De novo expression of MECA-79 glycoprotein-determinant on developing B lymphocytes in gut-associated lymphoid tissues

Abstract

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.

Figure 1

MECA-79 is induced in developing B lymphocytes in GALT. (a) Proportions of MECA-79⁺ IgM⁺ B cells were determined in different tissues at the indicated age by flow cytometry (mean ± SD; n = 3–4 at each time point). (b) Five-week appendix sections were stained with anti-rabbit IgM (red) and MECA-79 antibody or rat IgM isotype control antibody (green). The merged panels show overlap of staining for IgM and MECA-79. The upper panel is at lower magnification showing follicular and interfollicular areas, the box indicates the interfollicular area. The middle panel shows higher magnification of the boxed interfollicular area, the arrow shows an IgM⁺ MECA-79⁺ B cell. The lower panel is from a serial section showing the interfollicular area at higher magnification. There was complete absence of rat IgM isotype control antibody staining in the lower panel. Scale bars = 100 µm.

Figure 2

MECA-79 antibody recognizes a sodium chlorate (sulphation inhibitor)-sensitive ∼127 000 MW O-sialoglycoprotein. (a) A single glycoprotein band of ∼127 000 MW molecular weight was detected by Western blotting in the protein extracts of lymphoid-cell suspension (L) from 6-week-appendix but not from 1-week-appendix. Stromal-cell preparations (S) containing vascular endothelial cells from both 1- and 6-week-appendix reacted with MECA-79 antibody; molecular weight marker (M). (b) Appendix sections from a 2-week-old rabbit were treated with O-sialoglycoprotease (middle and lower panels) or with PBS (upper panel) and stained with MECA-79 antibody (upper and middle panels) or anti-rabbit IgM (lower panel). (c) Appendix cells from 3–6-week-old rabbits were cultured for 20 hr in-vitro in the absence or presence of different concentrations of sodium chlorate, a metabolic inhibitor of sulphation. After culture, cells were washed and proportions of IgM⁺ MECA-79⁺ B cell were determined (mean ± SD; n = 3). Scale bars = 100 µm.

Figure 3

MECA-79 determinant is expressed in immunized splenic germinal centres. Splenic sections from BGG immunized and unimmunized adult rabbits were stained with BGG-biotin followed by PE-streptavidin (red) and FITC-MECA-79 antibody (green). Scale bars = 100 µm.

Figure 4

FITC-labelled MECA-79⁺ B lymphocytes exit appendix and migrate into MLN. Appendix cells were labelled in-situ with FITC. After 18–20 hr, cells from appendix, MLN and peripheral blood were harvested and analysed by flow cytometry for MECA-79 and IgM expression on FITC⁺ cells. (a) Average percentages (n = 6) of MECA-79⁺ cells in the FITC⁺ lymphocytes gate in appendix, MLN and peripheral blood. Out of 11 rabbits injected with FITC, 6 were analysed for MECA-79 and IgM. (b) Three-colour flow cytometry results on an individual representative animal. Percentages shown in the upper right quadrant are MECA-79-PE⁺ lymphocytes within FITC⁺ population. The histogram shows anti-rabbit IgM-PerCP staining on FITC⁺ MECA-79⁺ lymphocytes. (c) The correlation between proportions of FITC⁺ lymphocytes within the lymphocyte gate in appendix and MLN was estimated by curve fit analysis. The correlation was highly significant (r² = 0·84; P < 0·001; n = 11); out of these 11 FITC-injected rabbits, 6 (closed circles) were analysed for MECA-79 in (a) (d) Most of the green FITC⁺ cells were found in B-cell areas (anti-IgM-PE; red) in a MLN section. Scale bar = 100 µm.

Figure 5

P-selectin is expressed in lymphatics of the T-dependent area. Five-week appendix sections were stained with anti-rabbit IgM (red; upper left panel) and a serial section with anti-P-selectin antibody (green; upper right panel). Solid box shows P-selectin⁺ lymphatics in an interfollicular thymus-dependent area and straight lymphatics (lower right panel at higher magnification). The dashed-box shows that perifollicular lymphatics are P-selectin-negative (lower left panel at higher magnification). Scale bars = 100 µm.