for this overlap was the autocrine action of IFN-alpha/beta secreted from LPS-treated macrophages. Changes in expression of specific proteins, designated p47b and p71/73, were found to correlate closely with the development and loss of the activated state. With the use of these proteins as markers, phenotypes could be constructed that distinguished unstimulated, LPS-treated, primed, and fully activated macrophages. Postcytolytic macrophages had a phenotype similar to unstimulated macrophages and could be reactivated by reexposure to inducing agents. They also reexpressed the protein markers that were characteristic of fully activated macrophages.

Animals↗
PubMed1986

Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach.

Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.

Amino Acid Sequence↗
PubMed1986

Prostaglandin E2 content of regressing and progressing Moloney sarcomas.

Regressing mouse Moloney sarcomas contain macrophages that are activated for tumor cell killing, while those found in progressively growing sarcomas either cannot kill or do so very poorly. Prostaglandin E2 (PGE2) has been shown to down-regulate macrophage activation in vitro. The study described here was designed, therefore, to ascertain and compare the concentrations of PGE2 in regressing and progressing Moloney sarcomas. Tumors were harvested for extraction and analyzed using conditions that minimized artifactual increases in PGE2 levels attributable to de novo synthesis. Concentrations of PGE2 were higher in progressing, compared to regressing, Moloney sarcomas during the early stages of tumor development. At nearly all time points, however, whether the neoplasms were of regressing or progressing type, the estimated concentrations of PGE2 in tumors exceeded the level that completely inhibits macrophage activation for tumor cell killing in vitro, that is, 10(-8) M. These data suggest either that PGE2 is not responsible for down-regulating activation in Moloney sarcomas or that, if PGE2 is responsible for the negative regulation of activation in progressing Moloney sarcomas, there must be something in regressing sarcomas that prevents the hormone from having its inhibitory effect.

Animals↗
PubMed1986

Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages.

Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.

Cell Line↗
PubMed1985

The strain of mouse and assay conditions influence whether MuIFN-gamma primes or activates macrophages for tumor cell killing.

Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MuIFN-gamma in macrophage activation. The findings suggest that the capacity of MuIFN-gamma to activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.

Animals↗
PubMed1985

Heterogeneity among macrophages cultured from mouse bone marrow. Morphologic, cytochemical and flow cytometric analyses.

The development of macrophages in culture from mouse bone marrow was followed for 14 days by light and electron microscopy, ultrastructural cytochemistry, and flow cytometric analysis. By 10 days greater than 97% of the cells in culture were mononuclear phagocytes, and by 12 days greater than 99% were identifiable as macrophages. Ultrastructurally, three subpopulations of mononuclear phagocytes were distinguished based on the appearance of cytoplasmic structures. Early in culture, cells containing large, membrane-bounded vesicles predominated. With increasing time in culture these cells were replaced to varying degrees first by cells that contained vesicles filled with relatively dense, osmiophilic material and, finally, by macrophages that contained granules of various sizes, shapes and staining densities. Cytochemical (peroxidase and acid phosphatase) and colloidal gold uptake studies at the ultrastructural level suggested that many, if not all, of these cytoplasmic structures arose by pinocytosis and subsequent fusion of pinocytic vesicles with lysosomes. Analysis of DNA content of propidium iodide-stained nuclei by flow cytometry, coupled with the examination of cells treated with colchicine to arrest mitosis in metaphase, suggested that cell cycling was a negligible contributor to heterogeneity within cultured populations. Thus, by waiting until 12-14 days after bone marrow cultures were initiated, with partial replenishment of the culture medium at 7 days, heterogeneity could be greatly reduced in cultured macrophage populations. Taking this fact into consideration could help to reduce the variability seen in functional studies of macrophage populations that are less homogeneous.

Acid Phosphatase↗
PubMed1985

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing.

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.

Animals↗
PubMed1984

Both the kind and magnitude of stimulus are important in overcoming the negative regulation of macrophage activation by PGE2.

Macrophages activated for tumor cell killing by bacterial lipopolysaccharide (LPS) were shown to lose their cytolytic activity if exposed to physiological levels of prostaglandin E2 (PGE2). Increasing the LPS stimulus more than 100-fold over the amount needed to activate the cells did not substantially increase their resistance to the negative regulatory effect of PGE2. By contrast, killing mediated by macrophages activated by a mixture of LPS and gamma interferon was maintained. The degree of resistance conferred was directly related to the magnitude of the stimulus employed, reaching the point where not even 10(-5) M PGE2 would diminish killing. Killing by both activated resident and inflammatory peritoneal macrophages could be maintained, but it was easier to do so if the cells had been elicited by an inflammatory stimulus. A preparation of type I interferons produced by cells of the macrophage cell line J774A. 1 behaved similarly, but was over 500 times less efficient at helping to maintain killing than gamma (type II) interferon was. Alpha interferon alone, i.e., without LPS, was capable both of activating macrophages and of maintaining the activated state in the presence of PGE2. The capacity for both activation and maintenance could be strikingly enhanced, however, by mixing alpha and gamma interferons together under conditions that were free of detectable LPS. The data reported here collectively suggest that induction and maintenance of macrophage activation may be separable mechanistically, and that the interferons are important to host defense not only because they participate in the induction of macrophage activation for tumor cell killing but also because they help to maintain the activated state once it has been induced.

Animals↗
PubMed1984

Gamma interferon interferes with the negative regulation of macrophage activation by prostaglandin E2.

Activation of mouse macrophages for tumor cell killing is negatively regulated by prostaglandin E2 (PGE2). The effect of this hormone is to shut off cytolytic activity that is expressed as a consequence of activation. A lymphokine in the culture supernates of concanavalin A stimulated spleen cells has been shown to change the sensitivity of activated macrophages to the negative regulatory effects of PGE2, thereby maintaining activation, as manifested by the continued expression of tumor cell killing by these cells. Using a highly specific polyclonal antiserum and gamma interferon produced either by a T-cell hybridoma or by recombinant DNA technology we show here that one lymphokine responsible for mediating the maintenance effect is gamma interferon.

Animals↗
PubMed1983

Macrophage activation: priming activity from a T-cell hybridoma is attributable to interferon-gamma.

Antiviral and macrophage-priming activities in the supernatant medium of a subclone of a concanavalin A-stimulated mouse T-cell hybridoma were investigated. The two activities were associated with a molecular weight of approximately 50,000 and could not be separated by various approaches. Both activities were eliminated by a highly specific neutralizing antibody against mouse interferon-gamma, but not by antibody against interferon-alpha and -beta. The ratio of priming to antiviral activity in the hybridoma culture supernate was indistinguishable from the ratio obtained with mouse interferon-gamma prepared by recombinant DNA technology. It was concluded from these data that the priming activity in hybridoma culture supernates was attributable to interferon-gamma and that this mediator is one form of the lymphokine macrophage-activating factor. Interferon-gamma was greater than 800 times more efficient at priming mouse macrophages for tumor cell killing than was a mixture of interferon-alpha and -beta. This finding contributes to growing awareness that type II interferon may have greater immunoregulatory potential than type I interferons.

Animals↗
PubMed1983

Two classes of mouse mast cells delineated by monoclonal antibodies.

A panel of rat monoclonal antibodies was used to classify mouse mast cells from four different sources on the basis of their surface determinants. Three populations of mast cells were derived by culturing normal bone marrow, spleen, and peripheral blood cells, whereas the fourth population was isolated directly from the peritoneal cavity. For each population, a surface antigen phenotype was determined by indirect immunofluorescence microscopy and compared to the phenotypes of the other populations of mast cells. The results showed that culture-derived mast cells, regardless of origin, had nearly identical antigen phenotypes, suggesting that these populations were comparable. By contrast, peritoneal mast cells had a different phenotype, suggesting that they represented a class distinct from culture-derived mast cells. These findings, which corroborate classification schemes based on nonimmunologic methods, demonstrate that subpopulations of mouse mast cells can be distinguished antigenically.

Animals↗
PubMed1983

Recombinant mouse gamma interferon induces the priming step in macrophage activation for tumor cell killing.

Mouse macrophages become activated to kill tumor cells by traversing a series of steps (1-3). The first of these does not cause the expression of cytolytic activity; instead, it primes macrophages to respond to a second signal(s) that then triggers the onset of killing (4-7). The mediator that is responsible for priming is contained, along with other lymphokines, in the culture supernatants of concanavalin A-stimulated spleen cells (5-7) cloned T lymphocytes (8), or some T cell hybridomas (9). Close association has been noted between macrophage priming activity and antiviral activity that is attributable to gamma interferon (10-12). However, unequivocal evidence that the two activities are products of the same molecule has not been available. We now how conclusively by using mouse gamma interferon (MulFN-gamma)3 produced by recombinant DNA technology that, in addition to antiviral activity, this lymphokine has the capacity to induce the priming step in the process of macrophage activation for tumor cell killing.

Animals↗
PubMed1983

Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon.

Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activating factor (MAF) produced by the murine T cell hybridoma clone 24/G1, which primes macrophages for nonspecific tumoricidal activity, is a form of gamma-interferon (IFN gamma). MAF and antiviral activities were generated by the clone in proportional amounts under a variety of culture conditions. Production of both activities showed identical dependence on cell density even when production precipitously ceased at cell concentrations greater than 1.2 X 10(6) cells/ml. MAF and antiviral activities displayed identical sensitivities to pH and temperature and were indistinguishable on the basis of binding to insolubilized polynucleotides. Dye-ligand chromatography of a stimulated hybridoma supernatant on a column of Matrex Gel Red A resulted in the 1500-fold purification and 100% recovery of the MAF activity. A qualitatively identical elution profile was also obtained for the antiviral activity; however, only 32% of the original activity was recovered. When subjected to gel filtration on a high performance liquid chromatography system (HPLC), the MAF and antiviral activities present in the Matrex Red pool displayed identical elution profiles and exhibited an apparent m.w. of 50,000. This technique resulted in another 10-fold purification and 50% recovery of the two activities. On HPLC chromatofocusing the MAF activity in the Matrex Red pool could be resolved into seven chromatographically distinct species yet could not be resolved from the antiviral activity on either a qualitative or quantitative basis. These results thus provide quantitative molecular evidence to support the concept that IFN gamma can act as a MAF.

Adsorption↗