Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice

We analyzed the T cell-independent production of IFN-gamma in the severe combined immunodeficiency (scid) mutant mouse. Spleen cells from scid mice secreted high levels of IFN-gamma in response to heat-killed Listeria monocytogenes (HKLM), but not to the T cell stimulus ConA. This response was ablated by prior removal of adherent macrophages. IFN-gamma secretion in vitro was preceded by the rapid production of TNF and was inhibited by addition of neutralizing mAb to TNF. Moreover, injection of scid mice with anti-TNF mAb increased the severity of infection with live Listeria and inhibited macrophage activation for class II-MHC expression. Finally, IFN-gamma secretion and class II-MHC expression were also inhibited by an antibody to asialoGM1, a reagent known to impair host NK cell function. These results suggest that TNF is a critical cytokine in the T cell-independent pathway of macrophage activation in scid mice.     

Differential induction of activation markers in macrophage cell lines by interferon-gamma

Macrophage cell lines were used in these studies as a model system to dissect the biochemical and functional mosaic of the macrophage activation process. In particular, the requirements for the induction of tumoricidal and bactericidal activity in the RAW 264.7 and WEHI-3 cell lines by interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) were determined. Changes in expression of a series of macrophage markers traditionally associated with macrophage activation were monitored during stimulation of the cells in order to determine whether a detectable pattern of activation-associated changes is associated with the development of a particular functional activity. These markers included changes in the cell surface expression of major histocompatibility complex-encoded Class I and Class II antigens and antigens in the Mac-1/LFA-1 family, alterations in the levels of membrane enzymes (5' nucleotidase and alkaline phosphodiesterase), and production of secretory products including hydrogen peroxide and the monokines interleukin-1, interferons-alpha/beta, and tumor necrosis factor-alpha. Our results demonstrate that a given homogeneous macrophage population expresses a distinct subset of functional activities in response to single, defined activating signals such as IFN-gamma and LPS. The display of a variety of macrophage surface antigens, enzymes, and secreted products is activated simultaneously by such treatment; however, the particular pattern of such activation-associated markers cannot reproducibly be used to predict the ability of an activated cell to perform a particular function. The results also suggest that macrophage cell lines expressing differential response patterns following IFN-gamma stimulation provide a valuable system for dissection of the molecular and cell biology of macrophage activation.     

Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism

We have studied the expression of Ia molecules by macrophages from mice with severe combined immunodeficiency (CB-17 scid) that lack demonstrable T cell and B cell functions. CB-17 scid mice had approximately normal numbers of Ia-bearing macrophages in the peritoneal cavity, spleen, and liver. Peritoneal macrophages responded in culture to T cell-derived lymphokines with enhanced expression of Ia molecules. However, unlike immunocompetent controls, SCID mice could not enhance Ia expression in an antigen-specific T cell-dependent manner after secondary challenge in vivo with a conventional protein antigen such as hemocyanin. Further demonstration of their T cell deficiency was the failure of CB-17 scid spleen cells to proliferate and produce IL 2 in response to the T cell mitogen, concanavalin A. Upon infection with Listeria monocytogenes, CB-17 scid mice developed chronically high loads of bacteria, whereas CB-17 control mice eliminated all viable bacteria and became resistant to secondary infection. However, Listeria-infected CB-17 scid mice did show, in parallel with the CB-17 controls, an unexpected and striking increase of Ia-positive macrophages. These data indicate that induction of Ia expression in macrophages can occur via a mechanism that is independent of mature T cells.     

Absolute macrophage dependency of T lymphocyte activation by mitogens.

A T lymphocyte subpopulation that contains only 0.3% macrophages and less than 2% B lymphocytes has been prepared from guinea pig lymph node cells by the use of two different types of adherence columns. This subpopulation does not porliferate in response to the mitogens Con A or PHA unless additional macrophages are added. The means by which macrophages restore T cell responsiveness to PHA has been investigated. Marcophages appear to function via two different distinct mechanisms in this experimental situation. The first mechanism involves the binding of PHA to the macrophage followed by the "presentation" of the mitogen to the T lymphocyte in a manner that induces cell activation. This presentation function requires that the macrophage be viable and metabolically active. The second mechanism by which macrophages function is by the elaboration of a soluble factor or factors. The presence of these factors has been reliably and reproducibly demonstrated by using a double-chambered, Marbrook-type tissue culture vessel. This soluble factor can induce activation of T lympohcytes with surface bound PHA in the apparent absence of any form of macrophage presentation. In contrast, the function of this factor is clearly distinct from that of the reducing agent, 2-mercaptoethanol, (2-ME) since 2-ME does not enable this T cell subpopulation to be activated by mitogens. On the basis of these observations, we propose that two distinct signals are required to activate this T lymphocyte subpopulation. One signal is delivered by the interaction of the mitogen with the T cell surface, and the second signal is delivered by a soluble factor(s) produced by macrophages. Whether all types of T lymphocytes require two signals to be activated, remains to be established.     

Effects of interferon-gamma on the activation of human T lymphocytes

The role of interferon (IFN)-gamma in the activation of human T cells was investigated. Addition of IFN-gamma to mixed-lymphocyte cultures (MLC) augmented both the proliferation and the development of T-cell-mediated cytotoxicity. IFN-gamma also augmented the early expression on CD8+ but not CD4+ lymphocytes of IL-2 receptor alpha chain (Tac antigen) and Class II major histocompatibility antigen (HLA-DR). This effect synergized with that caused by interleukin 2 and was not observed with IFN-alpha. The addition of neutralizing antibody against IFN-gamma to MLC suppressed the development of cytotoxicity and proliferation and the expression of activation antigens on CD8+ cells. In experiments in which highly purified CD8+ T cells were activated with cell-free stimuli, IFN-gamma slightly but significantly augmented proliferation, antibody to IFN-gamma suppressed proliferation, and excess IFN-gamma reversed this suppression. It is concluded that (i) IFN-gamma augmented activation of T cells in human MLC, (ii) IFN-gamma exerted effects directly on T cells, and (iii) IFN-gamma preferentially augmented CD8+ cell activation.     

Regulation of macrophage growth and antiviral activity by interferon-gamma

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.     

Down-regulation of macrophage CD9 expression by interferon-gamma.

CD9, a member of the tetraspanin family is a cell surface marker expressed on myeloid and nonmyeloid as well as on neoplastic cells. The present study has focused on the role of inflammation and macrophage activation in the regulation of CD9 expression. We report that the expression of CD9 on primary cultures of murine peritoneal macrophages was down regulated by Interferon-gamma, IFN-gamma. This down regulation was concentration-dependent and maximal by 48 h. The changes in surface expression were consistent with similar reductions in CD9 protein and message levels by Western and Northern blot analyses. The mechanism by which IFN-gamma decreases CD9 expression appears to be through the Stat1 signaling pathway as Stat1 knockout mice did not demonstrate any reduction in CD9 expression by IFN-gamma treatment. These results represent the first evidence for the down regulation of CD9 expression with macrophage activation.     

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.     

Inhibition of B lymphocyte activation by interferon-gamma

Helper/inducer T cell clones specific for protein antigens and class II MHC determinants consist of two nonoverlapping subsets. One (called Th1) secretes IL 2 and IFN-gamma and the other (Th2) produces BSF1 upon stimulation with antigen or polyclonal activators. By using hapten-binding normal B cells and the B lymphoma line WEHI-279 as assays for B cell helper (maturation) factors, we have shown that Th2 clone supernatants (SN) induce differentiation to antibody secretion, whereas Th1 SN do not. The failure of Th1 SN to activate B cells is due to inhibitory effects of IFN-gamma, because it can be reversed by a neutralizing monoclonal antibody specific for IFN-gamma. Thus, in the presence of this antibody, even Th1 SN stimulate B cell maturation maximally. Conversely, recombinant IFN-gamma inhibits proliferation and differentiation of B cells induced by active Th2 SN. These results demonstrate that IFN-gamma is a potent inhibitor of B lymphocyte activation and can be distinguished from growth and maturation-inducing helper factors that are produced by both subsets of helper T cells.     

Molecular mechanism of macrophage activation by Exopolysaccharides from liquid culture of Lentinus edodes

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. beta-Glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using exopolysaccharides prepared from liquid culture of Lentinus edodes. We found that fraction II (F-II), with large molecular weight protein polysaccharides, is able to strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production, cytokine expression, and morphological changes. F-II triggered the nuclear translocation of NF-kappaB and activated its upstream signaling cascades such as PI3K/ Akt and MAPK pathways, as assessed by their phosphorylation levels. The function-blocking antibodies to dectin-1 and TLR-2, but not CR3, markedly suppressed F-II-mediated NO production. Therefore, our data suggest that mushroom-derived beta-glucan may exert its immunostimulating potency via activation of multiple signaling pathways.     

Monoclonal antibodies against mouse gamma-interferon inhibit tumoricidal macrophage activation by T lymphocytes

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.     

Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma

In vitro, metabolism of modified forms of low density lipoprotein (LDL) by macrophages via the acetyl-LDL receptor pathway promotes the massive cellular accumulation of lipid. It has been postulated that in vivo this contributes to foam cell formation in the atherosclerotic lesion. Recent studies have shown that arterial wall cells in vitro can secrete a number of cytokines, several of which have been reported to modulate macrophage cell function. Thus, cytokines have the potential to modulate the acetyl-LDL receptor pathway and to influence the rate of foam cell generation. To study the regulation of this pathway by cytokines, the effect of cytokines on the degradation of acetyl-LDL protein by mouse peritoneal macrophages was examined. Initially, supernatant from stimulated lymphocytes was used as a source of cytokines. Macrophages preincubated with supernatants obtained after the stimulation of T-cell helper type 1 (Th1) clone HDK-1 or BALB/c spleen cells degraded acetyl-LDL at a slower rate, whereas supernatant from stimulated T-cell helper type 2 (Th2) clone D-10 had no effect. Comparison of the lymphokine profiles showed that spleen and HDK-1 cells secreted several lymphokines in common including significant levels of interferon-gamma. Interferon-gamma was then directly shown to be inhibitory; an anti-interferon-gamma monoclonal antibody blocked the HDK-1-mediated inhibition by 70% and the addition of recombinant interferon-gamma (IFN-gamma) to macrophages inhibited the specific degradation of acetyl-LDL in a dose- and time-dependent manner with a maximum suppression to approximately 40% of control. The inhibition was not accompanied by an increase in the amount of cell-associated acetyl-LDL and was not due to cell death nor could it be accounted for by the presence of endotoxin. To study the mechanism of the inhibition, the effects of IFN-gamma on the itinerary of acetyl-LDL and its receptor were examined. IFN-gamma decreased specific acetyl-LDL binding only to a small degree, and the rate of lysosome-mediated degradation was not affected. The principal alteration was in the rate of transport to the lysosome which was markedly slowed. Since the receptors eventually returned to the surface to maintain a steady state, and there was not an increase in cell-associated lipoprotein, there must be other changes in the itinerary that were not identified with the techniques used. Thus, the receptor cycle is being regulated at a discrete point. IFN-gamma also suppressed the LDL receptor pathway in macrophages, but this pathway was not affected by IFN-gamma in mouse fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contribution of lysophosphatidylcholine-treated nonadherent cells to mechanism of macrophage activation

Lysophosphatidylcholine (lyso-PC), a product of inflammation, stimulates (in vivo) mouse peritoneal macrophages to ingest target cells via Fc receptors. In vitro treatment of macrophages with lyso-Pc was unable to enhance ingestion activity. When a mixture of macrophages and nonadherent (B and T) cells was treated with 20 micrograms of lyso-Pc/ml for 30 min, a greatly enhanced Fc-mediated ingestion was observed at about 3 hr after treatment, suggesting that nonadherent cells contributed to activation mechanism of macrophages. The accumulated evidence suggests that treated B cells collaborated with untreated T cells in a stepwise fashion for the exchange of a signaling factor(s) for macrophage activation. When conditioned medium prepared by stepwise cultivation from treated B cells to untreated T cells was used for cultivation of untreated macrophages, a markedly enhanced Fc-mediated ingestion was observed. However, cultivation of macrophages with stepwise conditioned medium of treated T cells and untreated B cells produced no significant enhancement of phagocytic activity. Therefore, we concluded that lyso-Pc-treated B cells initiated the macrophage activation process by releasing and transmitting a signaling factor to T cells, and, in turn, the T cells modified the factor or supplied a new factor capable of the ultimate activation of macrophages for ingestion capacity. This lyso-Pc-induced factor(s) appears to be distinct from the established interleukins 1 and 2.     

Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.     

Modulation of macrophage activation by prostaglandins

The effect of prostaglandtn E(2), iloprost and cAMP on both nitric oxide and tumour necrosis factor-alpha release in J774 macrophages has been studied. Both prostaglandin E(2) and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-alpha. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E(2) and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-alpha generation, which in turn is likely to be mediated by cAMP.     

Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen

Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.     

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.     

Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism

Immunostimulatory DNA sequences (ISS) contain unmethylated CpG dinucleotides within a defined motif. Immunization with ISS-based vaccines has been shown to induce high antigen-specific cytotoxic lymphocyte (CTL) activity and a Th1-biased immune response. We have developed a novel ISS-based vaccine composed of ovalbumin (OVA) chemically conjugated to ISS-oligodeoxynucleotide (ODN). Protein-ISS conjugate (PIC) is more potent in priming CTL activity and Th1-biased immunity than other ISS-based vaccines. Cytotoxic lymphocyte activation by ISS-ODN-based vaccines is preserved in both CD4-/- and MHC class II-/- gene-deficient animals. Furthermore, PIC provides protection against a lethal burden of OVA-expressing tumor cells in a CD8+ cell-dependent manner. These results demonstrate that PIC acts through two unique mechanisms: T-helper-independent activation of CTL and facilitation of exogenous antigen presentation on MHC class I. This technology may have clinical applications in cancer therapy and in stimulating host defense in AIDS and chronic immunosuppression.