Microbicidal (killing) mechanisms employed by neutrophil and macrophage during phagocytosis

This lecture note is to give you a basic idea of how neutrophils and macrophages kill microorganism during phagocytosis.

Neutrophils are having greater antimicrobial activity when compared to macrophages. Neutrophils exhibit a larger respiratory burst than macrophages and as a result of that it is able to generate more reactive oxygen intermediates and reactive nitrogen intermediates. In addition, neutrophils express higher levels of defensins than macrophages.

Neutrophils and macrophages employ variety of microbicidal mechanisms and use multiple antimicrobial molecules stored in their granules for the activity.

1) Oxygen dependent Killing mechanism

Activated phagocytes produce large number of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) that have potent antimicrobial activity.

Method 1: Reactive oxygen species production

A) Reactive oxygen species (ROS) production – MPO Independent reactions

  • During phagocytosis, a metabolic process known as the respiratory burst/oxidative burst occurs in activated macrophages and neutrophils. This process produces various toxic oxygen products.
  • Respiratory burst leads to the activation of a membrane-bound oxidase called NADPH oxidase (phagocyte oxidase).
  • This enzyme catalyzes the reduction of oxygen to highly unstable superoxide anion that is extremely toxic to ingested microorganisms. During this reaction NADPH is oxidized to NADP+

(Activation of the phagocyte’s NADPH oxidase (Nox2) is a vital innate immune mechanism)

  • The NADPH required for this process is obtained by a large increase in pentose phosphate pathway activity.
  • The superoxide anion act as a precursor for other powerful oxidizing agents, including hydroxyl radicals and hydrogen peroxide

(Under the acidic conditions of the phagosome, O2.will rapidly dismutate into H2O2 and OH. are generated by the partial reduction of H2O2)

Reactive oxygen species released: Superoxide radical (O2.), Hydrogen peroxide (H2O2), hydroxyl radical (OH.)

 Oxygen dependent myeloperoxidase Independent reactions are shown below:

Additional point: Absence of NADPH oxidase will prevent the formation of reactive oxygen species and it will result in chronic granulomatous disease.

B) Reactive oxygen species (ROS) production – MPO dependent reaction

Some other enzymes present in the activated phagocytes can make use of this liberated reactive oxygen species to generate other toxic products.

Example: Myeloperoxidase enzyme catalyses the formation of hypochlorous acid (HOCl) which is a potent antimicrobial agent that will kill the ingested microorganism.

Myeloperoxidase (MPO) system of leukocyte

  • Mainly found in neutrophils (stored in azurophillic granules in resting state) and also in monocytes at a lesser degree. During degranulation of neutrophils these enzymes are released.
  • Heme cofactor containing peroxidase
  • Myeloperoxidase (MPO) is a critical enzyme in the conversion of hydrogen peroxide to hypochlorous acid in the presence of halides especially chloride.
  • It is the most effective microbicidal and cytotoxic mechanism of leukocyte
  • MPO is responsible for the characteristic green color of pus.

Action of HOCl: oxidation of cellular materials and destruction of vegetative bacteria and fungi

Action of hypochlorite (ClO ):powerful oxidising agent, oxidizes essential enzymes of the microbes

Method 2: Reactive nitrogen intermediates (RNI) production

Macrophages, neutrophils and mast cells form reactive nitrogen intermediates (RNIs).

 RNIs includes: Nitric oxide (NO) and its oxidized forms – nitrite (NO2) and nitrate (NO3), NO2 (nitrogen dioxide), HNO2 (nitrous acid)

  • RNIs are very potent cytotoxic agents. Nitric oxide is the most effective among them. NO also has antimicrobial activity.
  • Macrophages produce NO (nitric oxide) from the amino acid arginine. Activated macrophages express high levels of nitric oxide synthetase (NOS) They catalyse the oxidation of L-arginine to L-citrulline and nitric oxide (NO) gas

  • NO can combine with the superoxide anion to yield even more potent antimicrobial substances.

Example: Under the acidic conditions of the phagosome, O2. (Superoxide anion) will react with nitric oxide (NO) to form peroxynitrite (ONOO−) which is a potent cell damaging agent

 Method of action of NO: Block cellular respiration by complexing with the iron in electron transport proteins.

(Much of the antimicrobial activity of macrophages against bacteria, fungi, parasitic worms, and protozoa is due to nitric oxide and substances derived from it)

2) Oxygen Independent killing mechanism:

  • The lumen of the phagolysosome is an inhospitable environment for the ingested microorganism.
  • Activated macrophages and neutrophils synthesize various hydrolytic enzymes and antimicrobial proteins. Their degradative activities do not require oxygen.
  • An acidic vacuolar pH favors the activity of the hydrolases. Collectively these participate in the destruction of the entrapped microorganism.

Lysosomal hydrolases: lysozyme, phospholipase A2, ribonuclease, deoxyribonuclease, collagenase and proteases.

Functions of Different hydrolases

Lysozyme: Catalyzes the hydrolysis of β1-4 glycosidic linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine within peptidoglycan. Loss of membrane integrity leads to instability and bacterial cell lysis.

Phospholipase A2: digestive enzymes that hydrolyze phospholipids

Ribonuclease: digestive enzymes that degrade RNA

Proteases: Hydrolyze proteins in bacterial cell envelopes (Example: Cathepsin G and elastase)

Deoxyribonuclease: digestive enzymes that degrade DNA

Glycosylases:  Enzymes that hydrolyze glycosyl compounds

Antimicrobial Proteins and Peptides: Defensins, Lactoferrin, BPI (Specific for neutrophils), cathelicidins, major basic proteins and TNF – α (Only macrophages)

Functions

Cathelicidins: Cause damage to microbial cell membrane

Major basic proteins: Cytotoxic to parasites

Bactericidal/permeability increasing protein (BPI): Inhibits Gram negative bacterial growth

TNF – α: Cytotoxic for tumor cells

Defensins: Broad spectrum antimicrobial peptide (Cationic), Cathepsin G is an example for defensins

Defensins act against bacteria and fungi by permeabilizing their cell membranes. They form voltage-dependent channels in the membrane that allow ionic efflux.

Antiviral activity: Direct neutralization of enveloped viruses (Non enveloped viruses are not affected by defensins)

Lactoferrin: Sequester free iron available in the medium, which is an essential substrate required for bacterial growth

Neutrophil extracellular traps (NETs)

You all know that when an infection occurs, neutrophils are the first cell type arriving at the infection site. In the case of neutrophils death is not the end of their role in innate defense against pathogens. When living neutrophils come in contact with pathogens, some of the cells may transform themselves into extracellular fibers called neutrophil extracellular traps (NETs).

NETosis:  Specific type of programmed cell death by which NETs are formed

  • When NETosis occurs, the neutrophils become deformed, and the nuclear envelope and granule membranes disintegrate.
  • When the plasma membrane of the dying neutrophil breaks, the NET components (nuclear DNA and granule antimicrobial proteins) are mixed up.
  • At first the invading pathogens get caught in these NET fibers and then the antimicrobial agents of the NET degrade and kill the pathogens without the need for phagocytosis.

Components of NET: DNA, Histones (Nucleic acid contents) Neutrophil elastase, myeloperoxidase, histones, Lactoferrin, Gelatinase B, Cathepsin G etc (granular contents)

 

 

 

 

 

 

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