Hege S. Carlsen

Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease

BACKGROUND & AIMS Most macrophages in the normal intestinal mucosa have a mature phenotype. In inflammatory bowel disease (IBD), a monocyte-like subset (CD14+ L1+) accumulates. The aim of this study was to characterize its potential with regard to cytokines. METHODS Lamina propria mononuclear cells were adherence-separated, with or without depletion of CD14+ cells, and production of cytokines was investigated by bioassay, enzyme-linked immunosorbent assay, or immunocytochemistry. RESULTS Tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-1 receptor antagonist were found mainly in cells positive for myelomonocytic L1. In undepleted IBD cultures, TNF-alpha, IL-1alpha and beta, and IL-10 were markedly up-regulated by pokeweed mitogen stimulation; IL-1alpha and beta and IL-10 were also up-regulated by stimulation of interferon gamma and lipopolysaccharide in combination. The latter stimulation had no effect on normal control or CD14-depleted IBD cultures. Indomethacin caused a marked increase of TNF-alpha, particularly in undepleted IBD cultures, whereas IL-10 and IL-4 decreased TNF-alpha and IL-1beta in both CD14+ and CD14 macrophages. CONCLUSIONS In IBD mucosa, macrophages with a monocyte-like phenotype are primed for production of TNF-alpha and IL-1alpha/beta and may therefore be of significant pathogenic importance [corrected]. However, this CD14+ subset, as well as the mucosal resident macrophages, have preserved responsiveness to several down-regulatory factors such as the macrophage deactivators IL-10 and IL-4.

Naive T Cell Recruitment to Nonlymphoid Tissues: A Role for Endothelium-Expressed CC Chemokine Ligand 21 in Autoimmune Disease and Lymphoid Neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP+ cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP+ cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 ± 10%) and ulcerative colitis (66 ± 1%) expressed CCL21, and many perivascular CD45RA+ naive T cells were found in these tissues, but not in psoriasis, where CCL21+ vessels were rare (17 ± 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.

B cell attracting chemokine 1 (CXCL13) and its receptor CXCR5 are expressed in normal and aberrant gut associated lymphoid tissue

Background and aims: In mice, the B lymphocyte chemoattractant (BLC) CXC chemokine ligand 13 (CXCL13) is sufficient to induce a series of events leading to the formation of organised lymphoid tissue. Its receptor, CXCR5, is required for normal development of secondary lymphoid tissue. However, the human counterpart, B cell attracting chemokine 1 (BCA-1) has only been detected in the stomach and appendix and not in other parts of normal or diseased gut. Hence to elucidate the potential role of this chemokine and its receptor in human gut associated lymphoid tissue (GALT), we analysed their expression in normal intestine and ulcerative colitis (UC). Methods: Frozen sections of surgical specimens were studied by multicolour immunofluorescence staining, in situ mRNA hybridisation, and reverse transcription-polymerase chain reaction. Results: BCA-1 mRNA was detected in all normal colonic and UC specimens. BCA-1 was produced and accumulated in relation to peripheral dendritic elements of lymphoid follicles in Peyers patches and normal colon, as well as in irregular lymphoid aggregates in UC lesions. BCA-1 was partially associated with the traditional follicular dendritic cell phenotype but also with extracellular fibrils in GALT structures. CXCR5 protein was expressed by mantle zone B cells and appeared at a high level on scattered germinal centre T cells. Conclusions: BCA-1 and CXCR5 are expressed in normal GALT structures as well as in irregular lymphoid aggregates in UC. This strongly suggests that BCA-1 plays an important role not only in the formation of normal GALT but also in the generation of aberrant lymphoid tissue in inflammatory bowel disease.

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.

Should we use laparoscopic adrenalectomy for metastases? Scandinavian multicenter study

Laparoscopic adrenalectomy for metastases is considered controversial. Multicenter retrospective study was performed to gain new knowledge in this issue.

Chapter 32 – The Human Mucosal B-Cell System

This chapter deals with the activation of mucosal B cells and their unique immunoregulation that results in high levels of J-chain expression. The functional goal of this regulatory mechanism is the abundant generation of Ig polymers at mucosal effector sites, where these molecules can readily be subjected to external transport and become operative as secretory antibodies (S-IgA and S-IgM). Lymphoid cells are located in three histologically distinct tissue compartments at mucosal surfaces: immune-inductive organized mucosa-associated lymphoid tissue (MALT), the lamina propria or glandular stroma, and the surface epithelia. Although gut-associated lymphoid tissue (GALT) is the largest and best defined part of MALT, other potentially inductive sites for mucosal B-cell responses include—bronchus-associated lymphoid tissue (BALT) and nasopharynx-associated lymphoid tissue (NALT)—in humans, particularly the unpaired nasopharyngeal tonsil and the paired palatine tonsils. These organs make up most of the Waldeyers pharyngeal lymphoid ring and play a major role for mucosal immunity in human airways, because BALT structures are not present in normal lungs of adults and in only 40% of healthy adolescents and children.

The proportion of CD40+ mucosal macrophages is increased in inflammatory bowel disease whereas CD40 ligand (CD154)+ T cells are relatively decreased, suggesting differential modulation of these costimulatory molecules in human gut lamina propria

Background: Signal transduction through binding of CD40 on antigen‐presenting cells and CD40 ligand (CD154) on T cells appears to be crucial for mutual cellular activation. Antibodies aimed at blocking the CD40−CD154 costimulatory pathway dampen the severity of experimental colitis. To elucidate the microanatomical basis for signaling through this costimulatory pathway in human inflammatory bowel disease, we studied in situ the cellular distribution of these 2 molecules on lamina propria macrophages and T cells, respectively. Methods: Colonic specimens from 8 patients with ulcerative colitis and 8 with Crohns disease, 8 small bowel specimens of Crohns disease, and histologically normal control samples (6 from colon and 6 from small bowel) were included. Multicolor immunofluorescence in situ staining was performed to determine the percentage of subepithelial macrophages expressing CD40 and that of lamina propria T cells expressing CD154 while avoiding cells in lymphoid aggregates. Results: The proportion of subepithelial CD40highCD68+ macrophages was significantly increased in normal colon compared with normal small bowel and showed further elevation in both colon and small bowel afflicted with inflammatory bowel disease. In addition, on a per‐CD68+‐cell basis, CD40 expression was significantly increased in severely inflamed compared with moderately inflamed colonic specimens. Conversely, the proportion of CD154+ T cells was similar in colon and small bowel, and interestingly, it was significantly reduced in colonic inflammatory bowel disease. Conclusions: Our findings suggested that modulation of CD40 expression by subepithelial macrophages and CD154 by lamina propria T cells is inversely modulated in the human gut.

The traffic of mucosal lymphocytes to extraintestinal sites

Migration of lymphoid cells throughout the body is regulated by their expression of multiple adhesion molecules and receptors for chemoattractants (chemokine receptors) that interact with corresponding ligands on endothelial and stromal cells (1). Thus, primed T and B cells enter effector tissues in a site-specific manner (2). It was first believed that the dissemination of memory/effector B cells from mucosa-associated lymphoid tissue was completely integrated to ensure availability of a variety of secretory immunoglobulin A (SIgA) antibody specificities at every mucosal surface regardless of the immune-inductive site. The term ‘a common mucosal immune system’ has therefore been used for decades. However, even early indirect data suggested, instead, that mucosal B-cell homing is dictated by certain tissue-specific preferences (2). For ethical reasons it has not been possible to document this notion directly in humans by tracking of labeled immune cells, and there is no known marker on circulating mucosa-associated lymphoid tissue-induced B cells that allows precise identification of their region of origin. Therefore, uncertainty exists particularly with regard to the role of nasopharynx-associated lymphoid tissue (NALT), such as Waldeyer’s ring in humans (3), for the generation of plasma cells at regional and distant secretory effector sites. Knowledge to this end would be desirable for a future rational development of efficient mucosal vaccines applied via the nasal route. Because the vast majority of infectious agents use the mucosae as portal of entry, there is currently a great interest in the local production of IgA antibodies (4,5). We have used a DNA marker, namely C Ig heavychain gene deletion that identifies a surface (s)IgDIgM subset known to be generated in tonsillar germinal centers by nonclassic Ig class switching (6). This subset represents a minor fraction (2–5%) of tonsillar B cells but we found that its C deletion could be used as a signature of dissemination from human NALT. By polymerase chain reaction analysis of DNA from isolated cells or tissue samples, we were able to track specifically the presence of NALT-derived centroblasts or their J-chain positive IgD plasma cell progeny also at distant sites. We showed that these B cells preferentially home to regional secretory effector tissues, while entering the gut lamina propria to a much lesser extent, being virtually excluded from the small bowel and appendix. Such compartmentalized dispersion was maintained even in celiac disease and inflammatory bowel disease. This was surprising because extravasation in inflammatory bowel disease lesions is generally less restricted for cells of a mucosal phenotype as signified by the occurrence of IgA immunocytes with decreased J-chain expression, numerous IgG-producing cells, naïve T lymphocytes, and monocyte-like macrophages. The cervical lymph nodes displayed the highest C deletion frequency of all organized lymphoid tissues examined beyond Waldeyer’s ring. Notably, when sIgDIgM B cells leave this inductive site via draining lymph, they have to pass through the cervical nodes. However, we could not exclude that the deletion was induced directly in these nodes by microbial products derived from the nasopharynx, thus giving rise to sIgDIgM centrocytes with homing properties similar to NALT-derived B cells. Antigens reaching the regional lymph nodes via draining lymph, or being transported there by dendritic cells, may elicit mucosal immunity targeted at the same region. Importantly, the human nasal mucosa is very rich in various dendritic cell types, both within and beneath the epithelium. The leukointegrin 4ß7 is used by B cells derived from gut-associated lymphoid tissue to enter the intestinal mucosa through interactions with the endothelial mucosal addressin cell adhesion molecule (MAdCAM)-1 without the participation of L-selectin (CD62L) (1,2). Migration into the small intestinal lamina propria appears to be additionally dictated by the chemokine receptor CCR9 that interacts with the thymus-expressed chemokine (TECK, CCL25) selectively produced by the crypt epithelium in this part of the gut (1,7). Conversely, molecules used for preferential homing to human mucosal effector sites beyond the gut have remained elusive. Address correspondence and reprint requests to: Prof. Per Brandtzaeg, Head, Faculty Division, Rikshospitalet University Hospital Head, Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, Rikshospitalet, N-0027 Oslo, Norway; Fax: 47-23-07-15-11 (e-mail: per.brandtzag@labmed.uio.no). Journal of Pediatric Gastroenterology and Nutrition 39:S725–S726