Recombinant Mouse C-C motif chemokine 19 protein (Ccl19) (Active)

What is the primary function of mouse CCL19 in the immune system?

Mouse CCL19 functions as a chemotactic protein that primarily directs the migration of specific immune cells. It is strongly chemotactic for naive (L-selectinhi) CD4 T-cells and CD8 T-cells while exhibiting weaker attraction for resting B-cells and memory (L-selectinlo) CD4 T-cells . CCL19 plays essential roles in normal lymphocyte recirculation and homing, as well as in trafficking of T-cells in the thymus . It specifically binds to chemokine receptor CCR7, facilitating these cellular movements and interactions . Additionally, CCL19 serves to promote encounters between recirculating T-cells and dendritic cells, contributing to the orchestration of local and systemic immune responses .

Which tissues express the highest levels of CCL19?

Mouse CCL19 is expressed at varying levels throughout different tissues, with a distinct expression pattern. The highest expression levels are found in the lymph nodes, thymus, and appendix . Intermediate expression levels are detected in the colon and trachea . Lower expression levels are observed in the spleen, small intestine, lung, kidney, and stomach . This differential expression pattern aligns with CCL19's role in immune cell trafficking and secondary lymphoid organ function.

How does CCL19 differ from other chemokines in the CC family?

CCL19 belongs to the intercrine beta (chemokine CC) family characterized by two adjacent cysteines in their structure . While sharing this structural feature with other CC chemokines, CCL19 is distinguished by its specific binding to the CCR7 receptor and its involvement in lymphocyte recirculation and homing . Unlike some other chemokines that attract granulocytes and monocytes, CCL19 specifically attracts T-cells and B-cells but not granulocytes and monocytes . Additionally, CCL19 binds to atypical chemokine receptor ACKR4 and mediates the recruitment of beta-arrestin (ARRB1/2) to ACKR4, providing another mechanism for its biological activity .

What are the optimal conditions for using recombinant mouse CCL19 in chemotaxis assays?

When conducting chemotaxis assays with recombinant mouse CCL19, researchers should consider several methodological factors. Based on the protein's properties, optimal assay conditions typically involve:

• Concentration range: Use a titration between 10-100 ng/mL of recombinant CCL19, as this encompasses the chemotactic range for naive T-cells and B-cells

• Target cells: Freshly isolated naive CD4+ T-cells (L-selectinhi), CD8+ T-cells, or to a lesser extent, resting B-cells and memory CD4+ T-cells (L-selectinlo)

• Incubation time: 1-3 hours at 37°C with 5% CO2 is typically sufficient to observe migration

• Buffer system: A chemotaxis buffer containing RPMI-1640 with 0.5-1% BSA is recommended

• Positive controls: Include CCL21 (another CCR7 ligand) as a comparative control

• Transwell setup: Use 5-8 μm pore size depending on the cell type being assessed

Analyze results using flow cytometry for precise quantification of migrated cells, particularly when working with heterogeneous cell populations.

How should researchers validate the activity of recombinant mouse CCL19 protein?

Validating the activity of recombinant mouse CCL19 requires multiple complementary approaches:

• Functional assays:

  • Chemotaxis assays using naive T-cells or B-cells to confirm chemotactic potency

  • Calcium flux assays in CCR7-expressing cells to verify receptor engagement

  • Phosphorylation of ERK1/2 in target cells (observable within minutes of CCL19 stimulation)

• Biochemical validation:

  • SDS-PAGE to confirm purity (should be ≥95%)

  • Western blotting using specific anti-CCL19 antibodies

  • Mass spectrometry to verify molecular weight and sequence integrity

• Receptor binding studies:

  • Competitive binding assays with labeled CCL19 and CCR7-expressing cells

  • Binding to ACKR4 receptor and beta-arrestin recruitment assays

The most definitive validation combines both functional activity (chemotaxis) and biochemical confirmation of protein identity and purity.

What considerations are important when designing CCL19 stimulation experiments with adipocytes?

When stimulating adipocytes with CCL19, researchers should consider several methodological factors based on recent research:

• Cell model selection:

  • 3T3-L1 adipocytes are a validated model that responds to CCL19 stimulation

  • Primary adipocytes from different fat depots may respond differently

• Experimental design:

  • CCL19 upregulates CCR7 expression in adipocytes at both gene and protein levels

  • Include time-course experiments (4-24 hours) to capture both immediate and delayed responses

  • Monitor both ERK1/2 phosphorylation (increases) and AMPKα phosphorylation (decreases)

• Signaling pathway analysis:

  • Assess crosstalk between ERK1/2 and AMPKα pathways

  • Monitor downstream effects on PGC1α and UCP1 expression, particularly in brown adipocytes

  • Include assessments of inflammatory cytokine expression (MCP1, TNFα, CD11c)

• Controls:

  • Include CCR7 antagonists or neutralizing antibodies to confirm specificity

  • Consider fatty acid treatments (e.g., palmitic acid) as they may enhance CCL19 effects

This approach aligns with recent findings that CCL19 affects adipocyte metabolism through specific signaling pathways.

How can CCL19 knock-in mouse models be utilized to study metabolic disorders?

Adipocyte-specific CCL19 knock-in mouse models offer valuable insights into metabolic disorders. Based on recent research:

• Model characteristics:

  • Adipocyte-specific Ccl19 knock-in (KI) mice can be generated using CRISPR/Cas9-mediated homologous recombination without disrupting the endogenous Adipoq gene

  • These mice exhibit enlarged subcutaneous white and brown adipose tissue compared to wild-type mice

  • They show increased inflammatory markers in adipose tissue, particularly when challenged with high-fat diets

• Experimental protocols:

  • Challenge with different fat diets (40% or 60% fat diet) to model different degrees of obesity

  • The 40% fat diet shows the most remarkable phenotypic differences between genotypes

  • Monitor changes in inflammatory cytokines (MCP1, TNFα), immune cell infiltration, and metabolic parameters

• Signaling pathway analysis:

  • Assess ERK1/2 phosphorylation (increased) and AMPKα phosphorylation (decreased)

  • Evaluate PGC1α and UCP1 protein expression in brown adipose tissue (reduced in CCL19 KI mice)

  • Examine the correlation between CCL19 expression and insulin sensitivity measures

This model is particularly relevant for studying metabolic disorders in mild obesity or overweight conditions, such as those often observed in Asian populations .

What mechanisms underlie the effects of CCL19 on adipose tissue inflammation and metabolism?

The mechanisms linking CCL19 to adipose tissue inflammation and metabolism involve several interconnected pathways:

• Receptor-mediated signaling:

  • CCL19 stimulation upregulates CCR7 expression in adipocytes, creating a positive feedback loop

  • Binding to CCR7 activates ERK1/2 signaling pathways

  • Activated ERK1/2 inhibits AMPKα phosphorylation, a key regulator of energy metabolism

• Metabolic reprogramming:

  • Decreased AMPKα activation leads to impaired lipid metabolism and energy regulation

  • Reduced expression of PGC1α and UCP1 in brown adipose tissue impairs thermogenesis

  • These changes contribute to enlarged adipose tissue and altered energy expenditure

• Inflammatory cascade:

  • CCL19 overexpression increases proinflammatory cytokines MCP1 and TNFα in adipose tissue

  • Enhanced expression of CD11c suggests increased recruitment of inflammatory immune cells

  • Fatty acids, particularly palmitic acid, can further enhance these inflammatory responses

This mechanistic understanding explains how adipose-specific CCL19 overexpression contributes to a phenotype characterized by increased inflammation, impaired lipid metabolism, and reduced energy expenditure.

How do CCL19 and CCR7 interactions in different immune cells contribute to adipose tissue inflammation?

The interaction between CCL19 and CCR7 orchestrates a complex immune response in adipose tissue:

• Dendritic cell recruitment and activation:

  • CCL19 stimulation of bone marrow-derived dendritic cells (BMDCs) upregulates CCR7 expression

  • This creates a positive feedback loop enhancing dendritic cell migration to CCL19-rich adipose tissue

  • Activated dendritic cells can present antigens to T cells, perpetuating inflammation

• T cell recruitment and polarization:

  • CCL19 is strongly chemotactic for naive CD4+ and CD8+ T cells

  • Recruited T cells can become polarized toward pro-inflammatory phenotypes in the adipose microenvironment

  • Different T cell subsets (naive vs. memory) show differential responses to CCL19

• B cell involvement:

  • CCL19 attracts B cells, albeit less potently than T cells

  • B cell migration to the T-zone of secondary lymphoid tissues is influenced by CCL19

  • B cells can contribute to adipose tissue inflammation through antibody production and cytokine secretion

This complex interplay between CCL19/CCR7 and various immune cell populations creates a microenvironment conducive to chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction.

How should researchers interpret conflicting data on CCR7 deficiency in metabolic disease models?

The literature contains seemingly contradictory findings regarding the role of CCR7 in metabolic disease:

• Conflicting phenotypes in CCR7-null mice:

  • Some studies report that CCR7-null mice are protected against high-fat diet-induced obesity and insulin resistance

  • Other studies show that CCR7-null and wild-type mice similarly gain weight on high-fat diets

• Methodological considerations for resolving discrepancies:

  • Diet composition: Differences in fat content and source (40% vs. 60% fat diet) significantly impact outcomes

  • Genetic background: Strain variations can influence metabolic responses to CCR7 deletion

  • Sex differences: Male and female mice may respond differently to CCR7 deficiency

  • Age at intervention: The timing of high-fat diet introduction may affect outcomes

• Complementary approaches:

  • Compare results from loss-of-function (CCR7 knockout) and gain-of-function (CCL19 overexpression) models

  • Evaluate tissue-specific effects using conditional knockouts or overexpression models

  • Consider the influences of other CCR7 ligands (e.g., CCL21) in interpreting CCR7 knockout phenotypes

When interpreting conflicting data, researchers should carefully consider the specific experimental conditions, genetic backgrounds, and methodological approaches used in different studies.

What analytical techniques are most appropriate for quantifying CCL19-induced signaling changes in adipocytes?

To accurately quantify CCL19-induced signaling changes in adipocytes, researchers should employ several complementary techniques:

• Protein phosphorylation analysis:

  • Western blotting with phospho-specific antibodies for ERK1/2 and AMPKα

  • Phospho-flow cytometry for single-cell analysis of signaling events

  • Protein arrays to simultaneously assess multiple phosphorylation events

  • Temporal analysis (5 minutes to 24 hours) to capture both immediate and delayed responses

• Gene expression analysis:

  • Real-time PCR for quantifying changes in key genes (Ccr7, Mcp1, Tnfα, Cd11c)

  • RNA-seq for comprehensive transcriptional profiling

  • Single-cell RNA-seq to identify cell-specific responses within heterogeneous adipose tissue

• Metabolic assessments:

  • Seahorse analysis to measure mitochondrial respiration and glycolytic function

  • Lipidomics to quantify changes in lipid metabolism

  • Metabolic cage studies to assess whole-body energy expenditure in animal models

• Imaging techniques:

  • Confocal microscopy with fluorescently labeled antibodies to visualize protein localization

  • FRET-based approaches to analyze protein-protein interactions in live cells

  • In vivo imaging to track inflammatory cell recruitment to adipose tissue

These techniques, when used in combination, provide comprehensive insights into the molecular mechanisms underlying CCL19's effects on adipocyte signaling and metabolism.

How does the fatty acid environment influence CCL19 function in metabolic disease models?

The interaction between fatty acids and CCL19 signaling reveals important aspects of metabolic disease progression:

• Fatty acid effects on CCL19/CCR7 expression:

  • Palmitic acid (PA) promotes CCR7 expression in adipose tissue

  • CCL19 knock-in mice show elevated serum free fatty acid concentrations

  • PA administration further elevates serum fatty acid levels, with a more profound increase in CCL19 knock-in mice

• Synergistic effects on inflammation:

  • PA-treated CCL19 knock-in mice exhibit higher expression of CCR7 than wild-type mice

  • The combination of elevated CCL19 and PA increases expression of inflammatory markers (MCP1, TNFα, CD11c) in white adipose tissue

  • This synergy creates a feedforward loop of inflammation

• Mechanistic integration:

  • Fatty acids may alter membrane fluidity and receptor clustering, affecting CCL19-CCR7 interactions

  • Both CCL19 and fatty acids can activate ERK1/2 signaling through different initial pathways

  • The combined effect on AMPKα inhibition appears greater than either stimulus alone

These findings suggest that dietary fat composition significantly influences CCL19's effects on metabolism and inflammation, with implications for understanding how different diets influence metabolic disease progression in different populations.

What are the most promising therapeutic targets in the CCL19/CCR7 pathway for metabolic disorders?

Based on current understanding of the CCL19/CCR7 pathway, several therapeutic targets warrant investigation:

• Direct CCL19/CCR7 targeting:

  • CCR7 antagonists to block receptor activation

  • CCL19 neutralizing antibodies to reduce ligand availability

  • Small molecule inhibitors of downstream CCR7 signaling

• Pathway-specific interventions:

  • ERK1/2 inhibitors to prevent CCL19-induced AMPKα suppression

  • AMPKα activators to bypass CCL19's inhibitory effects on energy metabolism

  • PGC1α or UCP1 activators to restore thermogenic capacity in brown adipose tissue

• Cell-specific approaches:

  • Adipocyte-specific delivery systems to target CCL19/CCR7 signaling in fat tissue

  • Dendritic cell-targeted interventions to modulate CCL19-dependent immune cell recruitment

  • Combined approaches targeting both adipocytes and immune cells

The most effective therapeutic strategies may involve targeting the CCL19/CCR7 pathway in specific tissues or cell types rather than systemic inhibition, which could compromise normal immune function.

How might translational research bridge findings from mouse CCL19 models to human metabolic disorders?

Translating findings from mouse CCL19 models to human applications requires several strategic approaches:

• Comparative biology:

  • Analysis of CCL19 expression patterns in human vs. mouse adipose tissue samples

  • Correlation of adipose CCL19 levels with clinical parameters (BMI, HbA1c, C-reactive protein, HOMA-IR)

  • Identification of functional differences between human and mouse CCL19/CCR7 signaling

• Clinical correlation studies:

  • Assessment of serum CCL19 levels in individuals with varying degrees of obesity and insulin resistance

  • Adipose tissue biopsies to correlate CCL19 expression with inflammatory markers and metabolic parameters

  • Genetic association studies examining CCL19/CCR7 polymorphisms and metabolic disorder risk

• Human cellular models:

  • Primary human adipocyte cultures to validate CCL19 signaling mechanisms

  • Patient-derived iPSCs differentiated into adipocytes for personalized studies

  • Co-culture systems with human immune cells and adipocytes to model tissue interactions

• Biomarker development:

  • Evaluation of serum CCL19 as a potential biomarker for metabolic inflammation

  • Creation of composite biomarker panels including CCL19 and related inflammatory markers

  • Longitudinal studies tracking CCL19 levels during weight change or metabolic interventions

These translational approaches would help determine whether targeting the CCL19/CCR7 pathway could be effective in human metabolic disorders, particularly in early or mild conditions as observed in overweight Asian populations .