Recombinant Rhesus Macaque Interleukin-8 protein (CXCL8) (Active)

What is the structural composition of rhesus macaque IL-8?

Rhesus macaque IL-8 is a chemoattractant cytokine that functions primarily in neutrophil chemotaxis and activation . The gene encoding rhesus macaque IL-8 directs the translation of a 101 amino acid precursor protein . This protein shows remarkable conservation with human IL-8, demonstrating 94% sequence identity at the amino acid level . The high degree of homology explains the cross-reactivity observed between human and rhesus systems. The protein can be recombinantly expressed in bacterial systems such as Escherichia coli and purified to homogeneity using ion-exchange chromatography techniques . This purification approach yields biologically active protein that retains full functionality in both in vitro and in vivo experimental paradigms.

How does rhesus macaque IL-8 compare functionally to human IL-8?

Functionally, rhesus macaque IL-8 demonstrates strong similarity to human IL-8, although with some quantifiable differences in receptor binding affinity . Purified rhesus IL-8 binds specifically to rhesus IL-8 receptors with high affinity (Kd = 0.5 nM), while it binds to human IL-8 receptors with slightly lower affinity (Kd = 2 nM) . In chemotaxis assays, the protein effectively promotes migration of both rhesus neutrophils (EC50 = 2 nM) and human neutrophils (EC50 = 4 nM) . Importantly, monoclonal antibodies developed against human IL-8, such as DM/C7, cross-react with and neutralize rhesus IL-8 activity in vitro with an IC50 of 0.5-3.0 μg/ml . These properties facilitate translational research approaches where reagents and methodologies developed for human systems can often be directly applied to rhesus models with minimal modification.

What experimental models are suitable for studying rhesus macaque IL-8?

Several experimental models have been well-established for studying rhesus macaque IL-8. In vivo models using rhesus monkeys (Macaca mulatta) aged 2-4 years with body weights between 2.5-4.5 kg have been validated for IL-8 administration studies . These animals must be free of intestinal parasites, herpes B, simian T cell leukemia virus (STLV), and simian immunodeficiency virus (SIV) to ensure reliable results . Dermal inflammation models induced by phorbol myristoyl acetate application to rhesus ears have been used to study IL-8's inflammatory role . Additionally, in vitro models using purified rhesus neutrophils for chemotaxis assays and receptor binding studies provide complementary approaches for investigating IL-8 function at the cellular level . These experimental platforms collectively enable comprehensive investigation of IL-8 biology from molecular interactions to systemic effects.

What is the mechanism of IL-8-induced hematopoietic progenitor cell mobilization?

IL-8 induces rapid mobilization of hematopoietic progenitor cells (HPCs) through a mechanism that involves matrix metalloproteinase-9 (MMP-9/gelatinase B) . When administered intravenously to rhesus monkeys, IL-8 causes an immediate increase in plasma levels of MMP-9, followed closely by increases in circulating HPCs . This temporal relationship suggests a mechanistic link confirmed through inhibition studies. The proposed pathway involves IL-8 activation of neutrophils, which subsequently release MMP-9 . MMP-9 then degrades extracellular matrix components in the bone marrow to which stem cells are attached, facilitating their release into circulation . This mechanism is further supported by evidence that pretreatment with inhibitory anti-gelatinase B antibodies completely prevents IL-8-induced HPC mobilization without affecting MMP-9 production and secretion . The validation of this pathway provides important insights for designing stem cell mobilization strategies for clinical applications.

What dosing strategies are optimal when using recombinant IL-8 in primate models?

Optimal dosing of recombinant IL-8 in rhesus monkey models has been established through dose-response studies. For hematopoietic progenitor cell mobilization, a single intravenous bolus injection of 100 μg/kg body weight delivered over 30 seconds produces optimal results . This dosing regimen results in peak plasma IL-8 levels of approximately 5 μg/mL . Lower doses of 10 and 30 μg/kg have also been studied but show reduced efficacy . The calculated half-life of free IL-8 in circulation is approximately 9.9 ± 2.2 minutes, necessitating proper timing of sample collection in experimental protocols . For inflammation studies, similar dosing has been effective, though the precise regimen may vary depending on the specific experimental endpoint. When designing experiments requiring repeated IL-8 administration, intervals of 72 hours between doses prevent tachyphylaxis and maintain consistent mobilization responses .

HPC Mobilization Assessment

IL-8 activity can be effectively measured through quantification of hematopoietic progenitor cell mobilization. Following IL-8 administration, venous blood samples should be collected at strategic timepoints (1, 5, 15, 30, 45, 60, and 120 minutes post-injection) . Colony-forming unit-Mix (CFU-Mix) assays provide a functional readout of HPC numbers, typically showing increases from baseline levels of approximately 45 ± 48/mL to peak levels of 1,382 ± 599/mL at 30 minutes post-administration of 100 μg/kg IL-8 . Individual animals may show 10- to 100-fold increases in circulating HPCs, returning to near-baseline values (92 ± 52 CFU/mL) within 240 minutes .

MMP-9 Assessment

Zymographic analysis of plasma samples provides a complementary measure of IL-8 activity through quantification of MMP-9 release. Peak MMP-9 levels occur at 15-45 minutes post-IL-8 injection, reaching 5.5-6.5 μg/mL . Standard curves constructed using serial dilutions of purified human neutrophil-derived MMP-9 allow for quantitative comparison, assuming equivalent specific activities between monkey and human MMP-9 .

Neutrophil Response Profiling

Differential blood cell counts provide additional metrics of IL-8 activity. IL-8 administration initially causes neutropenia due to pulmonary sequestration (observable using 99mTc-labeled leukocytes), followed by neutrophilia with counts increasing up to 10-fold over baseline within 30 minutes . These parameters collectively form a comprehensive profile of IL-8 bioactivity in vivo.

What role does MMP-9 play in the IL-8 signaling pathway?

MMP-9 (gelatinase B) functions as a critical mediator in the IL-8 signaling pathway, particularly in the context of hematopoietic progenitor cell mobilization . Experimental evidence demonstrates that IL-8 stimulation induces rapid systemic release of MMP-9 from activated neutrophils . Zymographic analysis reveals that plasma MMP-9 levels increase dramatically and instantaneously following IL-8 administration, preceding the increase in circulating HPCs . The enzyme levels peak at 15-45 minutes post-injection and decrease by 2 hours, mirroring the kinetics of HPC mobilization . The causal relationship between MMP-9 and HPC mobilization has been conclusively demonstrated through antibody neutralization studies. Administration of a highly specific inhibitory monoclonal anti-gelatinase B antibody at doses of 1-2 mg/kg completely prevents IL-8-induced mobilization of HPCs, while lower doses (0.1 mg/kg) show only limited effect . Importantly, these antibodies specifically block MMP-9 activity without affecting its production and secretion in response to IL-8 . This mechanistic insight provides a molecular target for modulating stem cell mobilization in research and potential therapeutic applications.

Data: IL-8 Re-administration Response Patterns

How can IL-8 function be neutralized in experimental settings?

Neutralization of IL-8 function can be achieved through several validated approaches. Monoclonal antibodies represent the most specific method, with DM/C7 (a mouse monoclonal antibody against human IL-8) demonstrating effective neutralization of rhesus IL-8 activity both in vitro and in vivo . This antibody neutralizes IL-8 with an IC50 of 0.5-3.0 μg/ml in vitro . In vivo, systemic administration of DM/C7 completely inhibits dermal inflammation in rhesus ears induced by phorbol myristoyl acetate application . For studies focusing on downstream mediators of IL-8 signaling, targeted inhibition of specific pathway components provides an alternative approach. For instance, inhibitory monoclonal antibodies against gelatinase B (MMP-9) at doses of 1-2 mg/kg effectively block IL-8-induced HPC mobilization without affecting IL-8 receptor binding or neutrophil activation . These complementary approaches allow researchers to dissect the IL-8 signaling cascade at multiple levels, facilitating mechanistic studies of its diverse biological effects.

What are the critical parameters for IL-8 stability and activity in experimental protocols?

Maintaining IL-8 stability and activity requires careful attention to several experimental parameters. For in vivo administration, recombinant IL-8 should be diluted in endotoxin-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) to prevent protein adsorption to surfaces . The endotoxin concentration must be verified to be less than 0.05 unit/mg using the Limulus amebocyte lysate assay to avoid confounding immune responses . The short half-life of IL-8 in circulation (9.9 ± 2.2 minutes) necessitates precise timing in experimental protocols . For in vitro applications, storage conditions significantly impact protein stability. Purified IL-8 should be stored at -80°C in single-use aliquots to avoid freeze-thaw cycles that can compromise activity. Activity assessments using neutrophil chemotaxis assays should be performed periodically to confirm that the protein retains full functionality. Additionally, receptor binding assays using both rhesus and human IL-8 receptors provide quantitative measures of bioactivity, with expected Kd values of 0.5 nM and 2 nM, respectively .

What methodological advances have improved studies of IL-8 in primate models?

Recent technological advances have significantly enhanced the study of IL-8 in primate models. The development of organotypic models using neutrophils derived from human induced pluripotent stem cells allows for genetic modification to study neutrophil motility signaling and interactions with endothelium . These systems provide controlled environments for investigating IL-8 function without the complexities of whole-animal studies. Advanced omics approaches now enable comprehensive analysis of how IL-8 and its receptors are altered in various disease states . In experimental protocols, the refinement of zymographic techniques for quantifying MMP-9 release has facilitated the elucidation of key mechanistic relationships in IL-8 signaling . Additionally, improved fluorescence-activated cell sorting (FACS) analysis methods permit detailed immunophenotyping of circulating cell populations following IL-8 administration . These methodological advances collectively enhance our ability to dissect the complex biology of IL-8 and translate findings to human applications. The integration of these techniques with traditional approaches provides a more comprehensive understanding of IL-8 function across molecular, cellular, and systemic levels.