GRO-g/CINC-2b Rat

What is rat GRO-g/CINC-2β and how does it relate to other chemokines?

Rat GRO-g/CINC-2β is a member of the alpha (CXC) subfamily of chemokines that functions as a neutrophil chemoattractant. It belongs to a family of related proteins including CINC-1, CINC-2α, and CINC-3/MIP-2, which collectively represent rat counterparts to human GRO proteins. These chemokines share significant sequence homology, with protein sequences of CINC-2β showing 63-67% identity with CINC-1 and other family members . CINC-2β was originally identified alongside CINC-2α as novel neutrophil chemoattractants produced by granulation tissue in culture from carrageenin-induced inflammation in rats . The rat CINC family represents functional analogs to human GRO proteins, with CINC-2β specifically sharing approximately 71% identity with human GROβ .

What are the primary biological functions of CINC-2β in rats?

CINC-2β functions primarily as a potent neutrophil chemoattractant with multiple biological activities:

• Neutrophil chemotaxis: CINC-2β induces neutrophil migration in vitro at levels comparable to CINC-1 .

• Intracellular calcium mobilization: CINC-2β stimulates increased intracellular [Ca²⁺] in neutrophils in a dose-dependent manner .

• Degranulation: CINC-2β triggers the release of cathepsin G from rat neutrophils at levels comparable to CINC-1 .

• In vivo neutrophil recruitment: When injected into air pouches on rat backs, CINC-2β induces neutrophil infiltration similar to that caused by CINC-1 .

These functions establish CINC-2β as a key mediator in inflammatory responses, particularly in the early stages involving neutrophil recruitment and activation.

How is CINC-2β expression regulated in physiological and pathological conditions?

CINC-2β expression is regulated primarily during inflammatory responses. It was originally purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in rats . The expression pattern suggests regulation by inflammatory mediators, similar to other chemokines in this family. While specific data on CINC-2β regulation is limited in the search results, related research indicates that cytokines such as IL-1β, TNF-α, and bacterial products like LPS can induce expression of CINC family members . The temporal and spatial expression patterns likely reflect the progression of inflammatory responses, with expression potentially varying across different tissues and inflammation models.

What methods are used to produce recombinant rat CINC-2β for research?

Recombinant rat CINC-2β has been successfully produced in Escherichia coli expression systems:

• Expression system: E. coli has been established as an effective host for producing recombinant rat CINC-2β, as demonstrated in multiple studies .

• Protein characteristics: The recombinant protein typically includes amino acids Arg33-Ser100 of the full sequence .

• Purification approach: While specific purification details are not explicitly stated in the search results, standard approaches for recombinant chemokines typically involve affinity chromatography and other protein purification techniques to obtain functional protein.

• cDNA cloning: The cDNA for rat GRO/CINC-2β has been cloned by reverse transcription/PCR amplification using specific primers and total RNA extracted from lipopolysaccharide-stimulated rat macrophages .

The recombinant protein produced through these methods maintains biological activity, as demonstrated in multiple functional assays .

How can researchers detect and quantify CINC-2β in biological samples?

Researchers can detect and quantify rat CINC-2β in biological samples using the following methods:

• ELISA: Quantikine Rat CINC-2 alpha/beta ELISA kits provide a standardized method for measuring CINC-2β in cell culture supernatants, serum, and plasma samples . These assays typically offer:

  Performance Characteristic	Value/Range
  Assay Duration	4.5 hours
  Sample Types	Cell culture supernatants, serum, EDTA plasma, heparin plasma
  Measurement Range	Varies by kit (typical sensitivity in pg/mL range)
  Intra-Assay Precision CV%	3.4-10.8%
  Inter-Assay Precision CV%	3.9-14.6%

• Antibody-based detection: Monoclonal antibodies against rat CINC-2β (such as clone #83504) can be used in various immunoassay formats including sandwich immunoassays .

• Specificity considerations: When selecting detection methods, researchers should consider cross-reactivity. For example, some CINC-1 assays show ≤0.1% cross-reactivity with CINC-2β, allowing for specific detection of individual CINC family members .

What are the most effective in vitro assays for studying CINC-2β function?

Several validated in vitro assays can effectively evaluate CINC-2β biological activity:

• Chemotaxis assays:

  • The BaF3 mouse pro-B cell line transfected with human CXCR2 responds to recombinant rat CINC-2α in a dose-dependent manner and can be used to assess CINC-2β function .

  • Neutrophil chemotaxis assays using primary rat neutrophils in Boyden chamber-type apparatus provide direct measurement of CINC-2β chemotactic activity .

• Calcium mobilization assays:

  • Measuring increases in intracellular [Ca²⁺] in response to CINC-2β stimulation provides insight into receptor activation and early signaling events .

• Degranulation assays:

  • Measuring release of cathepsin G from rat neutrophils following CINC-2β stimulation assesses functional activation of neutrophils .

• Neutralization assays:

  • Anti-CINC-2β antibodies can neutralize chemotaxis in a dose-dependent manner, with typical ND₅₀ (neutralization dose for 50% inhibition) values of 0.5-2.5 μg/mL in the presence of 0.05 μg/mL recombinant protein .

What are the functional differences between CINC-2α and CINC-2β?

Despite their structural similarities, CINC-2α and CINC-2β exhibit both overlapping and distinct functional characteristics:

• Chemotactic activity: Both CINC-2α and CINC-2β demonstrate similar neutrophil chemotactic activities in vitro, comparable to CINC-1 .

• Calcium mobilization: When comparing intracellular calcium responses, both CINC-2α and CINC-2β induce increases in intracellular [Ca²⁺] in rat neutrophils in a dose-dependent manner, though specific potency differences between the two are not clearly defined in the available search results .

• Degranulation: Both CINC-2α and CINC-2β induce comparable responses to CINC-1 in triggering the release of cathepsin G from rat neutrophils .

• In vivo activity: When injected into air pouches on rat backs, both CINC-2α and CINC-2β induce neutrophil infiltration to a similar extent as CINC-1 .

While the available search results indicate many functional similarities between CINC-2α and CINC-2β, more detailed studies may reveal subtle differences in receptor binding preferences, signaling pathway activation, or tissue-specific effects that are not captured in the current data.

How do CINC-2β interactions with receptors differ from other CINC family members?

• Receptor usage: CINC family members, including CINC-2β, likely interact primarily with CXCR2 receptors, as demonstrated by the chemotactic response of CXCR2-transfected BaF3 cells to CINC-2α . This suggests CINC-2β similarly utilizes CXCR2 as its primary receptor.

• Cross-desensitization patterns: The search results indicate that CINC-3 was still able to induce increases in intracellular [Ca²⁺] in rat neutrophils previously stimulated with other CINCs, suggesting potential differences in receptor binding or desensitization patterns among CINC family members . Similar studies specific to CINC-2β would be needed to determine its unique receptor interaction profile.

• Binding affinity differences: While not explicitly stated in the search results, the biological functions of different CINC family members likely reflect differences in receptor binding affinities and downstream signaling pathway activation.

What are effective in vivo models for studying CINC-2β functions?

Several established in vivo models can effectively study CINC-2β function:

• Air pouch model: Injection of CINC-2β into preformed air pouches on the backs of rats induces neutrophil infiltration, providing a quantifiable measure of in vivo chemotactic activity . This model allows for:

  • Controlled delivery of defined CINC-2β concentrations

  • Measurement of temporal neutrophil recruitment

  • Assessment of neutralizing antibody effects

  • Comparison with other inflammatory mediators

• Carrageenin-induced inflammation: This model was used in the original identification of CINC-2β and provides a physiologically relevant inflammatory context in which to study CINC-2β production and function .

• Cytokine-stimulated tissue/cell models: Based on the relationship to other CINC family members, models using IL-1β, TNF-α or LPS stimulation of various rat tissues or cell lines could be relevant for studying CINC-2β expression and function .

What are the key considerations when using antibodies to neutralize CINC-2β activity?

When using antibodies to neutralize CINC-2β activity, researchers should consider:

• Antibody specificity: Select antibodies with demonstrated specificity for CINC-2β. For example, assay kits for CINC-1 show ≤0.1% cross-reactivity with CINC-2β, indicating high specificity is achievable .

• Effective neutralizing concentration: Antibodies such as Rat CXCL3/CINC-2α/β Monoclonal Antibody (Catalog # MAB516) typically show ND₅₀ values of 0.5-2.5 μg/mL in the presence of 0.05 μg/mL recombinant protein . Researchers should establish appropriate antibody concentrations for their specific experimental system.

• Validation of neutralization: Chemotaxis assays using BaF3 cells transfected with human CXCR2 or primary rat neutrophils provide functional validation of antibody neutralizing activity .

• Antibody format: Consider whether the research question requires monoclonal or polyclonal antibodies, and which antibody formats (whole IgG, Fab fragments, etc.) are most appropriate for the specific experimental system.

How can researchers accurately quantify CINC-2β in complex biological samples?

For accurate quantification of CINC-2β in complex biological samples, researchers should:

• Select appropriate assay platforms: Validated ELISA kits such as the Quantikine Rat CINC-2 alpha/beta Immunoassay provide reliable quantification in cell culture supernatants, serum, and plasma .

• Consider assay precision: When selecting quantification methods, evaluate both intra-assay and inter-assay precision:

  Sample	Intra-Assay CV%	Inter-Assay CV%
  Low concentration	10.8%	14.6%
  Medium concentration	5.0%	7.9%
  High concentration	3.4%	3.9%

  These values from the Quantikine assay demonstrate typical precision metrics that should be considered .

• Address matrix effects: Different sample types may contain substances that interfere with accurate measurement. Validate assays using spike-recovery experiments in the specific sample matrix being tested.

• Standardize sample collection and processing: For consistent results, establish standardized protocols for sample collection, processing, and storage to minimize pre-analytical variability.

What experimental controls are essential when studying CINC-2β in inflammatory models?

Essential experimental controls when studying CINC-2β in inflammatory models include:

• Positive controls: Include known inducers of CINC family expression such as IL-1β, TNF-α, or LPS to validate experimental systems .

• Related chemokine controls: Include other CINC family members (CINC-1, CINC-2α, CINC-3) as comparators to establish specificity of observed effects .

• Antibody controls: When using neutralizing antibodies, include isotype controls to distinguish specific from non-specific effects.

• Dose-response relationships: Establish dose-response relationships for CINC-2β-induced effects, as demonstrated in chemotaxis assays with CXCR2-transfected BaF3 cells .

• Temporal controls: Given the dynamic nature of inflammatory responses, include appropriate time-course measurements to capture both early and late effects of CINC-2β.

What are the current knowledge gaps in CINC-2β research?

Several important knowledge gaps exist in our understanding of CINC-2β:

• Receptor specificity: While CINC-2β likely acts primarily through CXCR2, detailed studies on receptor binding kinetics, potential interactions with other receptors, and comparisons with other CINC family members would enhance our understanding of its biological functions.

• Signaling pathway specificity: Greater characterization of downstream signaling pathways activated by CINC-2β and how these compare to pathways activated by other CINC family members would provide insight into potential functional differences.

• Pathophysiological roles: Further investigation into the specific roles of CINC-2β in various disease models would help distinguish its contributions from those of other chemokines.

• Regulation of expression: More detailed understanding of the transcriptional and post-transcriptional regulation of CINC-2β expression in different tissues and inflammatory conditions would provide insight into its physiological roles.

• Therapeutic targeting: Exploration of CINC-2β as a potential therapeutic target in inflammatory diseases requires further investigation of its specific contributions to pathological processes.

How can researchers distinguish between CINC-2α and CINC-2β effects in experimental systems?

To distinguish between CINC-2α and CINC-2β effects, researchers can implement several strategies:

• Specific antibodies: Develop and validate antibodies with specificity for either CINC-2α or CINC-2β, allowing selective neutralization or detection.

• Recombinant proteins: Use purified recombinant CINC-2α and CINC-2β in parallel experiments to directly compare their biological activities.

• Gene expression analysis: Design primers or probes that specifically detect CINC-2α or CINC-2β mRNA to distinguish expression patterns.

• Receptor binding studies: Perform competitive binding assays to determine potential differences in receptor binding affinities or preferences between CINC-2α and CINC-2β.

• Signaling pathway analysis: Compare signaling pathway activation downstream of CINC-2α and CINC-2β stimulation to identify potential differences in cellular responses.