IL5 Human, Sf9

What is IL5 Human and why is it produced in Sf9 cells?

IL5 Human is a cytokine that functions as a growth and differentiation factor for both B cells and eosinophils. It serves as a main regulator of eosinopoiesis, eosinophil maturation and activation. The elevated production of this cytokine has been reported in conditions such as asthma and hypereosinophilic syndromes . Human IL5 is a disulfide-linked homodimeric glycoprotein with 115 amino acids per monomer and adopts the typical four α-helical bundle "cytokine fold" structure seen in other cytokines .

Sf9 (Spodoptera frugiperda 9) insect cells are used for IL5 production because they provide several advantages for recombinant protein expression. The baculovirus expression system in Sf9 cells allows for proper folding and post-translational modifications of complex mammalian proteins. For IL5 specifically, Sf9 cells have been shown to produce biologically active protein with structural integrity comparable to native human IL5 . The structure of IL5 purified from Sf9 cells has been determined and confirmed to maintain its functional properties .

What is the amino acid sequence and glycosylation pattern of IL5 Human?

The amino acid sequence of IL5 Human produced in Sf9 cells includes: "ADPIPTEIPT SALVKETLAL LSTHRTLLIA NETLRIPVPV HKNHQLCTEE IFQGIGTLES QTVQGGTVER LFKNLSLIKK YIDGQKKKCG EERRRVNQFL DYLQEFLGVM NTEWIIESHH HHHH" . This sequence represents the complete IL5 protein with a C-terminal His-tag for purification purposes.

What are the storage and stability conditions for IL5 Human from Sf9 cells?

For optimal stability of IL5 Human from Sf9 cells, the following storage conditions are recommended:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• Store frozen at -20°C for longer periods of time

• For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles

The typical formulation consists of IL5 protein solution (0.5mg/ml) containing Phosphate Buffered Saline (pH 7.4) and 10% glycerol . This formulation helps maintain protein stability during storage and handling.

How does the IL5 receptor system function at the molecular level?

The IL5 receptor consists of two components that work in concert to mediate IL5 signaling:

• An IL5-specific α chain (IL-5Rα): A 60-kDa ligand-binding component that specifically binds IL5 with intermediate affinity

• A β chain (IL-5βR or βc): A 130-kDa component that associates with the IL-5/IL-5Rα complex, leading to a high-affinity, signal-transducing receptor complex

The formation of this receptor complex follows a sequential process. First, IL5 binds to IL-5Rα with intermediate affinity. This IL5/IL-5Rα complex then recruits the βc subunit, forming a high-affinity receptor complex capable of signal transduction . The βc subunit is shared with the receptors for interleukin 3 (IL3) and colony stimulating factor 2 (CSF2/GM-CSF), which explains some of the overlapping biological activities of these cytokines .

Sedimentation analysis has revealed a 1:1 association of purified epitope-tagged soluble receptor with its ligand, resulting in a 70-74-kDa complex . Circular dichroism analysis showed that the soluble α chain exists with a significantly ordered structure consisting of 42% β-sheet and 6% α-helix .

What methodologies are used for analyzing IL5 receptor interactions?

Several methodological approaches have been employed to study IL5-receptor interactions:

• Solid-phase binding assays: These involve coating plates with anti-hIgG and loading with soluble IL5Rα-hIgG3 fusion protein. Competition assays can then be performed using serial dilutions of IL5 muteins together with a fixed amount of 125I-labeled IL5 .

• Affinity cross-linking: This technique has been used to demonstrate specific binding of recombinant soluble α chains to 125I-IL5 .

• Scatchard plot analysis: Used with transfected COS-1 cells to determine binding affinities and receptor numbers .

• Surface plasmon resonance: This technique revealed a specific, competable interaction between purified α receptor ectodomains and immobilized rhIL5 with a dissociation constant of 9 nM .

• Bioassays using IL5-dependent cell lines: Two commonly used cell lines are:

  • FDCP1-CA1 cells: Derived from FDC-P1 cells by introducing hIL5Rα cDNA

  • TF1-hIL5Rα cells: A hIL5-dependent derivative of the human erythroleukemia cell line TF-1

These methodologies provide complementary approaches to characterize IL5-receptor interactions from different angles, offering insights into binding affinities, kinetics, and biological consequences.

How do mutations in IL5 affect receptor binding and biological activity?

Mutagenesis studies have provided valuable insights into the functional domains of IL5 and their role in receptor binding and activation. One extensive approach involved an "alanine scan" where charged residues in IL5 were individually replaced with alanine, followed by functional analysis .

What is the mechanism of IL5 antagonism by specific muteins?

The mechanism of IL5 antagonism by muteins like E13Q appears to be dependent on IL5Rα expression levels. Research using two FDC-P1 subclones expressing different numbers of α-subunits but similar numbers of murine βc-subunits demonstrated that:

• E13Q has biological activity comparable to wild-type IL5 only when a high number of α-chains are present on the cells

• Treatment with suboptimal doses of a neutralizing anti-IL5Rα antibody results in reduced activity of the E13Q mutant but not of wild-type IL5

These findings suggest that antagonism occurs through a mechanism related to the efficiency of βc-chain recruitment. When fewer IL5Rα molecules are available, the E13Q mutation appears to impair the ability of the IL5/IL5Rα complex to effectively recruit and engage the βc subunit required for signaling .

This understanding of how receptor density affects agonist/antagonist properties has important implications for the development of IL5-targeted therapeutics for allergic diseases, particularly asthma, where modulation of IL5 activity could provide therapeutic benefits.

How are soluble IL5 receptors generated and purified for research?

Soluble IL5 receptors have been engineered and expressed in baculovirus-infected Sf9 cells using the following approach:

• Engineering receptor constructs: Ectodomain constructs of the α chain (αRED) bearing C-terminal epitope tags are designed and cloned into appropriate expression vectors .

• Expression in Sf9 cells: The constructs are expressed in baculovirus-infected Sf9 cells, with maximum expression typically occurring 72 hours post-infection. The soluble receptor proteins are secreted into the medium .

• Purification: The epitope tag provides a straightforward purification method using immunoaffinity chromatography. This approach allows for the isolation of purified receptor material with an apparent molecular mass of 43 kDa, which is heterogeneously glycosylated .

The expressed soluble receptors have been shown to bind recombinant human 125I-IL5 specifically with an ED50 of 2-5 nM, demonstrating their biological activity . These epitope-tagged recombinant soluble IL5 receptors can be produced in large quantities and maintain their biological activity.

What are the potential therapeutic applications of IL5 pathway modulation?

The involvement of IL5 in allergic diseases, particularly bronchial asthma, has made it a prime target for therapeutic intervention. The understanding of IL5's role in eosinophil development, activation, and the pathogenesis of eosinophil-dependent inflammatory diseases has led to advances in therapeutic options .

Potential therapeutic approaches targeting the IL5 pathway include:

• Anti-IL5 monoclonal antibodies: Intravenous administration of humanized anti-IL5 monoclonal antibodies has been shown to reduce baseline bronchial mucosal eosinophils in mild asthma, providing important implications for treating asthma and other allergic diseases .

• Soluble IL5 receptors: Epitope-tagged soluble IL5 receptors have shown the ability to dose-dependently neutralize rhIL5-induced proliferation in IL5-dependent cell lines, suggesting potential therapeutic utility in modulating IL5-dependent eosinophilia .

• IL5 muteins as antagonists: Muteins like E13Q that can act as IL5 antagonists under specific conditions may offer therapeutic potential, although their activity depends on receptor expression levels .

These approaches target different aspects of IL5 biology and may provide options for treating various eosinophil-related allergic and inflammatory conditions.

What signaling pathways are activated by IL5 and how can they be studied?

IL5 signaling involves multiple intracellular pathways that collectively mediate its biological effects on target cells. The main signaling pathways activated by IL5 include:

• JAK-STAT pathway: Upon IL5 binding to its receptor complex, Janus Kinases (JAKs) are activated, leading to phosphorylation of Signal Transducers and Activators of Transcription (STATs), which then dimerize and translocate to the nucleus to regulate gene expression .

• Btk pathway: Bruton's tyrosine kinase (Btk) is activated following IL5 receptor engagement, contributing to downstream signaling events .

• Ras/Raf-ERK pathway: IL5 activates the Ras/Raf-ERK signaling cascade, which plays a role in cell proliferation and survival responses .

These pathways collectively lead to maintenance of survival and functions of B cells and eosinophils. To study these pathways, researchers employ various techniques:

• Phospho-specific antibodies and Western blotting: To detect activation of specific signaling molecules

• Pharmacological inhibitors: To block specific pathways and assess their contribution to IL5-mediated responses

• Gene knockdown/knockout approaches: To evaluate the role of specific signaling components

• Reporter gene assays: To monitor activation of transcription factors downstream of IL5 signaling

Understanding these signaling mechanisms provides insights into how IL5 exerts its biological effects and identifies potential points for therapeutic intervention.

Which cell types produce IL5 and under what conditions?

IL5 is produced by various cell types under different physiological and pathological conditions:

• T cells: IL5-producing T-cell hybridomas constitutively express 1.7-kb IL5 mRNA, which is augmented by stimulation with PMA plus calcium ionophore . T cells from tuberculosis-primed mice and nematode-infected mice express IL5 mRNA and produce IL5 upon stimulation with PPD and nematode-antigens, respectively .

• T helper 2 (Th2) cells: IL5 is produced by Th2 cells along with IL4 and IL10, distinct from Th1 cells that produce IL2, IFN-γ, and TNF-β .

• Other hematopoietic cells:

  • Eosinophils infiltrating into the mucosa of patients with active coeliac disease

  • Mast cells

  • γδT cells

  • NK and NKT cells

  • Reed-Sternberg cells in Hodgkin's disease with eosinophilia

• Non-hematopoietic cells: Epithelial cells can also produce IL5 under certain conditions .

The specific conditions that induce IL5 production vary by cell type but often involve antigen stimulation, inflammatory signals, or specific pathogen exposure. In Hodgkin's disease with eosinophilia, Reed-Sternberg cells show striking localization of IL5 mRNA, which may explain the associated eosinophilia .

How is IL5 receptor expression regulated in different cell types?

IL5 receptor expression, particularly the α subunit (IL5Rα), is regulated by complex transcriptional mechanisms that vary by cell type:

• In B cells: IL5Rα expression in activated B cells is regulated by a complex of transcription factors including E12, E47, Sp1, c/EBPβ, and Oct2 .

• In eosinophils: Human eosinophils express the IL5Rα subunit, and cDNAs for this receptor component have been isolated from both human eosinophils and the HL60 cell line . The expression of IL5Rα is typically higher on eosinophils compared to other cell types, contributing to the preferential responsiveness of these cells to IL5.

• Soluble receptor forms: Human eosinophils express, through differential splicing, two forms of soluble IL5Rα in addition to the membrane-bound receptor isoform from the same IL5Rα locus . These soluble forms arise from splicing to a soluble-specific exon that precedes the exon encoding the transmembrane domain.

The regulatory mechanisms controlling IL5Rα expression have important implications for understanding the cell-specific effects of IL5 and may offer targets for therapeutic intervention in IL5-mediated diseases.