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(Journal of Leukocyte Biology. 2000;68:807-814.)
© 2000 by Society for Leukocyte Biology

Expression of {alpha}4ß7 and E-selectin ligand by circulating memory B cells: implications for targeted trafficking to mucosal and systemic sites

Lusijah S. Rott*, Michael J. Briskin{ddagger} and Eugene C. Butcher{dagger}

* Laboratory of Immunology and Vascular Biology, Department of Pathology and the Digestive Disease Center, Stanford University, Stanford, California;
{dagger} Center for Molecular Biology and Medicine, Veterans Administration Medical Center, Palo Alto, California
{ddagger} LeukoSite, Inc., Cambridge, Massachusetts

Correspondence: Lusijah S. Rott, Veterans Administration Medical Center, 3801 Miranda Ave., 154B, Palo Alto, CA 94304. E-mail: lrott{at}cmgm.stanford.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have examined the expression of homing receptors on circulating memory B cells subsets. Blood IgD+ (naive) B cells homogeneously express a high level of intestinal homing receptor, {alpha}4ß7, but IgD- (putative memory) B cells comprise distinct {alpha}4ß7+ and {alpha}4ß7- subsets. Naive and {alpha}4ß7+ memory B cells but not {alpha}4ß7- cells bind MAdCAM-1, suggesting that {alpha}4ß7 expression may predict B cell intestinal homing. In contrast, {alpha}4ß7+ and {alpha}4ß7- B cells bind well to VCAM-1, possibly allowing recruitment of both subsets to extra-intestinal sites, including those tissues of the "common mucosal immune system" characterized by vascular VCAM-1 expression. sIgA+ B cells, which are associated with mucosal immunity in the gut and elsewhere, are heterogeneous in homing receptor expression—with discrete subsets expressing {alpha}4ß7, L-selectin, and cutaneous lymphocyte antigen (CLA). sIgA+ CLA+ B cells are enriched by binding to E-selectin, suggesting that CLA may participate in B cell homing to nonintestinal mucosal tissues characterized by vascular E-selectin expression, such as chronically inflamed bronchial or oral mucosal. We conclude that circulating human peripheral blood memory B cells, like T cells, consist of discrete homing receptor-defined subsets. This diversity in homing phenotypes is apparent even among sIgA (presumptive mucosal) memory B cells, implying heterogeneity in trafficking mechanisms to different target mucosal surfaces.

Key Words: mucosal immunity • cutaneous lymphocyte antigen (CLA) • MAdCAM-1 addressin • IgA • homing • trafficking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peripheral blood leukocytes express homing receptors (HR), which help determine tissue-specific patterns of trafficking to lymphoid and extra-lymphoid tissues and sites of inflammation [1 ]. Naive T cells express L-selectin (first identified as a peripheral lymph node HR) uniformly and moderate levels of the intestinal HR {alpha}4ß7. These HR are important for naive cell interactions with lymphoid organ high-endothelial venules (HEV) initiating their entry into organized lymphoid tissues, including Peyer’s patches (PP) of the gut wall [1 , 2 ]. Whereas naive lymphocytes are homogeneous in their trafficking properties, memory T cells are heterogeneous [3 ]. Memory T cells, which embody memory for intestinal antigens, express {alpha}4ß7 [4 ] and traffic to intestinal PP [5 ] and lamina propria. {alpha}4ß7+ T cells interact with the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) [6 ], which is displayed on HEV of gastrointestinal lymphoid organs (PP and mesenteric lymph node) and venules in the intestinal lamina propria and lactating mammary gland [reviewed in ref. 1 ]. Conversely, memory T cells for systemic antigens traffic to nonintestinal inflammatory sites, often utilizing {alpha}4ß1 integrin interaction with vascular cell adhesion molecule-1 (VCAM-1) [6 ], which decorates venules in diverse sites of inflammation but is observed rarely on endothelium in intestinal tissues [7 8 9 ]. T cells that embody memory for cutaneous antigens constitute a subset of systemic ({alpha}4ß7-) memory T cells and are identified by the expression of cutaneous lymphocyte antigen (CLA), a T cell receptor for vascular E-selectin [reviewed in refs. 1 10 ].

HR expression on B cells is less well-characterized, especially on circulating memory cell populations. B cell lines and peripheral blood B cells can interact with MAdCAM-1 via {alpha}4ß7 [6 , 11 ]. Immunohistologic studies reveal {alpha}4ß7 on immunoglobulin (Ig)A+ B cell blasts and plasma cells in the lamina propria and on some B cell blasts in gut-associated lymphoid tissue (GALT) such as PP and appendix [12 ]. It is interesting that systemic vs. oral immunization induces antibody-secreting cells (ASC) with different HR phenotypes. Use of magnetic-bead isolation procedures suggest significant differences in peripheral lymph node HR L-selectin and {alpha}4ß7 expression by ASC specific for systemically vs. orally administered antigens: Systemic antigen exposure results in a higher incidence of specific ASC among the anti-L-selectin-enriched population, whereas oral exposure results in a higher incidence of specific ASC among cells that bind beads coated with monoclonal antibody (mAb) to {alpha}4ß7 [13 , 14 ]. These studies suggest that there is significant heterogeneity in HR expression among effector B cells and their immediate precursors, circulating Ab-secreting plasmablasts. Another study has demonstrated staining of a subset of memory B cells with mAb HECA-452 [15 ], which recognizes E-selectin binding ligands on other cells [16 ] including the CLA on T lymphocytes. Preliminary studies indicate heterogeneity in {alpha}4ß7 expression by circulating small B cells [6 , 11 ] as well, but this has not been related to memory vs. naive subsets nor to expression of other HR.

Surface IgA expression on memory B cells is believed to be associated with mucosal immunity, including immunity to intestinal antigens. Intestinal PP contain a relatively high frequency of sIgA+ B cells [17 ], and it is now thought that these cells can embody "memory" in adoptive transfer experiments and can give rise to IgA-secreting plasma cells [18 ]. sIgA+ cells are frequent among PP germinal center cells but are not observed among peripheral lymph node germinal center cells following subcutaneous immunization [19 ]. Further evidence for the association of IgA with intestinal immunity is that oral ingestion of antigen induces the transient appearance of sIgA+ B cells in the blood [20 ]. However, oral and nasal immunizations generate IgA response in diverse sites of the "common mucosal immune system" including the gastrointestinal, genital, and bronchial tracts, and secretory glands such as salivary, parotid, lachrymal, cervical, and mammary glands [21 22 23 ]. IgA plasma cells are abundant in the bone marrow as well [24 ], and IgA has been identified in the prostate and urethral glands [25 ]. Among these sites, only gastrointestinal [26 , 27 ], lactating mammary [26 ], and, to a lesser extent, inflamed fallopian tubes [28 ] have been shown to express high levels of MAdCAM-1 in vessels involved in lymphocyte recruitment, suggesting that distinct molecular pathways and "recognition cascades" may control IgA+ lymphocyte subset homing to intestinal vs. other mucosal tissues.

In the present study, we examine the expression of the intestinal and cutaneous HR and of L-selectin by circulating B cells in man, and we correlate this with binding to vascular ligands and with sIg isotype expression, especially IgA. We demonstrate the existence of discrete HR-defined subsets of circulating memory B cells, including presumptive intestinal ({alpha}4ß7+) vs. nonintestinal ({alpha}4ß7-) B cells, and of a memory B cell subset that expresses CLA. We show further that B cell HR confer a specific subset binding function to vascular ligands MAdCAM-1, VCAM-1, and E-selectin. There is significant heterogeneity in {alpha}4ß7, CLA, and L-selectin expression even among sIgA+ B cells, suggesting that distinct mechanisms of lymphocyte-endothelial cell or adhesion/activation cascades may mediate sIgA+ B cell recruitment to diverse mucosal effector sites.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell preparation
Peripheral blood was collected from healthy adult volunteers, and mononuclear cells were obtained after a Ficoll-Hypaque (Histopaque 1077, Sigma Chemical Co., St. Louis, MO) gradient separation. Cells were washed twice with Hanks’ balanced saline solution (HBSS) without Ca/Mg and resuspended in phosphate-buffered saline (PBS) with 2% fetal calf serum (FCS) and 0.1% azide for fluorescein-activated cell sorter (FACS) staining or a binding buffer for adhesion assays. The binding buffer was prepared from powder RPMI without sodium bicarbonate supplemented with 2% FCS and 10 mM HEPES and adjusted to pH 7. In some FACS-staining experiments, B cells were purified with magnetic beads (Dynal, Great Neck, NY) followed by detachment of the antibody-bead complex with Dynal Detach-a-Bead, as per protocol. The average purity was 99.0%.

mAbs
FACS phenotype analysis was performed with the following mAbs. CD19 (B cell) fluorescein isothiocyanate (FITC) and HML-1 ({alpha}E) [29 ] were purchased from Immunotech (Westbrook, ME). In addition, we used another {alpha}E antibody (Beract-8) [30 ], which was a gift from Harold Stein (Free University, Berlin, Germany). FITC and biotin-conjugated goat anti-human Ig isotype reagents were obtained from CALTAG (South San Francisco, CA) except for anti-human IgD, which was purchased in purified form and conjugated in our own laboratory. Act-1 (anti-{alpha}4ß7 heterodimer) [31 ] was a kind gift from LeukoSite, Inc. (Cambridge, MA) and was used unconjugated or as Act-1 phycoerythrin (PE) after conjugation by Becton Dickinson (San Jose, CA). Rat anti-human ß1 (AIIBII) was a kind gift from Caroline Damsky at the University of California at San Francisco. Anti-L-selectin (DREG 56), anti-CLA (HECA 452) [32 ], rat IgM control Ab 79, and rat anti-ß7 (FIB504) [33 ] antibodies were produced in our laboratory and used as unconjugated, FITC, or biotinylated reagents. CD19 Allophycocyanin (APC), CD19 CyChrome, mouse isotype controls, streptavidin PE, and streptavidin cychrome were purchased from PharMingen (La Jolla, CA). Streptavidin PerCP was obtained from Becton Dickinson. Mouse antirat PE second stage was obtained from Chromoprobe (Mountain View, CA). Use of this mouse derived second-stage eliminated reactivity with other nonconjugated mouse-derived antibodies. Human-absorbed goat anti-mouse PE and FITC were from Biosource (Camarillo, CA). The following mAbs were used for sorting transfected cell lines: Mouse anti-human MAdCAM-1 (8C1) was produced in the laboratory of Michael Briskin at LeukoSite; anti-human VCAM-1 (TY1138) was a gift from Ted Yednock at Athena Neurosciences (South San Francisco, CA); and anti-E-selectin was purchased from PharMingen.

Adhesion assay
Stable human MAdCAM-1 [27 ], VCAM-1 (a gift from Ted Yednock at Athena Neurosciences), and E-selectin [34 ] transfectants were used for adhesion assays. The brightest 1–5% of cells were sorted periodically to maintain high-expression levels. The adhesion assay has been described previously [6 ]. Briefly, human peripheral blood mononuclear cells were added to the plates with previously plated transfectants and rotated at 50 rpm for 30 min at room temperature on a model 2G Gyrotary Shaker (New Brunswick Scientific, Edison, NJ). Nonadherent cells were removed from the plates and reserved for FACS staining as the unbound fraction. The bound cells were removed from the plate with a 1–2 min treatment with 5 mM ethylenediaminetetraacetate (EDTA), which removed only the bound mononuclear cells and not the adherent transfected cells. A reserved portion of the starting cells, transfectant bound, and unbound cell populations was then spun down and resuspended in PBS with 2.5% newborn calf serum (NCS) and 0.1% azide in preparation for FACS staining.

Cell staining and flow cytometric analysis
Cells (5x104–0.5x106) were used for each sample. Cells were stained on ice for 20 min and washed in the PBS buffer given above. Staining for multicolor analysis took place in four steps. When we were staining with mouse and rat unconjugated antibodies, 100 µl supernatant or 1 µg mouse antibody or control isotype was added to cells first. After incubation and washing, a second-stage goat F(ab')2 anti-mouse FITC or PE was added, followed by another wash. To avoid second-stage cross-reactivity with following Abs, the second stage was blocked for 10 min by the addition of 10 µl 10% normal mouse and rat serum as appropriate. Depending on the protocol, conjugated goat anti-human Ig isotype, mouse (including biotinylated), and unconjugated rat antibodies were then added and washed. Finally, streptavidin cychrome or PerCP and mouse F(ab')2 antirat PE (if unconjugated rat Abs had been used) were added, incubated, and washed. Samples were fixed with 1% paraformaldehyde before acquiring data.

FACS analysis was performed on standard FACScan or FACScaliber flow cytometers (Becton Dickinson). Set-up of the instrument was carried out by eye, optimizing settings for the unstained control samples. Compensation was adjusted with control samples’ stained antibodies reactive with mutually exclusive populations (CD19 FITC or APC, CD56 PE, and CD4 biotin plus PerCP). Contour plots represent data with 18% probability contour lines with one smooth.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{alpha}4ß7 expression by a subset of memory B cells
We examined the distribution of intestinal HR {alpha}4ß7 on memory and naive B cells initially using sIgD expression as a marker of naive phenotype [35 ]. Figure 1A illustrates representative FACS plots comparing expression of ß7 integrin on memory and naive B cells and CD4+ T cells. Memory (IgD-) B cells were subdivided clearly into ß7+ and ß7- subpopulations. On average, the ß7+ memory subpopulation was 10.2% ± 4.2 SD (N=11) of the total CD19+-gated cells, and the ß7- memory subpopulation was 9.1% ± 5.3 SD. IgD+ (naive) B cells are uniformly ß7+.



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  		Figure 1. (A) IgD- B cells are subdivided into distinct {alpha}4ß7+ and {alpha}4ß7- subpopulations. The representative contour plots show similarity of gated CD19+ B cell and CD4+ T cell phenotypes with respect to mucosal HR expression on the memory subsets (N=11). (B) The histograms demonstrate that FIB504 (anti-ß7) and Act-1 (anti-{alpha}4ß7) stain identical numbers of B cells. FACS staining of B cells with two different {alpha}E antibodies (unpublished results) demonstrates lack of detectable {alpha}E on B cells and justifies use of Act-1 or FIB504 to identify {alpha}4ß7+ cells. (C) B cells have little if any reactivity to anti-{alpha}E antibodies (N=5). The staining for {alpha}E and the isotype control were performed with primary {alpha}E or nonspecific {gamma}1 antibodies, followed by goat anti-mouse FITC.

 
ß7 can form heterodimers with {alpha}E. In peripheral blood, {alpha}Eß7 is expressed primarily on CD8+ T cells. We evaluated {alpha}E expression and association with ß7 in several ways and found few if any {alpha}Eß7-expressing peripheral blood B cells. As shown in histograms of gated memory B cells (Fig. 1B) , antibodies against {alpha}4ß7 (Act-1) and ß7 (FIB504) identify indistinguishable populations in peripheral blood. The presence of an {alpha}Eß7 population would be indicated as an increased FIB504 over Act-1 signal, because FIB504 reacts with {alpha}4ß7 and {alpha}Eß7, and Act-1 reacts only with {alpha}4ß7. In addition, we stained B cells with anti-{alpha}E antibodies (HML-1 and Beract-8) and found in five individuals examined that <1% of memory B cells expressed detectable {alpha}E (Fig. 1C) . Staining intensity of these positive cells was low and nondiscreet. In two of the five samples, staining was at isotype-control levels (<0.5%) or below. IgD- B cells with an increased forward light scatter, presumed immunoblasts, were similar except that one of five individuals had 5% {alpha}E+ cells among gated IgD- B cell (CD19+) blasts. We conclude that on circulating blood B cells, ß7 associates primarily with {alpha}4, and its expression defines a discrete {alpha}4ß7+ (Act-1+) subset of memory cells with levels of the intestinal HR similar to those on presumptive naive (IgD+) B cells and on {alpha}4ß7hi memory T cells.

Correlation of HR with sIgA and sIgG expression
Secreted IgA provides humoral protection at diverse mucosal surfaces, including the gastrointestinal tract. We asked whether intestinal HR expression might correlate with surface IgA display. Flow cytometric analysis revealed heterogeneity of isotype expression by {alpha}4ß7+ B cells, as shown in Figure 2 . Although most sIgA+ B cells were {alpha}4ß7+ (65%±16.2 SE, N=16), there was consistently a significant sIgA+ subset, which was {alpha}4ß7-. IgG+ B cells were likewise {alpha}4ß7+ and {alpha}4ß7-,with a minority being {alpha}4ß7+ (38.6%±9.6 SE, N=16). The ratio of {alpha}4ß7+ to {alpha}4ß7- cells among sIgG+ B cells was 0.7±0.2 SE, N=16. Thus, roughly two-thirds of sIgA+ cells but only one-third of sIgG+ B cells expressed {alpha}4ß7. sIgM+ B cells had a high level of {alpha}4ß7 expression generally, which is consistent with expression of this isotype by the predominant µ{delta}+ naive B cells. We did observe consistently, however, a small sIgM+ subset lacking {alpha}4ß7. The control, nonspecific, goat-derived antibody had <0.5% background-binding.



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  		Figure 2. Heterogeneity of ß7 expression by sIgA+, sIgG+, and sIgM+ B cells. Representative contour plots show staining of FIB504 vs. IgA, IgG, and IgM on gated CD19+ B cells (N=16).

 
We examined the expression of other HR on sIgA+ and sIgG+ B cells also. Figure 3 shows contour plots of {alpha}4ß7 vs. CLA and L-selectin expression on purified peripheral blood B cells, which were gated for IgA or IgG expression. A significant number of sIgA+ B cells expressed CLA (average 12.5%±2.1 SE, N=5). Among sIgA+-gated B cells, CLA+ cells were about fivefold more abundant among the {alpha}4ß7- than the {alpha}4ß7+ fraction (average 10.4% of {alpha}4ß7- compared with 2.1% of {alpha}4ß7+ IgA+ B cells). Although there was variability in the percentage of sIgG+ B cells expressing CLA (mean 5.5%±1.2 SE, N=4), most were in the {alpha}4ß7- subpopulation also (average 4.6%±1.9 SE of {alpha}4ß7- vs. 0.9%±0.2 SE of {alpha}4ß7+). In all individuals, sIgA+ B cells had a higher frequency of CLA+ cells than sIgG+ B cells (average 3.6-fold). Staining with a nonspecific FITC-labeled rat IgM, used as an isotype control for CLA FITC, demonstrated negligible background staining (<0.2%). [Examination of tonsillar lymphocytes revealed similar results (unpublished results, N=3) but with higher numbers of CLA+ cells and more equal distribution of CLA expression among IgA+ and IgG+ B cells. On average, 20.4%±4.6 SE of sIgA+ tonsil B cells coexpressed CLA, whereas 17.6%±2.4 SE (N=3) of sIgG+ B cells were CLA+.] Peripheral blood sIgA+ B cells displayed heterogeneous levels of L-selectin also. Those expressing the most L-selectin had a lower level of {alpha}4ß7, and {alpha}4ß7 bright cells had a broader range of L-selectin (top left plot in Fig. 3 ). In contrast, sIgG+ B cells were more uniformly L-selectin+ with a unimodal if broad pattern, independent of {alpha}4ß7 expression (lower left plot in Fig. 3 ). Because L-selectin expression is essential for efficient extravasation via lymphoid HEV, expression of L-selectin may allow circulating memory sIgA+ and sIgG+ B cell access to secondary lymphoid tissues, potentially including lymph nodes as well as PP and appendix.



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  		Figure 3. HR expression by gated sIgA+ and sIgG+ B cells. CLA (cutaneous lymphocyte HR) is expressed on a significant number of IgA+ B cells, primarily ß7-, and to a lesser extent on IgG+ B cells. Expression of L-selectin is brighter on the {alpha}4ß7- IgA+ subset generally and has a more varied expression level on the {alpha}4ß7+ subset. Gated IgG+ B cells display a unimodal of L-selectin. The three-color staining for these plots was performed on B cells, which had been purified with Dynal magnetic beads, from the same individual. Contour plots show IgA+-gated data on 80,000 B cells.

 
{alpha}4ß7 expression correlates with binding to MAdCAM-1, but {alpha}4ß7+ and {alpha}4ß7- B cells bind VCAM-1
Peripheral blood T cells bind to MAdCAM-1 via {alpha}4ß7, whereas binding to VCAM-1 is mediated predominantly by {alpha}4ß1 and in small part to {alpha}4ß7 [6 ]. In addition, even when normalized for {alpha}4ß7 levels, memory phenotype CD4+ T cells bind MAdCAM-1 better than naive phenotype cells [6 ]. To examine whether B cells behave similarly, we compared B cell binding with MAdCAM-1 and VCAM-1 transfectants. Figure 4A shows contour plots of B cell-starting and -bound fractions from a representative experiment. Cells that bound MAdCAM-1 transfectants exhibited a very high purity of {alpha}4ß7+ expression (98.7±0.4 SE, N=5). Binding of B cells to MAdCAM-1 transfectants was inhibited an average 94.3% ± SD 2.5 by anti-MAdCAM-1 Ab 8C1 and 98.2% ± SD 0.66 by anti-ß7 antibody FIB504 (N=2). Examination of IgD vs. {alpha}4ß7 expression on MAdCAM-1-bound B cells demonstrated that memory status (IgD- phenotype) is not a factor in binding avidity (in contrast to CD4+ T cells, among which memory {alpha}4ß7+ cells bind significantly better). The graph in Figure 4B gives a representation of relative changes in the naive IgD+, {alpha}4ß7+, and {alpha}4ß7- memory B cell subsets, presenting the percentage change in frequency of each subset in bound vs. starting populations. The IgD naive subset was not enriched significantly or depleted after binding MAdCAM-1 or VCAM-1. This suggests that B cell binding to MAdCAM-1 is dependent only on level of {alpha}4ß7 expression, which is similar for naive and memory subsets. As expected, sIgA cells were more prominent among the MAdCAM-1-bound (mean 3.6%, range 0.6–6.7, N=5, unpublished results) compared with the starting B cells (mean 2.0%, range 0.2–3.3, N=5), reflecting a twofold enrichment.



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  		Figure 4. (A) MAdCAM-1 binds {alpha}4ß7+ B cells selectively. IgD- {alpha}4ß7+ (memory) and IgD+ (naive) B cells bind equally well. Although {alpha}4ß7+ and {alpha}4ß7- memory B cells bind VCAM-1, the {alpha}4ß7- memory subset is somewhat enriched. (B) The line graph illustrates percentage of initial frequency of {alpha}4ß7+ and {alpha}4ß7- memory B and naive cell subsets following binding to VCAM-1 and MAdCAM-1 transfectants. The graph represents data from four experiments, and the error bars are SE.

 
We examined binding to VCAM-1 also as shown in Figure 4A . {alpha}4ß7+ and {alpha}4ß7- memory B cells, as well as naive B cells, bound to VCAM-1 transfectants. Similar results were observed previously for T cell subsets. The graph in Figure 4B illustrates that the {alpha}4ß7- subset was the most enriched among B cells that bind VCAM-1, but the {alpha}4ß7+ memory subset bound well to VCAM-1 also. The ability of {alpha}4ß7+ lymphocytes to interact with VCAM-1 may reflect their expression of significant levels of {alpha}4ß1 [6 ], which dominates lymphocyte binding to VCAM-1 [36 , 37 ]. Clearly, {alpha}4ß7- memory B cells were excluded from interaction with MAdCAM-1.

E-selectin binding B cells are enriched for expression of CLA
CLA is a carbohydrate-associated epitope that mediates binding of cutaneous memory T cells to E-selectin [16 , 38 ]. To determine if B cells also bind E-selectin, we evaluated the phenotype of B cells bound and eluted from E-selectin-transfected Chinese hamster ovary (CHO)-k cells. As shown in Figure 5 , the CLA+ B cell fraction was enriched drastically in the adherent population. An average of 3.7% ± SE 0.43 (N=7) B cells expressed CLA in the starting fraction. The percentage of B cells expressing CLA jumped to an average 32.0% ± SE 4.9 (N=7) after binding to E-selectin transfectants. We also examined binding to nontransfected control CHO-k cells. Although the background was significant (20.7%±SE 6.0, N=4), there was no enrichment of CLA+ cells; the CHO-k-bound profile looked identical to the starting fraction. As predicted by increased expression of CLA on IgA+ B cells, there was an enrichment of IgA+ B cells among B cells binding E-selectin transfectants (4.9%±0.9 SE in starting fraction compared with 12.9%±0.9 SE in E-selectin-bound population, N=7). The graph in Figure 5B illustrates the -fold increase of CLA+ cells comparing the starting peripheral blood B cells with E-selectin-adherent populations of CLA+ and CLA- B cells, and CLA+ and CLA- IgA+ B cells.



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  		Figure 5. (A) E-selectin binds CLA+ B cells preferentially. Dot plots illustrate enrichment for CLA-expressing B cells, notably those that are IgA+, after binding to CHO-k E-selectin transfectants. (B) The line graph illustrates the percentage of intitial frequency of selected lymphocyte subsets after binding CHO-k control and CHO-k E-selectin transfectants (N=7, with SE bars).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results reveal a division of circulating memory B cells into subpopulations based on patterns of HR expression. Of particular interest is the definition of discrete presumptive intestinal ({alpha}4ß7+) vs. nonintestinal ({alpha}4ß7-) subsets among memory IgD- B cells. Only {alpha}4ß7+ B cells bind well to MAdCAM-1, which may provide a basis for {alpha}4ß7+ B cell homing to (and {alpha}4ß7- memory B cell exclusion from) the gastrointestinal tract. In parallel studies in the mouse, we have examined recently the ability of HR-defined subsets of memory B cells to confer immunity to an intestinal pathogen, rotavirus [24 ]. Memory {alpha}4ß7+, memory {alpha}4ß7-, and naive splenocytes were transferred from orally immunized mice into immunodeficient Rag-2 recipients. Transfer of memory {alpha}4ß7+ B cells but not of naive or {alpha}4ß7- memory subsets led to productive intestinal immune responses including secretory IgA antibody in the stool, clearance of intestinal rotavirus, and immunity to reinfection [24 ]. Although it is not yet clear whether B cells specific for intestinal antigens are found exclusively in the {alpha}4ß7+-recirculating pool, these and the present studies of circulating memory B cell subsets in man indicate that there is a well-defined segregation of intestinal vs. extra-intestinal memory B cells among recirculating lymphocytes, with functional immunity for intestinal antigens associated with, and in part defined by, {alpha}4ß7 expression.

The pattern of {alpha}4ß7 expression observed on IgD- B cells parallels that of memory (CD45RA-) CD4+ T cells, which are subdivided into discrete {alpha}4ß7+ and {alpha}4ß7- subsets also. However, in contrast to naive CD4+ T cells, which display only moderate levels of {alpha}4ß7, naive IgD+ B cells display high levels of {alpha}4ß7+, nearly comparable with those of {alpha}4ß7+ memory B (and T) cells. The levels on naive B cells are in fact significantly higher (2–3x) than those on naive phenotype CD4 T cells. It is interesting to speculate that this difference between naive B and T cells may contribute to the observed relative preference in homing and distribution of naive B cells to PP and naive T cells to popliteal lymph nodes (PLNs) [39 ].

In contrast to the selective binding of {alpha}4ß7+ B cells to MAdCAM-1, we have shown here that {alpha}4ß7+ and {alpha}4ß7- memory B cells (as well as naive B cells) recognize and bind to VCAM-1. This suggests that intestinal {alpha}4ß7+ as well as {alpha}4ß7- memory cells may be able to enter extra-intestinal mucosal tissues, where VCAM-1 not MAdCAM-1 is the predominant vascular {alpha}4 integrin ligand. VCAM-1 is highly expressed, for example, in the inflamed genitourinary tract [28 , 40 ], salivary gland [41 ], and oral mucosa [42 ] and weakly on the vascular endothelium of inflamed conjunctiva [43 ]. Although the {alpha}4 integrins are only one of the components regulating lymphocyte trafficking, this ability of intestinal {alpha}4ß7-expressing memory cells to bind VCAM-1 may facilitate their cross-dissemination to other mucosal tissues and thus be one factor involved in unification of the "common mucosal immune system."

Of particular interest is our finding that circulating surface IgA+ B cells, presumed to be enriched for memory for mucosal antigens, are quite diverse in their patterns of HR expression, suggesting the involvement of distinct homing mechanisms in lymphocyte trafficking to different mucosal sites. Roughly two-thirds of sIgA+ cells expresses the intestinal HR {alpha}4ß7, but one-third is {alpha}4ß7-. Thus, many sIgA+ B cells presumably lack the ability to enter the intestinal lamina propria; these cells may be targeted preferentially to other mucosal tissues characterized by local IgA production but not by MAdCAM-1+ vessels (such as the conjunctiva, genitourinary tract, oral mucosa, bronchial lymphoid tissue, or tonsils and adenoids). They may traffic to or through the bone marrow, a major site of IgA production. {alpha}4ß7- B cells display high levels of {alpha}4ß1 [6 ], which they can use to interact with VCAM-1 [37 ]. As mentioned above, VCAM-1 is expressed (in lieu of MAdCAM-1) in several of these extra-intestinal mucosal sites and in bone marrow [44 ].

Our results confirm recent findings that a significant subset of sIgA+ B cells, especially the {alpha}4ß7- fraction, coexpresses CLA [15 ], an antigen used by circulating memory T cells as a HR for inflamed skin [16 ]. Moreover, we show that these CLA+ B cells are bound preferentially by E-selectin. It is reasonable to propose that these CLA+ B cells may also be targeted to cutaneous or related tissues lined by squamous epithelium, such as the oral mucosa or conjunctiva. Examination of keratinizing squamous mucosa around teeth and osseointegrated implants for expression of vascular cell adhesion molecules has revealed the CLA ligand E-selectin on vascular loops in close association with inflammatory infiltrates [42 ]. In this context, it is relevant that CLA is expressed purportedly on many CD3+ T cells infiltrating gingival mucosa [45 ], a tissue not associated with significant MAdCAM-1-expressing vessels. Thus, the CLA+ {alpha}4ß7- phenotype could be consistent with trafficking to the oral mucosa or similar tissues. IgA is known to be secreted in human sweat also [46 , 47 ], and IgA+ plasma cells have been observed in proximity to eccrine sweat glands in the skin as well [48 ].

In summary, our results demonstrate that circulating human memory B cells, like T cells, are heterogeneous in HR expression, consisting of relatively discrete HR-defined subsets. This heterogeneity is observed even among sIgA+ B cells, consistent with heterogeneity of trafficking mechanisms to diverse sites of mucosal IgA synthesis, such as the bronchial vs. oral vs. intestinal mucosa.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grants AI37832, AI37319, GM37734, AI47822, and HL57492, the Digestive Disease Center grant DK56339, and a merit award to E. C. B. from the Department of Veterans Affairs. We express gratitude to Mary Maniscalco for her assistance in preparing this manuscript.

Received May 13, 2000; revised August 9, 2000; accepted August 10, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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