nitrophenols and Endotoxemia

The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.

Topics: Amino Acid Substitution; Animals; Antibody Formation; Communicable Diseases; Endotoxemia; Epitopes; Immunochemistry; Lipopolysaccharides; Male; Metabolic Diseases; Mice; Mutation, Missense; Neoplasms; Nitrophenols; Self Tolerance; Tumor Necrosis Factor-alpha; Vaccines

The role of tyrosine phosphorylation in endotoxin-induced phagocytic and tumor necrosis factor secretory responses was studied in rat alveolar macrophages and lung-recruited neutrophils. Exploration of sexual dimorphism in some aspects of these functions was also a specific aim. Male and female rats were injected intratracheally with endotoxin or saline. Two and a half hours later the animals were subjected to bronchoalveolar lavage, and alveolar macrophages and lung-recruited neutrophils were isolated. Circulating neutrophils of endotoxin-treated rats were also isolated at this time. Phagocytosis and CD11b/c and CD18 expression were measured by flow cytometry; tumor necrosis factor was measured with a cytotoxicity assay. Using the protein tyrosine kinase inhibitor AG126 and phosphotyrosine immunoblotting, we demonstrated that tyrosine phosphorylation is an important signaling pathway in the activation of these cells by endotoxin and that it is coupled to phagocytosis and tumor necrosis factor secretion, but not to beta 2 integrin expression. Conditioned medium of alveolar macrophages of endotoxin-injected rats upregulates phagocytosis by blood neutrophils of naive rats and this upregulating activity is tyrosine phosphorylation dependent. The substrates for tyrosine phosphorylation are different in alveolar macrophages and lung neutrophils, as are their sensitivities to AG126. Significant gender differences exist in the modulation of phagocytosis by inhibition of tyrosine phosphorylation and in tumor necrosis factor secretion by endotoxin-stimulated alveolar macrophages.

Topics: Animals; CD18 Antigens; Culture Media, Conditioned; Endotoxemia; Endotoxins; Enzyme Inhibitors; Escherichia coli Infections; Female; Immunoblotting; Integrin alphaXbeta2; Lung; Macrophage-1 Antigen; Macrophages, Alveolar; Male; Neutrophils; Nitriles; Nitrophenols; Phagocytosis; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sex Factors; Tumor Necrosis Factor-alpha; Tyrphostins; Up-Regulation