Expression and purification of recombinant rat eosinophil-associated ribonucleases, homologues of human eosinophil cationic protein and eosinophil-derived neurotoxin, and their characterization.

BACKGROUND: Human eosinophils contain two eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats, 8 homologues of human ECP and EDN have been identified. To clarify the biological activity of rat eosinophil ribonucleases, we cloned rat eosinophil-associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribonuclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacterial expression system. METHODS: As we have already cloned the complete nucleotide sequence for rat EAR-1, we determined that for rat EAR-2 cDNA by the rapid amplification of cDNA ends procedure. Recombinant rat pre-EAR-1 and pre-EAR-2 were expressed in Escherichia coli as N-terminal 6 x histidine-tagged proteins, isolated from the insoluble fraction of the cell lysate and purified by a single-step method using an Ni-NTA resin column after solubilization with a 6 M guanidine solution. RESULTS: The deduced amino acid sequence revealed that the molecular weight of EAR-2 containing the signal peptide is 17.3 kD and the isoelectric point is 8.59. The homology in amino acid sequence between rat pre-EAR-2, and human pre-ECP and human pre-EDN is 51 and 53%, respectively. The purified and refolded recombinant rat pre-EAR-1 and pre-EAR-2 showed bactericidal activity against E. coli and Staphylococcus aureus. CONCLUSIONS: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats.

Analysis of biological activities of recombinant rat interleukin-5.

We analyzed the biological activities of recombinant rat interleukin-5 (IL-5). Purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in eosinophil viability was inhibited in a time- and concentration-dependent manner. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B cell growth factor II, eosinophil differentiation factor, and eosinophil survival-enhancing factor.

Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities.

Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.

Identification of cDNA for rat homologues of human major basic protein and eosinophil cationic protein.

We have determined the complete nucleotide sequence for cDNA of rat homologues of human eosinophil major basic protein (MBP) and eosinophil cationic protein (ECP) using the rapid amplification of cDNA ends (RACE) procedure. Nucleotide sequence of cDNA of rat MBP revealed that mRNA of rat MBP encodes a protein containing 227 amino acids which has three functional domains; namely, the signal peptide, the acidic peptide that contains numerous acidic amino acids and the mature MBP, as in human, guinea pig and mouse MBP. In addition, cDNA of a rat homologue of human ECP was also cloned. The deduced amino acid sequence revealed that this gene encodes a putative protein with a molecular weight of 15.5 kD which has ribonuclease activity. The homology of amino acid sequence between the rat homologue and the murine eosinophil-associated ribonucleases (EARs) was high (65%). Therefore, we named this rat homologue 'rat EAR-1'.

Effects of glucocorticoids on apoptosis of infiltrated eosinophils and neutrophils in rats.

The effects of glucocorticoids on the survival of rat eosinophils and neutrophils infiltrated into the peritoneal cavity were examined. Glucocorticoids including dexamethasone, prednisolone and hydrocortisone inhibited the survival of rat peritoneal eosinophils at 10(-6) M, whereas they prolonged survival of rat peritoneal neutrophils at 10(-8) M. Sex steroids including estradiol and progesterone did not affect cell survival. Dexamethasone decreased the viability of eosinophils after 3 days of incubation and maintained the viability of neutrophils until 4 days after incubation concentration dependently. The EC50 of dexamethasone for inhibition of the survival of eosinophils was 1.5 x 10(-8) M, and that for the spontaneous death of neutrophils was 6.4 x 10(-10) M, suggesting that glucocorticoids at concentrations that inhibit eosinophil survival prolong neutrophil survival. Analysis of DNA fragmentation of cultured eosinophils and neutrophils revealed that glucocorticoids enhance eosinophil apoptosis but inhibit neutrophil apoptosis. The effects of dexamethasone on viability and DNA fragmentation were counteracted by the glucocorticoid receptor antagonist, mifepristone, concentration dependently. These findings indicate that glucocorticoids induce contradictory effects via the glucocorticoid receptor on rat eosinophils and neutrophils extravasated to an inflammatory locus such as the peritoneal cavity by modulating apoptosis.

Increase of eosinophilic cell population in bone marrow of rats by immunization with Ascaris suum antigen.

Immunization of rats with the antigen, Ascaris suum extract, increased the number of peripheral eosinophils. Analysis by Western blot and reverse transcription-polymerase chain reaction revealed that the levels of major basic protein and its mRNA in the bone marrow were also increased, suggesting that eosinophilic cell population in the bone marrow is increased by the immunization. These findings indicate that immunization with this antigen stimulates differentiation of progenitor cells to eosinophils in the bone marrow, and induces blood eosinophilia.

Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease.

Using the rapid amplification of cDNA ends (RACE) procedure, we have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil cationic protein (ECP)/eosinophil-associated ribonuclease (EAR). The deduced amino acid sequence revealed that the molecular weight of rat preECP/EAR is 18.0 kDa and the isoelectric point is 9.85, indicating that rat ECP/EAR is highly cationic. The homology of amino acid sequence between rat ECP/EAR and human ECP is 54%, and that between rat ECP/EAR and human eosinophil-derived neurotoxin (EDN) is 51%. Rat ECP/EAR is also homologous to human ribonuclease k6 (homology 47%).

Cloning of cDNA for rat eosinophil major basic protein.

We have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil major basic protein (MBP) using the rapid amplification of cDNA ends (RACE) procedure. The deduced amino acid sequence revealed that the rat prepro-MBP has three functional domains, namely the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP, as in human and guinea pig MBP.

Isolation and partial characterization of eosinophil granule proteins in rats--eosinophil cationic protein and major basic protein.

Peritoneal eosinophilia was induced in rats using Ascaris suum extract as an antigen, and characteristics of granule proteins of eosinophils collected from the peritoneal cavity were investigated. Peritoneal eosinophilia was induced by injection of the antigen solution into the peritoneal cavity of the immunized rats that had been orally administered with cyclophosphamide. Peritoneal cells were collected 48 h after injection of the antigen solution, incubated in plastic dishes, and nonadherent cells were collected as an eosinophil-rich fraction, from which granule proteins were extracted. Granule proteins were then purified by cation exchange chromatography, gel filtration, copper chelate affinity chromatography, and reverse-phase HPLC. Two distinct basic proteins of which molecular weights are 18- and 17-kD were obtained. Partial N-terminal amino acid sequence analysis revealed that the 18-kD protein and the 17-kD protein were homologous to the human eosinophil cationic protein (ECP) and the human and guinea pig major basic protein (MBP), respectively. Both the two proteins showed strong bactericidal activity against Escherichia coli and Staphylococcus aureus. These results indicate that rat eosinophils also possess ECP and MBP in their granules as well as human and guinea pig eosinophils.