Effect of prostaglandin E2 on the gamma-interferon induction of antigen-presenting ability in P388D1 cells and on IL-2 production by T-cell hybridomas

The effect of prostaglandin E2 on the gamma-interferon (IFN-gamma)-mediated induction of Ia expression and antigen-presenting activity in macrophage cell lines was studied. Using a lymphokine preparation obtained from the T-cell hybridoma FS7-20.6.18 (known to produce interferon) to induce the expression of Ia in P388D1 cells, the influence of PGE2 on this phenomenon was studied. Screening of the cell cultures by indirect immunofluorescence using an anti-I-Ad monoclonal antibody confirmed the inhibitory effect of PGE2 in the induction of I-Ad. However, the inhibition of the antigen-presenting ability of these cells, as measured by their capacity to stimulate interleukin 2 (IL-2) production by antigen-specific, I-region-restricted (Ag/I) T-cell hybridomas, was more difficult to demonstrate and was only evident when using low concentrations of Ia-inducing lymphokines or when using "low avidity" T-cell hybridomas. The latter were distinguished by the limited response (in the form of IL-2 production) that was observed when they were tested with P388D1 cells that had been induced with IFN-gamma. By contrast, PGE2 had profound inhibitory effects on the ability of T-cell hybridomas to secrete IL-2 in response to Ag/I or concanavalin A. These results suggest that although PGE2 inhibits the full induction of Ia on macrophages, it has little effect on the induction of Ag/I presentation by the same cells, probably because most T cells require relatively low levels of Ia on the surface of presenting cells. T-cell responses to Ag/I are inhibited, however, because of the effects of PGE2 on the T cells themselves.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Opposing effects of glucocorticoids on interferon-gamma-induced murine macrophage Fc receptor and Ia antigen expression

Two macrophage markers associated with differentiation are the Fc receptor (FcR) and the Ia antigen. Expression of these markers is increased with IFN-gamma treatment, although some evidence suggests that the induction pathway for Fc receptor and Ia antigen expression may be dissociable. In this study, the effect of glucocorticoids on basal and IFN-induced levels of Fc-mediated phagocytosis and Ia antigen expression was investigated. Macrophages incubated for 2 days with glucocorticoids alone showed no change in basal levels of Fc-mediated phagocytosis. However, incubation with glucocorticoids plus IFN-gamma resulted in increased Fc-mediated phagocytosis and binding to a much greater extent than IFN-gamma treatment alone. This enhancement was specific for IFN-gamma, because the IFN-beta-induced increase in Fc-mediated phagocytosis and binding was not affected by glucocorticoids. In contrast to the expression of Fc receptor capacity, both basal and IFN-gamma-induced levels of Ia antigen expression were inhibited by glucocorticoids. The glucocorticoid effect on these two markers was not observed with other steroid hormones, nor was it altered by inhibitors of the arachidonic acid pathway. The findings of this study provide additional evidence that induction of Fc receptor and Ia antigen by IFN-gamma occurs by different mechanisms.     

Induction of macrophage Ia antigen expression by rIFN-gamma and down-regulation by IFN-alpha/beta and dexamethasone are mediated by changes in steady-state levels of Ia mRNA

In previous studies, the induction of Ia antigens on murine peritoneal exudate macrophages by recombinant IFN-gamma (rIFN-gamma) and the antagonism of rIFN-gamma-induced Ia expression by the inhibitors IFN-alpha/beta and glucocorticoids have been examined. In this report, these findings have been extended to an analysis of total or cytoplasmic mRNA from macrophage cultures treated with rIFN-gamma in the absence or presence of these two inhibitors. Recombinant IFN-gamma induced a 5.7- to 6.5-fold increase in steady-state levels of Ia (A alpha-specific) mRNA. Coordinate increases in steady-state mRNA for A beta, and E alpha were observed in response to rIFN-gamma. Maximum induction occurred 24 hr post-treatment and required the continued presence of rIFN-gamma. Induction of A alpha-specific mRNA was sensitive to the protein synthesis inhibitor cycloheximide. Simultaneous treatment of macrophage cultures with rIFN-gamma and IFN-alpha/beta or the glucocorticoid dexamethasone (DEX) resulted in a significant decrease in steady-state, A alpha-specific mRNA levels compared with treatment with rIFN-gamma alone. This analysis suggests that both the induction of Ia expression by rIFN-gamma, and the antagonism of rIFN-gamma-induced Ia gene expression by IFN-alpha/beta and DEX, are regulated by cognate changes in Ia mRNA.     

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.     

Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.     

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)     

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.     

Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation

Ia antigens seem to control immune responses on at least two levels. First, they influence the antigen recognition repertoire of the T cells. Second, their variable expression on certain antigen-presenting cells is a powerful regulatory mechanism for the local immune reaction. This is particularly important in the central nervous system (CNS) in which no Ia antigens are normally expressed. Recent experiments in this context have shown that astrocytes are able to express Ia antigens during interaction with T cells, and that they function as antigen-presenting cells. The Ia-inducing activity is produced by activated T cells, and can be replaced by immune interferon (IFN-gamma). In this study we report on the functional and kinetic relationship between Ia antigen expression on astrocytes and the immune-specific activation of T cells by astrocytes. Normal resting astrocytes were found to be negative for Ia antigens by immunofluorescence and by biochemical criteria. Moreover, they are only able to stimulate T cells after they have been induced to express Ia antigens by a signal from the T cells, which is probably mediated by IFN-gamma. In conclusion, the immune-specific interaction between astrocytes and T lymphocytes is a sensitively controlled system that might be pivotal to the development of immune responses in the brain. Malfunction of the system could be an important factor in the pathogenesis of aberrant immune reactions in the CNS, e.g., in multiple sclerosis.     

Inhibition of macrophage-induced antigen-specific T-cell proliferation by interferon-gamma

The effects of IFN-gamma on macrophage (M phi)-mediated antigen-specific T-cell proliferation was investigated. A well-defined assay system using purified resident populations of antigen-pulsed peritoneal M phi and immune T cells was used to measure M phi-induced antigen-specific T-cell proliferation. Antibody affinity purified or recombinant IFN-gamma inhibited M phi-induced T-cell proliferation when KLH-pulsed M phi from mice given IFN-gamma prior to KLH were cultured with KLH immune T cells from normal mice. Monoclonal rat anti-IFN-gamma antibody neutralized the inhibitory effect of IFN-gamma. This inhibition of T-cell proliferation occurred despite the fact that these M phi appeared to be activated by IFN-gamma treatment as measured by increased tumoricidal activity. The mechanism for the inhibition was unrelated to class II (Ia) expression, IL-1 secretion, and prostaglandin secretion. These results demonstrate the complex and sensitive role IFN-gamma has in regulating the immune response.     

Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.     

Effect of interferon-gamma and human alpha 2-macroglobulin on peritoneal macrophage morphology and Ia antigen expression.

While the primary role of the plasma protein alpha 2-macroglobulin (alpha 2M) appears to be related to its proteinase inhibitory activity, alpha 2M has been reported to regulate the immune response in vitro. Previous studies have demonstrated that, although native alpha 2M has no effect on macrophage function, proteinase- or CH3NH2-treated alpha 2M antagonize the IFN-gamma-induced expression of class II major histocompatibility complex (Ia) antigens on mouse peritoneal macrophages. In this investigation, we examined the effects of alpha 2M-CH3NH2 on the IFN-gamma-induced expression of macrophage Ia antigens by indirect immunofluorescence microscopy, radioimmunoassay, and immunoprecipitation of biosynthetically-labelled Ia. While alpha 2M-CH3NH2 suppressed the IFN-gamma induced increase in the percentage of Ia-positive macrophages detected by immunofluorescence microscopy, alpha 2M-CH3NH2 had no effect on the average of number of Ia molecules expressed per cell as detected by radioimmunoassay. In addition, alpha 2M-CH3NH2 had no effect on the ability of IFN-gamma to induce biosynthesis of Ia. Microscopic examination of IFN-gamma-treated macrophages revealed that treatment with alpha 2M-CH3NH2 prevented IFN-gamma-induced changes in macrophage morphology. IFN-gamma-treatment of elongated inflammatory macrophages was associated with the generation of round cells which possessed few cytoplasmic projections. By contrast, addition of alpha 2M-CH3NH2 to the incubation prevented the IFN-gamma-induced morphological changes, and the cells remained elongated with irregular cytoplasmic borders. We postulate that alpha 2M-CH3NH2 decreases the IFN-gamma-induced expression of Ia by preventing morphological changes in macrophages, resulting in the distribution of existing Ia over a larger surface area. As a consequence of this, the perceived fluorescence intensity of the bound antibody is lowered and the cells appear to be Ia-negative.     

Antitumor effects of human recombinant macrophage colony-stimulating factor against rat brain tumors

The tumoricidal effects of M-CSF were examined using two subcutaneously-transplanted rat brain tumor cell lines, 9L and T9 gliomas. In rats treated with high-dose M-CSF (16 million U/kg administered for 4 days a week for 3 weeks), 9L glioma growth was inhibited by 81.9% following subcutaneous (s.c.) injection and by 70.5% after intraperitoneal (i.p.) injection and T9 glioma growth was inhibited by 69.2% after i.p. injection. After short-term treatment with high-dose M-CSF (32 million U/kg administered s.c. for 6 consecutive days, 9L glioma growth was inhibited by 82.1%. All these inhibitory effects differed significantly compared with the respective untreated control groups. However, treatment with low-dose M-CSF (1.6 million U/kg administered s.c. for 4 days a week for 3 weeks) showed no significant effects against 9L and T9 glioma growth compared with the untreated controls. No significant effects of M-CSF against cell proliferation, measured as PCNA expression, were observed in any group. Significant hematopoietic effects on the leukocyte counts were observed only in the groups treated with high dose M-CSF. These results suggest that M-CSF at a high dose which produces hematopoietic effects on peripheral leukocytes inhibits the growth of gliomas. This inhibitory effect may have been due to a tumoricidal mechanism of M-CSF that depended on the production or release of some hematopoietic soluble factors, but was independent of PCNA expression by the tumors.Abbreviations BBB blood-brain barrier - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - hM-CSF human macrophage colony-stimulating factor - IFN interferon - IL-1 interleukin-1 - IL-6 interleukin-6 - M-CSF macrophage colony-stimulating factor - PCNA proliferating cell nuclear antigen - rhM-CSF recombinant human macrophage colony-stimulating factor - TNF tumor necrosis factor     

Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.     

The differential inhibition of hemopoietic growth factor activity by cytotoxins and interferon-gamma

The effects of recombinant human tumor necrosis factor (TNF), lymphotoxin (LT), and interferon-gamma (IFN-gamma) on the growth of human hemopoietic progenitor cells in clonal culture have been examined. Colony growth was induced by using granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). A suppressive effect of TNF, LT, and IFN-gamma on the development of granulocyte, macrophage, and mixed granulocyte/macrophage colonies was shown. Suppression of colonies formed after stimulation with G-CSF was greater than that observed after stimulation with GM-CSF. In the presence of a monoclonal antibody to TNF, or polyclonal antibodies to either LT or IFN-gamma, the inhibitory effect of the molecule to which the antibody was directed was abrogated. These findings suggest that progenitor cells responsive to G-CSF or GM-CSF have different sensitivities to the effects of TNF, LT, and IFN-gamma. Defining the interactions of growth factors and inhibitors should increase understanding of mechanisms underlying diseases associated with suppression of normal hemopoiesis, and in predicting the effects in vivo of these bioregulatory molecules in clinical medicine.     

A T cell-independent mechanism of macrophage activation by interferon-gamma

A primary interest in immunity to intracellular pathogenic microorganisms and tumors is to understand the mechanisms by which macrophages are activated for various functions. Two parameters of macrophage activation are the expression of the class II histocompatibility proteins or Ia molecules (1), and cytotoxic activity. The ability of T cells to induce these responses has been extensively documented and occurs via their secretion of interferon-gamma (IFN-gamma) after interaction with antigen (2-6). However, in a recent study using mice with the severe combined immunodeficiency (scid) mutation (7) which have no detectable T or B cell functions (7-9), we were surprised to find the induction of Ia expression on macrophages and the partial inhibition of bacterial growth after infection with Listeria monocytogenes (10). We have now utilized neutralizing monoclonal antibodies specific for murine IFN-gamma to investigate the mechanism of macrophage activation in scid mice. We show here that IFN-gamma can be produced by scid mice in the absence of lymphocyte-mediated immunity, and this IFN-gamma is important for macrophage activation during infection with Listeria. These results indicate the presence of an important T lymphocyte-independent mechanism of macrophage activation and IFN-gamma production in response to infection.     

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.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects

Studies were designed to investigate whether the cellular immunodeficiency state observed in human glioblastoma patients could be due to inhibitory factors released by the tumor cells. Cultured human glioblastoma cells were found to secrete an interleukin 1-like factor (m.w. 22,000) and a factor (m.w. 97,000) that inhibits interleukin 2 (IL 2)-dependent T cell mechanisms. This is demonstrated by its inhibitory effect on the IL 2-induced proliferation of T cell clones and on the induction of alloreactive cytotoxic T cells in mixed lymphocyte cultures. Additionally the glioblastoma cell-derived 97,000-m.w. factor inhibited growth of neuroblasts but not of fibroblasts and thus shares the characteristics of the neuroblast growth inhibition factor (NGIF) previously detected in the supernatant of fetal rat glia cell cultures. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.     

Regulation of IFN-gamma induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.