Recombinant Human C-C motif chemokine 3 protein (CCL3), partial (Active)

What is the molecular structure of recombinant human CCL3?

Recombinant human CCL3/MIP-1 alpha is an approximately 8 kDa chemokine derived from residues Ala27-Ala92 of the full protein. The mature protein forms complexes with sulfated proteoglycans in a reversible process, where CCL3 associates into noncovalently-linked dimers that can further form tetramers and high molecular weight polymers . This oligomerization is biologically significant as it affects receptor binding and signaling properties. When working with recombinant CCL3, researchers should consider that the protein's storage conditions and concentration may affect its oligomerization state, which in turn could influence experimental outcomes.

How does human CCL3 compare to orthologs in other species?

Mature human CCL3 shares approximately 70-74% amino acid sequence identity with mouse, rat, and cotton rat CCL3 . This high degree of conservation indicates the evolutionary importance of this chemokine, but researchers should be aware of these differences when translating findings between animal models and human studies. When designing cross-species experiments, sequence alignment analysis should be performed to identify conserved functional domains and potential species-specific variations that might influence receptor binding affinity or downstream signaling.

What are the known receptors for CCL3 and their cellular distribution?

CCL3 primarily signals through CCR5 and CCR1 receptors. The CCL3-CCR5 pathway is particularly important for macrophage function, enhancing their metabolism, increasing lysosomal activity, and promoting antigen uptake . Additionally, CCL3 can interact with the decoy chemokine receptor D6, which regulates CCL3 availability in tissues . When designing experiments to study CCL3-mediated effects, researchers should consider the receptor expression profile of their target cells and potential cross-reactivity with other chemokine receptors.

How does CCL3 modulate immune cell recruitment and function?

CCL3 plays a multifaceted role in immune cell recruitment and activation. It promotes chemoattraction, adhesion to activated vascular endothelium, and cellular activation of multiple hematopoietic cell types including activated T cells, NK cells, neutrophils, monocytes, immature dendritic cells, and eosinophils . In hepatocellular carcinoma models, targeted delivery of CCL3 reprograms the tumor immune microenvironment by promoting immune cell recruitment and tertiary lymphoid structure formation . For studying these processes, researchers can use transwell migration assays with dose ranges of 3-10 ng/mL of recombinant CCL3 to induce chemotaxis in CCR5-expressing cells .

What is the role of CCL3 in the tumor microenvironment?

CCL3 is typically suppressed in the tumor microenvironment, and its expression positively correlates with immune infiltration and inflammatory responses . Studies show that targeted liver delivery of rAAV-Ccl3 can reprogram the immune microenvironment in hepatocellular carcinoma, suppressing tumor growth via immune engagement. CCL3 enhances macrophage antigen uptake and activates cytotoxic T cells, facilitating T cell infiltration and upregulating MHC II expression on macrophages . When investigating CCL3 in cancer models, researchers should analyze both the cellular sources of CCL3 and its effects on different immune cell populations within the tumor microenvironment using techniques such as single-cell RNA sequencing, flow cytometry, and multiplex immunofluorescence.

How does CCL3 expression change during infection and inflammation?

During infections, CCL3 expression is dynamically regulated. In mouse models of T. cruzi infection, increased expression of CCL3 mRNA transcripts was detected in heart tissue during acute infection (28 days post-infection) and persisted during the chronic phase (120 days post-infection) . Similarly, CCL3 protein levels were elevated in cardiac tissue during acute infection and, though reduced, remained elevated during chronic infection . The temporal dynamics of CCL3 expression should be considered when designing infection studies, with appropriate timepoints selected to capture both acute and chronic phases.

What are the optimal conditions for studying CCL3-induced chemotaxis?

When studying CCL3-induced chemotaxis, researchers should consider using modified Boyden chamber assays with fibronectin-coated filters to allow cell adhesion and transmigration. The effective dose (ED50) for CCL3-induced chemotaxis of human monocytes typically ranges from 2-10 ng/mL, while for BaF3 mouse pro-B cells transfected with human CCR5, the ED50 is approximately 3-10 ng/mL . It's important to note that the chemotactic response may vary depending on the cell type and activation state. For example, peripheral blood mononuclear cells from mice with chronic T. cruzi infection respond differently to CCL3 compared to cells from acutely infected mice . When designing chemotaxis experiments, include appropriate positive and negative controls, and consider testing a range of CCL3 concentrations to establish dose-response relationships.

How can CCL3 protein and mRNA expression be accurately measured in tissues?

For measuring CCL3 protein levels in tissues, enzyme-linked immunosorbent assay (ELISA) can be used, with results typically expressed as ng of CCL3 per 100 mg of tissue . For mRNA expression analysis, reverse transcription polymerase chain reaction (RT-PCR) with primers specific for CCL3 is recommended, with expression standardized against housekeeping genes such as HPRT . Immunohistochemistry (IHC) can be employed to visualize CCL3 protein localization in tissue sections, particularly in relation to specific cell types like CD8+ T cells and F4/80+ macrophages . For comprehensive analysis, researchers should combine multiple techniques to correlate mRNA expression, protein levels, and cellular localization.

What approaches can be used to study CCL3's effects on antigen presentation?

To investigate CCL3's impact on antigen presentation, researchers can employ co-culture systems, confocal imaging, and flow cytometry to assess macrophage-T cell interactions. Studies have shown that the CCL3-CCR5 pathway enhances macrophage metabolism, increases lysosomal activity, and improves antigen uptake, thereby strengthening adaptive immune responses . Ex vivo assays with antigen-specific T cells can be used to measure the functional consequences of CCL3-enhanced antigen presentation. Additionally, the use of inhibitors targeting specific components of the CCL3-CCR5 signaling pathway can help elucidate the mechanisms involved in enhanced antigen presentation.

How can recombinant CCL3 be used in cancer immunotherapy research?

Recombinant CCL3 shows promise in cancer immunotherapy research, particularly for reprogramming the immunosuppressive tumor microenvironment. Studies with targeted liver delivery of rAAV-Ccl3 in hepatocellular carcinoma models demonstrated enhanced immune cell recruitment, tertiary lymphoid structure formation, and suppressed tumor growth . When incorporating CCL3 in immunotherapy studies, researchers should consider combination approaches with immune checkpoint blockade, as CCL3 has been shown to enhance immune checkpoint blockade efficacy in preclinical models . Design experiments to evaluate both direct effects on tumor cells and indirect effects mediated through immune cell recruitment and activation, using endpoints such as tumor growth, survival, and immune cell profiling.

What are the methodological considerations for CCL3 knockout or overexpression studies?

When conducting CCL3 knockout studies, researchers can use CCL3-deficient mice (e.g., B6.129P2-Ccl3tm1Unc/J) with appropriate wild-type controls (e.g., C57BL/6) . For overexpression studies, viral vectors such as recombinant adeno-associated virus (rAAV) expressing Ccl3 can be employed for targeted delivery to specific tissues . The phenotype of CCL3-deficient or overexpressing models should be comprehensively characterized, including baseline immune cell composition and functional responses to various stimuli. Control experiments should include genetic characterization to confirm knockout or overexpression status, and littermate controls should be used when possible to minimize genetic background effects.

How can single-cell RNA sequencing be utilized to understand CCL3's impact on the immune microenvironment?

Single-cell RNA sequencing (scRNA-seq) provides valuable insights into how CCL3 shapes the immune microenvironment by revealing cell type-specific responses. This approach can identify which immune cells express CCL3 receptors, how CCL3 alters their transcriptional programs, and the subsequent changes in cellular functions . When designing scRNA-seq experiments, researchers should consider:

• Including both CCL3-treated and untreated conditions

• Sampling at multiple timepoints to capture dynamic responses

• Using computational analysis methods such as trajectory inference to understand cellular state transitions

• Validating key findings with orthogonal methods like flow cytometry or multiplex immunofluorescence

This approach can reveal novel CCL3-responsive cell populations and previously unknown mechanisms of action.

How should researchers address variability in CCL3-induced responses between different cell types and experimental models?

Variability in CCL3-induced responses may stem from differences in receptor expression, signaling pathway components, or cellular activation states. To address this:

• Quantify CCR1 and CCR5 receptor expression on target cells using flow cytometry or qPCR

• Consider the activation state of cells, as this may affect receptor expression and signaling capacity

• Use dose-response experiments to determine the optimal CCL3 concentration for each cell type

• Include positive controls (cells known to respond to CCL3) and negative controls (receptor antagonists or cells lacking CCR1/CCR5)

• When comparing different models, account for species differences in CCL3 sequence and receptor binding properties

For example, peripheral blood mononuclear cells from chronically infected mice respond differently to CCL3 compared to cells from acutely infected mice, highlighting the importance of contextual factors .

What potential artifacts should researchers be aware of when working with recombinant CCL3?

When working with recombinant CCL3, researchers should be aware of several potential artifacts:

• Oligomerization state: CCL3 can form dimers, tetramers, and high molecular weight polymers, which may affect its biological activity . Protein concentration and storage conditions can influence oligomerization.

• Endotoxin contamination: Ensure that recombinant CCL3 preparations are endotoxin-free, especially when studying inflammatory responses, as endotoxin can independently activate immune cells.

• Protein aggregation: Improper handling or storage may lead to protein aggregation and loss of activity. Always follow manufacturer's recommendations for reconstitution and storage.

• Batch-to-batch variability: Different lots of recombinant CCL3 may exhibit varying levels of biological activity. Include internal controls to normalize for this variability.

• Carrier protein effects: Some recombinant CCL3 preparations include carrier proteins that might influence experimental outcomes. Consider using carrier-free formulations for sensitive applications .