Interferon gamma and lymphotoxin or tumor necrosis factor act synergistically to induce macrophage killing of tumor cells and schistosomula of Schistosoma mansoni.

Macrophages play a crucial role in the defense against tumors and parasites. Activation of tumoricidal and microbicidal effector mechanisms requires stimulation of macrophages with macrophage-activating factors (MAF). One such MAF is interferon gamma (IFN-gamma). In some assays, substantial activity of IFN-gamma on murine macrophages, however, is only observed in synergy with lipopolysaccharide (LPS) or other cytokines (1). In addition, certain cytokines have been shown to induce monocyte or macrophage activation in the absence of IFN-gamma (2-5). We previously described lymphokines in the supernatant of a murine T cell clone that synergized with IFN-gamma in the induction of tumoricidal and schistosomulicidal murine macrophages (1). We called this lymphokine(s) macrophage cytotoxicityinducing factor 2 (MCIF2)(1). A candidate for MCIF2 was lymphotoxin (LT), because the T cell clone supernatant contained high amounts of LT. LT is functionally homologous and structurally related to the macrophage product tumor necrosis factor (TNF). Therefore, we tested whether recombinant (r) LT or rTNF can function as MAF. We report here that rLT or rTNF synergize with rIFN-gamma in the induction of tumoricidal and schistosomulicidal murine macrophages.

Demonstration and partial characterization of the interferon-gamma receptor on human mononuclear phagocytes.

Radioiodinated recombinant human interferon-gamma (IFN gamma) bound to human monocytes, U937, and HL60 cells in a specific, saturable, and reversible manner. At 4 degrees C, the different cell types bound 3,000-7,000 molecules of IFN gamma, and binding was of comparable affinity (Ka = 4-12 X 10(8) M-1). No change in the receptor was observed after monocytes differentiated to macrophages or when the cell lines were pharmacologically induced to differentiate. The functional relevance of the receptor was validated by the demonstration that receptor occupancy correlated with induction of Fc receptors on U937. Binding studies using U937 permeabilized with digitonin showed that only 46% of the total receptor pool was expressed at the cell surface. The receptor appears to be a protein, since treatment of U937 with trypsin or pronase reduced 125I-IFN gamma binding by 87 and 95%, respectively. At 37 degrees C, ligand was internalized, since 32% of the cell-associated IFN gamma became resistant to trypsin stripping. Monocytes degraded 125I-IFN gamma into trichloroacetic acid-soluble counts at 37 degrees C but not at 4 degrees C, at an approximate rate of 5,000 molecules/cell per h. The receptor was partially characterized by SDS-polyacrylamide gel electrophoresis analysis of purified U937 membranes that had been incubated with 125I-IFN gamma. After cross-linking, the receptor-ligand complex migrated as a broad band that displayed an Mr of 104,000 +/- 18,000 at the top and 84,000 +/- 6,000 at the bottom. These results thereby define and partially characterize the IFN gamma receptor of human mononuclear phagocytes.

The effect of trypsin digestion on the structure and iron-donating properties of transferrins from several species.

The effect of trypsin digestion on iron-saturated and iron-free (apo) human, rabbit, bovine, pig and horse tranferrins has been studied. Iron-binding fragments were produced only from iron-saturated pig and bovine transferrins although some cleavage of the polypeptide chain occurred in all cases. The apo-transferrins were generally degraded to a greater extent than the corresponding iron-saturated proteins. The ability of the different transferrins to donate iron to rabbit reticulocytes varied in the order rabbit approximately pig greater than human approximately horse greater than bovine. Trypsin digestion considerably reduced the ability of pig and bovine transferrins to donate iron to rabbit reticulocytes, slightly reduced the iron-donating ability of rabbit transferrin, and had almost no effect on that of human or horse transferrins.

The interaction of bovine transferrin and monoferric transferrin fragments with rabbit reticulocytes.

1. The mechanism of interaction of transferrin with reticulocytes has been investigated using monoferric fragments derived by proteolysis from bovine Fe2-transferrin. 2. Rabbit reticulocytes readily took up iron from bovine transferrin, but only slight uptake occurred from the C-terminal fragment (S), and almost none from the N-terminal fragment (F). 3. The degree of binding of transferrin and fragments to the cells was in the order transferrin greater than fragment F greater than fragment S. 4. Binding of transferrin and fragment S, but not of fragment F, was reduced when incubation was performed at 4 degrees C instead of 37 degrees C, and all iron uptake was abolisehd. 5. Preincubation of reticulocytes with fragment S, but not with fragment F, somewhat reduced subsequent iron uptake from transferrin. 6. The presence of bovine serum albumin (40 mg/ml) in the incubation buffer inhibited iron uptake, but iron uptake nevertheless occurred from transferrin in bovine serum. 7. No differences were detected in the rate of 59Fe uptake from transferrin labelled asymmetrically by sequential additions of 59Fe and 56Fe to apotransferrin. 8. It is concluded that both halves of the transferrin molecule are involved, perhaps in different ways, in the interaction of transferrin with reticulocytes, and that rabbit reticulocytes do not take up iron preferentially from one of the binding sites of bovine transferrin.

The nature of iron released by resident and stimulated mouse peritoneal macrophages.

Mouse peritoneal macrophages were allowed to ingest 59Fe, 125I-labelled transferrin-antitransferrin immune complexes, and the release of 59Fe and degraded transferrin was studied. Some iron was released as ferritin, but a major portion was bound by bovine transferrin present in the culture medium, which contained fetal calf serum. If the medium was saturated with iron prior to incubation with the cells, little of the released iron was then bound by transferrin but appeared either as a high molecular weight fraction or, if nitrilotriacetate was present in the medium, some also appeared as a low molecular weight fraction. The release of non-ferritin iron was biphasic, the early, rapid phase being more prolonged with resident cells than with stimulated cells. The rate of release in the late phase did not differ significantly between resident and stimulated cells. Incubation at 0 degrees C completely suppressed the release of degraded transferrin, but iron release continued at about 30% of the rate seen in control cultures at 37 degrees C. A model for the intracellular handling of ingested iron is proposed to take account of the different release patterns of resident and stimulated macrophages.

Phylogeny of the third component of complement, C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species.

The third component of complement, C3, binds to several other complement proteins. To study the diverse reactivities of C3, we analyzed the conservation of structural and functional features in the C3 from different species. First, we developed a method to purify swine (Po), rabbit (Rb), mouse (Mo), cobra (Co), Xenopus (Xe), axolotl (Ax), and trout (Tr) C3 from plasma. This involved protein precipitation by polyethylene glycol, followed by anion-exchange, gel filtration, and cation exchange chromatography. All C3's tested were comprised of two chains (alpha/beta-chain) and contain a thiolester bond within the alpha-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved (as detected by ConA binding) in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the beta-chain and Po and Mo C3 only in the alpha-chain. Co C3, in contrast to cobra venom factor (CVF), lacks ConA binding carbohydrates in both chains. N-terminal amino acid sequence analysis of the alpha-, alpha'-, and beta-chains showed varying degrees of similarity within the different C3's. The N-termini of the Xe and Ax C3 beta-chains were found to be blocked. The conservation of binding sites in the different C3's for human complement receptors type one (CR1) and two (CR2) and for factors H and B was investigated due to the structural and functional similarities of these molecules and to the ability of some of them to bind to the same domain(s) in human C3.(ABSTRACT TRUNCATED AT 250 WORDS)

Preactivation of macrophages in mice acutely infected with Schistosoma mansoni.

This paper shows that peritoneal murine macrophages become preactivated in vivo during the course of a Schistosoma mansoni infection. Thus, less macrophage-activating factor (MAF) was required to induce in vitro tumoricidal and schistosomulicidal activity in macrophages from S. mansoni-infected mice than in macrophages from uninfected control animals. Moreover, the respiratory burst activity, as measured by chemiluminescence, was enhanced in macrophages from S. mansoni-infected mice as compared to controls, whether or not lymphokine (LK) was present in the macrophage cultures. This response appeared at 3 weeks and persisted at least until 12 weeks after infection. Interferon-gamma (IFN-gamma) is most likely involved in the mechanisms leading to such an increased cytolytic and oxidative activity, since in vitro experiments showed: 1) that less IFN-gamma was required to induce tumoricidal activity in macrophages from infected as compared to macrophages from uninfected animals, 2) that the activity of (2'-5')-adenylate synthetase (2'-5' A-synthetase), an enzyme strongly induced by IFN, was elevated in cells from livers of S. mansoni-infected mice.

Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators.

Adjuvant research is being oriented to TLR-agonists, but complement activation has been relatively unexplored. In previous studies it was demonstrated that poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (PVMA NPs) used as adjuvant differentially activate dendritic cells through toll like receptors (TLR) stimulation, however, a high dose of these NPs was used. Now, we demonstrated a dose-response effect, with a concentration as low as 20µg/mL able to stimulate TLR2 and TLR4 transfected dendritic cells. In addition, we investigated whether PVMA NPs are able to exploit also the immunomodulatory benefits of complement activation. Results indicated that the hydroxylated surface of these NPs highly activated the complement cascade, as measured by adsorption studies and a complement fixation bioassay. Stable binding of C3b to NPs was confirmed as indicated by lability to SDS treatment after washing resistance. Complement consumption was confirmed as the lytic capacity of complement exposed to NPs was abolished against antibody-sensitized sheep erythrocytes, with a minimal inhibitory concentration of 50µg NPs, equivalent to a surface of 1cm(2). On the contrary, nanoparticles prepared with poly(lactic-co-glycolic acid) (PLGA), used as a reference, did not consume complement at a concentration ≥3mg NPs (≥40cm(2)). Complement consumption was inhibited when PVMA NPs were cross-linked with diamino groups (1,3-diaminopropane), indicating the role of hydroxyl groups as responsible of the phenomenon. These results favour a model whereby PVMA NPs adjuvant activate complement on site to attract immature antigen presenting cells that are activated through TLR2 and TLR4.

Complexing capacity profiles of naturally occurring ligands in Tempranillo wines for Cu and Zn: an electroanalytical approach for cupric casse.

Complexing capacity of naturally occurring ligands in Vitis vinifera (Tempranillo variety) wines has been studied with respect to two target metals (Cu and Zn) by differential pulse anodic stripping voltammetry (DPASV). Eight commercial wines of two certified brands of origin (CBO) and a young wine along its vinification process were monitored. Conditional stability constants and total complexing ligand(s) concentration(s) have been calculated for both metals. Discussion of the particular electrochemical responses for Cu and Zn for all samples is presented. A follow-up of the Cu stripping response allowed differentiating a commercial wine from one under processing related to the cupric casse phenomenon. Interaction of Cu with two molecular forms of cyanidin has been theoretically modeled at natural wine pH.

Parameters affecting the immunogenicity of microencapsulated tetanus toxoid.

The immunogenic potential of tetanus toxoid (TT) was compared when either adsorbed to aluminium hydroxide (TT-alum) or entrapped in microparticles consisting of poly(D,L-lactic-co-glycolic acid) (PLA:PGA, 55:45) derived polymers. Furthermore, the effect of administering the microparticles in an aqueous buffer or water-in-oil emulsion on the TT immunogenicity was also investigated. When mice were immunized with the different formulations, similar levels of anti-TT antibodies were observed during the primary IgG response. The choice of the carrier seemed to play an important role for both the level and maintenance of the secondary IgG response, attained as a consequence of a booster immunization with TT-alum. The strongest secondary antibody response was obtained by priming with TT-containing microparticles, resuspended in water-in-oil emulsions. As expected, incomplete Freund's adjuvant (IFA) proved to be a more potent adjuvant than peanut oil, whereas resuspension of the microparticles in aqueous solution induced a relatively less efficient antibody response. Overall, microencapsulated TT primed the mice more effectively, since the secondary antibody response was higher and persisted longer compared with TT-alum priming. These results indicate that in addition to TT maintaining its antigenicity after microencapsulation, the microparticles also potentiate its immunogenic properties. This approach should prove very useful for designing more effective vaccines.

Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.

Release of iron by resident and stimulated mouse peritoneal macrophages following ingestion and degradation of transferrin-antitransferrin immune complexes.

A method of loading macrophages from normal and inflammatory mouse peritoneal exudates with 59Fe using 59Fe, 125I-transferrin-antitransferrin immune complexes is described and the subsequent release of iron and degraded transferrin to the incubation medium has been studied. Release of iron occurred more rapidly from resident macrophages than from thioglycollate broth-induced (stimulated) macrophages, but degradation of the 125I-transferrin in the immune complexes was faster in stimulated cells. A small percentage of the iron released was in the form of ferritin. Desferrioxamine (1 mM) increased the release of iron from both stimulated and resident macrophages, the effect being proportionally greater in the stimulated cells. Ascorbic acid (1 mM) had no effect on the release of iron, nor did the addition of apotransferrin (1 mg/ml) to the culture medium. These results support the concept of a blockade of iron release by reticuloendothelial cells in states of inflammation, and suggest that it may be a primary cause of the anaemia of chronic disease.

Interferon-gamma-dependent modulation of C3b receptors (CR1) on human peripheral blood monocytes.

Cell surface expression of the C3b receptor (CR1) was transiently down regulated on purified human monocytes exposed to purified recombinant human interferon-gamma(rIFN-gamma). Receptors were quantitated by using CR1-specific monoclonal antibody by radioimmunoassay or flow cytometry and by rosette analysis with the use of C3b-coated bovine erythrocytes. The reduction of CR1 was dependent on the dose of IFN-gamma used and the time of cellular exposure. Down-regulation was transient, with maximum loss occurring after 2 to 3 days of stimulation with IFN-gamma. However, after 6 days CR1 was re-expressed at the plasma membrane. This effect was observed with monocytes isolated by either centrifugal elutriation or adherence on fibronectin-coated dishes. IFN-gamma-dependent diminution of CR1 occurred concomitant with increased expression of Fc receptors and HLA-DQ antigen and unaltered expression of the C3bi receptor (CR3). The mechanism of CR1 loss from the monocyte cell surface was not due to internalization, because total cellular levels of CR1 (plasma membrane and intracellular pool) also decreased in response to IFN-gamma. These results indicate that IFN-gamma may be involved in regulating CR1 on mononuclear phagocyte surfaces.

Effect of interferon-alpha, interferon-gamma and tumour necrosis factor on African swine fever virus replication in porcine monocytes and macrophages.

Bovine interferon-alpha I1 (IFN-alpha I1) and porcine interferon-gamma (IFN-gamma) inhibited African swine fever virus replication in both porcine monocytes and alveolar macrophages. The most potent antiviral activity was observed with IFN-gamma-treated alveolar macrophages. The production of both a virulent (CC83) and a non-virulent (BA71) isolate of the virus was inhibited. Bovine tumour necrosis factor alpha did not show antiviral activity in either monocytes or alveolar macrophages. Rather, an increase of African swine fever virus production in tumour necrosis factor alpha-treated monocytes was found. An analysis of viral protein synthesis in IFN-alpha I1- and IFN-gamma-treated alveolar macrophages showed an inhibition of synthesis of some viral proteins. The inhibition of late proteins was very pronounced in IFN-gamma-treated cells, and it was probably a consequence of the inhibition of African swine fever virus DNA polymerase activity.

Isolation and properties of bovine colostral trypsin inhibitor.

The trypsin inhibitor of bovine colostrum was isolated by affinity chromatography, and impurities removed by trichloroacetic acid precipitation. The inhibitor showed electrophoretic microheterogeneity which was not due to sialic acid content. It inhibited bovine and rat trypsin, showed weak inhibition of bovine chymotrypsin and was inactive against rat chymotrypsin and bovine renin, kallikrein, thrombin and trypsinogen. The dynamics of secretion of the inhibitor in the first 8 milkings post-partum were very similar to those of colostral immunoglobulins.