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The Immune System's Use of Oxygen and Nitric Oxide

Biological Uses for Reactive Oxygen Species - ROS

ROS can be used in the body as defense mechanisms. They can be generated by immune cells that phagocytize (engulf) bacteria and other foreign substances. After uptake, these immune cells undergo an oxidative burst, increasing the amount of dioxygen that they consume, leading to production of superoxide and hydrogen peroxide which can be used to kill the engulfed bacteria. Recent articles by Wentworth et al. go even farther. They demonstrated that antibody molecules (protein which bind to foreign molecules and target them for clearance or further immune response) can also generate ROS when they bind to their target. Such an outcome was totally unexpected, but not inconsistent with the observation that antibodies can have catalytic activity if made against transition state analogs of substrates. Over 100 different antibodies were found to generate hydrogen peroxide through the reaction of singlet oxygen (possibly generated by phagocytotic neutrophils during oxidative bursts) with water to form H2O2. (We have previously discussed the idea that singlet O2 can be generated from ground state O2 by excitation through UV light or through collisional activation with an excited state chromophore - a conjugated alkene or aromatic molecule). They postulate the following reaction to form hydrogen peroxide. The oxygen in the peroxide comes from water, as shown by labeling water with 18O:

2H2O + 1O2 (singlet oxygen) →  H2O3 + H2O →  H2O2 + ?

Antibodies show this effect to a much greater extent than other proteins studied. Using UV irradiation as a source of energy, the rate of production of H2O2 was linear with time, but decreased from the initial rate as H2O2 concentrations increased. If it was removed with catalase, the initial rate returned to normal, suggesting that H2O2 was a product inhibitor of the reaction. The reactions also saturated with dioxygen as well. The maximal wavelength of UV irradiation to produce H2O2 was identical to the maximal wavelength of absorbance for tryptophan in the protein. The excited state tryptophan presumably deexcites by energy transfer to triplet dioxygen (3O2 ) in much the manner described earlier for photobleaching. A significant source of electrons needed to be found to account for the production of so much H2O2 which if it was made from singlet oxygen would require 2 electrons for each H2O2. These numbers of electrons could not come from photooxidation of aromatic amino acids in the protein. Hence they theorized that water would add as a nucleophile to the singlet oxygen, providing the source of electrons required to make H2O2. This reactions would go through the H2O3 intermediate.

In another recent study (Nov. 02), the same team discovered that antibodies can not only produce H2O2 but also ozone. O3 is used in killing pathogens but also might lead to inflammation. In their previous work described above, they generated singlet oxygen in a "nonphysiological" way - through UV irradiation of the antibodies. They also didn't show that the ROS produced can kill bacteria without any other immune mediators. They used a new singlet oxygen generating system which led to insufficient peroxide formation to account for the extensive killing of E. Coli that they observed (95%). This led them to look for the generation of a more potent oxidizing agent - which they believe is consistent with the formation of ozone. They also showed that neutrophils were the source of the required singlet oxygen as described above.

In an additional study (Dec 02), they showed that antibodies in the presence of singlet oxygen and in the absence of immune cells or other immune proteins (like complement proteins) can "destroy antigenic target". They also confirmed that ozone is produced by antibodies during bacterial killing by neutrophils (which produce sinlget oxygen) and in inflammatory states. Signaling through ozone might amplify other immune molecules that signal the activation (or hyperactivation) of the immune system.

Their latest study (Nov. 03) examined the involvement of ozone (generated from antibodies in the presence of singlet O2 from neutrophils) in the oxidation of lipoproteins, a process which has been associated with atherosclerosis in coronary arteries. First they tested if ozone could be generated by atherosclerotic lesions treated with phorbol myristate acetate. 14/15 plaques produced ozone, as evidenced by the photobleaching of the dye indigo carmen to isatin sulfonic acid. This conversion, known to be specific for ozone, is shown below.

Figure: Conversion of indigo carmen to isatin sulfonic acid

 

12cholesterolozone2.gif

They then looked for the telltale signature of the reaction of ozone with plaque cholesterol (cleavage of the D5,6 double bond to form 5,6-secosterol as shown below) and found it. This reaction is known not to occur with other oxygen oxidants, including both triplet and singlet oxygen, superoxide, and the hydroxyl free radical. A secondary reaction product aldol condensation product was also produced. They named these new products atheronals.

Figure: Cholesterol/ozone reaction

13cholesterolozone1.gif

Figure: Ozonolysis mechanism

 

14jpegozonolysismech.jpegFigure:

Significant increases in secosterol was found in plaques treated with the phorbal ester. 6 of 8 patients with atherosclerosis had elevated plasma levels of the secosterol, while only 1 of 15 of control patients did. They also showed that these ozone products caused normal macrophages to fill with cholesterol (presumably from oxidized LDL), and that the structure of the LDL apoprotein B100 changed. These findings might provide a link between the immune system and cholesterol in the generation of cardiovascular disease.

Just Say NO - The Chemistry of Nitric Oxide

In the last decade, the role of another gaseous free radical, nitric oxide (.NO) has become apparent. This molecule is synthesized biologically through the action of an inducible heme enzyme, nitric oxide synthase, which forms NO by the oxidation by dioxygen of the guanidino group of Arg, which gets converted to citrulline. .NO is soluble and can diffuse through cell membranes into the cytoplasm, where it has a myriad of effects in signal transduction pathways. However, it can also be metabolized to form reactive nitrogen intermediates (much as with dioxygen) which can be deleterious to the body, if they damage native biomolecules, or advantageous, when they are used by immune cells like macrophages in the destruction of engulfed bacteria.

A molecular orbital diagram of .NO shows it to have a bond order of 2.5 and one unpaired electron in a p2p* antibonding orbital (hence the notation .NO).

 

15MONO.gif

Here are some of the relevant reactions of NO and its reactive nitrogen intermediates (RNI):

  • The high energy electron p2p* can leave on oxidation of NO to form the nitrosyl ion, NO+. The bond order in NO+ is three suggesting strong interactions between the N and O atoms.

  • .NO (oxidation number of N of +2) is thermodynamically unstable and can disproportionate or dismutate (like superoxide) in a self redox reaction to form nitrous oxide (N2O) and nitrogen dioxide (.NO2), with oxidation number for N of +1 and +4, respectively.

  • In acidic condition .NO can be oxidized to nitrite (NO2-) which can be protonated to form nitrous acid (HNO2).

  • Nitrous acid can disproportionate or dismutate to form .NO and the radical .NO2 which can be protonated to ONOOH with a pKa of 6.5.

  • .NO can react with superoxide (.O2-) faster than any other molecule to form peroxynitrite, ONOO- . .NO and superoxide (.O2-) are both produced by cells during infllammation, leading to increased levels of peroxynitrite.

  • The O-OH bond in protonated peroxynitrite (ONO-OH) can be cleaved either heterolytically to produce NO3- and H+ (70%) or homolytically to produce the radical .NO2 and the highly reactive .OH free radical (30%).

  • The deprotonated form of peroxynitrite (ONOO-) can oxidize organic molecules, especially thiols and CO2. When it reacts with CO2, it can form either NO3- and CO2 (65%) or CO3.- and .NO2 (35%). These latter products can oxidize organic molecules in one electron steps. Peroxynitrite can also oxidize protein, DNA, and lipids.
  • .NO2 and peroxynitrite can interact with Tyr side chains in proteins to form nitrated proteins (Tyr-NO2), whose presence reflects inflammation.

Macrophages make use of RNIs in the killing of engulfed bacteria. How does one of humankinds greatest foes, Mycobacteria tuberculosis, the causative agent of tuberculosis, avoid this killing mechanism? It is estimated that the bacteria resides persistently in latent form in 2 billion people. If it becomes activated, it becomes one of the greatest killers. Consider the following table:

Killer Diseases through time (Scientist, June 2, 2003)

Historic Pandemics cases deaths
Justinian Plague (6th centr.) 142 million (on 70% mort) about 100 mill
China Plague (Bubonic) 3rd Pandemic (1896-1930) 30 milion 12 milion
Spanish flu 1918-19 1 billion 21 million

Pandemics Today

Pandemics Today cases/yr deaths/yr
Malaria 300-500 mil 1 million
TB 8 mill 2 mill
AIDS 6 million 3 mill

People infected with the bacteria but without clinical symptoms must mount a sufficient enough immune response to restrain proliferation of the bacteria, but not enough to clear it from the body. Immune-compromised people (transplant recipients taking anti-rejection drugs or AIDS patients) have more difficulty in holding the bacteria at bay. One immune response mediator which restrains bacterial growth is the soluble protein interferon g (a protein cytokine released by immune cells). This protein induces synthesis of nitric oxide synthase, producing .NO, which through the reactions listed above, can damage bacterial macromolecules.

Bacteria are "digested" in acidic phagosomes of the macrophage. Nitrite formed in the acid conditions from .NO (generated by inducible NO synthase) forms .NO2 and peroxynitrite which have antibacterial properties (better than anti-Tb drugs). Mutant mice that can not synthesize inducible NO synthase have little defense against the bacteria. Darwin e al. exposed the bacteria to sources of nitrite at pH conditions typical of macrophage phagosomes and found mutants sensitive to the RNIs. The mutations involved protein of the bacterial proteasome, a complex multi-protein complex which proteolyzes unwanted (presumably damaged) cellular proteins.

Bacterial genes encoding proteins associated with the bacterial proteasome seem to confer resistance to the effects of macrophage-inducted RNI production. The macrophage proteasome, like other eukaryotic proteasomes, is a cytoplasmic protein complex which degrades damaged cytoplasmic proteins. Although the mechanism is uncertain, the bacterial proteasome may rid the bacteria of nitrated or otherwise oxidized (damaged) proteins or remove the nitrate and facilitate refolding of the damaged protein.