IL 8 Rhesus Macaque

What is IL-8 in rhesus macaques and how does it compare to human IL-8?

Rhesus macaque IL-8 (also known as CXCL8) is a chemokine that functions primarily as a neutrophil chemoattractant and activator. It is structurally and functionally similar to human IL-8, with 94% amino acid sequence identity. The open reading frame directs the translation of a 101 amino acid precursor protein . When purified, mature rhesus IL-8 has a molecular weight of approximately 9.14 kDa (79 amino acids) .

The high degree of homology between rhesus and human IL-8 makes the rhesus macaque an excellent model for studying inflammatory processes relevant to human disease. The gene encoding rhesus IL-8 is located on chromosome 5, similar to its position in humans .

What are the primary functions of IL-8 in rhesus macaque models?

IL-8 in rhesus macaques serves several key functions that have been experimentally verified:

• Neutrophil chemotaxis: Rhesus IL-8 effectively promotes in vitro chemotaxis of both rhesus neutrophils (EC50 = 2 nM) and human neutrophils (EC50 = 4 nM) .

• Receptor binding: Purified rhesus IL-8 binds specifically to both rhesus (Kd = 0.5 nM) and human (Kd = 2 nM) IL-8 receptors .

• Hematopoietic progenitor cell mobilization: IL-8 induces rapid mobilization of hematopoietic progenitor cells (HPC) from the bone marrow into peripheral circulation, with increases observed within 30 minutes of IL-8 administration .

• Inflammation mediation: IL-8 plays a prominent role in inflammation in primates, as demonstrated by studies showing that neutralizing IL-8 can inhibit dermal inflammation induced by phorbol myristoyl acetate .

These functions make IL-8 a valuable target for studying inflammatory processes and developing anti-inflammatory therapeutics using rhesus macaque models.

What methods are used to measure IL-8 levels in rhesus macaque samples?

When measuring IL-8 in rhesus macaque samples, researchers typically employ several validated techniques:

• Enzyme-linked immunosorbent assays (ELISAs): Commercial or custom ELISAs specific for rhesus IL-8 are commonly used to measure cytokine levels in plasma samples. Studies have reported baseline plasma IL-8 levels in middle-aged monkeys at approximately 242 ± 179 pg/mL .

• Functional assays: Neutrophil chemotaxis assays can be used to indirectly measure IL-8 activity in biological samples. The EC50 values for rhesus neutrophils (2 nM) and human neutrophils (4 nM) provide a reference for such functional readouts .

• Receptor binding assays: Competitive binding assays using purified rhesus IL-8 can measure IL-8 receptor interactions, with documented binding affinities (Kd) of 0.5 nM for rhesus receptors and 2 nM for human receptors .

• Longitudinal sampling: To account for individual variation, multiple sampling over time is recommended, as inter-individual differences in IL-8 levels have been shown to be consistent over successive years, suggesting that IL-8 levels represent a stable, trait-like characteristic .

When designing studies, researchers should consider that baseline IL-8 levels differ significantly between age groups, with middle-aged monkeys showing lower levels compared to older animals .

How can researchers effectively neutralize IL-8 activity in rhesus macaque models?

Neutralizing IL-8 activity in rhesus macaque models can be achieved through several approaches:

• Monoclonal antibodies: The mouse monoclonal antibody DM/C7, which neutralizes human IL-8 activity, has been shown to also recognize and neutralize rhesus IL-8 in vitro with an IC50 of 0.5-3.0 μg/ml. Systemic administration of DM/C7 has been demonstrated to completely inhibit dermal inflammation in rhesus macaques induced by external application of phorbol myristoyl acetate . This cross-reactivity provides a valuable tool for IL-8 neutralization in rhesus models.

• Indirect inhibition approaches: Rather than targeting IL-8 directly, researchers can inhibit downstream mediators. For example, the administration of a highly specific inhibitory monoclonal anti-gelatinase B antibody (1-2 mg/kg) completely prevented IL-8-induced mobilization of hematopoietic progenitor cells, while a lower dose (0.1 mg/kg) had only limited effect .

• Dose considerations: When designing neutralization experiments, it is critical to establish appropriate dosing. The dose-dependent effects observed with anti-gelatinase B antibody highlight the importance of dose-response studies when developing inhibition protocols .

For optimal results, researchers should validate antibody cross-reactivity and establish appropriate dosing regimens specific to their experimental endpoints.

What is the relationship between IL-8 and MMP-9 in rhesus macaque models?

The relationship between IL-8 and matrix metalloproteinase-9 (MMP-9, also known as gelatinase B) in rhesus macaques represents a critical mechanism in stem cell mobilization and inflammation:

• Temporal relationship: Intravenous administration of IL-8 (0.1 mg/kg) in rhesus monkeys results in a dramatic instantaneous increase in plasma levels of MMP-9, followed by a 10- to 100-fold increase in circulating hematopoietic progenitor cells (HPC) within 30 minutes. Both MMP-9 and HPC levels decrease approximately 2 hours after IL-8 injection .

• Mechanistic dependency: Experimental evidence strongly suggests that MMP-9 functions as a mediator of IL-8-induced HPC mobilization. This has been demonstrated through inhibition studies where pretreatment with a specific inhibitory monoclonal anti-gelatinase B antibody (1-2 mg/kg) completely prevented IL-8-induced HPC mobilization .

• Proposed mechanism: IL-8 activates neutrophils, which release MMP-9. The released MMP-9 then degrades extracellular matrix molecules in the bone marrow, cleaving matrix components to which stem cells are attached and thereby facilitating their mobilization into circulation .

This mechanistic relationship provides researchers with multiple intervention points when studying stem cell mobilization and inflammatory processes in rhesus macaque models.

How does aging affect IL-8 levels and what are the neurological implications?

Aging has significant effects on IL-8 levels in rhesus macaques, with important neurological correlates:

• Age-related changes in IL-8 levels: Middle-aged monkeys have significantly lower IL-8 levels (242 ± 179 pg/mL) compared to older animals, indicating an age-associated increase in this proinflammatory cytokine .

• Neuroanatomical correlations: Higher circulating IL-8 levels predict smaller gray matter volumes in bilateral hippocampus, suggesting a relationship between peripheral inflammatory markers and brain structure .

• Dual effects of IL-8 on neural tissue: IL-8 can exert both neuroprotective/neurotrophic effects in certain culture conditions and neurotoxic actions through secondary mediators like amyloid-beta (Aβ). The neurotoxic effects are mediated by microglial activation and involve the release of reactive oxygen species (ROS) and neutrophil-mediated production of proteases .

• Proxy for neuroinflammation: Peripheral levels of IL-8 may indirectly gauge the degree of microglial activity in the brain, providing a potentially accessible biomarker for central inflammatory processes .

• Intervention potential: Calorie restriction (CR) significantly lowers IL-8 levels in aged rhesus macaques, suggesting a potential intervention strategy to mitigate age-related inflammatory processes .

These findings have important implications for researchers studying neuroinflammation, neurodegeneration, and brain aging in rhesus macaque models.

How can researchers control for individual variation in IL-8 levels in experimental designs?

Controlling for individual variation in IL-8 levels is critical for robust experimental design in rhesus macaque studies:

• Longitudinal sampling: Inter-individual differences in IL-8 are consistent over successive years, suggesting they represent stable, trait-like characteristics . Researchers should collect baseline measurements from individual animals over multiple time points before experimental manipulation to establish reliable baselines.

• Consideration of covariates: Several factors influence IL-8 levels and should be controlled for:

  • Age: Significant differences exist between middle-aged and old monkeys

  • Reproductive state: Hormonal status affects inflammatory markers

  • HPA axis activity: Concentrations of fecal glucocorticoid hormones correlate with plasma IL-8 levels

  • Body condition: May influence inflammatory status

  • Social rank: Can affect stress levels and immune function

• Screening for chronic elevation: Some individuals show permanently elevated cytokine levels or HPA axis activity, suggesting chronic stress or disease . These individuals may respond differently to experimental manipulations and should be identified during screening.

• Statistical approaches: Using each animal as its own control in pre/post designs or employing mixed-effects models that account for repeated measures and individual random effects can help control for baseline individual variation.

• Group balancing: When assigning animals to experimental groups, stratify based on baseline IL-8 levels to ensure comparable distributions across groups.

By implementing these strategies, researchers can enhance the sensitivity of their experiments to detect treatment effects against the background of individual variation.

What experimental designs are optimal for studying IL-8's role in neuroinflammation?

When designing studies to investigate IL-8's role in neuroinflammation using rhesus macaque models, several approaches have proven effective:

• Combined peripheral biomarker and neuroimaging approach: Collecting peripheral blood samples for IL-8 quantification in conjunction with MRI and diffusion tensor imaging (DTI) has successfully demonstrated associations between systemic IL-8 levels and both brain volume and microstructure . This non-invasive approach is particularly valuable for longitudinal studies where animals continue to be evaluated until natural mortality.

• Intervention designs with anti-inflammatory strategies: Calorie restriction (CR) has been shown to significantly lower IL-8 levels in rhesus macaques . Comparing CR animals to normally fed controls provides a model for studying how modulation of inflammatory markers affects neurological outcomes.

• Amyloid-beta challenge model: Injecting fibrillar Aβ into the brains of geriatric female rhesus monkeys produces lesion volumes and reactive oxygen species. This model can be enhanced by co-injecting inhibitory factors to investigate protection against IL-8-associated neuroinflammation .

• Consideration of bidirectional relationships: When interpreting results, researchers should consider the bidirectional relationship between systemic and central cytokine release. Peripheral levels of IL-8 may indirectly reflect the degree of microglial activity in the brain .

• Age-stratified designs: Due to significant differences in IL-8 levels between middle-aged and older monkeys, studies should either focus on specific age ranges or explicitly incorporate age as a variable in the analysis .

These design considerations will enhance the translational value of rhesus macaque studies investigating IL-8's role in neuroinflammation.

How does calorie restriction affect IL-8 expression and what are the implications for inflammatory research?

Calorie restriction (CR) has significant effects on IL-8 expression in rhesus macaques, with important implications for inflammatory research:

• Reduced IL-8 levels: Long-term 30% calorie restriction significantly lowers circulating levels of IL-8 in aged rhesus macaques compared to normally fed controls . This reduction is consistent with findings in humans showing that CR reduces IL-8 gene expression .

• Concurrent anti-inflammatory effects: CR not only reduces pro-inflammatory IL-8 but also raises levels of anti-inflammatory interleukin-10 (IL-10), suggesting a broad shift in the inflammatory profile .

• Neurological correlates: The CR-induced reduction in IL-8 is associated with preserved brain volume and microstructure, particularly in the hippocampus, where higher IL-8 predicts smaller gray matter volumes .

• Longitudinal benefits: The effects of CR on inflammatory profiles appear to be sustained over long periods, with studies examining animals after 13-19 years of calorie restriction showing persistent benefits .

• Experimental applications: The CR model provides researchers with a well-established intervention to modulate IL-8 levels experimentally. This can be particularly valuable for studies investigating:

  • Aging and neurodegeneration

  • Inflammatory disease processes

  • Metabolic influences on inflammation

  • Cognitive aging

Researchers can leverage the CR model to study mechanisms linking inflammation reduction to various health outcomes, including neurological, cardiovascular, and metabolic parameters.

What experimental considerations should be addressed when using IL-8 as a biomarker in longitudinal studies?

When employing IL-8 as a biomarker in longitudinal studies with rhesus macaques, researchers should address several key considerations:

By addressing these considerations, researchers can maximize the utility of IL-8 as a biomarker in longitudinal studies with rhesus macaques.

How do binding affinities and functional properties of IL-8 differ between rhesus macaques and humans?

Understanding the similarities and differences in IL-8 properties between rhesus macaques and humans is crucial for translational research. The comparative binding and functional characteristics include:

These comparative properties demonstrate that:

• The high degree of sequence homology (94%) between rhesus and human IL-8 translates to significant functional similarity, supporting the use of rhesus macaques as models for human inflammatory processes .

• Despite the high homology, there are quantitative differences in receptor binding and neutrophil activation, with rhesus IL-8 showing higher affinity for rhesus receptors (Kd = 0.5 nM vs. 2 nM) and greater potency in activating rhesus neutrophils (EC50 = 2 nM vs. 4 nM) .

• The cross-reactivity of neutralizing antibodies like DM/C7 facilitates translational studies, allowing the same tools to be used across species .

These comparative aspects should be considered when designing experiments and interpreting results from rhesus macaque models for potential human applications.

What are the key methodological considerations when translating IL-8 research from rhesus macaques to human applications?

When translating IL-8 research findings from rhesus macaque models to human applications, researchers should address several methodological considerations:

By addressing these methodological considerations, researchers can enhance the translational value of IL-8 research conducted in rhesus macaque models.

What are the current challenges in IL-8 detection and quantification in rhesus macaque samples?

Researchers face several challenges when detecting and quantifying IL-8 in rhesus macaque samples:

• Antibody specificity: While rhesus and human IL-8 share 94% sequence homology , not all anti-human IL-8 antibodies cross-react equally with rhesus IL-8. Researchers must validate antibody specificity for rhesus IL-8 before employing them in immunoassays.

• Individual variation: Significant inter-individual differences in IL-8 levels exist among rhesus macaques , necessitating larger sample sizes or repeated sampling to establish reliable baselines and detect treatment effects.

• Contextual factors: IL-8 levels are influenced by numerous factors including age, reproductive state, stress levels, and social rank . Controlling or accounting for these variables requires comprehensive documentation and potentially complex statistical models.

• Sample handling considerations: IL-8 stability in different sample types (plasma, serum, tissue homogenates) may vary, requiring optimization of collection, processing, and storage protocols to prevent degradation or artificial elevation due to ex vivo activation.

• Reference ranges: Limited published data on normal reference ranges for IL-8 across different age groups, sexes, and reproductive states in rhesus macaques complicates interpretation of results. Available data suggests middle-aged monkeys average 242 ± 179 pg/mL, significantly lower than older monkeys .

• Assay sensitivity: Detecting physiologically relevant changes in IL-8 levels, particularly in cerebrospinal fluid or tissue samples where concentrations may be lower, requires highly sensitive assays with appropriate detection limits.

Addressing these challenges requires rigorous method validation, careful experimental design, and consideration of contextual factors when interpreting results.

What emerging research directions are developing in IL-8 rhesus macaque models?

Several promising research directions are emerging in the field of IL-8 research using rhesus macaque models:

• Neuroinflammation and cognitive aging: The association between peripheral IL-8 levels and reduced hippocampal volume suggests a role for IL-8 in age-related cognitive decline . Future research is likely to explore:

  • Mechanistic pathways linking peripheral IL-8 to central inflammatory processes

  • Interventions targeting IL-8 to mitigate age-related cognitive decline

  • Combined biomarker approaches integrating IL-8 with other inflammatory markers and neuroimaging

• Calorie restriction and anti-inflammatory mechanisms: The finding that calorie restriction lowers IL-8 and raises anti-inflammatory IL-10 opens avenues for investigating:

  • Molecular mechanisms underlying CR's anti-inflammatory effects

  • Calorie restriction mimetics that may modulate IL-8 without dietary intervention

  • Optimized CR protocols for maximum anti-inflammatory benefit

• IL-8/MMP-9 axis in stem cell mobilization: The relationship between IL-8, MMP-9 release, and hematopoietic progenitor cell mobilization suggests applications in:

  • Optimizing stem cell collection protocols for transplantation

  • Developing targeted therapies for hematological disorders

  • Understanding fundamental mechanisms of stem cell niche interactions

• Individual variability as a research focus: The stability of inter-individual differences in IL-8 levels over time suggests genetic or epigenetic regulation that could be explored through:

  • Genomic and epigenomic profiling of high vs. low IL-8 producers

  • Longitudinal studies examining environmental influences on IL-8 expression

  • Personalized medicine approaches based on inflammatory profiles

• Advanced imaging correlations: Building on existing work linking IL-8 to brain structure , future studies may employ:

  • Functional MRI to explore relationships between IL-8 and brain activity

  • PET imaging with microglial activation tracers to directly link peripheral IL-8 to central inflammation

  • Multimodal imaging combined with longitudinal cytokine profiling