(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
-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.
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
(-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.
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 ()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.
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 is the initial enzyme
concentration, and t is time.
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 10M 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).
Figure 1:
The effect of anti-cathepsin G antibody
on neutrophil chemotaxis to 10M 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.
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 10M 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.
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 10M 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).
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 10M (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).
Figure 4:
The effect of leucine, methionine, and
phenylalanine (P1) antichymotrypsin on neutrophil chemotaxis to
10M fMLP. The histograms are mean with
standard error bars for six experiments. The significance of any
differences from the control value is
indicated.
Figure 5:
The effect of Z-Gly-Leu-Phe-CMK,
Cbz-Gly-Gly-Phe-CMK, and plasma antichymotrypsin on neutrophil
polarization to 10M 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.
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 -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 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 -chymotrypsin.
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 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.
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 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.
Previous
work has shown that the neutrophil surface serine proteinase has a K 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.
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.