Local hormone networks and intestinal T cell homeostasis

Science

Volumn 275, Issue 5308, 1997, Pages 1937-1939

  Wang, Jin ^a   Whetsell, Michael ^a   Klein, John R ^a  

^a UNIVERSITY OF TULSA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIGEN; CD4 ANTIGEN; CD45 ANTIGEN; CD8 ANTIGEN; CORTICOSTEROID; DNA; ENDOTOXIN; HORMONE; IODINE 125; MONOCLONAL ANTIBODY; PROTIRELIN RECEPTOR; T LYMPHOCYTE RECEPTOR; THYROID HORMONE; THYROTROPIN; THYROXINE;

ANIMAL CELL; ARTICLE; DNA SEQUENCE; ENZYME LINKED IMMUNOSORBENT ASSAY; FLOW CYTOMETRY; GENE; GENE EXPRESSION; HOMEOSTASIS; HORMONE SYNTHESIS; HYPOTHALAMUS HYPOPHYSIS THYROID SYSTEM; IMMUNE SYSTEM; IMMUNOREGULATION; INTESTINE; MOUSE; NONHUMAN; PRIORITY JOURNAL; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; SEQUENCE HOMOLOGY; T LYMPHOCYTE;

ANIMALS; HOMEOSTASIS; IMMUNITY, MUCOSAL; INTESTINAL MUCOSA; INTESTINE, SMALL; MICE; MICE, INBRED BALB C; MICE, INBRED C57BL; MICE, NUDE; POINT MUTATION; RECEPTORS, THYROTROPIN; RECEPTORS, THYROTROPIN-RELEASING HORMONE; T-LYMPHOCYTE SUBSETS; T-LYMPHOCYTES; THYROTROPIN; THYROTROPIN-RELEASING HORMONE;

EID: 0030964447     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5308.1937     Document Type: Article

References (36)

1. J. R. Klein and R. L. Mosley, in Mucosal Immunology: Intraepithelial Lymphocytes, H. Kiyono and J. R. McGhee, Eds. (Raven, New York, 1994), pp. 33-60; R. Hershberg et al., Proc. Natl. Acad. Sci. U.S.A. 87, 9727 (1990); H. Ohno et al., EMBO J. 12, 4357 (1993).
2. J. R. Klein and R. L. Mosley, in Mucosal Immunology: Intraepithelial Lymphocytes, H. Kiyono and J. R. McGhee, Eds. (Raven, New York, 1994), pp. 33-60; R. Hershberg et al., Proc. Natl. Acad. Sci. U.S.A. 87, 9727 (1990); H. Ohno et al., EMBO J. 12, 4357 (1993).
3. J. R. Klein and R. L. Mosley, in Mucosal Immunology: Intraepithelial Lymphocytes, H. Kiyono and J. R. McGhee, Eds. (Raven, New York, 1994), pp. 33-60; R. Hershberg et al., Proc. Natl. Acad. Sci. U.S.A. 87, 9727 (1990); H. Ohno et al., EMBO J. 12, 4357 (1993).
4. R. Boismenu and W. L. Havran, Science 266, 1253 (1994); K. Komano et al., Proc. Natl. Acad. Sci. U.S.A. 92, 6147 (1995).
5. R. Boismenu and W. L. Havran, Science 266, 1253 (1994); K. Komano et al., Proc. Natl. Acad. Sci. U.S.A. 92, 6147 (1995).
6. J. Wang and J. R. Klein, Science 265, 1860 (1994); Cell. Immunol. 161, 299 (1995).
7. J. Wang and J. R. Klein, Science 265, 1860 (1994); Cell. Immunol. 161, 299 (1995).
8. _, Int. Immunol. 8, 231 (1996).
9. BALB/c mice and C57BL/6 euthymic and congenitally athymic nude mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were raised at the University of Tulsa vivarium. Congenitally hypothyroid hyt/hyt mice and euthyroid BALB/ cBY (+/+) progenitor strain mice were purchased from the Jackson Laboratory or were raised at the University of Tulsa from hyt/+ breeder stocks obtained from the Jackson Laboratory. Confirmation of gene mutation in hyt/hyt mice was done by PCR and gene sequence analyses of the TSH-R gene across the mutation region using DNA from hyt/hyt mouse tissue samples. Use of laboratory animals conformed to institutional and NIH guidelines. Isolation of small intestine epithelia and IELs was done as described (6), except that the Percoll fractionation step was omitted so as to obtain all cell populations present within the epithelium. Thymocytes, spleen cells, lymph node cells, and Peyer's patch cells were isolated by dissociation of tissues through a 60-mesh stainless steel screen into Hanks' balanced salt solution.
10. R. L. Mosley and J. R. Klein, J. Immunol. Methods 156, 19 (1992).
11. J. R. Klein, R. L. Mosley, D. Kaiserlian, Proc. Soc. Exp. Biol. Med. 195, 329 (1990).
12. 6 cells were reacted with unlabeled LCA or G8.8 mAbs for 30 min at 4°C, washed, and reacted with PE-labeled antibody to rat Ig. For direct staining, cells were reacted with PE-labeled LCA mAb for 30 min at 4°C, washed, and fixed. Unlabeled isotype control antibody followed by PE-labeled antibody to rat Ig was used for control indirect staining; PE-labeled isotype control antibody was used for control direct staining. Cells were analyzed on an EPICS 751 flow cytometer interfaced to an MDADS II computer (Coulter Electronics, Hialeah, FL).
13. A. Farr, A. Nelson, J. Truex, S. Hosier, J. Histochem. Cytochem. 39, 645 (1991).
14. +; mean values ± SD of four experiments) (6). In the latter case, the reactivity of antibody to rat Ig to the residual LCA mAb used for depletion of IELs was ruled out using PE-labeled antibody to rat Ig in the absence of G8.8; <1% of enriched epithelial cells were reactive with antibody to rat Ig alone, whereas 98.3% were reactive after addition of G8.8. Thus, by physical criteria and by expression of tissue-specific marker, IELs and epithelial cells were very highly enriched populations, as also confirmed by the distinct patterns of gene expression in those cell populations (Fig. 2). RNA was extracted from each cell preparation (11, 29).
15. RNA extracted from IELs and epithelial cells was treated with deoxyribonuclease (BRL, Gaithersburg, MD) for 15 min at 25°C and converted into cDNA using reverse transcriptase (BRL) according to procedures previously reported by this laboratory (29). Primers were constructed from published sequences for TSHβ, TRH-R, and TSH-R, respectively (12, 13, 23): TSHβ upstream, 5′-GAGTGTGGCTACTGCCTGACC-3′; TSHβ downstream, 5′-ACACTTGCCACACTTGCAGCT-3′; TRH-R upstream, 5′-ATGTTGTGCCAATGATCCTG-3′; TRH-R downstream, 5′-TAGGGCCACACTGTAGTTAGC-3′; TSH-R upstream, 5′-GCGTCTCCACCCTGTGAGTGTCA-CC-3′; and TSH-R downstream, 5′-CATGTAAGGGTTGTCTGTGATTTC-3′. Amplification was done using a 40-cycle program consisting of 94°C for 45 s, 65°C for 45 s, and 72°C for 15 s. For DNA sequencing, the PCR-amplified products shown in Fig. 2 were diluted 1:5000 and reamplified using a nested upstream primer and the original downstream primer (11). DNA sequencing was done using fluorescent dye - labeled dideoxynucleotides with the nested primers with Taq polymerase. Products were analyzed on a Biosystems 370A automated DNA sequencer (Applied Biosystems). Nested upstream primers were as follows: TRH-R, 5′-AGGGCTGGAGAGAAATGAGTTGAC-3′; TSH-R, 5′-GACTCATCTGAAGACCATACCCAGTCTTGCA-3′; and TSHβ, 5′-CTGTTTCTTCCCAAATATGCACTC-3′. Sequences obtained from the PCR products using the primers described above were identical to published sequence regions for the appropriate genes: TSHβ, 291-413 (12); TRH-R, 970-1115 (13); and TSH-R, 215-491 (23).
16. J. Gurr, J. F. Cattarall, I. A. Kourides, Proc. Natl. Acad. Sci. U.S.A. 80, 2121 (1983).
17. R. Straub, G. C. Frech, R. H. Juho, M. C. Bershengorn, ibid. 87, 9514 (1990).
18. The ELISA assay for detection of TSH was performed according to a published protocol (15). Briefly, microtiter wells of ELISA plates were coated by incubating lymphoid cell culture supematants (30) overnight at 4°C. Plates were washed and wells were flooded with PBS containing 0.1% bovine serum albumin (BSA) for 1 hour at room temperature. Wells were washed three times with PBS-tween (0.5%), and rabbit antibody to mouse TSHβ (1:250; National Hormone and Pituitary Program, Rockville, MD) was added for 1 hour at room temperature. Wells were washed three times with PBS-tween, and biotinylated goat antibody to rabbit Ig (1:5000; Zymed Laboratories, South San Francisco, CA) was added for 30 min at room temperature. Cells were washed three times with PBS-tween, and streptavidin -horseradish peroxidase (1:5000; Zymed) was added for 30 min at room temperature. After washing, wells were reacted with O-phenylenediamine (Sigma) and reactions were quantified on a BT2000 Microkinetics ELISA Reader (Fisher Scientific, Dallas, TX).
19. D. Harbour, T. E. Kruger, D. Coppenhaver, E. M. Smith, W. J. Meyer, Mol. Cell. Endocrinol. 64, 229 (1989).
20. E. M. Smith, M. Phan, D. Coppenhaver, T. E. Kruger, J. E. Blalock, Proc. Natl. Acad. Sci. U.S.A. 80, 6010 (1983).
21. T. E. Kruger, L. R. Smith, D. V. Harbour, J. E. Blalock, J. Immunol. 142, 744 (1989).
22. K. Vidal, I. Grosjean, J.-P. Revillard, C. Gespach, D. Kaiserlian, J. Immunol. Methods 166, 63 (1993).
23. -9 M. Binding of hormone in the absence of cells was <100 cpm per tube.
24. S. Satoh, P. Feng, U. J. Kim, J. F. Wilber, Neuropeptides 27, 195 (1994).
25. W. Xia et al., Endocrinology 136, 3146 (1995).
26. S. A. Stein et al., Thyroid 1, 257 (1991).
27. S. A. Stein et al., Mol. Endocrinol. 8, 129 (1994).
28. D. Stickney, J. Wang, M. Hamad, J. R. Klein, Blood 84, 3034 (1994); M. Hamad and J. R. Klein, Immunology 82, 611 (1994).
29. D. Stickney, J. Wang, M. Hamad, J. R. Klein, Blood 84, 3034 (1994); M. Hamad and J. R. Klein, Immunology 82, 611 (1994).
30. F. Pekonen and B. D. Weintraub, Endocrinology 103, 1668 (1978).
31. R. P. Green et al., Proc. Natl. Acad. Sci. U.S.A. 85, 5592 (1988).
32. G. Pelletier, J. Cell. Biol. 62, 185 (1974).
33. F. Labrie, in Hormones, from Molecules to Disease, E. E. Basaulieu and P. A. Kelly, Eds. (Chapman & Hall, New York, 1990), p. 261.
34. R. L. Mosley et al., Int. Immunol. 6, 231 (1994).
35. 6 cells per milliliter) in serum-free RPMI 1640 supplemented with 1% BSA were cultured with or without synthetic TRH (Sigma, T-9146). Cultures were harvested after 1 and 6 hours or refed after 24 hours, and cell-free supernatants were harvested after 48 hours. Cell culture supernatants were used to coat ELISA microtiter plates as described in (14).
36. We thank A. Farr for mAb G8.8, D. Kaiserlian for the MODE-K cell line, and K. Miller and R. L. Mosley for critical review of the manuscript. Supported by NIH grant DK35566.