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Activation Mechanisms of Adherent Human Neutrophils
By Irene Ginis and Alfred I. Tauber
The mechanism by which unstimulated human neutrophils
initiate a respiratory burst on adherence t o a surface has
been examined. When neutrophils adhere t o a plastic
surface, they immediately generate a sustained burst of
superoxide (0,- 1. However, this respiratory burst is not
initiated by adherence alone, since neutrophils attached t o
fibronectin fail t o mount a response. Adhesion t o plastic is
calcium (Ca") independent, but 0,- production requires
Ca2+-containing buffer in the initiation phase, that is,
during adhesion and the early phase of 0,- production. The
Ca2+-dependentstep was shown t o involve protein kinase
C (PK-C) in that the 0,- production, but not adherence, was
blocked with 1-( 5-isoquinolinesulfonyll-2-methylpiperazine
(H-7). and PK-C was found t o translocate from the cytosol
t o the membrane on adhesion. Furthermore, it may be
inferred that this translocation results in the generation of
a Ca2+ independent form of PK-C. PK-M. since leupeptin.
which inhibits the generation of PK-M. also blocked 0,production. This finding was corroborated by showing that
after 5 minutes in a Ca2+-containingbuffer, enough time t o
initiate 0,- production and PK-C translocation, Ca2+is no
longer required for sustained 0,- release. These results, in
aggregate, demonstrate that neutrophils are activated by
adhesion t o plastic to generate 0,- , a PK-C-dependent
process that appears t o involve a Ca2+-independentform of
the kinase, PK-M. Why adherent neutrophils generate a
respiratory burst on plastic and not fibronectin surfaces
probably reflects activation of distinct receptors, whose
nature must still be defined. Another issue t o address is the
priming effect of adhesion, since cells adherent t o plasticor fibronectin-coated surfaces have an enhanced 0,response to formylmethionyl-leucine-phenylalanine(FMLP)
compared with neutrophils stimulated in suspension. This
may relate to increased Ca2+ mobilization, an important
mediator of priming for FMLP responses. Thus, adhesion as
a priming event does not necessarily initiate cell effector
function, and the further elucidation of the plastic and
fibronectin models suggests a means of characterizing the
crucial events that control neutrophil activation.
0 1990 by The American Society of Hematology.
N
Chemical Co, St Louis, MO. 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) was obtained from Seika GAKU
America, Inc, St Petersburg, FL. [as2P]adenosine triphosphate
(ATP) was purchased from New England Nuclear Research Products, Wilmington, DE. Pertussis toxin (FT) and cholera toxin (CT)
(azide free) were purchased from List Biological Labs, Campbell,
CA. Human plasma fibronectin and leupeptin were obtained from
Calbiochem, La Jolla, CA. Chlamydia trachomatis E/UW-S/OX
were grown and harvested as previously described.I2
Isolation of neutrophils. Neutrophils were isolated from normal
volunteers by erythrocyte sedimentation in dextran-citrate, followed
by hypotonic lysis and Ficoll-Paque (Pharmacia Find Chemicals,
Piscataway, NJ) densitity gradient centrifugation as previously
de.scribed.13The isolated neutrophils were then suspended in phosphate buffered saline (PBS'+) that contained 140 mmol/L NaCl, 3
mmol/L KC1, 1 mmol/L K,HPO,, 1 mmol/L CaCI,, and 1 mmol/L
MgCI, (pH 7.4), and was maintained at 4OC until used.
Respiratory burst. To assess spontaneous 0,-production of
neutrophils adherent to plastic and fibronectin-coated surfaces, the
amount of SOD-inhibitable reduction of cytochrome C by the
discontinuous assay was calculated using a modification of our
previously described method.I4 For these experiments, 5 x lo6
neutrophils in 1 mL PBS2+ (or another buffer as indicated) were
added to polystyrene 35 x 10 mm culture dishes (FALCON 3001)
together with cytochrome C (1.5 mg/mL) and incubated for various
EUTROPHILS, in vivo, perform their various functions (chemotaxis, phagocytosis, and cytotoxicity) usually adherent to either endothelial or interstitial tissue or cell
target surfaces.'-3Most in vitro models that attempt to assess
neutrophil-stimulated activities in these settings use cells
that have been activated with various agonists which both
augment adherence to the surface and initiate various
effector functions.4-"Such models might assume that adherence and activation are two aspects of the same process. The
results of these experiments are inconsistent: some investigators claim that neutrophil adherence enhances their responses to formylmethionyl-leucine-phenylalanine
(FMLP):4*" phorbol myristate acetate (PMA): and other
agents,' while others show that adherent neutrophils are less
responsive to these stimuli.'"
We have noted that of the diverse experimental protocols
applied to neutrophil adherence studies, the primary variable
is the sequence of experimental manipulation between the
adherence step and those assays of cellular response to the
agonist. In our studies, we have attempted to distinguish
between adherence and activation: adherence per se does not
necessarily initiate a respiratory burst, as shown by neutrophils binding to a fibronectin-coated surface, but, on adhesion to plastic, a rigorous burst was initiated. The design of
these experiments then allowed us to address the role of
calcium (Ca'+) and protein kinase C (PK-C) in the adhesion
process and the subsequent initiation of respiratory burst
activity. By showing different requirements of each event, we
have demonstrated the initial metabolic sequence of activation of neutrophils adherent to plastic. This stimulation
arises from activation of a pathway distinct from that used by
chemoattractants, but the nature of this receptor(s) still
requires definition.
MATERIALS AND METHODS
Materials. FMLP, PMA, zymosan, cytochrome C (type VI),
superoxide dismutase (SOD), histone, type 11-S, L-a-phosphatidyl-Lserine, and phorbol 12.13 dibutyrate were obtained from Sigma
Blood. V d 76, No 6 (September 15). 1990: pp 1233-1239
From the William E. Castle Hematology Research Laboratory
and Departments of Pathology and Medicine, Boston City Hospital,
and Boston University School of Medicine, Boston, MA.
Submitted August 1. 1989; accepted May 22,1990.
Supported by National Institutes of Health research grants no.
NHL-33565, POI-AID-24760. and AID-20064.
Address reprint requests to Arfred I. Tauber, MD. Boston
University School of Medicine, S Building-Room 301. 80 E
Concord St. Boston, MA 02118.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
8 1990 by The American Society of Hematology.
OOO6-4971/90/7606-0014$3.OO/0
1233
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1234
GlNlS AND TAUBER
times as indicated under the same conditions. The aspirated buffer
from each dish was then centrifuged (400g for 5 minutes) and
cytochrome C reduction was assessed by scanning between 570 and
530 nm in a Perkin Elmer Model 559 spectrophotometer.
The amount of 0,-production initiated in adherent cells by the
addition of unopsonized zymosan (2.0 mg/mL), chlamydia (50
organisms/cell), or FMLP (5 x IO-’ mol/L) followed the above
protocol except that the agonists were added 10 minutes after cells
were allowed to adhere and nonadherent cells were washed out.
The rate of 0,-production in adherent neutrophils stimulated
with FMLP was measured by the SOD-inhibitable reduction of
cytochrome C by continuously monitoring optical density at 550 nm
by minor modification of previously described methods.” A total of
5 x IO6 in 1 mL PBS2+ neutrophils was allowed to adhere for 5
minutes on the opaque wall of a 4-mL, two-sided polystyrene cuvette
(Fisher Scientific Co, Pittsburgh, PA), washed, and incubated with
1.8 mL PBS2+ and 1.5 mg/mL cytochrome C. Absorbance was
monitored under continuous stirring (Spectrocell, Inc, Oreland, PA)
and thermostatted at 37OC. After a preincubation period of 3
minutes, during which no spontaneous metabolic activity was observed, neutrophils were stimulated with FMLP at a final concentration of 5 x 10- mol/L. Note that cytochalasin B was not used in
production during the first 2 minutes
these studies. Rates of 0,following stimulation were calculated from the original tracings.
Neufrophil adherence. The number of adherent cells was measured individually for each experiment. Cuvettes or culture dishes
with adherent cells were washed three times with PBS2+to remove
nonadherent cells and incubated with Pierce BCA protein assay
reagent (Pierce, Rockford, IL ) for 30 minutes at 37OC. Each sample
was then read at 562 nm (Beckman DU-7 spectrophotometer,
Beckman Instruments, Fullerton, CA) and compared with a standard protein curve derived from known cell numbers. As shown in
Fig I A , in 10 minutes, 2.5 to 3.2 x IO6 cells adhered to the surface
(50% to 65% of the total number of cells adhered). In fibronectin
experiments, Petri dishes or cuvettes were coated with 3 pg/cm2 of
human plasma fibronectin according to Fehr et a1,6 allowed to
incubate for 45 to 60 minutes at room temperature, washed twice
with PBS2+,and immediately used, as described above. The number
of adherent cells was 2.7 to 3.4 x IO6.
In these experiments, a model was designed in which cells were
first allowed to adhere and were then activated with FMLP to assess
each process independently. In most previous studies, an agonist was
added to neutrophil suspensions, then cells were allowed to adhere,
which not only initiated neutrophil end function responses, but also
greatly enhanced adherence. Under such conditions, neutrophils
adhered in high p r o p ~ r t i o n . ~ .In
’ ~our
. ’ ~ preliminary experiments, we
showed that adherence of cells without stimulation is dependent on
release stimulated by FMLP was
the type of surface used, while 0,equal on each surface when calculations were corrected for the
number of adherent cells (data not shown), which alerted us to
generation) on the basis of
calculate neutrophil response (ie, 0,adherent cell number.
Measurement of intracellular calcium [ca’+],. [Ca2+,]levels
were determined using the fluorescent indicator (Fura-2).I4 Isolated
neutrophils were incubated with the acetoxy methyl ester of Fura-2
(2.5 pmol/L) in PBS2+ for 30 minutes at 37OC, washed, and
resuspended in fresh buffer; 2 mL ( 106/mL) of Fura-2 loaded cells
were constantly stirred in the cuvette for [Ca’+], measurements of
suspended cells, or cells were first allowed to adhere to the cuvette
wall as described for continuous 0,production and then incubated
with 2 mL of fresh buffer. In each case, cells were stimulated with 5
x I O - ’ mol/L FMLP. CaZt determinations were measured using
the ratio of the peak fluorescence at exitation wavelengths of 340 to
380 nm and emission wavelength of 510 nm (Perkin-Elmer [Nor-
’
4-1
A
T
l !
I
.
10
I
20
.
I
.
30
I
.
40
I
d
50
time [min]
91
-5fn
0
7
a
6
2
5
0
0
E
c
“
0
B
8
4
3
2
1
0
0
10
20
30
time [min]
40
50
Fig 1. Time course of neutrophil adherence (A) and spontaneous 0,- production in adherent cells (BI. Neutrophil adhered to
plastic dishes as detailed in Materials and Methods, and were not
further stimulated. Each point represents the mean SD of eight
experiments.
*
walk, CT] model LS5 spectrofluorometer). The Ca2+concentration
was calculated using a Kd of 224 nm.I4
PK-C translocation. A 4-mL neutrophil suspension ( 5 x IO6
mL) was preincubated for 10 minutes with 1 mmol/L leupeptin and
then added to two Petri dishes (100 x 15 mmol/L) and allowed to
incubate at 37OC for 5 minutes. Nonadherent cells were washed out,
and 1 mL of fresh PBS2+containing 1 mmol/L leupeptin was added.
Neutrophils were then removed with a cell scraper (Gibeoware,
GIBCO, Grand Island, NY ), paired samples were pooled, and the
distribution of PK-C in cytosol and membrane fractions was then
assessed according to the method of Melloni et a1.I6 In summary,
cells were centrifuged for 7 minutes at 4OC, 400g. and the pellet was
resuspended by vortexing in 1 mL of lysis buffer (10 pmol/L Hepes,
0.25 mol/L sucrose, 5 mmol/L EDTA, 1 mmol/L dithiothreitol)
containing 1 mmol/L leupeptin. The suspension was then gently
sonicated three times for 10 seconds at 4°C using a tip sonicator
(Ultrasonics, Maidstone, England ) at settings 3 to 5 and transferred
to a polycarbonate tube and centrifuged at 150,OOOg for 30 minutes,
thus obtaining cytosolic and membrane fractions. The membrane
fraction was solubilized by resuspending the pellet in 1 mL of lysis
buffer containing 1 mmol/L leupeptin and 0.1% Triton X-100,
thoroughly homogenized at 4OC, and then incubated in an ice bath
for 1 hour. The harvested cytosolic and solubilized fractions,
respectively, were loaded onto a 1.5-mL packed E-52 resin (What-
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1235
ACTIVATION OF ADHERENT NEUTROPHILS
man) previously equilibrated with 10 mmol/L Hepes, 5 mmol/L
EDTA, 1 mmol/L dithiothreitol, pH 7.5. The kinase activity was
eluted from resin with 1.5 mL of 100 mmol/L NaCl in Tris buffer
(pH 7.5).
For measurements of cytosolic and membraneous PK-C activity,
20 pL of each sample were incubated for 10 minutes at 3OoCwith 30
pL of the PK-C incubation mixture, containing 11.1 mmol/L Tris
buffer (pH 7 . 9 , 0.17 mmol/L ATP, 1.3 mg histone, 67 pg
phosphatidyl-L-serine, 2.0 pg phorbol dibutyrate, 16.7 mmol/L
MgCl,, 1.6 mmol/L Ca2+CI,,and 5 pmol/L [aJ2P]ATP.For control
experiments, 5 mmol/L EDTA substituted for Ca2+and phospholip
ids. After incubation at 3OOC for 10 minutes, 30-pL samples were
pipetted onto filter paper (1.5 cm2),which were then immediately
transferred to a solution containing 10% TCA and 10 mmol/L
sodium pyrophosphate. The filters were then washed four times in
this solution, dried, and counted in Scintiverse Universal LSC
cocktail (Fisher Scientific, Fairlawn, NY); the radioactivity was
measured with a TM Analytic Scintillation (Brandon, FL) counter.
Background radioactivity of the sample without 32Pwas subtracted
from the radioactivity of each probe. The difference between the
radioactivity of the probe incubated in the presence of phospholipids
and Ca2+and that of the control probe incubated without phospholipids and Ca2+was considered a measure of PK-C activity in each
sample. As all the samples were obtained from the same number of
cells (2 to 3 x lo6),it was assumed that the ratio of radioactivities
from membrane and cytosol aliquots of each sample reflected the
distribution of PK-C activity in the cells studied.
RESULTS
Adherent neutrophil 0,-production. When neutrophils
become adherent to plastic surfaces, "spontaneous" 0,is
generated. As shown in Fig lA, adhesion becomes stable by 5
to 10 minutes, while 0,production does not plateau until 40
minutes of incubation (Fig IB). Note that the magnitude of
0,-production is similar to that obtained with FMLPstimulated neutrophils in suspension (see Fig 4a). Cells
adherent to fibronectin, however, generated no spontaneous
0,(Table 1).
Adhesion on a plastic surface is Ca2+ independent, for
there was no difference observed in adhesion between cells
incubated in PBS2+,Ca2+-freePBS with EGTA (3 mmol/L)
(Fig 2), or with Ca2+-depletedcells (incubated for 2 hours in
Ca2+-free buffer)I4: 3.2 rt: 1.3 x lo6 cells for each case
(mean % SD for four experiments). However, 0,stimulated
by adherence is diminished in a Ca2+-free buffer (Fig 3).
During the first 10 minutes of incubation, 0,-production
was observed in Ca2+-freemedium, but the burst was not
sustained as observed in PBS2+.This finding suggested a role
for extracellular Ca2+for continued respiratory burst activity, and we thus directed our attention to PK-C, whose
sustained activation is dependent on an extracellular source
4-
c
C
2
T
a
c
'0-
m a
r
T
3-
O
O';
L-
d
E
a
2-
2
i !
1
.
10
I
'
20
I
'
I
'
40
30
I
i
50
time [min]
Fig 2. Effect of Ca" and H-7 on neutrophil adherence. Neutrophil adherence on plastic dishes in PBS++ buffer (open squares), in
Ca2+-freebuffer (closed squares), and in the presence of H-7 (100
pmollL) (diamonds). Each point represents the mean f SD of
three experiments.
of Ca2+.17Specifically, having shown that adherence was
Ca2+independent, but subsequent 0,generation was Ca2+
dependent, we next determined whether PK-C might regulate either function.
Predictably, PK-C would have no role in the adhesion
reaction, but might participate in the activation of the
respiratory burst. Our first approach was to use the kinase
inhibitor H-7.I7 Neutrophils were preincubated with various
concentrations of H-7 for 5 minutes and adhesion and
spontaneous 0,-production was then assessed. Neutrophil
adhesion was unaffected by the inhibitor (Fig 2), although
H-7 effectively inhibited 0,-production, with significant
inhibition obtained with 30 pmol/L (P i.04). When anproduction
other PK-C inhibitor, staurosporin, was used, 0,was inhibited more than 90% a t concentrations of approximately 40 to 80 nm, but adherence was unaltered a t this
concentration (data not shown). Because the staurosporine
inhibitory effect on 0,generation required higher concentraY l
Table 1. 0,- Production of Stimulated Human Neutrophils on
Plastic or Fibronectin-Coated Surfaces
SurfaceJStimulus
None
Zymosan
Plastic
5.08
+ 2.16 (11)'
7.56
Fibronectin
1.59
+ 1.05 (6)d
8.36
+ 1.52(6)'
+ .74 (3)"
Chlamydia
2.81
0.86
+ 1.24(5)'
+ 1.03 (3)'
Data are expressed as nmol 0,-/106 calls/l5 minutes (mean f SO). Numbers in
parenthesesindicate number of experiments.
;
vf,
Probabilityvalues: a vb. ~ 0 5a :vc, c.05: a vd. 1.01; d ve, ~ 0 1andd
not significant.
0
10
20
30
40
50
time [min]
Fig 3. Effect of Ca2+ on 0,- production by plastic-adherent
neutrophils measured as detailed in Materials and Methods.
Neutrophils were suspended in either Ca*+-free buffer (closed
squares) or in PBS2+ (open squares). Each point represents the
mean f SD of three experiments (P .06).
-=
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1236
GlNlS AND TAUBER
tions than expected as a result of its exclusive effects on
PK-C," we could only conclude that these data suggested
that adhesion was PK-C independent, but that resultant 0,generation was sensitive to kinase inhibition.
PK-C activation is a multistep process and only its first
stage, translocation of the phospholipid-dependent form of
PK-C from the cytoplasm to the cell membrane, requires
Ca2+.17 Following thiol proteinase (calpain) cleavage of
Table 3. PK-C Activity Distribution in Suspended and Adherent
Neutrophils
~
surpsndsd Cdls
Resting
Adhasm cdls
PMA-Stimulated
Fibrot"
Plestk
88.1 f 3.9 17). 21.0 f 2.3 ( 3 ~
28.5
W-d
Paticdate membrane 13.9 f 3.9 (7)b 79.0 f 2.3 (3bd 71.5
Data 6 n arprmwd as ~ m t a g of
e mtal FKC activity.
lpmd phmphorvlamd hinms/l mg protein): &ne cella. 14.03
6.1 i4ie 96.6
6.1 141'
* 2.3 1310
3.4 f 2.3 13Ih
f 10.79; PMA-stimubrsd
PK-C, a membrane-free form is generated that is both CaZ+ calls.5.51 .953:plastieKhasntcalls, 19.99 f 10.34;Imlfil"ctinadwnntcd!s. 17.56
f 7.201msan * Sol. Nu~nb~shpaamheaeaindieaenumbaof~imsms.
and phospholipid independent.I6With this schema, we might
Prhabilitv valug: a v b, -=.OW a v c, <.028; a w e , c.ooo9,a v g, not micart:b v f.
<.002:b vg. .02: e v g , <.W
and g vh, c.002.
expect that if 0,-production in adherent neutrophils is
mediated through PK-C activation, then by 5 minutes, when
the adherence process is completed and neutrophil 0,- suspension or allowed to adhere to plastic for 5 minutes,
exhibited significant differences in membrane-associated
production had been initiated, further respiratory burst
PK-C. The possibility that PK-C translocation might be due
activity might have different metabolic requirements. One
to cell disruption is excluded by the fact that in fibronectinpossibility is that continued PK-C activation might be
adherent cells, no PK-C translocation was observed (Table
necessary for sustained 0,-production; either continued
3). Thus, these results are consistent with the inhibitor and
activation of native PK-C or, alternatively, elaboration of the
Ca++depletion studies.
Ca2+-independentPK-M form might mediate this 0,-genComparison of adherent and suspended neutrophil activaerating activity. (PK-M activity would be independent of
on adhertion. Having shown that neutrophils generate 0,Ca2+and sensitive to the calpain inhibitor, leupeptin.")
ence, we then examined the effect of further stimulation with
To examine this hypothesis, we measured 0,production
in neutrophils that were first allowed to adhere in PBS2+, FMLP. To compare kinetics of FMLP stimulation in adherthen nonadherent cells were washed out and fresh Ca2+-free ent and suspended neutrophils, 0,-was measured in a
continuous assay, where activation conditions were the same
buffer was added to the dishes for a 10-minute incubation.
(Fig 4). The duration of the FMLP-stimulated response was
The results, shown in Table 2, demonstrate that preincubashort in both adherent and suspended cells (2 to 3 minutes),
tion of cells in PBS2+ buffer for 5 to 10 minutes (a), is
production, even in a Ca2+-free but the rate of 0,-production was significantly higher in
sufficient for subsequent 0,cells adherent to either plastic or fibronectin (Fig 4a, c, and
buffer, comparable to the case in which Ca2+ is present in
both steps (b). If the first preincubation was performed in
e).
Requirements for Ca2+by FMLP-stimulated cells and the
Ca2+-freebuffer (c), in a Ca'+-containing buffer with H-7
intracellular Ca2+mobilization response were next assessed.
(d), or in Ca2+-containingbuffer with the calpain inhibitorAdherent neutrophils showed less dependence on extracelluleupeptin (e), the ability of adherent cells to produce 0,,
lar Ca2+ than suspended cells. Neutrophils stimulated in a
even in the presence of Ca2+,was significantly diminished or
Ca2+-freebuffer (PBS without Ca2+plus 3 mmol/L EGTA)
completely abolished. Leupeptin had no significant effect on
exhibited a significant 0,-response, while suspended cells
adhesion (data not shown).
generated no significant quantities of 0,under these condiTo exclude the possibility of a nonspecific effect of H-7 or
tions (Fig 4b, d, and f).
leupeptin, and to directly confirm PK-C activation on adherence to plastic, direct measurements of PK-C activity in
membrane and cytosol of adherent neutrophils were undertaken. As shown in Table 3, neutrophils, either kept in
Table 2. Effects of Ca2+Chelation, H-7. or Leupeptin on 0,Production by Adherent Neutrophils
Preincubatii
Buffer.
Incubation
PBS2+(a)
PBSZ+(b)
PBS/EGTA (c)
PBS2+H-7 (100 pmol/L) (d)
PBS2+leupeptin (1 mmol/L (e)
PBS2+
PBS/EGTA
PBS2+
PBS2+
PBSz+
Buffert
T
10 -
02-ProductionS
(nmd/lOe cells/l5 min)
3.01
3.62
0.97
0.84
.99
i 1.04 (8)
i 2.93
(5)
0.57 (5)
i 0.28 (5)
i 0.55 (6)
f
Probability values: a v b, c.79:a v c, ~ 0 2a;v d, c.02; and a v e,
<.03.
*Cells were preincubated in Cazf-containingbuffer (PBS2+)with or
without inhibitorsor Ca2+-freabuffer (PBS/EGTA) for 5 minutes.
tNonadherent cells were washed out and fresh buffer added with
cytochrome c to measure 0,-production as detailed in Materials and
Methods.
#02-production data is expressed as mean + SD. Numbers in
parenthesesindicate number of experiments.
a
b
C
d
e
f
Fig 4. Rates of 0'- productionof suspended and adherent cells
stimulated with FMLP (mean i SD). (a) Suspended cells in PES'+;
(b) suspended cells in Ca2+-freebuffer; (c) plastic adherent cells in
PES"; (d) plastic adherent cells in Ca'+-free buffer: (e) fibronectinadherent calls in P&+; (f) fibronectin-adherent cells in Ca'+-free
buffer. Probability values: a versus b. <.02; a versus e. <.02: a
versus d, ~ 0 0 3b:versus d, c.07;and b versus f, ~ 0 0 9 .
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1237
ACTIVATION OF ADHERENT NEUTROPHILS
When FMLP-induced changes in intracellular Ca2+concentration were monitored in suspended and plastic-adherent
cells loaded with Fura-2 (Fig 5), the same pattern of response
was seen: the Ca2+increase in adherent cells was higher than
in suspended cells and did not depend significantly on the
presence of extracellular Ca2+.Further, while in suspended
cells, Caz+ rapidly returned to baseline levels; in adherent
cells, Ca2+remained elevated for a minimum of 10 minutes.
We note that the magnitude of the 0,-response to FMLP
of cells adherent to plastic for 30 minutes is less than that
elicited by FMLP when cells have been adherent on a surface
for 5 minutes (2.53 f 0.964 v 6.45 f 1.24). Since adhesion
actually leads to cell activation, this diminished response is
functionally deactivation,’ which, in this system, still requires definition. These data may well explain the results of
generation of stimulated neutroothers who found that 0,phils was diminished on
As noted above, when cells adhere to a fibronectin-coated
is noted, but when
surface, no significant production of 0,these fibronectin-adherent cells are stimulated with unopsonized zymosan after 5 minutes of adhesion, significant 0,generation ensues (Table 1). It is noteworthy that these
unopsonized particles elicit no 0,from human neutrophils
in suspension (data not shown and refs 22 and 23) and, in this
regard, adherence to either uncoated plastic or fibronectin is
functionally equivalent to priming.,’ Note, however, that
unopsonized chlamydia evoked no respiratory burst in adherent cells on fibronectin and significantly inhibited 0,production of plastic bound cells, as previously shown for
cells in suspension, confirming the organism’s ability to
inhibit the NADPH-oxidase.”
DISCUSSION
Neutrophils circulate in the blood for a short period and
then function primarily adherent to tissue surfaces,’” where
their toxic products may be directed at sites of attachment.%”
Most studies that have examined neutrophil activation char250
200
d
T
P
1
0 :
0
I
I
I
I
I
2
4
6
8
10
time
[min]
Fig 5. Intracellular Ca2+changes in plastic-adherent and suspended neutrophils stimulated with FMLP were measured as
detailed in Materials and Methods. Suspended cells, P6S2+:open
squares; suspended cells, Ca2+-free buffer: closed diamonds:
plastic-adherent cells, P6S”: closed squares; plastic-adherent
cells, Ca2+-freebuffer: open diamonds.
acteristics have been performed with cells in suspension;
however, we are concerned that cell responses may be altered
by the adhesion process itself. In fact, we have found that
when neutrophils adhere to plastic, they immediately initiate
02-production (Fig 1B). Responses to soluble or particulate
stimuli reflect differences of the activation process in adherent and suspended cells. Unopsonized zymosan, which elicits
no 0,-generation by suspended neutrophils, induces a
response in cells adherent to fibronectin (Table
rigorous 0,1). In this setting, adhesion to fibronectin may be viewed as
priming.” The priming effect of adherence was confirmed in
the recent studies of Neumann and Kownatzki on neutrophils adherent to the nylon fibers, which also showed higher
response to different activators of respiratory burst.” In
addition, fibronectin receptor activation on adherence might
play a specific role in this priming phenomenon.28Again,
with zymosan or FMLP stimulation of cells bound to plastic,
a less dramatic, but significant, stimulation is observed in the
early adhesive phase (Table 1 and Fig 4). (In the case of
chlamydia, inhibition is in fact noted, because the organism
directly inhibits the respiratory burst enzyme, NADPHoxidase.I2) However, when cells are allowed to adhere for
longer periods (ie, 30 minutes), less 0,-is generated by
FMLP stimulation, a case of functional deactivation. The
nature of the “priming” and “deactivation” phenomena was
not primarily the subject of these studies, but elucidation of
this observation is important in establishing the parameters
by which adherent neutrophils respond to physiologic stimuli, and is the focus of ongoing studies in our laboratory.
The mechanism of 0,-activation by adherence has been
defined in these studies as involving a Ca2+-independent
adherance step that is independent of kinase activation (ie,
insensitive to H-7or staurosporine), and then followed by a
Ca2+-dependent event that initiates 0,-production. This
Ca’+-dependent step appears to be the activation of PK-C, as
it is H-7 sensitive and correlates with the translocation of
PK-C from the cytosol to the membrane, as seen in neutrophil activation with PMA,29-32
FMLP,29*30*32
platelet-activating f a ~ t o r ,latex
~ ~ .beads,
~ ~ opsonized z y m o ~ a n ,bacteria,36
~~.~~
leukotriene B,,30and 1-oleoyl-2-acetylglycerol.3’In the subsequent period (ie, that following the 5-minute initiation phase
of these studies), a sustained generation of 0,-is observed
that is again Ca2+ independent, but continues to be H-7
generation is blocked by leupeptin, an inhibitor
sensitive. 0,of calpain (the proteinase that elaborates PK-M), the Caz+independent form of PK-C.37 We thus hypothesized that a
similar scheme of PK-C metabolism to that observed with
PMA-stimulated neutrophils in s u ~ p e n s i o n ~occurs
~ . ~ ~ -in~ ~
adherent neutrophils. Specifically,we hypothesize that PK-M
is responsible for the sustained 0,-production initiated by
the adhesion event.
Finally, what is the mechanism by which adhesion initiates
PK-C translocation leading to cell activation, ie, 0,production? By our model, adhesion alone initiates PK-C translocation, which, in other systems, is related to an increase in
cytosolic Ca2+ and, presumably, the elaboration of
diacylglycer01.l~However, the mechanism of this activation
has not yet been defined for adherent neutrophils. In our
model, when neutrophils were preincubated with PT (500
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1238
GlNlS AND TAUBER
p g / m L ) for 2 hours a n d CT (20 p g / m L ) for 1 hour, neither
toxin inhibited spontaneous 0,generation in adherent cells
(data not shown). Similar studies with PT, using suspended
neutrophils, inhibited full FMLP effector functions, but had
n o effect o n FMLP p r i m i n g , w h i c h involves Ca2+
m ~ b i l i z a t i o n . ~A' similar pathway for PT-insensitive CaZ+
mobilization has been demonstrated with influenza virus
~ t i m u l a t i o nThus,
. ~ ~ precedent for cell activation by a CaZ+dependent, PT-insensitive pathway has been well described,
and we are inclined to conclude that neutrophils activated by
adherence to plastic mobilize PK-C, presumably by cytosolic
Ca2+elevation, through a mechanism similar to that charted
by other studies from our laboratory. Preliminary studies
indicate t h a t this pathway involves t h e activation of phosphoThe question as to whether adhesion molecules,
lipase D.40*4'
or other receptors, directly trigger events leading to PK-C
activation still requires i n ~ e s t i g a t i o n . ~Future
~ . ~ ~ studies directed at this problem will focus on the stimulation cascade
of adherent cell activation and its physiologic control. In this
context, elucidation of priming and deactivation promise to
add another dimension to explaining physiologic parameters
of neutrophil function.
ACKNOWLEDGMENT
The authors thank Ann Marie Happnie for her expert secretarial
assistance.
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1990 76: 1233-1239
Activation mechanisms of adherent human neutrophils
I Ginis and AI Tauber
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