0022-1554/92/$3.30 The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.

Vol. 40, No. 10. pp. 1527-1534, 1992 Printed in UXA.

Original Article

I Detection of CDI mRNA in Paneth Cells of the Mouse Intestine by In Situ Hybridization’ JOCELYNE LACASSE and LUIS H. MARTIN2 National Research Council of Canaab, Biotechnology Research Institute, Montred Quebec, Canaab. Received for publication January 17, 1992 and in rwised form May 4, 1992; accepted May 8, 1992 (2A2562).

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s)remains unknown, it has been proposed that CD1 may present antigens to specific subsets of p e r i p h d T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 “A. These cells are thought to be involved in the immunological regulation of

Introduction A comparative sequence analysis of cluster of differentiation 1(CDI) genes from three species (i.e., human, mouse, and rabbit) suggests the existence of two classes of CD1 genes with a higher intraclass homology across species than interclass homology within species (1). One class comprises the human CDlA, CDIB, CDlC genes and the rabbit CDlB gene; the other class consists of the human, rabbit, and mouse CDlD genes; the human CDlE gene has somewhat intermediate position between the two families. This peculiar pattern of sequence conservation could well imply distinct biological functions reflected by different tissue distributions of the two classes of CD1 antigens. In humans, cortical thymocytes co-express CDla, CDlb, and CDlc antigens (2), whereas epidermal Langerhans cells predominantly express the CDla antigen with low levels of CDlc antigen (3-8). The CDlc antigen is also detected on dermal dendritic cells (5,8), some of which co-express CDla (5,8), on a subset of circulating B-cells ($),lo), and in the mantle zone of lymphoid follicles in lymph nodes and spleen ( 5 ) . The two human CDlD and CDlE genes are transcribed (11) and at least a proportion of the transcripts are properly spliced (1,12). Recently, a putative CDld protein has been identified and preliminary evidence suggests that it may differ in distribution from other human CDI molecules (13,14). Also

Supported by an NSERC post-doctoral fellowship UL). Correspondence and present address: Dr. L. H. Manin, Biomira, Inc., BiotechnologyResearch Institute, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2.

intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed. (JHisrochem Cyrochem 40:15271534, 1992) CDI; Paneth cells; In situ hybridization; mRNA expression; Non-isotopic probes; Mouse.


in contrast to human CDla, b, and c, a much wider tissue distribution of CD1 gene transcripts and CD1 antigens is observed in the mouse, whose genome contains only two highly homologous (95%) CD1 genes, CDlD.1and CDlD.2 (15). The expression of these genes has been detected by S1 nuclease protection experiments(15), Western blot analysis (16), or immunohistochemistry(17) in adult and fetal thymus, spleen, liver, lung, and intestine, as well as in fetal brain, kidney, and heart. Even though the CD1 genes are located outside the major histocompatibility complex (MHC) in humans (18,19) and mice (15,20), the CD1 family shares the following featureswith the MHC Class Ib molecules (TLa, Qa in mice): a similar structure (15,21); a low degree or absence of polymorphism (22); and a non-covalent association with the p2 microglobulin (23-25). However, there is as yet scant direct evidence as to CD1 function. Potential roles as modulators of T-cell function (26-29) or Langerhans-cellfunction (6,7) and as non-classical antigen-presenting molecules (18) have been suggested. The latter proposal is supported by the observed inhibitory effect of anti-CD1 monoclonal antibodies (MAb) on the cytolytic activity of two CD4- CDS-, alp or y/& human T-lymphocyte lines derived from peripheral blood (30). More recently, the in vivo oligoclonal expansion of human a /p intestinal intraepithelial lymphocytes (IEL) was demonstrated and an IEL T-cell line was isolated, which exhibited cytolytic activity against CD1 transfectants (31). The identification of the specific cell types expressing the murine CD1 antigens in various tissues should contribute to the elucidation of their functions for eventual comparative studies involving human CD1 antigens as well. We have now used in situ hybridization to investigatethe expression of CD1 genes in the mouse intes1527

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tine, where a preferential localization of y16 T-cells showing a limited T-cell receptor (TCR) diversity is known to occur (32).

Materials and Methods Tissue Processing. Four-week-old BALBlc mice (Charles River; StConstant, Quebec, Canada) were perfused under ketamine (50 mglkg body weight) and xylasine (10 mglkg body weight) anesthesia through the left ventricle and nascent aorta, first with 20 ml of 0.9% (wlv) NaCl and then with 4% (wlv) paraformaldehyde (Fisher Scientific; Nepean, Ontario, Canada) in sodium phosphate buffer 0.01 M, pH 7.4, for 10 min. Thymus lobes, fragments of small intestine including duodenum, jejunum, and ileum, and fragments of transverse colon were collected and kept for 24 hr at 4'C in the same fixative. The tissue fragments were then washed with sodium phosphate buffer and embedded in paraffin after graded ethanol dehydration. Tissue sections (5-6 pm thick) were mounted on glass slides previously treated with sulfochromic acid (Fisher; Fair Lawn, NJ) and coated with a gelatin mixture consisting of 0.2% (wlv) gelatin 275 Bloom (Fisher), 0.33% (wlv)chromium potassium sulfate (Anachemia; Montreal, Quebec, Canada) and 0.02% (wlv) sodium azide (Sigma; St Louis, MO) and with 0.01% (wlv) poly-L-lysine (Sigma). Subdoing of CD1 Probe. A 350 BP genomic DNA fragment derived from the murine CDlD.2 gene and cloned in pUC18 vector was generously provided by A. Bradbury (Laboratory of Molecular Biology, Cambridge, UK). This fragment, which covers the entire a3 coding domain of gene CDlD.2 and part of the adjacent introns, was subcloned into the BamHI and Hind111 restriction sites of the vector Bluescript KSII' (Stratagene;La Jolla, CA). The orientation of the insert, which should hybridize to transcripts from both CDlD.1 and CDlD.2 genes (15), was verified by sequencing with the dideoxy-termination method (33). The sequence was determined to be identicalto that previously reported (ref. 15, residues 1619-1969). The properly linearized Bluescript construction was used as template for the production of anti-sense and sense radiolabeled or biotinylated probes.

Preparation of CD1 RNA Probes. Radiolabeled RNA probes were prepared with a RNA transcription kit (Stratagene),according to the manufacturer's recommendations.The transcription mix consisted of 1 pg of linearized DNA template, 0.4 mM rAV, 0.4 mM rGTP, 0.4 mM rUTP, 0.4 mM rCTP, 62.5 pCi [a-35Sr]-CTP(800 Cilmmol), 0.03 M dithiothreitol, 10 U of human placental RNAse inhibitor (Gibco BRL, Gibco Canada; Burling ton. Ontario, Canada) and 50 U of either T3 (anti-senseprobe) or T7 (sense probe) RNA polymerase in transcription buffer (40 mM Tris-HC1. pH 8, 1 mM MgC12, 2 mM spermidine, 50 mM NaCI). in a total volume of 25 pl. Biotinylated RNA probes were prepared similarly except that the transcription mix contained 1 mM biotin-11-rUTP(Enzo Biochem; New York, NY)instead of 0.4 mM rUTP and only 1 pCi [ u - ~ ~ S ~ J - C toTmonitor P the incorporation of nucleotides. Transcription was performed for 2 hr at 37'C; subsequently,the DNA template was digested for 15 min at 37'C with added RNAse-free DNAse (10 Ulpg DNA template) (Pharmacia; Uppsala, Sweden).The RNA transcriptswere ethanol precipitated and re-suspended in 2 5 pl of TE (100 mM Tris-HC1, pH 7.5, 50 mM E m )containing 10 U of human placental RNAse inhibitor. The integrity of the full length probes was verified on autoradiographed 6% polyacrylamide minigels. The incorporation of the biotinlabeled UTP was monitored on dot-blots revealed with avidin-biotinylated peroxidase complex (ABC peroxidax) (Vector Laboratories; Missisauga, Ontario, Canada), using 0.02% (vlv) hydrogen peroxide (Fisher) and 0.1% (w/v)

diaminobenzidine tetrahydrochloride (DAB) (Sigma) in Tris-HC1 0.1 M, pH 7.2. In Situ Hybridization. Before hybridization the sectionswere deparaffinized in xylene (three times for 10 min). rinsed in absolute ethanol (four times for 10 min), dried, and permeabilized for 20 min, at 37'C with predigested proteinase K (1 pglml) (Stratagene) in 100 mM Tris-HCI, pH 8, SO mM EDTA.Negatively charged tissue proteins were blocked for 10 min with rapid stirring in fresh 0.1 M triethanolamine (Anachemia; Montreal, Quebec, Canada), pH 8, containing 0.004% (vlv) acetic anhydride. The sections were then washed in 2 x SSC (1 x SSC is: 0.15 M NaCI, 0.015 M trisodium citrate) (BDH; Toronto, Ontario, Canada) and dehydrated through graded ethanol concentrations. The sections were hybridized under sealed coverslips for 16-20 hr at S O T in a moist chamber. The hybridization buffer (HB) consisted of 50% (v/v) deionized formamide (BDH), 10% (wlv) dextran sulfate (Pharmacia), 2 x SSC, 1 x Denhart's solution [0.02% (wlv) Ficoll (MW 400,000) (Sigma), 0.02% (wlv) polyvinylpyrolidone( h w 360,000) (Sigma), 0.02% (wlv) bovine serum albumin (fraction V) (ICN Biochemicals; Cleveland, OH), 10 mM dithiothreitol (Sigma) and 0.1 mglml denatured salmon sperm DNA (Sigma)]. The probes were diluted in HB so that 1.7 x lo6 cpm of radiolabeled probe or 3 pl of biotinylated probe, in a total volume of 25 pl, were applied to each tissue section. After hybridization, the sections were washed for 5 min at 22'C. Unhybridized RNA probes were removed by treatment with DNAse-freeRNAse (10 Ulml) (Boehringer-Manheim Canada; L a d , Quebec, Canada) in 0.5 M NaCI, 10 mM Tris-HCI, pH 8, for 30 min at 37%. The sections were then washed at 50°C in 2 x SSC and in 1 x SSC for 10 min and at room temperature in 0.5 x SSC for another 10 min.

Visualization. The sections hybridized with radiolabeled probes were dehydrated through graded ethanol concentrations,dipped in water-diluted (1:l) NTB2 photographic emulsion (Kodak; Rochester, NY) and exposed for 17-20 days before development in Kodak D19 solution. The sectionshybridized with biotinylated probes were transferred from 0.5 x SSC to PBS [0.01M sodium phosphate buffer containing 0.9% (wlv) NaCI] for 10 min before a 2-hr incubation at 22°C with ABC-peroxidase diluted as recommended by the manufacturer. After a 10-minwash in PBS the sections were equilibrated for 10 min in the buffer used for the enzymatic development, which was done with the glucose oxidase-DAB-nickel method (34).

Results As a positive control, murine CD1 mRNA was first localized in thymic tissue sections. As expected,most cortical thymocytes transcribed CDl genes (Figures 1A and 1D) and, in contrast to the human thymus where CD1 expression is restricted to the cortex, murine medullary thymocytes also transcribed the CD1 genes (Figures 1B and 1E). This latter observation confirms the results obtained by ribonuclease mapping of total RNA from thymocytes fractionated with the PNA agglutination method (35). The eosinophilic epithelioreticular cells were not labeled over background levels (Figures 1B and 1E). In the small intestine, from duodenum to ileum, the only epithelial cells expressing CD1 mRNA were the Paneth cells (Figures 2, 3A, 3B, 4A, and 4B), clearly recognizable by their location at the b

Figure 1. Expression of CD1 mRNA in the mouse thymus. (A-C) Autoradiographs of thymus sections hybridizedwith CD1 anti-sense probe (A, cortex; B, medulla) or (C) CD1 Sense probe. C, cortex; m, medula. Hematoxylin-eosin counterstaining. (D-F) Thymus sections hybridized with biotinylated CD1 anti-sense probe (0,cortex; E, medulla) or biotinylated(F) CD1 sense probe. C, cortex; m, medulla. Visualization with ABC-peroxidase and glucose oxidase-DBA-nickel method. No counterstaining.Arrows, unlabeled medullary epithelioreticular cells. Original magnifications: A, B, D, E x 480; C, F x 400. Bars = 20 pm.

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trols did not produce any labeling above background levels for either the autoradiographic (Figures IC, 3C, and 3D) or the enzymatic (Figures 1F and 4C) reaction.


Figure 2. Expression of CD1 mRNA in Paneth cells of (A) duodenum and (e) jejunum. Autoradiographs of tissue sections hybridized with CD1 anti-sense probe. Hematoxylin-eosin counterstaining. Bg. Brunneh glands. Original m a g nifications: A x 325; B x 375. Bars = 20 pm.

very bottom of the Lieberkuhn crypts and their conspicuous eosinophilic and refractile secretory granules. The Brunner’s glands in the duodenum (Figure 2A), the monocytic or lymphoid elements of the lamina propria (Figures 2, 3A. 3B, 4A, and 4B) and Peyer’s patches (Figure 5) were not labeled over background levels. Neither the fibroblasts nor the smooth muscle cells of the small intestine wall were significantly labeled. CD1 mRNA was not detected in colon tissue sections (results not shown). Identical results were obtained with radioactive and biotinylated antisense probes in the various tissues examined. There was no apparent difference in sensitivity between the autoradiographic and the enzymatic method. Endogenous mRNA could be localized with biotinylated probes, which until now have mainly been used for the detection of viral mRNAs expressed at high levels. However, a better resolution was observed with the enzymatic detection; this was most evident in thymocytes, where only a very narrow rim of cytoplasm is available for hybridization. The specificity of the in situ hybridization reactions was assessed with the radioactive or biotinylated sense probes prepared from the same template as the anti-sense probes. These negative con-

In this study we have confirmed by in situ hybridization analysis that murine CD1 mRNA is expressed in both cortical and medullary thymocytes. Examination of the intestine indicated that CD1 mRNA was also present, but in a restricted pattern of expression. Specifically, only the Paneth cells situated at the base of Lieberkuhn crypts contained CDl mRNA. Identical results were obtained using either autoradiographic or enzymatic detection. In a recent immunohistochemical study, CD1 antigen expression was observed in the intestinal epithelium of the mouse (17); the cells expressing CD1 antigens covered the extrusion zone of the intestinal villi but no immunoreactive cells were observed either lower along the villi or in the Lieberkuhn crypts. However, Western blot analysis performed in our laboratory failed to reveal CD1 antigen expression in the adult mouse intestine (16). Since all the antibodies exhibit some crossreactivities, we decided to further investigate this discrepancy by utilizing in situ hybridization on tissue sections. The intestinal CD1 gene expression that escaped detection by the less sensitive Western blot analysis of whole-tissue lysates was observed in single Paneth cells on tissue sections even though these cells represent a minor cell subpopulation in the intestine. However, there was no detectable CD1 mRNA in the extrusion zone of the intestinal villi. Although Paneth cells have been extensively studied since their discovery by Schwalbe in 1872, their role is still unknown. Morphologically they resemble zymogenic cells such as the acinar exocrine cells of the pancreas, and it has been suggested that Paneth cells participate in food digestion as a diffuse exocrine gland scattered along the intestine. This proposal was based on differential secretory granule counts on fasting and re-feeding (36-38), on the observed increase in the number of Paneth cells after pancreatic duct ligation, which causes degeneration of the exocrine pancreas (39), and on the immunohistochemical or histochemical localization of various enzymes including anionic and cationic trypsins (40), carboxylic ester hydrolase (41). phospholipase A2 (42), P-galactosidase (43), and P-glucuronidase (43). However, an autoradiographic study revealed a continuous secretory activity in Paneth cells, even in fasted mice, with no dietary influence on the number or size of secretory granules (44). Paneth cell involvement in heavy metal elimination (45-47), monoamine absorption (48). and regulation of crypt cell growth (49) has also been suggested. The immunohistochemical localization of epidermal growth factor (50) and its enhancing factor (51) in Paneth cells supports the latter view. Paneth cells are now mostly regarded as being involved in the regulation of the intestinal flora owing to their ability to phagocytose microorganisms (52.53) and to the lysozyme (muramidase content of their secretory granules (54-59). The bacterial environment of the gut lumen has a direct effect on Paneth cell secretory activity (59-61). The immunohistochemical localization of IgA or IgG in Paneth cells was interpreted as probably reflecting an opsonization phenomenon preceding internalization of infec-

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Figure 3. Expression of CD1 mRNA in Paneth cells of ileum detected with radiolabeled probes. Autoradiographs of ileum sections hybridized with (A,B) CD1 anti-sense probe or (C,D) CD1 sense probe. A and C are darkfield photomicrographsat low m a g nification; Band Dare higher magnification views of the right side of A and the left side of C. respectively. Hematoxylin-eosin counterstaining. Original magnifications: A. C x 160; E, D x 375. Bars = 20 pm.


tious antigens rather than local synthesis or adluminal transcellular transport from the lamina propria (62,63). The hypothesis of an involvement of Paneth cells in immunological defense is further strengthened by the detection of tumor necrosis factor (64) and cryptdin (a member of the corticostatin/defensin family) (65) gene transcripts on tissue sections. Autoradiographic studies have shown that the mature mouse Paneth cells are not proliferative (66-68)and that their turnover rate (23.3 days) exceeds that of absorptive and mucous intestinal epithelial cells (3 days) (68).It was suggested that they might derive from precursor cells located outside the intestinal epithelium (66)or might arise from stem cells occupying a higher position in the crypt and migrating downward during differentiation (68).The expression by Paneth cells of CD1 gene transcripts, together with cytokine gene transcripts (64,65),reopens the debate on their origin. Even though there is no evidence supporting a bone marrow origin of Paneth cells, such a possibility should not be excluded, as exemplified by another cell type expressing MHC Class I1 molecules as well as CD1 antigens in humans, the epidermal Langer-


hans cells: these cells, first thought to be nerve cells because of their dendritic shape and histochemical properties, were subsequently shown to originate from the bone marrow and to act as antigenpresenting cells in delayed-type hypersensitivity reactions (69).However, unlike the epidermal Langerhans cells, the murine Paneth cells do not express the following leucocyte markers: the leucocyte common antigen (LCA, CD45); the Type I1 receptor for the constant region of immunoglobulins (FcRII, CDw32); the Type 3 receptor for complement (C3bi receptor, CD1lb); and the monocytelmacrophage antigen F4/80(64). The expression of CD1 antigens, TNF,and cryptdin by Paneth cells could also be related to the pathogenesis of gastric and colon tumors. Tumors of the small intestine are very rare, and many Paneth cells are frequently encountered in intestinal metaplasia areas adjacent to gastric and colon tumors (70,71).Their involvement in tumor growth control was long suspected, and it was suggested that they might secrete a protective factor against tumor growth (72). An increased number of Paneth cells is commonly observed in intestinal metaplasia associated with gastric and colon inflammatory

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Figure 5. Lack of CD1 mRNA expression in lymphoid cells of Peyer's patches. Autoradiographof a Peyer'spatch at the junction between duodenum and jejunum. Tissue section hybridized with CD1 anti-sense probe. B is a higher magnification view of the left side of the darkfield photomicrograph in A. Hematoxylin-eosin counterstaining. Arrows, labeled Paneth cells in jejunum; arrowhead, labeled Paneth cells in duodenum, not included in B; Pp. Peyer's patches. Original magnifications: A x 160; B x 300. Bars = 20 wm.



Figure4. Expression of CD1 mRNA in Paneth cells of ileum detected with biotinylated probes. Ileum sections hybridized with (A.B) CD1 anti-sense probe or (C) CDl sense probe. Visualization with ABC-peroxidase and glucose oxidase-DAB-nickel method. No counterstaining. Original magnification: x 375. Bars = 20pm.

diseases such as chronicatrophic gastritis,ulcerative colitis, and diverticulitis (49,73). It is also worth noting that the Vsl subset of human y16 T-cells was recently reported to increase in the intestine of patients with celiac disease (32,74). a pathological condition associated with Paneth cell deficiency in a-antitrypsin during the active state of the disease (75).

In the adult mouse, some y16 T-cell subpopulations show a predilection for epithelia (32). These subpopulations are characterized by a restricted usage of V gene segments, which is distinct from one epithelium to the other. This limited diversity of the TCRs expressed in epithelia is compatible with the recognition of nonpolymorphic molecules such as CD1 antigens and MHC Class Ib molecules, and has led to the suggestion that this mechanism may maintain the epithelial integrity at the interface with the external milieu (76.77). Interestingly, a human T-cell line that recognizes CDla, b, c. and d antigens has been recently derived from intestinal oligoclonal IEL (31). Therefore, the detection of CD1 gene transcripts in Paneth cells of the mouse intestine is not only relevant to the understanding of the function of CD1 antigens and the role

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of Paneth cells but might well be of significance to the elucidation of antigen-presentation mechanisms to IEL.

Acknowledgments We grateful4 acknowledge the cn2idreading andcomments on this munuscrigt by Drs S. Anderson and D.D.Morsel:

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Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Althou...
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