Inger Nina Farstad

Regional specialization in the mucosal immune system: what happens in the microcompartments?

Mucosal immunity is an important arm of the immune system because it operates in tissues involved in everyday infectious defence as well as in tolerance against innocuous environmental and dietary antigens. Here, Per Brandtzaeg and colleagues discuss compartmentalized regulation of mucosal B cells and mechanisms that might explain the strikingly regionalized effector disparity of the human mucosal immune system.

Regional specialization in the mucosal immune system: primed cells do not always home along the same track

According to the current paradigm of lymphocyte trafficking, primed B and T cells extravasate in the intestinal lamina propria chiefly by means of the mucosal homing receptor alpha4beta7, which interacts with the vascular addressin MAdCAM-1. However, as discussed here, this mechanism cannot explain the preferential homing of B cells with a high level of J-chain expression to mucosal effector sites outside the gut.

The B-cell system of human mucosae and exocrine glands.

Summary: The mucosae and exocrine glands harbour the largest activated B‐cell system of the body, amounting to some 80–90% of all immunoglobulins (Ig)‐producing cells. The major product of these immunocytes is polymeric (p)IgA (mainly dimers) with associated J chain. Both pIgA and pentameric IgM contain a binding site for the polymeric Ig receptor (pIgR), or secretory component (SC), which is a requirement for their active external transport through secretory epithelia. The pIgR/SC binding site depends on covalent incorporation of the J chain into the quaternary structure of the polymers when they are produced by the local immunocytes. This important differentiation characteristic appears to be sufficient functional justification for the J chain to be expressed also by most B cells terminating at secretory effector sites with IgD or IgG production; they probably represent a ‘spin‐off’ from sequential downstream CH switching on its way to pIgA expression, thus apparently reflecting a maturational stage of effector B‐cell clones compatible with homing to these sites. Observations in IgA‐deficient individuals suggest that the magnitude of this homing is fairly well maintained even when the differentiation pathway to IgA is blocked. Certain microenvironmental elements such as specific cytokines and dendritic cells appear to be required for induction of IgA synthesis, but it remains virtually unknown why this isotype normally is such a dominating product of local immunocytes and why they have such a high level of J chain expression. Also, despite the recent identification of some important requirements in terms of adhesion molecules (e.g. integrin α4β7 and MAdCAM‐1) that explain the “gut‐seeking” properties of enterically induced B cells, the origin of regionalized homing of B cells to secretory effector sites outside the gut remains elusive. Moreover, little is known about immune regulation underlying the striking disparity of both the class (IgD, IgM) and subclass (IgA1, IgA2, IgGI, IgG2) production patterns shown by local iinmttnocytes in various regions of the body, although the topical microbiota and other environmental stimuli might be important. Rational design of local vaccines will depend on better knowledge of both inductive and migratory properties of human mucosal B cells.

Microbial Colonization Drives Lymphocyte Accumulation and Differentiation in the Follicle-Associated Epithelium of Peyer’s Patches

Peyer’s patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4+CD86−), whereas mature DCs (CD86high) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.

Human Intestinal B‐Cell Blasts and Plasma Cells Express the Mucosal Homing Receptor Integrin α4β7

Interactions between homing receptors on circulating leucocytes and endothelial addressins regulate tissue‐specific cellular extravasation. Although integrin á4β7 appears to be the main receptor for guthoming T lymphocytes, less is known about molecules mediating mucosal B cell homing. Expression of integrin α4β7 on B lymphocytes, B cell blasts, and plasma cells in human gut‐associated lymphoid tissue (GALT; the Peyers patches and appendix) and lamina propria was studied by multi‐colour immunofluorescence applied on cryosections. Isolated mononuclear cells from the same tissue compartments were examined by flow cytometry and compared with peripheral blood B cells. Integrin α4β7 was expressed by IgA+ B cell blasts and plasma cells (CD38high) in the lamina propria, B cell blasts in GALT, and sIgD+ B lymphocytes in peripheral blood. In contrast, GALT sIgD+ B lymphocytes were negative or only weakly positive for α4β7. These results suggested that B lymphocytes down‐regulate αAβ7 upon extravasation in GALT but up‐regulate this integrin after antigen‐priming. Thus, α4β7 may be a homing receptor also for B cell blasts extravasating in the gut lamina propria, where this integrin is maintained on plasma cells, perhaps as a local retention factor.

Immunoglobulin A cell distribution in the human small intestine: phenotypic and functional characteristics

We compared B‐cell phenotypes in Peyers 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.

Immune functions and immunopathology of the mucosa of the upper respiratory pathways.

The specific defence of airway mucosae depends primarily on secretory immunity. The B cells involved are initially stimulated in organized mucosa-associated lymphoid tissue, apparently including the tonsils and adenoid. From these inductive sites, memory cells migrate to secretory effector sites where they differentiate terminally to immunoglobulin (Ig)-producing plasma cells. Locally produced Ig consists mainly of J chain-containing dimers and larger polymers of IgA (pIgA) that are selectively transported through glandular cells by an epithelial receptor called secretory component or the pIg receptor. IgG can participate in immune exclusion because it reaches the secretions by passive diffusion. However, its proinflammatory properties render IgG antibodies of local immunopathological importance when elimination of penetrating antigens is unsuccessful. T helper (Th) cells activated in this process may by a Th2 cytokine profile promote persistent inflammation with extravasation and priming of eosinophils. This development appears to be part of the late-phase allergic reaction, perhaps initially driven by interleukin-4 (IL-4) released from mast cells that are subjected to IgE-mediated activation, and subsequently also by Th2 cell activation. Eosinophils are potentially tissue-damaging, particularly after priming with IL-5. Various cytokines up-regulate adhesion molecules on endothelial and epithelial cells, thereby enhancing migration of eosinophils into the mucosa, and perhaps in addition causing aberrant immune regulation within the epithelium. Soluble antigens bombarding the epithelial surfaces normally seem to induce several immunosuppressive mechanisms, but mucosal homeostasis appears less patent in the airways than oral tolerance to dietary antigens operating in the gut.

Phenotypes of B and T cells in human intestinal and mesenteric lymph

BACKGROUND & AIMS Cells in lymph draining the human gut have not been characterized previously. The aim of this study was to phenotype B and T cells present in microlymphatics of Peyers pathces and in mesenteric lymph. METHODS The studies were conducted by multicolor immunohistochemistry, flow cytometry, and immunocytochemistry. RESULTS In decreasing order of frequency, microlymphatics in Peyers patches contained naive T (CD3+CD45RA+ alpha 4 beta 7low) and B (sIgD+CD20+ alpha 4 beta 7low) lymphocytes, memory T (CD45RO+ alpha 4 beta 7+) and B (sIgD-CD20+ alpha 4 beta 7+) lymphocytes, and B-cell blasts (CD19+CD38high alpha 4 beta 7high). Naive cells were usually positive for L-selectin, memory cells were either positive or negative, and B-cell blasts were usually negative. Mesenteric lymph contained naive T (approximately 60%) and B (approximately 25%) lymphocytes, memory T and B lymphocytes (approximately 10%), and B-cell blasts (approximately 2%). Cytospins confirmed these results and showed, in addition, that B-cell blasts contained cytoplasmic immunoglobulin (Ig) A, IgM, or IgG in overall proportions of 5:1: < 0.5. CONCLUSIONS Our results are similar to the phenotypes previously described in animal thoracic or mesenteric lymph. A fraction of the B cells stimulated in Peyers patches are near terminal differentiation (contain cytoplasmic Ig) before they enter peripheral blood. Many memory cells, and most if not all B-cell blasts entering lymph show an adhesion molecule profile (alpha 4 beta 7high L-selectin low) in keeping with the presumed phenotype of lymphoid cells destined for mucosal effector sites such as the gut lamina propria.

M cell pockets of human Peyer's patches are specialized extensions of germinal centers

M cells in follicle‐associated epithelium of Peyers patches (PP) mediate antigen entrance into the underlying lymphoid tissue. To investigate the functional potential of B cells in this unique microcompartment, the expression of co‐stimulatory molecules necessary for B‐T cell interaction was examined in histologically normal human PP by three‐color immunohistochemistry. In the M cell areas, CD80 / CD86 expression was much more frequent on memory (sIgD–CD20+) B cells than on naive (sIgD+CD20+) B cells. M cell areas identified by such co‐expression of CD20 and CD80 / CD86 were always spatially related to germinal centers (GC). Contrary to the GC B cell phenotype (sIgD–CD20+CD80 / 86hiCD10+Bcl‐2–), however, M cell‐associated B cells with a high level of CD80 / CD86 were CD20loCD10–Bcl‐2+, and adjacent memory T cells (CD3+CD45R0+) often expressed CD40L (CD154). Autologous peripheral blood B‐T cell cocultures with purified protein derivative as antigen showed that the sIgD–CD80 / CD86hiCD20lo phenotype could indeed be generated during cognate B‐T interactions, concurrent with CD40L up‐regulation on memory T cells. Thus, this M cell‐associated phenotype might result from B‐T cell interactions in the course of antigen presentation by memory B cells, with subsequent CD40 engagement by CD40L‐expressing cognate memory T cells. We propose that this M cell‐associated event contributes to memory B cell survival and diversification of intestinal immunity, representing a specialized limb of GC function.

Expression and Modulation of the Human Immunoglobulin A Fc Receptor (CD89) and the FcR γ Chain on Myeloid Cells in Blood and Tissue

CD89, the human immunoglobulin A (IgA) Fc receptor (FcR), is a potential target for antibody‐based therapeutics, but little is known about its expression and modulation in vivo. In this study, we examined the expression pattern of CD89 and its signalling subunit, the FcR γ chain, on circulating myeloid cells and in various tissues. Our results showed a wide tissue distribution of CD89+ cells. Thus, CD89+ cells were evident as clusters in tonsils and appendix and scattered in varying numbers in lymph nodes, kidney, liver, intestinal mucosa, bronchoalveolar lavage and peritoneal fluid. Most CD89+ cells were identified as neutrophils with high levels of CD89. A few recently emigrated macrophages (CD14low), weakly positive for CD89, were occasionally found in the tissues and more often in the peritoneal fluid. The level of CD89 on neutrophils in tissues and peripheral blood was similar, whereas on monocytes it was much lower in the tissues than in blood, and it was absent on CD14–/CD68+ intestinal lamina propria macrophages. Conversely, we detected much higher levels of the FcR γ chain in monocytes than in neutrophils, but the FcR γ chain was also downregulated in tissue macrophages as well as in in vitro‐differentiated monocyte‐derived macrophages and dendritic cells. The implications of our current findings on the biological functioning of CD89 are discussed.