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. 2006 Dec 1;119(4):461–469. doi: 10.1111/j.1365-2567.2006.02457.x

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

Rajesh K Sinha 1, Guibin Yang 1, Cornelius Alexander 1, Rose G Mage 1
PMCID: PMC2265829  PMID: 17177829

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 ∼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.

Keywords: MECA-79, sulphated O-linked glycoprotein, B-lymphocyte exit, appendix, rabbit

Introduction

Following activation of lymphocytes, the array of glycoproteins displayed on the cell surface is altered. These molecules participate in processes associated with cell functions after activation including modifying cell adhesion and migration. Compared to resting B cells, activated human B cells have been shown to express different carbohydrate epitopes.1,2 These newly expressed carbohydrate epitopes serve as ligands for selectins such as CD62E and CD62P.1,3 The role of selectins and their carbohydrate ligands in leucocyte recruitment across vascular endothelium is well established.4,5 Binding of lymphocyte CD62L to its carbohydrate ligands on high endothelial venules (HEV) in peripheral lymph nodes initiates lymphocyte rolling along the lumenal surface of HEV.5,6 The MECA-79 antibody detects a CD62L-ligand7 that contains a sulpho-sialyl-Lewis-x determinant that is present on different protein backbones.8 This group of glycoproteins, defined by reactivity with the antibody MECA-79, is termed peripheral lymph node addressin (PNAd). The MECA-79 antibody reactivity was initially reported to be restricted to postcapillary endothelial venules in lymphoid tissues and at the sites of chronic inflammation.5 Recently, however, human uterine epithelial cells were found to up-regulate MECA-79 reactive oligosaccharide-based CD62L-ligands during the phase of trophoblast implantation.5,9

Although the processes of leucocyte entry from the blood into the tissue are relatively well understood, exit of these cells from the tissue into the lymphatics has attracted much less attention. In the past few years, certain adhesion molecules such as the macrophage mannose receptor and common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) have been shown to support binding of lymphocytes to lymphatic endothelium.10,11

In young rabbits, early B-cell Ig-repertoire diversification occurs in the appendix and other gut associated lymphoid tissues (GALT).12–14 From peripheral blood, immunoglobulin M (IgM)+ B cells seed the appendix.15,16 In the presence of gut flora, massive clonal expansion, diversification, and selection occurs; B cells then exit the appendix to form the preimmune repertoire in the periphery.13,14,17,18 In this study we found that the MECA-79 epitope is expressed on developing B lymphocytes in Peyer's patches, appendix and on immunized splenic germinal centre B lymphocytes. MECA-79+ B lymphocytes exit appendix and migrate into draining mesenteric lymph nodes. Interaction of the MECA-79 epitope and CD62P on lymphatic endothelium may be involved in the exit of B cells from appendix into lymphatics.

Materials and methods

Animals

Rabbits of the 2R1 haplotype (VH1a2) were from the allotype-defined colony of the National Institute of Allergy and Infectious Diseases. Animal studies (ASP LI-6) were reviewed and approved by the animal care and use committees of NIAID and of Spring Valley Laboratories where the animals were bred and housed. Rabbits were killed at different ages and appendix, Peyer's patches, mesenteric lymph nodes (MLN), spleen and heparinized peripheral blood were collected. The appendices and Peyer's patches were removed and washed two to three times in chilled phosphate-buffered saline (PBS) to remove faecal material. The tissues were chopped into small pieces in PBS and pressed through a stainless steel mesh screen with a rubber-tipped syringe plunger. Debris and cell clumps were removed by passing the suspension through 70 µm nylon mesh. Viable lymphocytes were isolated from heparinized peripheral blood by density gradient centrifugation using Lympholyte-Rabbit lymphocyte separation medium (Cedarlane, Ontario, Canada). Red blood cells were removed by treating the spleen and blood lymphocytes preparation with ACK lysing buffer (Cambrex Bio Science, MD) for 3–4 min at room temperature.

Antibodies

The primary antibodies/reagents used were fluoroscein isothiocyanate (FITC)- or peroxidase-conjugated goat anti-rabbit IgM (Southern Biotechnology Associates Inc., Birmingham, AL), rat anti-PNAd (MECA-79, BD Pharmingen, San Diego, CA), FITC rat anti-PNAd (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human P-selectin (NPL44-10, Takara Bio Inc., Shiga, Japan) that cross-reacts with rabbit P-selectin and bovine gammaglobulin (BGG)-biotin. Secondary antibodies/reagents were peroxidase goat anti-rat IgM, peroxidase goat anti-mouse IgG (Jackson Immunoresearch Laboratories Inc., West Grove, PA), phycoerythrin (PE) goat anti-rat IgM, PE goat anti-mouse IgG (Southern Biotechnology Inc.), streptavidin PerCP and streptavidin PE (BD Pharmingen).

Flow cytometry

Cells were resuspended in 100 µl PBS with 2% heat-inactivated fetal calf serum (FCS) (PBS-FCS), incubated with 1–2 µg of primary antibodies on ice for 40 min and then washed twice with PBS-FCS. The cells were further incubated for 40 min in 100 µl PBS-FCS containing fluorescent-tagged secondary antibodies and 0·25 µg of fluorochrome labelled goat anti-rabbit IgM antibody. The cells were again washed twice and analysed immediately using a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA). Appropriate isotype controls were included.

Immunohistochemistry and -fluorescence analyses

Appendices of 5–6-week-old rabbits were cut into 2-mm slices, submerged in O.C.T. compound, quickly frozen in a slurry of dry ice and 2-methylbutane (Sigma-Aldrich, St Louis, MO), and preserved at −70°. Six µm serial sections were cut at −20° using an IEC cryostat microtome (Sakura Finetechnology Co., Tokyo, Japan) and stored at −25°. Sections were fixed in acetone for 10 min and rehydrated with PBS for 5 min at room temperature. Non-specific binding was blocked with 10% normal goat serum incubation for 30 min. One µg of the primary antibody was diluted in PBS containing 10% normal goat serum and incubated on the sections for 30 min. The sections were then washed with PBS four to five times and further incubated with 0·5 µg secondary antibody in blocking serum. When peroxidase-conjugated antibodies were used, cells were visualized either with Vector Blue or DAB substrate (Vector Laboratories Inc., Burlinghame, CA). Slides were coverslipped with aqua mount (ThermoShandon, Pittsburgh, PA). In experiments where fluorochrome-labelled secondary antibodies were used, sections were coverslipped with Prolong antifade mounting medium (Roche Diagnostics, Mannheim, Germany).

O-sialoglycoprotease treatment of appendix tissue sections

Appendix serial sections from 2-week-old rabbits (6 µm thickness) were treated with O-sialoglycoprotease enzyme (48 µg/100 µl PBS; Cedarlane Laboratories Ltd) at 37° for 2 hr in a humidified chamber. The sections were washed in PBS and stained with MECA-79 or anti-rabbit IgM antibodies separately as described above.

Immunoblotting with MECA-79

Appendices from a 1- and 6-week-old rabbit were cleaned with chilled PBS, cut into small pieces and pressed through a stainless steel mesh screen with a rubber-tipped syringe plunger until most of the loosely held lymphocytes were separated. Total proteins were separately extracted from fibrous stromal and vascular elements, and from lymphocyte suspensions by using T-PER tissue protein extraction reagent and Halt protease inhibitor cocktail kit (Pierce, Rockford, IL). Eighteen µg of protein per lane were electrophoresed in 5–14% linear sodium dodecyl sulphate–polyacrylamide electrophoresis gel under reducing condition (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to a nitrocellulose membrane using standard methods. The membranes were blocked overnight in 2% blocking buffer (Roche Diagnostics, Mannheim, Germany). The membranes were probed with rat anti-PNAd (MECA-79) antibody or with rat IgM isotype control (40 µg in 10 ml of 0·5% blocking buffer) for 1 hr with shaking and washed twice, 15 min each, with 0·1% Tween-20 Tris-buffered saline (TTBS) and then twice, 15 min each, with 0·5% blocking buffer in TBS. The membranes were incubated with anti-rat IgM-peroxidase (18 µg in 10 ml of 0·5% blocking buffer) for 30 min with shaking and washed four times, 15 min each, with TTBS. Blots were developed using a chemiluminescence kit (Roche Diagnostics) and exposed to Biomax photographic plates (Eastman Kodak Co., New York, NY).

Inhibition of expression of MECA-79 determinant by sodium chlorate

Appendix cells (3 × 106) were incubated for 20 hr at 37° in 1 ml of complete RPMI-1640 medium (Sigma-Aldrich) containing 30% heat-inactivated FCS in absence or presence of different concentrations (10–40 mm) of sodium chlorate (Sigma-Aldrich), a metabolic inhibitor of sulphation. At the end of the culture period, cells were harvested, washed with PBS-FCS and stained with anti-rabbit IgM and MECA-79 antibodies. Samples of freshly isolated appendix cells were similarly stained.

Bovine gamma globulin (BGG) immunization

Rabbits were immunized subcutaneously with BGG (0·5 mg in complete Freund's adjuvant) followed by an intravenous boost (0·5 mg in PBS) 10 days later. Spleens were collected 11 days after the boost and stored in O.C.T compound at −70°.

Labelling of appendix cells with FITC

A longitudinal incision of approximately 5 cm was made in the skin and muscles below the umbilicus to reach the appendix. Using a 30-gauge needle, a total of 1 ml FITC (0·5 mg/ml, Sigma-Aldrich) was injected into the lumen of the appendix distributed at five to six different sites. The exact amount of FITC injected into appendix varied in experiments done on different dates in part because FITC did not completely dissolve in PBS. To avoid leaking of FITC from the appendix, the lumen of the appendix was closed for 5 min by holding the base of appendix firmly between the thumb and index fingers. Incisions were sutured, and the rabbits were kept warm on heated pads during recovery. At 18–20 hr postinjection, appendix, Peyer's patches, MLN, spleen and heparinized peripheral blood were collected for analyses by flow cytometry. Tissues were also frozen in Tissue-Tek O.C.T compound (Sakura Finetek, Torrance, CA) for immunofluorescence analysis.

Statistics

Student's paired-t-test was employed and P-values less than 0·05 were considered statistically significant. Curve fit analysis and correlation coefficient were determined using KaleidaGraph software (Synergy Software, Reading, PA).

Results

MECA-79 is induced in developing B lymphocytes in GALT

From 3 days to 11 weeks of age, we determined the percentages of B lymphocytes expressing MECA-79 in appendix, Peyer's patches, MLN, spleen and peripheral blood by flow cytometric analyses using anti-rabbit IgM and MECA-79 (anti-PNAd) antibodies (n = 3–4 at each time point). There was a gradual increase of MECA-79+ B lymphocytes from < 0·5% at 3 days to 77·3 ± 1·8% at 6 weeks in appendix. A similar trend was observed in Peyer's patches. In contrast to GALT, spleen and peripheral blood had very low (< 2%) proportions of MECA-79+ B lymphocytes at all time points. In MLN, there were moderate proportions (5–24%) of MECA-79+ B cells (Fig. 1a).

Figure 1.

Figure 1

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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.

MECA-79 is expressed on B cells in follicular and interfollicular areas

Five-week appendix sections were stained with anti-rabbit IgM (red) and MECA-79 (green) antibodies. Both the follicular B cells (Fig. 1b; upper panel) and B cells in the T-cell area (Fig. 1b; middle panel) were MECA-79+. A serial section was stained with anti-rabbit IgM (red) and rat IgM isotype control for MECA-79 antibody (green). There was no staining with the rat IgM isotype control and there was no spillover from the red into the green channel (Fig. 1b; lower panel). The merged panels show overlap of staining for IgM and MECA-79.

MECA-79 antibody recognizes an ∼127 000 MW O-sialoglycoprotein on B cells

By Western blotting using MECA-79 antibody we detected a single glycoprotein band of approximately 127 000 MW in the protein extract from stromal elements of both 1- and 6-week-appendix. Single-cell suspension preparations, mainly containing lymphocytes, derived from 6-week appendix but not from 1-week appendix showed reactivity with the antibody (Fig. 2a). To further characterize the MECA-79 reactive epitopes, frozen sections from 2-week appendix were treated with O-sialoglycoprotease and then stained with MECA-79 antibody. The enzyme treatment abolished MECA-79 staining on B cells. The enzyme had no effect on IgM staining of B cells (Fig. 2b).

Figure 2.

Figure 2

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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.

Expression of MECA-79 is inhibited by sodium chlorate

MECA-79 antibody binds to a sulphated carbohydrate epitope of PNAd. To test whether MECA-79 antibody binding on appendix cells is sensitive to inhibition of sulphation, cells were cultured in the absence or presence of different concentrations of sodium chlorate, a metabolic inhibitor of sulphation, and analysed by flow cytometry 20 hr later. Figure 2(c) shows a dose dependent decrease in proportions of B lymphocytes expressing the MECA-79 epitope. The observed effect of sodium chlorate suggests that the MECA-79 epitope on these B cells is indeed sulphated. The percentages of live cells after the 20 hr in culture, as estimated by trypan blue exclusion, were similar in 40 mm sodium chlorate treated (47·8 ± 20·1%) and untreated samples (44·7 ± 13·5%; n = 3; P = 0·47).

MECA-79 determinant is expressed in germinal centres of spleen

The above flow cytometry data showed that in contrast to GALT, very few splenic B cells in healthy unimmunized rabbits expressed MECA-79 determinant. To investigate whether expression of MECA-79 determinant is GALT specific or is also expressed in germinal centres of peripheral lymphoid organ such as spleen, rabbits were immunized with BGG. Splenic sections from immunized and unimmunized controls rabbits were stained with BGG-biotin followed by PE-streptavidin and FITC-MECA-79 antibody. In immunized rabbits, BGG+ germinal centres were strongly MECA-79+. In contrast, unimmunized rabbits' spleens had only a few small clusters of MECA-79+ B cells (Fig. 3).

Figure 3.

Figure 3

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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.

O-linked sulfated glycoprotein+ (MECA-79 reactive) B lymphocytes exit appendix and migrate into MLN

MECA-79+ B cells originate in GALT (Fig. 1a). Although less than 2% of B cells were MECA-79+ in the periphery (spleen and blood), moderate proportions were present in MLN (5–24%). To test the hypothesis that MECA-79 is expressed on appendix B lymphocytes while they migrate through efferent lymphatics into MLN, we labelled appendix cells with FITC in situ. 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. The proportions of FITC+ MECA-79+ cells (within the FITC+ lymphocyte gate) were 54·1 ± 6·4, 9·1 ± 2·4 and 0·3 ± 0·2 (mean ± SD; n = 6) in appendix, MLN and peripheral blood, respectively (Fig. 4a). Almost all of these FITC+ MECA-79+ cells were IgM+ B cells (Fig. 4b). Further, the mean fluorescence intensity of MECA-79 staining was significantly lower in MLN FITC-labelled B cells (7·6 ± 1·3) compared to FITC-labelled appendix B cells (16·8 ± 2·6; P = 0·0008). To rule out the possibility that cells in the MLN were locally labelled by FITC draining through efferent lymphatics into MLN, we checked the correlation between proportions of FITC+ cells within lymphocyte gate in appendix and MLN by curve fit analysis (Fig. 4c). The correlation was highly significant (r2 = 0·84; P < 0·001; n = 11) indicating that at least 84% of variability in proportions of FITC+ MLN cells is explained by variability in proportions of FITC+ appendix cells. In a few experiments, proportions of FITC-labelled lymphocytes were very low. This could be caused by variability in total amount of FITC injected into appendix. In MLN tissue sections, most of the FITC+ cells were found in IgM+ B-cell follicles (Fig. 4d). If free FITC drained into MLN, we would have expected to see almost equal distribution of FITC labelled cells in B-cell follicular and T-dependent areas.

Figure 4.

Figure 4

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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.

P-selectin is expressed in lymphatics of interfollicular regions

We had shown that B cells in the interfollicular area were expressing O-linked sulphated glycoprotein (MECA-79+) and that MECA-79+ B cells were migrating from appendix into draining MLN. Thus, we investigated the expression of selectins on lymphatics that might be receptors for glycoprotein. Appendix sections were stained with anti-P-selectin antibody. We could detect expression of P-selectin on lymphatics in interfollicular T-cell areas and straight lymphatics (Fig. 5). P-selectin staining was not observed on the perifollicular lymphatics.

Figure 5.

Figure 5

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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.

Discussion

Rabbit is among a group of animals in which early development and maturation of B lymphocytes occurs in GALT. The CD62L-ligand, PNAd as detected by monoclonal antibody MECA-79, is expressed on HEV in appendix and supports recruitment of immature CD62L+ IgM+ B cells into rabbit appendix.16 These immature IgM+ B cells undergo further proliferation, immunoglobulin repertoire diversification and selection in appendix and other GALT such as Peyer's patches and sacculus rotundus [reviewed in 19]. Mature B cells then exit the appendix to form the preimmune repertoire in the periphery.13,14,17,18

We reported earlier16 that MECA-79+ HEV in appendix increased between 1 day and 1 week, remained relatively high until 2 weeks, and decreased rapidly thereafter. Whereas PNAd+ HEV were in close association with developing B-cell follicles in young appendix, by 6 weeks PNAd+ HEV were restricted to T-cell areas.16 Here we report that in contrast to PNAd+ HEV, a MECA-79 epitope appeared on B cells developing in GALT and the proportions of MECA-79+ B cells gradually increased after birth (<0·5% at 3 days to >70% at 6 weeks in appendix). The MECA-79 epitope was also detected on splenic germinal centre B cells after immunization. Western blotting, immunohistochemistry following O-sialoglycoprotease treatment of appendix sections, and partial inhibition of expression of the MECA-79 epitope by sodium chlorate indicated that the MECA-79 epitope on B cells is an approximately 127 000 MW O-linked sulphated glycoprotein. The Western blot results indicate that the MECA-79 reactive glycoprotein is of the same molecular weight in B lymphocytes and in stromal cell preparations containing endothelial cells. Although it is of interest to identify the protein backbone in B lymphocytes compared to HEV, attempts to obtain pure material for sequencing have thus far been unsuccessful. The MECA-79 antibody recognizes sulphated-N-acetyllactosamine.5 Although MECA-79 antibody reactivity towards lymphocytes has not been reported, other sulphated-glycans have been shown to be expressed by granulocytes and various carcinomas.20 These naturally occurring sulphated-glycans on granulocytes and carcinomas as well as in-vitro transfectants expressing a sulpho-Le(x) trisaccharide served as P-selectin ligands.20,21

Two of our observations indicate that the MECA-79 determinant might be involved in exit of B cells from appendix. First, almost all the B cells in the interfollicular thymus-dependent area were MECA-79+ and second, lymphatic endothelium in the thymus-dependent area expressed P-selectin. Lymphocytes were frequently observed migrating through lymphatic endothelial walls in the thymus-dependent area, but not through the walls of the perifollicular sinuses.22 This earlier study suggested that lymphocytes exit from the follicles into the lymphatics via the lymphatic plexus in the thymus-dependent area. Consistent with this anatomical study of rabbit appendix, we found that P-selectin was expressed in the thymus-dependent area and not in the perifollicular lymphatics. In situ labelling of appendix lymphocytes with FITC followed by flow cytometric analysis of FITC-labelled B cells in draining MLN and peripheral blood showed that at least 20% of B cells migrating from appendix into MLN were MECA-79+. The estimate of FITC-labelled MECA-79+ B cells in MLN is very conservative. We have observed that the MECA-79 determinant is down-regulated once B cells leave the appendix microenvironment and migrate into MLN. Further, the absence of detectable MECA-79 determinant on FITC-labelled B cells in peripheral blood supports the proposal that the MECA-79 determinant is gradually lost once B cells exit the appendix.

The other possibility is that the presence of MECA-79 determinant on B cells serves some function in enabling CD62L+ T cells and/or dendritic cells to interact with MECA-79 bearing B cells. Developing appendix follicles have been shown to harbour both CD4+ and CD8+ T lymphocytes23 and most of the T lymphocytes in appendix express CD62L.15 Recently, it has been shown that in nude rats, the peripheral phase of B-lymphocyte maturation and appropriate expression of surface molecules require T-lymphocyte help.24

In conclusion, developing B cells in GALT express an O-linked sulphated proteoglycan uniquely recognized by the monoclonal antibody MECA-79. This surface molecule was found to be expressed by activated B cells and may be involved in exit of developed B cells from GALT into draining MLN and/or function in enabling T–B-cell interaction.

Acknowledgments

We thank Drs K. Schueler and R. Schueler, and P. DeGrange for assistance with animal survival surgery and FITC injection into appendix. We thank Drs A. Anderson, M. Mage, B. Newman, R. Pospisil, G. Rai, S. Ray and N. Puliyath for helpful advice during the conduct of these studies and for comments about this manuscript. This research was supported by the intramural research program of the NIH (NIAID).

Abbreviations

BGG

bovine gamma globulin

FCS

fetal calf serum

GALT

gut-associated lymphoid tissues

HEV

high endothelial venules

MLN

mesenteric lymph nodes

PBS-FCS

phosphate-buffered saline with 2% heat-inactivated fetal calf serum

PNAd

peripheral lymph node addressin

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