Immunoglobulin A cell distribution in the human small intestine: phenotypic and functional characteristics - PubMed (original) (raw)
Immunoglobulin A cell distribution in the human small intestine: phenotypic and functional characteristics
I N Farstad et al. Immunology. 2000 Nov.
Abstract
We compared B-cell phenotypes in Peyer's patches and solitary lymphoid follicles (organized gut-associated lymphoid tissue, GALT) with those in jejunal or ileal lamina propria. In situ, immunostaining showed that small B cells of naive [surface immunoglobulin D-positive (sIgD+) CD27-] and memory (sIgD+/- CD27+) phenotypes occurred almost exclusively in GALT, whereas the lamina propria contained only scattered sIgA+ CD27+ memory cells. In contrast, B-cell blasts and plasma cells negative for CD20 and often also for CD19 but with strong expression of CD38, CD27 and cytoplasmic IgA (cIgA), dominated in the lamina propria but were scarce in GALT. By flow cytometry, the proportion of dispersed CD19+ B lymphocytes varied from 4 to 42% among jejunal mucosal samples; between 5 and 50% of these were sIgD+, suggesting a variable contamination with GALT cells. B-cell blasts and plasma cells, identified by their large size and strong expression of CD38, were regularly found (25-35% of the total mononuclear cell population). Distinction between B-cell blasts and mature plasma cells was made by the presence or absence of human leucocyte antigen (HLA) class II molecules, CD45RA, CD19 and surface immunoglobulin. No CD19+ B cells outside GALT expressed CD5, but a very small portion of the lamina propria B-cell blasts were positive for CD28. Dispersed sIgA+ lamina propria cells expressed low levels of CD40, proliferated on CD40 ligation and constitutively secreted IgA in vitro. We concluded that the lamina propria B-cell compartment consists mainly of B-cell blasts and plasma cells but also has scattered, small sIgA+ cells that can proliferate in response to CD40 ligation and may therefore function as local memory cells for recall antigens.
Figures
Figure 1
Distribution of B-cell phenotypes in a Peyer's patch (PP) compared with adjacent lamina propria (LP) demonstrated by immunofluorescence staining. Photomicrographs depicting the periphery of the same Peyer's patch with adjacent villous lamina propria in serial sections (a–f) of a normal ileal biopsy specimen (original magnification × 250). Colour code of markers is indicated in each panel. Cytokeratin is stained blue to visualize the epithelium. Yellow indicates coexpression of markers. The topographic hallmarks are labelled in (a): D, dome; GC, germinal centre; M, mantle zone; MZ, marginal zone; and T, T-cell area. Note that the GC is only partially represented in these sections. Two M-cell areas in the follicle-associated epithelium above the dome are also shown. (a) Yellow cells expressing both CD19 and CD20 are present only in the follicle, dome area and M-cell pockets; occasional weakly stained red cells corresponding to B-cell blasts and plasma cells are seen in adjacent LP. (b) Yellow cells expressing CD20 and sIgD are present only in the follicle (section through mantle zone); green cells outside the follicle represent memory B cells in the marginal zone. Note that very few yellow (sIgD+ CD20+ naive) or green (IgD– CD20+ memory) cells are seen in the lamina propria. (The asterisk indicates area where the epithelium is accidentally overlaid on the follicle.) (c) Virtually all CD20+ cells coexpress CD45RA (yellow); occasional single CD45RA+ cells corresponding to the CD19+ cells in (a) are present in LP (horizontal arrow), and red cells at lower left represent naive T cells in PP (vertical arrow). (d) Co-staining for CD38 and CD20 shows that these markers are almost reciprocally expressed, in that most intrafollicular B cells bear no or low levels of CD38, whereas it is strongly expressed on large lamina propria cells representing B-cell blasts and plasma cells, as well as on a few germinal centre cells. (e)
l
-selectin is coexpressed on a fraction of CD20+ cells in the follicle as well as in M-cell pockets; red cells represent mainly naive T cells in PP (lower left). Note that very few
l
-selectin+ cells (CD20+ B cells or CD20– T cells) are present in LP. (f) Co-staining for CD19 and CD5 demonstrates lack of overlap between these markers, indicating that no intestinal B cells (i.e. only T cells) express CD5 as detected by this method. An intraepithelial CD5+ T cell is indicated (arrow). (g) Co-staining for CD19 and CD27 to visualize the distribution from PP to LP in the same biopsy specimen (note that this section as well as (h) were cut at a deeper level than those illustrated above). (h) Green separation of the same field. In the germinal centre and mantle zone, CD27 is only expressed by very few cells and therefore most of the follicle appears red when double-exposed. The follicle periphery, corresponding to the marginal zone, as well as the T-cell area (T), contain many CD27+ cells. By contrast, strongly CD27+ cells are present in adjacent LP, mostly representing plasma cells; CD19 expression on these cells is faint and they therefore appear brightly green.
Figure 2
Flow-cytometric definition of R1 and R2 cells in small intestinal mucosal samples. Dispersed mononuclear mucosal cells were obtained from organ donors and subjected to immunofluorescence staining for the respective markers by an indirect method as decribed in the Materials and Methods. Upper left panel shows gates (R1 and R2 cells) that were set on the basis of surface marker expression (_y_-axis; CD3, CD19, CD38) in relation to cell size (FSC, _x_-axis) on ungated populations. The cells falling outside R1 and R2 represent epithelial cells and dead cells. R1 cells (small) mainly consist of T cells (CD3+, upper right panel) with few cells expressing CD19 (lower right panel). R2 cells (large) mainly consist of B-cell blasts and plasma cells (CD38hi) with very few T cells (right panels); however, many large cells express CD19 (lower left). Some relatively small cells show rather strong CD38 expression and thus the distinction of R1 from R2 cells is not exact (lower right).
Figure 3
Flow-cytometric analysis of R1 and R2 B-cell isotypes in small intestinal mucosal samples. Numbers in quadrants represent percentage positive cells. Left panels represent controls obtained with irrelevant primary antibodies conjugated to Tricolor (containing Cy5 and therefore similar to Cy-Chrome; _y_-axis) or FITC (_x_-axis). (a) Profiles of a sample with few B cells (10% CD19+ cells in R1): the main proportion of R1 CD19+ cells express sIgA while very few bear sIgD or sIgM. R2 cells are mainly sIgA+ and most cells in this sample also express CD19. In both R1 and R2 distinct fractions of cells without CD19 express surface immunoglobulin; this probably indicates that some of the immunoglobulin secreted from mature plasma cells is transiently membrane-bound [this also applies to (b)]. (b) Profiles of a sample with many B cells (42% CD19+ cells in R1): although sIgA+ cells dominate among the CD19+ cells, significant fractions of sIgD+ and sIgM+ cells are also present. R2 cells are mainly sIgA+ but fewer of these cells express CD19 than in (a).
Figure 4
Flow-cytometric examination of CD45RO, HLA-DR and CD38 on R1 and R2 B cells in B-cell-poor small intestinal mucosal sample. Numbers in quadrants represent percentage positive cells. Dispersed mononuclear cells were examined by flow cytometry and small (R1) versus large (R2) cells compared for the markers indicated. The fourth and fifth panels in the rows represent controls obtained with relevant versus irrelevant primary antibodies followed by similar secondary antibody conjugates. In the first panel row, very few R1 or R2 CD19+ cells express CD45RO, indicating that most cells expressing this marker are T cells. Conversely, most CD19+ cells in R1 express HLA-DR while very few R2 CD19+ cells are positive (second panel row). Few CD19+ HLA-DR– cells in R1 might reflect that distinction of R1 from R2 cells is not exact when based on size and CD38 (see Fig. 2), and these cells could in fact represent relatively small B-cell blasts. In the third panel row, CD38+ R1 cells are negative for HLA-DR while HLA-DR+ cells are negative or weakly positive for CD38. The latter cells represent CD19+ cells while the former represent a mixture of relatively small T- and B-cell blasts. In R2, HLA-DR is mainly present on very few CD38hi cells; the single HLA-DR+ cells in the lower right quadrant represent macrophages (see Results section).
Figure 5
Examination of isolated sIgA+ cells. (a) Enrichment of sIgA+ cells from dispersed starting population (left) and after bead selection for sIgA+ cells (right) shown by flow cytometry as described in the Materials and Methods. Numbers in upper right corners indicate percentage positive cells (ungated). (b) Immunoglobulin production (µg/ml) by bead-selected and sorted versus unsorted sIgA+ cells as described in the Materials and Methods, subjected to different stimuli indicated by numbers. Note that the scale for sorted* and unsorted** cells is different (× 10 ng/ml) from that for bead-selected cells (µg/ml). (c) Proliferation (c.p.m. values) by bead-selected and sorted versus unsorted sIgA+ cells subjected to different stimuli indicated by numbers.
References
- Dunn-Walters DK, Isaacson PG, Spencer J. Sequence analysis of human IgVH genes indicates that ileal lamina propria plasma cells are derived from Peyer's patches. Eur J Immunol. 1997;27:463–7. -PubMed
- Kroese FG, de Waard F, Bos NA. B-1 cells and their reactivity with the murine intestinal microflora. Semin Immuno. 1199;6:11–18. -PubMed
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