Recombinant Mouse C-C motif chemokine 8 protein (Ccl8) (Active)

What is Recombinant Mouse C-C Motif Chemokine 8 Protein (CCL8)?

Recombinant Mouse C-C Motif Chemokine 8 Protein (CCL8) is a full-length protein produced using E.coli expression systems with greater than 97% purity as determined by SDS-PAGE and HPLC analysis . The protein belongs to the CC chemokine family and is also known by several synonyms including Monocyte chemoattractant protein 2 (MCP-2) and Small-inducible cytokine A8 . CCL8 has a molecular weight of approximately 8.5 kDa and comprises the complete sequence of 74 amino acids spanning positions 24-97 of the mature protein . The protein functions primarily in the immunology research area, acting as a chemoattractant that regulates cell migration and inflammatory responses.

How does CCL8 function within the immune system?

CCL8 functions as a chemokine that primarily mediates the recruitment and migration of immune cells, particularly monocytes, during inflammatory responses. The biological activity of recombinant CCL8 is typically determined through chemotaxis bioassays using human peripheral blood monocytes, with effective concentration ranges of 10-100 ng/ml . In mouse models, CCL8 serves as one of two ligands for the chemokine receptor CCR8, alongside CCL1, highlighting its role in immune cell signaling and trafficking . Research indicates that CCL8 is predominantly produced by specialized CD169+SIGN-R1+ macrophages located in lymph node medullary areas and interfollicular regions, suggesting a role in coordinating immune responses within secondary lymphoid organs . This spatial regulation of CCL8 expression contributes to its function in orchestrating immune cell movement and interactions during both homeostatic and inflammatory conditions.

What are the optimal storage and reconstitution conditions for CCL8?

For optimal storage and reconstitution of Recombinant Mouse C-C Motif Chemokine 8 Protein, researchers should follow specific protocols to maintain protein integrity and activity. The protein is typically supplied as a lyophilized powder prepared from a 0.2 μm filtered PBS buffer at pH 7.4 . Prior to reconstitution, it is recommended to briefly centrifuge the vial to bring all contents to the bottom, especially important for small volumes of lyophilized protein . The reconstitution process should use deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL, with the addition of glycerol (5-50% final concentration) recommended for long-term storage stability . For optimal preservation, reconstituted protein should be aliquoted to minimize freeze-thaw cycles and stored at -20°C or preferably -80°C, as repeated freeze-thaw cycles can significantly degrade protein quality and biological activity.

How is CCL8 expression regulated in different physiological and pathological contexts?

CCL8 expression demonstrates significant context-dependent regulation across various physiological and pathological conditions. In normal lymph nodes, CCL8 protein is detectable adjacent to lymphatic endothelium in the medullary region and within subcapsular sinus (SCS) regions, particularly at the junction with the medullary region . Under Th2-immunization conditions, the number of discrete CCL8-expressing cells significantly increases in wild-type mice but not in CCL8-deficient mice, indicating specific immune-dependent regulation . In pathological contexts, CCL8 shows marked upregulation in sera from patients with Graft-versus-host disease (GVHD), with concentrations ranging from 52.0 to 333.6 pg/mL compared to non-GVHD samples containing less than 48 pg/mL . Particularly severe cases of fatal GVHD demonstrate extremely elevated CCL8 levels (333.6 and 290.4 pg/mL), suggesting its potential value as a diagnostic biomarker . In diffuse large B-cell lymphoma (DLBCL), CCL8 expression is significantly higher compared to normal lymph node tissues, with variable expression across different DLBCL subtypes and clinical stages .

How can researchers effectively measure CCL8 activity in experimental models?

Measuring CCL8 activity in experimental models requires specific methodological approaches that account for both protein concentration and functional capacity. The biological activity of recombinant CCL8 is primarily assessed through chemotaxis bioassays using human peripheral blood monocytes, with active concentrations typically ranging between 10-100 ng/ml . Researchers should consider implementing multi-parameter assays that evaluate not only the presence of CCL8 but also its functional impact on target cells. For in vivo tracking of CCL8 expression, transgenic reporter systems such as the REC8 mouse model, which incorporates eGFP at the start codon of the CCL8 gene, provide valuable tools for visualizing dynamic expression patterns in different tissues and under various immune stimulation conditions . When analyzing serum or tissue samples, quantitative approaches such as ELISA can detect CCL8 with diagnostic significance, as demonstrated in GVHD studies where thresholds of >48 pg/mL showed potential clinical relevance . For comprehensive functional assessment, calcium flux assays and receptor binding studies using labeled ligands like fluorescently-tagged human CCL1 (which binds murine CCR8) can provide insight into receptor-ligand interactions and downstream signaling events .

What are the implications of using CCL8 in DLBCL research and potential therapeutic applications?

The implications of CCL8 in diffuse large B-cell lymphoma (DLBCL) research extend beyond basic characterization to potential diagnostic and therapeutic applications. CCL8 has been identified as a key component in the tumor microenvironment (TME) of DLBCL, with expression levels significantly higher in DLBCL tissues compared to normal lymphoid tissues (p=0.016) . Research indicates that CCL8 expression correlates positively with markers of M2 macrophages (CD163, MS4A4A, and VSIG4), suggesting a potential role in facilitating an immunosuppressive microenvironment that may contribute to immune escape mechanisms . Gene enrichment analyses reveal that CCL8-related differentially expressed genes are associated with immune-related processes and secretory granule membranes linked to cytokine and chemokine activity . The prognostic value of CCL8 has been demonstrated through Cox regression analyses, which identified CCL8 expression level as an independent prognostic factor alongside age, ECOG performance status, clinical stages, and LDH ratio . Furthermore, CCL8 expression varies by DLBCL subtype and anatomical site of origin, with testicular DLBCL containing more CCL8 than central nervous system and nodal DLBCL, providing potential for subtype-specific therapeutic targeting .

How do CCL8 expression patterns differ across lymphoid tissues and what methodologies best capture these differences?

CCL8 expression patterns demonstrate significant spatial and cellular heterogeneity across lymphoid tissues, requiring sophisticated methodological approaches to accurately characterize this distribution. In lymph nodes, CCL8 protein localizes to two primary regions: adjacent to gp38-/+ lymphatic endothelium in the medullary region and within subcapsular sinus (SCS) regions, particularly at the junction with the medullary region . Cell-specific expression analysis through advanced enzymatic digestion techniques and flow cytometric sorting has identified CD169+SIGN-R1+CD11c+CD11b+ macrophages as the primary source of CCL8 within lymph nodes . This finding was further validated through alternative sorting schemes that confirmed CCL8 production by CD11c+CD103+CD11bhighCD169+F4/80+ macrophages . Methodologically, transgenic reporter systems like the REC8 mouse model provide powerful tools for visualizing CCL8 expression in vivo, incorporating eGFP at the CCL8 start codon to enable direct fluorescent visualization . Complementary approaches include clodronate-mediated depletion of CD169+SIGN-R1+F4/80+ macrophages, which abolishes Siglec1 and CCL8 expression within draining lymph nodes, confirming the cellular source through loss-of-function . For clinical tissue analysis, quantitative PCR combined with immunofluorescence imaging offers dual validation of expression patterns, as demonstrated in studies comparing CCL8 levels between DLBCL subtypes (germinal center B-cell vs. non-germinal center B-cell) .

What technical considerations are important when using CCL8 as a biomarker for GVHD?

When implementing CCL8 as a biomarker for Graft-versus-host disease (GVHD), several critical technical considerations must be addressed to ensure reliable and clinically meaningful results. Establishing appropriate reference ranges is fundamental, as studies indicate that non-GVHD samples typically contain less than 48 pg/mL of CCL8 (mean ± SE: 22.5 ± 5.5 pg/mL, range: 12.6-48.0 pg/mL), while GVHD sera demonstrate significantly elevated levels ranging from 52.0 to 333.6 pg/mL (mean ± SE: 165.0 ± 39.8 pg/mL) . Sample collection timing is crucial since CCL8 may serve as an early diagnostic marker, necessitating standardized protocols for when samples should be collected relative to transplantation or symptom onset . Analytical methods must be validated for clinical laboratory use, with consistent sensitivity, specificity, and reproducibility across different testing sites and personnel . Researchers should consider potential confounding factors that might affect CCL8 levels independently of GVHD, such as concomitant infections (particularly Gram-positive sepsis, which has been associated with increased CCL8 levels) . Integration with other biomarkers and clinical parameters may enhance diagnostic accuracy, as CCL8 alone might not capture the full complexity of GVHD pathophysiology across diverse patient populations .

What are the recommended protocols for CCL8 protein purification and quality assessment?

The purification and quality assessment of Recombinant Mouse C-C Motif Chemokine 8 Protein (CCL8) involve multiple critical steps to ensure high purity and biological activity. For E.coli-expressed recombinant CCL8, purification typically employs a multi-stage process beginning with cell lysis under optimized buffer conditions, followed by primary capture using affinity chromatography if a tag system is employed . For tag-free CCL8 production, ion exchange chromatography combined with size exclusion chromatography serves as the preferred method to achieve the high purity levels exceeding 97% that are standard for research applications . Quality assessment should include SDS-PAGE to verify molecular weight (8.5 kDa for CCL8) and purity, complemented by HPLC analysis for more precise purity determination . Endotoxin testing using the Limulus Amebocyte Lysate (LAL) method is essential, with acceptable levels being less than 1.0 EU/μg for most research applications . Biological activity assessment through chemotaxis bioassays using human peripheral blood monocytes provides functional validation, with effective concentration ranges typically between 10-100 ng/ml . Mass spectrometry analysis can provide additional confirmation of protein identity and sequence integrity, particularly important for verifying the complete sequence from positions 24-97 of the mature protein .

How should researchers design experiments to study CCL8-mediated cell migration?

Designing robust experiments to study CCL8-mediated cell migration requires careful consideration of multiple technical parameters to ensure physiologically relevant and reproducible results. The experimental setup should incorporate appropriate positive and negative controls, including comparisons with other chemokines such as CCL1, which also binds to the CCR8 receptor in mice . Concentration gradients should be carefully established, noting that recombinant CCL8 demonstrates biological activity in chemotaxis assays at concentrations between 10-100 ng/ml, which provides a starting range for dose-response studies . Cell selection is critical, with human peripheral blood monocytes serving as standard respondent cells for chemotaxis assays, though experiments may be expanded to include other immune cell populations known to express CCR8 or other relevant receptors . For in vitro migration assays, Transwell systems with appropriate pore sizes based on the studied cell type offer quantifiable data on directional migration, while real-time cell imaging systems provide dynamic information about migration velocities and patterns . For in vivo tracking of CCL8-responsive cells, adoptive transfer experiments using labeled cells combined with two-photon microscopy or intravital imaging in models such as the REC8 transgenic mouse can reveal physiologically relevant migration dynamics within lymphoid tissues .

What analytical methods best characterize CCL8 interactions with immune cells in lymphoid tissues?

Characterizing CCL8 interactions with immune cells in lymphoid tissues requires sophisticated analytical methods that capture both spatial organization and molecular interactions. Immunofluorescence microscopy with multi-color labeling provides crucial insights into the precise localization of CCL8 relative to specific lymphoid tissue structures and cell populations, as demonstrated in studies showing CCL8 expression adjacent to lymphatic endothelium and within subcapsular sinus regions . For identifying CCL8-producing cells, enhanced enzymatic digestion techniques combined with magnetic bead-based positive selection and flow cytometric sorting have successfully identified CD169+SIGN-R1+ macrophages as the primary source of CCL8 in lymph nodes . Functional validation through depletion studies, such as clodronate-mediated elimination of CD169+SIGN-R1+F4/80+ macrophages, provides confirmatory evidence by demonstrating the abolishment of CCL8 expression following targeted cell removal . For receptor-ligand interaction studies, fluorescently-labeled ligands such as human CCL1 (which binds to murine CCR8) enable visualization of receptor expression on specific cell populations and assessment of binding dynamics . Transgenic reporter systems like the REC8 mouse model, which incorporates mCherry at the CCL1 start codon and eGFP at the CCL8 start codon, offer powerful tools for simultaneous tracking of multiple chemokine ligands in vivo under various immunization conditions .

How can CCL8 be effectively utilized in GVHD research and potential diagnostic applications?

Utilization of CCL8 in Graft-versus-host disease (GVHD) research and diagnostic applications requires strategic integration of this biomarker into comprehensive research and clinical protocols. Longitudinal serum sampling in transplant recipients allows for tracking CCL8 dynamics before and after transplantation, potentially identifying early elevation patterns that precede clinical GVHD manifestations . Correlation analyses between CCL8 levels and GVHD severity grades help establish whether the particularly high levels observed in fatal GVHD cases (333.6 and 290.4 pg/mL) represent a consistent pattern that could inform prognostic assessments . For diagnostic implementation, validation studies should establish precise cutoff values, expanding on the preliminary observation that non-GVHD samples contain less than 48 pg/mL while GVHD sera range from 52.0 to 333.6 pg/mL . Multivariate analysis incorporating CCL8 alongside other established GVHD biomarkers and clinical parameters may enhance diagnostic accuracy and patient stratification . Mechanistic studies exploring how CCL8 contributes to GVHD pathophysiology, particularly its role in immune cell recruitment and activation within target tissues, could identify potential therapeutic targets beyond diagnostic applications . Development of point-of-care testing for CCL8 would facilitate rapid clinical decision-making, especially important in acute GVHD where early intervention significantly impacts outcomes .

What are the experimental approaches for studying CCL8's role in tumor immunology?

Studying CCL8's role in tumor immunology requires comprehensive experimental approaches that address both mechanistic understanding and potential therapeutic applications. Tissue microarray analysis comparing CCL8 expression between malignant and corresponding normal tissues provides foundational data, as demonstrated in DLBCL studies showing significantly higher CCL8 expression in tumor samples . Correlation analyses between CCL8 expression and tumor-associated macrophage markers (particularly CD163, MS4A4A, and VSIG4 for M2 macrophages) help elucidate the relationship between CCL8 and key immune cell populations within the tumor microenvironment . Functional assays including macrophage migration, polarization, and tumor cell co-culture experiments can determine whether CCL8 directly influences macrophage recruitment and phenotype in the context of malignancy . Pathway analysis through Gene Ontology enrichment and Gene Set Enrichment Analysis identifies broader biological processes associated with CCL8 expression, such as immune-related processes and secretory granule membranes linked to cytokine and chemokine activity . In vivo manipulation through CCL8 knockdown/knockout or overexpression in tumor models, combined with detailed immune phenotyping, provides causal evidence for CCL8's role in tumor progression and immune evasion . Clinical correlation studies linking CCL8 expression to patient outcomes across different cancer types and stages establish its potential value as a prognostic biomarker, as demonstrated in DLBCL where CCL8 expression correlates with survival time and clinical stage .

What role does CCL8 play in dendritic cell migration and immune response regulation?

CCL8 plays a multifaceted role in dendritic cell migration and immune response regulation, influencing both cellular trafficking and subsequent immune activation. The chemokine functions through interaction with the CCR8 receptor, which in mice has two ligands: CCL1 and CCL8 . Research using fluorescently-labeled human CCL1 (hCCL1), which binds to murine CCR8, has demonstrated that CD11c+CD11b+CD301b+ dendritic cells (CD301b+ DCs) express detectable CCR8 that increases after Th2-immunization . This CCR8 expression pattern suggests a specific role for CCL8-CCR8 signaling in regulating dendritic cell responses to Th2-associated stimuli . The spatial distribution of CCL8-producing cells within lymphoid tissues is strategically positioned to influence dendritic cell migration, with CD169+SIGN-R1+ macrophages located in the interfollicular regions and lymph node medullary areas serving as the primary source of CCL8 . This localization creates chemokine gradients that likely direct dendritic cell movement within lymph nodes, facilitating interactions with other immune cells and contributing to the orchestration of adaptive immune responses . The induction of CCL8 expression following Th2-immunization, as confirmed by both quantitative PCR and transgenic reporter mouse models, indicates a specific role in type 2 immune responses, potentially regulating dendritic cell functions in allergic or helminth-associated immunity .