Recombinant Mouse C-C motif chemokine 9 protein (Ccl9) (Active)

What is Recombinant Mouse CCL9 protein and what are its key identifiers?

Recombinant Mouse CCL9 is an 11 kDa secreted monomeric polypeptide belonging to the beta (or CC) intercrine family of chemokines. It is classified specifically as a member of the NC6 or six cysteine-containing CC subfamily of chemokines. This protein is also known by several alternative names including macrophage inflammatory protein-1 gamma (MIP-1γ), macrophage inflammatory protein-related protein-2 (MRP-2), small-inducible cytokine A9, and CCF18 in rodents .

The commercially available recombinant form typically refers to E. coli-derived mouse CCL9 protein spanning amino acids Gln22-Gln122 of the full-length protein sequence. The protein is generally provided in lyophilized form, either with or without bovine serum albumin (BSA) as a carrier protein .

What are the structural characteristics of CCL9 protein?

CCL9 is synthesized as a 122 amino acid precursor that contains a 21 amino acid signal sequence and a 101 amino acid mature region. The mature protein has six cysteine residues, which is distinctive compared to other CC family members that typically have four cysteines. This expanded structure allows CCL9 to form a third intrachain disulfide bond with its two extra cysteines .

Mouse CCL9 shares approximately 75% amino acid identity with rat CCL9/10. Within the NC6 subfamily, there are no direct human-to-rodent interspecies orthologs, making CCL9 a rodent-specific chemokine. The full-length CCL9 circulates as a complete molecule, while in inflammatory environments, it can undergo proteolytic processing to generate multiple isoforms .

What is the tissue distribution and expression pattern of CCL9?

CCL9 is expressed primarily in the liver, lung, and thymus, although some expression has been detected across a wide variety of tissues with the notable exception of the brain . It is constitutively secreted and circulates at relatively high concentrations in the blood of healthy animals .

During pathological conditions such as pancreatic inflammation or cancer development, CCL9 expression can be significantly upregulated. In models of pancreatic ductal adenocarcinoma (PDAC), CCL9 expression is increased in areas undergoing acinar-to-ductal metaplasia (ADM). Interestingly, some infiltrating macrophages (F4/80-positive cells) surrounding the ADM areas have been observed to express CCL9, suggesting both autocrine and paracrine sources of this chemokine .

How should recombinant CCL9 be stored and reconstituted for experimental use?

Storage Recommendations:

• Lyophilized protein remains stable for up to 12 months when stored at -20°C to -80°C

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Upon receipt, store immediately at the recommended temperature

Reconstitution Guidelines:

The choice between carrier-containing and carrier-free formulations should be based on experimental requirements. For cell or tissue culture experiments and ELISA standards, the carrier protein (BSA) version is generally recommended. The carrier-free protein is preferable for applications where the presence of BSA could potentially interfere with experimental outcomes .

What are the primary receptors for CCL9 and what signaling pathways does it activate?

CCL9 primarily signals through two G protein-coupled receptors: CCR1 and CCR3. Both receptors are found to be upregulated during Kras G12D-mediated pancreatic acinar-to-ductal metaplasia, suggesting their importance in this pathological process .

CCR1 is particularly notable as it is the most abundant chemokine receptor found on osteoclasts. Binding of CCL9 to its receptors activates calcium release in neutrophils and can initiate multiple downstream signaling pathways. In osteoclasts, CCL9-CCR1 interaction promotes receptor-activator-of-NF-κB-ligand (RANKL)-induced osteoclast formation and survival .

When designing experiments to study CCL9 signaling, researchers should consider:

• The expression levels of CCR1 and CCR3 in their target cells

• The potential cross-reactivity with other chemokine ligands that share these receptors

• The differential activation of signaling pathways depending on whether full-length or proteolytically processed CCL9 is used

What is the role of CCL9 in pancreatic acinar-to-ductal metaplasia and cancer progression?

Recent research has identified CCL9 as a novel downstream target of oncogenic Kras G12D in promoting pancreatic ductal adenocarcinoma (PDAC) initiation through acinar-to-ductal metaplasia (ADM). This represents a significant mechanistic insight into how oncogenic Kras drives the early stages of pancreatic cancer development .

In experimental models:

• Knockdown of CCL9 in Kras G12D-expressing acini reduced Kras G12D-induced ADM in 3D organoid culture systems

• Exogenously added CCL9 or overexpression of CCL9 alone was capable of driving pancreatic ADM

• In transgenic p48-cre:Kras G12D mice, depletion of CCL9 using neutralizing antibodies significantly reduced ADM formation and pancreatic intraepithelial neoplasia (PanIN) structures

The molecular mechanism involves upregulation of reactive oxygen species (ROS) through the NADPH oxidase system and increased expression of metalloproteinases including MMP14, MMP3, and MMP2. These findings suggest that targeting CCL9 could potentially represent a novel therapeutic approach for preventing PDAC initiation and progression .

How does CCL9 regulate reactive oxygen species and what are the downstream effects?

CCL9 plays a critical role in regulating intracellular reactive oxygen species (ROS) levels, particularly in the context of pancreatic acinar-to-ductal metaplasia. The relationship between CCL9 and ROS has been experimentally demonstrated through several approaches:

• Overexpression of Kras G12D increases levels of total intracellular ROS in primary acini

• Knockdown of CCL9 using shCCL9 lentivirus significantly diminishes Kras G12D-induced intracellular ROS levels

• Overexpression of CCL9 alone in primary murine pancreatic acini with wildtype Kras dramatically increases intracellular ROS levels up to 4-fold compared to control cells

The source of CCL9-induced ROS has been identified as the NADPH oxidase system. Knockdown of p22phox, an essential subunit of the NADPH oxidase complex, completely reduces ROS production even below basal levels and blocks CCL9-induced ADM of the pancreas .

Functionally, depletion of ROS using the general ROS scavenger N-acetyl-L-cysteine (NAC) almost completely abolishes CCL9-induced acinar-to-ductal metaplasia in 3D organoid culture, without affecting cell viability. This demonstrates that ROS production is a necessary downstream mediator of CCL9's effects on pancreatic ADM .

How does CCL9 contribute to osteoclast activation and bone resorption?

CCL9 plays a significant role in bone metabolism through its ability to activate osteoclasts via CCR1, which is the most abundant chemokine receptor found on these cells. Research has demonstrated that CCL9 promotes receptor-activator-of-NF-κB-ligand (RANKL)-induced osteoclast formation and survival .

This activity has important implications for understanding bone resorption mechanisms in both physiological and pathological conditions. CCL9's role in osteoclast biology suggests it may be a potential therapeutic target in bone-related disorders such as osteoporosis .

When studying CCL9's effects on bone metabolism, researchers should consider:

• The interaction between CCL9 and RANKL signaling pathways

• The differential effects of full-length versus proteolytically processed CCL9 on osteoclast activation

• The potential confounding effects of other chemokines that may signal through CCR1

Cell Culture Systems:

• 3D organoid culture systems using primary acini provide an excellent model for studying CCL9's role in acinar-to-ductal metaplasia

• For studying osteoclast effects, bone marrow-derived macrophage cultures treated with RANKL can be used to assess CCL9's impact on osteoclastogenesis

• The effective concentration range for CCL9 in most cell culture applications is typically 0.0900-0.900 μg/mL

Genetic Manipulation:

• shRNA-mediated knockdown of CCL9 has been successfully demonstrated in primary acinar cells

• Adenovirus delivery methods can be used for expressing genes of interest (such as Kras G12D) in primary acini

• Lentiviral systems are effective for stable knockdown of CCL9 or related pathway components

In Vivo Approaches:

• CCL9 neutralization can be achieved using specific neutralizing antibodies in mouse models

• Transgenic mouse models such as p48-cre:Kras G12D can be used to study CCL9's role in pancreatic cancer development

• Assessment of bone phenotypes in models with altered CCL9 expression can reveal its role in skeletal homeostasis

Detection Methods:

• Immunohistochemistry using antibodies against CCL9, along with markers like CK-19 (ductal marker) and F4/80 (macrophage marker)

• Real-time qRT-PCR for quantification of CCL9 and receptor mRNA levels

• ROS measurement assays to assess one of the key downstream effects of CCL9 signaling

• Matrix metalloproteinase expression and activity assays

What are the differences between full-length CCL9 and its proteolytically processed forms?

NC6 chemokines like CCL9 are typically only marginally active at full length but are converted to highly active forms upon N-terminal truncation. In inflammatory environments, mature CCL9 undergoes natural truncation by 28, 29, or 30 amino acids at the N-terminus, generating a highly active, 8 kDa, 71-73 amino acid CCR1 ligand .

This behavior contrasts with other CCR1 ligands such as CCL3/MIP-1 alpha and CCL5/RANTES, which actually lose their potency when proteolytically processed. Under normal conditions, CCL9 circulates as a full-length molecule, but during inflammation with subsequent enzyme release, local processing can generate early, potent leukocyte chemoattractants .

For researchers, this has important implications:

• When designing experiments, consider whether full-length or truncated forms of CCL9 are most relevant to your biological question

• The proteolytic processing of CCL9 may represent an important regulatory mechanism in vivo

• Different experimental outcomes may be observed depending on whether inflammatory conditions that promote CCL9 processing are present

How can the biological activity of recombinant CCL9 be measured?

Several functional assays can be employed to assess the biological activity of recombinant CCL9:

• Calcium Mobilization Assays: Since CCL9 activates calcium release in neutrophils and other CCR1/CCR3-expressing cells, calcium flux assays using fluorescent indicators can measure immediate receptor activation.

• Chemotaxis Assays: CCL9 has chemokinetic properties and can induce migration of specific cell populations. Transwell migration assays using neutrophils or other responsive cells can quantify this activity.

• ROS Production Measurement: As CCL9 significantly increases ROS levels in target cells like pancreatic acini, measurement of ROS using fluorescent probes can serve as a functional readout.

• 3D Organoid Culture Systems: For measuring CCL9's ability to induce morphological changes such as acinar-to-ductal metaplasia, 3D organoid cultures of primary pancreatic acini provide a physiologically relevant model system .

• Osteoclast Formation Assays: CCL9's ability to promote RANKL-induced osteoclast formation can be assessed using bone marrow-derived macrophage cultures and subsequent osteoclast quantification.

• MMP Expression Analysis: Quantifying the expression and activity of metalloproteinases (MMP14, MMP3, MMP2) can serve as downstream markers of CCL9 activity, particularly in the context of tissue remodeling .

What are common technical challenges when working with recombinant CCL9 and how can they be addressed?

What experimental controls should be included when studying CCL9 function?

• Positive Controls:

  • Known CCR1/CCR3 ligands to confirm receptor functionality

  • Known inducers of the biological process being studied (e.g., TGF-α for pancreatic ADM)

• Negative Controls:

  • Heat-inactivated CCL9 to control for non-specific protein effects

  • Unrelated chemokines that do not act through CCR1/CCR3

• Specificity Controls:

  • CCL9 neutralizing antibodies to confirm observed effects are CCL9-specific

  • CCR1/CCR3 antagonists to confirm receptor involvement

• Pathway Controls:

  • ROS scavengers like NAC when studying CCL9-induced oxidative stress

  • Inhibitors of metalloproteases when studying MMP-dependent effects

  • NADPH oxidase inhibitors or p22phox knockdown to control for ROS-mediated effects

What are emerging areas of CCL9 research that require further investigation?

• Therapeutic Targeting: Given CCL9's role in pancreatic cancer initiation and progression, research into specific inhibitors or neutralizing approaches could yield novel therapeutic strategies for PDAC and potentially other cancers.

• Receptor-Specific Signaling: Further delineation of CCL9 signaling through CCR1 versus CCR3, and the distinct downstream pathways activated, remains an important area for investigation.

• Proteolytic Regulation: The enzymes responsible for CCL9 processing in vivo, and how this processing is regulated in different tissue microenvironments, requires additional characterization.

• Immune Cell Interactions: The role of CCL9 in recruiting and activating specific immune cell populations, particularly in the context of cancer and inflammation, represents an exciting frontier for research.

• Cross-Species Functional Equivalents: Since CCL9 lacks direct human orthologs, identifying functional equivalents in human systems would facilitate translation of rodent findings to human disease.

How might CCL9 research contribute to understanding human disease mechanisms?

While CCL9 itself is a rodent-specific chemokine without direct human orthologs, research on its mechanisms of action can provide valuable insights into fundamental biological processes relevant to human diseases:

• The role of chemokines in cancer initiation and progression, particularly in pancreatic cancer

• Mechanisms of acinar-to-ductal metaplasia as a precursor to pancreatic cancer

• ROS regulation in cellular transformation and cancer development

• Chemokine regulation of osteoclast function in bone homeostasis and disease

• Proteolytic processing as a regulatory mechanism for chemokine activity