LATENT ADENOVIRAL INFECTION IN THE PATHOGENESIS OF
EMPHYSEMA
James C. Hogg and Shizu Hayashi
University of British Columbia Pulmonary Research Laboratory,
St. Paul’s Hospital
Vancouver, British Columbia, Canada V6Z 1Y6
Chronic obstructive lung disease (COPD) was ranked the 4th leading
cause of death in the US in 1993 (Div. Of Vital Statistics, National Center for
Health Statistics, CDC) and 12th among conditions that contribute to the global
burden of disease in 1996 (Murray and Lopez, 1996). Emphysema is an
important component of this syndrome and is defined by abnormal permanent
enlargement of the airspaces distal to the terminal bronchioles accompanied by
destruction of alveolar walls but without obvious fibrosis (Snider et al, 1985).
This destructive process is widely believed to be due to an imbalance between
proteolytic enzymes and their inhibitors present in the cigarette smoke-induced
alveolar inflammation although the cell types, enzymes and inhibitors involved
remain controversial (Janoff et al, 1977; Janoff, 1983; D’Armiento et al, 1992;
Finkelstein et al, 1995; Findlay et al, 1997; Hautakemaki et al, 1997).
An important feature of the role of cigarette smoking in the pathogenesis
of emphysema is that it induces airway and alveolar inflammation in all smokers
but only 10-15% of heavy smokers develop emphysema and airways obstruction
(Fletcher et al, 1976). This suggests that the underlying inflammatory process
responsible for emphysema is influenced by other risk factors and environmental
air pollution, occupational exposure to dusts and fumes, reduced socioeconomic
status, genetic factors, particularly 1-antitrypsin deficiency, low birth weight,
allergies and pulmonary infections have all been proposed as independent risk
factors for the development of COPD (Pride and Burrows, 1995). This brief
review will concentrate on the risk introduced by lower respiratory tract infections
with particular reference to recent observations that latent adenoviral infection is
capable of amplifying cigarette smoke induced lung inflammation.
A British Medical Research Council report in 1965 recommended that for
epidemiological purposes, the clinical spectrum of chronic bronchitis could be
divided into simple hypersecretion, purulent hypersecretion and chronic
obstructive bronchitis (Medical Research Council, 1965). The underlying
hypothesis was that the progressive nature of obstructive airways disease was
based on infection of the respiratory tract where host defense had been
compromised by cigarette smoking. Subsequent studies did not support this
view because they provided evidence that chronic airways obstruction could
develop in smokers who had few if any symptoms of chronic sputum production
(Fletcher et al, 1976). Furthermore, excess sputum production during acute
exacerbations of chronic bronchitis was not associated with an irreversible
decline in lung function following the acute illness and an increased frequency of
these acute exacerbations did not accelerate the long-term decline in lung
function (Higgins et al, 1982). The response to antibiotics suggests that some
acute exacerbations are due to bacterial infections while others are related to
mycoplasma and viruses. In a study of separate well documented exacerbations
where viral etiology was proven, Smith et al (1980) found that influenzal viral
infections were associated with the greatest changes in FEV1 and that smaller
statistically significant changes were associated with parainfluenza virus,
rhinovirus and adenovirus. However, they like others could find no long-term
effects when the acute exacerbations were caused by viral infection.
An alternative hypothesis that childhood respiratory infections accelerate
the decline in lung function associated with cigarette smoking in adulthood was
suggested by epidemiological studies linking data from childhood with adult
disease (Samet et al, 1983). Acute lower respiratory tract infection in childhood
(ALRI) is a major worldwide health problem which ranked first among conditions
contributing to the global burden of disease (Murray and Lopez, 1996). Viral
infections contribute about 20-30% of all cases of ALRI (Avila et al, 1989;
Blanding et al, 1989) and in a survey of a closed community, approximately
53.5% of the cases of viral etiology were attributable to RSV compared to 13.9%
to adenovirus, 7.0% to influenza, 4.7% to parainfluenza, 2.3% to more than one
virus (Avila et al, 1989). In studies of cases being admitted to a pediatric
hospital, viral infection accounted for 36.3% of ALRI with RSV and adenovirus
accounting for the bulk of cases (Videla et al, 1998).
An important feature of the adenovirus compared to the other common
respiratory viruses responsible for ALRI is that its genome is made up of DNA
rather than RNA. A DNA genome provides the adenovirus with the advantage
that portions of its DNA can persist in host cells as either a circular extra
chromosome (plasmid) or by integration into the host DNA after the complete
viral replication has stopped. The ability to persist could be responsible for the
fact that ALRI caused by adenovirus is known to have serious sequelae that
include the Swyer James syndrome (or unilateral hyperlucent lung), permanent
airways obstruction, bronchiectasis, bronchiolitis obliterans and steroid-resistant
asthma (Becroft, 1971; Kollee et al, 1975; Simila et al, 1981; Wenman et al,
1982; Sly et al, 1984; Macek et al, 1994). Recent work suggests that latent
adenoviral infection may amplify cigarette induced airway inflammation and
contribute to the pathogenesis of emphysema.
The agents now known as adenoviruses were first isolated from primary
cell cultures derived from human adenoids (Rowe et al, 1953) and from
secretions obtained from an epidemic of respiratory disease in military recruits
(Hilleman et al, 1954). The convergence of information from these two separate
sources resulted in their being named adenoviruses in 1956 (Enders et al, 1956).
This adenovirus family was rapidly shown to be responsible for a wide range of
infections in mammalian and avian hosts and the discovery that human
adenovirus type 12 produced malignant tumors in newborn hamsters sparked an
early and sustained interest in their molecular biology (Trentin et al, 1962). The
adenovirus was one of the first microorganisms to have its genome sequenced
and although they have not been clearly implicated in the pathogenesis of human
tumors, new knowledge from these studies resulted in a rapid progression in our
understanding of how the adenovirus adheres to host cells, penetrates into the
nucleus and takes over the host cell transcriptional and translational machinery
for the purpose of replicating itself.
The adenoviruses are icosahedral particles that are 70-100 nm in
diameter and consist of a protein shell surrounding a double stranded DNA core
(Norrby, 1966). The genomes of the more than 100 types isolated so far have
the same general features in that there are two origins for DNA replication, 5
early transcription units, two delayed early units and one major late unit which is
processed to generate 5 different families of mRNA (Shenk, 1996). The
replication of the adenovirus is dependent on transcriptional activation which is
initiated by activation proteins encoded by one of the early transcription units
referred to as the E1A gene (Shenk and Flint, 1991; Nevins, 1992). These E1A
proteins dramatically increase the rate of transcription of both the viral genome
and host cell genes by binding to specific proteins in the host cell transcriptional
machinery (Shenk, 1996). These interactions between E1A protein and host
transcriptional elements create conditions that are favourable to viral replication
by inducing quiescent cells to enter the S phase of the cell cycle, modulating
transcription of host genes and either inducing or inhibiting host cell apoptosis
(Shenk, 1996). Early studies in humans using classical hybridization techniques
showed that the viral DNA could be present in tonsils (Green et al, 1979;
Neumann et al, 1987) and peripheral blood lymphocytes (Horvath et al, 1986)
after viral replication had stopped. More recent studies have shown that the
adenoviral DNA from the E1A gene is present in human lungs where it might
amplify the transcription of host genes expressed during cigarette smoke induce
lung inflammation.
The opportunity to use in situ hybridization to study lung tissue from a
group of children who died of ALRI of proven adenovirus etiology showed that
the virus targets epithelial cells in both the conducting airways and the gas
exchanging surface of the lung where it primarily infected the type II cells (Figure
1). The type II cells cover approximately 7% of the alveolar surface, produce
surfactant and are the progenitors of the type I cell that cover the remaining 93%
(Crapo et al, 1982). The recognition that inflammatory cells migrate out of
vessels and into the alveolar airspace by passing between the type I and type II
cells (Walker et al, 1995) puts the type II cell in an ideal position to influence the
inflammatory response in alveolar tissue (Figure 2). Over the past 11 years, we
have conducted experiments to determine if the persistence of latent adenoviral
infection in these cells might amplify cigarette smoke-induced alveolar
inflammation.
In studies designed to determine if E1A DNA persisted in adult human
lungs (Matsuse et al, 1992), we used PCR to look for E1A DNA in the lungs from
patients undergoing lung resection for tumor. We found more E1A DNA in lung
tissue from patients with COPD than in tissue from age and gender matched
controls with similar smoking histories. Subsequent studies with Elliott et al
(1995) using immunohistochemistry demonstrated the E1A protein in both
conducting airway epithelial cells and the type II cells on the gas exchanging
surface of the lung (Figure 3). These human studies encouraged us to initiate an
investigation of type II-like cells in culture to determine if they expressed
mediators that influence inflammatory cell migration and whether this expression
might be upregulated by the presence of E1A.
The A549 cell line is derived from a peripheral lung carcinoma that has
many type II cell characteristics. When these cells are transfected with E1A
DNA, they demonstrate lamellar bodies and tight junctions consistent with a type
II alveolar cell phenotype (Elliott et al, 1996). When challenged with
inflammatory stimuli, the cells transfected with E1A produced excess ICAM-1
(Keicho et al, 1997a) and IL-8 (Keicho et al, 1997b) compared to control cells
that were transfected with the same cassette that did not contain E1A DNA.
Subsequent studies of these cells have demonstrated that the presence of E1A
protein leads to the activation of the transcription factor NF- B (Siebenlist et al,
1994) to initiate these changes (Keicho et al., 1997c) and very recent studies
have shown that the excess ICAM-1 and IL-8 production induced by TNF- is
steroid resistant when E1A is present (Jamil et al, 1999). The observations
suggest that latent adenoviral infection of type II cells in situ might provide a
mechanism that could amplify cigarette smoke-induced lung inflammation by
inducing an overexpression of the genes that regulate inflammation.
To test this hypothesis, we used a method developed some years ago for
exposing guinea pigs to cigarette smoke (Simani et al, 1974). This exposure
causes an acute cigarette smoke-induced lung inflammation involving the lower
airways and gas exchanging surface (Hulbert et al, 1981). Wright et al (1990)
have subsequently shown that chronic exposure using this method results in the
development of centriacinar emphysema. We are currently pursuing
experiments designed to determine whether or not latent adenoviral infection of
the guinea pig lung would result in excess alveolar inflammation and a more
rapid onset of emphysema in cigarette smoke-exposed animals.
In a series of experiments with Vitalis et al (1996) where human
adenovirus 5 was introduced into the nasal passages of guinea pigs, we found
that the virus entered the lung and replicated. In situ hybridization studies of lung
tissue during this acute infection showed that the replicating adenovirus was
located in the nucleus of both conducting airway and alveolar type II epithelial
cells. The animals seroconverted to the presence of the virus and PCR studies
conducted on DNA from their lung tissue 35-40 days later when the virus was no
longer replicating showed that the E1A DNA and protein continued to be present
in lung tissue. In subsequent studies when these animals with latent adenoviral
infection were exposed to a single dose of cigarette smoke, they developed a
much greater lung inflammatory response than the sham infected control group
(Vitalis et al, 1998). Studies currently underway on animals chronically exposed
to cigarette smoke will determine if the latent infection also accelerates the
development of emphysema.
A collaboration with Dr. Robert Rogers and his group from Pittsburgh has
allowed us to compare lung tissue from patients with severe emphysema
undergoing lung volume reduction surgery to lung tissue from patients requiring
resection for tumor. Our preliminary findings (Elliott et al, 1998) suggest that
when patients are matched for age, gender and smoking history, the progression
from normal lung function with very minimal emphysematous destruction to
severe functional abnormality where there is advanced emphysema is associated
with a greater inflammatory response in the lung and a greater prevalence of
E1A protein in the type II alveolar cell. This association is consistent with a
causal relationship between latent adenoviral infection and the type II cell, an
excess cigarette smoke-induced inflammatory response and the progressive
development of emphysema.
The cigarette smoking habit is well established as the number one risk
factor for the development of emphysema and chronic airways obstruction.
Respiratory tract infections are postulated to increase this risk but there is no
evidence to support the concept that either viral or other causes of the acute
exacerbations irreversibly reduce lung function or that the frequency of these
exacerbations have any effect on long-term decline in lung function (Higgins et
al, 1982). The epidemiological studies showing that childhood lower respiratory
tract infections add additional risk of developing COPD by the cigarette smoking
habit have not been followed by clear explanations of how this might happen.
The studies reviewed here indicate that the adenovirus accounts for a substantial
percentage of childhood ALRI produced by viruses and suggest a mechanism by
which this virus may persist in type II cells and express proteins capable of
amplifying cigarette smoke-induced lung inflammation.
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