Intravascular neutrophil activation due to carbon monoxide poisoning.
ABSTRACT We hypothesized that platelet-neutrophil interactions occur as a result of acute carbon monoxide (CO) poisoning, and subsequent neutrophil activation triggers events that cause neurologic sequelae.
To identify platelet-neutrophil interactions and neutrophil activation in patients and in animal models, and to establish the association between these intravascular events and changes linked to CO-mediated neurologic sequelae in an animal model.
Blood was obtained from 50 consecutive patients. Abnormalities were variable depending on the carboxyhemoglobin level at study admission and duration of CO exposure. Platelet-neutrophil aggregates were detected and plasma myeloperoxidase (MPO) concentration was significantly elevated in those with confirmed CO poisoning. Among patients exposed to CO for over 3 h, flow cytometry scans of neutrophils revealed increased surface expression of CD18 and, in some groups, MPO on the cell surface. Animal models revealed consistent evidence of platelet-neutrophil aggregates, neutrophil activation and surface MPO, and plasma MPO elevation. MPO was deposited along the brain vascular lining and colocalized with nitrotyrosine. CO poisoning caused abnormalities in the charge pattern of myelin basic protein (MBP), changes linked to adaptive immunologic responses responsible for neurologic sequelae in this model. Changes did not occur in thrombocytopenic rats, those receiving tirofiban to inhibit platelet-neutrophil interactions, or those receiving L-nitroarginine methyl ester to inhibit nitric oxide synthesis. Alterations in MBP did not occur in CO-poisoned knockout mice lacking MPO.
Acute CO poisoning causes intravascular neutrophil activation due to interactions with platelets. MPO liberated by neutrophils mediates perivascular oxidative stress, which is linked to immune-mediated neurologic sequelae.
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ABSTRACT: This study was conducted with rats to assess the involvement of leukocytes in a model of CO-mediated brain injury. Myeloperoxidase activity, measured as an index of leukocyte sequestration, was found to be increased 10-fold in brain microvessel segments prepared from rats immediately or 90 min after exposure to CO. Fluorescence and light microscopic examinations revealed leukocytes in microvessels taken from CO-poisoned rats, but not in that from control rats. Studies were then conducted with rats that had been made leukopenic or treated with monoclonal anti-CD-18 F(ab')2 fragments to inhibit leukocyte adherence to the vasculature. Neither of these groups of animals exhibited the biochemical changes observed in the brains of sham-treated rats: conversion of xanthine dehydrogenase (XD) to sulfhydryl-irreversible xanthine oxidase (XO), and lipid peroxidation, at 90 min following CO poisoning. Treatment with a synthetic serine protease inhibitor, gabexate mesylate, also prevented these biochemical changes if administered immediately after CO poisoning, but the agent did not inhibit leukocyte sequestration. Rats depleted of XD and XO by a tungsten diet, and those treated with allopurinol to inhibit XD and XO, also exhibited at least a 10-fold increase in myeloperoxidase activity in microvessels immediately after CO poisoning, but only a 5-fold increase at 90 min. In vitro studies demonstrated that B2 integrin-dependent polymorphonuclear leukocyte adherence was impaired immediately following CO poisoning although the adherence molecules were expressed on the membrane surface. Adherence function normalized by 45 min. The results suggest that leukocytes are responsible for the development of biochemical changes in brain following CO poisoning, and the sequence of events is as follows: leukocyte sequestration in the microvasculature, B2 integrin-dependent adherence, protease-mediated conversion of XD to XO, O2 radical-dependent lipid peroxidation.Toxicology and Applied Pharmacology 01/1994; 123(2):234-47. · 4.45 Impact Factor
Article: Functional inhibition of leukocyte B2 integrins by hyperbaric oxygen in carbon monoxide-mediated brain injury in rats.[show abstract] [hide abstract]
ABSTRACT: Exposure to hyperbaric oxygen [3 atmospheres absolute (ATA) for 45 min] inhibited carbon monoxide (CO)-mediated lipid peroxidation in the brains of rats by preventing the conversion of xanthine dehydrogenase to oxidase, a conversion process known to be due to the action of leukocytes. The effect was the same whether treatment was given 24 hr before or up to 45 min after poisoning. Hyperbaric oxygen did not inhibit the initial interaction of leukocytes with brain microvasculature, based on measurements of myeloperoxidase (MPO) in microvessel segments, but persistent adherence, which is due to B2 integrins, did not occur. Exposing rats to 3 ATA pressure (0.21 ATA O2) after CO poisoning had no significant effects. A progressive reduction in brain microvessel MPO titers occurred with exposure to O2 at 1, 2, or 3 ATA after CO poisoning, but 1 ATA O2 treatment did not significantly inhibit xanthine oxidase formation or lipid peroxidation. In vitro studies with polymorphonuclear leukocytes (PMN) from rats exposed to hyperbaric oxygen corroborated the absence of PMN B2 integrin function, but when these cells were stimulated they exhibited normal B2 integrin expression on their surface and also normal elastase release and superoxide radical production. Adherence functions of PMN that do not require B2 integrins appeared to remain intact after exposure to hyperbaric oxygen, as peritoneal neutrophilia in response to a glycogen challenge was not inhibited. B2 integrin function could be restored by incubating cells with 8 bromo cGMP, and incubation with phorbol ester stimulated the adherence function of both control and hyperbaric oxygen-exposed PMN. These results provide a clear mechanism for the inhibition of CO-mediated brain lipid peroxidation by hyperbaric oxygen and indicate that hyperoxia causes a discrete disturbance of PMN adherence function.Toxicology and Applied Pharmacology 01/1994; 123(2):248-56. · 4.45 Impact Factor
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ABSTRACT: Platelet-neutrophil interactions play an important role in thrombotic and inflammatory responses. Although it is well known that adhesion of platelets to neutrophils requires interactions of adhesion molecules on platelets such as P-selectin, or GPIIb/IIIa with their counterparts on neutrophils, little is known on the role of lipid mediators in this response. Here we studied involvement of thromboxane (TX) A2, platelet activating factor (PAF) and cysteinyl leukotrienes (cysLTs) in the mechanisms of platelet-neutrophil adhesion that was induced by thrombin (10-100 mU/ml), fMLP (0.01-1 microM) or LPS (0.001-100 microg/ml). All three stimulators in a concentration- and time-dependent manner induced platelet-neutrophil adhesion as quantified by the method of Jungi et al. [Blood 67(3) (1986) 629]. Platelet-neutrophil adhesion induced by each of the three activators was inhibited by blocking antibodies towards P-selectin, GPIIb/IIIa or CD18, but it was not affected by anti-E selectin antibody. Moreover, platelet-neutrophil adhesion induced by thrombin, fMPL or LPS was inhibited by the inhibitor of cyclooxygenase (aspirin), by TXA2 synthase inhibitor (camonagrel), by PAF receptor antagonist (WEB 2170), by the inhibitor of FLAP (MK 886) and by cysLTs receptors antagonist (MK 571). On the other hand, the selective inhibitor of COX-2 (rofecoxib) as well as the inhibitor of cytochrome P450-dependent monoxygenase (17-ODYA) were ineffective. In summary, adhesion of platelets to neutrophils is regulated not only by specific interaction between adhesion molecules on platelets and neutrophils, but also by lipid mediators such as TXA2, PAF and cysLTs released upon activation of platelets or/and neutrophils.Thrombosis Research 07/2003; 110(5-6):287-92. · 2.44 Impact Factor
Intravascular neutrophil activation due to carbon monoxide poisoning
Stephen R. Thom1,2, Veena M. Bhopale1, Shih-Tsung Han1,3, James M. Clark1, and
Kevin R. Hardy1,2
Institute for Environmental Medicine1, and Department of Emergency Medicine2
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068; and
3Emergency Department, Chang-Gung Memorial Hospital, Taoyuan, Taiwan, R.O.C.
Running title: Platelet-neutrophil adhesion and CO
Subject Category: (33) Poisoning and toxicologic issues in the ICU
Word Count: 3,386 (characters + spaces 23,066)
This publication was made possible by grant number P50AT000428 from the National Center for
Complementary and Alternative Medicine.
This article has an online supplement which is accessible from this issue's table of content
online at www.atsjournals.org
To whom correspondence should be addressed:
Stephen R. Thom, M.D., Ph.D.
Institute for Environmental Medicine, University of Pennsylvania
1 John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104-6068
AJRCCM Articles in Press. Published on August 24, 2006 as doi:10.1164/rccm.200604-557OC
Copyright (C) 2006 by the American Thoracic Society.
Rationale: We hypothesized that platelet-neutrophil interactions occur as a result of acute carbon
monoxide (CO) poisoning and subsequent neutrophil activation triggers events that cause
Objectives: To identify platelet-neutrophil interactions and neutrophil activation in patients and
in animal models, and to establish the association between these intravascular events and
changes linked to CO-mediated neurological sequelae in an animal model.
Measurements and Main Results: Blood was obtained from 50 consecutive patients.
Abnormalities were variable depending upon the admission carboxyhemoglobin level and
duration of CO exposure. Platelet-neutrophil aggregates were detected and plasma
myeloperoxidase (MPO) concentration was significantly elevated in those with confirmed CO
poisoning. Among patients exposed to CO for over 3 hours flow cytometry scans of neutrophils
revealed increased surface expression of CD18 and in some groups, MPO on the cell surface.
Animal models revealed consistent evidence of platelet-neutrophil aggregates, neutrophil
activation and surface MPO, and plasma MPO elevation. MPO was deposited along the brain
vascular lining and co-localized with nitrotyrosine. CO poisoning caused abnormalities in the
charge pattern of myelin basic protein (MBP), changes linked to adaptive immunological
responses responsible for neurological sequelae in this model. Changes did not occur in
thrombocytopenic rats, those receiving tirofiban to inhibit platelet-neutrophil interactions, or L-
nitroarginine methyl ester to inhibit nitric oxide synthesis. Alterations in MBP did not occur in
CO-poisoned knock-out mice lacking MPO.
Conclusions: Acute CO poisoning causes intravascular neutrophil activation due to interactions
with platelets. MPO liberated by neutrophils mediates perivascular oxidative stress that is linked
to immune-mediated neurological sequelae.
Abstract word count: 253; Key words: myeloperoxidase, neuropathology, myelin basic protein
Carbon monoxide (CO) is the leading agent of injury and death by poisoning worldwide (1). An
elevated carboxyhemoglobin (COHb) level can precipitate tissue hypoxia due to direct effects of
CO on hemoglobin and because of impaired perfusion from cardiac dysfunction. High COHb
levels impair mitochondrial electron transport because CO binds to cytochrome c oxidase. In
brain, this has been shown to impair ATP synthesis and increase mitochondrial production of
reactive oxygen species (3,4). Energy production and mitochondrial function are restored after
COHb levels decrease, but the transient changes can cause neuronal necrotic or apoptotic death
(5-7). These processes are likely to be responsible for neurological abnormalities that are
manifested during initial hospitalization of some patients (8-10).
Neurological dysfunction may occur in a delayed fashion. Impairments of concentration and
learning, dementia, cog wheel rigidity, amnesia and/or depression develop in 23 to 76 % of
patients from several days to approximately 4 weeks after poisoning (10-16). The
pathophysiology for these sequelae remains unclear. COHb values correlate poorly with clinical
outcomes and even when CO poisoning appears to be relatively mild, delayed neurological
sequelae may still occur (1, 11-19). Therefore, additional pathophysiological mechanisms
beyond COHb-mediated hypoxia are thought to exist.
The goal of this investigation was to determine whether CO poisoning triggers physical
interactions between circulating platelets and neutrophils, and whether this association initiates
events that can be related to neuropathology. Consideration that CO may stimulate heterotypic
(platelet-neutrophil) aggregation was fueled by observations that CO increases the flux of nitric
oxide (.NO) released from platelets, apparently because CO and .NO compete for hemoprotein
targets (20). When stimulated platelets synthesize .NO or when platelets are artificially loaded
with .NO-donating compounds, platelets and neutrophils aggregate (21). This process is thought
to be related to production of reactive .NO-associated species, such as peroxynitrite, due to
neutrophil-derived superoxide reacting with .NO produced by platelets. Platelet adhesion
molecules can be activated by direct addition of peroxynitrite to platelet suspensions (22). Once
a physical linkage between platelets and neutrophils is established, neutrophils exhibit a marked
increase in oxidative burst and synthesis of additional reactive .NO-derived species (21).
Physical associations between neutrophils and platelets can also precipitate neutrophil
degranulation (23). We hypothesized that these relatively complex platelet-neutrophil
interactions are triggered by CO exposure, and that intravascular neutrophil degranulation will
cause additional perivascular changes that mediate neuropathology.
CO can inhibit homotypic (platelet-platelet) adhesion. At concentrations of 10 to 600 µM CO
inhibits aggregation of platelets in purified suspensions through pathways involving cyclic GMP,
impaired calcium entry, and inhibition of cytochrome P-450 (24-26). The effect of CO on
heterotypic adhesion between platelets and leukocytes has not been investigated in depth.
Cigarette smoke, although clearly a more complex stimulus than pure CO, was reported to
induce aggregates between leukocytes and platelets in hamsters (27). In experimental settings
where homotypic adhesion is inhibited, leukocyte-platelet aggregation is accelerated when
platelets are activated (28).
In this paper, we show that exposure to CO triggers intravascular platelet-neutrophil interactions
that lead to neutrophil degranulation in both experimental animals and patients suffering acute
CO poisoning. In the animal model, we show that the MPO concentration in brain increases due
to CO poisoning, MPO is deposited along the vascular lining, and MPO appears to cause
vascular oxidative stress based on co-localization between MPO and nitrotyrosine. MPO can
catalyze the reaction between nitrite and H2O2to form nitrogen dioxide (.NO2), which will nitrate
local protein tyrosine residues, cause lipid peroxidation, and stimulate expression of endothelial
adhesion molecules (29-32). Co-localization of MPO and nitrotyrosine along the subendothelial
lining has been found in human tissues after several inflammatory diseases (33).
Recently, we demonstrated that acute CO poisoning triggers an immunological response that
causes functional neurological deficits (34). Adduct formation between products of lipid
peroxidation and myelin basic protein (MBP) cause alterations in the three-dimensional structure
and charge pattern of MBP that initiate an adaptive immunological response. In this paper, we
show that neutrophils and platelets are required for CO-mediated charge alterations in MBP. We
have directly linked the alterations in myelin basic protein with MPO by examining responses in
knock-out mice lacking MPO. From these data, we conclude that platelet-neutrophil interactions
and neutrophil degranulation are major components of CO toxicity.
CONDENSED METHODS (word count: 542):