Identification of a Novel Eosinophil Chemotactic Cytokine (ECF-L) as a Chitinase Family Protein

A novel eosinophil chemotactic cytokine (ECF-L) was purified from the culture supernatant of splenocytes of mice by a combination of anion-exchange chromatography, Procion red-agarose affinity chromatography, size exclusion high performance liquid chromatography (HPLC), and reverse phase HPLC. The NH2-terminal amino acid sequence was determined by direct protein sequencing. An ECF-L cDNA clone of 1,506 nucleotides was isolated from a cDNA library, and the nucleotide sequence predicted a mature protein of 397 amino acids. A recombinant ECF-L showed a level of eosinophil chemotactic activity comparable with that of natural ECF-L, and the activity was inhibited by a monoclonal antibody to ECF-L. ECF-L also attracted T lymphocytes and bone marrow polymorphonuclear leukocytes in vitro, whereas it caused selective extravasation of eosinophils in vivo. ECF-L mRNA was highly expressed in spleen, bone marrow, lung, and heart. A comprehensive GenBank data base search revealed that ECF-L is a chitinase family protein. ECF-L retains those amino acids highly conserved among chitinase family proteins, but Asp and Glu residues essential for the proton donation in hydrolysis were replaced by Asn and Gln, respectively. Although ECF-L contains a consensus CXC sequence near the NH2 terminus akin to chemokine family proteins, the rest of ECF-L shows poor homology with chemokines. A novel eosinophil chemotactic cytokine (ECF-L) was purified from the culture supernatant of splenocytes of mice by a combination of anion-exchange chromatography, Procion red-agarose affinity chromatography, size exclusion high performance liquid chromatography (HPLC), and reverse phase HPLC. The NH2-terminal amino acid sequence was determined by direct protein sequencing. An ECF-L cDNA clone of 1,506 nucleotides was isolated from a cDNA library, and the nucleotide sequence predicted a mature protein of 397 amino acids. A recombinant ECF-L showed a level of eosinophil chemotactic activity comparable with that of natural ECF-L, and the activity was inhibited by a monoclonal antibody to ECF-L. ECF-L also attracted T lymphocytes and bone marrow polymorphonuclear leukocytes in vitro, whereas it caused selective extravasation of eosinophils in vivo. ECF-L mRNA was highly expressed in spleen, bone marrow, lung, and heart. A comprehensive GenBank data base search revealed that ECF-L is a chitinase family protein. ECF-L retains those amino acids highly conserved among chitinase family proteins, but Asp and Glu residues essential for the proton donation in hydrolysis were replaced by Asn and Gln, respectively. Although ECF-L contains a consensus CXC sequence near the NH2 terminus akin to chemokine family proteins, the rest of ECF-L shows poor homology with chemokines. eosinophil chemotactic factor(s) splenocyte-derived ECF phosphate-buffered saline fetal bovine serum regulated on activation normal T cell expressed growth-related oncogene monocyte chemoattractant protein high performance liquid chromatography monoclonal antibody base pair polyacrylamide gel electrophoresis 4-morpholinepropanesulfic acid macrophage inflammatory protein polymorphonuclear leukocyte Many parasites, especially tissue-invasive helminths, cause local eosinophilia as well as systemic eosinophilia in mammalian hosts. For example, Schistosoma mansoni (1.Boros D.L. Warren K.S. J. Exp. Med. 1970; 132: 488-507Crossref PubMed Scopus (387) Google Scholar) Schistosoma japonicum (2.Warren K.S. Boros D.L. Hang L.M. Mahmoud A.A.F. Am. J. Pathol. 1975; 80: 279-290PubMed Google Scholar), Toxocara canis (3.Sugane K. Oshima T. Parasite Immunol. 1982; 4: 307-318Crossref PubMed Scopus (32) Google Scholar), andMesocestoides corti (4.Todd Jr., K.S. Simon J. Dipietro J.A. Lab. Anim. 1978; 12: 51-53Crossref PubMed Scopus (12) Google Scholar) form eosinophilic inflammatory lesions in host tissue at the site where they invade or lay their eggs. The mechanisms by which eosinophils are recruited to local inflammatory sites via the circulation have been elucidated at least in part using eosinophil chemotactic factors (ECFs)1 (5.Owhashi M. Kitagawa K. Horii Y. Ishii A. Curr. Topics Vet. Res. 1994; 1: 37-41Google Scholar). Increased eosinopoiesis in bone marrow is observed prior to the tissue eosinophilia in parasitic infections (6.Spry C.J.F. Cell Tissue Kinet. 1971; 4: 351-364PubMed Google Scholar, 7.Owhashi M. Maruyama H. Nawa Y. Int. J. Parasitol. 1996; 26: 705-711Crossref PubMed Scopus (10) Google Scholar). Indeed, most multipotent hemopoietic stem cells are located in bone marrow (8.Maruyama H. Higa A. Asami M. Owhashi M. Nawa Y. Exp. Hematol. 1991; 19: 77-80PubMed Google Scholar). On the other hand, kinetic studies suggest that the spleen is the site where eosinophils mature (9.Spry C.J.F. Cell Tissue Kinet. 1971; 4: 365-374PubMed Google Scholar), indicating eosinophil influx from the bone marrow to spleen at the maturation stage. Previous studies have shown that a splenocyte-derived ECF (ECF-L) is produced in an antigen-specific manner during the course of parasitic infections accompanying systemic and local eosinophilia (7.Owhashi M. Maruyama H. Nawa Y. Int. J. Parasitol. 1996; 26: 705-711Crossref PubMed Scopus (10) Google Scholar), and CD4−CD8+ T cells play an important role in ECF-L production (10.Owhashi M. Arita H. Niwa A. Parasitol. Res. 1998; 84: 136-138Crossref PubMed Scopus (26) Google Scholar). Although ECF-L can be discriminated from other ECFs in terms of its physicochemical and immunochemical properties (11.Owhashi M. Nawa Y. Int. Arch. Allergy Immunol. 1987; 83: 290-295Crossref Scopus (4) Google Scholar), the molecular structure of ECF-L still remains unclear. To obtain further insight into the immunological functions of ECF-L at the molecular level, cloning of cDNA encoding ECF-L is needed. In this paper, we present the isolation of cDNA encoding ECF-L, the molecular expression of ECF-L, and a structure comparison of ECF-L with related proteins. The results clearly show that ECF-L is a novel eosinophil chemotactic cytokine that belongs to the chitinase protein family. Female C57BL/6 or ddY mice were obtained from Japan SLC (Hamamastu, Japan) and used at 8–10 weeks of age. They were infected intraperitoneally with 30 cercariae of S. japonicum (Japanese strain) or 100 tetrathyridiae of M. corti and were killed by cervical dislocation under ether anesthesia. Soluble egg antigen of S. japonicum was prepared as described previously (12.Owhashi M. Ishii A. J. Immunol. 1982; 129: 2226-2231PubMed Google Scholar). In short, the eggs were harvested from the intestines of S. japonicum-infected mice by enzymatic digestion using Pronase (Kaken, Tokyo, Japan) and collagenase (type I, Sigma Chemical Co., St. Louis, MO). Eggs were suspended in 5.8 mm phosphate-buffered saline (PBS, pH 7.4) and homogenized with a Teflon homogenizer in an ice-chilled water bath. The mixture was centrifuged at 100,000 × g for l h. The supernatant was used as S. japonicum egg antigen. Purification of a major 260-kDa antigen (J1) was performed as described previously (13.Owhashi M. Horii Y. Imai J. Ishii A. Nawa Y. Int. Arch. Allergy Immunol. 1986; 81: 129-135Crossref Scopus (3) Google Scholar). For preparation of soluble M. cortiantigen, tetrathyridiae of M. corti were homogenized with a Teflon homogenizer in an ice-chilled water bath and centrifuged at 100,000 × g for 30 min. The supernatant was used asM. corti antigen. For collection of eosinophil-rich peritoneal exudate cells, ddY mice were infected intraperitoneally with 100 larvae of M. corti. 3 weeks later, peritoneal lavage was harvested. The cells were washed twice with PBS and suspended in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 2% FBS (Life Technologies, Inc.). To remove adherent cells, the cell suspension was incubated in a tissue culture flask (Nunc) at 37 °C for 30 min. The nonadherent cells were used for eosinophil chemotaxis experiments. By this method, more than 1 × 107 eosinophils were collected from one mouse, and the purity of the eosinophils was 50–70%. Major contaminants were macrophages and neutrophils. Neutrophil-rich or macrophage-rich peritoneal exudate cells were obtained from normal ddY mice that received an intraperitoneal injection of 2 ml of 0.1% oyster glycogen (Nacalai Tesque, Kyoto, Japan) 2 h or 48 h before use, for neutrophils or macrophages, respectively. Neutrophil-rich (>90%) or macrophage-rich (40–50%) cell suspensions from three animals were pooled and used for the respective chemotaxis experiments. T lymphocytes for chemotaxis indicator cells were prepared as a T cell line. Female C57BL/6 mice were immunized with 20 μg of ovalbumin (Sigma) in complete Freund's adjuvant. 12 days later, the spleen was removed, ground to single cell suspensions, and washed twice with PBS. The splenocytes were cultured with 30 μg/ml OVA in RPMI 1640 containing 2% FBS. Blast cells were separated on a Ficoll (Amersham Pharmacia Biotech) density gradient and then cultured without antigen for 7 days in RPMI 1640 containing 2% FBS and 5% rat growth factor (supernatant of rat splenocytes cultured with 1 μg/ml concanavalin A in RPMI 1640 containing 2% FBS for 2 days). The cells (5 × 105/ml) were restimulated with 10 μg/ml ovalbumin in the presence of 5 × 106/ml irradiated splenocytes. The blast cells were cultured in RPMI 1640 containing 2% FBS and 5% rat growth factor for 7 days. After washing twice with PBS, they were used for chemotaxis experiments. Chemotactic activities in vitrowere measured as described previously (10.Owhashi M. Arita H. Niwa A. Parasitol. Res. 1998; 84: 136-138Crossref PubMed Scopus (26) Google Scholar) with slight modifications. Briefly, multiwell microchemotaxis chambers (Neuro Probe, Bethesda, MD) were equipped with Millipore membrane filters (Millipore Co., Bedford, MA) with a pore size of 3 μm for eosinophil and neutrophil chemotaxis or 8 μm for macrophage and T lymphocyte chemotaxis. The of indicator cells was to 1 × 106/ml with RPMI 1640 medium containing 2% FBS. was performed at 37 °C for 1 h in neutrophil chemotaxis or for 2 h in or T lymphocyte chemotaxis. The were to M. Am. J. Pathol. Google Scholar). The indicator cells that into the membrane were from a level from the to the The from the was as a and μm from the to of than 20 high at The of cells was in 10 high The chemotaxis were in and at least or was used as chemotaxis were on the of ddY mice that been with 100 tetrathyridiae of M. corti 3 weeks 2 h later, mice were and the injection sites were The tissue were in in at and with and The spleen was from normal or S. japonicum-infected mice 8 weeks and in The cell suspensions were washed with and suspended in RPMI was at 37 °C for h in a 5% and in the presence of purified egg antigen medium was obtained by at × g for 10 min. The supernatant was by a membrane (Millipore and at °C anion-exchange chromatography was at °C using a × with pH was performed with a gradient of in the at a of 10 and were Procion red-agarose (Life Technologies, was in a × and was with pH were the and to the Procion red-agarose The was washed with ml of and then was performed with a gradient of in the at a of 10 and were A was to ml and to a size exclusion × Tokyo, Japan) with PBS. was performed at a of and of ml were A was to ml and to an × was performed with a gradient of in 0.1% acid at °C and a of and were The amino acid sequence was determined by on a protein Female were immunized twice at a in with a of ml of a of the purified ECF-L in PBS and 2 weeks the they were and the serum was of a monoclonal antibody to ECF-L was to a described previously J. Immunol. 1991; PubMed Scopus Google Scholar) with modifications. In short, were immunized twice with the purified ECF-L in adjuvant. The were 2 weeks the and were to A clone was and the was purified from the culture supernatant of by gel chromatography on (Amersham Pharmacia Biotech) for The of was determined as by a The of bone marrow cells was isolated by the J. A. PubMed Scopus Google Scholar). was by (Amersham Pharmacia cDNA was performed using a cDNA (Amersham Pharmacia The were into the site of was then in by of recombinant × were using × antibody as an antibody with the antibody were isolated from the cDNA and into were prepared by using a (Amersham Pharmacia The of were determined by the using an A site was of the and an site of the to obtain a cDNA by and the cDNA was with and was then to The recombinant was cells with a and the was collected days cells were cultured in medium with fetal bovine serum (Life Technologies, at To recombinant proteins, cells at a density of were infected with the recombinant The cells were harvested h washed with a of in pH homogenized in a pH and centrifuged at for 20 min. ECF-L was purified from the supernatant using Procion the as for the of natural ECF-L from the culture supernatant of splenocytes. was performed on polyacrylamide gel by using the of 1970; PubMed Scopus Google Scholar) with protein molecular (Amersham Pharmacia a (Amersham Pharmacia Biotech) was performed as described H. T. J. S. A. PubMed Scopus Google Scholar). were with × and × were with containing and in 100 mm pH from was isolated using Tokyo, Japan) from C57BL/6 mice infected with 100 tetrathyridiae of M. corti 2 weeks 10 μg of from was in gel in and a membrane (Amersham Pharmacia After for 3 h in 5 × and 100 μg/ml at were with for h under were washed three with 1 × containing 0.1% at °C and then to of ECF-L and were used for activity was measured by a using mm (Sigma) in pH as the from (Sigma) was used as a (Life Technologies, chromatography × was performed with PBS at a of and were For of ECF-L the medium by spleen cells obtained from S. japonicum-infected mice was and to a anion-exchange ECF activity was at the of the gradient 1 were by and then to the Procion red-agarose affinity are shown in 1 The major ECF activity was at were and by 1 were pooled and purified further on a reverse phase A single at with the of ECF The purified ECF-L showed on 1 with a molecular of and the eosinophil chemotactic activity was 100 that of the supernatant 1 The cDNA of bone marrow of mice was with an antibody or one was to be The clone was and purified three of antibody The was purified and into shown in sequence revealed that the cDNA was 1,506 with a that a protein 397 amino acids The amino acid sequence the NH2-terminal acid by the NH2-terminal sequence of natural ECF-L as determined by a protein The molecular of mature ECF-L, from the amino acid was is to the one determined by 1 The was than that determined by (11.Owhashi M. Nawa Y. Int. Arch. Allergy Immunol. 1987; 83: 290-295Crossref Scopus (4) Google Scholar). The sequence consensus sites K. J. PubMed Google Scholar). In the mRNA contains an with a the ECF-L was prepared by a expression and purified by a combination of anion-exchange chromatography on affinity chromatography on Procion gel chromatography on and reverse phase HPLC. The purified recombinant protein showed a single on with a molecular of The ECF activity of the recombinant ECF-L was comparable with that of the natural ECF-L and was inhibited by to ECF-L as well as natural ECF-L the of ECF-L as a the chemotactic activity of and was at in the of ECF-L was for the chemotaxis shown in natural and recombinant ECF-L chemotactic activity for T lymphocytes and bone marrow cells as well as for of the bone marrow cells in the membrane showed with In ECF-L a chemotactic activity for mature neutrophils and chemotactic activity for of ECF-L was to that of On the other hand, showed chemotactic activity for mature neutrophils but for bone marrow to ECF-L most of the chemotactic activity of ECF-L for T and bone marrow cells but the chemotactic activities of or To ECF-L eosinophils to into the inflammatory recombinant ECF-L was on the of mice infected with M. corti. shown in of eosinophils were observed at the site where ECF-L was of the leukocytes the were eosinophilic of leukocytes in the were eosinophils in the The of eosinophils with ECF-L. The in eosinophil was observed at as as 5 × of the ECF-L production by splenocytes were in M. shown in production of eosinophil chemotactic activity by splenocytes was as as 2 weeks by in an antigen-specific manner and with The of ECF-L was with the eosinophil chemotactic activity of the culture supernatant of splenocytes the ECF-L mRNA expression of was at 2 weeks M. corti high level mRNA expression was in spleen, bone marrow, lung, and of mRNA were also in and A for ECF-L was in normal mice by whereas a level of mRNA was by reverse in bone marrow, spleen, and in normal mice of the reverse from the spleen, and bone marrow mRNA of ddY were to the sequence of the isolated cDNA clone from the A comprehensive search of GenBank or acid data revealed that ECF-L homology with and and chitinase family in An of 20 or chitinase family was performed using the sequence residues are highly conserved in more than of the 20 chitinase family proteins, and residues are conserved in chitinase family ECF-L ECF-L retains residues of the highly 7 shows an of a where the of the is located T. K. K. T. H. M. H. J. PubMed Google Scholar). essential for activity T. K. K. T. H. M. H. J. PubMed Google Scholar), is conserved in of the and chitinase family other than ECF-L. Although the other essential acid for activity T. K. K. T. H. M. H. J. PubMed Google Scholar), is conserved in of the it is replaced by or in chitinase family that have activity J. PubMed Google Scholar, K. J. 1996; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar, Y. Y. PubMed Scopus Google Scholar, 1994; Google Scholar, L.M. J. PubMed Scopus Google Scholar). In ECF-L, the acid and the acid are by and respectively. 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The of is in terms of the that in the of chemotactic on ECF-L. that eosinophils to cells, and their are by of and their J. Allergy Immunol. 1994; PubMed Scopus Google Scholar). ECFs produced in the inflammatory site play a role in the of eosinophils from and their to the inflammatory In the present we showed that ECF-L the extravasation of eosinophils into the site at as as 1 that be caused by ECF-L an of ECF-L is produced the The in the of ECF-L in in studies be the of mechanisms of in the of J. Allergy Immunol. 1994; PubMed Scopus Google Scholar). to ECF-L is to cause tissue eosinophilia the of this is of ECF-L mRNA was to the where the M. corti larvae as we spleen, bone marrow, lung, and are the of production of mRNA encoding ECF-L. and also that ECF-L on of bone cells ECF-L chemotactic activity for bone marrow results with that ECF-L the chemotactic of eosinophils located in bone marrow (10.Owhashi M. Arita H. Niwa A. Parasitol. 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