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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 684-693
By
From the Department of Pathology, Tumor Immunology, University of
Regensburg, Regensburg, Germany; and the Department of Clinical
Immunology, Hannover Medical School, Hannover, Germany.
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
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 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
Fc 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 Fc 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 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-Fc 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.
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).
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.
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).
Characterization of MWReg30.
MWReg30 strongly and exclusively bound to platelets
(Fig 3A) and megakaryocytes
(Fig 3B). The antibody did not recognize
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.
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.
Mechanisms of the MWReg30-induced reaction: importance of
Fc
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).
In this study, we show that the in vivo administration of the MoAb
MWReg30 that is directed to the platelet-specific integrin 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.
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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TOP
ABSTRACT
INTRODUCTION
RII/III
and was absent in Fc
-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).
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.
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 
) or in untreated controls (
) and (B) in
Fc
B upregulation rendering possible target cells as for example
endothelial cells refractory for damaging effects of
platelets,