Fractalkine Human, His

What is the structural composition of human fractalkine and how does it differ from other chemokines?

Human fractalkine (CX3CL1) is the only member of the CX3C subfamily of chemokines, characterized by a unique structural organization. Mature human fractalkine consists of an N-terminal chemokine domain containing the distinctive CX3C motif, followed by a mucin-like stalk region in the extracellular domain (ECD), a transmembrane segment, and a short cytoplasmic domain . This structure differs from other chemokines as fractalkine exists in both membrane-tethered and soluble forms, allowing it to function both as an adhesion molecule and as a chemoattractant .

The soluble form is generated through proteolytic cleavage by enzymes such as ADAM10 and ADAM17 . In experimental settings, recombinant human fractalkine is often produced with tags such as His-tag or Avi-tag to facilitate purification and detection, resulting in a protein of approximately 88-103 kDa under SDS-PAGE analysis .

How is CX3CR1 involved in fractalkine signaling and what cell types express this receptor?

CX3CR1 is the exclusive receptor for fractalkine, making this ligand-receptor pair unique among chemokines, which typically exhibit promiscuous binding patterns . This receptor is predominantly expressed on various immune cells including NK cells, T cells, monocytes, and macrophages . Within the central nervous system, CX3CR1 is primarily expressed by microglia.

The receptor mediates both the adhesive and chemoattractant functions of fractalkine . When fractalkine binds to CX3CR1, it can trigger receptor internalization, as demonstrated by the significant reduction in CX3CR1+ NK cells following treatment with recombinant fractalkine (from 89.73% to 6.24% after 2 hours and 2.1% after 24 hours of treatment) . This interaction not only influences cell migration but also modulates cellular phenotypes and cytokine production patterns, particularly in NK cells and microglia .

What are the optimal conditions for reconstituting and storing recombinant human fractalkine for experimental use?

Recombinant human fractalkine should be reconstituted in sterile, filtered PBS or serum-free cell culture medium at concentrations between 0.1-1.0 mg/ml. For His-tagged versions, avoid using buffers containing imidazole, which can interfere with the His-tag. After reconstitution, the protein should be aliquoted to minimize freeze-thaw cycles and stored at -80°C for long-term stability or at -20°C for use within 1-2 months .

For experimental treatments, fractalkine is typically used at concentrations ranging from 10-100 ng/ml, with 30 ng/ml being commonly employed to simulate physiological levels found in tissue microenvironments such as those observed in esophagogastric adenocarcinoma omentum . When designing experiments, it's critical to account for the rapid internalization of the CX3CR1 receptor, which can occur within 2 hours of exposure to fractalkine .

What methods are most effective for detecting fractalkine expression and measuring its activity in experimental systems?

Several complementary approaches can be used to effectively detect and measure fractalkine:

• Protein detection methods:

  • Western blotting using antibodies directed against the N-terminal chemokine domain can detect fractalkine as an approximately 95-kDa band in whole-cell lysates

  • ELISA can quantify soluble fractalkine in conditioned media, with a typical detection limit of around 320 pg/ml

  • Immunofluorescence staining can visualize cellular localization

• Binding activity assessment:

  • When Human CX3CL1/Fractalkine MAb is immobilized at 1 μg/mL, biotinylated recombinant human CX3CL1/Fractalkine His-tag Avi-tag binds with an ED50 of 0.15-0.9 μg/mL

• Functional assays:

  • Chemotaxis assays measuring migration of CX3CR1-expressing cells

  • Cytokine production assays after fractalkine treatment, particularly measuring IFN-γ and TNF-α production

  • CX3CR1 surface expression analysis by flow cytometry to detect receptor internalization following fractalkine binding

How does fractalkine influence NK cell phenotype and function, and what are the implications for cancer immunotherapy?

Fractalkine significantly influences NK cell phenotype and function through multiple mechanisms that have important implications for cancer immunotherapy:

• Chemotactic effects: Fractalkine drives NK cell migration toward fractalkine-rich tissues. In esophagogastric adenocarcinoma (EAC), antagonism of CX3CR1 significantly reduces NK cell migration to omental adipose tissue but has less impact on migration toward tumor tissue . This suggests a mechanism by which NK cells are diverted from tumors to the omentum in obese EAC patients.

• Phenotypic conversion: Treatment with fractalkine induces a conversion from CX3CR1+CD27− to CX3CR1−CD27+ NK cells. After 24 hours of fractalkine exposure, there is a significant decrease in CX3CR1+ NK cells (from 85.5% to 34.45%, p=0.0021) and an increase in CD27+ NK cells (from 5.34% to 9.03%, p=0.0174) . This phenotypic shift is significant because:

  • CD27+ NK cells are predominantly cytokine-producing rather than cytotoxic

  • This pattern mirrors the dominant NK cell phenotype observed in EAC omentum

• Cytokine production modulation: Fractalkine treatment for 24 hours significantly increases the proportion of NK cells producing proinflammatory cytokines:

  • IFN-γ: from 19.27% to 42.77% (p=0.0479)

  • TNF-α: from 28.05% to 39.75% (p=0.0253)

These findings suggest that CX3CR1 antagonism could be a novel therapeutic strategy in obesity-associated cancers to prevent NK cell trafficking to the omentum and subsequent dysfunction. Treatment timing appears critical, with optimal timing suggested to be after neoadjuvant chemoradiotherapy .

What experimental models best demonstrate the dual roles of fractalkine in inflammation and tissue protection?

The dual roles of fractalkine in both promoting inflammation and providing tissue protection are best demonstrated through several complementary experimental models:

• Neuron-microglial co-culture systems:

  • These models reveal how fractalkine released from neurons communicates with CX3CR1-expressing microglia

  • Co-cultures of wild-type neurons with CX3CR1-null glia demonstrate that fractalkine fails to protect against toxicity when the receptor is absent

  • These systems have shown that fractalkine can protect striatal neurons from synergistic morphine and Tat toxicity

• Obesity-associated cancer models:

  • EAC patient samples and in vitro models demonstrate how fractalkine influences NK cell trafficking between circulation, tumor, and omentum

  • These models show simultaneous pro-inflammatory effects (increased IFN-γ and TNF-α) and potentially detrimental effects on anti-tumor immunity through NK cell diversion

• CX3CR1 knockout models:

  • Comparisons between wild-type and CX3CR1-null models demonstrate the receptor-dependence of fractalkine effects

  • These models reveal that CX3CR1-null systems typically show increased microglial numbers (>300% increase) and greater apparent neuron losses

Each model provides unique insights into fractalkine's context-dependent functions, with effects varying based on tissue type, disease state, and presence of other inflammatory mediators.

How can His-tagged fractalkine be utilized for studying receptor-ligand interactions and developing targeted therapeutics?

His-tagged recombinant human fractalkine offers several advantages for studying receptor-ligand interactions and developing targeted therapeutics:

• High-affinity purification and detection:

  • The His-tag allows for metal affinity chromatography purification, resulting in >95% purity preparations

  • Tagged proteins can be detected using anti-His antibodies, enabling tracking in complex biological systems

  • When combined with other tags (like Avi-tag), it enables multiple detection methods and controlled immobilization

• Binding kinetics and structure-function studies:

  • His-tagged fractalkine can be immobilized on biosensor chips for surface plasmon resonance (SPR) analysis to determine binding kinetics with CX3CR1

  • The resolved 2 μg/lane of biotinylated recombinant human fractalkine His-tag Avi-tag protein shows bands at 88-103 kDa under both reducing and non-reducing conditions , providing insights into structural conformations

• Therapeutic development applications:

  • Creating bispecific molecules by fusing His-tagged fractalkine with other bioactive molecules

  • Designing fractalkine antagonists based on structure-activity relationships identified using the tagged protein

  • Developing CX3CR1 antagonists as potential therapeutic strategies in obesity-associated cancers to prevent NK cell trafficking to the omentum

For studying the pharmacokinetics of potential therapeutics, researchers should account for the significant receptor internalization that occurs following fractalkine binding, with surface CX3CR1 expression decreasing from 89.73% to 6.24% on NK cells within just 2 hours of exposure .

What is the significance of fractalkine's role as an HIV co-receptor, and how can this be investigated experimentally?

Fractalkine's receptor, CX3CR1, can serve as an HIV-1 co-receptor with CD4, highlighting a critical role in viral pathogenesis . This function has significant implications for understanding HIV infection mechanisms and developing potential therapeutic interventions:

• Mechanistic significance:

  • CX3CR1 functions alongside CD4 to facilitate HIV-1 entry into target cells

  • This alternative co-receptor pathway may contribute to HIV infection of cells expressing low levels of the classical co-receptors CCR5 and CXCR4

  • The interaction may facilitate the spread of HIV-1 infection throughout the body, particularly in the CNS where fractalkine-CX3CR1 signaling is prominent

• Experimental investigation approaches:

  • Cell-based infection assays: Using cells expressing CD4 and CX3CR1 to assess HIV entry efficiency

  • Competitive binding studies: Determining whether recombinant fractalkine can block HIV envelope binding to CX3CR1

  • Receptor mutagenesis: Identifying the specific domains of CX3CR1 involved in HIV binding versus fractalkine binding

  • In vivo models: Examining whether CX3CR1 genetic variants correlate with HIV susceptibility or disease progression

• Therapeutic implications:

  • Development of entry inhibitors targeting the CX3CR1-HIV interaction

  • Potential use of modified fractalkine derivatives to competitively inhibit HIV binding

  • Investigation of whether fractalkine signaling modulates HIV latency or reactivation in reservoir sites

Understanding this alternative co-receptor pathway may be particularly important for developing strategies to limit HIV neuroinvasion and associated neurocognitive disorders.

What are the critical quality control parameters for validating recombinant human fractalkine preparations?

When validating recombinant human fractalkine preparations for research use, several critical quality control parameters should be assessed:

• Purity assessment:

  • SDS-PAGE analysis under both reducing and non-reducing conditions should show a predominant band at 88-103 kDa

  • High-performance liquid chromatography (HPLC) should indicate >95% purity for research applications

  • Mass spectrometry can confirm the expected molecular weight and identify any truncations or modifications

• Biological activity verification:

  • Binding assays: When properly immobilized, high-quality fractalkine should bind to anti-fractalkine antibodies with an ED50 in the range of 0.15-0.9 μg/mL

  • Functional assays: The preparation should induce CX3CR1 internalization on NK cells within 2 hours of exposure

  • Cytokine induction: Treatment should increase IFN-γ and TNF-α production in NK cells after 24 hours

• Endotoxin testing:

  • Limit of <1.0 EU/μg protein is essential for cell culture applications

  • Limulus Amebocyte Lysate (LAL) assay is the standard method for endotoxin detection

• Stability assessment:

  • Accelerated stability studies at various temperatures

  • Assessment of activity after multiple freeze-thaw cycles

  • Long-term storage stability at recommended conditions (-80°C)

For His-tagged variants, additional controls should include verification of tag integrity and confirmation that the tag does not interfere with biological activity by comparison to untagged variants.

How do different experimental conditions influence fractalkine shedding and what methods accurately quantify this process?

Fractalkine shedding, the process by which membrane-bound fractalkine is cleaved to release its soluble form, is influenced by various experimental conditions and can be quantified through several methods:

• Factors influencing fractalkine shedding:

  • Constitutive versus induced shedding: Fractalkine is constitutively cleaved and released at relatively high levels (approximately 2.50 ± 0.15 ng/ml in control culture medium)

  • Cell type variations: The rate of shedding differs between neurons and glia, with neurons being a major source of fractalkine when co-cultured with glia

  • Inflammatory stimuli: Exposure to inflammatory cytokines can increase ADAM10/ADAM17 activity and enhance shedding

  • Cell density: The ratio of neurons to glia (e.g., 1:20 ratio used to mimic in vivo conditions) affects the concentration of released fractalkine

• Quantification methods:

  • ELISA: Can detect soluble fractalkine in conditioned medium with a typical detection limit of around 320 pg/ml

  • Western blotting: Using antibodies against the N-terminal chemokine domain can detect the ~95 kDa full-length protein in cell lysates and the soluble form in media

  • Flow cytometry: Can measure surface expression on cells to indirectly assess shedding rates

  • Fluorogenic substrate assays: Can measure ADAM10/ADAM17 activity as a proxy for potential shedding activity

• Experimental considerations:

  • Time-course experiments are essential as shedding kinetics vary considerably (significant effects observable within 2 hours)

  • Inclusion of protease inhibitors as controls can help distinguish between specific cleavage events and non-specific degradation

  • The culture medium should be serum-free during shedding experiments to avoid interference from serum proteases

Understanding and accurately quantifying fractalkine shedding is crucial for interpreting experimental results, particularly in co-culture systems where multiple cell types may contribute to the soluble fractalkine pool.

How does fractalkine-mediated neuron-microglial communication influence neuroinflammation and neuroprotection?

Fractalkine serves as a critical mediator in neuron-microglial communication, with complex effects on both neuroinflammation and neuroprotection:

• Signaling mechanism:

  • Neurons predominantly express membrane-tethered fractalkine, while microglia express the CX3CR1 receptor

  • This arrangement establishes a direct communication channel between neurons and microglia

  • Fractalkine can be cleaved from neuronal membranes to generate a soluble form that can diffuse and signal to distant microglia

• Neuroprotective effects:

  • Fractalkine can protect neurons against toxicity from various insults, including the synergistic toxicity of morphine and HIV Tat protein

  • This protection appears to be mediated through CX3CR1-expressing microglia, as fractalkine fails to protect wild-type neurons co-cultured with CX3CR1-null glia

  • One mechanism of protection involves the normalization of microglial motility, which is elevated by neurotoxic agents like Tat and morphine

• Microglial regulation:

  • Fractalkine signaling can limit aggressive microglial surveillance that may lead to toxic effects on neurons

  • CX3CR1 knockout models show >300% increase in microglial numbers, suggesting fractalkine signaling normally restrains microglial proliferation

  • The absence of fractalkine-CX3CR1 signaling can lead to heightened microglial activation and potentially exacerbate neurotoxicity

This neuron-microglial communication system represents a promising target for therapeutic interventions in neuroinflammatory and neurodegenerative conditions, where modulating microglial activation states could provide neuroprotection.

What are the methodological considerations for investigating fractalkine's role in different neurological disease models?

When investigating fractalkine's role in neurological disease models, researchers should consider several methodological approaches and potential challenges:

• Model selection considerations:

  • In vitro co-culture systems: Neuron-microglial co-cultures allow for controlled manipulation of fractalkine signaling but lack the complexity of in vivo systems

  • CX3CR1 reporter models: Mice with CX3CR1-GFP knockin alleles enable visualization of receptor-expressing cells but may have altered receptor function

  • Conditional knockout models: Cell-specific deletion of fractalkine or CX3CR1 can help distinguish direct versus indirect effects

• Experimental design considerations:

  • Timing of interventions: The temporal dynamics of fractalkine signaling vary by disease context

  • Dosing of recombinant fractalkine: Typical experimental concentrations range from 10-100 ng/ml, with 30 ng/ml approximating physiological levels

  • Measurement of multiple endpoints: Changes in microglial morphology, motility, phagocytic activity, and cytokine production should be assessed alongside neuron survival

• Disease-specific considerations:

  • Neurodegenerative diseases: Examine age-dependent changes in fractalkine expression and CX3CR1+ cell distribution

  • Neuroinfectious diseases: Assess how pathogens (like HIV) interact with the fractalkine-CX3CR1 axis

  • Neuro-oncology: Investigate how tumors alter fractalkine signaling in the CNS microenvironment

• Technical challenges:

  • Distinguishing between effects on resident microglia versus infiltrating peripheral immune cells

  • Accounting for region-specific variations in fractalkine expression and CX3CR1+ cell density

  • Managing the rapid internalization of CX3CR1 following fractalkine exposure, which can complicate receptor expression analysis