Recombinant Mouse C-C motif chemokine 3 protein (Ccl3) (Active)

What is the structure and function of recombinant mouse Ccl3 protein?

Recombinant mouse Ccl3 is an approximately 8 kDa chemokine that belongs to the beta (CC) subfamily of chemokines. The commercially available E. coli-derived mouse Ccl3 protein typically spans amino acids Ala24-Ala92 of the native sequence . Functionally, Ccl3 forms complexes with sulfated proteoglycans and can assemble into noncovalently-linked dimers that further organize into tetramers and high molecular weight polymers .

These structural characteristics are essential for its biological functions, which include:

• Chemoattraction of activated T cells, NK cells, neutrophils, monocytes, immature dendritic cells, and eosinophils

• Promoting adhesion to activated vascular endothelium

• Cellular activation of various hematopoietic cell types

• Inhibition of hematopoietic progenitor cell proliferation (hence its alternative name as stem cell inhibitor)

The protein exerts its effects through interactions with multiple receptors, primarily CCR1, CCR3, and CCR5 .

How is Ccl3 involved in immune responses during viral infections?

Ccl3 plays a critical role in the early immune defense against viral infections, particularly in cytomegalovirus (CMV) infection models. Recent research using CCL3-EASER mice has revealed that natural killer (NK) cells are both the principal producers and sensors of Ccl3 during early murine cytomegalovirus (mCMV) infection .

This creates an auto/paracrine communication system that amplifies the NK cell response, which is essential for early defense against the virus. When Ccl3-producing cells are depleted (as demonstrated in CCL3-EASER mice treated with diphtheria toxin), mice show approximately 1,000-fold higher mCMV loads in the liver compared to controls, along with increased infection foci and immune infiltration .

The importance of Ccl3 in viral defense is further emphasized by the fact that mice lacking Ccl3-producing cells succumb to mCMV infection approximately one week after challenge, consistent with the known higher susceptibility of Ccl3-deficient mice to this virus .

Which cell types produce and respond to Ccl3 during homeostasis and infection?

Under homeostatic conditions, NK cells transcribe Ccl3 but translation is limited. During infection, particularly with viruses like mCMV:

Ccl3-producing cells:

• NK cells (primary early source during viral infection)

• Macrophages (contribute at later timepoints)

• Various hematopoietic cells (upon induction)

• Fibroblasts

• Smooth muscle cells

• Epithelial cells

Ccl3-responding cells:

• NK cells (primary responders during early viral infection)

• NKT cells

• Activated T cells (variable response)

• Neutrophils

• Monocytes

• Immature dendritic cells

• Eosinophils

Interestingly, experiments with CCL3-EASER mice revealed that NK cells act as sentinels, prepared to rapidly produce and secrete Ccl3 during infections. NK and NKT cells only internalized Ccl3 when isolated from infected mice, suggesting that responsiveness to this chemokine requires activation, possibly by type I interferons or other infection-induced cytokines .

How does type I interferon signaling regulate Ccl3 production and function?

Type I interferon (IFN-I) signaling plays a crucial role in regulating Ccl3 production, primarily at the posttranscriptional level. Studies using CCL3-EASER reporter mice revealed that NK cells constitutively produce Ccl3 transcripts even in uninfected mice, but translation primarily occurs after viral infection .

When IFN-I signaling was blocked, researchers observed a reduced slope in the linear regression line between the channel intensities of reporters for Ccl3 transcription and translation, indicating that IFN-I enhances the efficiency of translating Ccl3 transcripts into protein .

The relationship between IFN-I and Ccl3 follows this pattern:

• NK cells maintain Ccl3 transcripts in a ready state

• Viral infection induces IFN-I production

• IFN-I stimulates the translation of existing Ccl3 transcripts

• Local IFN-I in infected organs increases Ccl3 production over time

• IFN-I may also enhance cellular responsiveness to Ccl3

This regulation mechanism ensures rapid Ccl3 production during early infection, supporting the sentinel role of NK cells in the initial immune response .

What are the molecular mechanisms of Ccl3-mediated NK cell auto/paracrine communication?

The discovery that NK cells both produce and respond to Ccl3 reveals a sophisticated auto/paracrine communication system that amplifies antiviral responses. The molecular mechanisms involve:

The dual role as both producer and recipient implies that Ccl3 creates a rapid response amplification system, critical for controlling early viral replication before adaptive immunity develops .

How do proteoglycans and oligomerization affect Ccl3 bioactivity and stability?

Ccl3 forms complexes with sulfated proteoglycans, which significantly impacts its bioactivity and stability. These interactions have several important consequences:

• Protection from proteolytic degradation:

  • Ccl3 complexes are protected from proteolytic digestion by insulin degrading enzyme (IDE)

  • Monomeric Ccl3 is susceptible to IDE cleavage

  • This protection extends the biological half-life of the chemokine

• Oligomerization dynamics:

  • Ccl3 forms noncovalently-linked dimers in a reversible process

  • These dimers further assemble into tetramers and high molecular weight polymers

  • The equilibrium between monomers, dimers, and higher-order structures affects receptor binding and activity

• Gradient formation:

  • Interaction with proteoglycans creates chemokine gradients essential for directional cell migration

  • The diffusion rate and local concentration of Ccl3 are regulated by these interactions

  • This gradient is critical for the precise recruitment of immune cells to infection sites

• Receptor interaction modulation:

  • Proteoglycan binding can enhance or inhibit receptor activation depending on the context

  • Different oligomeric states may preferentially activate specific receptor subtypes

These structural characteristics must be considered when designing experiments with recombinant Ccl3, as they directly impact functional outcomes in both in vitro and in vivo systems.

What are the optimal conditions for using recombinant mouse Ccl3 in migration assays?

When designing migration assays with recombinant mouse Ccl3, researchers should consider the following methodological guidelines:

Optimal concentration ranges:

• For general neutrophil chemotaxis: 5-25 ng/mL (ED50)

• For BaF3 mouse pro-B cells transfected with human CCR5: 0.4-2.0 ng/mL (ED50)

Assay parameters:

• Buffer composition: Use chemically defined media with low serum (0.5-1%) to reduce background migration

• Incubation time: 2-4 hours is typically sufficient to observe directed migration

• Cell preparation: Fresh isolation and minimal manipulation maintain responsive receptor expression

• Controls: Include both negative (buffer-only) and positive (known chemoattractant) controls

• Quantification method: Either direct cell counting or fluorescence-based detection systems

Chamber selection:

• Transwell systems (3-5 μm pore size depending on cell type)

• μ-Slide chemotaxis chambers for real-time visualization

• 3D collagen matrix systems for tissue-like environments

Important considerations:

• Pre-activation of certain cell types (like NK cells) may be necessary, as CCL3-EASER mice studies showed that NK cells only internalized Ccl3 when isolated from infected mice

• The oligomerization state of Ccl3 can affect activity; fresh preparations or stabilized formulations should be used

• Document the exact source and preparation of recombinant Ccl3, as E. coli-derived and mammalian cell-derived proteins may have different activities

How can reporter systems be used to track Ccl3 expression and signaling in vivo?

The development of CCL3-EASER (ErAse, SEnd, Receive) mice demonstrates a sophisticated approach to tracking Ccl3 expression and signaling. Based on this model, researchers can implement several methodologies:

Dual fluorescence reporter systems:

• Transcription reporters:

  • Fluorescent proteins (like tdTomato) directly driven by the Ccl3 promoter

  • Allow identification of cells actively transcribing Ccl3

• Translation reporters:

  • Fusion proteins combining Ccl3 with fluorescent tags (e.g., Venus)

  • Enable tracking of Ccl3 protein production and secretion

• Uptake tracking:

  • Fluorescently tagged Ccl3 can identify cells that internalize the chemokine

  • Particularly valuable for identifying responding cells in complex tissues

Experimental approaches:

• Mixed bone marrow chimeras:

  • Create chimeric animals with both reporter and non-reporter cells

  • Allows discrimination between cell-autonomous and non-autonomous effects

  • Critical for identifying true responder cells when producers and responders overlap

• Ex vivo co-culture systems:

  • Co-culture potential producer and responder populations

  • Use transwell systems to distinguish between direct uptake and cell engulfment

  • Apply specific stimuli to activate Ccl3 production

• Intravital microscopy:

  • Perform real-time imaging of fluorescent reporter activity in live animals

  • Track cellular dynamics and chemokine gradients in tissues

  • Correlate with functional outcomes like pathogen clearance

These reporter systems provide powerful tools to address fundamental questions about the spatiotemporal dynamics of Ccl3 production and response during immune reactions that cannot be answered using traditional methods like ELISA or PCR.

What are the key methodological considerations for studying Ccl3 in mouse models of viral infection?

When designing studies to investigate Ccl3 in mouse models of viral infection, researchers should consider the following methodological approaches:

Mouse model selection:

• Wild-type C57BL/6J: Standard for baseline responses

• Ccl3-knockout mice: To assess loss-of-function effects

• CCL3-EASER mice: For tracking Ccl3 production and sensing

• Receptor-deficient models: Ccr1−/−, Ccr3−/−, or Ccr5−/− to distinguish receptor-specific effects

• Cell-specific conditional knockout mice: For tissue or cell-type restricted deletion

Viral infection protocols:

• Virus selection: mCMV is well-established for studying Ccl3 responses

• Route of administration: Intraperitoneal, intravenous, or tissue-specific delivery

• Viral dose: Titrate to achieve desired pathology without overwhelming the system

• Timepoints: Include very early timepoints (6-24h) to capture initial NK cell-mediated Ccl3 production

Assessment methods:

• Viral load quantification:

  • qPCR for viral genome copies

  • Plaque assays for infectious particles

  • Immunohistochemistry for infected cell visualization

• Immune response analysis:

  • Flow cytometry for cellular identification and activation status

  • Multiplex cytokine/chemokine assays

  • Single-cell RNA sequencing for comprehensive profiling

  • Histopathology for tissue damage and cellular infiltration

• Functional assays:

  • Ex vivo NK cell cytotoxicity

  • Chemotaxis assays with isolated cells

  • Adoptive transfer experiments

  • Antibody blocking of specific receptors

Critical controls:

• Depletion controls (anti-NK1.1 or anti-Ly49H) to confirm NK cell dependence

• Type I interferon blocking or IFNAR-/- mice to assess IFN-I dependency

• Time-course experiments to distinguish early vs. late Ccl3 sources

• Heat-inactivated virus to distinguish between infection and particle recognition

Based on the CCL3-EASER mice studies, researchers should be particularly attentive to the dual role of NK cells as both producers and sensors of Ccl3, which requires specialized experimental designs like mixed chimeras to properly evaluate .

How should researchers interpret changes in Ccl3 transcription versus translation during immune responses?

The analysis of Ccl3 transcription versus translation presents unique challenges that require careful interpretation. Based on studies with CCL3-EASER mice, researchers should consider:

Analytical approaches:

• Correlation analysis:

  • Plot transcription (e.g., tdTomato signal) against translation (e.g., Venus-Ccl3 signal)

  • Calculate linear regression to determine the relationship between transcription and translation

  • Monitor changes in slope as an indicator of translational efficiency

• Temporal analysis:

  • Track transcription and translation markers over time

  • Identify lag phases between transcription and protein production

  • Determine how quickly translation begins after transcriptional activation

• Single-cell analysis:

  • Characterize cell-to-cell variability in transcription versus translation

  • Identify potential subpopulations with different regulatory patterns

  • Correlate with cellular activation states

Interpretation guidelines:

• Baseline dissociation: NK cells may maintain Ccl3 transcripts in homeostasis without significant translation, representing a "ready state"

• Post-transcriptional regulation: Changes in the ratio of translation to transcription suggest alterations in post-transcriptional control, as observed with IFN-I signaling

• Reporter considerations:

  • Different half-lives of the transcript and protein reporters may affect interpretation

  • tdTomato reporter may persist longer than actual Ccl3 transcripts

  • This could lead to overestimation of transcription relative to translation during resolution phases

• Functional correlation:

  • Correlate translational efficiency with functional outcomes

  • Higher translational efficiency may indicate heightened biological significance

  • Consider tissue-specific differences in translation efficiency

This analytical framework helps researchers distinguish between constitutive and induced Ccl3 expression patterns and identify the key regulatory checkpoints during immune responses.

What statistical approaches are appropriate for analyzing Ccl3-dependent cell migration data?

Analyzing Ccl3-dependent cell migration requires appropriate statistical methods to account for the complex nature of chemotactic responses. Researchers should consider these approaches:

Data collection considerations:

• Replicate structure:

  • Technical replicates: Multiple wells/chambers per condition

  • Biological replicates: Cells from different donors/animals

  • Experimental replicates: Independent experiments on different days

• Controls:

  • Negative control: Random migration (no chemokine)

  • Positive control: Known potent chemoattractant

  • Dose-response: Multiple Ccl3 concentrations to establish ED50

• Normalization methods:

  • Migration index: (Cells migrated to test/Cells migrated to control)

  • Chemotactic index: (Directed migration - Random migration)/Random migration

  • Percent of input: (Migrated cells/Total cells) × 100

Statistical analysis framework:

• For simple comparisons:

  • Student's t-test (two conditions)

  • One-way ANOVA with appropriate post-hoc tests (multiple conditions)

  • Use paired tests when comparing responses from the same cell source

• For dose-response relationships:

  • Non-linear regression to determine ED50 values

  • Compare ED50 values between different cell types or conditions

  • Four-parameter logistic regression for complete dose-response curves

• For time-course experiments:

  • Two-way ANOVA with time and treatment as factors

  • Repeated measures analysis when tracking the same population

  • Area under the curve (AUC) calculations for cumulative response

• For complex datasets:

  • Mixed-effects models to account for donor/animal variability

  • MANOVA when measuring multiple outcome variables

  • Principal component analysis to identify patterns in multiparameter data

• For in vivo migration:

  • Survival analysis for time-to-recruitment data

  • Spatial statistics for tissue distribution patterns

  • Cell tracking algorithms for intravital microscopy data

Reporting results:

These statistical approaches enable robust analysis of Ccl3-mediated migration, accounting for the biological variability inherent in chemotaxis assays.

How can researchers distinguish between direct and indirect effects of Ccl3 in complex immune responses?

Distinguishing direct from indirect effects of Ccl3 in complex immune responses requires careful experimental design and analysis. Based on current research methods, including those used with CCL3-EASER mice, researchers should implement these strategies:

Experimental approaches:

• Receptor antagonism:

  • Use specific receptor antagonists for CCR1, CCR3, and CCR5

  • Compare effects of blocking individual versus multiple receptors

  • Employ receptor-deficient cells in reconstitution experiments

• Cell-specific manipulations:

  • Conditional knockout models targeting specific cell populations

  • Mixed bone marrow chimeras with defined cellular compositions

  • Adoptive transfer of wild-type cells into Ccl3-deficient hosts (or vice versa)

• Temporal separation:

  • Time-course experiments with frequent sampling

  • Sequential blocking or depletion at different timepoints

  • Correlate Ccl3 production with subsequent cellular responses

• Ex vivo validation:

  • Isolate cells from in vivo experiments for functional testing

  • Compare phenotypes of cells that did or did not encounter Ccl3

  • Perform ex vivo receptor expression analysis

Analytical frameworks:

• Pathway analysis:

  • Map signaling events downstream of Ccl3 receptor engagement

  • Identify unique versus overlapping pathways with other stimuli

  • Use phospho-flow cytometry or mass cytometry for high-dimensional analysis

• Secondary mediator assessment:

  • Measure induction of secondary cytokines and chemokines

  • Block potential secondary mediators to isolate Ccl3-specific effects

  • Create network models of cytokine/chemokine cascades

• Transcriptional profiling:

  • Compare gene expression signatures of direct Ccl3 stimulation versus in vivo responses

  • Identify Ccl3-dependent transcriptional programs

  • Use single-cell approaches to capture cellular heterogeneity

• Visualization techniques:

  • Multiplex immunofluorescence to co-localize Ccl3 with responding cells

  • Intravital microscopy to track cell-cell interactions in real-time

  • Spatial transcriptomics to map Ccl3 responses in tissue contexts

Case study from CCL3-EASER experiments:

The CCL3-EASER mouse model demonstrated an approach to distinguish direct Ccl3 sensing in a system where producer and responder cells overlapped. By creating mixed chimeras where only some NK cells could produce Ccl3, researchers identified cells that internalized Ccl3-Venus without producing it (tdTomato-negative, Venus-positive) . This approach conclusively demonstrated direct Ccl3 sensing by NK cells rather than secondary effects.

Similarly, transwell experiments with macrophages revealed that apparent uptake signals were actually due to cell engulfment rather than direct Ccl3 sensing, highlighting the importance of proper controls .

What are common pitfalls when working with recombinant Ccl3 and how can they be addressed?

Researchers working with recombinant mouse Ccl3 may encounter several challenges that can impact experimental outcomes. Understanding and addressing these pitfalls is essential for generating reliable data:

Protein stability and activity issues:

• Aggregation and oligomerization:

  • Ccl3 naturally forms dimers, tetramers, and higher-order oligomers

  • Solution: Add carrier proteins (0.1% BSA) to dilute preparations

  • Test multiple concentrations to account for variable active fractions

  • Prepare fresh working solutions or store single-use aliquots at -80°C

• Loss of activity during storage:

  • Minimize freeze-thaw cycles (maximum 1-2)

  • Store concentrated stock solutions

  • Validate activity periodically with functional assays

• Adsorption to labware:

  • Use low-binding microcentrifuge tubes and pipette tips

  • Pre-coat plates with BSA for migration assays

  • Include carrier protein in all dilution buffers

Experimental design challenges:

• Receptor desensitization:

  • High Ccl3 concentrations can cause receptor internalization

  • Titrate carefully to find optimal concentration range

  • Use pulse-chase designs for prolonged experiments

• Species-specific activity differences:

  • Mouse Ccl3 shares only 73% amino acid identity with human Ccl3

  • Use species-matched reagents when possible

  • Validate cross-species activity before mixed-species experiments

• Inconsistent cellular responsiveness:

  • NK cells only respond to Ccl3 when activated by infection or cytokines

  • Pre-activate cells when appropriate (e.g., with IFN-I)

  • Include positive control populations known to respond consistently

Technical troubleshooting:

• Poor migration in chemotaxis assays:

  • Verify chemokine activity with bioassay

  • Check cell viability and receptor expression

  • Optimize incubation time and temperature

  • Ensure appropriate pore size for cell type

  • Confirm absence of air bubbles in migration setup

• Background issues in binding/uptake studies:

  • Include competition controls with unlabeled chemokine

  • Test for non-specific binding with receptor antagonists

  • Use appropriate blocking reagents

  • Consider fluorescence compensation when using multiple reporters

• Variability between experiments:

  • Standardize cell isolation and culture conditions

  • Use the same recombinant protein lot when possible

  • Include internal standards for normalization

  • Document environmental variables (temperature, CO2, humidity)

By anticipating these common challenges and implementing appropriate controls and optimizations, researchers can improve the reliability and reproducibility of experiments involving recombinant mouse Ccl3.

How can researchers address conflicting data regarding Ccl3 function in different experimental systems?

Conflicting data regarding Ccl3 function across different experimental systems is not uncommon. Researchers can employ several strategies to reconcile discrepancies and develop a more comprehensive understanding:

Systematic analysis approaches:

• Direct comparative studies:

  • Test multiple Ccl3 sources side-by-side

  • Use standardized readouts across systems

  • Maintain consistent experimental conditions

  • Create a matrix of variables to identify key differences

• Meta-analysis techniques:

  • Systematically review published literature

  • Extract standardized effect sizes where possible

  • Identify moderating variables that explain heterogeneity

  • Create forest plots to visualize effect consistency

• Collaboration networks:

  • Establish multi-laboratory validation studies

  • Exchange protocols, reagents, and samples

  • Implement blinded analysis to reduce bias

  • Create shared data repositories

Reconciliation frameworks:

• Context-dependent functionality:

  • Map conditions where Ccl3 shows consistent versus variable effects

  • Consider microenvironmental factors (cytokine milieu, tissue context)

  • Evaluate temporal aspects of Ccl3 function

  • Example: NK cells exhibit context-dependent responses to Ccl3 based on activation state

• Cell type-specific responses:

  • Characterize receptor expression profiles across cell types

  • Determine downstream signaling pathway differences

  • Assess how cell differentiation/activation state affects response

  • Example: NK cells but not T cells showed Ccl3 uptake in co-culture experiments

• Dose-response relationships:

  • Generate complete dose-response curves rather than single concentrations

  • Consider biphasic response patterns

  • Evaluate receptor occupancy requirements

  • Example: Different ED50 values for neutrophil versus BaF3 cell chemotaxis

Technical reconciliation strategies:

• Method standardization:

  • Develop detailed standard operating procedures

  • Create reference standards for key assays

  • Establish minimal reporting requirements

  • Implement quality control metrics

• Reagent validation:

  • Authenticate cell lines and primary cells

  • Verify recombinant protein activity and identity

  • Validate antibody specificity

  • Consider developing community-wide reference standards

• Computational modeling:

  • Create mechanistic models incorporating conflicting data

  • Identify parameter spaces that explain divergent results

  • Test model predictions with targeted experiments

  • Refine models iteratively based on new data

Case study approach:
When faced with specific conflicting data, researchers should:

• Document all methodological differences between studies

• Systematically test each variable individually

• Develop a unified model that accommodates apparently conflicting results

• Design critical experiments to test this unified model

For example, if studies disagree on whether Ccl3 activates T cells, researchers might discover this depends on T cell activation state, subset, or the presence of secondary signals - as demonstrated by the finding that NK cells only internalize Ccl3 when previously activated by infection .

What strategies can improve the reproducibility of Ccl3-based experimental systems?

Ensuring reproducibility in Ccl3-based experimental systems requires systematic approaches to standardization, documentation, and validation. Researchers can implement these strategies to enhance reproducibility:

Reagent standardization:

• Recombinant protein quality control:

  • Use validated lots with defined specific activity

  • Implement batch testing before experimental use

  • Consider creating laboratory reference standards

  • Document source, catalog number, and lot in publications

• Cell source consistency:

  • Maintain detailed records of animal strains and housing conditions

  • Standardize isolation protocols with defined parameters

  • Characterize cell populations by flow cytometry before use

  • Consider cryopreserving large batches of primary cells

• Assay component standardization:

  • Use calibrated instruments with regular maintenance

  • Prepare master mixes for complex reagent combinations

  • Implement positive and negative controls in each experiment

  • Consider using automated liquid handling when available

Protocol optimization and validation:

• Robust protocol development:

  • Perform parameter sensitivity analysis

  • Identify critical steps that impact variability

  • Implement quality control checkpoints

  • Create detailed step-by-step protocols with troubleshooting guides

• Protocol validation:

  • Test reproducibility across multiple operators

  • Conduct inter-laboratory comparisons when possible

  • Perform power analysis to determine appropriate sample sizes

  • Validate across different equipment models if relevant

• Statistical rigor:

  • Pre-register experimental designs and analysis plans

  • Implement randomization and blinding procedures

  • Report all exclusion criteria and outlier handling

  • Include all relevant controls in statistical analysis

Data management practices:

• Comprehensive documentation:

  • Maintain detailed laboratory notebooks (electronic preferred)

  • Record all deviations from standard protocols

  • Document environmental conditions and timing

  • Preserve raw data files in their original format

• Data analysis transparency:

  • Use version-controlled analysis scripts

  • Document all data transformations and normalizations

  • Make analysis code available with publications

  • Consider pre-registering analysis plans for complex studies

• Data sharing:

  • Deposit raw data in appropriate repositories

  • Share detailed protocols via platforms like protocols.io

  • Provide comprehensive methods sections in publications

  • Consider open lab notebook approaches for ongoing work

Specific considerations for Ccl3 systems:

• Biological variability awareness:

  • NK cell responsiveness to Ccl3 depends on activation state

  • Ccl3 oligomerization affects function and may vary between preparations

  • Type I interferon levels influence Ccl3 translation efficiency

  • Account for these variables in experimental design

• Reporter system standardization:

  • For fluorescent reporter systems like CCL3-EASER, establish signal calibration standards

  • Document imaging parameters and analysis thresholds

  • Include fluorescence minus one (FMO) controls for gating

  • Consider photobleaching and reporter stability over time

• Cross-validation approaches:

  • Confirm key findings using complementary methodologies

  • Validate reporter results with independent protein measurements

  • Correlate in vitro findings with in vivo observations

  • Use genetic approaches (knockout/knockin) to confirm specificity