Recombinant Mouse Interleukin-5 protein (Il5) (Active)

What is the molecular structure of mouse IL-5 and how does it differ from human IL-5?

Mouse IL-5 consists of a polypeptide chain of 133 amino acid residues, including an N-terminal signal sequence of 20 residues and a secreted core polypeptide with a molecular mass of 12.3 kDa. It contains three putative N-glycosylation sites and three cysteine residues that are important for its structural integrity. Human IL-5 encodes a polypeptide of 134 residues with a 19-residue N-terminal signal peptide. The nucleotide and amino acid sequence homologies between human and mouse IL-5 are 77% and 70%, respectively, indicating significant conservation but with notable species-specific differences . These structural differences can be important considerations when using mouse models to study IL-5 biology with potential human applications.

How does the IL-5 receptor complex function in mouse cells?

The high-affinity receptor for IL-5 is a complex consisting of the ligand-binding IL-5 receptor alpha (IL-5Rα, CD125) and the transmembrane common beta chain (βc/CD131), which is shared with receptor complexes for IL-3 and GM-CSF. Initially, IL-5Rα binds IL-5 at low affinity and then associates with preformed βc oligomers to form the signaling-competent receptor complex . The mouse IL-5Rα consists of a 322 amino acid extracellular domain (ECD) with a WSxWS motif and a four-cysteine motif, a 22 amino acid transmembrane segment, and a 54 amino acid cytoplasmic domain . This complex structure is essential for proper signal transduction and biological activity of IL-5 in mouse experimental models.

What are the primary signaling pathways activated by mouse IL-5?

Mouse IL-5 primarily signals through three major pathways: JAK-STAT, Btk, and Ras/Raf-ERK signaling cascades . Upon binding to its receptor, IL-5 initiates receptor dimerization, leading to activation of JAK kinases associated with the cytoplasmic domains of the receptor. This results in STAT protein phosphorylation, particularly STAT5, which dimerizes and translocates to the nucleus to regulate gene expression. Additionally, IL-5 activates the Btk pathway, which is particularly important in B cell responses, and the Ras/Raf-ERK pathway, which contributes to cell proliferation and survival . Understanding these signaling mechanisms is crucial for researchers developing experimental approaches to modulate IL-5 activity in mouse models.

What are optimal storage and handling conditions for maintaining recombinant mouse IL-5 activity?

To maintain optimal biological activity of recombinant mouse IL-5, researchers should store the protein at -80°C in small aliquots to avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of activity. When working with the protein, it should be kept on ice and diluted in buffers containing carrier proteins such as BSA (0.1-1%) to prevent non-specific binding to laboratory plasticware. For experiments requiring longer incubation periods, researchers should consider supplementing culture media with fresh IL-5 every 24-48 hours due to the protein's potential degradation at physiological temperatures. Implementing these practices helps ensure consistent and reliable results when using recombinant mouse IL-5 in experimental settings.

What concentration ranges of recombinant mouse IL-5 are effective for in vitro studies with different cell types?

Effective concentration ranges of recombinant mouse IL-5 vary depending on the target cell type and experimental endpoint. For eosinophil differentiation studies using bone marrow-derived hematopoietic progenitors, concentrations of 1-10 ng/mL are typically effective . For mature eosinophil activation and survival assays, concentrations of 0.1-5 ng/mL are often sufficient. When studying B cell responses, particularly B-1 cells, higher concentrations (5-20 ng/mL) may be required to observe significant effects on proliferation and antibody secretion . For macrophage activation studies, concentrations of 10-50 ng/mL have shown effects on cytokine secretion, phagocytosis, and survival . Researchers should conduct preliminary dose-response experiments to determine optimal concentrations for their specific experimental systems.

How can IL-5 transgenic and IL-5Rα knockout mouse models be used to study eosinophil-mediated pathologies?

IL-5 transgenic mice, which overexpress IL-5, show marked increases in eosinophil numbers in peripheral blood and eosinophil infiltration in various tissues, making them valuable models for studying eosinophilic disorders such as asthma and allergic inflammation . These mice also exhibit increased B-1 cell numbers with concomitant hypergammaglobulinemia and autoantibody production, providing insights into IL-5's role in humoral immunity . Conversely, IL-5Rα knockout mice show reduced numbers of B-1 cells and eosinophils (approximately one-third of wild-type levels), decreased serum levels of IgM and IgG3, and reduced frequency of IgA-producing cells in mucosal tissues . These models are particularly useful for evaluating the effectiveness of anti-IL-5 therapeutics and for dissecting the specific contributions of IL-5 signaling to disease pathogenesis in conditions such as asthma, where IL-5Rα knockout mice show ameliorated airway hyperreactivity in ovalbumin challenge models .

What is the role of recombinant mouse IL-5 in sepsis models and what are the implications for therapeutic applications?

Recombinant mouse IL-5 has demonstrated unexpected protective effects in polymicrobial sepsis models. Loss of IL-5 increases mortality, tissue damage in the lung, IL-6 and IL-10 production, and bacterial burden during sepsis, while therapeutic administration of IL-5 improves survival . Interestingly, IL-5 administration results in neutrophil recruitment in vivo, and IL-5 overexpression in the absence of eosinophils decreases mortality from sepsis and increases circulating neutrophils and monocytes . Novel data have shown IL-5 receptor expression on neutrophils and monocytes during sepsis, and IL-5 augments cytokine secretion, activation, phagocytosis, and survival of macrophages . Macrophage depletion before sepsis onset eliminates IL-5–mediated protection, highlighting the importance of macrophages in IL-5's protective effects . These findings suggest an eosinophil-independent role for IL-5 in sepsis and potential therapeutic applications beyond allergic diseases.

How do findings from mouse IL-5 studies translate to human clinical applications, particularly in asthma therapy?

Research with recombinant mouse IL-5 has provided valuable insights that have directly translated to human clinical applications, particularly in the treatment of asthma and other eosinophilic disorders. Studies in mice demonstrating IL-5's central role in eosinophil development and tissue recruitment led to the development of humanized anti-IL-5 monoclonal antibodies for clinical use . These biologics, including benralizumab, which targets IL-5Rα, have shown remarkable efficacy in treating severe asthma by reducing eosinophilic inflammation . Benralizumab works by binding to isoleucine-61 residue of domain 1 of human IL-5Rα, preventing IL-5 binding to target cells and subsequent activation of IL-5-dependent signaling pathways . Additionally, through its Fc region, benralizumab binds to FcγRIIIa on natural killer cells, inducing eosinophil apoptosis via antibody-dependent cell-mediated cytotoxicity . Clinical data indicate that more than half of patients treated with anti-IL-5 biologics for severe asthma achieve a complete response to treatment, becoming free from exacerbations .

What are the methodological considerations for detecting IL-5 expression in mouse tissues and cell populations?

Accurate detection of IL-5 expression in mouse tissues and cells requires careful methodological considerations. Researchers have developed IL-5/Venus knock-in mice that allow direct visualization of IL-5-expressing cells through fluorescence microscopy . Using these models, IL-5 expression has been detected in T cells cultured under TH2-skewing conditions and in innate IL-5-producing cells in various tissues including the intestines, peritoneal cavity, and lungs . For researchers without access to these specialized mouse models, techniques such as flow cytometry with intracellular cytokine staining following brefeldin A treatment can identify IL-5-producing cells, although sensitivity may be limited due to rapid secretion. RT-PCR and in situ hybridization have successfully detected IL-5 mRNA in mouse tissues, with in situ hybridization providing information about cellular localization . Importantly, strain differences between C57BL/6 and BALB/c mice affect IL-5 production patterns, with innate IL-5-producing cells residing mainly in the lung of C57BL/6 mice and IL-5-producing TH cells predominating in the peritoneal cavity of BALB/c mice . These strain differences are relevant for asthma pathogenesis studies and should be considered when designing experiments.