IL1A Human, His Active

What is IL1A and what are its basic functions?

IL1A (Interleukin 1-alpha) is a cytokine of the interleukin 1 family encoded by the IL1A gene in humans. It is responsible for the production of inflammation and the promotion of fever and sepsis. IL1A binds to the interleukin-1 receptor (IL-1R) and functions in the pathway that activates tumor necrosis factor-alpha. This cytokine is also known by multiple alternative names including hematopoietin 1, fibroblast-activating factor (FAF), lymphocyte-activating factor (LAF), and endogenous pyrogen (EP), reflecting its pleiotropic biological functions discovered over decades of research . IL1A possesses metabolic, physiological, and hematopoietic activities, and plays a central role in regulating immune responses, particularly as an "alarmin" that signals tissue damage and initiates inflammatory cascades .

How is IL1A expressed and regulated in human cells?

IL1A is constitutively expressed in epithelial, endothelial, and stromal cells and can be upregulated in both hematopoietic and non-hematopoietic cells by various stimuli. These stimuli include Toll-like receptor (TLR) agonists, inflammatory cytokines (including IL1A itself), oxidative stress, fatty acid-induced mitochondrial uncoupling, and hormones . The Il1a promoter lacks canonical TATA and CAAT box regulatory regions but contains binding sites for the transcription factor Sp1, which mediates expression during homeostasis. During inflammatory stimulation, binding sites for AP1 and NF-κB transcription factors upregulate IL1A expression . Additionally, in myeloid cells, a long noncoding anti-sense Il1a transcript (AS-IL-1α) is required for Il1a transcription, though the exact mechanism remains unclear . Translation of mRNA for IL1A is highly dependent upon mTOR activity, and IL1A and NF-κB mutually induce each other in a positive feedback loop .

What is the difference between precursor and mature forms of IL1A?

A distinctive feature of IL1A is its bioactivity in both precursor (pro-IL1A) and mature forms, unlike IL1B which requires inflammasome activation for maturation and bioactivity. The IL1A precursor has a molecular weight of approximately 30,000 Da . Both pyroptotic and necrotic cell death can release bioactive IL1A. Studies comparing the biological activity of recombinant IL1A precursor to its mature form have demonstrated that the precursor is indeed biologically active . This unique property makes IL1A bioavailable in a broader range of cellular scenarios compared to other cytokines that require specific processing for activation . The mature form results from cleavage by inflammatory proteases, though the functional significance of this cleavage is not fully understood .

How can His-tagged recombinant IL1A be used in experimental designs?

His-tagged recombinant IL1A provides researchers with a purified protein that can be used in various experimental applications. The histidine tag allows for easier purification through metal affinity chromatography and for tracking the protein in experimental systems. When designing experiments with His-tagged IL1A, researchers should consider the following approaches:

• Receptor binding assays: Using His-tagged IL1A to study its binding kinetics with IL-1R through techniques such as surface plasmon resonance

• Cell stimulation experiments: Applying purified His-tagged IL1A to cell cultures to analyze downstream signaling cascades

• In vivo models: Administering His-tagged IL1A to animal models to study inflammatory responses

• Structural studies: Using the purified protein for crystallography or other structural biology approaches

Researchers should validate that the His-tag does not interfere with the biological activity of IL1A in their specific experimental context, as the tag could potentially affect protein folding or receptor binding in some applications .

What methodologies are recommended for studying IL1A-dependent inflammatory responses?

To study IL1A-dependent inflammatory responses, researchers can employ multiple complementary approaches:

• Cell culture models: Stimulate relevant cell types (macrophages, epithelial cells, endothelial cells) with recombinant IL1A and measure production of pro-inflammatory mediators such as COX-2, IL-6, and TNF through ELISAs, qPCR, or protein arrays .

• Sterile injury models: Administer necrotic cells to peritoneal cavity in animal models and assess neutrophil infiltration. IL1A-dependent responses can be confirmed using IL1A-neutralizing antibodies or IL1A-deficient animals .

• Infection models: Challenge cells or animals with pathogens such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, or Staphylococcus aureus and analyze IL1A-dependent host defense mechanisms using gene knockout models or neutralizing antibodies .

• Transcriptional profiling: Use RNA-sequencing or microarray analysis to identify genes regulated by IL1A signaling.

• Neutrophil recruitment assays: Measure IL1A-induced neutrophil infiltration in tissues through flow cytometry or immunohistochemistry, particularly following infections with organisms like Candida albicans .

These methodologies should be selected based on the specific research question, with appropriate controls to distinguish IL1A-specific effects from other inflammatory mediators.

How does IL1A interact with other cytokines in experimental systems?

IL1A exhibits synergistic interactions with multiple cytokines, most consistently and clinically relevant being its synergism with TNF. This synergism has been demonstrated in numerous biological processes including radioprotection, the Shwartzman reaction, PGE2 synthesis, sickness behavior, nitric oxide production, nerve growth factor synthesis, insulin resistance, body mass changes, and chemokine synthesis . When designing experiments to study IL1A, researchers should consider:

• Combinatorial stimulation experiments: Treating cells with both IL1A and TNF to observe synergistic effects compared to individual cytokine treatments.

• Cytokine network analysis: Employing protein arrays or multiplex assays to measure multiple cytokines simultaneously following IL1A stimulation.

• Blocking studies: Using neutralizing antibodies against potential interacting cytokines to delineate specific versus cooperative effects.

• Temporal analysis: Examining the timeline of cytokine production, as IL1A expression often precedes IL1B expression during infections (as observed with Cryptococcus neoformans) .

Understanding these interactions is crucial as they can dramatically amplify inflammatory responses beyond what would be expected from individual cytokines alone.

How does IL1A contribute to the pathogenesis of inflammatory diseases?

IL1A functions as an apical instigator of inflammation in several major human diseases. As an alarmin or danger-associated molecular pattern (DAMP), IL1A released from dying cells drives inflammatory processes in various conditions:

• Sterile inflammation: IL1A released during necrotic cell death initiates inflammatory responses by signaling through IL-1R and inducing pro-inflammatory mediators like COX-2, IL-6, and TNF. This creates an amplification loop that can exacerbate tissue damage .

• Autoinflammatory disorders: IL1A plays a role in certain autoinflammatory conditions, acting in both hematopoietic and non-hematopoietic compartments .

• Cancer-associated inflammation: IL1A can contribute to tumor progression by promoting angiogenesis, invasiveness, and metastasis through its pro-inflammatory functions .

• Microbial diseases: IL1A cooperates with TNF signaling to promote granuloma formation during Mycobacterium tuberculosis infection. It also protects from gut-derived sepsis induced by Pseudomonas aeruginosa colonization .

Understanding these pathogenic mechanisms has led to the development of IL1A inhibitors as potential therapeutic agents for these conditions .

What regulatory molecules control IL1A activity in biological systems?

Several regulatory molecules control IL1A activity in biological systems:

• IL-1Ra (IL-1 receptor antagonist): The most important regulatory molecule for IL1A activity, typically produced in a 10- to 100-fold molar excess. IL-1Ra competitively binds to IL-1R without inducing signaling, thereby inhibiting IL1A activity .

• Soluble IL-1R type I: This receptor has high affinity for IL1A and is produced in a 5-10 molar excess, serving as a decoy receptor that sequesters IL1A without inducing signaling .

• IL-10: This anti-inflammatory cytokine inhibits IL1A synthesis .

• IL-1R2: Binding of cytosolic IL1A by IL-1R2 may serve to sequester IL1A during inflammatory signaling .

• Nuclear sequestration: During apoptosis, pro-IL1A can be sequestered in the nucleus, followed by clearance of the nuclei-containing apoptotic bodies by phagocytes, thus preventing IL1A release and downstream inflammatory responses .

These regulatory mechanisms are crucial for maintaining immune homeostasis and preventing excessive inflammation.

What are the methodological approaches to study IL1A in cancer research?

IL1A plays complex roles in cancer development and progression. Researchers studying IL1A in cancer contexts should consider these methodological approaches:

• Expression analysis: Examine IL1A expression in tumor tissues versus normal tissues using immunohistochemistry, qPCR, or RNA-sequencing.

• Functional studies in cancer cell lines: Manipulate IL1A expression through knockdown or overexpression approaches to assess effects on proliferation, migration, invasion, and resistance to apoptosis.

• Tumor microenvironment analysis: Investigate how IL1A affects the tumor microenvironment, including recruitment of immune cells, angiogenesis, and fibroblast activation.

• In vivo models: Utilize genetic approaches (IL1A knockout or overexpression) or pharmacological approaches (IL1A neutralization) in animal models of cancer to assess tumor growth, metastasis, and survival.

• IL1A inhibition strategies: Test IL1A-targeting therapies alone or in combination with conventional cancer treatments to identify potential synergistic effects .

• SASP factor analysis: Examine how IL1A contributes to the senescence-associated secretory phenotype (SASP) through mTOR activity, which may influence the tumor microenvironment .

These approaches can help elucidate the context-dependent roles of IL1A in promoting or inhibiting cancer development.

What are the optimal storage and handling conditions for recombinant IL1A?

For optimal research outcomes when working with recombinant IL1A Human, His Active protein, researchers should follow these storage and handling guidelines:

• Storage temperature: Store lyophilized protein at -20°C and reconstituted protein in aliquots at -80°C to avoid repeated freeze-thaw cycles.

• Reconstitution: Reconstitute in sterile buffer (commonly PBS or similar physiological buffer) with carrier protein (typically 0.1% BSA) to prevent protein loss through adsorption to tubes.

• Working concentration: IL1A is biologically active at picomolar to femtomolar concentrations . Prepare working dilutions immediately before use.

• Stability considerations: Avoid repeated freeze-thaw cycles which can lead to protein degradation and loss of activity. For long-term storage, make small aliquots.

• Quality control: Periodically validate protein activity using established bioassays, such as IL-8 secretion from responsive cell lines.

Following these guidelines will help maintain the biological activity of IL1A for experimental applications.

How can researchers distinguish between IL1A-specific and general inflammatory effects?

Distinguishing IL1A-specific effects from general inflammatory responses requires rigorous experimental design:

• Specific neutralizing antibodies: Use IL1A-specific neutralizing antibodies in parallel with isotype controls to block IL1A-mediated effects. In vivo neutrophilic responses to unfractionated supernatants containing IL1A can be blocked by IL1A-specific neutralizing antibodies .

• Genetic approaches: Utilize IL1A knockout cells/animals compared to wild-type controls. Additionally, compare with IL1B knockout models to distinguish between these related cytokines.

• Receptor antagonists: Apply IL-1Ra to block IL-1R signaling and determine if observed effects are dependent on IL-1R.

• Recombinant protein controls: Include irrelevant recombinant proteins with similar tags as controls when using His-tagged IL1A.

• Dose-response studies: Perform careful dose-response experiments, as IL1A is bioactive at very low concentrations (picomolar to femtomolar range) .

• Temporal analysis: Examine early versus late responses, as IL1A often functions as an apical initiator of inflammatory cascades with distinct temporal patterns compared to other inflammatory mediators .

These approaches help ensure that observed effects are truly IL1A-dependent rather than reflecting general inflammatory processes.

What bioassays are recommended for confirming IL1A activity?

To confirm the biological activity of recombinant IL1A Human, His Active, researchers can employ several established bioassays:

• Fibroblast proliferation assay: Measure proliferation of fibroblasts following IL1A stimulation, as IL1A induces fibroblast proliferation both in vitro and in vivo .

• Pro-inflammatory cytokine induction: Quantify the production of IL-6, TNF, or IL-8 from responsive cell types such as keratinocytes, macrophages, or endothelial cells following IL1A treatment .

• PGE2 production assay: Measure prostaglandin E2 release from stimulated cells, as IL1A induces cyclooxygenase synthesis and PGE2 release .

• Acute phase protein induction: Assess the stimulation of acute phase protein secretion from hepatocytes .

• Neutrophil recruitment: In vivo assays measuring neutrophil infiltration following local administration of IL1A .

• NF-κB activation reporter assay: Use cells transfected with an NF-κB responsive reporter gene to measure IL1A-induced signaling.

These bioassays provide functional confirmation of IL1A activity and can be used for quality control of recombinant protein preparations.

What are the current knowledge gaps in IL1A biology?

Despite increasing appreciation of IL1A's importance in human diseases, several critical knowledge gaps remain:

• Subcellular targeting mechanisms: The mechanisms controlling the subcellular targeting of IL1A and their relative biological functions remain poorly understood .

• Secretion regulation: Mechanisms that regulate IL1A secretion from cells under different conditions need further investigation .

• Proteolytic processing function: The functional significance of IL1A cleavage by inflammatory proteases remains unclear .

• Context-dependent roles: How IL1A functions differently in various disease contexts, sometimes promoting protective immune responses and other times driving immunopathology, requires further elucidation .

• Interaction with the microbiome: The relationship between IL1A and microbiome composition in inflammatory diseases represents an emerging area of investigation.

• Epigenetic regulation: The epigenetic mechanisms controlling IL1A expression in different cell types and disease states remain to be fully characterized.

Addressing these knowledge gaps will enhance our understanding of IL1A biology and potentially reveal new therapeutic targets.

How might emerging technologies advance IL1A research?

Emerging technologies offer exciting opportunities to advance IL1A research:

• Single-cell technologies: Single-cell RNA sequencing and CyTOF can reveal cell-specific patterns of IL1A expression and response in complex tissues.

• CRISPR-Cas9 genome editing: Precise genetic manipulation can create more sophisticated cellular and animal models to study IL1A function.

• Intravital imaging: Real-time visualization of IL1A release and subsequent immune cell recruitment in living organisms.

• Organoid models: Three-dimensional tissue cultures provide more physiologically relevant systems to study IL1A function in tissue-specific contexts.

• Computational modeling: Systems biology approaches can help predict IL1A network interactions and identify potential therapeutic targets.

• Structural biology advances: Cryo-electron microscopy and other structural approaches might provide new insights into IL1A-receptor interactions and conformational changes.

These technologies will likely facilitate more nuanced understanding of IL1A's role in health and disease.