Experimental immunology
The role of proteinase-activated receptor 3 (PAR3) in mouse hybridoma studied with monoclonal antibody generated against thrombin cleavage site

Introduction

Inflammation is a critical event preceding and affecting the specific immune response. Correspondingly, lymphocytes express receptors for a variety of inflammatory substances and cytokines. Thrombin produced upon tissue damage and inflammation exerts its cellular effects through proteinase-activated receptors (PARs), which mediate cell activation upon receptor cleavage. The cleavage unmasks a peptide motif to function as a tethered ligand for the receptor. Three members of PAR family, PAR1, PAR3 and PAR4, are activated by thrombin, while PAR2 is cleaved by trypsin and tryptase [1]. Human T lymphocytes were shown
to express PARs 1, 2 and 3 [2-3], PAR1 being functionally connected with T cell antigen receptor [4]. The roles
of PAR2 and PAR3 in lymphocytes are currently unknown. A consensus of data regards PAR3 as a cofactor for PAR4 in mouse platelets [5]. However, a differential expression of PAR3 and PAR4 genes in mouse tissues assumes that PAR3 can play an independent physiological role [6]. Unlike other members of the PAR family, PAR3 was not found to be activated by tethered ligand-derived peptide [1]; therefore the development of alternative tools, such as functionally active antibodies is of great importance.
The aim of our experiments was to generate monoclonal antibodies to the fragment 31-47 of human PAR3, which contains the thrombin cleavage site 38-39. Difficulties met upon mAb generation encouraged us studying the expression and function of PAR3 in mouse hybridoma cells.

Results

Mice immunized with hemocyanin-conjugated PAR3 (31-47) peptide produced both IgG and IgM serum peptide-specific antibodies. However, generation of corresponding monoclonal antibodies appeared problematic. Almost all PAR3 peptide-specific hybridoma clones produced IgM, were unstable and lost their activity after several passages in culture. We succeeded to select a single stable clone 8E8 producing mAb of IgG2b subclass, which specifically bound PAR3 peptide with high affinity (Figure 1). This mAb also bound mouse platelets previously reported for PAR3 expression [7] and slightly delayed their aggregation stimulated with thrombin (Figure 2). The latter finding agreed with the results obtained using rabbit PAR3-specific antibodies [7] and PAR3–/– mice [8] and indicated that
mAb 8E8 recognized the native receptor and could interfere with the thrombin effect.
The difficulties in generating mAbs made us suggesting that PAR3 is expressed in hybridoma and can be involved in regulating its vital functions. To test this we studied mAb 8E8 interaction with unrelated hybridoma 1D6. As shown in Figure 3, mAb 8E8 stained hybridoma in flow cytometry, the binding being inhibited by either pre-treating cells with thrombin or pre-incubating the mAb with BSA-coupled PAR3-peptide. The apparent inhibitory effect of BSA alone was due to partial inactivation of mAb in the course of overnight incubation necessary to establish equilibrium in the mAb-peptide binding; however, the effect of peptide-BSA conjugate was significantly stronger than that of BSA.
Thrombin inhibited hybridoma 1D6 proliferation and so did mAb 8E8 by its own (Figure 4). The effect of mAb 8E8 was additional to that of thrombin upon low, but not high thrombin concentrations indicating that thrombin and mAb 8E8 bound the same receptor. Neither PAR4 activating peptide AYPGKF [9] nor its partly inverted analogue YAPGKF influenced hybridoma proliferation, whereas PAR3 antigenic peptide did it similarly to thrombin and mAb 8E8.

Discussion

The results presented demonstrate that mAb 8E8 generated against human PAR3 peptide recognized mouse PAR3. Corresponding human and mouse PAR3 fragments are identical in the N-terminal portion (Figure 1), therefore, the epitope recognized by mAb 8E8 seems to include the AKPTL sequence close to thrombin cleavage site. Such fine specificity underlies the mAb 8E8 thrombin-like activity found. The ligand-like effect of receptor-specific antibodies, which either cross-link receptors [10] or even cleave target proteins [11] is documented. There is no direct data whether mAb 8E8 cleaved or not PAR3 in hybridoma cells. However, some evidence on the mechanism of PAR3/mAb 8E8 functioning was obtained using PAR3/4 peptides.
The thrombin effect on hybridoma proliferation could be mimicked by PAR3 (31-47) peptide, but not by PAR4 activating peptide. Therefore, unlike in platelets, PAR4 was not involved in PAR3-mediated effect in hybridoma cells, while PAR3 tethered ligand (TFRGAP) contained within PAR3 (31-47) was. This fragment was shown to activate PAR1 or PAR2 on human T lymphocytes [2-3]. If it is the case in hybridoma cells, mAb 8E8 should be catalytic to cleave PAR3 and to generate the tethered ligand. If mAb 8E8 is not catalytic, it should be recognized that PAR3 aggregation on hybridoma cell surface is sufficient to trigger receptor signaling. Additional experiments are required to distinguish between these two hypotheses.
In conclusion, we succeeded in generating functionally active PAR3-specific mAb 8E8, which can be used to study PAR3 functions in human and mouse cells. Our data for the first time demonstrate the presence of PAR3 in mouse
B lymphocyte-derived hybridoma and show that this receptor mediates the inhibitory effect of thrombin on cell proliferation. Such finding explains difficulties in generating PAR3-specific hybridomas and suggests that thrombin is able to influence the B lymphocyte-dependent branch of immunity.
Material and Methods

Animals

BALB/c mice used for hybridoma generation were kept in the animal facility of Palladin Institute of Biochemistry, Kyiv. SV129 mice used for platelet isolation were born and bred at DLAR facility at SUNY, Stony Brook, NY. For retroorbital bleeding mice were anesthetized with Metofane, while for spleen removal they were sacrificed by cervical dislocation in accordance with approved protocols of SUNY and Palladin Institute of Biochemistry IACUC.

Cell lines

Mouse hybridoma 1D6 specific to a3 subunit of nicotinic acetylcholine receptor was generated by us previously [12]. The SP-2/0 hybridoma used as a fusion partner was from the stocks of Palladin Institute of Biochemistry. Cell lines including newly obtained hybridomas were grown in RPMI 1640 medium supplemented with 20 mM HEPES, 20 mM L-glutamine, 5 × 10–5 M b-mercaptoethanol, 40 µg/ml gentamicin and 5-10% fetal calf serum (Atlanta, USA)
at 37°C.

Thrombin and synthetic peptides

The bovine thrombin preparation (“Sigma”, T4648) with the established proteolytic activity of 50 to 175 NIH units/mg has been used. The thrombin activity of 0.1, 1 and 10 NIH units/ml approximately corresponded to 1, 10 and 100 nM of thrombin.
The peptides Ac-AKPTLPIKTFRGAPPNS-amide corresponding to 31-47 fragment of human PAR3, AYPGKF and YAPGKF, corresponding to activating PAR4 peptide and its inverted analogue, respectively, and ESKATNATLDPRSFL-LRNPNDKYEPFWEDEEKNESGLTEYRLVSINKSSPLQ-KQLPAFISEDASGYLTSS corresponding to 30-99 fragment of human PAR1 were synthesized at CASM facility at SUNY, Stony Brook. For immunizations and antibody testing PAR3 peptide was coupled to keyhole limpet hemocyanin or BSA using glutaraldehyde as described elsewhere [12].
Hybridoma production and mAb characterization
BALB/c mice were immunized intraperitoneally with 50 µg of PAR3 peptide conjugated to hemocyanin in complete Freund’s adjuvant. The second immunization was performed one month later with the same dose of the antigen emulsified in incomplete Freund’s adjuvant. The sera were tested by ELISA. Mice having high antibody titers were boosted with 50 µg of the antigen without adjuvant four days before the spleen removal.
Hybridomas were generated by fusing the splenocytes of immunized mice with SP-2/0 cells as described [13] and were re-cloned three times by limiting dilutions. The antibodies were purified from the hybridoma culture medium by affinity chromatography on Protein A-agarose (Pharmacia, Sweden) and were biotinylated by a standard procedure using N-hydroxysuccinimido-biotin (Sigma) as a coupling reagent.
The mAb specificity, affinity and isotype were tested by ELISA, the bound antibodies being revealed with goat anti-mouse Fab-specific peroxidase conjugate (Sigma) or with mouse isotype-specific goat antibodies (Sigma) followed by anti-goat peroxidase conjugate. The bound peroxidase activity was developed with o-phenylendiamine-containing substrate solution. Dissociation constants were calculated according to [14] with Stevens’s correction for bivalent antibodies [15].

Flow cytometry

Platelets were purified from the citrated murine blood by centrifugation followed by gel filtration over a Sepharose 2B column equilibrated with HEPES-buffered modified Tyrodes buffer (138 mM NaCl, 2.7 mM KCl, 0.4 mM NaHPO4, 12 mM NaHCO3, 0.2% BSA, 0.1% dextrose,
10 mM HEPES, pH 7.5). The platelets eluted in void volume were diluted with the buffer to a count of 1.3 × 10⁸/ml. Purified platelets or hybridoma 1D6 cells were stained with biotinylated mAb 8E8 followed by Streptavidin-Phycoerythrin conjugate (PharMingen BD). In competition experiments, either the cells were pre-treated with thrombin for 15 min, or mAb 8E8 was pre-incubated with PAR3-BSA conjugate overnight prior to staining. Flow cytometry was performed on FACS Calibur (Beckton Dickinson) in Stony Brook and on EPICS XL (Beckman-Coulter) in Kyiv.

Platelet aggregation

The rested platelets were pre-incubated with mAb 8E8 or mouse IgG2b in a buffer supplemented with 60-100 µg/ml of human fibrinogen and 2 mM MgCl2, and then placed in a whole blood aggregometer (Chronolog, model 570 with Aggrolink software). Aggregation was induced with a range of thrombin doses and was monitored optically at 37°C
for 5 minutes. Control and test samples were run simultaneously for a direct comparison.

Testing hybridoma proliferation

The colorimetric methylthiasol tetrasolium assay [16] was used to study 1D6 hybridoma cell proliferation. Cells were seeded into 96-well culture plates at 5 × 10³ cells per well in the culture medium containing purified antibodies, peptides or thrombin. Methylthiasol tetrasolium solution was added 3 days after for a final concentration of
0.4 mg/ml and cells were incubated for 4 hours at 37°C. The resulting formazan crystals were dissolved in 100 µl/well of dimethyl sulphoxide. Then 0.1M glycine in 0.1M NaCl, pH 10.5, 25 µl/well was added for 2 min and the absorbance at 540 nm was measured using Stat Fax-2100 microplate reader (Awareness Technology INC). Cell numbers that corresponded to the measured absorbance values were calculated using a calibration curve.


Acknowledgements

The work was supported with Civilian Research and Development Foundation (CRDF) grant UB1-2433-KV-02.

References

1. Steinhoff M, Buddenkotte J, Shpacovitch et al. (2005): Proteinase-activated receptors: transducers of proteinase-mediated signaling in inflammation and immune response. Endocr Rev 26: 1-43.
2. Bar-Shavit R, Maoz M, Yongjun Y et al. (2002): Signalling pathways induced by protease-activated receptors and integrins in T cells. Immunology 105: 35-46.
3. Hansen KK, Saifeddine M, Hollenberg MD (2004): Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells. Immunology 112: 183-190.
4. Joyce DE, Chen Y, Erger RA et al. (1997): Functional interactions between the thrombin receptor and the T-cell antigen receptor in human T-cell lines. Blood 90: 1893-1901.
5. Nakanishi-Matsui M, Zheng YW, Sulciner DJ et al. (2000): PAR3 is a cofactor for PAR4 activation by thrombin. Nature 404: 609-613.
6. Hollenberg MD, Compton SJ (2002): International Union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev 54: 203-217.
7. Ishihara H, Zeng D, Connolly AJ et al. (1998): Antibodies to protease-activated receptor 3 inhibit activation of mouse platelets by thrombin. Blood 91: 4152-4157.
8. Kahn ML, Zheng YW, Huang W et al. (1998): A dual thrombin receptor system for platelet activation. Nature 394: 690-694.
9. Major CD, Santulli RJ, Derian CK, Andrade-Gordon P (2003): Extracellular mediators in atherosclerosis and thrombosis. Lessons from thrombin receptor knockout mice. Artherioscler Thromb Vasc Biol 23: 931-939.
10. Kahn CR, Baird KL, Jarrett DB, Flier JS (1978): Direct demonstration that receptor cross-linking or aggregation
is important in insulin action. Proc Natl Acad Sci USA 75: 4209-4213.
11. Xu Y, Yamamoto N, Janda KD (2004): Catalytic antibodies: hapten design strategies and screening methods. Bioorg Med Chem 12: 5247-5268.
12. Skok MV, Voitenko LP, Voitenko SV et al. (1999): Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies. Neuroscience 93: 1437-1446.
13. Harlow E, Lane D (eds.): Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1988.
14. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME (1985): Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 77: 305-319.
15. Stevens FJ (1987): Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody. Mol Immunol 24: 1055-1060.
16. Mossman T (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods 65: 55-63.