Recombinant Mouse C-X-C motif chemokine 16 protein (Cxcl16), partial (Active)

What is recombinant mouse CXCL16 protein and what is its basic structure?

Recombinant mouse CXCL16 protein is a fragment protein typically spanning amino acids 27 to 114 of the full sequence, expressed in expression systems such as Escherichia coli with high purity (>98%) and low endotoxin levels (<1 EU/μg) . The protein belongs to the intercrine alpha (chemokine CxC) family and undergoes post-translational modifications including glycosylation . The amino acid sequence includes specific motifs characteristic of the CXC chemokine family with a conserved structure that enables receptor binding and biological activity .

What are the major biological functions of CXCL16?

CXCL16 exhibits dual functionality in biological systems. First, it induces strong chemotactic responses and calcium mobilization through binding to its receptor CXCR6/Bonzo, facilitating directed cell migration . Second, it acts as a scavenger receptor on macrophages, specifically binding to oxidized low-density lipoprotein (OxLDL), suggesting potential involvement in pathophysiological processes such as atherogenesis . Additionally, CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria, contributing to innate immune responses .

Where is CXCL16 primarily expressed in normal tissues?

CXCL16 demonstrates tissue-specific expression patterns. High levels of CXCL16 mRNA and protein have been detected in lung tissue, with substantial amounts produced by alveolar macrophages and bronchial epithelial cells . The protein is also expressed in liver tissue, where it influences uptake and subcellular localization processes . In lymphoid organs, CXCL16 is produced by dendritic cells located in T cell zones and by cells in the red pulp of the spleen . Additionally, CXCL16 and its receptor CXCR6 are physiologically expressed by cells of the brain parenchyma, including astrocytes, microglia, and neurons .

What cells respond to CXCL16 signaling?

CXCL16 primarily acts on cells expressing its receptor CXCR6. Cell populations that bind and migrate in response to CXCL16 include several subsets of T cells and natural killer T (NKT) cells . In the nervous system, CXCL16 affects neurons through modulation of neurotransmitter release at both GABA-ergic and glutamatergic synapses . The interaction between CXCL16 and responsive cells plays crucial roles in immune cell trafficking, inflammatory responses, and tissue homeostasis across multiple organ systems.

What are the optimal methods for analyzing CXCL16 expression in tissues and cells?

Analysis of CXCL16 expression can be performed at both mRNA and protein levels using complementary techniques:

For mRNA detection:

• Quantitative PCR using validated primers is the gold standard. For mouse CXCL16, researchers have successfully used primers: forward 5′-AAA CAT TTG CCT CAA GCC AGT-3′, reverse 5′-GTT TCT CAT TTG CCT CAG CCT-3′ .

• For human CXCL16: forward 5′-GCA GCG TCA CTG GAA GTT GTT AT-3′, reverse 5′-TGC GGT GAG GAT GAA GAT GAT GA-3′ .

For protein detection:

• ELISA for quantification in biological fluids such as serum or bronchoalveolar lavage .

• Western blotting for tissue or cellular extracts.

• Flow cytometry for cell surface expression using nonenzymatic cell dissociation methods to preserve membrane integrity .

How should in vitro stimulation experiments be designed to study CXCL16 regulation?

For optimal in vitro stimulation experiments examining CXCL16 regulation:

• Cell preparation:

  • Grow cells to confluency (approximately 1 × 10^6 epithelial cells or 200 × 10^3 fibroblasts/smooth muscle cells) in appropriate multi-well plates .

  • Prior to stimulation, starve cells overnight in serum-free medium to reduce background signaling .

• Stimulation conditions:

  • Use physiologically relevant cytokines known to regulate CXCL16, including IFN-γ (5 ng/mL), TNF-α (5 ng/mL), IL-4 (10 ng/ml), or combinations thereof .

  • Culture with stimulants for 24 hours in fresh growth medium to allow sufficient time for gene expression changes .

• Analysis methods:

  • Collect conditioned medium for secreted CXCL16 detection by ELISA.

  • Process cells for RNA extraction using TRIzol reagent for expression analysis.

  • Prepare cell lysates using appropriate buffers (e.g., Laemmli buffer) for Western blotting .

What are the technical considerations for functional studies using recombinant CXCL16?

When conducting functional studies with recombinant CXCL16, researchers should consider:

• Protein handling:

  • Recognize that this is an active protein that may elicit biological responses in vivo, requiring cautious handling .

  • Use appropriate reconstitution buffers and storage conditions to maintain protein integrity.

• Dosage determination:

  • For neurophysiological studies, effective concentrations around 10 nM have been documented .

  • Dose-response experiments should be performed to determine optimal concentrations for specific cell types.

• Functional readouts:

  • For chemotaxis assays, establish appropriate positive controls and migration parameters.

  • For calcium mobilization, optimize fluorescent indicator loading and measurement conditions.

  • For electrophysiological studies, standardize recording parameters for consistent results .

• Controls:

  • Include heat-inactivated protein controls to confirm specificity of biological effects.

  • Where appropriate, use receptor antagonists or cells lacking CXCR6 expression as negative controls.

How does CXCL16 modulate neurotransmission in the central nervous system?

CXCL16 exerts sophisticated modulatory effects on synaptic transmission in the brain, particularly in the hippocampal CA1 region. Electrophysiological studies have revealed that CXCL16 differentially regulates inhibitory and excitatory neurotransmission:

• Effects on GABAergic transmission:

  • CXCL16 (10 nM) reduces the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) to approximately 72.7% of control values within 20 minutes of application .

  • The treatment significantly increases the paired-pulse ratio (PPR) from 1.67 to 2.43, suggesting a presynaptic mechanism involving decreased probability of GABA release .

• Effects on glutamatergic transmission:

  • Conversely, CXCL16 increases the peak amplitude of evoked excitatory postsynaptic currents (eEPSCs) and reduces the PPR (from 1.41 to 1.26), indicating increased probability of glutamate release .

  • These effects suggest CXCL16 may enhance excitatory neurotransmission while simultaneously reducing inhibitory inputs.

• Signaling mechanisms:

  • The modulatory actions of CXCL16 in the CNS require functional adenosine receptor type 3 (A3R) .

  • The pathway involves cross-communication between neurons, astrocytes, and microglia, with the chemokine CCL2 playing a downstream role in the signaling cascade .

What is the role of CXCL16 in intestinal inflammation and how is it regulated?

CXCL16 plays a significant role in intestinal inflammation, particularly in Crohn's disease:

• Expression patterns:

  • CXCL16 mRNA levels are altered in colonic biopsies from Crohn's disease patients compared to healthy controls .

  • Similar dysregulation is observed in experimental models including the TNF∆ARE mouse model of ileitis and murine cytomegalovirus (MCMV)-induced colitis .

• Regulatory mechanisms:

  • Pro-inflammatory cytokines IFN-gamma and TNF-alpha strongly induce CXCL16 expression in intestinal epithelial cells .

  • This cytokine-mediated upregulation suggests CXCL16 production increases during active inflammation.

• Functional significance:

  • CXCL16 acts as a potent chemoattractant for CXCR6+ T cells, potentially contributing to lymphocyte recruitment to sites of intestinal inflammation .

  • The protein's dual function in bacterial recognition and immune cell recruitment positions it as a critical mediator in the pathogenesis of inflammatory bowel diseases, with particular relevance to Crohn's disease .

• Signaling pathways:

  • CXCL16 activates specific intracellular signaling cascades in intestinal epithelial cells, including MAP kinases and Akt pathways .

  • These signaling events likely contribute to altered epithelial barrier function and inflammatory responses.

What are the technical challenges in studying CXCL16-CXCR6 interactions in complex tissues?

Investigating CXCL16-CXCR6 interactions in complex tissues presents several technical challenges:

• Receptor-ligand specificity:

  • While CXCL16 is the only known ligand for CXCR6, confirming specificity in tissue contexts requires careful controls including receptor antagonists or genetic models.

  • The presence of soluble CXCL16 (cleaved from membrane-bound form) complicates interpretation of binding studies.

• Cell-type heterogeneity:

  • In tissues like brain and intestine, multiple cell types express CXCL16 and/or CXCR6, including neurons, glia, immune cells, and epithelial cells .

  • Single-cell approaches or cell sorting strategies are necessary to delineate cell-specific effects.

• Temporal dynamics:

  • CXCL16 expression can be constitutive in some tissues (e.g., lungs) but inducible in others .

  • Capturing the temporal aspects of CXCL16-CXCR6 signaling requires time-course experiments with appropriate sampling intervals.

• Functional redundancy:

  • Other chemokines may compensate for CXCL16 deficiency in knockout models.

  • Approaches using conditional and tissue-specific gene manipulation help address this limitation.

How does CXCL16 contribute to neuroprotection in brain injury models?

CXCL16 demonstrates significant neuroprotective properties in models of brain injury:

• Protective mechanisms:

  • CXCL16/CXCR6 signaling plays a crucial role in counteracting brain glutamate excitotoxic damage, particularly following cerebral ischemia .

  • The neuroprotective effects involve complex intercellular communication between microglia, astrocytes, and neurons .

• Signaling pathway:

  • The protective mechanism requires adenosine receptor type 3 (A3R) activation .

  • This leads to the release of CCL2 by glial cells, which appears to be a downstream mediator of the neuroprotective effects .

  • The pathway helps maintain homeostasis of excitatory and inhibitory neurotransmission, potentially preventing excitotoxicity.

• Experimental evidence:

  • In electrophysiological studies, CXCL16 modulates both inhibitory and excitatory synaptic transmission in the hippocampal CA1 region, suggesting a role in maintaining synaptic balance .

  • These findings indicate that CXCL16 may be a potential therapeutic target for conditions involving glutamate excitotoxicity, such as stroke or traumatic brain injury.

What is the current understanding of CXCL16's role in respiratory diseases?

CXCL16 plays significant roles in respiratory physiology and pathology:

• Constitutive expression:

  • The lung demonstrates high levels of CXCL16 mRNA expression .

  • Relatively high concentrations of CXCL16 are detected in bronchoalveolar lavage from both normal subjects and patients with respiratory conditions .

• Cellular sources:

  • Alveolar macrophages produce substantial amounts of CXCL16 in vitro .

  • Bronchial epithelial cells express CXCL16, potentially contributing to T cell recruitment to the lung .

• Pathological implications:

  • Elevated CXCL16 levels have been documented in asthma and sarcoidosis patients .

  • The constitutive expression in respiratory tissues suggests a homeostatic role that may be dysregulated in disease states.

  • As a scavenger receptor and chemoattractant, CXCL16 may influence both innate immunity and lymphocyte trafficking in respiratory conditions.

How might CXCL16 be targeted therapeutically in inflammatory conditions?

Based on current understanding, several approaches for therapeutic targeting of CXCL16 in inflammatory conditions can be considered:

• Potential intervention strategies:

  • Neutralizing antibodies against CXCL16 to block its interaction with CXCR6

  • Small molecule antagonists of CXCR6 to prevent downstream signaling

  • Inhibitors of proteases that generate soluble CXCL16 from the membrane-bound form

  • Modulation of upstream regulators like IFN-gamma and TNF-alpha that induce CXCL16 expression

• Disease-specific considerations:

  • In Crohn's disease: Targeting CXCL16 may reduce pathogenic T cell recruitment to intestinal tissues .

  • In neuroinflammation: Augmenting CXCL16 signaling might enhance neuroprotection against excitotoxicity .

  • In respiratory conditions: Modulating CXCL16 could alter T cell trafficking and macrophage function in the lungs .

• Challenges and considerations:

  • CXCL16's dual role as both inflammatory mediator and protective factor requires careful contextual assessment.

  • Tissue-specific delivery systems would help target relevant anatomical locations while minimizing systemic effects.

  • The constitutive expression in some tissues suggests physiological roles that should be preserved while targeting pathological activity.

What are promising areas for future investigation of CXCL16 biology?

Several promising research directions emerge from current CXCL16 knowledge:

• Structural and functional studies:

  • Detailed structural analysis of CXCL16-CXCR6 interactions to facilitate drug design

  • Investigation of structure-function relationships through targeted mutagenesis of specific protein domains

  • Exploration of potential additional receptors or binding partners beyond CXCR6

• Tissue-specific functions:

  • Further characterization of CXCL16's roles in the central nervous system, particularly in synaptic plasticity and learning

  • Deeper investigation of CXCL16 in mucosal immunity and barrier function in intestinal and respiratory systems

  • Exploration of potential roles in other tissues where expression has been detected but functions remain unclear

• Regulatory mechanisms:

  • Identification of transcription factors and epigenetic regulators controlling CXCL16 expression

  • Investigation of post-translational modifications affecting CXCL16 activity

  • Characterization of proteolytic processing pathways that generate soluble CXCL16 from membrane-bound forms

• Therapeutic applications:

  • Development and testing of CXCL16-targeted interventions in preclinical disease models

  • Exploration of CXCL16 as a biomarker for inflammatory conditions

  • Investigation of CXCL16 in combination therapies with established treatment approaches

What methodological advances would enhance CXCL16 research?

Advancing CXCL16 research would benefit from several methodological improvements:

• Improved detection systems:

  • Development of highly specific antibodies distinguishing membrane-bound from soluble CXCL16

  • Creation of reporter systems to monitor CXCL16-CXCR6 interactions in real-time

  • Advanced imaging techniques to visualize CXCL16 trafficking and localization in live cells

• Genetic models:

  • Generation of conditional knockout models allowing tissue-specific and temporal control of CXCL16 or CXCR6 expression

  • CRISPR-engineered cell lines with modified CXCL16/CXCR6 to study structure-function relationships

  • Humanized mouse models to better translate findings to human disease contexts

• High-throughput approaches:

  • Application of proteomics to identify CXCL16-interacting proteins

  • Transcriptomic analyses to comprehensively assess CXCL16-induced gene expression changes

  • Systems biology approaches integrating multiple data types to model CXCL16 signaling networks

• Translational approaches:

  • Development of standardized bioassays for CXCL16 activity applicable across research laboratories

  • Establishment of sample collection and processing protocols for clinical studies

  • Creation of databases integrating CXCL16-related findings across multiple disease models and experimental systems

What are the key considerations when designing experiments with recombinant CXCL16?

When designing experiments with recombinant mouse CXCL16, researchers should consider:

• Protein quality and characterization:

  • Verify protein activity before experiments using established bioassays

  • Confirm purity (>98%) and low endotoxin levels (<1 EU/μg) to avoid confounding results

  • Consider the specific fragment being used (e.g., aa 27-114) and how it relates to the full-length protein

• Experimental controls:

  • Include appropriate positive controls (known CXCL16-responsive systems)

  • Implement negative controls (receptor antagonists, CXCR6-deficient cells)

  • Consider time-course experiments to capture both immediate and delayed responses

• Physiological relevance:

  • Use concentrations that approximate those found in biological fluids

  • Consider the microenvironment of the tissue being modeled

  • Account for potential differences between soluble and membrane-bound forms

• Documentation and reporting:

  • Clearly specify the exact protein used (species, fragment, tag, etc.)

  • Document reconstitution methods and storage conditions

  • Report detailed methodology to facilitate replication and comparison across studies