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Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A Alter Cell Shape
and F-Actin Distribution and Inhibit Stimulus-Dependent Increases in
Cytoskeletal Actin of Human Neutrophils
By Peter Kreienbuhl, Hansuli Keller, and Verena Niggli
The phosphatase inhibitors okadaic acid and calyculin A
were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (21 pmol/L)
that were effective on neutrophil functions (shape changes
and marginal stimulation of pinocytosis) were shown to
stimulate the incorporation of 32P04 into many neutrophil
proteins several-fold. Calyculin A was effective at 50-fold
lower concentrations. In the presence of the inhibitors, the
cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both
phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase
activity is thus involved in maintaining shape and F-actin
localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin.
The increase in cytoskeletal actin observed at 30 minutes of
stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by
okadaic acid or calyculin A, suggesting an important role of a
phosphatase. In contrast, the early increase in cytoskeletonassociated actin observed at 1 minute of stimulation with
peptide was not affected. This finding indicates that the
increased association of actin with the cytoskeleton in the
early and the later stages of neutrophil activation may be
mediated by different signalling pathways.
o 1992by The American Society of Hematology.
P
HOSPHORYLATION and dephosphorylation reactin.1° In summary, the results available so far suggest that
increased actin polymerization can be elicited via at least
tions play a major role in regulating biologic rethree different pathways: (1) activation of neutrophils by
sponses. This applies also to leukocyte activation. Activachemotactic peptides via an unknown PKC-independent
and inhibitors7-10 of protein kinases, particularly
protein kinase C (PKC), have the capacity to elicit or
pathway; (2) direct activation of PKC by phorbol ester; and
(3) inhibition of the basal activity of an as yet unidentified
modify various leukocyte responses, including leukocyte
staurosporine-sensitive enzyme not identical with PKC.
motility. These modifications of neutrophil functions are
Okadaic acid has been suggested as a new probe for the
associated with increased actin polymerization in response
study of cellular regulation.% Okadaic acid is a polyether
to activators or inhibitors of PKC and accumulation of
fatty acid isolated from the marine sponges Halichondna
F-actin in newly formed p s e ~ d o p o d s . ~Cytoskeletal
,~-l~
proOkadaic and Halichondna melanodocia. Okadaic acid inhibteins have been shown to be targets of protein kinases,
its preferentially protein phosphatase 2A at lower concenespecially PKC. Smooth muscle myosin light chain kinase,
trations and also, at higher concentrations, phosphatase 1.
as well as myosin light chain from human platelets are
It thus blocks the dephosphorylation of protein kinase
phosphorylated by PKC and the reaction can be induced by
substrates, thereby increasing the amount of actual phosphorbol esters.15-17Other cytoskeletal protein kinase subphate bound to protein. Thus, okadaic acid may become a
strates include myotube intermediate filaments such as
tool in analyzing the role of protein phosphorylation
desmin and vimentin,’* vinculin, talin, and p r ~ f i l i n . ~ ~ - ~useful
l
in relation to biologic activity. We show here that okadaic
Chemoattractants were also found to increase myosin
acid alone has a number of biologic effects on leukocytes
phosphorylation in DictyosteliumZ2 and phosphorylation
and is also capable of modifying the effects of leukocyte
changes correlated temporarily with cyclic adenosine monoactivators such as chemotactic peptides or phorbol esters.
phosphate (CAMP)-induced cell shape changesz3A 47,000-d
To complete our studies, we have in addition used another
protein derived from human platelets may modulate actin
polymerization through phosphorylation, as shown in vitro,24 phosphatase inhibitor, calyculin A (structurally unrelated
to okadaic acid), which inhibits in vitro both phosphatase 1
and PKC-mediated phosphorylation of tau was found to
and 2A at nanomolar concentrations with equal potency.27
reduce its ability to promote tubulin polymerization and to
Calyculin A affected neutrophil functions in the same way
cross-link actin filaments.25 Shape changes, motility, and
as okadaic acid, but at 50-fold lower concentrations. A
actin polymerization may thus be controlled by protein
control substance, methyl okadaate, which is inactive on
kinases via regulation of different cytoskeletal proteins that
are involved in actin filament contraction, membrane linkage, and control of polymerization.
From the Department of Pathology, University of Bem, Bem,
Little is known regarding the signals involved in eliciting
Switzerland.
increased actin polymerization in neutrophils upon activaSubmitted October 30, 1991; accepted August IO, 1992.
Supported by the Swiss National Science Foundation.
tion with chemotactic peptide. Direct activation of PKC by
Address reprint requests to Verena Niggli, PhD, Department of
phorbol ester has been shown to stimulate actin polymerizaPathology, University of Bern, Murtenstr. 31, PostJach, 3010 Bern,
tion.12 We have recently found that the potent but unspeSwitzerland.
cific protein kinase inhibitor staurosporine increases the
The publication costs of this article were defrayed in part by page
level of actin associated with the Triton X-100-insoluble
charge payment. This article must therefore be hereby marked
cytoskeleton in neutrophils, similar to the reaction caused
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
by phorbol ester, whereas a derivative more specific for
indicate this fact.
PKC was inactive.10The latter inhibitor did not prevent the
0 1992 by The American Society of Hematology.
chemotactic peptide-induced increase in cytoskeletal ac0006-4971l92/801I -0004$3.00/0
Blood, Vol80. No 11 (December 1). 1992: pp 291 1-2919
291 1
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2912
phosphatases in vitro,28 had, in contrast, no significant
KREIENBUHL, KELLER, AND NlGGLl
guished between the following types of shapes. (1) Spherical
neutrophils were unstimulated control cells characterized by a
circular contour and a smooth surface. (2) Spherical neutrophils
with unifocal projections were basically spherical cells with a
MATERIALS AND METHODS
smooth surface and a circular outline, except for an unifocal
projection on one side. (3) Polarized cells were elongated cells
Reagents and suppliers were as follows: N-formyl-L-norleucyl-Lshowing front-tail polarity. (4) Nonpolar cells with surface projecleucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-lysine
(fNLPNTL;
tions were cells without morphologic front-tail polarity, but with
Bachem, Bubendorf, Switzerland); human serum albumin (HSA,
numerous major projections distributed all over the cell surface. In
Behringwerke, Marburg, Germany); Metrizoate (Nycomed AS,
the present study two subtypes have been distinguished. One is the
Oslo, Norway); Methocel (Pro Chem AG, Zurich, Switzerland);
type observed after stimulation with PMA or diacylglycerols
neutrophil isolation medium (Los Alamos Diagnostics, Los Alamos,
characterized by multiple surface lamella, which often show a
NM); okadaic acid (Moana Bioproducts, Honolulu, Hawaii);
pointed end. The other shape type, the shape produced by okadaic
calyculin A and methyl okadaate (LC Services Corporation,
acid or calyculin A, is new. The surface projections have a more
Woburn, MA); phorbol 12-myristate 13-acetate (PMA), fluoreswavy appearance. Shape changes in living cells after stimulation
cein isothiocyanate-dextran (FITC-dextran, FD-70), paraformaldewere recorded by videomicroscopy within the first 50 minutes.
hyde and lysolecithin (L-a-lysophosphatidylcholine)
(Sigma ChemThe localization of F-actin by staining with NBD-phallacidin. The
ical Corp, St Louis, MO); N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
intracellular localization of F-actin was determined by NBDphallacidin (NBD-phallacidin; Molecular Probes Inc, Junction
City, OR); HEPES and glutaraldehyde (Serva Feinbiochemica,
phallacidin staining of paraformaldehyde-fixed cells, as previously
Heidelberg, Germany); dimethylsulfoxide(DMSO) and diisopropyldescribed by Howard and M e ~ e r , with
~ ] modifications as detailed
fluorophosphate (Fluka AG, Buchs, Switzerland); [32P]orthophosin Roos et
phate (in aqueous solution, HC1-free, carrier-free) (Amersham,
Determination of cytoskeleton-associatedactin. Cells were resusBuckinghamshire, UK); Triton X-100 (a 10% solution in H20,
pended in basic medium without HSA or EGTA, pH 7.4. To block
stored under nitrogen) (Pierce, Rockford, IL). Water-insoluble
endogenous proteases, the isolated neutrophils were preincubated
with diisopr~pylfluorophosphate~~
compounds were dissolved in DMSO. The corresponding amount
before the assays, as described
previously.1°The amount of cytoskeletal actin, as the percentage of
of DMSO was always included in the controls. Concentrations of
actin insoluble in 1% Triton X-100, was determined as described
DMSO up to 0.4%, the maximal amount used, did not affect
neutrophil functions. Moreover, calyculin A had the same effects
previously," with small modifications.I0 Briefly, aliquots (450 pL)
of the cell suspension were incubated in the presence of 10 mmol/L
on neutrophils that okadaic acid had, but at a SO-fold lower
EDTA and with 0.4% DMSO or phosphatase inhibitors (stock
concentration, in the presence of only 0.02% to 0.04% DMSO. The
solutions in DMSO). Stimuli were added in 10 pL medium to the
basic medium (pH 7.2) was prepared as follows: 138 mmol/L NaCI,
cell suspension of 450 pL. EDTA was added to minimize cell
6 mmol/L KCI, 1.1 mmol/L EGTA, 1 mmol/L Na2HP04, 5
aggregation and adhesion and to block proteolysis after solubilizammol/L NaHC03, 5.5 mmol/L glucose, 20 mmol/L HEPES, 2%
tion. The omission of EDTA did not alter the results. The adhesion
(wtlvol) HSA. EGTA was included to minimize cell aggregation
of cells to Eppendorf tubes was minimal, as 94% ? 6% (mean 2 SD,
and adhesion. The omission of EGTA did not affect our results.
n = 3) of cells could be recovered from the tubes after 40 minutes
Cell preparation. Human blood was obtained by venepuncture
of incubation at 37°C in the presence of
mol/L fNLPNTL. For
from healthy volunteers and heparinized (10 UlmL). Neutrophil
the quantification of cytoskeletal actin, the reaction was stopped by
granulocytes were separated in a first step with Metrizoatethe addition of 500 IJ.Lof a twofold-concentrated ice-cold lysis
M e t h ~ c e and
l ~ ~in a second step with neutrophil isolation medibuffer containing 2% Triton X-100, 160 mmol/L KCI, 20 mmol/L
~1111.~"
The cells (97% to 100% neutrophils) were then washed and
Tris, pH 7.5, 20 mmol/L EDTA, and 0.02% NaN3.33 The tubes
resuspended in basic medium.
were incubated on ice for 10 minutes, followed by centrifugation
Shape change and fluid pinocytosis assays. The cell suspension
and washing of the pellets as described.I0 The amount of actin in
(lo6cells/mL in basic medium) was preincubated in a reciprocatpellets and supernatants was analyzed by sodium dodecyl sulfateing water bath in plastic tubes with FITC-dextran (5 mg FITCpolyacrylamide gel electrophoresis (SDS-PAGE) as detailed in
dextran/mL cell suspension) and with 0.4% DMSO or okadaic acid
Niggli and Keller.Io Differences between data were analyzed with
at the concentrations indicated at 37°C for 10 minutes. Incubation
the Student's t-test for paired data, with a P value of <.05
was then continued with or without additional stimuli at uniform
considered significant.
concentrations for 30 minutes. Under these conditions, cell adheProtein phosphorylation in intact neutrophils. Neutrophils were
sion to the tubes was minimal, as 93% ? 8% (mean ? SD of 3
isolated, treated with diisopropylfluorophosphate (see previous
experiments) of the cells were recovered from the plastic tubes
section) and washed several times with a phosphate-free buffer
after 40 minutes at 37°C in the shaking water bath. The reaction
(138 mmol/L NaCI, 6 mmol/L KCI, 20 mmol/L HEPES, pH 7.4,
was terminated by fixation in 1% glutaraldehyde (vol/vol; final
5.5 mmol/L glucose). The cells were resuspended in this buffer
concentration) at 37°C for 30 minutes. Fixed cells were washed five
(20 x loh cells/mL) and were incubated with 0.5 mCi of
times and resuspended in phosphate-buffered saline (PBS) contain[32P]orthophosphate/mL for 1 hour at 37°C in a shaking water
ing 1 mg NaN3/mL. The net uptake of FITC-dextran was assessed
bath. At the end of the incubation period, the cells were washed
by means of flow cytometry (Epics Profile 11; Coulter Corporation,
twice with a 0.5-fold volume of the above buffer, and then
Hialeah, FL). The mean channel was determined.
resuspended in this buffer containing, in addition, 10 mmol/L
Cell shape of the fixed cells was determined in Sykes-Moore
EDTA (3 x lo6 cells/mL). The omission of EDTA did not alter
chambers using a Zeiss IM 35 microscope (X 100 oil immersion
our results. The cells were subsequently exposed to 0.4% DMSO or
objective) and differential interference contrast microscopy (Nomarphosphatase inhibitors and stimuli as described in the previous
ski Optics) (Carl Zeiss, Oberkochen, Germany). Photographs were
section. The reaction was stopped by the precipitation of protein
taken using a Kodak T Max 400 film (Eastman Kodak, Rochester,
with trichloroacetic acid as described.I0 The precipitates were
NY).
electrophoresed through 7.5% to 20% gradient gels, followed by
The criteria for the classification of neutrophil shape (200
autoradiography.1°
cells/sample) have been described previo~sly.~
Briefly, we distin-
effects at 1 p,mol/L.
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PHOSPHATASES AND NEUTROPHIL FUNCTIONS
RESULTS
Effect of phosphatase inhibitors on protein phosphorylation
in intact neutrophils. We were interested in obtaining
information on the role of phosphatases in neutrophil
functions. It was important to know whether okadaic acid,
which inhibits phosphatases 2A and 1 in vitro, is also active
in intact neutrophils. Therefore, we have analyzed the
effect of okadaic acid on protein phosphorylation in neutrophils preloaded with 32P04.Incubation of cells with 1O-x
mol/L PMA for 5 minutes resulted in increased incorporation of 32P04into several proteins, among them bands of 47
Kd and 70 Kd (Fig 1, lane 2). PMA also induced dephosphorylation of a 20-Kd band. These findings arc comparable to
results previously obtained in human neutr0phiIs.3~Incubation of the cells with 1O-y mol/L fNLPNTL for 5 minutes
resulted mainly in increased phosphorylation of the 70-Kd
band and dephosphorylation of the 20-Kd band (Fig 1, lane
3). Preincubation of cells for 30 minutes with 1 pmol/L
Fig 1. Effect of okadaic acid on protein phosphorylationin intact
neutrophils. Cells were prelabeledfor 1 hour with 3nP04and washed
twice. Cells were preincubated for 30 minutes at 37°C in the presence
of 10 mmol/L EDTA and 0.4% DMSO or okadaic acid (1 pmol/L, final
concentration). Subsequently, medium or PMA (lO-8 mol/L) or
fNLPNTL (lO-9 mol/L) were added. After 5 minutes, the reaction was
stopped by the addition of ice-cold trichloroacetic acid (final concentration, 10% [wt/vol]).'o The precipitated proteinswere separated on
a 7.5% to 20% gradient gel. The corresponding autoradiogram is
shown. The cells (6.6 x 105 cells per lane) were treated with: lane 1,
DMSO and medium; lane 2, DMSO and PMA; lane 3, DMSO and
fNLPNTL; lane 4, okadaic acid and medium; lane 5, okadaic acid and
PMA; lane 6, okadaic acid and fNLPNTL. Molecular masses of standard proteins in kilodaltons are shown on the right side of the
autoradiogram. (a) A phosphorylatedband of 70 Kd; (b) a band of 47
Kd; (e) a band of 20 Kd.
2913
okadaic acid, followed by a further incubation either with
medium or PMA or fNLPNTL, resulted in a marked
increase in incorporation of 32P04into at least 24 bands
(Fig 1, lanes 4 through 6). For example, in the experiment
shown in Fig 1, incorporation of '*PO4 into major phosphoproteins of 80 to 90 Kd was stimulated twofold by okadaic
acid, and into bands of 52 to 54 Kd fivefold. Comparable
results have been found recently for electropermeabilized
human neutrophils equilibrated with [32P]ATP and 2
pmol/L okadaic acid.3s Apparently, kinases are constitutively active in resting cells, and phosphatases control
phosphorylation of a variety of substrates. We have also
studied the time course of the effects of okadaic acid on
protein phosphorylation in resting cells. Increases in phosphorylation occurred slowly. The first effects were observed
15 minutes after the addition of the inhibitor (1.3- t 0.2fold increase in phosphorylation of the 52 to 54-Kd band).
Phosphorylation of this band was 1.7- t 0.3-fold increased
after 20 minutes, 2.6- t 0.5-fold after 30 minutes, and
3.8- 2 0.7-fold after 40 minutes (mean t SD,n = 3 to 4).
Phosphorylation was still increasing after 40 minutes. This
slow time course may at least partly be due to slow
penetration of okadaic acid into the cells, as 2 pmol/L
okadaic acid has been shown to induce in electroporated
human neutrophils a 1.5-fold increase in phosphorylation
of a 59-Kd band within 3 minutes.35 Calyculin A at 0.02
pmol/L markedly increased phosphorylation of the same
bands, just as okadaic acid did (6- to 7-fold increase in
phosphorylation of the 52- to 54-Kd band after 40 minutes).
Figure 1 thus shows that okadaic acid can enter the
neutrophils and is active on cellular phosphatases. Questions on specific protein phosphatase substrates possibly
involved in the effects of okadaic acid on neutrophils
described below will be addressed in future studies.
Shape changes induced by okadaic acid and calyculin A.
Okadaic acid produced dose-dependent shape changes at
concentrations of 0.5 pmol/L and higher. Almost 100% of
the cells showed a nonpolar configuration at 1 pmol/L.
Nonpolar cells with surface projections induced by okadaic
acid were different from those observed in nonpolar cells
induced by PMA (Fig 2). Okadaic acid-induced projections
were less pointed and more wavelike. This morphologic
difference is so characteristic that one can tell on the basis
of the morphologic alterations whether okadaic acid was
present in active concentrations. The dose-response curve
is shown in Fig 3A. Furthermore, okadaic acid was found to
suppress front-tail polarity induced by lO-9 mol/L
MLPNTL in a dose-dependent fashion (Fig 3B). Cells
treated with 1O-y mol/L fNLPNTL and 1 or 2 pmol/L
okadaic acid showed a similar morphology as cells treated
with the respective concentrations of okadaic acid alone
(Fig 2B and B+). The cells consisted almost completely of
nonpolar cells with surface projections. Neutrophils stimulated with 1O-y mol/L PMA plus increasing concentrations
of okadaic acid were mainly nonpolar with surface projections (Fig 3C). However, in the absence or at low concentrations of okadaic acid ( < O S pmol/L), the cells showed the
morphology characteristic for PMA, whereas at high concentrations (1 and 2 pmol/L), the morphology was very similar
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KREIENEUHL, KELLER, AND NIGGLI
2914
Fig 2. Effect of okadaic acid on neutrophil morphology. Shape changes of human neutrophils after stimulation in suspension with 1 pmol/L
okadaic acid, lO-9mol/L fNLPNTL, or lO-9 mol/L PMA. Neutrophils were incubated in basic medium (containing 1.1 mmol/L EGTA and 2% HSA)
with the following additions: (A) control in basic medium with 0.4% DMSO; (A+) 1 pmol/L okadaic acid; (B) 0.4% DMSO lO-9 mol/LfNLPNTL;
(B+) 1 pmol/L okadaic acid 10-9mol/L fNLPNTL; (C) 0.4% DMSO 10-9mol/L PMA; (C+) 1 pmol/L okadaic acid 10-9mol/L PMA. Cells
were preincubated at 37°C for 10 minutes with 0.4% DMSO or with 1 pmol/L okadaic acid, followed by incubation for another 30 minutes with or
without 1O-9 mol/ L fNLPNTL or lo-gmol/L PMA. The total incubation time for all samples was 40 minutes. The cells were subsequently fixed for
30 minutes with glutaraldehyde and washed. Photographs were taken using Nomarski Optics. Scale bar, 10 pm.
+
Fig 3. Concentration-dependent effects of okadaic acid on neutrophi1 shape changes. (0-0) Spherical cells; (m-m) nonpolar cells
with surface projections; (&--A) polarized cells. Neutrophils were
incubated inbasic medium (containing 1.1 m m o
EGTA
l/~
and 2%
HSA) with increasing concentrations of okadaic acid alone at 37°C for
40 minutes (A), or with okadaic acid for 10 minutes and then
incubation was continued for another 30 minutes after the addition of
10-9mollLfNLPNTL (E), or with okadaic acid for 10 minutes and then
incubation was continued for another 30 minutes after the addition of
10-9mol/L PMA (C). DMSO was always 0.4% (final concentration).
Controls without okadaic acid were incubated for 40 minutes with
0.4% DMSO, without or with subsequent addition of PMA or
fNLPNTL. At the end of the incubation period of a total of 40 minutes,
cells were fixed with glutaraldehyde and examined using Nomarski
Optics. Values are the mean 2 SD of five experiments.
+
+
+
to that observed with okadaic acid alone (Fig 2C and C+).
Calyculin A had effects on cell morphology comparable to
those of okadaic acid, but at 50- to 100-fold lower concentrations (Fig 4F). Half-maximal effects of calyculin A on cell
shape occurred at 0.010 2 0.003 Fmol/L (mean f SD,
n = 5). Up to 100% of the cells showed a nonpolar
morphology similar to that induced by okadaic acid, at 0.02
Fmol/L calyculin A. As shown for okadaic acid (Fig 2),
preincubation of cells with calyculin A before the addition
of PMA or “LPNTL also induced the morphology typical
for calyculin A alone. The control substance methyl
okadaate, which does not inhibit phosphatases in vitro,Z8
did not significantly affect cell morphology at 1 pmol/L. At
high concentrations of okadaic acid ( > 2 pmol/L) or
calyculin A ( > 0.02 Fmol/L), neutrophils assumed a multilobed appearance, because of the formation of multiple
small and large bleb-like protrusions (not shown).
The time course of the response has been studied by
continuousvideorecording of neutrophils stimulated with 1
mol/^ okadaic acid. The typical morphologic c.anges
developed after about 15 minutes of stimulation. Comparable results were obtained with cells incubated in plastic
tubes and fixed after different time points; half-maximal
effects were observed at 15 to 20 ,,,inUtes and maximal
effects were observed at 20 to 25 minutes of incubation with
Okadaic acid- The shape changes performed bY individua1
cells were so slow that they could not be detected without
time-lapse recording. Thus, the shape changes induced by
okadaic acid are much slower than those induced by
chemotactic peptides or PMA. The time courSe Of appearance of shape changes correlates with the slow increase in
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2915
PHOSPHATASES AND NEUTROPHIL FUNCTIONS
Fig 4. Effects of okadaic acid or calyculin A on shape and F-actin distribution in human neutrophils. Shape and F-actin distribution in human
neutrophils after stimulation in suspension with okadaic acid, calyculin A, fNLPNTL, or PMA. Neutrophils were incubated in basic medium
(containing 1.1 mmol/L EGTA and 2% HSA) with the following additions: (A and a) control in basic medium with 0.4% DMSO; (Band b) 1pmol/L
okadaicacid; (Candc)0.4% DMSO 10-9mol/LPMA; (Dandd)0.75pmol/Lokadaicacid 10-9mol/LfNLPNTL; (Eande)0.75pmol/Lokadaic
acid 10-9 mol/L PMA; (F and f) 0.012 pmol/ L calyculin A. Cells were preincubated at 37°C for 10 minutes with 0.4% DMSO or with phosphatase
inhibitors and then incubated for another 30 minutes with or without l O - 9 mol/L fNLPNTL or l O - 9 m o l / L PMA. DMSO was 0.02% (final
concentration) in (F and 1)and 0.4% in all other samples. The total incubation time for all samples was 40 minutes. Cells were subsequently fixed in
paraformaldehyde and stained with NED-phallacidin. Photographs were taken using Nomarski Optics (A through F) and fluorescence microscopy
(a through 1). Scale bar, 10 pm.
+
+
protein phosphorylation observed in the presence of okadaic acid (see above).
Reoeanization of F-actin induced by okadaic acid and
calyculinA. Unstimulated neutrophils show a diffuse cytoplasmic distribution of F-actin. After stimulation with 1 or 2
Fmol/L okadaic acid, or with 0.01 to 0.02 pmol/L calyculin
A, for 40 minutes, neutrophils showed a shift of F-actin,
which now appeared to be associated with the cell outline
(Fig 4B, b, F, and f). Close examination of the cells showed
that occasional fluorescence over the cell body was actually
+
associated with plasma membrane projections appearing in
the focal plane (Fig 4B and b). This contrasts with the
pictures obtained with PMA in which F-actin accumulated
in distinct thin surface lamella (Fig 4C and c). Combined
stimulation of neutrophils with PMA and phosphatase
inhibitors, or fNLPNTL and phosphatase inhibitors, resulted in an F-actin distribution similar to that observed
with inhibitors alone (Fig 4D,d, E, and e). Incubation of
the cells for 30 to 40 minutes with 1 pmol/Lokadaic acid or
0.02 pmol/L calyculin A, concentrations that had maximal
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2916
KREIENBUHL,KELLER, AND NIGGLI
Table 2. Effects of Calyculin A and Methyl Okadaate on the
effects on cell morphology (Fig 3), did not significantly
Association of Actin With the Neutrophil Cytoskeleton
affect the level of cytoskeleton-associated actin (Tables 1
and 2).
Cytoskeletal
First
Second
Actin (relative
Effect of okadaic acid on fluid pinocytosis (net uptake of
to control)
Addition
Addition
FZTC-dextran). PMA (W9 mol/L) alone produces a
None
None
(30
min)
1.00
2 0.44 (n = 5)
Okadaic
marked stimulation of uptake of FITC-de~tran.~
0.02
pmol/L
CA
None
(30
min)
0.90
f 0.06 (n = 3)
acid alone had relatively little effect. It produced a marginal
None
mol/LfNLPNTL (1 min)
2.05 2 0.48 (n = 3)
dose-dependent increase in fluid pinocytosis up to almost
0.02 pmol/L CA
mol/LfNLPNTL (1 min) 2.02 f 0.59 (n = 3)
twofold the activity of the control cells. The stimulating
None
mol/LfNLPNTL (30 min) 1.75 f 0.43 (n = 5)
activity of fNLPNTL or low9mol/L PMA on pinocytosis
0.02 pmol/LCA
mol/LfNLPNTL (30 min) 1.01 f 0.25 (n = 5)
was not significantly modified by increasing concentrations
None
mol/L PMA (30 min)
2.29 2 0.83 (n = 4)
of okadaic acid up to 2 kmol/L (Fig 5).
0.02 pmol/L CA
mol/L PMA (30 min)
0.94 f 0.23 (n = 4)
Effect of okadaic acid and calyculin A on the stimulus2.25 ? 0.61 (n = 4)
1 pmol/L MOA lo-@mol/L PMA (30 min)
dependent association of actin with the cytoskeleton. fNLPNTL
Neutrophils were preincubated in the presence of EDTA (10 mmol/L)
mol/L) induced a rapid transient increase in cytoskelwith either DMSO (0.2%. final concentration) or CA, or MOA (first
eta1 actin, followed by a decrease (Tables 1 and 2). This
addition). Preincubationtime was 10 minutes for all samples, except for
time course is comparable with previously published data
the samples subsequently incubated for 1 minute with fNLPNTL. The
obtained with the same technique.33The chemotactic peplatter two samples were preincubated with CA or DMSO for 30 minutes.
Subsequently, basic medium, fNLPNTL, or PMA was added (second
tide-dependent increase in cytoskeletal actin also correlates
addition) and the incubation was continued for the time specified.
with a decrease in G-actin, and an increase in F-actin as
determined with an NBD-phallacidin-binding assay.11,36-38 Cytoskeletal actin was then determined as described in Materials and
Methods (mean 2 SD). In control cells incubated with DMSO, 6% to
After 30 minutes, the percentage of cytoskeletal actin was
17% of the total cellular actin was associated with the cytoskeleton.
lower than after 1 minute, but it was still significantly
Abbreviations: CA, calyculin A; MOA, methyl okadaate.
increased above controls (Tables 1 and 2, P < .005; see also
Niggli and Kellerlo Fig 4). A comparable small increase in
due to activation of cells by adhesion to plastic tubes as a
F-actin persisting for at least 20 minutes after the addition
result
of long-term incubation, for the following reasons.
of stimulus has also been reported by investigators using the
Adhesion
is certainly not a major event under our condiNBD-phallacidin a ~ s a y .This
~ ~ ,persisting
~~
increase is not
tions, as 94% 2 6% (mean f SD, n = 3) of the cells could
be recovered from the tubes after 40 minutes of incubation
Table 1. Effect of Okadaic Acid on the Association of Actin With the
in plastic tubes. Moreover, we analyzed in separate experiNeutrophil Cytoskeleton
Cytoskeletal
First
Addition
Second
Addition
Actin (relative
to control)
Experiment 1 (10-min preincubation)
None
None (30 min)
1 pmol/L OA
None (30 min)
None
10-9 mol/LfNLPNTL (1 min)
1 pmol/L OA
mol/LfNLPNTL (1 min)
None
10-9 mol/L fNLPNTL (30 min)
1 pmol/L OA
mol/L fNLPNTL (30 min)
None
10-8 mol/L PMA (30 min)
1 pmol/L OA
10-8 mol/L PMA (30 min)
1.00 f 0.19
0.75 2 0.12
2.75 f 0.66
3.12 f 0.86
1.61 f 0.09
0.89 f 0.21
2.19 f 0.61
0.99 f 0.21
Experiment 2 (30-min preincubation)
None
None (1 min)
1 pmol/L OA
None (1 min)
None
lO-gmol/LfNLPNTL (1 min)
1 pmol/L OA
10-9 mol/LfNLPNTL (1 min)
None
mol/L fNLPNTL (5 min)
1 pmol/L OA
10-9 mol/L fNLPNTL (5 min)
1.OO
0.72
2.70
2.38
2.21
1.02
T
T
T
f 0.54
f 0.22
f 0.62
f 0.67
f 0.86
f 0.30
Neutrophils were preincubated for 5 minutes with EDTA (10 mmol/L,
final concentration), followed by a further preincubation with DMSO
(0.4%, final concentration) or okadaic acid (first addition) for 10
(experiment 1) or 30 minutes (experiment 2). Subsequently, basic
medium, fNLPNTL, or PMA was added (second addition) and the
incubation was continued for the time specified. Cytoskeletal actin was
then determined as described in Materials and Methods (mean 2 SD of
three independent experiments). In control cells incubated with DMSO,
7% to 15% of the total cellular actin was associated with the cytoskeleton.
Abbreviation: OA, okadaic acid.
Concentration of okadaic acid (pM)
Fig 5. Effect of okadaic acid on fluid pinocytosis. Fluid pinocytosis
was determined by flow cytometry. Cells were stimulated with
increasing concentrations of okadaic acid alone (O),okadaic acid and
mol/L PMA (U).
mol/L fNLPNTL (A),or okadaic acid and
Neutrophils were incubated with 0.4% DMSO or with increasing
concentrations of okadaic acid (first stimulus) at 37°C for a total of 40
minutes. DMSO was always 0.4% (final concentration). After 10
minutes of incubation with DMSO or with okadaic acid, the second
stimulus was added and incubation was continued for another 30
minutes. Cells were fixed in glutaraldehyde and the relative fluorescence intensity was determined using flow cytometry. Values are the
mean k SDM of five experiments.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHOSPHATASES AND NEUTROPHIL FUNCTIONS
merits the amount of cytoskeletal actin in only those cells,
which were removable from the tubes after 40 minutes of
incubation at 37°C. These cells, incubated for 30 minutes
with
mol/L fNLPNTL, still showed a 1.7- ? 0.4-fold
(mean 2 SD, n = 3) increase in cytoskeletal actin compared with controls ( P < .05). The latter experiment has
been performed in the presence of less than 0.001%
DMSO. These results show that this persistent increase in
cytoskeletal actin is due neither to adhesion nor to the
presence of DMSO.
The effect of 1 kmol/L okadaic acid on cytoskeletal actin
was studied using either 10 or 30 minutes of preincubation
with the inhibitor before the addition of stimulus. The latter
conditions were used especially for subsequent short-time
incubation with stimuli, to ensure maximal entry of okadaic
acid into the cells. Preincubation of cells for 10 or 30
minutes with okadaic acid had no significant effect on the
peptide-induced rapid initial increase in cytoskeletal actin
(1 minute), but inhibited the increase obtained at 5 and 30
minutes after addition of fNLPNTL by at least 80% (Table
1, P < .05). Preincubation of cells with okadaic acid also
completely prevented the PMA-induced increase in cytoskeletal actin (Table 1,P < .025). Comparable results were
obtained with 0.02 pmol/L calyculin A (Table 2). Calyculin
A inhibited the increase in cytoskeletal actin induced by 30
minutes of incubation with fNLPNTL by at least 75%
(P < .0025) and that by PMA by at least 88% (P < .0125).
In contrast, the initial increase at 1 minute was not
significantly affected (0% to 14% inhibition). Longer preincubation times up to 40 minutes with calyculin A or okadaic
acid still did not result in inhibition of the early event (not
shown). The inactive control substance methyl okadaate
did not significantly affect the increase induced, for instance, by PMA (Table 2).
Thus, okadaic acid and calyculin A counteract the
increases in cytoskeletal actin induced in neutrophils by
either 30 minutes of incubation with PMA or long-time (5
to 30 minutes) incubation with fNLPNTL. However, the
peptide-dependent rapid initial increase in cytoskeletal
actin appears to be refractory to the action of phosphatase
inhibitors. The concentrations used had maximal effects on
cell morphology of resting cells, and completely prevented
fNLPNTL-induced development of cell polarity (Figs 2
through 4).
DISCUSSION
Incubation of human neutrophils with micromolar concentrations of okadaic acid or nanomolar concentrations of
calyculin A alone results in characteristic shape change and
shift of F-actin to the plasma membrane, whereas the level
of cytoskeletal actin is not significantly altered. This is in
contrast to findings with chemotactic peptide or PMA, in
which altered shape is associated with a marked increase in
cytoskeletal actin and F - a ~ t i n . ~ JShape
~ J ~ changes and
membrane association of F-actin may thus occur without an
increase in cytoskeleton-associated actin. However, it is
important to note that the shape changes induced by
okadaic acid are very slow and are much slower than those
induced by fNLPNTL or PMA. Furthermore, we cannot
2917
exclude that okadaic acid acts by increasing the turnover of
actin filaments without increasing the level of cytoskeletal
actin. Endotoxin has also been reported to induce changes
in shape and F-actin localization in neutrophils without
changing the level of F - a ~ t i n . ~ ~
Okadaic acid and calyculin A are thought to act by
inhibiting protein phosphatases, thereby increasing net
phosphate incorporation into proteins. Indeed, both inhibitors have been shown to markedly stimulate overall levels of
protein phosphorylation in intact cells and in permeabilized
n e ~ t r o p h i l s . ~We
~ , ~found
~ , ~ ~that micromolar concentrations of okadaic acid (Fig 1) or 0.02 pmol/L calyculin A
induce a marked increase in protein phosphorylation of at
least 25 bands in intact neutrophils. The present results,
obtained using two phosphatase inhibitors with different
structure and potency, show that phosphatases have an
important regulatory function in neutrophils. Increased
phosphorylation of one or several substrates as a result of
phosphatase inhibition appears to lead to changes in cell
morphology and F-actin location. A correlation between
effects on phosphorylation and shape changes is confirmed
by the comparably slow time course of both events. The
finding that calyculin A acts on neutrophils at 50-fold lower
concentrations than okadaic acid suggests that the effects of
these inhibitors are due to inhibition of a phosphatase
1-type enzyme, which is inhibited by calyculin A in vitro at
low nanomolar concentration^.^^ However, these findings
could also be explained by differences in the efficiency of
penetration of these inhibitors into the neutrophils. In
macrophages, okadaic acid was found to produce a dosedependent increase in the phosphorylation of the 20-Kd
light chain of myosin, with half-maximal effects occurring in
the micromolar range.4l
Interestingly, preincubation of neutrophils with micromolar concentrations of okadaic acid or 0.02 p,mol/L calyculin
A completely prevents increased association of actin with
the cytoskeleton induced by PMA or 5 to 30 minutes of
incubation with fNLPNTL (Tables 1 and 2). In a recent
study, the effect of okadaic acid on the PMA-induced
increase in F-actin has been tested in intact human neutrophils, using NBD-phallacidin
Downey et a142
found only a small partial inhibition induced by okadaic
acid. The reason for the discrepancy between our data and
theirs may be the short (5 minutes) preincubation time with
the drug used by Downey et a1,4*which in our hands would
not be sufficient for maximal effects. Interestingly, it has
been shown recently that pretreatment of neutrophils with
okadaic acid produces a time-dependent inhibition of the
phorbol ester-induced respiratory burst that becomes maximal after 30 minutes of preincubation with okadaic acid.35
Both phorbol ester and chemotactic peptide may activate
PKC in ne~trophils.3~9~~
Inhibition of phosphatases by the
inhibitors would be expected to increase the phosphorylation of PKC substrates even further. Our findings suggest
that overphosphorylation of a substrate(s) of PKC or other
kinases inhibits the stimulus-dependent increase in cytoskeletal actin. Possibly, activation of a phosphatase, and the
resulting dephosphorylation of a protein, plays a crucial
role in phorbol ester-dependent signalling for increased
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2918
KREIENBUHL, KELLER, AND NlGGLl
actin polymerization, implicating a novel mechanism. PMA
may act on this phosphatase indirectly, via activation of
PKC, or possibly even directly. A direct effect of PMA on a
phosphatase would explain the finding that various PKC
inhibitors do not prevent PMA-induced actin assembly.42In
our hands, the PKC-specific inhibitor CGP 41 25144 also
does not inhibit the PMA-dependent increase in cytoskeletal actin (V. Niggli, unpublished data, April 1991). Moreover, our results suggest that a comparable signal is operative in the late stage of activation by fNLPNTL. In this
context, our recent observation that staurosporine, a potent
inhibitor of several protein kinases, stimulates actin association with the cytoskeleton at low (nanomolar) concentrations is of interest.1° A decrease in protein phosphorylation,
induced either by activation of a phosphatase or inhibition
of a kinase, may thus be able to trigger actin reorganization.
How inhibition of a phosphatase leads to changes in actin
localization and prevents stimulus-dependent increases in
the level of cytoskeleton-associated actin is not known. A
82-Kd PKC substrate in neutrophils may be involved. This
protein only associates with membranes in the dephosphorylated form and okadaic acid (1 kmol/L) has been shown
to prevent dephosphorylation and membrane association of
this protein in chemotactic peptide-stimulated neutrop h i l ~ Thelen
.~~
et al propose a role of this protein in
reversible actin-membrane linkage.45
In contrast to the above findings, preincubation of cells
with okadaic acid or calyculin A does not prevent the rapid
increase in cytoskeletal actin occurring 1 minute after the
addition of fNLPNTL. This suggests different signalling or
regulatory pathways for increased association of actin with
the cytoskeleton in the early and late stages of neutrophil
activation .
The effects of okadaic acid and calyculin A on the
morphology and F-actin distribution of PMA- and fNLPNTLstimulated cells are rather complex. On the one hand, these
inhibitors prevent the association of actin with the cytoskeleton. On the other hand, they prevent fNLPNTL- or
phorbol ester-induced shape changes (polarization or formation of pseudopods) and, instead, impose the typical
morphology induced by the inhibitors alone. Therefore,
they do not just restore the resting state of the neutrophils,
but rather activate and disturb the typical morphology of
cells activated by other stimuli. We have noted that the
effect of PMA on cell morphology overrides that of
fNLPNTL? Okadaic acid and calyculin A were now found
to override the effects of both PMA and MLPNTL. The
increase in phosphorylation shows a corresponding hierarchy (fNLPNTL < PMA < < okadaic acid) (Fig 1). This
may indicate that the morphologic effects could be related
to the degree of phosphorylation. Suppression of cell
polarity (Fig 2 ) by okadaic acid is one, but not the only,
explanation for inhibition of macrophage chemotaxis induced by micromolar concentrations of the
Okadaic acid had no effect on the marked increase in
pinocytosis induced by fNLPNTL or PMA under conditions
in which it has been shown to prevent stimulus-dependent
increases in cytoskeletal actin (Fig 5). Thus, pinocytosis can
occur in the absence of a significantly increased association
of actin with the cytoskeleton. The two events appear to be
dissociated.
In summary, the results presented here suggest that the
steady-state activity of a phosphatase (possibly type 1) is
instrumental in maintaining cell shape and F-actin localization in resting human neutrophils. Moreover, we have
evidence for a novel signalling or regulatory pathway
involved in stimulus-dependent increases in cytoskeletal
actin. This pathway, requiring the activity of a phosphatase
sensitive to okadaic acid and calyculin A, appears to be
operative in the later stage of neutrophil activation by
chemotactic peptide and in activation by phorbol ester.
ACKNOWLEDGMENT
We thank A. Damschen and K. Mujynya for excellent technical
assistance, and Dr K. Malmstrom for helpful discussions.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1992 80: 2911-2919
Protein phosphatase inhibitors okadaic acid and calyculin A alter cell
shape and F-actin distribution and inhibit stimulus-dependent
increases in cytoskeletal actin of human neutrophils
P Kreienbuhl, H Keller and V Niggli
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