Charles G. Cochrane

Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.

Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000.

A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.

Oxidant-induced DNA damage of target cells.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.

Studies on the Pathogenesis of the Adult Respiratory Distress Syndrome

Bronchoalveolar lavage (BAL) fluid was obtained from 24 sequentially studied patients with adult respiratory distress syndrome (ARDS) for assessment of potential activating and mediating factors. Proteolytic activity of the fluids was observed by measuring cleavage of radiolabeled proteins of the contact (Hageman factor) and complement systems. Proteolytic activity was observed in 17 of 24 (71%) patients with ARDS, and BAL fluid of the 7 ARDS patients without demonstrable, active, enzyme exhibited inhibitory activity for the proteolytic activity. The enzymes cleaved Hageman factor, prekallikrein, plasminogen, high molecular weight kininogen, C4, C3, C5, and Factor B of the complement system. Cleavage of the contact system proteins producted fragments similar or identical in size to the fragments observed during activation of these molecules, although continued incubation invariably reduced the protein to small peptide fragments. None of 7 normal individuals, and 29 of 99 patients (29%) with other forms of pulmonary disease contained measurable enzymes. The proteolytic activity in BAL fluid of ARDS patients was blocked by diisopropylphosphofluoridate (0.1 mM), Trasylol, soybean trypsin inhibitor, and normal plasma, or plasma deficient in inhibition of the first component of complement. Alpha(1)-proteinase inhibitor (alpha1-PI)-deficient plasma failed to inhibit the proteolytic activity and addition of alpha1-PI to the deficient plasma reconstituted the inhibition. MUCH OF THE PROTEOLYTIC ACTIVITY OF THE BAL FLUID FROM ARDS PATIENTS WAS IDENTIFIED AS NEUTROPHIL ELASTASE: the fluids cleaved elastin and synthetic peptide substrate of neutrophil elastase, neutrophil elastase antigen was present in the BAL fluids as determined immunologically using antineutrophil elastase, alpha1-PI was the major inhibitor in plasma, and the enzyme was inhibited by diisopropylphosphofluoridate but not chelation. In addition, purified neutrophil elastase produced cleavage fragments of proteins of the contact system similar to those of the BAL fluids. In each of the seven BAL fluids of ARDS patients that did not reveal active elastase, alpha1-PI was present in active form (as determined by (125)I-trypsin binding). In 9 of the 17 patients with active elastase in the BAL fluid, alpha1-PI antigen was present in the fluid, but was inactive (no binding of (125)I-trypsin). Immunoelectrophoretic analysis of elastase and alpha1-PI throughout proteins in these BAL fluids revealed the presence of both elastase and alpha1-PI that migrated with the same R(f), suggesting the presence of an enzyme-inhibitor complex. Free, inactive alpha1-PI was also observed in these fluids. The data reveal that in BAL fluids from all 24 patients with ARDS, leukocytic elastase and/or alpha1-PI exist. A complex of elastase and alpha1-PI was observed in BAL fluids, and in some cases where active enzyme and alpha1-PI coexisted, free, but inactive alpha1-PI was present.

Pathogenesis of the adult respiratory distress syndrome. Evidence of oxidant activity in bronchoalveolar lavage fluid.

Evidence is presented indicating that oxidants are generated in lungs of patients with the adult respiratory distress syndrome (ARDS). The evidence was derived from observations that alpha-1-PI, recovered in bronchoalveolar lavage (BAL) fluid, had been inactivated by oxidation, presumably oxidation of the methionyl residue in the reaction site of the molecule. This was indicated by findings that activity of the alpha-1-PI could be restored by exposure to the reducing agent, dithiothreitol in the presence of methionyl sulfoxide peptide reductase. The amount of activity restored was proportional to the amount of inactive alpha-1-PI present at 52,000 D. Oxidation of the 52,000-D alpha-1-PI was also revealed by the finding that the inactive molecule was subject to proteolytic cleavage to 47,000 D when exposed to porcine pancreatic elastase, a characteristic of alpha-1-PI with oxidized methionyl residues in the reactive site. Inactivation of the alpha-1-PI in vivo also resulted from complexing to an active enzyme, shown previously to be neutrophil elastase, and from proteolytic cleavage in vivo, that produced a fragment of 47,000 mol wt. In contrast to that in BAL fluids, the alpha-1-PI in plasma of patients with respiratory distress syndrome was found to be greater than 90% active in 14 of 22 cases and 50-90% active in 8 cases. This suggested that for the most part, alpha-1-PI was inactivated after leaving the vessels and entering the lung. The circulating alpha-1-PI in patients with the respiratory distress syndrome was found to be equally susceptible to oxidative inactivation as alpha-1-PI from normal individuals. It seems improbable therefore that patients develop ARDS because of labile alpha-1-PI inhibitor.

Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects.

Kininogenase activity was detected by cleavage of radiolabeled substrate (125I-high molecular weight kininogen [HMWK]) in 22 of 24 bronchoalveolar lavage (BAL) fluid samples from 17 asthmatics who either responded to aerosolized allergen challenge or had symptoms of active asthma. In contrast, six of seven normal controls lacked enzymatic activity. Levels of free immunoreactive kinin found in BAL fluid correlated with the presence of kininogenase activity (P = 0.002). The cleavage pattern of 125I-HMWK by the BAL fluid kininogenase (a dominant 65,000-mol wt fragment), and synthetic inhibitor profile (phe-phe-arg-CH2Cl and phenylmethylsulfonyl fluoride) were compatible with a tissue kallikrein. Peak kininogenase activity eluted at an apparent molecular weight of 20,000-34,000 by HPLC gel filtration. Its antigenic identity was established by immunoblotting with anti-human urinary kallikrein antibody and its activity was inhibited by this antibody. Lysylbradykinin was generated during incubation of fractionated BAL fluid and purified HMWK, the characteristic cleavage product of the tissue kallikreins. We conclude that elevated amounts of tissue kallikrein and kinin are present in the bronchoalveolar spaces of asthmatic subjects. Kinin generation may contribute to the asthmatic response directly through edema formation and smooth muscle contraction and by augmenting release and/or production of preformed (histamine) and secondary mediators such as leukotrienes and platelet-activating factor.

Cellular injury by oxidants

Oxidants, generated by stimulated leukocytes, induce a variety of distinct biochemical changes in target cells. Hypochlorous acid (HOCl), produced by the action of peroxidase on hydrogen peroxide (H2O2) in the presence of chloride ions, acts at low molar concentrations (10-20 microM) to damage proteins on cell membranes and destroy their function. H2O2 rapidly permeates cells and causes inhibition of adenosine triphosphate (ATP) synthesis via both glycolytic and oxidative phosphorylation (mitochondrial) pathways. In the glycolytic pathway, damage is limited to the step involving glyceraldehyde-3-PO4 dehydrogenase (GAPDH). This results from both an attack of H2O2 on GAPDH and, indirectly, by a reduction in concentration of the GAPDH cofactor, nicotinamide adenine dinucleotide (NAD). This latter effect was found to result from activation of the enzyme, poly(adenosine diphosphate) (ADP)-ribose polymerase, an enzyme involved in deoxyribonucleic acid (DNA) repair. DNA damage in target cells was found at low concentrations of H2O2 (20-80 microM) in many cell types. Strand breaks and base hydroxylation were observed, resulting in the generation of hydroxyl radicals (.OH) from H2O2, in the presence of a transition metal. DNA damage resulted in either cell injury and death or mutations of the base sequence and amino acid residues. These latter effects led to malignant transformations in cultured cells in both tissue cultures of the cells, and in vivo in athymic mice. Exposure of a proto-oncogene, K-ras 4B, also led to the development of a malignant transformation by virtue of mutations in codon positions 12 and 61. Thus, oxidant effects on target cells can damage multiple functional pathways inside the cells, as well as give rise to malignant transformation via DNA damage.

Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury.

To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage-like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors.

Mechanisms of hypochlorite injury of target cells.

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.

Immunologic Tissue Injury Mediated by Neutrophilic Leukocytes

Publisher Summary This chapter discusses the neutrophilic leukocyte because of growing evidence for an important role for this cell type. It discusses the significance of the various mechanisms to human disease and the mediation of acute immunologic injury of tissues in which certain proteins from the plasma, together with certain cellular factors, notably the neutrophilic leukocytes, play significant and interdependent roles. This pathway of mediation in present state of knowledge is distinguishable from other systems of mediation by the principal cell involved, the neutrophile. The pathways of mediation that eventuate in injury of a particular structure in tissues are discussed. Evidence of immunologically induced, neutrophile-mediated injury in human disease is circumspect at present. Direct evidence implicating antigens and antibodies as inciting agents in human disease is accumulated, for example, in the pathogenesis of various forms of glomerulonephritis. In acute and chronic human glomerulonephritis, the deposition of γ-globulin and complement has been well-recorded.