CHAPTER 5. MECHANISMS BY
         WHICH CIGARETTE
         SMOKE ALTERS THE
         STRUCTURE AND
         FUNCTION OF THE
         LUNG

251


CONTENTS

EFFECT OF CIGARETTE SMOKING ON
INFLAMMATORY AND IMMUNE
PROCESSES IN THE LUNG

Introduction

Effect of Smoking on Numbers and Types of
Inflammatory Cells

Effect of Smoking on the Morphology and Function of
Inflammatory Cells

Emphysema
Populations Deficient in Alpha,-antitrypsin
  Alpha,-antitrypsin
Proteolytic Enzymes Inducing Emphysematous
   Change
    Papain
     Pancreatic Elastase
    Polymorphonuclear Leukocyte Elastase
    Alveolar Macrophage Elastase
  Protease-Antiprotease Hypothesis
    Increased Elastase Owing Lo the Cellular
     Response to Smoke
       Number of Cells
       Elastase Content
       Release
     Proximity
       Milieu
    Decreased Antiprotease Owing to Oxidation
    Explanation for Upper Lobe Distribution
    Animal Models of Emphysema
     Spontaneous Emphysema
     Experimentally Induced Emphysema
      Oxides of Nitrogen
      Cadmium Salts
     Cigarette Smoke

253


The Effects of Smoking on Cellular and Immune Defense
Mechanisms

In Vitro Effects of Cigarette Smoke on Inflammatory
and Immune Effector Cells
The Effect of Cigarette Smoke on Antibody
Production

EFFECTS OF CIGARETTE SMOKE ON
AIRWAY MUCOCILIARY FUNCTION

Introduction

Normal Mucociliary Function
  Cilia
  Mucus
Mucociliary Interaction

Effects of Cigarette Smoke on Mucociliary Function
  Short-Term Exposure
     Cilia
     Mucus
    Mucociliary Interaction
Long-Term Exposure
     Cilia
     Mucus
    Mucociliary Interaction
Fractionation and Filtering of Cigarette Smoke
  Effects of Filters

Mucociliary Function in Chronic Bronchitis
  Cilia
  Mucus
Mucociliary Interaction

Summary and Conclusions

References

254


EFFECT OF CIGARETTE SMOKING ON
INFLAMMATORY AND IMMUNE PROCESSES
IN THE LUNG

Cigarette smoke is a complex mixture of several thousand
different constituents that may produce physiologic and pathologic
changes. This discussion focuses on the cellular and immune
responses of the lung to cigarette smoke, the mechanism by which
smoking can cause emphysema, and the impact of smoking on
mucociliary clearance. The last 20 years have witnessed dramatical-
ly increased understanding of cigarette-induced lung injury, particu-
larly emphysema, thus enhancing our understanding of the process
by which cigarette smoking can lead to emphysema.

Introduction

Inhalation of cigarette smoke markedly alters the inflammatory
and immune processes in the lung, leading to increases in the total
number of inflammatory cells and to changes in cell type and
function. These effects of cigarette smoke on lung inflammatory cells
may play a role in decreased pulmonary host defenses against
various microorganisms and the development of lung cancer, chronic
bronchitis, and pulmonary emphysema (USPHS 1971, 1972, 1973,
1974,1975; USDHHS 1981).

Effect of Smoking on Numbers and Types of Inflammatory
Cells

One of the most consistently observed effects of cigarette smoking
on the lung is a marked increase in the numbers of inflammatory
cells, especially at sites of disease. Increased numbers of inflammato-
ry cells have been seen in pathological studies of the lungs of
cigarette smokers, as well as in lungs of animals exposed to cigarette
smoke. In addition, increased numbers of inflammatory cells occur in
bronchoalveolar lavage fluid of cigarette smokers and in lavage fluid
of animals exposed to cigarette smoke.

Spain and Kaufman (1953) noted inflammatory changes in the
lung bronchi of cigarette smokers. Later, Anderson and Foraker
(1961) described the presence of an alveolitis, and McLean (1959)
described the presence of a bronchiolitis in these patients. In an
autopsy study of patients with early emphysema (McLaughlin and
Tueller 19711, numerous abnormal, brownish-pigmented alveolar
macrophages were found in adjacent, otherwise intact parenchyma,
but none were found in normal lungs. Identical pigmented macro-
phages were found in the sputum of patients obtained from
apparently healthy cigarette smokers. The frequency of occurrence
of these macrophages in the tissue appeared to be related to the

255


number of cigarettes consumed. Niewoehner et al. (1974) evaluated
the lungs of young smokers and controls of comparable age from a
population that had experienced sudden nonhospital deaths. In
smokers, a characteristic lesion occurred in the form of respiratory
bronchiolitis associated with clusters of pigmented alveolar macro-
phages. This lesion was present in the lungs of all smokers studied,
but was rarely seen in nonsmokers. Lungs of smokers also showed
small, but significant, increases in mural inflammatory cells and
denuded epithelium in the membranous bronchioles as compared
with controls. The researchers suggested that this respiratory
bronchiolitis may be a precursor of emphysema and may be
responsible for the subtle functional abnormalities that are observed
in young smokers. Mitchell et al. (1976) also noted the presence of
significant amounts of inflammation in the small airways of
cigarette smoker lungs, and Cosio et al. (1978) suggested that the
primary lesion in the small airways was a progressive inflammatory
reaction, leading to fibrosis with connective tissue deposition in the
airway walls. These lesions were closely correlated with abnormali-
ties in pulmonary function.

As noted above, most early investigators concentrated on the role
of the increased numbers of pigmented alveolar macrophages
present at disease sites in cigarette smokers. These pigmented
macrophages, because of their numbers and prominent coloration on
histologic sections, were initially the sole focus of research on the
inflammatory response in these patients. More recently, however,
Ludwig et al. (1983) evaluated the relationship between cigarette
smoking and the accumulation of neutrophils in the lungs of
smoking and nonsmoking humans. Human lungs were obtained from
autopsies of 10 cigarette smokers and 5 nonsmokers who experienced
nonhospital death. These studies indicated a marked increase in
neutrophil infiltration in the lungs of cigarette smokers compared
with nonsmokers, and identified the site of the accumulation as the
alveolar septa. Neutrophils were found in the alveolar walls of
smokers both with and without emphysema. The researchers con-
cluded that a marked neutrophil accumulation occurs in the lungs of
cigarette smokers, that it precedes the development of emphysema,
and that it continues once emphysema is established. They further
suggested that the neutrophils may play a role in the destruction of
the alveolar septa of the lungs in cigarette smokers. The presence of
increased numbers of neutrophils in cigarette smokers' lungs has
also been documented by extracting inflammatory cells from open
lung biopsies of smokers and nonsmokers (Hunninghake and Crystal
1983). A higher percentage of these inflammatory cells were
neutrophils in smokers compared with nonsmokers. Finally, the
association between cigarette smoking and increased numbers of
inflammatory cells, including neutrophils. at disease sites has also

256


been confirmed in numerous animal studies (Frasca et al. 1971;
Dahlgren et al. 1972; Rylander 1974; Park et al. 1977).
Increased numbers of inflammatory cells in the lungs of smokers,
as compared with nonsmokers, have also been observed by all
investigators performing bronchoalveolar lavage studies (Davis et al.
1976; Demarest et al. 1979; Harris et al. 1970, 1975; Hunninghake et
al. 1979a, 1980a; Hunninghake and Crystal 1983; Hunninghake and
Gadek 1981-1982; Hunninghake and Moseley, in press; Reynolds et
al. 1977; Reynolds and Newball 1974, 1976; Rodriquez et al. 1977;
Warr et al. 1976,1977; Warr and Martin 1974,1978). Such increases
have been detected additionally in lavage fluid of animals chronical-
ly exposed to cigarette smoke (Davies et al. 1977; Flint et al. 1971;
Holt et al. 1973). The majority of these studies have demonstrated
increases in both the number of macrophages and the number of
neutrophils, although Hoidal and Niewoehner (1982) found increases
only in the former.

The presence of neutrophils in the lungs of cigarette smokers is of
interest because these cells contain elastase, an enzyme believed to
be important in the pathogenesis of emphysema (Lieberman 1976;
Karlinsky and Snider 1978; Kuhn and Senior 1978; Carp and Janoff
1978; Snider and Korthy 1978; Schuyler et al 1978; Janoff et al. 197'1;
Hunninghake et al. 1979a; Hunninghake and Crystal 1983; Hun-
ninghake and Gadek 1981-1982; Hunninghake and Mosley 1984;
Laurel1 and Eriksson 1963). Alveolar macrophages have also been
implicated as a source of an elastase-like metalloprotease (Harris et
al. 1975; Rodriguez et al. 1977). This enzyme is not inhibited by
alpha,-antitrypsin (a,AT) (Banda and Werb 19811, the major anti-
elastase in the lower respiratory tract (Gadek et al. 1981). Although
macrophages are clearly present in large numbers in the alveolar
structures of smokers (Niewoehner et al. 1974; Harris et al. 19751,
several lines of evidence suggest that neutrophils may play a
significant and perhaps more important role in increasing the
elastase burden of the lungs.

First, neutrophils store and release significantly more elastase
than do alveolar macrophages (Barrett 1977; Rodriguez et al. 1977;
Levine et al. 1976). Comparative estimates of elastase production by
human neutrophils and alveolar macrophages suggest that neutro-
phils are at least 1,000 times more potent elastase producers (Janoff
et al. 1979).

Second, although alveolar macrophages of cigarette smokers have
been shown to release elastase in vitro (Rodriguez et al. 19771, it is
not clear whether the elastase was produced by these cells or was
secreted by other types of cells, such as neutrophils, and subsequent-
ly ingested by the macrophages (Janoff et al. 1977). In this regard,
recent studies by Campbell et al. (1979) and McGowan et al. (1983)
have shown that alveolar macrophages are capable of phagocytosing

257


neutrophil elastase via a receptor-mediated mechanism; some of the
elastase remains enzymatically active for up to 48 hours. These
findings suggest that alveolar macrophages may, in fact, be capable
of both decreasing and increasing the protease burden of the lung.

Third, once a neutrophil has left its vascular space, its lifespan is
only a few hours; when the neutrophil dies, it may release at least a
portion of its preformed enzymes, including elastase. Thus, when a
neutrophil is present within a tissue, it is possible that the tissue will
be exposed not only to the elastase secreted by the neutrophil while
it is functional, but also to the elastase stored by the neutrophil and
released when the neutrophil disintegrates. In this context, the
finding that neutrophils represent only a small percentage of all
inflammatory and immune effector cells in the smoker's lungs would
not preclude the smoker's exposure to a large chronic burden of
neutrophil elastase. In contrast, the alveolar macrophage has a half-
life of months to years (Thomas et al. 19761, and it stores little, if any,
elastase (Rodriguez et al. 1977; Levine et al. 1976).

Macrophages may also play an important role in this process by
secreting a potent chemotactic factor for neutrophils (Hunninghake
and Crystal 1983). This hypothesis is supported by the following
observation: alveolar macrophages of cigarette smokers spontaneous-
ly release a chemotactic factor for neutrophils, whereas alveolar
macrophages of nonsmokers do not. In addition, in vitro exposure to
cigarette smoke particulates results in the release of a chemotactic
factor from the alveolar macrophages of nonsmokers. The migration
of neutrophils to the lung in response to the chemotactic factor may
be augmented by factors in cigarette smoke. In this regard, McCusk-
er et al. (1983) have shown that nicotine is a potent chemokinetic
factor for neutrophils, enhancing the migration of these cells to
other chemotactic factors. Once neutrophils are present in the lung,
they may release elastase, because both cigarette smoke (Blue and
Janoff 1978) and the macrophage-derived chemotactic factor stimu-
late these cells to release the enzyme (Gadek et al. 1979a, b).

The postulated release of elastase by neutrophils could also partly
explain how the number of macrophages are increased in this
disorder. Fragment,s of elastin (which are probably generated by the
release of neutrophil elastase at sites of disease activity) are potent
chemoattractants for blood monocytes, the precursors of alveolar
macrophages (Senior et al. 1980; Hunninghake et al. 1981). These
fragments of elastin possess no chemotactic activity for neutrophils.

Effect of Smoking on the Morphology and Function of
inflammatory Cells

No size differences have been observed between alveolar macro-
phages from smokers and those from nonsmokers when the cells are

258


fixed in suspension immediately after bronchoalveolar lavage (Table
1). Harris and coworkers (1970) observed a mean size of 23.3 pm
(range, 10 to 47 pm) for nonsmokers and 26.4 pm (range, 12 to 53 pm)
for smokers. Reynolds and Newball (1974), using similar methods,
did not find any size differences between smoker and nonsmoker
alveolar macrophages.
The morphology of smoker macrophages clearly differs, however,
from that of nonsmokers (Table 1). Macrophages of smokers show
increased numbers of large lysosomes, phagolysosomes, endoplasmic
reticulum, ribosomes, and Golgi vesicles (Golde 1977; McLemore et
al. 1977; Martin 1973; Warr and Martin 1978; Rasp et al. 1978; Pratt
et al. 1971; Brody and Craighead 1975). These findings are generally
associated with activated mononuclear phagocytes, and these macro-
phages have probably become activated by the ingestion of the
particulates present in cigarette smoke. Smoker macrophages have
pigmented inclusions that appear to have platelike or needlelike
configurations when seen by electronmicroscopy (Golde 1977; Warr
and Martin 1978; Pratt et al. 1971; Brody and Craighead 1975).
Studies of the nature of these inclusions by X-ray analysis suggest
they may be, at least in part, particulates of aluminum silicate
(Brody and Craighead 1975). Together with in vitro studies showing
that alveolar macrophages are activated following phagocytosis of
particulates (Hunninghake et al. 1980a), these findings are compat-
ible with the notion that macrophages of smokers are activated in
vivo.
Alveolar macrophages from cigarette smokers have an increased
ability to generate superoxide anion (Hoidal et al. 1979a, 1980,1981),
the functional effects of which include an increased capacity to kill
lung fibroblasts. These observations suggest that alveolar macro-
phages from cigarette smokers are increasingly able to injure lung
parenchymal cells, and that they may contribute to the observed loss
of lung cells in the alveoli of patients with pulmonary emphysema.
A variety of other effector functions of smokers' alveolar macro-
phages have also been evaluated (Table 1). Alveolar macrophages
from cigarette smokers appear to have a normal or increased ability
to migrate in response to chemotactic factors (Demarest et al. 1979;
Warr and Martin 1974). They differ, however, from normal alveolar
macrophages in several other respects: for example, increased
glucose utilization has been reported in some studies (Harris et al.
1970), but was normal in others (Hoidal et al. 1979a). Oxygen
consumption has been reported to be normal (Hoidal et al. 1979a),
but the protein content of these cells has been increased (Harris et
al. 1975; Warr and Martin 1978). Alveolar macrophages from
smokers release less PGE, and thromboxane B, than normal
macrophages (Laviolette et al. 1981), suggesting that cigarette
smoking induces a lesion in phospholipid hydrolysis or the mecha-


TABLE l.-Cigarette-smoking-induced abnormalities in the
      inflammatory and immune effector systems >
       within human alveolar structures

Parameter                    Findings in smokers

cell types present
Total number of cells
Percent polymorphonuclear leukocytes
Percent T lymphocytes
Percent B lymphocytes
Lymphocyte function
Response to mitogens
Macrophage structure
Diameter
Ruffling of cell surface
Number and size of cytoplasmx structures
Abnormal cytoplasmlc inclusions

Macrophage propertws and function
Surface receptors
  1gGFc
  C3b
Phagwy&ls and killing of microorganisms
  Bacteria
  Fungi
Effector and accessory cell function
responsiveness to chemotactic factors
  Casein
  Activated serum
Function as act-ry cell to lymphocytes
Responsiveness to MIF
Production of neutrophll chemotactlc factor
SecretIon of superoxIde anion
Secretlon of elastase
Release of prostaglandm E, and
  thromboxane B,
Miscellaneous properties and function
Glucose utdlvltion
Oxygen consumption
Protem content
Content of various enzymes
  Eklstase
  Acid protease
  Neutral protease
  Esterrise
  Acid phosphatase
  &glucuromdase
  Lysozyme
  Aryl hydrocarbon hydroxylase
  An@otensmsonvertmg enzyme
Spreading and adherence properties

Plllocytosls
Content of a,-antlproteinase

IIlCEased
Increased
Increased or normal
Normal

Decreased

Normal
Decreased
Increass
Pigmented inclusions,
particulates with plate or
needle-like confwratlon.
presence of aluminum silicate

NOM"1
Decreased

Kormal or decreased
Normal

Increased
Normal
Decreased
Decreased
Increased
Increased
Increased

Decreased

Increased or normal
Normal
Increased

Increased
IllCreased
Normal
Increased
Increased
Increased
Normal or increased
Increased
Increased
Increased in presence of serum.
decreased nylon adherence
Decreased
Increased

SOURCE Adapted from Hunnmghake et al 119791

260


nism regulating hydrolysis. Smoker macrophages also appear to
have increased amounts of various enzymes, including acid protease
(Harris et al. 1975), neutral protease (Harris et al. 1975), esterase
(Harris et al. 1975), acid phosphatase (Martin 1973!, angiotensin-
converting enzyme (Hinman et al. 1979), P-glucuronidase (Martin
1973), lysozyme (Martin 1973), and arylhydrocarbon hydrolase
(Cantrell et al. 1973; Harris et al. 1978; McLemore et al. 1977b, c,
1978; McLemore and Martin 1977). The functional significance of
increased amounts of these enzymes is not entirely clear.

In addition to its effects on the inflammatory and immune effector
cells in the lung, cigarette smoke may also affect the composition of
epithelial surface fluid. For example, some investigators have found
that the amount of immunoglobulin G (IgG) present in lavage fluid is
increased (Reynolds and Newball 1974); others have noted normal
levels (Warr et al. 1977). Interestingly, cigarette smoking appears to
cause a significant decrease in the secretory component of immuno-
globulin A (IgA) in the lavage fluid of some people who smoke
cigarettes (Merrill et al. 1980). This effect most likely indicates a
subtle injury to the epithelium of the lung that produces this factor.
The only additional factors that have been reported to be abnormal
in lavage fluid of cigarette smokers are an increase in the amounts of
fibronectin (Villiger et al. 1981) and a decrease in the function, but
not the amount, of a,AT (Gadek et al. 1979; Janoff et al. 1979). This
latter finding has been disputed by others (Stone et al. 1983).

Emphysema

A number of lines of evidence link the cellular changes described
above with the development of emphysema. They include observa-
tions in populations deficient in a,AT, in animal models of emphyse-
ma, and most important, in human cigarette smokers.

Populations Deficient in Alpha,-antitrypsin

Eriksson (1965) described the characteristic features of a,AT-
deficiency-associated lung disease. Approximately 60 percent of
affected individuals develop symptoms of airways obstruction by age
40, and 90 percent by age 50. Excluding the influence of cigarette
smoking, there is no sexual predominance of disease. Kueppers and
Black (1974) found that dyspnea occurred a decade earlier in
cigarette smokers (35 years in smokers versus 44 years in nonsmok-
ers), and estimated that 70 to 80 percent of all PiZZ persons (where
Pi= protease inhibitor) will develop lung disease. Larsson (1978) has
projected that nearly 60 percent of PiZZ people will ultimately die of
lung-related disease.

Ore11 and Mazodier (1972) reviewed the morphologic features of
a,ATdeficiency-associated emphysema and found primarily the

261


panacinar or panlobular form. Emphysematous lesions may be
distributed uniformly throughout the lungs (Ore11 and Mazodier
1972), but frequently show a predominant lower lobe distribution
(Greenberg et al 1973).

In people genetically deficient in alAT, the increased numbers of
inflammatory cells found in the lungs of smokers probably present
an increased elastase burden to the lung and magnify the protease-
antiprotease imbalance. This may explain the deleterious effects of
cigarette smoke in this population. Kueppers and Black (1974)
reviewed data on the impact of cigarette smoking in people severely
deficient in a,AT and concluded that, in addition to experiencing
earlier onset of respiratory symptoms and pulmonary function
abnormalities, cigarette smokers die at an earlier age from respira-
tory failure than similiarly afflicted nonsmokers. The increased
prevalence of emphysema in populations deficient in alAT, plus the
exacerbation of this lung disease by smoking, suggests that protease-
antiprotease imbalance may also play a role in the development of
emphysema by smokers who are not deficient in a,AT. This
suggestion has resulted in a substantial body of research that has
characterized a,AT, defined the nature of elastase-induced emphyse-
ma, and clarified and supported the protease-antiprotease hypothe-
sis of cigarette-induced emphysematous lung injury.

Alpha,-antitrypsin

The deficient constituent of a,-globulin was initially described by
Schultze et al. (1955) as a,-3,5-glycoprotein but later renamed a,-
antitrypsin (a,AT) when it was found to inhibit trypsin activity
(Schultze et al. 1962). Subsequently, a,AT has been shown to inhibit
a variety of proteolytic enzymes including neutrophil elastase
(Ohlsson 1971), neutrophil collagenase (Tokoro et al. 1972; Ohlsson
1971), cathepsin-G (Travis et al. 1978), chymotrypsin (Travis et al.
1978; Rimon et al. 1966), plasmin (Rimon et al. 1966), thrombin
(Rimon et al. 1966), Hageman factor cofactor (Crawford and Ogston
1974), coagulation factor XI (Heck and Kaplan 1974), acrosin and
kallikrein (Fritz et al. 1972a, b), urokinase (Crawford and Ogston
1974; Clemmensen and Christensen 1976), and renin (Scharpe et al.
1976). Although the range of proteases inhibited by a,AT appears
broad, the association rate constants of these enzymes for a,AT
differ (leukocyte elastase > chymotrypsin > cathepsin-G > trypsin
> plasmin > thrombin) (Beatty et al. 1980), and the inhibitory role
of a,AT against enzymes with low association rate constants, such as
trypsin, may be negligible. The names a,-protease inhibitor or al-
proteinase inhibitor better describe this broader range of inhibitory
functions and are preferred by some authors. In deference to
historical usage and in accord with the recommendations of the

262


Nomenclature Meeting for this substance (Cox et al. 1983), the name
a,AT has been retained in this discussion.
The inhibitor a,AT is a polymorphic plasma protein (Fagerhol and
Cox 1981; Cox and Celhoffer 1974; Cox et al. 1980; Cox 1981;
Fagerhol and Braend 1965) encoded by two codominant autosomal
alleles and inherited as a single Mendelian trait. The basal serum
concentration is genetically determined (Eriksson 1964; Kueppers et
al. 1964; Fagerhol and Gedde-Dahl 1969; Talamo et al. 1966). More
than 31 allelic variants or Pi types (where Pi, or protease inhibitor, is
the symbol assigned the genetic locus of the a,AT allele) have been
identified (Cox and Celhoffer 1974; Cox et al. 1980; Cox 1981). The
variants are designated by capital letters, B through Z, correspond-
ing to their approximate electrophoretic mobility, relative to the
anode, in acid starch gel electrophoresis or their relative positions on
polyacrylamide isoelectric focusing. New variants are named accord-
ing to the conventions established by the Fifth International
Workshop on Gene Mapping and the Nomenclature Meeting for
a,AT (Cox et al. 1980).
The M allele (PiM) has a gene frequency of about 0.9 and is the
most common Pi type in all populations tested (Kueppers 1978). The
a,AT serum concentration in PiMM homozygotes is between 1.3 and
2.2 g/liter (depending on the method of measurement and the purity
of standard) (Kueppers 1968; Jeppsson et al. 1978a) and, by conven-
tion, defines normal. Pi types with decreased circulating levels of
a,AT include (serum concentration expressed as percent normal)
null 0% (Feldman et al. 1975; Talamo et al. 1973), Mmalton and
Mduarte 12% (Cox 1976; Lieberman et al. 1976), Z 15% (Laurel1 and
Eriksson 1963; Fagerhol and Laurel1 1970), P 30% (Fagerhol and
Hauge 1969), S 60% (Fagerhol 1969), and I 68% (Arnaud et al. 1978).
PiZ was the first variant recognized (Laurel1 and Eriksson 1963)
and is the Pi type most frequently associated with a serum deficiency
of a,AT (Kueppers 1978). Its allele frequency varies markedly
between different ethnic and racial groups. In the United States, the
allele frequency is greater than 0.010 in whites but nearly zero in
blacks (Kueppers 1978). Approximately 1 in 2,000 whites is homozy-
gous for the Z gene (Laurel1 and Sveger 1975).
Although a decrease in hepatic synthesis is probably the major
mechanism for quantitatively significant reductions in serum a,AT,
the factors that modulate such synthesis are only partially under-
stood (Morse 1978). Impaired hepatic secretion, as evidenced by the
presence of intrahepatic cytoplasmic inclusions containing accumu-
lations of a,AT polypeptides (Blenkensopp and Haffenden 1977),
occurs in persons with the PiZ genotype. It is uncertain if these
intrahepatic inclusions exert a negative feedback inhibition on the
hepatocyte and thereby retard biosynthesis of a,AT. Intrahepatic
inclusions are not found with the S and null Pi types (Carrel1 et al.


19821, suggesting that decreased synthesis, independent of impaired
secretion, is primarily responsible for the reduced serum levels of
a,AT. Catabolic studies of the PiM and PiZ proteins have identified
similar half-lives in the circulation, 6 to 7 days and 5 days,
respectively (Laurel1 et al. 1977; Jeppsson et al. 1978b). It is therefore
unlikely that accelerated peripheral catabolism contributes signifi-
cantly to serum deficiencies in a,AT.

In addition to quantitative deficiencies in serum a,AT, a reduction
in serum inhibitory capacity could also result from a loss in the
functional activity of a,AT. Most genetic variants, however, are
functionally equivalent to normal a,AT (PiMM) in their capacities to
inhibit both trypsin and elastin (Billingsley and Cox 1982).

The inhibitor a,AT is a low molecular weight (51,000 daltons)
(Mega et al. 1980; Carrel1 et al. 1981; Chan et al. 1976; Pannell et al.
1974; Jeppsson et al. 19781 protein comprised of a single polypeptide
chain containing 394 amino acid residues. Three carbohydrate side
chains are attached, each containing terminal sialic acid residues
(Mega et al. 1980; Carrel1 et al. 1981). The a,AT reacts stoichiometri-
tally with free protease in a ratio of 1:l; one mole of a,AT inhibits
one mole of protease and yields a stable complex (Cohen 1973). An in
vitro study (James and Cohen 1978) found, however, that complete
inhibition of elastase requires molar ratios of a,AT to elastase
greater than 2.2:1. This phenomenon may be explained by elastase
having two major sites of attack on a,AT. Attack against one site
leads to a conformational change in a,AT and inhibition of elastase,
whereas attack against the other site results in cleavage and
inactivation of a,AT. The a,AT-protease complexes that form during
protease inhibition are not reutilized by the body (Balldin et al.
19781, and the body supplies of a,AT are replenished via de novo
synthesis by the liver.

In addition to hepatic biosynthesis, a,AT is synthesized by at least
two other endogenous sources. Both human peripheral lymphocytes
and rat alveolar macrophages have been shown to synthesize a,AT.
Ikuta et al. (1982) demonstrated that concanavalin A-stimulated
monocytes interact with human peripheral lymphocytes, causing a
threefold increase in a,AT synthesis. White et al. (1981) cultured rat
alveolar macrophages and recovered newly synthesized radio-labeled
FS)a,AT from the cell culture medium. Macrophages and lympho-
cytes, by virtue of their close physical proximity to the sites of
connective tissue injury, may play a significant role in defense
against proteolytic destruction. The physiologic significance of
extrahepatic synthesis of a,AT remains speculative, however.

While certain chemical and physiological aspects of a,AT are
clear, the exact biochemical mechanism by which it causes protease
inhibition is uncertain. It is generally agreed that the reactive center
of a,AT is located on a single serine-methionine segment peptide

264


bond on the carboxyl-terminal end (Carrel1 et al. 1982; Kurachi et al.
1981).

Proteolytic Enzymes Inducing Emphysematous Change
Proteolytic enzymes have been a major focus of investigation
following the demonstration by Gross et al. (1965) of papain's ability
to induce emphysematous changes in rats.

Papain

Papain, a proteolytic enzyme with a broad range of substrate
specificities (Bergmann and Fruton 1941; Kimmel and Smith 1953),
reproducibly causes emphysema-like lesions in a variety of experi-
mental animals following aerosolization or intracheal instillation
(Gross et al. 1965; Palecek et al. 1967; Goldring et al. 1968; Caldwell
1971; Pushpakom et al. 1970; Marco et al. 1969). A number of studies
have helped to clarify the critical importance of elastolysis in
papain-induced emphysema.

Snider et al. (1974) tested amorphous and crystalline forms of
papain and found that the emphysema-inducing properties of these
preparations were directly proportional to their abilities to degrade
and solubilize elastin. Heat inactivation of papain destroyed its
emphysema-inducing capabilities. Similarly, intratracheal pretreat-
ment of hamsters with human a,AT, an inhibitor of papain
elastolytic activity, ameliorates papain-induced emphysematous
changes (Martorana and Share 1976). Furthermore, Blackwood et al.
(1973) showed that the elastolytic activities of several microbial
enzymes, rather than their nonspecific protease activities, correlate
best with the enzyme's ability to induce emphysematous changes
following intravenous administration to rats. Snider et al. (1977)
showed that enzymes lacking elastolytic activity, such as collagenase
or trypsin, do not produce emphysema in hamsters.

Whereas these studies support the notion that the early histologic
changes induced by papain are a direct consequence of its elastolytic
activity, they do not preclude the possibility that endogenous factors
may contribute to subsequent disease progression. Snider and
Sherter (1977) noted a gradual increase in static lung volumes in
hamsters following a single intratracheal injection of pancreatic
elastase. Stone et al. (1979) followed the fate of tritium-labeled
pancreatic elastase and found that enzymatically active prepara-
tions are retained longer within the lung than inactive preparations,
and that 1qGguanidated elastase remains bound to lung matrix for at
least 96 hours. This suggests that tissue-bound elastase may continue
to digest elastin for extended periods of time. Martorana et al. (19821
found no progression in the mean linear intercept measurements or
internal surface areas in the lungs of papain-treated dogs between 3

265

480-144 0 - 85 - 13


and 6 months after treatment. However, the mean pulmonary
arterial pressure and pulmonary arteriolar resistance did increase
during this interval.

Papain-treated animals exhibit the expected physiologic changes
of emphysema: increased RV, FRC, and TLC, decreased elastic recoil,
increased static lung compliance at middle and low lung volumes,
and reduced diffusing capacity (DLcoVA and Delco) (Caldwell 1971;
Pushpakom et al. 1970; Marco et al. 1969; Giles et al. 1970; Johanson
and Pierce 1973). Studies by Kobrle et al. (1982) have shown that
following papain administration the elastic fibers are disrupted and
that the elastin content of the lung initially decreases, but later
returns to normal after a period of accelerated synthesis. The newly
synthesized fibers are disordered; however (Kuhn and Senior 1978;
Kuhn and Starcher 1980).

Pancreatic Elastase

The ability of porcine pancreatic elastase to rapidly hydrolyze
insoluble elastin (Partridge and Davis 1955) and its commercial
availability in a highly purified crystalline form have led to its
extensive use as an experimental agent for inducing emphysema in
animals (Karlinsky and Snider 1978). Lesions resembling human
panacinar emphysema can be induced in hamsters within 2 hours of
intratracheal instillation of pancreatic elastase (Kaplan et al. 1973).
The severity of the lesions, as assessed by histologic or physiologic
criteria, is dose related (Raub et al. 1982), with adult animals being
more susceptible to pancreatic elastase than young animals (Lucey
and Clark 1982). Within a few hours of intratracheal instillation in
hamsters, hemorrhagic lesions develop and an influx of polymorpho-
nuclear leukocytes is seen (Hayes et al. 1975; Kuhn and Tavassoli
1976). Digestion of elastin fibers is apparent in the pleura and in the
alveolar walls by 4 hours, but is more extensive at 24 and 48 hours
(Kuhn et al. 1976). By day 4, there is a diminution in the number of
polymorphonuclear leukocytes (PMNs), but many macrophages
remain (Morris et al. 1981). The hemorrhage and cellular infiltration
resolves within 3 weeks, and the ensuing lesions resemble panacinar
emphysema (Kuhn et al. 1976). Over 95 percent of the detectable
urinary excretion of desmosine and isodesmosine, amino acid
markers of in vivo elastolysis, appears within 2 days of elastase
instillation; only small amounts can be detected by day 3 (Goldstein
and &archer 1977). Kucich et al. (1980) developed a hemagglutina-
tion inhibition assay to measure elastinderived peptides in serum,
and found that elastinderived peptides could be detected in the
serum of dogs for a period of 12 days following administration of a 25
to 50 mg dose of porcine pancreatic elastase and for 40 days following
a 100 mg dose. Janoff et al. (1983b) found increases in urinary
desmosine excretion during the first 48 hours following endobronchi-


al instillation of pancreatic elastase to sheep; increases in mean
linear intercepts and decreases in lung ventilation and perfusion
were found after 4 weeks. All changes correlated positively with the
elastase dose. Studies have shown a decrease in the lung elastin
content within the first 24 hours of intratracheal injection of
elastase (Kuhn et al. 1976; Ip et al. 1980; Goldstein and Starcher
1977). Physiologic studies (Snider and Sherter 1977; Snider et al.
1977) of experimental animals after pancreatic elastase administra-
tion have shown increases in the lung compliances and in the volume
of air within the lungs at specified transpulmonary pressures (25 and
-20 cm H*O). These physiologic alterations appear to progress in
severity for about 26 weeks following exposure to elastase (Snider
and Sherter 1977).

In spite of substantial experimental verification of ability of
pancreatic elastase to induce emphysematous changes in animals
following intratracheal instillation, there is little evidence implicat-
ing endogenous pancreatic elastase in the pathogenesis of pulmonary
emphysema in humans. A serine endopeptidase of pancreatic origin
(elastase 2) has been shown to circulate in human blood (Geokas et
al. 1977). However, the enzyme is rapidly bound to serum inhibitors
a,AT and a,-macroglobulin (a,M) and inactivated (Gustavsson et al.
1980). Although a,Melastase complexes retain enzymatic activity
against low molecular weight synthetic elastin substrates (N-succi-
nyl-I-.-alanyl-Lalanyl-Lalanine-4-nitroanilide) (Twumasi and Liener
1977; Barrett and Starkey 1973); high molecular weight proteins
such as elastin are prevented from reaching the enzyme and are not
hydrolyzed (Barrett and Starkey 1973).

Attempts to induce emphysematous changes via the intravenous
injection of elastase have met with limited success. Hamsters
injected intravenously with nonfatal doses of pancreatic elastase fail
to show histologic changes characteristic of emphysema (Schuyler et
al. 1978) and do not manifest detectable reductions in lung elastin (Ip
et al. 1980). However, elastic recoil is lost at low lung volumes
(Schuyler et al. 1978). Fierer et al. (1976) has noted enlargements in
the airspaces of rats treated intravenously with large doses (330 U) of
pancreatic elastase. They also found increases in the mean linear
intercepts and rarefication of the amorphous components of elastin
within the lungs. It is doubtful, however, if proportionally similar
intravenous levels of pancreatic elastase occur in humans with
pulmonary emphysema.

Polymorphonuclear Leukocyte Elastase

Polymorphonuclear leukocytes (PMN) appear to be a more plausi-
ble source of endogenous elastase in the human lung than the
pancreas, and are more likely to be incriminated in the pathogenesis
of naturally occurring pulmonary emphysema. PMNs contain elasto-

267


lytic enzymes (Janoff 1973; Ohlsson and Ohlsson 1974; Rindler-
Ludwig et al. 1974) that can be released in active form within the
lung. Experimental studies have clearly demonstrated the ability of
PMN elastase to degrade lung elastin and to induce emphysematous
lesions in animals.

Marco et al. (1971) and Mass et al. (1972) induced experimental
emphysema in dogs by the administration of aerosolized crude
leukocyte homogenates. Using purified human leukocyte elastase,
Janoff et al. (1977) demonstrated the ability of the enzyme to digest
dog lung elastin in vitro and to cause significant dilation of terminal
respiratory structures when instilled into isolated perfused dog
lungs. The in vivo intratracheal instillation of human leukocyte
elastase in dogs produces foci of alveolar destruction within 90
minutes of administration (Janoff et al. 1977). Senior et al. (1977)
studied the effects of intratracheally injected human leukocyte
elastase on hamsters and found a reduction in lung elastin in treated
animals, as well as mild patchy airspace dilation. Sloan et al. (1981)
were able to show that purified dog leukocyte elastase could also
produce emphysematous lesions in dogs when instilled endobronchi-
ally.

Guenter et al. (1981) developed a dog model of experimentally
induced emphysema that avoided the necessity of intratracheal
instillation of enzymes. They repetitively injected E. coli endotoxin
intravenously, thereby inducing extensive leukocyte sequestration
within the lungs of the dogs. A previous study had shown that the
sequestered cells degranulate and disintegrate within the vascular
bed (Coalson et al. 1970). Histologic studies of these dogs revealed
mild airspace destruction and prominent intra-alveolar fenestra-
tions.

Alveolar Macrophage Elastase

In a widely cited article (Mass et al. 1972), dog alveolar macro-
phage homogenates (obtained by the method of Brain 19701, adminis-
tered to two mongrel dogs produced "some dilatation and nonuni-
formity in the size of the airspaces accompanied by some alveolar
wall destruction" in one of the dogs. The other dog showed no
evidence of emphysema.

In spite of the paucity of animal data, the pulmonary alveolar
macrophage (PAM) has been the focus of much investigation. Both
experimental and clinical evidence is available that implicates this
cell in the pathogenesis of pulmonary emphysema.
Two possible mechanisms by which macrophages may mediate
tissue injury are being actively studied. One mechanism involves the
release of elastolytic enzymes followed by unrestrained proteolysis.
The second mechanism involves either a direct or an indirect injury

268


following the release of toxic forms of partially reduced oxygen such
as superoxide anions, hydroxyl radicals, and hydrogen peroxide.
The ability of human alveolar macrophages to synthesize and
secrete an elastolytic enzyme distinct from PMN elastase is the
subject of controversy. Although human alveolar macrophages have
been shown to synthesize a metalloprotease distinct from the serine
protease (elastase) of the PMNs (DeCremoux et al. 19781, its
hydrolytic activity against insoluble elastin substrate has not been
conclusively demonstrated (Hinman et al. 1980; Levine et al. 1976).
Interpretation of the observation that human alveolar macrophages
raised in cell culture systems secrete an enzyme with true elastolytic
activity against insoluble elastin (Rodriguez et al. 1977; DeCremoux
et al. 1978) is complicated by the fact that alveolar macrophages bind
and internalize PMN elastase (Campbell and Greco 1982; White et
al. 1982; Campbell and Wald 1983). Hinman et al. (1980) detected a
calcium-dependent metalloprotease in the culture medium and in
the cell lysates of human alveolar macrophages and initially
demonstrated elastolytic activity against synthetic elastin substrate
and soluble elastin by both the culture medium fluid and the cell
lysates. However, after 3 and 5 days of culture, no detectable activity
against insoluble elastin was evident. The authors calculated that
the initial elastolytic activity observed could be quantitatively
explained by PMN contamination. The recognition that human
alveolar macrophages internalize human PMN elastase (Campbell
and Greco 1982; White et al. 1982; Campbell et al. 1979; Campbell
and Wald 1983) and that the internalized PMN elastase retains
enzymatic activity for at least 48 hours (McGowan et al. 1983)
suggests an alternative explanation.
Green et al. (1979) subcultured human alveolar macrophages for 3
months and found measurable elastase activity against solubilized
elastin during the entire period. They concluded that the elastase
activity appeared to be synthesized continuously rather than being
internalized from external sources.
In summary, human alveolar macrophages release elastolytic
enzymes capable of digesting connective tissue. Whether the elastase
released by these cells represents an enzyme synthesized de novo or
a previously internalized PMN elastase is uncertain and requires
further study.
Human alveolar macrophages, especially from cigarette smokers,
secrete highly reactive oxygen species (Hoidal et al. 1979a) that are
:apable of directly injuring endothelial cells (Sacks et al. 1978) and
`ibroblasts (Hoidal et al. 1981) and of inactivating a,AT (Carp and
Ianoff 1979, Janoff 1979a).
Whole cigarette smoke inhibits PMN chemotaxis in vitro in a dose-
ependent manner (Bridges et al. 1977). However, when alveolar
lacrophages are exposed to cigarette smoke either in vitro or in


vivo, they release a PMN chemotaxic factor (Hunninghake et al.
198Oc) (see above).

Protease-Antiprotease Hypothesis

The protease-antiprotease hypothesis proposes that enzymatic
digestion of lung parenchyma occurs as a direct consequence of a
genetic or acquired imbalance of the protease-antiprotease system
and that the subsequent repair of connective tissue is unable to
return the structures to normal. This hypothesis derives principally
from two observations: (1) people genetically deficient in a,AT
(Laurel1 and Eriksson 1963), the major antielastase of the lower
respiratory tract of humans (Gadek et al. 1981a), are at greatly
increased risk of developing pulmonary emphysema, and (2) proteo-
lytic enzymes produce physiologic and anatomic lesions resembling
emphysema when administered to experimental animals (Gross et
al. 1965). Attempts to integrate the clearly established relationship
of cigarette smoking and pulmonary emphysema with the protease
antiprotease hypothesis have led in-Testigators to search for ways in
which smoking perturbs this balance.

Increased Elastase Owing to the Cellular Response to Smoke

At least five variables, aside from the genetically determined level
of antiprotease activity, could influence the elastase burden of the
lungs. These variables include (1) an increase in the number of
elastase-containing cells within the lung, (2) an increase in the
quantity of prepackaged or newly synthesized elastase per cell, (3)
the quantity of elastase released from the cells, (4) the proximity of
the elastase to suitable substrate, and (5) the extracellular milieu
(i.e., pH, ionic strength, and factors such as platelet factor 4).

Number of Cells

As discussed earlier, the human cigarette smoker has increased
numbers of alveolar macrophages in the bronchoalveolar lavages
compared with nonsmokers (Rodriguez et al. 1977; Harris et al. 1975;
Reynolds and Newball 1974; Hoidal and Niewoehner 1982). Holt and
Keast (1973b) found sustained elevations of pulmonary macrophages
in mice exposed to cigarette smoke. Cigarette smoke has been shown
to recruit PMNs into the airways (Kilburn and McKenzie 1975;
Rylander 1974) and to induce alveolar macrophages to release a
chemotactic factor for PMNs (Hunninghake et al. 198Oc). The
circulating PMNs are reported to be increased in cigarette smokers
(Corre et al. 1971; Galdston et al. 1977). Hunninghake et al. (198Oc)
and Reynolds and Newball (1974) found increased numbers of PMNs
in the lavage fluid of smokers, but Hoidal and Niewoehner (1982)
reported similar numbers of PMNs in the lavages of cigarette

270


smokers and nonsmokers. Hunninghake and Crystal (1983) obtained
isolated cell suspensions from the bronchoalveolar lavage fluids and
from open lung biopsies of nonsmokers and cigarette smokers with
both normal lung parenchyma and sarcoidosis. They found a
significantly increased number of neutrophils and macrophages in
the lavage fluid and in the biopsy specimens from cigarette smokers
as compared with nonsmokers, both in patients with normal lung
parenchyma and in those with sarcoidosis.

Elastase Content

Harris et al. (1975) found an increase in the elastase-like esterase
and protease activity of macrophages obtained from smokers as
compared with nonsmokers. Galdston et al. (1977) found the PMN
elastase levels of circulating PMNs to be elevated in patients with
chronic obstructive lung disease and suggested that the intracellular
elastase levels may be genetically determined (Galdston et al. 1973).
Other investigators (Lam et al. 1979; Rodriquez et al. 1979) reported
similar findings, but Kramps et al. (1980) failed to find any
correlation between the PMN elastase levels and obstructive lung
disease in PiZZ patients, although they did note a difference in
PiMM patients. Lanky et al. (1980) demonstrated that dogs infected
with Type 3 pneumococcus had increased PMN elastase-like esterase
activity within their cells, suggesting an acute phase reaction.

Release

A variety of mechanisms may lead to the extracellular release of
lysosomal contents. These include cell lysis, regurgitation during
phagocytosis, reverse endocytosis, humoral mediation, and cytocha-
lasin B treatment of cells (Klebanoff and Clark 1978). Wright and
Gallin (1979) showed that migration of PMNs is associated with the
leakage of various enzymes. Sandhaus (1983) found that migrating
human neutrophils degrade elastin in vitro in the presence or
absence of human a,AT. A similar mechanism may occur during
neutrophil migration in vivo. Hutchison et al. (1980) found that the
soluble fraction of cigarette smoke suppressed the release of lysoso-
ma1 enzymes (acid phosphatase and acid ribonuclease) from PMNs
obtained from healthy persons, but not from the PMNs of emphy-
sematous patients. Blue and Janoff (1978) demonstrated that the
water-insoluble fraction of cigarette smoke has a cytotoxic effect on
PMNs in vitro and causes them to release their lysosomal contents,
including beta-glucuronidase, acid phosphatase, and elastase. Eliraz
et al. (1977) found that canine alveolar macrophages and PMNs,
when stimulated with the water-soluble fraction of cigarette smoke,
secrete elastase. Abboud et al. !1983), however, compared the release
of elastase and 8-glucoaminodase from PMNs obtained from ciga-

271


rette smokers and with that from nonsmokers and found no
differences. In vitro stimulation of these cells by ?ither phagocytosis
or chemotactic polypeptides did not alter the results. These research-
ers concluded that chronic smoking does not affect neutrophil
elastase release in vitro and that among smokers there is no
significant relationship between in vitro neutrophil elastase release
and abnormalities in lung function. They speculated that some of the
differences between studies may be related to experimental condi-
tions, such as the concentrations of cigarette smoke.

Because the mechanisms involved in the release of intracellular
contents are complex and the representativeness of in vitro condi-
tions to in vivo events is uncertain, definite conclusions await
further studies.

Proximity

Elastolytic activity is conditioned by the absorption of elastase
onto elastin substrate (Robert and Robert 1970); the adsorption, in
turn, results from the electrostatic attraction between negatively
charged carboxylate groups of elastin and positively charged groups
of elastase (Hall and Czerkowski 1961; Gertler 1971). Campbell et al.
(1982) found that a,AT has less inhibitory activity against PMN
elastase derived from cells in contact with substrate than against
PMN elastase free in solution. They reasoned that the partial
exclusion of protease inhibitors from the PMN-connective tissue
interface may account for this phenomena and may be an important
factor in elastase-mediated injury. Focusing more on the macroenvi-
ronment within the lung, Janoff et al. (1983c) found that the
bronchoalveolar lavage fluids of young asymptomatic cigarette
smokers contain significantly more elastase activity than the lavage
fluids from nonsmokers. Kucich et al. (1983) found that the serum
lung em&in-derived peptides were elevated in some smokers and
most patients with COLD, suggesting that elastolysis may be taking
place in smokers and COLD patients.

Milieu

A number of in vitro experiments have examined the chemical and
physical conditions that modify neutrophil elastase kinetics. Lesti-
enne and Bieth (1980) demonstrated that human leukocyte elastase
activity is activated in the presence of substrate excess, hydrophobic
solvents, and increasing ionic strength. The adsorption of sodium
dodecyl sulfate (SDS), a hydrophobic, anionic ligand, onto the surface
of elastin enhances the elastolytic activity of pancreatic elastase
(Kagan et al. 1972). Lonky et al. (1978) showed that platelet factor 4
(PF,) in physiologic concentrations is capable of in vitro stimulation
of human neutrophil elastase (HLE) against lung elastin. Low doses


of HLE instilled intratracheally in hamsters failed to induce
physiologic, morphologic, or biochemical changes, but following the
addition of PFI, a significant injury was evident, and the elastin
content of the lung was lowered by 20 percent (Lonky et al. 1978).
Boudier et al. (1981) demonstrated that human leukocyte cathepsin-
G, an enzyme in the azurophilic granules that possesses little
intrinsic elastolytic activity, stimulates the rate of solubilization of
human lung elastin by HLE. The elastolytic activity increased by
more than five times the HLE rate when the HLE-cathepsin-G
mixture was present in equimolar concentrations. The relevance of
these findings to the physiologic conditions that prevail in vivo
requires further study.

Laurent et al. (19831 recently discovered that the water-soluble
components of filtered cigarette smoke suppress, in a dose-dependent
manner, the lysyl oxidase-catalyzed oxidation of the epsilon-amino
groups of lysine residues in tropoelastin. This step is essential for the
formation of covalent cross-links between neighboring elastin poly-
peptide chains that, in turn, are necessary for normal elastic
strength within the lung.

Decreased An tiproteme Ou<ing to Oxidation

A comprehensive review of the role of oxidative processes in
emphysema has recently been published by Janoff et al. (1983a). In
vitro studies have revealed that oxidants such as chloramine T
(Abrams et al. 1381) or ozone (Johnson 19801 cause a loss in the
inhibitory capacity of a,AT for neutrophil elastase. The mechanism
of inactivation has been identified as the oxidation of methionine
and tyrosine residues (Johnson and Travis 1979; Cohen 1979; Carp
and Janoff 1978) within the a,AT molecule. Chloramine T, when
administered differentially to dogs, reduces EIC .`I TIC of serum and
also results in emphysema (Abrams et al. 1981). Cigarette smoke is
also known to contain oxidants (Stedman 1968; Pryor et al. 19831.
Aqueous solutions of cigarette smoke reduce the elastase inhibitory
capacity of human serum (Carp and Janoff 1978) and result in less
binding of elastase to a,AT in vitro (Carp and Janoff 19781. Some
investigators have found that the a,AT activity is reduced in the
lavage fluids (BALFI obtained from human cigarette smokers and
from rats exposed to cigarette smoke (Gadek et al. 1979; Carp et al.
1982; Janoff et al. 1979a). Stone et al. (1983) reported similar levels of
functional a,-antitrypsin in the bronchoalveolar lavage fluids of
human smokers and nonsmokers. Janoff and Chan (1984) have
suggested that this difference in results may reflect the timing of the
lavage in these studies, as rats chronically exposed to cigarette
smoke had rapid inactivation of u,-antitrypsin following smoke
exposure, but also had a more rapid recovery of a,-antitrypsin
activity than did rats acutely exposed to smoke. Stone et al. also


recognized that their study may not have detected a reduction in
alpha,-antitrypsin activity if it was accompanied by a rapid recovery
to normal levels. Methionine sulfoxide peptide reductase, an enzyme
present in human PMNs, can reactivate a,AT oxidized by chlora-
mine T or by the myeloperoxidase system, but a,AT exposed to
cigarette smoke plus peroxide (Carp et al. 1983) has been shown to be
either resistant to reactivation by the myeloperoxidase system (Carp
et al. 1983) or incompletely reactivated (James et al. 1984).

Human ceruloplasmin has been shown to prevent myeloperoxi-
dase mediated oxidation of a,AT under specified conditions of pH
and solvency (Taylor and Oey 19821, although the role played by
ceruloplasmin in limiting oxidation by phagocytes in vivo is unclear.
Taylor et al. (1983) examined plasma and leukocyte lysosomal
samples from a group of COLD patients and measured the ability of
these samples to inhibit lipid peroxidation. While they reasoned that
inhibitors of peroxidation could protect a,AT from inactivation by
neutralizing lipid free radicals, they found that inhibition required
factors from both plasma and lysosomal extracts and that the factor
was not ceruloplasmin. Two of ten emphysematous patients had
reduced plasma factor activity, and one of these patients also had
reduced lysosomal factor. Controls had normal values for both of
these factors.

Galdston et al. (1984) examined serum ceruloplasmin concentra-
tions and antioxidant activity in male and female smokers. Smokers
of both sexes had higher serum ceruloplasmin concentrations than
did nonsmokers; women in both smoking categories had higher
concentrations than their male counterparts. Serum antioxidant
activity showed a significant positive correlation with serum cerulo-
plasmin levels; however, for comparable ceruloplasmin concentra-
tions, serum antioxidant activity was significantly lower in smokers
than in nonsmokers of both sexes. The researchers suggest that
cigarette smoking may cause partial inactivation of serum antioxi-
dant activity that is accompanied by an insufficient increase in
ceruloplasmin concentration.

Endogenous phagocytes are also capable of generating oxidants
(Babior 1978; Klebanoff and Clark 1978). The phagocytic enzyme
myeloperoxidase, in the presence of hydrogen peroxide and halide
ions, oxidatively inactivates a,AT (Matheson et al. 1979, 1981).
Smoking, as described above, elevates the oxidative metabolism in
lung macrophages (Hoidal and Niewoehner 1982; Fox et al. 1979;
1980; 1981).
Thus, it is clear that the oxidants present in cigarette smoke and
the lung macrophages of smokers can inactivate a,AT. This inactiva-
tion, coupled with the increased elastase burden that may result
from the inflammatory cell response of the lung to smoke, could tip

274


the balance of the protease-antiprotease system in the direction of
elastin degradation.

Explanation for Upper Lobe Distribution

In accord with the protease-antiprotease hypothesis, emphysema-
tous lesions result from the unrestrained proteolytic digestion of
connective tissue elements. The regional distribution of lesions
within the lung is thought to be conditioned by both biochemical and
physiological variables,

The predilection of lower lobe involvement in persons with a,AT
deficiency is hypothesized to result from an increased number of
elastasecontaining cells because of the higher vascular perfusion to
this area in erect man. This excess could occur because of the
deposition of senescent leukocytes in these areas of higher blood flow
(Guenter et al. 1981). In addition, inhaled particulates preferentially
deposit in the lower lobes (Milic-Emili et al. 1966; Dollfuss et al.
1967), and the leukocytes release their enzyme extracellularly
during the ingestion of these particulates. Because of the genetic
deficiency of a,AT, the proteolytic activity is unopposed and
destruction occurs.

The predominance of upper lobe lesions in cigarette smokers with
normal systemic levels of a,AT is again thought to result from
variations of ventilation and perfusion within the lung (Cockcroft
and Horne 1982). However, in normal individuals the proteolytic
activity due to the excess particulate deposition in the bases is
inhibited by a,AT that is replenished by the increased vascular
perfusion also occurring in the bases. The upper lobes, although less
well ventilated than the lower lobes, nevertheless have higher
ventilationqrerfusion ratios because of the proportionately greater
fall in perfusion. The oxidative inactivation of a,AT by cigarette
smoke in the upper lobes therefore may not be compensated by
vascular repletion of the inactivated a,AT, and an imbalance of
protease-antiprotease may occur. The upper lobe injury may then be
magnified by mechanical stresses caused secondary to the negative
intrapleural pressures generated by gravitational forces in erect
man (West 1971).

Animal Models of Emphysema
Spontaneous Emphysema

Emphysema occurs spontaneously in animals in forms resembling
the types seen in human disease (Karlinsky and Snider 1978).
However, the low incidence and unpredictable occurrence of disease
in animals greatly limit their utility as experimental models.