Garbiñe Roy

Human small-intestinal epithelium contains functional natural killer lymphocytes

BACKGROUND & AIMS CD3(-) non-T lymphocytes constitute the second most abundant lymphoid subset in the human small-bowel epithelium, and these CD3(-) intraepithelial lymphocytes are virtually absent in active celiac disease. Phenotypically, they resemble natural killer cells and have been termed natural killer-like intraepithelial lymphocytes. Because of the limited availability of appropriate human samples, functional studies have not yet been reported, and it is not yet clear whether these are true natural killer cells. METHODS We used magnetic bead-based purification and flow cytometry to study several aspects of normal human small-bowel natural killer-like intraepithelial lymphocytes: intracellular cytokine content (basally and after activation); ability to lyse natural killer-sensitive K562 target cells; and expression of perforins, Fas ligand, and other functional markers. RESULTS CD3(-) intraepithelial lymphocytes cultured in interleukin-2 showed a higher lymphokine-activated killer activity than CD3(+) intraepithelial lymphocytes (48%-83% lysis exerted by CD3(-) intraepithelial lymphocytes at an effector-target cell ratio of 2:1 vs. 8%-18% by CD3(+) intraepithelial lymphocytes). Perforin content correlated with this lytic potential (75% +/- 4% in CD3(-) vs. 5% +/- 4% in CD3(+) intraepithelial lymphocytes). Both CD3(-) and CD3(+) cells displayed a type I cytokine profile (interferon-gamma > tumor necrosis factor-alpha > interleukin-2; undetectable interleukin-4 and interleukin-10). In addition to their activated phenotype, subsets of natural killer-like intraepithelial lymphocytes expressed CD8alphaalpha and intracellular CD3epsilon chain, showing the existence of heterogeneity within this cell lineage. CONCLUSIONS This is the first demonstration of functional natural killer cells within the human gut epithelium. These cells might play an important role in innate mucosal immunity (host defense and tumor surveillance) and tolerance.

Susceptibility of different leukocyte cell types to Vaccinia virus infection

BackgroundVaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus.ResultsWe have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells.ConclusionsWhen infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.

Intraepithelial lymphocytes and coeliac disease: permanent changes in CD3−/CD7+ and T cell receptor γβ subsets studied by flow cytometry

Permanent changes in intestinal intraepithelial lymphocytes have been observed in coeliac patients. The aim of this investigation was to study small intestinal intraepithelial lymphocytes by using flow cytometry and to evaluate its diagnostic value in coeliac disease. Three‐colour flow cytometry analyses were performed on isolated epithelial cells of 117 intestinal biopsies obtained from 113 children (54 coeliac disease, 4 other enteropathies, 18 Helicobacter pylori associated gastritis and 37 normal controls). A multiple logistic regression model was developed to select the best intraepithelial lymphocytes subset predictor of coeliac disease. Coeliac patients had significant higher levels of T cell receptor yS intraepithelial lymphocytes than control patients (p < 0.01), H. pylori patients (p < 0.01) and other enteropathies (p < 0.05). The density of CD3∼/CD7+ intraepithelial lymphocytes, a intraepithelial lymphocyte subset poorly characterized by immuno‐histochemical methods, was significantly lower in coeliac patients than in the control group (p < 0.01), H. pylori group (p < 0.01) and other enteropathies (p < 0.01). Both changes remained altered independent of the coeliac patients diet. The data were used on a logistic regression analysis in order to calculate sensitivity [94.4%; 95% confidence interval (CI) 83.7‐98.6%], specificity (94.9%; 95% CI 84.9‐98.7%) and likelihood ratio for a positive test 18.5 (95% CI 6.1‐55.8) in the diagnosis of coeliac disease.

Helicobacter pylori infection and insulin-dependent diabetes mellitus

Helicobacter pylori is associated with different diseases: duodenal ulcer, rosacea, ischaemic heart disease and gastric cancer. Given the abnormal immunological response and the high prevalence of gastrointestinal symptoms in diabetic patients, we conducted a study on H. pylori prevalence among these patients. We designed a case control study of a population-based cohort. Eighty insulin-dependent diabetes mellitus (IDDM) patients with an average age (24.05 +/- 8.3 years), and 100 control subjects (25 +/- 7.1 years) were selected to verify the seroprevalence of Helicobacter pylori in these populations. One serum sample was obtained from each subject for evaluation of antibodies against Helicobacter pylori, parietal cells (APA) and pancreatic islets cells (ICA). The seroprevalence of H. pylori among IDDM patients aged less than 24 years was significantly higher than among control subjects; the corresponding rate among IDDM aged greater than 24 years was significantly lower than among control subjects. Antibodies against parietal cells (APA) and islet cells (ICA) among H. pylori positive diabetic patients were significantly higher than among H. pylori negative diabetic patients. IDDM patients were subdivided on the basis of the evolutive course of diabetes. Seroprevalence of H. pylori as well as prevalence of ICAs decreased with IDDM duration. Nevertheless, no variation in the prevalence of APAs during the course of diabetes was observed. We observed an association between the seroprevalence of Helicobacter pylori and the duration of IDDM. The seroprevalence of H. pylori and ICA decreased with the evolutive course of diabetes mellitus among IDDM. The prevalence of ICA and APA in IDDM H. pylori positive subjects was higher than among controls.

Description of an enzyme-linked immunosorbent assay for the detection of protein tyrosine kinase.

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu.Na,Tyr) 4:1 to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to the color at 492 nm developed in each well.

Cytokine Production by Intestinal Intraepithelial Lymphocyte Subsets in Celiac Disease

One of the earliest signs of mucosal immune activation in celiac disease (CD) is an increase in the intraepithelial lymphocyte (IEL) count in the small intestinal epithelium. Though most of those IELs express T cell receptor (TcR)-αβ chains, CD is characterized by an increase in TcR-γ δ+ IELs and by the loss of CD3− IELs. There is currently little evidence that these changes in IEL subset distribution are of relevance in the pathogenesis of CD. We aimed to determine the pattern of cytokine production by IEL subsets isolated from duodenal biopsy specimens from control subjects and CD patients at different stages of the disease. We quantified the capacity of IEL subsets to produce IFN-γ, TNF-α, IL-2, IL-4, and IL-10 by intracellular staining by flow cytometry. All IEL subsets studied displayed a type I cytokine profile in both CD and control subjects, with TcR-αβ+ IELs being the main IFN-γ producers. Untreated CD exhibited a trend toward a superior accumulation of IFN-γ per cell but a reduced proportion of INF-γ+ cells in vitro in association with a significantly increased apoptotic rate of IELs. IL-4 was almost undetectable in all cases and IL-10 showed a tendency to increase in treated and “silent” celiac patients. IEL subsets have a similar Th1 profile in controls and CD patients, and the superior in vitro apoptosis of IELs from CD patients may reflect their superior in vivo activation. The induction of IL-10-dependent regulatory Tr1 responses may be of potential clinical significance in this disease and merits further investigation.

Clinical and immunological features of adult-onset generalized autoimmune gut disorder.

OBJECTIVES:Autoimmune enteropathy is a rare disorder of unknown pathogenesis, characterized by protracted diarrhea, villous atrophy, and enterocyte autoantibodies. Its association with extended inflammation of the whole gastrointestinal tract is termed as generalized autoimmune gut disorder (GAGD), generally a pediatric disease of difficult management due to its association with immunodeficiency. The aim of our work is to describe the mucosal immunological basis of an adult-onset case of GAGD.METHODS:We studied an adult female with a severe inflammatory involvement of the gastrointestinal tract (stomach, small and large bowel, and liver) and antienterocyte autoantibodies. She had antibody deficiency and a predisposition to systemic autoimmunity. We analyzed, by immunohistochemistry and flow cytometry, the phenotypic and functional characteristics of her intestinal intraepithelial and lamina propria (LP) lymphocytes.RESULTS:We observed the prominent and constant presence of an unusual CD4+αE/β7− Tc subset in the jejunal epithelium. Signs of the lymphocyte activation as well as the prominent lymphoid TNF-α production observed in the rectal mucosa support the involvement of a cell-mediated pro-inflammatory response in the pathogenesis of GAGD.CONCLUSIONS:We report the second case of an adult fulfilling all diagnostic criteria for GAGD. We propose that the activated LP CD4+ T lymphocytes, as well as those atypically located in the epithelium, may play a pathogenic role. The αE/β7− IEL could constitute a diagnostic marker of intestinal autoimmunity in the cases when autoantibodies are not evidenced, and mucosal TNF-α might represent a novel therapeutic target in this severe disease.

Age-related variation of intraepithelial lymphocytes subsets in normal human duodenal mucosa.

The enumeration of intestinal intraepithelial lymphocytes (IELs), and the phenotyping of CD3+CD103+ (TcRαβ, TcRγδ) and CD3−CD103+ IEL subsets constitute useful diagnostic tools for the correct interpretation of the mucosal histology of duodenal/jejunal biopsies in many pathological conditions of the small intestine, particularly celiac disease (CD). This work evaluates the ranges of duodenal IEL counts by flow cytometry in healthy mucosa from pediatric and adult controls, establishing normal reference values for CD3+ TcRγδ and CD3− subsets and their variation with age. Seventy-four pediatric controls and 36 adult controls were identified on the basis of their normal histology from more than 1,000 duodenal diagnostic biopsies performed in Caucasian subjects. Total IEL counts and IEL subsets (“IEL lymphogram”) were analyzed by four-color flow cytometry (FCM). IEL represent 7.7%±0.4 (mean±SE) and 8.5%±0.5 of the cells isolated from the epithelium in the pediatric and adult series, respectively. The upper normal range, considered as the 97 percentile, is 14% in pediatrics and 15% in adults. No significant difference was observed between TcRγδIEL percentages in children (6.9%±0.5 of the total IELs) and adults (6.6%±0.8). However, the density of CD3− IELs is significantly higher (p < 0.001) in the mucosa from controls under 3 years (50.2%±2.6) than in adults (25.5%±2.1). IEL lymphogram by flow cytometry is an easy, quick and reliable analysis performed in one of the biopsy specimens obtained during a diagnostic endoscopy, and confers specificity to the histopathological findings. IEL counts below 14% in children and 15% in adults should be considered within a normal range in the evaluation of duodenal mucosa by FCM. No differences with age were observed with respect to TcRγδIEL, while the CD3− IEL fraction was significantly higher on children under 3 years, with a trend to increase again in the elderly.

Isolation of human small bowel intraepithelial lymphocytes by annexin V-coated magnetic beads.

Human intestinal intraepithelial lymphocytes (IEL) are important effector cells of the mucosal immune system and their study is hampered by the difficulty of their isolation. The molecular study of enriched samples of IEL is mandatory in the diagnosis of enteropathy-associated T-cell lymphoma and refractory celiac sprue. In order to isolate human small bowel IEL, we took advantage of the stress that intestinal epithelial cells (IEC) suffer during the conventional initial steps of IEL isolation, which induces their apoptosis but not that of IEL. After cell individualization by dithiothreitol and ethylenediamine tetraacetic acid, two-thirds of human IEC can be stained with Annexin-V due to their surface exposure of phosphatidyl serine, a sign of apoptosis. This percentage increases to 95% after performing a density gradient to enrich for IEL. This allows for the use of Annexin-V-coated magnetic beads, originally designed for the removal of dead cells from cell cultures, to obtain >95% pure, 99% viable and untouched IEL after two rounds of depletion. This simple procedure has proven useful for the isolation of human IEL for functional and molecular studies and can conceivably facilitate the diagnosis of intestinal lymphoid malignancies that rely upon the study of pure IEL preparations.

Distal duodenum versus duodenal bulb: intraepithelial lymphocytes have something to say in celiac disease diagnosis.

Background and AimAfter clinical screening and the serological test, many patients still require a duodenal biopsy for celiac disease diagnosis. Mild histological lesions, unspecific findings and patchiness are frequent outcomes of this mandatory diagnostic tool, thus complicating clinical decisions.MethodsWe analyzed the lymphoid components [number of total intraepithelial lymphocytes (IELs), TcR-γδ and CD3−IELs] of the duodenal epithelium by flow cytometry in samples obtained from bulb and distal duodenum during upper gastrointestinal endoscopies performed for diagnostic purposes.ResultsIEL counts and IEL subset distribution (IEL lymphogram) remain invariant along duodenal mucosa revealing a specific profile (immunophenotype) that characterizes either a healthy mucosa or a celiac mucosa. The celiac immunophenotype persists regardless of the biopsy’s anatomical location or the corresponding histological findings.ConclusionsWe propose the IEL lymphogram by flow cytometry as an immunological parameter to discern celiac condition from healthy mucosa. This obviates not only misinterpretation of minor histological changes, but also patchiness and the concerns about the location and number of biopsies.