Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine at Mount Sinai Medical Center, Miami Beach, Florida 33140
ASTHMA IS AN INFLAMMATORY disease of
the airways. Prominent effector cells of this inflammatory response
include mast cells, eosinophils, and neutrophils, and, as such,
understanding the contribution of secreted mediators from these cells
to the pathophysiology of asthma has been the subject of intense
investigation. Human mast cell tryptases (EC 3.4.21.59) comprise a
family of trypsin-like neutral serine proteinases that are
predominantly expressed in mast cells (31). There are Tryptase has effects on peptides, proteins, cells, and tissues, and
many of these actions can ultimately contribute to asthma symptoms. Tryptase degrades vasoactive intestinal peptide, an endogenous bronchodilator (35); activates prekallikrein
and generates kinins (19, 21, 29), important mediators in
the development of bronchoconstriction and airway hyperresponsiveness (13, 32); and activates and regulates mast cell secretion both in vivo (25, 26) and in vitro (15, 17).
Injection of tryptase into the skin causes an acute wheal and flare in
sheep (25) and a late inflammatory cell response in guinea
pigs (16). While these late inflammatory events could
naturally progress from mast cell activation, a second mechanism may
involve the release of the potent granulocyte chemotaxin interleukin-8
(7, 10). Finally, tryptase can increase airway smooth
muscle responsiveness to histamine in vitro (33) and
airway hyperresponsiveness in vivo (26), although this may
not occur in all species (18). The concept that tryptase
is a major contributor to allergic airway disease is supported further
by in vivo studies in experimental animals and in humans that show that
small molecule tryptase inhibitors block allergen-induced early- and
late-phase bronchoconstriction and airway hyperresponsiveness (9,
22, 36). These results indicate that inhibiting tryptase
activity in the airways may have an acute beneficial effect on asthma.
In addition to these acute inflammatory processes, tryptase may induce
more long-term effects. Tryptase is a known mitogen for dog tracheal
smooth muscle cells (6), human smooth muscle cells
(2), and human lung and dermal fibroblasts (1,
14). Both smooth muscle hyperplasia and fibrotic changes
contribute to thickening of the airway wall that can lead to a
permanent reduction in airway caliber. These structural changes are
part of the process referred to as airway remodeling (4).
Although the pathogenetic mechanisms of airway remodeling are poorly
understood, the clinical consequences of this process include an
irreversible component of airway obstruction and heightened airway
hyperresponsiveness, both of which contribute to increased severity and
frequency of asthma exacerbations. Part of this phenomenon is a
consequence of smooth muscle shortening due to the thickened airway
wall. Thus compared with normal airway smooth muscle, asthmatic airway smooth muscle needs to shorten by only 40% of its resting length to
occlude the airway lumen (20).
The biological actions of tryptase outlined above seem to require an
active catalytic site since tryptase-induced responses can be
blocked by inactivating the enzyme by heating or by use of reversible
tryptase inhibitors such as
N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride (APC-366), leupeptin, benzamidine, or
bis(5-amidino-2-benzimidazolyl)methane (8). One putative
mechanism by which tryptase is thought to stimulate cellular responses
is through activation of protease-activated receptors (PARs). PARs are
G protein-coupled receptors that are self activated by specific enzymes
or by synthetic peptides (24). In the case of enzymatic
activation, the NH2 terminus of the extracellular domain of
the receptor is cleaved, and the remaining peptide then binds to
itself, thereby initiating transmembrane-signaling events. PAR
stimulation can also be mimicked by synthetic peptides that bind to the
appropriate domain of the receptor, but, in this case, activation
occurs without proteolytic cleavage. Of the four PARs identified (PAR-1
through PAR-4), tryptase (and trypsin) activates PAR-2
(27). In the lung, immunolocalization of PAR-2 expression has been found on mast cells (12) and human airway smooth
muscle cells (11).
The localization of PAR-2 on mast cells is consistent with the
aforementioned physiological actions of tryptase in the airways, and,
in a recent study, Berger and coworkers (2) provide
evidence that tryptase-induced mitogenic activity in human airway
smooth muscle cells is dependent on PAR-2 activation. In their study, tryptase-induced mitogenic activity was measured by thymidine incorporation. The maximum response was seen 24 h after incubating the cells with tryptase (30 mU/ml), recombinant tryptase, or
PAR-2-activating peptide and trypsin (which also activates PAR-2).
Incubation of tryptase with the protease inhibitors leupeptin,
benzamidine, and APC-366 reduced tryptase enzymatic activity by 98%,
45%, and 44%, respectively, and reduced the tryptase-induced
mitogenic activity in the smooth muscle cell cultures by 113%, 81%,
and 79% compared with the response seen with no inhibitor present. Heat inactivation of the enzyme also inhibited the mitogenic response, and there was no mitogenic response after incubation of the cultures with scrambled (inactive PAR-2) peptide. Tryptase-induced thymidine incorporation was abolished by treating the cells with pertussis toxin,
which suggests that a pertussis toxin-sensitive G protein is involved
in the proliferative response. Collectively, these findings indicate
that the mitogenic effects of tryptase on human airway smooth muscle
cells require PAR-2 stimulation by the enzyme with an active catalytic
site. Berger et al. (3) also arrived at similar
conclusions after examining the effects of tryptase on cytoplasmic
calcium concentrations in human airway smooth muscle.
In the study by Brown et al., one of this issue's articles in focus
(Ref. 5, see p. L197), Brown and colleagues provide evidence to the
contrary. These investigators confirm that tryptase, at physiologically
relevant concentrations, acts as a mitogen for human airway smooth
muscle cells. Muscle cell mitogenic activity was measured by
bromodeoxyuridine incorporation 24-48 h after exposure to
mitogens. In this preparation, tryptase's mitogenic activity is
comparable to classic peptide growth factors such as recombinant
platelet-derived growth factor-BB and recombinant human basic
fibroblast growth factor. The tryptase response, however, is much
greater than other serine protease mitogens, including thrombin, factor
Xa, and factor XII. Comparable experiments were done in dog cells with
similar results, except that in the dog cells recombinant human insulin
growth factor 1, which had no effect on human cells, had a marked
proliferative effect. The unique and now controversial finding is that
inhibiting tryptase's proteolytic activity did not block its mitogenic
effects in human airway smooth muscle cells, even though these cells
appeared to express PAR-2 by PCR. For this study, Brown and colleagues
used amidino phenylmethanesulfonyl fluoride (p-APMSF), an
irreversible inhibitor, rather than the reversible inhibitors
used by Berger et al. (2). The p-APMSF reduced tryptase
enzymatic activity by 99%, similar to the degree of inhibition seen
with leupeptin in the study by Berger et al., but the tryptase-induced
mitogenic effect was unaffected. Interestingly, the results were
different in the dog tracheal smooth muscle cell cultures in which
inhibiting tryptase activity with p-APMSF significantly reduced, but
did not completely block, the tryptase-induced mitogenic response. These results indicate that tryptase catalytic activity is not required
for mitogenic activity in human cells, but it is required to achieve a
complete mitogenic response in dog cells. Thus Brown and coworkers
demonstrate a proliferative response to inactivated tryptase in cells
from two species, although to different degrees. These results suggest
that the tryptase-induced mitogenic effect in human airway smooth
muscle cells is, for the most part, nonproteolytic and therefore most
likely not mediated through PAR-2 activation. One putative mechanism
suggested by the investigators is the binding of glycosolated residues
on the tryptase molecule to cell surface mannose receptors
(28) since these receptors were shown to mediate bovine
smooth muscle cell proliferative responses (23).
It is difficult to explain the divergent results of these two studies.
Obviously, the differences can be attributed, in part, to experimental
conditions and methodology. Could the differences in the tryptase
inhibitors used explain the findings? Possibly. However, differences in
inhibitors cannot explain the mitogenic effects produced by the
PAR-2-activating peptide seen by Berger and colleagues
(2). Brown and coworkers demonstrated that their cultures
expressed PAR-2 but did not test the effect of the PAR-2-activating peptide in their system. This could prove to be an interesting experiment in light of the different findings from the two groups. Could it be that the tryptase-p-APMSF complex activates PAR-2 directly
or somehow generates a PAR-2-activating peptide?
Barring these potential explanations, the possibility that tryptase
acts via PAR-2-dependent and -independent mechanisms is an intriguing
hypothesis. As indicated by Brown and coworkers (5), such
findings may impact the development of tryptase antagonists designed to
treat the various disorders in which this enzyme plays a role. Thus the
present inhibitors that modulate the more acute tryptase-mediated
responses to allergen exposure (e.g., bronchoconstriction and airway
hyperresponsiveness) may fail to control aspects of airway remodeling
not completely dependent on PAR-2 activation. Future studies will be
required to determine the relative importance of tryptase-induced
PAR-2-dependent and -independent mechanisms as they relate to smooth
muscle cell hyperplasia and increased collagen deposition in the
asthmatic airway. Such studies will be important for determining the
contribution of tryptase to the remodeling process. Does
tryptase continue to have a role as remodeling progresses, and if
it does, can we block or reverse this effect? Answers to such questions
will be important not only for understanding pathophysiology of asthma
but possibly other mast cell-mediated diseases.
ARTICLE
TOP
ARTICLE
REFERENCES
-
and
-forms of the enzyme, but the present discussion will concern
itself with the
-isoenzymes (
I,
II, and
III), which appear
to be activated intracellularly and stored in secretory granules
(34). Tryptase is enzymatically active in the form of a
noncovalently linked tetramer. Upon dissociation, the monomers lose
activity. Tryptase is stabilized by heparin and by other negatively
charged proteoglycans, and this mechanism is thought to govern tryptase
activity in vivo (30, 34). Upon mast cell activation, the
-tryptases, bound to heparin, are secreted in parallel with
histamine. Thus, historically, tryptase has often been used as a marker
of mast cell activation. More recent in vivo and in vitro studies have,
however, suggested a far more prominent role for this enzyme in both
the acute and chronic inflammatory processes that contribute to the
pathophysiology of asthma.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. M. Abraham, Dept. of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140 (E-mail: Abraham{at}msmc.com).
10.1152/ajplung.00429.2001
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