Nitric oxide-mediated bactericidal pathway

L-arginine metabolism in macrophages

The fate of the amino acid L-arginine inside phagocytes directly depends on the microenvironment and signaling molecules acting on the cell. There are two independent pathways of its enzymatic cleavage that lead to completely different biological effects.

1. Inducible NO synthase (iNOS) pathway. Activated by pro-inflammatory cytokines IFNγ and TNFα. The enzyme cleaves L-arginine to produce citrulline and nitric oxide (NO). This specific process provides a powerful bactericidal effect and also causes local blood vessel dilation (vasodilation).
2. Arginase pathway. Triggered by interleukins IL-4 and IL-13. In this case, arginine is converted into L-ornithine. Ornithine is subsequently used to synthesize proline (necessary for collagen formation) and polyamines (which stimulate cell proliferation).

iNOS activation and the action of nitric oxide

For the iNOS enzyme to actively produce nitric oxide, the combined (synergistic) action of the cytokines IFNγ and TNFα is required. An additional induction factor is tetrahydrobiopterin, whose activity also increases in the presence of TNFα.

Nitric oxide itself (synthesized in the cytosol) possesses microbicidal and antitumor activity, but its derivatives inflict significantly greater damage on pathogens. The primary weapon of macrophages becomes peroxynitrite ($OONO^-$). It is formed when an NO molecule meets a superoxide radical.

The mechanism of action of peroxynitrite involves the oxidation of sulfhydryl (-SH) groups in microbial proteins and other molecules, instantly stripping them of their functionality. This tool is most effective against complex intracellular pathogens: mycobacteria tuberculosis, protozoa, fungi, and viruses. The end products of these reactions are nitrite anions and radicals, as well as nitrate anions.

Interaction of the oxygen and nitrogen pathways

Phagocytes possess alternative defense mechanisms based on the generation of toxic radicals. Bactericidal systems operate with maximum efficiency when the nitrogen and oxygen cascades combine.

• Oxygen pathway: The enzyme NADPH oxidase reduces oxygen to a superoxide anion ($O_2^{\bullet-}$), which then cascades into hydrogen peroxide and a hydroxyl radical.
• Nitrogen pathway: The enzyme NO synthase generates a nitric oxide radical ($NO^{\bullet}$).

The actual strike against the microbe occurs upon their contact. The superoxide anion and nitric oxide merge, forming peroxynitrite. Subsequently, it converts into peroxinitrous acid, which spontaneously decays into two extremely aggressive components: nitrogen dioxide ($NO_2^{\bullet}$) and a hydroxyl radical ($OH^{\bullet}$).

Over the course of evolution, microorganisms have learned to defend themselves against this combined attack. The main bacterial shield against oxygen radicals and nitrogen-containing compounds is the enzyme bacterial superoxide dismutase.

Acidification of the phagolysosome internal environment

Successful bacterial digestion is impossible without the acidification of granule contents—this is a mandatory condition for their fusion with phagosomes. The process occurs stepwise: in early endosomes, the environment is mildly acidic (pH 6.0–6.5), in late endosomes it drops to 5.5–6.0, and in mature phagolysosomes it reaches 4.5–5.0. Such a drop in pH directly exerts bacteriostatic and bactericidal action.

The main tool of acidification is the V-ATPase protein complex. It actively pumps hydrogen protons from the cytosol into the vesicle interior. Initial phagosomes lack this enzyme; it is delivered there only upon fusion with lysosomes. In neutrophils, V-ATPase is present in all granules except specific ones (which is why their environmental reaction is neutral).

Interestingly, the pH level in neutrophil phagolysosomes is slightly higher than in macrophages. This is due to two reasons. First, the potent neutrophil NADPH oxidase actively consumes free protons to synthesize hydrogen peroxide. Second, the generated reactive oxygen species partially suppress the activity of the V-ATPase itself.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Detailed mechanism of NADPH oxidase and the oxygen pathway.
• Biochemical role of polyamines and proline in regeneration processes.
• Mechanisms of action of bacterial superoxide dismutase.
• Structural organization of the V-ATPase proton pump.

Other topics in the "Innate Immunity" section

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