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,
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Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Sainte-Foy (Québec), Canada;
Central Laboratory of The Netherlands Red Cross Blood Transfusion Services, Amsterdam, The Netherlands; and
Department of Clinical Chemistry, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin, secretory leukocyte
protease inhibitor, and elafin, showed any effect on granzyme A or B
activity. In addition, granzyme A was shown to be fully active in BAL
fluids. Hence, these data show that granzyme activity may be poorly
controlled by protease inhibitors in inflamed tissues. Thus, granzymes
could contribute to tissue remodeling and inflammation characterizing
HP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of granzymes in the extracellular milieu could significantly modulate the inflammatory response. For example, granzyme A is believed to participate in target cell lysis, migration, and extravasation of T lymphocytes; activation of prourokinase-type plasminogen activator; regulation of B cell growth; and control of viral infection (6). Moreover, granzyme A stimulates the production of different cytokines (7, 8) and is an IL-1ß-converting enzyme (9). Finally, extracellular granzymes A and B may also contribute to tissue destruction and remodeling by degrading various extracellular matrix proteins (10, 11, 12).
Numerous inflammatory lung diseases such as hypersensitivity pneumonitis (HP)3 are characterized by the presence of increased numbers of activated CTL and NK cells in the alveolar space (13). Alveolar lymphocytes are able to show increased granzyme B gene expression (14). Therefore, increased levels of granzymes could be generated into the alveolar space during a lymphocytic pulmonary inflammation and contribute to the disease process.
The lung is protected from the detrimental destructive potential
of proteases by protease inhibitors (15). The main serine
protease inhibitors present in the lung are
1-antitrypsin, secretory leukocyte protease
inhibitor (SLPI), and elafin (15, 16). It is not yet clear
whether these inhibitors can inhibit granzymes. Some have reported that
1-antitrypsin inhibits granzyme B
(17), while we (C.E.H., unpublished observations) and
others (P. Bird, personal communication) have not observed such
inhibition. Since elafin suppresses by 75% the degradation of
insoluble elastin by purified peripheral blood T cells
(18), and granzyme A is elastolytic (19), it
has been suggested that elafin inhibits granzyme A (18).
To our knowledge, there are no data available on granzyme inhibition
by SLPI.
The present study was done to measure levels of granzymes A and B
in bronchoalveolar lavage (BAL) fluids obtained from normal subjects
and patients with HP. Finally, we verified whether granzymes are
inhibited by 1-antitrypsin, SLPI, and elafin,
and whether they are active in BAL fluids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BAL fluid samples were obtained from 14 unexposed healthy subjects and 12 patients with active HP. All subjects were nonsmokers. The diagnostic of HP (including farmer’s lung, peat moss lung, humidifier lung, and pigeon breeder’s disease) was based on published criteria that include a documented history of exposure to sensitizing Ag, symptomatic acute febrile episodes, dyspnea, and abnormal pulmonary functions characterizing this disease (13).
BAL was done with a 300-ml (6 x 50 ml) instillation of isotonic saline (0.9% NaCl). Total cell count was determined with a hemocytometer and differential cell count on Diff-Quik (Dade Diagnostics, Aguada, PR)-stained cytocentrifuge cell preparations. After centrifugation, cell-free aliquots of BAL fluids were frozen at -70°C until analysis. For two normal and nine HP subjects, serum was collected from venous blood and stored at -70°C until analysis.
Measurement of granzyme A and B levels
BAL fluid levels of granzymes A and B were measured with two previously described and validated ELISA using specific anti-granzyme mAbs showing no cross-reactivity with other serine proteases (3).
Inhibition of granzymes by serine protease inhibitors and determination of active granzyme levels in BAL samples
Effect of serine protease inhibitors on granzyme A
activity was done using a method based on the interaction of active
granzyme A with antithrombin III (20). Briefly, granzyme
A, purified as described previously (20), was bound to
anti-granzyme A mAb-28 immobilized onto an ELISA plate at 3 ng/ml,
and incubated with increasing concentrations of up to 10,000-fold molar
excess of 1-antitrypsin (Calbiochem, La Jolla,
CA), SLPI (R&D Systems, Minneapolis, MN), or elafin (Peptides
International, Louisville, KY), with biotinylated antithrombin III in
the presence of heparin. Bound biotinylated antithrombin III was
detected with streptavidin-coupled polymerized HRP. Purified
antithrombin III (Sigma-Aldrich Chemicals, St. Louis, MO) was
tested as a positive control.
Effect of serine protease inhibitors on granzyme B activity was
performed according to a protocol from Enzyme Systems Products
(Livermore, CA). Briefly, granzyme B (Enzyme Systems Products) and
inhibitors were resuspended in 100 mM Tris, 500 mM NaCl, pH 7.5, at a
working concentration of 150 nM. Then, 5 pmol of granzyme B was
incubated with 5, 20, or 80 pmol of
1-antitrypsin, SLPI, or elafin for 30 min at
37°C in a final volume of 50 µl. This volume was added to 150 µl
of 270 µM BOC-Ala-Ala-Asp-S-Bzl (Enzyme Systems Products) in 200 mM
HEPES, 300 mM NaCl, 1 mM EDTA, 0.05% (v/v) Triton X-100, pH 7,
containing 410 µM of 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman’s
reagent). Kinetic changes in absorbance were measured at 405 nm with a
THERMOmax microplate reader (Molecular
Devices, Sunnyvale, CA).
These two methods for granzyme A and B activity were also used to determine the presence of active granzyme A and B concentrations in the BAL fluid samples.
Statistical analysis
BAL total and differential cell counts are expressed as mean ± SEM and were analyzed using a paired Student’s t test. Levels of granzymes A and B are expressed as median (range) and were analyzed by a Mann-Whitney test. Spearman’s coefficient of rank correlation was used to assess the degree of association between BAL cell numbers and serum granzyme levels. In all cases, p < 0.05 was considered as significant (21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BAL fluid recovery was similar for each group of subjects.
Of the 300-ml instilled, 181 ± 21 and 170 ± 14 ml were
recovered from normal and HP subjects, respectively. Total cell number
in BAL fluid was 97.1 ± 12.2 x 103
cells/ml for normal subjects. Cell number was significantly increased
in HP patients to 785.3 ± 103.5 x 103
cells/ml (t test, p = 0.0001). All
BAL cell types were increased in HP patients compared with control
subjects (Table I
). Macrophages
represented about 85% of BAL cells in normal subjects. In contrast,
lymphocytes were the predominant cells (>60%) in HP patients.
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BAL fluids from normal subjects showed minimal levels of
granzymes A and B (Figs. 1 and 2). In these subjects, the median (range)
granzyme A and B concentrations were 4 (0–37) and 0 (0–6) pg/ml of
BAL fluid, respectively. It is noteworthy that, of 14 normal subjects
studied, 5 and 10 had no detectable levels of granzymes A and B,
respectively.
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) and 10 (0–78) pg/ml of granzyme B (Fig. 2). These
values were significantly higher than those of the normal subjects
(Mann-Whitney test, p < 0.001 and p =
0.007 for granzymes A and B, respectively). Granzyme A and B levels
were correlated with each other (rs =
0.69, p = 0.004). In addition, both granzyme levels
were correlated with the number of lymphocytes/ml
(rs = 0.794, p <
0.001 and rs = 0.735,
p = 0.002 for granzymes A and B, respectively). Serum concentrations of granzymes A and B
Granzymes A and B were only measured in serum from two
normal and nine HP subjects; samples were not available from the other
subjects. In the two normal subjects, concentrations of granzymes A and
B were 37 and 134 pg/ml, and 10 and 13 pg/ml, respectively. In HP
patients, serum levels of granzymes A and B were 50 (27–262) and 6
4–368(4–368) pg/ml, respectively. These concentrations are similar to
previously published serum concentrations of granzymes
(3). There was no correlation between BAL and serum levels
of granzymes A or B (Fig. 3).
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Neither 1-antitrypsin, SLPI, nor
elafin, even in molar excess, showed any inhibitory activity against
granzyme A or B. To ensure that the three inhibitor preparations
were fully active, they were shown to inhibit human sputum elastase
(Elastin Products Company, Owensville, MO) using the chromogenic
substrate N-methoxysuccinyl-Ala-Ala-Pro-Val
p-nitroanilide (Sigma-Aldrich Chemicals), according to a
procedure described at www.elastin.com.
Determination of granzyme A and B activities in BAL fluid samples
Having shown the presence of granzymes A and B in BAL fluid
samples and that serine protease inhibitors, present in BAL fluid in
vivo, do not inhibit both granzymes in vitro, we next addressed the
question as to whether granzymes A and B in BAL fluids were active.
Usually, the BLT assay is used to measure active granzyme A levels.
However, in our hands, this assay has a sensitivity of about 3 ng/ml.
Therefore, we determined the levels of active granzyme A with a novel
and more sensitive assay, which is based on the binding of antithrombin
III to active granzyme A in the presence of heparin (20).
The sensitivity of this assay, as it was run, was approximately 50
pg/ml of active granzyme A. Since only seven HP patients had more than
50 pg/ml of granzyme A, as measured by ELISA, granzyme A activity was
also determined in subjects with other lung conditions having high
levels of granzyme for a total of 15 subjects. There was a highly
significant correlation between BAL granzyme A levels determined by
ELISA, on one hand, and active granzyme A levels, on the other (Fig. 4). The slope of the regression line was
0.962, a value quite close to 1. Taken together, these results show
that all granzyme A present in BAL fluid is fully active. Accordingly,
we did not measure complexes between granzyme A and its natural
inhibitor antithrombin III in any of the BAL fluid samples. Finally, we
were unable to show any active granzyme B in BAL fluids, even in the
samples with the highest concentrations of this granzyme. However, this
negative result could be due to the lack of sensitivity of the
esterolytic assay used to detect granzyme B activity. Considering this
lack of sensitivity, the presence of active granzyme B in BAL fluid
samples cannot be ruled out.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin, SLPI, and elafin, and 4)
granzyme A is fully active in BAL fluid. Normal BAL fluid is characterized by a proportion of lymphocytes of 5–15% (22), which includes activated (23, 24) and granzyme B gene-expressing (14) lymphocytes. The presence of such activated alveolar lymphocytes in the normal setting could explain the presence of low levels of granzymes in some healthy subjects.
There was a correlation between BAL levels of granzymes A and B. This makes sense considering that both granzymes are stored in the same cytoplasmic granules and are released together following exocytosis (6, 25). The increase in granzyme concentrations in HP is more than likely locally derived since there was a correlation between granzyme A and B levels, on one hand, and lymphocyte numbers, on the other hand. Also, the fact that serum granzyme levels were within normal range in HP patients (except for one subject) and were not correlated to BAL levels supports the concept that granzyme release is restricted to the lung and that serum levels are probably not influenced by local lung production. Published normal plasmatic concentrations of granzymes A and B are 33.5 1–121(1–121) and 11.5 1–113(1–113) pg/ml, respectively (3). In contrast, circulating granzyme concentrations can increase well over 1000 pg/ml in patients with EBV or HIV-1 infection (3), or rheumatoid arthritis (5).
In vitro, we did not observe any significant inhibitory activity
of 1-antitrypsin, SLPI, or elafin on granzyme
A or B. The present results, as well as previous results from our group
(20), are in contradiction with those of others who have
reported that 1-antitrypsin inhibits both
granzyme A (6) and B (17), but are in
accordance with those of one group that was unable to substantiate the
inhibition of granzyme B by 1-antitrypsin (P.
Bird, personal communication). Regarding SLPI and elafin, our results
clearly show that they do not inhibit either granzyme A or B. Based on
indirect observations, Cowan and colleagues suggested that elafin could
inhibit granzyme A (18). Our results do not support this
concept.
An important observation from the present study is that granzyme
A activity strongly correlated with antigenic levels in BAL fluid,
indicating that all granzyme A in the lung is active. In support for
this, we did not measure a detectable inhibitory effect of
1-antitrypsin, SLPI, or elafin, the main
serine protease inhibitors in the lungs (15, 16), on
granzyme A or B activity in vitro. We did not measure any active
granzyme B in the BAL fluids tested. Considering the low concentrations
of granzyme B in BAL fluids, the lack of sensitivity of the assay used
to determined granzyme B activity, and that neither
1-antitrypsin, nor SLPI, nor elafin inhibits
granzyme B in vitro, the presence of active granzyme B in the alveolar
space cannot be ruled out.
Beside 1-antitrypsin, SLPI, and elafin,
other serine protease inhibitors obviously do not contribute
significantly to the inactivation of active granzyme A in the lung
since this granzyme is fully active in BAL fluids.
2-Macroglobulin, present in small
concentration (0.01 µM) in the normal alveolar epithelial lining
fluid (15), is reported to inhibit both granzymes A and B
(6, 17). We recently showed that
2-macroglobulin is a major inhibitor of
granzyme A in the blood compartment (20), in which this
inhibitor is present at much higher concentrations than in the alveolar
space, i.e., 2.5–5 µM (15). The assay we used to
determine granzyme A activity in the present study, based on the
interaction of the protease with antithrombin III, is the same as we
previously used to quantify the same activity in the blood
(20). It is important to point out that such assay
excludes the possibility that granzyme A is entrapped, and therefore
inhibited, by 2-macroglobulin
(20), since entrapped proteases are not able to interact
with high m.w. substrates such as antithrombin III in the case of
granzyme A. Moreover, we have clearly shown that all granzyme A in BAL
fluid is active. From this, we can conclude that
2-macroglobulin does not play any important
regulatory role in granzyme A activity in the lung alveolar space.
1-Antichymotrypsin is also present in the
alveolar space, but it does not inhibit granzyme A (6).
Finally, proteinase inhibitor 9 (PI-9), a member of the OVA
serpin family, is an efficient inhibitor of granzyme B, but this
inhibitor is not secreted by cells and restricts its action to the
cytoplasm of lymphocytes, protecting these cells from granzyme
B-induced apoptosis (26).
The presence of increased levels of active granzyme A in the
alveolar space in HP is of particular interest. Indeed, granzyme A
stimulates the production of TNF-, IL-6, and IL-8 (7, 8). These cytokines are important signal molecules coordinating
the lung inflammatory and immune responses (27). Not
surprisingly, BAL levels of these cytokines are increased in HP
(28). Therefore, it is tempting to speculate that granzyme
A contributes, at least in part, to their up-regulation. Moreover,
being an IL-1ß-converting enzyme (9), granzyme A could
further contribute to the local inflammatory response characterizing
HP. Last but not least, the proteolytic action of granzymes A and B on
collagen and proteoglycans (10, 11, 12) may significantly
contribute not only to tissue remodeling, but also to the migration of
lymphocytes into the alveolar space in HP, perpetuating, therefore, the
chronic state of this lung disease. It remains to clarify whether
increased BAL levels of granzymes are specific to HP or if this is also
a characteristic of other lymphocytic lung diseases.
In conclusion, we provide evidence that BAL fluid from patients with HP contains high levels of granzymes A and B that are not inhibited by the main serine protease inhibitors of the lung, and that granzyme A is fully active in the alveolar space. These data not only suggest that these proteases may contribute to the pathogenesis of HP, but also show that high concentrations of soluble active granzymes may occur locally in inflamed tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Guy M. Tremblay, Hôpital Laval, Room M2691, 2725 chemin Sainte-Foy, Sainte-Foy, Québec, Canada G1V 4G5.
3 Abbreviations used in this paper: HP, hypersensitivity pneumonitis; BAL, bronchoalveolar lavage; SLPI, secretory leukocyte protease inhibitor.
Received for publication February 22, 2000. Accepted for publication July 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thrombin. J. Immunol. 156:2585.[Abstract]
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