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Serum albumin induces iNOS expression and NO production in RAW 267.4 macrophages
Michael Poteser1, Ichiro Wakabayashi
1Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Abstract:
1. We investigated the effects of serum albumin on inducible nitric oxide synthase (iNOS) expression in RAW 267.4 macrophages. Crude fraction-V type albumin as well as bovine serum albumin filtrated for endotoxin induced concentration-dependent iNOS expression in macrophages. Accordingly, NO production (estimated by supernatant nitrite) was markedly (up to 10-fold) increased in the presence of albumin. 2. Albumin-induced expression of iNOS protein was inhibited by cycloheximide and NO production was abolished after incubation of the cells with an iNOS inhibitor, N(G)-monomethyl-l-arginine (LNMMA). 3. An inhibitor of the NF-kappaB pathway, pyrrolidine dithiocarbamate (PDTC), as well as inhibitors of JAK2/STAT and ERK, AG490 and U0126, respectively, significantly reduced albumin-induced iNOS expression and NO production, while an inhibitor of the p38 pathway, SB203580, did not significantly affect NO production induced by albumin. 4. Both types of serum albumin were contaminated with traces of endotoxin. The endotoxin levels were found not to be sufficient for the observed induction of nitrite production in RAW 267.4 cells. In addition, the albumin-stimulated induction of iNOS was not reduced by preincubation of albumin-containing media with polymyxin B, a LPS inhibitor. 5. Polymerised albumin fractions were detected in the commercially available albumin tested in this study. A monomeric albumin-rich fraction, separated by ultrafiltration, showed a potent inducing effect on iNOS expression and NO production, while a polymer-rich fraction showed a smaller effect. 6. Advanced glycation endproducts (AGE) of albumin were not formed by interaction with glucose in incubation medium, as AGE was not increased even after long-time (4 weeks) incubation in albumin-containing media [3.2-4.4 microg ml(-1) (basal) vs 4.8-5.6 microg ml(-1) (in glucose-containing media)]. However, the duration of albumin exposure to glucose influenced the basal stimulatory properties of albumin. 7. Our results suggest that serum albumin fractions, as gained by cold alcoholic extraction, may include determinants that stimulate or further enhance stimulation of RAW 267.4 cells and are different from endotoxin, polymeric albumin and AGE.
Insights
Serum albumin, particularly monomeric fractions, significantly enhances nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in macrophages. These effects are independent of endotoxin, polymeric albumin, or advanced glycation endproducts.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Serum albumin is a crucial protein in biological systems.
- Its role in modulating immune responses, particularly in macrophages, requires further elucidation.
- Previous studies have suggested potential immunomodulatory effects of albumin.
Purpose of the Study:
- To investigate the impact of serum albumin on inducible nitric oxide synthase (iNOS) expression in RAW 267.4 macrophages.
- To identify the specific components of albumin responsible for these effects.
- To differentiate albumin's effects from known inflammatory stimuli like endotoxins.
Main Methods:
- RAW 267.4 macrophages were treated with different fractions of serum albumin.
- iNOS expression and nitric oxide (NO) production were measured.
- Inhibitors of key signaling pathways (NF-κB, JAK2/STAT, ERK, p38) and endotoxin inhibitors were employed.
Main Results:
- Both crude and endotoxin-filtered serum albumin induced concentration-dependent iNOS expression and markedly increased NO production in macrophages.
- Albumin-induced iNOS expression was dependent on protein synthesis and NO production pathways, and modulated by NF-κB and ERK signaling.
- Monomeric albumin fractions showed potent induction of iNOS and NO production, distinct from endotoxin, polymeric albumin, or advanced glycation endproducts.
Conclusions:
- Serum albumin contains specific determinants, distinct from endotoxin, polymers, or AGEs, that stimulate iNOS expression and NO production in macrophages.
- Monomeric albumin fractions are particularly potent inducers of these immune responses.
- These findings highlight a novel immunomodulatory role for serum albumin in macrophage activation.
