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The Journal of Immunology, 2001, 166: 2179-2185.
Copyright © 2001 by The American Association of Immunologists

The Microenvironment of Human Peyer’s Patches Inhibits the Increase in CD38 Expression Associated with the Germinal Center Reaction

Mark J. Guilliano*, Amy E. Foxx-Orenstein{dagger} and Deborah A. Lebman*

* Department of Microbiology and Immunology and {dagger} Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer’s patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin {alpha}4{beta}7, is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-{gamma}. However, coculture of tonsil naive B cells with an Ab directed against integrin {beta}7 inhibits IFN-{gamma}-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of {beta}7 can inhibit the induction of CD38 expression, suggests that ligation of {alpha}4{beta}7 in vivo may contribute to a PP-specific GC phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Circulating, mature IgD+ IgM+ naive B cells extravasate into secondary lymphoid tissue. Following activation in the T cell areas, B cells migrate to follicles and ultimately form a germinal center (GC).3 In GCs B cells interact with follicular dendritic cells (FDCs) and T cells resulting in differentiation into precursors of memory B cells or plasma cells. For the most part, developmental stages of secondary B cells have been defined using human tonsils. These analyses demonstrated that CD38 expression can be used to distinguish B cell populations (1). GC and plasma cells express high levels of CD38, whereas memory and naive cells do not. CD38+ GC B cells can be further divided into two developmental stages, the centroblast and the centrocyte stages. The kinetics of the GC reaction has been studied in the tonsil, revealing that somatic mutation occurs at the centroblast (Bm3) stage (2), and isotype switching occurs at the centrocyte (Bm4) stage (3).

Based on studies with tonsil B cells, CD38 expression has been used to follow the development of B cells in vitro. The signals required for naive B cells to acquire a GC phenotype are identified by their ability to induce CD38 expression (4). Similarly, the loss or retention of CD38 can be used to track the in vitro development of CD38+ GC cells into memory or plasma cells, respectively (1). CD38 can also be used to define the stages of murine B cell development, but the pattern of expression differs from that in humans. In the mouse, CD38 is expressed on primary follicular B cells, follicular mantle B cells, and, recently, isotype-switched cells, but not GC B cells in Peyer’s patches (PP) and spleen. It is not expressed on mature murine plasma cells (5). Although CD38 has been regarded as merely a marker of differentiation, the fact that it possesses enzymatic activity and acts both as an adhesion and signal transduction molecule suggests that it functions in cell development (6, 7). This concept is further supported by the observation that ligation of CD38 inhibits apoptosis of GC B cells (8).

The expression pattern of integrins and their ligands contributes to the recruitment of lymphocyte subsets into specific tissues. The integrin heterodimer {alpha}4{beta}7 is expressed on both IgD+-naive B cells and activated cells that home to intestinal effector sites. The major ligand for {alpha}4{beta}7 is mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Because the ability of {alpha}4{beta}7-positive lymphocytes to home to mucosal tissue appears to correlate with the restricted expression of MAdCAM-1, it has been described as the intestinal homing receptor. For example, MAdCAM-1 is expressed on lamina propria vasculature as well as on high endothelial venules (HEVs) in both PP and mesenteric lymph nodes. It is also expressed in GC of human appendix but not peripheral lymph nodes (9). Similarly, FDCs in murine PP express MAdCAM-1, but lymph node FDCs do not (10). Oral mucosa and tonsils lack significant MAdCAM-1 expression (9), indicating that intestinal trafficking cannot be equated with mucosal trafficking. The restriction of receptor expression can, to some extent, explain a common intestinal immune response as well as the separation of intestinal and nonintestinal immune mechanisms. Variation in homing receptor expression in secondary lymphoid tissue may also contribute to the regulation of lymphocyte differentiation in a tissue-specific manner. In this regard, these studies, which demonstrate that the pattern of CD38 expression in PP and tonsil differ, support the concept that there are tissue-specific aspects to B cell development. In addition, the observation that signaling through {beta}7 inhibits CD38 up-regulation suggests that extravasation into intestinal sites may alter the developmental program.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation

All tissue was obtained using protocols approved by either the Virginia Commonwealth University Institutional Review Board or Western Institutional Review Board. Biopsies of human PP and lamina propria were obtained during routine endoscopic evaluation of patients with visually normal terminal ileum. Right colon and terminal ileum specimens were also obtained from patients undergoing colectomy for cancer. Intestinal tissue was incubated with dispase grade II for 30–45 min to produce a cell suspension, washed once with RPMI 1640, and resuspended in 1% BSA/PBS at 0.25–1 x 106 cells/20 µl. Dispase has been previously shown to selectively digest lymphoid follicles (11). Tonsils were obtained from patients undergoing tonsillectomy. Tonsil tissue was either treated with dispase as above or disrupted mechanically and passed through a tissue sieve. The resulting suspension was layered over Ficoll to isolate the lymphocyte population.

Flow cytometry analysis

Anti-CD19 PE (HIB19), anti-CD19 FITC (HIB19), anti-CD3 FITC (UCHT1), anti-CD38 FITC (HIT2), anti-IgD FITC (IA6-2), anti-IgD PE (IA6-2), PE- and FITC-mouse IgG1, {kappa} isotype controls (MOPC-21), and Streptavidin Cy-Chrome were purchased from BD PharMingen (San Diego, CA). Biotin-conjugated peanut agglutinin (PNA) was purchased from Pierce (Rockford, IL) and anti-human CD38 FITC (AT13/5) was purchased from Dako (Carpinteria, CA). Single cell suspensions, which were >95% viable by trypan blue exclusion, were immunostained essentially as described (12). Briefly, 0.5–1 x 106 cells were incubated for 30 min at 4°C with a mixture of FITC-, PE-, and biotin-conjugated Abs, washed three times, and incubated for 30 min at 4°C with Streptavidin Cy-Chrome (1 µg/ml). The incubation and wash buffer consisted of PBS containing 0.5% BSA and 0.1% sodium azide. The stained cells were fixed in 0.8% paraformaldehyde, and data was acquired using a FACScan (Becton Dickinson, Mountain View, CA) and analyzed using Cyclops 2000, version 4 (Cytomation, Fort Collins, CO and Palo Verde Software, Tucson, AZ).

Cell culture

B cells were isolated using anti-CD19-conjugated magnetic beads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. IgD+ cells were isolated by incubating a single cell suspension with biotinylated anti-IgD (Southern Biotechnology Associates, Birmingham, AL) and positively selected using streptavidin-conjugated magnetic beads (Miltenyi Biotec). Cells were resuspended at 1 x 106 cells/ml in B cell medium (5% FCS, 50 µg/ml Apotransferrin, 5 µg/ml insulin, 0.05 mM 2-ME in IMDM; Sigma, St. Louis, MO). A combination of anti-{lambda} and anti-{kappa} Abs was used to cross-link the B cell receptor (BCR). Affinity-purified goat anti-human {lambda} and anti-human {kappa} Abs (Rockland, Gilbertsville, PA) were used at 50 ng/ml each in PP B cell cultures. Tonsil IgD+ cells were cultured in 1 µg/ml of anti-{kappa} and anti-{lambda}. Recombinant IFN-{gamma} was purchased from R&D Systems (Minneapolis, MN) and used at 104 U/ml. Stimulation through CD40 was accomplished using anti-CD40 (0.5 µg/ml) in the presence of CDw32-L cells (1 x 105 cells/ml) (13). Alternatively, 293 cells transfected with CD40 ligand (CD40L; provided by Dr. Lori Covey, Rutgers University, Piscataway, NJ) were added at 0.5 x 106 cells/ml. Purified anti-human integrin {beta}7 (FIB504) and goat anti-rat IgG2a (RG7/1.30) were purchased from BD PharMingen (San Diego, CA) and used at 10 and 5 µg/ml, respectively.

RT-PCR

IFN-{gamma} gene-specific primers, including 18S control primers, were purchased from Ambion (Austin, TX). The RT reaction was performed on 2 µg of tonsil or PP RNA using a RetroScript kit (Ambion). Initial experiments were performed to make sure that amplification was not saturated at the time of quantification. To ensure that both 18S and IFN-{gamma} were measured in the linear range it was necessary to perform two rounds of PCR amplification. The first round of PCR amplification was performed on 5 µl of the RT reaction using a Clontech (Palo Alto, CA) AdvanTaq Plus PCR system with only the IFN-{gamma} primers. An initial denaturation step of 94°C for 3 min was followed by 15 cycles at 95°C for 30 s and 68°C for 3 min. For the second round of amplification, 5 µl of the first PCR was subjected to 28 cycles with both IFN-{gamma} and 18S primers. PCR was performed in a MJ Research (Cambridge, MA) PTC-100 thermocycler. The PCR products were analyzed on an 8% polyacrylamide gel and stained with Sybr-Green 1 (Molecular Probes, Eugene, OR). The bands corresponding to IFN-{gamma} and 18S PCR products were quantified by densitometry using a digital camera and Alpha Imager 2000 imaging system with Alpha Ease version 3.3a software (all obtained from Alpha Innotech, San Leandro, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD38 expression in the tonsil and PP

To confirm the identity of PP visualized during ileo-colonoscopy, biopsies from putative PP were compared with those obtained from colon. PP can be distinguished from lamina propria flow cytometrically by several features including the forward/side scatter profile and ratio of B to T cells (14, 15). Analysis of biopsies obtained during routine endoscopic procedures demonstrated that tissue visually identified as PP contain a lymphoid population that is relatively uniform in size with approximately equal numbers of CD19- and CD3-expressing cells. The percentage of CD19+ B cells in a PP sample ranged from 33 to 59% and CD3+ T cells ranged from 23 to 51% with an average B to T cell ratio of 1.32 ± 0.38 (n = 15). In contrast, lamina propria-derived biopsies have two lymphoid populations with the majority of cells expressing CD3 (Fig. 1Go). The percentage of CD19+ B cells in a lamina propria sample ranged from 13 to 34% and the percentage of CD3+ T cells ranged from 43 to 63% with an average B to T cell ratio of 0.41 ± 0.11 (n = 4). Immunohistochemistry of paraffin-embedded biopsy tissue confirmed the presence of prominent follicles and dome area, consistent with the structure of PP (data not shown).



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  		FIGURE 1. Identification of PP. Dual color FACScan analysis of cell suspensions stained with PE anti-CD19 and FITC anti-CD3 from either PP or lamina propria biopsy samples. Data are representative of 15 (PP) and four (lamina propria) experiments.

 
To determine the relative proportion of GC, memory, and naive cells, PP and tonsils were stained with anti-CD19, anti-CD38, and anti-IgD. Tonsillar CD19+ B cell subpopulations can be distinguished by their expression of IgD and CD38: naive (IgD+CD38-), GC (IgD-CD38+), and memory (IgD-CD38-) (4, 16, 17). However, in PP, CD38 and IgD expression distinguished only two populations (Fig. 2Go, Table IGo). To confirm that the isolation procedure did not eliminate GC cells, an alternative GC marker was used. The ability to bind high levels of PNA is a characteristic of GC cells (18, 19) and can be used in conjunction with IgD expression to distinguish between naive, GC, and memory B cell populations in both tonsil and PP (Fig. 3Go, Table IIGo). In tonsil, anti-CD38 costains with PNA on GC B cells. Analysis of four tonsils revealed that 92 ± 4.5% of PNAhigh CD19+ cells expressed CD38, confirming that both methods of detection identify the same population. However, in PP no CD38+ CD19+ PNAhigh population was found. The absence of a PNAhigh CD38+ population in PP indicates that CD38 is not up-regulated during the GC reaction in PP (Fig. 4Go). Previous studies have demonstrated that CD38 is also expressed at high levels on human plasmablasts, plasma cells, and activated B cells (14, 15). To confirm that the isolation procedure did not remove CD38, single cell suspensions derived from colon lamina propria were double-stained for CD19 and CD38. Approximately half of lamina propria B cells express CD38, and the majority (87 ± 13%) of those were found in the population of large cells (Fig. 5Go). The fact that we could identify CD19+CD38+ plasmablasts and activated B cells in the lamina propria indicates that sample treatment did not affect CD38 detection. In addition, both a CD38-expressing cell line (Daudi) and tonsillar tissue were incubated with dispase and continued to express CD38 (data not shown). The difference in CD38 expression does not appear to be age dependent. Tonsil from a 20-year-old patient contained ~30% IgD+ cells and >60% of B cells expressed CD38, whereas PP B cells from a 20-year-old patient were CD38 negative. The relative proportions of B cell populations in young patients were similar to older patients although the proportion of B cells with a GC phenotype was variable (5–40%). Furthermore, immunohistochemistry of PP and tonsil tissue confirmed the absence of CD38 on PP GC B cells (data not shown).



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  		FIGURE 2. Human PP B cells do not express CD38. Cells from PP and tonsil were triple-stained with PE anti-CD19, FITC anti-CD38, and biotinylated anti-IgD, followed by CyChrome-streptavidin. CD38 and IgD expression on the CD19+ population was determined by FACScan analysis. Data are representative of four (PP) and three (tonsil) experiments.

 

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  		Table I. Definition of B cell populations using CD38 and IgD expression1

 


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  		FIGURE 3. Identification of three CD19+ B cell populations. PP cell suspensions were stained with PE anti-CD19, FITC anti-IgD, and biotinylated PNA, followed by CyChrome-streptavidin. IgD expression and ability to bind PNA by the CD19+ population was determined by FACScan analysis. Data are representative of five experiments.

 

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  		Table II. Definition of B cell populations using IgD expression and ability to bind PNA1

 


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  		FIGURE 4. Tonsil, but not PP GC B cells both express CD38 and bind high levels of PNA. Cells from PP and tonsil were triple-stained with PE anti-CD19, FITC anti-CD38, and biotinylated PNA, followed by CyChrome-streptavidin. CD38 expression and ability to bind PNA by the CD19+ population was determined by FACScan analysis. Data are representative of five (PP) and four (tonsil) experiments.

 


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  		FIGURE 5. CD38 expression in colonic lamina propria. Cells from lamina propria biopsy samples were dual stained with PE anti-CD19 and FITC anti-CD38. The level of CD38 expression on CD19+ cells in small (R1) and large (R2) populations was determined by FACScan analysis. Data are representative of five experiments.

 
The observation that PP GC B cells do not express high levels of CD38 may be due to an inability of PP B cells to up-regulate CD38. To examine this, CD19+CD38- PP B cells were cultured in the presence of CD40L, anti-BCR, and IFN-{gamma}. Previous studies demonstrated that IFN-{gamma} was required for optimal induction of CD38 expression on activated CD38- tonsil B cells (4). BCR cross-linking of PP B cells was achieved by treatment with low levels of anti-{kappa} and anti-{lambda} (50 ng/ml of each). Because high levels of BCR cross-linking has been shown to negatively select GC B cells (20), BCR was stimulated at low levels to maintain the survival and proliferation of naive, memory, and GC B cells. Culture for 4–6 days resulted in a 2- to 3-fold increase in the number of cells. FACScan analysis of cultured CD19+ cells revealed a 3.3 ± 1.38-fold increase in CD38 expression after 4–6 days (Fig. 6Go), demonstrating that PP B cells retain the potential to express CD38. It is likely that the cells that proliferate are naive cells because other investigators have demonstrated a similar proliferative response in cultures of naive cells (20). In addition, although GC cells survive, their proliferative activity is limited under these conditions (20). Regardless, IFN-{gamma} induces an increase in CD38 expression on all B cells that survive or proliferate in response to CD40L and low level BCR stimulation, demonstrating that PP B cells retain the ability to express CD38. The susceptibility of PP B cells to stimuli that increase CD38 expression is also demonstrated by the fact that lamina propria plasma cells and B cell blasts, which are derived from PP, express CD38 (Ref. 21 and Fig. 5Go). Taken together, these data strongly suggest that the microenvironment of the PP inhibits the up-regulation of CD38 associated with the human GC reaction but it does not prevent B cells from responding to stimuli that can up-regulate CD38 expression.



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  		FIGURE 6. PP B cells can be induced to express CD38. CD19+ PP cells were cultured for 6 days with CD40L, anti-{kappa}, anti-{lambda} ± IFN-{gamma}. CD38 expression on CD19+ cells was determined by FACScan analysis. Mean fluorescent intensity is displayed in brackets. Data are representative of three experiments.

 
IFN-{gamma} expression in the tonsil and PP

Because PP B cells cultured in the presence of IFN-{gamma} are capable of expressing high levels of CD38 (see Fig. 6Go), the lack of CD38 expression in the PP could result from a disparity in IFN-{gamma} expression between the tonsil and PP. If the tonsil expresses significantly more IFN-{gamma} than PP, CD38 may be preferentially induced at that site. To address this, IFN-{gamma} message levels in the tonsil and PP were determined by RT-PCR. RNA was isolated from both tonsil and PP. Relative RT-PCR using IFN-{gamma} specific primers and an 18S internal control was performed, and the resulting PCR products were quantified in a polyacrylamide gel (Fig. 7Go). To ensure that comparisons could be made between samples, initial experiments were performed to visualize product accumulation with respect to cycle number, and the PCR protocol was designed such that both IFN-{gamma} and 18S amplification was in the linear range when quantified. The observed ratios of IFN-{gamma} to 18S are similar in the tonsil and PP, indicating that tonsils do not have a significantly higher level of IFN-{gamma} and suggesting that increased IFN-{gamma} in tonsils is not responsible for the observed difference in CD38 expression. No correlation between IFN-{gamma} and CD38 expression was observed.



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  		FIGURE 7. Tonsil and PP express similar levels of IFN-{gamma} mRNA. Relative RT-PCR was performed on RNA from tonsil (Ton) or PP. The amount of IFN-{gamma} RNA is expressed as a ratio of 18S RNA to IFN-{gamma} mRNA. M = DNA marker ({phi}X174/HinfI).

 
Cross-linking {beta}7 on naive B cells inhibits IFN-{gamma}-induced CD38 expression

Phenotypic differences could result from signaling through differentially expressed surface molecules. Because naive IgD+ B cells in both blood and tonsil express {alpha}4{beta}7 (15, 22), the disparity between tonsil and PP GC phenotypes may be a reflection of the restricted distribution of the ligand for the integrin heterodimer {alpha}4{beta}7. MAdCAM-1 is expressed on the HEVs of PP, but not on tonsil HEVs. Integrin {alpha}4{beta}7 binds MAdCAM-1 and transduces a signal in B cells (23). Signaling through this integrin during extravasation or during the GC reaction could contribute to a PP-specific phenotype. To investigate this, the effect of {beta}7 cross-linking on CD38 expression was ascertained. Initial experiments using an assay measuring the cleavage of MTT as an indication of the activity of mitochondrial dehydrogenase enzymes and as a measure of proliferation demonstrated that cross-linking {beta}7 did not impact proliferation (data not shown). Naive B cells from PP, peripheral blood, and tonsil express a similar, intermediate level of {alpha}4{beta}7 (Refs. 14, 15, 22, 24 and data not shown). Naive IgD+ tonsil cells were cultured with CD40L, anti-{kappa}, anti-{lambda}, and IFN-{gamma} with or without the addition of cross-linked anti-{beta}7 for 6 days. Regardless of stimulation, there was a comparable increase (2- to 4-fold) in cell number following 6 days of culture. As shown previously, IFN-{gamma} treatment causes a 1.9-fold (average) increase in the level of CD38 expression on CD40L/BCR-stimulated IgD+ B cells. Addition of anti-{beta}7 to the culture inhibits this effect (Fig. 8Go, Table IIIGo). These data suggest that signaling through {alpha}4{beta}7 on naive B cells can prevent up-regulation of CD38. Two additional lines of evidence indicate that it is unlikely that the difference in CD38 expression reflects a selective proliferation of CD38+ cells in the absence of {beta}7 cross-linking. BCR stimulation with 1 µg/ml of anti-{kappa} and anti-{lambda} has been shown to induce CD38+ cells to undergo apoptosis in 3–6 days (Ref. 20 and data not shown). In experiment 3, in which tonsil B cells were subjected to an initial CD38 depletion step and contained <1% CD38+ B cells on day 0, a similar effect of stimuli was observed on day 6. Thus, signaling through {alpha}4{beta}7 on naive B cells inhibits up-regulation of CD38 expression in response to CD40L, IFN-{gamma}, and BCR.



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  		FIGURE 8. Coligation of {beta}7 inhibits IFN-{gamma}-induced CD38 hyperexpression. Tonsil IgD+ cells were cultured for 6 days with 293/CD40L cells, anti-{kappa}, and anti-{lambda} in the presence or absence of IFN-{gamma} or anti-{beta}7 plus goat anti-rat IgG. CD38 expression was determined by FACScan analysis. Mean fluorescence intensity is displayed in brackets. Data are representative of three experiments.

 

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  		Table III. {beta}7 cross-linking inhibits induction of CD38 expression

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Based on analysis of B cell populations in human tonsil, five stages of development have been described. Each stage is characterized by expression of surface molecules whose receptors or ligands are also present in tonsil. Analysis of these interactions will ultimately lead to greater understanding of B cell development. PP play an integral role in the regulation of intestinal immunity and occupy a unique niche in the immune system. They differ from other secondary lymphoid organs in two major ways. First, they are not supplied by afferent lymphatics. A specialized epithelial cell, the M cell, delivers native Ag from the gut to the underlying lymphoid tissue. Second, efferent lymphatics from the PP drain to the mesenteric lymph nodes, another secondary lymphoid tissue, before entering the circulation. The primary function of the PP appears to be a site for the initial exposure of naive lymphocytes to intestinal Ags. After Ag priming, a secretory IgA response is initiated and disseminated throughout the intestine (12, 21, 25). Despite the anatomic and functional differences between PP and tonsil, discussions of PP B cell development have generally been extrapolated from tonsil analysis. This could be due in some part to the difficulty in obtaining PP specimens and isolating viable GC B cells. GC cells die by apoptosis within a few hours unless they are activated through BCR and CD40 (26). In previous studies, PP samples were treated with dispase at 37°C for up to 5 h, followed by a subsequent incubation at 37°C for 6–10 h (11, 14). Prolonged incubation at 37°C may lead to GC cell apoptosis, and therefore skew subsequent analysis. In this study, the biopsy samples are treated with dispase for a relatively short time (30–45 min) to preserve GC populations.

Although CD38 expression is regarded as a hallmark of human GC B cell development, PP GC do not express CD38. There are several possible explanations for the difference between PP and tonsil GC. Because the majority of tonsils are obtained from children, whereas PP are primarily obtained from adults, it is possible that decreased CD38 expression is a function of age. However, studies in the tonsil have shown that the number of follicles is reduced with age, but CD38 expression levels are not diminished (27, 28). Although the relative proportions of B cells in PP of young and old patients were similar, the proportion of B cells with a GC phenotype was variable. CD38 expression in PP was not affected by age, and comparison of CD38 expression in tonsil and PP from individuals of the same age revealed the tissue-specific difference. Thus, the lack of CD38 expression in PP is unlikely to be age related. Another consideration for differential expression is that tonsils are obtained from patients with tonsillitis or hypertrophy and represent tissue undergoing an inflamed or altered local immune response. Monocytes in inflamed tissue secrete a number of inflammatory cytokines, including IFN-{gamma} (29). In vitro analysis demonstrates that IFN-{gamma} is required for the induction of high levels of CD38 expression on CD38- tonsil B cells raising the possibility that ongoing inflammatory responses could account for high levels of CD38 expression on tonsil GC B cells. However, IFN-{gamma} message levels in the tonsil and PP do not correlate with the observed discrepancy in CD38 expression in the two sites.

Finally, PP and tonsil differ in addressin expression patterns. Integrin ligation by these addressins can deliver developmental signals that are only encountered in specific tissues. Integrin {alpha}4{beta}7 on naive B cells can bind MAdCAM-1 in the PP but not in the tonsil. Naive B cells entering tonsil do not encounter MAdCAM-1 and up-regulate CD38 expression during the GC reaction. These studies demonstrate that ligation of {beta}7 inhibits the induction of CD38 expression, suggesting that ligation of {alpha}4{beta}7 in vivo may contribute to a PP-specific GC phenotype. Importantly, cross-linking {beta}7 did not effect B cell proliferation in response to CD40L and BCR. Although {beta}1 integrin cross-linking can increase the proliferative response of T cells to TCR stimulation (30), it is not clear whether this reflects a property of integrin stimulation or T cells. In B cells the early signal transduction events following ligation of {beta}1 and {beta}7 integrins are similar (23). However, cytoplasmic tails of {beta} integrins are not interchangeable and do not interact with the same proteins, suggesting that the outcome of ligation is specific to each {beta} integrin (31, 32, 33). These findings suggest that ligands encountered during extravasation may alter the developmental path of lymphocytes.

The observation that CD38 is not expressed at high levels on PP GC B cells clearly defines phenotypic differences between the tonsil and PP, and suggests alternative developmental mechanisms at these sites. In the mouse, CD38 expression is also down-regulated on PP GC B cells (5). However, the difference in CD38 expression patterns between mouse and human suggests that CD38 plays a different role in B cell development in the two species. Unlike human plasma cells, murine plasma cells do not express CD38. Murine CD38 is expressed on stem cells, defining a self-renewing population. In contrast, human primitive hemopoietic cells do not express CD38 (34). CD38 functions as an ectoenzyme that catalyzes the conversion of NAD+ to nicotinamide and cyclic ADP-ribose, and also the hydrolysis of cyclic ADP-ribose to ADP-ribose. The particular physiological relevance of this function has yet to be determined. In addition to its function as an ectoenzyme, CD38 ligation increases intracellular Ca2+, cell cycling, and protection from apoptosis (6, 7, 8). Although the precise role of CD38 in GC B cell development is not clear, there are several possibilities. In vitro studies indicate that CD38 signaling prevents apoptosis of tonsillar GC B cells (8). Because stimulation through BCR and CD40 also prevents centrocytes from entering apoptosis, the anti-apoptotic effect of CD38 may not be essential to GC B cell survival. Nonetheless, it may contribute to maintenance of the GC population. A second possibility is that CD38, which can act as a BCR coreceptor (35), alters the sensitivity of GC B cells to Ag. Consequently, differences in CD38 expression may be a protective mechanism that relates to the relative level of Ag exposure. Regardless, the absence of CD38 expression in PP GC indicates that B cells undergo tissue-specific differentiation pathways, which are potentially directed by differences in expression patterns of adhesion molecules.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants R21 AI44762 and K04 AI01344. Back

2 Address correspondence and reprint request to Dr. Deborah A. Lebman, Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298. Back

3 Abbreviations used in this paper: GC, germinal center; FDCs, follicular dendritic cells; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PP, Peyer’s patches; HEVs, high endothelial venules; PNA, peanut agglutinin; BCR, B cell receptor; CD40L, CD40 ligand. Back

Received for publication July 21, 2000. Accepted for publication November 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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