March 29, 2012
So, here we go. The first installment in this series is a manuscript about non-muscle myosins found in human leukocytes. This paper showed that PMN and monocytes from patients with head and neck cancer contain myosin II and also MLCK. Myosin levels were decreased in leukocytes from cancer patients compared to those from normal subjects, whereas MLCK levels were similar in PMN and monocytes from both patient groups. After in vitro exposure to formylpeptide chemoattractant, myosin levels decreased significantly in the PMN and monocytes obtained from normal subjects but were not altered in cancer patient leukocytes.
This suggested that myosin was abnormally regulated in cancer patient leukocytes and that leukocytes from those patients may have been 'pre-activated' in situ. Such pre-activation might indicate that the host had been actively repulsing an advanced cancer. However, the long-recognized chemotactic inhibition seen in the leukocytes from such patients suggests just the opposite, that the leukocytes had been activated in situ, had then expended their efforts, had down-regulated their surface receptors, and then became more or less refractile to further stimulation both in situ and in vitro.
Below is the full paper and a link to the PDF:
Myosin II and Myosin Light Chain Kinase in Neutrophils and Monocytes from Cancer Patients
Myosin II and Myosin Light Chain Kinase in
Neutrophils and Monocytes from Cancer Patients
by
Robert J. Walter,
Tzu-Chieh Chao 1, Amelia H. Janeczek 1,
John R. Danielson, and
Hernan M. Reyes
Department
of Surgery, Cook County
Hospital and Hektoen Institute for
Medical Research, Chicago, IL
1 Department of Anatomy
and Cell Biology, University of Illinois at Chicago, Chicago, IL
Address
all correspondence to:
Robert J. Walter, Ph.D.
Department
of Surgery
Hektoen
Institute for Medical Research
625 South Wood Street
Chicago, IL 60612
Telephone: (312) 633-7237
FAX:
(312) 732-3102
Running Title: Myosin in Tumor Patient Leukocytes
Keywords: chemotaxis, neutrophils, monocytes, myosin,
myosin light chain kinase, cancer, polarization
Abbreviations:
cAMP,
3',5'-cyclic adenosine monophosphate; CT, control; CA, cancer; PMN, polymorphonuclear
leukocyte; MLCK, myosin light chain kinase; BSA, bovine serum albumin; FITC,
fluorescein isothiocyanate; FMLP, N-formyl-methionyl-leucyl-phenylalanine;
HEPES, N-2-hydroxyethylpiperazine-N-2'-ethanesulfonic acid; HBS, Hank's
balanced salt solution
ABSTRACT
In
vitro chemotaxis of monocytes obtained from tumor patients is often severely
depressed but the mechanism of this inhibition is not known. The actomyosin cytoskeleton is thought to
play a pivotal role in generating the forces required for cells to perform
activities such as chemotaxis. This
study has employed flow cytometry and fluorescence microscopy to examine two
components of the actomyosin machinery, i.e., myosin II and myosin light chain
kinase (MLCK) in unstimulated and in formylpeptide-stimulated neutrophils and
monocytes isolated from normal (CT) and cancer patient (CA) peripheral blood
samples. Decreased amounts of myosin
were observed in unstimulated CA neutrophils and monocytes compared to
unstimulated CT leukocytes. Upon
stimulation with formylpeptide chemoattractant, the amount of myosin detected
in CT leukocytes decreased markedly but in CA leukocytes was altered very
little. Similar amounts of MLCK were
observed in unstimulated CT and CA leukocytes and in formylpeptide-stimulated
cells. In the fluorescence microscope,
adherent monocytes and PMN showed diffuse cytoplasmic myosin and MLCK
fluorescence throughout the cytoplasm with some increase in intensity at the
trailing end (uropod) in formylpeptide-stimulated cells. Since the actomyosin cytoskeleton is
intricately involved in leukocyte chemotaxis, alterations in the cytoskeleton
may dramatically affect cell motility.
CA leukocytes exhibited decreased baseline myosin levels and a vastly
different response to formylpeptide stimulation as compared to CT
leukocytes. Unstimulated CA monocytes
also exhibited increased spontaneous polarization but FMLP-stimulated
polarization was inhibited. These
cytoskeletal alterations and changes in the response of CA leukocytes to
formylpeptide stimulation may result in decreased chemotaxis by these cells.
INTRODUCTION
The
actomyosin cytoskeleton is thought to play a prominent role in nonmuscle cell
motility. In leukocytes and single cell
eukaryotes such as Dictyostelium
discoideum and Acanthamoeba castellanii, cytoplasmic actin and
myosin (myosin II or conventional myosin) have been studied extensively and are
known to generate the forces that drive chemotaxis (1,2). Two types of models have been developed to
describe the mechanism of force generation in these cells. The first of these is the actin thin
filament-based model which focuses upon the extensive actin remodeling known to
occur during chemotactic activation.
Within seconds of exposure to a chemoattractant, extensive actin
polymerization occurs followed over the next few minutes by a much slower
depolymerization (3,4). While these
events are occurring, actin-rich pseudopods are extended by the cell and, in time,
the entire cell becomes polarized structurally with actin concentrated in both
the lamellipod and uropod (4-7). This
model proposes that the formation and breakdown of a three dimensional actin
lattice is responsible for the cytoplasmic protrusive and contractile
activities associated with cell locomotion (2).
The regulation of actin polymerization and depolymerization is thought
to involve a range of known actin binding proteins (filamin, acumentin,
profilin, gelsolin, etc.) and cytoplasmic calcium (2,8,9).
Myosin
II, one of these actin binding proteins, is central in the second model of
force generation, the myosin thick filament-based model. According to this, after chemotactic
activation and concurrent with actin remodeling, myosin heavy chain is
transiently dephosphorylated and myosin light chain is phosphorylated (10,11)
triggering myosin heavy chain assembly into thick filaments (2,12,13). Subsequently, myosin heavy chain
phosphorylation occurs, impeding further assembly of myosin thick filaments
(14,15), and leading to depolymerization of existing myosin filaments
(16). During thick filament assembly in Dictyostelium, myosin shifts in position from the endoplasm into
the cortical cytoplasm underlying the plasma membrane and also becomes
associated with the posterior uropod (15,17).
In lower eukaryotes and 3T3 fibroblasts, myosin II is largely excluded
from the lamellipod during periods of intense membrane ruffling (16,18,19) but
in motile leukocytes and HeLa cells, myosin was localized in the lamellipod not
the uropod (20). The reason for this
discrepancy is not known. Nonetheless,
when colocalized in the uropod, it is thought that myosin thick filaments may
interact with actin by means of myosin ATPase to generate the forces required
to drive cell migration and assist in tail retraction (13,21). These actomyosin interactions are probably
regulated by calcium (22) and the phosphorylation of the myosin light chain by
myosin light chain kinase (MLCK) (23-25).
The
relative importance of myosins I and II in cell locomotion has been the subject
of much recent study. Genetically
altered Dictyostelium deficient in
myosin light or heavy chains exhibited markedly decreased rates of locomotion
and defective pseudopod formation (26).
In Acanthamoeba,
microinjection of monoclonal anti-myosin II slows but does not stop locomotion
(19). Similarly, myosin I deficient Dictyostelium exhibited slowed
chemotaxis and decreased phagocytic rates, but no other phenotypic changes
(27). Thus, in lower eukaryotes, it
seems likely that both forms of myosin are required for normal cell
motility. However, in vertebrate
leukocytes, no role for myosin I can be assigned because it has yet to be
demonstrated in these cells (28). It is presumed that the
activation sequence described above is necessary to bring about normal
chemotaxis and that alterations in any of the steps of this sequence may result
in defective or reduced chemotaxis. A
severe defect in monocyte chemotaxis has been demonstrated in many cancer
patients, but the cause of this defect is not known (29,30). Monocytes, macrophages, and neutrophils are
thought to participate directly in tumor destruction (31,32) and studies of
experimental animal tumor models have consistently demonstrated that these
leukocytes are potent anti-tumor effectors (33). Monocyte and neutrophil accumulation at sites
of tumor growth is accomplished by means of chemotaxis and this response
appears to be an essential aspect of the antitumor response (34). Due to the significance of the leukocyte
cytoskeleton in chemotactic activation, we have examined cell polarization as
well as the myosin II and MLCK content and distribution in normal leukocytes
and in leukocytes from patients with advanced cancer. As compared to normal leukocytes,
unstimulated PMN and monocytes from these patients expressed reduced levels of
myosin, monocytes showed reduced cell polarization, and increased spontaneous
polarization. After formylpeptide
stimulation, normal PMN and monocytes exhibited reduced myosin levels, but
leukocytes from cancer patients exhibited no change in myosin content and
reduced cell polarization. This abnormal
modulation of myosin in cancer patient leukocytes may contribute to the
chemotactic and polarization defect observed in leukocytes from these patients.
MATERIALS AND METHODS
Selection of Patients
Patients
selected for this study were recently diagnosed as having advanced tumors
(sarcoma or carcinoma) but were not yet receiving chemotherapy, radiation
therapy, or other medications at the time of venipuncture. Non-cancer patient controls were selected to
give age and sex distributions comparable to the cancer patient group.
Cells and Incubation
Conditions
PMN
and mononuclear leukocytes were isolated from venous blood collected in EDTA or
heparin as described previously (30,35).
Erythrocytes were sedimented at 1xg in 1.25% dextran (pyrogen-free) and
the leukocyte-rich plasma drawn off. Two
ml of plasma were diluted 1:1 with a buffer (HBS) containing 140 mM NaCl, 10 mM
KCl, 10 mM HEPES, 5mM glucose, 2 mg/ml bovine serum albumin (BSA), pH 7.4 and
layered onto one ml of Lymphocyte Separation Medium (Organon Teknika, Durham,
NC). This gradient was then centrifuged
at 500xg for 5 min at room temperature, the mononuclear cells at the interface
removed, and the PMN pellet resuspended.
PMN were rinsed in HBS containing 2 mM EDTA and contaminating
erythrocytes lysed by hypotonic shock.
The mononuclear fraction was diluted with 5 ml of HBS with 2 mM EDTA and
2.5% dextran and centrifuged for 5 min at 500xg. The platelet-rich supernatant was removed and
the dextran centrifugation procedure repeated twice on the resuspended cell
pellet. Cells were resuspended in HBS
containing 1 mM MgCl2 and 0.2 mM CaCl2, counted using a
hemacytometer, and stored until use at 4ºC.
Differential counts were also performed on these cell preparations.
Chemotaxis Assays
Cells
were tested for their chemotactic capability in multiwell chemotaxis chambers
as previously described (30,35).
Briefly, formyl-methionyl-leucyl-phenylalanine (FMLP) over a range of
concentrations (10”-11M to 10”-6M) was placed into the lower wells of a 48 well
microchemotaxis chamber (Neuroprobe, Cabin John, MD) and purified neutrophils
or monocytes were loaded into the upper wells (25 µl; 20,000 cells). A polyvinylpyrrolidone-free polycarbonate
filter (Nucleopore, Pleasanton,
CA) with 5 µm pores separated the
upper from the lower wells and the chamber was incubated at 37ºC in a
humidified incubator for 2 hours.
Samples were run in triplicate and nuclei in 5 (400X) microscope fields
from each well were counted. Monocytes
from all tumor patients studied here exhibited a marked chemotaxis defect
(65-81% inhibition) compared to non-cancer controls.
Polarization Assay
Patient
and control monocytes suspended in HBS/BSA at 37ºC in siliconized tubes were
incubated for various times in the presence or absence of FMLP. At the completion of each incubation,
buffered paraformaldehyde was added to a final concentration of 3%, cells were
stained with Hoechst 33258 to aid in cell identification, and monocyte
polarization determined for 100 cells using light microscopy. Monocytes were considered to be polarized if
they exhibited a change in the normal, rounded morphology such they became
elongated or triangular in shape or exhibited asymmetric lamelipodial
extension.
Antibodies and Staining
Procedures
Cells
were exposed to either buffer alone or to FMLP for increasing periods of time
(1 to 10 min) at 37ºC. For PMN, a
uniform field of 1 nM FMLP was employed as stimulant whereas 0.1 nM FMLP was
used for monocytes. Antimyosin and
anti-MLCK antibodies were the generous gift of Dr. P. deLanerolle (University of Illinois
at Chicago) and were used as described
previously with some modifications (36).
Unstimulated or FMLP-treated cells were fixed at 4ºC in 2%
paraformaldehyde in phosphate buffered saline (PBS), washed, and then lysed in
acetone (-20ºC). After washing in 50%
ethanol, autofluorescence and unreacted fixative were quenched using sodium
borohydride (1 mg/ml) in 50% ethanol.
Cells were then rinsed in PBS containing 1% normal goat serum and 0.1%
BSA and non-specific binding blocked using 5% normal goat serum. Cells were then reacted with primary antibody
for 1 hr at room temperature. Either
rabbit anti-human platelet myosin serum (1:40 dilution) or affinity purified
rabbit anti-turkey gizzard MLCK (1:100 dilution) were used as primary
antibodies. Anti-human platelet myosin
antibody reacts strongly with mammalian and avian non-muscle myosin and to a
lesser degree with mammalian and avian smooth muscle myosin (37). Polyclonal rabbit anti-turkey gizzard MLCK
antibody reacts with MLCK of smooth muscle but not with actin, myosin,
tropomyosin, alpha-actinin, or filamin (36).
Subsequent to this incubation, cells were washed and further incubated
in FITC-goat anti-rabbit IgG (1:60 dilution; Cappel) for 1 hr. Cells were then washed using three changes of
PBS. All buffers and antibodies were
passed through a 0.22 µm pore size micropore filter before use to remove
aggregates. For flow cytometry, some
cell preparations were stained with phycoerythrin-conjugated MY-9 only (Coulter
Immunodiagnostics, Hialeah,
FL). MY-9 is a monoclonal antibody that binds to
normal peripheral blood monocytes but not to granulocytes, erythrocytes,
platelets, or lymphocytes (38).
Flow Cytometry
Stained
cells were filtered through nylon mesh (25 µm pore size; TETKO Inc., Elmsford, NY) and
analyzed using an EPICS C flow cytometer (Coulter Electronics, Hialeah, FL)
equipped with a 500 mW argon ion laser emitting at 488 nm. Forward-angle and right-angle light scatter,
fluorescein (535 nm), and phycoerythrin (580 nm) fluorescence were acquired for
at least 5000 cells in each experimental group.
Fluorescence was collected using a three-decade logarithmic amplifier.
Some
cells were fixed in 2% buffered paraformaldehyde and stained with either
phycoerythrin-conjugated MY-9 (Coulter Immunology, Hialeah, FL;
diluted 1:30) or with phycoerythrin-conjugated nonspecific mouse immunoglobulin
(diluted 1:30). MY-9 was used to aid in
determining the exact monocyte and neutrophil gate settings and to ascertain
the proportion of monocytes counted in the monocyte gate.
Polarization Assay
The
time course and concentration dependence of the monocyte polarization response
was assessed for monocytes suspended in HBS in siliconized tubes. Cells were either exposed to a range of
concentrations of FMLP for 20 min at 37ºC, to 1 nM FMLP for 0 to 60 min, or to
buffer alone for 0 to 60 min. Incubations were terminated by the addition of
paraformaldehyde to a final concentration of 4%, and cells were stained with
Hoechst 33258. Monocytes were identified
by nuclear morphology and size using fluorescence microscopy and cell
polarization evaluated for 100 cells at each data point. Cells were considered to be polarized if they
exhibited a distinct shape change including triangular shape, asymmetric
protuberance formation, or lamellipodial extension.
Fluorescence Microscopy
and Photography
For
fluorescence microscopy, purified PMN and monocytes were remixed and allowed to
adhere to clean glass coverslips at 37ºC for 5 min. After fixation, extraction, and staining as
described above, labeled leukocytes were further stained with Hoechst 33258 (10
µg/ml) in PBS for 10 min at room temperature.
After washing, cover slips were mounted on slides in PBS with 90%
glycerol containing paraphenylenediamine (1 mg/ml) (39). These preparations were examined using a
Nikon Optiphot microscope with a Leitz 100 W mercury epiiluminator and
photographed using Kodak Tri-X Pan (ASA 400) or Ektachrome (ASA 1600).
Statistical Evaluation
of Data
Mean
channel numbers for each time or treatment were treated as individual
parametric values and groups of these values were compared using unpaired
t-tests. Chi-square was employed to
evaluate non-parametric cell polarization data.
Mean channel numbers are the average of three separate experiments (mean
± SD) performed on blood samples from different CT and CA subjects as starting
material. A probability value < 0.05
was considered significant.
RESULTS
Myosin Redistributes
into the Uropod upon FMLP Stimulation
Unstimulated,
adherent CT leukocytes were generally uniformly spread except for a few cells
(< 5%) that were spontaneously polarized.
Antimyosin staining disclosed diffuse cytoplasmic fluorescence in both
rounded and spontaneously polarized cells (Figure 1a). Increased amounts of fluorescence were seen
in the central regions of the cells but very little fluorescence was associated
with the cortical regions of the cell.
Unstimulated PMN and monocytes from CA patients were similar in overall
appearance to CT leukocytes, but exhibited somewhat decreased levels of fluorescence
(Figures 1b, c). In all groups,
monocytes displayed less intense fluorescence than did PMN.
Formylpeptide-stimulated CT leukocytes were
structurally polarized exhibiting a distinct leading lamellipod and trailing
uropod (Figures 1d-g). Antimyosin staining was more concentrated beneath the plasma
membrane than that seen in unstimulated cells.
PMN showed distinct concentrations of fluorescence in the trailing
uropod region of polarized cells and monocytes showed slight increases in the
the amount of fluorescence in the uropod.
Formylpeptide-stimulated PMN and monocytes from CA patients (Figure 1f,
g) were similar in overall appearance to stimulated CT leukocytes (Figure 1d,
e), but in general exhibited decreased levels of fluorescence. Differences in fluorescence intensity were
clearly evident in specimens examined in the flow cytometer.
MLCK is Diffusely
Distributed
Unstimulated,
adherent CT and CA PMN and monocytes stained for MLCK exhibited faint diffuse
cytoplasmic fluorescence (Figures 2a-d).
Formylpeptide-stimulated CT and CA
leukocytes displayed MLCK staining that was in most cases diffuse throughout
the cytoplasm, but in some cells was unmistakably concentrated in the uropod
region (Figures 2e, f).
Myosin Staining Differs
in CT and CA Leukocytes
Phycoerythrin-conjugated
MY-9 staining was used to confirm the location and cell type found within the
monocyte gate (Figure 3). This was
necessary because the extraction protocol used prior to staining for myosin or
MLCK resulted in less discrete monocyte distributions as seen in scatterplots
(right angle versus forward light scatter).
MY-9 staining of the gated monocyte population revealed that 85% of the
cells were monocytes. Due to their low
cytoplasmic granularity, the remainder were presumed to be large
lymphocytes. This same proportion of
monocytes was seen in both CA and CT samples.
Unstimulated
CT PMN stained for myosin were seen as discreet unimodal distributions. As a group (Table 1), unstimulated CT PMN had
an average mean channel number of 80 ± 4 (Figure 4) whereas unstimulated PMN
from CA patients had a mean channel number of 48 ± 9 (p < 0.005 compared to
unstimulated CT). Upon exposure to 1 nM
FMLP, the mean channel number observed for CT PMN declined to 63 ± 5 after 1
minute of exposure and to 61 ± 5 after 4 minutes of exposure to FMLP. In contrast, under these same conditions the
mean channel number for CA PMN increased to 49 ± 10 and 53 ± 11, respectively.
Unstimulated
CT monocytes stained for myosin were seen as broad unimodal distributions
having a mean channel number of 77 ± 4 whereas unstimulated monocytes from CA
patients were seen as broad unimodal distributions having an average mean
channel number of 22 ± 6. Upon exposure
to 0.1 nM FMLP, the mean channel number observed for CT monocytes declined to
47 ± 3 after 1 minute of exposure and to 30 ± 5 after 4 minutes of exposure to
FMLP. Under these same conditions the
mean channel number for CA monocytes remained virtually unchanged at 21 ± 5 and
20 ± 3, respectively.
MLCK Staining is Similar
in CT and CA Leukocytes
Unstimulated
CT PMN stained for MLCK (Table 2) were seen as a unimodal distributions having
a mean channel number of 63 ± 10, whereas unstimulated PMN from CA patients
displayed mean channel numbers of 62 ±10.
Upon exposure to 1 nM FMLP, the mean channel number observed for CT PMN
was 67 ± 14 after 1 minute of exposure to FMLP and 64 ± 17 after 4
minutes. Under these same conditions,
the mean channel number for CA PMN increased slightly to 63 ±10 and 65 ±10,
respectively.
Unstimulated
CT monocytes stained for MLCK were seen as a broad unimodal peak having a mean
channel number of 61 ± 3 whereas unstimulated monocytes from CA patients
exhibited unimodal peaks with an average mean channel number of 55 ± 3. Upon exposure to 0.1 nM FMLP, the mean
channel number observed for CT monocytes increased to 70 ± 3 and 72 ± 2 after 1
and 4 minutes of exposure to FMLP, respectively. Under these same conditions, the mean channel
number for CA monocytes increased to 62 ± 5 and 65 ± 3, respectively.
Spontaneous and
FMLP-Stimulated Cell Polarization is Altered in CA Monocytes
CT
and CA patient monocytes were exposed to buffer alone or to different
concentrations of FMLP for 20 min at 37ºC (Figure 5). In the presence of FMLP, polarization was
significantly reduced in CA as compared to CT monocytes with maximal
polarization evident at 10-9 M FMLP.
However, in buffer alone, polarization was significantly increased in CA
as compared to CT monocytes.
As
seen in Figure 6, CT and CA patient monocytes were incubated with 10-9
M FMLP or in buffer alone for times ranging from 0 to 60 min. In buffer alone, the fraction of polarized CA
monocytes was significantly greater at all times (except 0 time) than that seen
for CT monocytes. FMLP-stimulated CA
monocytes exhibited significantly decreased polarization for incubation times
between 5 and 30 min but significantly increased polarization after 60 min of
incubation as compared to that seen for CT monocytes.
DISCUSSION
We
have examined myosin staining, MLCK staining, and cell polarization in
unstimulated and formylpeptide-stimulated PMN and monocytes isolated from CT
and CA patients. We have observed: 1)
decreased myosin staining in PMN and monocytes from CA patients as
compared to leukocytes taken from CT patients, 2) that stimulation with formylpeptide decreases
myosin staining in CT leukocytes but not in leukocytes from CA patients,
3) comparable levels of MLCK in CT and
CA leukocytes, and similar levels of MLCK staining in both cell groups when
stimulated with formylpeptide, 4) that
unstimulated CA monocytes exhibited increased polarization but that
formylpeptide-stimulated polarization was inhibited as compared to CT
monocytes.
Myosin
staining of monocytes (CT and CA) was less intense than that of PMN (CT and CA)
as seen in the fluorescence microscope and in the flow cytometer. After formylpeptide stimulation of CT patient
leukocytes, distinct morphological alterations became evident. Myosin staining became more concentrated
beneath the plasma membrane especially in the uropod region and markedly
reduced amounts of myosin staining were seen.
Amounts of MLCK staining remained unchanged but staining was sometimes
concentrated in the uropod. Previous
reports on the distribution of myosin in chemotactically-activated cells are
somewhat contradictory and there have been few reports on myosin distributions
in higher eukaryotic cells such as leukocytes.
Most of the recent studies in lower eukaryotes have shown myosin II
within cellular protrusions or in the endoplasm but have not described its transcellular
location (anterior, middle, or posterior) in the motile cell.
In
unstimulated cells (see Table 3), myosin II is found diffusely distributed in
the endoplasm and often absent from the cell cortex. In chemotactically activated amoebae, myosin
II is consistently found in the uropod and the cortical cytoplasm. Similarly, myosin II co-caps with surface Ig
on human lymphocytes to a region analogous to the uropod of a motile cell and
is absent or depleted from the leading edge of carcinoma cells. Using fluorescence photobleaching recovery,
DeBiasio et al. (40) have also found that myosin is present but immobile in the
leading edge of motile 3T3 cells.
However, in rabbit PMN responding to a gradient of complement fragments,
myosin II was localized to the lamellipod (20).
Our findings differ from those of Valerius et al. in that we find myosin
most often in the uropod of human PMN exposed to FMLP. The contradiction may be due to differences
in cell types, species, chemoattractants, or methods of applying the attractant
(yeast-generated gradient versus uniform field). However, our findings concur closely with
those obtained with lower eukaryotes, lymphocytes, and cultured fibroblasts and
therefore serve to clarify our understanding of myosin distribution in a range
of motile cell types.
The
quantitative changes in myosin levels observed here during leukocyte
activation, i.e. decreases in myosin staining, were somewhat unexpected in view
of some previous reports. White et al.
(41) have shown for rabbit PMN that the levels of myosin associated with the
Triton-insoluble cytoskeleton remained unchanged when they were assessed 15
seconds after FMLP stimulation and Feinstein et al. (42) have shown a
substantial thrombin-induced increase in the myosin content of the
Triton-insoluble platelet cytoskeleton.
However, the present study has utilized preparative and analytic
procedures that differ considerably from those employed in the aforementioned
studies. Most notably the time of
exposure to FMLP was greater here (1 and 4 min) as compared to the study of
White et al. (15 seconds) and the fixation/extraction procedures employed here
involved chemical fixatives and organic solvents (ethanol, acetone) rather than
detergents as were used by others. On
the other hand, Dharmawardhare et al. (16) have shown that the myosin II
content of the Dictyostelium
cytoskeleton peaks at 25-30 seconds after cAMP stimulation and reaches a
minimum, about 50% below baseline levels, 40 seconds after cAMP
stimulation. Levels of
cytoskeleton-associated myosin remained significantly below baseline throughout
the remainder of the 70 second time course studied there. The myosin content of human leukocytes seen
in the present study at both 1 and 4 minutes after FMLP stimulation was correspondingly
low.
In
Acanthamoeba, Baines and Korn (43)
have shown that plasma membrane associated myosin IC is labile to saponin
treatment, whereas contractile vacuole associated myosin IC is preserved. Thus, it seems that myosin I may be more or less
extractable depending on its subcellular location. Baines and Korn have suggested that this
differential vulnerability to extraction may be related to the extent to which
myosin interacts with membrane lipids.
In this study, we are localizing and quantitating myosin II, not myosin
IC, but the principle of differential extractability or solubility may also
apply here. Such differential
extractability may be related to putative interactions between myosin II and membrane
lipids or to the state of myosin phosphorylation. When myosin heavy chain is phosphorylated,
myosin thick filaments depolymerize, and myosin may be lost from the
cytoskeleton (16,44). Chemoattractant
stimulation of leukocytes from CT patients may cause myosin heavy chain to be
phosphorylated or otherwise redistributed and less tightly associated with the
cell constituents that are preserved under the fixation/extraction conditions
used here.
We
have also observed that MLCK expression remained relatively unchanged after
FMLP stimulation. This suggests that the
markedly decreased myosin staining observed after FMLP stimulation was not
simply the result of increased nonspecific protein extraction during specimen
preparation. The decrease in myosin
staining seen in CT PMN and monocytes upon FMLP exposure appears to be
selective for myosin.
In
unstimulated leukocytes from CA patients, we have observed dramatically reduced
myosin staining compared to that seen in unstimulated leukocytes from CT
patients. On the other hand, myosin in
CA leukocytes was virtually unaffected by exposure to FMLP such that these
cells did not exhibit the expected decrease in myosin staining upon FMLP
stimulation. Since myosin
phosphorylation apparently determines the extent of the association between
myosin and the cytoskeleton, these findings suggest that myosin
phosphorylation/ dephosphorylation may be abnormal in CA leukocytes. We hypothesize that leukocytes from CA
patients may be pre-activated by exposure to circulating factors present in the
blood and this may reduce cytoskeleton-associated myosin even before in vitro
stimulation with FMLP. Upon addition of
FMLP, myosin levels remain unchanged because cells had become refractory to
further stimulation.
As
further evidence of this, CA monocyte samples not pretreated with FMLP (i.e.,
unstimulated) exhibit increased numbers of polarized cells as compared to
unstimulated CT monocytes. Furthermore,
CA monocyte samples stimulated by FMLP exhibit decreased numbers of polarized
cells at all times less than 40 min and at all FMLP concentrations used
here. Others have studied cell
polarization in normal monocytes treated with CA patient effusions (45),
tumor-derived low molecular weight factors (46), and fragments of retroviral
p15E peptide analogues such as the peptide LDLLFL (47), but little data on
polarization in CA patient leukocytes has been reported. Results from previous studies are generally
in agreement with those described here.
Interestingly, Cianciolo et al. (45) showed that cancer patient pleural
or peritoneal effusions contained high molecular weight factors that were
stimulatory and low molecular weight factors that were inhibitory to normal
monocyte polarization. The data
presented here suggest that similar effects may be evident in leukocytes
isolated from CA patients. The high
levels of polarization evident in unstimulated CA monocytes indicate that these
cells are pre-activated as they are obtained from the patient. This further suggests that the reductions in
cell polarization seen in FMLP-stimulated CA monocytes may result from
desensitization of these cells toward FMLP.
The
cellular mechanism of the chemotactic defect in CA patient monocytes is
unknown. However, it is thought that a
serum-borne cell-directed inhibitor may be responsible for the defect
(48,49). This inhibitor causes decreased
monocyte polarization in response to chemoattractant (50,51) [unpublished
data], alterations in formylpeptide receptor expression (35), suppression of
the respiratory burst (52), and inhibition of protein kinase C-related cell
functions (53). Myosin heavy and light
chains are known substrates for protein kinase C (44), but the role of protein
kinase C in regulating myosin phosphorylation in non-muscle cells is not known
(Wilson and DeLan, 1992). The
alterations in myosin content of leukocytes in CA patients described here may
be directly involved in the inhibition of cell polarization and the chemotactic
deficiency seen in cells from these patients.
This may contribute to the inability of these immune effectors to deter
the growth and spread of neoplasia and to the susceptibility of these patients
to life-threatening bacterial infections (54).
ACKNOWLEDGMENTS
The
authors would like to thank Dr. Abraham Mark and Robert Novak for their
assistance with flow cytometry and John Krewer for technical assistance. This work was supported in part by the
American Cancer Society, Illinois Division.
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FIGURES
Figure 1
Leukocytes
were fixed, lysed in acetone, quenched in sodium borohydride, and nonspecific
binding blocked with BSA and NGS. Cells
were exposed to rabbit anti-chicken gizzard myosin serum, FITC-labeled goat
anti-rabbit IgG, and stained with Hoechst 33258 to aid in the identification of
monocytes. (a) unstimulated, adherent CT leukocytes showed
diffuse cytoplasmic fluorescence in both rounded and spontaneously polarized
(arrow) cells. Very little fluorescence
was associated with the plasma membrane.
(b, c) Unstimulated PMN and
monocytes from CA patient. Nuclear
fluorescence (c) clearly distinguishes monocytes from PMN (arrowhead). Monocytes usually displayed less intense,
more diffuse fluorescence than did PMN (d-g).
Formylpeptide-stimulated CT PMN and
monocytes exhibited a distinct leading lamellipod and trailing uropod (d,e;
arrowheads). Formylpeptide-stimulated
PMN and monocytes from CA patients (f,g).
Antimyosin staining was concentrated beneath the plasma membrane for
both PMN and monocytes and prominently in the trailing uropod region of
polarized PMN. (a) Magnification bar = 20 µm; (b-g) Magnification bar = 10 µm.
Figure 2
Leukocytes
were fixed, lysed in acetone, quenched in sodium borohydride, and nonspecific
binding blocked with BSA and NGS. Cells
were exposed to affinity-purified rabbit anti-turkey gizzard MLCK, FITC-labeled
goat anti-rabbit IgG, and stained with Hoechst 33258 to aid in the
identification of monocytes.
Unstimulated, adherent CT (a, b) and CA (c, d) PMN and monocytes
exhibited faint diffuse cytoplasmic fluorescence. Formylpeptide-stimulated
CT (e) and CA (f) leukocytes displayed MLCK staining that was in
most cases diffuse throughout the cytoplasm, but in some cells (arrows) was
concentrated in the uropod region. (a-d)
Magnification bar = 10 µm; (e, f) magnification bar = 20 µm.
Figure 3
Right
angle versus forward light scatter (a) of CT PMN, monocytes, and
lymphocytes. Monocytes were enriched,
fixed, extracted with acetone, treated with antimyosin followed by
FITC-conjugated goat antirabbit IgG and finally with phycoerythrin-conjugated
MY-9. The upper right gate surrounds the
region containing residual PMN contaminating the monocyte suspension. Due to their distinct granularity and size
this region is easily distinguished in most preparations. The lower left gate surrounds the region
typically occupied by monocytes.
Lymphocytes, red blood cells, and platelets are seen to the far
left. MY-9 staining (b) of the
myosin-positive cells found in this gate indicated that 85% of the cells
observed were monocytes.
Figure 4
Right
angle versus forward light scatter (A), a representative plot showing purified
CT PMN gate and cells. Cells were fixed,
extracted, and stained for myosin. Log
green fluorescence versus cell count histograms (B) showed a sharp, unimodal
distribution having a mean channel number (for this sample) of 78 ± 9 (SD).
Figure 5
CT
(filled circles) and CA (open circles) patient monocytes were exposed to buffer
alone or to different concentrations of FMLP for 20 min at 37ºC. At each concentration, monocyte polarization
in CA samples was significantly different (p<0.01) than that seen in CT
samples. Data are expressed as mean ± SD
(n = 6).
Figure 6
CT
(filled circles) and CA (open circles) patient monocytes were incubated with
10-9 M FMLP (solid lines) or in buffer alone (dotted lines) for times varying
from 0 to 60 min. In buffer alone, the
fraction of polarized CA monocytes was significantly greater (p<0.05) at all
times (except 0 time) than that seen for CT monocytes. FMLP-stimulated CA monocytes exhibited
significantly decreased (p<0.05) polarization for incubation times between 5
and 30 min but significantly increased (p<0.05) polarization after 60 min of
incubation as compared to that seen for CT monocytes. Data are expressed as mean ± SD (n = 6).
TABLE 1
Averaged Mean Channel Numbers for PMN and Monocytes
from CT and CA Subjects Anti-myosin Staining
PMN MONOCYTES
CT CA CT CA
_______________________________________________________________________
Unstimulated 80 ± 4 a 48 ± 9 77 ± 16 a 22 ± 6
FMLP-treated
1 min 63 ± 5 b 49 ± 10 47 ± 3 b 21 ± 5
4 min 61
± 5 53 ± 11 30 ± 5 b 20 ± 3
_______________________________________________________________________
Leukocytes
from 3 CT and 3 CA subjects were studied.
The mean channel numbers for each group were averaged (mean ± SD).
a
p<0.005 when compared to unstimulated CT group.
b p<0.05 when compared to FMLP stimulated CT group.
TABLE 2
Averaged Mean Channel Numbers for PMN and Monocytes
from CT and CA Subjects Anti-MLCK Staining
PMN MONOCYTES
CT CA CT CA
_______________________________________________________________________
Unstimulated 63 ± 10 a 62 ± 10 61 ± 3 a 55 ± 3
FMLP-treated
1
min 67 ± 14 a 63
± 13 70 ± 3 a 62 ± 5
4
min 64 ± 17 a 65
± 10 b 72 ± 2 ab 65 ± 3
_______________________________________________________________________
Leukocytes
from 3 CT and 3 CA subjects were studied.
The mean channel numbers for each group were averaged (mean ± SD).
a
Not
significantly different from paired CT group.
b p<0.05 when compared
to unstimulated group.
TABLE 3
Myosin II Localization in Unstimulated and Chemotactically
Activated Cells
Cell type Myosin II distribution Stimulant Reference
Dictyostelium discoideum excluded
from cortex None (55)
Dictyostelium discoideum rods
in endoplasm None (17)
Acanthamoeba castellanii cell
cortex None (43)
Acanthamoeba castellanii diffuse
in cytoplasm None (56)
V2
rabbit carcinoma absent
from lamellipod None (57)
Rabbit
PMN diffuse None (20)
Human
PMN diffuse None (58)
Dictyostelium discoideum rods
in ectoplasm cAMP (17)
Dictyostelium discoideum posterior
cortex cAMP (5)
Dictyostelium discoideum uropod
only cAMP
(59)
Dictyostelium discoideum uropod
only cAMP
(60)
3T3
fibroblasts absent from
protrusions Wound (40)
immobile in
lamellipod
Human
lymphocytes uropod Ig capping (61)
Human
lymphocytes uropod capping (62)
Mouse
T lymphocyte uropod capping (63)
Rabbit
PMN, HeLa cells lamellipod complement (20)
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