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(Circulation Research. 2001;88:430.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Antigraft Antibody-Mediated Expression of Metalloproteinases on Endothelial Cells

Differential Expression of TIMP-1 and ADAM-10 Depends on Antibody Specificity and Isotype

Gwénola Boulday, Stéphanie Coupel, Flora Coulon, Jean-Paul Soulillou, Béatrice Charreau

From INSERM U437 "Immunointervention en allo et xénotransplantation " and Institut de Transplantation et de Recherche en Transplantation, C.H.U., Nantes cedex, France.

Correspondence to Pr. Jean-Paul Soulillou, MD, PhD, INSERM U437, 30 bd J. Monnet, 44093 Nantes cedex 01, France. E-mail jps{at}nantes.inserm.fr

	Abstract

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Abstract—Endothelial cell (EC) interaction with antigraft antibodies (Abs) mediates EC injury and activation involved in vascular graft rejection. The aim of this study was to identify EC genes regulated in response to antigraft Ab binding that contribute to the endothelium alterations implicated in graft rejection or survival. By means of RNA differential display, 13 cDNA fragments corresponding to genes differentially expressed in ECs incubated with antigraft Abs were identified. Among these cDNAs were found the tissue inhibitor of metalloproteinase-1 (TIMP-1) and a desintegrin and metalloproteinase (ADAM-10). We demonstrated that TIMP-1 and ADAM-10 mRNA and protein expression was rapidly upregulated in ECs in response to antigraft Ab binding. Our data showed that TIMP-1 was upregulated in response to human IgG but not IgM and anti-galactosyl (Gal) 1-3Gal human xenogeneic Abs. In contrast, upregulation of ADAM-10 in ECs was shown to be mostly mediated by anti–Gal1-3Gal IgM Abs. Specific effects of human IgG and IgM xenogeneic Abs on endothelial transcripts indicate that different isotypes and specificities of Abs may mediate different EC changes. Our results suggest that interaction of ECs with antigraft Abs, according to their specificity, selectively induces synthesis and release of metalloproteinases and inhibitors, controlling proteolytic processes and immunological events that respectively contribute to graft rejection or survival.

Key Words: gene expression • endothelial cells • metalloproteinases • mRNA • transplantation

	Introduction

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The endothelium provides a structural barrier between the circulation and surrounding tissues by controlling hemostasis and leukocyte recruitment. Endothelial cell (EC) injury or activation promotes alterations of EC phenotype and function involved in major inflammatory processes, such as vasculitis and vascular graft rejection.^{1 2} Activated ECs exhibit procoagulant activity, express major histocompatibility complex (MHC) antigens, and regulate a broad panel of inflammatory molecules in response to various stimuli, including lipopolysaccharide (LPS), tumor necrosis factor- (TNF-), interleukin-1ß (IL-1ß), interferon-, and complement.^{2 3}

The vascular endothelium plays a central role in antibody (Ab)-mediated graft rejection in allotransplantation^{4 5} as well as in xenotransplantation.^{6 7} Ab reactivity with antigenic determinants on donor vascular ECs results in various processes in the graft vasculature. Detrimental effects of anti-EC Abs on graft survival may result from EC damage through complement-mediated and Ab-dependent cell-mediated cytotoxicity initiating acute vascular rejection of the graft. Ab-mediated complement activation promotes endothelial expression of adhesion molecules and coagulation factors through the synthesis of IL-1.^{8 9} Nevertheless, Ab reactivity with the endothelium can trigger EC activation even in the absence of complement.^{10 11}

Although MHC molecules are presently considered to be the major antigens in alloantibody reactivity with graft ECs, non-MHC alloantigens are also involved in rejection processes.^{12 13 14} Non–MHC-related antigraft Abs are the main players in xenograft rejection processes occurring between discordant species, such as pig to primate.^{7 15} Preformed human natural xenogeneic Abs, mostly IgM directed against the galactosyl (Gal) 1-3Gal epitope on the EC surface, bind to the graft endothelium and activate the complement system, initiating the hyperacute rejection of vascularized xenograft.^{15 16} When hyperacute rejection is prevented through inactivation of the complement system, antigraft Ab–mediated EC activation remains associated with acute vascular xenograft rejection.^{6 7}

Although not fully elucidated, interaction of either alloreactive or xenoreactive Abs with the graft endothelium seems to promote proinflammatory gene expression.^{1 4 11} However, in some situations, a graft can survive in the presence of both antigraft Abs and complement, a situation referred to as accommodation.¹⁷ This phenomenon has been shown to result from the expression of protective genes by the accommodated graft ECs or smooth muscle cells, including the antiapoptotic A20, bclXL, Bcl2, and heme oxygenase-1 genes.¹⁸ It has been shown that binding of IgG antigraft Abs to graft ECs could trigger the expression of these genes.^{17 19} However, effectors and mechanisms allowing graft accommodation remain unclear. Understanding both the specificity of antigraft Abs that lead to endothelial protection and the pattern of genes associated with accommodation could provide new insight for the prevention of graft rejection. In this context, the identification, cloning, and characterization of differentially expressed genes after EC exposure to antigraft Abs should provide relevant and important insight into the molecular events related to graft rejection and survival.

In the present study, we performed RNA differential display (DD) on porcine ECs treated with human serum as a source of anti-EC Abs, LPS, or TNF-. Comparison of the patterns of gene expression between resting and stimulated ECs allowed us to identify, among 70 specifically regulated mRNA species, the tissue inhibitor of metalloproteinase-1 and a disintegrin and metalloproteinase, ADAM-10, as molecules induced after interaction with antigraft Abs. Rapid induction of the tissue-inhibitor of metalloproteinase-1 (TIMP-1) gene and protein expression in the endothelium after Ab binding to ECs has been confirmed both in vitro and in vivo. We also demonstrated that TIMP-1 was upregulated in response to human IgG but not IgM Abs. In contrast, upregulation of ADAM-10 in ECs was shown to be mostly mediated by anti–Gal1-3Gal IgM Abs. To our knowledge, this is the first report showing a selective effect of IgG and IgM human isotypes on endothelial gene expression. These data suggest that antigraft Abs, which, depending on their isotype and specificity, induce specific pattern of metalloproteinase expression on ECs, may contribute to graft survival or rejection.

	Materials and Methods

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Endothelial Cell Culture and Activation
Porcine ECs were isolated and cultured as previously described.²⁰ Activation experiments were performed on confluent EC monolayers using 50 ng/mL LPS (Sigma), 100 U/mL recombinant human TNF- (provided by Professor Neuman, Ludwigshafen, Germany), or heat-inactivated human serum as a source of anti-EC Abs. Four individual human sera (Blood Transfusion Center) were used for RNA DD analysis, and a pool of human AB sera (20 donors, Bioatlantic) was used for all other experiments. These sera do not contain anti-MHC Abs. Absence of LPS and TNF- in the sera was confirmed by E-toxate (Sigma) and ELISA assays (Quantikine, human TNF- immunoassay, R&D System) (data not shown). Human anti–Gal1-3Gal Abs were depleted by repeated adsorptions of pooled human sera onto pig red blood cells (1:1). Purified human IgG and IgM were purchased from Sigma. Pig serum was collected from Large White pigs.

Animal Model
Care and use of animals in the present study were in compliance with institutional guidelines. Male Sprague-Dawley rats (300 to 400 g body weight) purchased from Charles River (Saint-Aubin, les Elbeuf, France) were injected intravenously under anesthesia, with either 10 µg/kg of recombinant rat TNF- (PreproTech) or 1 mL of a pool of human sera. Animals were killed for organ collection 1 hour or 4 hours after treatment.

mRNA Analysis
Total RNA was isolated as previously described²⁰ and treated with DNase I (Boehringer Mannheim). DD reverse transcriptase–polymerase chain reaction (RT-PCR) was performed as previously described²¹ using the RNA image kit (GenHunter Corp). Twenty-four PCR combinations were performed using three 1-base anchored oligod(T) 3' primers and eight random 13-mer 5' primers (GenHunter Corp). Radiolabeled PCR products were separated by electrophoresis through a 6% denaturing polyacrylamide gel. PCR products were eluted from the gel and subcloned using the TOPO TA-cloning kit (Invitrogen) according to the manufacturer’s instructions. cDNAs were sequenced by Appligene (Appligene Oncor Center).

Semiquantitative RT-PCR was performed as previously described.²⁰ Real-time quantitative RT-PCR was performed as previously described²² using an ABI prism 7700 Perkin-Elmer sequence detection system.

Flow Cytometry and Immunochemistry
Rabbit polyclonal anti–TIMP-1 and anti–ADAM-10 Abs were purchased from Chemicon. Cross-reactivity of these Abs with porcine and rat proteins was checked by Western blot analysis (data not shown).

For flow cytometry, ECs were first incubated with either 10% human sera or anti–ADAM-10 Ab (Chemicon Inc) before incubation with FITC-labeled anti-human IgG- or IgM-µ mAbs (Jackson Laboratory) or FITC-conjugated anti-rabbit Abs (Jackson Laboratory), respectively.

For immunofluorescence, ECs were incubated with anti–TIMP-1 Ab (Chemicon Inc), followed by incubation with FITC-conjugated anti-rabbit Ab (Jackson Laboratory) in the presence of 0.5 µg/mL propidium iodide (Sigma). Fluorescence was observed using a laser confocal microscope (TCS SP, Leica).

Immunostaining was performed on heart sections with a standard 3-step indirect immunoperoxidase technique, as previously described,²³ using anti–TIMP-1 (Chemicon Inc) as the primary Ab. Tissue sections were then incubated with biotin-conjugated horse anti-rabbit IgG (Vector). Human IgG binding was revealed using a biotin-labeled goat F(ab')2 anti-human IgG (Fc) Ab (Jackson Laboratory).

	Results

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In Vitro Binding of Human Antigraft Abs to ECs
To investigate endothelial gene regulation in response to antigraft Ab binding, we developed an in vitro model of Ab-EC interaction using human serum as a source of human natural antigraft Abs and cultured primary porcine ECs as target cells. Preformed natural human antigraft Abs included both IgG and IgM directed against pig ECs.¹⁵ For this study, 4 individual sera and a pool of human sera were used. As demonstrated in Figure 1, titration of human antigraft natural Abs from the sera, performed by FACS analysis, showed significant binding of both human IgG and IgM to ECs for the 4 tested sera (S₁, S₂, S₃, S₄), with individual variations as previously demonstrated.²⁴ On average, reactivity of both IgG and IgM from the 4 sera was similar to the reactivity of a pool of human sera (Figure 1). For all experiments, human sera were used after heat inactivation to avoid complement-mediated EC lysis.

View larger version (30K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of human natural antigraft antibody binding to ECs. Porcine ECs (2x105 cells/well) were incubated with 4 individual human sera (S1, S2, S3, and S4) or with a pool of human sera (dilution 1:10). Binding of human natural Abs to ECs was revealed using an FITC-labeled mAb to either human IgG or IgM (black histogram). Controls were performed by omitting the first antibody (white histogram). Fluorescence was measured on 10 000 cells/sample using a FACScalibur. Data were depicted in histograms plotting mean fluorescence intensity on a logarithmic scale (x-axis) vs cell number (y-axis).

RNA DD Analysis
DD RT-PCR was used to identify vascular EC genes differentially regulated in response to human antigraft Ab binding. For RNA DD analysis, ECs were treated by either LPS, human TNF-, or 4 individual human sera for 2, 12, and 24 hours. RNA was isolated from resting and treated ECs, reverse-transcribed, and amplified by PCR using 24 random primer sets. The cDNAs were selected on the basis of their differential pattern of expression using cells incubated with medium supplemented with 10% FCS as a control (resting ECs). To prevent the inclusion of false-positives, only cDNAs regulated by at least 3 of the 4 human sera were selected. In this way, we selected 70 cDNAs differentially expressed on activated ECs. Among them, 17 were specifically regulated by LPS, 22 were specifically regulated by TNF-, and 18 were regulated by both LPS and TNF- (G.B., S.C., F.C., J.-P.S., B.C., unpublished data, January 2000). Furthermore, 13 cDNAs were regulated in ECs incubated with human anti-EC Abs. Most of these genes were also responsive to LPS and TNF (Table). The 13 selected cDNAs were then subcloned and sequenced, and the sequences were submitted to the National Center for Biotechnology Information (NCBI), European Molecular Biology Laboratory, and Expressed Sequence Tags GenBank databases for sequence comparison (http://www.ncbi.nlm.nih.gov; http://www.embl-heidelberg.de).

View this table: [in this window] [in a new window]   Table 1. Identification of Endothelial Genes Differentially Expressed in Response to Human Antigraft Antibodies

Differential Endothelial Gene Expression in Response to Antigraft Ab Binding
Of 13 cDNAs, 6 corresponded to yet-unidentified molecules (data not shown). Seven cDNAs showed a high homology with previously reported molecules (Table). Among the identified sequences, 2 belonged to the metalloproteinase family: TIMP-1 and ADAM-10. According to DD RT-PCR, both ADAM-10 and TIMP-1 expression was induced in ECs treated by LPS, TNF-, and human serum. RT-PCRs, using primers specific for ADAM-10 and TIMP-1, were performed, and PCR products were sequenced to confirm the sequence homologies. The 673-bp PCR product for porcine ADAM-10 showed 94% homology to 829 to 1501 bp of the bovine ADAM-10 sequence (NCBI GenBank accession number Z21961), and the 652-bp PCR product for porcine TIMP-1 showed 99% homology to 48 to 699 bp of the swine collagenase inhibitor (NCBI GenBank accession number S96211).

Differential Expression of TIMP-1 and ADAM-10 on ECs
First, differential expression of TIMP-1 and ADAM-10 transcripts in ECs was confirmed by Northern blot and RT-PCR. Figure 2 shows that TIMP-1 mRNA levels were strongly increased in porcine ECs treated for 2 hours with human sera, with a 15-fold increase in mRNA levels compared with mRNA levels in resting cells. By comparison, upregulation of TIMP-1 transcripts in ECs activated for 2 hours with either LPS or TNF was lower (a 5- and 4-fold increase compared with TIMP-1 expression in resting cells for LPS and TNF-, respectively). In contrast, upregulation of ADAM-10 mRNA levels observed on ECs in response to serum at 2 hours was similar (a 2.5-fold increase compared with ADAM-10 expression in resting cells) to that obtained in ECs activated by LPS and TNF- (corresponding to a 2.7-and 2-fold increase compared with mRNA levels in resting cells for LPS and TNF-, respectively) (Figure 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Upregulation of TIMP-1 and ADAM-10 mRNA levels in ECs. ECs were treated for 2 hours with DMEM 10% FCS as a control or with DMEM 10% FCS containing either 50 ng/mL LPS, 100 U/mL TNF-, or 20% human sera in DMEM. A, Quantitative RT-PCRs were performed using specific primers for porcine TIMP-1 (5' to 3': 5'-GCTGCTGCTGTGGCTGACT, 3'-AC- AGGTGGGAGCGGGAAGA) and ß-actin (5'-ATGTTTGAGACCT- TCAACAC and 3'-CACGTCACACTTCATGATGCA). An aliquot of 100 ng of reverse-transcribed RNA from samples or from standard dilutions was amplified using SYBR green PCR core reagent. PCR was carried out under the following conditions: 1 step of 2 minutes at 55°C followed by 10 minutes at 95°C and 40 cycles of 15 seconds at 95°C followed by 1 minute at 60°C. The exact number of copies was deduced from a comparison of the measured fluorescence with the standard curve. Each sample was analyzed in duplicate. Results are expressed as a ratio of TIMP-1/ß-actin transcript number (x100) in a histogram. B, Semiquantitative RT-PCR was carried out using the following specific primers (5' to 3'): porcine ADAM-10 5'-GGTGAAACG- CATAAGAATC, 3'-TCTGAATCATCCCGACACT and ß-actin 5'-ATGTTTGAGACCTTCAACAC, 3'-CACGTCACAC TTCATGA- TGCA. Hybridization of PCR products (673 bp) was performed using -[32P]-dCTP–labeled probe for ADAM-10. Relative mRNA levels for ADAM-10 were normalized to ß-actin and expressed as arbitrary units. Semiquantitative analysis was conducted by densitometry using Molecular Analyst software.

Early Induction of TIMP-1 and ADAM-10 Mediated by Anti-EC Abs
Kinetic analysis on Ab-treated ECs showed that TIMP-1 mRNA level was maximal at 2 hours and returned to basal levels at 12 hours, whereas TIMP-1 expression on TNF-–activated ECs increased gradually between 2 and 12 hours and reached a maximal level at 24 hours (data not shown). Ab-mediated early induction of TIMP-1 protein expression was also confirmed by immunofluorescence performed on ECs incubated with human serum for 0, 1, 2, 8, 12, or 24 hours. Confocal microscopy scanning showed a strong increase of intracellular TIMP-1 protein levels in ECs incubated for 2 hours with human sera compared with resting cells (0 hours) (Figure 3). TIMP-1 protein expression decreased at 8 hours and returned to basal levels at 12 hours. In contrast, TIMP-1 expression was maximal at 24 hours in TNF-–treated cells (data not shown).

View larger version (76K): [in this window] [in a new window]   Figure 3. Kinetics of antibody-mediated expression of TIMP-1 in ECs. EC monolayers were cultured for 0, 1, 2, 8, 12, and 24 hours in the presence of 20% human serum. TIMP-1 expression was revealed using an anti–TIMP-1 Ab and investigated by indirect immunofluorescence using a confocal laser microscope (magnification x630). Nuclei were stained red with propidium iodide.

TIMP-1 expression on EC activation was also evaluated in vivo in rats 1 or 4 hours after injection of human serum or rat TNF-. First, TIMP-1 mRNA levels in hearts from rats injected with human Abs or TNF- and from control rats were compared by RT-PCR (Figure 4). At 1 hour, the TIMP-1 mRNA steady state was markedly stronger in rats treated with human serum compared with TNF-–treated rats. This returned to a basal level at 4 hours, which corroborates our in vitro findings.

View larger version (99K): [in this window] [in a new window]   Figure 4. Induction of TIMP-1 expression after anti-EC Ab binding to cardiac rat ECs in vivo. A, Semiquantitative RT-PCR. Southern blot after RT-PCR (18 cycles of amplification) analysis of mRNA levels in heart from rats treated for 1 and 4 hours with 10 µg/kg of rat TNF-, 1 mL of human serum, or PBS as a control. RT-PCRs were performed using the following specific primers (5' to 3'): rat TIMP-1 5'-CCAACCCACCCACAGACA, 3'-CCCCACAGCCAGCACTAT and HPRT 5'-TGCTGGATTACA- TTAAAGCGC, 3'-CTTGGCTTTTCCACTTTCGC. B, Immunohistology. Frozen heart sections from rats treated with human sera for 4 hours or with PBS were fixed and stained using a standard 3-step indirect immunoperoxidase technique. Immunostaining of serial tissue sections was performed with an anti–TIMP-1 Ab as primary antibody, followed by an incubation step with a FITC-labeled anti-rabbit Ab. Human IgG binding was revealed using a biotinylated-labeled anti-human IgG antibody. Negative controls were performed by omitting the first antibody (not shown). Tissue sections were counterstained with H&E (magnification x40).

Specific immunostaining on serial cardiac sections clearly showed that TIMP-1 protein was induced in hearts from rats injected with human sera compared with PBS-treated rats and was mostly colocalized with deposits of human IgG on the cardiac vascular endothelium. Therefore, anti-EC Ab interactions with endothelium, both in vitro and in vivo, promote an earlier and stronger expression of TIMP-1 than TNF-. FACS analysis (Figure 5) also confirmed the rapid induction of ADAM-10 expression on EC surface after exposure to antigraft Abs.

View larger version (32K): [in this window] [in a new window]   Figure 5. FACS analysis of ADAM-10 expression on ECs. EC monolayers were treated for 0, 2, 24, 36, and 48 hours with human TNF- or 20% human serum. The cells were then incubated with an anti–ADAM-10 Ab, as first antibody, followed by an incubation step with a FITC-labeled anti-rabbit Ab. Controls were performed by omitting the first antibody (thin histogram). Fluorescence was then measured on 10 000 cells/sample using a FACScalibur. Data were depicted in histograms plotting mean fluorescence intensity on a logarithmic scale (x-axis) vs cell number (y-axis).

Selective Upregulation of ADAM-10 and TIMP-1 in ECs According to Anti-EC Ab Isotype
Although not well understood, human natural IgM and IgG are thought to promote divergent biological changes in porcine ECs.^{11 19} The specific effect of anti-EC Abs on TIMP-1 and ADAM-10 mRNA expression was investigated after a 2-hour incubation of porcine ECs in the presence of purified human IgG or IgM (Figure 6). Human IgG and IgM were used at relevant concentrations (similar to the physiological IgG and IgM concentration in serum diluted 1:5). ECs incubated for 2 hours with medium containing FCS were used as controls. We observed by RT-PCR that human IgG strongly promoted TIMP-1 expression, whereas no expression was observed in ECs incubated with IgM (6.2±1.1- and 1.52±0.8-fold increase in mRNA levels compared with medium alone for IgG and IgM, respectively) (Figure 6). As opposed to TIMP-1, mRNA levels for ADAM-10 increased in ECs incubated with human IgM but were not significantly affected by human IgG (3.1±0.2- and 1.85±0.55-fold increase compared with resting cells for IgM and IgG, respectively).

View larger version (20K): [in this window] [in a new window]   Figure 6. Selective effect of anti-EC Ab isotype on ADAM-10 and TIMP-1 mRNA expression. Semiquantitative RT-PCRs were performed on RNA from ECs incubated for 2 hours with medium or medium containing purified human IgG or IgM. PCR products were blotted onto membranes before hybridization with specific -[32P]-dCTP–labeled cDNA probes encoding ADAM-10, TIMP-1, and ß-actin, respectively. Quantification was conducted by PhosphorImager analysis. Relative mRNA levels for TIMP-1 (black histogram) and ADAM-10 (white histogram) were normalized to ß-actin values and expressed as n-fold increase compared with cells treated with medium. Results are mean±SD of 3 independent experiments. *P<0.05 vs cells treated with medium alone.

Effect of Human Natural Anti–Gal1-3Gal Abs on TIMP-1 and ADAM-10 Expression
In vitro data provide evidence for the direct proinflammatory activation of ECs by human xenogeneic natural Abs.¹¹ These Abs are primarily directed against Gal1-3Gal, the major xenoantigen in the pig-to-primate xenotransplant model. To test whether human anti–Gal1-3Gal Abs could mediate TIMP-1 and ADAM-10 expression, porcine ECs were incubated for 2 hours with the same human serum but either with or without depletion of anti–Gal1-3Gal Abs. As shown in Figure 7, TIMP-1 induction was obtained even in the absence of anti–Gal1-3Gal Abs (5.6±1.2- and 3.9±0.5-fold increase compared with medium-treated cells for anti–Gal1-3Gal–depleted and normal sera, respectively). Statistical analysis demonstrated no significant difference (NS, P>0.05) between normal and anti-Gal–depleted serum for the induction of TIMP-1, confirming that TIMP-1 regulation is not an anti-Gal–dependent process. In contrast, upregulation of ADAM-10 mRNA levels was only observed in the presence of anti–Gal1-3Gal Abs (2.4±0.3-fold increase compared with resting cells, P<0.05). Compared with the induction of ADAM-10 mRNA levels mediated by anti-Gal–containing serum, ADAM-10 mRNA levels in ECs treated with anti-Gal–depleted serum was similar to basal levels observed in resting cells treated with medium. In addition, to discriminate the specific effect of antigraft human Ig to nonspecific serum-dependent effect, porcine artery endothelial cells incubated with porcine serum (prepared in similar conditions as for human and also diluted 1:5 in culture medium) have been used as a negative control. Our data showed no significant increase of transcript level for TIMP-1 and ADAM-10 on cells treated with porcine serum compared with untreated cells (P>0.05). Together, these data suggest that induction of TIMP-1 by human serum specifically resulted from IgG binding but was not an anti–Gal1-3Gal–dependent process. In contrast, the regulatory effect of serum on ADAM-10 was mostly mediated by the anti–Gal1-3Gal human IgM. Taken together, these data suggest that TIMP-1 expression could reflect an IgG non-anti–Gal1-3Gal–mediated EC accommodation pathway, whereas ADAM-10 may be related to IgM anti–Gal1-3Gal–induced EC activation and injury.

View larger version (19K): [in this window] [in a new window]   Figure 7. Selective effect of anti-EC Ab specificity on ADAM-10 and TIMP-1 mRNA expression. Semiquantitative RT-PCR was performed on RNA from ECs incubated for 2 hours with medium or medium containing 20% normal or anti–Gal1-3Gal–depleted human serum or pig serum. PCR products were blotted into membranes before hybridization with specific -[32P]-dCTP–labeled cDNA probes encoding ADAM-10, TIMP-1, and ß-actin, respectively. Quantification was conducted by PhosphorImager analysis. Relative mRNA levels for TIMP-1 (black histogram) and ADAM-10 (white histogram) were normalized to ß-actin values and expressed as n-fold increase compared with cells treated with medium. Results are mean±SD of 3 independent experiments. Statistical significance was determined using Student’s t test. *P<0.05 vs cells treated with medium alone. **P<0.05 vs cells treated with anti-Gal–depleted human serum.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we developed an in vitro model using cultured ECs to mimic the Ab-EC interactions that occur in graft ECs during vascular rejection. By means of RNA differential display, we studied endothelial genes specifically regulated after exposure to antigraft Abs. This interaction is most likely important in the generation of vascular lesions in allotransplantation^{4 14} and in xenotransplantation.^{7 16} We identified a collection of 70 cDNAs differentially expressed compared with resting ECs, depending on the stimuli. Most of the cDNAs (57 of 70) were specifically regulated by LPS or TNF but not by antigraft Abs.

In this study, we focused on 13 of 70 cDNAs that corresponded to mRNA species differentially expressed after Ab-mediated EC stimulation. The selected molecules were secondarily confirmed by RT-PCR, to be regulated on ECs incubated with antigraft Abs. Of 13 cDNAs, 6 corresponded to yet-unidentified molecules, including 4 sequences specifically regulated (mostly downregulated) by antigraft Abs.²⁵ Seven cDNAs showed a high homology with previously reported molecules (Table). These cDNAs encode molecules responsive to Abs but also to TNF- and LPS. These inducible molecules include a transcription factor, ubiquitin-conjugating E2 enzyme variant-1,²⁶ and 2 ribosomal proteins (L9 and L27a) that probably reflect the increase in transcriptional and translational activity of ECs during activation. Two of the cDNAs upregulated by human Abs belong to the metalloproteinase family: TIMP-1, 1 of the 4 inhibitors of matrix metalloproteinases (MMP),²⁷ and ADAM-10, also called MADM,²⁸ a member of a protein family characterized by a disintegrin and a metalloproteinase domain.^{29 30}

TIMP-1 is the inhibitor of MMP-9 (gelatinase B), which contributes to matrix degradation. Under normal physiological conditions, there is a balance between MMP-9 and TIMP-1 that can be modified during the remodeling of basal membranes. Several studies have shown the implication of endothelial TIMP-1 in processes such as angiogenesis³¹ and cell invasion.³² We have demonstrated here that TIMP-1 mRNA and protein are upregulated by antigraft Abs on ECs with different kinetics compared with LPS and TNF-. The induction of TIMP-1 mRNA and protein in the endothelium has also been observed in vivo. In this animal model, induction of TIMP-1 in the cardiac endothelium in response to human antigraft antibody binding was roughly similar in intensity and kinetics to that observed in vitro. In vivo experiments also confirm that the induction of TIMP-1 results from IgG Abs rather than from IgM. IgG xenoreactive antigraft Abs have been shown to induce changes in EC phenotype, including a downregulation of vascular cellular adhesion molecule-1 and MHC class I expression, which can be correlated to the protection of EC against complement-mediated lysis and activation, and to graft accommodation.¹⁹

That induction of TIMP-1 expression on graft ECs could be related to an antibody-mediated protective process is suggested by recent findings showing an inhibitory effect of TIMP-1 on cytokine processing and apoptosis. First, TIMP-1 was shown to inhibit the processing of IL1-ß by MMP-9.³³ IL-1 is known to be the mediator of inflammatory gene expression, including chemokines, on ECs after exposure to human natural Abs and complement.⁸ Therefore, besides having effects on microvasculature structure and permeability, we hypothesize that TIMP-1 could also inhibit the secretion of IL-1ß in activated ECs and, therefore, contribute to the downregulation of inflammation near the activated ECs. Second, recent data demonstrating protective³⁴ and antiapoptotic effects of TIMP-1 overexpression³⁵ suggest that inhibitors of the plasminogen activator/MMP system may contribute to or initiate the regulation of protective genes associated with graft survival, such as bcl_XL and A20, as previously reported.¹⁸ The possible contribution of TIMP-1 to graft accommodation is now being investigated in our laboratory.

As opposed to TIMP-1, upregulation of ADAM-10 expression by IgM anti-Gal Abs may reflect EC activation. Indeed, upregulation of ADAM-10 in response to antigraft Abs was similar in intensity and in kinetics to that obtained in the presence of LPS and TNF-. ADAMs have been implicated in a variety of processes, such as cell-cell and cell-matrix adhesion and proteolysis of the extracellular matrix in a wide variety of cell types.²⁹ ADAM-10 was first identified as a transmembrane protein from bovine brain that was able to degrade myelin and cleave type IV collagen.²⁹ Moreover, ADAM-10, as well as the TNF-–converting enzyme (ADAM-17), has a role in TNF- processing by cleaving the transmembrane pro-TNF to the active soluble form TNF-.²⁸ To our knowledge, neither the expression nor the function of ADAM-10 in ECs has been previously described. We hypothesize that the upregulation of ADAM-10 in activated ECs could play a role in the processing of cytokines or their receptors, such as TNF, IL-1, or IL-6, or in the cleavage of transmembrane proteins, such as FasL, as reported for other metalloproteinases.^{29 30 33} Furthermore, because ADAM-10 was implicated in the notch-signaling pathway, its overexpression in ECs could also promote T-cell differentiation and dendritic cell maturation.²⁹ Whether antibody binding to porcine ECs directly mediates TIMP-1 and ADAM-10 upregulation or leads to the rapid synthesis or release of inflammatory mediators that may contribute to TIMP-1 and ADAM-10 expression remains to be investigated.

In conclusion, our finding that 2 members of the metalloproteinase family, TIMP-1 and ADAM-10, were vigorously and rapidly overexpressed after exposure of ECs to human antigraft Abs suggests that, according to their specificity, Ab binding to the endothelium can selectively modify the expression of numerous endothelial genes, far more than reported to date. Specific effects of human IgG and IgM xenogeneic Abs on some endothelial transcripts also suggest that different isotypes and specificities of Abs may mediate different EC changes. IgM and IgG could therefore give rise to different activation pathways leading to, respectively, a deleterious or protective endothelial phenotype. Whether typical activation pathways mediated by Ab-EC recognition could initiate graft accommodation or rejection is being investigated.

	Acknowledgments

 
This work was supported by a European Economic Community grant (BIO4-CT97-2242) and by the Etablissement Français des Greffes. G.B. received a grant from La Fondation pour la Recherche Médicale. The authors thank Françoise Goret and Yann Godfrin for technical assistance and advice on differential display RT-PCR and Joanna Ashton for editorial assistance.

	Footnotes

 
Original received August 3, 2000; revision received December 18, 2000; accepted January 3, 2001.

	References

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