S100A8 Human

What is human S100A8 and what are its primary biological functions?

S100A8 (also known as MRP8 and calgranulin A) is a 10 kDa member of the S100 family, belonging to the EF-hand superfamily of Ca²⁺ binding proteins. It is produced predominantly by neutrophils and monocytes and plays multiple roles in inflammatory processes. S100A8 is constitutively expressed in myeloid cells as a calcium sensor, participating in cytoskeleton rearrangement and arachidonic acid metabolism . During inflammation, S100A8 is actively released and functions as a damage-associated molecular pattern (DAMP) molecule, stimulating leukocyte recruitment and inducing cytokine secretion . S100A8 has both intracellular and extracellular actions, with its extracellular functions often mediated through pattern recognition receptors, particularly toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) .

How does S100A8 interact with S100A9 to form heterodimers?

S100A8 forms calcium-dependent heterodimer/heterotetramer complexes (termed calprotectin) with S100A9, in addition to forming homodimeric complexes . The heterodimer formation is predominant, with homodimers existing in much smaller quantities due to stability issues . The calcium dependency is critical for this dimerization process, as demonstrated by studies showing that the biological effects of S100A8 and S100A9 are calcium-dependent . The interaction between S100A8 and S100A9 involves conformational changes triggered by calcium binding, which exposes hydrophobic surfaces necessary for protein-protein interactions. This calcium-dependent formation of dimers appears to be a prerequisite for many of their biological activities, including their effects on endothelial barrier function .

What is the distribution of S100A8 expression in human tissues and cell types?

S100A8 is predominantly expressed in myeloid cells, with neutrophils and monocytes being the primary sources . Different monocyte subpopulations express varying levels of S100A8; the human CD14⁺CD16⁻ monocyte population expresses more S100A8 than the CD14⁺CD16⁺ population . This pattern is similar to the elevated expression of S100A8 in mouse Ly-6C⁺ monocytes compared to Ly-6C^lo monocytes . Within human atherosclerotic lesions, S100A9 immunoreactivity (which often correlates with S100A8) is associated with macrophages, microvessels, and calcified areas . The percentage of S100A8/A9-positive macrophages is higher in rupture-prone atherosclerotic lesions compared to stable ones . In inflammatory conditions, increased expression and infiltration of S100A8/A9-positive neutrophils are observed in affected tissues .

How does calcium binding affect the structure and function of S100A8?

S100A8 contains EF-hand calcium-binding motifs that undergo significant conformational changes upon calcium binding . These changes are essential for S100A8's biological activities and its ability to form homo- and heterodimers. The calcium dependency is particularly crucial for S100A8's effects on endothelial barrier function; studies have shown that S100A8's ability to induce F-actin and ZO-1 disorganization in HUVECs and increase monolayer permeability depends on the presence of calcium .

Experiments investigating the calcium dependency of S100A8-evoked endothelial responses have demonstrated that calcium binding to S100A8 may be the prerequisite for its functional alterations of endothelial cells . This calcium dependency likely relates to the conformational changes that expose hydrophobic surfaces necessary for protein-protein interactions and receptor binding. The specific calcium-binding affinity of S100A8 and the precise nature of the conformational changes induced are important factors determining its activity under different physiological and pathological conditions.

What roles do S100A8 and S100A8/A9 play in cardiovascular pathology?

S100A8 and S100A8/A9 have emerged as important factors in cardiovascular pathology, functioning as damage-associated molecular pattern (DAMP) molecules . Plasma levels of S100A8/A9 predict cardiovascular events in humans, with each increasing quartile associated with higher risk of recurrent cardiovascular events in clinical trials . Deletion of these proteins has been shown to protect Apoe⁻/⁻ mice from atherosclerosis, indicating their causal role in disease pathogenesis .

At the cellular level, S100A8, S100A9, and S100A8/A9 can induce F-actin and ZO-1 disorganization in human umbilical venous endothelial cells (HUVECs) and increase endothelial monolayer permeability in a dose- and time-dependent manner . These effects depend on the activation of p38 and ERK1/2 signal pathways through TLR4 and RAGE receptors . In atherosclerotic plaques, the percentage of S100A8/A9-positive macrophages is higher in rupture-prone lesions compared to stable ones, and increased serum levels and expression of S100A8/A9 have been observed in infiltrated neutrophils in plaques of patients with unstable angina .

What are the optimal methods for detecting and quantifying S100A8/A9 in human samples?

For quantitative measurement of S100A8/A9 heterodimer in human samples, enzyme-linked immunosorbent assay (ELISA) is the most widely used and validated method. Commercial ELISA kits are available for detecting human S100A8/S100A9 heterodimer in various sample types, including cell culture supernatants, tissue lysates, serum, plasma, saliva, urine, and human milk . Researchers should be aware of the significant variations in normal concentration ranges across different sample types:

For tissue analysis, immunohistochemistry using specific antibodies against S100A8, S100A9, or the heterodimer can localize expression in different cell types. Western blotting can be used for semi-quantitative analysis and to distinguish between monomeric, homodimeric, and heterodimeric forms . For cell culture studies, flow cytometry can detect intracellular S100A8/A9 in specific cell populations.

How should experiments be designed to study S100A8 in inflammatory disease models?

When designing experiments to study S100A8 in inflammatory disease models, several key considerations should be addressed:

• Selection of appropriate cell models: THP-1 human acute monocytic leukemia cells can be used to study S100A8/A9 secretion, with PMA stimulation increasing production from 11.6 ng/mL to 38.0 ng/mL . Human umbilical venous endothelial cells (HUVECs) are valuable for studying endothelial responses to S100A8/A9, particularly effects on barrier function .

• Physiologically relevant concentrations: Based on the sample values provided, researchers should use concentrations of S100A8/A9 that reflect levels observed in pathological conditions. Serum levels can reach 6540 ng/mL in some individuals, while levels in other biological fluids vary widely .

• Distinguishing between protein forms: Experiments should clearly differentiate between effects of S100A8 homodimers, S100A9 homodimers, and S100A8/A9 heterodimers, as they may have distinct functions .

• Calcium dependency: Since S100A8 functions are calcium-dependent, calcium concentrations should be carefully controlled and reported in experimental protocols .

• Receptor involvement: The roles of TLR4 and RAGE should be assessed using specific inhibitors or blocking antibodies to determine receptor-mediated effects, considering the preferential binding of S100A8 to TLR4 and S100A9 to RAGE .

What approaches can differentiate between intracellular and extracellular functions of S100A8?

Differentiating between intracellular and extracellular functions of S100A8 requires strategic experimental approaches:

• For intracellular functions, genetic manipulation through siRNA knockdown, CRISPR/Cas9 knockout, or overexpression systems can be used to modulate intracellular S100A8 levels without directly affecting extracellular S100A8.

• For extracellular functions, recombinant S100A8 protein can be added to cell cultures, and receptor-mediated effects can be studied using receptor antagonists or blocking antibodies against TLR4 and RAGE .

• To specifically block extracellular S100A8 without affecting intracellular protein, neutralizing antibodies can be added to the culture medium.

• Subcellular fractionation followed by western blotting can determine the distribution of S100A8 between cytoplasmic, membrane, and nuclear compartments.

• Cell-impermeable crosslinking agents can be used to identify extracellular binding partners of S100A8 at the cell surface.

• Calcium chelators can be employed to distinguish calcium-dependent functions, which are particularly important for extracellular activities and dimerization .

How reliable is S100A8/A9 as a biomarker for inflammatory conditions?

S100A8/A9 has demonstrated significant potential as a biomarker for various inflammatory conditions. In cardiovascular disease, plasma levels of S100A8/A9 predict the risk of future nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death among apparently healthy individuals . S100A8/A9 has been identified as an early marker for detection of acute coronary syndromes, and the risk of recurrent cardiovascular events increases with each increasing quartile of S100A8/A9 levels .

In community-acquired pneumonia (CAP), serum S100A8/A9 levels in patients were found to be 1.5 times higher than in controls, suggesting its value as a diagnostic biomarker . The reliability of S100A8/A9 as a biomarker is supported by its well-characterized role in inflammatory processes and its significant elevation in various pathological states.

What are the challenges in translating S100A8 research to clinical applications?

Several challenges exist in translating S100A8 research to clinical applications:

• Functional duality: S100A8 and S100A8/A9 exhibit both pro-inflammatory and anti-inflammatory properties depending on context, making therapeutic targeting complex .

• Sample type variability: The significant differences in S100A8/A9 concentrations across sample types (serum, plasma, tissue, etc.) complicate the establishment of standardized clinical cutoff values .

• Heterodimer vs. homodimer functions: The distinct functions of S100A8 homodimers versus S100A8/A9 heterodimers need to be considered when developing targeted interventions .

• Receptor complexity: The interactions of S100A8 and S100A8/A9 with multiple receptors (TLR4, RAGE) with different affinities and downstream effects present challenges for receptor-targeted therapeutics .

• Disease-specific relevance: While S100A8/A9 is elevated in many inflammatory conditions, determining its specific pathogenic role in each disease is necessary for effective therapeutic targeting.

These challenges necessitate careful experimental design and interpretation when developing S100A8-targeted diagnostics or therapeutics for clinical use. The blockade of S100A8/A9 activity using small-molecule inhibitors or antibodies has shown promise in improving pathological conditions in murine models, suggesting potential as a therapeutic target despite these challenges .

How do fecal calprotectin (S100A8/A9) levels compare with other inflammatory markers in intestinal diseases?

Fecal calprotectin (S100A8/A9) has become an important biomarker for intestinal inflammation. Fecal extracts can contain highly variable concentrations of S100A8/A9, ranging from 8.71 ng/mL to 1175 ng/mL in samples analyzed using standardized extraction methods . In tissue analyses, normal human colon lysates show approximately 14,700 ng/mL of S100A8/A9, while cancerous colon tissue shows slightly lower levels at 11,215 ng/mL .

S100A8/A9 offers several advantages over traditional inflammatory markers like C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in intestinal diseases:

• Specificity for intestinal inflammation: Fecal calprotectin directly reflects intestinal neutrophil migration and activation, providing more specific information about intestinal inflammation than systemic markers.

• Correlation with disease activity: S100A8/A9 levels in fecal samples correlate well with endoscopic and histological measures of intestinal inflammation.

• Non-invasive assessment: Fecal calprotectin provides information about intestinal inflammation without requiring invasive procedures like endoscopy or biopsy.

• Stability in samples: S100A8/A9 is relatively stable in fecal samples, allowing for convenient collection and processing.

What are common pitfalls in S100A8/A9 research and how can they be avoided?

Researchers studying S100A8/A9 should be aware of several common pitfalls:

• Protein form confusion: Failing to distinguish between effects of S100A8 homodimers, S100A9 homodimers, and S100A8/A9 heterodimers. Solution: Clearly specify which form is being studied and use appropriate detection methods that can discriminate between these forms .

• Calcium concentration variability: Not controlling for calcium concentrations, which significantly affect S100A8/A9 structure and function. Solution: Standardize calcium concentrations in experimental buffers and report them in methods sections .

• Recombinant protein quality issues: Using improperly folded or contaminated recombinant proteins. Solution: Verify protein quality through functional assays before use and consider using carrier-free preparations for certain applications to avoid interference from carrier proteins .

• Sample collection variability: Inconsistent sample collection and processing affecting S100A8/A9 measurements. Solution: Standardize collection protocols, particularly for blood samples where release from neutrophils during clotting can affect levels .

• Receptor specificity oversight: Ignoring the receptor preferences of S100A8 for TLR4 and S100A9 for RAGE. Solution: Consider both receptors when studying either protein and use specific inhibitors or blocking antibodies to distinguish receptor-mediated effects .

How should researchers interpret contradictory findings regarding S100A8 function?

When encountering contradictory findings regarding S100A8 function, researchers should consider several factors:

• Experimental context: S100A8 functions are highly context-dependent, with the protein exhibiting both pro-inflammatory and regulatory roles depending on the microenvironment .

• Protein form: Determine whether studies examined S100A8 alone, S100A9 alone, or the S100A8/A9 heterodimer, as these have distinct functional properties .

• Concentration effects: S100A8/A9 can exhibit dose-dependent effects, with different concentrations potentially leading to different biological outcomes .

• Receptor expression: The cellular response to S100A8/A9 depends on receptor expression patterns (TLR4, RAGE), which may vary across cell types and activation states .

• Post-translational modifications: Modifications can alter S100A8 function significantly, and should be characterized when possible.

• Temporal factors: Acute versus chronic exposure to S100A8/A9 may yield different outcomes, reflecting adaptation mechanisms.

To reconcile contradictory findings, researchers should thoroughly describe their experimental conditions, specify the form of S100A8 used, report concentrations, characterize the receptor expression in their model systems, and consider the temporal aspects of their observations.

What are key considerations when comparing S100A8 data across different disease models?

When comparing S100A8 data across different disease models, researchers should consider:

• Baseline levels: Different diseases may have different baseline levels of S100A8/A9. The fold change relative to appropriate controls may be more informative than absolute values when comparing across diseases .

• Cellular source: The cellular source of S100A8/A9 may vary across diseases. For example, neutrophils versus monocytes/macrophages may contribute differently in different pathologies .

• Sample type differences: Compare like with like—serum levels in one disease should not be directly compared to tissue levels in another. Normal ranges vary significantly between sample types (e.g., serum: 481-6540 ng/mL vs. EDTA plasma: 127-1395 ng/mL) .

• Heterodimer vs. individual proteins: Some disease models may alter the ratio of S100A8/A9 heterodimer to individual proteins. Measure both the heterodimer and individual proteins when possible .

• Disease stage: S100A8/A9 levels may vary with disease progression. Compare data from similar disease stages or time points .

• Analytical methods: Different detection methods may yield different absolute values. Standardize measurement techniques or focus on relative changes rather than absolute values when comparing across studies using different methodologies .