1Division of Enzyme Chemistry, Institute for Enzyme Research,
The University of Tokushima, Tokushima, Japan;2Department
of Gastroenterology, Affiliated Hospital, Nantong Medical
College, Nantong 226001, China;and 3Department of Laboratory
Medicine, The University of Tokushima School of Medicine,
Tokushima, Japan
Abstract: The pathogenesis of influenza encephalopathy or
encephalitis is poorly understood. This review summarizes
our recent studies of the roles played by inflammatory cytokines,
inducible nitric oxide synthase (iNOS), adhesion molecules
and mini-plasmin in influenza encephalitis. After the intranasal
infection of newborn mice with the non-neurotropic strain
of influenza A virus (IAV) Aichi/2/68/H3N2, encephalitis
and severe brain edema were observed within 3-5 days. IAV-RNA
and abnormalities in the blood-brain barrier permeability
were detected in association with an increase in the mRNA
expressions of endothelin-1, iNOS, and tumor necrosis factor-α.
Furthermore, the accumulation in the brain capillaries of
mini-plasmin, which proteolytically induces the viral envelope
fusion activity and allows the virus to enter the cells,
changes the brain from non-susceptible to susceptible to
non-neurotropic IAV multiplication. The accumulation of
mini-plasmin was markedly greater in newborn mice with an
impaired mitochondrial fatty acid metabolism. These inflammatory
mediators and the accumulation of mini-plasmin in the brain
may play an important role in the onset and progression
of IAV encephalitis. J. Med. Invest. 50:1-8, 2003
Keywords:influenza encephalitis;cytokines;nitric oxide synthase;mini-plasmin
INTRODUCTION
The influenza A virus (IAV) is one of the most common infectious
pathogens in human, causing considerable morbidity and mortality
in infants and older persons (1). IAV readily infects and
replicates in the airway epithelial cells, though occasionally
replicates in the central nervous system, particularly in
children younger than 6 years of age. There appears little
doubt as to the encephalitis of patients infected with a
neurovirulent strain of IAV, such as the 1918/1919 pandemic
(2). There are also rare, though often fatal, cases of encephalopathy
due to infection by non-neurovirulent IAV in Reye's syndrome
children treated with antipyretics (3-7). A systemic disorder
of mitochondrial β-oxidation and degeneration of the
liver with fatty infiltration are suspected to be involved
in acute encephalitis or encephalopathy of patients suffering
from Reye's syndrome. However, the pathological roles of
an impaired mitochondrial β-oxidation in the influenza
encephalitis and the mechanism of cerebral IAV invasion
remain unclear. This review focuses on the pathogenesis
of influenza-virus-associated encephalitis or encephalopathy,
and discusses the roles of inflammatory cytokines, inducible
nitric oxide synthase (iNOS) and mini-plasmin in the development
of encephalitis.
INFLUENZA A VIRUS AND ITS PATHOGENESIS
Influenza viruses are enveloped, negative-stranded RNA viruses,
which belong to the family of Orthomyxoviridae, and are
subclassified into A, B and C, of which the A viruses are
the most important pathogens (8). IAV-RNA is composed of
eight segmented genes, which encode for ten different proteins,
including envelope glycoproteins hemagglutinin (HA) and
neuraminidase (NA), matrix protein M1, nucleoprotein, three
polymerases (PB1, PB2 and PA), ion channel protein M2, and
nonstructural proteins (NS1 and NS2). Anti-HA antibodies
inhibit viral attachment, and are classified (H1-H15) according
to their antigenicity. Anti-NA antibodies restrict the viral
replication and are distinguished as nine (N1-N9) subtypes.
Viruses with HA types, H1, H2 and H3, and NA types, N1 and
N2, have been identified in human and H3N2 subtype of IAV
is predominant in Japan (4, 5, 9). During the years 1989-1999,
H3N2 and H1N1subtypes of IAV and B type influenza viruses
were co-circulating in Chinese populations (Fig. 1), while
IAV H3N2 was the predominant strain (76%), associated with
a morbidity as high as 10% in the Beijing area (10). Infection
with IAV affects the upper respiratory tract first, followed
by an acute and diffuse inflammation of the broncho-alveolar
tract. Clinically, IAV infections are accompanied by the
production of endogenous pyrogens, high fever, malaise and
pulmonary complications. The pathogenesis and organ tropism
of the virus are primarily determined by genetic polymorphisms
of its subtypes, and by organ-specific trypsin-type processing
protease(s) for viral envelope glycoprotein HA, i.e. tryptase
Clara, ectopic anionic trypsin and mini-plasmin in the airways,
which induce the membrane fusion activity of a virus in
vivo and allow the viral genome to enter the cells (11-14).
In influenza encephalitis, the heavy immunoreactive deposits
of both virus antigen and mini-plasmin, which amplify the
multiplication of IAV and destructs the blood-brain barrier
(BBB) in vivo, have been exclusively detected in the brain
capillaries of newborn mice infected by IAV. These changes,
as well as cerebral edema, were more prominent in newborn
juvenile visceral steatosis (JVS) mice, which have an inherited
defect of the sodium-dependent carnitine transporter, OCTN2,
in the plasma membrane, than in newborn wild type (WT) mice.
Large amounts of mini-plasmin are generated from plasmin
or plasminogen by granulocyte elastase in pulmonary inflammatory
loci, along with an infiltration of granulocytes and monocytes
(14). Since there was little infiltration of inflammatory
cells in the brain capillaries even after IAV infection,
and mini-plasmin was not detected in the capillaries before
infection, accumulated mini-plasmin in the brain capillaries
might be transferred from lungs with pneumonia. Mini-plasmin
accumulated in the brain capillaries may increase the permeability
of endothelial cells by its proteolytic activity, as well
as transform capillary endothelial cells from non-susceptible
to susceptible to the multiplication of IAV (Yao et al.,
manuscript submitted for publication). Studies of the molecular
mechanisms of mini-plasmin accumulation in the brain capillaries
of mice with an impaired mitochondrial fatty acid metabolism
are currently in progress.
INFLUENZA-ENCEPHALITIS AND REYE'S SYNDROME
Reye's syndrome is characterized by encephalitis and fatty
degeneration of the liver due to an impaired free fatty
acid metabolism and β-oxidation in mitochondria in
children treated with aspirin, ibuprofen and diclofenac,
and, in over 85% of cases, infected with influenza or varicella
(15, 16). To determine whether a disorder of mitochondrial
β-oxidation is a risk factor for influenza encephalopathy
or encephalitis, we infected newborn JVS mice and acquired
carnitine deficiency mice with non-neurotropic IAV/Aichi/2/68
(H3N2), which has been classified as a dominant and epidemic
influenza subtype since 1968. Carnitine is an obligatory
amino acid for the transfer of long-chain fatty acids from
the cytosol to mitochondria. These fatty acids contribute
the major source of energy in the mitochondria, particularly
in patients with high fever, vomiting and anorexia during
the newborn/suckling periods. Antipyretics, such as aspirin,
ibuprofen and diclofenac, have potent anti-inflammatory
effects, though impair the mitochondrial fatty acid metabolism
and generation of ATP. Furthermore, influenza virus proteins,
such as M protein, PB2 and PB1-F2, also cause mitochondrial
damage and inhibition of β-oxidation (17, 18). Therefore
influenza virus infection in combination with antipyretic
treatment may cause a systemic disorder of fatty acid metabolism,
particularly in the newborn/suckling period (19). Newborn
JVS mice have significantly higher numbers of virus-genome
in the brains, accumulation of virus antigen in the capillaries,
and an increased blood-brain barrier permeability after
intranasal infection with non-neurotropic IAV. Mini-plasmin
was prominently accumulated with virus antigen in the brain
capillaries of JVS mice, but only mildly in WT mice. Although
the mechanisms of IAV-associated encephalopathy by antipyretics
have not been clarified, Funato et al. have recently described
a single-base mutation of the CYP2C9 gene, the major cytochrome
P450 gene product that catalyzes diclofenac in human liver,
in one of thirty healthy subjects (20). This mutation in
the CYP2C9 gene may be related to diclofenac-induced influenza-virus-associated
encephalitis or encephalopathy.
AVIAN IAV INFECTION AND ENCEPHALITIS
It is noteworthy that, during the influenza surveillance
of 1998-1999 in China, five strains of avian IAVs were isolated
from patients with influenza-like illness (10). To examine
the infectivity of avian influenza virus in mammalian species,
4-week old ddY mice were infected intranasally with the
H5N1 A/Hong Kong/156/97 (HK156) and A/Hong Kong/483/97 (HK483)
influenza viruses isolated from humans. HK156 and HK483
required 200 and 5 plaque forming units of virus, respectively,
to administer a 50% lethal dose to the mice in a 10 µl
volume of inoculum. Both viruses caused encephalitis and
severe bronchopneumonia (21). While the severity of lung
lesions caused by the viruses was similar, lesions in the
brain caused by HK483 were more extensive than those caused
by HK156. This was concordant with the measurements of brain
homogenates virus titers, which were over 100-fold higher
in HK483- than in HK156-infected mice, while virus titers
in lung homogenates were nearly identical. Both viruses
were detected in heart, liver, spleen and kidney homogenates,
and in the blood of the infected mice. Virus antigen was
sporadically detected by immunohistochemical staining, though
was associated with no degenerative changes in the heart
and liver. The antigen was not detected in the thymus, spleen,
pancreas, kidney or gastrointestinal tract. In contrast,
it was found regularly in adipose tissue attached to these
organs. The adipose tissue showed severe degenerative changes,
and contained high virus titers, similar to those measured
in lungs. Thus, infection with HK156 and HK483 in this mouse
model was pneumo-, neuro- and adipo-tropic, not pantropic.
Furthermore, the neurotropism of HK483 was higher than that
of HK156, which may explain its higher lethality.
CYTOKINE LEVELS AFTER IAV INFECTION
Analyses of the host immune responses are required to study
the molecular pathologic mechanisms of IAV. The infected
epithelial cells and inflammatory leukocytes produce a variety
of chemotactic, proinflammatory, and other immunoregulatory
cytokines. The increase in expression of cytokine genes
is associated with the activation of NF-κB, AP-1,
STAT and IRF signal-transducing molecules in the infected
cells (22-26). We found a significant increase in the expression
of E-selectin, vascular cellular adhesion molecule-1 (V-CAM-1),
macrophage inflammatory protein-2 (MIP-2), inducible nitric
oxide synthase (iNOS) and endothelin-1 (ET-1) mRNAs, along
with an increase in IAV-RNA in the brain of WT mice after
IAV infection, whereas these mRNAs were undetectable in
absence of infection (Fig. 2). Among the mRNAs tested, the
greatest increases in the expressions of ET-1 and iNOS mRNAs
were detected in the brain. These proteins have been reported
to trigger the formation of edema in various tissues. TNF-α
is a pro-inflammatory cytokine that causes apoptotic tissue
injury and a potent inhibitor of mitochondrial respiration.
It prominently induces the mitochondrial permeability transition
(MPT) in living cells, resulting in a necrotic and apoptotic
cell death. Thus, an abrupt increase in TNF-α concentrations
after virus infection in the systemic circulation induces
systemic MPT in multiple organs, and MPT in cerebral capillary
cells cause brain edema in acute IAV encephalopathy or encephalitis
(27). We have found that the concentrations of TNF-α
in the brain of IAV-infected WT newborn mice were significantly
increased on day 5 after inoculation, and that treatment
with diclofenac further increased these concentrations (Fig.
3). Since some cytokines, such as IFN-α/β, IFN-r,
and IL-2, are protective against influenza infection, whereas
others, such as IL-1, TNF-α and IL-6, are involved
in the progression of inflammation (26), further studies
of the concentrations of other cytokines in the brain, and
studies of their mutual interactions are required to elucidate
the pathogenesis of IAV encephalitis.
ROLE OF NITRIC OXIDE
Nitric oxide (NO) has complex and diverse functions in physiologic
and pathophysiologic processes in vivo. NO and oxygen radicals
appear to be key molecules explaining the pathogenesis of
various infectious diseases, including influenza (28). IAV
infection promotes the release of reactive oxygen (ROS)
and nitrogen (RNS) species, such as NO, into the extracellular
space. ROS and RNS induce tissue injury, and treatment with
exogenous antioxidants attenuates tissue damage and lowers
mortality in IAV-infected mice. NO, a product of iNOS, is
produced by activated macrophages, neutrophils, type II
pneumocytes, and airway epithelial cells after virus infection
(29). In the brain of newborn WT mice, the concentrations
of iNOS-mRNA increased over time after virus inoculation
(Fig. 4A), and significant differences in these concentrations
(P<0.05) were measured on day 5 between the infected
and non-infected brains (Fig. 4B). NO contributes to the
antimicrobial host defense. However, these oxygen and nitrogen
reactive intermediates cannot discriminate between exogenous
invading pathogens and the host itself and function as mediators
of nonspecific innate defense against various microbes.
Reactive NO can affect bacteria rather selectively, while
the surrounding normal tissue remains intact (Fig. 4C).
In contrast, in viral infections, free radical mediators
cause nonspecific oxidative injury as well as oxidative
stress to the virus-infected tissues.
ENDOTHELIN ISOPEPTIDES AS INFLAMMATORY MEDIATORS
The endothelins [ETs:ET-1, ET-2 and ET-3 (1-21)], a family
of 21-residue peptide were first isolated from the culture
medium of porcine endothelial cells and shown to be vasoconstrictors
(30). Recent studies indicated that ETs are distributed
in various tissues and cells and serve a variety of physiological
and pathological functions, not only as smooth-muscle constrictors,
but also as inflammatory mediators. Furthermore, the bioactive
ET peptide family has expanded. New, smooth-muscle-constricting,
31-amino acid endothelins [ETs(1-31)], have been discovered
by our group (31, 32). Attention has been attracted to the
new functions of these ET isopeptides, such as chemokine
function for eosinophils, monocytes and neutrophils (33,
34), and mediators of brain edema formation (35). In the
brain, ETs and their receptors localize in neurons, glial
cells and smooth muscle cells, and microvessel endothelial
cells play pathophysiologic roles by modulating neuronal
functions and cerebral blood flow. We found that, after
5 days of IAV inoculation, the concentrations of ET-1 (1-21)
and ET-1 (1-31) in the brain of WT newborn mice were significantly
higher than in non-infected brains. In the lungs, changes
in the concentrations of ET-1 (1-21) were insignificant,
whereas the concentrations of ET-1 (1-31) were significantly
increased. Treatment with diclofenac had no effect on cerebral
ET-1 concentrations. Putting these observations together,
an increase in mRNA concentrations of ET-1 (Fig. 2) and
in cerebral protein concentrations of ET-1 (1-21) and ET-1
(1-31) (Fig. 5) cause the formation of edema in the brain
after IAV infection.
CONCLUSIONS
The pathologicalmechanisms of influenza encephalitis associated with an
increase in the expressions of inflammatory mediators and
accumulation of mini-plasmin in the brain capillaries have
been presented in the light of current information. The
proinflammatory cytokine TNF-α and IAV proteins cause
mitochondrial injury, which, in turn, may stimulate the
gene expression of adhesion molecules, such as V-CAM-1 and
E-selectin, in the brain capillaries, causing the accumulation
of mini-plasmin on the surface of cerebral capillaries.
Mini-plasmin is a processing enzyme of HA of IAV, which
induces the viral envelope fusion activity and allows the
viral genome to enter the cells. This accumulation may potentiate
the multiplication of IAV in the brain capillaries. In addition,
increases in the mRNA expressions of iNOS and ET-1, which
cause brain injury and an increase in the BBB permeability,
lead to severe brain edema in IAV encephalitis or encephalopathy.
A greater understanding of the changes in the expression
of inflammatory cytokines and of the molecular mechanism
of mini-plasmin accumulation in cerebral capillaries will
provide profound insights into several components of influenza
encephalitis.
ACKNOWLEDGMENT
This study was supported in
part by Grants-in-Aid from the Ministry of Education, Science
and Culture (13557014), and Research on Brain Science (H12-Brain-015)
from the Ministry of Health Labor and Welfare of Japan.
REFFERENCES
1. Cox NJ, Subbarao K:Influenza. Lancet. 354:1277-1282,
1999
2. Ward AC:Neurovirulence of influenza A virus. J Neurovirol
2:139-151, 1996
3. Fujimoto S, Kobayashi M, Uemura O, Iwasa
M, Ando T, Katoh T, Nakamura C, Maki N, Togari H, Wada Y:PCR
on cerebrospinal fluid to show influenza- associated acute
encephalopathy or encephalitis. Lancet 352:873-875, 1998
4. Sugaya N:Influenza-associated encephalopathy in Japan:Pathogenesis
and treatment. Pediatrics Int 42:215, 2000
5. Okabe N, Yamashita K, Taniguchi K, Inouye S:Influenza surveillance system of
Japan and acute encephalitis and encephalopathy in the influenza
season. Pediatrics Int 42:187-191, 2000
6. Belay ED, Bresee JS, Holman RC, Khan AS, Shahriari A, Schonberger LB:Reye's
syndrome in the United States from 1981 through 1997. N
Engl J Med 340:1377-1382, 1999
7. Butterworth RF:Antipyretics and Reye's syndrome. Clin Invest Med 21:209-210, 1998
8. Lamb RA, Krug RM:Orthomyxoviridae:the viruses and their
replication. In:Fields BN, Knipe DM, Howley PM, eds. Fields
virology, 3rd ed. Lippincott-Raven Publishers. Philadelphia,
Pa. 1996 pp. 1353-1395.
9. Takahashi M, Tango T:Estimation of excess mortality associated with influenza-epidemics
by age and cause specific death in Japan, 1975-1999. Nippon
Eiseigaku Zasshi (in Japanese) 57:571-584, 2002
10.Guo YJ:Influenza activity in China:1998-1999. Vaccine 20:S28-S35, 2000
11.Kido H, Yokogoshi Y, Sakai K, Tashiro M, Kishino Y, Fukutimi
A, Katunuma N:Isolation and characterization of a novel
trypsin-like protease found in rat bronchiolar epithelial
Clara cells:A possible activator of the viral fusion glycoprotein.
J Biol Chem 267:13573-13579, 1992
12.Towatari T, Ide M, Ohba K, Chiba Y, Murakami M, Shiota M, Kawachi M, Yamada
H, Kido H:Identification of ectopic anionic trypsin I in
rat lungs potentiating pneumotropic virus infectivity and
increased enzyme level after virus infection. Eur J Biochem
269:1-9, 2002
13.Kido H, Murakami M, Oba K, Chen Y, Towatari
T:Cellular proteinases trigger the infectivity of influenza
A and Sendai viruses. Mo Cells 9:235-244, 1999
14.Murakami M, Towatari T, Ohuchi M, Shiota M, Akao M, Okumura Y, Parry
MAA, Kido H:Mini-plasmin found in the epithelial cells of
bronchioles triggers infection by broad-spectrum influenza
A viruses and Sendai virus. Eur J Biochem 268:2847-2855,
2001
15.Crocker JFS, Digout SC, Lee SH, Rozee KR, Renton
K, Field CA, Acott P, Murphy MG:Effect of antipyretics on
mortality due to influenza B virus in a mouse model of Reye's
syndrome. Clin Inves Med 21:192-202, 1998
16.Smith TCG:Reye's syndrome and the use of aspirin. Scott Med J 41:409, 1996
17.Mazo EL, Rusiaev VA, Fedorov AN, Iaroslavtseva NG, Kharitonenkov
IG:Effect of the matrix protein on the influenza virus on
oxidative phosphorylation in preparations of isolated liver
mitochondria from white mice. Vopr Virusol 33:153-157, 1998
18.Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik
I, Basta S, O'Neill R, Schickli J, Palese P:A novel influenza
A virus mitochondrial protein that induces cell death. Nature
Med 7:1306-1312, 2001
19.Murphy MG, Crocker JFS, Her H:Abnormalities
in hepatic fatty-acid metabolism in a surfactant/influenza
B virus mouse model for acute encephalopathy. Biochim Biophys
Acta 1315:208-216, 1996
20.Funato T, Kozawa K, Kaku M:Relationship
of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced
influenza-virus-associated encephalopathy. Rinsho Byori
(in Japanese) 50:140-145, 2002
21.Nishimura H, Itamura S, Iwasaki T, Kurata T, Tashiro M:Characterization of human
influenza A (H5N1) virus infection in mice:neuro-, pneumo-
and adipotropic infection. J Gen Virol 81:2503-2510, 2000
22.Julkunen I, Melen K, Nyqvist M, Pirhonen J, Sareneva
T, Matikainen S:Inflammatory responses in influenza A virus
infection. Vaccine 19:S32-S37, 2001
23.Kaufmann A, Salentin
R, Meyer RG, Bussfeld D, Pauligk C, Fesq H, Hofmann P, Nain
M, Gemsa D, Sprenger H:Defense against influenza A virus
infection:essential role of the chemokine system. Immunobiology
204:603-613, 2001
24.Aiba H, Mochizuki M, Kimura M, Hojo
H:Predictive value of serum interleukin-6 level in influenza
virus-associated encephalopathy. Neurology 57:295-299, 2001
25.Kaiser L, Fritz RS, Straus SE, Gubareva L, Hayden FG:Symptom
pathogenesis during acute influenza:interleukin-6 and other
cytokine responses. J Med Virol 64:262-268, 2001
26.Ito Y, Ichiyama T, Kimura H, Shibata M, Ishiwada N, Kuroki H,
Furukawa S, Morishima T:Detection of influenza virus RNA
by reverse transcription-PCR and proinflammatory cytokines
in influenza-virus-associated encephalopathy. J Med Virol
58:420-425, 1999
27.Kallas EG, Reynolds K, Andrews J, Treanor
JJ, Evans TG:Production of influenza-stimulated tumor necrosis
factor-alpha by monocytes following acute influenza infection
in humans. J Interferon Cytokine Res 19:751-755, 1999
28.Akaike T, Maeda H:Nitric oxide and virus infection. Immunology
101:300-308, 2000
29.Akaike T:Role of free radicals in viral
pathogenesis and mutation. Rev Med Virol 11:87-101, 2001
30.Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi
M, Mitsui Yazaki Y, Goto K, Masaki T:A novel potent vasoconstrictor
peptide produced by vascular endothelial cells. Nature 332:411-415,
1988
31.Nakano A, Kishi F, Minami K, Wakabayashi H, Nakaya
Y, Kido H:Selective conversion of big endothelins to tracheal
smooth muscle-constricting 31-amino acid-length endothelins
by chymase from human mast cells. J Immunol 159:1987-1992,
1997
32.Kido H, Nakano A, Okishima N, Wakabayashi H, Kishi
F, Nakaya Y, Yoshizumi M, Tamaki T:Human chymase, an enzyme
forming novel bioactive 31-amino acid length endothelins.
Biol Chem 379:885-891, 1998
33.Sharmin S, Shiota M, Murata E, Cui P, Kitamura H, Yano M, Kido H:A novel bioactive 31-amino
acid ET-1 peptide stimulates eosinophil recruitment and
increases the levels of eotaxin and IL-5. Inflamm Res 51:195-200,
2002
34.Cui P, Tani K, Kitamura H, Okumura Y, Yano M, Inui
D, Tamaki T, Sone S, Kido H:a novel bioactive 31-amino acid
endothelin-1 is a potent chemotactic peptide for human neutrophils
and monocytes. J Leukoc Biol 70:306-312, 2001
35.Sato M, Noble LJ:Involvement of the endothelin receptor subtype
A in neuronal pathogenesis after traumatic brain injury.
Brain Res 809:39-49, 1998
Received for publication November 14, 2002;accepted December 9, 2002.
Address correspondence and reprint requests to Hiroshi Kido, M.D., & Ph.D., Division
of Enzyme Chemistry, Institute for Enzyme Research, The
University of Tokushima, Kuramoto-cho 3-18-15, Tokushima
770-8503, Japan and Fax:+81-88-633-7424.
|
