Recombinant Rat C-C motif chemokine 4 protein (Ccl4) (Active)

What is Recombinant Rat CCL4 and what are its basic structural characteristics?

Recombinant Rat CCL4, also known as macrophage inflammatory protein-1 beta (MIP-1 beta), is a member of the beta (C-C) subfamily of chemokines that functions primarily as a chemoattractant for monocytes and basophils, but not for eosinophils or neutrophils . It is typically secreted as a 14 kDa glycoprotein monomer, though noncovalent dimers are likely to occur in physiological conditions . The protein contains 126 amino acid residues with the mature form spanning from Ala24 to Asn92 (according to accession number P50230) . Structurally, the first five amino acids of the mature protein are essential for its biological activity, and deletion of the N-terminal glutamine, which is pyrrolidone carboxylic acid-modified, dramatically decreases its activity on basophils while surprisingly stimulating eosinophil chemotaxis . Rat CCL4 shares significant sequence homology with other mammalian CCL4 proteins, particularly 86% amino acid identity with mouse CCL4 and 80% with human CCL4 .

How does Rat CCL4 compare to CCL4 from other species?

Rat CCL4 exhibits varying degrees of homology with CCL4 from other species, reflecting both conserved functions and species-specific adaptations. The rat CCL4 propeptide shares 82% amino acid identity with mouse CCL4 over the 148 amino acid sequence . When compared to other mammalian species, rat CCL4 shows more moderate homology: 57% with equine CCL4, 52% with human CCL4, 52% with porcine CCL4, 52% with canine CCL4, and 52% with guinea pig CCL4, respectively, over the first 100 amino acids . A notable structural difference is that rat and mouse CCL4 have a 49 amino acid extension at the C-terminus compared to human CCL4, which may contribute to species-specific functions or interactions . These sequence variations must be considered when designing cross-species experiments or interpreting results from animal models in relation to human systems. Despite these differences, the core chemotactic functions and receptor interactions appear to be evolutionarily conserved, making rat CCL4 a valuable model for understanding fundamental chemokine biology .

What are the primary cellular sources and targets of Rat CCL4 in physiological conditions?

In physiological conditions, Rat CCL4 is produced by a diverse array of cell types that contribute to immune and inflammatory responses. Fibroblasts, tumor cells, smooth muscle cells, endothelial cells, and mononuclear phagocytes can produce CCL4 either constitutively or upon mitogenic stimulation . Additionally, activated leukocytes, lymphocytes, vascular endothelial cells, and pulmonary smooth muscle cells secrete CCL4 at sites of inflammation . Within the bone marrow environment, CCL4 is released from osteoblast cells to restore the homeostasis of hematopoietic stem cells during bone marrow activation . The primary cellular targets of Rat CCL4 include monocytes and basophils, which are attracted to sites of microbial infection and other pathologic inflammation such as allergic asthma and ischemic myocardium . CCL4 also enhances the recruitment of preosteoclasts to bone during the early stages of osteoclast differentiation, playing a role in bone remodeling and homeostasis . Notably, BaF3 mouse proB cells transfected with human CCR5 show chemotactic responses to recombinant rat CCL4, demonstrating the conservation of receptor-ligand interactions across species .

What are the recommended methods for reconstituting and storing Recombinant Rat CCL4 for optimal activity?

Recombinant Rat CCL4 is typically supplied in lyophilized form, requiring proper reconstitution to maintain its biological activity for experimental use . For reconstitution, it is recommended to briefly centrifuge the vial before opening to collect all material at the bottom. The lyophilized protein should be reconstituted in sterile, ultra-pure water or a buffer solution appropriate for the downstream application, typically at a concentration of 0.1-1.0 mg/mL, though specific reconstitution instructions may vary by manufacturer and should be consulted in the lot-specific Certificate of Analysis . After reconstitution, the solution should be gently mixed rather than vortexed to prevent protein denaturation. For storage of reconstituted protein, it is advisable to divide the solution into single-use aliquots to avoid repeated freeze-thaw cycles which can compromise protein activity. Long-term storage of reconstituted CCL4 should be at -20°C or preferably -80°C, while the lyophilized form can be stored at -20°C for extended periods . When handling the protein, researchers should work under sterile conditions and minimize exposure to room temperature, as chemokines can be sensitive to degradation. For experiments requiring maximal biological activity, freshly reconstituted protein is generally preferred over repeatedly thawed aliquots.

How can researchers validate the biological activity of Recombinant Rat CCL4 in vitro?

Researchers can employ several complementary assays to validate the biological activity of Recombinant Rat CCL4 in vitro, with chemotaxis assays being the gold standard. The most direct functional assessment is through chemotaxis experiments using cells that express the CCR5 receptor, such as BaF3 mouse proB cell lines transfected with human CCR5 . In these assays, migration is typically quantified using Resazurin or similar viability reagents to measure the number of cells that migrate through a membrane toward the chemoattractant . A dose-response curve should be established to determine the optimal concentration range, which is typically in the nanogram to microgram per milliliter range. The specificity of CCL4-induced chemotaxis can be confirmed using neutralizing antibodies against CCL4, where the neutralization dose (ND50) is typically 0.1-0.5 μg/mL in the presence of 0.075 μg/mL recombinant rat CCL4 . Additionally, researchers can validate CCL4 activity by measuring its effects on specific cell populations, such as monocytes or basophils, or by assessing downstream signaling pathways activated by CCR5 engagement using phospho-specific antibodies against key signaling molecules. Western blot analysis using cell lysates from activated cells (such as LPS-treated NR8383 rat alveolar macrophage cells) can also confirm the expression and secretion of endogenous CCL4 for comparison with recombinant protein .

What experimental controls should be included when working with Recombinant Rat CCL4 in cell-based assays?

When conducting cell-based assays with Recombinant Rat CCL4, several critical controls should be included to ensure experimental validity and accurate interpretation of results. First, a vehicle control containing the same buffer and additives used for CCL4 reconstitution (without the protein) is essential to distinguish CCL4-specific effects from those caused by buffer components. Second, a dose-response curve using at least five concentrations of CCL4 should be established to determine the optimal working concentration and to ensure that the observed effects fall within the linear range of the biological response. Third, specificity controls using neutralizing antibodies against CCL4 (such as Sheep Anti-Rat CCL4/MIP-1 beta Antigen Affinity-purified Polyclonal Antibody) can confirm that the observed effects are directly attributable to CCL4 activity . Fourth, positive controls using other well-characterized chemokines that act on the same cell type can provide comparison points for the magnitude of response. Fifth, for chemotaxis assays specifically, random migration controls (without a chemokine gradient) should be included to establish baseline migration rates. Finally, cell viability assessments should be performed in parallel to ensure that any observed effects are not due to cytotoxicity, particularly when using higher concentrations of recombinant proteins which may contain preservatives or endotoxin contaminants that could affect cell health independently of chemokine activity.

How does CCL4 interact with its receptor CCR5, and what are the downstream signaling pathways activated?

CCL4 primarily exerts its biological functions through interaction with the CCR5 receptor, a G protein-coupled receptor expressed on various immune cells. When CCL4 binds to CCR5, it triggers conformational changes in the receptor that activate multiple intracellular signaling cascades. The interaction between rat CCL4 and CCR5 leads to chemotaxis of CCR5-expressing cells, as demonstrated by migration assays using BaF3 mouse proB cells transfected with human CCR5 . At the molecular level, CCR5 activation by CCL4 initiates signaling through G proteins, particularly Gαi, leading to inhibition of adenylyl cyclase and reduction in cyclic AMP levels. This is followed by activation of phospholipase C, resulting in the generation of inositol trisphosphate and diacylglycerol, which respectively mobilize intracellular calcium and activate protein kinase C. CCL4-CCR5 interaction also activates multiple downstream kinase pathways, including MEK and JNK, which have been implicated in the downregulation of CCR5 expression during osteoclastogenesis induced by RANKL . Furthermore, CCR5 signaling activates phosphoinositide 3-kinase (PI3K) and small GTPases like Rac and Rho, which regulate cytoskeletal rearrangements necessary for directed cell migration. It's worth noting that while full-length CCL4 primarily activates CCR5, N-terminally truncated forms of human CCL4 (not specifically documented for rat CCL4 in the provided sources) can also interact with CCR1 and CCR2, potentially activating alternative signaling pathways .

What are the roles of Rat CCL4 in bone homeostasis and osteoclastogenesis?

Rat CCL4 plays a significant role in bone homeostasis by regulating the recruitment of preosteoclasts and influencing osteoclastogenesis through complex cellular interactions. Research has demonstrated that CCL4 enhances the migration and viability of preosteoclast cells, thereby facilitating their recruitment to bone tissue during the early stages of bone remodeling . This chemotactic function contributes to the maintenance of hematopoietic stem cell homeostasis within the bone marrow microenvironment, where CCL4 is released from osteoblast cells during bone marrow activation . Interestingly, while CCL4 promotes preosteoclast migration, it does not directly affect receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in mouse preosteoclast cells . A key regulatory mechanism involves the expression of CCR5, the primary receptor for CCL4, which is rapidly reduced by RANKL treatment during osteoclastogenesis—a process that can be reversed by interferon-gamma (IFN-γ) . Mechanistically, the downregulation of CCR5 by RANKL is mediated through the MEK and JNK signaling pathways in preosteoclast cells, and this reduction in CCR5 expression actually promotes osteoclastogenesis when RANKL is abundant . These findings suggest a biphasic role for the CCL4-CCR5 axis in bone metabolism: initially promoting the recruitment of preosteoclast precursors, followed by a reduction in CCR5 signaling that facilitates RANKL-driven osteoclast differentiation.

How can researchers use Recombinant Rat CCL4 to study inflammation and immune cell recruitment in rodent models?

Researchers can leverage Recombinant Rat CCL4 as a powerful tool to investigate inflammation and immune cell recruitment in rodent models through several methodological approaches. First, direct administration of recombinant CCL4 via subcutaneous, intraperitoneal, or site-specific injection can be used to induce localized inflammation and study the subsequent immune cell infiltration patterns, particularly of monocytes and basophils, which are primary targets of CCL4 chemotaxis . The recruitment dynamics can be analyzed using flow cytometry, immunohistochemistry, or intravital microscopy to track labeled immune cells in real-time. Second, CCL4 can be used in combination with other inflammatory stimuli to study how chemokine networks synergize during complex inflammatory responses, such as in models of allergic asthma, ischemic injury, or microbial infection . Third, neutralizing CCL4 with specific antibodies in rodent models of inflammation can help delineate the specific contribution of this chemokine to disease pathogenesis compared to other chemokines. Fourth, the use of recombinant CCL4 in ex vivo assays with cells isolated from rodent models of disease can provide insights into how pathological conditions affect cellular responsiveness to chemokine signals. Finally, CCL4 can be administered in bone-related studies to investigate its role in preosteoclast recruitment and subsequent effects on bone remodeling, particularly in models of inflammatory bone diseases where the CCL4-CCR5 axis may contribute to pathological bone loss . When designing these studies, researchers should carefully consider dosing based on published literature, as well as the timing and route of administration to best model the physiological or pathological process under investigation.

What are common challenges in working with Recombinant Rat CCL4 and how can these be addressed?

Researchers working with Recombinant Rat CCL4 may encounter several challenges that can affect experimental outcomes if not properly addressed. First, protein aggregation during storage or reconstitution can significantly reduce the biological activity of CCL4. This can be minimized by reconstituting the lyophilized protein in appropriate buffers (avoiding extreme pH values), using gentle mixing rather than vortexing, and storing reconstituted protein in single-use aliquots at -80°C to prevent repeated freeze-thaw cycles. Second, endotoxin contamination in recombinant protein preparations can confound results, particularly in inflammation studies, as it can independently activate immune cells. Researchers should select endotoxin-tested grade proteins or consider additional purification steps if necessary. Third, varying receptor expression levels on target cells can lead to inconsistent responses to CCL4 stimulation. This variability can be addressed by characterizing CCR5 expression on target cells before experiments and maintaining consistent culture conditions to minimize fluctuations in receptor expression. Fourth, cross-reactivity with other chemokine receptors or species variants may occur, as rat CCL4 shows approximately 8% cross-reactivity with recombinant mouse CCL4/MIP-1 beta in direct ELISAs . Specificity can be confirmed using receptor antagonists or cells lacking specific receptors. Fifth, the chemotactic activity of CCL4 follows a bell-shaped dose-response curve, where too high concentrations can actually inhibit migration due to receptor desensitization. Establishing a complete dose-response relationship for each experimental system is therefore crucial for determining the optimal working concentration.

How can researchers accurately quantify Recombinant Rat CCL4 in biological samples?

Accurate quantification of Recombinant Rat CCL4 in biological samples requires appropriate analytical techniques tailored to the sample type and expected concentration range. Enzyme-linked immunosorbent assay (ELISA) represents the gold standard for CCL4 quantification in cell culture supernatants, serum, or tissue homogenates. For ELISA development, sheep anti-rat CCL4/MIP-1 beta antigen affinity-purified polyclonal antibodies have been validated for direct ELISA applications, though researchers should be aware of potential cross-reactivity with mouse CCL4 (approximately 8%) . Western blotting offers an alternative approach for CCL4 detection, particularly useful when sample purity is a concern or when distinguishing between different forms of the protein. Using this method, CCL4 can be detected at approximately 12 kDa in reducing conditions, as demonstrated in lysates from LPS-treated NR8383 rat alveolar macrophage cell lines . For more complex biological matrices, immunoprecipitation may be required prior to Western blotting to enrich for CCL4. Mass spectrometry-based approaches provide the highest specificity and can identify post-translational modifications, though they require specialized equipment and expertise. When quantifying CCL4 in in vivo samples, researchers should consider the short half-life of chemokines in circulation and potentially implement sample collection strategies that use protease inhibitors to prevent ex vivo degradation. For all quantification methods, standard curves using purified recombinant rat CCL4 should be included, and the limit of detection and quantification should be established for the specific biological matrix being analyzed.

What are important considerations when designing in vivo experiments using Recombinant Rat CCL4?

Designing in vivo experiments with Recombinant Rat CCL4 requires careful consideration of multiple factors to ensure meaningful and reproducible results. First, researchers must establish appropriate dosing regimens based on the specific biological question and target tissue. While specific dosing information for rat CCL4 wasn't provided in the search results, studies with related recombinant proteins like rhCygb used 2 mg/kg administered subcutaneously daily in rat models . Second, the route of administration significantly impacts CCL4 distribution and activity—local injections (subcutaneous, intra-articular, or site-specific) are appropriate for studying localized inflammation, while systemic administration (intravenous or intraperitoneal) allows examination of broader effects on circulating immune cells or distant tissues. Third, the timing of CCL4 administration relative to other experimental manipulations is critical, as chemokine actions are often context-dependent and temporally regulated. Fourth, researchers should include appropriate controls, including vehicle-treated animals and, ideally, animals treated with heat-inactivated CCL4 to control for potential endotoxin or other contaminant effects. Fifth, validation of CCL4 activity in vivo can be challenging; therefore, biomarkers of CCL4 activity should be established, such as monitoring the recruitment of CCR5-expressing cells to target tissues or assessing changes in downstream signaling pathways. Sixth, researchers should consider the half-life of recombinant CCL4 in circulation, which may necessitate repeated dosing or the use of slow-release delivery systems for sustained effects. Finally, strain differences in rats may affect responses to CCL4, as genetic background can influence chemokine receptor expression patterns and downstream signaling efficacy, so strain selection should be consistent across experiments.

How is Recombinant Rat CCL4 being used to study interactions between inflammatory pathways and bone metabolism?

Recombinant Rat CCL4 has emerged as a valuable tool for investigating the complex interplay between inflammatory signaling and bone metabolism, revealing novel therapeutic targets for inflammatory bone disorders. Recent research has established that CCL4 enhances the recruitment of preosteoclasts to bone in the early stages of bone remodeling, functioning as a chemotactic factor that promotes migration and viability of preosteoclast cells . This recruitment process represents a critical initial step in the bone remodeling cascade that precedes osteoclast differentiation. Intriguingly, while CCL4 facilitates preosteoclast recruitment, it does not directly influence RANKL-induced osteoclast differentiation, suggesting a stage-specific role in osteoclastogenesis . The dynamic regulation of the CCL4 receptor CCR5 during osteoclastogenesis provides further insights into the temporal control of bone metabolism—RANKL treatment rapidly reduces CCR5 expression through MEK and JNK signaling pathways, and this downregulation actually promotes osteoclastogenesis when RANKL is abundant . Conversely, CCR5 expression can be recovered by IFN-γ during osteoclastogenesis, providing a potential regulatory checkpoint . These findings highlight how inflammatory mediators like IFN-γ can modulate bone metabolism by influencing the CCL4-CCR5 axis. Researchers are now using recombinant CCL4 in combination with pro-inflammatory cytokines to model inflammatory bone diseases and test potential therapeutic approaches that target specific steps in this pathway, such as CCR5 antagonists or inhibitors of the downstream signaling cascades that regulate CCR5 expression.

How can advanced techniques like CRISPR-Cas9 gene editing be combined with Recombinant Rat CCL4 for mechanistic studies?

Combining CRISPR-Cas9 gene editing technology with Recombinant Rat CCL4 offers powerful new approaches for dissecting the molecular mechanisms underlying CCL4 signaling in immune and inflammatory responses. Researchers can employ CRISPR-Cas9 to create precise modifications in the genes encoding CCL4, CCR5, or downstream signaling components in primary rat cells or cell lines to investigate specific structure-function relationships and signaling pathways. For example, guided by the knowledge that the first five amino acids of mature CCL4 are essential for its activity , CRISPR-Cas9 can be used to generate targeted mutations in these residues to create cells expressing CCL4 variants with altered receptor binding or signaling properties. Similarly, the finding that RANKL-induced downregulation of CCR5 is mediated by MEK and JNK pathways and promotes osteoclastogenesis suggests that CRISPR-Cas9-mediated knockout or modification of these kinases could provide insights into the regulation of CCR5 expression and its impact on osteoclast differentiation. Another advanced application involves creating reporter cell lines where fluorescent or luminescent proteins are knocked into endogenous CCL4 or CCR5 loci to monitor their expression dynamics in real-time under various stimuli. These engineered cells can then be treated with Recombinant Rat CCL4 to study receptor internalization, recycling, and downstream signaling events with high temporal resolution. Furthermore, CRISPR-Cas9 can be used in vivo in rat models to create tissue-specific knockouts of CCL4 or CCR5, which can then be complemented with exogenous recombinant CCL4 administration to parse the relative contributions of local versus systemic chemokine signaling in inflammatory conditions or bone homeostasis.

What are the key takeaways for researchers working with Recombinant Rat CCL4?

Researchers working with Recombinant Rat CCL4 should recognize several key aspects of this chemokine to maximize the value and reliability of their experimental work. First, rat CCL4 functions primarily as a chemoattractant for monocytes and basophils, but not for eosinophils or neutrophils, making it particularly relevant for studying specific subsets of immune cell recruitment in inflammation models . Second, the protein's structural features, including its 14 kDa monomeric form and the critical importance of its first five N-terminal amino acids for biological activity, should inform experimental design and interpretation . Third, researchers should be aware of the cross-species variations in CCL4 sequence and structure, including the 49 amino acid C-terminal extension present in rat and mouse CCL4 compared to human CCL4, which may impact translational aspects of their research . Fourth, the CCL4-CCR5 signaling axis exhibits context-dependent functions, such as its dual role in bone metabolism—promoting preosteoclast recruitment initially while its receptor CCR5 is downregulated during RANKL-induced osteoclastogenesis to facilitate differentiation . Fifth, technical considerations for working with recombinant CCL4 include proper reconstitution and storage procedures, validation of biological activity through chemotaxis assays, and the inclusion of appropriate controls to distinguish CCL4-specific effects from non-specific responses . Finally, researchers should leverage the accumulating knowledge about CCL4's roles in various physiological and pathological processes, from bone homeostasis to inflammatory diseases, to design experiments that address meaningful biological questions while being aware of potential confounding factors such as endotoxin contamination or receptor desensitization at high concentrations.

What are promising future directions for research using Recombinant Rat CCL4?

Future research using Recombinant Rat CCL4 holds significant promise across several scientific domains, with opportunities to advance both basic understanding and therapeutic applications. One promising direction involves deeper investigation of the molecular mechanisms governing CCL4-mediated immune cell recruitment and activation in tissue-specific microenvironments, particularly using advanced imaging techniques to visualize chemokine gradients and cell migration in real-time. The finding that CCL4 enhances preosteoclast recruitment to bone while its receptor CCR5 is dynamically regulated during osteoclastogenesis opens avenues for exploring targeted interventions in inflammatory bone disorders such as rheumatoid arthritis and periodontitis. Additionally, the observation that CCL4 deficiency promotes autoantibody development, possibly due to compromised regulatory T cell recruitment , suggests that modulating the CCL4-CCR5 axis could represent a novel approach for treating autoimmune conditions. From a methodological perspective, integrating recombinant CCL4 studies with emerging technologies like single-cell RNA sequencing, spatial transcriptomics, and CRISPR-Cas9 gene editing will enable unprecedented resolution in mapping chemokine response networks and identifying novel signaling components. Furthermore, the development of modified CCL4 variants with enhanced stability, receptor specificity, or signaling properties could yield valuable research tools and potential therapeutic agents. Translational research efforts might focus on establishing the relevance of findings from rat models to human systems, given the 80% sequence homology between rat and human CCL4 , potentially leading to new diagnostic biomarkers or therapeutic strategies targeting chemokine networks in human disease.