Assembly of contact-phase factors on the surface of the human

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1994 84: 474-482
Assembly of contact-phase factors on the surface of the human
neutrophil membrane
LM Henderson, CD Figueroa, W Muller-Esterl and KD Bhoola
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Assembly of Contact-Phase Factors on the Surface
of the Human Neutrophil Membrane
By Lydia M. Henderson, Carlos D. Figueroa, Werner Muller-Esterl, and Kanti D. Bhoola
H-kininogen (HK), a major factor involved in contact-phase
activation, was recently immunolocalized on the external
surface of human neutrophils. Experiments were, therefore,
designed to consider the question of whether the complete
assembly of contact factors occurs on the outer surface of
the neutrophil membrane. By immunolocalization techniques, and using specific antibodies directed against the
various contact factors, we now demonstrate that plasma
prekallikrein (PK), factor XI (FXI), and factor XI1 (FXII) are
present on the exterior face ofthe human neutrophil. Failure
to localize HK, PK, or FXI by monoclonal antibodies directed
to their reciprocal binding sites, and displacement of PK/FXI
by peptide HK31, which mimics the relevant binding site(s)
of HK, suggested that prekallikrein and FXI are anchored to
the neutrophil membrane through attachment to thekininogen molecule. Probing of the kinin moiety by a specific antibody showed that kininogen molecules bound to theneutrophil cell membrane contain the kinin sequence, which can
be released by plasma kallikrein or by tissue kallikrein. Our
results led us to the novel conclusion that neutrophils provide a circulating platform for the components of the contact-phase system.
0 1994 by The American Society of Hematology.
T
rected to the mutual binding sites of both PK and FXI on
the HK molecule, and a MoAb to the kinin
We
show that the complete array of contact factors is sited on
the surface of the neutrophil plasma membrane. Our results
provide us with an insight into the possible involvement of
the neutrophil-borne contact-phase system in the release of
the vasoactive peptides, kinins, at the sites of inflammation.
HE MAJOR CONSTITUENTS of the contact-phase activation system’ are plasma prekallikrein (PK), factor
XI (FXI), factor XI1 (FXII), and H-kininogen (HK; highmolecular weight kininogen).’ They are synthesized and secreted by hepatocytes. In plasma, HK circulates as a complex
with PK and FXI, re~pectively.~.~
This particular property is
mediated by the light-chain segment of HK.s L-kininogen
(LK), the low-molecular weight member of the kininogen
family, is devoid of the specific light chain sequences that
reside in HK.6 Recently, we immunolocalized HK and PK in
human hepatocyte^,^ and HK and LK on human
neutrophils.*
Furthermore, HK binding to platelets,’”’ endothelial
cell^,^^"^ and neutrophils’6 hasbeen demonstrated. These
findings prompted the question whether all of the circulating
contact-phase proteins were bound to the neutrophil surface,
and, if so, what was the spatial relationship between these
proteins (PK, FXI, FXII) to the HK molecule, known to be
attached to the neutrophil cell membrane.
To test the hypothesis that a circulating cell, namely, the
neutrophil, might provide on its surface a platform for the
assembly of the contact-phase proteins, we undertook a number of experiments using several immunolocalization techniques. Specific antibodies against the contact phase proteins
were applied including monoclonal antibodies (MoAbs) diFrom the Departments of Biochemistry and Pharmacology, University of Bristol, UK; Institute of Histology and Pathology,Austral
University, Vuldivia, Chile; Department
of Pathobiochemistry, Institute of Physiological Chemistry, Universityof Mainz, Germany; and
the Department of Experimental and Clinical Pharmacology, Medical School, University of Natal, Durban, South Africa.
Submitted May 12, 1993; accepted March 15, 1994.
Supported by grants fromthe Medical Research Council, the Arthritis and Rheumatism Council (UK), Fondo National de Desarrollo
Cietijico y Technologico Direccion de Investigacion y Desarrollo,
Universidud Austral de Chile (Chile), the Deutsche Forschungsgemeinschaft, the Volkswagen Foundation, and the Fonds der Chemischen Industrie (Germany).
Address reprint requests to Kanti D. Bhoola, MD, PhD, Department of Experimental and Clinical Pharmacology, Medical School,
University of Natal, PO Box 17039, Congella 4013, South Africa.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8402-0003$3.00/0
474
MATERIALS AND METHODS
Neutrophil isolation. Human neutrophils were isolated from anticoagulatedwhole blood provided by volunteersatTheMedical
School, University of Bristol,England, UK andatTheRegional
Hospital, Valdivia, Chile. Eighteen milliliters of blood, collected in
2 mLof105-mmol/Lsodiumcitrate,wasmixedwith
20 mLof
dextran (6% wt/vol; average molecular weight, 266 kD and 60 rnL
of phosphate-buffered saline (PBS; I O mmol/L sodium phosphate,
2.7 mmoW KCI, 137 mmol/L NaC1, pH 7.4) containing 0.4% wt/
v01 trisodium citrate. The mixture was left to stand for 45 minutes
at room temperature to allowred blood cells to sediment. The supernatant was spun through Lymphoprep (Nycomed, Birmingham, UK)
at 800s for 20 minutes. Remaining red blood cells were removed
by
two rounds of hypotonic lysis (erythrocyte lysis buffer: 155 mmol/
L NH,CI, 2.7 mmol/L KHCO,, 3.7 mmol/L EDTA, pH 7.4), and
rebuffered to restore the osmolarity. The cells were centrifuged
at
50013 for I O minutes and the pellet was washed three times in PBStrisodium citrate buffer. This procedure yielded a cell preparation
that contained approximately 98% neutrophils; the remaining 2% of
the cellsisolated by thistechniquecomprisedmainlyeosinophils
and some monocytes, but no platelets ( < . l %). Failure to observe
considerable superoxide production by the isolated neutrophils, and
lack of a substantial degranulation of the cells indicated that our
isolation procedures did not significantly activate the neutrophils.
Theisolatedneutrophilswere
floated ontocircularcoverslips
in
tandem, with one set left unfixed and the other fixed with 4% (wt/
vol) paraformaldehyde in PBS.
Binding of kininogens I O isolated neutrophils. The amount and
rate of loss of cell surface proteins by washing with buffer during
cell isolation procedures depends on the nature and mechanism of
attachment of the proteins to the cell surface, and is indicatedby the
affinity of the protein forits binding site. In the present experiments,
neutrophils were washed at4°C to minimize such a release.
To judge
the losses of neutrophil-bound proteins [‘251]-labeled HK” or LK
(100%)were incubated for 45 minutes at 4°C with neutrophils. The
cellswereextensivelywashed
with coldHanks’buffer,andthe
radioactivitybound to theneutrophilsdetermined by ay-counter
(Packard, Dorset, UK). A fraction of 13% (1 8%) of ‘251-HK(LK)
remained bound to the cells even after several washes.
Cytoplast preparation. Cytoplasts,
which are right-side out, single-compartment, enucleated cells” 22 were prepared by incubating
Blood, VOI 84, N O 2 (July 15). 1994: pp 474-482
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CONTACTPHASEFACTORS
ON HUMAN NEUTROPHILS
9
Fig 1. Localization of the components of the contact-phase systemon neutrophils. Nonfixed neutrophils were incubatedwith la) anti-HK
[I-IMIdilution
,
1:l.OOO). (b) anti-PK(AS176,1:1,OOO), (c) anti-FXI(I:l,OOO), or (d) anti-FXII (1:l.OOO). The immunostainedcells were imaged under
the confocal optical scanning microscope. The intensity of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow,
moderate; red, maximal. The barin each row isequivalent to 4 pm.
neutrophilsat37°C
for 10minutes in 12.5% ( d v o l ) Ficoll-70
(Sigma Chemical CO, Poole, Dorset,UK) containing 5 pg/mL cytochalasinB(Sigma), 5.5 mmovL glucose,beforeloadingontoa
discontinuous Ficoll gradient (cushions of 12.5%,16%,and25%
d v o l ) . Using a Ficoll-70 stepgradient, neutrophils were resolved
into cytoplasts and karyoplasts. The cytoplasts were harvested
from
the12.5% and 16%Ficoll interfacefollowingcentrifugation at
81,OOOg for 60 minutes at 37OC in a prewarmed Sorvall centrifuge
(Sorvall, Newtown, CT). Isolated cytoplasts were floated onto circular coverslips in tandem, with one set left unfixed, and the other
fixed with 4% paraformaldehyde in PBS.
Antibodies. Neutrophils were separately incubated with the followingantibodies:affinity-purifiedpolyclonalantibodies
to HK,
mouse MoAbs to the heavy chain (HKH4),
from sheep (I-108);23
light chain (HKLIZ), and PK bindingsite (HKL16) of HK19 rabbit
antiserum to PR (AS176); mouse MoAb to the HK binding site of
PK (PK6);I8 mouse MoAb to bradykinin (SBK1); mouse MoAb to
the HKbinding site of FXI (FEHl);" rabbitantiserum to FXk
rabbit antiserumto Fxu,rabbit antiserum to atrial natriuretic peptide
Fig 2. Ultrastructural double immunolabeling of HK and PK on
(ANF, Peninsula Laboratories, Belmont, CA); rabbit antiserum to
neutrophils. Affinity-purified antibodiesto HK (1-106,from sheep) preoxytocin (UCB Bioproducts, Brussels, Belgium); rabbit antiserum
viously coupled to 15-nm gold particles(smallarrows), and antiserum
to residues 389 to409 of the LK light chain (R7);= rabbit antiserum to PK IAS176. from rabbit) followed by goat anti-rabbit IgG coupled
to tissue kallikrein (KDBl)." Note that the antibodies were elicited to 30-nm gold particles (large arrows) were applied (g, granule; N.
nucleus. (Original magnification x 85,OOO.)
to human antigen sequences throughout.
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47 6
Immunostaining for confocalmicroscopy. The isolated neutrophils (fixed or nonfixed) and cytoplasts (fixed) were washed three
times with PBS (pH 7.4) containing 1% (wt/vol) bovine serum albumin (Sigma), 1% (wt/vol) human IgG (Blood Transfusion Service,
Southmead Hospital, Bristol, UK), and 0.2% (wt/vol) sodium azide.
Before immunostaining, isolated neutrophils or cytoplasts were incubated with 1% (wt/vol) of human IgG in PBS to exclude the nonspecific binding of antibodies to Fc receptors present on the neutrophil
membrane. Sodium azide was included in all the washes to prevent
internalization of the antibodies by the nonfixed neutrophils. Incubation with the primary antibody lasted for 3 hours. The cells were
washed and then incubated for 30 minutes with fluorescein-isothiocyanate (FITC)-conjugated Fab2 fragments of antispecies IgG (diluted 1:140, vol/vol), (Sigma). The irnmunostained cells were observed with a confocal fluorescence microscope. To visualize
intracellularly located antigens, fixed neutrophils were permeabilized
by treatment with 0.2% (wt/vol) of Triton X-100 before to the primary antibody application.
Confocal scanning laser microscopy. Neutrophils were observed
under a confocal fluorescence microscope (Bio-Rad, Hemel Hempstead, Hertfordshire, UK) equipped with an excitatory argon laser.
Confocal optical scanning microscopy permits noninvasive sectioning of cells, asit collects the emitted fluorescent light from
withinthe focal plane ofthe objective lens. This property of the
confocal microscope allows visualization of cell antigens by specific
antibodies, within discrete regions of cells or tissue by a noninvasive
optical sectioning of intact, fixed and nonfixedcells.2sIn the absence
of the primary antibody, no fluorescent image was obtained from
the cells.
lmmunoelectronmicroscopy.
The isolated, washed neutrophils
were incubated with affinity-purified sheep anti-HK (1-108, in a
dilution of 1:100, voYv01) previously coupled to 15-nm gold partiAfter incubation for 45 minutes at room temperature, the cells
were washed with PBS, and then incubated for a further 45 minutes
with rabbit anti-PK (AS176, in a dilution of 1:500, vol/vol). The
neutrophils were washedwith PBS and incubated for 30 minutes
with goat antirabbit IgG labeled with 30-nm gold particles (Amersham; Arlington Heights, IL) in a dilution of 1:4 (vol/vol). The cells
were washed threetimes with PBS for 5 minutes each and fixed with
3% (vol/vol) glutaraldehyde in PBS for 1 hour at room temperature.
Finally, the cells were postfixed with I%(vol/vol) osmium tetroxide
and embedded in epon-araldite as previously described.24Ultrathin
sections were counterstained with lead citrate and examined under a
Philips EM-300 electron microscope (Philips, Eindhoven, Holland).
Displacement of P U F X I from neutrophils. A synthetic peptide
of 3 1 residues (HK3 1, positions 565 to 595 of the HK sequence),
which covers the entire PK binding site of the HK light chain and
overlaps the corresponding binding site for FXI,” was synthesized
as previously described.” Isolated neutrophils were incubated for
15 minutes inthe absence or presence of 100 pg/mL of HK31
in PBS, pH 7.4, washed withthe same buffer, and subjected to
imrnunostaining.
Kinin releasefromneutrophil-boundkininogens.
Isolated human neutrophils were resuspended inPBS containing 5 mmol/L
glucose, and tissue kallikrein or plasma kallikrein was added to a
final concentration of 100 ng/mL. For control, cells were incubated
without enzyme. Aliquots from the incubation mixture were taken
at 0-, 15-, and 30-minute intervals, The cells were spun rapidly in
an Eppendorf centrifuge, fixed, and immunostained with an antibradykinin antibody (SBK 1).
Immunocytochemicalcontrols.
Controls included replacement
of the primary antibody by nonimmune serum or IgG of the same
species, also by omission of the primary antibody, and by the use
of nonkinin peptide antibodies (anti-oxytocin, anti-ANF). Additional
controls were prepared by preabsorption of theprimary antibody
with 50 p.g/rnL of the respective purified antigen (HK, LK,PK,
HENDERSON ET AL
Table 1. Differential Staining of Site-Directed MoAbs
Neutrophils
Antibodv
Nonfixed
Fixed
FEHI
HKH4
HKL12
-
f
Abbreviations: -,
staining.
+
+S
+
+
no staining; +, weak staining; ++, moderate
tissue kallikrein, bradykinin, ANF, oxytocin). For immunoelectron
microscopy, omission or replacement of rabbit anti-PK by nonimmune rabbit serum resulted in the absence of a specific gold labeling
of the neutrophil membrane (not illustrated).
RESULTS
Conventional light microscopy is limited in its ability to
demonstrate many of the details of cellular structure. We
therefore used thenew powerful imaging technique of confocal scanning laser microscopy applying FITC-labeled F(ab)z
secondary antibodies, with the pseudocolor gradient from
blue(nil)tored(maximal)providinganestimateofthe
amount of antigen on the cell.
A sheep polyclonal anti-HK antibody directed exclusively
against the light chain portion of HK gave positive staining
circular pattern of
onnonfixedneutrophils(Figla).The
staining at or near the midoptical section of the cells was
HK on the outer surface of
indicative of the presence of
the neutrophil membrane. A distinct annular localization of
labeling was also obtained when nonfixed neutrophils were
incubated with the rabbit polyclonal anti-PK antibody (Fig
lb). In the absence of the primary antibody, no fluorescent
cells (data not shown), thus
image was obtained from the
demonstrating the specificity of the immunolocalization of
HK and PK on neutrophils. The results are consistent with
thefindingthat
a fraction(13%)ofexogenouslyapplied
radiolabeled HK remained bound to the neutrophils even
after several washes with Hanks’ buffer (see Materials and
Methods) similar results have been reported for cell adhesion
molecules exposed on the surface of the neutrophil^.^^^^*
Thenextquestionweaddressedwaswhethertheremaining factors of the contact-phase assembly, namely FXII
and FXI, also reside on the neutrophil membrane. Both proteins immunolocalized on the nonfixed neutrophil, indicating
attachment of these coagulation factors to the external surface of theneutrophilcellmembrane(FigICandd).On
electronmicrographs,immunoreactiveHKlocalizedas
a
cluster of gold particles restricted to the external surface of
the neutrophil cell membrane (not shown). Ultrastructural,
double immunolabeling of HK and PK confirmed the coexistence of the enzyme and its substrate sequestered on the
external surface of the neutrophil plasma membrane (Fig 2 ) .
Because HK and PK circulate in plasma as a complex,
the colocalization of both these proteins on the external surface of nonfixed neutrophils prompted the question: is PK
a kininogenbridgeor is
linkedtotheneutrophilthrough
bound directly to a recipient site on the neutrophil membrane? This question was investigated using two site-directed
antibodies: (1) antibody HKL16 directed to the PK binding
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CONTACTPHASEFACTORS
477
ON HUMAN NEUTROPHILS
I.
: ,
-.;
c
r-
'3d
U
Fig 3. Contact-phase factors on the surface of neutrophils probed by binding site-directed entibodiw. (a) Nonfixed neutrophils, MoAb
antibody HKL16 directed to the PK binding site of HK; (b) nonfixed neutrophils; mouse MoAb PK6 directed to the HK binding site of P& (c)
fixed neutrophils, HKLl6; (dl fixed neutrophils, PK6; (e) fixed cytoplasts, HKL16; (R fixed cytoplasts, PK6. Antibodies (from escitas] were epplied
in a dilution of 1:lOO. The lntendty of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow, moderate; red, maximal,
The bar in each row isequivalent to 4 pm.
site of the HK light chain (positions 569 to 595), and (2)
antibody PK6 directed at the
HK binding region of the heavy
chain of PK. Our attempts failed to detect HK with HKL16
(Fig 3a) or PK with PK6 (Fig 3b) on nonfixed neutrophils,
whereas the location
of HK and PK on the plasma membrane
of noniixed cells had been demonstrated with the polyclonal
antibodies 1-108 (HK) and AS176 (PK), respectively (see
Fig l a and b). Together, these results may suggest that the
accessfor HKL16 and PK6 totheirrespectiveepitopes,
which represent the mutual binding sitesof HK and PK, is
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470
HENDERSON ET AL
I
Fig 4. Displacement of PK and FXI from HK by
peptide HK31. Isolated neutrophils were incubated
for 15 minutes in the absence (a, c) or presence (b.
d)oflWpg/mLofHK3l.Atthoendoftheincubation
period, the nonfixed cells were immunostainedwith
(a, b) anti-PK(AS176;
1:.lOOO) or (c, d) anti-FXI
(l:l,OW), andimagedunder
the confocal microscope. The intensity of immunoreactivity is indicated
by the color strip: blue, nil; green, minimal; yellow,
moderate;red,maximal.
The bar in each row is
equivalent to 4 Cm.
I-
Fig 7. Kinin liberation from neutrophil-bound kininogens followed by confocal microscopy.Isolated
neutrophils were incubated with (ad) plasma kallikrein or (e-h) tissuekallikrein for 0 (a, e), 15 (b. f),and
30 minutes (c,g). Immunostaining was done with
antibradykinin (SBKl, 1:lW) followed by FITGlabeled (Fab); fragments of goat antimouse IgG
[1:140), and confocal microscopy. For controls, the
first antibody was omitted (d) or replaced
by an antibody t o an unrelated antigen (h). The intensity of
immunoreactivity is indicated by the color strip:
blue, nil; green, minimal; yellow, moderate;red,
equivalent t o lpm.
0
maximal. The bar in each row is
L
m
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CONTACTPHASEFACTORS
ON HUMAN NEUTROPHILS
479
I
Fig 6. Probing for the kinin moiety of neutrophil-bound kininogens. Nonfixed cells were incubated with (a) MoAb to bradykinin
(SBK1,1:1001 or lb) antiserum to oxytocin (1:100l. The immuno
stained cells were analyzed by confocal microscopy.The intensity
of immunoreactivity is indicated bythe color strip: blue, nil; green,
minimal; yellow, moderate; red, maximal. The bar in aach row is
equivalent to 4 pm.
these site-specific antibodies. Demonstration
of the immunoreactivity of both m 1 6 and PK6 on fixed neutrophils (Fig
3c and d) and on fixed cytoplasts (Fig 3e and f) provides
further evidence of the membrane localization of HK and
PK on the neutrophil and shows that the relevant antigenic
Fig 5. Specificity of the cellular localization probed by anti-tissue
epitopes are exposed upon fixationof the cells.
Cells were incubatedwith (a) antitissue kallikallikrein and anti".
To furtheranalyze this phenomenon,weappliedtwo
krein lnonfixed cells, KDB1,l:l,OOO),
(b) antitirue kallikrein (fixed
MoAbs, m 4 and HKL12, which bind to well-defined epipermeabilizedcells, KDB1, l:l,OOO), or (c) anti-u (nonfixed cells,R7.
1:l.OOO). and examined under
the confocal microscope. The
intensity
topes ofHK that are distinct from PK
thebinding region, and
of immunoreectivii is indicated bythe color strip: blue, nil; green,
tested for their differential binding to nonfixed neutrophils.
A
minimal; yellow, moderate; red, maximal. The bar in each row is
weak,
though
distinct,
immunostaining
of
nonfixed
neutroequivalent to 4 pm.
phils, and a positive staining after fixation of the cells was
observed (Table l), indicating that epitopes other than the
blocked on nonfixed neutrophils by the complex formation
PWHK binding sites are readily accessible on kininogens
between the PK heavy chain and the HK light chain. To
bound to nonfixedneutrophils. In a similarapproach,the
spatial relationship between FXI and HK was probed with
address this question, we first fixed the cells to allow dissociation and random cross-linking of the complex components MoAb FEHl directed to the HK binding site on FXI. Fixed
to the cell structures, and probed the individual proteins with
or nonfixed neutrophils were incubated with
FEHl in a dilu-
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480
tion 1:100,and imaged on the confocal microscope. No immunostaining was observed on nonfixed neutrophils, indicating that the access of FEHl to the binding site is blocked,
whereas after fixation the binding site became accessible to
the antibody (Table 1). These results are compatible with
the notion that PK and FXI are present on the neutrophil
surface in form of complexes with HK, which dissociate
under the conditions of our fixation procedure.
To further explore the anchor sites of PK and FXI on the
neutrophil surface we determined whether either of these
proteins could be displaced by synthetic peptide HK3 1, a
molecular surrogate of the PK/FXI binding site(s) on HK.
Isolated neutrophils were incubated for 15 minutes with or
without 100 pg/mL of HK31. At the end of the incubation
period, the cells were immunostained with antikallikrein
(AS176) or anti-FXI antiserum, and imaged on the confocal
microscope (Fig 4a and c, controls). Inclusion of peptide
HK3 1 effectively displaced PK and FXI from their HK binding site as evidenced by the almost complete absence of
staining (Fig 4b and d). Our experiments with peptides that
mimic the binding sites for PK and FXI on HK, and with
antibodies directed to these sites support the view that PK
and FXI are anchored to the outer surface of the neutrophil
via HK.
To show the specificity of our approach, we localized an
enzyme of the kinin system, tissue kallikrein, which is located in intracellular stores of the neutrophil cell.” In nonfixed cells, with the plasma membrane intact, and the access
of antibodies to the inside of the cells blocked, no immunostaining was observed with antitissue kallikrein (Fig 5a).
When neutrophils were fixed and permeabilized by the use
of a nonionic detergent (0.2%Triton X-100 in PBS), tissue
kallikrein localized within the neutrophil (Fig 5b). In contrast, the preferred substrate of tissue kallikrein, LK was
localized on the outer surface of the intact neutrophil membrane using a specific antiserum directed to the unique light
chain portion ofLK (Fig 5c). Therefore, access of tissue
kallikrein to LK or HK can only occur when the neutrophil
either degranulates or secretes tissue kallikrein in response
to stimuli.
It was also important to determine whether the kininogens
on the isolated, but nonstimulated, neutrophils contain their
kinin moiety, thereby providing an endogenous substrate for
the enzymic action of the kallikreins after neutrophil activation or degranulation. Using a monoclonal antibradykinin
antibody (SBKl), we obtained annular localization patterns
(Fig 6a), indicative of the presence of the kinin moiety in
the kininogen domain D4. In control experiments, antisera
against the unrelated peptides, oxytocin or ANF, failed to
produce staining (exemplified for anti-oxytocin, Fig 6b).
Hence, at least a fraction, if not all of the neutrophil-bound
kininogen, is present in the native, kinin-containing form.
The ability of plasma kallikrein and tissue kallikrein to
release kinin from kininogens on the neutrophil surface was
examined by incubating human neutrophils for 30 minutes
at 37°C with either tissue kallikrein or plasma kallikrein at a
concentration of 100 ng/mL. Aliquots of the cell suspensions
were taken at 0-, 1 5 , and 30-minute intervals. The cells
were recovered by centrifugation and immunostained with
a monoclonal antibradykinin antibody, SBK1. Cells treated
HENDERSON ET AL
with plasma kallikrein showed a considerable loss of staining
by 15 minutes, which seemed to be complete by 30 minutes
(Fig 7a, b, and c), whereas the response time to tissue kallikrein was longer with loss of staining commencing at 30
minutes (Fig 7e, f, and g). Antibody controls were done in
the absence of the first antibody (Fig 7d) or in the presence
of an unrelated first antibody (Fig 7h); controls where cells
had been maintained for 30 minutes in buffer only did not
show significant changes in the staining patterns compared
with the samples withdrawn at zero time (not shown). These
results demonstrate that the kinin moietyof neutrophil-bound
kininogens is readily accessible for the kallikreins, and at
least a major part of the available kinin is released upon
exposure of the cells to active kallikreins. The presently
available antibody probes for kinins do not distinguish between the precursor protein holding the kinin sequence. Furthermore, in purified systems, plasma kallikrein and tissue
kallikrein each cleave the two types of kininogens though
at different rates (not shown). Hence, we are unable to discriminate between the kininogen sources that deliver kinins
on the neutrophil surface.
DISCUSSION
This study demonstrates that all the essential components
of the contact-phase system assemble on the surface of the
neutrophil cell membrane. Our data suggest that HK and
FXII bind to recipient sites exposed on the external face of
the neutrophil membrane, whereas PK is secured to the cell
surface indirectly through its docking protein, HK. Likewise
FXI may be tethered through HK to the neutrophil surface.
Hence, an array of functional proteins is assembled on the
surface of neutrophils, thereby providing a unique circulating
platform (solid phase) for the contact activation system. A
similar assembly has been reported for cultured human endothelial cells,29implying that immobile platforms for the contact system may exist as well.
Our observation of a cell-bound contact system on neutrophils suggests the involvement of a novel mechanism for the
formation of kinins from kininogens. The sequence of events
for such a phenomenon would include the discrete and circumscribed formation of kinins (bradykinin, kallidin) from
HK and/or LK on the surface of the neutrophil either by the
FXII-mediated activation of PK, and/or by the release of
Sic
Fig 8. Assemblyof the contact-phase fectors on the neutrophil
surface. 82. bradykinin B2 receptor. Dotted lines indicate the poltuIated contraction of endothelial cells following stimulation of their
B2 receptor by kinin.
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CONTACTPHASEFACTORS
ONHUMAN NEUTROPHILS
weight kininogen on stimulated washed human platelets. Biochemisneutrophil-borne tissue kallikrein in its active form. We protry 23:6863, 1984
pose that the locally released kinin may, in a paracrine man11. Gustafson El, Schutsky D, Knight LC, Schmaier AH: High
ner, enhance the passage of the neutrophils into the extracelmolecular weight kininogen binds to unstimulated platelets. J Clin
lular space by causing the endothelial cells to retract (Fig
Invest 78:310, 1986
8). Such a mechanism permits the transudation of plasma
12. Schmaier AH, Smith PM, Purdon AD, White JG, Colman
content by controlling vascular permeability, and the passage
RW:
High molecular weight kininogen. Localization in the unstimuof circulating neutrophils into the interstitial tissue space
lated and activated platelet and activation by a platelet calpain(s).
surrounding the site of injury or inflammati~n.~'
Our notion
Blood 67:119, 1986
is compatible with the long-known effects of kinins in the
13. Schmaier AH,KuoA, Lundberg D, Murray S , Cines DB:
microcir~ulation.~'
The expression of high molecular weight kininogen on human umbilFurthermore, kinins generated by the neutrophil-borne
ical vein endothelial cells. J Biol Chem 263:16327, 1988
contact system might act in an autocrine manner. This hy14. Van Iwaarden F, de Groot PG, Sixma JJ, Berrettini M, Bouma
pothesis is consistent withthe finding that activated PK
BN: High molecular weight kininogen is present in cultured human
causes the release of elastase from neutrophil^.^^ Our data
endothelial cells: localization, isolation and characterization. Blood
shed a new light on the observation that the response of
71:1268, 1988
neutrophils to a degraded form of kallikrein, p-kallikrein,
15. Van Iwaarden F, de Groot PG, Bouma BN: The binding of
lacking the HK binding properties is markedly a t t e n ~ a t e d . ~ ~high molecular weight kininogen to cultured human endothelial cells.
In essence, our findings support the previous conclusion that
J Biol Chem 263:4698, 1988
attachment to HK is a major requirement for the observed
16. Gustafson EJ, Schmaier AH, Wachtfogel YT, Kaufman N,
effect of plasma kallikrein on neutrophil^.^^ Clearly, addiKucich U, Colman RW: Human neutrophils contain and bind high
molecular weight kininogen. J Clin Invest 84:28, 1989
tional experiments are needed to provide further evidence
17. Henvald H, Jahnen-Dechent W, Abd Alla S , Hock H, Bouma
for these intriguing suggestions. To this end, studies on the
BN, Muller-Ester1 W:Mapping of the high molecular weight kininoimmunolocalization of the bradykinin B2 receptor on neutrogen binding site of prekallikrein. Evidence for a discontinuous epiphils and endothelial cells are being undertaken.
ACKNOWLEDGMENT
We are indebted to Profs B. Chappell and 0. Jones (Bristol) for
the use of the Confocal Optical Scanning Microscope, funded by
the Wellcome Trust. We thank Drs B. Bouma (Utrecht) for a gift
of antibody FEHI, M. Phillips (London) for antibody SBK1, J. Hock
(Marburg) for antiserum to FXI, and A. Schmaier (Ann Arbor) for
antibody to FXII. We also thank C. Jeal for preparing the color
montages.
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