Recombinant Human Pro-interleukin-16 (IL16) (Active)

What is the primary function of recombinant human IL-16 in immune regulation?

Recombinant human IL-16 functions as a proinflammatory cytokine that modulates immune cell responses primarily through interaction with CD4 receptors. It serves as a potent chemoattractant for CD4+ T lymphocytes and plays a key role in inflammatory conditions like asthma by recruiting CD4+ cells to disease sites . Additionally, IL-16 stimulates the expression and production of other proinflammatory cytokines (IL-1β, IL-6, IL-15, and TNF-α) from CD14+CD4+ monocytes and macrophages, thus amplifying inflammatory responses . These effects suggest that IL-16 acts as an important initiator and sustainer of inflammatory processes.

Which cell types respond to IL-16 stimulation and how can researchers identify responsive cells?

IL-16 primarily affects CD4-expressing cells, with different response patterns based on cell type:

• CD14+CD4+ monocytes and maturing macrophages secrete IL-1β, IL-6, IL-15, and TNF-α upon stimulation

• CD4+ T lymphocytes do not secrete these cytokines in response to IL-16 despite expressing CD4

• Dendritic cells and macrophages demonstrate altered HIV susceptibility when exposed to IL-16

• Adipocytes respond to IL-16 by modulating markers of adipogenesis, lipid metabolism, and inflammatory signaling

When identifying responsive cells, researchers should assess CD4 expression using flow cytometry and confirm functional responses through cytokine secretion assays or specific marker expression analysis depending on the cell type of interest.

What is the optimal concentration range for recombinant IL-16 in experimental settings?

Optimal concentrations of recombinant IL-16 vary by experimental endpoint and cell type. Based on research data, the following concentration guidelines are recommended:

Researchers should note that higher concentrations (>1000 ng/ml) may actually inhibit cytokine production rather than enhance it, indicating a biphasic dose-response curve .

How should researchers design time-course experiments to measure IL-16-induced cytokine expression?

Time-course experiments for IL-16 should include both early and late timepoints to capture the full response profile. Based on published data:

• mRNA expression analysis:

  • Include 2, 4, and 8-hour timepoints after IL-16 stimulation

  • Extract RNA using standard protocols

  • Perform RT-PCR for target cytokines (IL-1β, IL-6, IL-15, TNF-α)

  • Expected results: No increase at 2 hours, significant increases at 4 hours (especially with 50 ng/ml IL-16)

• Protein secretion analysis:

  • Include 8, 24, and 48-hour timepoints

  • Collect supernatants and perform ELISAs for target cytokines

  • Expected results: No significant increases at 8 hours, significant secretion by 24 hours, with levels potentially plateauing by 48 hours

For validation, include appropriate controls such as unstimulated cells and positive controls (e.g., LPS stimulation, which typically induces higher cytokine levels than IL-16) .

What isolation techniques are recommended for studying IL-16 effects on specific immune cell subpopulations?

When isolating specific immune cell subpopulations to study IL-16 effects, consider these methodological approaches:

• For monocyte/macrophage isolation:

  • Magnetic-activated cell sorting (MACS) using CD14 beads provides a practical approach with good purity

  • Culture isolated CD14+ cells with rhIL-16 (50-500 ng/ml) for 24-48 hours

  • Analyze supernatants for IL-1β, IL-6, IL-15, and TNF-α by ELISA

• For CD4+ T lymphocyte isolation:

  • MACS separation using CD4 beads is effective

  • Note that purified CD4+ T cells typically do not secrete cytokines in response to rhIL-16 alone

• For adipocyte studies:

  • Use established models like differentiated 3T3-L1 preadipocytes

  • Confirm differentiation by measuring lipid droplet accumulation and expression of adipocyte markers (Plin1, Fabp4, AdipoQ, and Pparg)

  • Treat mature adipocytes with IL-16 (optimal at 10 ng/ml)

Include appropriate purity checks by flow cytometry to ensure cell population homogeneity before IL-16 stimulation experiments.

How does IL-16 structure relate to its diverse biological functions?

IL-16's structure-function relationship is complex, with different domains mediating distinct biological activities:

• C-terminal domains critical for chemoattractant activity:

  • The minimal peptide sequence RRKS (corresponding to Arg106 to Ser109) mediates inhibition of IL-16 chemoattractant activity

  • Arg107 is especially critical, as substitution with alanine eliminates chemoattractant function

  • Point mutations in this region can selectively impair chemoattractant activity while preserving other functions

• N-terminal regions influence MLR inhibition:

  • MLR (mixed lymphocyte reaction) inhibition remains intact even with deletion of C-terminal regions through Arg106

  • Deletion of 12 or 22 N-terminal residues reduces MLR inhibition without affecting chemoattractant activity

  • Combined deletion of both N-terminal (12 residues) and C-terminal (16 residues) domains abolishes both chemoattractant and MLR-inhibitory functions

These structural insights suggest IL-16 engages in different receptor interactions for its diverse functions, with both N-terminal and C-terminal domains participating in receptor binding or activation . Researchers investigating specific IL-16 functions should consider using domain-specific mutants or peptide fragments rather than the full protein.

What are the mechanisms of IL-16-mediated HIV-1 suppression in antigen-presenting cells?

IL-16 demonstrates potent HIV-suppressive activity in monocyte-derived macrophages and dendritic cells through multiple mechanisms:

• Early intervention required:

  • IL-16 must be present early after infection to exert its anti-HIV effects

  • Pre-treatment of cells for 24 hours before infection is not effective

• Blockade of viral entry:

  • IL-16 added during the infection period effectively blocks virus entry

  • This leads to reduced proviral DNA levels in antigen-presenting cells

  • The effect works against both macrophage-tropic and dually tropic primary HIV isolates

• Mechanism independent of β-chemokines:

  • Unlike other HIV-suppressive cytokines, IL-16's anti-HIV activity is not linked to the induction of virus-suppressive concentrations of β-chemokines

  • It also does not appear to work by inhibiting HIV-enhancing cytokines

These findings suggest IL-16 protects antigen-presenting cells through direct interference with viral entry processes, potentially by interacting with CD4 (the primary receptor for HIV) or by modulating additional factors involved in viral entry.

What is the role of IL-16 in adipose tissue function and metabolic disease?

IL-16 plays a complex role in adipose tissue biology and metabolic disorders:

• Differential expression in obesity:

  • IL-16 expression is higher in visceral white adipose tissue (vWAT) compared to subcutaneous white adipose tissue (sWAT) in individuals with obesity

  • Serum IL-16 levels are elevated in patients with obesity compared to normal-weight individuals

  • Interestingly, IL-16 levels increase 6 months after bariatric surgery before returning to baseline by 12 months

• Effects on adipogenesis and metabolism:

  • IL-16 modulates markers of adipogenesis (Pref1)

  • It affects lipid metabolism gene expression (Plin1, Cd36, and Glut4)

  • IL-16 treatment at 10 ng/ml decreases Plin1 expression, which has been linked to increased proinflammatory responses

  • Decreased Glut4 expression suggests IL-16 may impair glucose uptake

• Impact on fibrosis and inflammation:

  • IL-16 decreases Hif1a and Vegf expression, potentially affecting tissue remodeling

  • It increases the Mmp9/Timp1 ratio, suggesting enhanced extracellular matrix remodeling

  • In palmitate-treated adipocytes (mimicking obesity), IL-16 promotes expression of Ccl2 and Il6, indicating a role in early inflammatory signaling

These findings suggest IL-16 may contribute to metabolic dysfunction by altering adipocyte function, promoting inflammation, and impairing glucose metabolism in the context of obesity.

How can researchers address variability in IL-16 response between different donor samples?

When working with primary cells from different donors, variability in IL-16 response is common. To address this:

• Standardize isolation protocols:

  • Use consistent isolation techniques for peripheral blood mononuclear cells (PBMC)

  • Assess CD4 and CD14 expression levels by flow cytometry before experiments

  • Consider standardizing for cell viability and activation state

• Include internal controls:

  • Always run dose-response curves for IL-16 (5-500 ng/ml) for each donor

  • Include positive controls (e.g., LPS stimulation) to normalize responses between donors

  • Consider using a reference donor sample across experiments

• Increase sample size:

  • Include sufficient biological replicates (minimum n=3 donors)

  • Report donor-to-donor variability transparently

  • Consider pooled analysis approaches for heterogeneous responses

• Validate IL-16 activity:

  • Use neutralizing anti-IL-16 antibodies to confirm specificity of observed effects

  • Test multiple IL-16 concentrations as the optimal dose may vary between donors

What are the recommended controls for IL-16 activity assays?

To ensure robust experimental design when working with IL-16, incorporate these controls:

• Positive biological controls:

  • LPS stimulation for cytokine production (typically induces higher levels than IL-16)

  • SDF-1/CXCL12 for chemotaxis assays

  • PHA or anti-CD3/CD28 for T cell activation studies

• Negative controls:

  • Heat-inactivated IL-16 (protein denatured at 95°C for 10 minutes)

  • Irrelevant recombinant proteins of similar size

  • Vehicle/diluent only

• Specificity controls:

  • Neutralizing anti-IL-16 antibody to block IL-16 effects

  • Isotype control antibodies

  • Domain-specific IL-16 mutants to distinguish functional regions

• Biological validation:

  • Compare effects across multiple cell types (monocytes vs. T cells)

  • Use both primary cells and cell lines when possible

  • Include time-course and dose-response analyses

How might IL-16 signaling be exploited therapeutically for inflammatory and metabolic diseases?

IL-16 represents a potential therapeutic target with several promising avenues for intervention:

• For inflammatory conditions:

  • C-terminal peptide antagonists targeting the RRKS motif could inhibit IL-16 chemoattractant activity without affecting other functions

  • Neutralizing antibodies against IL-16 may reduce inflammatory cytokine cascades in conditions like asthma

  • Selective inhibitors of IL-16-induced cytokine production could provide targeted anti-inflammatory effects

• For HIV infection:

  • IL-16 or IL-16-mimetic compounds could potentially block HIV entry into antigen-presenting cells

  • Combined approaches targeting IL-16 receptors alongside traditional antiretroviral therapy might enhance protection of macrophages and dendritic cells

• For metabolic disorders:

  • Modulating IL-16 activity might alter adipose tissue inflammation and remodeling

  • Targeting the IL-16 pathway could potentially improve glucose metabolism and adipocyte function

  • Considering the complex dynamics of IL-16 levels after bariatric surgery, temporal modulation of IL-16 might complement weight loss interventions

Future research should focus on developing selective modulators of IL-16 activity that can target specific functions while preserving others, potentially providing more precise therapeutic approaches for diverse pathological conditions.

What techniques are recommended for investigating IL-16 interactions with its receptor complexes?

Investigating IL-16 receptor interactions requires sophisticated approaches:

• Protein-protein interaction methods:

  • Surface plasmon resonance (SPR) to measure binding kinetics between IL-16 and CD4 or other potential receptors

  • Co-immunoprecipitation using tagged IL-16 variants to identify interaction partners

  • Proximity ligation assays to visualize receptor interactions in intact cells

• Receptor identification strategies:

  • Competitive binding assays using domain-specific IL-16 peptides

  • Crosslinking studies with photoreactive IL-16 analogs

  • Genetic approaches using CRISPR-Cas9 to knock out candidate receptors

• Functional validation:

  • Calcium mobilization assays to measure receptor activation

  • Signaling pathway analysis using phospho-specific antibodies

  • Receptor blocking antibodies to confirm specificity of interactions

• Advanced structural biology:

  • Cryo-electron microscopy of IL-16-receptor complexes

  • X-ray crystallography of IL-16 bound to receptor fragments

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

These approaches can help elucidate how different domains of IL-16 engage with receptor components to mediate its diverse biological functions, including chemoattractant activity and MLR inhibition .