Margret C. M. Vissers

Living with a killer: the effects of hypochlorous acid on mammalian cells.

The production of hypochlorous acid (HOCl) by the myeloperoxidase‐H2O2‐Cl system of phagocytes plays a vital role in the ability of these cells to kill a wide range of pathogens. However, the generation of a potent oxidant is not without risk to the host, and there is evidence that HOCl contributes to the tissue injury associated with inflammation. In this review, we discuss the biological reactivity of HOCl, and detail what is known of how it interacts with mammalian cells. The outcome of exposure is dependent on the dose of oxidant, with higher doses causing necrosis, and apoptosis or growth arrest occurring with lower amounts. Glutathione (GSH) and protein thiols are easily oxidized, and are preferred targets with low, sublethal amounts of HOCl. Thiol enzymes vary in their sensitivity to HOCl, with glyceraldehyde‐3‐phosphate dehydrogenase being most susceptible. Indeed, loss of activity occurred before GSH oxidation. The products of these reactions and the ability of cells to regenerate oxidized thiols are discussed. Recent reports have indicated that HOCl can activate cell signaling pathways, and these studies may provide important information on the role of this oxidant in inflammation.

Oxidative damage to fibronectin. I. The effects of the neutrophil myeloperoxidase system and HOCl.

Exposure of purified human plasma fibronectin to the myeloperoxidase-H2O2-Cl- system of neutrophils or to reagent HOCl resulted in extensive changes to its primary and tertiary structures. When 1.14 microM fibronectin was exposed to 50-400 microM HOCl or 50-400 microM H2O2 plus myeloperoxidase and Cl-, there was progressive loss of tryptophan fluorescence and cysteines, and an increase in bityrosine fluorescence and carbonyl content. Analysis by SDS-PAGE indicated extensive crosslinking of the fibronectin, the crosslinks being stable under reducing conditions. The coincident increase of bityrosine fluorescence suggests that crosslinking may be largely due to intermolecular bityrosines rather than disulfides. All changes observed with the myeloperoxidase system were inhibited by azide or methionine, and were dependent upon the presence of chloride, indicating that they are mediated by HOCl. The reaction between HOCl and fibronectin resulted in the formation of long-lived chloramines. Exposure to increasing amounts of oxidant resulted in an increase in the susceptibility of fibronectin to proteolytic attack by purified neutrophil elastase. Analysis by SDS-PAGE showed a different fragmentation pattern for oxidant-treated fibronectin compared with the native protein. This suggests that regions of the molecule which were previously resistant to proteolysis were denatured to create susceptible sites for elastase. This demonstration that fibronectin is extensively modified by the myeloperoxidase system has implications for the mechanism of tissue injury by neutrophils in inflammation, since a loss of functional fibronectin would result in cell detachment and a distortion of normal tissue organization.

Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases released from neutrophils.

Human neutrophils, when stimulated with phorbol myristate acetate or fMet-Leu-Phe in the presence or absence of cytochalasin B, released metalloproteinases that catalytically inactivated the plasma serine proteinase inhibitor, alpha 1-antitrypsin. Inactivation, measured as loss of elastase inhibitory capacity, was accompanied by cleavage of a Mr 4,000 peptide from the COOH-terminus. Cleavage of alpha 1-antitrypsin by cell supernatants was inhibited by EDTA, o-phenanthroline, and DTT, but not by inhibitors of serine or thiol proteinases. Gelatinase and collagenase were separated from the medium of stimulated neutrophils. Both preparations cleaved and inactivated alpha 1-antitrypsin, with cleavage occurring close to the reactive center, at the Phe-Leu bond between positions P7 and P6. Cleavage by purified gelatinase was very slow and could account for only a minor fraction of the activity of neutrophil supernatants. The collagenase preparation was more active. However, the unusual cleavage site, and the ability of fMet-Leu-Phe-stimulated neutrophils to cleave alpha 1-antitrypsin without releasing collagenase, suggests that collagenase is not responsible for cleavage by the cells, which, by implication, is due to an as yet uncharacterized metalloenzyme. Our results demonstrate that by releasing metalloproteinases, neutrophils could proteolytically inactivate alpha 1-antitrypsin at sites of inflammation. This provides an alternative to the previously documented mechanism of inactivation by neutrophil-derived oxidants.

Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease

The removal of neutrophils from inflammatory sites is essential for the resolution of inflammation. Surface changes, including phosphatidylserine exposure, label neutrophils for phagocytosis by macrophages. Here, we demonstrate that externalization of phosphatidylserine and uptake by monocyte‐derived macrophages occurred in human neutrophils ingesting Staphylococcus aureus. Both processes were dependent on oxidant production from the neutrophil NADPH oxidase. There was no requirement for myeloperoxidase, and H2O2 was identified as the most likely trigger for PS exposure. We hypothesize that clearance of stimulated neutrophils would be delayed in chronic granulomatous disease (CGD) neutrophils, which lack a functional NADPH oxidase. To explore this possibility, heat‐killed S. aureus were injected into the peritoneum of CGD and normal mice. Elevated neutrophil numbers were observed in the inflammatory exudate of the CGD animals, consistent with impaired recognition and clearance.

Membrane changes associated with lysis of red blood cells by hypochlorous acid

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidenced by ektacytometry, and an increase in K(+)-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, but complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.

Modification of red cell membrane lipids by hypochlorous acid and haemolysis by preformed lipid chlorohydrins

Hypochlorous acid (HOCl), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCl with the double bonds of unsaturated lipids produces alpha, beta-chlorohydrin isomers. We have exposed red cell membranes to HOCl and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10(6) cells were treated with > or = 0.16 nmoles HOCl. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by 14C scintillation counting. Addition of HOCl-treated oleic acid to red cells resulted in rapid lysis of a fraction of the cells in a concentration dependent manner. HOCl-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCl-treated cholesterol-containing liposomes.

A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils.

We have developed a method that enables the rates of phagocytosis and killing of bacteria by neutrophils to be measured in a single assay. Neutrophils were incubated with bacteria, and at specific intervals were separated from uningested bacteria by low speed centrifugation. Rates of phagocytosis and killing were calculated from the decrease in number of extracellular bacteria and change in the number of intracellular bacteria. Both phagocytosis and killing were shown to follow first‐order kinetics, and rate constants were calculated without having to separate the assay into two phases. In contrast to two‐step methods, our method measures killing from the moment the neutrophils start ingesting the bacteria, and also eliminates the need to halt neutrophil activity temporarily and restart the assay after the extracellular bacteria have been removed. We obtained reproducible results for the phagocytosis and killing of Staphylococcus aureus (t1/2 = 9 min and 6 min respectively) and Escherichia coli (t1/2 = 10 min and 2 min respectively). We also were able to detect a 56% impairment in the rate of killing of S. aureus by neutrophils from an individual with a low level of myeloperoxidase. J. Lcukoc. Biol. 55: 147–152; 1994.

Ascorbate deficiency results in impaired neutrophil apoptosis and clearance and is associated with up-regulation of hypoxia-inducible factor 1α

Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L‐gulono‐γ‐lactone oxidase (Gulo)−/− mice, which are unable to synthesize ascorbate, to generate ascorbate‐deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate‐deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate‐induced inflammation in Gulo−/Ȓ mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate‐deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo−/− mice had elevated levels of hypoxia‐inducible factor (HIF)‐1α, a transcription factor regulated by Fe2+‐dependent hydroxylases which require ascorbate for optimal activity. HIF‐1α has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up‐regulation of HIF‐1α blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells.

Degradation of glomerular basement membrane by human neutrophils in vitro.

The glomerular basement membrane is susceptible to immunologic injury when immune complexes or anti-basement-membrane antibodies become lodged in its network. We have studied the digestion of glomerular basement membrane prepared from normal human kidney by isolated neutrophils. In the absence of immunoglobulin aggregates or immune complexes, there was little evidence of neutrophil adherence to the membrane, of release of lysosomal enzymes, or of digestion. However, when the basement membrane contained immunoglobin G (IgG) aggregates generated in situ by heating the membrane impregnated with IgG to 63 degrees C, electron micrographs showed neutrophils adherent to the basement-membrane surface and phagocytosis of smaller fragments. Lysosomal enzymes were detectable in the extracellular medium, and measurements of either total protein or hydroxyproline solubilized showed digestion of 80 micrograms basement membrane/h per 10(7) cells. Hydroxyproline solubilization was almost totally inhibited by phenylmethylsulphonyl fluoride, indicating that the neutrophil serine proteinases, elastase and cathepsin G are responsible for degradation. These findings provide direct evidence for the digestion of extracellular matrix by neutrophils stimulated in situ by deposited immune complexes as a contributor to inflammatory tissue damage.

Different effects of hypochlorous acid on human neutrophil metalloproteinases: Activation of collagenase and inactivation of collagenase and gelatinase

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.