Immunohistochemical characterization of lymphocytes in microscopic colitis.

BACKGROUND AND AIMS: Microscopic colitis (MC), encompassing the subgroups collagenous colitis (CC) and lymphocytic colitis (LC), is characterized by macroscopically normal or near-normal colonic mucosa, and an increased number of intraepithelial lymphocytes (IELs) and mononuclear cell infiltration in the underlying lamina propria (LP), in addition to an increased collagen layer in CC. This study aimed to characterize the inflammatory cells involved in mucosal inflammation, using immunohistochemistry. METHODS: Paraffin-embedded biopsies from 23 untreated patients with MC (CC=13, LC=10) and 17 controls were stained with antibodies against CD3, CD4, CD8, CD20, CD30, Foxp3, CD45RO and Ki67. Computerized image analysis was used to calculate areas of stained lymphocytes in the surface and crypt epithelia as well as in the LP. RESULTS: In CC and LC, an increase of predominantly CD8(+) lymphocytes was seen in both the epithelium and the lamina propria, whereas a decreased amount of CD4(+) lymphocytes was found in the lamina propria. CD45RO(+) and Foxp3(+) cells were more abundant in all areas in both patient groups compared to controls, as were CD20(+) areas, although more scarce. Ki67(+) areas were only more abundant in the epithelium, whereas CD30(+) areas were more abundant in the lamina propria of both patient groups compared to controls. CONCLUSIONS: This study confirms an increased amount of CD8(+) lymphocytes in the epithelium. Lymphocytic proliferation and activation markers were more abundant, whereas a decreased amount of CD4(+) lymphocytes was seen in the LP. Further studies are needed to reveal the underlying mechanism(s).

A novel large granular lymphocyte-like cell isolated from IL-2-supplemented murine intestinal lamina propria lymphocyte cultures with potent inhibitory action on lymphocyte proliferation.

Regulation and control of the local immune system in the gut mucosa are poorly understood phenomena. Recently we observed in whole alpha-CD3-driven, IL-2-supplemented, lamina propria (LP) T cell cultures that pronounced anergy developed concomitant with the appearance of large granular lymphocyte (LGL)-like cells. The LGL-like cells did not appear in the cultures in the absence of alpha-CD3 activation. These cells expressed Thy 1.2, NK1.1, AsGM1, CD3, and most often CD8 but were always negative for CD4. They exclusively required IL-2 to proliferate and survive in culture and several cell lines were established. Analysis of the regulatory ability of these cells on immune responses revealed that the gut LGL-like cells strongly inhibited T as well as B cell proliferation by releasing a soluble factor(s). Subsequent detection in the culture supernatants of cytokines with reported inhibitory properties on lymphocyte proliferation, interferon-gamma, and transforming growth factor beta correlated poorly to the inhibitory action of the supernatants. Despite the NK-like nature of these cells no or weak cytotoxic activity could be detected against Yac-1 target cells. The LGL-like cells expressed Fc receptors for IgE and demonstrated properties in EM and cytochemical analysis which resembled mucosal mast cells, but unlike such cells the LGL-like cells did not contain histamine or serotonin. The lamina propria of normal mouse small intestine was found to contain cells with morphology and staining pattern similar to those of cultured LGL-like cells, i.e., double-positive for Thy 1.2 and NK 1.1. We propose that this novel intestinal LGL-like cell, probably of T cell origin, may act as a potent suppressive cell in mucosal immune responses exerting a regulatory function on T cell activities and preventing adverse inflammatory reactions in the intestinal mucosa. Therefore, we suggest that these cells may be referred to as granulated inhibitory lymphocytes (GIL) cells. The lamina propria GIL cells exhibit many similarities to the recently described immunosuppressive decidual LGL cells in the placenta.

Antigen-specific and polyclonal CD4+ lamina propria T-cell lines: phenotypic and functional characterization.

Surface phenotype and function of lamina propria CD4+ T cells have been evaluated. In addition, long-term, antigen-specific and polyclonal lamina propria CD4+ T-cell lines have been generated and characterized. Lamina propria CD4+ T cells represent approximately 30% of lamina propria lymphocytes and are responsive to a variety of T-cell mitogens, including anti-CD3, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In each case, however, lamina propria T cells are less responsive to these mitogens than spleen T cells. Freshly isolated lamina propria T cells produce substantial amounts of interleukin-2 (IL-2), interleukin-4 (IL-4), gamma interferon and to a lesser extent interleukin-5 (IL-5). Antigen-specific lamina propria CD4+ T-cell lines were generated by orally immunizing animals with antigen (KLH) in conjunction with cholera toxin as an oral adjuvant. Polyclonal lamina propria CD4+ T-cell lines were generated from unimmunized animals using anti-CD3 as a polyclonal stimulus. Both antigen-specific and polyclonal CD4+ T-cell lines were Thy-1+, alpha beta TCR+ and CD8-. The antigen-specific CD4+ T-cell line when stimulated by anti-CD3 and PMA produces predominantly IL-2, IL-4 and gamma interferon, with very little IL-5. In contrast, the polyclonal CD4+ T-cell line when similarly stimulated produces predominantly IL-4 and IL-5, with very little IL-2 and no detectable gamma interferon. In summary, lamina propria CD4+ T cells have been evaluated and in vitro conditions have been determined for successful generation of lamina propria CD4+ T-cell lines.

Host defense against cholera toxin is strongly CD4+ T cell dependent.

This study investigates the role of CD4+ T cells in host defense against cholera enterotoxin-induced diarrhea. Antitoxin immunoglobulin A formation and gut protection against cholera toxin (CT) following oral immunizations with CT were evaluated in normal mice and mice that had been depleted of CD4+ T cells by in vivo treatment with specific anti-CD4 monoclonal antibodies. Flow cytometer analysis demonstrated that anti-CD4 monoclonal antibody effectively eliminated CD4+ T cells in the spleen, mesenteric lymph nodes, and Peyer's patches. In contrast, lamina propria lymphocytes demonstrated only some decrease in CD4+ T-cell numbers following antibody treatment. However, CD4 expression of individual lamina propria lymphocytes was strongly down-regulated. Depletion of CD4+ T cells performed prior to oral immunization with CT completely inhibited the ability to respond to CT. No antitoxin production, as detected at the single-cell level by the ELISPOT technique, was found in the spleen, mesenteric lymph nodes, or Peyer's patches, nor did we observe serum antitoxin responses in these mice. Control mice demonstrated strong antitoxin responses in all locations following oral immunization with CT. Anti-CD4 antibody treatment also effectively inhibited the antitoxin immunoglobulin A response in the lamina propria to CT as well as blocked the ability to develop gut protection against CT challenge of ligated intestinal loops after oral CT immunization. Thus, in vivo CD4+ T-cell depletion rendered these mice unable to develop protective immunity in the gut following oral immunization with CT. Moreover, CD4+ T-cell depletion effectively inhibited the antitoxin immune response in the gut lamina propria, mesenteric lymph nodes, Peyer's patches, and spleen when performed prior to both priming and booster immunizations with CT. This study clearly demonstrates the requirement of functional CD4+ T cells in the gut immune system for the development of host defense against CT-induced disease. Our data also reinforce the concept of a strong association between gut protection against CT and local production of neutralizing immunoglobulin A antitoxin.