Respiratory syncytial virus protects against the subsequent development of Japanese cedar pollen-induced allergic responses.

Respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans still remain inconclusive. The association between RSV infection and allergic diseases may be dependent on atopic background and previous history of RSV infection. In this study, the influence of the timing of RSV infection on the development of Japanese cedar pollen (JCP)-induced allergic responses was examined. BALB/c mice were intranasally infected with RSV before or after sensitization to JCP. Production of cytokines in the culture fluid of lung parenchyma cells and the level of antigen-specific antibodies in the serum were determined. It became clear that JCP was a strong inducer for the elicitation of Th2-type responses, characterized by production of interleukin (IL)-4 and IL-5 in the lung and JCP-specific IgE antibody in the serum. RSV infection, however, suppressed JCP-induced allergic responses by decreasing the production of Th2-like cytokines and Th2-type antibodies. This phenomenon was observed more clearly in the groups that were infected with RSV, 2 weeks or 2 days before sensitization to JCP. The inhibitory mechanism of RSV infection seems to be due to RSV-induced Th1 type dominant environment, which down-regulated the Th2-type responses subsequently induced by allergen sensitization. On the other hand, JCP-inoculation altered RSV-induced immune responses to shift from Th1- to Th2-type dominance, by inhibiting RSV-induced Th1-like cytokine production. These data provide evidence that under a certain condition, RSV infection may play a protective role in JCP-induced allergic responses.

Molecular cloning, expression, and purification of pig interleukin-5.

Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation. Pig IL-5 was cloned, sequenced, and expressed to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions. These pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated. Total RNA was extracted and reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase chain reaction product. 3' rapid amplification of cDNA ends (3'RACE) and 5'RACE procedures were applied to identify the 3' and 5' ends, respectively. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification. The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M(r) and forms a biologically active homodimer of 28 000 M(r). Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep, horse, mouse, and rat counterparts.

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.

Characterization of a bioassay for detection of recombinant and native ovine interleukin-5.

In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.

Sex-determined resistance against Leishmania mexicana is associated with the preferential induction of a Th1-like response and IFN-gamma production by female but not male DBA/2 mice.

Female DBA/2 mice are relatively resistant to infection with Leishmania mexicana compared with male mice. Following subcutaneous infection with 5 x 10(6) L. mexicana, amastigotes lesion growth in male and female DBA/2 mice was measured and the developing immune responses were monitored both in vitro and in vivo. Over the 10 week duration of the experiment all male DBA/2 mice developed rapidly growing non-healing lesions while female mice either developed no lesions whatsoever or developed smaller slower growing lesions than males. Both male and female mice produced parasite specific IgG2a during the course of the disease. However, significant titres of parasite specific IgG1 antibodies could be detected only in male mice indicating a Th2-influenced response in this sex. Furthermore, female mice, unlike male mice, developed significant parasite induced cutaneous delayed-type hypersensitivity footpad responses, indicating a Th1-influenced response in female mice. Although both male and female DBA/2 mice infected with L. mexicana displayed a significant increase in the number of cells in their draining lymph nodes at week 10 post-infection, no significant differences could be observed in the numbers of CD4+, CD8 + T cells as well as B cells between male and female DBA/2 mice. However. following in vitro stimulation, the lymph node cells from female mice displayed significantly higher antigen specific proliferative responses than the males and produced significant amounts of IFN-gamma which could not be detected in the equivalent culture supernatants from male mice. There were no significant differences in the levels of Th2-associated cytokines IL-4 and IL-5, produced by the lymph node cells of both sexes. Treatment of female DBA/2 mice with IFN-gamma neutralizing antibody following L. mexicana infection resulted in lesion growth equivalent to male mice. Conversely, intralesional injections of murine recombinant IFN-gamma significantly inhibited lesion growth in male mice.

Cytokines in nasal fluids from school children with seasonal allergic rhinitis.

Allergic rhinitis is a particularly good model for studies of cytokine production in vivo. In this study the occurrence of the cytokines IL-4, IL-5, IL-10 and IFN-gamma as well as the soluble receptor for IL-4 in nasal lavage fluids were assayed in 38 school children, with seasonal allergic rhinitis, and 19 healthy age-matched, non-atopic controls, using highly sensitive enzyme immunoassays. IL-4 levels in patients with seasonal allergic rhinitis were markedly increased in comparison with those in non-atopic controls or in atopic patients before the start of the pollen season. In controls, but not in the atopic patients, levels of IFN-gamma and IL-5 were significantly higher in specimens obtained during the pollen season than in those obtained outside the season. The IL-4/IFN-gamma ratios were significantly higher in atopic than in non-atopic subjects and further increased in atopic patients during the season. In addition to IL-4, elevated levels of IL-10 were observed in association with seasonal rhinitis. Following treatment with a topical steroid (budesonide) there was a statistically significant increase of the levels of soluble IL-4 receptor. These findings indicate that nonatopic and atopic individuals react to pollen exposure with distinct cytokine patterns in agreement with the Th1/Th2 concept. Topical steroids may possibly decrease inflammation by increasing the formation of soluble IL-4 receptor.

Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells.

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant baculovirus-infected insect cells expressed high levels of rhIL-5 (5-15 mg/liter culture) of which > 90% was processed to the mature form and secreted into the culture medium. After removing cells by centrifugation, rhIL-5 was purified by first adjusting the culture medium to the calculated pI value of mature IL-5 (pI 7.44) and then passing the conditioned medium through tandem linked anion- and cation-exchange columns. The resulting pass-through fraction contained the rhIL-5 and was devoid of contaminating proteins. An optional hydrophobic-interaction chromatography step effectively concentrated the pure homodimeric N-glycosylated rhIL-5 with a high overall yield (> 90%). N-terminal amino acid sequence determination indicated that cleavage of the human IL-5 leader sequence in insect cells occurred between Ala19 and Ile20. Recombinant human IL-5 prepared by this procedure bound to the high-affinity IL-5 receptor present on an eosinophilic leukemia cell line and elicited a proliferative response in the IL-5-dependent murine B-cell line BCL1. This rapid and simple procedure for the expression and purification of mature rhIL-5 should therefore enable studies requiring large amounts of this cytokine.

Purification, crystallization and preliminary X-ray analysis of murine interleukin-5.

Wild-type and mutant forms of murine interleukin-5 (mIL-5) have been expressed in the baculovirus expression system, purified, and used in crystallization trials. Attempts to obtain diffraction quality crystals of wild-type protein were unsuccessful. The substitution of glutamine for Asn75 preserved biological activity, while removing one of two predicted N-linked glycosylation sites, and the resulting protein was crystallized from polyethylene glycol 8000 at pH 7.8 in two crystal forms. The orthorhombic crystals, which belong to space group P2(1)2(1)2 with cell dimensions a = 55.9 A, b = 83.0 A and c = 52.3 A, diffract to beyond 2.5 A resolution. The second crystal form belongs to a trigonal space group, either P3(1)21 or P3(2)21, with cell dimensions a = b = 62.1 A, c = 129.9 A, and diffracts to about 3.8 A resolution. Each crystal form probably contains one mIL-5 dimer per asymmetric unit.

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 A.

Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin-5 from Escherichia coli.

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.

Role of the C-terminus in the biological activity of human interleukin 5.

Alterations of the C-terminal amino acid sequence of recombinant human interleukin 5 (rhIL-5) caused significant changes in its biological activity. Removal of eight amino acids from the C-terminus of rhIL-5 by CNBr treatment led to a complete loss of biological activity as determined by the BCL1 cell IgM-inducing assay. Oxidation of Met residue located at C-terminus also resulted in a loss of activity. These results suggest that the C-terminal amino acids of rhIL-5 are crucial for its biological actions.

High-level production of murine interleukin-5 (IL-5) utilizing recombinant baculovirus expression. Purification of the rIL-5 and its use in assessing the biologic role of IL-5 glycosylation.

We describe here a recombinant baculovirus expression system useful for high level production of murine recombinant interleukin-5 (rIL-5). In addition, we describe a single-step technique of purification of the rIL-5 from the baculovirus-infected Sf9 cell supernatants, using an anti-IL-5 affinity column. The baculovirus-derived rIL-5 has physical properties and functional activities in various lymphoid cell assays similar to those of natural T cell-derived IL-5 and reacts with anti-IL-5 antibodies. Finally, the rIL-5 is similar to natural T cell-derived IL-5 in manifesting heterogeneous glycosylation; however, glycosylation does not appear to be necessary for biologic function, at least in a lymphoid cell proliferation assay.

Production and purification of recombinant human interleukin-5 from yeast and baculovirus expression systems.

A cDNA for human interleukin-5 (hIL-5) was created from the hIL-5 gene using site-directed mutagenesis to splice out the introns in vitro. This cDNA was expressed in yeast and baculovirus systems, utilizing in both cases an in-frame fusion to the pre sequence of the alpha-mating-type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum-containing medium (2.7 mg/l), whereas in serum-free medium ten times less hIL-5 was produced. In the yeast system much lower levels of hIL-5 were produced (12.5 micrograms/l). Recombinant hIL-5 was purified to homogeneity from serum-free baculovirus cultures. The rhIL-5 consisted of a 30-kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL1 cells of 1.5 x 10(4) U/mg, of 3 x 10(5) U/mg in the murine eosinophil differentiation factor assay, and 2.4 x 10(7) U/mg in a human peripheral eosinophil maintenance assay.

Structural comparison of murine T-cell (B151K12)-derived T-cell-replacing factor (IL-5) with rIL-5: dimer formation is essential for the expression of biological activity.

T-cell-replacing factor (TRF)/IL-5 is a T-cell-derived glycoprotein which has pleiotropic activity on lymphoid and myeloid cells. IL-5 polypeptide translated into Xenopus oocytes are heterogeneous in molecular size (40,000 to 60,000 under nonreducing conditions) and yields a monomeric form (Mr of 25,000 to 30,000) under reducing conditions (J. Immun., 140, 1175-1181, 1988). We purified T-cell-derived TRF and rIL-5 using anti-TRF/IL-5 antibody-coupled affinity column from supernatants of a T-cell hybridoma B151K12 and supernatants of HeLa cells, respectively, which had been transfected with murine IL-5 cDNA, and determined their partial N-terminal amino acid sequence (27 residues for B151-TRF and 13 residues for rIL-5). A single amino acid sequence of each sample obtained beginning from methionine that was identical to that predicted from IL-5 cDNA. This finding supports the notion that secreted B151-TRF polypeptide consists of 113 amino acids. Purified B151-TRF supported eosinophilopoiesis of human bone marrow cells as effective as mouse rIL-5 and human rIL-5. B151-TRF competitively inhibited 35S-labeled rIL-5 binding to target cells to the same extent at rIL-5. Treatment of purified rIL-5 and B151-TRF with reducing reagents such as 2-ME, sodium borohydride or dithiothreitol produced a monomeric form of IL-5 which did not exert a biological activity. Reduction and alkylation of rIL-5 caused the loss of binding to its target cells. These results strongly suggest that B151-TRF exists as a homodimer and its primary structure and secondary structures are identical to those of rIL-5. Moreover, the formation of inter-molecular disulfide bond(s) linked by two pairs of cystein residues is essential for the expression of the biological activity of mouse IL-5.

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells.

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.