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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 684-693
Acute Systemic Reaction and Lung Alterations Induced by an Antiplatelet
Integrin gpIIb/IIIa Antibody in Mice
By
Bernhard Nieswandt,
Bernd Echtenacher,
Frank-Peter Wachs,
Josef Schröder,
J. Engelbert Gessner,
Reinhold E. Schmidt,
Georges
E. Grau, and
Daniela N. Männel
From the Department of Pathology, Tumor Immunology, University of
Regensburg, Regensburg, Germany; and the Department of Clinical
Immunology, Hannover Medical School, Hannover, Germany.
 |
ABSTRACT |
Shock is frequently accompanied by thrombocytopenia. To investigate
the pathogenic role of platelets in shock, we examined the in vivo
effects of monoclonal antibodies (MoAbs) against mouse platelet
membrane proteins. Injection of the platelet-specific MoAb MWReg30 to
the fibrinogen receptor (gpIIb/IIIa) rendered mice severely hypothermic
within minutes. Isotype-matched control antibodies, even if they also
recognized platelet surface antigens, did not induce comparable signs.
MWReg30 induced early signs of acute lung injury with increased
cellularity in the lung interstitium and rapid engorgement of alveolar
septal vessels. Despite this in vivo activity, MWReg30 inhibited rather
than stimulated platelet aggregation in vitro. MWReg30-binding to
platelets led to phosphorylation of gpIIIa, but did not induce
morphological signs of platelet activation. The MWReg30-induced
reaction was abolished after treatment with MoAbs 2.4G2 to Fc RII/III
and was absent in FcRIII-deficient mice, clearly demonstrating the
requirement for FcRIII on involved leukocytes. Simultaneous
administration of tumor necrosis factor exacerbated, whereas a
tolerizing regimen of tumor necrosis factor or bacterial
lipopolysaccharide completely prevented the reaction. These data
suggest that platelet surface-deposited MWReg30-immune complexes lead
to an acute Fc-mediated reaction with pulmonary congestion and
life-threatening potential that could serve as an in vivo model of
acute lung injury.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ACUTE LUNG INJURY occurring in
association with septic shock appears to result from an inflammatory
reaction involving particularly the lung microvasculature. At the same
time, many other regulatory systems in the body become activated by
inflammatory stimuli. In extreme situations such as septic shock, the
molecular mechanisms leading to microvascular injury, tissue damage,
and organ failure, therefore, become very complex. Most forms of shock, particularly after a variety of diseases, such as sepsis, major trauma,
and malignancy, can be accompanied by thrombocytopenia and disseminated
intravascular coagulation. Thrombocytopenia is most commonly regarded
as a secondary complication, resulting from endothelial injury.
Alternatively, platelets could also play an active role, because
depletion of platelets resulted in reduced morbidity or mortality in
different experimental settings related to shock.1-4 The
interactions of platelet surface molecules with leukocyte or
endothelial cell adhesion molecules can lead to platelet activation and
to the release of biologically important mediators. The facts
that platelets are so numerous and that degranulation is a rather rapid
event may cause a very effective yet locally restricted
reaction.5 Such a role for platelets can be
predicted in some types of hypersensitivity reactions and immune
complex-mediated diseases.
Hyperactive polymorphonuclear neutrophils play a central role in the
pathogenesis of acute lung injury. Neutrophils from patients with
severe bacterial infection display enhanced responses when stimulated
in vitro.6 In vitro investigations and studies using the
extracorporeal rat lung perfusion model of acute lung injury demonstrated that capillary leakage results from two independent insults, one leading to priming events with the result of sequestration of polymorphonuclear neutrophils and a second activating
signal.7 Observations from the isolated lung perfusion
model in the absence of blood constituents suggest the possibility that
the priming effect can act on resident cells in the lung, eg,
macrophages or endothelial cells.8 Such a priming for
enhanced platelet activating factor (PAF) production by tumor necrosis
factor (TNF) on macrophages, endothelial cells, and neutrophils has
been demonstrated in vitro.9 In the transfusion-related
acute lung injury (TRALI), the second signal can be provided by Igs
directed against recipient granulocytes10 or by
biologically active compounds generated during storage of cellular
blood components involving the action of lipid mediators and triggering
of the PAF receptor.7
The role of TNF mediating pathogenic effects in bacterial
lipopolysaccharide (LPS)-induced shock has clearly been
established.11-13 A trend towards protection by
neutralizing TNF in patients with severe sepsis rather than septic
shock has been reported.14 However, especially when
administered late in full-blown shock, this treatment did not result in
any beneficial effect, clearly indicating that TNF action alone does
not account for the shock syndrome.15 Another important
pathogenic element is the activation of Fc receptor III (FcRIII,
CD16) on granulocytes and macrophages, as described for the
anaphylactic shock.16,17 Among numerous other pathways not
mentioned here, an excessive activation of the complement system can
also lead to severe inflammation and tissue destruction.18
We present here evidence that specific binding of an anti-gpIIb/IIIa
monoclonal antibody (MoAb) MWReg30, but not of other antibodies to the
platelet surface, formed immune complexes in situ that led to an
FcRIII-mediated shock-like reaction with clear signs of acute lung
injury, the extent of which was modulated by inflammatory signals.
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MATERIALS AND METHODS |
Animals.
Specific-pathogen-free mice (NMRI, C3H/HeN, C3H/HeJ, and BALB/c) 5 to
7 weeks of age were obtained from Charles River (Sulzfeld, Germany) and
kept in the animal facilities of the University of Regensburg
(Regensburg, Germany) for the experiments. Mice deficient in FcRIII
were generated in collaboration with the group of J.S. Verbeek
(Utrecht, The Netherlands) as described.19 These mice were
bred and maintained under dry barrier conditions in the animal facilities at the Hannover Medical School (Hannover, Germany) and were
studied at 2 to 4 months of age.
Reagents.
Recombinant mouse TNF (rmTNF) was prepared and purified in our
laboratory with a specific activity of 8 × 107 U/mg
as determined in the TNF bioassay. LPS (LPS-W from Salmonella minnesota 9700) was obtained from Difco
Laboratories (Detroit, MI); methylene blue, Mianserin, cyproheptadine,
and acid glycoprotein (bovine  -1 AGP) were from Sigma
(Deisenhofen, Germany). Cobra venom factor preparations
(CVF) were kindly provided by J. Köhl (Hannover, Germany);
soluble complement receptor I (sCRI) was kindly provided by M. Kirschfink (Heidelberg, Germany); the peptides GRGDS and YIGSR were
synthesized and kindly provided by R. Frank (Heidelberg, Germany).
Antibodies.
MoAbs were generated and purified following standard procedures from
the following hybridomas: rat-antimouse-LFA-1 MoAb
(H35.89.9),20 rat-antimouse-intercellular adhesion
molecule-1 (ICAM-1) MoAb (YN1/1),21
anti-granulocyte MoAb (R14),22 and
rat-antimouse-FcRII/III MoAb (2.4G2).23 The MoAbs
anti-CD18 and anti-CD13 were obtained from Pharmingen (Hamburg,
Germany). For in vivo application, 100 µg of the MoAb were injected
intraperitoneally (IP) in 200 µL phosphate-buffered
saline (PBS) 24 hours before the challenge. Rat antimouse platelet
MWReg30 (IgG1) and MWReg31 (IgG2a) MoAbs were generated in our
laboratory and screened as described.24
Induction of thrombocytopenia.
Rabbit antimouse platelet serum was produced by immunization of rabbits
with purified platelets as described.25 Injection of 100 µL of this serum IP reduced the number of circulating platelets to
less than 0.05 × 106/µL (normal value, 1.0 to
1.2 × 106/µL) within 1 hour and kept it
at this level for at least 48 hours. Thrombocytopenic mice were taken
into the experiments 24 hours after injection of the antiplatelet serum.
Hypothermia measurement.
Body temperature was measured at the indicated times with a rectal probe.
Platelets preparation and counting.
Mice were bled under ether anesthesia from the retroorbital plexus.
Blood from 10 mice was pooled into a tube containing 0.5 mL 0.1 mol/L
sodium citrate, and platelet-rich plasma (PRP) was obtained by
centrifugation at 300g for 10 minutes at room temperature. The
platelets were washed 3 times with PBS by centrifugation at 1,300g for 10 minutes and were used immediately. Isolated
platelets did not show any signs of activation as tested by staining
for P-selectin or morphological signs of degranulation. For
determination of platelet counts, blood (20 µL) was obtained from the
retroorbital plexus of anesthetized mice using siliconized
microcapillaries and immediately diluted 1:100 in Unopette Kits (Becton
Dickinson, Heidelberg, Germany). The diluted blood sample was allowed
to settle for 20 minutes in an improved Neubauer hemocytometer, and platelets were counted under phase contrast at 400× magnification.
Flow cytometry.
MWReg30 MoAb was fluoresceinated at a fluorescein:protein ratio of 3:1
by standard procedures with fluorescein isothiocyanate (FITC; Sigma)
and separated from free FITC by gel filtration on a PD-10 column
(Pharmacia, Uppsala, Sweden). Cells were stained at a
final concentration of 5 µg/mL fluoresceinated antibody in PBS for 20 minutes on ice. For determination of membrane-bound MWReg30, mice were
bleed 3 hours after MWReg30 injection. Platelets were washed with PBS
twice and stained with FITC-labeled goat antirat Ig (Pharmingen) for 20 minutes at 4°C in the dark. All samples were analyzed on a FACscan
(Becton Dickinson), and platelets were gated by forward/side scatter characteristics.
Aggregometry.
To determine platelet aggregation, transmission was measured using PRP
(200 µL with ~106 platelets/µL). Ten microliters of
rat IgG (300 µg/mL), MWReg30 (300 µg/mL), or rat antiserum to mouse
gpIIb/IIIa (10 µL) and 10 µL of Tyrode's buffer (Sigma) were mixed
with PRP for 1 minute at 37°C before 10 µL of ADP (110 µmol/L;
Sigma), phorbol 12-myristate 13-acetate (PMA; 1 µg/mL; Sigma), or
collagen A (1 mg/mL; Biochrom, Berlin, Germany) as aggregation-inducing
agents were added. For negative controls 10 µL of apyrase (100 U/mL;
grade III; Sigma) was added. Transmission was recorded in a Fibrintimer
2 channel aggregometer (APACT Laborgeräte + Analysensysteme,
Hamburg, Germany) over 10 minutes and was expressed as arbitrary units
with 100% transmission adjusted with plasma.
Histology.
For histology, lung, liver, and kidney samples were fixed by immersion
in 4% PBS-buffered formaldehyde and embedded in paraffin. Sections (2 to 3 µm) were stained with hematoxlin and eosin. Additional lung
samples were fixed in cacodylate-buffered Karnovsky fixative (2.5%
glutaraldehyde and 2% paraformaldehyde), postfixed in 1% osmium
tetroxide, and embedded in EMbed-812 epoxy resin, and semithin sections
(0.8 µm) were stained with 1% toluidine blue.
For immunohistochemistry, acetone-fixed cryosections of mouse spleens
were stained with MWReg30 at a final concentration of 5 µg/mL for 20 minutes on ice, washed with PBS, and incubated for 1 hour at room
temperature with biotinylated goat polyclonal antibodies antirat IgG
(Southern Biotechnology, Bioreba, Reinach, Switzerland) followed by the
addition of horseradish peroxide (HRPO)-avidin. Color
reaction was obtained by the addition of AEC substrate-chromogen (Dako,
Glostrup, Denmark), and the slides were counterstained
with toluidine blue. Bone marrow cells (106) or tumor cells
were stained with FITC-labeled MWReg30 at a final concentration of 5 µg/mL for 20 minutes on ice, washed with PBS, and investigated under
the fluorescence microscope.
Immunoprecipitation.
Immunoprecipitation was performed as described
previously.26 Briefly, 108 washed platelets
were surface-labeled with PBS containing 100 µg/mL
6-(+)-biotinylamidohexanoic acid N-hydroxysulfosuccimide ester sodium
salt (NHSS-LC-biotin; Serva, Heidelberg, Germany) and subsequently
solubilized in 1 mL lysis buffer (Tris-buffered saline containing 20 mmol/L Tris/HCl, pH 8.0, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 0.5 µg/mL
leupeptin, and 0.5% Nonidet P-40 (all from Boehringer Mannheim,
Mannheim, Germany). Cell debris was removed by
centrifugation (12,000g for 20 minutes). After preclearing (8 hours), 10 µg MWReg30 or control MoAb was added together with 25 µL
of protein G-Sepharose (Pharmacia), and precipitation took place
overnight at 4°C. Samples were run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
along with a biotinylated molecular weight marker and transferred onto
a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with streptavidin-horseradish peroxidase (1 µg/mL) for 1 hour after
blocking. After extensive washing, biotinylated proteins were
visualized by ECL (Amersham, Arlington Heights, IL).
Sequencing.
The antigen of 1011 unbiotinylated platelets was
immunoprecipitated with MWReg30. After electrophoresis on SDS-PAGE and transfer to PVDF membrane, specific bands were cut out and digested with trypsin by standard methods. High-performance liquid
chromatography (HPLC)-purified tryptic fragments were
subjected to an Applied Biosystems (Foster City, CA)
protein sequencer (model 477A) with an online PTH-analyzer (model 120A).
Tyrosine phosphorylation of gpIIIa.
Platelets were stimulated with MWReg30 (30 µg/mL) for 3 minutes and
taken up in Laemmli sample buffer containing sodium vanadate as
described.27 For each reaction, 200 µL of PRP
(~109/mL) was treated and lysed immediately by the
addition of 200 µL of 2× sample buffer (to give a final
concentration of 37 mmol/L Tris, pH 6.8, 11.8% glycerol, 2.36% SDS, 2 mmol/L sodium vanadate, and 0.002% bromphenol blue).
Immunoprecipitation with protein G-Sepharose was performed for 1 hour
at 4°C with the addition of MWReg30 to platelets either 3 minutes
before or immediately after (control) platelet lysis with lysis buffer
(see above). Samples were boiled for 3 minutes, run on a 7.5% SDS-PAGE
along with a molecular weight marker, and transferred to PVDF membrane. After blocking, the membrane was incubated with antiphosphotyrosine antibody PY-20 (Santa Cruz Biotechnology, Santa Cruz, CA)
at a final concentration of 2 µg/mL for 1 hour. Bound PY-20 was
detected by horseradish peroxidase-conjugated sheep antimouse IgG
(Dianova, Hamburg, Germany) and visualized by ECL (Amersham).
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RESULTS |
Induction of hypothermia by a platelet-specific MoAb, MWReg30.
To investigate the role of platelets in hypersensitivity reactions and
shock, antisera and MoAbs against mouse platelet membrane proteins were
generated. Rats were immunized with purified platelets and the
resultant antisera were tested for their ability to induce thrombocytopenia and shock signs in mice. All antisera markedly reduced
the platelet counts. One antiserum induced, in addition, significant
hypothermia in mice. Fusion of the spleen cells from this rat with
mouse myeloma cells resulted in a number of hybridomas. Two different
clones were identified to produce MoAb (MWReg30, IgG1 and MWReg31,
IgG2a) directed against two distinct mouse platelet membrane antigens.
Purified MoAb from these clones were tested for their ability to induce
hypothermia in mice. Injection of 100 µg MWReg30
(Fig 1A) but not MWReg31 (Fig 1C) rapidly
induced a strong hypothermia in normal mice. Mice rendered
thrombocytopenic by injection of 100 µL of a rabbit antiplatelet
serum 24 hours before MWReg30 challenge failed to develop hypothermia
(Fig 1B). A rabbit antiserum to mouse platelets, ie, platelet depletion itself, did not show any effect on body temperature (Fig 1D). Isotype-matched (rat IgG1) control MoAb to aminopeptidase (anti-CD13) and to LFA-1 (anti-CD18), respectively, did not induce any hypothermia, even when 100 µg were injected intravenously (data not shown). The
CD18 epitope recognized by the anti-CD18 MoAb is expressed on mouse
platelets, as determined by flow cytometry, whereas aminopeptidase (CD13) is not. Injection of 100 µg rat IgG or rabbit IgG did not induce hypothermia.
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| Fig 1.
Induction of hypothermia by MWReg30 but not other
MoAbs requirement of platelets. Normal (A) or thrombocytopenic (B)
male NMRI mice received 100 µg of affinity-purified MWReg30 IP in 100 µL sterile PBS. As control for platelet-specific antibodies, normal
mice received 100 µg affinity-purified MWReg31 (C) or 100 µL of a
rabbit antimouse platelet serum (D). The mean values of the body
temperatures of 3 mice per group at the indicated times after
injection of the antibodies are given ± SD.
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Absence of linkage between hypothermia and thrombocytopenia.
Upon intravenous (IV) injection of MWReg30, the central temperature
decreased within 10 minutes, to reach a nadir at 60 minutes, with a
return to normal within 2 to 3 hours. The hypothermic response after
MWReg30 injection was not dose-dependent but rather occurred with a
threshold between 3 and 10 µg (Fig 2A).
The lowest dose required to exert hypothermia (defined as a temperature
decrease of 5°C or more) in naive, healthy mice was 10 µg per
mouse. In contrast, there was a clear dose-dependency in the
MWReg30-induced thrombocytopenia (Fig 2B) and in the number of in vivo
MWReg30-labeled platelets in mice determined after 3 hours (Fig 2C).
The maximal thrombocytopenia, presenting as a 70% decrease in platelet
counts, was reached with 30 µg. MWReg31 had a comparable
thrombocytopenia-inducing activity to MWReg30, but failed to cause
hypothermia, as shown in Fig 1C. Injection of a rabbit antimouse
platelet antiserum led to an even more pronounced thrombocytopenia,
with more than a 95% decrease in platelet counts, but not to
hypothermia (Fig 1D).
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| Fig 2.
Hypothermia is independent of thrombocytopenia induction.
Dose-dependency of hypothermia (A), thrombocytopenia (B), and
platelet-bound MWReg30 levels upon IV injection of the indicated amount
of MWReg30 in 200 µL PBS. The hypothermia is expressed as the
decrease in degrees Celcius measured 60 minutes after injection,
whereas platelet counts and platelet antirat Ig staining were performed
3 hours after injection. Results are expressed as the means from 3 mice
per group ± SD.
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Characterization of MWReg30.
MWReg30 strongly and exclusively bound to platelets
(Fig 3A) and megakaryocytes
(Fig 3B). The antibody did not recognize
v 3 integrin-positive tumor cells (bEnd3),
and immunohistochemical staining of different organ sections and tumor
cells (endothelioma, fibrosarcoma, thymoma, monocytic cell lines)
demonstrated the platelet/megakaryocyte specificity of the MoAb.
Immunoprecipitates obtained from surface-biotinylated platelets were
analyzed by SDS-PAGE under nonreducing (Fig 3C, lanes 1 and 2) and
reducing conditions (Fig 3C, lanes 3 and 4). MWReg30 precipitated a
complex of two membrane proteins of 135 and 80 kD. Under reducing
conditions, the 135-kD protein resolved into two bands of 110 and 25 kD
(the latter being determined in gels of higher density; not shown), whereas the apparent molecular mass of the 80-kD chain slightly increased. Amino acid sequences of tryptic fragments identified the
80-kD protein (5'-DASHLLVFT-3') as gpIIIa (integrin
3/CD61)28 and the 135-kD protein
(5'-LRGEQMASYF-3', 5'-PQALSTPTL-3',
5'-DGYNDIAV-3') as gpIIb (integrin
aIIb/CD41),29 identifying the immunoprecipitated protein as
the fibrinogen receptor (gpIIb/IIIa).
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| Fig 3.
Characterization of the MWReg30 MoAb. Binding
characteristics of the MWReg30 MoAb were investigated (A) by flow
cytometric analysis, which shows that all circulating mouse platelets
were intensively stained with FITC-labeled MWReg30. The shaded area
indicates platelets stained with an FITC-labeled control rat MoAb. (B)
Mouse spleen sections were stained with MWReg30. (C) Immunoprecipitates
with MWReg30 (lanes 1 and 3) or control rat IgG (lanes 2 and 4)
obtained from lysates of surface-biotinylated platelets were analyzed
by SDS-PAGE under nonreducing (lanes 1 and 2) and reducing conditions
(lanes 3 and 4) by staining of the blotted proteins.
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Histopathology of the MWReg30-induced reaction.
IV injection of MWReg30 caused within 15 minutes a peripheral
vasodilatation, as evidenced by red extremities (tail and feet) and
uncoordinated movements. These signs persisted for 60 to 90 minutes.
Proportional to the injected dose of MWReg30, mice showed intestinal
hemorrhages, ruffled fur, and reduced flow rate during blood sampling.
Lung, kidney, and liver were sampled at different times after MWReg30
injection for histological examination. Histopathology of the whole
lung performed 60 minutes after injection showed enhanced thickness of
alveolar septa, due to edema and to increased cellularity, consisting
of polymorphonuclear and mononuclear cell accumulation in capillaries,
as compared with lungs from mice injected with PBS
(Fig 4) or control IgG (not shown). This
change was found to be uniform, not focal, in the two lungs (n = 6). After 24 hours, lungs showed a further enhancement of septa thickness, due to mononuclear, but not polymorphonuclear, cell accumulation in
capillaries and interstitium, as well as plasma leakage into the
alveolar space (Fig 4C). No changes were observed in peribronchiolar areas. As early as 20 minutes after IV injection, semithin sections showed that the vascular engorgement was essentially due to red blood
cells, with very few leukocytes or platelets (Fig 4D). Twenty-four hours after injection, some mice showed centrolobular necrosis in the
liver as well as glomerular shrinkage and enlargement of Bowman spaces
in the kidney, compatible with hemodynamic changes of shock, but
without evidence of acute tubular necrosis. Thrombi or
microthrombi were not seen in any of the examined organs. Platelets recovered from the blood of mice 30 minutes after injection of MWReg30
did not express P-selectin as determined by FACS analysis. In sera from
mice obtained 90 minutes after injection of MWReg30, no TNF was
detectable.
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| Fig 4.
Lung histopathology. Aspect of alveolar walls 60 minutes
after IV injection of PBS (A) or 10 µg of MWReg30 (B); (C) 24 hours
after IV injection of 10 µg of MWReg30; (D) vascular engorgement of
septal capillaries with erythrocytes, 20 minutes after IV injection of
100 µg of MWReg30. (A through C) Hematoxylin/eosin staining of
paraffin-embedded section (original magnification × 400). (D)
Toluidine blue staining of Epoxy resin-embedded sections (original
magnification × 1,000).
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In vitro effects of MWReg30 on platelet aggregation and activation.
MWReg30 did not have any aggregating effect by itself in vitro when
tested up to a concentration of 30 µg/mL but interfered with platelet
aggregation, as determined by aggregometry. MWReg30 and the antiserum
to gpIIb/IIIa completely prevented PMA-induced platelet aggregation
after 1 minute of preincubation of the platelets in PRP.
Collagen-induced aggregation was only inhibited by 14% with MWReg30 as
compared with the 50% inhibition with the anti-gpIIb/IIIa antiserum.
ADP-induced aggregation, on the other hand, was stabilized by MWReg30,
whereas the antiserum to gpIIb/IIIa completely prevented aggregation.
Exposure of mouse platelets in suspension to MWReg30 did not induce any
sign of activation. Neither degranulation nor pseudopodia formation was
observed 10 minutes after binding of MWReg30, as established by
electron microscopic examination. No P-selectin expression or
surface-bound fibrinogen was detectable by cytofluorometric analysis.
Phosphorylation of a 80-kD protein occurred upon exposure of platelets
to MWReg30 (Fig 5A). The electrophoretic
mobility of this tyrosine-phosphorylated protein from platelet lysates
was indistinguishable from gpIIIa (CD61). Immunoprecipitation with MWReg30 enriched for the phosphorylated 80-kD band that was much stronger when the platelets had been pre-exposed to MWReg30 before lysis (Fig 5B), indicating identity with the gpIIIa chain of the fibrinogen receptor trimer.
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| Fig 5.
Protein phosphorylation upon platelet activation by
MWReg30. Tyrosine-phosphorylated proteins from mouse platelets were
separated by gel electrophoresis. (A) Plain platelet lysates without
(lane 1) or after 5 minutes of pre-exposure of the platelets to MWReg30
(30 µg/mL) (lane 2) stained for phosphotyrosin after blotting. (B)
Immunoprecipitates of gpIIb/IIIa from platelet lysates with MWReg30
without (lane 1 and 3) and after 5 minutes of pre-exposure of the
platelets to MWReg30 (30 µL/mL) stained for protein detection with
Coomassie (lanes 1 and 2) or for phosphotyrosin after blotting (lanes 3 and 4).
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Mechanisms of the MWReg30-induced reaction: importance of
FcRIII triggering.
In vivo blocking of FcRII/III by pretreatment with the 2.4G2 MoAb
completely prevented the MWReg30-induced hypothermia
(Fig 6A). Mouse platelets were not stained
by the anti-FcRII/III 2.4G2 MoAb, as determined by FACS analysis.
Unlike human platelets, mouse platelets do not express FcRII.
Granulocyte-depletion with MoAbs to mouse granulocytes before MWReg30
injection partly inhibited the hypothermic response. The body
temperature decrease in granulocyte-depleted mice was shorter and not
as marked as in control animals (maximal decrease of 2.5°C ± 0.8°C after 30 minutes in granulocyte-depleted v 4.7°C ± 0.4°C after 45 minutes in normal mice). More importantly, MWReg30 did not induce any hypothermia in FcRIII-deficient mice, clearly identifying this as the FcR responsible for the hypothermic response (Fig 6B). Even though no hypothermia developed in these mice,
MWReg30 injection reduced their platelet numbers significantly. The
FcRIII-deficient mice a priori have an elevated platelet count
(1.6 ± 0.1 × 106/µL, n = 6) that was reduced
by 25% (1.2 ± 0.1 × 106/µL, n = 6) within 2 hours after MWReg30 injection to platelet counts of normal mice.
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| Fig 6.
Involvement of FcR triggering in the MWReg30-induced
reaction. Hypothermia induced by IV injection of MWReg30 (100 µg) was
determined (A) in mice after blocking their FcRII/III with 2.4G2
MoAb (100 µg; IP) ( ) or in untreated controls ( ) and (B) in
FcRIII-deficient mice () compared with untreated control animals
(). The mean values of the body temperatures of 3 mice per group at
the indicated times after injection of the antibodies from 1 of 2 experiments are given ± SD.
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Sensitization/desensitization by TNF and LPS.
To test whether the hypothermia induced by MWReg30 was modulated by
inflammatory stimuli, mice received 5 µg rmTNF 90 minutes before
MWReg30 injection. These mice (n = 6) became hypothermic even faster
(decrease of >4°C within 15 minutes) than nonsensitized controls,
and all animals died between 30 and 40 minutes after the MWReg30
injection. Control mice not pretreated with rmTNF recovered from
MWReg30-induced hypothermia within 90 minutes and survived (n = 6).
Also, rmTNF-pretreated mice (n = 4) did not develop any hypothermic
reaction upon injection of either 100 µg anti-CD18 or 100 µg
anti-CD13 MoAb as MWReg30-isotype control antibodies (data not shown).
In addition, the hypothermic effect of MWReg30 injection was
indistinguishable in LPS-responder (C3H/HeN) and LPS-low responder
(C3H/HeJ) mice, indicating that possibly contaminating LPS did not
contribute to the MWReg30 effect (data not shown).
Injection of small amounts of rmTNF (600 ng) or LPS (10 µg) per mouse
24 hours before MWReg30 challenge, which by itself had no hypothermic
effect, resulted in complete prevention of MWReg30-induced hypothermia
(Fig 7). This protection was neither due to
thrombocytopenia induced by the LPS or TNF pretreatment because
platelet counts at the time of challenge were unchanged nor due to
altered binding characteristics of MWReg30 to platelets, as determined
by flow cytometry.
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| Fig 7.
Desensitization for the MWReg30-induced hypothermia by
LPS or TNF pretreatment. Hypothermia induced by IP injection of MWReg30
(100 µg) was determined in mice after pretreatment with (A) PBS (200 µL), (B) LPS (10 µg), or (C) rmTNF (600 ng) IP 24 hours before
challenge. The mean values of the body temperatures of 3 mice per group
at the indicated times after injection of the antibodies from 1 of 2 experiments are given ± SD.
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| |
DISCUSSION |
In this study, we show that the in vivo administration of the MoAb
MWReg30 that is directed to the platelet-specific integrin IIb3-(gpIIb/IIIa, fibrinogen receptor)
induces acute hypothermia and pathological changes, especially in the
lung. To exert these effects, an engagement of the MoAb with its
antigen on platelets was required, speaking against a nonspecific
activity of this MoAb. Hypothermia is one of the signs that
characterize endotoxic shock in mice.30,31
The lung congestion, as well as the increased cellularity of the lung
septa, due to mononuclear and polymorphonuclear cells, are akin to
LPS-induced lung pathology. The vascular engorgement, essentially due
to erythrocytes, was more pronounced than in mice injected with 1,000 µg LPS (not shown). Histological examination showed that these
changes were neither focal nor associated with atelectasis: the
increased cellularity was uniform and was observed in both lungs. In
contrast, control lungs from mice injected either with PBS or with
normal IgG were consistently free of accumulated cells in the septa. In
MWReg30-injected animals, the increased cellularity appeared to be due
to cell accumulation in both vascular and interstitial compartments,
although electron microscopic studies are required to clarify this
point. Preliminary studies show enrichment of pinocytotic vesicles
(Männel et al, manuscript in preparation). Altogether, the picture is compatible with the very early steps of
acute lung injury.
The syndrome described here might be the result of an in situ-formed
immune complex reaction, with the antigen being the platelet surface
gpIIb/IIIa. The reason why the MWReg30-induced effect took place so
rapidly in vivo could be the immediate access of the MoAb to its target
antigen in the vascular system. A mere opsonization of the platelets
may not explain the effect, because different platelet-specific control
antibodies, although inducing comparable thrombocytopenia, did not lead
to the same reaction. Even the isotype-matched anti-CD18, which also
reacts with a platelet surface protein and other newly developed MoAb
to different platelet antigens (manuscript in
preparation), did not induce hypothermia. One reason
could obviously be that these antibodies find fewer epitopes on the
platelet surface than the MWReg30, because the gpIIb/IIIa constitutes
the most abundant platelet membrane protein. Apart from the antigen
specificity of the antibody involved, the in situ immune complexes
formed by identical isotypes may also differ in their FcR binding
capacity.32 At this point, it is still not clear whether
platelets, besides serving as carriers for the immune complexes in
situ, contribute actively to the pathology.
In our in vitro studies, the observed tyrosine phosphorylation of the
integrin subunit gpIIIa upon MWReg30 binding is the only indication
for a signal mediated directly via gpIIb/IIIa. Such tyrosine
phosphorylation of the integrin subunit of the fibrinogen receptor
has been suggested to be an important event in initiating outside-in
signaling cascades into the platelet.27 However, electron
microscopic analysis of MWReg30-stimulated platelets did not show any
morphological evidence for platelet activation (intact and dense
granules, absence of pseudopod formation).
Unlike in the human system, in which antibodies to gpIIb/IIIa led to
secondary responses such as secretion, second wave of platelet
aggregation, and release of procoagulant membrane
vesicles,33 MWReg30 directed to gpIIb/IIIa did not induce
any measurable platelet activation or aggregation per se. Rather,
stimulus-induced platelet aggregation was either inhibited or
stabilized as in the case of ADP-stimulation. This might be explained
by a steric interference once MWReg30 is bound to gpIIb/IIIa.
Additionally, neither P-selectin expression nor surface-bound
fibrinogen was detectable by flow cytometric analysis, nor was
ATP-release measurable in a whole blood test system after the addition
of MWReg30. Autocrine or juxtacrine activation with antibodies to
membrane proteins of human platelets is a well-recognized phenomenon
either by direct receptor activation or by cross-linking of the
receptors, by FcRII-mediated interplatelet or intraplatelet
activation,34 or by complement activation.35 It
has been suggested that the complement system plays an important role
in this platelet activation.36 However, FcRII/III have
not been demonstrated on mouse platelets. In contrast to human
platelets, which carry the FcRII (A),37 this isoform is
not produced in mice. Our staining attempts with the antibody for the
FcRII/III (2.4G2) on mouse platelets were also negative. Taken
together, these findings do not support a platelet activation function
of MWReg30.
Based on these data and especially on the results with the
FcRIII-deficient mice, FcRIII on cell types other than platelets must be involved. The alleviation of the MWReg30-induced syndrome in
mice depleted of granulocytes supports this concept. Besides granulocytes, FcRIII-bearing monocytes, macrophages, natural killer
cells, and mast cells are also likely candidates for the pathogenesis
of this syndrome. In the FcRIII-deficient mice, the syndrome did not
develop, even though the platelet numbers were reduced. The capacity of
MWReg30 to lower the platelet count is somewhat in contrast to the
reported resistance of such mice to develop experimental immune
thrombocytopenia induced by an autoimmune antimouse platelet MoAb of
the same isotype, IgG1.38 Whether the MWReg30-induced
hypothermia and reduction of platelet numbers involves different
mechanisms than a regular FcRIII-dependent thrombocytopenia, as
described by these investigators, requires further studies.
Neither inhibition of hypotension with methylene blue, an inhibitor of
the NO-induced activation of the cytosolic guanylate cyclase, nor the
acute-phase protein 1-acid-glycoprotein (AGP),39 nor
inhibition of integrin binding to RGD or YSIGR sequences containing sites, nor MoAb to LFA-1 or ICAM-1 conferred protection. Drugs used to
antagonize serotonin or histamine effects were equally ineffective. The
finding that neither decomplementation of the mice by CVF nor
pretreatment with soluble CRI prevented the syndrome does not support
the view of a major complement involvement in our system. On the other
hand, the injection of CVF itself led to a similar hypothermic reaction
as MWReg30 injection. Clearly, more investigations are needed to
ascertain the precise role of complement in the development of the
observed pathology.
The concept that several pathways have to act in concert for the
development of the MWReg30-induced syndrome and that one pathway
involves FcRIII-positive cells is supported by the synergistic effect of TNF and MWReg30. The observation that sublethal amounts of
TNF injected shortly before MWReg30 led to the death of the animals
within 40 minutes demonstrates a strong sensitizing effect of
inflammatory stimuli for the MWReg30-induced reaction. This priming
effect of TNF for the MWReg30-induced reaction parallels the findings
in experimental models of LPS-induced shock as well as of acute lung
injury.8 It has been shown that concomitant injections of
LPS and TNF,40 or IL-1 and TNF,41 lead to
exacerbated pathological consequences upon a subsequent challenge with
LPS. Also, the in vivo effect of MWReg30 injection alone is not the result of a synergistic action of MWReg30 with traces of LPS
contamination, because the reactions of LPS responder and nonresponder
mice are indistinguishable. For acute lung injury, it has been
postulated that two sequential insults are required: one provided by an
underlying predisposing clinical condition, eg, recent surgery or
active infection leading to neutrophil priming and activation of the pulmonary endothelium and a second triggering stimulus. In
transfusion-related acute lung injury, Igs that either bind to the
recipient granulocytes or recognize HLA determinants on recipient or
donor leukocytes10 or biologically active lipids produced
during blood storage7 trigger the organ failure. In our
model, this second signal could be provided by the MWReg30 injection.
Development of a refractory state by desensitization with LPS or TNF
for an LPS challenge, on the other hand, requires time between the
injections: mice are desensitized when low doses of LPS or TNF are
administered at least 24 hours before the challenge.42 A
similar desensitizing effect of TNF and LPS was also seen for the
challenge with MWReg30. This could indicate that similar regulatory mechanisms govern our antibody-induced syndrome, the widely used LPS-induced experimental model for shock, and acute lung injury. The
regulatory mechanisms accounting for these LPS or TNF effects remain to
be analyzed. Transient upregulation of FcRIII expression on
monocytes or other cells43 could be considered as a cause for sensitization and, afterwards, active downregulation as cause for
desensitization.44 It remains to be investigated whether a
counteracting mechanism of the body to cope with the degree of insult,
eg, NF- B upregulation rendering possible target cells as for example
endothelial cells refractory for damaging effects of
platelets,45-47 is the reason for desensitization.
No direct effects of LPS or TNF on platelets have been described so
far; however, the participation of platelets in shock, either directly
or via mediators, such as PAF, PF4, thromboxane A2, or
-thromboglobulin, has been shown.5,7,8,48,49 Although we
could not demonstrate any MWReg30-induced platelet activation, our data
do not rule out any platelet release reactions occurring after
interaction with FcRIII-carrying cells. So far, clinical
intervention studies based on findings in available experimental models
on shock have been largely disappointing.50 The
MWReg30-induced syndrome combining the findings from LPS-shock models
and the results from investigations on acute lung injury might provide a new in vivo model that reproduces features of very early steps of
shock development, thus setting the stage for the detrimental action of
inflammatory mediators. Investigations of the regulatory mechanisms
governing the extent of the pathological consequences can easily be
performed in this model. Also, results from studies aiming at
alleviating the MWReg30 action might be useful for future strategies
designed to interfere with septic lung injury in humans.
| |
ACKNOWLEDGMENT |
The authors thank J. Köhl for CVF, M. Kirschfink for sCRI, R. Deutzmann for sequencing, E. Heinmöller for help in aggregometry, and R. Straub, C. Galanos, R. Urbaschek, and G. Bein for helpful discussions.
| |
FOOTNOTES |
Submitted September 4, 1998; accepted March 12, 1999.
Supported in part by DFG (SFB265 Project No. B01) to R.E.S. and by BMBF
(01KI9473 Project No. A3) to D.N.M.
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.
Address reprint requests to Daniela N. Männel, PhD,
Department of Pathology, University of Regensburg, D-93042 Regensburg,
Germany.
| |
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[Abstract]
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J. L. Teeling, T. Jansen-Hendriks, T. W. Kuijpers, M. de Haas, J. G. J. van de Winkel, C. E. Hack, and W. K. Bleeker
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[Abstract]
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B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.-P. Cazenave, P. Ohlmann, C. Gachet, and H. Zirngibl
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[Abstract]
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S. M. Taylor, M. P. Reilly, A. D. Schreiber, P. Chien, J. R. Tuckosh, and S. E. McKenzie
Thrombosis and shock induced by activating antiplatelet antibodies in human Fcgamma RIIA transgenic mice: the interplay among antibody, spleen, and Fc receptor
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[Abstract]
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B. Nieswandt, W. Bergmeier, K. Rackebrandt, J. E. Gessner, and H. Zirngibl
Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice
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[Abstract]
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P. F. Piguet, C. Da Laperrousaz, C. Vesin, F. Tacchini-Cottier, G. Senaldi, and G. E. Grau
Delayed Mortality and Attenuated Thrombocytopenia Associated with Severe Malaria in Urokinase- and Urokinase Receptor-Deficient Mice
Infect. Immun.,
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[Abstract]
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B. Nieswandt, W. Bergmeier, V. Schulte, K. Rackebrandt, J. E. Gessner, and H. Zirngibl
Expression and Function of the Mouse Collagen Receptor Glycoprotein VI Is Strictly Dependent on Its Association with the FcRgamma Chain
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[Abstract]
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Y. Kimura, A. Hart, M. Hirashima, C. Wang, D. Holmyard, J. Pittman, X.-L. Pang, C. W. Jackson, and A. Bernstein
Zinc Finger Protein, Hzf, Is Required for Megakaryocyte Development and Hemostasis
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION