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(Received for publication, May 5, 1995) From the
Neutrophil chemotaxis plays an important role in the
inflammatory response and when excessive or persistent may augment
tissue damage. The effects of inhibitors indicated the involvement of
one or more serine proteinases in human neutrophil migration and shape
change in response to a chemoattractant. Monospecific antibodies,
chloromethylketone inhibitors, and reactive-site mutants of
The pathogenesis of lung diseases such as adult respiratory
distress syndrome and emphysema is thought to result from an imbalance
between leukocyte serine proteinases (such as neutrophil elastase,
cathepsin G, and proteinase III) and the proteinase inhibitors of the
lung(1, 2, 3) . There are a variety of these
inhibitors, but most important are the serpins, a family of serine
proteinase inhibitors, typified by antitrypsin
( The physiological
mechanisms controlling neutrophil chemotaxis are unclear, although Ward
and Becker (7) suggested that this process may be under the
control of a surface serine proteinase. They showed that the inhibition
of this enzyme by organophosphorus compounds reduced neutrophil
chemotaxis (8, 9) but were unsure as to the identity
of the natural substrate or inhibitor(10) . Other workers have
reported that chloromethylketone inhibitors of chymotrypsin-like
proteinases were able to suppress human neutrophil chemotaxis (11, 12, 13) and superoxide production (14) as well as attenuating membrane potential changes in rat
neutrophils(15) . Furthermore King and co-workers (16) reported that a monoclonal antibody against human
neutrophils inhibited superoxide anion generation in response to the
chemotactic peptide fMLP ( Human neutrophil cathepsin G and immunopurified polyclonal
anti-cathepsin B antibodies were from Dr. D. Buttle (Strangeways
Laboratory, Cambridge, UK), human neutrophil elastase was provided by
D. Bruce (Department of Haematology, University of Cambridge), and
sheep anti-human cathepsin G and anti human-elastase immunoglobulin
were obtained from the Binding Site Ltd. (Birmingham, UK).
Cbz-Gly-Gly-Phe-CMK, tosyl-Lys-CMK, Cbz-Phe-Ala-DMK, and
MeOSuc-Ala-Ala-Pro-Val-CMK were from Bachem Feinchemikalien AG
(Bubendorf, Switzerland), and Z-Gly-Leu-Phe-CMK was from Enzyme System
Products. Recombinant methionine (P1) antitrypsin was obtained from Dr.
H. P. Schnebli, (Ciba-Geigy, Basel, Switzerland), arginine (P1)
antitrypsin and the pKV50 plasmid were from Delta Biotechnology,
(Nottingham, UK).
where E All values were determined on
2-3 separate occasions with the results quoted as weighted mean
with standard error.
The chemotactic response
was based on the multiple blind well assay system(34) . The
lower well contained 27 µl of 10
Figure 1:
The effect of anti-cathepsin G antibody
on neutrophil chemotaxis to 10
It was possible that the
inhibition was nonspecific and resulted from the interaction of the
antibody Fc fragment with neutrophil receptors (38) . To assess
this possibility, the cells were incubated with an immunopurified
antibody to the macrophage protein cathepsin B, which is not present in
human neutrophils(39) . The antibody had no effect on
neutrophil chemotaxis (control 24.2 ± 2.3 cpf; anti-cathepsin B
antibody 23.3 ± 1.9 cpf at 5.7 µg/ml: n =
4), whereas similar concentrations of anti-cathepsin G antibody
inhibited cell migration by 64%. Furthermore, polyclonal
anti-elastase antibodies also had no effect on the chemotactic response
of neutrophils to fMLP, suggesting that the effect was specific to
cathepsin G.
Figure 2:
The effect of Z-Gly-Leu-Phe-CMK,
Cbz-Gly-Gly-Phe-CMK (a) and MeOSuc-Ala-Ala-Pro-Val-CMK (b) on neutrophil chemotaxis to 10
The chloromethylketone inhibitor of
trypsin (tosyl-Lys-CMK; (43) ), an enzyme that is not found in
the neutrophil, had no effect on neutrophil chemotaxis at
concentrations of up to 100 µM (control 33.4 ± 3.1
cpf; 100 µM tosyl-Lys-CMK 29.2 ± 4.0 cpf: n = 6). In addition Cbz-Phe-Ala-DMK, a specific inhibitor of
the thiol proteinase cathepsin B, had no effect on chemotaxis at
concentrations up to 100 µM (control 30.8 ± 4.7
cpf; 100 µM Cbz-Phe-Ala-DMK 26.5 ± 2.7 cpf; n = 4). Z-Gly-Leu-Phe-CMK (45 µM) suppressed
the neutrophil chemotactic response over a range of fMLP concentrations (Fig. 3a). In contrast, similar concentrations of both
Cbz-Gly-Gly-Phe-CMK and MeOSuc-Ala-Ala-Pro-Val-CMK produced a shift in
the peak chemotactic response to fMLP. Cbz-Gly-Gly-Phe-CMK, at a
concentration of 45 µM, produced a peak cell migration of
46.6 ± 4.1 cpf in response to 10
Figure 3:
The effect of Z-Gly-Leu-Phe-CMK (45
µM; a), Cbz-Gly-Gly-Phe-CMK (45 µM; b) and MeOSuc-Ala-Ala-Pro-Val-CMK (65 µM; b) on the fMLP dose-response curve. The x axis
represents increasing concentrations of fMLP, and the y axis
represents the chemotactic response. The results are mean with standard
error bars.
Similarly, the peak chemotactic response of neutrophils incubated
with 65 µM MeOSuc-Ala-Ala-Pro-Val-CMK was at an fMLP
concentration of 10
Figure 4:
The effect of leucine, methionine, and
phenylalanine (P1) antichymotrypsin on neutrophil chemotaxis to
10
Figure 5:
The effect of Z-Gly-Leu-Phe-CMK,
Cbz-Gly-Gly-Phe-CMK, and plasma antichymotrypsin on neutrophil
polarization to 10
There was no effect on cell
viability, and the proteins were not themselves chemoattractants.
Furthermore the effects were not reduced by the endotoxin sequester
polymyxin B, and the inhibition was apparent over a range of fMLP
concentrations (data not shown). Arginine (P1) antichymotrypsin had no
effect on the chemotactic response at active site concentrations
(against human The inhibitory effect of antichymotrypsin was also apparent with
a second chemoattractant, zymosan-activated serum. Using this agent,
the peak chemoattractant response was observed at a dilution of 7%
(v/v). Leucine (P1) antichymotrypsin reduced the control value from
38.6 (±6.0) to 30.6 (±7.2) cpf at 0.22 µM and to 23.8 (±3.3) cpf at 0.44 µM active site.
Arginine (P1) antichymotrypsin had no effect at active site
concentrations of up to 1.5 µM (control 24.0 ± 4.4
cpf; arginine (P1) antichymotrypsin 1.5 µM active site
29.4 ± 6.5; n = 4).
Despite the obvious importance of neutrophil migration,
little is known about the mechanisms available to control or reduce
this response at a site of inflammation after the initiating insult has
been removed. We have shown that inhibitors of cathepsin G (antibodies,
chloromethylketones, and active site mutants of antichymotrypsin) are
able to attenuate neutrophil migration in vitro. The
chloromethylketones and antichymotrypsin that retard neutrophil
chemotaxis were also rapid inhibitors of bovine Immunopurified antibodies to cathepsin G were able to reduce
neutrophil migration to the chemoattractant fMLP, presumably by binding
to this enzyme on the surface of the neutrophils. The results of King et al.(16) also support the hypothesis that cathepsin
G plays a role in neutrophil activation; antibodies against a
chymotrypsin-like enzyme on the surface of neutrophils were able to
inhibit superoxide anion production induced by fMLP. The absence of an
effect with anti-elastase and anti-cathepsin B antibodies suggested a
specific role for cathepsin G in mediating the cellular response to
fMLP. In order to confirm this role for cathepsin G synthetic
chloromethylketone inhibitors of neutrophil elastase, cathepsin G,
chymotrypsin and trypsin were assessed for their ability to attenuate
neutrophil chemotaxis. Those that inhibited predominantly cathepsin G
(Z-Gly-Leu-Phe-CMK) and chymotrypsin (Cbz-Gly-Gly-Phe-CMK) were potent
inhibitors of chemotaxis, whilst the specific inhibitor of elastase
(MeOSuc-Ala-Ala-Pro-Val-CMK) exerted its only effect at a higher
concentration. This is despite Z-Gly-Leu-Phe-CMK and
Cbz-Gly-Gly-Phe-CMK having significantly lower association rate
constants with cathepsin G (51.2 M Active site mutants were used to probe the specificity
of the neutrophil surface enzyme involved in the chemotactic response.
The results were assessed using active concentrations of proteins as
this allowed a more useful comparison between proteins and avoided
inaccuracies and distortions when using preparations with different
specific activities. Recombinant antitrypsin preparations had no effect
on neutrophil chemotaxis at active site concentrations of up to 0.48
µM (20 µg/ml). Indeed the concentration of methionine
(P1) antitrypsin had to be raised to 0.98 µM (40
µg/ml) before there was a significant fall in the chemotactic
response. Nevertheless this response still occurred at physiological
concentrations of antitrypsin found within the plasma or at sites of
inflammation(45) . Clearly the inhibition of neutrophil
elastase alone (which was efficiently obtained with valine (P1)
antitrypsin) was insufficient to inhibit the chemotactic response at
concentrations up to 0.48 µM. Active site mutants of
antichymotrypsin, the cognate inhibitor of cathepsin G, were then
assessed for their ability to inhibit chemotaxis. The kinetic analysis
confirmed that those mutants, which were efficient inhibitors of
cathepsin G and chymotrypsin (leucine, methionine, and phenylalanine
(P1) antichymotrypsin) were also able to attenuate neutrophil
chemotaxis to the peptide fMLP. Once again, this occurred at
concentrations well below those obtained in the plasma or at sites of
inflammation (46) The importance of inhibitor specificity was
confirmed by arginine (P1) antichymotrypsin, which has a 10-fold lower
association rate constant and forms a 10-fold less stable complex (as
determined by the K Previous
work has shown that the neutrophil surface serine proteinase has a K The results suggest that the serpins antitrypsin and
antichymotrypsin can play an important role in modulating neutrophil
migration by interacting with cathepsin G or a chymotrypsin-like enzyme
on the surface of the neutrophil. This contrasts with the effect of
antichymotrypsin on neutrophil superoxide anion production, which
appears to be independent of the active site (24) and may be
mediated by enzyme-inhibitor complex formation(47) .
Interestingly, both complexed (48, 49) and cleaved (50, 51) serpins are able to stimulate neutrophil
chemotaxis. Thus native and cleaved proteins may interact to modulate
neutrophil migration at sites of inflammation, the former reducing
chemotaxis during periods of health, and the latter promoting cell
migration to sites of inflammation.
Volume 270,
Number 40,
Issue of October 06, pp. 23437-23443, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-antitrypsin and -antichymotrypsin
were used to probe the specificity of the proteinases involved in
chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis.
Moreover, rapid inhibitors of cathepsin G and -chymotrypsin
suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine
(fMLP) and zymosan-activated serum in multiple blind well assays and to
fMLP in migration assays under agarose. The concentrations of
antichymotrypsin mutants that reduced chemotaxis by 50% would
inactivate free cathepsin G with a half-life of 1.5-3 s, whereas
the concentrations of chloromethylketones required to produce a similar
inhibition of chemotaxis would inactivate cathepsin G with a half-life
of 345 s. These data suggest different modes of action for these two
classes of inhibitors. Indeed the chloromethylketone inhibitors of
cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin
(Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape
change in the purified neutrophils exposed to fMLP. Antichymotrypsin
did not affect shape change in response to fMLP even at concentrations
that were able to reduce neutrophil chemotaxis by 50%. These results
support the involvement of cell surface proteinases in the control of
cell migration and show that antichymotrypsin and chloromethylketones
have differing modes of action. This opens the possibility for the
rational design of anti-inflammatory agents targeted at neutrophil
membrane enzymes.
-proteinase inhibitor) and
antichymotrypsin(4) . Each of the family members has a unique
inhibitory specificity, but they all share a similar overall molecular
structure(5) . Neutrophil chemotaxis is an important component
of the inflammatory response, and the recruitment of neutrophils to the
lung may play a pivotal role in the pathogenesis of
emphysema(6) . If neutrophil migration into the lungs is
excessive, there will be enhanced delivery and release of proteolytic
enzymes, which may overwhelm the native inhibitors causing lung damage
and eventually the development of emphysema.)by binding to a surface
chymotrypsin-like enzyme. Recent work (17) has demonstrated
that serine proteinase inhibitors (particularly antichymotrypsin) are
able to reduce neutrophil chemotaxis in response to the same
chemoattractant. This has led to the suggestion that these agents exert
their effects by binding and inhibiting a cognate surface serine
proteinase, possibly cathepsin G, involved in receptor-mediated cell
activation. This hypothesis is supported by the demonstration that
neutrophil elastase, and probably cathepsin G, can bind to the cell
membrane after secretion and therefore play a role in cell
migration(18) . Moreover, the human neutrophil contains
isoforms of elastase and cathepsin G that may have differing
roles(19) . The current study uses inhibitors to assess the
role of serine proteinases in modulating the neutrophil chemotactic
response. This was achieved by examining the effects of antibody and
chloromethylketone enzyme inhibitors along with P1 mutants of
antitrypsin and antichymotrypsin, which have a range of inhibitory
profiles and thus could be used to probe the specificity of the serine
proteinases controlling chemotaxis.
Purification of Anti-cathepsin G Antibodies
Cathepsin G (10 mg) was coupled to cyanogen bromide-activated
matrix according to the manufacturer's instructions. The gel (3.5
ml) was then packed into a column at 20 ml/h, washed with 0.05 M Tris, pH 8.0, and 1 ml of anti-cathepsin G antibody was applied at
the same flow rate. Immunopurified antibody was eluted from the column
with 0.2 M glycine, pH 2.5, and fractions of 2 ml were
collected into tubes containing Tris to restore neutral pH. The column
was reequilibrated with 0.05 M Tris, pH 8.0, and the procedure
was repeated. The IgG concentration of the eluate was determined
spectrophotometrically ( =
14.5), and the specificity of the antibody was confirmed by radial
immunodiffusion.
Construction, Expression, and Purification of Antitrypsin
Mutants
Active site mutants of antitrypsin were produced by
site-directed mutagenesis and expressed in Saccharomyces cerevisiae strain AB 116 as described previously(20) . The
recombinant protein was extracted and purified to homogeneity according
to the method of Travis et al.(21) . Purity was
assessed by 10-20% (w/v) SDS-polyacrylamide gel electrophoresis,
and total antitrypsin concentration was determined by rocket
immunoelectrophoresis (22) with a yeast recombinant methionine
(P1) antitrypsin standard that had been calibrated by amino acid
analysis. The recombinant P1 antitrypsin mutants migrated as several
bands on SDS-polyacrylamide gel electrophoresis; amino-terminal
sequencing confirmed these to be due to amino-terminal, but not
reactive center loop, cleavage. The specific activities of these
mutants against bovine -chymotrypsin were arginine (P1)
antitrypsin 77%, methionine (P1) antitrypsin 89%, valine (P1)
antitrypsin 67%, and lysine (P1) antitrypsin 81%.Construction, Expression, and Purification of
Antichymotrypsin Mutants
Recombinant antichymotrypsin with leucine, methionine,
phenylalanine, and arginine at the P1 (358) position were expressed in Escherichia coli and purified to homogeneity as described
previously(23, 24) . Purity was confirmed by
10-20% (w/v) SDS-polyacrylamide gel electrophoresis, and total
protein concentration was determined by Bradford assay (25) against a recombinant leucine (P1) antichymotrypsin
standard that had been calibrated by amino acid analysis. The specific
inhibitory activities of leucine, methionine, arginine, and
phenylalanine (P1) antichymotrypsin against bovine -chymotrypsin
were 66, 69, 37, and 32%, respectively.Inhibition Kinetics
Active Site Determination of Serine Proteinases and
Recombinant Inhibitors
The active site titration of bovine
-chymotrypsin and human -thrombin were performed according to
the method of Chase and Shaw (26) using the suicide substrates p-nitrophenyl acetate and p-nitrophenyl p`-guanidino benzoate, respectively. Bovine -chymotrypsin
was used to determine the activity of recombinant antitrypsin and
antichymotrypsin using the substrate Suc-Ala-Ala-Pro-Phe-pNA as
detailed previously(27) . These inhibitors were in turn used to
determine the activity of cathepsin G and neutrophil elastase using the
substrates Suc-Ala-Ala-Pro-Phe-pNA and MeOSuc-Ala-Ala-Pro-Val-pNA,
respectively.Determination of Second Order Association Rate
Constants
Equimolar active site concentrations of enzyme and
inhibitor were incubated at 20 °C as described
previously(28) . The value of the association rate constant (k) was determined by nonlinear
regressionanalysis according to the following equation(29) ,
is the initial enzyme
concentration, and t is time.Determination of Pseudo-first Order Association Rate
Constants
7-35-fold active site molar excess of inhibitor
was incubated with the enzyme for varying time intervals at 20 °C.
The quantity of free enzyme was then determined by adding reaction
buffer containing substrate, and the association rate constant was
calculated as described previously(30, 31) .Determination of Association Rate Constants and K
Kinetic
parameters for the interaction of recombinant antitrypsin and
antichymotrypsin with human neutrophil elastase and cathepsin G were
determined at 20 °C as detailed previously(27) . All
buffers contained 0.1% (v/v) Triton X-100, and the substrates
MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA were present at
a concentration of 400 µM and 6 mM for elastase
and cathepsin G, respectively. The K
Values Using Progress Curve Analysis
values for
neutrophil elastase with MeOSuc-Ala-Ala-Pro-Val-pNA and cathepsin G
with Suc-Ala-Ala-Pro-Phe-pNA were determined to be 0.1 ± 0.01
and 1.9 ± 0.23 mM, respectively.Isolation of Neutrophils
Venous blood (10 ml) was collected from healthy volunteers
into lithium heparin tubes, and the neutrophils were isolated on
percoll gradients according to the method of Jepsen and
Skottun(32) . The cells (>95% pure, >95% viable by trypan
blue exclusion) were washed, counted, and resuspended at the required
concentrations in Hepes-buffered RPMI 1640 medium/bovine serum albumin
(2 mg/ml). All reagents were endotoxin-free (<20 pg/ml) as
determined by a Limulus assay. Following isolation, the cells were
spherical, indicating that they had not been activated during
harvesting(33) .Neutrophil Chemotaxis in the Multiple Blind Well Assay
System
The chloromethylketones and diazomethylketone were dissolved
in a minimum quantity of dimethylsulfoxide and then diluted to the
required concentration with Hepes-buffered RPMI 1640 medium. The effect
of these inhibitors on chemotaxis was assessed by incubating them with
the isolated neutrophils for 30 min at room temperature prior to
placing the cells in the chemotactic chamber. Similarly the effect of
the antitrypsin and antichymotrypsin mutants was assessed after
incubating with the neutrophils for 30 min, and that of the antibodies
was assessed after incubating for 60 min.M fMLP
or 7% (v/v) zymosan-activated serum as the
chemoattractant(35) , and the upper chamber was filled with 50
µl of the neutrophil suspension (final concentration, 3
10
cells per ml) with or without antibody or inhibitor. The
two chambers were separated by a 2.0-µm pore size polycarbonate
filter. Negative control wells contained medium but no chemoattractant
in the lower chamber. The assay plates were incubated at 37 °C for
20 min, and the membrane was then washed, fixed, and stained. The cells
were counted from 5 areas of each membrane at 400 magnification,
and the mean value was obtained. Each experiment was performed in
triplicate, and the mean value of the three membranes was taken as the
result. All experiments were repeated on six different cell
preparations unless otherwise stated. In the event of the negative
control exceeding 5% of the positive control, the membrane was
discarded. All values are expressed as mean cells/high power field
(cpf) ± S.E., and differences between groups of six subjects
were assessed by the Wilcoxon-Signed Rank test. Under these conditions,
the chemotactic assay had a control within-batch co-efficient of
variation of 4.0% (n = 5).Neutrophil Chemotaxis under Agarose
Human neutrophils were isolated as described previously and
incubated at 5 10
cells/ml in Hepes-buffered RPMI
1640 medium with 10% (v/v) fetal calf serum with increasing
concentrations of the chloromethylketone or antichymotrypsin at room
temperature. The cells were then allowed to migrate under agarose to a
chemoattractant (10M fMLP) or
buffer(36) , which allows the measurement of both chemotaxis
and chemokinesis. The value for chemokinesis was then subtracted from
that of chemotaxis to give a final result. The coefficient of variation
was 9% (n = 6).
Neutrophil Polarization to fMLP
Neutrophils were isolated on percoll gradients, washed twice
with sterile calcium-free buffer (normal saline with 0.1% (w/v) human
albumin, 8 mM glucose, and 10 mM Hepes), counted, and
resuspended at 1 10 cells/ml in Hanks'
buffered saline solution containing 0.1% (w/v) albumin and 8 mM glucose. Cells were incubated with chloromethylketones
(0-100 µM) or antichymotrypsin (0-0.5
µM active site) at room temperature for 30 min prior to
the addition of fMLP to a final concentration of 10M. The suspensions were then left at room temperature
for a further hour before fixing with 10 volumes 2% (v/v) formaldehyde.
Polarization was measured by flow cytometry on a Becton Dickenson
FACScan with Lysis II software after gating on the neutrophil
population.
The Effect of Anti-cathepsin G, Anti-elastase, and
Anti-cathepsin B Antibodies on Neutrophil Chemotaxis
The anti-cathepsin G antibodies reduced the chemotactic
response of neutrophils to 10M fMLP in a
dose-dependent manner (Fig. 1) from a control value of 30.5
(±3.1) to 11.0 (±2.2) cpf at 5.7 µg/ml (p < 0.025). The antibody did not affect cell viability (as
measured by trypan blue exclusion) or cause neutrophil agglutination
(as visualized under light microscopy at 400
magnification), and
the inhibition could not be prevented by 5 µg/ml polymyxin B
sulfate, a sequester of endotoxin(37) . Furthermore, the
antibody was able to exert its effect over a range of fMLP
concentrations (10-10
M) and was not itself chemotactic when placed in the
lower chamber (positive control, 10
M fMLP
44.5 cpf; negative control, 1.3 cpf; 5.7 µg/ml anti-cathepsin G
antibody, 1.1 cpf: n = 2).
M fMLP. The x axis represents increasing concentrations of antibody, and
the y axis the represents the average number of neutrophils
counted per high power field. The histogram is mean with standard error
bars. The significance of any differences from the control value is
indicated.
The Effect of Chloromethylketones on Neutrophil
Chemotaxis
The specific inhibitor of cathepsin G,
Z-Gly-Leu-Phe-CMK(40) , was highly effective at reducing
neutrophil chemotaxis to fMLP. Cell migration was reduced from a
control value of 39.5 ± 4.4 to 3.9 ± 2.6 cpf (90.2%
inhibition; p < 0.025) at 55 µM (Fig. 2a). Similarly, Cbz-Gly-Gly-Phe-CMK, an
inhibitor of chymotrypsin (41) and to a lesser extent of
cathepsin G(40) , reduced chemotaxis from a control value of
36.6 ± 2.7 to 1.5 ± 1.1 cpf (97.0% inhibition; p < 0.025) at 55 µM (Fig. 2a).
MeOSuc-Ala-Ala-Pro-Val-CMK, a specific inhibitor of neutrophil elastase
that has no effect on cathepsin G(40, 42) , also
inhibited neutrophil chemotaxis although less effectively. At 65
µM this chloromethylketone reduced the chemotactic
response to fMLP from a control value of 31.8 ± 6.6 to 14.5
± 5.5 cpf (54% inhibition; p < 0.025), although
lower concentrations (55 µM and below) had no effect (Fig. 2b). None of these agents at concentrations of up
to 100 µM had any effect on cell viability as determined
by trypan blue exclusion or by lactate dehydrogenase release.
Furthermore, none of the chloromethylketones was chemotactic when
placed in the lower chamber.
M fMLP. The x axis represents increasing concentrations of
inhibitor, and the y axis represents the average number of
neutrophils counted per high power field. The histograms are mean with
standard error bars. The significance of any differences from the
control value is indicated.
M fMLP. This was significantly greater (p < 0.025) than
the value obtained at 10
M fMLP (29.1
± 6.1 cpf) but not significantly less than the peak response in
the control curve (53.0 ± 2.7 cpf) that occurred, as expected,
at 10
M fMLP (Fig. 3b).
M (42.8 ± 1.5
cpf). This was significantly greater than the response to
10
M fMLP (34.2 ± 3.9 cpf; p < 0.025) but less than the chemotactic response of the control
cells to 10
M fMLP (53.0 ± 2.7 cpf; p < 0.025; Fig. 3b).
Recombinant P1 Antitrypsin Mutants
Association Rate Constants of P1 Antitrypsin
Mutants
The association rate constants of the antitrypsin
mutants with bovine -chymotrypsin, cathepsin G, and neutrophil
elastase are shown in Table 1. Methionine (P1) antitrypsin has
been reported to be labile, losing inhibitory activity upon standing at
4 °C(21) . To avoid this, the protein was harvested in the
presence of 0.01 M 
-mercaptoethanol, and full inhibitory
activity was confirmed by dialysis against this agent (0.05 M Tris, 0.01 M -mercaptoethanol, pH 8.0), which had no
effect on the association rate constant with bovine chymotrypsin (2.5
± 0.1 10M s
prior to dialysis; 2.4 ± 0.2
10M s
following dialysis). The progress curve kinetic analysis allowed
the determination of the K
value for methionine
and arginine (P1) antitrypsin with cathepsin G as 5.8 ± 0.5 and
143 ± 5 nM, respectively.
The Effect of Antitrypsin Mutants on Neutrophil
Chemotaxis
None of the recombinant antitrypsin preparations
inhibited neutrophil chemotaxis at concentrations of up to 0.48
µM active site (data not shown). However when the
concentration of methionine (P1) antitrypsin was increased to 0.95
µM, there was a 30% reduction in chemotactic response to
fMLP (p < 0.05).Recombinant P1 Antichymotrypsin Mutants
Association Rate Constants of P1 Antichymotrypsin
Mutants
The association rate constants of the antichymotrypsin
mutants and bovine chymotrypsin, neutrophil elastase, and cathepsin G
are shown in Table 2. Progress curve analysis allowed the
determination of K values for leucine (P1)
antichymotrypsin, methionine (P1) antichymotrypsin, phenylalanine (P1)
antichymotrypsin, and arginine (P1) antichymotrypsin with cathepsin G
as 62 ± 9 pM, 79 ± 10 pM, 208 ±
11 pM, and 1.9 ± 0.09 nM, respectively.
The Effect of Antichymotrypsin Mutants on Neutrophil
Chemotaxis
Those proteins that were efficient inhibitors of
cathepsin G were also able to inhibit neutrophil chemotaxis to the
peptide fMLP. Leucine (P1) antichymotrypsin produced a
concentration-related fall in neutrophil chemotaxis from a control
value of 51.5 (±5.6) to 23.6 (±3.8) cpf at 0.44
µM active site (Fig. 4). Similarly, phenylalanine
(P1) antichymotrypsin produced a concentration-related fall in
neutrophil chemotaxis from a control value of 36.8 (±2.5) to
16.2 (±2.0) cpf at 0.44 µM active site, and
methionine (P1) antichymotrypsin reduced the chemotactic response from
41.4 (±1.9) to 18.8 (±2.0) cpf at 0.44 µM active site (Fig. 5).
M fMLP. The histograms are mean with
standard error bars for six experiments. The significance of any
differences from the control value is
indicated.
M fMLP. The curves are
representative of the results obtained on cells from three subjects. A, negative control; B, positive control
(10
M fMLP); C, Z-Gly-Leu-Phe-CMK
100 µM; D, Z-Gly-Leu-Phe-CMK 50 µM; E, Z-Gly-Leu-Phe-CMK 10 µM; F,
Z-Gly-Leu-Phe-CMK 1 µM; G, Cbz-Gly-Gly-Phe-CMK
100 µM; H, plasma antichymotrypsin 0.5
µM.
-thrombin) of up to 1.5 µM when a
slight but significant (p < 0.025) fall was observed
(control 32 ± 3.6 cpf; arginine (P1) antichymotrypsin 1.5
µM active site 26.6 ± 3.4 cpf: n =
6).The Effect of Inhibitors of Cathepsin G on Neutrophil
Migration under Agarose
There has been some debate about the
ability of the multiple blind well assay system to differentiate
between neutrophil chemotaxis and random movement, chemokinesis.
Inhibitors of cathepsin G were therefore assessed, by a different
observer, for their effect on neutrophil migration to fMLP under
agarose. The peak leading front response was apparent at
10M fMLP and was reduced in a dose-related
manner by inhibitors of cathepsin G and chymotrypsin. Z-Gly-Leu-Phe-CMK
reduced the leading front from a control value of 8.3 ± S.E. 0.7
to 5.3 ± 0.3 mm at 1 µM, 3.7 ± 0.3 mm at 10
µM, 2.7 ± 0.7 mm at 50 µM, and 1.7
± 0.3 mm at 100 µM. Similarly Cbz-Gly-Gly-Phe-CMK
reduced the control value from 8.3 ± 0.7 to 8.0 ± 1 mm at
1 µM, 4.7 ± 0.3 mm at 10 µM, 4.3
± 0.4 mm at 50 µM, and 1.7 ± 0.3 mm at 100
µM. Plasma antichymotrypsin-reduced neutrophil chemotaxis
from the control value to 7.0 ± 0.6 mm at 0.1 µM,
6.7 ± 0.3 mm at 0.3 µM and 5.7 ± 0.3 mm at
0.5 µM. All values are based on results of cells isolated
from 3 individuals.
The Effect of Inhibitors of Cathepsin G on Neutrophil
Polarization
Z-Gly-Leu-Phe-CMK abolished neutrophil polarization
at 100 µM in a dose-dependent manner (Fig. 5).
Cbz-Gly-Gly-Phe-CMK was less effective at inhibiting neutrophil shape
change and at higher concentrations activated the cells inducing
polarization above the value of the negative control. Antichymotrypsin
had no effect on polarization at concentrations of 0.5 µM (Fig. 5).
-chymotrypsin. s
and 4.1 M
s
, respectively(40) ) than
MeOSuc-Ala-Ala-Pro-Val-CMK has with elastase (922 M
s
(40) ). The
concentrations of chloromethylketones used here were similar to those
used by other workers to inhibit chemotaxis (11, 12, 13) and attenuate antibody-dependent
cellular cytotoxicity(44) . Strikingly, the reduction in
chemotaxis by both Cbz-Gly-Gly-Phe-CMK and MeOSuc-Ala-Ala-Pro-Val-CMK
was mitigated by increasing the concentration of chemoattractant. This
suggests that these agents were not irreversible inhibitors of
chemotaxis. In contrast, the effect of Z-Gly-Leu-Phe-CMK, which is
significantly more efficient in inactivating cathepsin G than
chymotrypsin (51.2 M
s
and 3.0 M
s
,
respectively (40) ), was apparent over a range of fMLP
concentrations. The results with synthetic inhibitors led to the
assessment of variants of the naturally occurring proteinase
inhibitors, antitrypsin, and antichymotrypsin, on neutrophil
chemotaxis.
value) with cathepsin G than
the other active site mutants. This protein had only a small effect on
chemotaxis at concentrations over 3-fold greater than those required by
the other mutants to reduce the chemotactic response by over 50% (Fig. 4). The effect of inhibitors of cathepsin G on neutrophil
migration was confirmed in assays under agarose. Such assay systems
also take into account random migration or chemokinesis. value for a synthetic substrate closer to
chymotrypsin than cathepsin G(24) , suggesting that the enzyme,
although cathepsin G-like, is not cathepsin G. The present data,
however, suggest that the enzyme involved in the control of chemotaxis
is unlikely to have a specificity identical to chymotrypsin, as
methionine (P1) antitrypsin is able to inhibit chymotrypsin more
efficiently than the antichymotrypsin mutants and yet has little effect
on chemotaxis until higher concentrations of inhibitor are used. The
association rate constants reported here allow the determination of
half time for the inhibition of cathepsin G at a given inhibitor
concentration. For the antichymotrypsin mutants tested, there was a
correlation between a short half-life for the inhibition of cathepsin G
and a reduction in the chemotactic response. The concentrations of
antichymotrypsin mutants that reduced chemotaxis by 50% would
inactivate cathepsin G with a half-life of approximately 1.5-3 s.
The correlation between a short half-life for inactivation of cathepsin
G and the inhibition of chemotaxis could not be extended across
different classes of inhibitors. Z-Gly-Leu-Phe-CMK reduced chemotaxis
by 50% at a concentration that would inactivate cathepsin G with a
half-life of 345 s. This is significantly longer for the same
inhibition of chemotaxis by the antichymotrypsin mutants and suggests
that the enzyme may not be cathepsin G or that, by virtue of their
size, chloromethylketones are more able to inactivate membrane bound
cathepsin G than larger proteins. A third possibility is that the
uncharged chloromethylketones are able to cross the cell membrane and
inhibit an intracellular cathepsin G that is released from
intracellular granules during chemotaxis. The differential effects of
antichymotrypsin and the chloromethylketone inhibitors is underscored
by their effect on neutrophil shape change in response to fMLP. The
cathepsin G inhibitor Z-Gly-Leu-Phe-CMK abolished neutrophil shape
change in a dose-dependent manner, Cbz-Gly-Gly-Phe-CMK, a less potent
inhibitor, had less effect, whereas antichymotrypsin had no effect at
all. Thus chloromethylketone inhibitors mediate their effect, at least
partly, by blocking cell shape change in response to a chemoattractant,
but the point of action of antichymotrypsin occurs subsequent to
polarization.
)
We wish to thank Dr. H. P. Schnebli for providing the
methionine (P1) antitrypsin standard and the Department of
Biochemistry, University of Cambridge for performing N-terminal
sequencing and amino acid analysis on the recombinant proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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