Trond S. Halstensen

Intraepithelial T Cells of the TcRγ/δ+CD8− and Vδ1/Jδ1+ Phenotypes are Increased in Coeliac Disease

Expression of the γ/δ T‐Cell receptor (TcR) for antigen on CD3+ intraepithelial lymphocytes (IEL) was studied in situ by two‐colour immunofluorescence on jejunal tissue secretions from 24 patients with coeliac disease and 17 controls. The proportion of intraepithelial TcRγ/δ+ cells was significantly increased (P<0.002) in untreated (median 20%, range 11–53%) as well in treated (gluten‐free diet) coeliac disease (median 23%, range 16–55%) compared with controls (median 2%, range 0–39%). Although TcRγ/β+ IEL dominated both in controls and coeliac disease. T cells expressing the TcRα/δ were preferentially located within the epithelium rather than in the lamina propria. Paired staining for TcRγ/δ and CD8 revealed that most (∼90%) intraepithelial TcRγ/δ+ lymphocytes in coeliac disease were CD8−. A remarkably large fraction (median 67%, range 58–94%) of intraepithelial TcRγ/δ+ cells expressed the Vδ1/Jδ1‐encoded epitope revealed by monoclonal antibody δTCS1. Our results suggested that increase of the intraepithelial TcRγ/δ+ CD8− subset of T cells is particularly related to coeliac disease.

Increased macrophage subset in inflammatory bowel disease: apparent recruitment from peripheral blood monocytes.

Mucosal specimens from active Crohns disease (ileum, n = 6; colon, n = 6), active ulcerative colitis (n = 9), normal ileum (n = 6), and normal colon (n = 6) were subjected to paired immunofluorescence staining for characterisation of macrophage subsets in situ. In the normal state, only few CD68+ macrophages (< 10%) expressing the myelomonocytic L1 antigen (calprotectin) were seen. In inflamed mucosa, especially near small vessels, the CD68+L1+ fraction increased with the degree of inflammation, near ulcers to median 65% (range 35-91%). Cells reactive with the monoclonal antibody RFD7 were also increased in inflammation but less than 5% of them costained for L1 antigen. It is concluded that L1 producing macrophages are distinct from the RFD7+ subset and probably recently recruited from peripheral blood monocytes. Like granulocytes, L1+ macrophages may be important in non-specific defence, providing calprotectin with putative anti-microbial and anti-proliferative properties.

Epithelial Deposition of Immunoglobulin G1 and Activated Complement (C3b and Terminal Complement Complex) in Ulcerative Colitis

The epithelial destruction seen in ulcerative colitis remains unexplained. Complement activation has been proposed to be involved, but no definite evidence has been available to this end. In the present study, we examined immunohistochemically ulcerative colitis lesions with monoclonal antibodies to activation neoepitopes in the complement component C3b and in the cytolytically active terminal complement complex. Colonic tissue specimens from 23 patients with ulcerative colitis were examined by indirect two-color immunofluorescence staining with monoclonal antibodies to the four human immunoglobulin G subclasses and to activated complement C3b or terminal complement complex. All except two patients had activated C3b deposited apically on the surface epithelium of involved mucosa. Immunoglobulin G1 was found on the epithelium in extensively prewashed specimens from 7 of 11 patients, and a striking colocalization of immunoglobulin G1, C3b, and terminal complement complex was observed in 4. Immune deposits were not observed in 31 noninflamed specimens from the same ulcerative colitis patients. Only 1 of 44 histologically normal mucosae from 17 controls and 1 of 10 colonic adenomas contained some epithelial complement deposits. It is concluded that activated complement is often deposited along the brush border of the surface epithelium in active ulcerative colitis lesions and may be associated with immunoglobulin G1 autoantibody.

Distribution of macrophages and granulocytes expressing L1 protein (calprotectin) in human Peyer's patches compared with normal ileal lamina propria and mesenteric lymph nodes.

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 Peyers 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 Peyers 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 Peyers patches, where CD68+L1- macrophages were abundant. Also subepithelial and interfollicular CD68- interdigitating dendritic cells in Peyers 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 Peyers patches may support the immunostimulatory function of these macrophage rich structures, which are held to be crucial for induction of specific mucosal immunity.

Epithelial expression of HLA, secretory component (poly-Ig receptor), and adhesion molecules in the human alimentary tract.

Epithelial HLA class II is differentially expressed (DR >> DP) only after birth in salivary glands and small intestinal mucosa, in contrast to class I determinants and secretory component (SC) which appear early in gestation. However, there is a brisk postnatal increase in SC expression along with the class II induction, suggesting stimulation by cytokines from activated immune cells. T lymphocytes remain quite scanty in postnatal salivary glands, and the striking SC and class II expression might reflect a synergistic effect of IFN-gamma and TFN-alpha on immature epithelial cells. Enhanced epithelial expression of both SC and class II in salivary glands from sudden infant death victims could be the effect of immunostimulation caused by an infectious agent. Strikingly upregulated SC and epithelial class II expression (DR > DP > DQ) is seen in various inflammatory lesions such as obstructive sialadenitis, Sjögrens syndrome, chronic gastritis, and celiac disease. IFN-gamma and TNF-alpha are most likely involved as the expression patterns can be reproduced with these cytokines in vitro on colonic epithelial cell lines. However, these molecules of the Ig supergene family do not show a selective response in epithelia of inflammatory lesions because increased expression is also seen for lysozyme, lactoferrin and some other proteins. ICAM-1 can be upregulated on epithelial cells by various cytokines in vitro although the situation remains uncertain in mucosal inflammation. The expression pattern in IBD is complicated by dysplastic epithelial changes leading to reduced SC levels which may thus, in turn, jeopardize the poly-Ig transport mechanism. Epithelial class II molecules appear to have antigen-presenting properties, but the immunopathologic role of their increased expression in inflammatory disease in terms of induction of autoimmunity and/or abrogation of oral tolerance is a matter of continuing dispute.

Gluten stimulation of coeliac mucosa in vitro induces activation (CD25) of lamina propria CD4+ T cells and macrophages but no crypt-cell hyperplasia.

Jejunal biopsy specimens from 10 patients with treated coeliac disease and seven non‐coeliac controls were challenged in vitro with peptic‐tryptic gluten digest. Mucosal T cells were examined in situ by three‐colour immunofluorescence staining for expression of the activation marker CD25 (the p55 α‐chain of intcrlcukin‐2 receptor) and the nuclear proliferation marker revealed by monoclonal antibody Ki‐67. Intraepithelial T cells expressed CD25 rarely whereas the proportion of activated lamina propria T cells increased (P< 0.002) from median 2.8% (cultured with 20% fetal calf serum alone for 24–48 h) to 10.0% after 24h with gluten (n= 10; range 1.1–17.4%) and to 10.4% after 48 h (n = 7; range 1.4–17.5%). Such gluten‐induced increase of CD25+ T cells was not observed in specimens from non‐coeliac control subjects. Crypt‐cell hyperplasia and T‐cell proliferation (Ki‐67+) were observed neither in the coeliac nor in the control mucosae after gluten stimulation. Three‐colour staining combining a polyclonal antibody reagent to CD3 and a monoclonal antibody to CD25 with a monoclonal antibody to CD45RO, CD4. CDS, the p75 β‐chain of intcrleukin‐2 receptor, integrin χEβ7, or HLA‐DR showed that most of the CD25+ T cells (> 90%) were CD4+ CDS, co‐expressed CD45RO and the p75 β‐chain. and often also the integrin χEβ7 but not HLA–DR. In addition to these activated T cells, a dominating population of CD25+ CD3‐CD4+ subepithelial pan‐HLA–class I+ macrophages (CD68+) with variable expression of the p75 β‐chain was often induced by gluten challenge.

Upper airway inflammation in waste handlers exposed to bioaerosols

Aims: To examine work associated upper airway inflammation in 31 waste handlers, and to correlate these findings with personally monitored exposure to different bioaerosol components. Methods: Cell differentials, interleukin 8 (IL-8), myeloperoxidase (MPO), and eosinophilic cationic protein (ECP) were examined in NAL (nasal lavage), and swelling of the nasal mucosa was determined by acoustic rhinometry before work start on Monday and the following Thursday. Bioaerosol exposure was determined by personal full shift exposure measurements on Monday, Tuesday, and Wednesday and analysed for total bacteria, fungal spores, endotoxin, and β(1→3)-glucans. Results: The increased percentage of neutrophils from Monday (28%) to Thursday (46%) correlated with increases in ECP (rS = 0.71, p < 0.001) and MPO (rS = 0.38, p < 0.05), and showed a close to significant correlation with nasal swelling (rS = −0.55, p = 0.07). The Thursday levels of neutrophils, MPO, and IL-8 were associated with the exposure to fungal spores (range 0–2.0 × 106/m3) and endotoxin (range 4–183 EU/m3) measured the day before, and the median exposure to β(1→3)-glucans (range 3–217 ng/m3), respectively (rS = 0.47–0.54, p < 0.01). Swelling of the nasal mucosa was associated with the fungal spore and β(1→3)-glucan exposure (rS = 0.58–0.59, p < 0.05). Conclusion: These results are based on a relatively small population, and conclusions must be drawn with care. The results suggested that a moderate exposure to fungal spores, endotoxins, and β(1→3)-glucans during waste handling induced upper airway inflammation dominated by neutrophil infiltration and swelling of the nasal mucosa.

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.

Airway inflammation in waste handlers exposed to bioaerosols assessed by induced sputum

Work-associated lower airway inflammation in waste collectors was examined by induced sputum and correlated with the bioaerosol exposure. Organic waste collectors (n=25) underwent induced sputum collection and spirometry before work on Monday and the following Thursday. Total cells, cell differentials, interleukin (IL)-8 and eosinophilic cationic protein were determined. Personal full-shift exposure measurements were performed Monday, Tuesday and Wednesday and analysed for total bacteria, fungal spores, endotoxins and β(1–3)-glucans. The percentage of neutrophils (46–58%) and the IL-8 concentration (1.1–1.4 ng·mL−1) increased from Monday to Thursday. Forced expiratory volume in one second (FEV1) was significantly reduced on Thursday, and the decrease in FEV1/forced vital capacity correlated with the increase in the percentage of neutrophils. The median exposure to endotoxin (range 7–180 EU·m−3) and β(1–3)-glucan (range 5–220 ng·m−3) was correlated with the increase in IL-8. Bioaerosol exposure during waste collection induced an inflammatory response in the lower airways, characterised by neutrophils and interleukin-8 secretion, that influenced the lung function. The inflammatory response was related to microbial components in the bioaerosol and was more pronounced for endotoxin than β(1–3)-glucan exposure. No associations were found for mould spores or bacteria.

Surface epithelium related activation of complement differs in Crohn's disease and ulcerative colitis.

IgG1 and activated complement are colocalised on the colonic epithelial brush border in active ulcerative colitis. To investigate whether such deposition is specific for ulcerative colitis, we examined ethanol fixed mucosal specimens from 18 patients with Crohns colitis and 14 with terminal ileitis by indirect two colour immunofluorescence staining. Monoclonal antibodies to the IgG subclasses and to neoepitopes of activated complement C3b and the terminal complement complex were used in combination with rabbit antiserum to C1q, C4c or cytokeratin. Granular deposition of C3b and terminal complement complex were observed at the luminal face of the surface epithelium in 10 of 18 patients with Crohns colitis. Specimens from eight of 14 patients with ileal involvement were intensely stained for activated complement (primarily C3b) within the surface mucus layer. No epithelial IgG, C1q or C4c deposition was observed. The results suggest that early and late phase complement activation takes place at the luminal face of the epithelium in Crohns disease. The absence of colocalised IgG and complement components involved in the classical activation pathway (C1q and C4c), however, suggest that other immunopathological mechanisms (the alternative pathway?) are primarily involved in Crohns disease in contrast with ulcerative colitis.