CD163 Monoclonal Antibody

What is CD163 and what cellular populations express this marker?

CD163 is a 130-140 kDa membrane glycoprotein that functions as an acute phase-regulated transmembrane protein and belongs to the scavenger receptor cysteine-rich superfamily. It primarily serves as a receptor for hemoglobin-haptoglobin complexes. CD163 is exclusively expressed on the cell surface of human monocytes and macrophages, with differential expression patterns: monocytes exhibit lower surface expression compared to tissue macrophages and histiocytes, which demonstrate high expression levels .

Specifically, CD163 is present on all circulating monocytes and most tissue macrophages except those found in the mantle zone and germinal centers of lymphoid follicles, interdigitating reticulum cells, and Langerhans cells. At the molecular level, CD163 is present on all CD14-positive monocytes, most CD64-positive monocytes, and shows particularly elevated expression on CD16-positive monocytes . The marker can also be used to identify several specialized macrophage populations including Type 3 Dendritic (DC3) Cells, Large Intestine Macrophages, and various subsets of Alveolar Macrophages .

How does CD163 expression change during inflammation and what factors regulate its expression?

CD163 expression undergoes significant modulation during inflammatory processes, reflecting its role in resolving inflammation and tissue homeostasis. The protein is upregulated on mononuclear phagocytes in response to anti-inflammatory cytokines including IL-10 and IL-6, as well as by glucocorticoids such as dexamethasone . This upregulation typically occurs predominantly in the late phase of inflammation, suggesting CD163's involvement in the resolution phase of inflammatory responses.

Conversely, pro-inflammatory stimuli such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) induce a different response - they trigger shedding of CD163 from the cell surface into plasma or cell supernatant . This shedding mechanism likely represents a regulatory process that modulates CD163 function during acute inflammatory responses. The balance between upregulation and shedding of CD163 may serve as a potential biomarker for monitoring inflammatory disease progression and response to therapy.

What is the biological function of CD163 in macrophage physiology?

CD163 plays multiple critical roles in macrophage biology, primarily centered around hemoglobin clearance and iron homeostasis. Its principal function is to mediate the endocytosis of haptoglobin-hemoglobin complexes, thereby protecting tissues from free hemoglobin-mediated oxidative damage . This protective mechanism is particularly important during hemolysis when free hemoglobin is released into circulation.

Beyond hemoglobin scavenging, CD163 contributes to iron recycling through the endocytosis and subsequent breakdown of heme. The binding affinity of CD163 for hemoglobin-haptoglobin complexes is both calcium-dependent and pH-dependent, with higher affinity observed for complexes containing multimeric haptoglobin of HP1F phenotype compared to dimeric haptoglobin of HP1S phenotype .

CD163 also functions as a signaling molecule, inducing a cascade of intracellular signals upon ligand binding. This cascade involves tyrosine kinase-dependent calcium mobilization, inositol triphosphate production, and ultimately leads to the secretion of cytokines including IL-6 and CSF1 . These signaling functions suggest CD163 may modulate macrophage activation states and contribute to tissue homeostasis beyond its scavenging capabilities.

What are the optimal applications for different CD163 monoclonal antibody clones?

Different CD163 monoclonal antibody clones demonstrate varying efficacy across experimental applications, making clone selection crucial for experimental success. Based on the available data, clone GHI/61 (exemplified by antibody MA5-17716) has been validated for multiple applications including flow cytometry (FACS), frozen section immunohistochemistry (IHC-F), immunoprecipitation (IP), and Western blotting (WB) . This versatility makes it suitable for researchers requiring multi-platform confirmation of results.

The R20 clone has demonstrated particular efficacy in flow cytometry applications for detecting CD163 on human peripheral blood monocytes, making it an excellent choice for immunophenotyping studies . For researchers focusing on tissue macrophage identification in histological specimens, clone M130/1210 has shown superior specificity for macrophages compared to CD68 antibodies, particularly in contexts such as rheumatoid arthritis where distinguishing between synovial macrophages and synovial intimal fibroblasts is challenging .

When selecting an antibody clone, researchers should consider the specific cellular population under investigation, the preservation method of the sample, and whether quantitative (flow cytometry) or qualitative (immunohistochemistry) assessment is required, as no single clone performs optimally across all applications and tissue types.

How can CD163 monoclonal antibodies be utilized in flow cytometry to identify macrophage subpopulations?

Flow cytometric analysis using CD163 monoclonal antibodies provides a powerful approach for identifying and characterizing heterogeneous macrophage populations. To optimize this application, researchers should implement a multi-parameter staining protocol that combines CD163 with other macrophage-associated markers for comprehensive phenotyping.

A validated protocol involves co-staining human peripheral blood monocytes with anti-CD14 PE-conjugated monoclonal antibody and anti-CD163 monoclonal antibody, followed by detection using an APC-conjugated secondary antibody . This approach allows discrimination between monocyte subsets based on their CD163 expression levels. For more comprehensive phenotyping, researchers can expand this panel to include CD16 (to identify non-classical monocytes), CD64 (to identify classically activated macrophages), and markers of activation status.

When analyzing tissue-resident macrophages, enzymatic tissue digestion protocols must be carefully optimized to preserve CD163 epitopes, as aggressive digestion may cleave surface CD163. Single-cell suspensions should be filtered to remove debris before antibody staining, and viability dyes should be included to exclude dead cells that can bind antibodies non-specifically. For quantitative comparison between samples, calibration beads should be used to standardize fluorescence intensity measurements.

What are the considerations for using CD163 monoclonal antibodies in immunohistochemistry applications?

CD163 immunohistochemistry presents unique methodological considerations for optimal results in tissue sections. For formalin-fixed, paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval is typically necessary to expose CD163 epitopes that may be masked during fixation. Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) retrieval solutions have both been successfully employed, though optimal conditions may need to be determined empirically for specific antibody clones.

The specificity of CD163 monoclonal antibodies for macrophages in immunohistochemical applications has been demonstrated to be superior to that of anti-CD68 antibodies, particularly in contexts such as rheumatoid arthritis where distinguishing between synovial macrophages and synovial intimal fibroblasts is challenging . This makes CD163 a preferred marker for histological identification of tissue macrophages in various inflammatory conditions.

For dual or multiple immunostaining, sequential antibody application with intervening blocking steps is recommended to prevent cross-reactivity. When quantifying CD163-positive cells in tissue sections, digital image analysis using appropriate software can provide more reproducible results than manual counting, particularly in tissues with complex architecture or high macrophage infiltration.

How can CD163 monoclonal antibodies be modified for targeted drug delivery systems?

The development of CD163 monoclonal antibody-modified drug delivery systems represents an advanced application particularly relevant for targeted cancer immunotherapy. A methodologically robust approach involves the chemical conjugation of CD163 monoclonal antibodies to polymeric nanoparticle drug carriers through bioorthogonal chemistry techniques.

One validated protocol employs a "Click" chemistry strategy, where dibenzocyclooctynyl-coupled CD163 monoclonal antibody (mAb-CD163-DBCO) is prepared through amidation between NHS-PEG4-DBCO and the CD163 monoclonal antibody at a precisely controlled molar ratio of 1:30 . The reaction is conducted by incubating 100 μL of mAb-CD163 solution (0.5 mg·mL−1 in phosphate buffer pH 7.4) with 8 μL of NHS-PEG4-DBCO in DMSO (1.0 mg·mL−1) for 12 hours at 25°C. Following this conjugation, unreacted NHS-PEG4-DBCO is removed by centrifugation using an ultrafiltration tube (MWCO 10,000 Da, 7000 g, 5 min) with three subsequent washes in phosphate buffer .

For integration with drug-loaded nanoparticles, the mAb-CD163-DBCO is reacted with azide-functionalized nanoparticles through strain-promoted azide-alkyne cycloaddition. After the coupling reaction, ultracentrifugation (210,000 g, 4°C, 50 min) effectively separates the antibody-modified nanoparticles from unbound antibody conjugates . The extent of antibody modification can be quantified using BCA protein assay of the supernatant, while the morphology and size distribution of the resulting nanoparticles should be characterized using transmission electron microscopy and dynamic light scattering.

This targeted delivery approach has demonstrated remarkable efficacy in preclinical models, with tumor inhibition rates reaching 81% , highlighting the potential of CD163-targeted therapeutic strategies in cancer immunotherapy.

What methodological approaches can be used to study CD163-mediated signal transduction in macrophages?

Investigating CD163-mediated signal transduction requires sophisticated methodological approaches to capture the complex intracellular events following receptor engagement. A comprehensive protocol would begin with macrophage isolation and purification, followed by controlled CD163 ligation and subsequent analysis of signaling cascades.

Primary human monocyte-derived macrophages or CD163-expressing cell lines can be stimulated with hemoglobin-haptoglobin complexes to trigger physiological CD163 signaling. Alternatively, antibody-mediated cross-linking using anti-CD163 antibodies can be employed to induce receptor clustering and activation. For temporal analysis of signaling events, time-course experiments with cell lysis at defined intervals following stimulation are essential.

Phosphorylation events in the tyrosine kinase-dependent cascade can be detected using phospho-specific antibodies in Western blot or phospho-flow cytometry. Calcium mobilization, a key early event in CD163 signaling, can be monitored in real-time using calcium-sensitive fluorescent dyes and live-cell imaging or plate reader systems. For assessment of downstream inositol triphosphate (IP3) production, radioimmunoassay or mass spectrometry-based approaches provide quantitative measurement.

Cytokine secretion (particularly IL-6 and CSF1) resulting from CD163 activation should be quantified using ELISA or multiplex bead-based assays of cell culture supernatants. For comprehensive pathway analysis, phosphoproteomic profiling using mass spectrometry can identify novel phosphorylation targets, while pharmacological inhibitors targeting specific kinases can help delineate the hierarchy of signaling events.

How can researchers differentiate between membrane-bound and soluble CD163 in experimental systems?

Distinguishing between membrane-bound and soluble CD163 is methodologically challenging but essential for understanding CD163 biology in different physiological and pathological contexts. A multi-platform approach is recommended for comprehensive analysis.

For in vitro studies, flow cytometry using non-permeabilizing conditions allows specific detection of membrane-bound CD163, while ELISA of cell culture supernatants can quantify soluble CD163 released through shedding. When analyzing patient samples, membrane-bound CD163 on circulating monocytes can be assessed by flow cytometry of fresh whole blood or isolated peripheral blood mononuclear cells, while soluble CD163 in serum or plasma is typically measured using commercial or laboratory-developed ELISA protocols.

To directly investigate CD163 shedding mechanisms, researchers can stimulate cells with shedding inducers such as LPS or PMA and measure both surface CD163 (by flow cytometry) and soluble CD163 (by ELISA) in time-course experiments . Inhibitors of metalloproteases can be employed to determine which proteolytic enzymes are responsible for CD163 cleavage from the cell surface.

Western blot analysis using antibodies recognizing different epitopes can help distinguish intact membrane-bound CD163 (~130-140 kDa) from cleaved soluble forms. For advanced analysis of soluble CD163 fragments, immunoprecipitation followed by mass spectrometry can characterize the precise cleavage sites and fragmentation patterns resulting from different shedding stimuli.

What are common technical challenges when working with CD163 monoclonal antibodies and how can they be addressed?

Researchers working with CD163 monoclonal antibodies frequently encounter several technical challenges that require specific troubleshooting approaches. One common issue is epitope masking in fixed tissues, which can be addressed through optimized antigen retrieval protocols. For formaldehyde-fixed samples, extended heat-induced epitope retrieval (20-40 minutes) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) typically improves antibody accessibility to CD163 epitopes.

Non-specific background staining, particularly in immunohistochemistry applications, can be minimized by implementing more stringent blocking protocols using combinations of serum, bovine serum albumin, and commercial blocking reagents. For tissues with high endogenous peroxidase activity, dual hydrogen peroxide blocking steps (one before and one after primary antibody incubation) may be necessary.

For flow cytometry applications, reduced CD163 detection may result from proteolytic cleavage during sample preparation. This can be mitigated by including protease inhibitors in all buffers and minimizing the time between sample collection and analysis. When analyzing tissues, enzymatic digestion conditions should be carefully titrated to balance between achieving single-cell suspensions and preserving CD163 epitopes.

Blue fluorescent dye conjugates (such as CF®405S and CF®405M) are not recommended for detecting CD163 due to lower fluorescence intensity and higher non-specific background compared to other fluorophores . Instead, longer-wavelength fluorophores like APC or PE typically provide better signal-to-noise ratios for CD163 detection.

False-negative results may occur due to CD163 shedding induced by sample handling. Immediate fixation of samples with paraformaldehyde (1-4%) can help preserve surface CD163 for flow cytometric analysis.

How should researchers interpret variability in CD163 expression levels across different macrophage populations?

Interpreting heterogeneous CD163 expression across macrophage populations requires consideration of both biological variation and methodological factors. Biologically, CD163 expression levels reflect macrophage polarization status, with anti-inflammatory M2-like macrophages typically expressing higher CD163 levels than pro-inflammatory M1-like macrophages. Therefore, expression variability may represent functional heterogeneity within the macrophage population under study.

When comparing CD163 expression between different tissue macrophage populations, researchers should recognize the tissue-specific regulation of CD163. For example, alveolar macrophages typically display moderate and heterogeneous CD163 expression, while Kupffer cells in the liver and dermal macrophages generally exhibit more uniform high expression . These differences likely reflect tissue-specific microenvironmental factors that regulate CD163 transcription and protein stability.

Methodologically, standardization is essential for meaningful comparisons. For flow cytometry, fluorescence intensity should be reported using standardized metrics such as molecules of equivalent soluble fluorochrome (MESF) or antibody binding capacity (ABC) rather than arbitrary units. Including biological reference samples with known CD163 expression in each experiment provides internal calibration for inter-experimental comparisons.

For immunohistochemistry, semi-quantitative scoring systems that account for both staining intensity and percentage of positive cells provide more reproducible assessments than binary positive/negative categorization. Digital image analysis using consistent thresholding parameters offers the most objective approach for quantitative comparison across tissue samples.

What controls should be included when using CD163 monoclonal antibodies in various experimental setups?

Rigorous experimental design with appropriate controls is essential for generating reliable data with CD163 monoclonal antibodies. For flow cytometry applications, an isotype control antibody matched to the CD163 primary antibody (same species, isotype, and concentration) is mandatory to establish thresholds for positive staining . Fluorescence-minus-one (FMO) controls, which include all fluorochromes except anti-CD163, help identify spillover effects in multicolor panels.

Biological positive controls should include cell populations known to express CD163, such as monocytes for peripheral blood analysis or specific tissue macrophage populations for tissue studies. When available, cell lines with stable CD163 expression provide consistent positive controls across experiments. Conversely, CD163-negative populations such as lymphocytes or CD163-knockout cell lines serve as biological negative controls.

For immunohistochemistry, section-to-section or run-to-run variability should be controlled using tissue microarrays containing reference tissues with known CD163 expression patterns. Secondary antibody-only controls help identify non-specific binding of detection reagents, while blocking peptide controls (where the antibody is pre-incubated with excess CD163 antigen) can confirm binding specificity.

In functional studies involving CD163 targeting, knockdown controls using CD163-specific siRNA or CRISPR-mediated gene editing provide evidence that observed effects are specifically mediated through CD163 rather than off-target mechanisms. For therapeutic applications such as antibody-modified nanoparticles, non-targeted equivalents (lacking the CD163 antibody) are essential controls to demonstrate targeting specificity .

What are the latest advances in using CD163 monoclonal antibodies for targeted cancer immunotherapy?

Recent advances in CD163-targeted cancer immunotherapy have focused on exploiting the presence of CD163-positive tumor-associated macrophages (TAMs), which generally exhibit M2-like, tumor-promoting phenotypes. A particularly promising approach involves antibody-drug conjugate technology to deliver cytotoxic payloads specifically to these cells.

Cutting-edge research has demonstrated successful development of CD163 monoclonal antibody-modified doxorubicin-polymer prodrug nanoparticles (mAb-CD163-PDNPs) with pH responsiveness and targeted delivery capabilities . These nanoparticles are engineered through a stepwise process involving Schiff base reaction to form amphiphilic polymer prodrugs capable of self-assembly, followed by conjugation with CD163 antibodies through bioorthogonal "Click" chemistry . This sophisticated delivery system allows for controlled drug release specifically in the acidic microenvironment of endosomes following receptor-mediated endocytosis.

In vivo studies with these modified nanoparticles have demonstrated remarkable efficacy with tumor inhibition rates reaching 81% . The dual mechanism of action involves both direct depletion of tumor-promoting TAMs and delivery of cytotoxic agents to the tumor microenvironment. This approach represents a paradigm shift from conventional cancer immunotherapies that typically focus on T cell activation rather than macrophage modulation.

Future directions in this field include developing combination strategies that pair CD163-targeted approaches with checkpoint inhibitors or other immunomodulatory agents to achieve synergistic anti-tumor effects through comprehensive remodeling of the tumor immune microenvironment.

How can single-cell technologies enhance our understanding of CD163 biology in heterogeneous macrophage populations?

Single-cell technologies have revolutionized our ability to dissect heterogeneity within macrophage populations, offering unprecedented insights into CD163 biology. Single-cell RNA sequencing (scRNA-seq) enables comprehensive transcriptomic profiling of individual macrophages, revealing distinct subpopulations based on gene expression patterns that may not be apparent using bulk analysis methods.

These approaches have identified previously unrecognized heterogeneity in CD163 expression and co-expression with other markers across tissue-resident macrophage populations. For example, single-cell analyses have revealed that CD163 expression often correlates with specific functional gene modules rather than with classical M1/M2 polarization signatures, suggesting more complex regulation than previously appreciated.

Mass cytometry (CyTOF) allows simultaneous analysis of over 40 protein markers at the single-cell level, enabling deep phenotyping of macrophage subsets based on surface receptor expression, signaling pathway activation, and functional markers. This approach can correlate CD163 expression with specific functional states across diverse tissue environments.

For spatial context, multiplexed immunofluorescence or imaging mass cytometry provides information about the microanatomical localization of CD163-positive macrophages relative to other cell types within tissues. These techniques have revealed that CD163-positive macrophages often occupy specific niches within tissues and tumors, suggesting microenvironmental regulation of CD163 expression.

Integration of these complementary single-cell technologies through computational approaches offers the most comprehensive view of CD163 biology. Future applications will likely include longitudinal single-cell profiling to understand dynamic changes in CD163 expression during disease progression and therapeutic intervention.

What are the potential applications of CD163 monoclonal antibodies in monitoring and treating inflammatory diseases?

Beyond cancer applications, CD163 monoclonal antibodies hold significant potential for monitoring and treating various inflammatory diseases. From a diagnostic perspective, immunohistochemical detection of CD163-positive macrophages in tissue biopsies provides valuable information about the local inflammatory milieu, as increased CD163-positive macrophage infiltration is associated with the resolution phase of inflammation and tissue repair processes.

In rheumatoid arthritis, CD163 immunostaining has proven superior to CD68 for distinguishing synovial macrophages from synovial intimal fibroblasts , offering improved accuracy in assessing macrophage infiltration as a biomarker of disease activity. Similar applications in inflammatory bowel disease, dermatological conditions, and neuroinflammatory disorders are being actively investigated.

Therapeutic approaches targeting CD163 for inflammatory diseases are in early developmental stages but show promise. One strategy involves inducing a phenotypic shift in macrophages from pro-inflammatory to resolution-promoting phenotypes through targeted delivery of immunomodulatory compounds to CD163-positive cells. Alternatively, engineered antibodies that enhance CD163's hemoglobin-scavenging function could reduce oxidative damage in conditions involving hemolysis.

For monitoring disease activity and treatment response, quantifying soluble CD163 in serum represents a non-invasive approach. Levels of soluble CD163 are elevated in various inflammatory conditions including sepsis, liver disease, and macrophage activation syndrome, making it a potential biomarker for disease activity. Development of standardized assays using well-characterized monoclonal antibodies would enhance the clinical utility of soluble CD163 measurements.