We initially analyzed ASC from spleen, a central filtering and collecting organ for circulating immune cells that comprises plasma cells of each isotype. Spleen cells were isolated, placed in an upper Transwell chamber, and allowed to migrate to individual chemokines placed in the bottom well. ASC in the starting population and in the bottom well after migration to specific chemokines or control media were enumerated by conventional ELISPOT assays, and the percent of ASC migrated was determined. The migration of naive follicular B cells, a population that circulates through secondary lymphoid tissues but not the gut wall, is presented for comparison.
A shows that IgM-ASC failed to respond well to any chemokine tested, although they migrated above background to SDF-1α, (CXCL12), a widely expressed chemokine ligand for CXC chemokine receptor (CXCR)4 that is active on many lymphocyte subsets. IgG–ASC ( A) displayed a detectable but weak SDF-1α response, as well, but also migrated significantly to MIG (CXCL9), a ligand for CXCR3. CXCR3 and its ligands have been implicated in inflammatory T cell migration (13
); thus, migration to MIG may help IgG–ASC enter diverse tissue sites of inflammation.
Figure 1. TECK selectively attracts IgA ASC. (A) Mouse splenocytes were migrated to optimal chemokine concentrations as described in Materials and Methods. Follicular B lymphocytes were identified by flow cytometry as B220+/CD3−/IgMlo/IgDhi or (more ...)
The most dramatic chemotactic responses, however, were displayed by IgA–ASC ( A, right panel). These IgA-producing cells respond well to SDF-1 α (a chemokine whose receptor is expressed by most leukocytes), but also to TECK (CCL25). In control experiments, incubation with TECK had no effect on the numbers of IgA–ASC detected or on the amount of IgA produced per ASC during overnight cultures (data not shown), indicating that the ASC arrived in the bottom chemoattractant wells by migration. A representative ELISPOT well illustrating IgA–ASC migration to TECK is presented as B. Consistent with their efficient response to TECK, sorted IgA+ (but not IgG+) ASC express abundant messenger RNA for the TECK receptor, CCR9 ( C; note the presence of two bands, indicating expression of both CCR9 splice variants).
Unlike most circulating naive and memory lymphocytes including follicular (IgD+
) B cells (5
) (illustrated in ), neither IgA– nor IgG–ASC migrated to the lymphoid tissue-expressed CCR7 ligands ELC/CCL19 () or SLC/CCL21 (data not shown), chemokines implicated in homing to lymphoid organs. These results are consistent with recent studies showing that ASC phenotype (B220int
) splenocytes from alum/nitrophenyl chicken γ-globulin-immunized mice migrate to SDF-1α but not ELC or SLC (14
). Similarly, Wehrli et. al. has suggested that early ASC phenotype cells can migrate to ELC and SDF-1α, but that they largely lose these responses in association with exit from their lymphoid sites of generation (15
). Thus most ASC may be programmed to migrate to extralymphoid sites, rather than recirculate through secondary lymphoid tissues.
IgA–ASC are thought to be induced in the intestine-associated Peyer's patch and mesenteric lymph nodes. They then travel via the lymphatic system through the thoracic duct into the bloodstream. Many localize to or pass through the spleen on their way to populating the intestinal LP (16
). Some IgA-ASC also migrate to and reside in the BM, where they contribute to systemic IgA production (18
). As shown in , TECK responsiveness is a common feature of IgA–ASC harvested from Peyer's patches and mesenteric lymph nodes, as well as spleen. On average, BM IgA–ASC responded relatively less well to TECK (although the difference observed is not statistically significance) (). In fact, migration of BM IgA–ASC to TECK and SDF was variable and in some experiments was significantly less than migration by IgA–ASC from MLN or spleen. These differences may reflect variability in the proportion of terminally differentiated plasma cells in the BM (see below), where mature plasma cells are expected to be present at a higher frequency than in lymphoid organs.
Figure 2. IgA–ASC in Peyer's patch, mesenteric lymph nodes, and spleen migrate well to TECK. Lymphocytes were harvested from the indicated lymphoid organs and the migration of IgA–ASC present in these organs to medium (Basal) or medium containing (more ...)
The development of naive B cells into differentiated IgA–ASC is associated with the gain of surface IgA expression and a progressive decrease in B220 levels; few if any ASC express B220 at the high levels seen on naive or germinal center B cells, and many terminally differentiated tissue plasma cells are B220neg
). The gut LP is a major site of IgA–ASC recruitment and the relative high percentage of IgA–ASC in this organ allowed us to address the B220 levels of the TECK-responsive IgA–ASC. (The cytological and histochemical terms “plasmablasts” and “plasma cells” have been used inconsistently in reference to ASC expressing varying levels of B220 [references 14
, and 20
), and will not be used here). A illustrates that both IgA+
) and IgA+
large lymphocytes are found in the LP, and we have confirmed that both of these populations comprise IgA–ASC (unpublished data). B demonstrates that the IgA+
LP IgA–ASC population responds much better to TECK than IgA+
ASC. The LP IgA+
ASC population had also lost its responsiveness to SDF-1α (similar to a B220neg
lymph node population described by Wehrli et. al., reference 15
). Splenic TECK-responsive IgA–ASC were also restricted to the IgA+
but not IgA+
ASC populations (data not shown). IgA+
ASC may represent cells that have terminally homed to the site of their final residence with no further need for migratory receptors.
Figure 3. B220int but not B220−IgA+ ASC phenotype cells in LP migrate to TECK. LPLs were stained for B220, IgA, and for excluded lineage markers (a dump cocktail or anti-Thy1.1, NK1.1, IgD, and CD11c). (A) Large lymphocytes were gated for dump negativity (more ...)
We next asked whether cells producing antibody in response to a well-defined intestinally restricted pathogen could also migrate to TECK. RV, a double-stranded RNA virus, exclusively infects the small intestine villous epithelium in humans and mice, causing diarrhea (21
). RV is responsible for up to a million childhood deaths per year. Protection against RV in immunodeficient mice can be conferred by transfer of immune B cells, and immunity correlates with anti-RV IgA but not anti-RV IgG levels in the mouse model (22
). Mesenteric lymph node cells were harvested from mice 10 d after RV infection, and cells migrating to TECK or SDF-1α were tested for the isotype of anti-RV antibody production by modified (antigen-specific) ELISPOT assay (8
). RV-specific IgA-ASC represent ~10–12% of total IgA ASC in mesenteric nodes at this time (9
). shows that these RV-specific IgA–ASC migrate strongly to TECK. In contrast, in an experiment in which mesenteric node IgG–ASC were assessed in parallel, RV-specific IgG-ASC did not respond to TECK above background (data not shown). The frequency of RV-specific IgA–ASC migration to TECK was comparable to that of the total IgA–ASC population in the same experiments. We conclude that IgA–ASC induced by small intestinal infection with RV display efficient chemotaxis to TECK.
Figure 4. RV-specific IgA–ASC migrate to TECK. Mesenteric lymph node cells from RV-infected mice were harvested, enriched for ASC, and allowed to migrate to medium (Basal) or medium containing optimal chemotactic concentrations of mTECK (300 nM) or SDF-1α (more ...)
Migration to the widely expressed chemokine SDF-1α is a property shared with most mature leukocytes, and many other cell types as well (24
). Such a general response may contribute to motility within tissues or retention within a tissue (14
), but is unlikely to control differential tissue and subset-specific cell homing. In contrast, recent studies of TECK tissue expression patterns reveal that it is critically positioned to contribute to IgA–ASC localization in the gut. TECK mRNA is highly expressed in the thymus, where it is hypothesized to participate in T cell development (25
). In the periphery however, TECK mRNA is largely restricted to the gastrointestinal tract, especially the small intestinal mucosa (27
). In situ hybridization and immunohistochemistry reveal TECK expression by epithelial cells in the jejunum, duodenum and ileum, especially but not exclusively in the crypt regions near the base of intestinal villi (28
). This region is enriched in MAdCAM-1+
venules supporting extravasation of homing lymphocytes, as well. Additional chemoattractants, possibly including SDF-1α (30
), may support further migration and dispersal of ASC throughout the intestinal LP. In contrast, TECK is poorly expressed by nonintestinal epithelial tissues (28
). Thus, the studies presented here lead to the hypothesis that selective expression of TECK by intestinal epithelial cells may serve to efficiently recruit IgA–ASC to the gastrointestinal LP, and it will be of interest to assess the role of TECK in circulating ASC interactions with LP venules, and in diapedesis into the LP, in future in situ studies. Interestingly, TECK is expressed at lower levels in the colon than in the small intestines. It could contribute to ASC migration there as well, but other chemoattractants may play parallel roles in the homing of IgA–ASC to the colon and other mucosal tissues.
Our results provide the first demonstration that ASC respond to chemokines and demonstrate further the specialization of chemokine responses can be associated, at the population level, with ASC specialization in terms of isotype expression. The selective recruitment of IgA–ASC by the intestinal chemokine TECK not only provides a mechanism for the recruitment and/or retention of mucosal ASC in the intestinal wall as originally hypothesized by Lamm (4
), but may also provide a paradigm in which tissue-selective chemokine expression by specialized epithelia helps determine the character of local humoral and cellular immune responses.