The handling of Listeria monocytogenes by macrophages: the search for an immunogenic molecule in antigen presentation

The activation of T lymphocytes for immunity to the intracellular pathogen Listeria monocytogenes requires that Ia-positive macrophages ingest the bacteria. The subsequent handling of Listeria by macrophages was examined in this report and related to antigen presentation to T cells. Macrophages pulsed with radiolabeled Listeria, besides releasing acid-soluble radioactivity--an indication of extensive catabolism of the Listeria-derived proteins--were also found to release acid-insoluble peptides. The rate of release of the peptides was not markedly affected by treatment with chloroquine, ammonia, or monensin and was independent of the state of activation and the level of Ia expression of the macrophage. The peptides were not associated with fragments of membranes and were represented by several molecular species. Listeria-derived peptides were also found associated with the macrophage plasma membrane. The membrane-associated peptides behaved like integral membrane proteins and could be released by proteases or detergents. Their expression was independent of the dose of Listeria and the level of Ia expression of the macrophage, and their presence could not be inhibited by protease inhibitors or chloroquine. The Listeria peptides released by the macrophages were very weakly immunogenic in a T cell proliferation assay. Purified plasma membranes from Listeria-pulsed macrophages, which contained membrane-associated Listeria peptides, were not immunogenic by themselves but could be reprocessed by additional macrophages to subsequently stimulate T cells. Trypsin treatment of Listeria-pulsed macrophages did not cause a significant reduction in their ability to stimulate T cells. No association was found between Ia molecules and either the membrane-associated or the released peptides with the use of several technical approaches. Hence, after internalization of Listeria, potentially immunogenic material can be found at the cell surface as well as in the culture fluid. The release of soluble peptides is a clear indication that proteins can be recycled after their internalization in vesicles.     

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.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Alveolar macrophage activation in experimental legionellosis.

Legionella pneumophila is a facultative intracellular parasite of alveolar macrophages. In vitro studies have shown that lymphokine-activated mononuclear phagocytes inhibit intracellular replication of L. pneumophila. To determine if recovery from legionellosis is associated with activation of alveolar macrophages in vivo to resist L. pneumophila, we studied an animal model of Legionnaires' disease. Rats were exposed to aerosolized L. pneumophila and alveolar macrophages were harvested during the recovery phase of infection. We compared these alveolar exudate macrophages with normal resident alveolar macrophages for the capacity to support or inhibit the intracellular growth of L. pneumophila. We also measured Ia expression as a marker of immunologic activation, and studied binding of bacteria, superoxide release, and the expression of transferrin receptors as potential mechanisms of resistance to L. pneumophila. For perspective on the specificity of these responses, we also studied alveolar exudate cells elicited by inhalation of heat-killed L. pneumophila, live Listeria monocytogenes, and live Escherichia coli. We found that alveolar exudate macrophages elicited by live L. pneumophila, but not heat-killed L. pneumophila, resisted the intracellular growth of L. pneumophila. Exudate macrophages in resolving legionellosis exhibited increased Ia expression, diminished superoxide production, and downregulation of transferrin receptors. Binding of L. pneumophila to exudate macrophages was indistinguishable from that to resident macrophages in the presence of normal serum, and augmented in the presence of immune serum. Alveolar exudate macrophages elicited by E. coli also inhibited growth of L. pneumophila, and exhibited a modest increase in Ia expression without change in transferrin receptors. Exudate cells induced by L. monocytogenes exhibited up-regulation of Ia without diminution of superoxide release. Alveolar cells harvested after inhalation of heat-killed L. pneumophila did not differ from resident alveolar macrophages in the expression of surface markers. These findings suggest that alveolar macrophages are immunologically activated in vivo to serve as effector cells in resolving legionellosis, and that live bacteria are required to induce this expression of immunity. The mechanism of resistance to parasitism by L. pneumophila may entail restriction of the intracellular availability of iron, but does not involve diminished bacterial binding or an augmented respiratory burst.     

Induction of macrophage Ia expression by lipopolysaccharide and Listeria monocytogenes in congenitally athymic nude mice

Experiments were performed to analyze the mechanism by which lipopolysaccharide (LPS) modulates the expression of Ia by murine peritoneal macrophages in vivo. We investigated the effect of LPS on Ia expression in T cell deficient mice by using the congenitally athymic nude mouse model. Injection (i.p) of LPS into athymic (nu/nu) mice resulted in a dramatic increase in the expression and biosynthesis of Ia by peritoneal macrophages 7 days after injection. The magnitude and kinetics of this induction were equivalent to increases observed after LPS injection of euthymic (nu/+) mice. Viable Listeria monocytogenes also increased Ia expression in athymic mice, but in contrast to the induction observed in euthymic mice at 3 and 7 days after injection, increased Ia expression was not seen until 7 days. Ia induction by either LPS or L. monocytogenes in athymic mice was not due to the presence or development of mature T cell function as defined by assays for T cell mitogenesis and interleukin 2 production. We conclude that increased macrophage Ia expression by LPS and L. monocytogenes in vivo can occur in the absence of mature functioning T cells.     

Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia expression in vivo

Experiments were performed to analyze the modulation of macrophage Ia expression and biosynthesis by Salmonella minnesota-derived lipopolysaccharide (LPS) in vivo. The i.p. injection of LPS into LPS-responder mice caused a dramatic increase in the Ia expression of the peritoneal macrophage population harvested 1 wk after injection. As little as 1 ng of lipid-rich Re595 LPS per mouse caused a significant I-Ak increase, and 1 microgram was optimal; wild-type S. minnesota LPS was less active. No I-Ak induction by LPS was observed in the LPS-nonresponder strain C3H/HeJ. LPS-induced macrophages showed a 6- to 16-fold increase in I-Ak expression by radioimmunoassay (RIA), a 3- to 10-fold increase in the proportion of I-Ak-positive cells, and a 10- to 15-fold increase in I-Ak biosynthetic capacity. The magnitude of this induction by LPS was comparable to increases observed after injection of live Listeria monocytogenes. The kinetics of I-Ak induction by LPS and by L. monocytogenes were different: LPS caused an initial decrease in I-Ak expression 1 day after injection, and I-Ak induction by LPS occurred more slowly and maintained heightened expression longer. Several H-2 gene products (H-2Kk, I-Ak, and I-Ek) were augmented in LPS-induced macrophages. In keeping with increased I-A and I-E expression, LPS-induced macrophages were more effective than normal macrophages in presenting antigen to T lymphocytes. We suggest that the modulation of macrophage Ia expression is one important mechanism contributing to the immunoregulatory activity of LPS.     

Membrane vesicles shed by murine melanoma cells selectively inhibit the expression of Ia antigen by macrophages

We previously demonstrated that membrane vesicles shed by the F10 variant of the murine B16 melanoma cell line inhibited the induction by interferon-gamma (IFN) of murine macrophage immune response region-associated (Ia) antigen expression. In this paper we present evidence that the inhibition of macrophage Ia antigen expression is a selective effect of vesicles and characterize its temporal requirements. Membrane vesicles shed from F10 cells did not affect the expression of macrophage H-2K or H-2D antigens under conditions shown to profoundly inhibit Ia antigen expression. Similarly, the induction of plasminogen activator and interleukin 1 from macrophages was not inhibited by the vesicles. The vesicles did not measurably decrease total cellular RNA or protein synthesis. Macrophages were sensitive to the inhibitory effects of the vesicles during the induction and maintenance phases of Ia expression. Pretreatment of macrophages with vesicles before culture with IFN did not reduce the induction of Ia. The rate of decline of Ia expression after removal of IFN was unaffected by the presence of vesicles. Removal of vesicles from cultures of IFN-treated macrophages resulted in only a partial recovery of Ia expression, suggesting that the inhibition of Ia expression may be a slowly reversible process. The selective and partially reversible inhibition of Ia expression by vesicles shed from the plasma membrane of tumor cells is a possible mechanism whereby tumor-bearing hosts may become immunocompromised.     

Control of macrophage Ia expression in neonatal mice--role of a splenic suppressor cell

The control of macrophage expression of I region-associated antigens (Ia) in neonatal mice was studied by comparing responses of neonatal and adult mice to immune vs nonimmune stimuli. Adults generated peritoneal exudates rich in Ia-bearing macrophages in response to i.p. injection of live Listeria monocytogenes, Listeria-immune T cells, and heat-killed Listeria, or a soluble mediator termed macrophage Ia-recruiting factor (MIRF). Neonates failed to respond to these stimuli. In contrast, both neonates and adults generated Ia-negative peritoneal exudates when stimulated with thioglycollate. A neonatal spleen cell that blocked the response of adults both to immune T cells and heat-killed Listeria and to MIRF was identified and characterized. Some of the suppressor cells appeared to be early precursors of the phagocytic lineage that develop into mature monocyte-macrophages. Suppression was apparently mediated by metabolites of arachidonic acid since indomethacin and aspirin in vivo blocked the effect. Similar suppressor activity was found in adult bone marrow and in adult resident peritoneal exudate cells. Thus, the phagocytic line autoregulates its surface expression of Ia in both neonatal and adult mice. This mechanism becomes particularly pointed during early development and could contribute to the lack of immunity during ontogeny.     

Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes

T lymphocytes and macrophages from Listeria-infected mice were used to evaluate the processing and presentation of live Listeria monocytogenes in vitro. Antigen presentation to T cells was quantitated by interleukin-2 production. In contrast to inert antigens such as heat-killed Listeria, live bacteria were processed and presented poorly. To evaluate the role of hemolysin (Hly), we used isogenic pairs of Hly+ and Hly- Listeria as antigens. In contrast to live Hly- bacteria, which were presented as well as heat-killed Listeria, live Hly+ bacteria were presented poorly. Hly+ bacteria also inhibited the presentation of heat-killed Listeria. This effect was apparent with as few as 10 bacteria/macrophage and was not due to loss of macrophage viability or decreased Ia expression after exposure to the live bacteria. With respect to murine listeriosis, the LD50 values for the Hly- strains were at least 1000 times higher than those for the Hly+ strains. These results suggest that the ability of Hly+ bacteria to inhibit antigen processing and presentation may be an important determining factor in Listeria infection and immunity.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Dendritic cells acquire antigens from live cells for cross-presentation to CTL

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.     

Studies on antigens associated with the activation of murine mononuclear phagocytes: kinetics of and requirements for induction of lymphocyte function-associated (LFA)-1 antigen in vitro

Macrophages activated and primed in vivo, although not resident or responsive macrophages, express the lymphocyte function associated (LFA)-1 antigen. By contrast, the biochemically related Mac-1 antigen is expressed on all populations of macrophages. In the present paper, we studied regulation of the LFA-1 antigen in vitro. LFA-1 could be induced in vitro on thioglycollate (TG)-elicited but not on proteose peptone (PP)-elicited or resident macrophages. Specifically, macrophage-activating factor (MAF), interferon-gamma (IFN-gamma), or picogram amounts of endotoxin (LPS) induced LFA-1 on TG-elicited macrophages following overnight incubation. Interferon, -alpha or -beta, fucoidin, and colony-stimulating factor were not effective. While some levels of LFA-1 could be detected as soon as 10 hr, peak expression was observed after 16 to 32 hr of incubation. The induction could be completely abrogated by cycloheximide, suggesting that protein synthesis was required. These results indicate that the induction of LFA-1 on mononuclear phagocytes is closely regulated and that the requirements for such induction are distinct from but share certain similarities with induction of cytotoxic functions and expression of Ia antigen.     

Species-dependent regulation of monocyte/macrophage Ia antigen expression and antigen presentation by prostaglandin E

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.     

Inhibition of recombinant interferon-gamma-induced Ia antigen expression by shed B16 F10 melanoma cell membrane vesicles

The expression of immune region-associated (Ia) antigens by macrophages is a prerequisite for antigen presentation, which is necessary for the activation of T helper cell function. A decrease in macrophage Ia expression is associated with a decrease in immune function in vitro. However, the effect of diseases accompanied by immunosuppression, such as cancer, on macrophage Ia expression has not been studied. The expression of Ia antigen was induced by the culture of murine peritoneal macrophages with recombinant interferon-gamma (IFN). Maximal expression was achieved after 4 days of culture. Membrane vesicles shed from the murine B16 F10 melanoma cell line inhibited the in vitro induction of Ia expression by 40 to 90% in allogeneic and syngeneic systems. Inhibition was not due to toxicity, a reduction in IFN activity, phagocytosis or contamination of the vesicle preparation with endotoxin, which is an inhibitor of Ia expression. Inhibition exerted by vesicles was prostaglandin-dependent and was over-come by increasing concentrations of IFN. It is possible that the reduction of macrophage Ia antigen expression by tumor cell products, such as shed membrane vesicles, contributes to the immunosuppression of tumor-bearing hosts. Employing IFN to reverse the inhibition provides a strategy for improving the therapy of patients with cancer.     

Sodium periodate-induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1

T lymphocytes oxidized with the mitogen sodium periodate undergo a proliferative response when cultured in the presence of Ia+ accessory cells. However, the exact role(s) the accessory cells play in such a response has not been clearly defined. We have evaluated the role of Ia and the requirement for interleukin 1 (IL 1) in periodate mitogenicity by using the Ia+ cloned tumor cell lines P388AD (Ia+, IL 1 inducible) and P388NA (Ia+, IL 1 noninducible) as accessory cells. P388AD but not P388NA was able to supply accessory cell function to periodate-treated T cells, suggesting that Ia expression alone was not sufficient to reconstitute a response. Monoclonal anti-I-Ad and anti-I-Ed antibody blocked the accessory cell function of P388AD. In addition, monoclonal antibody GK 1.5, directed against the T cell determinant L3T4a, blocked the P388AD/periodate-treated T cell interaction, confirming that this interaction was restricted by class II molecules. Although Ia expression was required, the response was not major histocompatibility complex (MHC) restricted, because allogeneic as well as syngeneic macrophages were capable of supplying accessory cell function to periodate-treated T cells. Exogenous IL 1 alone was able to trigger periodate-treated T cells, suggesting that Ia was required for the induction of IL 1 synthesis by the accessory cells. Furthermore, purified IL 2, devoid of IL 1 activity, was able to fully reconstitute the proliferative response of accessory cell-depleted oxidized T cells to a level equal to that of whole spleen accessory cells or P388AD. These data suggest that periodate-treated T cells can proliferate with IL 1 alone and that Ia+ accessory cells in periodate-mediated T cell mitogenicity may function in the release of IL 1 and the induction of IL 2 synthesis by the T cells.     

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.     

Macrophage-T cell interactions involving Listeria monocytogenes--role of the H-2 gene complex.

This study reports on the in vitro interactions between T cells from Listeria-immunized mice, macrophages from normal mice, and heat-killed Listeria organisms. This interaction was assayed either by determing the amount of thymocyte mitogen in culture fluids after 24 hr, or by estimating the degree of T cell proliferation after 96 hr. Each assay depended on critical concentrations of macrophages, T cells, and heat-killed Listeria, points that were evaluated in a number of experiments. Both assays required specific Listeria-immune T cells. For an effective interaction, the T cells and the macrophages had to share the I-A region of the H-2 gene complex. Macrophages bearing Ia, which represented a minor population of macrophages, were essential for the proliferative response to macrophage-associated Listeria. Also, Ia-bearing macrophages were an important component in the interactions leading to increased secretion of mitogen. The immunogenic moiety associated with Listeria was short-lived, disappearing 24 hr after uptake of Listeria by macrophages. The interactions were not blocked by anti-Listeria antibodies but were partially sensitive to trypsinization.     

Antigen processing and presentation by macrophages

The classical macrophage is one of the most important cells involved in presenting antigen to helper T cells, because of its ability to regulate its expression of Ia molecules and to encounter and process particulate and soluble antigens. We have summarized in this report studies examining the handling by macrophages of two different antigens, the bacteria Listeria monocytogenes and the protein hen egg white lysozyme (HEL). The purpose was to identify potential sources of immunogenic peptides. Presentation of Listeria required an intracellular processing stage sensitive to lysosomotropic drugs. The Listeria required internalization and processing, after which immunogenic molecules were recognized by T cells on the macrophage surface. Metabolic studies showed that Listeria-derived peptides were released by macrophages that had phagocytosized the bacteria. The release of these peptides was a temperature-dependent process, unaffected by inhibiting lysosomal catabolism by treatment with chloroquine. Listeria-derived peptides were also detected on the surface of the macrophage. These peptides behaved like integral membrane proteins, some of which persisted for at least 24 hr at the macrophage surface. When tested for immunogenicity, the released peptides were very weakly immunogenic. The membrane-associated peptides alone could not stimulate Listeria-specific T cells, but could be reprocessed by additional macrophages and subsequently stimulate the T cells. A defined antigen system using HEL-specific T-cell hybridomas was used to examine the processing of HEL. Presentation of HEL required a chloroquine-sensitive intracellular processing stage. In examining two T-cell hybridomas, a differential requirement for antigen processing was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine modulation of immune activation associated suppression of macrophage cyclooxygenase activity in vivo.

Intraperitoneal infection with Listeria monocytogenes (LM) results in activation of the peritoneal macrophage population which displays increased surface expression of major histocompatibility (MHC) Class II (Ia) antigen and markedly suppressed prostaglandin (PG) synthesis. We demonstrate here that this decrease in PG production is also seen after treatment by mitogen (Con A) and endotoxin (LPS), and can be explained by reduced cyclooxygenase activity in these cell populations. We show that, whereas Ia expression was augmented at all doses of LM and Con A tested, it displayed a biphasic response to LPS in vivo: increase at the lowest dose and inhibition at higher doses. In order to identify possible endogenous mediators of these responses, we used highly purified preparations of recombinant murine (rMu) cytokines and neutralizing cytokine specific monoclonal antibodies (MAbs) to examine whether interferon-gamma (IFN-gamma) and/or tumor necrosis factor (TNF) down-regulate macrophage cyclooxygenase activity in vivo. We found that IFN-gamma induced Ia expression but had no effect on PG secretion. In contrast, TNF-alpha suppressed PG synthesis and inhibited Ia surface expression. Similarly, in our model of Con A-induced peritoneal macrophage activation, pretreatment of animals with a neutralizing MAb to rMuIFN-gamma completely blocked the induction of Ia positive macrophages by Con A but did not affect Con A-dependent suppression of PG synthesis. Pretreatment with MAb to TNF had no effect on Con A-induced Ia levels, but significantly inhibited suppressed PG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide

Murine peritoneal exudate cells (PEC), analyzed immediately after isolation, did not express detectable IL 1 activity or IL 1-specific mRNA. Stimulation of these cells by adherence induced the expression of intracellular, membrane, and extracellular IL 1 activities within 4 hr. Analysis of mRNA from these cells showed a concurrent induction of both IL 1 alpha and IL 1 beta mRNA within 1 hr. However, this stimulation of IL 1 expression was transient, since PEC cultured for 5 days no longer expressed IL 1 bioactivity or specific mRNA. Stimulation of these quiescent cells with bacterial lipopolysaccharide induced the re-expression of intracellular, membrane, and extracellular IL 1 activities as well as IL 1 alpha and IL 1 beta mRNA. We found no qualitative difference in the degree or rate of induction of IL 1 alpha compared with IL 1 beta mRNA. These results indicate that resting macrophages are IL 1 negative, and that the IL 1 inducing stimuli used in this study act transiently to increase the levels of IL 1 alpha and IL 1 beta mRNA.