Isolation of murine small bowel intraepithelial lymphocytes

A method for the preparation of cellular suspensions of the epithelium of murine small bowel for the purpose of isolation and recovery of intraepithelial lymphocytes employing intraluminal hyaluronidase digestion is described. Discontinuous Percoll centrifugation of these suspensions yielded a 76-82% pure population of intraepithelial lymphocytes. Between 1.6 and 4.0 X 10(7) viable intraepithelial lymphocytes were obtained per gram of gut. The isolate contained approximately equal numbers of T, B and null lymphocytes.     

Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine.

A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.     

Pathological and clinical significance of increased intraepithelial lymphocytes (IELs) in small bowel mucosa

Intestinal intraepithelial lymphocytes (IELs) belong to a unique T-cell population interspersed between epithelial cells of both the small and large intestine. It is becoming increasingly recognised that an increased number of IELs with a normal villous architecture is within the wide spectrum of histological abnormalities observed in coeliac disease. An increased number of IELs is the earliest pathological change following gluten challenge and a high IEL count may be the only sign of gluten sensitivity. Therefore, the finding of a raised IEL count with normal villous architecture is of sufficient clinical importance to be reported in routine small bowel biopsies. However, it is evident that not all small intestinal biopsy specimens showing increased IELs are explained by gluten sensitivity. Increased IELs in small bowel mucosa have also been associated with autoimmune disorders, tropical sprue, food protein intolerance, Helicobacter pylori-associated gastritis, peptic duodenitis, parasitic and viral infections, as well as the development of intestinal lymphoma. Histological examination of a biopsy specimen of the small bowel remains the diagnostic gold standard for coeliac disease. There will be an ever increasing demand for histological confirmation of gluten sensitivity in patients in whom the classic microscopic appearance of flattened villi may not have fully developed. The more widespread recognition by histopathologists of the pattern of injury manifested by increased numbers of IELs in intestinal biopsy specimens will certainly help in early diagnosis of coeliac disease, lessen diagnostic confusion and influence the modern practice of gastrointestinal tract medicine. This review discusses some of the recent developments in clinical pathology pertaining to increased IELs in small bowel mucosal biopsies.     

肠上皮淋巴细胞与炎症性肠病相关性的研究进展

肠上皮淋巴细胞（iIEL）定位于消化道黏膜上皮细胞间，与肠上皮细胞紧密接触并相互作用，介导黏膜局部免疫防御和维持肠黏膜组织稳定性。iIEL通过其细胞毒活性及所分泌的细胞因子调节肠上皮细胞的凋亡及再生，在炎症性肠病的发生及发展中起一定作用。     

Phenotypic and functional assessment of intraepithelial lymphocytes (IEL) isolated from rat colon and small bowel

We have compared the proliferative potential of IELs isolated from rat colon (CIEL) and small bowel (SBIEL), and compared this with that observed using spleen lymphocytes. Unless additional irradiated spleen cells were added as a source of accessory cells, both IEL populations show poor proliferation in response to Con A stimulation. The CD4/CD8 ratio in spleen, SBIEL and CIEL was markedly different (3:1, 1:3, and 1:1, respectively). Cells expressing surface markers characteristic of macrophages were not routinely found in SBIELs. Both IEL preparations inhibited spleen cell proliferation in response to Con A or immobilized anti-CD3, and produced a soluble factor(s) capable of causing similar inhibition. For CIEL this inhibition was dependent upon a proliferation-independent but cell-cell contact dependent event.     

Non-gluten sensitivity-related small bowel villous flattening with increased intraepithelial lymphocytes: not all that flattens is celiac sprue

Seven patients (mean age, 37.6 years; 5 women) had several weeks of gluten sensitivity (GS)-like symptoms; 2 had flu-like symptom prodromes. The 7 cases had morphologically similar biopsy specimens; all tissue fragments had uniform injury--increased lymphoplasmacytic lamina propria inflammation, moderate to complete villous flattening, numerous crypt mitoses, and markedly increased villous intraepithelial lymphocytes (IELs). All patients were diagnosed with GS and prescribed a gluten-free diet; all returned 9 to 38 weeks later questioning their diagnosis because symptoms had resolved substantially or completely. Clinical improvement was unrelated to gluten abstinence or ingestion. Repeated endoscopy and colonoscopy performed 4.1 to 16 months later showed normal mucosa in all 7 patients. Diseases other than GS can cause marked villous flattening and increased villous IELs in adults. The cause of small bowel mucosal injury is unknown. A similar non-GS-associated clinicopathologic complex, assumed to be due to a protracted viral enteritis or slow regression of a virus-induced immune reaction, occurs in children. The temporal aspects of symptom improvement and mucosal restitution in these 7 patients are similar to "acute self-limited colitis." An overly exuberant immune response to an infectious agent is possible.     

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8αα TCRαβ IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required.Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (> 85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.     

Phenotypic heterogeneity of intraepithelial T lymphocytes from mouse small intestine.

We have used two-colour flow cytometry to examine the heterogeneity of intraepithelial lymphocytes (IEL) from mouse small intestine. We have confirmed the predominance of CD3+ Thy 1- CD8+ IEL and show that a substantial but variable proportion of CD8+ IEL does not express the alpha beta T-cell receptor (TcR) for antigen. Simultaneous analysis of the co-expression of the alpha and beta chains of the CD8 heterodimer and of the alpha beta TcR revealed three populations of CD8+IEL. The first of these expressed both CD8 alpha and beta chains and had normal expression of V beta families and so represented conventional CD8+ alpha beta TcR+ T cells. The second population comprised alpha beta TcR- T cells (presumed gamma delta TcR+) which expressed only the alpha chain of the CD8 molecule. Finally, we identified a second, unique population of alpha beta TcR+ CD8+ IEL which were also CD8 beta-. Gamma delta + IEL predominated in mice aged less than 8 weeks, but there was a rapid increase in both populations of alpha beta TcR+ CD8+ IEL in older mice. CD8+ IEL were similar to peripheral CD8+ T cells in having high expression of the CD45RB molecule, but CD4+ IEL had generally lower expression of CD45RB than their peripheral counterparts, despite having normal expression of TcR. These findings emphasize the heterogeneity of IEL and underline the need to study phenotypically defined populations.     

IL-4 down-regulates the responsiveness of human intraepithelial lymphocytes

IL-4 is an important regulator of intestinal inflammation, yet little is known regarding its action on intestinal lymphocytes. Intestinal lymphocytes were isolated from jejunal mucosa of patients undergoing gastric bypass operations for morbid obesity. The impact of IL-4 was measured by its effects on proliferation using ³H-thymidine incorporation, IL-2 production using the CTLL assay, and IL-2 receptor generation using immunofluorescence. The production of IL-4 was measured by ELISA. IL-4 significantly inhibited the proliferation of intraepithelial lymphocytes (IEL) to a variety of stimuli as well as the development of lymphokine-activated killer (LAK) cells. In contrast, it had no effect on the proliferation of CD8⁺ T cells from peripheral blood. The inhibitory effect of IL-4 on IEL did not occur during activation, since IL-2 production and receptor expression were not altered. Rather, it occurred during cell cycling, since over 50% inhibition resulted whether IL-4 was added at the initiation of an IL-2-stimulated culture or after 24 or 48 h incubation. IL-4 was secreted by activated lamina propria lymphocytes (LPL) but not by IEL. IL-4,produced by activated LPL, may enter the epithelial compartment and down-regulate responsiveness of IEL.     

Activation of human intraepithelial lymphocytes reduces CD3 expression

The aim of this study was to examine in detail the low functional capacity of human intraepithelial lymphocytes (IELs) in response to phytohaemagglutinin (PHA) and CD3 ligation. Human IELs were extracted from jejunal mucosa obtained from patients undergoing gastric bypass operations for morbid obesity and compared to peripheral blood (PB) lymphocytes composed predominantly of CD8+ T cells. Calcium influx ([Ca2+]i) was analysed using Fura-2-loaded cells; IL-2 receptor expression was measured by immunofluorescence and flow cytometry; IL-2 binding was determined using radiolabelled IL-2; IL-2 production was quantified by ELISA; and apoptosis was detected with Apo 2.7 staining. Compared to naive PB CD8+ T lymphocytes, calcium influx by IELs was only transient with CD3 ligation and low in amplitude with PHA. IL-2 receptor expression was reduced after CD3 ligation, yet normal in numbers and affinity after PHA stimulation. Both cell types secreted similar amounts of IL-2. CD3 expression on IELs, but not PB CD8+ T cells, declined upon activation, due partly to incomplete reexpression after modulation. Little apoptosis was found. The partial activation of IELs in response to PHA and CD3 ligation, as manifested by diminished [Ca2+]i, resulted in a decline in CD3 expression.     

Proliferative kinetics of large and small intraepithelial lymphocytes in the small intestine of the mouse

The proliferative kinetics of the intraepithelial lymphocytes (IL) of the mouse intestine have been evaluated. By inducing mitotic arrest it was found that large IL — constituting about 50% of the IL — showed a mitotic rate of 2.3. Autoradiographic results obtained after two different schedules of ³H-thymidine injections showed that 30% of the large IL were in DNA synthesis, and that the large IL were renewed at a rate comparable to that of blast cells from Peyer's patches, mesenteric lymph nodes and thoracic duct lymph. The small IL were renewed very rapidly compared to small lymphocytes of peripheral lymphoid tissues, although small lymphocytes with lifespans of several weeks were also present in the epithelial sheet. By the use of intestinal perfusion, in vivo, it was estimated that the loss of lymphocytes from intestinal villi into the lumen of the gut was negligible, and it is concluded that the most probable kinetic model for the majority of IL is: B and T lymphoblasts invade the epithelium and undergo mitosis. B lymphoblasts give rise predominantly to plasma cells, and T lymphoblasts give rise to small lymphocytes — probably long-lived — which reenter the circulation.     

Lymphokine secretion and proliferation of intraepithelial lymphocytes from murine small intestine.

Compared to other peripheral lymphocytes, intestinal intraepithelial lymphocytes (IEL) have previously been shown to have low proliferative capabilities. However, there are two main populations of IEL in the small intestine of mice. First, there is the thymus-dependent CD3+,Thy-1+ population, most of which expresses the alpha beta T-cell receptor (TcR), and second there is the thymus-independent CD3+,Thy-1- population, most of which expresses the gamma delta TcR. In this study Thy-1-enriched and Thy-1-depleted lymphocytes from murine intestinal epithelium were studied separately for their ability to proliferate and secrete lymphokines in vitro after mitogenic stimulation, after stimulation via the TcR-CD3 complex and after stimulation with the superantigen Staphylococcus aureus B (SEB). Here we show that Thy-1-enriched IEL are not an immunocompromised population of cells but are functionally competent T cells that are capable of proliferation and lymphokine secretion after stimulation with concanavalin A (Con A), phorbol myristate acetate (PMA) and anti-CD3 monoclonal antibody (mAb). Furthermore, Thy-1-enriched IEL proliferate and secrete lymphokines after 'superantigenic' stimulation with SEB. In contrast, the majority of Thy-1-depleted IEL do not proliferate, and secrete only minimal levels of lymphokine to any of the stimuli tested in this study.     

Isolation of human spontaneous killer lymphocytes by bacterial adherence.

Human lymphocyte subpopulations (B, T1, T2, T3, and T4 our denomination) have been identified previously by bacterial adherence and differences between them in mitogen responses and specific cytotoxic activity have been found. In this study another aspect has been investigated in order to find functions associated with these subpopulations, namely the spontaneous killing (SK) ability. Freshly isolated human peripheral blood lymphocytes (PBL) were separated into adherent and non-adherent cells following centrifugation against various bact:rial monolayers. The PBL and the resulting subpopulations of PBL were tested alone or in combination as effector cells in a 4 hr cytotoxicity assay against human lymphoblastoid cel- lines of B or T cell origin. The T3 + T4 cells or T4 cells alone showed a significantly higher SK activity against both B and T target cell lines when compared with unseparated PBL, T1 + T2, or T3 cells alone. Whe Fc portion of IgG, contain the lymphocytes responsible for SK activity and that SK cells can be purified by negative selection using bacterial adherence.     

Analysis of natural killer effector and suppressor activity by intraepithelial lymphocytes from mouse small intestine.

Intraepithelial lymphocytes (IEL) are morphologically similar to NK cells in other tissues and we have studied the NK activity of IEL isolated from mouse small intestine. In contrast to spleen NK cells, IEL showed little activity against YAC-1 over 4 h but had high levels of NK activity when the assay was extended to 18 h. IEL from nude mice did not show the enhanced NK activity found in other tissues. IEL were also found to suppress the NK activity of spleen cells and this suppressor function was not mediated by T lymphocytes or macrophages. The results indicate that the intestinal epithelium contains a population of potent NK cells which may represent a type of NK cell different to that found in other tissues. In addition, there are also cells capable of regulating NK cell function in the epithelial layer.     

Isolation of human splenic macrophages and lymphocytes by countercurrent centrifugal elutriation

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.     

Isolation of normal human IgA,IgM and IgG fragments by polyacrylamide beads immunoadsorbents

Polyacrylamide beads antibody immunoabsorbents were used in order to isolate human IgA, IgM and fragments of papain-digested human IgG. The proteins obtained were pure as judged by immunochemical techniques. The antisera raised with these purified proteins were monospecific. The binding capacity and the yield were satisfactory. These antibody immunoadsorbents offer several advantages: a) highly purified antigens can be quickly obtained in a one-step procedure from body fluids; b) their handling is easy especially when using a column; c) they can be used for a long period of time and for many experiments without any noticeable loss in their binding capacity.     

Adhesion molecules utilized in binding of intraepithelial lymphocytes to human enterocytes

The expression of adhesion molecules by human duodenal intraepithelial lymphocytes (IEL) was examined by two-color flow cytometry. Resting IEL expressed LFA-1, HML-1, CD44. Stimulation with phytohemagglutinin (PHA) resulted in down-regulation of expression of these molecules with induction of expression of ICAM-1 and VLA-4. VLA-4 expression was also found on non-activated IEL from patients with celiac disease. In addition, IEL expressed an antigen recognized by a novel monoclonal antibody D2.1. The molecular mass of D2.1 is heterogenous: 82 kDa in peripheral blood lymphocytes and 44 kDa in an IEL line. Expression of this antigen was also up-regulated by PHA. To determine the involvement of these antigens in binding of IEL to human enterocytes, we developed a system based on adherence of an IEL cell line to the I407 fetal intestinal cell line. Monoclonal antibodies VLA-4, D2.1 and to a lesser extent ICAM-1 blocked adherence of IEL to I407 cells. These data suggest that VLA-4 and D2.1 may be involved in adherence of IEL to human enterocytes or secreted matrix molecules in vivo.