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Combined immunohistochemical protocols to differentiate macrophages within the mononuclear-phagocyte system
G Lazzarini1, F Abramo1, F Albanese2
1Department of Veterinary Sciences, University of Pisa, Pisa, Italy.
Background:
The "mononuclear phagocyte system" (MPS) refers to dispersed mononuclear monocytes and macrophages and is used to distinguish them from polymorphonuclear cells. The term "histiocyte" indicates large cells with voluminous granulated cytoplasm, sometimes containing engulfed particles, recognized as fully differentiated end cells of the MPS. Dendritic cells (DC) represent another diversified population whose inclusion in the MPS is still debated. The diverse cells of the MPS cannot all be characterized by single antigen markers or unique functions expressed at all stages of cell differentiation or activation. Nevertheless, in a diagnostic setting, their reliable identification plays a major role when a specific therapy must be established. Understanding the heterogeneity among MPS cell populations is indeed relevant to define different therapeutic approaches that can range from the use of antibiotics to immunomodulatory agents. For this reason, we attempted to establish a protocol to reliably identify the proportion of macrophages within the mononuclear phagocyte system in a tissue and/or in a given inflammatory population.
Methods:
the Tafuri method was used in different double immunofluorescence protocols using an anti-Iba-1, anti-MAC387, and anti-CD11b-CD68-CD163-CD14-CD16 antibody.
Results And Discussion:
in normal canine skin the anti-Iba-1 antibody stained an epidermal cell population (i.e. Langerhans cells) and scattered cells within the dermal compartment. MAC387 was unable to stain cells containing Leishmania amastigotes in leishmaniasis-diagnosed samples as the anti-CD11b-CD68-CD163-CD14-CD16 antibody did. By using a combination of staining protocols to differentiate macrophages within the whole histiocytic infiltrate we validated the use of a cocktail of rabbit monoclonal antibodies raised against CD11b, CD68, CD163, CD14, CD16 to stain skin macrophages.
Insights
This study developed a reliable method to identify macrophages within the mononuclear phagocyte system (MPS). The validated antibody cocktail effectively stains skin macrophages, aiding in diagnosing inflammatory conditions and guiding therapy.
Area of Science:
- Immunology
- Cell Biology
- Veterinary Pathology
Background:
- The mononuclear phagocyte system (MPS) comprises monocytes and macrophages, crucial for immune responses.
- Differentiating MPS cell populations, like macrophages and dendritic cells (DCs), is challenging due to marker and functional heterogeneity.
- Accurate identification of MPS cells is vital for targeted therapies in inflammatory diseases.
Purpose of the Study:
- To establish a reliable protocol for quantifying macrophages within the mononuclear phagocyte system in tissue and inflammatory infiltrates.
- To differentiate macrophages from other histiocytic cells for improved diagnostic accuracy.
Main Methods:
- Utilized the Tafuri method with double immunofluorescence protocols.
- Employed antibodies including anti-Iba-1, anti-MAC387, and a cocktail of anti-CD11b, anti-CD68, anti-CD163, anti-CD14, and anti-CD16.
Main Results:
- Anti-Iba-1 stained epidermal Langerhans cells and dermal cells in normal canine skin.
- MAC387 and the CD11b-CD68-CD163-CD14-CD16 antibody cocktail did not stain Leishmania-infected cells.
- A cocktail of rabbit monoclonal antibodies against CD11b, CD68, CD163, CD14, and CD16 successfully stained skin macrophages.
Conclusions:
- Validated a cocktail of CD11b, CD68, CD163, CD14, and CD16 antibodies for accurate identification of skin macrophages.
- This protocol aids in differentiating macrophages within histiocytic infiltrates.
- The findings support improved diagnostic strategies and therapeutic interventions for inflammatory conditions involving MPS cells.
