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The antibody MY4 recognizes CD14 on porcine monocytes and macrophages
H W Ziegler-Heitbrock1, B Appl, E Käfferlein
1Institute for Immunology, Klinikum Innenstadt, Munich, Germany.
Abstract:
Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74-22-15. Two-colour immunofluorescence reveals that My4 and 74-22-15 antigens are coexpressed on the same cells, and cell sorter-purified My4+ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4+ monocytes amongst all leucocytes is 5.8% with 580 cells/microliters. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four-fold lower level on AM. Treatment of cells with phosphatidylinositol-specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74-22-15. Immunoprecipitation with the My4 antibody from surface labelled pig mononuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide- (LPS)-induced interleukin-6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.
Insights
The My4 antibody specifically targets porcine monocytes and macrophages, identifying the CD14 antigen. This antibody is crucial for studying immune responses in pigs and blocking lipopolysaccharide-induced interleukin-6 production.
Area of Science:
- Immunology
- Veterinary Science
- Cell Biology
Background:
- Monoclonal antibodies against human CD14 are established tools.
- Porcine immune cell markers require further characterization for comparative immunology.
Purpose of the Study:
- To determine if the My4 antibody recognizes porcine CD14.
- To characterize the expression and function of CD14 in porcine monocytes and macrophages.
Main Methods:
- Immunofluorescence staining of porcine peripheral blood mononuclear cells (PBMC) and macrophages with My4 and 74-22-15 antibodies.
- Flow cytometry and cell sorting.
- Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment.
- Immunoprecipitation and Western blotting.
- Lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production assay.
Main Results:
- My4 antibody stained 14.6% of porcine PBMC, similar to the anti-porcine monocyte antibody 74-22-15.
- My4 and 74-22-15 antigens were coexpressed on monocytes.
- My4 stained porcine peritoneal and alveolar macrophages, with lower levels on alveolar macrophages.
- PI-PLC treatment reduced My4 staining, suggesting a GPI-anchored protein.
- Immunoprecipitation revealed a 54 kDa band, and Western blotting showed bands similar to human soluble CD14.
- My4 antibody blocked LPS-induced IL-6 production in PBMC.
Conclusions:
- The My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.
- This finding provides a valuable tool for porcine immunology research.
- My4 antibody's ability to block LPS-induced IL-6 suggests a role in regulating inflammatory responses.