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S W Russell

Publications and source records attributed to S W Russell.

At least 37 records · Page 2Linked to original sources
PubMed1994

Gestation-related expression of the interferon-gamma receptor gene in mouse uterine and embryonic hematopoietic cells.

Macrophages and natural killer (NK)-like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-gamma (IFN-gamma), tissues taken from cycling and pregnant mice were tested for IFN-gamma receptor (IFN-gamma R) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-gamma R mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IFN-gamma R mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-gamma R mRNA were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by g.d. 7 and in NK cells by g.d. 9. IFN-gamma R expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-gamma R mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study suggest relationships between IFN-gamma R expression and ovarian hormones as well as cell maturation and support the postulate that IFN-gamma receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation.

Animals↗
PubMed1994

Necessity and sufficiency of beta interferon for nitric oxide production in mouse peritoneal macrophages.

Bacterial lipopolysaccharide and some cytokines can activate macrophages to secrete nitric oxide. Macrophage-derived nitric oxide is a key cytotoxic factor for microbicidal and tumoricidal processes. We report here that a monoclonal antibody specific for beta interferon inhibited lipopolysaccharide-induced nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages and macrophage-like cell line RAW 264.7. In addition, exogenous added beta interferon enabled lipopolysaccharide-hyporesponsive thioglycolate-elicited C3H/HeJ peritoneal macrophages to produce nitric oxide in response to lipopolysaccharide. These data support the concept that beta interferon provides an essential signal(s) for lipopolysaccharide-triggered nitric oxide production by mouse macrophages. Heat-killed Staphylococcus aureus, a gram-positive bacterium which was unable to initiate nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages in vitro, promoted nitric oxide formation in the presence of beta interferon, suggesting that beta interferon may be a general cofactor necessary for bacterium-derived stimulus-induced nitric oxide production in these macrophages. However, neither beta interferon nor tumor necrosis factor alpha, alone or in combination, triggered nitric oxide production in thioglycolate-elicited mouse peritoneal macrophages, demonstrating that these macrophage-derived cytokines, while necessary, were not sufficient by themselves for the induction of nitric oxide production in these cells. On the other hand, gamma interferon and tumor necrosis factor alpha acted together to induce nitric oxide production in vitro in the absence of lipopolysaccharide in thioglycolate-elicited mouse peritoneal macrophages, indicating that these two types of interferons provided different signals during the activation of these macrophages.

Animals↗
PubMed1993

Binding site on the murine IFN-gamma receptor for IFN-gamma has been identified using the synthetic peptide approach.

We have studied the structural parameters involved in the binding of murine IFN-gamma (MuIFN-gamma) to its receptor. Ten synthetic overlapping peptides corresponding to the extracellular domain of the MuIFN-gamma receptor (MuIFN-gamma R) were synthesized. In direct binding studies, biotinylated MuIFN-gamma bound specifically to receptor peptide (95-120). Further, the NH2-terminal IFN-gamma peptide, MuIFN-gamma (1-39), also specifically bound to receptor peptide (95-120). Binding of both labeled MuIFN-gamma and MuIFN-gamma (1-39) to MuIFN-gamma R peptide (95-120) was inhibited by either unlabeled molecule. The COOH-terminal receptor binding peptide, MuIFN-gamma (95-133), neither bound to any receptor peptides nor blocked the binding of intact MuIFN-gamma or MuIFN-gamma (1-39) to receptor peptide (95-120). Polyclonal antibodies to each of the peptides were then produced. Each of the anti-peptide antisera recognized its corresponding peptide and bound denatured cloned soluble receptor by Western blotting. Furthermore, the antisera to peptides representing the inner region of the extracellular domain of the receptor bound to nondenatured soluble MuIFN-gamma R. Specifically, antisera to the receptor peptides (73-97), (95-120), (118-143), (142-163), and (161-182) bound to soluble MuIFN-gamma R, whereas antisera to peptides (1-21), (20-49), (46-74), (178-203), and (202-227) did not bind. Most important, antisera to peptides (95-120) and (118-143) competed with [125I]MuIFN-gamma for binding to soluble receptor. These results show that the region of the MuIFN-gamma R encompassing amino acid residues (95-120) is a binding site on the receptor for the NH2-terminal of MuIFN-gamma by direct binding, and that the larger region (95-143) on the receptor may play a role in binding of intact MuIFN-gamma based on blocking of binding by site-specific antibodies.

Amino Acid Sequence↗
PubMed1993

Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide.

The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

Amino Acid Oxidoreductases↗
PubMed1993

Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide.

Macrophages can become activated to kill both tumor cells and a variety of microbes. Results here show that synthesis of nitric oxide (NO), a mediator of many macrophage cytotoxic functions, was greatly increased when cells of the mouse macrophage cell line RAW 264.7 were costimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), compared to LPS alone. This increase paralleled increases in cytotoxicity. Northern analysis showed that increased production of NO was preceded by markedly enhanced expression of mRNA for the inducible form of macrophage NO synthase (mac-NOS), which catalyzes the synthesis of NO. Cycloheximide inhibited the induction of mac-NOS mRNA by IFN-gamma and LPS, indicating that expression of this mRNA required de novo protein synthesis. Elevated expression of mac-NOS mRNA was not due to an increase in its stability. Rather, the combination of IFN-gamma and LPS induced a much higher rate of transcription of the mac-NOS gene than did stimulation with LPS alone. These results provide one explanation of why priming and triggering stimuli, such as IFN-gamma and LPS, respectively, synergistically activate macrophages and may be applicable to explaining how IFN-gamma augments NO-dependent microbicidal activity in macrophages as well.

Amino Acid Oxidoreductases↗
PubMed1993

The mouse macrophage activation-associated marker protein, p71/73, is an inducible prostaglandin endoperoxide synthase (cyclooxygenase).

The inducible protein p71/73 marks the response of mouse macrophages to one of several stimuli (e.g., bacterial lipopolysaccharide or poly I:C) that trigger the expression of cytolytic activity when these cells have previously been primed for tumor cell killing by interferon-gamma (IFN-gamma). The results reported here identify this marker protein as the inducible prostaglandin endoperoxide synthase (PES), TIS10/PES-2. Identification was based on four findings: (1) p71/73, like the TIS10/PES-2 protein, was associated with cellular membranes; (2) the sequence of amino acids in the NH2 terminus of both p71 and p73 was 96% identical to the predicted NH2-terminal sequence of the TIS10/PES-2 protein; (3) a polyclonal antiserum raised against the COOH-terminal region of the TIS10/PES-2 gene product recognized p71/73 in immunoblots; and (4) dexamethasone, which blocks induction of TIS10/PES-2 expression, inhibited the induction of both p71/73 synthesis and tumoricidal activity in macrophage. Several regulatory roles for this protein in the activation process are possible.

Amino Acid Sequence↗
PubMed1992

Regulation by transforming growth factor-beta 1 of expression and function of the receptor for IFN-gamma on mouse macrophages.

Transforming growth factor-beta (TGF-beta) is known phenomenologically as a negative regulator of several functions of mouse bone marrow macrophages. The studies reported here extend this list by showing that TGF-beta can suppress cytolytic activity of mouse bone marrow culture-derived macrophages that already have become activated by IFN-gamma and LPS for tumor cell killing, as well as confirm that this cytokine can interfere with the induction of activation. Suppression was caused by a shift in the dose response curve for IFN-gamma rather than absolute inhibition; the 50% effective dose for IFN-gamma was increased approximately fourfold by treatment with TGF-beta. TGF-beta also decreased the absolute number of IFN-gamma R on the surfaces of pretreated macrophages by approximately 30 to 35%, without altering the affinity with which IFN-gamma bound. The increased concentration of IFN-gamma needed to produce the higher level of receptor occupancy explained the observed shift in the IFN-gamma dose response curve. These results suggest that TGF-beta mediates its negative regulatory effects on macrophage activation by interfering with coupling of the IFN-gamma R to the pathways that induce and maintain macrophage activation for tumor cell killing. Such effects are consistent with the view that TGF-beta is a negative regulator of macrophage activation for tumor cell killing. Because of this fact, neoplastic cells that secrete this cytokine may have a distinct survival advantage.

Animals↗
PubMed1992

The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor.

The mechanism of binding of murine IFN-gamma to its receptor has not been determined. We have studied this mechanism by examining the binding of overlapping synthetic peptides of IFN-gamma to cloned soluble murine IFN-gamma R. IFN-gamma (1-39) and IFN-gamma (95-133) were able to compete with [125I]IFN-gamma for binding to cloned soluble receptor. Peptides corresponding to the inner region of IFN-gamma--IFN-gamma (36-60), IFN-gamma (54-91), and IFN-gamma (78-107)--showed a markedly reduced ability to compete with [125I]IFN-gamma for receptor binding relative to the N-terminal and C-terminal peptides. In direct binding studies, the binding of [125I]-IFN-gamma (1-39) to soluble receptor could only be competed by IFN-gamma (1-39) and IFN-gamma and not by any of the other peptides including IFN-gamma (95-133). This suggests that the N- and C-termini of IFN-gamma bind to different regions of the receptor. These data in conjunction with previous structure/function studies and x-ray crystallographic data have allowed us to formulate a "velcro-key" model of IFN-gamma binding to receptor that involves both the N- and C-terminal domains. The N-terminus binds in the classical "lock-and-key" manner characterized by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct from the N-terminus likely through the polycationic region, which is conserved across species barriers. Binding of this type would exhibit high affinity and low specificity similar to a piece of velcro. This interaction becomes specific when the C-terminus is in the context of the whole IFN-gamma molecule and may act to increase the affinity of receptor binding and/or facilitate signal transduction.

Amino Acid Sequence↗
PubMed1992

Characterization and use of monoclonal and polyclonal antibodies against the mouse interferon-gamma receptor.

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-gamma) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 microM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-gamma) blocked binding of the ligand and, as expected, prevented induction by IFN-gamma of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (greater than 50%) blocked binding of IFN-gamma. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.

Animals↗
PubMed1992

A specific sequence of stimulation is required to induce synthesis of the antimicrobial molecule nitric oxide by mouse macrophages.

Nitric oxide production by macrophages required either simultaneous or sequential exposure to gamma interferon and lipopolysaccharide; exposure to lipopolysaccharide followed by exposure to gamma interferon gave little response. The apparently evanescent nature of the lipopolysaccharide signal, necessitating persistent stimulation, could be essential to down-regulating nitric oxide production after bacteria are cleared in vivo.

Animals↗
PubMed1991

Stable expression of a secreted form of the mouse IFN-gamma receptor by rat cells.

The biologic effects of IFN-gamma are mediated through a receptor that is expressed in relatively low abundance on normal mammalian cells. As a consequence, investigations of the physicochemical and ligand-binding properties of the purified receptor have been limited. The work reported here characterizes a secreted form of the receptor for mouse IFN-gamma, made by deletion of the nucleotides that code for the anchoring domain from a cDNA that encodes the receptor binding protein and its related signal peptide. When transfected into rat XC cells, this construct produced up to approximately 1 mg/liter of a secreted protein that had the characteristics of the native receptor. Both the secreted protein and its mRNA were of sizes that were consistent with loss of the transmembrane region. The protein was detectable by a mAb that is specific for an epitope that is found in the ligand binding site of the receptor for mouse IFN-gamma, as well as by a goat polyclonal IgG that is monospecific for the mouse IFN-gamma R. Supernates that contained the secreted protein blocked binding of IFN-gamma to mouse IFN-gamma R and inhibited in a dose-dependent manner the IFN-gamma-mediated priming of mouse bone marrow culture-derived macrophages for tumor cell killing. Availability of relatively large amounts of a secreted protein that retains ligand-binding activity should facilitate purification and basic studies of the receptor binding protein and could provide new approaches to the treatment/prevention of diseases that arise due to inappropriate response of cells to IFN-gamma. In addition, because this secreted receptor, unlike others, consists of both the extracellular and intracellular domains, it is likely that it will be useful in determining how the cytoplasmic portion of the receptor is involved in receptor function.

Amino Acid Sequence↗
PubMed1991

Activation-associated marker proteins: peptide mapping and their expression in macrophage cell lines.

The activation-associated markers, p47b and p71/73, have been recognized as minor proteins in peritoneal and bone marrow culture-derived macrophages activated for tumor cell killing. Proteins with identical characteristics of migration on 2-dimensional gels and comparable Cleveland peptide maps are described here in macrophage cell lines that could be activated for tumor cell killing (J774A.1, RAW 264, UNC-2). Macrophage cell lines that could not be activated (P388D1 and PU5-1.8) did not express the markers. The expression of these markers in activatable macrophage cell lines strengthens their association with the activation process and provides a bulk source of the proteins for purification studies.

Animals↗
PubMed1990

Induction of macrophage-mediated tumor cytotoxicity by a hamster monoclonal antibody with specificity for lipopolysaccharide receptor.

Experiments have been carried out to assess the immunostimulatory activity of a hamster IgM mAb (mAb5D3) with specificity for an 80-kDa LPS-binding protein expressed on murine macrophages and monocytes. The addition of mAb5D3 to cultures of murine bone marrow-derived macrophages activated these cells to become tumoricidal for mastocytoma cells in vitro. The activity of mAb5D3 was enhanced in the presence of IFN-gamma. Neither mAb5D3 nor LPS were able to activate macrophages from the LPS-hyporesponsive C3H/HeJ mouse, although these cells responded normally to heat-killed Listeria monocytogenes. The results of several experiments establish that the observed LPS-like activity of mAb5D3 was not due to contaminating endotoxin: 1) the activity of mAb5D3 but not LPS was heat labile at 100 degrees C; 2) the activity of LPS but not mAb5D3, was inhibited by addition of polymyxin B; and 3) quantitative estimates of endotoxin contamination by Limulus amoebocyte lysate reactivity. These experiments thus demonstrate that mAb5D3 can serve as an agonist for LPS-dependent macrophage responses and, when considered with those of our companion paper showing specificity of mAb5D3 for the 80-kDa LPS-binding protein, provide strong support for the concept that the 80-kDa LPS-binding protein previously identified serves as a functional receptor for LPS on murine macrophages.

Animals↗
PubMed1990

Interferons exhibit temporally distinct regulation of two bovine macrophage Fc receptors.

Interferon (IFN)-induced modulation of two distinct types of Fc receptors (FcR) on bovine bone marrow culture-derived macrophages was quantified by flow cytometry. We have established the presence of separate FcR for monomeric and aggregated IgG on these cells, equivalent to the FcRI and FcRII of mice, respectively. These two kinds of FcR differed in protease sensitivity, hierarchy of preferential binding of immunoglobulin sub-class, and cross-inhibition. Treatment of macrophages with either recombinant bovine IFN-gamma (rBoIFN gamma) or rBoIFN alpha I1 produced a dose-dependent increase in the expression of both types of FcR; however, expression of the FcR for aggregated IgG was increased a full 24 h prior to that for monomeric antibody. Further, expression of the FcRII-equivalent declined substantially by 48 h of IFN exposure, while the presence of the FcRI-equivalent receptor was still enhanced. Finally, low doses (1 unit/ml) of either rBoIFN gamma or rBoIFN alpha I1 failed to alter FcR expression, yet mixtures of IFNs at this concentration were able to potentiate FcR expression. This is the first time that a differential time course of induction of FcR types by IFNs and the effects of mixtures of IFNs on FcR expression has been described in any species.

Animals↗
PubMed1989

Macrophage activation-associated proteins. Characterization of stimuli and conditions needed for expression of proteins 47b, 71/73, and 120.

The expression of cellular proteins was analyzed by two-dimensional gel electrophoresis during and after exposure of mouse macrophages to either mouse rIFN-gamma or natural MuIFN-beta sufficient to prime macrophages for tumor cell killing. The reversible inhibitor of protein synthesis, cycloheximide (CY), was included in some experiments during exposure to IFN. While it was present, CY suppressed protein synthesis by greater than 90%, but did not affect priming for tumor cell killing that was induced by either kind of IFN, as measured in cytotoxicity assays. Further analysis showed that, after CY and IFN were removed, protein synthesis recovered fully within 1 h. p47b, a protein that has been associated closely with the induction of the primed state in mouse macrophages, was then substantially expressed despite no new stimulation by IFN. Thus, macrophages in which protein synthesis had been reversibly inhibited delayed full processing of a signal delivered by IFN, until after protein synthesis had resumed. Such a delay in processing may explain how macrophages subsequently became activated, despite treatment with CY. The expression of the protein doublet, p71/73, was induced, regardless of which of three dissimilar agents (LPS, heat killed Listeria monocytogenes, poly I:C) was used to trigger the expression of cytolytic activity by primed macrophages. Therefore, the likelihood was increased that p71/73, expressed with p47b, is a valid phenotypic marker for fully activated, cytolytic macrophages. By contrast, p120, another protein that has been proposed as a marker of full activation in peritoneal macrophages, was expressed by bone marrow culture-derived macrophages regardless of whether or not they were cytolytic for tumor cells. It cannot be regarded as a reliable marker of macrophage activation in all circumstances, therefore.

Animals↗
PubMed1989

Culture of macrophages from bovine bone marrow.

Macrophages perform important immunoregulatory and host defense functions. Examination of this cell type in the bovine has been restricted because of lack of a means to obtain pure bovine macrophage populations reproducibly. We have developed a system for production of large numbers of macrophages from this species with greater than 99% purity. Stem cells were obtained from the bone marrow of neonatal calves and cultured in vitro in the presence of macrophage-colony-stimulating factor. Bovine bone marrow culture-derived macrophages were esterase-positive, expressed Fc receptors for aggregated IgG, and bovine macrophage differentiation markers. In addition, they displayed class I and class II major histocompatibility (MHC) antigens. The level of MHC antigen expressed could be further enhanced by treatment with recombinant bovine interferons. The macrophages exhibited expected functions, for example, Fc-mediated ingestion of opsonized sheep red blood cells. Augmentation of phagocytic capacity by either alpha or gamma interferon could also be demonstrated. The data reported here confirm that bone marrow culture is a convenient, reliable source of macrophages for investigations of this bovine cell type.

Animals↗
PubMed1989

Causal association of interferon-gamma with tumor regression.

Mouse interferon-gamma (MuIFN-gamma) can cause the rejection of malignant cells in vivo. The evidence presented here in support of this claim includes, first, that spontaneous regression of MSC sarcomas was associated with high intratumoral concentrations of endogenously-produced MuIFN-gamma. By contrast, progressively growing, lethal neoplasms of the same kind invariably contained very little IFN-gamma. Second, spontaneously regressing MSC sarcomas were converted into progressively growing, lethal neoplasms by injecting mice with a monoclonal antibody that neutralized the biological effects of endogenous IFN-gamma. Another monoclonal antibody that bound to, but did not neutralize, mouse IFN-gamma had no effect on the course of tumor regression. Together, these data causally relate MuIFN-gamma to the successful rejection of malignant cells in vivo. They also suggest that findings of poor therapeutic efficacy for IFN-gamma are probably attributable to problems other than an intrinsic lack in the biological activity of the lymphokine.

Animals↗