Related Experiment Videos
Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell
M A Hoijer1, M J Melief, J Calafat
1Department of Immunology, Erasmus University Rotterdam, The Netherlands.
Abstract:
N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.
Insights
N-acetylmuramyl-L-alanine amidase (NAMLAA) degrades inflammatory peptidoglycan. This study maps NAMLAA in human tissues and immune cells, finding it in granulocytes and macrophages, aiding acute myeloid leukemia (AML) classification.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- N-acetylmuramyl-L-alanine amidase (NAMLAA) degrades peptidoglycan, a bacterial cell wall component with inflammatory properties.
- Previous research indicated that NAMLAA degrades peptidoglycan, reducing its inflammatory effects.
Purpose of the Study:
- To determine the tissue and cellular localization of NAMLAA.
- To evaluate the utility of a monoclonal antibody recognizing NAMLAA for discriminating between myeloid lineages in acute myeloid leukemia (AML).
Main Methods:
- Immunohistochemistry, immunoelectron microscopy, flow cytometry, and Western blotting were used to localize NAMLAA.
- Monoclonal antibodies against purified NAMLAA were generated.
Main Results:
- NAMLAA was detected in human spleen, liver, lymph nodes, and arthritic synovial tissues.
- Neutrophilic and eosinophilic granulocytes, mature macrophages, and thrombocytes expressed NAMLAA.
- NAMLAA expression was identified in AML-M2 myeloblasts and AML-M7 megakaryoblasts, but not in more immature AML subtypes (M0, M1).
Conclusions:
- NAMLAA is present in various human tissues and specific immune cell types, including granulocytes and macrophages.
- The expression pattern of NAMLAA in myeloid cells suggests its potential as a marker for distinguishing granulocytic from monocytic AML subtypes.