Recombinant Human Interleukin-5 protein (IL5) (Active)

What is Recombinant Human Interleukin-5 and what are its primary biological functions?

Recombinant human IL-5 (rhIL-5) is a laboratory-produced version of the natural cytokine interleukin-5. Initially identified for its ability to support mouse B cell growth and terminal differentiation into antibody-secreting cells, rhIL-5 exhibits pleiotropic activities on various target cells . Its most characterized function is regulating eosinophil development, activation, and survival.

The primary documented biological activities of rhIL-5 include:

• Selective stimulation of eosinophil morphological changes and functional activation

• Induction of eosinophil differentiation from precursors in both human and mouse bone marrow

• Contribution to eosinophil chemotaxis and calcium flux

• Support of B cell survival and function through specific signaling pathways

Functionally, purified rhIL-5 has been demonstrated to induce comparable eosinophilia in BALB/c mice as observed during parasitic infection with Mesocestoid corti .

How is the structure of Recombinant Human IL-5 characterized?

Human IL-5 is encoded by a cDNA that produces a polypeptide consisting of 134 amino acid residues, including a 19-residue N-terminal signal peptide . The mature secreted protein has a molecular mass of approximately 12.3 kDa before glycosylation. Critical structural features include:

• Three putative N-glycosylation sites

• Three conserved cysteine residues important for tertiary structure

• Significant homology with mouse IL-5 (70% amino acid sequence homology)

The functional form of IL-5 is a homodimer, which is crucial for its biological activity. The structural integrity of this dimeric configuration is essential for receptor binding and subsequent signaling.

Which cells produce IL-5 and what cell populations respond to it?

Production sources:

• T helper type 2 (Th2) cells (primary adaptive immune source)

• Innate IL-5-producing cells (lineage-negative, c-Kit+, Sca-1+, T1/ST2+ cells)

• Granulocytes

• Natural helper cells

• Macrophages in coronary artery plaque

Notably, strain differences exist in the localization of innate IL-5-producing cells, with C57BL/6 mice showing higher proportions in the lung compared to BALB/c mice, which may explain strain differences in asthma pathogenesis .

Responsive cell populations:

• Eosinophils (primary target, expressing IL-5Rα)

• Basophils

• Mouse B-lineage cells (including BCL1 lymphoma cells and IL-5-dependent B cell lines like LyH7.B13)

• Human airway smooth muscle cells (express IL-5 receptors when passively sensitized with atopic serum)

Interestingly, while rhIL-5 affects mouse B cell lines, it demonstrates no measurable activity on human tonsillar B cells in terms of CD23 expression, anti-μ costimulated proliferation, or immunoglobulin production .

What are the optimal methods for measuring IL-5 in biological samples?

Accurate measurement of IL-5 in biological samples presents several methodological challenges. For sputum samples specifically, the following optimized protocol increases recovery:

• Add protease inhibitors (PI) to sputum samples during processing (increases IL-5 recovery by approximately 24%)

• Process sputum using the selection method with dithiothreitol (DTT) dispersion

• Analyze using commercial ELISA systems calibrated with appropriate standards

Important considerations:

• Storage of IL-5-spiked sputum significantly reduces recovery rates

• Adding blocking protein does not further increase recovery beyond PI addition

• Adding PI to DTT-processed sputum does not affect total cell count, viability, or differential cell counts

For serum/plasma measurements, commercial ELISA kits provide reliable quantification, though sensitivity limits should be considered when analyzing samples from healthy subjects where IL-5 may be present at extremely low concentrations.

How can researchers validate the bioactivity of recombinant human IL-5?

Bioactivity validation of rhIL-5 can be performed through several complementary assays:

• Cell proliferation assays:

  • Using the mouse IL-5/3-dependent B cell line LyH7.B13, which is at least 10-fold more sensitive than BCL1 mouse lymphoma cells

  • This cell line responds specifically to IL-5 but not to IL-1, IL-2, IL-3, IL-4, IL-6, IFN-γ, or GM-CSF

• Eosinophil differentiation assay:

  • Measuring eosinophil differentiation from precursors in human or mouse bone marrow cultures

  • Quantifiable through morphological assessment and cell surface marker analysis

• Functional eosinophil assays:

  • Eosinophil activation (shape change, adhesion molecule expression)

  • Degranulation responses

  • Chemotaxis assays

• In vivo bioactivity:

  • Induction of blood eosinophilia in mice (e.g., BALB/c) following 7 consecutive days of rhIL-5 injection

  • Assessment of tissue eosinophil infiltration

What are key considerations for experimental design using recombinant human IL-5?

When designing experiments with rhIL-5, researchers should consider:

How does IL-5 contribute to asthma pathophysiology and what therapeutic implications does this have?

IL-5 plays a central role in asthma pathophysiology through several mechanisms:

• Eosinophil-mediated inflammation:

  • Drives eosinophil differentiation, activation, and tissue infiltration

  • Prolongs eosinophil survival in tissues

  • Contributes to airway remodeling through eosinophil-derived mediators

• Airway hyperresponsiveness mechanisms:

  • IL-5 elicits cholinergic-type hyperresponsiveness in normal human bronchus and smooth muscle

  • This effect can be blocked by IL-5 receptor antibodies

  • Human airway smooth muscle expresses IL-5 receptors when sensitized by serum from atopic patients

• Systemic vs. local IL-5 effects:

  • The main determinant of airway inflammation appears to be the level of circulating IL-5, not locally produced IL-5

  • This provides rationale for systemic anti-IL-5 therapies

Therapeutic implications:

• Anti-IL-5 biologics have demonstrated efficacy in severe asthma

• In real-world clinical settings, 58% of patients treated with anti-IL-5 biologics achieve complete response after 12 months

• Complete responders experience greater improvements in forced expiratory volume in 1 second (FEV1) and Asthma Control Questionnaire scores compared to non-complete responders

Predictors of complete response include age at onset, less severe disease at baseline (no maintenance oral corticosteroids, lower ACQ score), and higher blood eosinophil counts .

What is the role of IL-5 in cardiovascular diseases?

Recent research has revealed an unexpected role for IL-5 in cardiovascular diseases:

What are the current therapeutic approaches targeting IL-5 and their comparative efficacy?

Several therapeutic approaches targeting the IL-5 pathway have been developed for treating eosinophilic conditions:

• Anti-IL-5 antibodies:

  • Directly neutralize circulating IL-5

  • Examples include mepolizumab, which has been shown to reduce baseline bronchial mucosal eosinophils in mild asthma

  • Administration to asthmatics significantly reduces both blood and sputum eosinophil counts

• Anti-IL-5 receptor antibodies:

  • Benralizumab: a humanized monoclonal antibody against IL-5Rα (interleukin 5 receptor alpha subunit) present on eosinophils and basophils

  • Blocks IL-5 binding to its receptor and induces antibody-dependent cell-mediated cytotoxicity of eosinophils and basophils

• Comparative clinical outcomes:

  • In real-world clinical settings, more than half (58%) of patients treated with anti-IL-5 biologics achieve a complete response after 12 months

  • Complete responders show superior effects on lung function and symptoms compared to non-complete responders

  • Real-life experience suggests that anti-IL-5 biologics may have similar or even superior effects to those shown in randomized controlled trials

• Response predictors:

  • Complete response to anti-IL-5 therapy can be predicted by several factors:

    • Age at asthma onset

    • Less severe disease at baseline

    • Higher blood eosinophil counts

What are the primary technical challenges when working with recombinant human IL-5?

Researchers face several technical challenges when working with rhIL-5:

• Stability and storage considerations:

  • Protein degradation during storage affects bioactivity

  • Presence of proteases in biological samples can reduce IL-5 recovery

• Measurement difficulties:

  • Methodological problems affect IL-5 measurement in sputum samples

  • Dithiothreitol (DTT), commonly used for sputum processing, may affect protein structure and detection

  • Low circulating levels in healthy individuals challenge detection limits

• Species differences:

  • While rhIL-5 affects both human and mouse eosinophil precursors, its effects on B cells differ significantly between species

  • These differences must be considered when translating findings between model systems

• Receptor heterogeneity:

  • Expression of IL-5Rα varies between cell types and activation states

  • IL-5Rα expression in activated B cells is regulated by a complex of transcription factors including E12, E47, Sp1, c/EBPβ, and Oct2

How might IL-5 research contribute to understanding immune system evolution?

IL-5 research provides insights into immune system evolution through several avenues:

• Comparative genomics:

  • Human and mouse IL-5 show 77% nucleotide and 70% amino acid sequence homology

  • Conserved structural features suggest evolutionary pressure to maintain function

• Functional divergence:

  • Species differences in IL-5 responsiveness (particularly in B cells) highlight evolutionary adaptation

  • Understanding how and why these differences evolved may reveal fundamental principles of immune system development

• Cross-species receptor-ligand interactions:

  • rhIL-5 functions across species barriers for certain cell types (eosinophils) but not others (B cells)

  • This selective cross-reactivity provides insights into receptor-ligand co-evolution

• Innate lymphoid cell biology:

  • IL-5-producing innate cells in the intestine, peritoneal cavity, and lung (innate IL-5-producing cells) represent an evolutionary ancient immune mechanism

  • These cells respond to IL-25 and IL-33 and enhance mucosal IgA production, connecting innate and adaptive immunity

Beyond asthma and allergy, what other conditions might benefit from IL-5-targeted therapies?

Several emerging applications for IL-5-targeted therapies include:

• Eosinophilic gastrointestinal disorders:

  • Eosinophilic esophagitis

  • Eosinophilic gastroenteritis

  • Hypereosinophilic syndromes

• Cardiovascular applications:

  • Given the finding that IL-5 levels are decreased in coronary artery disease patients

  • IL-5's inhibitory effect on oxidized low-density lipoprotein-induced Th1 and Th17 differentiation suggests potential cardioprotective properties

• Parasitic infections:

  • IL-5's role in eosinophil development makes it relevant for helminth infections

  • Understanding IL-5 biology could lead to novel approaches for treating parasitic diseases

• Eosinophilic granulomatosis with polyangiitis (EGPA):

  • Anti-IL-5 therapies have shown promise in this rare systemic vasculitis

  • EGPA patients were included in real-world studies of anti-IL-5 biologics

How do genetic variations in IL-5 or its receptor affect biological responses?

Genetic variation in IL-5 and IL-5 receptor genes impacts biological responses in several ways:

• IL-5 polymorphisms:

  • Single nucleotide polymorphisms in the IL-5 gene have been associated with:

    • Asthma susceptibility and severity

    • Atopic dermatitis

    • Eosinophil count variations

• IL-5Rα variations:

  • Polymorphisms in IL-5Rα affect receptor expression levels and signaling efficiency

  • These variations may predict response to anti-IL-5 therapies

• Strain differences in animal models:

  • Different mouse strains show variations in:

    • IL-5-producing cell localization (BALB/c vs. C57BL/6)

    • Pathophysiology of allergic airway diseases

    • Underlying mechanisms of inflammation

• Pharmacogenomics implications:

  • Genetic testing may eventually guide selection of patients most likely to benefit from anti-IL-5 therapies

  • Predictive biomarkers of response continue to be an active area of research