Examining the Role of Paneth Cells in the Small Intestine by Lineage Ablation in Transgenic Mice

The Paneth cell lineage is one of four epithelial lineages derived from the adult mouse small intestine's multipotent stem cell. Mature Paneth cells secrete antimicrobial peptides (cryptdins), growth factors, as well as two gene products, a secreted phospholipase A2 and matrilysin, that has been implicated as modifiers of adenoma formation in mice containing a mutation in the tumor suppressor Apc. Immature Paneth cells are located just above and below the cell layer, in intestinal crypts, that has been proposed to contain the multipotent stem cell. Paneth cells differentiate during a downward migration to the crypt base. The location and direction of Paneth cell migration, their high density and long residency time at the crypt base, and the nature of their secreted gene products, suggest that they may influence the structure and/or function of the stem cell niche. Paneth cell ablation can therefore be viewed as an experimental manipulation of the cellular microenvironment that purportedly contains the stem cell and its immediate descendants. Two types of ablation experiments were performed in transgenic mice. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene (CR2) were used to express an attenuated diphtheria toxin A fragment. Light and electron microscopic immunohistochemical analyses of several pedigrees of postnatal day 28 to 180 animals established that ablation of Paneth cells is accompanied by an increase in the proportion of undifferentiated crypt base columnar cells. These cells normally co-exist with Paneth cells. The ablation does not produce a detectable effect on the proliferation or terminal differentiation programs of the other three lineages or on host-microbial interactions. The last conclusion is based on the ability of crypts to remain free of microbes detectable by Gram and Warthin-Starry stains and by retention of the normal crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. CR2-directed expression of simian virus 40 large T antigen also results in a loss of mature Paneth cells but produces a marked amplification of crypt cells having a morphology intermediate between Paneth and granule goblet cells. EM immunohistochemical analyses suggest that intermediate cells can differentiate to mature goblet cells but not to Paneth cells, as they migrate up the crypt-villus axis. Our findings suggest that (i) stemness in the crypt is not defined by instructive interactions involving the Paneth cell; (ii) expressing a Paneth cell fate may require that precursors migrate to the crypt base; (iii) antimicrobial factors produced by Paneth cells are not required to prevent colonization of small intestinal crypts; and (iv) this lineage does not function to maintain the asymmetric crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. The Paneth cell lineage is one of four epithelial lineages derived from the adult mouse small intestine's multipotent stem cell. Mature Paneth cells secrete antimicrobial peptides (cryptdins), growth factors, as well as two gene products, a secreted phospholipase A2 and matrilysin, that has been implicated as modifiers of adenoma formation in mice containing a mutation in the tumor suppressor Apc. Immature Paneth cells are located just above and below the cell layer, in intestinal crypts, that has been proposed to contain the multipotent stem cell. Paneth cells differentiate during a downward migration to the crypt base. The location and direction of Paneth cell migration, their high density and long residency time at the crypt base, and the nature of their secreted gene products, suggest that they may influence the structure and/or function of the stem cell niche. Paneth cell ablation can therefore be viewed as an experimental manipulation of the cellular microenvironment that purportedly contains the stem cell and its immediate descendants. Two types of ablation experiments were performed in transgenic mice. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene (CR2) were used to express an attenuated diphtheria toxin A fragment. Light and electron microscopic immunohistochemical analyses of several pedigrees of postnatal day 28 to 180 animals established that ablation of Paneth cells is accompanied by an increase in the proportion of undifferentiated crypt base columnar cells. These cells normally co-exist with Paneth cells. The ablation does not produce a detectable effect on the proliferation or terminal differentiation programs of the other three lineages or on host-microbial interactions. The last conclusion is based on the ability of crypts to remain free of microbes detectable by Gram and Warthin-Starry stains and by retention of the normal crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. CR2-directed expression of simian virus 40 large T antigen also results in a loss of mature Paneth cells but produces a marked amplification of crypt cells having a morphology intermediate between Paneth and granule goblet cells. EM immunohistochemical analyses suggest that intermediate cells can differentiate to mature goblet cells but not to Paneth cells, as they migrate up the crypt-villus axis. Our findings suggest that (i) stemness in the crypt is not defined by instructive interactions involving the Paneth cell; (ii) expressing a Paneth cell fate may require that precursors migrate to the crypt base; (iii) antimicrobial factors produced by Paneth cells are not required to prevent colonization of small intestinal crypts; and (iv) this lineage does not function to maintain the asymmetric crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. The structural and functional organization of the adult mouse small intestinal epithelium lends itself to studying both the regulation and integration of cellular proliferation, differentiation, and death programs. The epithelium contains four principal cell types: absorptive enterocytes (comprising >80% of the total population), enteroendocrine cells, mucus-producing goblet cells, and Paneth cells. All four lineages are derived from a multipotent stem cell that is functionally anchored near the base of each of the small intestine's 1.1 million crypts of Lieberkühn (1Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 537-562Crossref PubMed Scopus (1120) Google Scholar, 2Bjerknes M. Cheng H. Am. J. Anat. 1981; 160: 51-63Crossref PubMed Scopus (182) Google Scholar, 3Bjerknes M. Cheng H. Am. J. Anat. 1981; 160: 65-75Crossref PubMed Scopus (56) Google Scholar, 4Loeffler M. Birke A. Winton D. Potten C. J. Theor. Biol. 1993; 160: 471-491Crossref PubMed Scopus (136) Google Scholar). Cell division is confined to these crypts (5Potten C.S. Loeffler M. Development. 1990; 110: 1001-1020Crossref PubMed Google Scholar). Enterocytes, enteroendocrine, and goblet cells migrate out of the crypt and up an adjacent villus. Migration is highly ordered and associated with terminal differentiation. Cell death occurs near the villus tip where cells are exfoliated into the lumen (6Gavrieli Y. Sherman Y. Ben-Sasson S.A. J. Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9167) Google Scholar, 7Hall P.A. Coates P.J. Ansari B. Hopwood D. J. Cell Sci. 1994; 107: 3569-3577Crossref PubMed Google Scholar). Proliferation, differentiation, and death take place in a spatially well-organized continuum that extends from the crypt to the apex of a villus. This sequence is completed rapidly (2–5 days in the case of enterocytes, enteroendocrine, and goblet cells; Refs. 1Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 537-562Crossref PubMed Scopus (1120) Google Scholar and8Cheng H. Am. J. Anat. 1974; 141: 481-502Crossref PubMed Scopus (173) Google Scholar, 9Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-480Crossref PubMed Scopus (545) Google Scholar, 10Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 503-520Crossref PubMed Scopus (247) Google Scholar) and is recapitulated throughout the lifespan of the mouse. The Paneth cell lineage differs from the others in a number of notable ways. It is the only lineage that executes its terminal differentiation program during a downward migration from the stem cell zone to the crypt base (11Cheng H. Am. J. Anat. 1974; 141: 521-536Crossref PubMed Scopus (159) Google Scholar). It is the longest lived lineage, and the only one that exists entirely within the proliferative compartment. Each crypt contains 30–50 mature Paneth cells that survive for 18–23 days before degenerating and undergoing phagocytosis by their neighbors (11Cheng H. Am. J. Anat. 1974; 141: 521-536Crossref PubMed Scopus (159) Google Scholar, 12Cheng H. Merzel J. Leblond C.P. Am. J. Anat. 1969; 126: 507-525Crossref PubMed Scopus (76) Google Scholar, 13Troughton W.D. Trier J.S. J. Cell Biol. 1969; 41: 251-268Crossref PubMed Scopus (105) Google Scholar). Paneth cell age correlates with position in the crypt; the most mature cells are located at or near the crypt base (2Bjerknes M. Cheng H. Am. J. Anat. 1981; 160: 51-63Crossref PubMed Scopus (182) Google Scholar). The size of the Paneth cell's apical secretory granules also correlates with age; larger granules are produced by older cells (2Bjerknes M. Cheng H. Am. J. Anat. 1981; 160: 51-63Crossref PubMed Scopus (182) Google Scholar, 11Cheng H. Am. J. Anat. 1974; 141: 521-536Crossref PubMed Scopus (159) Google Scholar). The function of the Paneth cell has not yet been clearly defined. Residency at the crypt base places this lineage in a position to release products from its apical granules that could affect establishment and/or maintenance of the stem cell's niche or influence the properties of the stem cell's descendants. A number of factors exported by Paneth cells could regulate epithelial proliferation and differentiation programs. They include tumor necrosis factor-α (14Tan X. Hsueh W. Gonzalez-Crussi F. Am. J. Path. 1993; 142: 1858-1865PubMed Google Scholar), guanylin (15de Sauvage F.J. Keshav S. Kuang W.J. Gillett N. Henzel W. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9089-9093Crossref PubMed Scopus (132) Google Scholar), and epidermal growth factor (16Poulsen S.S. Nexo E. Olsen P.S. Hess J. Kirkegaard P. Histochemistry. 1986; 85: 389-394Crossref PubMed Scopus (194) Google Scholar). Two Paneth cell products have been implicated as modifiers of adenoma formation in mice heterozygous for a mutation in the adenomatous polyposis coli gene,Apc Min (17Su L.-K. Kinzler K.W. Vogelstein B. Preisinger A.C. Moser A.R. Luongo C. Gould K.A. Dove W.F. Science. 1992; 256: 668-670Crossref PubMed Scopus (1355) Google Scholar). Production of matrilysin, a matrix metalloproteinase, is limited to the Paneth cell lineage in the adult mouse intestine (18Wilson C.L. Heppner K.J. Rudolph L.A. Matrisian L.M. Mol. Biol. Cell. 1995; 6: 851-869Crossref PubMed Scopus (132) Google Scholar). The protein is expressed in a high percentage of early stage human colorectal neoplasms and Min adenomas (19Wilson C.L. Heppner K.J. Labosky P.A. Hogan B.L.M. Matrisian L.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1402-1407Crossref PubMed Scopus (545) Google Scholar). Min/+ mice homozygous for a null allele of the matrilysin gene have 60% fewer adenomas than animals with the wild type allele, suggesting that the enzyme functions as suppressor of tumorigenesis (19Wilson C.L. Heppner K.J. Labosky P.A. Hogan B.L.M. Matrisian L.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1402-1407Crossref PubMed Scopus (545) Google Scholar). Pla2 g2a encodes a phospholipase A2 that is secreted from Paneth cells (20Mulherkar R. Desai S.J. Rao R.S. Wagle A.S. Deo M.G. Histochemistry. 1991; 96: 367-370Crossref PubMed Scopus (28) Google Scholar, 21Mulherkar R. Rao R.S. Wagle A.S. Patki V. Deo M.G. Biochem. Biophys. Res. Commun. 1993; 195: 1254-1263Crossref PubMed Scopus (38) Google Scholar, 22Harwig S.S.L. Tan L. Qu X. Cho Y. Eisenhauer P.B. Lehrer R.I. J. Clin. Invest. 1995; 95: 603-610Crossref PubMed Google Scholar). This gene is a strong candidate for Mom1, a semi-dominant modifier of adenoma size and multiplicity in Min/+ animals (23MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 24Gould K.A. Dietrich W.F. Borenstein N. Lander E.S. Dove W.F. Genetics. 1996; 144: 1769-1776Crossref PubMed Google Scholar, 25Gould K.A. Luongo C. Moser A.R. McNeley M.K. Borenstein N. Shedlovsky A. Dove W.F. Hong K. Dietrich W.F. Lander E.S. Genetics. 1996; 144: 1777-1785Crossref PubMed Google Scholar). Paneth cells also export lysozyme (26Ghoos Y. VanTrappen G. Histochem. J. 1971; 3: 175-178Crossref PubMed Scopus (60) Google Scholar, 27Peeters T. VanTrappen G. Gut. 1975; 16: 553-558Crossref PubMed Scopus (132) Google Scholar) and a family of defensin-related anti-microbial peptides known as cryptdins (28Ouellette A.J. Selsted M.E. FASEB J. 1996; 10: 1280-1289Crossref PubMed Scopus (240) Google Scholar). The intestine contains a complex microflora. Components of this microflora are able to establish stable niches at particular positions along the duodenal-ileal axis (29Savage D.C. Annu. Rev. Microbiol. 1977; 31: 107-133Crossref PubMed Scopus (1697) Google Scholar). The fact that different cryptdins exhibit distinct developmental and spatial patterns of expression along this axis (30Darmoul D. Ouellette A.J. Am. J. Physiol. 1996; 271: G68-G74PubMed Google Scholar) suggests that Paneth cells could play a role in modulating the composition of the microbiota or contribute to mucosal barrier functions. We have examined the contribution of this lineage to epithelial and microbial homeostasis by generating two types of transgenic mice in which mature Paneth cells have been eliminated. A 2.7-kb 1The abbreviations used are: kb, kilobase pairs; SV40 TAg, simian virus 40 large T antigen; CR2, nucleotides −6500 to +34 of the mouse cryptdin-2 gene; DT-A, diphtheria toxin A fragment; tox176, an attenuated DT-A containing a Gly128 → Asp substitution; hGH, human growth hormone; P, postnatal day; BrdU, 5-bromo-2′-deoxyuridine; PAS, periodic acid-Schiff stain; EPX, endogenous peroxidases; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; Cy3, indocarbocyanine; Cy5, indodicarbocyanine; SFB, segmented filamentous bacterium; GALT, gut-associated lymphoid system; TCR, T-cell receptor; PBS, phosphate-buffered saline; DBA,Dolichos biflorus agglutinin; UEA-1, Ulex europaeus agglutinin 1. DNA fragment containing simian virus 40 large T antigen (SV40 TAg) was excised from pIF-TAg-hGH (31Hauft S.M. Kim S.H. Schmidt G.H. Pease S. Rees S. Harris S. Roth K.A. Hansbrough J.R. Cohn S.M. Ahnen D.J. Wright N.A. Goodlad R.A. Gordon J.I. J. Cell Biol. 1992; 117: 825-839Crossref PubMed Scopus (57) Google Scholar) with BamHI and subcloned into theBamHI site of pCR-H1 (32Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (217) Google Scholar). This yielded pCR2-TAg which contained SV40 TAg under the control of nucleotides −6500 to +34 of the mouse cryptdin-2 gene (33Huttner K.M. Selsted M.E. Ouellette A.J. Genomics. 1994; 19: 448-453Crossref PubMed Scopus (70) Google Scholar). Complete digestion of pIF-TAg-hGH with BamHI, followed by partial digestion with EcoRI, allowed purification of a DNA fragment containing pBluescript SK+ (Stratagene) with nucleotides +3 to +2150 of the human growth hormone gene (hGH; Ref.34Seeburg P.H. DNA ( N. Y. ). 1982; 1: 239-249Crossref PubMed Scopus (213) Google Scholar). This fragment and a BamHI/EcoRI fragment containing cryptdin-2−6500 to +34 and SV40 TAg from pCR2-TAg were ligated together, producing pCR2-TAg-hGH. pCR2-TAg-hGH was subsequently cut with BamHI, treated with Klenow, and ligated to a 0.6-kb HincII fragment containing an attenuated diphtheria toxin A fragment (tox176; Ref. 35Maxwell F. Maxwell I.H. Glode L.M. Mol. Cell. Biol. 1987; 7: 1576-1579Crossref PubMed Scopus (71) Google Scholar). The resulting plasmid, pCR2-tox176-hGH, contained tox176 immediately downstream of cryptdin-2−6500 to +34 and immediately upstream of hGH+3 to +2150. tox176 was placed in exon1 of hGH to enhance the chances of efficiently expressing the toxin. hGH will not be produced from the RNA transcript of cryptdin-2−6500 to +34-tox176-hGH+3 to +2150: the initiator Met and the first stop codon are from the open reading frame of tox176, and there is no ribosomal re-entry site to re-initiate translation at the downstream initiator ATG of hGH. A 9.2-kb fragment containing cryptdin-2−6500 to +34 and SV40 TAg (CR2-TAg) was released from pCR2-TAg by digestion with NotI andEcoRI. pCR2-tox176-hGH was digested with NotI andXhoI to liberate a 9.4-kb DNA fragment containing cryptdin-2−6500 to +34-tox176-hGH (CR2-tox176). An 8.3-kb fragment containing cryptdin-2−6500 to +34 linked to hGH+3 to +2150 (CR2-hGH) was released from pCR-H1 withEcoRI (32Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (217) Google Scholar). Each fragment was purified by agarose gel electrophoresis followed by glass bead extraction (Geneclean, Bio 101) and used for pronuclear injection of FVB/N oocytes. Oocytes were subsequently transferred to pseudopregnant Swiss Webster females using standard techniques (36Hogan, B., Constantini, F., and Lacy, E. (eds) (1986) Cold Spring Harbor Laboratory Laboratory, Cold Spring Harbour, NY.Google Scholar). Live born mice were screened for the presence of transgenes by extracting tail DNA and performing polymerase chain reactions using primers that anneal to hGH DNA (CR2-tox176; CR2-hGH; 5′-AGGTGGCCTTTGACACCTACCAGG-3′ and 5′-TCTGTTGTGTTTCCTCCCTGTTGG-3′) or to SV40 TAg DNA (CR2-TAg; 5′-ATGAATGGGAGCAGTGGTG-3′ and 5′-GCAGACACTCTATGCCTGTGTGG-3′). The polymerase chain reaction mixtures (final volume = 25 μl) contained 50 mm KCl, 20 mm Tris, pH 8.4, 2 mm MgCl2, 200 μm dNTPs, primers (1 μm each), 0.7 unit of Taq DNA polymerase (Boehringer Mannheim), and approximately 0.5 μg of genomic DNA. The following cycling conditions were used to amplify an hGH fragment from CR2-tox176 and CR2-hGH DNAs: denaturation, 1 min at 94 °C; annealing, 1.5 min at 55 °C; and extension, 2 min at 72 °C for 30 cycles. For CR2-TAg, denaturation was performed at 95 °C and annealing at 58 °C. Four CR2-hGH founders were identified from 38 live born mice, 2 CR2-tox176 founders from 67 mice, and 10 CR2-TAg founders from 87 animals. Pedigrees were established from each of the CR2-hGH and CR2-tox176 founders and from 8 of the CR-TAg founders. All pedigrees were maintained by crosses to normal FVB/N littermates. Pedigree 61, containing CR2-hGH, has been described in an earlier publication (32Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (217) Google Scholar). Mice were housed in microisolator cages under a strictly controlled light cycle (lights on at 0600 h and off at 1800 h) and given a standard irradiated chow dietad libitum (Pico rodent chow 20, Purina Mills). Routine screens for hepatitis, minute, lymphocytic choriomeningitis, ectromelia, polyoma, sendai, pneumonia, and MAD viruses, enteric bacterial pathogens, and parasites were negative. Specific pathogen-free transgenic animals and their nontransgenic littermates were sacrificed between postnatal days 28 (P28) and P180. Immediately after sacrifice, the small intestine was removed en bloc, flushed with ice-cold phosphate-buffered saline (PBS), fixed in 10% buffered formalin (Fisher) for 4–6 h, and then washed in 70% ethanol overnight at room temperature. The intestine was embedded in plastic (JB-4 Embedding Kit, Polysciences), and 1–2-μm thick sections (“thin sections”) cut from its proximal, middle, and distal thirds (these segments were arbitrarily designated duodenum, jejunum, and ileum, respectively). Alternatively, after washing in 70% ethanol, the intestine was cut open along its duodenal-ileal axis, rolled into a circle, and held in this circular configuration by mounting agar (2% agar (Sigma) in 5% buffered formalin). Each of the resulting “Swiss rolls” was then placed in a tissue cassette, embedded in paraffin, and 5 μm-thick serial sections were prepared. Plastic- or paraffin-embedded sections were stained with hematoxylin and eosin, phloxine/tartrazine, or with Alcian blue and periodic acid Schiff (PAS) using standard protocols (37). Goblet cells were quantitated by counting Alcian blue/PAS-positive cells in all well-oriented jejunal crypt-villus units present in at least two non-adjacent sections cut from Swiss rolls (sections were prepared from three P28 transgenic animals and three normal littermates per pedigree). Paneth cells were likewise quantitated by counting phloxine/tartrazine-positive cells in jejunal crypts. To analyze the distribution of components of the microflora along the crypt-villus units of specific pathogen-free transgenic animals and their normal littermates, mice from the various pedigrees were sacrificed at P28, P42, and P120–P180. Their small intestines were fixed 4–6 h in 10% buffered formalin without prior flushing and then cut into 1–2-cm segments. Each segment was embedded in paraffin, 4–6-μm thick sections were cut, and the sections treated with Warthin-Starry or Gram stains (37). Transgenic mice and their normal littermates were sacrificed at P28, P42, and P120–180 (n = 3–5/group/pedigree/time point). Some animals received an intraperitoneal injection of an aqueous solution of 5′-bromo-2′-deoxyuridine (120 mg/kg, BrdU) and 5′-fluoro-2′-deoxyuridine (12 mg/kg) 1.5 to 72 h before sacrifice. The small intestine was then removed from each animal, flushed with cold PBS, fixed in Bouin's solution for 8 h at room temperature, treated with 70% ethanol, and 4–6-μm thick sections cut from paraffin-embedded Swiss rolls. Sections were then deparaffinized, rehydrated, and placed in PBS-blocking buffer (1% bovine serum albumin, 0.3% Triton X-100 in PBS) for 20 min at room temperature. Slides were incubated overnight at 4 °C with the following antibodies: (i) rabbit antiserum raised against residues 4–35 of cryptdin-1 (the antisera reacts with purified cryptdins 1, 2, 3, and 6 (32Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (217) Google Scholar, 38Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (281) Google Scholar, 39Roth K.A. Hertz J.W. Gordon J.I. J. Cell Biol. 1990; 110: 1791-1801Crossref PubMed Scopus (79) Google Scholar, 40Bry L. Falk P.G. Midtvedt T. Gordon J.I. Science. 1996; 273: 1380-1383Crossref PubMed Scopus (514) Google Scholar), was supplied by Michael Selsted, University of California, Irvine, and was diluted 1:500 in PBS-blocking buffer); (ii) rabbit antiserum to the secreted phospholipase A2 encoded byPla2g2a (also known as enhancing factor; Refs. 20Mulherkar R. Desai S.J. Rao R.S. Wagle A.S. Deo M.G. Histochemistry. 1991; 96: 367-370Crossref PubMed Scopus (28) Google Scholar and 21Mulherkar R. Rao R.S. Wagle A.S. Patki V. Deo M.G. Biochem. Biophys. Res. Commun. 1993; 195: 1254-1263Crossref PubMed Scopus (38) Google Scholar) was obtained from Rita Mulherkar, Cancer Research Institute, Tata Memorial Center, Bombay, India; dilution = 1:40,000); (iii) rabbit anti-human lysozyme (Dako, Santa Barbara, CA; specificity in the FVB/N intestine described in Ref. 32Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (217) Google Scholar; dilution = 1:500); (iv) rabbit anti-serotonin (Incstar, Stillwater, MN; 39; 1:8000); (v) rabbit anti-chromogranin A (Incstar; 1:10,000); (vi) rabbit anti-hGH (Dako; 39; 1:2000); (vii) rabbit anti-SV40 TAg (a generous gift of Doug Hanahan, University of California, San Francisco; 40; 1:2000); and (viii) goat anti-BrdU (Ref. 41Cohn S.M. Lieberman M.W. J. Biol. Chem. 1984; 259: 12456-12462Abstract Full Text PDF PubMed Google Scholar; 1:1000). Antigen-antibody complexes were detected with indocarbocyanine (Cy3)- or indodicarbocyanine (Cy5)-conjugated donkey anti-rabbit or anti-goat immunoglobulins (Ig; Jackson Immunoresearch; 1:500). Sections were also incubated with a series of fluorescein isothiocyanate (FITC)-conjugated lectins (all obtained from Sigma, all used at a final concentration of 5 μg/ml PBS blocking buffer): (i)Ulex europaeus agglutinin 1 (UEA-1; carbohydrate specificity = Fucα1,2Gal epitopes; lineage specificity in P28-P180 FVB/N small intestine = Paneth, goblet, and enteroendocrine cells; Ref. 42Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar); (ii) peanut (Arachis hypogaea) agglutinin (PNA, Galβ3GalNAc; all four epithelial lineages; Ref. 42Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar); and (iii) Dolichos biflorusagglutinin (DBA; GalNAcα3GalNAc and GalNAcα3Gal epitopes; Paneth and goblet cells plus enterocytes; Ref. 42Falk P. Roth K.A. Gordon J.I. Am. J. Physiol. 1994; 266: G987-G1003Crossref PubMed Google Scholar). The spatial distribution of components of the diffuse gut-associated lymphoid system (GALT) was examined in P42 CR2-tox176 mice and their normal littermates (n = 3 animals/group/pedigree) using the following panel of monoclonal antibodies from PharMingen (each diluted 1:1000 in PBS-blocking buffer): (i) rat anti-mouse CD4 (clone H129.19); (ii) rat anti-mouse α chain of CD8 (clone 53–6.7); (iii) hamster anti-mouse β-subunit of the αβ T-cell receptor (TCR; clone H57–597); (iv) hamster anti-mouse γδ TCR (clone GL3); and (v) rat anti-mouse CD45R/B220 (a B-cell marker; clone RA3–6B2). Mice were sacrificed, and the middle third of the small bowel was flushed with PBS and then frozen in OCT (Miles). Serial sections were cut, fixed in methanol (−20 °C for 15 min), washed 3 times (3 min/cycle) in PBS, and treated with PBS-blocking buffer (15 min at room temperature). A variety of methods that are traditionally used for eliminating endogenous peroxidase (EPX) activities from cryostat sections of the intestine either failed to adequately remove EPX or to preserve the antigens that we were studying (cf. Ref. 43Hunyady B. Mezey É. Pacak K. Palkovits M. Histochem. Cell Biol. 1996; 106: 447-456Crossref PubMed Scopus (18) Google Scholar). However, we found that endogenous biotin levels were below the limits of detection with tyramide signal amplification protocols that employed horseradish peroxidase (HRP)-conjugated streptavidin. Therefore, cells with EPX activity were labeled by incubating frozen sections of intestine for 8–10 min at room temperature with FITC-conjugated tyramide (obtained from NEN Life Science Products and diluted 1:100 in 1 × amplification diluent from the same manufacturer). Following 3 washes in PBS (5 min each), the sections were incubated overnight at 4 °C with one of the primary antibodies and then washed in TNT buffer (0.1 m Tris, pH 7.5, 0.15 m NaCl, 0.05% Tween 20; 3 cycles with 5 min/wash). Two secondary antibodies were used to visualize antigen-antibody complexes. (i) If HRP-conjugated goat anti-rat Ig (Kirkegaard and Perry Labs) was used, it was first diluted 1:100 in TNB buffer (TNB = 0.1 m Tris, pH 7.5, 0.15m NaCl, 0.5% blocking reagent from NEN Life Science Products) and then placed on the section for 30 min (this and all subsequent steps were performed at room temperature). After three washes with TNT buffer, biotinyl-tyramide was added (diluted 1:100 in 1 × amplification diluent). Following a 8–10-min incubation, the sections were washed several times in TNT buffer and overlaid with Cy3-conjugated streptavidin (Jackson Immunoresearch; diluted 1:500 in TNB) for 30 min. (ii) If a biotinylated anti-hamster Ig (diluted 1:100 in TNB) was used as the secondary antibody, it was detected with HRP-conjugated streptavidin (NEN Life Science Products; diluted 1:1000 in TNB) followed by amplification with biotinyl-tyramide and application of Cy3-streptavidin, as described above. Two controls were performed for each experiment employing a given primary antiserum: (i) direct amplification of EPX alone (see above) and (ii) omission of primary antibodies. The latter control involved direct amplification of EPX followed by application of an HRP-conjugated secondary antibody and subsequent indirect tyramide signal amplification with biotinyl tyramide and Cy3-streptavidin. Alternatively, when biotinylated secondary antibodies were employed, there would be a control to establish whether there was any labeling of endogenous biotin. This control consisted of direct tyramide amplification of EPX with FITC-tyramide followed by HRP-streptavidin but without addition of the biotinylated secondary antibodies or the primary antibodies. (Note that adding Cy3-streptavidin alone to jejunal sections did not produce any cellular staining.) A Molecular Dynamics Multiprobe 2001 inverted confocal microscope system was used to scan sections subjected to single and/or multi-label immunohistochemistry. Sections were also viewed and photographed using a Zeiss Axioscope. Apoptotic cells were scored in adjacent sections of Swiss rolls, prepared from normal, CR2-tox176, and CR2-TAg mice, using the terminal deoxynucleotidyltransferase-mediated, dUTP nick end-labeling assay, and by their morphologic appearance after staining with hematoxylin and eosin (6Gavrieli Y. Sherman Y. 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