Inhibition of lymphocyte homing by carbohydrate-binding proteins, lectins
Lab/Group: Fukuda Lab (Burnham institute)
Related Journal & Article Information
Journal: Nature Immunology
Article Title: Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment
Introduction
Lymphocyte homing assay consists of intravenous injection of fluorescent-labeled lymphocytes and measuring the number of labeled lymphocytes recovered in secondary lymphoid organs. One h (for short homing assay) to 24 h (for long term homing assay) after the injection of the labeled lymphocytes, lymph nodes, Peyer’s patches and other lymphoid organs are isolated and the number of fluorescent lymphocytes over total lymphocyte number is determined. This process can be inhibited by pre-injection of lectins that specifically bind to glycans that carry L-selectin ligands
Materials
Reagents
1 mM Chloromethyl fluorescence diacetate (CMFDA) (Molecular Probe- Invitrogen)
Erythroagglutinating phytohemagglutin (E-PHA) (EY Labs)
Tomato lectin (LEA) (EY Labs)
Concanavalin A (Con A) (EY Labs)
40 μm or 70 μm mesh (BD-Biosciences)
Red blood cell lysing buffer (8.8 g/l ammonium chloride in 0.01 M Tris-HCl buffer, pH 7.5, Sihma)
Equipment
Fluorescent-activated cell sorter (FACsort-3 colors, BD-Bioscience)
Procedure
1. Isolate mesenteric lymph nodes and spleen from sacrificed wild-type mice.
2. Squeeze these lymph nodes and spleen between two coverslips and lymphocytes are dropped into plastic 6-well containing RPMI-1640 medium.
3. After passing through 70 μm mesh, centrifuge the medium to collect cells.
4. The cells are incubated with red blood cell lysis buffer at room temperature for 3 – 5 min, then add 10 times volume of RPMI-1640 medium containing 10 % fetal bovine serum and incubate at 37 °C for 30 min.
5. After centrifuge at 1,000x g for 5 min, pellet lymphocytes are suspended in 5 ml of RPMI-1640 containing 1μM CMFDA solution, and incubated at 37 °C for 30 min.
Alternatively orange tracker Orange CMRA is used insead of CMFDA using the ame conditions.
6. CMFDA-labeled lymphocytes are recovered by centrifugation at 1000 x g for 5 min, suspended in RPMI-1640 containing 10 % fetal bovine serum and incubated at 37 °C for 30 min. Cell pellet is washed with RPMI-1640 twice and cell suspension is passed through 70 μm mesh, reconstituted in 0.9 % NaCl to obtain two hundred fifty thousand cells per 100 μl.
7. E-PHA (150 to 400 μg), LEA (150 to 300 μg) or Con A (150 μg) dissolved in 150 μl of PBS is intravenously injected through a tail vein. One hour after the injection of a lectin, CMFDA-labeled lymphocytes (twenty five million cells) are injected.
8. One to 24 h after lymphocyte injection, the mice are sacrificed and the peripheral ( cervical and lateral axillary) lymph nodes, mesenteric lymph nodes, Peyer’s patches, spleen, and thymus are collected. Lymphocytes are squeezed out as described in 2 and 3, debris is removed by passing through 70 μm mesh and lymphocytes are suspended in 2 ml of PBS.
9. The cell suspension (hundred to hundred fifty thousand cells) is analyzed by flow cytometry. The number of fluorescent cells is compared to the number of total cells. Control experiments are carried out by injecting only PBS. The ratio of control and lectin-inhibited experiments are compared.
Troubleshooting
High concentration of lectins may be toxic and may have overt reactions by the mice. Such a high dose injection needs to be avoided.
Critical Steps
The solution of lectins and CMFDA-labeled lymphocytes need to be injected entirely. For the second injection of CMFDA-labeled lymphocytes, a tail vein that was not used for lectin-injection should be used. Alternately, CMFDA-lymphocyte injection can be done at a site different from that was used for lectin injection
Anticipated Results
Con A should have a minimum effect on lymphocyte homing since L-selectin ligands are not present on Con A-reactive N-glycans. E-PHA and LEA have a significant inhibitory effect on wild-type mice and may almost completely inhibit lymphocyte homing in Core2GlcNAcT/Core1-βGlcNAc double knockout mouse. The effect of lectins is highest in lymphocyte homing to peripheral lymph nodes, moderate to mesenteric lymph nodes and very moderate to Peyer’s patches.
References
Mitoma, J., Bao, X., Petryanik, B., Schaeril, P., Gauguet, J-M., Yu.S-Y., Kawashima, H., Saito H., Ohtsubo, K., Marth, J.D., Khoo, K-H., von Andrian, U. H., Lowe, J. B., and Fukuda, M. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment. Nature Immunology 8, 409-508 (2007).
Acknowledgements
Keywords
lymphocyte homing
L-slectin ligands
secondary lymphoid organs
N-glycans
6-sulfo sialyl Lewis x
Figure 1
Lymphocyte homing with or without pretreatment of N-glycan-specific lectins
One hour after intravenous injection of LEA (300 μg), E-PHA (200 μg for DKO and 400 μg for WT) or ConA (200 μg), orange CMRA-positive lymphocytes were injected into tail veins of DKO (left) or WT (right) mice. CMRA-lymph nodes were recovered 1 h later and were measured by flow cytometer. DKO mice are deficient in bith Core2GlcNAcT-1 and Core1-β3GlcNAcT. * , P < 0.05; ** , P < 0.01; *** , P < 0.001 compared to PBS control.
