Search

Antibodies to the cytosolic leucocyte L1 protein (or calprotectin) were examined for reactivity with macrophages, neutrophils, and eosinophils identified by paired immunofluorescence staining in sections of normal human ileal mucosa, including Peyer's patches. Macrophages were recognised by expression of the myelomonocytic antigen CD68 (monoclonal antibody KP1). Neutrophilic granulocytes were identified by their content of neutrophil elastase, and eosinophilic granulocytes by monoclonal antibody EG2. Virtually all CD68+ macrophages in normal lamina propria and Peyer's patches were L1- and the same was true for most extravasated macrophages in normal peripheral lymph nodes. Some mesenteric lymph nodes, however, and all peripheral lymph nodes with overt pathological processes (malignant lymphoma) contained many CD68+L1+ macrophages. Numerous L1+ cells were also localised to the crypt region and to some extent beneath the villous epithelium in normal lamina propria, but they were mainly identified as EG2+ eosinophils. Such cells were remarkably scarce or absent beneath the follicle associated epithelium in the dome region of Peyer's patches, where CD68+L1- macrophages were abundant. Also subepithelial and interfollicular CD68- interdigitating dendritic cells in Peyer's patches (recognised by antibody to S-100 protein) were usually unreactive with L1 antibody. The L1 protein shows a broad spectrum of antimicrobial activities in vitro, and its putative antiproliferative properties are interesting in relation to the immunosuppression postulated to take place in lamina propria. The virtual absence of L1 producing cells beneath the follicle associated epithelium in Peyer's patches may support the immunostimulatory function of these macrophage rich structures, which are held to be crucial for induction of specific mucosal immunity.

SUMMARYTwo-colour immunofluorescencc staining for intracellular J chain and IgA (or J chain and IgG) was performed on tissue sections of normal human ileal mucosa (eight adult kidney donors), mesenteric lymph nodes (MLN), peripheral lymph nodes, and palatine tonsils. The most prominent J chain positivily was seen for IgA (97.3%) and IgG (81.7%) immunocytes in the ileal lamina propria (LP). Moreover, the proportion of J chain-expressing exlrafollicular immunocytes was significantly higher (P < 0.05) in MLN than in peripheral lymph nodes for the IgA class (58.5%versus 25.6%); the same proportion for the IgG class was 45.9%versus 30.4%. In clinically normal palatine tonsils of adults, extrafollicular J chain expression was much lower than in peripheral lymph nodes; 14.2% for IgA cells and 5.5% for IgG cells. When related to subclass production, J chain expression was found to be higher for IgA2 than for IgA 1 cells in all tissues examined (palatine tonsils excluded because of a small number of lgA2 cells), the difference being significant in M LN and ileal LP (P < 005). The J chain positivity tended to be higher for all IgG subclasses in MLN than in peripheral lymph nodes; this difference was significant (P

The relative distribution of IgA and IgG subclass-producing immunocytes was examined by two-colour immunohistochemistry in normal human distal ileum including Peyer's patches (PP), regional mesenteric lymph nodes (MLN), and peripheral lymph nodes. IgA2 cells predominated slightly over IgA1 cells in the PP dome area. There was a decreasing median proportion of IgA2 cells in the order of PP (52%), distant ileal lamina propria (40%), MLN (32%), and peripheral lymph nodes (11%). The reverse was true for IgA1 cells in independent enumerations. These results support the notion that PP-derived B cells after stimulation are seeded mainly to the lamina propria of the distal gut, but that there is a substantial retention and terminal differentiation of this migrating population in regional MLN. The median subclass proportions of IgG-producing cells in the PP dome area were in independent determinations 68% IgG1, 23% IgG2, 8% IgG3, and 9% IgG4. This distribution was fairly similar to that seen in other tissue categories, except for a trend towards increased IgG1 and reduced IgG2 proportions in peripheral lymph nodes and reduced IgG1 along with increased IgG3 in normal palatine tonsils. The data suggested an association between the expression of IgG2 (and possibly IgG4) and IgA2 in intestinal mucosal immune responses.

The secretory antibody system depends on local production and selective epithelial transport of polymeric immunoglobulin A (pIgA) and immunoglobulin M (pIgM). To this end there is a fascinating co-operation between the local immune system and the glandular epithelium by their production of two key factors, J chain and secretory component (SC), respectively. Mucosal immune regulation is normally geared for generation of early memory B-cell clones with prominent J-chain expression and preference for IgA production. Moreover, cytokines released from activated T cells and macrophages probably stimulate the secretory epithelium to perform enhanced SC-dependent transport of J chain-containing pIgA and pIgM when their local production is increased. There is also circumstantial evidence to suggest that interactions between the gut epithelium and T cells are involved in mucosal downregulation of systemic types of immunity to non-replicating luminal antigens. IgG (and IgE) responses and delayed-type hypersensitivity may thereby be minimized to avoid undue complement activation, increased mucosal penetrability, inflammation, and tissue damage. It is possible that abrogation of such hyporesponsiveness to usually harmless luminal antigens contributes to the pathogenesis of various mucosal disorders. Enhanced stimulation of local T lymphocytes, increased epithelial expression of HLA class II determinants, and disproportionate local overproduction of IgG, are features that may reflect such a deleterious development leading to disease. Expansion of a particular TCRγ/δ + intraepithelial T-cell subset may also be involved in coeliac disease.

The monocyte-derived cell lineages include classical phagocytic macrophages and various antigen-presenting dendritic cell types. The latter are poorly phagocytic but play an important role as accessory antigen-presenting cells in the induction of immune responses. S-100 protein (a formalin-resistant cytoplasmic antigen) is a useful marker for the specialized dendritic cells in the T-cell areas of lymphoid tissues, the so-called interdigitating cells or IDC [1], We have recently observed that reactive macrophages can be selectively labelled by staining for the formalin-resistant myelomonocytic LI antigen [2]. As a monoclonal antibody (Mac 387) to the latter marker is now available [3], we were able to perform paired staining for macrophages and putative IDC on formalin-fixed biopsy material from normal human distal ileum, including Peyer’s patches (PP).

The main function of secretory IgA is to exert immune exclusion; that is, by intimate cooperation with innate non-specific defence mechanisms, it dampens down penetration of soluble antigens and inhibits epithelial colonisation of bacteria and viruses. Secretory IgM may exert a similar protective function in the gut as its local synthesis sometimes is markedly increased, especially in selective IgA deficiency. IgG should not be considered a secretory immunoglobulin because its external translocation depends on passive intercellular diffusion. By activating complement, antibodies of this isotype may cause increased mucosal permeability and tissue damage. IgG may thus contribute to persistent immunopathology in mucosal lesions. The same is true for IgE antibodies which, in atopic individuals, may be carried into the gut mucosa by mast cells and cause their degranulation with histamine release. Secretory IgA and secretory IgM are the products of two cell types: plasma cells synthesise IgA dimers and IgM pentamers which, by non-covalent association, become complexed with the secretory component (SC) which is synthesized by serous-type glandular cells. The adsorption of the Ig polymers to the SC-expressing epithelial cells depends on J chain-determined binding sites. This fact gives biological significance to the striking J chain expression shown by mucosal immunocytes regardless of the Ig class they produce. The immunocytes populating the gut mucosa apparently belong to relatively early memory B cell clones. The obvious functional goal of J chain expression at this stage of clonal differentiation is local generation of SC-binding IgA and IgM polymers. In various gut diseases, altered immune regulation results in a disproportionately increased number of J chain-negative IgG-producing cells in the mucosa. Such altered immunological homeostasis may contribute to perpetuation of inflammatory bowel diseases.

The densities of IgG-, IgA-, IgM- and IgD-producing immunocytes were determined by paired immunofluorescence staining and morphometric analysis in the lamina propria of normal appendix specimens. Normal colon specimens were used as reference material, mostly paired from individual subjects. The density (median of cells/mm2 lamina propria area) of IgA immunocytes tended to be slightly higher in the appendix than in the colon (1259 vs 962) and the same held true for IgM cells (71 vs 55). Conversely, the overall density of IgG immunocytes was much higher in the appendix than in the colon (95 vs 38). A striking feature was the fact that almost 50% of all immunocytes were of the IgG isotype adjacent to lymphoid follicles. It seemed justified to conclude, therefore, that the abundance of such follicles explains the overall enrichment of IgG-producing cells in normal appendix mucosa. These immunocytes most likely represent follicle derived B cells that have reached terminal maturation locally, whereas precursors generated from less mature memory clones probably emigrate and home ubiquitously to distant sites of the gut lamina propria where they develop into IgA-producing immunocytes.

The intestinal secretory IgA system is quantitatively the most important humoral immune system of the body — about 1010 Ig-producing cells being present per metre of gut (1). The precursors of these immunocytes are apparently generated largely in the Peyer’s patches owing to specialized mechanisms of antigen uptake and immune regulation (2,3). Lymphoid follicles of the appendix and solitary follicles scattered throughout the small and large bowel mucosa, show comparable features and may function as a part of the gut-associated lymphoid tissue (GALT). A comparative study of the immunoglobulin (Ig) isotype expression of B cells associated with lymphoid follicles of the appendix, colon and ileum mucosa and in Peyer’s patches was therefore carried out. Cytoplasmic J chain was included as a marker of clonal immaturity (4) to study the extent of terminal B cell maturation in situ.

M cells of follicle-associated epithelium (FAE) transport luminal antigens into Peyer’s patches (PP), but the complete role of these cells in local immunity has not been defined. To this end it would be of interest to evaluate their spatial relationship to T cells and their possible capacity to present antigens to T cells.