Recombinant Mouse Interleukin-1 alpha protein (Il1a)

What is mouse IL-1α and how does it function in the immune system?

Mouse IL-1α is a pleiotropic cytokine and critical amplifier of inflammation in response to both infection and sterile cellular insults. As a key member of the IL-1 family (which comprises 11 members), it functions as an "alarmin" or danger-associated molecular pattern (DAMP) when released during cell death .

IL-1α signals through the IL-1 receptor (IL-1R) to trigger multiple immune responses including:

• Production of inflammatory mediators like cyclooxygenase-2 (COX-2), IL-6, and TNF

• Activation of NF-κB and MAPK pathways

• Regulation of neutrophil-chemotactic factors, particularly the chemokine KC (CXCL1) in mice

• Self-amplification through positive feedback mechanisms

• Physiological manifestations including fever, hypotension, and increased pain sensitivity

The commercially available recombinant mouse IL-1α protein (typically covering amino acids Ser6-Ser161) reproduces these biological activities in experimental systems .

How does IL-1α differ structurally and functionally from IL-1β?

Despite signaling through the same receptor (IL-1R), IL-1α and IL-1β exhibit important differences:

These differences explain why IL-1α and IL-1β can have distinct roles in disease processes. For example, clinical studies showed that anakinra (blocking both IL-1α and IL-1β) improved outcomes in COVID-19, while canakinumab (IL-1β-specific blocker) did not .

What regulates IL-1α expression and activity?

IL-1α expression is regulated at multiple levels:

Transcriptional regulation:

• The Il1a promoter lacks canonical TATA and CAAT box regulatory regions

• Contains binding sites for Sp1 (mediates homeostatic expression)

• Contains binding sites for AP1 and NF-κB (upregulate during inflammation)

• In myeloid cells, requires a long noncoding anti-sense Il1a transcript (AS-IL-1α)

Upregulating stimuli include:

• Toll-like receptor (TLR) agonists

• Inflammatory cytokines (including IL-1α/β, creating positive feedback)

• Oxidative stress

• Fatty acid-induced mitochondrial uncoupling

• Hormones

Post-translational modifications:

• Myristoylation

• Acetylation on Lys82

• Phosphorylation at Ser90

Subcellular localization control:

• Nuclear shuttling via nuclear localization signal (NLS)

• Interaction with HS-1-associated protein X (HAX)-1 promotes nuclear localization

• Cytosolic retention via binding to intracellular IL-1R2

What methodological approaches distinguish IL-1α from IL-1β functions in mouse models?

To distinguish IL-1α-specific functions from IL-1β:

• Genetic approaches:

  • Use IL-1α knockout mice (Il1a-KO) versus IL-1β knockout mice (Il1b-/-)

  • Important: Two different Il1a-KO lines exist (line1 and line2) with different phenotypes regarding IL-1β production

• Pharmacological approaches:

  • Use IL-1α-specific neutralizing antibodies versus IL-1β-specific antibodies

  • Compare effects of recombinant IL-1α versus IL-1β administration

• Temporal analysis:

  • Examine early versus late responses (IL-1α-KO line1 shows reduced IL-1β production only at early timepoints)

• Cell type-specific analyses:

  • Investigate responses in cells primarily expressing IL-1α (epithelial/endothelial) versus IL-1β (immune cells)

• Functional readouts:

  • Measure KC (CXCL1) production (IL-1α-dependent) versus TNF (not IL-1α-dependent)

  • Assess nuclear effects using transcriptomic analysis

How should researchers select between different IL-1α knockout mouse models?

Two distinct IL-1α knockout mouse lines have been described, with important differences:

Selection guidelines:

• For investigating purely IL-1α-specific functions, use Il1a-KO line2

• For chronic inflammation models, either line may be appropriate

• For acute inflammation studies, be aware that Il1a-KO line1 has confounding effects on IL-1β

• Always report which knockout line was used in publications

What are optimal protocols for detecting IL-1α in mouse samples?

For detecting mouse IL-1α in experimental samples:

• Bioassay approach:

  • Measure KC (CXCL1) production in response to samples, as this is specifically IL-1α-dependent

  • Compare responses in wild-type versus IL-1α-deficient cells to confirm specificity

• Subcellular localization:

  • Use cellular fractionation followed by western blotting to distinguish nuclear versus cytoplasmic IL-1α

  • Immunofluorescence with antibodies against the N-terminal domain (containing NLS)

• Stimulation conditions:

  • LPS plus ATP stimulation provides a reliable protocol for measuring IL-1α-dependent responses

  • For sterile inflammation models, use necrotic cells to trigger IL-1α release

• Controls:

  • Include both IL-1α-/- and IL-1β-/- samples to distinguish specific responses

  • Use recombinant IL-1α (typically at 3-7 pg/mL ED50) as positive control

How does IL-1α function as an alarmin in sterile inflammation?

IL-1α serves as a primary alarmin in sterile inflammation through several mechanisms:

• Constitutive expression and immediate bioavailability:

  • IL-1α is constitutively expressed in many cell types

  • Unlike IL-1β, it does not require processing for bioactivity

  • Released immediately upon cell death caused by sterile injury

• Experimental evidence:

  • Neutrophil infiltration into the peritoneal cavity after administration of necrotic cells is IL-1α-dependent

  • This response requires IL-1R signaling in radio-resistant cells

• Regulation during different forms of cell death:

  • During apoptosis: IL-1α is sequestered in the nucleus to prevent release

  • During necrosis: IL-1α is released and triggers inflammatory responses

  • During pyroptosis: Both IL-1α and IL-1β are released

• Methodological approaches to study alarmin function:

  • Use necrotic cell transfer models

  • Compare neutrophil recruitment in wild-type versus IL-1α-/- mice

  • Measure IL-1α-dependent versus IL-1β-dependent inflammatory mediators

What roles does IL-1α play in neuroinflammation and neuroprotection?

IL-1α has demonstrated both neuroinflammatory and neuroprotective functions:

• Neuroprotective effects:

  • Administration of IL-1α is neuroprotective and neuro-restorative following experimental ischemic stroke in mice

  • This was demonstrated by KE Salmeron et al. (2019) using recombinant IL-1α in vivo

• Experimental approaches:

  • Ischemic stroke models using middle cerebral artery occlusion

  • Recombinant IL-1α administration (timing, dose, and route are critical variables)

  • Assessment of neurological outcomes and tissue preservation

  • Measurement of inflammatory mediator production

• Dual roles consideration:

  • IL-1α may have different effects depending on:

    • Timing of intervention (acute versus chronic phase)

    • Concentration of IL-1α (low versus high dose)

    • Type of neurological injury (ischemic, traumatic, neurodegenerative)

How does IL-1α contribute to autoinflammatory disorders?

IL-1α plays critical roles in several autoinflammatory conditions:

• Mouse models of autoinflammation:

  • In the Ptpn6 spin mouse model (develops inflammatory skin disease):

    • Disease development is IL-1α-dependent but IL-1β-independent

    • Neutrophil infiltration is IL-1α-dependent

    • IL-1α is the primary driver of inflammation

• Human autoinflammatory conditions:

  • IL-1α is implicated in skin disorders like systemic sclerosis (SSc)

  • Increased expression of IL-1α is observed in fibroblasts from SSc patients

  • IL-1α is localized in the nucleus, cytoplasm, and cell membrane in affected tissues

• Microbiome influences:

  • Gut microbiota modulation by diet or antibiotics affects IL-1α and IL-1β expression

  • This can influence extra-intestinal autoinflammatory disorders

  • Suggests therapeutic potential in manipulating gut microbiota to modulate IL-1α-driven inflammation

How do findings from IL-1α mouse models translate to human disease?

Translating mouse IL-1α findings to human disease requires careful consideration:

• Comparative effectiveness of IL-1 targeting therapies:

  • Anakinra (blocks both IL-1α and IL-1β) reduced clinical progression in COVID-19 patients

  • Canakinumab (specific IL-1β blocker) failed to improve COVID-19 patient survival

  • This suggests specific and potentially dominant roles for IL-1α in some human inflammatory conditions

• Methodology for translational research:

  • Validate findings using human cells and tissues

  • Compare mouse and human IL-1α expression patterns and regulation

  • Consider species differences in IL-1R distribution and downstream signaling

• Specific human diseases with IL-1α involvement:

  • Systemic sclerosis: Increased IL-1α expression in fibroblasts from skin lesions

  • COVID-19: Differential response to IL-1α/β versus IL-1β-specific blockade

  • Various autoinflammatory conditions where IL-1α may function as the primary instigator

What approaches can distinguish nuclear versus receptor-mediated functions of IL-1α?

Distinguishing IL-1α's nuclear transcription factor activity from its IL-1R signaling requires specialized approaches:

• Nuclear function analysis:

  • Use IL-1α constructs with mutated nuclear localization signal

  • Perform chromatin immunoprecipitation to identify IL-1α binding to DNA

  • Analyze interaction with nuclear proteins (p300, PCAF, GCN5)

  • Employ transcriptomic analysis with nuclear-restricted versus secreted IL-1α

• Receptor-mediated function analysis:

  • Use IL-1R knockout cells/animals while preserving IL-1α expression

  • Compare effects of wild-type IL-1α versus mutants lacking receptor binding

  • Block IL-1R with antagonists while maintaining intracellular IL-1α

• Cell-specific approaches:

  • In vascular smooth muscle cells, IL-1α is retained in cytosol (IL-1R2 interaction)

  • In epithelial cells, IL-1α is primarily nuclear

  • These cell type differences can be exploited to distinguish functions

What are the critical quality control parameters for recombinant IL-1α protein?

When working with recombinant mouse IL-1α:

• Biological activity assessment:

  • The ED50 for IL-1α activity is typically 3-7 pg/mL in standard bioassays

  • Activity should be verified using:

    • Lymphocyte activation assays

    • KC (CXCL1) production in responsive cells

    • IL-1R-dependent signaling activation

• Preparation considerations:

  • Available as carrier-containing (400-ML) or carrier-free (400-ML/CF) preparations

  • E. coli-derived mouse IL-1α typically contains amino acids Ser6-Ser161

  • Storage and reconstitution conditions significantly affect activity

• Application-specific validation:

  • For neuroinflammation studies: verify blood-brain barrier penetration or use intracerebroventricular delivery

  • For infection models: ensure preparation is endotoxin-free

  • For chronic inflammation models: validate stability over experimental timeframe

What experimental controls are essential when studying IL-1α functions?

Essential controls for IL-1α experiments include:

• Genetic controls:

  • Compare responses in wild-type, IL-1α-/-, and IL-1β-/- samples

  • Specify which IL-1α knockout line is being used (line1 vs. line2)

  • Include IL-1R-/- controls to verify receptor dependency

• Temporal controls:

  • Early versus late timepoints (critical due to different kinetics of IL-1α versus IL-1β)

  • IL-1α-KO line1 shows differences in IL-1β production only at early timepoints

• Stimulation controls:

  • Compare sterile versus pathogen-induced inflammation

  • Include pathway-specific controls (e.g., NLRP3 inflammasome activators)

  • Use defined stimulation protocols (LPS+ATP is recommended as a standard approach)

• Readout specificity:

  • KC (CXCL1) production is specifically IL-1α-dependent

  • TNF production is not IL-1α-dependent

  • These differential responses help confirm IL-1α-specific effects