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*
R&D Department, Abbott Laboratories, Granada, Spain and
Department of Biochemistry and Molecular Biology, University of Granada, Spain
2To whom correspondence should be addressed. E-mail: manuel.manzano{at}abbott.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, CD8ß, CD22 and CD45R) in those lymphocyte populations was analyzed by flow cytometry. The percentages of cells expressing T-cell antigens, such as CD3, were significantly higher in LPL during weaning compared to d 0. The percentages of cells expressing CD8 and CD8ß increased in both IEL and LPL. However, the percentage of CD4+ cells tended (P = 0.07) to decrease in IEL and to increase in LPL. The percentages of cells expressing B-cell antigens, such as CD22 or CD45R in PPL increased. Changes in the specific phenotypes of intestinal lymphocyte populations at weaning are apparently related to the maturation of the intestinal immune system during early life. Thus, B cells increase in PPL and T cell increase in IEL and LPL.
KEY WORDS: • intestinal lymphocytes • epithelium • lamina propria • Peyer’s patches • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). During absorption, the intestine is exposed to a wide variety of antigens derived from foods, resident bacteria and invading microorganisms and thus requires a powerful and selective defense system. Gut-associated lymphoid tissue has long been considered a main body lymphoid tissue (2
), such as those in liver, spleen and the phagocytic mononuclear system. Within the small intestine, there are three main lymphocyte populations, which reside in the epithelium, the lamina propria and Peyer’s patches.
Intraepithelial lymphocytes (IEL)3
are cells located above the epithelial basement membrane between mucosal columnar epithelial cells. Most IEL are CD8+ T cells (90% in mice and 50 to 80% in humans), and therefore they basically have a cytotoxic function (3
). Lamina propria T cells are mainly CD4+ T cells (60–70%), the vast majority of which express the ß T cell receptor (95%), just as in the peripheral blood (4
). However, lamina propria T cells differ from peripheral blood T cells in that most (95%) display a mature or memory phenotype (5
–10
). A quite prominent cell type also present in the lamina propria (20 to 40%) is the highly differentiated B cell, which is in the plasma-cell stage of development (11
). Peyer’s patches lymphocytes (PPL) play a crucial role in the induction of intestinal immune responses; lectins or microorganisms that invade the Peyer’s patches (PP) adhere to the follicle-associated epithelium and induce an antibody response in secretions (12
). The predominant role of PPL is to cooperate in the differentiation of B cells towards IgA-producing cells (13
), a process involving CD4+ T cells (14
), probably through the secretion of modulatory cytokines.
There is considerable literature describing the systemic immune system at birth; however, only limited information is available on the gastrointestinal immune system at early stages of life. This system, which matures gradually during the postnatal period, is highly influenced by antigenic stimulation, which is especially intense at weaning. We examined the changes in T and B cells subsets (CD3+, CD4+, CD8+, CD8ß+, CD22+ and CD45R/B220+) in intestinal lymphocyte populations (intraepithelial, Peyer’s patches and lamina propria) of mice during the weaning period.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fifty male BALB/c mice (Iffa Credo, Lyon, France) 3-wk-old were housed in groups of five per cage, under 12-h cycles of light and dark in a conventional climatizated room at 22°C. Mice had free access to standard mouse nonpurified diet and water. Mice (n = 10) were killed at 0, 4, 7, 12 and 18 d after the beginning of the experiment. The Animal Welfare Committee of the University of Granada approved the protocol of this study.
Sample preparation
Intestinal lymphocytes were isolated following a modified procedure of Gautreaux et al. (15
). The small intestine segment from the Treitz ligament to the ileocecal valve was isolated, and the luminal content was flushed with Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO). Visible Peyer’s patches were excised, and the intestine was opened longitudinally and cut into small pieces. To isolate the small intestinal epithelium, the pieces were incubated for 15 min at 37°C in 25 mL of HBSS with 0.005 mol/L dithiothreitol (DTT; Roche Molecular Biochemicals, Indianapolis, IN), 0.002 mol/L EDTA (Sigma-Aldrich) and 0.025 mol/L Tris buffer (Sigma-Aldrich) in a shaking water bath (100 strokes per min). The supernatant was discarded, fresh HBSS-DTT-EDTA-Tris was added, and the incubation procedure was repeated. Supernatants containing the epithelial cells from both incubation steps were pooled, and the cells were washed by centrifugation with RPMI 1640 culture medium containing 5% (v/v) heat-inactivated fetal calf serum (Sigma-Aldrich), 0.02 mol/L HEPES (Sigma-Aldrich), 0.002 mol/L L-glutamine, 500 U of penicillin and 0.1 g/L streptomycin (Sigma-Aldrich) (complete medium). Lamina propria lymphocytes (LPL) were liberated from the remaining sediment by placing the intestinal debris in 25 mL of complete medium with 20 U/L collagenase, 120 U/L dispase and 2 x 105 U/L Dnase I (Roche Molecular Biochemicals) for 90 min in a 37°C shaking water bath at 100 strokes per min. Excised Peyer’s patches were placed in complete medium and dissected with scalpels and were also subjected to enzymatic digestion for 30 min.
Each cell type isolated from the epithelium, the lamina propria and Peyer’s patches were subjected to discontinuous Percoll (Pharmacia, Uppsala, Sweden) density-gradient centrifugation to enrich them in lymphocytes. The commercial Percoll solution was diluted 9:10 with 9% NaCl yielding an isotonic Percoll solution that was diluted with complete medium to obtain three solutions with several Percoll percentages (75, 40 and 30%), which were used in descending order. Cells were resuspended in 2 mL of complete medium and placed over the 30% fractions. After centrifugation at 650 x g for 20 min, the interfaces between the 75 and 40% layers were removed, and the cells were washed by centrifugation in 25 mL of complete medium. Cells were then resuspended in 2 mL of 40% Percoll and centrifuged at 650 x g. The cell pellets, enriched for lymphocytes (IEL, LPL and PPL), were collected and washed by centrifugation with phosphate-buffered saline (PBS; Sigma-Aldrich).
Staining with monoclonal antibodies and flow cytometry
Lymphocyte preparations (1 x 106 cell/L, 100 µL) were placed in 3-mL tubes with different concentrations of monoclonal antibodies (anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD8b, anti-CD22 and anti-CD45R/B220), previously measured according to the recommendations of the manufacturer (Pharmingen, San Diego, CA) and were incubated for 30 min in the dark at 4°C. Cells were pelleted by centrifugation (500 x g, 5 min), washed with PBS and resuspended in 100 µL of 4% (v/v) formaldehyde. Following an incubation of 20 min at 4°C, the cells were washed with PBS and further fixed with 300 µL of 1% (v/v) formaldehyde.
Fluorescence-activated cell sorter analysis of cell preparations was performed on an ORTHO-cytoron flow cytometer (Ortho Diagnostic Systems, Westwood, MA), acquiring between 1500 and 3000 gated events, based on the forward light scatter and side light scatter properties of the cell preparations. Nonspecific fluorescence was determined by two controls (for fluorescein isothiocyanate and phycoerythrin) prepared for each cell preparation.
Statistical analysis
Total number of CD45+ cells was calculated for each population (IEL, LPL and PPL). Specific lymphocyte subsets were expressed as absolute values and as mean percentages of CD45+ cells ± SD of the mean. Although cells were not costained with CD45 or any other markers, the relationship between the percentage of CD45+ and other cell markers was assumed. To evaluate differences due to the weaning period for the different lymphocyte subsets, we performed one-way ANOVA (SPSS 9.0). Data were previously normalized by multiplying each datum by the inverse of the variance of its group. A posteriori comparisons between different times and time 0 were performed by the Dunnet test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). There were decreases in CD45+ cells in IEL and LPL, whereas they increased in PPL during the study.
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. However, when results were expressed as percentages of CD45+ cells, most of the lymphocytes at d 0 were CD3+ (Fig. 1
). There were increases in the percentages of cells expressing CD8 and CD8ß and a trend (P = 0.07) for a decrease in those expressing CD4. The behavior of B cells was opposite that of T cells, with the percentages of cells expressing CD22 and CD45R/B220 decreasing during the study (Fig. 1)
.
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. However, when the lymphocyte subpopulations were expressed as percentages of CD45+ cells the percentage of T cells (CD3 cells) decreased at some points post-weaning, whereas those of B cells (CD22 and CD45R/B220) increased (Fig. 2
). The decrease in the percentage of T cells was apparently due mainly to cytotoxic T cells, because the proportions of CD8 and CD8ß also declined during the study (Fig. 2)
.
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. The percentage of T cells (CD3+) increased, due primarily to the cytotoxic subpopulations (CD8 + and CD8ß+). The proportion of CD4+ cells tended (P = 0.07) to increase during weaning. However, the percentage of B cells (CD22) decreased (Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Thus, the increase of CD45+ cells in Peyer’s patches could be due to the intense antigenic stimulation that usually takes place at weaning. However, it is important to point out there were also changes in the distribution (percentage of CD45+ cells) of the lymphocyte subpopulations, independent of the evolution of these subpopulations in terms of absolute numbers. These changes reflected relative increases in lymphocytes for the most abundant subpopulations in IEL, PPL and LPL.
T cells have been shown to be the most abundant subpopulation in adult IEL (16
,17
). Here, we observed T cells to represent the highest percentage of total IEL. In agreement with others (3
,18
), the increase was due mainly to the CD8+ subpopulation. The increase in the expression of CD8 by IEL also agrees with Ter-Steege et al. (19
), who described the neonatal development of IEL in the murine small intestine. They found a large increase in the percentage of CD3+ and CD8+ IEL, especially between 20 and 25 d of life. On the other hand, in the present study the expression of the B cell antigens, CD22 and CD45R/B220, was opposite to the behavior of the T cells. Since T cells constitute the main subpopulation in IEL, this suggests that the decrease in the percentage of B cells could result from the increase of CD3+ cells during the maturation of this lymphocyte population. It is important to point out that CD45R/B220 is expressed by a high percentage of IEL, being the sum of CD3 + cells plus CD45R/B220+ cells, which exceeds 100%. However, CD45R/B220 expression has previously been observed not only in B cells but also in CD8+ IEL T cells (20
). This would explain the high proportion of CD45R/B220+ IEL found in our work.
IEL serve the primitive function of keeping potential pathogens out of the intestinal epithelium and ignoring all other elements. This is accomplished by generating T cells that interact with unique antigen-presenting molecules (thymus leukemia antigen, CD1) on intestinal epithelial cells that bind a limited array of peptides or glycolipids (21
). IEL have also been found to be involved in a number of different activities, including surveillance of the intestinal epithelial layer for the detection of microbial pathogens, removal of damaged or transformed epithelial cells, maintenance of epithelial integrity via secretion of trophic factors important for epithelial cell growth and differentiation and regulation of local cell-mediated or humoral immune responses (21
).
In Peyer’s patches, the percentage of T lymphocytes decreased during the study, reflected by less CD3 expression (Fig. 2)
. This decrease was mainly due to T-cytotoxic lymphocytes (CD8+ and CD8ß+). The lower percentage of T lymphocytes may be the result of the higher percentage of B cells, given that Peyer’s patches are primarily a typical B cell lymphoid organ (22
–24
). In the present study, we found an increase in the percentage of those cells showing an increase in the expression of CD22 and CD45R. The greater proportion of B cells in PPL can be explained by the continuous differentiation undergone by these cells in the germinal centers, due principally to the postnatal stimulation by different types of antigens (25
,26
). The predominant role of T cells in PP is to aid in the differentiation of B cells, which is the main subpopulation of this level, toward IgA-producing cells through the production and secretion of several cytokines involved in the process known as IgA-switching, and the generation of suppressor mechanisms responsible for oral tolerance (10
).
In LPL, the most abundant subpopulation was T lymphocytes. Several investigators have reported that CD4+ cells predominate over CD8+ cells within this population (11
,27
,28
), but in the present study, we found no dominance of CD4+ cells. Furthermore, the proportion of this subpopulation of T-helper lymphocytes tended to be greater than d 0 only at d 7 and 12 of the study, whereas the CD8+ subpopulation in the lamina propria increased throughout the study. We cannot explain the lower percentages of CD4+ cells in LPL than found by others. We may have had a selective loss of this subpopulation during lymphocyte isolation, which is especially aggressive for this population (LPL). However, it is important to emphasize that high percentages of CD8+ cells in LPL during the neonatal period have been found previously (19
). Although CD3+ CD8+ cells constituted the main subpopulation in LPL as well as in IEL, T cells located in the lamina propria can play a different role than those located in the epithelium. In the lamina propria, lymphocytes interact freely with each other and with macrophages, and alternative activation pathways (CD2) are preferentially used because macrophages carefully control the expression of important stimulatory ligands. Maintenance of an adequate equilibrium among the different cytokines produced by CD4+ cells is important because intestinal inflammation, such as inflammatory bowel disease (10
), can be induced if such an equilibrium is disrupted. These observations are consistent with the conclusion that T cells in the lamina propria are pleomorphic but are highly enriched in subpopulations of activated memory cells that are geared for effector functions such as helper function. These functions are likely to be critical in maintaining normal host defense in the mucosal environment (29
,30
).
In summary, our results indicate a rapid development of IEL, LPL and PPL populations during the weaning period. These changes reflect mainly a higher lymphocyte percentage of the most abundant subpopulations, so that in PPL the main change was a higher percentage of B cells, whereas the most notable change in the IEL and LPL was a higher percentage of T cells, especially in IEL. How different nutrients and non-nutrients can influence the process of intestinal-lymphocyte maturation requires future investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CD, cluster of differentiation; DTT, dithiothreitol; HBSS, Hanks’ balanced salt solution; IEL, intraepithelial lymphocytes; LPL, lamina propria lymphocytes; PBS, phosphate-buffered saline; PP, Peyer’s patches; PPL, Peyer’s patches lymphocytes.
Manuscript received 6 December 2001. Initial review completed 21 January 2002. Revision accepted 26 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Field, M. & Frizell, R. A. (1991) Intestinal absorption and secretion. Schultz, S.G. eds. The Gastrointestinal System IV American Physiology Society Bethesda, MD. .
2. Alverdy, J. C. (1990) Effects of glutamine supplemented diets on immunology of the gut. J. Parenter. Enteral Nutr. 14:109S-113S.
3. Lefrancois, L. (1991) Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire?. Immunol. Today 12:436-438.[Medline]
4. Brandtzaeg, P. (1985) Research in gastrointestinal immunology. State of arts. Scand J. Gastroenterol. 20:137-156.
5. James, S. P., Fiocchi, C., Graeff, A. S. & Strober, W. (1986) Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytotoxic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Chron’s disease and control patients. Gastroenterology. 91:1483-1489.[Medline]
6. McGhee, J. R., Strober, W., Fujihashi, K. & Kiyono, H. (1993) T cell and cytokine regulation of mucosal antibody responses with emphasis on intraepithelial lymphocyte helper functions. Kiyono, H. McGhee, T. R. eds. Mucosal Immunology: Intraepithelial Lymphocytes 1993:1-20 Raven Press New York, NY. .
7. Schieferdecker, H. L., Ullrich, R., Hirseland, H. & Zeitz, M. (1992) T-cell differentiation antigens on lymphocytes in the human intestinal lamina propria. J. Immunol. 149:2816-2822.[Abstract]
8. Schieferdecker, H. L., Ullrich, R., Weiss-Breckwoldt, A. N., Schwartling, R., Stein, H., Riecken, E. O. & Zeitz, M. (1990) The HML-1 antigen of intestinal lymphocytes is an activation antigen. J. Immunol. 144:2541-2549.[Abstract]
9. Hurst, S. & Barrett, T. A. (1996) T-cell responses to enteric antigens. Semin. Gastrointest. Dis. 7:118-123.[Medline]
10. Abreu-Martin, M. T. & Targan, S. R. (1996) Regulation of immune responses of the intestinal mucosa. Crit. Rev. Immunol. 16:277-309.[Medline]
11. Kelsall, B. & Strober, W. (1999) Gut-associated lymphoid tissue: antigen handling and T-lymphocyte responses. Ogra, P. L. Mestecky, J. Lamm, M. E. Strober, W. Bienenstock, J. McGhee, J. R. eds. Mucosal Immunology 2nd edition 1999:293-317 Academic Press, Inc San Diego, CA. .
12. Elson, C., Woogen, S., Gaspari, M. & Ealding, W. (1987) Induction of secretory IgA responses to protein antigens. Adv. Exp. Med. Biol. 216B:877-885.
13. Strober, W. & Harriman, G. R. (1992) The regulation of IgA-B cell differentiation. Gastroenterol. Clin. North Am. 20:473-494.
14. MacDonald, T. T. & Spencer, J. (1994) Gut-associated lymphoid tissue. Ogra, P. L. Mestecky, J. Lamm, M. E. Strober, W. Bienenstock, J. McGhee, J. R. eds. Mucosal Immunology 2nd edition 1994:415-424 Academic Press, Inc San Diego, CA. .
15. Gautreaux, M. D., Deitch, E. A. & Berg, R. D. (1994) T lymphocytes in host defense against bacterial translocation from the gastrointestinal tract. Infect. Immun. 62:2874-2884.
16. Mosley, R. L., Styre, D. & Klein, J. R. (1990) CD4+CD8+ murine intestinal intraepithelial lymphocytes. Int. Immunol. 2:361-365.
17. Rocha, B., Vassalli, P. & Guy, G. D. (1991) The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J. Exp. Med. 173:483-486.
18. Guy-Grand, D. N., Cerf-Bensussan, N., Malissen, B., Malassis, C., Briottet, C. & Vassalli, P. (1991) Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173:471-481.
19. Ter-Steege, J., Buurman, W. A. & Pierre-Philippe, F. (1997) The neonatal development of intraepithelial and lamina propria lymphocytes in the murine small intestine. Dev. Immunol. 5:121-128.[Medline]
20. Barret, T. A., Gajewski, T. F., Danielpour, D., Chang, E. B., Beagley, K. W. & Bluestone, J. A. (1992) Differential function of intestinal intraepithelial lymphocyte subsets. J. Immunol. 149:1124-1130.[Abstract]
21. Aranda, R., Sydora, B. C. & Kronenberg, M. (1999) Intraepithelial lymphocytes: function. Ogra, P. L. Mestecky, J. Lamm, M. E. Strober, W. Bienenstock, J. McGhee, J. R. eds. Mucosal Immunology 2nd edition 1999:429-437 Academic Press, Inc San Diego, CA. .
22. Reynolds, J. D. & Morris, B. (1983) The evolution and involution of Peyer’s patches in fetal and postnatal sheep. Eur. J. Immunol. 13:627-635.[Medline]
23. Reynolds, J. D. (1986) Evidence of extensive lymphocyte death in sheep Peyer’s patches. I. A comparison of lymphocyte production and export. J. Immunol. 136:2005-2010.[Abstract]
24. Reynolds, J. D., Kennedy, L., Peppard, J. & Pabst, R. (1991) Ileal Peyer’s patch emigrants are predominantly B cells and travel to all lymphoid tissues in sheep. Eur. J. Immunol. 21:283-289.[Medline]
25. Spencer, J. & MacDonald, T. T. (1991) Ontogeny of human mucosal immunity. MacDonald, T. T. eds. Ontogeny of the Immune System of the Gut 1991:23-50 CRC Press Boca Raton, FL. .
26. Brandtzaeg, P., Nilssen, D. E., Rognum, T. O. & Thrane, P. S. (1991) Ontogeny of the mucosal immune system and IgA deficiency. Gastroenterol. Clin. North Am. 20:397-439.[Medline]
27. Selby, W. S., Janossy, G., Bofill, M. & Jewell, D. P. (1984) Intestinal lymphocyte subpopulations in inflammatory bowel disease: an analysis by immunohistological and cell isolation techniques. Gut 25:32-40.
28. Hirata, I., Berrebi, G., Austin, L. L., Keren, D. F. & Dobbins, W. O. (1986) Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease. Dig. Dis. Sci. 31:593-603.[Medline]
29. James, S. P. & Kiyono, H. (1994) Gastrointestinal lamina propria T cells. Ogra, P. L. Lamm, M. E. McGhee, J. R. Mestecky, J. Strober, W. Bienenstock, J. eds. Handbook of Mucosal Immunology 1994:293-317 Academic Press, Inc San Diego, CA. .
30. Mackay, C. R. & von Andrian, U. H. (2001) Memory cells. Local heroes in the struggle for immunity. Science (Wash DC) 291:2323-2324.
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