(Received for publication, July 19, 1995; and in revised form, October 11, 1995)
From the
In response to invading microorganisms, neutrophils produce
large amounts of superoxide and other reactive oxygen intermediates
(ROI) by assembly and activation of a multicomponent enzyme complex,
the NADPH oxidase. While fulfilling a microbicidal role, ROI have also
been postulated to serve as signaling molecules, because activation of
the NADPH oxidase was found to be associated with increased tyrosine
phosphorylation (Fialkow, L., Chan, C. K., Grinstein, S., and Downey,
G. P.(1993) J. Biol. Chem. 268, 17131-17137). The
mechanism whereby ROI induces phosphotyrosine accumulation was
investigated using electroporated neutrophils stimulated with guanosine
5`-O-3-thiotriphosphate in order to bypass membrane receptors. In vitro immune complex assays and immunoblotting were used to
identify five tyrosine kinases present in human neutrophils. Of these,
p56/59, p72
, and
p77
were activated during production of ROI.
Interestingly, the in vitro autophosphorylation activities of
p53/56
and p59
were found
to decline with ROI production. The mode of regulation of
p56/59
was explored in detail. Oxidizing agents
were unable to activate p56/59
in vitro and, once activated in situ, reducing agents failed to
inactivate it, suggesting that the effects of ROI are indirect.
Tyrosine phosphorylation of p56/59
paralleled
its activation, and dephosphorylation in vitro reversed the
stimulation. We therefore conclude that tyrosine phosphorylation is
central to the regulation of p56/59
and likely
also of p72
, which is similarly phosphorylated
upon activation of the oxidase. Because ROI have been shown to reduce
the activity of tyrosine phosphatases, we suggest that this inhibition
allows constitutively active kinases to auto/transphosphorylate on
stimulatory tyrosine residues, leading to an increase in their
catalytic activity. Enhanced phosphotyrosine accumulation would then
result from the combined effects of increased phosphorylation with
decreased dephosphorylation.
Neutrophils play a central role in host protection against
infection, killing pathogens by a series of rapid and highly regulated
responses. These include chemotaxis, phagocytosis, secretion of
anti-microbial agents, and generation of reactive oxygen intermediates
(ROI) ()(reviewed by Sha'afi and Molski(1988)).
Production of ROI is mediated by a multicomponent enzyme complex, the
NADPH oxidase, present in the membranes of neutrophils and other
leukocytes (Morel et al., 1991). Functional assembly of the
oxidase facilitates the transfer of one electron from cytosolic NADPH
to molecular oxygen, producing superoxide. Dismutation of superoxide in
turn generates hydrogen peroxide, and both of these molecules can
further generate other reactive oxygen intermediates, including
hypochlorous acid, hydroxyl radical, and peroxynitrite ion (Halliwell
and Gutteridge, 1990). Although the mechanisms whereby NADPH
oxidase-derived ROI attack microbial targets are not completely
understood, their importance in host defence is highlighted by a rare
genetic disorder, chronic granulomatous disease. Patients afflicted
with this disorder lack the ability to produce ROI and, as a result,
suffer from chronic and recurring infections that can be lethal (Smith
and Curnutte, 1991).
Although neutrophils are probably the most
efficient source of superoxide, virtually all eukaryotic cells produce
ROI, primarily as side products of electron transfer reactions in
mitochondria and the endoplasmic reticulum (Halliwell and Gutteridge,
1985). In addition to their microbicidal role in phagocytes, ROI have
been suggested to act as signaling molecules in other cells (Schreck
and Baeuerle, 1991). In principle, ROI constitute good candidate
signaling molecules because they are small, rapidly diffusible, and
highly reactive. Moreover, both intra- and extracellular concentrations
of ROI can be rapidly scavenged by several enzymes, including
superoxide dismutase, catalase, and the glutathione peroxidase system,
allowing tight control of ROI concentrations and rapid termination of
signals. The notion that reactive, small inorganic molecules can
function as intracellular signals is supported by the well established
role of nitric oxide in the regulation of vascular tone,
neurotransmission, and cell-mediated immune responses (Nathan and Xie,
1994). By comparison, much less is known about the role of ROI in
signaling, but suggested targets include the transcription factor
NFB (Schreck et al., 1991), tyrosine phosphatases (Hecht
and Zick, 1992, Fialkow et al., 1994), and phospholipase
A
(Zor et al., 1993).
Recent observations suggested a role for ROI in neutrophil signal transduction (Fialkow et al., 1993). Neutrophils stimulated to produce ROI were reported to undergo increased tyrosine phosphorylation of several proteins. Exogenous oxidants were able to mimic this response, whereas anti-oxidants could block it. Several lines of evidence suggested that ROI generated by the NADPH oxidase were responsible for the effect, including the finding that the increased tyrosine phosphorylation failed to occur in neutrophils from patients with chronic granulomatous disease. Inasmuch as tyrosine phosphorylation is an important mediator in the regulation of anti-microbial responses (Berkow and Dodson, 1990; Grinstein and Furuya, 1991), ROI may play an important role in the control of auto/paracrine signaling at sites of inflammation.
The extent of tyrosine phosphorylation is determined by the activity of two competing enzyme families, tyrosine kinases and phosphatases. Earlier in vitro (Hecht and Zick, 1992) and in vivo (Zor et al., 1993) studies have suggested that ROI can inhibit the activity of certain tyrosine phosphatases by oxidation of a conserved cysteine residue within their catalytic domain. Although the inhibition of tyrosine phosphatases may account for the elevated tyrosine phosphorylation induced by ROI, increased activity of tyrosine kinases could conceivably contribute to the response. Indeed, tyrosine kinases have been reported to be activated in lymphocytes by oxidizing agents (Bauskin et al., 1991; Nakamura et al., 1993). For these reasons, we investigated whether endogenous ROI generated by the NADPH oxidase affected the activity of tyrosine kinases in human neutrophils.
The kinase activity of immune complexes was
determined essentially as described (Burkhardt and Bolen, 1993). In
brief, immunoprecipitates were washed with 1 ml of kinase buffer (5
mM MnCl, 20 mM MOPS, pH 7.0), and
autophosphorylating activity was assayed by incubation with 25 µl
of kinase buffer containing 12.5 µCi of
[
-
P]ATP and 1 µM K-ATP. Where
specified, 1 µg of rabbit muscle enolase was included as an
exogenous substrate. Samples were incubated at 25 °C in an
Eppendorf Thermomixer, and reactions were stopped by the addition of
boiling 2
concentrated Laemmli sample buffer. The samples were
subjected to SDS-PAGE, and the gels were stained with Coomassie Blue
and dried in gel wrap (Biodesign Inc.). Dried gels were used for direct
quantitation of radioactivity with a Molecular Dynamics PhosphorImager
using Imagequant software or were subjected to radiography with an
intensifying screen.
To study the effect of oxidizing agents on the
tyrosine kinase activity of hck, immunoprecipitates of this
kinase were isolated from untreated, electroporated cells. After
washing, immune complexes were treated for 30 min with 1 mM diamide or 1 mM hydrogen peroxide at 30 °C while
shaking in a Thermomixer. As above, identical aliquots were used in
parallel for immunoblotting and in vitro kinase assay. To
study the effect of reducing agents on hck, immune complexes
from GTPS-stimulated cells were treated with 20 mM NAC or
1 mM dithiothreitol for 30 min at 37 °C and processed as
above.
To study the role of tyrosine phosphorylation in hck activation, immunoprecipitates obtained from GTPS-treated
cells were incubated at 37 °C for 30 min with or without 2
µg/ml of T-cell phosphatase. Aliquots of the beads were used for
kinase assays and for immunoblotting with anti-phosphotyrosine
antibodies to confirm the effectiveness of dephosphorylation by T-cell
phosphatase.
As shown in Fig. 1A, the addition of GTPS and NADPH to
permeabilized cells induced the accumulation of phosphotyrosine on a
number of proteins, as determined by immunoblotting (cf. lanes 1 and 4). Treatment of the electroporated cells with
GTP
S and NADPH alone was found to have little effect (lanes 2 and 3). The stimulatory effect of GTP
S or NADPH was
moderated by the presence of active tyrosine phosphatases. This is
indicated by the pronounced enhancement in phosphotyrosine accumulation
noted when vanadate, a phosphatase inhibitor, was included during
stimulation (Fig. 1A). For this reason, 10 µM sodium orthovanadate was included routinely in subsequent assays
to minimize dephosphorylation, thereby magnifying the responses. At the
concentration used, vanadate itself had negligible effects on tyrosine
phosphorylation (see lanes 1 and 2 in Fig. 1C), consistent with earlier findings (Bourgoin
and Grinstein, 1992). Moreover, whereas vanadate increased the extent
of phosphotyrosine accumulation, the phosphorylated substrates and the
time course of phosphorylation were similar in the presence and the
absence of the phosphatase inhibitor. As illustrated in Fig. 1B, phosphotyrosine accumulation induced by
GTP
S stimulation was rapid (evident after 1 min) and
time-dependent, with a maximal response seen after 10 min.
Figure 1:
Effect of GTPS on tyrosine
phosphorylation. A, NADPH dependence and potentiation by
sodium vanadate. Electroporated neutrophils were incubated at 37 °C
without(-) or with (+) the following agents for 5 min: 10
µM GTP
S, 2 mM NADPH, and 10 µM NaV, as indicated. Cells were then rapidly sedimented, boiled in
sample buffer, and subjected to SDS-PAGE. Analysis was performed by
immunoblotting with a monoclonal antibody to phosphotyrosine. B, time course of phosphotyrosine accumulation. Electroporated
neutrophils were treated without(-) or with 10 µM GTP
S, 2 mM NADPH, and 10 µM NaV for the
indicated time (min) and processed as in A. C,
dependence of tyrosine phosphorylation on NADPH oxidase-derived ROI.
Electroporated neutrophils were treated without(-) or with
(+) 10 µM GTP
S and/or 2 mM NADPH for 5
min at 37 °C. Where specified, the cells were treated with 2 mM diphenylene iodonium (DPI) or 2 mM NAC for 2 min
at 37 °C prior to GTP
S stimulation. The presence of 10
µM NaV during treatment is indicated. The results shown
are representative of three separate
experiments.
The
effect of GTPS on tyrosine phosphorylation was entirely dependent
on the presence of NADPH. As shown in Fig. 1C (as well
as in Fig. 1A), treatment of electroporated cells with
GTP
S had little effect when the nucleotide was omitted (cf.
lanes 3 and 4). This finding suggests that generation of
superoxide by the NADPH oxidase is required for the increase in
tyrosine phosphorylation following stimulation with GTP
S. In
support of this hypothesis, we found that NAC, a powerful anti-oxidant
that has been shown to scavenge ROI and increase cytosolic levels of
reduced glutathione (Halliwell and Gutteridge, 1985), effectively
attenuated the tyrosine phosphorylation produced by GTP
S in the
presence of NADPH. Moreover, diphenylene iodonium, an inhibitor of the
flavoprotein component of the NADPH oxidase (Ellis et al.,
1988), had a comparable effect (lane 6). These findings are in
agreement with those of Fialkow et al.(1993) and indicate that
NADPH oxidase-derived ROI promote tyrosine phosphorylation in
neutrophils.
Figure 2:
Identification of tyrosine kinases present
in neutrophils. A, immune complex kinase assays were performed in vitro using immunoprecipitates of the tyrosine kinases
indicated, prepared from lysates of electroporated neutrophils treated
with 10 µM GTPS, 2 mM NADPH, and 10
µM NaV for 2 min. Kinase reactions were stopped, and the
material was subjected to SDS-PAGE and autoradiography of the dried
gel. The assay was also performed using a rabbit nonimmune serum (cont). B, whole neutrophil lysates were
immunoblotted with antisera to the tyrosine kinases indicated. The closed arrowheads point to the tyrosine kinase. The open
arrowhead indicates an unidentified protein of
65 kDa that
cross-reacts with the syk antibody.
In
good agreement with the kinase assays of Fig. 2A, the
presence of lyn, hck, fgr, syk, and btk in neutrophils was confirmed by immunoblotting whole cell
lysates with the same antisera used for precipitation (Fig. 2B). Both the full-length (72 kDa) syk protein as well as its 40-kDa degradation product were
observed upon immunoblotting (Fig. 2B, closed
arrowheads). A band of
65 kDa was also recognized by the syk anti-serum. It is not presently clear whether this
polypeptide is related to syk or is merely a fortuitously
cross-reacting protein. It is noteworthy, however, that a band of
similar mobility was often found to be phosphorylated in syk immune complex assays (Fig. 3A), suggesting that
the 65-kDa polypeptide co-immunoprecipitates and can be phosphorylated
by syk.
Figure 3:
Modulation of tyrosine kinase activity by
ROI. A, immune complex kinase assays were performed in
vitro using immunoprecipitates of the tyrosine kinases indicated,
prepared from lysates of electroporated neutrophils treated without
(-) or with (+) 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 1 min. B,
immunoprecipitates of hck were prepared from lysates of
electroporated neutrophils treated without or with 10 µM GTP
S, 2 mM NADPH, and 10 µM NaV for the
indicated time (min) and subjected to immune complex kinase assays in
the presence of enolase. The kinase reactions were stopped, and the
samples were subjected to SDS-PAGE followed by autoradiography of the
dried gels. A representative experiment is shown in the inset.
Bands that correspond to autophosphorylation (closed arrow)
and enolase phosphorylation (open arrow) were quantified with
a PhosphorImager, and the results are presented as the percentage of
maximal response in the main panel. C, in vitro autophosphorylation and enolase phosphorylation activities of lyn were determined as in B for hck. The
data in B and C are the means ± S.E. of three
experiments.
Although
production of ROI stimulated some tyrosine kinases, others were
seemingly inhibited. The autophosphorylating abilities of lyn and fgr were diminished (73 ± 14 and 48 ±
16% of control activity, respectively; n = 3) following
1 min of GTPS stimulation (Fig. 3A). As for hck, the detailed time course of the effects of ROI on lyn activity was analyzed with enolase as substrate (see Fig. 3C). Interestingly, quantitation of the auto- and
enolase-phosphorylating activities of lyn immune complexes
revealed a discrepancy. Phosphorylation of the exogenous substrate was
markedly increased, whereas autophosphorylation decreased. These
findings suggest that nonradioactive phosphate is incorporated into lyn in the cells, prior to immunoprecipitation, precluding
subsequent incorporation of radiolabel into these sites. The reduced
autophosphorylation is therefore an inaccurate indication of the
enzymatic activity of lyn, which is at least transiently
stimulated by GTP
S.
Figure 4:
Effect of oxidizing/reducing agents on hck activity. hck immunoprecipitates were obtained
from lysates of electroporated neutrophils treated without (-) or
with (+) 10 µM GTPS, 2 mM NADPH, and 10
µM NaV (5 min) were incubated at 30 °C for 30 min with
either 1 mM diamide, 1 mM H
O
,
20 mM NAC, 1 mM dithiothreitol, or with buffer alone (none), as indicated. An aliquot of the immunoprecipitate was
used for in vitro kinase assay (A), and another was
used for anti-phosphotyrosine immunoblotting (B). The closed arrows indicate the position of immunoprecipitated hck, whereas the open arrow indicates the position of
the exogenous substrate, enolase.
The
inability of oxidants and reducing agents to affect hck autophosphorylation was confirmed by immunoblotting the
immunoprecipitates with anti-phosphotyrosine antibodies (Fig. 1B). The kinase was found to be
tyrosine-phosphorylated only after stimulation of the cells with
GTPS, and the phosphotyrosine content was unaffected by oxidizing
and reducing agents. Together, these results suggest that ROI do not
directly activate hck in GTP
S-stimulated neutrophils.
Figure 5:
Phosphotyrosine-associated kinase
activity. Anti-phosphotyrosine immunoprecipitates were obtained from
lysates of electroporated neutrophils treated without(-) or with
(+) 10 µM GTPS, 2 mM NADPH, and 10
µM NaV (2 min) and used to perform in vitro kinase assays as described under ``Experimental
Procedures.'' The closed arrowheads indicate bands that
displayed a prominent increase in phosphorylation following
stimulation. The results are representative of three separate
experiments.
The size of some of the phosphoproteins in
phosphotyrosine immunoprecipitates correspond to that of the active
kinases detailed in Fig. 2. To establish more directly whether
the active kinases are tyrosine phosphorylated, immunoprecipitates of lyn, hck, fgr, syk, and btk were prepared from control and GTPS-treated cells and probed
by immunoblotting with anti-phosphotyrosine antibodies. As illustrated
in Fig. 6A, endogenous generation of ROI is accompanied
by tyrosine phosphorylation of all the kinases studied (indicated with closed arrowheads), with the notable exception of btk, which remained unaffected. The figure also shows that
both the intact form of syk as well as its 40-kDa proteolytic
fragment (open arrowhead) was phosphorylated on tyrosine.
Figure 6:
A, phosphorylation of tyrosine kinases in situ. Anti-phosphotyrosine immunoblotting was performed on
immunoprecipitates of the specified tyrosine kinases, obtained from
electroporated neutrophils treated without(-) or with (+) 10
µM GTPS, 2 mM NADPH, and 10 µM NaV for 5 min. The solid arrows indicate the position of
the immunoprecipitated kinase. The open arrowhead indicates
the 40-kDa proteolytic fragment of syk, also observed in
anti-syk immunoblots (see Fig. 2B). B, time course of tyrosine phosphorylation. Cells were
stimulated for the times indicated (min), and immunoprecipitates of the
indicated kinases were obtained and subjected to SDS-PAGE and
anti-phosphotyrosine immunoblotting. The extent of tyrosine
phosphorylation was quantified in each case by densistometry and is
presented as the percentage of the maximum. The data are the means
± S.E. for three experiments.
The correlation between the occurrence of tyrosine phosphorylation
and the activation of the tyrosine kinases is further stressed by the
similarity of the time courses of both events. In Fig. 6B, the degree of tyrosine phosphorylation was
quantified in immunoprecipitates from cells stimulated for varying
periods of time with GTPS. All four kinases undergo rapid and
progressive phosphorylation, which is detectable by 1 min and maximal
between 5 and 10 min. This pattern closely resembles the time course of
activation of hck determined in Fig. 3C using
enolase as the substrate. It therefore appears likely that tyrosine
phosphorylation of the kinases regulates their activity.
This notion
was directly addressed by treatment of immunoprecipitates with an
active tyrosine phosphatase, the truncated T-cell phosphatase. hck was precipitated from GTPS-stimulated neutrophils and
incubated for 30 min in the presence or the absence of T-cell
phosphatase. The effectiveness of the phosphatase was ascertained by
immunoblotting the precipitates with anti-phosphotyrosine antibodies.
Exposure to T-cell phosphatase led to complete dephosphorylation of hck (indicated by the solid arrowhead in Fig. 7A). Immunoblotting confirmed that equal amounts
of hck were present before and after treatment with T-cell
phosphatase (data not shown). The autophosphorylating (solid
arrowhead) and exogenous kinase activity (open arrowhead)
of stimulated and dephosphorylated hck was next compared. Fig. 7B demonstrates that treatment with T-cell
phosphatase eliminated the kinase activity of hck stimulated
by ROI.
Figure 7:
Effect of tyrosine dephosphorylation on hck activity. hck was immunoprecipitated from lysates
of neutrophils treated with 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 5 min. Immunoprecipitates
were then treated for 30 min without(-) or with (+)
recombinant T-cell phosphatase (TC-PTP) at 30 °C while
shaking. An aliquot of the immunoprecipitated material was subjected to
anti-phosphotyrosine immunoblotting (A), and another was used
to perform the in vitro kinase assay (B). The closed arrowheads indicate immunoprecipitated hck,
whereas the open arrow indicates the location of the exogenous
substrate, enolase. The results shown are representative of three
separate experiments.
In this report, we analyzed the mechanism leading to
increased phosphotyrosine accumulation following ROI production in
neutrophils. In electroporated cells treated with GTPS, we
detected an elevated activity of several kinases, measured in
vitro. The activation of these kinases was rapid and correlated
well with the increase in tyrosine phosphorylation observed under these
conditions.
Kinases of three separate families were found to be
activated by ROI, as determined by autophosphorylation and
phosphorylation of an exogenous substrate, enolase. hck, a
member of the src family of tyrosine kinases and highly
expressed in granulocytes and macrophages (Ziegler et al.,
1987), displayed little activity in untreated cells but was rapidly
stimulated following the addition of GTPS. syk, which
belongs to a separate family of kinases, also displayed increased
activity following ROI production. In contrast, the closely related
ZAP-70 tyrosine kinase, thought to be important in B- and T-cell
receptor signaling (Sefton and Taddie, 1994) was not detectable in
active neutrophils using our immune complex kinase assay. btk,
a member of the tec family of tyrosine kinases, is expressed
in cells of myeloid and lymphoid lineage (Yamada et al., 1993)
and was also activated by ROI. To our knowledge, activation of btk in neutrophils had not been reported previously.
Although ROI
production led to the activation of some tyrosine kinases, it appeared
to have an opposite effect on the activity of others when estimated
from autophosphorylation in immune complex kinase assays. Thus, lyn and fgr displayed high activities in untreated,
electroporated neutrophils, which decreased following GTPS
stimulation. However, at least in the case of lyn, the
apparent decrease in activity likely reflected occupancy of substrate
sites by nonradioactive phosphate, which may have occurred in
situ, prior to immunoprecipitation.
Indeed, the
ability of the enzyme to phosphorylate exogenous substrates was increased following stimulation of the respiratory burst.
Therefore, caution must be exercised when equating the
autophosphorylating and catalytic activities of tyrosine kinases.
None of the other tyrosine kinases tested were found to be activated following generation of ROI. These included yes, which is reported to be present in neutrophils, where it can be stimulated by granulocyte macrophage colony-stimulating factor (Corey et al., 1993). Clearly, although 11 different antisera were used, our survey was incomplete, because other tyrosine kinases are likely to exist in neutrophils.
The mechanism underlying the activation of the
kinases by ROI was explored in some detail using hck as a
prototype. Although ROI production in situ appeared to
activate hck, oxidizing agents could not mimic this effect
when applied to hck immunoprecipitates in vitro.
Moreover, reducing agents failed to reverse the activation of hck isolated from GTPS-treated cells. We conclude that hck activity is not regulated directly by ROI but rather by some other
post-translational modification. Though not tested directly, we suggest
by extension that activation of the other kinases is similarly
indirect.
Because tyrosine phosphorylation of lyn, hck, fgr, and syk was found to occur upon
stimulation by GTPS, this post-translational modification was
considered as a possible mechanism of regulation. This notion was
evaluated using T-cell phosphatase to dephosphorylate activated hck. This procedure was found to eliminate the activity of the
kinase, suggesting that tyrosine phosphorylation mediates the effect of
ROI on hck activation.
Tyrosine kinase activity of src family members is thought to be suppressed by phosphorylation of a C-terminal residue, conserved among family members (Cooper, 1988; Liu et al., 1993; Cooper and Howell, 1993). Dephosphorylation of this residue has been shown to increase the activity of src family kinases (Cooper and King, 1987), in apparent conflict with our findings with hck, where complete dephosphorylation of the enzyme led to its inactivation. However, recent evidence has questioned this simple model of regulation. This includes the finding that a T-cell line lacking CD45 (the phosphatase that activates the src family member lck) was found to have higher lck activity even though its inhibitory C-terminal tyrosine residue was hyperphosphorylated (Burns et al. 1994). Although dephosphorylation of the C terminus may be important for the derepression of src family members, a number of unique tyrosine residues have been reported to be phosphorylated upon activation. These include the so-called autophosphorylation site within the kinase domain (Smart et al., 1981; Patchinsky et al., 1982) and sites within the N-terminal domain of some src family members (Souda et al., 1993). Evidence exists that phosphorylation of these residues is essential for kinase activity, possibly by stabilization of the active kinase (Veillette and Fournel, 1990; Mustelin, 1994). Recent evidence has also implicated serine phoshorylation in the regulation of src family members (Winkler et al., 1993; Watts et al., 1993). Whereas the regulation of src family kinases remains incompletely understood, our findings imply that tyrosine phosphorylation is necessary to maintain the activity of hck following activation by endogenous ROI.
The steps that follow ROI generation and lead to
kinase phosphorylation are unknown, but some insight is provided by
recent reports that (a) critical conserved cysteine residues
exist in the catalytic domain of many tyrosine phosphatases (Fisher et al., 1991) and (b) that both exogenous (Zor et
al., 1993, Fialkow et al., 1994) as well as endogenous
oxidants ()can inactivate tyrosine phosphatases, likely by
targeting their critical sulfhydryl moieties. In view of these
considerations, the following scenario can be envisaged. Under basal
conditions, the accumulation of tyrosine phophoproteins and the
autophosphorylation and stimulation of tyrosine kinases, some of which
are constitutively active, are prevented by the offsetting action of
tyrosine phosphatases. This delicate balance can be disrupted when ROI
diminish the rate of dephosphorylation by reaction with sulfhydryl side
chains in the catalytic domain of one or more tyrosine phosphatases.
Indeed, in neutrophils, CD45 has been shown to be susceptible to
inactivation by oxidants (Fialkow et al., 1994), and other
phosphatases present in these cells (e.g. PTP-1C) (
)are likely to be similarly affected.
It is noteworthy
that modulation of tyrosine kinase activity has been reported in
lymphoid cells exposed to HO
(Schieven et
al., 1993). Treatment with the oxidant was found to activate syk but not lyn, resembling our observations in
neutrophils. Like most other cells, lymphocytes can potentially
generate ROI by electron transfer reactions in mitochondria and the
endoplasmic reticulum. However, the magnitude of the oxidative response
is far greater in phagocytes, which express high levels of the NADPH
oxidase (see the Introduction). In this regard, it is important that in
the present experiments activation of phosphorylation was elicited by endogenously generated ROI, lending credence to the
physiological significance of the observations. It is possible to
envisage that stimulation of the NADPH oxidase, one of the earliest
effectors of neutrophils, could promote phosphotyrosine accumulation by
the combined inhibition of phosphatases and activation of kinases. This
could in turn have important consequences on more slowly developing
responses such as phagocytosis and degranulation. In this context, hck has been suggested to have a role in phagocytosis (Lowell et al., 1994), and syk has also been proposed to be
essential to the anti-microbial response (Asahi et al., 1993).
It is also conceivable that ROI secreted by neutrophils may have
paracrine effects, stimulating neighboring quiescent neutrophils or
other cells present in the inflammatory milieu, including lymphocytes
and macrophages.