Oxidative metabolism of murine macrophages.

This unit describes two simple and straightforward microassays that can be used to measure the levels of NO(2)(-) and O(2)(-), respectively that are generated by a small number of immunologically-stimulated macrophages. Detection of these products may be used to identify cytokine(s), microbe(s), or microbial products(s) that regulate oxidative metabolism and effector activity. Although a number of other reliable and sensitive methods are available for assaying these two oxidative metabolites, the microassays described here require little time, technical expertise, or materials. It is not clear at present whether human monocytes/macrophages can also produce NO(2)(-). These protocols are therefore restricted to mouse macrophages.

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. coli J5 during serum incubation.

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirement for inner membrane damage and cell death of Gram-negative bacteria.

Interaction of anti-cholesterol antibodies with human lipoproteins.

Inoculation of mice with cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A results in the production of antiserum containing IgM Ab to cholesterol. The specificity of the Ab was to cholesterol and structurally similar sterols containing a 3 beta-hydroxyl group. Anti-cholesterol binding activity was significantly diminished if the 3 beta-hydroxyl was altered by either epimerization, substitution, oxidation, or esterification. A similar specificity for 3 beta-hydroxy-sterols was observed for an anti-cholesterol IgM mAb. Both hyperimmune serum and the mAb reacted with intact human very-low-/intermediate-density lipoprotein (VLDL/IDL) and low-density lipoproteins (LDL), but not high-density lipoproteins (HDL), in an ELISA, but could react with total lipid extracts containing cholesterol that were prepared from all three lipoprotein classes. Functionally, immune serum or the mAb aggregated and induced a fusion-like reaction with VLDL/IDL and LDL at low temperatures: these aggregates result in spherical structures visible with light microscopy. Similarly, binding of anti-cholesterol A to small cholesterol-rich liposomes resulted in the appearance of vesicular structures with approximately 20- to 200-fold increased diameters. These data demonstrate that the anti-cholesterol Ab recognize unesterified cholesterol in VLDL/IDL and LDL; high-density lipoprotein cholesterol in the intact lipoprotein, however, appears to be protected from reaction with these Ab.

Role of atheroma liposomes and malondialdehyde-modified low-density lipoproteins in complement activation.

We investigated the ability of atheroma-associated liposomes and malondialdehyde (MDA)-modified low-density lipoproteins (MDA-LDL) to activate complement. Complement activation markers C3a, Bb, C4d and SC5b-9 were measured in both normal and complement-deficient sera. We found that MDA-LDL was able to generate C3a and SC5b-9, predominantly by the alternative pathway. High-density lipoproteins modified with MDA were also capable of C3a generation although to a lesser degree. The presence of atheroma-associated liposomes did not result in detectable levels of complement activation markers. We conclude that MDA-modified lipoproteins may represent a possible source for complement activation within atherosclerotic lesions.

Analysis of anticholesterol antibodies using hydrophobic membranes.

An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.

Induction of nitric oxide synthase protects against malaria in mice exposed to irradiated Plasmodium berghei infected mosquitoes: involvement of interferon gamma and CD8+ T cells.

Exposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mRNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon gamma (IFN-gamma) or in vivo depletion of CD8+ T cells, but not CD4+ T cells, at the time of challenge blocked expression of iNOS mRNA and ablated protection in immunized mice. These results show that both CD8+ T cells and IFN-gamma are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-gamma, likely provided by malaria-specific CD8+ T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells.