Olav Fausa

Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease

BACKGROUND & AIMS Celiac disease appears to be a T cell-mediated enteropathy induced by gluten in genetically predisposed individuals. Duodenal biopsy specimens from patients with celiac disease and histologically normal controls were investigated to see if cytokine expression is related to disease activity. METHODS Cytokine messenger RNA (mRNA) expression was determined by quantitative reverse-transcription polymerase chain reaction and in situ expression by immunohistochemistry. RESULTS In normal controls, mRNA levels were usually below the quantitative limit, even after in vitro gluten stimulation. By contrast, interferon (IFN)-gamma mRNA was increased more than 1000-fold in untreated disease. In vitro gluten stimulation of specimens from treated patients (gluten-free diet) increased IFN-gamma mRNA to the levels of untreated patients. In addition, increased mRNA levels for interleukin (IL)-2, IL-4, IL-6, and tumor necrosis factor alpha were found after such stimulation, whereas mRNA for IL-5, IL-10, and IL-12p40 was usually below the quantitative level. Biopsy specimens from untreated patients contained on average 10-fold more lamina propria cells positive for IFN-gamma than normal controls, whereas cells containing IL-4 were rare in both subject groups. CONCLUSIONS The results show that mucosal gluten exposure in patients with celiac disease rapidly elicits high levels of IFN-gamma expression and lower levels of IL-2, IL-4, IL-6, and tumor necrosis factor alpha even in the virtual absence of IL-12.

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.

T cells from the small intestinal Mucosa of a DR4, DQ7/DR4. DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8

CD is an immunologic disease of the small intestine which is precipitated by ingestion of wheat gliadin. Most patients carry the HLA-DQ (alpha 1*0501, beta 1*0201) (DQ2) heterodimer. We recently reported that a preponderance of gliadin-specific T cells from the small intestinal mucosa of DQ2-positive CD patients were restricted by this DQ heterodimer. However, a small percentage of CD patients do not carry this DQ heterodimer, and most of them instead carry DQ (alpha 1*0301, beta 1*0302) (DQ8). Here we report that a majority of gliadin-specific T cells from the small intestinal mucosa of a DR4,DQ7/DR4,DQ8 heterozygous CD patient are restricted by DQ8. Thus, preferential presentation of gliadin-derived peptides to T cells by the CD-associated DQ2 and DQ8 molecules may be an initial and important immunopathogenic step in CD.

Immunohistochemical Characterization of Local Immunoglobulin Formation in Ulcerative Colitis

Compared with 6 control patients without overt inflammation, 8 patients with active ulcerative colitis exhibited the following characteristics in tissue specimens from the large intestine. (1) Persisting glands seemed to mediate a normal external transport of IgA and IgM via secretory component-producing columnar epithelial cells. (2) The average total numbers of mucosal IgA and IgM immunocytes in areas with persisting glands were raised 2.2 and 5.2 times, respectively. (3) The average total number of mucosal IgG immunocytes in such areas was raised about 30 times, and the submucosa, in addition, contained a fairly dense immunocyte population with 84% IgG cells. This study suggests that a primary defect in the local secretory immunoglobulin system cannot explain the development of ulcerative colitis. A pronounced local IgG immunocyte response is associated with the pathogenesis of this disease. Regardless of the initiating etiological factor(s), locally produced IgG antibodies may directly, or in cooperation with lymphocytes, be responsible for chronicity and recurrences.

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.

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.

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.

Intestinal B-cell isotype response in relation to local bacterial load: Evidence for immunoglobulin A subclass adaptation☆

BACKGROUND & AIMS In experimental animals, the indigenous microbiota modulates mucosal immunity. In humans, such direct evidence is scarce. The aim of this study was to examine the effect of intestinal bacteria on the local immunoglobulin (Ig) response. METHODS The numbers of IgA-, IgM-, and IgG-producing immunocytes per defined mucosal length unit were determined, and the local IgA subclass response was studied using immunohistochemistry in jejunal segments from adults with bacterial overgrowth and in sterile ileal urinary conduits from children. RESULTS The ileal bladder mucosa showed atrophy, but the number of immunocytes only tended to be decreased. The jejunal segments with bacterial overgrowth showed minor histological changes; the numbers of IgA and IgG immunocytes were fairly normal, whereas the number of IgM immunocytes was significantly reduced (P < 0.05) (12 cells/U) compared with control mucosa (24 cells/U). The number of IgA2 immunocytes was significantly decreased (P < 0.01) in ileal conduits (7 cells/U or 30% of total IgA) but increased (P < 0.05) in jejunal segments with bacterial overgrowth (42 cells/U or 43% of total IgA) compared with normal ileum (15 cells/U or 40% of total IgA) and jejunum (24 cells/U or 23% of total IgA). CONCLUSIONS An association exists between bacterial load and IgA subclass production. An increase in IgA2 may enhance mucosal protection and probably reflects immunomodulation caused by lipopolysaccharides.

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.

Expression of major histocompatibility complex class II subregion products by jejunal epithelium in patients with coeliac disease.

The MHC class II subregion product (HLA‐DR), HLA‐DP, and HLA‐DQ) were located by immunofluorescence in serial sections of ethanol‐fixed, paraffin‐embedded jejunal mucosa from control subjects and patients with coeliac disease (CD). DR staining was seen in a granular luminal distribution and basolaterally on surface epithelial was positive for DR almost to the bottom of the glands. This contrasted with virtually absent glandular DR standing in controls and weak staining including only the upper part of the crypts in 5 out of 11 treated patients. HLA‐DQ appeared only in three untreated patients and was restricted to patches of surface epithelium. The number of intraepithelial T lymphocytes per millimetre of surface epithelium was significantly higher in untreated than in treated CD patients or controls; it was also significantly higher in specimens with epithelial DP expression than in those without. This suggested that intraepithelial lymphocytes modulate epithelial class II expression.