Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFNa) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFNa. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFNa inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261410 (P2) of the extracellular domain of subunit 1 of the interferona receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDSPAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFNaB on Daudi cells, but did not block IFNaB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFNaB on Hep 2/c cells challenged with encephalomyocarditis virus. Correspondence


Introduction
Interferons (IFNs) are biologically active proteins produced by cells in response to a variety of stimuli such as microbes, tumors and antigens.IFNs exert antiviral, antimicrobial, antitumor, and immunomodulatory ef-fects.These effects are mediated by the binding of IFN to specific receptors on the cell surface, signaling transduction through the nucleus and activating specific genes (1)(2)(3).IFNs are assigned to two classes, type I and type II IFN, according to their interaction with specific receptors (4)(5)(6)(7).IFNs =, ß and S. Yoon et al.
M share a common receptor called IFN-= receptor (IFNAR), while IFN-C interacts with a distinct receptor (IFN-C receptor).IFN-= comprises at least 13 subclasses of structurally similar glycoproteins encoded by related genes, whereas IFNs-ß, -C and -M have only 1 subclass (3).Abnormal production of IFN-= has been reported in systemic lupus erythematosus (SLE), type I diabetes and in graft-versus-host disease (GVHD) (8)(9)(10).Preble et al. (8) have shown that an acidlabile type of IFN-= is specifically elevated in about 50% of SLE patients.Increased levels of IFN-= were observed in the pancreases of patients with type I diabetes, while other cytokines (TNF-=, IL-1ß, IL-2, IL-4, and IL-6) were not increased (9).The development of autoimmune diseases and severe GVHD complication has been reported to occur after IFN-= therapy in patients with carcinoid tumors or chronic hepatitis C (10)(11)(12)(13)(14). It was shown that irradiated mice undergoing GVHD after allogeneic bone marrow transplant develop high levels of IFN-= activity.In contrast, mice given T-cell-depleted allogeneic bone marrow failed to develop GVHD and had no detectable serum IFN-= activity (15).This evidence indicates that IFN-= may contribute to the pathogenesis of autoimmune diseases and plays an important role in allograft rejection of bone marrow transplants (16)(17)(18).Therefore, the development of agents antagonistic to IFN-= would greatly contribute to new alternatives for the trreatment of these patients.Indeed, injection of polyclonal anti-mouse IFN-=/ß antibodies has reduced the resistance to allogeneic bone marrow transplantation in mice, demonstrating their potential inhibition of IFN-= activity in vivo.However, anti-IFN-= antibodies were able to neutralize only some subclasses of IFN-=.One approach to the inhibition of the action of IFN-= would be the production of anti-IFNAR antibodies that can block the effects of all IFN-= subclasses.Uzé et al. (19) showed that polyclonal antibodies raised against cells expressing the subunit 1 of IFNAR (IFNAR 1) can neutralize the activity of several IFN-= subclasses.Similarly, Benoit et al. (20) observed that monoclonal antibodies directed against the extracellular domain of the IFNAR, which was expressed in Escherichia coli and COS 7 cells, neutralize the biological activity of rIFN-=2, rIFN-=B, rIFN-ß and rIFN-M and of IFN-= purified from leukocytes or Namalwa cells, as well as the IFN present in the serum of AIDS patients.Nguyen et al. (21) have also expressed the extracellular domain of the IFNAR 1 in Escherichia coli as a fusion protein with glutathione-S-transferase and have demonstrated that this recombinant protein inhibits the antiviral and antiproliferative activities of rIFN-=B and competes with the cell surface receptor for IFN-= binding.This information led us to search for inhibitors of IFN-= activity by producing polypeptide fragments of IFNAR 1 and raising antibodies to the fragments.
We report here the production of two polypeptide fragments encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of IFNAR 1 expressed in Escherichia coli.The biological activity of these polypeptides and of their respective antibodies was evaluated using antiproliferative and antiviral assays.

Cell lines
Human Daudi lymphoblastoid cells were provided by Dr. Kath C. Zoon (Food and Drug Administration, Bethesda, MD, USA) and maintained in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS).Hep 2/c cells of the epidermoid carcinoma cell line were provided by Dr. Consuelo L.F. Dias (Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil) and maintained in RPMI 1640 medium supplemented with 5% heatinactivated FBS.

Interferon
Recombinant IFN-=B, with a specific activity of 2 x 10 8 IU/mg, was provided by Dr. David Gangemi (Clemenson State University, SC, USA).

Expression
The two cDNA fragments of IFNAR 1 encoding insert 1 from nucleotides 277 to 780 and insert 2 from nucleotides 781 to 1230 were produced by the polymerase chain reaction using cDNA of IFNAR 1 as template.Specific oligonucleotide primers containing the restriction enzyme sites EcoRI and BamHI for P1 and EcoRI and NdeI for P2 were used.cDNA fragments were inserted into plasmid pRSET A (Invitrogen, San Diego, CA, USA) and the clones were sequenced with a Sequenase TM v2.0 instrument (Amersham, Buckinghamshire, England) (22).Expression of the two fragments of IFNAR 1 cDNA was achieved by transformation into E. coli BL21(DE3)pLysS cells, followed by IPTG induction for 3 h at 37 o C in L-broth containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol.Polypeptide 1 was synthesized with a polyhistidine linker and an enterokinase cleavage site.

Purification
Polypeptides P1 and P2 produced in E. coli as insoluble inclusion bodies were extracted twice by sonication (30 s), using sonication buffer (1 mM EDTA, 100 mM NaCl, 1 mM PMSF, 2 mM DTT, and 100 mM Tris-HCl, pH 9.2, for P1 and pH 7.5 for P2) and once more with sonication buffer plus 1% Triton X-100.The final fraction was solubilized in 8 M urea containing 1 mM DTT, 1 mM EDTA and 100 mM Tris-HCl, pH 9.2, for P1 and pH 7.5 for P2.The ureasolubilized fractions containing polypeptides P1 or P2 were purified by gel filtration chromatography using Sephadex G-100 and G-50 for P1 and P2, respectively.The purified polypeptides were refolded by dialysis against decreasing concentrations of urea (6, 4, 2, 1.5, 1, and 0 M) and against RPMI 1640 medium supplemented with 1% FBS at 4 o C. The protein content of the purified fractions was determined by Lowry's method (23).

Preparation of polyclonal anti-P1 and anti-P2 antibodies
Mice were immunized by sc injection with 200 µg of polypeptides P1 or P2 in complete Freund adjuvant.After 15 days, incomplete Freund adjuvant was also injected sc.Ten days later, the level of specific serum antibodies was assayed by ELISA and the good responders were boosted with polypeptides by sc injection in incomplete Freund adjuvant.Five days later, the serum of the animals was obtained and used as polyclonal antibody after IgG purification through a protein A column.

In vitro inhibition assay of antiproliferative activity of IFN-=B
Antiproliferative assays were performed using Daudi cells.Daudi cells were collected by centrifugation and resuspended in warmed (37 o C) RPMI 1640 medium at a density of 4 x 10 5 cells/ml.Cultures were allowed to equilibrate for 2 h at 37 o C and 100 µl of cell culture was added to each well of a 96-well plate.For the inhibition assay of P1 and P2, 6.25 nM IFN-=B was pre-incubated at 37 o C for 1 h with varying concentrations (0-250 µM) of the polypeptides.The mixtures were added to the Daudi cells and incubated at 37 o C for 3 days.For the inhibition assay of polyclonal antibodies, serial dilutions (10 -1 -10 -5 ) of the polyclonal antibodies were pre-incubated with Daudi cells at 4 o C for 1 h, followed by the addition of 6.25 nM IFN-=B and incubation at 37 o C for 3 days.Cell proliferation was determined by an MTT colorimetric assay (24).

In vitro inhibition assay of antiviral activity of IFN-=B
Antiviral assays were performed using a combination of Hep 2/c cells and encephalomyocarditis virus (EMCV).Polypeptides (0-1,250 µM) were pre-incubated with 2.5 nM IFN-=B at 37 o C for 1~2 h.The mixture was added to 100 µl of Hep 2/c cells (5 x 10 4 cells/100 µl/well) on a 96-well plate and incubated at 37 o C for 1 day.Following incubation, 200 µl of viral suspension was added to all wells and incubated at 37 o C for 25~26 h.The complete (100%) cytopathic effect (CPE) of EMCV on unprotected virus was used as control.The CPE was determined by staining with amido blue black and absorbance measured at 620 nm in a Microelisa reader (25).For the inhibition assay of polyclonal antibodies, serial dilution and preincubation with Hep 2/c cells at 37 o C for 2 h in a 96-well plate were performed.One IU/ 100 µl (2.5 nM) IFN-=B was added to this mixture and incubated at 37 o C for 1 day.Addition of the viral suspension and determination of the CPE were performed as mentioned above.

Statistical analysis
The non-parametric Spearman correlation test and Student t-test were used to detect differences as indicated in the Results section (26).The level of significance was set at P<0.05.

Expression and purification of polypeptides P1 and P2
The cloned plasmids with the two cDNA fragments of the extracellular domain of IFNAR 1 were purified and cleaved with the restriction enzymes to check the presence of the inserted cDNA fragment.The high expression of polypeptides P1 (22 kDa) and P2 (17 kDa) in E. coli BL21(DE3)pLysS was demonstrated by SDS-PAGE under reducing conditions (Figure 1).
Polypeptides P1 and P2, produced as inclusion bodies in E. coli, were submitted to serial sonication steps for the isolation of recombinant polypeptides from bacterial proteins.As shown in Figure 1, the third sonication removed most, but not all, contaminant bacterial proteins.Sonication partially purified the polypeptides and facilitated the next purification step by gel filtration chromatography.The amount of purified and refolded polypeptides obtained by dialysis was 25 µg/ ml for P1 and 15 µg/ml for P2.

Inhibition of the antiproliferative activity of IFN-=B
Based on the sensitivity of Daudi cells to IFN-=B we initially used 25 nM and 12.5 Anti-P1 antibodies inhibited the antiproliferative activity of IFN-=B at dilutions of 10 -3 and 10 -1 and anti-P2 antibodies at dilutions of 10 -3 , 10 -2 and 10 -1 (Student t-test, P<0.05); this inhibition was also dose-dependent (Spearman test, r = 1.0000,P = 0.0028 for anti-P1 and anti-P2 antibodies) (Figure 2).As shown in Figure 3, there were no significant differences in CPE between controls and cells treated with the polypeptides.Polypeptides P1 and P2 inhibited the antiviral activity of 2.5 nM IFN-=B by less than 3%.The polyclonal anti-P1 and anti-P2 antibodies did not block the antiviral activity of IFN-=B, showing the same CPE for IFN-=B on control cells and cells treated with antibodies.The increased CPE at 0.1 antibody dilution observed at the same dilution of a negative serum may have been due to the interference of serum with IFN-= activity.

Discussion
Our main interest in the production of IFN-= inhibitors stems from the finding of the important role of IFN-= in the development of autoimmune diseases and GVHD.In this study, we have expressed two recombinant polypeptide fragments of the extracellular domain of IFNAR 1 in E. coli and their polyclonal antibodies in order to evaluate their in vitro inhibition activity on IFN-=.The recombinant polypeptides P1 and P2 were expressed in the E. coli BL21(DE3) pLysS strain, using the pRSET expression plasmid vector, a pET derivative (plasmid for expression by T7 RNA polymerase) which permits high levels of expression of heterologous gene products and easy purification of recombinant insoluble protein by centrifugation (27).The expression system BL21(DE3)pLysS/pRSET yielded a significant amount of polypeptides even when protein was lost during the purification process.
The purification of the recombinant polypeptides involved three important steps: 1) isolation by serial sonication to separate the insoluble recombinant polypeptides from soluble bacterial proteins, 2) purification of the recombinant polypeptides by chromatography under denaturing conditions, and 3) refolding of the polypeptides by dialysis against decreasing concentrations of denaturant in order to gradually remove the denaturant.To avoid aggregation during dialysis, refolding experiments were performed with lower concentrations of the polypeptides (<100 µg/ml).P1 and P2 inhibited 6.25 nM IFN-=B in a dose-dependent manner in the antiproliferative assays.P1 showed stronger inhibition than P2 at 250 nM, 2.5 µM, and 25 µM.However, the polypeptides did not inhibit the antiproliferative activity of higher concentrations of IFN-=B (12.5 and 25 nM IFN-=B) (data not shown) although they were used at 10,000-fold molar excess over IFN-=B.
It has been reported that the two subunits of IFN-= receptor, IFNAR 1 and IFNAR 2, participate in the binding of IFN-=.The IFN-= binding affinity for the receptor was increased by the co-expression of IFNAR 1 and IFNAR 2, which itself binds weakly to IFN-= (28)(29)(30).Therefore, the need of a high concentration of recombinant polypeptides for inhibition of IFN-=B activity and the absence of response to high concentrations of IFN-=B in our experiments could be due to the lack of synergism of IFNAR 2 in the binding process.In other words, the natural IFNAR 1 on the surface of Daudi cells has more affinity for IFN-= because of the cooperative action of IFNAR 2 than the recombi-nant polypeptide.In our experiments, each inhibition test of the antiproliferative activity of IFN-=B was carried out by incubation for 3 days at 37 o C.During this period, a reversible reaction of IFN-=B with recombinant polypeptides and natural IFNAR may occur.The higher affinity of IFN-=B for natural IFNAR on cells probably allowed IFN-=B to display the antiproliferative activity.Moreover, Raz et al. (31) have suggested the participation of a third factor (accessory elements) encoded by a gene located on chromosome 21q22.2for full IFN-= receptor activity.Therefore, the absence of these factors in the assays could result in the low affinity of the recombinant polypeptides.
Although the two recombinant polypeptides showed low inhibitory activity on IFN-=B in our in vitro experiments, they open the possibility of practical antagonist production in the cytokine research area.In pathological processes associated with cytokine disorders, such as GVHD and autoimmune diseases, the production of a cytokine or antibody has been investigated for therapeutic purpose.
The polyclonal anti-P1 and anti-P2 antibodies inhibited 6.25 nM IFN-=B in a dosedependent manner in the antiproliferative assays.Anti-P1 and anti-P2 antibodies showed similar inhibition of IFN-=B activity (Figure 2), but did not block IFN-=B activity at higher concentrations.These antibodies were able to bind IFNAR on the surface of Daudi cells competing with IFN-=B for receptor binding and proved to be useful in studies of IFNAR binding for the isolation of natural IFNAR and accessory components related to natural IFNAR on the cell surface.
In the inhibition assay of IFN-=B activity by an antiviral test, we used Hep 2/c cells and EMCV.This combination showed high sensitivity to the antiviral activity of IFN-=B, being able to inhibit more than 80% of the viral activity at less than 6.25 nM (1 IU/ 100 µl) IFN-=B.IFN-=B (125 nM) inhibited Daudi cell proliferation by 80% (50 IU/100 µl) and 80% of the viral CPE on Hep 2/c cells at only 6.25 nM, showing its powerful antiviral activity.For this reason, the standard procedure for the identification of neutralizing anti-IFN antibodies is the antiviral neutralization assay according to the recommendations of the WHO Expert Committee on Biological Standardization (32).Nevertheless, it has been shown that the antiproliferative assay is easy to perform, reproducible, and more sensitive than the antiviral neutralization assay for the detection of neutralizing IFN-= antibodies (33).In our experiments, the antiviral assay did not show good sensitivity for the inhibition assay of two recombinant polypeptides and their respective polyclonal antibodies.The blocking activity of the recombinant polypeptides and polyclonal antibodies in the antiviral assay was lower when compared with the antiproliferative assay.This difference in sensitivity between the antiproliferative test and the antiviral test may be due to the different cell lines used presenting different IFNAR and consequently different IFNAR affinity for antibodies or IFN-=B.
The possibility of new therapeutic approaches to autoimmune diseases and GVHD is very important.We have studied the production of IFN-= inhibitors.However, the two recombinant polypeptides showed a low ability to act as IFN-= inhibitors.In further studies, we will investigate the role of IFNAR 2 and their antibodies.The anti-IFNAR 1 and anti-IFNAR 2 antibodies may be used together to totally inhibit the activity of all IFN-= subclasses.

Inhibition of the antiviral
activity of IFN-=B IFN-=B inhibited the CPE of Hep 2/c cells challenged with EMCV in a dose-dependent manner.The CPE of the virus was less than 10% with 125 nM IFN-=B (50 IU/ 100 µl) and 15-25% with 2.5 nM (1 IU/100 µl) IFN-=B, showing the extremely high antiviral activity of IFN-=B on Hep 2/c cells challenged with EMCV.

Figure 3 -
Figure 3 -Effect of polypeptides (A) and of their respective antibodies (B) on the antiviral activity of IFN-=B.Hep 2/c cells (3 x 10 5 /ml) were incubated with 0.125~1250 µM polypeptides or serial dilutions of polyclonal antibodies in the presence of 2.5 nM IFN-=B for 1 day before challenge with encephalomyocarditis virus.The cytopathic effect was determined by staining with amido blue black and absorbance reading at 620 nm.