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Department of Pathology and Division of Vascular Biology, Weill Medical College of Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of these anti-PECAM reagents on chronic inflammation in vivo has not been addressed. This has not been for lack of good models, but for technical problems associated with chronic administration of the reagents. Immunocompetent animals will make Abs against foreign Igs, including the human Ig tail of PECAM-IgG. Furthermore, it is difficult to prepare sufficient quantities of these reagents in batches of similar specific activity and free of traces of contaminating endotoxin for repetitive injections over many months.
One approach to study the role of a molecule in long-term physiology or pathophysiology is to genetically delete it. However, the response of the organism to this targeted deletion is often the expression of compensatory mechanisms conducted by molecules that normally do not participate in that function or make relatively minor contributions to it, with the result that there is no significant change in phenotype.
This, in fact, appears to be the case for the recently described PECAM "knockout" mouse (10). Although anti-PECAM reagents administered to wild-type mice block leukocyte emigration by up to 80–90% (1, 2, 3, 4, 8, 9), there is always a considerable residual leukocyte response that cannot be blocked by any concentration or combination of these agents. We demonstrated that mice with a targeted deletion in the PECAM-1 gene have a very mild phenotype, with no discernible quantitative reduction in response in several acute inflammatory models (10). We postulated that the PECAM-independent routes of transmigration, which normally account for only 10–20% of diapedesis in these inflammatory models in wild-type mice, have been expanded in the knockout mice to support the full wild-type levels of leukocyte migration.
In this report, we chose an alternative approach: to engineer transgenic mice expressing a soluble form of murine PECAM as an IgG chimera (mPECAM-IgG). This same molecule has been shown to block acute inflammation in vivo in the thioglycollate peritonitis model when administered exogenously (4). The cDNA encoding mPECAM-IgG was subcloned behind the apolipoprotein A1 (ApoA1) promoter (11). Our goal was to produce mice that had circulating levels of mPECAM-IgG that would not affect normal immune surveillance or wound healing but which would be sufficient to block the enhanced leukocyte emigration that would normally ensue following an inflammatory challenge. These mice would provide us with a continuous source of genetically identical animals continuously dosing themselves with the transgene product. By using the human IgG Fc domain for these constructs, we could monitor soluble mPECAM levels and "tag" the PECAM-IgG with reagents that recognized human IgG without interfering with its ability to bind mPECAM. Because the mice begin expressing the transgene in utero, they would not recognize the human IgG as foreign. Such mice could be used to study the role of PECAM in chronic inflammatory conditions as well as to study whether they would become resistant to the anti-inflammatory effects of PECAM-IgG when administered chronically.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthesis of transgenic construct
To generate soluble mPECAM-IgG chimeric protein, the extracellular domain of mPECAM was fused to the Fc region of human IgG1 (see Ref. 4 for details), and the resulting fragment was then inserted into the HindIII and XbaI sites of a plasmid vector pcDNA I (Invitrogen, San Diego, CA) in which its CMV promoter had been replaced with human ApoA1 promoter (11). The NdeI site of the CMV promoter was converted into HindIII site by linker ligation, and the PCR-generated ApoA1 promoter was inserted into this HindIII site.
Transgenic mouse production and detection
The pcDNAI/mPECAM-IgG transgenic plasmid was isolated and
purified by double CsCl gradient centrifugation. The purified DNA was
linearized by SfiI digestion, and 
5 ng of DNA was
microinjected into BALB/c-fertilized oocytes from FVB/n mice, and
implanted into pseudopregnant FVB/n mice at the Rockefeller University
transgenic mouse facility. All mice were bred and maintained at the
Rockefeller University Laboratory Animal Research Center (New York, NY)
in a pathogen-free environment. Genomic DNA was isolated from mouse
tails biopsied after 3 wk of age, and offspring containing the
integrated transgene were identified by Southern blot analysis.
Soluble chimeric protein in the sera was quantitated by ELISA using purified human IgG as standards, similar to the procedure described by Liao et al. (3). In brief, 96-well polyvinyl microtiter dishes were coated with 25 µg/ml of purified goat anti-human Fc Ab (Pierce, Rockford, IL), nonspecific binding was blocked with PBS containing 0.1% OVA, and dilutions of the test sera were then incubated on the treated plates, which were then washed extensively. Bound chimera was detected with alkaline phosphatase-conjugated goat anti-human Fc polyclonal Ab (Pierce) and substrate (p-nitrophenyl phosphate) in Attophos substrate buffer (JBL Scientific, San Luis Obispo, CA). Fluorescence was quantified on a Cytofluor 3500 (PerSeptive Biosystems, Framingham, MA) (3).
The molecular size of the transgenic protein was determined by Western blot: 10 µl of sera was fractionated on 4–16% SDS-PAGE. Proteins were immobilized onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) and probed with HRP-conjugated goat anti-human IgG Ab. Transgene-positive mice were then crossed to FVB/n mice to establish founder lines, from which heterozygous mice were intercrossed to obtain mice homozygous for the transgene.
In an additional approach, tissues were homogenized in an electric tissue homogenizer (Omni International, Waterbury, CT) in 0.1% Nonidet P-40 (Sigma, St. Louis, MO) and protease inhibitors. Equal amounts of protein, as determined by BCL protein assay (Pierce), were separated by SDS-PAGE and subjected to immunoblot as above.
Hematological parameters
All of the mice, from three separate founder lines, were weighed, and peripheral bloods were taken from age- and sex-matched transgenic-positive and -negative littermates. Complete white blood cell counts and differential counts were measured by the Diagnostic Laboratory of the Weill Medical College Research Animal Resource Center.
Quantitative PCR
A variety of tissues were excised from age-matched transgenic and nontransgenic mice. Approximately 100 mg of fresh tissue was immersed in 1 ml of Trizol solution (Life Technologies, Gaithersburg, MD) and total RNA was isolated and purified according to the manufacturer’s standard protocol. Contaminating DNA in the resulting RNA samples was eliminated by digestion with RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN). RT-PCR was performed by using the primers listed below and TaqMan probes (Perkin-Elmer, Foster City, CA) using previously published methods (12, 13). The copy number of RNA message of both transgenic and endogenous PECAM genes were quantitated by using plasmids containing the transgenic construct and murine full-length PECAM cDNA as standards. Values are expressed as copies of mRNA per 50 ng of total RNA, normalized with house-keeping gene, GAPDH.
The nucleotide sequences of primers and TaqMan probes are as follows: Endogenous mPECAM cytoplasmic domain: forward primer, 5'-CTGAACTCCAACAGCGAGAAGCT-3'; reverse primer, 5'-TCAAGGGAGGACACTTCCACTT-3'; and TaqMan probe, 5'-TGTGGAAGCCAACAGCCATTACGGTT-3'. Human IgG1 Fc: forward primer, 5'-GTGAGCCACGAAGACCCTGA-3'; reverse primer, 5'-GACCTTGCACTTGTACTCCTTGC-3'; and TaqMan probe, 5'-ACAGCACGTACCGTGTGGTCAGCGT-3'. Murine GAPDH: forward primer, 5'-GCATCTTCTTGTGCAGTGCCAGCC-3': reverse primer, 5'-TTGCCGTGAGTGGAGTCATACT-3'; and TaqMan probe, 5'-TGCAGTGGCAAAGTGGAGATTGTTGC-3'.
Flow cytometry
Approximately 25 µl of heparinized mouse blood was diluted in 100 µl of PBS containing 20 mM EDTA and 0.1% human serum albumin. RBCs were lysed by adding 2 ml of lysis buffer (1.66% ammonium chloride) and incubated for 10 min at room temperature. After a single centrifugation (1200 rpm for 5 min) the white blood cells were resuspended in cold HBSS. Single cell suspensions were incubated for 30 min at room temperature with primary Abs, washed twice as above, and incubated for an additional 30 min with FITC-conjugated secondary Abs, if necessary. Analysis was performed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA).
For some experiments using mPECAM-IgG, fibroblasts transfected with mPECAM were subjected to flow cytometry and analyzed as in Ref. 4 .
Histochemistry
Organs from freshly killed mice were surrounded with OCT (Miles Laboratories, Elkhard, IN) and snap-frozen by immersion in liquid nitrogen. Frozen sections were cut 5 µm thick, picked up on gelatin-coated glass slides, fixed in acetone for 5 min before storage at -20oC. Immunoperoxidase histochemistry was performed as described (2) by using rat mAb against murine PECAM-1, VCAM-1, ICAM-1, and ICAM-2 (all from PharMingen, San Diego, CA) and peroxidase-labeled rabbit anti-rat IgG (Dako, Carpintera, CA).
To detect endogenous mPECAM in tissues in the presence of a vast excess of soluble mPECAM-IgG, immunohistochemistry was performed using a rabbit antiserum generated against the cytoplasmic tail of mPECAM (generously provided by Dr. André Veillette, McGill Cancer Center, Montreal, Canada) diluted 1:500 and detected with peroxidase-labeled swine anti-rabbit IgG (Dako). Reaction product was developed with diaminobenzidine-H2O2. Slides were counterstained with hematoxylin, dehydrated in graded ethanols, and mounted in Permount (Fisher Chemical, Fairlawn, NJ).
Thioglycollate broth-induced peritonitis
These studies were performed and analyzed as previously described (2). Measurements (animal weight; peritoneal lavage volume, cell density, and differential count; peripheral blood count and differential; and general autopsy) were generally performed 18 h after i.p. injection of 1 ml of 4% thioglycollate broth. In each experiment, age- and sex-matched littermates were used as controls. Over the course of these studies, mice ranging in age from 8 wk to 6 mo were used in individual experiments. There was no change in the inflammatory response of any strain as a function of age within the range tested.
Isolation and purification of mPECAM-Ig from transgenic sera and serum transfer experiment
The transgenic protein was purified from 10 ml of serum drawn from Tg11 mice by affinity chromatography using CNBr-activated Sepharose coupled to rabbit anti-human Fc Abs (Pierce), preabsorbed with mouse serum. The bound protein was eluted with 0.1 M glycine (pH 2.5) and neutralized with 1/10 vol of 1 M Tris-HCl. After dialysis, the pure protein was dissolved in PBS and filter-sterilized before injecting animals.
Appropriately diluted serum from Tg11 mice (10-fold or 4-fold diluted) or comparable quantities of purified transgenic protein injected i.v. into wild-type mice several hours before thioglycollate injection i.p. The inflammatory response of the recipient mice was analyzed 18 h later. Serum from age-matched wild-type mice was used as a control.
PECAM-IgG dose response
To determine whether PECAM-IgG lost its ability to block TEM at high concentrations, assays were conducted in vitro using human PECAM-IgG (hPECAM-IgG), HUVEC, and human monocytes. These assays were performed as previously described (4) except that the concentrations of PECAM-IgG ranged from 1 µg/ml up to 1 mg/ml.
Statistics
Nonparametric data were evaluated by the Mann-Whitney U test using JMP software (SAS, Research Triangle Park, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three founder lines were generated by using the transgene
construct containing the ApoA1 promoter (Fig. 1
a). Mice of all three lines
were the same size and weight as their wild-type littermates. They were
healthy and fertile and had normal ratios of male and female offspring.
The mice were maintained in a clean (but not specific pathogen-free)
environment, had normal life spans, and had no apparent increase in
susceptibility to infectious diseases from environmental organisms or
tendency for wounds and lacerations to become infected (data not
shown).
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). The mice
showed no spontaneous bleeding tendencies, and their hematologic
profiles were similar to their wild-type littermates (Table II).
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10 µg/ml as determined by ELISA; homozygotes had levels
of 20 µg/ml. Mice of the Tg5 and Tg11 lines (high producers)
heterozygous for the transgene generally expressed between 500 and 800
µg/ml. Lower levels (100–400 µg/ml) were produced with advanced
age. Homozygous mice of these lines produced approximately double these
levels, but the phenotype observed was the same as for the
heterozygotes (see below). The higher levels of circulating PECAM-IgG
in Tg11 correlated with increased message levels for the transgene,
especially in the liver and lung (Table I
) but not with intensity of
hybridization signal on Southern blot (data not shown). Immunoblot of
transgenic serum demonstrated that the PECAM-IgG was running as a
single band of 230 kDa, the expected size of the intact dimerized form
(Fig. 1b), which was quantitatively converted to a 115-kDa
band when 2-ME was used to reduce interchain disulfide bonds between
the Fc portions of the chimeras (Fig. 1c).
Endogenous PECAM expression on the murine leukocytes was not changed by
transgene expression. Fig. 2 shows flow
cytometric analysis of leukocytes separately gated on mononuclear cells
(PBMC) and neutrophils. As previously reported (4, 10),
murine neutrophils had low but consistently detectable levels of PECAM,
whereas mononuclear cells bore considerably more. Similarly, both types
of leukocytes expressed wild-type levels of CD11a, CD11b (data not
shown), CD18, and CD62L (L-selectin).
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a), as previously found for
human leukocytes and hPECAM-IgG in vitro, presumably reflecting the low
density of PECAM molecules on these cells (4). This result
also demonstrates that the transgenic protein was not circulating bound
via leukocyte Fc receptors. In fact, for unknown reasons, PECAM-IgG
purified from the plasma of Tg11 mice and incubated at a concentration
of 1 mg/ml with isolated washed murine leukocytes did not bind to the
cells (Fig. 3, e--h). Control experiments
demonstrated that human IgG could be readily detected if it were indeed
bound by murine leukocyte Fc receptors. When heat-aggregated human IgG
was incubated with mouse blood, it bound to the leukocytes (presumably
via low affinity Fc receptors) and remained bound through the
preparation for FACS analysis (Fig. 3, b and
d).
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b) and of binding stably in a homophilic manner
to cells transfected with higher levels of mPECAM than expressed by
leukocytes (Fig. 3, i--l) This does not mean that
PECAM-IgG does not interact in a homophilic manner with leukocytes;
it only means that due to the low affinity of PECAM-PECAM interaction,
the PECAM-IgG does not bind stably enough to the leukocytes to survive
the stringent washes in the FACS procedure (Ref. 4 and
W. A. Muller, unpublished data). In the plasma of transgenic mice,
where the leukocytes are constantly surrounded by PECAM-IgG, any bound
molecules that dissociated could be rapidly replaced by others in the
environment.
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). 2) Detergent
extracts of lungs (a tissue particularly enriched for endothelium)
containing equal amounts of total protein were subjected to immunoblot
analysis using a polyclonal Ab against the cytoplasmic tail of mPECAM.
This Ab specifically identified equivalent amounts of endogenous
cellular PECAM in these samples (Fig. 4).
Control experiments probing the same samples with an Ab against human
IgG identified only PECAM-IgG in the samples from transgenic mice (Fig. 1d). Note the absence of a band at 135 kDa, the size of
endogenous PECAM, in these samples (Fig. 1d). 3) The same Ab
was used in immunohistochemical studies on frozen sections of kidneys
from these mice. The staining pattern of all vascular structures was
identical in the wild-type mice and all three transgenic lines. Even
the relative intensity of staining (arterioles = venules >
glomeruli > peritubular capillaries) was the same in all lines
(data not shown).
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Response to inflammatory challenge
Wild-type FVB/n mice and all strains of transgenic mice had
similar numbers of mononuclear cells and virtually no polymorphonuclear
cells (PMN) resident in their peritoneal cavities when unstimulated
(Figs. 5 and
6). When wild-type FVB/n mice were
challenged with an i.p. injection of thioglycollate broth and
sacrificed 18 h later, a dramatic inflammatory exudate including
large numbers of neutrophils as well as monocytes was seen (Fig. 5). In
contrast, age-matched Tg8 (moderate producer) littermates showed a
marked blunting of the inflammatory response. Heterozygous mice that
have circulating mPECAM-IgG levels of 10 µg/ml showed a 50–70%
reduction in PMN numbers; monocytes were reduced to near basal levels
(Fig. 5 and Table IV). In homozygous mice
producing 20 µg/ml, PMN infiltration was blocked by >80% (Fig. 5
and Table IV). This level of plasma mPECAM-IgG was similar to the
levels that were achieved in experiments in which exogenously
administered mPECAM-IgG was found to block PMN emigration into the
peritoneal cavities of wild-type mice by 80% (4).
Circulating leukocyte counts in Tg8 mice receiving thioglycollate broth
were elevated at 18 h, as had been seen previously with wild-type
mice receiving anti-PECAM reagents in the face of a thioglycollate
challenge (2, 4) consistent with the notion that
leukocytes could be mobilized normally but could not enter the site of
inflammation.
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summarizes the results
obtained in all of the experiments performed over the course of this
study. Too much of a good thing
A surprising result came when evaluating the high producer lines
of transgenic mice. The response to i.p. thioglycollate was virtually
the same as for wild-type mice at both 3 and 6 h (data not shown)
and at 18 h (Fig. 6). Peritonitis in response to thioglycollate
broth was blocked, as expected, in the high producer strains by Abs
against CD11a and CD11b (Fig. 6). This result indicates that these mice
were using leukocyte ß2 integrins for adhering
to the vascular wall.
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a) or
purified transgenic protein (Fig. 7b) from these mice
transferred to wild-type mice blocked inflammation well and in a
dose-dependent fashion. Fig. 7 shows data using mPECAM-IgG from the
Tg11 strain; similar results were obtained using sera from the Tg5
strain (data not shown). Many chemoattractants, chemokines, and Abs whose actions rely on cross-linking their ligands exhibit a bell-shaped dose-response curve. If the blocking effect of PECAM-IgG exhibited such properties, it was possible that the high levels of mPECAM-IgG in the plasma of Tg5 and Tg11 mice were well beyond the optimal inhibitory point and were, in fact, no longer effective. Alternatively, if PECAM-IgG bound to itself at high concentrations producing aggregates in which the binding sites of PECAM were already occupied, a decrease in efficacy as concentration rose above a certain threshold might be expected.
We initially tried to test these hypotheses in vivo by infusing mice
i.v. with 200 µl of mPECAM-IgG from Tg11 mice at a concentration of 6
mg/ml (the highest concentration we could achieve without running into
viscosity problems) 1 h before administration of thioglycollate
broth. The serum half-life of PECAM-IgG is 24 h. With this dose,
we were able to achieve a serum concentration that was still 250
µg/ml at the time of sacrifice. This concentration blocked influx of
leukocytes as well as the lower concentrations did (data not shown).
Because we could not reasonably achieve higher concentrations of
PECAM-IgG, we decided to investigate this question in a system that we
could better manipulate. Thus, experiments were conducted in vitro
using human leukocytes and endothelium and using hPECAM-IgG in an assay
that we had previously demonstrated to rely on homophilic PECAM-PECAM
interactions in the same manner as the in vivo peritonitis assay (Fig. 8).
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, demonstrate that
the plateau blockade by PECAM-IgG is maintained up to the highest dose.
Therefore, it is unlikely that the failure of Tg5 and Tg11 leukocytes
to be blocked by the high concentrations of PECAM-IgG circulating in
their plasma is due to a decreased response to these
concentrations. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These mice are presently being mated to introduce the PECAM-IgG transgene into strains that are susceptible to atherosclerosis (ApoE-deficient), collagen-induced arthritis (DBA/1), and experimental allergic encephalitis (SJL) to determine whether chronic exposure to therapeutic levels of circulating PECAM-IgG will effectively inhibit leukocyte emigration, and hence disease, in those models.
Because two lines of mice produced from this same transgene construct
did not have a noticeable defect in acute inflammation, one might
wonder whether the block of acute inflammation seen in the Tg8 mice
producing 10–20 µg/ml of mPECAM-IgG was unique and nonreproducible.
In fact, however, we have produced an independent line of transgenic
mice expressing mPECAM-IgG behind a different promoter. These mice
constitutively express mPECAM-IgG at 25 µg/ml and display a
phenotype similar to that of Tg8. That is, they are healthy and have
normal levels of circulating leukocytes but have a severely blunted
response to i.p. injection of thioglycollate broth (data not
shown).
Our data strongly suggest that it is the level of circulating
mPECAM-IgG to which the transgenic animals are chronically exposed
that determines the phenotype. When the plasma levels are below a
certain threshold, transmigration can proceed at a basal level that is
sufficient for normal wound repair and homeostasis in a pathogen-free
environment. However, the levels of mPECAM-IgG in these mice is
sufficient to block transmigration in an inflammatory response when the
mice are acutely challenged. On the other hand, chronic exposure to
supratherapeutic levels (
400 µg/ml) of mPECAM-IgG apparently lead
to unresponsiveness to its anti-inflammatory effects. Exposure of
the transgenic mice began in utero; however, it is possible that
chronic exposure to such high levels of anti-PECAM reagents
beginning later in life could produce similar effects. This result
would have important implications for dosing anti-PECAM
therapy.
Those transgenic mice constitutively expressing high levels of
PECAM-IgG were apparently resistant to its effects. The mechanism for
this resistance is not clear but was not due to 1) down-regulation of
PECAM-1 on either leukocytes or endothelial cells, 2) binding of
mPECAM-IgG to leukocyte Fc receptors, 3) production of an ineffective
transgene product, or 4) intrinsic ineffectiveness of PECAM-IgG
inhibition at high doses. At high concentrations, PECAM will bind to
itself homophilically (14). However, we do not think that
homophilic aggregation, rendering PECAM domains 1 and 2 unavailable for
interaction with leukocytes or endothelial cells (14, 15),
is responsible for the inability of mPECAM-IgG to block in these mice:
First, mPECAM-IgG at 6 mg/ml injected into wild-type mice or hPECAM-IgG
at 1 mg/ml in vitro were still effective at blocking PECAM
interactions. Second, such aggregates of PECAM-IgG would most likely be
bound by leukocyte Fc receptors, which we did not observe (Fig. 3).
Third, mPECAM-IgG in Tg11 serum and purified PECAM-IgG at
concentrations 1 mg/ml did not show any tendency to aggregate in
vitro, as seen on polyacrylamide gels (Fig. 1b and data not
shown). Fourth, the concentration of mPECAM-IgG in the unstimulated
peritoneal cavities of Tg11 mice was equivalent to the plasma level
(our unpublished data). If large aggregates formed in the blood, these
would have to disassociate spontaneously to cross the vasculature into
the peritoneal cavity. Such spontaneous disassociation would also make
the individual mPECAM-IgG molecules accessible to leukocytes and
endothelium at the same venules during the inflammatory response.
Rather, we postulate that these mice were rendered unresponsive to
PECAM and used PECAM-independent pathways of transmigration.
Leukocytes freshly prepared from transgenic mice did not bear PECAM-IgG
(Fig. 3). This was not surprising. Previous experiments using human
leukocytes and hPECAM-IgG demonstrated that the density of PECAM on
leukocytes was too low to support homophilic PECAM-PECAM-IgG
interactions (Ref. 4 and data not shown). It also was not
too surprising that PECAM-IgG was not bound by high affinity Fc
receptors of leukocytes. Because the concentration of endogenous murine
IgG is 10–20 mg/ml, this would out-compete the human IgG of the
transgene product present at 
1 mg/ml. Therefore, binding of PECAM-IgG
to murine high affinity Fc receptors is unlikely to explain the
phenotype of these mice in any case. What was surprising was that
purified PECAM-IgG did not bind to high affinity Fc receptors of
isolated, extensively washed leukocytes (Fig. 3, e--h). This may be due to denaturation of the IgG
portion during its isolation on protein A. However, the purified
protein could be re-isolated on protein A and was functionally active
(Fig. 7b). It is possible that there was some inherent
misfolding or abnormal posttranslational modification of the human IgG
Fc domain when made by murine liver and lung cells.
In wild-type mice and in vitro, anti-PECAM reagents routinely block
transmigration by up to 70–90% depending on the model, but never
block it completely (1, 2, 3, 4, 5, 7, 9, 16). The
PECAM-independent pathways of transmigration used by the high producer
strains may be those that normally account for this 20% residual
leukocyte emigration. If so, these mice will be an excellent
experimental system to identify these molecules and how they function.
It would be very difficult to identify molecules responsible for the
low percentage of PECAM-independent transmigration in wild-type mice,
especially if more than one were involved.
Alternatively, mice that have developed in the continuous presence of such high plasma concentrations of mPECAM-IgG may have learned to employ truly novel adhesion molecules and/or pathways for emigration. Indeed, we may have made "functional PECAM knockouts." CD31-null mice develop and live in the absence of PECAM function and have only a subtle defect in acute inflammation (10). It will be interesting to compare the pathways used by these mice to those used by the PECAM null mice generated by targeted deletion.
The mechanism of resistance of these mice to the anti-inflammatory effects of their own circulating PECAM-IgG is not known, but it is not due to absence of PECAM molecules on either the leukocytes or endothelial cells. Experiments are underway to determine whether the cells are desensitized to PECAM at the signal transduction level, how long this desensitization lasts if they are removed from the high-dose mPECAM-IgG environment of the mouse, or whether they simply override or bypass the block in PECAM function by using alternative molecular pathways.
The transgenic mice expressing moderate levels of mPECAM-IgG will be useful in testing the efficacy of long-term anti-PECAM therapy in models of chronic inflammation. The transgenic mice expressing high levels of mPECAM-IgG will be useful tools for investigating the PECAM-independent pathways of TEM and, perhaps, PECAM signaling. This technique has broader implications, however, because it can be applied to other adhesion molecules and other extracellular proteins that can be inhibited by soluble decoys. The anti-inflammatory reagents are not limiting and (due to constitutive production beginning in embryogenesis) nonimmunogenic. Thus, similar transgenic mice could be made to study the roles of other (adhesion) molecules in large populations of genetically identical mice over periods as long as the life span of the animal.
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Acknowledgments |
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Footnotes |
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2 Current address: Imclone Systems, Inc., 180 Varick Street, New York, NY 10014
3 Address correspondence and reprint requests to Dr. William A. Muller, Department of Pathology, C-420, Weill Medical College, 1300 York Avenue, New York, NY 10021. E-mail address:
4 Abbreviations used in this paper: TEM, transendothelial migration; PECAM, platelet/endothelial cell adhesion molecule-1; mPECAM-IgG, fusion protein consisting of the complete extracellular region of murine PECAM fused to the Ch2 + Ch3 domains of human IgG1; hPECAM-IgG, human PECAM-IgG; PMN, polymorphonuclear cells; ApoA1, apolipoprotein A1; Tg, transgenic; CAM, cell adhesion molecule.
Received for publication June 4, 1999. Accepted for publication September 2, 1999.
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References |
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 N. ILAN, A. MOHSENIN, L. CHEUNG, and J. A. MADRI PECAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics FASEB J, February 1, 2001; 15(2): 362 - 372. [Abstract] [Full Text] [PDF]
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 J Sun, C Paddock, J Shubert, H. Zhang, K Amin, P. Newman, and S. Albelda Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization J. Cell Sci., January 4, 2000; 113(8): 1459 - 1469. [Abstract] [PDF]
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