Recombinant Human C-C motif chemokine 15 protein (CCL15), partial (Active)

What is the molecular structure of CCL15 and how does it differ from other CC chemokines?

CCL15, also known as Leukotactin-1 (LKN-1), MIP-5, HCC-2, and NCC-3, is a unique CC chemokine that contains six conserved cysteine residues instead of the typical four found in most CC chemokines. The full-length precursor protein consists of 113 amino acid residues with a 21-residue signal peptide that is cleaved to generate a 92 amino acid mature protein. The additional two cysteine residues in CCL15 form a third disulfide bond, distinguishing it structurally from conventional CC chemokines. This structural variation likely contributes to the chemokine's specific functional properties and receptor interactions .

What are the primary biological functions of CCL15 in normal physiological conditions?

CCL15 functions primarily as a potent chemoattractant for monocytes and T-lymphocytes. It also attracts eosinophils and induces calcium flux in human CCR1-transfected cells. Under normal physiological conditions, CCL15 is constitutively expressed in the gut and liver, suggesting tissue-specific regulatory functions. Additionally, CCL15 plays a role in regulating hematopoiesis by suppressing colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by growth factors . Recent research also indicates that CCL15 modulates hematopoietic progenitor cell (HPC) motility and adhesion, suggesting a role in stem cell trafficking and homing under normal conditions .

How do truncated forms of CCL15 differ functionally from the full-length protein?

N-terminally truncated forms of CCL15 show dramatically enhanced biological activity compared to the full-length protein. The 68 amino acid truncated form is approximately 50-fold more active than the 92 amino acid full-length CCL15 as a chemoattractant for THP-1 cells . Specifically, CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) amino acids are the main proteolytic products found in hemofiltrate and display significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes compared to full-length CCL15 . These truncated forms also demonstrate enhanced ability to induce adhesiveness of mononuclear cells to fibronectin, suggesting proteolytic processing serves as a critical activation mechanism for CCL15 in vivo .

What are the recommended reconstitution and storage conditions for recombinant CCL15 to maintain optimal bioactivity?

For carrier-containing recombinant human CCL15 (with BSA):

• Reconstitute lyophilized protein at 10 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For storage, use a manual defrost freezer and avoid repeated freeze-thaw cycles as these significantly reduce protein activity

For carrier-free recombinant human CCL15:

• Reconstitute at a higher concentration of 100 μg/mL in sterile PBS

• Storage conditions remain the same - use a manual defrost freezer and minimize freeze-thaw cycles

Both formulations are typically shipped at ambient temperature but should be stored immediately at recommended temperatures upon receipt to preserve bioactivity .

What assays are most effective for measuring CCL15 activity in experimental settings?

Several validated assays effectively measure CCL15 bioactivity:

• Chemotaxis assays: Measuring migration of BaF3 mouse pro-B cells transfected with human CCR1 in response to CCL15 (ED₅₀ = 0.6-3 ng/mL)

• Calcium flux assays: Particularly effective with CCR1-transfected cell lines to measure receptor activation by different CCL15 isoforms

• Cell adhesion assays: Measuring CCL15-induced adhesiveness of mononuclear cells to fibronectin under various conditions

• CFU-A colony assays: Evaluating the impact of CCL15 on hematopoietic progenitor cells, where CCL15(27-92) dose-dependently affects colony size

• Competitive repopulation assays: For investigating CCL15's effects on hematopoietic stem cell engraftment and repopulation potential in vivo

For quantification in biological samples, enzyme-linked immunosorbent assays (ELISA) provide reliable measurement of CCL15 concentrations in serum and bronchoalveolar lavage fluid (BALF) .

What proteases are involved in the processing of CCL15 and how can researchers experimentally simulate this activation?

Neutrophil serine proteases play a critical role in processing full-length CCL15 into more active truncated forms. Specifically:

• Cathepsin G: Identified as the principal protease producing Δ23 and Δ26 CCL15 isoforms (CCL15(24-92) and CCL15(27-92))

• Elastase: Produces a Δ21 isoform (CCL15(22-92))

Researchers can experimentally simulate this activation through:

• Incubation of full-length CCL15(1-92) with purified neutrophil cathepsin G or elastase at physiological concentrations

• Co-culture with activated neutrophils, which release these proteases in their pericellular space

• Treatment with neutrophil supernatants containing released proteases

For verification of processing, chromatographic separation can identify the resulting isoforms, with CCL15(27-92) eluting at approximately 36 minutes and CCL15(24-92) at 38 minutes using standardized methods .

How can researchers distinguish between full-length and truncated forms of CCL15 in biological samples?

Distinguishing between full-length and truncated forms of CCL15 in biological samples requires specific analytical techniques:

• Chromatographic separation: High-performance liquid chromatography (HPLC) can separate different CCL15 isoforms based on their hydrophobic and hydrophilic properties, with distinct retention times (tᵣ) for different forms: full-length CCL15(1-92) and truncated forms like CCL15(27-92) and CCL15(24-92)

• Mass spectrometry: For precise identification of N-terminal truncations by determining the exact molecular weight of isolated CCL15 isoforms

• Isoform-specific antibodies: Where available, antibodies recognizing specific epitopes present or absent in truncated forms

• Functional bioassays: Comparing activity levels, as truncated forms exhibit significantly higher potency in inducing calcium flux and chemotaxis compared to full-length CCL15

• Combined approach: For comprehensive analysis, researchers should employ a combination of chromatographic separation followed by immunological detection and functional characterization

How does CCL15 influence hematopoietic progenitor cell (HPC) function, and what experimental models best demonstrate these effects?

CCL15, particularly in its truncated form CCL15(27-92), significantly influences HPC function through several mechanisms:

• Enhanced migration: CCL15(27-92) significantly enhances CXCL12-induced transwell migration of Lin-/Sca1+ HPCs, functioning as a co-stimulator of HPC migration

• Increased adhesion: CCL15(27-92) strengthens shear stress-dependent adhesion to vascular cell adhesion molecule-1 (VCAM-1), a critical interaction for HPC homing to bone marrow

• Colony formation modulation: In CFU-A assays, CCL15(27-92) dose-dependently reduces colony size when performed with murine bone marrow and Lin-/Sca1+ HPCs

• Improved engraftment: Pretreatment of bone marrow with CCL15(27-92) significantly increases competitive repopulation in murine models

The most effective experimental models include:

• Transwell migration assays with Lin-/Sca1+ HPCs

• Shear stress adhesion assays to VCAM-1

• CFU-A colony formation assays

• Competitive repopulation assays in murine models

These models collectively demonstrate that CCL15 modulates adhesive and migratory properties of HPCs with potential to improve short-term engraftment in stem cell transplantation .

What is the relationship between G-CSF treatment, CCL15 activation, and HPC mobilization?

During hematopoietic progenitor cell (HPC) mobilization with granulocyte colony-stimulating factor (G-CSF), blood plasma contains significantly increased concentrations of activated CCL15(27-92) (1.1 ± 0.1 ng/ml) compared to controls (0.4 ± 0.1 ng/ml, p = 0.02) . This relationship involves multiple mechanisms:

• G-CSF induces neutrophil activation: G-CSF treatment strongly stimulates neutrophil proliferation and activation, leading to release of serine proteases elastase and cathepsin G

• Proteolytic processing: These neutrophil proteases cleave full-length CCL15(1-92) to generate activated forms including CCL15(27-92)

• Enhanced HPC function: Activated CCL15 then modulates HPC adhesion and migration capabilities, potentially contributing to mobilization and subsequent engraftment efficiency

This relationship creates a positive feedback mechanism where G-CSF treatment leads to neutrophil activation, which generates activated CCL15, which in turn enhances HPC mobilization and function, ultimately improving transplantation outcomes .

How might CCL15 be utilized to improve hematopoietic stem cell transplantation outcomes?

CCL15, particularly in its activated form CCL15(27-92), offers several potential strategies to improve hematopoietic stem cell transplantation outcomes:

• Ex vivo priming: Pretreatment of bone marrow with CCL15(27-92) before transplantation significantly increases competitive repopulation in murine models, suggesting this approach could enhance engraftment efficiency in clinical settings

• Enhanced homing: CCL15(27-92) strengthens integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and enhances CXCL12-induced migration, both critical processes for successful stem cell homing to bone marrow niches

• Combinatorial approaches: Combining CCL15 with other hematopoietic modulators could create synergistic effects. Research indicates CCL15 works cooperatively with CXCL12, suggesting potential combinatorial strategies

• Targeted release systems: Developing controlled release systems that deliver active CCL15 to specific bone marrow niches could enhance localized effects while minimizing systemic exposure

Implementation of these approaches requires careful titration of CCL15 concentrations, as dose-dependent effects have been observed in colony formation assays, suggesting optimal dosing will be critical for successful clinical translation .

What role does CCL15 play in inflammatory lung diseases and what are the implications for biomarker development?

CCL15 has emerged as a potentially important biomarker in inflammatory lung diseases, particularly Chronic Hypersensitivity Pneumonitis (CHP):

• Elevated expression: Immunohistochemical investigations revealed high CCL15 expression in the lungs of CHP patients

• Serum biomarker potential: Serum CCL15 levels in CHP patients (29.1 ± 2.1 μg/mL) were significantly higher than those in idiopathic pulmonary fibrosis patients (19.7 ± 1.3 μg/mL, p = 0.01) and healthy subjects (19.5 ± 1.7 μg/mL, p = 0.003)

• Correlation with disease parameters: BALF CCL15/Albumin ratio showed significant inverse correlations with:

  • Forced vital capacity (β = -0.47, p = 0.0006)

  • Percentage of predicted carbon monoxide diffusion capacity (β = -0.41, p = 0.0048)

  • BALF lymphocyte count (β = -0.34, p = 0.01)

• Prognostic value: Multivariate Cox proportional hazards analysis revealed that high BALF CCL15/Albumin ratio was independently associated with poor prognosis in CHP patients (HR 1.1, 95% CI 1.03-1.18, p = 0.004)

These findings suggest CCL15 could serve as a valuable diagnostic and prognostic biomarker for CHP, potentially helping to address the current challenges in accurate diagnosis of this condition. Further validation studies with larger cohorts would be necessary to establish standardized cutoff values and confirm clinical utility .

What are the common challenges in working with recombinant CCL15 and how can they be overcome?

Researchers working with recombinant CCL15 frequently encounter several challenges:

• Stability issues:

  • Challenge: CCL15 can lose activity during storage and handling

  • Solution: Store at recommended temperatures, minimize freeze-thaw cycles, and use carrier proteins (BSA) for dilute solutions

• Variability between isoforms:

  • Challenge: Different N-terminal truncated forms have vastly different potencies

  • Solution: Clearly document which specific isoform is being used in experiments and standardize to a specific truncated form for consistent results

• Proteolytic degradation during experiments:

  • Challenge: Endogenous proteases in biological samples may further process CCL15

  • Solution: Consider adding protease inhibitors when appropriate, though note this may prevent natural activation processes in some experimental designs

• Receptor specificity overlaps:

  • Challenge: CCL15 acts primarily through CCR1 but also binds CCR3, potentially confounding results

  • Solution: Use receptor-specific antagonists or receptor-deficient cells to parse specific signaling pathways

• Species differences:

  • Challenge: Human CCL15 may have different activities in murine systems

  • Solution: Consider species-specific differences when translating between human and mouse models; test both human CCL15 and murine CCL9 (orthologue) when appropriate

What analytical methods provide the most accurate quantification of CCL15 in complex biological samples?

For accurate quantification of CCL15 in complex biological samples, researchers should consider these analytical approaches:

• Enzyme-linked immunosorbent assay (ELISA):

  • Most commonly used for quantifying CCL15 in serum and BALF

  • For optimal results, normalize BALF CCL15 to albumin levels (CCL15/Alb ratio) to account for dilution effects

• Chromatographic separation with immunodetection:

  • Provides distinction between different CCL15 isoforms

  • Can separate full-length CCL15 from N-terminally truncated forms based on retention time differences

• Mass spectrometry:

  • For precise identification and quantification of specific CCL15 isoforms

  • Particularly valuable for detecting novel truncated forms in complex biological mixtures

• Bioactivity assays:

  • Using CCR1-transfected cell lines to measure functional activity through calcium flux

  • Provides functional quantification complementary to protein concentration measurements

• Multiplexed immunoassay platforms:

  • Allow simultaneous detection of CCL15 alongside other chemokines/cytokines in the same sample

  • Valuable for studies investigating complex inflammatory networks

For maximal accuracy, researchers should employ orthogonal approaches combining at least two different detection methods, particularly when analyzing samples with potential proteolytic processing activity .