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Immunocytochemical analysis of mitogen responses of carp (Cyprinus carpio L.) peripheral blood leucocytes
J C Koumans-van Diepen1, E G Harmsen, J H Rombout
1Department of Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, Netherlands.
Insights
This study investigated carp immune cell responses to phytohaemagglutinin (PHA) and lipopolysaccharide (LPS). Results show PHA stimulates surface immunoglobulin-negative (sIg-) cells, while LPS primarily activates dull surface immunoglobulin-positive (sIg+) cells.
Area of Science:
- Immunology
- Fish immunology
- Cell biology
Background:
- Peripheral blood leucocytes (PBL) play a crucial role in fish immune responses.
- Surface immunoglobulin (sIg) expression is a key marker for B cell identification and function.
- Understanding differential immune cell activation is vital for assessing fish health and disease resistance.
Purpose of the Study:
- To investigate the differential responses of surface immunoglobulin-positive (sIg+) and surface immunoglobulin-negative (sIg-) carp PBL to mitogens.
- To characterize the specific cell populations activated by phytohaemagglutinin (PHA) and lipopolysaccharide (LPS).
- To elucidate the role of sIg expression in mitogenic stimulation of carp leucocytes.
Main Methods:
- Isolation of sIg+ and sIg- enriched carp PBL populations using magnetic cell sorting (MACS).
- In vitro mitogenic stimulation with PHA and LPS, measuring 3H-thymidine incorporation.
- Simultaneous detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) and sIg using fluorescence microscopy and flow cytometry.
Main Results:
- PHA stimulation significantly enhanced the number of sIg- cells, indicating their primary role in PHA response.
- LPS stimulation led to an increase in sIg+ cells, with flow cytometry revealing activation mainly in dull sIg+ cells, not sIg- cells.
- Conflicting results between MACS sorting and flow cytometry highlighted the challenge in distinguishing dull sIg+ from sIg- cells.
Conclusions:
- Carp peripheral blood leucocytes exhibit distinct responses to PHA and LPS based on sIg expression.
- PHA primarily activates sIg- cells, while LPS stimulates dull sIg+ cells, suggesting differential B cell activation pathways.
- Further refinement of cell sorting and identification techniques is needed to accurately differentiate sIg subpopulations in carp immune studies.
Abstract:
Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) responses of surface immunoglobulin-positive (sIg+) and surface immunoglobulin-negative (sIg-) carp peripheral blood leucocytes (PBL) were studied. sIg+ cell-enriched and depleted carp PBL populations (sIg+ and sIg- cell fractions, respectively) were obtained by magnetic cell sorting (MACS) and mitogenic stimulation in vitro was measured by 3H-thymidine incorporation. The mitogen responses of sIg+ and sIg- cells in non-separated carp PBL cultures were analysed by simultaneous detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) and sIg with the fluorescence microscope and flow cytometer. Flow cytometric determination of the percentage of sIg+ cells in combination with absolute cell counting, revealed an increase of sIg+ cells but not of sIg- cells after LPS stimulation while the number of sIg- cells and not of sIg+ cells was enhanced after PHA stimulation. LPS stimulation showed an increased 3H-thymidine incorporation in the sIg- cell fraction compared with non-separated cells and BrdU incorporation was observed in sIg- cells from LPS-stimulated cultures by fluorescence microscopy. However, flow cytometric analysis showed that mainly dull sIg+ cells and not sIg- cells are stimulated by LPS. These dull sIg+ cells were not sorted from sIg- cells with MACS and could apparently not be distinguished from sIg- cells by light microscopy. PHA stimulates sIg- cells and not sIg+ cells as was estimated by all techniques used.
