The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.     

Antibody inhibition of suppressor cell induction

The IJ genetic restrictions of suppressor T (Ts) cells are controlled by H-2-related determinants that are expressed on antigen-presenting cells. This has led to the hypothesis that Ts cells carry receptors for a self H-2-related ligand that is expressed on specialized antigen-presenting cells. We refer to this H-2-related ligand as the IJ interacting molecule. This report evaluates the ability of rabbit antibodies directed against idiotypes on monoclonal anti-IJ antibodies (the latter are presumably reactive with the Ts cell receptor) to bind IJ interacting molecule and to inhibit antigen presentation to Ts cells. Such anti-idiotypic reagents were prepared against T cell-reactive monoclonal anti-IJk and anti-IJd antibodies. The F(ab')2 fragments of these anti-idiotypic reagents blocked Ts cell induction. The inhibition was haplotype specific and mapped to the IJ region. The anti-idiotypic antibodies blocked the generation of Ts1, Ts2, and Ts3 cells. The cellular target of the blocking activity mediated by these anti-idiotypic antibodies is a macrophage. This was shown by using a cloned macrophage hybridoma line for both Ts induction and absorption of antibody activity. The combined data support the concept that macrophages express IJ interacting determinants that are responsible for Ts cell induction.     

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.     

Modulation of suppressor T cell induction with gamma-interferon

The ability of antigen-coupled splenic adherent cells to induce suppressor T cells (Ts) is dependent on the presence of I-J determinants on antigen-presenting cells. After 4 days of in vitro culture, antigen-coupled adherent cells lose the capacity to induce Ts. Supernatants from Con A-stimulated lymphocyte cultures and purified interferon-gamma can sustain accessory function for the induction of Ts. Furthermore, after in vitro culture of splenic adherent cells, there is an apparent correlation between the loss of I-A determinants and the decrease in I-J-restricted Ts induction. Stimulation of Ia expression with interferon-gamma results in a simultaneous increase in the ability to induce Ts. Finally, elimination of I-A-bearing splenic adherent cells with antibody + C eliminates I-J-restricted Ts induction. The combined data imply a co-regulation of I-A and I-J on the antigen-presenting cells involved in the induction of both the Ts1 and Ts3 suppressor T cell subsets.     

Control of suppressor cell activation via endogenous prostaglandin synthesis: The role of T cells and macrophages

Splenic macrophages were cultured in the inner chamber of a Marbrook flask and their ability to induce suppressor activity in a glass-wool-adherent T-cell population was determined. Nontreated, lipopolysaccharide-, or phytohemagglutinin-treated macrophages, all induced comparable levels of suppressor activity in glass-wool-adherent T cells. Suppression induced by untreated macrophages was reversible by the addition of a prostaglandin synthetase inhibitor. In addition, macrophages enhanced non-glass-adherent T-cell activation of glass-wool-adherent cells. The results indicate that macrophage in concert with T cells may activate T suppressor cells via a prostaglandin-dependent pathway.     

The role of macrophages in the generation of T-helper cells. I. The requirement for macrophages in helper cell induction and characteristics of the macrophage-T cell interaction.

The role of macrophages in the induction of helper cells in vitro was investigated. Using either soluble or particulate antigens, macrophages were found to be essential. This was true regardless of the anatomical source of the T cells (spleen, lymph node or the cortisone resistant pool of the thymus), or of the method of macrophage depletion, (adherence to polystyrene or nylon wool, or by the use of carbonyl iron). There were some differences, however, depending on the physical nature of the antigen used. With soluble antigen, 2-mercaptoethanol or allogeneic macrophages would not overcome the macrophage deficit, whereas they did with particulate antigen. The nature of the interaction between macrophages and T cells was investigated using flasks with double chambers, separated by a nucleopore membrane with 0.2 μm pores. Since there was effective interaction, contact between T cells and macrophages is not essential for helper cell induction.     

Characterization of the accessory cells involved in suppressor T cell induction

The ability of UV-treated splenic adherent cells (SAC) to induce T cell-mediated immunity and suppressor T cells was analyzed in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. UV irradiation of 0.88 KJ/m2 decreased the capacity of NP-coupled SAC to induce delayed-type hypersensitivity (DTH) responses by about 50%. The ability of uncoupled UV-treated SAC to induce allogeneic DTH response was also imparied, indicating that UV-treated SAC are inefficient at inducing DTH in these systems. TS1 induction by UV-treated NP-SAC was evaluated TS1 induction by UV-treated NP-SAC was evaluated by using adherent cells that were subjected to the same dose of UV irradiation that impaired DTH induction. Intravenous administration of 10(3) or 10(4) UV-treated NP-coupled SAC induced TS1 cells with the same efficiency as non-UV-irradiated cells. The TS1 cells induced in this fashion were antigen specific. Furthermore, to establish that the antigen was not reprocessed by the host, I-J-mismatched, UV-treated NP-SAC were unable to induce TS1 cells. The population of antigen-presenting cells responsible for TS1 induction appear to express both I-A and I-J determinants. TS2 induction by UV-treated accessory cells was also analyzed. TSF1 inducer suppressor factor was pulsed onto graded numbers of either normal or UV-treated adherent cells. The same levels of antigen-specific suppression were induced with normal and UV-treated cells. Finally, TS3 induction by UV-treated NP-SAC was analyzed. UV-treated and normal NP-SAC (3 X 10(3] induced antigen-specific suppression of NP DTH responses. I-J-mismatched, UV-treated NP-SAC failed to induce suppression, suggesting that the hapten was not reprocessed by the host under these experimental conditions. The accessory cell population responsible for TS3 induction appears to express both I-A and I-J determinants. Thus, there are at least two functional distinctions between the antigen-presenting cells that induce immunity vs those that induce suppressor cells. First, UV treatment selectively impairs the antigen-presenting cells, which activate the positive limb of the immune response. Second, I-J determinants appear to be specifically associated with the SAC, which induce suppressor T cells. Although these criteria can be used to distinguish the accessory cells involved in suppressor cell pathways from those controlling helper T cell induction, there were no discernible phenotypic differences among the accessory cells that induce the TS1, TS2, and TS3 subsets.     

A role for the myoglobin redox cycle in the induction of endothelial cell apoptosis

This study investigates the potential role of the ferric/ferryl redox cycle of myoglobin (Mb) in the development of endothelial cell injury. Bovine aortic endothelial cells were incubated with ferric Mb (0.5-100 micro M) in the presence or absence of low steady states of H(2)O(2) (3-4 micro M) generated by glucose oxidase (GOX). The reaction of ferric Mb with H(2)O(2) generated ferryl Mb as monitored spectrophotometrically. Ferryl Mb formation correlated with the induction of apoptosis as indicated by morphological criteria, caspase 3 activation, phosphatidylserine (PS) externalization, and nuclear condensation by Hoechst 33342 staining. The addition of ascorbate or catalase inhibited the formation of ferryl Mb and the onset of apoptosis, whereas apoptosis was enhanced in cells depleted of intracellular glutathione by pretreatment with buthionine sulfoximine. Mb and Mb/GOX suppressed cell cycle progression, but only Mb/GOX produced significant cell loss revealed by the accumulation of sub G1 events. These results suggest a role for the Mb redox cycle in the induction of endothelial cell apoptosis, which may be relevant in the pathophysiology of diseases characterized by the release of Mb from damaged muscle.     

T cell induction of membrane IL 1 on macrophages

We have studied the role of T cells in the induction of a membrane-associated form of interleukin 1 (mIL 1) in murine macrophages. T helper cell clones and a T cell hybridoma induced macrophages to express mIL 1 after an antigen-specific, Ia-restricted interaction. Induction of mIL 1 was proportional to antigen concentration and was increased in the early course of the response in macrophages pretreated in culture with interferon-gamma. mIL 1 activity was detectable 4 hr after interaction with T cells. mIL 1 induction was inhibited by antibodies to either class II molecules or the T cell receptor. Two pathways of T cell-mediated mIL 1 induction could be defined. In the first, T cells, whose protein synthesizing capacity was completely eliminated by pretreatment with the irreversible protein synthesis inhibitor emetine, induced levels of mIL 1 expression indistinguishable from controls. In the second, T cells stimulated by paraformaldehyde-fixed macrophages in the presence of concanavalin A or antigen secreted a soluble factor that induced macrophage mIL 1 expression. Thus, it appears that T cells may induce macrophages to express mIL 1 both by direct cell-cell contact mediated through binding of T cell receptor to the Ia/antigen complex, and through the release of a lymphokine after activation. This lymphokine does not appear to be IL 2, IFN-gamma, BSF-1, or CSF-1.     

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.     

Serum factors required for arginase induction in macrophages

Two nondialyzable factors in the sera of several species were required for the induction of arginase in resident peritoneal macrophages in vitro. One factor stimulated PGE₂ production by macrophages, was inhibited by indomethacin, and could be replaced by the addition of PGE₂ or DBcAMP. Thirty percent serum was required for maximal activity of this factor. DBcAMP reduced the requirement for serum by 10-fold, suggesting the requirement of a second factor which required less than 3% serum for maximal activity. A factor with similar activity was found in the supernatant of tumor cells grown in vitro.     

A paradoxical role for myeloid-derived suppressor cells in sepsis and trauma

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature myeloid cells whose numbers dramatically increase in chronic and acute inflammatory diseases, including cancer, autoimmune disease, trauma, burns and sepsis. Studied originally in cancer, these cells are potently immunosuppressive, particularly in their ability to suppress antigen-specific CD8(+) and CD4(+) T-cell activation through multiple mechanisms, including depletion of extracellular arginine, nitrosylation of regulatory proteins, and secretion of interleukin 10, prostaglandins and other immunosuppressive mediators. However, additional properties of these cells, including increased reactive oxygen species and inflammatory cytokine production, as well as their universal expansion in nearly all inflammatory conditions, suggest that MDSCs may be more of a normal component of the inflammatory response ("emergency myelopoiesis") than simply a pathological response to a growing tumor. Recent evocative data even suggest that the expansion of MDSCs in acute inflammatory processes, such as burns and sepsis, plays a beneficial role in the host by increasing immune surveillance and innate immune responses. Although clinical efforts are currently underway to suppress MDSC numbers and function in cancer to improve antineoplastic responses, such approaches may not be desirable or beneficial in other clinical conditions in which immune surveillance and antimicrobial activities are required.     

A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.     

A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration

Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration.     

Immunoregulatory activity of culture-induced suppressor macrophages

Rat splenic cells precultured in vitro for 5 days exhibited marked suppressive activity on the secondary cytotoxic T lymphocyte (CTL) response to a Gross virus-induced lymphoma. Suppressive activity was produced by macrophages (MØ) rather than lymphocytes and as low as 1% MØ content was sufficient to achieve completely inhibited CTL responses. Aspirin, indomethacin, and d,l-6-chloro-2-methylcarbazole-2-acetic acid prevented cultured splenic MØ from exerting their inhibitory effect, thereby suggesting a role for prostaglandins in suppression. Events which occurred within the first 24 to 48 hr of the CTL response were susceptible to the suppressive action of MØ since normal CTL responses were obtained if suppressive MØ were added later than Day 2 or if indomethacin was added within the first 24 to 48 hr of culture. Two processes of lymphocyte activation, namely blast transformation and DNA synthesis, were inhibited in the presence of suppressive MØ. However, suppression of these processes did not result in the loss of CTL progenitor cells since CTL responses that were inhibited in the presence of suppressive MØ proceeded normally following their removal.     

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.