Inhibition of interferon gamma-induced macrophage microbicidal activity against Leishmania major by liposomes: inhibition is dependent upon composition of phospholipid headgroups and fatty acids.

Multilamellar liposomes of phosphatidylcholine and phosphatidylserine at a 7:3 molar ratio significantly inhibited activation of murine resident peritoneal macrophages by recombinant murine interferon-gamma for cytotoxicity against amastigotes of the protozoan parasite Leishmania major; other macrophage effector functions, such as particle phagocytosis or tumoricidal activity, were unaffected. This inhibition was not due to direct toxic effects of liposomes against parasite or macrophage, was fully reversible, and was directed at one or more early events in macrophage-LK interactions which ultimately induce microbicidal activity. Liposomes containing some natural phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidic acid or diphosphatidyl glycerol), but not phosphatidylcholine, phosphatidylglycerol, or several synthetic saturated phospholipids, prevented the induction of macrophage microbicidal activity. Inhibition by liposomes of various composition was not related to the efficiency with which these vesicles were ingested by macrophages. Inhibitory activity was directly influenced by changes in the phospholipid head group, as well as by the number of unsaturated bonds in phospholipid fatty acids: for a given phospholipid in liposomes, inhibition was directly related to the number of unsaturated bonds among the fatty acids. These data support a role for phospholipids in postbinding regulation of macrophage activation and add to our understanding of how liposome delivery systems can be designed to avoid potential microbicidal suppressive effects.

Macrophage activation factor from EL-4, a murine T-cell line: antigenic characterization by hamster monoclonal antibodies to murine interferon-gamma.

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.

Inhibition of lymphokine-induced macrophage microbicidal activity against Leishmania major by liposomes: characterization of the physicochemical requirements for liposome inhibition.

Resident peritoneal macrophages from untreated mice develop potent microbicidal activity against amastigotes of Leishmania major after in vitro treatment with lymphokine (LK) from mitogen-stimulated spleen cells. LK-induced macrophage microbicidal activity was completely and selectively abrogated by treatment with phosphatidylcholine-phosphatidylserine (PC/PS) liposomes. Other macrophage effector functions (phagocytosis, tumoricidal activity) were unaffected, as was cytotoxicity by macrophages activated in vivo or by LK in vitro before liposome treatment. Activation factors in LK were not adsorbed or destroyed by liposomes. Liposome-induced inhibition was unaffected by indomethacin and was fully reversible: macrophages washed free of liposomes developed strong microbicidal activity with subsequent LK treatment. Changes in liposomal lipid composition markedly altered suppressive effects, but inhibition was not dependent on liposome size, cholesterol content, charge, or number of lamellae. Liposomes composed of PC alone or in combination with any of five different phospholipids were not suppressive. In contrast, inhibition was directly dependent on PS concentration within PC/PS liposomes. Phosphoserine was not inhibitory nor was dimyristoyl PS (synthetic saturated PS). However, the lysophospholipid metabolite of PS, lysoPS, was strongly suppressive. These studies suggest that the reversible and selective inhibition of LK-induced macrophage microbicidal activity by PC/PS liposomes is mediated by PS and its lysoPS metabolite.

Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes.

Resident peritoneal macrophages from untreated mice develop microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica (current nomenclature = Leishmania major) after in vitro exposure to LK from antigen-stimulated leukocyte culture fluids. This LK-induced macrophage microbicidal activity was completely abrogated by addition of 7:3 phosphatidylcholine: phosphatidylserine liposomes. Liposome inhibition was not due to direct toxic effects against the parasite or macrophage effector cell; factors in LK that induce macrophage microbicidal activity were not adsorbed or destroyed by liposome treatment. Other phagocytic particles, such as latex beads, had no effect on microbicidal activity. Moreover, liposome inhibition of activated macrophage effector function was relatively selective: LK-induced macrophage tumoricidal activity was not affected by liposome treatment. Liposome inhibition was dependent upon liposome dose (5 nmoles/culture) and time of addition of leishmania-infected, LK-treated macrophage cultures. Addition of liposomes through the initial 8 hr of culture completely inhibited LK-induced macrophage microbicidal activity; liposomes added after 16 hr had no effect. Similarly, microbicidal activity by macrophages activated in vivo by BCG or Corynebacterium parvum was not affected by liposome treatment. Liposome treatment also did not affect the increased resistance to infection induced in macrophages by LK. These data suggest that liposomes interfere with one or more early events in the induction of activated macrophages (macrophage-LK interaction) and not with the cytotoxic mechanism itself (parasite-macrophage interaction). These studies add to the growing body of data that implicate cell lipid in regulatory events controlling macrophage effector function.

Differential inhibition of macrophage microbicidal activity by liposomes.

In vitro culture of murine resident peritoneal macrophages with lymphokine (LK)-rich leukocyte culture fluids induces enhanced microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica. Macrophages infected with Leishmania and treated with LKs after infection acquire the capacity to kill the intracellular parasite within 72 h. When compared with control macrophage cultures treated with medium lacking LKs, 80 to 90% fewer macrophages treated with LKs contained amastigotes. In experiments designed to test liposome delivery of LKs to infected macrophages, addition of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) completely abrogated LK-induced microbicidal activity. Liposomes containing only phosphatidylcholine were not inhibitory. Inhibition of LK activity by the liposomes occurred regardless of whether the liposomes contained LKs. Liposomal inhibition of activated macrophage effector activity was limited to intracellular killing; LK-induced macrophage extracellular cytolysis (i.e., tumor cytotoxicity) was not affected by liposome treatment. These data indicate that elucidation of the effects of liposome composition on acquired host defense mechanisms may be useful for the design of drug delivery systems that allow expression or augmentation of immunologically induced mechanisms for the intracellular destruction of infectious agents.

Deficiency of Con A-induced suppressor cell activity in peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax.

Con A-pretreated mononuclear (MNC) cells from Thai adults with naturally acquired P. falciparum or P. vivax malaria were significantly less effective in suppressing the responsiveness of autologous or normal allogeneic responder cells to mitogenic lectins or allogenic stimulator cells than pretreated cells from healthy donors. Serial studies of three patients demonstrated that reduced suppressor cell activity was present early in malaria infection but returned to normal soon after treatment. These studies demonstrate that the loss of T cells previously observed in patients with malaria, in part may functionally represent a loss of suppressor T cells.

Cold-reactive immunoglobulin M antilymphocyte antibodies directed against B cells in Thai children with dengue hemorrhagic fever.

Antilymphocyte antibodies were found in 51 of 83 serum specimens from Thai children with dengue hemorrhagic fever (DHF). The lymphocytotoxic activity was complement dependent, and cytotoxicity was detected in the 19S immunoglobulin M-associated serum fractions at a temperature optimum of 15 degrees C. Sera with lymphocytotoxic activity were cytotoxic to autologous as well as allogeneic lymphocytes from patients and healthy adult donors and were directed primarily against B cells, with some T cell cross-reactivity. This study suggests that infection with DHF induces predominately cold-reactive antilymphocyte antibodies in DHF patients that could potentially interact with peripheral blood cells of patients and modulate the humoral immune responses of patients during infection.

Subpopulations of T cells (Tg and Tm) in patients with malaria.

In the present study we utilized rosetting techniques to enumerate the putative suppressor (Tg) and helper (Tm) T-cell subpopulations in the peripheral blood of adult Thais with malaria. A lower percentage of both Tg and Tm subpopulations and a lower number and percentage of total T cells was found in these patients during the acute period of infection than in the peripheral blood of healthy donors. However, the percentages of total T, Tg and Tm cells were higher during the convalescent period and were comparable to the values found in the peripheral blood of healthy donors. The significance of these findings are discussed. No correlations were found between the percentage of these T-cell subpopulations and the level of parasitemia or the hematocrit.

Characterization of cold reactive lymphocytotoxic antibodies in malaria.

Characterization of cold reactive lymphocytotoxic antibodies present in sera from Thai adults with malaria revealed that the antibodies are predominantly 19S (IgM), directed against both autologous and allogeneic mononuclear cells, complement-dependent, present in titres ranging from 1:2 to 1:16, and exhibit greater lymphocytotoxic activity during the acute stage of malarial infection than during the convalescent stage. The lymphocytotoxic antibodies were primarily directed against B cell targets or both B as well as T cell targets. In addition some sera were reactive with enriched monocyte/macrophage indicator cells at 15 degrees C, but not 37 degrees C. Antibodies directed against B cell targets were lymphocytotoxic both at 15 degrees C as well as 37 degrees C. The results indicate that IgM lymphocytotoxic antibodies in the sera of patients with malaria are directed primarily against B cells with reactivity to a lesser extent against T cells and macrophages and thus may play an immunoregulatory function in the humoral immune response to malaria infection.

Deficient spontaneous cell-mediated cytotoxicity and lectin-induced cellular cytotoxicity by peripheral blood mononuclear cells from Thai adults naturally infected with malaria.

To assess general cytotoxic effector cell capabilities by peripheral blood mononuclear cells from patients with active malaria infections, we examined antibody-dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and lectin-induced cellular cytotoxicity by using human and chicken erythrocyte, Chang cell line, and K562 cell line targets. By using human erythrocyte and Change cell line targets, we found that Thai adults naturally infected with malaria had significantly impaired lectin-induced cellular cytotoxicity. In addition, spontaneous cell-mediated cytotoxicity was deficient with K562 but not with Chang cell line targets. Finally, no change in antibody-dependent cellular cytotoxicity was observed when chicken erythrocyte or Chang cell line targets were used. These observations, coupled with our previous observations of a physical loss of peripheral blood T cells, the presence of lymphocytotoxic serum antibodies, and defective T suppressor cell generation in patients with malaria, indicate that major alterations in the cellular immune system occur in patients with active malaria infections.

Effect of anti-coagulants on cold-reactive anti-lymphocyte activity in the blood of patients with malaria.

The effect of three different anti-coagulants on the level of cold-reactive anti-lymphocyte activity (ALA) in the peripheral blood (PB) of malarious individuals was assessed to determine if plasma could be substituted for serum in assays designed to characterize ALA. Results show that plasma obtained by treating PB with acid-citrate dextrose or ethylenediamine tetraacetic acid can be used instead of serum in these assays but that plasma obtained from heparin-treated blood cannot.

Suppression of mitogenic lectin-induced blast transformation of human peripheral blood mononuclear cells by pyrimethamine.

The anti-malarial drug pyrimethamine suppresses in vitro mitogenic lectin-induced blast transformation by human peripheral blood mononuclear cells (MNC) when the drug is added to cells (1 X 10(-5) M/culture). Sulphadoxine, a second widely used anti-malarial drug has no suppressive effect on the MNC. MNC responsiveness in the mixed leucocyte reaction and cellular viability are not altered by either pyrimethamine or sulphadoxine. In addition, no significant suppression is found when serum obtained from individuals on pyrimethamine-sulphadoxine chemoprophylaxis is added to MNC in the assays. The data, however, do not totally rule out any clinically significant suppressive effect by the anti-malarial drugs on human cellular immune responses.

Inhibition of mitogenic lectin induced blast transformation in human peripheral blood mononuclear cells by mefloquine.

The effect of mefloquine-HCL, a new 4-quinoline methanol anti-malarial compound, on in vitro blast transformation of human peripheral blood mononuclear cells (MNC) was studied. Mefloquine significantly suppressed lectin-induced blast transformation of MNC from healthy Thai adults but MNC responsiveness in the mixed leucocyte reaction (MLR) and cellular viability were not reduced by the concentrations of mefloquine studied. Both T and non-T MNC responsiveness was lower in cultures containing the drug than in normal control cultures. The addition of serum from individuals on mefloquine chemoprophylaxis caused no significant suppression in the blast transformation assays or the MLR but the data do not rule out any clinically significant in vivo suppressive effect by mefloquine on human cellular immune response.

Differences in attachment antigens of gonococci in reinfection.

On the basis of bacterial typing, pyocin typing, and antibiotic sensitivity tests, two consorts appeared to have been reinfected 34 and 41 days later, respectively, with the same gonococcal strain, suggesting a failure in these patients to develop effective immunity to reinfection. However, these tests do not measure antigens which mediate attachment, a function which may correlate with virulence. When the above infecting strains were retested in an inhibition-of-attachment assay using rabbit gonococcal antisera, the antigens mediating attachment were found to be different. Homologous antisera inhibited attachment of the homologous strain at a high titer. Absorbing the antisera with the initial infecting strain did not remove any of the blocking activity of the antisera raised to the reinfecting strai, but the latter strain did share some attachment antigens in common with the initial infecting strain. Homologous antisera also bound preferentially to pili purified from the homologous strains.