Release Of Toxic Oxygen Metabolites Research Articles

Injection of ANCA-No Neutrophils, No Glomerular Damage: The Role of Neutrophils in the Induction of Glomerulonephritis by Anti-Myeloperoxidase Antibodies. Am J Pathol 167: 39-45, 2005.

Since the seminal observations of Davies (1) and later of van der Woude (2), it is well known that antineutrophil cytoplasmic antibodies (ANCA) are found in approximately 80% of patients with pauci-immune necrotizing/crescentic glomerulonephritis and with systemic small vessel vasculitis. The most common epitopes recognized by ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3) (3), the former more frequently, but not uniquely, in Wegener’s granulomatosis, the latter mostly in microscopic polyangiitis (4). ANCA stimulate cytokine-primed neutrophils and monocytes, causing for instance respiratory burst, release of toxic granule constituents such as oxygen metabolites and proteinases, as well as endothelial cell injury (5). However, it had long remained controversial whether ANCA are just a marker of disease or have a causal role in the genesis of necrotizing and crescentic glomerulonephritis. The elegant animal model of Xiao et al. (6) had provided definite proof that ANCA were both necessary and sufficient to cause necrotizing and crescentic glomerulonephritis, as well as systemic necrotizing arteritis and hemorrhagic pulmonary capillaritis. For this purpose the authors used MPO knockout mice for which mouse MPO is a “foreign” substance. They immunized these mice with mouse MPO to obtain mouse MPO antibodies. In a second step, mice which were unable to launch an immune response against such mouse MPO antibodies, i.e., knockout mice with a deletion of the recombinase activating gene 2 and therefore devoid of functional B and T cells, received purified mouse anti-MPO IgG or control IgG by intravenous injection. This maneuver reproduced the features of microscopic polyangiitis, i.e., focal necrotizing and crescentic glomerulonephritis with fibrinoid necrosis, crescent formation, and absence or paucity of glomerular IgG deposits. The same outcome was seen when mouse anti-MPO splenocytes were injected. These experiments with injection of mouse anti-MPO IgG or adoptive transfer of mouse anti-MPO splenocytes left no doubt that MPO antibodies alone were able to cause the disease in the absence of B or T cells. What had remained unclear, however, was the cellular target through which MPO antibodies mediated glomerular and vascular injury. To chase down the culprit and to provide evidence that indeed neutrophils—and not for instance endothelial cells, as postulated by others—were the target of the MPO antibodies and the key effector cells, the authors now recently carried out an additional experiment. They used the model of the neutropenic mouse. After injection of NIMP-R14, a rat monoclonal antibody, the mice were selectively depleted of circulating neutrophils, the number decreasing from 14% to 1%. Neutropenic mice or control mice received a low (50 μg/g body weight) or high (50 μg/g body weight on day 0 and day 3) dose of anti-MPO IgG by intravenous injection. Control mice received an injection of bovine serum albumin. Five days after injection of anti-MPO IgG, mice without neutrophil depletion developed hematuria, proteinuria, and leucocyturia. Renal histology documented focal glomerular necrosis and glomerular crescents. Glomerular neutrophil phenotyping revealed glomerular infiltration by neutrophils in foci of inflammation and necrosis as well as in a few afferent arterioles, while monocytes/macrophages were mainly observed in glomerular crescents. With the higher dose, blood urea nitrogen was increased as well, but interestingly necroses were seen on average only in 17.8 ± 7.8% of glomeruli. All these lesions were attenuated or even absent in neutropenic mice receiving the anti-MPO IgG. The observation of no glomerular injury in the absence of neutrophils points to the neutrophil as the key effector cell in the induction of the acute glomerular injury of MPO-induced glomerulonephritis, although an ancillary role of monocytes is not completely excluded. The smoking gun points to the neutrophil, and this fits in nicely with past observations documenting the ability of human MPO ANCA and PR3 ANCA to activate and degranulate neutrophils causing the release of toxic oxygen metabolites (7), proteases, nitric oxide (8) and inflammatory cytokines (9), facilitating attachment and killing of endothelial cells (10,11). Why is the observation of Xiao et al. important? It points to maneuvers interfering with ANCA-induced neutrophil recruitment and activation as potential therapeutic targets, if this can be achieved safely and without inducing harm.

Pulmonary oedema after venous air embolism during craniotomy

Pulmonary oedema after venous air embolism during craniotomy

A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils.

To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-. production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1, 2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-. production. This increase was associated with the translocation of cytosolic factors p47(phox) and p67(phox) of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-. production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

Crohn's disease monocytes are primed for accentuated release of toxic oxygen metabolites

Background: Inflammatory bowel disease occurs in regions of the intestine characterized by a bowel content high in bacteria. Intestinal bacteria synthesize cell wall products such as lipopolysaccharide; when normal monocytes or macrophages come in contact with these products, they can be primed to release a number of inflammatory mediators. Mediators such as toxic oxygen metabolites released as part of the respiratory burst may contribute to inflammatory tissue damage. The aim of this study was to determine if monocytes from patients with Crohn's disease are primed by lipopolysaccharide for a greater respiratory burst. Methods: The generation of superoxide anion was measured by superoxide dismutase inhibitable reduction of ferricytochrome c. Results: Freshly isolated monocytes from active untreated Crohn's disease patients (n = 8) showed enhanced stimulated release of superoxide anion when compared with normal monocytes (n = 15; 3.80 ± 0.12 vs. 1.02 ± 0.06 nmol/5 min; P < 0.001). We tested the hypothesis that the monocyte priming factor in Crohn's disease serum may be lipopolysaccharide by showing that Crohn's disease serum lost its ability to prime normal monocytes after lipopolysaccharide was removed (0.25 ± 0.25 nmol/5 min, P < 0.001). Conclusions: These studies indicate that bacterial cell wall products may be important proinflammatory molecules involved in the initiation and/or perpetuation of Crohn's disease.

The function of granulocytes and alveolar macrophages following a single dose of endotoxin in rabbits. Effects of methylprednisolone

It is a generally held opinion that steroids attenuate the activation of phagocytes. However, this statement has its limitations; in rabbit endotoxemia, for instance, steroids enhance the procoagulant activity of monocytes. The present study aimed to investigate the release of toxic oxygen metabolites (TOM) from granulocytes and alveolar macrophages 24 h after endotoxin injection in rabbits, and the effect of concomitant injection of methylprednisolone (MP). Release of TOM was assessed by peak chemiluminescence (CLP). Expression of thromboplastin activity by alveolar macrophages was determined as well, employing a recognized method for assessment of activity. In terms of mean +/- s.e.mean, endotoxin increased granulocyte count from a baseline value of 1.8 +/- 0.2 X 10(6) cells/ml to 3.7 +/- 1 X 10(6) cells/ml, which increased further to 9.8 +/- 2.5 X 10(6) cells/ml following administration of MP. Whereas endotoxin given alone caused no significant change in granulocyte CLP, additional administration of MP increased CLP from 1723 +/- 389 to 16610 +/- 8428 counts. On the other hand, MP attenuated an endotoxin-induced increase in both CLP and thromboplastin activity of alveolar macrophages. Thus, MP appears to have a proinflammatory effect on circulating granulocytes in rabbit endotoxemia, simultaneously depressing the function of stationary macrophages. This may suggest an injurious effect of MP in rabbit endotoxemia.

Alterations of the Respiratory Burst of Polymorphonuclear Leukocytes from Diabetic Children A Chemiluminescence Study

The respiratory burst of polymorphonuclear leukocytes was investigated in 24 children with insulin dependent diabetes mellitus and 24 healthy controls. This oxygen dependent, membrane associated process generates a number of toxic oxygen metabolites which are implicated in the pathogenesis of endothelial damage. The activity of polymorphonuclear leukocytes was studied in terms of luminol amplified chemiluminescence. It was found that the resting luminol amplified chemiluminescence activity of isolated polymorphonuclear leukocytes from diabetic children was significantly higher than that of controls (342,000 +/- 174,000 cpm vs. 165,000 +/- 82,000 cpm, p less than 0.01). The addition of respiratory burst inhibitors caused a significant reduction of basal chemiluminescence (greater than 80%). When the ratio of phorbol myristate acetate stimulated activity to basal activity was calculated and used as an activation index, it was found to be significantly reduced in diabetics relative to controls (4.29 +/- 2.46 vs. 8.34 +/- 3.21, p less than 0.01). These observations suggest that increased release of toxic oxygen metabolites from polymorphonuclear leukocytes in diabetic subjects may play a role in the development of diabetic angiopathies.

The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively.

Occupancy of specific receptors on neutrophils by adenosine or its analogues diminishes the stimulated release of toxic oxygen metabolites from neutrophils, while paradoxically promoting chemotaxis. We now report evidence that two distinct adenosine receptors are found on neutrophils (presumably the A1 and A2 receptors of other cell types). These adenosine receptors modulate chemotaxis and O2- generation, respectively. N6-Cyclopentyladenosine (CPA), a selective A1 agonist, promoted neutrophil chemotaxis to the chemoattractant FMLP as well as or better than 5'N-ethylcarboxamidoadenosine (NECA). In contrast, CPA did not inhibit O2- generation stimulated by FMLP. Pertussis toxin completely abolished promotion of chemotaxis by CPA but enhanced inhibition by NECA of O2- generation. Disruption of microtubules by colchicine or vinblastine also abrogated the enhancement by NECA of chemotaxis whereas these agents did not markedly interfere with inhibition by NECA of O2- generation. FMLP receptors, once they have bound ligand, shift to a high affinity state and become associated with the cytoskeleton. NECA significantly increased association of [3H]FMLP with cytoskeletal preparations as it inhibited O2-. Disruption of microtubules did not prevent NECA from increasing association of [3H]FMLP with cytoskeletal preparations. Additionally, CPA (A1 agonist) did not increase binding of [3H]FMLP to the cytoskeleton as well as NECA (A2 agonist). These studies indicate that occupancy of one class of adenosine receptors (A1) promotes chemotaxis by a mechanism requiring intact microtubules and G proteins whereas engagement of a second class of receptors (A2) inhibits O2- generation. Signalling via A2 receptors is independent of microtubules, insensitive to pertussis toxin and is associated with binding of [3H]FMLP to cytoskeletal preparations.

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H2O2, O2- or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O2- generation and O2 consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, O2- generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of O2- generation is similar in the presence or absence of cytochalasin B (IC50 = 53 +/- 19 and 16 +/- 5 nM, respectively, P = NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O2 consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O2 consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O2- release and O2 consumption (10 +/- 5 and 5 +/- 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O2 consumption in parallel with inhibition of O2- release.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide production by neutrophils in a model of adult respiratory distress syndrome.

Neutrophils (polymorphonuclear leukocytes [PMNs]) are thought to contribute to the pathophysiology of adult respiratory distress syndrome (ARDS) by the release of toxic oxygen metabolites. This study investigated superoxide production by circulating and bronchoalveolar lavage (BAL) PMNs in a rat model of ARDS induced by chronic Escherichia coli (lipopolysaccharide) endotoxemia. Superoxide production was stimulated by fmet-leu-phe, opsonized zymosan, and phorbol myristate acetate. Circulating and BAL PMNs from lipopolysaccharide-infused rats compared with PMNs from control rats are primed for nonselective increased superoxide production. The BAL PMNs are not only partially primed to release superoxide on adherence, they concomitantly have a depressed superoxide response to a phagocytic (opsonized zymosan) stimulus. These PMN responses may partially explain both the pulmonary injury and the increased susceptibility to pulmonary infection seen in patients with ARDS.

Role of activation in alveolar macrophage-mediated suppression of the plaque-forming cell response.

Alveolar macrophages (AM) are highly suppressive of the in vitro plaque-forming cell (PFC) response of spleen cells obtained from mice primed with sheep erythrocytes. Comparison of macrophage populations obtained from disparate anatomical sites revealed that although in both cases there was a cell-concentration-dependent suppression of the PFC response, resident AM or AM activated as a result of intravenous injection of Mycobacterium bovis BCG were equally suppressive at the doses examined. Although there was a similar dose-dependent suppression with peritoneal macrophages, BCG-activated cells were more suppressive of the PFC response than were resident cells. In contrast, splenic macrophages at comparable concentrations were not at all suppressive. Resident AM exhibited significantly lower levels of 5'-nucleotidase activity than did resident peritoneal macrophages. Macrophage-mediated suppression of the in vitro PFC response could not be attributed to the release of toxic oxygen metabolites (H2O2, O2- ,and .OH) or prostaglandins, since the addition of catalase, superoxide dismutase, 2-mercaptoethanol, or indomethacin did not completely reverse suppression. These results suggest that the lung microenvironment may maintain AM in an activated state which contributes to their potential immunoregulatory functions.

Role of the adherence-promoting receptors, CR3, LFA-1, and p150,95, in binding of Histoplasma capsulatum by human macrophages.

The principal host cell of H. capsulatum (Hc) is the M phi within which the pathogenic yeast phase of the fungus multiplies during active disease. The initial interaction between Hc yeasts and M phi therefore is a crucial step in the pathogenesis of histoplasmosis. In the present study, we have identified the major receptor mechanism that mediates the attachment of unopsonized Hc yeasts to human monocyte-derived M phi from peripheral blood. Binding of Hc yeasts by M phi is rapid, temperature dependent, and requires both Ca and Mg ions for optimum activity. Recognition of Hc yeasts does not require Fc receptors, mannosyl/fucosyl receptors, beta-glucan receptors, or secretion of C3 by M phi. Studies were performed on the effect of down regulating specific receptors of the CR3/LFA-1/p150,95 adherence-promoting protein family from the apical portion of M phi to determine the effects upon binding of Hc yeasts. Anti-beta chain mAbs that recognize all three of these proteins blocked binding of yeasts. However, removal of individual receptors with antibodies against the alpha polypeptides caused negligible depression of binding, and removal of any pair caused only modest depression. Thus, each of the members of the CR3/LFA-1/p150,95 family is independently capable of binding Hc. The delineation of this new mechanism for nonopsonic recognition by M phi that is exploited by Hc yeasts will aid in future studies to identify the Hc ligand, to elucidate the stoichiometry of CR3/LFA-1/p150,95 binding, and to determine triggering mechanisms for release of toxic oxygen metabolites.

Human endothelial cells modulate granulocyte adherence and chemotaxis.

Although human endothelial cells (EC) and granulocytes interact in several ways, the factors that regulate such interactions are not well defined. In this study we found that EC and their products directly altered granulocyte adherence (GA) and chemotactic activity. The spontaneous adherence of granulocytes to human umbilical vein EC monolayers was significantly reduced at 5, 15 and 30 min if the granulocytes were preincubated with EC that were stimulated by rocking. At 30 min the spontaneous adherence of EC-preincubated granulocytes was 36% of that of control granulocytes (P = 0.004). The augmented adherence stimulated by FMLP was also decreased (54% of control) by preincubation of the granulocytes with rocked EC. The ability of stimulated EC to inhibit GA was attenuated when the EC monolayers that were used for preincubation with the granulocytes were pretreated with the cyclooxygenase inhibitor indomethacin. GA to unstimulated EC was not significantly altered by indomethacin or aspirin, suggesting that cyclooxygenase products do not influence GA under resting conditions. Preincubation of granulocytes with rocked EC monolayers or supernatant media from rocked EC monolayers diminished their chemotactic response to FMLP by 45 to 65. This inhibition was also attenuated by pretreatment of the EC with indomethacin. EC supernatant medium caused a rapid increase in granulocyte intracellular cyclic AMP, with a maximum increase to 200% of control at 1 min. These data indicate that stimulated EC release one or more arachidonic acid products that alter spontaneous and inflammatory mediator-stimulated granulocyte activity. Prostacyclin, a major cyclooxygenase product of EC arachidonate, depressed inflammatory mediator-augmented GA to EC monolayers and chemotaxis when present in nanomolar concentrations. We conclude that EC-derived prostacyclin, alone or in combination with other EC products, alters GA and chemotaxis stimulated by inflammatory mediators. This provides a mechanism by which EC may modulate granulocyte distribution as well as granulocyte responses that are influenced by adherence, such as the release of toxic oxygen metabolites and granular enzymes.

White Blood Cell Count and Haematocrit as Predictors of Coronary Recurrence after Myocardial Infarction

Baseline white blood cell count (WCC) and haematocrit were examined in relation to recurrent coronary events and to all-cause mortality in 2026 persons enrolled in the first Persantin-Aspirin Reinfarction Study (PARIS-1) 2-60 months after myocardial infarction. WCC was strongly related to coronary recurrence (relative risk 3.5 for men with WCC greater than or equal to 9 X 10(9)/l vs men with WCC less than 5 X 10(9)/l) and total mortality (relative risk 2.6). No such relationships were found for haematocrit. WCC correlated also with cigarette-smoking, diuretic use, serum cholesterol and uric acid; however, the associations with coronary recurrence and total mortality persisted on multiple linear and logistic regression analysis including these variables and treatment group (p less than 0.001). WCC is therefore an easily-measured prognostic variable in survivors of myocardial infarction. Furthermore, we suggest that white blood cells may promote myocardial ischaemia by capillary plugging and/or release of toxic oxygen metabolites.

Dissociation of phagocytosis from stimulation of the oxidative metabolic burst in macrophages.

We explored the relationship between phagocytosis and the triggering of oxidative metabolism using resident, lipopolysaccharide (LPS)-elicited, and bacille Calmette-Guérin (BCG)-activated murine peritoneal macrophages. Sheep erythrocytes (E) coated with IgG [E(IgG)], E coated with IgM and complement [E(IgM)C], and E treated with 1% glutaraldehyde (GE) were used as stimuli. All three types of macrophages released superoxide anion (O2-) during phagocytosis of E(IgG). All macrophage types phagocytosed E(IgM)C and GE but none were stimulated to release O2- during phagocytosis of these particles. Vigorous consumption of oxygen was also stimulated by the ingestion of E(IgG) but not by ingestion of E(IgM)C or GE. E(IgM)C did not scavenge the O2- released from macrophages during phagocytosis of E(IgG) or during exposure to phorbol myristate acetate, and further addition of IgG anti-E antibody to E(IgM)C or GE permitted optimal stimulation of macrophage O2- release by these particles. The capacity of macrophages to ingest E(IgM)C and GE without stimulating the respiratory burst raises the possibility that clearance of particulate matter not opsonized with specific IgG might be achieved without stimulation of the release of toxic oxygen metabolites, and, therefore, without the risk of oxidative damage to the phagocytic cell or surrounding tissue.

Priming of macrophages for enhanced oxidative metabolism by exposure to proteolytic enzymes.

Preincubation for 10-30 min with trypsin, pronase, chymotrypsin, or papain primed macrophages to undergo a twofold to sixfold increase in oxidative metabolism, measured as release of superoxide anion or hydrogen peroxide, during stimulation by phorbol myristate acetate or ingestion of Candida parapsilosis. Preincubation of macrophages with inactivated proteases, nonenzyme proteins, or neuraminidase did not affect their oxidase response. Exposure of macrophages to proteases generated at sites of inflammation could prime these cells for a more effective oxidase response to phagocytosis or for greater tissue damage from release of toxic oxygen metabolites.

Effect of anti-inflammatory drugs and agents that elevate intracellular cyclic AMP on the release of toxic oxygen metabolites by phagocytes: Studies in a model of tissue-bound IgG

Human phagocytes in contact with fixed aggregated IgG emit a burst of oxygen-dependent chemiluminescence and release superoxide anion (O 2 −) and hydrogen peroxide. These oxygen metabolites are highly reactive and could contribute to inflammatory tissue damage. Methylprednisolone and prednisolone inhibited the generation of O 2 − and chemiluminescence by neutrophils and monocytes in this model. Indomethacin and aspirin had no definite effects in either assay. Chloroquine had no effect on O 2 release but inhibited chemiluminescence. Agents that elevate intracellular levels of cyclic AMP (dibutyryl cyclic AMP, prostaglandins E 1 and E 2, and histamine) also decreased elaboration of O 2 − and chemiluminescence, especially when combined with theophylline. Agents that elevate intracellular cyclic GMP had no effect. Prednisolone and mediators of increased cyclic AMP also inhibited the production of O 2 − and chemiluminescence by eosinophils. These results are consistent with the possibility that the anti-inflammatory effects of corticosteroids are due in part to their ability to suppress the release of toxic oxygen metabolites from phagocytic cells at the sites of inflammation. They also suggest that elevation of intracellular levels of cyclic AMP could constitute a natural means of limiting inflammatory tissue damage.