Recombinant Mouse C-X-C motif chemokine 5 protein (Cxcl5), partial (Active)

What are the biochemical specifications of commercially available Recombinant Mouse CXCL5?

How should Recombinant Mouse CXCL5 be reconstituted and stored for optimal stability?

For optimal reconstitution, the protein vial should be briefly centrifuged prior to opening to bring the contents to the bottom. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . To enhance stability for long-term storage, it is recommended to add glycerol to a final concentration of 5-50%, with 50% being the default concentration used by many manufacturers .

Storage recommendations include:

• Store the unopened vial at -20°C/-80°C upon receipt

• For multiple use applications, create working aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as these can damage protein integrity

How does CXCL5 regulate neutrophil recruitment and chemokine scavenging during inflammation?

CXCL5 exhibits a complex and seemingly paradoxical role in neutrophil recruitment during inflammation. Despite being a neutrophil chemoattractant itself, CXCL5 can actually impair neutrophil recruitment to inflammatory sites through its interactions with erythrocyte Duffy Antigen Receptor for Chemokines (DARC) .

The underlying mechanism involves:

• CXCL5 binding to erythrocyte DARC, which impairs DARC's ability to scavenge other chemokines from circulation

• This results in elevated plasma concentrations of other neutrophil chemoattractants like CXCL1 and CXCL2

• The elevated systemic levels of these chemokines leads to:

  • Attenuation of chemokine gradients between circulation and tissues

  • Desensitization of CXCR2 on neutrophils

  • Reduced neutrophil chemotactic responsiveness

In a model of E. coli pneumonia, CXCL5-deficient mice demonstrated increased neutrophil influx to the lung and decreased bacterial burden, despite CXCL5's known role as a neutrophil chemoattractant . This counterintuitive finding highlights CXCL5's role in regulating chemokine gradients necessary for efficient neutrophil trafficking to sites of infection.

Calcium flux assays confirmed that neutrophils from CXCL5-deficient mice maintain better CXCR2 responsiveness (with indo-1 violet/blue ratio peaking at 541±27) compared to wild-type mice, supporting the model of CXCR2 desensitization by elevated ligand concentrations in normal conditions .

What are the key signaling pathways activated by CXCL5 and its receptor CXCR2?

CXCL5 exerts its biological effects primarily through binding to its receptor CXCR2, activating multiple downstream signaling pathways that regulate various cellular processes:

• STAT3 Signaling Pathway:

  • CXCL5 binding to CXCR2 activates STAT3 phosphorylation

  • This activation promotes cell proliferation and survival

  • Knockdown of either CXCL5 or CXCR2 reduces STAT3 signaling

• AKT/PI3K Pathway:

  • CXCL5/CXCR2 interaction triggers AKT phosphorylation

  • Activated AKT promotes cell survival, proliferation, and metastatic potential

  • In cancer cells, CXCL5 knockdown attenuates AKT signaling

• MMP2/9 Regulation:

  • CXCL5 stimulation increases MMP2/9 secretion

  • These matrix metalloproteinases facilitate cell invasion and migration

  • Experimental data shows reduced MMP2/9 secretion following CXCL5 or CXCR2 knockdown

• PXN-AKT-PD-L1 Signaling Cascade:

  • Recent research has identified a positive feedback loop involving CXCL5

  • This signaling cascade regulates PD-L1 expression, impacting T cell immunity

  • The pathway represents a potential therapeutic target in cancer

Functional assays have demonstrated that disruption of CXCL5/CXCR2 signaling significantly suppresses malignant cellular phenotypes including proliferation, clonogenesis, apoptosis resistance, migration, and invasion .

How can researchers effectively measure CXCL5 activity in neutrophil chemotaxis assays?

The biological activity of Recombinant Mouse CXCL5 is commonly assessed through neutrophil chemotaxis assays, which provide quantifiable measurements of its functional potency. Below is a methodological approach for conducting these assays:

Neutrophil Chemotaxis Assay Protocol:

• Neutrophil Isolation:

  • Isolate human or mouse neutrophils from peripheral blood using density gradient centrifugation

  • Verify neutrophil purity (>95%) using flow cytometry with neutrophil markers

• Transwell Migration Setup:

  • Use a modified Boyden chamber with 3-5μm pore size

  • Add CXCL5 at various concentrations (typically 1-100 ng/ml) to the lower chamber

  • Add neutrophils (1-5×10^5 cells) to the upper chamber

• Activity Measurement:

  • Incubate for 1-2 hours at 37°C, 5% CO2

  • Count migrated cells by flow cytometry or microscopy after fixation and staining

  • Calculate migration index as the ratio of cells migrating toward CXCL5 versus medium control

• Verification of Receptor Specificity:

  • Include CXCR2 antagonist controls to confirm receptor-mediated effects

  • Use checkerboard analysis to distinguish between chemotaxis and chemokinesis

The biological activity of CXCL5 is typically observed in the concentration range of 10-100 ng/ml, with maximal activity often seen at 50 ng/ml . When analyzing results, a dose-response curve should be constructed to determine the EC50 value, which can serve as a measure of potency.

What methodologies are available for studying CXCL5's role in disease models?

Researchers have employed various experimental approaches to investigate CXCL5's role in disease pathophysiology:

• Genetic Manipulation Models:

  • CXCL5 knockout mice to evaluate loss-of-function effects

  • CXCL5 overexpression models to assess gain-of-function effects

  • Conditional knockout systems for tissue-specific CXCL5 deletion

• Therapeutic Administration Studies:

  • Intravenous administration of recombinant CXCL5 to disease models

  • Pharmacokinetic studies to determine optimal dosing regimens

  • Assessment of short-term (10 weeks) and long-term (2 years) outcomes

• Cellular Functional Assays:

  • Wound healing assays to assess cell migration capacity

  • Transwell invasion assays to measure invasiveness

  • Cell proliferation and clonogenesis assays to evaluate growth potential

  • Apoptosis assays to determine cell survival effects

• Molecular Signaling Analysis:

  • RNA sequencing to identify transcriptional changes

  • Western blotting to monitor signaling pathway activation

  • Luminex assays to measure cytokine/chemokine levels

  • Flow cytometry to assess cellular phenotypes and activation status

• Combination Therapy Approaches:

  • Co-administration of CXCL5 with standard therapeutics

  • Anti-CXCL5 antibodies combined with checkpoint inhibitors

  • Factorial design experiments to assess synergistic effects

For example, in lupus studies, researchers administered CXCL5 to Fas^lpr mice and monitored various disease parameters including survival, autoantibody levels, proteinuria, nephritis indices, complement deposition, and neutrophil extracellular trap formation .

How does CXCL5 contribute to autoimmune disease pathogenesis and potential treatment?

CXCL5 demonstrates significant therapeutic potential in autoimmune diseases, particularly systemic lupus erythematosus (SLE), through its immunomodulatory effects on multiple pathways:

CXCL5 Levels in Autoimmune Disease:

• SLE patients show significantly lower serum CXCL5 levels compared to healthy individuals (p<0.0001)

• CXCL5 levels negatively correlate with disease activity (p=0.004)

• In lupus-prone Fas^lpr mice, disease severity progression inversely correlates with plasma CXCL5 levels

Therapeutic Effects of CXCL5 Administration:
Intravenous administration of CXCL5 to Fas^lpr mice resulted in:

• Restored endogenous circulatory CXCL5 levels

• Improved survival rates

• Reduced anti-dsDNA antibody titers

• Decreased proteinuria

• Improved lupus nephritis activity and chronicity indices

• Reduced renal complement deposition

• Decreased neutrophil extracellular trap formation

Immunomodulatory Mechanisms:
CXCL5 treatment modulates immune responses through:

• Regulation of neutrophil trafficking and suppression of neutrophil activation

• Reduction of neutrophil degranulation, proliferation, and renal infiltration

• Promotion of energy production in renal-infiltrated immune cells

• Activation of certain T cell populations

• Reduction of tissue fibrosis, granulocyte extravasation, complement components, and interferons

Interestingly, factorial design experiments indicated that CXCL5 may enhance host tolerability to cyclophosphamide in vulnerable individuals, suggesting potential benefits as an adjunctive therapy .

What is the role of CXCL5 in cancer progression and its potential as a therapeutic target?

CXCL5 plays a multifaceted role in cancer biology and presents as both a biomarker and potential therapeutic target:

CXCL5 as a Cancer Biomarker:

• Elevated serum CXCL5 levels are associated with tumor progression in multiple cancer types

• Preoperative serum CXCL5 levels are markedly higher in cancer patients compared to healthy individuals (p=0.001)

• CXCL5 expression correlates with its receptor CXCR2 in cancer tissues

Oncogenic Functions of CXCL5/CXCR2 Signaling:
Experimental data from knockdown studies demonstrate that CXCL5/CXCR2 signaling promotes:

• Cancer cell proliferation and clonogenic capacity

• Resistance to apoptosis

• Enhanced migration (measured by wound healing assays)

• Increased invasion (measured by transwell invasion assays)

• Activation of oncogenic signaling pathways including STAT3 and AKT

• Increased MMP2/9 secretion facilitating extracellular matrix degradation

Immune Evasion Mechanisms:
Recent research has uncovered a critical role for CXCL5 in immune evasion through:

• Upregulation of PD-L1 expression in cancer cells

• Creation of an immunosuppressive tumor microenvironment

• Impairment of CD8+ T cell anti-tumor immunity

• Mobilization of immunosuppressive neutrophils

• Establishment of a PXN-AKT-PD-L1 positive feedback loop

Therapeutic Targeting Approaches:
Preclinical studies have shown promising results with:

• Anti-CXCL5 antibody therapy

• Combination therapy with anti-PD-L1 immune checkpoint inhibitors

• Significant tumor growth inhibition in vivo with combination approaches

The dual targeting of CXCL5 and PD-L1 represents a potentially synergistic approach that addresses both tumor cell-intrinsic growth pathways and immune evasion mechanisms.

How do structural differences between CXCL5 and other CXC chemokines influence their functional diversity?

The structural nuances between CXCL5 and other CXC chemokines contribute significantly to their functional specialization:

Structural Comparison Analysis:
While CXCL5 maintains the canonical chemokine fold similar to CXCL1, CXCL2, CXCL7, and CXCL8, several key structural differences exist:

• The organization of β-sheets and α-helices remains conserved across these chemokines

• Higher structural variability is observed in the N-terminal residues, N-terminal loop, and 30s turn

• These variable regions coincide with functionally important domains and show the largest sequence differences

When superimposing backbone residues (positions 11-76) of CXCL5 with other CXC chemokines:

These structural variations likely contribute to:

• Differential receptor binding affinities and specificities

• Distinct protein-protein interaction profiles

• Varied susceptibility to proteolytic processing

• Different oligomerization properties

• Specialized functional roles in neutrophil recruitment versus retention

Understanding these structure-function relationships is crucial for rational drug design targeting specific chemokine functions while preserving others.

What are the critical considerations when designing experiments to study CXCL5-mediated effects in complex disease models?

When designing experiments to investigate CXCL5 in disease models, researchers should address several critical considerations:

1. Proteolytic Processing and Isoform Specificity:

• CXCL5 undergoes proteolytic processing after secretion from fibroblasts and epithelial cells

• Multiple N-terminal (processed between positions 41-48) and C-terminal (processed between positions 118-132) forms exist

• GCP-2(1-78) and GCP-2(9-78) are the most prominent forms, with GCP-2(9-78) showing higher potency

• Experimental design should specify which isoform is being studied or measured

2. Expression System Considerations:

• CXCL5 produced in different expression systems (E. coli vs. HEK 293) may exhibit different post-translational modifications

• These differences can affect biological activity and should be considered when interpreting results

• Endotoxin contamination must be minimized (<0.005 EU/μg) to prevent confounding inflammatory effects

3. Receptor Complexity:

• While CXCR2 is the primary receptor, CXCL5 may interact with other receptors including atypical chemokine receptors

• DARC (Duffy Antigen Receptor for Chemokines) interactions critically affect CXCL5 bioavailability

• Experiments should account for receptor expression patterns in the model system studied

4. Context-Dependent Effects:

• CXCL5 exhibits apparently contradictory functions in different disease contexts (pro-inflammatory in some, anti-inflammatory in others)

• Time-dependent effects should be assessed with both short-term (10 weeks) and long-term (2 years) endpoints

• Dose-response relationships should be thoroughly characterized

5. Methodological Approaches:

• Combined in vitro and in vivo approaches provide complementary insights

• Genetic approaches (knockdown, knockout, overexpression) should be coupled with pharmacological interventions

• Appropriate controls for specificity, including receptor antagonists and neutralizing antibodies

• Advanced analytical methods including RNA sequencing, mass spectrometry, and multiplexed cytokine analysis provide comprehensive assessment

By addressing these considerations, researchers can design robust experiments that advance our understanding of CXCL5's complex roles in health and disease.

What are promising new approaches for targeting the CXCL5/CXCR2 axis in inflammatory and autoimmune diseases?

The therapeutic potential of CXCL5 in inflammatory and autoimmune diseases opens several promising research directions:

1. Selective CXCL5 Modulation Strategies:

• Development of isoform-specific CXCL5 modulators that target pathogenic functions while preserving beneficial immune surveillance

• Design of selective CXCR2 agonists/antagonists that specifically modulate CXCL5 signaling without affecting other ligands

• Exploration of biased signaling modulators that activate beneficial downstream pathways while inhibiting harmful ones

2. Precision Medicine Approaches:

• Identification of biomarker profiles that predict patient responsiveness to CXCL5-targeted therapies

• Development of circulating CXCL5 level assays as companion diagnostics

• Patient stratification strategies based on CXCL5/CXCR2 expression patterns

3. Novel Delivery Systems:

• Targeted delivery of recombinant CXCL5 to specific tissues using nanoparticle formulations

• Cell-based delivery systems using engineered cells programmed to secrete CXCL5 in response to inflammatory cues

• Sustained-release formulations to maintain therapeutic CXCL5 levels while minimizing systemic exposure

4. Combination Therapy Optimization:

• Further investigation of CXCL5 as an adjunct to standard immunosuppressive therapies

• Factorial design studies to identify optimal dosing regimens and drug combinations

• Exploration of synergistic combinations targeting multiple aspects of autoimmune pathogenesis

Research priorities should include detailed mechanistic studies to fully elucidate CXCL5's immunomodulatory effects in specific disease contexts, development of more predictive preclinical models, and early-phase clinical studies to establish safety and preliminary efficacy.

How can advanced structural biology approaches enhance our understanding of CXCL5-receptor interactions?

Advanced structural biology approaches offer transformative potential for understanding CXCL5-receptor interactions at molecular resolution:

1. Cryo-Electron Microscopy (Cryo-EM) Applications:

• Determination of CXCL5-CXCR2 complex structures in different activation states

• Visualization of oligomeric assemblies and higher-order signaling complexes

• Elucidation of conformational changes during receptor activation and signaling

2. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Mapping protein dynamics and conformational changes upon binding

• Identification of allosteric communication networks within the CXCL5-CXCR2 complex

• Characterization of binding interfaces with other interaction partners

3. Single-Molecule Fluorescence Resonance Energy Transfer (smFRET):

• Real-time monitoring of CXCL5-induced conformational changes in CXCR2

• Investigation of the dynamics of receptor activation and signaling

• Determination of the kinetics of ligand binding and dissociation

4. Molecular Dynamics Simulations:

• In silico modeling of CXCL5-receptor interactions in membrane environments

• Prediction of binding energetics and identification of key interaction residues

• Virtual screening of potential modulators targeting specific interaction interfaces

5. Structure-Based Drug Design:

• Rational design of small molecules targeting specific CXCL5-CXCR2 interaction sites

• Development of peptide mimetics based on critical binding epitopes

• Creation of bispecific molecules targeting both CXCL5 and complementary pathways