ACKR1 Antibody

What is ACKR1 and what are its main biological functions?

ACKR1 (Atypical Chemokine Receptor 1), previously known as the Duffy Antigen Receptor for Chemokines (DARC), is a widely conserved cell surface protein expressed primarily on erythrocytes and the endothelium of post-capillary venules. This multifunctional receptor has several key biological roles. First, it serves as the receptor for the parasite causing malaria. Second, it plays a crucial role in regulating innate immunity by displaying and trafficking chemokines. Intriguingly, a common mutation in the ACKR1 promoter leads to loss of the erythrocyte protein while leaving endothelial expression unaffected, highlighting its tissue-specific regulation mechanisms .

For experimental approaches, researchers should note that endothelial ACKR1 is rapidly down-regulated when cells are extracted and cultured from tissue, which has historically limited investigation. Recent methodological advances have shown that exposure to whole blood can induce ACKR1 expression in cultured primary human lung microvascular endothelial cells, providing new opportunities for functional studies .

What applications are suitable for ACKR1 antibodies in laboratory research?

ACKR1 antibodies have diverse applications in research settings, with specific methodologies established for detection and functional analysis. Based on validated protocols, ACKR1 antibodies can be effectively used in:

• Western Blot (at concentrations of 0.3-1 μg/mL)

• Immunohistochemistry (at 3 μg/mL)

• Flow Cytometry (at 10 μg/mL)

• Peptide ELISA (at 1:6000 dilution)

These applications allow for detection of both reported isoforms of human ACKR1 (NP_001116423.1 and NP_002027.2). When designing experiments, it's important to note that commercially available antibodies are often generated against synthetic peptides consisting of specific amino acid sequences, such as HRAELSPSTENSSQLDFED-C . This knowledge is essential for understanding potential epitope recognition and cross-reactivity patterns.

How can endogenous ACKR1 expression be induced in cultured endothelial cells?

A significant methodological challenge in ACKR1 research has been the rapid downregulation of both ACKR1 transcript and protein when endothelial cells are extracted and cultured from tissue. Recent research has established a protocol to overcome this limitation:

• Grow Human Pulmonary Microvascular Endothelial Cells (HPMECs) to confluency in 6-well plates

• Incubate with 1ml of whole blood (or complete media as control) for 24 hours

• Alternatively, incubate with isolated cellular components (monocytes, polymorphonuclear leukocytes, or erythrocytes) for 6 hours

• Wash cells with PBS after incubation

• Process for immunoblotting or qPCR as needed

This approach has revealed that contact with neutrophils specifically induces ACKR1 expression, and the process is regulated by NF-κB. Following induction and removal of blood, ACKR1 protein is rapidly secreted via extracellular vesicles, a finding with implications for experimental design and timing .

How does ACKR1 contribute to cellular signaling and what methodologies are appropriate for studying these pathways?

Despite its structural similarity to G protein-coupled receptors, ACKR1 does not initiate classical chemokine receptor signaling. Research protocols to confirm this non-signaling characteristic include calcium flux assays in ACKR1-expressing cells:

• Load cells with Fura2-AM (10 ng/μl) in HBSS for 30 minutes at 37°C

• Wash three times with HBSS and incubate for 15 minutes

• Transfer coverslips to a cell chamber and measure fluorescence using ratiometric microscopy

• Record baseline calcium concentration for 5 minutes

• Treat with IL-8 (100 ng/ml) and monitor for 10 minutes

• Compare with positive controls such as VEGF (40 ng/ml) or calcium ionophore (5μM)

Studies have confirmed that endogenous ACKR1 does not signal upon stimulation with IL-8 or CXCL1. Instead, ACKR1 functions primarily as a chemokine transporter and presenter. This methodology allows researchers to distinguish ACKR1's non-signaling properties from classical chemokine receptors when validating function in experimental models .

What is the role of anti-ACKR1 autoantibodies in post-COVID vascular dysfunction and how can they be detected?

Recent research has identified anti-ACKR1 autoantibodies as potential mediators of vascular dysfunction in COVID-19 survivors. For researchers investigating this phenomenon, two validated detection methodologies have been established:

• Microarray-based detection:

  • Design custom microarray kits with immobilized recombinant ACKR1 protein on glass slides

  • Incubate with patient plasma samples

  • Quantify binding of autoantibodies to the immobilized ACKR1

• Flow cytometry-based detection:

  • Utilize K562 cells (human erythroleukemic cell line) ectopically overexpressing ACKR1

  • Incubate cells with patient plasma

  • Measure bound autoantibodies via flow cytometry

  • This approach preserves post-translational modifications crucial for protein conformation and proper cellular localization

Research has shown significantly elevated levels of anti-ACKR1 autoantibodies in COVID-19 survivors compared to non-infected controls. These autoantibodies positively correlate with inflammatory cytokines and circulating endothelial cell counts, suggesting a mechanistic link between anti-ACKR1 autoantibodies and vascular injury .

How can researchers study the functional impact of anti-ACKR1 autoantibodies on endothelial cells?

To investigate the functional consequences of anti-ACKR1 autoantibodies on endothelial health, several experimental approaches have been validated:

• Endothelial-PBMC transwell co-culture assay:

  • Culture endothelial cells on transwell inserts

  • Add PBMCs to the upper chamber

  • Measure PBMC transmigration across the endothelial barrier

  • This assesses whether anti-ACKR1 affects leukocyte recruitment

• Antibody-dependent cellular cytotoxicity assay:

  • Treat endothelial cells with purified IgG from patient samples

  • Add patient PBMCs to assess immune cell-mediated responses

  • Measure lactate dehydrogenase released from damaged endothelial cells

  • Include ACKR1 blocking peptide or liposome ACKR1 as controls to confirm specificity

Research has demonstrated that purified IgG or patient PBMCs can lead to significantly higher levels of antibody-dependent and immune cell-mediated cytotoxicity, respectively. When both IgG and PBMCs are introduced together, they pronouncedly enhance antibody-dependent cellular cytotoxicity. Importantly, blocking peptides targeting the N-terminal extracellular domain of ACKR1 can avert this cytotoxicity, providing evidence for the specificity of anti-ACKR1 autoantibodies in mediating endothelial damage .

What are the key considerations when designing experiments to study ACKR1 in endothelial cells?

When designing experiments to study ACKR1 in endothelial cells, researchers should address several critical factors:

• Expression maintenance: Since ACKR1 is rapidly downregulated in cultured endothelial cells, experiments should incorporate whole blood or isolated neutrophils to induce and maintain expression. The timing is crucial - a 24-hour incubation with whole blood or 6-hour incubation with neutrophils is recommended based on validated protocols .

• Protein trafficking considerations: After induction and removal of blood components, ACKR1 protein is rapidly secreted via extracellular vesicles. Therefore, experimental timelines must account for this rapid trafficking when planning protein extraction or functional assays .

• Signaling controls: When studying ACKR1 signaling, appropriate positive controls (VEGF or calcium ionophore) should be included alongside experimental treatments like IL-8 or CXCL1. This helps distinguish between ACKR1's non-signaling properties and potential experimental artifacts .

• Expression verification: All experiments should include verification of ACKR1 expression through immunoblotting or qPCR, as expression levels can vary considerably between experimental conditions and cell sources .

What approaches can be used to study the role of ACKR1 in aortic dissection and macrophage regulation?

Recent single-cell transcriptomic analyses have revealed increased endothelial cells with high ACKR1 expression in type A aortic dissection (TAAD) tissues. For researchers investigating this area, several experimental approaches have proven valuable:

• Single-cell transcriptomic analysis:

  • Collect human aortic tissues from TAAD patients and controls

  • Process samples for single-cell RNA sequencing

  • Analyze cellular heterogeneity with focus on ACKR1-expressing populations

  • Identify co-expressed genes and pathways in ACKR1hi endothelial cells

• Genetic modulation approaches:

  • Perform ACKR1 knockdown experiments to suppress NF-κB signaling and SPP1 expression

  • Utilize ACKR1 overexpression models to confirm exacerbation of TAAD

  • Assess effects on macrophage migration and polarization in each condition

• Potential therapeutic targeting:

  • Conduct molecular docking studies to identify compounds that may interact with ACKR1

  • Test candidate molecules (such as amikacin) in vitro and in vivo for effects on ACKR1 function

  • Monitor TAAD progression in animal models with pharmacological ACKR1 modulation

These approaches have collectively demonstrated that endothelial cells with high ACKR1 expression contribute to TAAD progression by regulating macrophage migration and proinflammatory polarization through NF-κB signaling pathways .

How can researchers differentiate between ACKR1 isoforms and ensure antibody specificity?

Ensuring antibody specificity and isoform differentiation is crucial for reliable ACKR1 research. Researchers should implement the following approaches:

• Isoform validation: When selecting antibodies, verify recognition of both reported human ACKR1 isoforms (NP_001116423.1 and NP_002027.2). Commercial antibodies typically provide this information, but validation in your experimental system is recommended .

• Epitope mapping: Understanding the epitope recognized by your antibody (such as the HRAELSPSTENSSQLDFED-C sequence) is essential for interpreting results, especially when studying mutant forms or truncated variants of ACKR1 .

• Specificity controls:

  • Include ACKR1 knockout or knockdown controls

  • For autoantibody studies, use blocking peptides targeting specific domains (particularly the N-terminal extracellular domain) to confirm binding specificity

  • When studying anti-ACKR1 autoantibodies, validate findings with multiple detection methods (such as both microarray and flow cytometry-based approaches)

• Cross-reactivity assessment: If working across species, note that ACKR1 shows considerable sequence divergence, especially in the N-terminal extracellular domain. The highest conservation is observed in other regions, which may affect antibody performance in cross-species applications .

How should researchers interpret conflicting data regarding ACKR1 expression patterns and regulation?

When encountering conflicting data on ACKR1 expression and regulation, researchers should consider several key factors:

• Tissue-specific expression patterns: ACKR1 expression differs between erythrocytes and endothelial cells, with distinct regulatory mechanisms. A common mutation affects expression only on erythrocytes while preserving endothelial expression, suggesting independent regulatory pathways .

• Rapid downregulation in culture: The well-documented rapid downregulation of ACKR1 in cultured endothelial cells may account for inconsistent findings. Induction with whole blood or neutrophils may be necessary to observe physiologically relevant expression patterns in vitro .

• NF-κB regulation: Evidence shows that NF-κB regulates ACKR1 expression. Contradictory findings might reflect differences in NF-κB activation status across experimental conditions .

• Secretion via extracellular vesicles: After induction, ACKR1 protein is rapidly secreted via extracellular vesicles. This dynamic trafficking may lead to conflicting observations depending on measurement timing and techniques .

Robust experimental design should account for these variables by carefully controlling timing of inductions, confirming expression at both transcript and protein levels, and considering the dynamic regulation of ACKR1 in different cellular contexts.

What are the emerging links between anti-ACKR1 autoantibodies and post-viral vascular complications?

Research has identified novel connections between anti-ACKR1 autoantibodies and post-viral vascular dysfunction, particularly in COVID-19 survivors. Key findings include:

• Prevalence in COVID-19 survivors: Using both microarray and flow cytometry-based detection methods, studies have demonstrated significantly elevated levels of anti-ACKR1 autoantibodies in COVID-19 survivors compared to non-infected controls .

• Correlation with inflammatory markers: Anti-ACKR1 antibody levels positively correlate with several inflammatory cytokines, suggesting a mechanistic link between proinflammatory factors and the generation of these autoantibodies .

• Association with endothelial damage: A significant positive correlation exists between anti-ACKR1 autoantibody levels and circulating endothelial cell counts, implicating these antibodies in vascular injury processes .

• Mechanisms of damage: Experimental evidence supports multiple pathways for anti-ACKR1-mediated endothelial damage:

  • Direct antibody-dependent cytotoxicity

  • Immune cell-mediated responses

  • Enhanced antibody-dependent cellular cytotoxicity when both mechanisms act together

These findings represent the first report highlighting anti-ACKR1 autoantibodies as potential drivers of subclinical vascular dysfunction, suggesting their possible role in predisposing individuals to cardiovascular complications following viral infections. This emerging field warrants further investigation in larger cohort studies to establish clinical significance and develop potential protective strategies .

What are the most promising research avenues for therapeutic targeting of ACKR1?

Several promising research directions for therapeutic targeting of ACKR1 have emerged:

• Blocking peptides targeting ACKR1: Research has demonstrated that peptides targeting the N-terminal extracellular domain of ACKR1 can effectively counteract the effects of anti-ACKR1 autoantibodies. Further development of these peptides could lead to therapeutic strategies for preventing antibody-dependent cellular cytotoxicity in post-viral vascular complications .

• Small molecule modulators: Molecular docking studies have identified potential compounds that interact with ACKR1, such as amikacin. Further investigation of these and similar molecules may yield therapeutic options for conditions involving dysregulated ACKR1 expression or function .

• NF-κB pathway modulation: Given that NF-κB regulates ACKR1 expression, targeting this pathway may provide indirect means to modulate ACKR1 levels in pathological conditions like aortic dissection, where high ACKR1 expression contributes to disease progression .

• Diagnostic applications: Development of assays to identify individuals with elevated anti-ACKR1 autoantibodies could help identify patients requiring more rigorous vascular-protective management after viral infections or other inflammatory conditions .

• Epitope mapping: Further studies with comprehensive peptide screening panels to characterize epitopes would provide insights into the binding mechanisms and heterogeneities of human anti-ACKR1 autoantibodies, potentially leading to more targeted therapeutic approaches .

Each of these directions represents a promising avenue for translational research that could ultimately lead to clinical applications in vascular medicine, particularly in post-infectious and inflammatory vascular conditions.