Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes

doi:10.1016/0022-1759(96)00052-X

Journal of Immunological Methods

Volume 194, Issue 1, 17 July 1996, Pages 35-48

https://doi.org/10.1016/0022-1759(96)00052-X Get rights and content

Abstract

The study of intestinal intraepithelial lymphocytes (IEL) has been hindered by the difficulty of isolating a population of lymphocytes which is free of epithelial cell or lamina propria cell contaminants and representative of the in vivo population of IEL in both phenotype and function. We describe an improved technique for the extraction and purification of IEL from the proximal small intestine of the rat. This technique rapidly and reproducibly isolates 5–10×10⁶ IEL/rat with 90–95% purity and viability without the use of enzymes which affect lymphocyte function. The resulting cell population, which is 75% αβ T cell receptor (TCR)⁺, 70% CD8⁺, and 33% CD4⁺ T cells, and only 5% B cells and 2% macrophages, is of suitable purity to allow for flow cytometric analysis of the entire population of cells without requiring gating on lymphocytes. IEL are comprised of a unique T cell repertoire in that 27% of cells co-express the CD4 and CD8 molecules, but only 11% of CD4⁺ cells co-express CD45RC. All CD4⁺ cells express the αβTCR, but 9% of IEL are CD8⁺CD4⁻αβTCR⁻. The adhesion molecules α₄ integrin and l-selectin are expressed on 57% and less than 1% of IEL, respectively. The isolated IEL population contains mRNA for IL-1α, IL-1β, IL-1R, IL-1RA, IL-2, IL-6R, IFN-γ, TGF-α, TGF-β₁, and TNF-α. Mesenteric lymph node cells (MLNC) were examined in parallel. This technique allows for the isolation of rat IEL appropriate for phenotypic analysis by flow cytometry and for cytokine analysis by reverse transcription/polymerase chain reaction.

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SJZD has a stronger therapeutic effect on intestinal immune and GI hormone secretion in SDS rats, while the efficacy of NPS and S-3 showed slight differences. NPS mainly regulated the secretion of GI hormones in SDS rats and directly improved intestinal immunity by increasing the expression of T lymphocyte cells, while S-3 mainly enhanced intestinal immune function by increasing the expression of T lymphocyte cells and repairing the intestinal barrier in both direct and indirect ways. Additionally, experiments in pGF and GMD rats have proven that the immune-enhancing effects of SJZD, NPS, and S-3 on SDS rats and the regulation of GI hormones of S-3 are related to modulation of the gut microbiota composition, while the regulation of GI hormones by SJZD and NPS is not completely dependent on this modulation. In particular, Lactobacillus, SMB53, Blautia, Dorea, Collinsella and Adlercreutzia were significantly modulated by SJZD, and 3 genera (including Lactobacillus, Dorea and SMB53) were also remarkably regulated by NPS. S-3 significantly increased the abundance of Butyricimonas and Collinsella, which were different from altered genera in the SJZD group.
This study uncovered that NPS and S-3 are inseparable effective substances for SJZD in the treatment of SDS rats, in which NPS mainly improves intestinal motility dysfunction and S-3 mainly enhances intestinal immunity. The mediation effect of the gut microbiota is extremely important, but the regulating effect of NPS on gastrointestinal hormones has nothing to do with the gut microbiota.
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Unfortunately, comparison of specific frequencies between studies proved difficult because previous studies reported percentages of total lymphocytes rather than total intestinal cells. Such difficulties comparing results between gut immune cell phenotype studies that utilize different isolation protocols has been a topic of discussion for some time (Van der Heijden and Stok, 1987; Kearsey and Stadnyk, 1996). Indeed, the current study demonstrates that significant differences can be obtained simply by altering the isolation protocol (Fig. 4).
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To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14 days were investigated. IELs, particularly TCR-γδ⁺ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover.
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In the afternoon we administered 0.1 M acetic acid (diluent group) or acetic acid containing 1, 3 or 5 mg chitosan in a final volume of 200 μl per os by using a plastic pipette tip. Sixteen hours later [17,18], we obtained the small gut, we strictly removed and discarded all PPs and we purified IECs according to standard procedures [19]. Briefly, the small gut was rinsed in Ca2+ and Mg2+ free PBS and washed in PBS-dithiothreitol (200 μg/ml) to eliminate residual mucus and then shaken in RPMI 1640 (Sigma–Aldrich, Saint Louis, MO, USA) containing 0.03 M EDTA to remove the epithelium at 37 °C [16].
Chitosan is a copolymer of N-acetylglucosamine and glucosamine derived from chitin with several applications in pharmaceutical and medical fields. This polysaccharide exhibits adjuvant properties in mucosal immune responses of humans, rats and mice. Characterization of signals elicited by chitosan at the intestinal epithelium could explain its immunomodulatory activity and biocompatibility. We fed normal rats with single doses of chitosan and 16 h later, we purified intestinal epithelial cells (IECs) to assess immune and biochemical parameters. Following chitosan administration, mRNA expression and release of several cytokines and chemokines increased, injury markers maintained constitutive levels and MHC type II molecule expression was augmented. IEC supernatants showed higher levels of IL-10, IL-6 and TGF-β. Arginase activity of IECs increased upon chitosan interaction in vivo and in vitro. Together, after chitosan feeding, mild activation of IECs occurs in vivo, with production of regulatory factors that could be relevant for its biocompatibility and immunomodulatory effects.
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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.
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Research reportIsolation and characterization of highly purified rat intestinal intraepithelial lymphocytes

Abstract

Anal. Biochem.

J. Immunol. Methods

J. Immunol. Methods

Immunobiology

Differential function of intestinal intraepithelial lymphocyte subsets

J. Immunol.

Differences in intraepithelial lymphocyte T cell subsets isolated from murine small versus large intestine

J. Immunol.

Expression and regulation of adhesion molecules by γδ T cells from lymphoid tissues and intestinal epithelium

Eur. J. Immunol.

Human colonic intraepithelial and lamina proprial lymphocytes: cytotoxicity in vitro and the potential effects of the isolation method on their functional properties

Gut

Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine

Gut

Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family: Evolution of a cytokine control mechanism

Unusual phenotype of intestinal intraepithelial lymphocytes in the rat: predominance of T cell receptor α/β+/CD2− cells and high expression of the RT6 alloantigen

Eur. J. Immunol.

Function of αβ TCR+ intestinal intraepithelial lymphocytes: Th1- and Th2-type cytokine production by CD4+ CD8− and CD4+ CD8+ T cells for helper activity

Int. Immunol.

αβ T cell receptor-positive intraepithelial lymphocytes with CD4+, CD8− and CD4+, CD8+ phenotypes from orally immunized mice provide Th2-like function for B cell responses

J. Immunol.

Research report
Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes

Unusual phenotype of intestinal intraepithelial lymphocytes in the rat: predominance of T cell receptor α/β⁺/CD2⁻ cells and high expression of the RT6 alloantigen

Function of αβ TCR⁺ intestinal intraepithelial lymphocytes: T_h1- and T_h2-type cytokine production by CD4⁺ CD8⁻ and CD4⁺ CD8⁺ T cells for helper activity

αβ T cell receptor-positive intraepithelial lymphocytes with CD4⁺, CD8⁻ and CD4⁺, CD8⁺ phenotypes from orally immunized mice provide Th2-like function for B cell responses