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INTRODUCTION |
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TOP
INTRODUCTION
REFERENCES
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The purpose of this review is to relate developments in lung
cell biology to our understanding and treatment of pulmonary diseases. The authors' careers in pulmonary biology began
when we came to the Heart Institute of the National Institutes
of Health in 1968 (R.J.M.) and 1970 (R.G.C.). This was before
the creation of the Heart, Lung and Blood Institute in 1973. At that time, there was also no active basic or clinical investigation of pulmonary disease in the intramural Heart Institute
at NIH. The focus of the pulmonary investigation had been on
tuberculosis and had turned to pulmonary physiology, and investigations of pulmonary cell biology were just beginning.
This review will focus on surfactant and alveolar type II cell
biology and on the molecular basis of 
1-antitrypsin (1AT)
deficiency. The type II cell has been chosen to represent advances in cell biology and 1AT to represent advances in genetics and molecular biology. These recollections highlight the
collaboration and achievements in the intramural and extramural programs of the NHLBI.
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ALVEOLAR TYPE II CELLS AND PULMONARY SURFACTANT |
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In 1921, von Neergaard discovered that there was a marked
difference in the elastic recoil properties of the lung depending if the lung were filled with air or saline (1). He deduced that the surface tension in the lung was extremely low, much
lower than normal biologic fluids. However, this observation
lay dormant until the 1950s. Clements and Pattle independently demonstrated that the extracts of lung could lower surface tension and that the materials responsible for reducing
surface tension were lung phospholipids (2, 3). Within a few
years of this discovery, Avery and Mead reported that a deficiency in surface active material was the cause of respiratory
distress syndrome of the newborn (4). Soon thereafter, the
lipid component of surface active material that produced low
surface tension was identified as dipalmitoylphosphatidylcholine (DPPC) (5). Very rapidly, a clinical trial was undertaken
by Chu and colleagues to aerosolize pure DPPC into the lungs
of premature infants with respiratory distress syndrome (6).
Unfortunately, this trial was not successful. The reason this
trial failed was not apparent at the time, and the explanation
had to wait another 15 years for a better understanding of the
importance of the proteins of pulmonary surface active material. Successful replacement therapy not only requires the ability of a lung extract or phospholipids to generate a low surface
tension but also depends on the rate of absorption of the phospholipids to the air-liquid interface. It is essential for surface
active material to get into the air-liquid interface during each
breath. Premature babies breathe at a rapid rate, and therefore the rate of absorption has to be very rapid
within seconds. It became apparent that the rate of absorption of liposomal phospholipid was greatly enhanced by the addition of the
surfactant proteins (7). Therefore, these proteins had to be
discovered and characterized before successful surfactant replacement therapy was possible. Thus, although there was
rapid translation of the discovery that surfactant was deficient
in respiratory distress syndrome of the newborn, the clinical
science preceded the understanding of the physiologic effect
of surfactant in vivo.
Soon after the discovery of the phospholipid composition
of surface active material, the scientific community began to
address the question of how surface active material was synthesized and secreted. It became clear that the traditional biochemical approaches of isotopic labeling and subcellular fractionation to isolate enzymes and organelles could not be
applied to the lung because lung cells were heterogeneous.
The lung is composed of many different cell types, and type II
epithelial cells that produce surface-active material constitute
less than 15% of the total lung cells. A microsomal or mitochondria fraction isolated from whole lung was composed of
components from many diverse cell types and had limited
value. Hence, a major effort was undertaken to isolate alveolar type II cellsthe cells that make and secrete pulmonary
surfactant.
The initial approach was to enzymatically dissociate the entire lung into individual cells (10). However, type II cells could not be isolated by this approach because the physical means to separate individual cell populations on the basis of cell size and density did not have the resolving power to isolate type II cells from a crude population of dissociated lung cells. Kikkawa and colleagues made the first important breakthrough (11). They utilized crude trypsin to partially digest the lung, barium sulfate as a phagocytic particle to increase the density of macrophages, and separated lung cells on a density gradient. In addition, they developed a method to identify type II cells in a smear with a modified Papanicolaou stain (Figure 1A). This pivotal study showed that type II cell isolation was possible by limiting the enzymatic digestion of the lung and facilitated subsequent achievements. Identification of type II cells with the modified Papanicolaou stain was a major advance that led to the development of other methods to isolate type II cells. Previously, researchers had to rely on transmission electron microscopy, which was not possible for routine use (Figure 1B). Mason and colleagues modified this method to selectively dissociate epithelial cells by intratracheal instillation of enzymes which provided direct access to the epithelium. They also utilized elastase instead of trypsin, and fluorocarbon emulsion as a phagocytic particle and used differential adherence to purify type II cells in primary culture (12). A panning method with dishes coated with IgG or monoclonal antibodies to macrophages provided reproducible methods to eliminate most macrophages and purify the initial cell preparation (17, 18). Different methods for isolating type II cells have been reviewed recently (19). Isolated type II cells have made possible extensive studies on phospholipid synthesis, secretion and uptake, proliferation and gene regulation. The dramatic impact of these studies in the 1970s and 1980s can be seen by the number of articles about type II cells in general (Figure 2A) or isolated type II cells (Figure 2B) collected from a general Medline search.
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In the 1970s and 1980s the surfactant proteins were isolated and characterized and the genes responsible for those proteins were sequenced (8, 20, 21). Surfactant proteins (SP) A, B, and C were isolated from preparations of surface active material obtained by lavage, whereas SP-D was identified in the search for extracellular matrix proteins secreted by type II cells (22, 23). It became clear that SP-A, -B, and -C were critical components of surfactant and that DPPC alone was ineffective because of its extremely low rate of absorption (9). SP-A and SP-B were found to be necessary for the formation of tubular myelin (24, 25). Hence, natural surfactant became the agent of choice for surfactant therapy. Fujiwara and coworkers reported the successful use of natural surfactant in the treatment of hyaline membrane disease in the newborn (26). Surfactant replacement therapy is now used to improve gas exchange in premature infants throughout the world. There has been a dramatic improvement of survival. More recently, natural surfactant has been shown to improve gas exchange abnormalities in patients with adult respiratory distress syndrome (27). The cloning of surfactant proteins has also allowed astute clinicians to identify a new form of respiratory distress syndrome in infants caused by SP-B deficiency (28).
Molecular cloning of SP-A and SP-D have shown them to
be remarkably similar to mannose binding protein and to bovine conglutinin, respectivelyproteins involved in host defense or innate immunity. When SP-A and SP-D were originally isolated, it was not known that these proteins were an
important part of the host defense system. SP-A was thought
to be an important component of surfactant. This protein
bound lipid very tightly, facilitated the adsorption to the air-liquid interface, was required for tubular myelin formation, and
modulated the uptake and secretion of phospholipid with type II cells in vitro. However, there remained the unexplained
presence of SP-A in Clara cells. In addition, the structural similarity of SP-A to mannose binding protein required a reconsideration of its function and probable physiologic role. Subsequently, there have been numerous studies on the binding of
SP-A to viruses, bacteria, mycobacteria, and pneumocystis.
All of these reports strongly suggest an important role in host
defense, and these observations recently have been comprehensively reviewed (29). The current use of knockout technology allows investigators to inactivate a specific gene and to
evaluate the ability of the remaining gene products to maintain physiologic functions. Knockout of SP-B causes respiratory distress and failure of gas exchange in the newborn, as expected (30). However, the phenotype of the SP-A knockout
mouse was surprising. Although the expected deficiencies in
host defense properties of the lung do occur, there is relatively
little alteration in surfactant homeostasis in the normal animal
(31). Future studies will have to determine if, at times of
increased surfactant utilization during lung injury, the other
surfactant proteins can compensate for the loss of SP-A, whether these might be another similar unidentified protein in the mouse that compensates for the loss of SP-A, or whether
the major physiologic role of SP-A is host defense and not the
trafficking of surfactant phospholipid.
In vitro studies of type II cells have also improved the understanding of lung physiology. Most initial studies focused on phospholipid synthesis and secretion, which was difficult to do in the intact lung. However, some new concepts also have
emerged from studies with type II cells in vitro. The most important unanticipated observation was the discovery that alveolar type II cells in primary cultures form domes, which are
pockets of fluid underneath the epithelial monolayer (34, 35)
(Figure 3A, B). This observation implied that there was net
alveolar transport of fluid, and this proved to be transepithelial sodium transport from the apical to the basolateral surface. At the time of this discovery, there were no reports of active transport by the alveolar epithelium. These observations
meant that the Starling equationcommonly used to characterize fluid movement in the lungwas incomplete, because it
assumed no active transport in the lung. Although net sodium absorption and pharmacologic alterations to this process have been demonstrated in normal lung in vivo and in some states
of lung injury, it has not yet been possible to translate manipulation of this active transport system into practical clinical medicine to treat pulmonary edema in humans. Part of the problem
is that sodium transport requires an intact alveolar epithelium,
which is lost in adult respiratory distress syndrome because of
extensive epithelial injury.
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Isolated type II cells also allowed the discovery of potent growth factors for the alveolar epithelial cells. Whereas it is very difficult to study the role of individual growth factors in vivo, type II cells proliferate in vitro, and their response to growth factors can be studied in primary culture (36). Panos and colleagues demonstrated that keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are mitogens for type II cells (39). Subseqently, KGF has been shown to be a very potent mitogen for rat type II cells in vivo and has been successfully used to protect against oxygen, HCl, and bleomycin injuries in vivo (40). Whether the use of epithelial growth factors will be useful in the treatment of adult respiratory distress syndrome (ARDS) or other lung diseases remains to be determined. However, the use of epithelial specific growth factors adds a new dimension to potential therapeutic interventions to supplement the anti-inflammatory cytokine strategies that are currently being explored.
Recently it has been shown that the alveolar epithelial cells
are an important source of cytokines and growth factors that alter the inflammatory cascade. The alveolar epithelium is
more than a physical barrier and a source of pulmonary surfactant. Alveolar type II cells have been reported to synthesize components of the complement system, produce growth
factors such as HB-EGF, TGF- and TGF-
, and cytokines
such as MIP-2, MCP-1 and GM-CSF (43). However, it is not
known if the alveolar epithelium is a physiologically important
source of cytokines and growth factors in lung diseases.
Although pulmonary cell biology has made recent advances in this area of research, there remains much to be learned about the surfactant system and function of type II cells in health and disease. For the surfactant system, it is not known how the phospholipids are transported from the endoplasmic reticulum, sorted, and packaged in lamellar bodies. The intersection and regulation of the endocytic pathways and the direct synthetic pathways for the phospholipid and protein components of surface active material are not understood. The pathways of SP-A secretion are not known, and there may be a route independent of lamellar body secretion. Phosphatidylglycerol is decreased in numerous disease states and in animal models of lung injury, but neither the mechanism nor the physiologic consequences is known. Type II cell hyperplasia is the hallmark of most interstitial lung diseases in pulmonary fibrosis, but it is not known if these cells limit the fibrotic response by providing a barrier to fibroblast migration or if they enhance the inflammatory process by the elaboration of inflammatory cytokines.
Alveolar type II cell hyperplasia is critical for reforming the epithelium and preventing the migration of fibroblasts into the inflammatory coagulum in the alveolar spaces. This concept also has been supported by some in vivo studies (47). However, an argument can also be made that the type II cells are fibrogenic and enhance pulmonary fibrosis with which they are commonly associated. Although there has been progress in understanding the cell biology of alveolar epithelium, numerous unanswered questions remain. Mechanistic in vivo studies of selected cytokines and other proteins and their receptors are now possible through transgenic and knockout technology. Another major opportunity is the alveolar epithelium as a site for drug delivery and gene therapy. Nowhere else in the body is such a thin and simple epithelium readily accessible. The 100 square meters of epithelial surface in the lung provide a unique opportunity for drug absorption.
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1-ANTITRYPSIN DEFICIENCY: MODERN BIOLOGY
CONQUERS A COMMON HEREDITARY DISORDER |
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1-antitrypsin (1AT) deficiency is a common autosomal recessive hereditary disorder characterized by a marked reduction
in serum levels of 1AT, and a high risk for the development
of emphysema by the fourth to fifth decades of life (48).
Less commonly, 1AT deficiency is associated with hepatitis,
cirrhosis, and liver cancer, and in rare cases, with relapsing
panniculitis (61). The story of 1AT deficiency, from its
identification as a clinical entity by Laurell and Eriksson in
1963 (66) through the 1987 therapy trial that led to the approval of 1AT augmentation therapy (67), is a remarkable
example of the power of modern biology.
Basic Concepts
1AT is a serum protein produced by the liver (68). It functions primarily as an anti-protease that inhibits neutrophil elastase (NE), an omnivorous protease capable of destroying
the connective tissue matrix that defines the architecture of
the alveolar walls (69). In normal individuals, 1AT produced
by the liver diffuses into the lung, where it protects the fragile alveoli from the chronic burden of NE released by neutrophils that have gained access to the lower respiratory tract (51, 54,
55, 57, 60). The basic concept of the pathogenesis of the emphysema of 1AT deficiency is that mutations in the two parental 1AT genes cause reduced secretion of 1AT by the
liver, and thus a marked reduction of 1AT levels in blood
and throughout the body, including the lung (51, 53, 57,
60). This leaves the fragile alveolar walls vulnerable to proteolytic destruction of the alveolar walls by NE. Over many
years, the unfettered NE slowly destroys alveoli, a process
that is accelerated in cigarette smokers (51, 53, 60). By the
ages of 30 to 40 yr the lung destruction becomes clinically apparent, with the progressive loss of lung function causing a 10- to 15-yr reduction in the life span compared with the general
population (52).
Early Studies
In 1963, Laurell and Eriksson (66) described five individuals
with a marked reduction in the 1-globulin band in an electrophoretic analysis of serum. Within a short time, Eriksson recognized that the 1-globulin deficiency (known to be composed primarily of an inhibitor of trypsin, hence the name
"1-antitrypsin"), was inherited and associated with an increased risk for lung disease (70, 71). Studies during the same
period by Gross and colleagues (72, 73) demonstrated that intratracheal instillation of the proteolytic enzyme papain into
the lungs of experimental animals resulted in lung destruction.
The concept of genetic variants of 1AT was established when
strategies were refined to identify normal and deficient variants of 1AT by electrophoretic analysis of serum (74, 75).
This established the autosomal codominant inheritance pattern from the two parental 1AT genes (74). In 1968, NE
was discovered by Janoff, who was investigating the mechanisms of blood vessel damage in vasculitis (78). The relevance of the papain studies of Gross to 1AT deficiency was
clarified by studies of the late 1960s showing that instillation
of homogenates of neutrophils into the lungs of experimental
animals also produced lung destruction with features of emphysema (81, 82).
The next several years were a period of increasing sophistication and consolidation of concepts. The hypothesis that NE
was the primary proteolytic agent in 1AT deficiency solidified as studies with intracellular instillation of highly purified
preparations of NE were shown to cause emphysema in experimental animals (83, 84). The role of 1AT as an anti-protease
began to focus on NE, with studies demonstrating that 1AT
inhibited NE (85, 86); the studies of Beatty, Bieth, and Travis
(87) quantified the kinetics of association of NE and other
serine proteases with 1AT. These studies proved to be critical in the theoretical conceptualization of the role for 1AT in
protecting the lung in vivo, where the concentration and activity of the antiprotease must be considered (88). Thus, by 1980, within 17 years after the discovery of the 1AT deficiency state,
the basic concepts of the pathogenesis of the emphysema associated with 1AT deficiency had been established; i.e., 1AT
deficiency in humans was a hereditary disorder associated with
an increased risk for emphysema, 1AT likely served to protect the lung from neutrophil-derived proteolytic enzymes, and
the pathogenesis of the disease was linked to the 1AT deficiency state allowing uninhibited neutrophil elastase destruction of the lower respiratory tract.
Molecular Pathogenesis
By 1982, studies of the 1AT protein had demonstrated that
the common "Z" form of the 1AT protein was associated
with a single amino acid substitution (89) and thus was likely
caused by a single mutation in the 1AT gene. In 1984, Woo
and colleagues cloned and sequenced the 1AT gene (90) (see
Figure 4). At that time, the Pulmonary Branch of NHLBI was
very involved in applying the methodology of molecular biology to human lung disorders, and all of the necessary technology was then available to focus on the molecular pathogenesis
of 1AT deficiency. The initial studies relating to abnormal
1AT genes were carried out by Nukiwa, who cloned and sequenced the coding regions of the common Z mutation of the
1AT gene (91). Interestingly, these studies not only definitively proved that the abnormal Z protein resulted from a single mutation in exon V of the 1AT gene (Glu342 GAG
Lys342 AAG) (Figure 4), but also demonstrated a second mutation (Val213 Ala213) that was a normal variant (91). This
led to a series of studies by the Pulmonary Branch that defined the genetic basis of most of the normal 1AT gene variants (51, 53, 68, 76, 92). In parallel, cloning and sequencing of the coding exons of the 1AT gene of individuals
with 1AT gene deficiency associated with other than Z mutation led to the identification of a number of rare mutations
that caused the deficiency state (51, 53, 60, 93, 98).
Most of these rare mutations represented single base pair variants in the 1AT coding sequence, including null mutations
(101), but also including an example of an abnormal 1AT
gene in which most of the gene was deleted (108). In parallel with these studies, a pseudo-1AT gene was identified 3' to
the normal gene (109). Finally, Nukiwa and Ogushi cloned
and sequenced the chimpanzee and gorilla 1AT genes (110)
and compared them with the baboon 1AT gene characterized by Woo and colleagues (111) and with the known human
variants of the human 1AT gene. Interestingly, they found
only 11 nucleotide differences in the coding regions of the
chimpanzee and the normal M1 (Ala213) variant of the human 1AT gene (the oldest known normal 1AT variant), and only 19 nucleotide differences between the gorilla and human 1AT genes. On the basis of the assumption that humans
and baboons diverged evolutionarily 30 million years ago,
these data suggest the divergent time between humans and
chimpanzees to be 6 million years, and that of the human and
gorilla to be 8 million years (110). These data and studies of
mutations representing normal variants of the 1AT gene led
to the construction of an "evolutionary tree of the 1AT
gene" (110).
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Once the mutations of the 1AT gene had been identified,
investigations of the Pulmonary Branch shifted their focus to
two areas related to molecular pathogenesis of 1AT deficiency: diagnosis of the mutations and understanding how the
mutations lead to the deficiency state. Studies by Nukiwa
(110) showed that the common Z and S 1AT mutations
could be identified by gel techniques (these studies were prior
to the development of polymerase chain reaction [PCR] methods); once PCR methods became available, Okayama and colleagues (112) developed a strategy to rapidly identify mutations of the 1AT gene using allele-specific amplification. This
is a PCR method using primers where the 3' end is placed at
the mutation site. To understand how mutations led to the deficiency state, several strategies were used. First, based on the
knowledge that 1AT was expressed in small amounts by
mononuclear phagocytes, monocytes and alveolar macrophages
from normals and individuals with 1AT deficiency were evaluated for 1AT mRNA levels, rates of 1AT synthesis and secretion, and size of the newly synthesized 1AT (113). Second, studies were carried out to model the genetic repertoire
of cells secreting 1AT by using replication incompetent retroviral vectors to transfer genes to cells in vitro. This technology was adapted to transfer the normal 1AT cDNA and mutated forms of 1AT cDNAs to fibroblasts, cells that do not
normally express the 1AT gene, and to compare the levels of
1AT mRNA and the kinetics of secretion and size of the
1AT proteins produced by the cells. These studies provided
insight into the molecular pathogenesis of the deficiency state
of a variety of 1AT mutations (100, 114, 115). Finally,
Brantly and colleagues combined plasmid transfection studies
with mutagenesis strategies to demonstrate that the inability
to form an internal salt link in the 1AT protein (normally
Lys290 Glu342) plays a critical role in the reduced secretion
of the common Z mutation (Glu342 Lys342) (116).
Pulmonary Anti-Neutrophil Elastase Defenses
in 1AT Deficiency
While the general concept was that the emphysema associated
with 1AT deficiency was caused by a deficiency of anti-NE
defenses in the lower respiratory tract, by 1980 this hypothesis
had still not been proven in the human lung. Gadek and coworkers used bronchoalveolar lavage to demonstrate that this
concept was indeed true. Studies of normal respiratory epithelial lining fluid showed that 1AT represented more than 95%
of the anti-NE protection of the lower respiratory tract, with
the remainder provided by secretory leucoprotease inhibitor
and 2-macroglobulin (117). This was followed by studies by
Ogushi and coworkers comparing the association rate constants of inhibition of NE by the normal 1AT protein and the
common Z and S mutations of the 1AT protein (118, 119).
These studies revealed that the concentration of 1AT was
markedly reduced in the epithelial lining fluid of individuals
with 1AT deficiency and that the association rate constant
for NE was decreased as well. Put into the context of the theoretical conceptual studies of Bieth (88, 120) of the pathophysiologic consequences of reduced kinetic constants of protease
inhibitors, these studies dramatically demonstrate why the individual with the homozygous inheritance of the Z mutation
of the 1AT gene is at great risk for the development of emphysema. It also emphasizes the vulnerability of the active site
Met358 residue to oxidation in cigarette smokers, and the reduced activity of the anti-NE defenses in the lower respiratory
tract of normals who smoke (121). These observations helped
to explain why individuals with 1AT deficiency who smoke cigarettes are at much higher risk for the development of emphysema than individuals with 1AT deficiency who are non-smokers; i.e., the 1AT deficient individual is at risk because
of the deficiency state, but placed at a much higher risk when
the 1AT that is present is inactivated by cigarette smoke and
can no longer function to efficiently inhibit NE (122, 123).
Development of 1AT Augmentation Therapy
The series of studies culminating with the development of
augmentation therapy for 1AT deficiency began as a series
of events unrelated to 1AT deficiency per se. In 1977, James
Gadek joined the Pulmonary Branch laboratory as a Pulmonary Fellow after completing a fellowship with Michael Frank
in the Intramural Program of the National Institute of Allergy
and Infectious Disease. The focus of that laboratory was on
hereditary angioedema, an antiprotease deficiency disorder
caused by mutations in the C1-esterase inhibitor gene (124,
125). Gadek had been involved in studies using two approaches to C1-esterase inhibitor deficiency that were directly applicable to 1AT deficiency: (1) using the impeded androgen danazol to augment serum antiprotease levels; and (2) purifying the antiprotease from the plasma of normal volunteers
and administering it to the deficient individuals.
The concept of using danazol to augment serum 1AT levels was based on the knowledge that impeded androgens enhance the release of C1 esterase from the liver, which could
lead to clinical improvement in C1 esterase deficiency (124,
125). Unfortunately, while danazol provided a modest increase in serum 1AT levels in individuals with 1AT deficiency, these increases were well below the serum concentration of 1AT needed to protect the lung (126, 127). Another
strategy to augment liver production of 1AT was to use tamoxifen, the anti-estrogen component used to treat breast
cancer. While tamoxifen enhanced serum 1AT in some individuals with 1AT deficiency, the increases in 1AT levels of
most deficient individuals was modest, and insufficient to effectively treat the disorder (128, 129).
While the modified sex hormone concept was not successful in increasing 1AT concentration, the use of purified
1AT was theoretically compelling as a possible therapy for
1AT deficiency. The challenges to developing augmentation
therapy were formidable. What serum levels of 1AT were
necessary to protect the lung? How much 1AT would have
to be administered and how often to maintain these protective
levels? What was the best way to purify the 1AT from serum,
and where could we get enough human serum to accomplish this? Would the infused 1AT diffuse into the lung? Was it
safe to repetitively administer 1AT in this fashion?
The choice of 11 µM (80 mg/dl) as the theoretical "protective" serum 1AT level was based on the knowledge that this
was in the lower range for individuals with the SZ 1AT phenotype, and that most individuals with the SZ phenotype did
not develop emphysema (67, 130). On the basis of this target,
and studies suggesting that the serum half-life of 1AT was 4 to 5 d, it was estimated that approximately 4 g (60 mg/kg) of
purified 1AT would have to be administered intravenously
weekly to maintain serum 1AT levels above 11 µM. The NIH
Clinical Center Blood Bank provided the normal human serum, and a purification strategy was worked out based on
known plasma fractionation techniques. Studies were initiated
using four weekly infusions to five volunteers with 1AT deficiency and emphysema. The study followed serum 1AT concentrations and bronchoalveolar lavage to assess whether the
infused 1AT diffused across the alveolar interstitium and increased 1AT levels in the epithelial lining fluid of the lower
respiratory tract.The results followed the theoretical predictions, demonstrating that weekly infusions of 4 g were safe,
maintained serum concentrations > 11 µM, and that 1AT
diffused into the lung, increasing the anti-NE levels in the alveolar epithelial lining fluid (130).
The next challenge was to obtain enough purified 1AT to
provide therapy on a long-term basis. This was solved when
industry became interested in the problem and provided sufficient purified 1AT to carry out a trial in 21 individuals. Wewers and coworkers (67) confirmed that chronic therapy in a
larger group of individuals with 1AT resulted in the effect
predicted from prior, more limited studies (130) (Figure 5).
Importantly, augmentation therapy was shown to be safe, even
when administered to individuals who are "null homozygous,"
i.e., have mutations both parental 1AT genes such that their
immune systems have not been exposed to 1AT (67, 68). On
the basis of these data, the Food and Drug Administration approved 1AT augmentation therapy for general use, and it is now used worldwide to treat > 2,000 individuals with this disorder.
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Other studies regarding 1AT augmentation therapy for
1AT deficiency carried out in the Pulmonary Branch included a study by Hubbard and colleagues of monthly infusions
of 240 mg/kg of 1AT (this approach to therapy is widely used,
but has not been approved by the FDA) (131). In another approach, based on the concept that intravenous infusion of 1AT
"wastes" much of the 1AT (the lung is only 2% of the body
weight, and the infused 1AT diffuses throughout the body),
Hubbard hypothesized that aerosol administration might be a
more efficient approach to 1AT adminstration. He demonstrated this was feasible with 1AT purified from human plasma
(132) and with recombinant human 1AT (133, 134). Although
these studies have not been proven to be effective in the treatment of 1AT deficiency (135), this approach was adapted by
McElvaney and colleagues to suppress the NE burden on the
airway epithelial surface in cystic fibrosis (136), a strategy that
has been expanded into larger trials worldwide.
Gene Therapy
In the mid-1980s, the Pulmonary Branch began to investigate
the use of gene therapy to deliver proteins for treatment of
lung disorders. The initial studies were carried out using an ex
vivo approach with the retrovirus gene transfer vectors. At the
time, the NHLBI Intramural Program was evolving as a major
center for the early concepts of gene therapy, with French
Anderson focusing on cancer and immunodeficiency disorders, Arthur Neinhuis on hematologic disorders, and the Pulmonary Branch on 1AT deficiency, and later, cystic fibrosis.
Garver and colleagues demonstrated that fibroblasts could be
genetically modified to produce human 1AT (137). Using this strategy, they then showed that peritoneal implantation of these modified fibroblasts in experimental animals caused detectable human 1AT in serum and lung (138). A few years
later, Rosenfeld and coworkers used replication deficient adenovirus to transfer the human 1AT cDNA to experimental
animals with in vivo gene transfer to the respiratory epithelium (139). Jaffe and colleagues demonstrated high serum human 1AT concentration in experimental animals after adenovirus-mediated gene transfer to the liver (140), and Setoguchi
and colleagues showed that adenovirus could do the same following adenovirus-mediated transfer of the human 1AT cDNA to the peritoneal mesothelium (141). Thus, gene therapy for 1AT is feasible, although the necessity of providing
high levels of gene expression on a persistent basis continues
to be a major challenge.
Current Status
Despite the development of an approved therapy for this disorder, there continues to be multidisciplinary interest in 1AT and its deficiency state. The promoter of the 1AT gene has
been studied in detail, more mutations have been identified
and characterized, the neutrophil elastase gene has been
cloned and characterized, the three-dimensional crystallographic structure of both 1AT and NE have been solved,
1AT gene expression in hepatocytes and other cell types
continues to be characterized, transgenic animals have been
developed to overexpress the normal and mutated forms of
1AT, and the 1AT promoter has been used to direct gene
expression in the liver of transgenic animals (see Reference 60 for details). The National Heart, Lung and Blood Institute
Registry of 1-Antitrypsin Deficiency has followed more than
1,000 individuals with 1AT deficiency in 37 centers since
1989. The data evolving from the Registry have gone a long
way to definitively characterize the disorder and demonstrate
that augmentation therapy prolongs life. Finally, the low molecular weight inhibitors of NE are being developed, and liver
and lung transplantation continue to be evaluated as therapy
for some individuals with the 1AT deficiency state (see Reference 60).
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Footnotes |
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Dr. Crystal is supported, in part, by the National Heart, Lung and Blood Institute P01 HL51746; R01 CA75192; R21 CA75153; P01 HL59312; R01 HL59861-01; the Cystic Fibrosis Foundation, Bethesda, MD; the Will Rogers Memorial Fund, White Plains, NY; and GenVec, Inc., Rockville, MD. Dr. Mason is supported, in part, by the National Heart, Lung and Blood Institute Specialized Center of Research in Pulmonary Fibrosis P01 HL56556 and HL29891.
Correspondence and requests for reprints should be addressed to Robert J. Mason, M.D., National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. E-mail: masonb{at}njc.org
Acknowledgments: The authors thank Kathy Ryan Morgan and Nahla Mohamed for help in preparation of the manuscript.
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References |
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TOP
INTRODUCTION
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