Recombinant Human C-X-C motif chemokine 2 (CXCL2), partial (Active)

Basic Research Questions

• What is CXCL2 and what are its basic structural characteristics?

  Recombinant Human C-X-C Motif Chemokine 2 (CXCL2) is a small secreted cytokine belonging to the CXC chemokine family. When expressed in E. coli systems, human CXCL2 typically encompasses amino acids Thr39-Asn107 of the full protein. It has a molecular weight of approximately 7.67 kDa and shares 90% amino acid sequence identity with the related chemokine CXCL1 . The gene encoding CXCL2 is located on human chromosome 4 in a cluster with other CXC chemokines, indicating evolutionary relationships and potentially coordinated expression patterns .

• What cells primarily produce CXCL2 and under what conditions?

  CXCL2 is primarily secreted by activated monocytes, macrophages, and neutrophils at sites of inflammation . Interestingly, unlike many chemokines that have multiple cellular sources, CXCL2 has been found to be almost exclusively derived from neutrophils in certain inflammatory contexts . Production is typically stimulated by inflammatory mediators, with IL-1β serving as a predominant signal controlling the synthesis and secretion of CXCL2 in multiple cell types including pancreatic β-cells . In experimental settings, CXCL2 protein release can be induced 132-fold by IL-1β at 3 hours and can rise to 464-fold at 6 hours post-stimulation .

• What are the primary receptors and signaling mechanisms of CXCL2?

  CXCL2 primarily interacts with the G-protein coupled receptor CXCR2 on target cells . Upon binding to CXCR2, CXCL2 activates multiple downstream signaling cascades. Most notably, it can activate the CXCR2/PKC/NOX4 pathway in neutrophils . CXCL2 binding to CXCR2 induces calcium release and β-arrestin recruitment, which can be measured as functional readouts of receptor activation . The potency (EC50) of CXCL2 for calcium release has been reported to be approximately 9 nM, showing higher potency than CXCL1 (EC50 57 nM) and the CXCL1-CXCL2 heterodimer (EC50 121 nM) .

• How does CXCL2 compare functionally to other chemokines, particularly CXCL1?

  Despite their high structural homology (~90% amino acid sequence identity), CXCL1 and CXCL2 exhibit distinct functional properties and expression kinetics:

  • Expression timing: CXCL1 gene expression peaks at 1 hour after IL-1β stimulation, while CXCL2 peaks at 3 hours

  • Functional sequence: In neutrophil migration through venular walls, CXCL1 and CXCL2 act sequentially rather than redundantly

  • Cellular localization: During inflammation, CXCL1 and CXCL2 are compartmentalized differently, with CXCL2 often localized within endothelial cell junctions

  • Migration guidance: CXCL1 supports luminal neutrophil adhesion and crawling, while CXCL2 mediates transmigration from the luminal to abluminal side of endothelial cells

• How is CXCL2 gene expression regulated at the transcriptional level?

  CXCL2 gene transcription is tightly regulated by specific transcription factors, particularly NF-κB proteins which are critical for its expression . The CXCL2 promoter contains several predicted κB sequences that serve as binding sites for NF-κB. Notably, there is a conserved consensus κB site with an identical sequence in both CXCL1 and CXCL2 promoters at positions -641 and -640 respectively (relative to the transcriptional start site) . The table below shows the predicted κB sequences in CXCL1 and CXCL2 promoters:

  CXCL1	CXCL2	Position (CXCL1/CXCL2)
  GGGAATTTCCC	GGGGCTTTTCC	-83/-54
  GGGAAACACCC	GGGGATTTCCC	-102/-73
  GGAAGTTCCC	GGAAGTTCCC	-641/-640
  TGGACTTTCC	ND	-709/ND
  GGGATTTGCT	ND	-1299/ND

  Mutation of the consensus κB sequence decreases CXCL2 gene transcription by approximately 53% in response to IL-1β stimulation .

• What are the main functional roles of CXCL2 in physiological immune responses?

  CXCL2 plays several crucial roles in physiological immune responses:

  • Neutrophil chemotaxis: CXCL2 is a potent chemoattractant for neutrophils, guiding their migration to sites of inflammation

  • Directional migration guidance: CXCL2 provides directional cues for neutrophils within endothelial cell junctions, mediating persistent migration from the apical to basolateral aspect of endothelial cells

  • Hematopoietic regulation: CXCL2 functions as a hematoregulatory chemokine that can suppress hematopoietic progenitor cell proliferation

  • Osteoclast differentiation: CXCL2 can enhance the proliferation of osteoclast precursor cells through the activation of ERK and stimulate adhesion and migration of bone marrow-derived macrophages during osteoclastogenesis

  • Self-guided migration: Neutrophil-derived CXCL2 creates a self-guided migration response through endothelial cell junctions

Advanced Research Questions

• What are the methodological approaches to measuring CXCL2 activity in vitro and in vivo?

  Several established methodologies can be used to measure CXCL2 activity:

  In vitro assays:

  • Calcium release assay: Using mouse bone marrow neutrophils incubated with calcium-sensitive dye to measure fluorescence changes upon CXCL2 stimulation

  • β-Arrestin recruitment assay: Using the mCXCR2 PathHunter kit with mouse CXCR2 CHO.K1 cells to measure β-galactosidase-induced luminescence after CXCL2 binding

  • Chemotaxis assays: Using Transwell filters with CXCL2 in bottom chambers to assess neutrophil migration through filters coated with BSA or other chemokines

  • ELISA: To quantify CXCL2 protein secretion in cell culture supernatants

  In vivo assays:

  • Peritoneal recruitment assay: Administering CXCL2 intraperitoneally at different doses (0.1, 1, and 10 μg) and measuring neutrophil numbers in peritoneal lavage using cytospin and differential cell counts

  • High-resolution confocal intravital microscopy (IVM): Using fluorescently labeled neutrophils and endothelial cell junctions to track CXCL2-mediated neutrophil migration through venular walls in live animals

• How do CXCL1 and CXCL2 coordinate neutrophil migration through vascular walls?

  Despite their structural similarity, CXCL1 and CXCL2 perform distinct and sequential functions in guiding neutrophil migration through inflamed venular walls:

  1. Luminal adhesion and crawling: CXCL1 primarily mediates initial neutrophil adhesion to the luminal surface of endothelial cells and supports intraluminal crawling

  2. Transendothelial migration (TEM): CXCL2, almost exclusively derived from neutrophils themselves, creates a self-guided migration pathway through endothelial cell junctions

  3. Directional guidance: CXCL2 provides critical directional cues within endothelial cell junctions, mediating persistent migration from the apical to basolateral side of endothelial cells

  4. Junctional retention: Endothelial atypical chemokine receptor 1 (ACKR1), enriched within junctions, retains extrinsic CXCL2, creating a junctional chemokine "depot" required for efficient unidirectional luminal-to-abluminal neutrophil migration

  This non-redundant, sequential mechanism ensures efficient neutrophil extravasation during inflammatory responses.

• What role does CXCL2 play in pathological conditions like cancer and inflammatory diseases?

  CXCL2 is implicated in several pathological conditions:

  Cancer:

  • CXCL2 can contribute to tumor progression by promoting tumor cell proliferation, macrophage recruitment, and M2 polarization

  • In oral squamous cell carcinoma (OSCC), CXCL2 mediates pro-cancer effects and enhances macrophage recruitment and M2 polarization

  • CXCL2 knockdown can attenuate these pro-cancer effects, suggesting potential therapeutic targeting

  Inflammatory diseases:

  • In sepsis, CXCL2 on macrophage extracellular vesicles (EVs) recruits neutrophils and activates the CXCR2/PKC/NOX4 pathway, potentially contributing to tissue damage

  • CXCL2 is considered a novel therapeutic target for inflammatory bone destructive diseases due to its role in osteoclast differentiation and activity

  • CXCL2 can promote proinflammatory reactions, immune regulation, and angiogenesis in various disease contexts

• What is the role of CXCL2 in extracellular vesicle-mediated intercellular communication?

  CXCL2 plays a significant role in extracellular vesicle (EV)-mediated intercellular communication, particularly in inflammatory conditions:

  • EV enrichment: CXCL2 is highly expressed in EVs isolated from macrophages (such as mouse Raw264.7 macrophages) or from the serum of patients with sepsis

  • Neutrophil recruitment: CXCL2-containing EVs released from LPS-induced macrophages can recruit neutrophils both in vitro and in vivo

  • Signaling pathway activation: These CXCL2-containing EVs activate the CXCR2/PKC/NOX4 pathway in neutrophils

  • Tissue damage mediation: The activation of neutrophils by CXCL2-containing EVs can lead to tissue damage, particularly in the context of sepsis

  This EV-mediated CXCL2 delivery represents a novel intercellular communication pathway between macrophages and neutrophils during inflammatory responses, potentially contributing to both beneficial antimicrobial responses and detrimental tissue damage in conditions like sepsis.

• How can heterodimers of CXCL2 with other chemokines affect neutrophil recruitment and activation?

  CXCL2 can form heterodimers with other chemokines, particularly CXCL1, with distinct functional properties:

  • Formation: CXCL1 and CXCL2 can form heterodimers with properties distinct from either homodimer

  • Receptor activation potency: For calcium release (a measure of G protein activation), the CXCL1-CXCL2 heterodimer shows intermediate potency (EC50 121 nM) compared to CXCL1 (EC50 57 nM) and CXCL2 (EC50 9 nM)

  • Neutrophil recruitment profile: The heterodimer exhibits a unique neutrophil recruitment profile distinct from either chemokine alone:

    • At low doses (0.1 μg), the heterodimer is less active than either CXCL1 or CXCL2 alone

    • At higher doses, the heterodimer shows a robust recruitment profile that differs from the individual chemokines

  • Functional implications: The existence of CXCL1-CXCL2 heterodimers adds another layer of complexity to neutrophil recruitment regulation, potentially allowing fine-tuning of inflammatory responses depending on the relative expression levels of each chemokine

• What methods are most effective for CXCL2 gene silencing in research models?

  Based on the search results, several approaches to CXCL2 gene silencing have been successfully employed in research:

  • siRNA-mediated knockdown: Small interfering RNA (siRNA) targeting CXCL2 has been effectively used in multiple cell types

    • In cancer cell lines (e.g., cal27 cells), siRNA knockdown of CXCL2 has been shown to reverse the promoting-proliferation and promoting-migration effects in tumor models

    • In THP-1 macrophages, CXCL2 silencing can be used to evaluate the role of CXCL2 in macrophage-mediated pro-cancer activity

    • Testing multiple siRNA constructs (e.g., siRNA-1, siRNA-2, siRNA-3) and selecting the most effective one based on knockdown efficiency is a recommended approach

  • Validation methods: Confirmation of CXCL2 knockdown should be performed using:

    • RT-qPCR for mRNA levels

    • Western blot or ELISA for protein expression

    • Functional assays to confirm biological effect (e.g., migration assays, proliferation assays)

  When designing CXCL2 knockdown experiments, researchers should consider potential compensation by other chemokines, particularly CXCL1 given their high homology, and may need to perform double knockdowns in some experimental contexts.

• How can recombinant CXCL2 be optimally prepared and stored for experimental use?

  Based on the available information, the following guidelines can be recommended for preparation and storage of recombinant human CXCL2:

  • Expression system: E. coli expression systems are commonly used to produce recombinant human CXCL2 encompassing amino acids Thr39-Asn107

  • Formulation: Recombinant CXCL2 is typically lyophilized from a 0.2 μm filtered solution of 20mM TrisHCl, 400mM NaCl, pH 8.5

  • Storage conditions:

    • Lyophilized protein should be stored at -20°C, though it remains stable at room temperature for short periods

    • Once reconstituted, aliquoting is recommended to avoid repeated freeze-thaw cycles

  • Working concentrations:

    • For in vitro assays: Concentrations ranging from 0.1 nM to 10 nM have been used in chemotaxis assays

    • For in vivo experiments: Doses of 0.1, 1, and 10 μg have been used for peritoneal recruitment assays

  • Activity verification: Before experimental use, the activity of recombinant CXCL2 should be confirmed using:

    • Calcium flux assays with neutrophils

    • β-arrestin recruitment assays

    • Chemotaxis assays with CXCR2-expressing cells

• What are the cutting-edge approaches to studying CXCL2 function in complex inflammatory settings?

  Several advanced techniques are emerging as powerful tools for studying CXCL2 function in complex inflammatory settings:

  • High-resolution confocal intravital microscopy (IVM): This technique allows real-time visualization of CXCL2-mediated neutrophil migration through venular walls in live animals

    • Using transgenic mice expressing fluorescent proteins in specific cell types (e.g., Lyz2-EGFP-ki for myeloid cells, Acta2-RFPcherry-Tg for smooth muscle cells and pericytes)

    • Labeling endothelial cell junctions in vivo using non-blocking Alexa Fluor 647-anti-CD31 mAb

    • This approach has revealed the distinct roles of CXCL1 and CXCL2 in neutrophil extravasation

  • Extracellular vesicle isolation and characterization: Isolating EVs from various sources (macrophages, serum) and characterizing their CXCL2 content using:

    • Chemokine chip assays to detect CXCL2 in EVs

    • Functional assays to assess the ability of CXCL2-containing EVs to recruit neutrophils

  • Genetic approaches: Combining CXCL2 manipulation with advanced genetic tools:

    • Conditional knockout models to delete CXCL2 in specific cell types

    • CRISPR/Cas9 genome editing to modify CXCL2 or its regulatory elements

    • Reporter systems to monitor CXCL2 expression in real-time

  • Heterodimer studies: Investigating the formation and function of CXCL1-CXCL2 heterodimers using:

    • Protein engineering to create stable heterodimers

    • Biophysical methods to characterize heterodimer formation

    • Neutrophil recruitment assays to compare heterodimer function to monomers

• How can CXCL2 be targeted therapeutically in inflammatory and cancer contexts?

  Based on the research findings, several strategies for therapeutic targeting of CXCL2 show promise:

  • Direct CXCL2 inhibition:

    • Neutralizing antibodies against CXCL2

    • RNA interference approaches (siRNA, antisense oligonucleotides) to reduce CXCL2 expression

    • Small molecule inhibitors that disrupt CXCL2-CXCR2 interaction

  • Receptor targeting:

    • CXCR2 antagonists to block CXCL2 signaling

    • Modulation of ACKR1 (atypical chemokine receptor 1) which regulates CXCL2 availability in endothelial junctions

  • Signaling pathway intervention:

    • Inhibitors of the CXCR2/PKC/NOX4 pathway activated by CXCL2

    • NF-κB inhibitors to reduce CXCL2 transcription

  • Context-specific applications:

    • In cancer: Targeting CXCL2 to reduce tumor cell proliferation, macrophage recruitment, and M2 polarization

    • In inflammatory bone diseases: Targeting CXCL2 to inhibit osteoclast differentiation and activation

    • In sepsis: Targeting CXCL2 on macrophage extracellular vesicles to limit excessive neutrophil recruitment and tissue damage

  The therapeutic potential of CXCL2 targeting must be balanced against its important physiological roles in immune defense, requiring careful consideration of timing, tissue specificity, and potential compensatory mechanisms.

• What are the best experimental designs to study CXCL2 in the context of neutrophil-endothelial cell interactions?

  Optimal experimental designs for studying CXCL2 in neutrophil-endothelial interactions include:

  • In vitro transendothelial migration assays:

    • Culture endothelial cells on Transwell filters to form monolayers

    • Apply CXCL2 to either the upper or lower chamber

    • Measure neutrophil migration across the endothelial monolayer

    • Variations can include pre-treating endothelial cells with inflammatory cytokines (e.g., TNF) to mimic inflammation

  • Chemotaxis assays with manipulated presentation:

    • Coat Transwell filters with immobilized CXCL1 or CXCL2

    • Add soluble CXCL2 or CXCL1 to bottom chambers

    • This approach has revealed that neutrophils can respond to CXCL2 gradients regardless of prior exposure to immobilized CXCL1

  • Intravital microscopy of the cremaster muscle:

    • Use transgenic mice with fluorescently labeled neutrophils

    • Label endothelial cell junctions with fluorescent antibodies

    • Apply inflammatory stimuli (e.g., TNF)

    • Track neutrophil behavior (adhesion, crawling, transmigration) in real-time

    • Use blocking antibodies against CXCL2 or CXCR2 to assess functional roles

  • Ex vivo neutrophil-endothelial interaction studies:

    • Isolate primary neutrophils and endothelial cells

    • Co-culture under various conditions (with/without inflammatory stimuli)

    • Use confocal microscopy to visualize CXCL2 localization at endothelial junctions

    • Measure neutrophil migration parameters

  These approaches have revealed the critical role of CXCL2 in providing directional cues for neutrophils within endothelial cell junctions and mediating persistent migration from the apical to basolateral aspect of endothelial cells.