Recombinant Rat C-X-C motif chemokine 5 protein (Cxcl5) (Active)

What is the molecular structure of rat CXCL5 and how does it compare to other chemokines?

Rat CXCL5 adopts the typical chemokine fold characterized by a six-stranded antiparallel β-sheet with two overlying α-helices. The structure shows several distinct differences in the 30s loop and N-terminal residues compared to other chemokines. The CXCL5 dimer (excluding side chains) measures approximately 33 Å long, 26 Å wide, and 16 Å deep. The dimer interface is stabilized by six β-strand backbone hydrogen bonds and numerous packing interactions. While CXCL5 shares structural similarities with CXCL1, CXCL2, CXCL7, and CXCL8, superposition of backbone residues reveals root-mean-square deviation (rmsd) values between 1.1 and 2.2 Å, with higher variations in the N-terminal residues, N-terminal loop, and 30s turn regions .

What is the primary receptor for rat CXCL5 and how does this interaction mediate signaling?

Rat CXCL5 primarily interacts with the CXCR1 receptor, which differs from human CXCL5 that mainly binds to CXCR2 . This G-protein-coupled receptor interaction initiates downstream signaling cascades that mediate neutrophil chemotaxis and activation. One significant pathway activated by CXCL5 is the JAK2/STAT5/SOCS2 pathway, which has been implicated in blocking insulin signaling . The receptor binding mechanism involves a unique glycosaminoglycan (GAG) geometry where GAG-binding amino acid residues form a continuous surface layer that participates in receptor interactions, distinguishing it from other chemokines like CXCL1 and CXCL8 .

What are the main physiological functions of CXCL5 in normal rat tissues?

In normal physiology, rat CXCL5 functions as a neutrophil chemoattractant involved in leukocyte homeostasis . It plays crucial roles in regulating:

• Neutrophil trafficking and activation

• Leukocyte recruitment during inflammatory responses

• Angiogenesis regulation in tissue development and repair

• Pulmonary host defense against bacterial infections

Additionally, CXCL5 serves as an important mediator in the crosstalk between innate and adaptive immunity, as it affects both neutrophil infiltration and B lymphocyte accumulation in tissues, particularly during infectious challenges .

How is CXCL5 expression regulated in different cell types and under various stimuli?

CXCL5 is expressed in various epithelial cell lines alongside CXCL8 (IL-8) and CXCL1, primarily in response to pro-inflammatory stimuli. Key regulatory mechanisms include:

• Upregulation: Expression is increased in response to stimulation by IL-1 or TNF-alpha, common inflammatory mediators

• Downregulation: Interferon-gamma (IFNG) acts as a negative regulator of CXCL5 expression

• Tissue-specific regulation: Expression patterns vary across different tissue types, with significant expression in epithelial cells

• Pathological regulation: CXCL5 expression increases in response to muscle injury and various inflammatory conditions

Research methodologies for studying CXCL5 expression include real-time polymerase chain reaction, Luminex assays, and immunofluorescent staining, which can effectively track expression changes in both healthy and pathological states .

What experimental approaches are most effective for detecting and quantifying CXCL5 in rat tissue samples?

For optimal detection and quantification of CXCL5 in rat tissue samples, researchers should consider the following methodological approaches:

For comprehensive analysis, researchers should combine multiple techniques. For instance, in studies of CXCL5 in lupus models, investigators successfully employed a combination of Luminex, RT-PCR, and immunofluorescent staining to establish correlations between CXCL5 levels and disease activity .

How does CXCL5 function in immune-mediated disorders such as SLE, and what are the implications for therapeutic development?

In systemic lupus erythematosus (SLE), CXCL5 plays a complex immunomodulatory role. Serum CXCL5 levels are significantly lower in SLE patients compared to healthy individuals and negatively correlate with disease activity (p < 0.0001) . This counterintuitive finding suggests CXCL5 may have protective effects in SLE.

Therapeutic implications include:

• Supplementation approach: Administration of CXCL5 to lupus-prone MRL/lpr mice restored endogenous circulatory CXCL5, improved survival, and reduced multiple disease markers including anti-dsDNA antibodies, proteinuria, and lupus nephritis indices

• Mechanism of action: CXCL5 dictates neutrophil trafficking and suppresses neutrophil activation, degranulation, proliferation, and renal infiltration

• Metabolic effects: RNA sequencing revealed that CXCL5 mediates immunomodulation by promoting energy production in renal-infiltrated immune cells while reducing tissue fibrosis, granulocyte extravasation, complement components, and interferons

• Combination potential: Factorial design results indicate that CXCL5 may enhance host tolerability to cyclophosphamide in vulnerable individuals, suggesting potential as an adjuvant therapy

These findings highlight the need for careful experimental design when studying CXCL5 in autoimmune contexts, as its effects may be disease-stage specific and involve multiple cellular targets.

What is the role of CXCL5 in cancer progression and metastasis based on current research?

CXCL5 demonstrates complex functions in cancer biology, often exhibiting context-dependent effects. Current research indicates:

• Expression patterns: CXCL5 is frequently dysregulated in malignant tumors, with altered expression associated with tumor metastasis and angiogenesis

• Prostate cancer implications: CXCL5 overexpression promotes prostate cancer cell proliferation, migration and invasion through both autocrine and paracrine pathways

• Mechanistic actions: CXCL5 promotes tumor progression through:

  • Formation of immunosuppressive microenvironments

  • Enhancement of tumor angiogenesis

  • Facilitation of tumor cell migration and invasion

• Regulatory pathways: In prostate cancer, NDRG3 overexpression increases CXCL5 expression, promoting tumor cell growth through CXCR2 receptor interaction

When studying CXCL5 in cancer models, researchers should employ multiple detection methods across tissue types and correlate findings with clinical parameters to establish meaningful connections between CXCL5 expression and malignant phenotypes.

How does CXCL5 influence pulmonary immune responses during viral infections?

CXCL5 exhibits multifaceted roles in pulmonary viral infections, particularly influenza. Research using CXCL5-/- mouse models has revealed:

• Dual regulation of leukocyte recruitment: CXCL5 not only mediates neutrophil infiltration into infected lungs during innate immune responses but also affects B lymphocyte accumulation by regulating CXCL13 expression

• Macrophage-mediated effects: Inhibition of the CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs

• Suppressive activity: In vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge

• Adaptive immune modulation: CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to enhanced B cell immune responses and facilitating induced bronchus-associated lymphoid tissue formation during late infection and recovery stages

These findings highlight CXCL5's regulatory roles beyond simple neutrophil recruitment, suggesting it functions as an important bridge between innate and adaptive immunity during respiratory viral infections.

What are the critical considerations when designing experiments using recombinant rat CXCL5 protein?

When designing experiments with recombinant rat CXCL5, researchers should consider several critical factors:

• Protein stability and storage: Store desiccated at -20°C to maintain biological activity

• Biological activity assessment: Validate activity using chemotaxis bioassays with human peripheral blood neutrophils at concentrations of 10-100 ng/ml

• Species-specific differences: Consider that rat CXCL5 has been referred to as rat CXCL6 and is an ortholog of human CXCL5 and CXCL6, as well as murine CXCL5

• Receptor specificity: Account for the fact that rat CXCL5 binds primarily to CXCR1, which differs from human CXCL5's preference for CXCR2

• GAG interaction effects: Consider the unique GAG-binding properties of CXCL5, which may affect its bioavailability and activity in different experimental contexts

• Dose-response relationships: Thoroughly establish dose-response curves as CXCL5 may exhibit biphasic effects depending on concentration

• Experimental readouts: Employ multiple readouts (e.g., cell migration, signaling pathway activation, gene expression) to comprehensively assess CXCL5 effects

How should researchers approach contradictory findings regarding CXCL5's role in different disease models?

Contradictory findings regarding CXCL5's role in different disease models present a significant challenge. To address this complexity, researchers should:

• Consider context-dependent functions: CXCL5 exhibits opposing roles in different diseases - protective in SLE but potentially pathogenic in cancer

• Analyze temporal dynamics: Examine CXCL5 effects across different disease stages, as its function may change during disease progression

• Account for cellular heterogeneity: Analyze cell type-specific responses using techniques like CyTOF or single-cell RNA sequencing to resolve apparent contradictions

• Integrate pathway analyses: Use systems biology approaches to understand how CXCL5 interacts with broader signaling networks in different disease contexts

• Design rigorous controls: Include appropriate genetic controls (e.g., CXCL5-/- models) and receptor antagonists to establish causality

• Validate across models: Confirm findings across multiple model systems and correlate with human data when possible

• Consider compensatory mechanisms: Examine potential redundancy with other chemokines that may mask or alter CXCL5 effects

What novel therapeutic approaches are being explored based on CXCL5 biology?

Current research suggests several promising therapeutic directions based on CXCL5 biology:

• Autoimmune disease applications: Direct CXCL5 administration has shown beneficial effects in lupus models, improving survival and reducing pathology through modulation of neutrophil function and metabolic reprogramming of immune cells

• Cancer-targeting strategies: Since CXCL5 promotes tumor progression in many contexts, targeting the CXCL5-CXCR2 axis may represent a viable approach for cancer therapy by disrupting the immunosuppressive microenvironment, inhibiting angiogenesis, and reducing metastasis

• Infection and inflammation modulation: CXCL5's role in regulating both innate and adaptive immune responses during infections suggests potential applications in controlling excessive inflammation while maintaining protective immunity

• Combination therapy approaches: Evidence suggests CXCL5 may enhance tolerability to conventional therapies like cyclophosphamide, indicating potential as an adjuvant treatment

• Targeted delivery systems: Development of tissue-specific delivery systems could harness CXCL5's beneficial effects while minimizing potential off-target consequences

These approaches require careful experimental validation with consideration of disease-specific contexts and potential compensatory mechanisms within the chemokine network.

How can advanced imaging and analytical techniques enhance our understanding of CXCL5 function in complex biological systems?

Advanced imaging and analytical techniques offer powerful approaches to elucidate CXCL5 function in complex biological systems:

• Intravital microscopy: This technique allows real-time visualization of CXCL5-mediated leukocyte trafficking in living tissues, providing insights into the dynamics of cell recruitment and interaction

• Mass cytometry (CyTOF): As demonstrated in influenza infection studies, CyTOF enables comprehensive profiling of immune cell populations and their responses to CXCL5, revealing previously unappreciated effects on B cell accumulation

• Single-cell RNA sequencing: This approach can identify cell-specific transcriptional responses to CXCL5 stimulation or deficiency, helping resolve apparently contradictory findings across different cell types

• Spatial transcriptomics: Combining tissue localization with gene expression analysis can map CXCL5 effects within tissue microenvironments

• CRISPR-based functional genomics: Systematic perturbation of CXCL5-associated pathways can identify critical mediators and modifiers of CXCL5 function

• Computational modeling: Integration of experimental data into mathematical models can predict CXCL5 behavior across different contexts and generate testable hypotheses

• Proteomics approaches: Analysis of post-translational modifications and protein-protein interactions can reveal regulatory mechanisms affecting CXCL5 function

Implementation of these advanced techniques should be guided by clear hypotheses and complemented by traditional functional assays to establish biological relevance.

What are the most promising unexplored areas in CXCL5 research that could yield significant scientific insights?

Several promising research directions for CXCL5 remain relatively unexplored:

• Metabolic regulation: Further investigation into how CXCL5 influences energy metabolism in immune cells could reveal novel mechanisms of immunomodulation, particularly given its effects on energy production in renal-infiltrated immune cells in lupus models

• Tissue-specific functions: Systematic comparison of CXCL5 functions across different tissues and organ systems may reveal specialized roles beyond current understanding

• Developmental biology: The role of CXCL5 in embryonic development and tissue maturation remains poorly characterized

• Neural-immune interactions: Potential crosstalk between CXCL5-mediated immune responses and neurological functions deserves exploration

• Microbiome interactions: How CXCL5 influences and is influenced by the microbiome, particularly in mucosal tissues, represents an important research frontier

• Comparative biology: Evolutionary conservation and divergence of CXCL5 function across species could provide insights into fundamental biological processes

• Chronobiology: Temporal regulation of CXCL5 expression and function may reveal important circadian influences on immune responses

These areas represent opportunities for groundbreaking research that could substantially advance our understanding of CXCL5 biology and its therapeutic potential.

How might emerging computational and systems biology approaches transform our understanding of CXCL5 function?

Emerging computational and systems biology approaches offer transformative potential for CXCL5 research:

• Network analysis: Mapping CXCL5 within broader signaling networks can identify unexpected functional connections and explain context-dependent effects

• Multi-omics integration: Combining genomics, transcriptomics, proteomics, and metabolomics data can provide comprehensive views of CXCL5's biological effects

• Machine learning applications: Pattern recognition in large datasets may reveal novel CXCL5-associated biomarkers and disease correlations

• Quantitative systems pharmacology: Modeling CXCL5 interventions across biological scales can predict therapeutic outcomes and optimize dosing strategies

• Virtual screening approaches: Computational screening of small molecule libraries could identify novel CXCL5-CXCR1/2 modulators with therapeutic potential

• Pathway flux analysis: Quantitative analysis of signaling pathway dynamics can reveal rate-limiting steps in CXCL5-mediated responses

• Patient stratification algorithms: Computational approaches could identify patient subgroups most likely to benefit from CXCL5-targeted interventions