In vivo role of macrophages in transmission of dengue virus-induced suppressor signal to T lymphocytes

Dengue type 2 virus (DV)-induced suppressor T cells (Ts1) produce a soluble suppressor factor (SF) which stimulates a subpopulation of T lymphocytes (Ts2) to produce prostaglandin which suppresses DV-specific IgM antibody plaque forming cells (PFC) in vivo and in vitro. The present study was undertaken to investigate the intermediary role of macrophages in transmission of signal from Ts1 to Ts2. The SF is adsorbed on live or heat-killed mouse peritoneal macrophages and transmits DV-specific PFC in spleen after i.p. or i.v. inoculation in DV-primed mice. The suppression is antigen-specific. SF or SF-adsorbed heat-killed macrophages failed to transmit suppression in silica-treated macrophage-depleted mice when injected i.p., while SF-adsorbed live macrophages could transmit suppression. In macrophage-depleted mice suppression could be transmitted by SF-adsorbed heat-killed macrophages on i.v. inoculation. It was therefore concluded that live macrophage-killed cells are also essential for transmission of suppressor signal by SF from Ts1 to Ts2 in vivo.     

In vivo role of macrophages in transmission of dengue virus-induced suppressor signal to T lymphocytes.

Dengue type 2 virus (DV)-induced suppressor T cells (Ts1) produce a soluble suppressor factor (SF) which stimulates a subpopulation of T lymphocytes (Ts2) to produce prostaglandin which suppresses DV-specific IgM antibody plaque forming cells (PFC) in vivo and in vitro. The present study was undertaken to investigate the intermediary role of macrophages in transmission of signal from Ts1 to Ts2. The SF is adsorbed on live or heat-killed mouse peritoneal macrophages and transmits DV-specific PFC in spleen after i.p. or i.v. inoculation in DV-primed mice. The suppression is antigen-specific. SF or SF-adsorbed heat-killed macrophages failed to transmit suppression in silica-treated macrophage-depleted mice when injected i.p., while SF-adsorbed live macrophages could transmit suppression. In macrophage-depleted mice suppression could be transmitted by SF-adsorbed heat-killed macrophages on i.v. inoculation. It was therefore concluded that live macrophage-killed cells are also essential for transmission of suppressor signal by SF from Ts1 to Ts2 in vivo.     

Antigenic competition between dengue and Coxsackie viruses for presentation to B cells by macrophages

Macrophages (M phi) pulsed with dengue type 2 (DV) and Coxsackie B4 (CoxB) viruses present antigen to B lymphocytes leading to their clonal expansion as detected by counting antigen-specific IgM antibody plaque-forming cells (PFC). The present study was undertaken to investigate the site for competition in M phi between the two heterologous antigens, DV and CoxB, for their presentation to B cells. It was observed that DV-pulsed M phi presented antigen to B cells in mice depleted of T cells by treatment with anti-Thy 1.2 monoclonal antibodies. The B cells could not be stimulated in absence of M phi in mice treated with silica. The PFC counts for both the antigens were inhibited when M phi were pulsed simultaneously with DV and CoxB. PFC counts were increased by 53-120% by predigesting the antigens by trypsin. Inhibition of DV-specific response by CoxB was abrogated by predigesting CoxB. A marked reduction in DV-specific PFC response was observed when CoxB was superimposed on M phi pulsed with DV 24 h earlier. CoxB-specific PFC counts were not affected by superimposing DV on M phi pulsed with CoxB 24 h earlier. PFC response to the antigen given to M phi before glutaraldehyde fixation was not affected while that for the antigen given to glutaraldehyde-fixed M phi was markedly depressed. It is concluded that the competition between DV and CoxB for antigen presentation to B cells occurs in M phi at the level of antigen processing.     

Characterization of the dengue virus-induced helper cytokine.

Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mouse spleen to secrete a soluble helper cytokine (HF) which enhances the DV-specific IgM antibody plaque forming cells (PFC). The present study undertaken to purify and characterize HF shows that it can be purified by low pressure liquid chromatography (LPLC) using Sephacryl S-200 column. HF consisted of two subunits, having a M(r) of 65-68 kDa on SDS-PAGE, and both had similar activity. The isoelectric point of HF was 6.5. HF-specific antisera (HFAS) raised in mice neutralized the activity of HF in mice, reacted with it in a Western blot assay, and bound HF in an immunosorbent column. HF bound to DV-antigen in an immunosorbent column and enhanced only the DV-specific PFC. HF had no effect on PFC against heterologous antigens such as Japanese encephalitis virus, Coxsackie B4 virus or sheep red blood cells. HF generated in mice of H-2k haplotype, enhanced DV-specific PFC in the same strain of mice but had no effect on that in the H-2d or H-2q haplotype strains of mice. Thus, DV-induced HF with a M(r) of 65-68 kDa, antigen-specificity and genetic-restriction differs from most of the similarly acting cytokines but appears similar to the cell-free form of T cell receptor alpha beta dimer.     

Inhibition of the presentation of dengue virus antigen by macrophages to B cells by serine-protease inhibitors.

It has been shown that macrophages (M phi) process dengue type 2 virus (DV) antigen and present it to B cells leading to their clonal expansion as shown by DV-specific IgM antibody plaque-forming cell (PFC) count in spleen. The present study was undertaken to find out the nature of enzymes responsible for the processing of DV antigen in M phi. DV-pulsed M phi were treated with seven different protease inhibitors and then assayed for antigen presentation to B cells. It was observed that maximum inhibition occurred by treatment of M phi with PMSF, a serine-protease inhibitor. The effect of PMSF was dose dependent and was abolished by using predigested antigen. PMSF inhibited presentation of DV and sheep RBC antigens but had no effect on presentation of bovine serum albumin which does not require processing. The results thus identify the serine group of proteases as the main enzymes involved in processing the DV antigen in M phi.     

Transmission of dengue virus-induced suppressor signal from macrophage to lymphocyte occurs by cell contact

We have observed that live macrophages play an obligatory role in transmission of dengue type 2 virus (DV)-induced suppressor signal to a subpopulation of T lymphocytes. The present study was undertaken to resolve whether transmission of the suppressor signal is mediated by a soluble factor or by direct cell-to-cell contact. It was observed that the suppressor factor (SF) remains adsorbed on the surface of macrophages and can be retrieved from them completely by DV-stimulated spleen cells kept in contact with them. Suppression of DV-specific IgM plaque-forming cells (PFC) in spleen-cell cultures does not occur when SF-adsorbed macrophages are separated from them by cell-impermeable membranes. Culture fluid of SF-adsorbed macrophages have no suppressor activity. The suppression of PFC occurs only when SF-adsorbed macrophages remain in contact with DV-stimulated spleen-cell cultures. Thus transmission of suppressor signal from SF-adsorbed macrophages to lymphocytes occurs only by physical contact of the plasma membranes of the interacting cells.     

Transmission of dengue virus-induced suppressor signal from macrophage to lymphocyte occurs by cell contact.

We have observed that live macrophages play an obligatory role in transmission of dengue type 2 virus (DV)-induced suppressor signal to a subpopulation of T lymphocytes. The present study was undertaken to resolve whether transmission of the suppressor signal is mediated by a soluble factor or by direct cell-to-cell contact. It was observed that the suppressor factor (SF) remains adsorbed on the surface of macrophages and can be retrieved from them completely by DV-stimulated spleen cells kept in contact with them. Suppression of DV-specific IgM plaque-forming cells (PFC) in spleen-cell cultures does not occur when SF-adsorbed macrophages are separated from them by cell-impermeable membranes. Culture fluid of SF-adsorbed macrophages have no suppressor activity. The suppression of PFC occurs only when SF-adsorbed macrophages remain in contact with DV-stimulated spleen-cell cultures. Thus transmission of suppressor signal from SF-adsorbed macrophages to lymphocytes occurs only by physical contact of the plasma membranes of the interacting cells.     

Macrophage functions during dengue virus infection: antigenic stimulation of B cells.

This study was undertaken to investigate the function of dengue type 2 virus (DV)-infected mouse peritoneal macrophages (M phi) regarding the antigenic stimulation of B lymphocytes of the spleen. It was observed that a variable proportion of M luminal diameter show DV-specific immunofluorescent antigen, which depended upon the route of administration of the virus, being higher in i.p.-inoculated mice and in vitro-infected M luminal diameter monolayers. The DV-infected M luminal diameter presented the DV antigen to B cells in vitro and in vivo, leading to their clonal expansion as shown by counting the virus-specific IgM antibody plaque-forming cells (PFC). The PFC response depended upon the number of DV-infected M luminal diameter. The antigen was presented equally well both by I-A-negative and I-A-positive M luminal diameter. Superimposition of a heterologous antigen (Coxsackie B4 virus) in a Mackaness type of experiment depressed the capacity of M luminal diameter to present both the homologous as well as heterologous antigen.     

Obligatory role of macrophages in dengue virus antigen presentation to B lymphocytes.

The study was undertaken to investigate the role of dengue type 2 virus (DV)-infected mouse peritoneal macrophages (M phi) in presentation of the DV antigen to B lymphocytes as shown by counting virus-specific IgM antibody plaque-forming cells (PFC). It was observed that heat-killed or glutaraldehyde-fixed M phi did not present the antigen. Pretreatment of M phi with the lysosomotropic compounds ammonium chloride and chloroquine inhibited the antigen presentation. Depletion of M phi from the spleen cell cultures abrogated the immune response to DV. The tryptic-digested DV antigen could stimulate immune responses in B-lymphocyte enriched (depleted of M phi and T cells) spleen cell cultures, and the digested antigen could be presented by glutaraldehyde-fixed M phi. Pretreatment of M phi with a trypsin inhibitor abrogated antigen presentation. The findings thus show that even for presentation to B cells the DV antigen must be processed by M phi by a trypsin-like protease.     

Characterization and expression of H-21 region gene products on bone marrow-derived macrophages

Antigen-presenting macrophages (M phi) were derived from day 7 cultures of bone marrow stem cells using L cell conditioned medium. The adherent bone marrow-derived macrophages (BMM phi) were 100% esterase-positive, 95% positive for C3 receptors, 93% positive for Fc receptors, and 95% actively phagocytic. Indirect immunofluorescence using anti-Ia monoclonal antibodies resulted in 60% Ia-positive BMM phi on day 7 of stem cell culture. BMM phi could stimulate mixed lymphocyte reaction (MLR) proliferation across an I-A subregion difference, but not across I-J subregion differences. This contrasted with splenic M phi which stimulated MLR proliferation across both an I-A and I-J subregion difference. The apparent lack of I-J subregion determinants on BMM phi correlated with their ability to function as antigen-presenting cells. In these experiments, BMM phi effectively reconstituted the trinitrophenyl-specific IgM plaque-forming cell (PFC) response of B cells but not the primary burro red blood cell (BRBC)-specific IgM-PFC response of M phi-depleted spleen cells. When BMM phi were added to BRBC-primed T and B cells, they reconstituted the secondary IgG PFC response to levels obtained using splenic M phi. These experiments relate the differential expression of H-21 region determinants on antigen-presenting cells with their functional capacity.     

Macrophage transmission of suppressor signal for suppression of delayed hypersensitivity and humoral response in JEV-infected mice.

Japanese encephalitis virus (JEV) infection induces suppressor T-cells (Ts1) which suppress both the humoral (Ts-PFC) and cell mediated (Ts-DTH) immune response by producing soluble suppressor factors. This study shows that in the JEV model, both TS-PFC and Ts-DTH mediate suppression by recruiting a second subpopulation of suppressor T-cells, the Ts2-PFC and Ts2-DTH. The signal between Ts1 and Ts2 is transmitted by macrophages (M phi). The suppressor factors are adsorbed by peritoneal or splenic M phi. Both heat-killed and live M phi are capable of adsorbing suppressor factors but only live M phi are capable of presenting the signal to T-cells. Thus these are at least two generations of suppressor T-cells in the JEV-specific suppressor pathway and the presence of M phi is obligatory for transmission of the signal.     

Macrophage transmission of suppressor signal for suppression of delayed hypersensitivity and humoral response in JEV-infected mice

Japanese encephalitis virus (JEV) infection induces suppressor T-cells (Ts1) which suppress both the humoral (Ts-PFC) and cell mediated (Ts-DTH) immune response by producing soluble suppressor factors. This study shows that in the JEV model, both TS-PFC and Ts-DTH mediate suppression by recruiting a second subpopulation of suppressor T-cells, the Ts2-PFC and Ts2-DTH. The signal between Ts1 and Ts2 is transmitted by macrophages (M phi). The suppressor factors are adsorbed by peritoneal or splenic M phi. Both heat-killed and live M phi are capable of adsorbing suppressor factors but only live M phi are capable of presenting the signal to T-cells. Thus these are at least two generations of suppressor T-cells in the JEV-specific suppressor pathway and the presence of M phi is obligatory for transmission of the signal.     

Immune defects in chronic renal impairment: evidence for defective regulation of lymphocyte response by macrophages from patients with chronic renal impairment on haemodialysis.

Cellular mechanisms contributing to impaired lymphocyte proliferative responses in chronic renal impairment (CRI) were investigated using peripheral blood mononuclear cells (PBMC) from 25 patients receiving haemodialysis. Impaired T cell proliferative responses to phytohaemagglutinin were demonstrated. The hyporeactive PBMC from patients with CRI suppressed the responses of PBMC from normals to a greater degree than did control PBMC. This immunosuppression was reversed significantly by depleting adherent monocytes (M phi). To further determine if these impairments might be critically dependent on cell-cell contact, M phi from an additional 10 patients on haemodialysis were examined for ability to support B and T cell colony formation in semi-solid cultures stimulated by Staphylococcus protein A (SpA). When compared to normal controls, significantly fewer B and T cell colonies were observed with M phi from CRI patients than when autologous M phi were used. Also, T cells from patients were significantly less effective than controls in supporting B cell colony growth. Decreased T and B cell colony responses in patients were not due to a primary abnormality of these cells, since allogeneic mixing experiments showed that B and T cells from patients were able to form a sufficient number of colonies when control M phi or T cells from normals were used as accessory and helper cells. These findings suggest that although M phi-mediated suppressor activity is an important mechanism contributing to impaired lymphocyte responsiveness in patients with chronic renal impairment on haemodialysis, additional or related abnormalities in M phi 'accessory' function may also exist.     

Endothelial cells modulate both T-cell-dependent and T-cell-independent plaque-forming cell generation in vitro

The effects of live endothelial cells (EC), paraformaldehyde fixed EC, and EC supernatant were measured on pokeweed mitogen (PWM)-induced T-cell-dependent plaque-forming cell (PFC) generation by peripheral blood mononuclear cell (PBM). At low doses (less than or equal to 2 x 10(4) cells/culture) live EC helped PFC generation. At higher doses (greater than or equal to 10 x 10(4) cells/culture) the effect of live EC was always marked suppression (less than 10% of baseline PFC). In contrast both fixed EC and EC supernatant provided help exclusively over a wide dose range. The EC-helper effect enhanced the sensitivity of PBM to suboptimal PWM doses and also accelerated the rate of PFC generation during culture. EC influences on PFC could not be modified by gamma-interferon induction of surface DR which is known to modify EC accessory cell ability. There was also only minimal helper activity of live EC and fixed EC on the PFC generation by Epstein-Barr virus-induced cultures of purified B cells (which had been depleted of both T cells and monocytes). In contrast, suppression (greater than 97%) of PFC in isolated B-cell cultures was found even when EC constituted less than 1% of cultured cells. These results imply EC have the potential of providing multiple regulatory signals which modulate in vitro antibody production. EC-derived mechanisms are independent of their accessory cell function and require interaction with non-B cells for help, but suppression may occur directly at the B-cell level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Profiles of cell-to-cell interaction of Mycobacterium intracellulare-induced immunosuppressive macrophages with target T cells in terms of suppressor signal transmission

Previously, we have found that immunosuppressive macrophages (M(phi)s) induced by Mycobacterium intracellulare-infection (MI-M(phi)s) required cell contact with target T cells to express their suppressor activity against concanavalin A (Con A)-induced T cell mitogenesis. In this study, we examined the profiles of cell-to-cell interaction of MI-M(phi)s with target T cells. First, MI-M(phi)s displayed suppressor activity in an H-2 allele-unrestricted manner, indicating that MHC molecules are not required for cell contact. The suppressor activity of MI-M(phi)s was reduced markedly by paraformaldehyde fixation or treatment with cytochalasin B or colchicine, indicating that vital membrane functions are required for their suppressor activity. Secondly, the suppressor activity of MI-M(phi)s was independent of cell-to-cell interaction via CD40 ligand/CD40 and M(phi)-derived indoleamine 2,3-dioxygenase, which causes rapid degradation of tryptophan in T cells. Thirdly, precultivation of splenocytes with MI-M(phi)s, allowing cell-to-cell contact, reduced Con A- or anti-CD3 antibody-induced mitogenesis but not phorbol myristate acetate/calcium ionophore A23187-elicited proliferation of T cells. In addition, co-cultivation of T cells with MI-M(phi)s caused marked changes in profiles of the tyrosine phosphorylation of 33 kDa, 34 kDa and 35-kDa proteins and, moreover, the activation of protein kinase C and its translocation to the cell membrane. It thus appears that suppressor signals of MI-M(phi)s, which are transmitted to the target T cells via cell contact, principally cross-talk with the early signalling events before the activation of PKC and/or intracellular calcium mobilization.     

Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes

The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells. The results show a marked contrast between cytolytic and helper T cells in their growth and activation requirements. Thus, while Lyt-2+ cytotoxic T lymphocyte precursors grow exponentially in IL 2 after a short pulse with soluble Con A in the absence of accessory cells, Lyt-2- helper cell precursors completely fail to proliferate under the same conditions and require the continuous presence of lectin-pulsed M phi for significant growth. Furthermore, addition of IL 2 to M phi-stimulated cultures of Lyt-2- cells has no effect. T cells which produce IL 2 have the same growth characteristics as helper cells. In both cases, effector helper functions could be expanded more than 10-fold on a per cell basis by a 5-day-culture period under those growth supporting conditions. The development of effector helper functions, however, was strongly inhibited by the presence of Lyt-2+ T cells.     

Regulation of the specific plaque-forming cell response in man: restraint of B cell responsiveness.

Specific in vitro plaque-forming cell (PFC) responses of normal human lymphocytesare antigen dose-dependent, with bell-shaped dose response patterns. Both the bell-shaped antigen dose response characteristics as well as optimal PFC responseamplitudes are dictated by two distinct and separable T lymphocyte subpopulationswhich act on a nonlimiting set of PFC precursor B cells. Specific theophylline-sensitive suppressor and theophylline-resistant helper T cells are activated by antigen in adose-dependent fashion. The initially small suppressor cell pool expands exponentially following antigen contact, whereas the larger helper cell population does not. This leaves a restricted range of antigen concentrations where net helper activity issufficient to promote a PFC response. Due to coinduced suppressor activity, PFCresponses showed an overall negative restraint at all antigen concentrations. Limitingdilution analysis suggested that it is the numerical balance between the two alternative T cell subsets which determines the extent of this negative restraint. These datadelineate a cellular mechanism for the translation of an antigenic stimulus into a B cellantibody response; theophylline-sensitive suppressor cells are able to expand inresponse to this stimulus and represent the main regulative vector. Thus, in mixedlymphocyte populations, antigen concentration is a valuable probe for studies of thebalance of discrete T cell subpopulations but not for absolute B cell responsiveness.     

Lymph node macrophage heterogeneity: the phenotypic and functional characterization of two distinct populations of macrophages from rat lymph node

The isolation and characterization of lymph node macrophages (M phi) has shown a hitherto unknown heterogeneity. Two types of M phi were distinguished by morphology, monoclonal antibody staining and functional assays. The type I M phi failed to express surface Ia even when activated, a characteristic which has only previously been reported for splenic marginal zone M phi; despite studies suggesting an antigen presentation role for the M phi, the failure to express surface Ia would seem to eliminate an interaction with T helper cells for the type I M phi in the lymph node. In contrast, the type I M phi, other characteristics of clustering with activated B cells in vitro, the colocalization of the type I M phi and activated B cells in situ, the specific uptake of thymus-independent type 2 antigens and the failure to undergo respiratory burst activity all suggest a M phi-B cell interaction, possibly of a trophic nature. The defective microbicidal activity of the type I M phi may have been compensated for by the type II M phi, which expresses both strong respiratory burst activity and surface Ia expression when freshly isolated. However, unlike the inflammatory M phi the activated phenotype of the type II M phi did not appear to be interferon-gamma dependent because type II M phi could also be isolated from nude rat lymph nodes.     

Con A-propagated, auto-reactive T cell clones that secrete factors promoting high IgA responses

Four BALB/c T cell clones from among a set propagated in the presence of concanavalin A (Con A) were selected on the basis of their ability to produce supernatant factors promoting high IgA plaque-forming cell (PFC) responses by 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin (TNP-KLH)-primed splenic B cells in the presence of TNP-SRBC. Such clones could be derived from cultures containing T cells not only from gut-associated lymphoid tissue, but also from the spleen. The selected clones all proliferated well in the presence of syngeneic, irradiated APC without either Con A or exogenous IL-2, but required both APC and Con A to produce helper factors. Factors from three of the clones helped B cells both to proliferate and to differentiate into IgM, IgG and IgA PFC. Factors from the fourth clone helped B cells differentiate into IgA and IgG PFC and may have promoted switching to these isotypes but did not support either B cell proliferation or generation of IgM PFC. Cross-linking of B cell receptors for antigen was not required for the response to the helper factors since TNP-SRBC were unnecessary and high concentrations of them were actually inhibitory.     

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.