Aggregates of denatured proteins stimulate nitric oxide and superoxide production in macrophages.
OBJECTIVE
Denatured proteins are deposited in damaged tissues, around implanted biomaterials, during natural aging as well as in a heterogeneous group of amyloid diseases, such as Alzheimer's disease. There is evidence that tissue damage observed in amyloidosis is mediated mainly by factors released from activated macrophages, such as superoxide and nitric oxide (NO), as opposed to direct interaction between amyloid fibrils and nonimmune cells.


METHODS
Mouse resident peritoneal macrophages were stimulated in serum-free medium with different preparations of nonamyloidogenic proteins: alcohol dehydrogenase (AD), bovine serum albumin (BSA) or fibrinogen (FG). Intra- and extracellular superoxide production was measured by, respectively, nitro blue tetrazolium (NBT) reduction and lucigenin-enhanced chemiluminescence. Levels of nitrite (reflecting NO release) were measured in culture supernatants.


RESULTS
Aggregates of denatured, nonamyloidogenic proteins, but not their native or denatured but not aggregated counterparts, stimulated superoxide and/or NO production in macrophages. The NO production was mediated by beta(1) and beta(2) integrins, with a possible contribution of receptor for advanced glycation end products (RAGE). It was catalyzed by inducible NO synthase (iNOS), enhanced synergistically by interferon-gamma (IFN-gamma), and inhibited by covalently modified proteins-components of advanced glycation end products. Although intracellular superoxide production was stimulated significantly by denatured BSA and AD, but not by FG, both denatured BSA and FG strongly enhanced zymosan-stimulated extracellular release of reactive oxygen species.


CONCLUSIONS
Our results point at similarities in macrophage responses to denatured nonamyloidogenic proteins and to amyloid fibrils. Thus, the tissue injury observed in amyloidosis may result from overstimulation of mechanisms that, under physiological conditions, enable macrophages to recognize and remove denatured proteins.
Jozefowski
Szczepan
S
Department of Immunology, Jagiellonian University School of Medicine, Cracow, Poland. s.jozefowski@nencki.gov.pl
Marcinkiewicz
Janusz
J
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D019070:Cell Lineage / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D005340:Fibrinogen / Q000737:chemistry / Q000494:pharmacology; D006358:Hot Temperature; D016023:Integrins / Q000502:physiology; D015850:Interleukin-6 / Q000378:metabolism; D049449:Luminescence; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D009569:Nitric Oxide / Q000096:biosynthesis*; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*; D012709:Serum Albumin / Q000737:chemistry / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*
D016023:Integrins; D015850:Interleukin-6; D011506:Proteins; D012709:Serum Albumin; D013481:Superoxides; D009569:Nitric Oxide; D005340:Fibrinogen; D000426:Alcohol Dehydrogenase