Differential antigen presentation by cloned populations of mouse splenic macrophages.

Soft agar colonies of mouse splenic macrophages differ in their ability to process and present complex Ag to T cell hybridomas. To determine if the basis for this differential activity was the synthesis of molecules that might interfere with the activity of either the hybridoma or the indicator cells used for the bioassay of IL-2, culture supernatants were compared from Ag-presenting and nonpresenting cultures for their content of suppressor activity, using mitogen-treated mouse SC. No correlation was found between a colony's Ag-presenting activity and its secretion of suppressor factors, nor did colonies unable to present Ag release factors that interfered with the detection of IL-2. In a second approach, paired subcultures from individual colonies were tested for their ability to present, to the same hybridoma, both native Ag and the "preprocessed" peptide of the Ag. The presentation of native Ag was restricted to the progeny of a minority of the cloned macrophage progenitors, but all of the progeny cultures presented the peptide. Together, these results suggest that the basis for differential Ag presentation may be in the manner in which the cloned macrophages degrade and process ingested Ag.

Monocyte suppressor factor is plasminogen activator inhibitor inhibition of membrane bound but not soluble IL-1.

Human monocytes cultured in monolayer for 6 days were found to secrete a factor that suppressed the T cell proliferative response to soluble Ag and to alloantigens. The elaboration of this monocyte suppressor factor (MSF) was not inhibited by indomethacin. It has an apparent Mr of 50 to 60 kDa. It does not inhibit soluble IL-1 in the murine thymocyte costimulator assay but does inhibit the activity of membrane bound IL-1, which we observed to be almost exclusively IL-1 alpha. MSF contains elevated amounts of plasminogen activator inhibitor (PAI) when measured either as bioactivity or in an ELISA. Its immunosuppressive properties are inhibited by anti-PAI antibody. Furthermore, the eluate but not the effluent of an anti-PAI immunoabsorbent column contains all of the immunosuppressive activity. Based on these data we suggest that MSF is, in fact, PAI and postulate that the mechanism of action is inhibition of the plasmin cascade, thereby preventing the release of membrane bound IL-1. This suggests that monocytes possess an autoregulatory circuit that may have implications for the kinetics of the inflammatory response.

Regulation of lymphokine response during reinfection by influenza virus. Production of a factor that inhibits lymphokine activity.

Mononuclear cells, obtained from the spleens and lungs of influenza virus-seropositive C57BL/6 mice at 2 to 4 days after re-infection with homologous virus (strain A/Bangkok/1/79), produced a low m.w. factor in vitro that prevents the biologic expression, but not production, of the lymphokine, leukocyte migration inhibition factor (LIF). The low m.w. factor inhibited LIF activity without destroying the LIF molecule inasmuch as simple dialysis restored lymphokine activity to culture supernatants. Production of the low m.w. factor was observed from 2 to 4 days after re-infection, at which time the delayed-type hypersensitivity response to viral Ag was suppressed. In contrast, LIF was produced by splenocytes and lung mononuclear cells obtained at all times tested after re-infection (from 2 to 30 days). Production of the low m.w. factor required re-infection of influenza A virus-seropositive mice with type A virus; re-infection with influenza B virus failed to induce production. Ag specificity was also required in vitro for splenocytes to produce the factor; cells from type A virus-re-infected mice required type A Ag stimulation. Cell depletion studies with mAb plus C revealed that macrophages and T cells along with Ag stimulation were required for factor production by spleen cells. However, mononuclear cells obtained within 4 days from the lungs of re-infected mice did not require in vitro Ag stimulation for production of the low m.w. factor, and factor production was dependent upon the presence of CD4+ (L3T4) cells in the culture. Fractionation of culture supernatants over a Sephadex G-50 column indicated that the factor had a molecular mass of 2 to 3 kDa, and by FPLC chromatofocusing over a Mono P column, the factor eluted at a pH of approximately 8.2. Thus, re-exposure of influenza virus-seropositive mice to homologous virus resulted in the production of a low m.w. factor that prevented the biologic expression of LIF, but not its production. Lymphokines are an important component of the delayed-type hypersensitivity response; the presence of mononuclear cells secreting a low m.w. factor and LIF concomitantly at the site of virus replication (lungs) and the capacity of the factor to block the biologic expression of LIF in vitro suggest that the factor may have a role in the regulation of a delayed-type hypersensitivity response in vivo during re-infection.

Dual immunoregulatory effects of monoclonal IgG-antibodies: suppression and enhancement of the antibody response.

Nine monoclonal IgG-anti-TNP antibodies were investigated for their ability to modulate anticarrier responses in mice immunized with sheep red blood cells-2,4,6-trinitrophenyl (SRBC-TNP) or keyhole limpet haemocyanin-TNP (KLH-TNP). The antibodies enhanced the anticarrier response when KLH-TNP was used as antigen but suppressed it when SRBC-TNP was used. The enhancing and suppressive effects were not exerted by entirely the same sets of antibodies. The suppression was correlated to efficient antigen binding, but not complement activation, haemagglutination, or isotype of the monoclonal antibodies. In contrast, enhancement was correlated to isotype and complement activation but not to antigen binding capacity. Both the enhancing and the suppressive effects seem to require Fc-mediated functions of the IgG molecules since they modulate the anti-carrier response although they recognize hapten determinants. Thus, one and the same monoclonal hapten-specific IgG-antibody can enhance the anti-KLH response up to 38-fold whereas it suppresses the anti-SRBC response by more that 10-fold.

Leukotriene C4 produced by a human T-T hybridoma suppresses Ig production by human lymphocytes.

Multiple signals are involved in the regulation of Ig production by human B lymphocytes. Leukotrienes, especially LTB4, have been shown to inhibit Ig production by increasing the number and function of suppressor lymphocytes. Production of leukotrienes has been demonstrated by mast cells, basophils, eosinophils, polymorphonuclear leukocytes, monocytes, and macrophages. In this paper we demonstrate that a human T-T hybridoma grown at 5 x 10(5) cells/ml constitutively produces 5 ng/ml of LTC4. Furthermore, we demonstrate that either the supernatant from this hybridoma containing 0.5 to 10 ng/ml LTC4 or purified LTC4 in the range of 0.5 to 5 ng/ml can suppress 50 to 70% of Ig production by unfractionated human mononuclear cells, by normal human cells stimulated with Staphylococcus aureus Cowan I and B cell differentiation factors, and by the EBV-transformed B cell line SKW.6 in the presence of B cell differentiation factors. Thus, LTC4 can have direct effects on B cells and may have a role in normal B cell regulation.

Monoclonal antibodies against the major zymosan-induced chemotactic factor in rabbit serum.

The major zymosan-induced chemotactic factor in rabbit serum was purified by a two-step ion exchange chromatography procedure. The purified chemoattractant was used as antigen for production of murine monoclonal antibodies against the major chemotactic factor. The primary screening of the hybridoma cultures was an indirect ELISA using purified chemotactic factor as antigen. The final selection among ELISA-positive clones was performed according to the results obtained in a chemotactic inhibition assay. Six monoclonal antibodies were raised. These antibodies completely abrogated or substantially reduced the chemotactic activity in crude zymosan-activated serum. The chemotactic factor(s) could be absorbed onto an immunosorbent column containing monoclonal antibody and subsequently be specifically eluted with acid. By Western blot analysis the molecular weight of the major chemotactic factor was estimated to be approximately 15,000, and isoelectric focusing indicated a pI of about 9.4.

Interaction of a lymphokine with normal human macrophages results in release of a suppressor factor for mitogen-induced immunoglobulin synthesis.

Normal human macrophage/monocyte cultures exposed to a suppressor factor produced by concanavalin A-activated T cells (T-SF), respond by releasing after 72 h a macrophage-derived suppressor factor (M phi-SF). The M phi-SF inhibits pokeweed mitogen-induced Ig synthesis but not T- or B-cell proliferation. Cycloheximide treatment of the macrophages does not interfere with generation of the M phi-SF, suggesting that de novo synthesis is not required. The factor is not preformed, for virgin macrophages do not contain M phi-SF, but it appears in macrophage cell lysates after exposure to T-SF. The production of the M phi-SF is inhibited by the presence of 2-mercaptoethanol. Both T-SF and M phi-SF are L-rhamnose inhibitable, and the M phi-SF appears to be released as a high molecular weight complex which is dissociable into a low molecular weight form of a size similar to the T-SF, i.e. approximately 20,000. The T-SF induced M phi-SF has some similarities with soluble immune response suppressor (SIRS) but differs from this factor in its lack of effect upon lymphocyte proliferation failure to induce conversion of T-SF to M phi-SF by treatment with H2O2.

Spontaneous production of a suppressor factor by the human macrophage-like cell line U937. I. Suppression of interleukin 1, interleukin 2, and mitogen-induced blastogenesis in mouse thymocytes.

The human macrophage-like cell line U937 spontaneously produced a nondialyzable factor that inhibited interleukin 1 (IL 1), interleukin 2 (IL 2), and phytohemagglutinin (PHA)-induced blastogenesis in mouse thymocytes. The suppression by U937 supernatant factor occurred independently of the concentration of IL 1 or PHA, indicating that it was noncompetitive. The U937 suppressor factor was not cytotoxic for thymocytes, nor did it affect the spontaneous proliferation of T lymphoblastoid cell lines and U937. Physicochemical characterization showed that the U937 suppressor factor was nondialyzable, partially inactivated by heat treatment (56 degrees C), ammonium sulfate (67% saturation) precipitable, sensitive to pH 2.5, and resistant to freeze-thawing. Molecular weight of the factor inhibiting co-mitogenic IL 1 activity was approximately 85,000, as estimated by gel filtration. The U937 cell line may provide a model for the study of mechanisms and mediators of immunosuppression by mononuclear phagocytes.

FcR epsilon+ lymphocytes and regulation of the IgE antibody system. III. Suppressive factor of allergy (SFA) is produced during the in vitro FcR epsilon expression cascade and displays corollary physiologic activity in vivo.

Exposure of lymphoid cells to IgE induces the expression of Fc receptors for IgE (FcR epsilon) and the production of soluble mediators, termed IgE-induced regulants (EIR). Conventional suppressive factor of allergy (SFA) and enhancing factor of allergy (EFA), derived from mouse ascites fluids, both inhibit IgE-induced FcR epsilon expression in vitro in cultures of unfractionated and T cell-enriched, but not B cell-enriched, lymphoid cells. This indicates that the inhibitory activities of both entities are T cell dependent, and distinguishes them from the inhibitory EIRI, which inhibits FcR epsilon induction in the absence of T cells. Moreover, SFA and EFA can be distinguished from one another by differences in the T cell subsets required for the inhibitory activity of each respective mediator on in vitro IgE-induced FcR epsilon expression. Thus, SFA requires the presence of Lyt-1+ T cells, whereas EFA requires the presence of Lyt-2+ T cells. Supernatant fluids from IgE-stimulated unfractionated lymphoid cell cultures suppress in vivo IgE synthesis in mice, indicating that SFA is produced along with the other species of EIR. To define conditions required for SFA production in vitro, EIR-rich supernatant fluids were tested for the presence of SFA by using Lyt-2+ cell-blocked indicator cells in the in vitro FcR epsilon induction assay system (this eliminates the inhibitory activity of EFA). SFA production in vitro by IgE-stimulated lymphoid cells was shown to result from cooperative interactions between B cells and Lyt-1+ T cells. In addition, as observed with the induction of FcR epsilon in general, induction of SFA requires the initial interaction of B cells with IgE, and the release of the B cell-selective EIRB. Once produced, EIRB can directly stimulate Lyt-1+ cells, but not Lyt-2+ cells, to produce SFA. The physiologic significance of the in vitro induction of SFA by the action of EIRB on Lyt-1+ cells was confirmed by the demonstration that EIRB, devoid of detectable SFA, selectively suppressed in vivo IgE synthesis after administration to intact mice. This indicates that EIRB can stimulate resident T cells of irradiated SJL mice to produce SFA. Finally, as shown previously with conventional ascites-derived SFA, the SFA produced in vitro after stimulation of lymphoid cells with IgE is devoid of IgE-binding properties, because its inhibitory effects on in vivo IgE antibody synthesis are not removed by passage over IgE affinity columns.

FcR epsilon+ lymphocytes and regulation of the IgE antibody system. IV. Delineation of target cells and mechanisms of action of SFA and EFA in inhibiting in vitro induction of FcR epsilon expression.

SFA and EFA are derived from distinct mouse T cell hybridomas secreting one or the other (but not both) factor, and although both are capable of inhibiting FcR epsilon expression by unfractionated spleen cells induced by monomeric IgE, neither was inhibitory for EIRT-induced FcR epsilon expression by T cells in the same cell population. This suggests that the final target cell for the inhibitory effects of SFA and EFA is the FcR epsilon+ B lymphocyte. T cells are required for both SFA- and EFA-mediated FcR epsilon inhibition, and more precisely, as shown in this study, SFA stimulates Lyt-1+ cells in the presence or absence of IgE to produce a suppressive effector molecule (SEM), and EFA together with IgE stimulates Lyt-2+ cells to produce an enhancing effector molecule (EEM), both of which can directly inhibit FcR epsilon expression by B cells. SFA and SEM can inhibit both IgE- and EIRB-induced FcR epsilon expression by B cells, indicating that SFA may act by blocking the EIRB-mediated expansion of the FcR epsilon+ B cell population. EFA and EEM, in contrast, can inhibit IgE-induced but not EIRB-induced FcR epsilon expression, indicating that EFA may act at some point before the release of EIR, perhaps involving those FcR epsilon+ B cells that respond to IgE and produce EIRB. Finally, although neither SFA and EFA display IgE binding properties, both SEM and EEM, in contrast, are IgE binding factors (IgE-BF) and may be homologous to the suppressive IgE binding factor and potentiating IgE binding factor described by other investigators. The possible interrelationships between these various cells and factors are discussed.

Formation of rat IgE-binding factors by rat-mouse T cell hybridomas.

Mesenteric lymph node (MLN) cells of rats infected with Nippostrongylus brasiliensis (Nb) were obtained 8 days after infection and were incubated overnight with rat IgE for the formation of IgE-binding factors. These cells were then fused with BW 5147 mouse T lymphoma cells. Two hybrid clones (A4 and B6) obtained by the fusion formed IgE-binding factors upon incubation with IgE. It was found that 1-hr incubation with 3 micrograms/ml of rat IgE was sufficient to induce hybridoma cells to form IgE-binding factors. Both hybrid clones express rat T cell markers; the A4 clone bears Fc epsilon R, whereas the B6 clone bears Fc gamma R on their surface. The IgE-binding factors formed by both clones bound to rat IgE-coated Sepharose and could be eluted from the beads at pH 3.0. The factors also have affinity for mouse IgE but not human IgE nor rat IgG. IgE-binding factors formed by the A4 clone had a m.w. between 26,000 and 30,000; the B6 clone formed IgE-binding factors of 26,000 and 13,000 daltons. The factors of both high and low m.w. failed to bind to lentil lectin and Con A, but had affinity for peanut agglutinin. Purified IgE-binding factors of 13,000 daltons selectively suppressed the IgE-forming cell response of DNP-ovalbumin-primed MLN cells to homologous antigen, whereas the factor of 26,000 to 30,000 daltons neither suppressed nor enhanced the IgE response.

Preparation of soluble immune response suppressor and macrophage-derived suppressor factor.

Concanavalin A-activated murine suppressor T cells act via the mediator, soluble immune response suppressor (SIRS) which non-specifically suppresses IgM and IgG antibody responses to a variety of antigens, cytotoxic T lymphocyte responses and proliferative responses to alloantigens and mitogens in vitro. SIRS is a protein with an apparent MW of 45,000-55,000; the target of SIRS is the macrophage (M phi). M phi following treatment with SIRS release a second factor, M phi-derived suppressor factor (M phi-SF), which is directly responsible for the observed suppression of responses. Moreover. M phi-SF appears to be modified SIRS by all criteria used to date; M phi-SF can be obtained by reacting SIRS with low concentrations of H2O2 in the absence of M phi. Thus, M phi appear to serve only as a source of H2O2 and the mechanism of M phi-SF action action appears to have an oxidative basis. M phi-SF activity is lost following treatment with sulfhydryl reagents such as 2-mercaptoethanol, dithiothreitol or cysteine, reducing agents such as NaBH4 and a variety of peroxidase substrates such as pyrogallol, phenylenediamine, and ascorbic acid. Additionally, M phi-SF-mediated inhibition can be reversed by high concentrations of 2 mercaptoethanol or dithiothreitol under appropriate conditions. Since M phi-SF appears to be modified SIRS, oxidized by peroxide, and not a distinct second mediator produced by M phi in response to SIRS, we propose eliminating the term M phi-SF and using SIRSox to denote the active form of SIRS produced either by the SIRS-H2O2 reaction or SIRS-treated M phi.

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.

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor.

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.

[Analogies between "fraction P" with immunosuppressive properties in pregnant females and a protein fraction induced by estradiol treatment in the amphibian Salamandra salamandra L]. / Analogies entre la "fraction P" à propriétés immunosuppressives chez les femelles gestantes, et une fraction protéique induite par un traitement à l'oestradiol, chez l'Amphibien Urodèle, Salamandra salamandra L.

A "fraction P" analogue possessing immunosuppressive properties during pregnancy in the Salamandra, can be induced by treating males and females with oestradiol. Both fractions possess the same physiochemical characteristics, and equally react against a Rabbit immune serum anti "fraction P" of pregnant Salamandra. But the proteic fraction induced by hormonal treatment does immunosuppressive properties.

[Regulatory role of bone marrow in immunogenesis. III. Humoral factors stimulating and suppressing antibody formation produced by bone marrow cells in in vitro cultures]. / Reguliatornaia rol' kostnogo mozga v immunogeneze. Soobshchenie III. Gumoral'nye faktory supressii i stimuliatsii antitelogeneza, produtsiruemye kletkami kostnogo mozga v kul'ture in vitro.

As revealed by the method of cultivation of bone marrow and spleen cells, separated by nucleopore membrane, in two-chamber bottles, the bone marrow cells were capable of producing humoral factor stimulating antibody genesis by the spleen cells. A direct contact of the bone marrow cells with the actively proliferating antigen-stimulated cells of the spleen led to production of a spleen humoral factor suppressing the antibody genesis by the spleen cells. The suppressive action of the bone marrow cells on the antibody genesis in the culture of the spleen cells was mediated through the suppression of the spleen cells proliferation; proliferation of the bone marrow cells is enhanced.