IL 16 Human, (121 a.a.)

What is IL-16 Human (121 a.a.) and what are its key structural characteristics?

IL-16 Human (121 a.a.) is a recombinant form of the human interleukin-16 cytokine, also known as lymphocyte chemoattractant factor (LCF), consisting of 121 amino acid residues with a molecular weight of approximately 12.4 kDa . The protein is derived from a larger 631 amino acid precursor protein that undergoes cleavage at residue 511 to generate the 121-residue C-terminal peptide, which is released as the bioactive mature form of IL-16 . Structurally, IL-16 contains a characteristic PDZ domain, which typically features a defined globular structure with a peptide-binding groove located between αB and βB structural elements and a highly conserved carboxylate-binding loop .
Unlike other PDZ domains, IL-16's solution structure reveals a distinctive feature: a tryptophan residue that obscures the recognition groove . This structural characteristic has significant implications for IL-16's function and interaction with binding partners. The protein shares approximately 85% amino acid sequence identity with murine IL-16, indicating evolutionary conservation of its structure and function .

Which cell types produce IL-16 and what is its receptor interaction mechanism?

IL-16 is predominantly secreted by several immune cell types, including lymphocytes, epithelial cells, eosinophils, and CD8+ T-cells . This diverse cellular origin contributes to its presence at various inflammatory sites, particularly those characterized by TH1-mediated inflammation such as those observed in autoimmune diseases, ischemic reperfusion injury, and tissue transplant rejection .
The primary signaling mechanism of IL-16 occurs through interaction with the CD4 receptor . This interaction is crucial for its chemoattractant activity and immunomodulatory functions. The binding mechanism involves the PDZ domain, though interestingly, when an inhibitory antibody (such as mAb 14.1) binds to IL-16, it induces a significant conformational change in the PDZ domain . This change involves rotation of the αB-helix, movement of the tryptophan residue that normally obscures the peptide-binding groove, and consequent opening of the binding site . This structural flexibility may be integral to IL-16's diverse functional capabilities.

What are the primary biological functions of IL-16 in immune regulation?

IL-16 serves multiple functions in immune regulation. It acts as a chemoattractant cytokine, recruiting CD4+ cells to sites of inflammation . Additionally, IL-16 induces the expression of IL2Rα on T-cells, potentially modulating T-cell activation and proliferation responses .
A particularly significant function of IL-16 is its ability to suppress human immunodeficiency virus (HIV) replication . This antiviral activity positions IL-16 as a potential endogenous regulator of HIV infection. Conversely, research has demonstrated that IL-16 enhances influenza A virus (IAV) infection in both human lung epithelial A549 cells and mouse embryonic fibroblasts (MEFs) . This seemingly contradictory role in different viral infections highlights the complexity of IL-16's immunomodulatory activities.
IL-16 also inhibits T-cell antigen receptor/CD3-mediated T-cell stimulation in mixed lymphocyte reactions , suggesting a regulatory role in adaptive immune responses. Furthermore, IL-16 can inhibit interferon (IFN)-β and IFN-stimulated genes (ISG) expression, potentially affecting the antiviral state of cells during infection .

How can researchers effectively express and purify recombinant IL-16 for experimental studies?

For researchers studying IL-16, obtaining pure, biologically active protein is essential. Based on established methodologies, recombinant human IL-16 can be effectively expressed and purified using the following protocol:

• Expression System Selection: Use the Escherichia coli strain BL21 (DE3) transformed with a pLEICS-01 vector containing the IL-16 sequence (residues 502-631, corresponding to the mature secreted form) . This system has been demonstrated to produce soluble IL-16 with appropriate post-translational modifications.

• Expression Conditions: For standard protein production, grow bacteria at 37°C in appropriate media. For isotope-labeled protein (necessary for NMR studies), use modified Spizizen minimal medium containing specific isotopes such as 15NH4SO4 (4 g liter−1) and/or [13C6]glucose (2 g liter−1) . For deuterated samples, prepare media in 100% D2O.

• Purification Strategy:

  • Initial purification: Affinity chromatography using a nickel-nitrilotriacetic acid column (for His-tagged protein) .

  • Tag removal: Incubate with tobacco etch virus protease overnight at 4°C to remove the His tag .

  • Final purification: Gel filtration chromatography (Superdex 75 16/60) .

  • Buffer conditions: 25 mM phosphate, pH 7.0, 100 mM NaCl, 100 μM EDTA, 1 mM DTT, 2 mM MgCl2, and 1 mM imidazole .

• Quality Control: Verify protein purity through SDS-PAGE and HPLC analyses, aiming for >95% purity . Functional validation through bioactivity assays is also recommended before experimental use.
  This methodology ensures production of high-quality IL-16 protein suitable for structural, biochemical, and cell-based studies.

What experimental approaches best elucidate IL-16's role in viral infection models?

To investigate IL-16's role in viral infection models, researchers should consider the following methodological approaches:

• Cell-Based Infection Models:

  • Human lung epithelial A549 cells and mouse embryonic fibroblasts (MEFs) have been successfully used to study IL-16's effects on influenza A virus (IAV) infection .

  • Madin-Darby canine kidney (MDCK) cells can be employed for studying viral binding .

  • Experimental design should include:

    • Transfection with IL-16-expressing plasmids or vector controls

    • Infection with virus at appropriate MOI (e.g., MOI=0.1 for replication studies, MOI=1-10 for entry studies)

    • Time-course analysis (12h, 24h points for viral protein expression; 15min, 30min, 1h for entry studies)

• Genetic Approaches:

  • Compare wild-type (WT) and IL-16 knockout (KO) cells or animals to assess the impact of IL-16 deficiency on viral infection parameters .

  • Confirm knockout efficiency via immunoblot analyses before proceeding with experiments .

• Viral Parameters Analysis:

  • Measure viral protein expression (e.g., NP, NS, HA) via immunoblot analysis .

  • Quantify viral gene expression (NA, HA, M genes) through qRT-PCR .

  • Assess viral progeny production using plaque assays or TCID50 assays .

• Mechanistic Studies:

  • For viral binding studies: Incubate cells with virus at 4°C (prevents internalization), followed by surface-staining for viral proteins and flow cytometry analysis .

  • For entry studies: Allow virus binding at 4°C, then shift to 37°C for various time periods, followed by qRT-PCR analysis of viral RNA levels .

  • For replication studies: Monitor viral protein and RNA levels at later time points (12-24h post-infection) .
    This comprehensive experimental approach allows for detailed characterization of how IL-16 impacts different stages of viral infection, from entry to replication and progeny virus production.

How does IL-16 modulate interferon responses during viral infection?

IL-16 has been demonstrated to inhibit interferon responses during viral infection, representing a key mechanism by which it may enhance susceptibility to certain viral pathogens. Researchers investigating this phenomenon should implement the following methodological approaches:

• Gene Expression Analysis:

  • Measure IFN-β and ISG15 mRNA levels through qRT-PCR in cells with manipulated IL-16 expression (overexpression or knockout) following viral infection .

  • Track temporal changes by analyzing samples at multiple time points post-infection .

• Protein Production Assessment:

  • Quantify IFN-β protein levels in cell culture supernatants or tissue homogenates (e.g., lung homogenates and bronchoalveolar lavage fluid from infected animals) using ELISA .

  • Compare IFN-β production between wild-type and IL-16 KO conditions .

• Promoter Activity Studies:

  • Utilize luciferase reporter assays with IFN-β or ISRE (Interferon-Stimulated Response Element) promoter constructs to directly assess the impact of IL-16 on interferon signaling pathways .

  • Co-transfect cells with the reporter constructs and either empty vector or IL-16-expressing plasmid, followed by appropriate stimulation .

• In Vivo Verification:

  • Compare IFN-β production in wild-type versus IL-16 KO mice infected with virus (e.g., PR8 influenza virus) .

  • Harvest tissues at day 2 and day 3 post-infection for optimal detection of interferon responses .
    Experimental data from these approaches reveals that IL-16 overexpression significantly reduces IFN-β and ISG15 expression in virus-infected cells, while IL-16 deficiency enhances these responses . In the murine model, IL-16 KO mice demonstrate significantly higher IFN-β mRNA levels in lung tissue and increased IFN-β protein in lung homogenates and bronchoalveolar lavage fluid compared to wild-type mice following influenza infection .
    The mechanistic basis for this inhibition appears to involve suppression of IFN-β and ISRE promoter activities, as demonstrated by reduced luciferase activity in reporter assays when IL-16 is overexpressed . This identifies IL-16 as a negative regulator of type I interferon responses, which may contribute to its enhancement of influenza virus infection.

What contradictions exist in IL-16 activities across different viral infection models?

A significant research challenge in IL-16 biology is reconciling its seemingly contradictory roles in different viral infection models. Researchers should be aware of the following paradoxical findings:

• Opposing Effects on Different Viruses:

  • IL-16 suppresses HIV-1 replication, functioning as an antiviral factor in this context .

  • Conversely, IL-16 enhances Influenza A Virus (IAV) infection in both human lung epithelial cells and mouse embryonic fibroblasts, promoting viral replication and pathogenesis .

• Mechanisms Behind Differential Activities:

  • The antiviral activity against HIV may be related to IL-16's interaction with CD4, which serves as a receptor for HIV entry .

  • The proviral activity for IAV appears unrelated to viral entry (as IL-16 does not affect IAV binding or entry) but is instead linked to inhibition of interferon responses .

• Experimental Considerations for Resolving Contradictions:

  • When designing experiments to address these contradictions, researchers should:

    • Carefully control for virus-specific factors, including receptor usage and replication mechanisms

    • Consider cell type-specific responses to IL-16

    • Examine concentration-dependent effects

    • Investigate temporal aspects of IL-16 activity during infection

• Technical Approaches to Resolve Contradictions:

  • Parallel comparative studies using both viruses in the same experimental system

  • Domain mutation studies to identify regions of IL-16 responsible for virus-specific effects

  • Receptor blocking studies to determine if CD4 interaction is essential for all IL-16 activities
    Understanding these contradictions is essential for accurately interpreting experimental results and developing targeted interventions based on IL-16 biology.

How do structural changes in IL-16 affect its function and interaction with antibodies?

The structural dynamics of IL-16, particularly its PDZ domain, present both challenges and opportunities for researchers. Current evidence reveals:

• Conformational Flexibility:

  • IL-16's PDZ domain undergoes significant conformational changes upon antibody binding .

  • The binding of the 14.1Fab antibody fragment triggers rotation of the αB-helix, movement of the tryptophan residue that typically obscures the peptide-binding groove, and opening of this binding site .

• Implications for Function:

  • This structural flexibility suggests IL-16 may potentially modify its activity through cryptic peptide binding sites that become accessible only under specific conditions .

  • Researchers should consider that static structural models may not capture the full functional repertoire of IL-16.

• Methodological Approaches to Study Conformational Changes:

  • X-ray crystallography of IL-16 in complex with binding partners (as demonstrated with the 14.1Fab fragment)

  • Nuclear Magnetic Resonance (NMR) studies using 13C/15N/2D-labeled IL-16 to observe dynamic structural changes in solution

  • Hydrogen-deuterium exchange mass spectrometry to map regions of conformational flexibility

  • Molecular dynamics simulations to model potential structural transitions

• Design Considerations for IL-16 Targeting Molecules:

  • Researchers developing inhibitors should consider targeting both the active and cryptic conformations of IL-16

  • Antibodies or small molecules that stabilize specific conformational states might offer selective modulation of IL-16 functions

  • The unusual tryptophan residue that obscures the peptide-binding groove represents a potential target for structure-based drug design
    Understanding these structural dynamics is crucial for researchers interpreting binding and functional studies and represents an important consideration when developing therapeutic strategies targeting IL-16.

What methodological considerations are critical for IL-16 neutralization studies?

When designing and interpreting IL-16 neutralization studies, researchers should consider several methodological factors:

• Antibody Selection and Characterization:

  • Monoclonal antibodies like mAb 14.1 have demonstrated efficacy in neutralizing IL-16 and reducing TH1-type inflammatory responses .

  • Before conducting neutralization experiments, researchers should fully characterize antibody binding properties:

    • Epitope mapping to determine precise binding site

    • Binding affinity measurements (KD values)

    • Assessment of potential cross-reactivity with related proteins

    • Verification that the antibody recognizes the relevant species homolog (human vs. murine IL-16)

• Experimental Readouts for Neutralization:

  • Cell migration assays with dendritic cells or epidermal cells can demonstrate functional neutralization .

  • Analysis of TH1-type inflammatory responses provides evidence of immunological neutralization .

  • In viral infection models, measurement of viral load and interferon responses following antibody treatment demonstrates efficacy .

• In Vivo Model Considerations:

  • Rodent models of acute kidney injury have successfully demonstrated mAb 14.1 efficacy in vivo .

  • When designing animal studies, researchers should consider:

    • Dosing regimen (timing relative to disease onset)

    • Administration route

    • Antibody half-life and tissue distribution

    • Species-specific differences in IL-16 biology (85% sequence identity between human and murine IL-16)

• Controls and Validation:

  • Include isotype control antibodies to account for non-specific effects

  • Use IL-16 knockout models as positive controls for complete IL-16 neutralization

  • Consider complementary approaches (genetic knockdown, small molecule inhibitors) to validate antibody findings
    These methodological considerations ensure robust and reproducible results when evaluating IL-16 neutralization strategies for potential therapeutic applications.

What therapeutic approaches targeting IL-16 show promise for inflammatory and autoimmune conditions?

IL-16 has emerged as a potential therapeutic target for various inflammatory and autoimmune conditions. Several promising approaches warrant further investigation:

• Monoclonal Antibody Therapeutics:

  • The 14.1 antibody has demonstrated efficacy in reducing TH1-type inflammatory responses and shows promise in rodent models of acute kidney injury .

  • Future research should focus on:

    • Humanization of promising murine antibodies for clinical development

    • Development of antibodies targeting specific conformational states of IL-16

    • Exploration of antibody formats (Fab, F(ab')2, IgG subtypes) for optimal tissue penetration and half-life

• Small Molecule Inhibitors:

  • The structural data on IL-16's PDZ domain and its conformational changes suggest opportunities for small molecule development .

  • Research approaches should include:

    • Structure-based drug design targeting the peptide-binding groove

    • Allosteric inhibitors that stabilize the closed conformation of the peptide-binding site

    • Fragment-based screening to identify novel chemical starting points

• Target Disease Areas:

  • Evidence supports IL-16 as a therapeutic target in:

    • Autoimmune diseases (rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus)

    • Ischemic reperfusion injury

    • Tissue transplant rejection

    • Potentially influenza infection, where IL-16 enhances viral pathogenesis

• Combination Therapy Approaches:

  • Given IL-16's role in inhibiting interferon responses during viral infection , combining IL-16 inhibition with interferon-boosting strategies may offer synergistic benefits in certain contexts.

  • Dual targeting of IL-16 and related inflammatory cytokines could provide more comprehensive control of inflammatory cascades.
    These therapeutic approaches represent promising avenues for translating our understanding of IL-16 biology into clinical interventions for inflammatory and autoimmune conditions.

How can structural knowledge of IL-16 guide the development of novel inhibitors?

The unique structural features of IL-16, particularly its PDZ domain and conformational dynamics, provide specific opportunities for structure-guided inhibitor development:

• Targeting the Cryptic Peptide-Binding Groove:

  • The structure of the 14.1Fab-IL-16 complex reveals that antibody binding opens a typically obscured peptide-binding groove .

  • Researchers can exploit this insight by:

    • Designing peptidomimetics that stabilize the open conformation

    • Developing small molecules that compete for binding in this groove

    • Creating allosteric modulators that favor the open conformation, thereby disrupting normal IL-16 function

• Structure-Based Drug Design Strategies:

  • With available crystal structures, computational approaches can be employed:

    • Virtual screening of compound libraries against the open peptide-binding site

    • Molecular dynamics simulations to identify transient binding pockets

    • Fragment-based approaches targeting specific structural elements

• Targeting the Tryptophan Switch Mechanism:

  • The tryptophan residue that obscures the recognition groove represents a unique structural feature .

  • Compounds that interfere with the movement of this residue could potentially lock IL-16 in an inactive conformation.

• Exploiting Species Differences:

  • With 85% sequence identity between human and murine IL-16 , structural differences could be exploited for selective targeting.

  • A comparative analysis of the structures could identify unique pockets or conformational states for species-selective inhibitor design.

• Rational Design of Improved Antibodies:

  • Structural data on the 14.1Fab-IL-16 interaction can guide engineering of improved antibodies:

    • Affinity maturation targeting specific contact residues

    • Stabilization of the antibody-IL-16 complex

    • Development of bispecific antibodies incorporating IL-16 binding domains These structure-guided approaches offer promising avenues for developing novel therapeutics targeting IL-16, potentially leading to more effective treatments for autoimmune diseases, inflammatory conditions, and certain viral infections.