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Molecular basis of macrophage activation. Expression of the low potential cytochrome b and its reduction upon cell
Abstract:
The expression of the novel b-type cytochrome, which is part of the superoxide anion (O2-)-generating system in phagocytes, has been investigated in population of mouse peritoneal macrophages heterogeneous in their capability to produce O2-). Reduced minus oxidized difference spectra of intact cells showed the appearance of a b-type cytochrome with major peaks in the alpha region at 558 to 559 nm and in the gamma region at 426 to 428 nm. Resident peritoneal macrophages, as well as thioglycollate broth-elicited and Corynebacterium Parvum-activated macrophages and neutrophils expressed about 50 pmol cytochrome b/10(7) cells. In intact macrophages and neutrophils, Na-dithionite reduced greater than 75% of the cytochrome b measurable in disrupted cells. No correlation was found between capability to produce O2-) by different population of macrophages and their content of cytochrome b. When stimulated in strictly anaerobic conditions with phorbol myristic acetate, macrophages activated in vivo by i.p. injection of Corynebacterium Parvum reduced approximately 40% of their total cytochrome b. In resident peritoneal macrophages that produced five times lower amounts of O2-, cytochrome b reduction was instead undetectable. Potentiometric properties of cytochrome b was investigated in macrophage subcellular particles. Both resident and Corynebacterium Parvum-activated macrophages revealed the presence of b chromophores with very low potentials of -255 and -244 mV, respectively, whose content was not different in the two populations. These results show that resident and activated macrophages express the same amount of cytochrome b, but upon stimulation with PMA, activated macrophages recruit a higher number of cytochrome b molecules in parallel with an enhanced production of O2-.
Insights
Activated macrophages recruit more cytochrome b for superoxide anion production. Resident and activated macrophages have similar cytochrome b levels, but activated cells show increased recruitment upon stimulation, enhancing superoxide anion generation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Phagocytes generate superoxide anion (O2-) using a b-type cytochrome system.
- Understanding cytochrome b expression is crucial for phagocyte function.
Purpose of the Study:
- To investigate the expression and function of novel b-type cytochrome in mouse peritoneal macrophages with varying superoxide anion production capabilities.
- To correlate cytochrome b content and recruitment with superoxide anion generation in different macrophage populations.
Main Methods:
- Spectroscopic analysis (reduced minus oxidized difference spectra) of intact cells and subcellular particles.
- Measurement of cytochrome b content in resident, elicited, and activated macrophages and neutrophils.
- Assessment of cytochrome b reduction upon stimulation with phorbol myristic acetate (PMA) under anaerobic conditions.
- Potentiometric analysis of cytochrome b in macrophage subcellular particles.
Main Results:
- A b-type cytochrome with characteristic spectral peaks was identified in phagocytes.
- Resident, elicited, and activated macrophages and neutrophils expressed similar basal levels of cytochrome b (approx. 50 pmol/10(7) cells).
- Activated macrophages, upon PMA stimulation, recruited a higher percentage of their total cytochrome b for reduction compared to resident macrophages, correlating with enhanced O2- production.
- Potentiometric studies revealed low redox potentials for cytochrome b in both resident and activated macrophages, with no significant difference in content.
Conclusions:
- Macrophages express similar basal amounts of cytochrome b, irrespective of activation state or O2- production capacity.
- Macrophage activation enhances the recruitment of existing cytochrome b molecules into the functional O2- generating system upon stimulation.
- This increased recruitment of cytochrome b is a key mechanism for the enhanced superoxide anion production observed in activated phagocytes.