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Intracellular staining of Mx proteins in cells from peripheral blood, bone marrow and skin
A N al-Masri1, T Werfel, D Jakschies
1Department of Clinical Immunology, Medical School Hannover, Germany.
Aim/Background:
The Mx proteins are known to be specifically and dose dependently induced in mononuclear cells (MNC) by type I interferons (IFN). The aim of this study was to establish a staining method for the human intracellular Mx proteins, MxA and MxB, in leucocytes and bone marrow and skin cells.
Methods:
Several monoclonal antibodies directed against the MxA and MxB proteins were generated. These antibodies were used to stain Mx proteins in both frozen and paraffin wax sections using the standard alkaline phosphatase anti-alkaline phosphatase (APAAP) method.
Results:
Granulocytes, monocytes and lymphocytes extracted from freshly collected blood from 21 healthy subjects did not stain. After incubating MNC from these subjects with IFN alpha 2b for 48 hours, Mx proteins were detected in monocytes and lymphocytes. Within two days of starting treatment with subcutaneous IFN alpha 2b, granulocytes, monocytes and lymphocytes of 16 patients with cancer stained strongly for Mx proteins. The intensity of staining was correlated with the Mx content of whole blood measured using a specific ELISA. Prior to IFN treatment, cells from bone marrow and skin tissue specimens were negative for Mx proteins with the exception of endothelial cells. During treatment with IFN alpha 2b, nearly all cells from bone marrow and skin stained intensely.
Conclusions:
These new monoclonal antibodies facilitate the detection of Mx positive cells in peripheral blood and in frozen or paraffin wax specimens. The advantage of this staining method is that individual cells which have responded to viruses or biologically active IFN alpha, beta or omega can be identified.
Insights
New staining methods detect human Mx proteins (MxA and MxB) in immune cells and tissues. This allows identification of cells responding to interferon therapy, crucial for viral infections and cancer treatment.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Mx proteins are induced by type I interferons (IFN) in mononuclear cells (MNC).
- A reliable staining method for intracellular MxA and MxB proteins in various human cells is needed.
Purpose of the Study:
- To establish a staining method for intracellular human MxA and MxB proteins.
- To detect these proteins in leukocytes, bone marrow, and skin cells.
Main Methods:
- Generation of monoclonal antibodies against MxA and MxB proteins.
- Standard alkaline phosphatase anti-alkaline phosphatase (APAAP) staining method used on frozen and paraffin-embedded tissues.
Main Results:
- Mx proteins were not detected in unstimulated blood cells (granulocytes, monocytes, lymphocytes).
- IFN alpha 2b treatment induced Mx proteins in monocytes and lymphocytes in vitro and in vivo.
- IFN treatment also induced intense Mx protein staining in bone marrow and skin cells, including endothelial cells.
Conclusions:
- Novel monoclonal antibodies enable detection of MxA and MxB proteins in various human cells.
- This staining method identifies individual cells responding to interferon stimulation.
- Useful for monitoring viral infections and interferon-based therapies in cancer patients.