(Received for publication, August 4, 1995; and in revised form, October 13, 1995)
From the
Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5`-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19-24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.
During human fetal organogenesis the intestine undergoes a dramatic transformation, characterized by morphological changes of mucosal epithelial cells and the establishment of a crypt/villus axis (1) . Once the epithelium of the small intestine is mature, there is continuous cellular renewal, with evidence of all epithelial cell types arising from common progenitor stem cells(2) . This dynamic epithelium has many physiological functions, including a role in host defense. Of the epithelial cell types, Paneth cells, intensely eosinophilic cells located at the bases of intestinal crypts, have ultrastructural hallmarks of secretory cells and are most abundant in the ileum(3, 4, 5, 6) . Several lines of evidence suggest that an important physiological role of Paneth cells is the synthesis of host defense effector molecules such as lysozyme(7, 8, 9) , phospholipase A2(10, 11, 12) , and antimicrobial peptides(13, 14, 15, 16, 17, 18, 19) .
Antimicrobial peptides are a prevalent mechanism of host defense utilized by phylogenetically diverse animal species, from insects to humans (for reviews see (20, 21, 22) ). Defensins are a large family of antimicrobial peptides, identified originally in leukocytes of rabbits and humans (for reviews see (23) ). These cationic peptides are 30-35 amino acids in length and are distinguished by a conserved cysteine motif(23) . Defensins are membrane active and have microbicidal activity toward a wide range of microorganisms in vitro(23) . In leukocytes, these peptides are stored in cytoplasmic granules and are released into phagolysosomes where they contribute to the killing of engulfed microorganisms(24) .
More recently, molecular studies have identified a distinct group of enteric defensin genes expressed in Paneth cells of the mouse(13, 25) and humans(18, 19) . In the mouse, there is evidence for the expression of 16 or more defensin genes in the small intestine(26) , whereas only two human homologues have been identified at this site(18, 19) . Mature enteric defensin peptides have been isolated from the murine small bowel, but the homologous human peptides have not yet been isolated (15, 16, 17, 26) . The murine peptides were found to have antibiotic activity comparable with the previously isolated myeloid counterparts from other species. Activity of two mouse enteric defensins against the intestinal parasite Giardia lamblia was also reported (27) .
It has been suggested that immaturity of local intestinal defenses may contribute to the increased susceptibility of neonates to infections from luminal flora and to necrotizing enterocolitis(28, 29) . Therefore, we sought to more clearly characterize defensin expression in Paneth cells of the human small intestine, with a focus on fetal expression. We observed that defensin expression coincides temporally with Paneth cell detectability and may be a useful marker of these cells. The developmental profile observed suggests that low level defensin expression in fetal development may be characteristic of immature enteric mucosal defense. This work also addresses the number of defensin isoforms expressed in human intestine and characterizes the genomic structure of the enteric defensin gene HD-6.
Reagents and general methodology for cloning, sequencing, probe labeling, and PCR amplification were described(18) . Human fetal intestinal tissue from second trimester abortuses was obtained and used in accordance with guidelines established by the Institutional Review Board at The Children's Hospital of Philadelphia and with permission from the Central Oxford Research Ethics Committee. Sequence data were analyzed using MacVector software (IBI, New Haven, CT).
Figure 3:
3` RACE analysis of defensin cDNA in human
small bowel. A, nucleotide sequences of the PCR primers DEF15s
and DEF15sI aligned with defensin sequences from human, guinea pig,
mouse, rat, and rabbit. The vertical lines indicate identity. B, anchored RT-PCR products from human intestinal RNA using
either DEF15s (15s) or DEF15sI (15sI) as an upstream PCR primer were
analyzed by agarose gel electrophoresis and ethidium bromide detection.
The molecular size standard (M) phiX174 digested with HaeIII. C, dot blot of 78 samples of plasmid DNA from
recombinant clones containing RACE-PCR products hybridized with a P-labeled probe from the common region of defensin mRNA,
SIG68a (COMMON PROBE), with a defensin 5 specific
oligonucleotide probe, HSIA-309a (HD-5), and with a defensin
6-specific oligonucleotide probe, HSIB-309a (HD-6). Clones 57
and 61 show hybridization to both HD-5 and HD-6 probes. Direct sequence analysis of the plasmid inserts reveal a
tandem ligation of both HD-5 and HD-6 cDNA in each of
these clones.
The following gene-specific primers were
used: 1) for HD-5, DEF5a (CCCAGCCATGAGGACCATCG) and DEF5b
(TCTATCTAGGAAGCTCAGCG), generating a 304-bp product; 2) for HD-6, primers HD-6/102s (CCACTCCAAGCTGAGGATGATC) and HD-6/405a
(TGATGGCAATGTATGGGACACACAC), generating a 326-bp product; and 3) for
-glucocerebrosidase, GD67A (CAGATACTTTTGTGAAGTTCC) and GDMID9B
(GACTGTCGACAAAGTTACGC), generating a 572-bp product. The PCR products
were resolved by electrophoresis in 1.5% agarose gels. The specific DNA
fragments generated by each PCR reaction were verified by direct
sequence analysis. RT-PCR primer pairs will amplify a product from
genomic DNA, but the product will be substantially larger than from
cDNA, because the forward and reverse primers lie in different exons.
Controls with no reverse transcriptase in each set of reactions were
found not to amplify a product from genomic DNA that might have
contaminated the RNA preparations.
Northern analysis was performed
as described(18, 19) . For HD-6 mRNA
detection, HSIB-309a was employed, and for HD-5, HSIA-309a was
used. In parallel, nylon membranes spotted with plasmid DNA containing
inserts encoding HD-5 and HD-6 were hybridized and
washed simultaneously with the Northern blot filters to control for
stringency. The control glyceraldehyde-3-phosphate dehydrogenase probe
was hybridized using an identical hybridization solution at 42 °C,
and the conditions of the final wash were 0.1 SSC/0.1% SDS at
50 °C for 30 min. The washed filters were exposed to film using an
intensifying screen at -70 °C for 2 weeks. The blots were
then stripped of probe by washing in 0.1
SSC/0.1% SDS at 65
°C for 1 h. Efficient stripping of the probe was documented by
autoradiographic exposure for 2 weeks.
Figure 1: Nucleotide sequence of HD-6 gene and flanking regions. A, a partial restriction map of HD-6 and flanking sequences. Pst, PstI; Bam, BamHI; Hin, HindIII. The thickened lines show the positions of both exons (exon 1, 211 bases; exon 2, 229 bases) that flank a 914-base intron. B, the nucleotide sequence of HD-6 and flanking regions. Numbering begins arbitrarily at the most 5` nucleotide of the sequence. Exon sequences are shown in uppercase letters, and the deduced amino acid sequence of the coding region is shown in three-letter code. The TATA box and CAAT box are underlined. The consensus splice junction residues are shown in bold. The polyadenylation signal is boxed. The Genbank accession number for the HD-6 genomic sequence is U33317.
Comparison of the genomic (Fig. 1) and the HD-6 cDNA sequences (19) indicated that the gene consists of two exons, separated by a 914-bp intron. The nucleotide sequence of the putative exons in the genomic clone are in complete agreement with those in the cDNA sequence. There are consensus sequences for splice junctions (Fig. 1, bold) and polyadenylation (Fig. 1, boxed). There is a TATA box at nucleotides 1341-1347, beginning 28 nucleotides upstream from the 5` terminus of the two most extended cDNAs identified by RACE-PCR (see below, Fig. 1, underlined). A CAAT box is seen at position 1278-1283 (Fig. 1, underlined).
In order to define the 5` transcription start site of the HD-6 gene, small intestinal cDNA (18) was amplified using the 5` RACE-PCR technique(32) . The downstream primer (HSIB-309a) was chosen from the second exon, and the upstream primer was complementary to the anchor sequence. Amplified products were subcloned and sequenced. The amplified cDNA sequence was identical to the corresponding region of the genomic sequence (data not shown). Four products were found to extend 5` to the putative initiating methionine codon. Three of these terminated 41 nucleotides upstream of the methionine codon. This site of transcription initiation is designated +1. The fourth RACE product terminated at +3 and may represent a minor site of transcription initiation or a premature termination of the reverse transcriptase. The identical termination points of products 1-3 indicate that this is the major transcription start site.
A dot
matrix analysis of nucleotide similarity of HD-5 and HD-6 is shown in Fig. 2A. Various degrees of sequence
identity were seen along a diagonal throughout the entire gene
sequences. The most striking identity was observed in the proximal 5`
region encompassing the first half of exon 1 and nucleotides of the
proximal 5`-flanking region (Fig. 2B). Several
consensus sequences corresponding to transcription factor binding sites
were identified in the flanking region, including two AP2 sites (38) (-784 and -1344) and six nuclear factor
interleukin-6 sites (39) (-244, -305, -650,
-788, -863, and -1292). Several of these sites are
found in the same location within the HD-5 flanking region,
such as AP2(-781) and nuclear factor interleukin-6 (-651
and -1284), suggesting these sites may prove to be functionally
significant. ()
Figure 2: Gene sequence analysis. A, a Pustell analysis of human enteric defensin genes HD-5 and HD-6. Sequence similarity was analyzed using a window of 14 nucleotides and was scored positive for a match of 10 of 14 (hash value = 1). A nearly identical pattern of nucleotide similarity was obtained when the analysis was scored positive for a match of 9 of 14, although the background was higher (data not shown). The axes indicate the approximate position of the exons (solid boxes) and show the major sites of transcription initiation (arrows). The approximate positions of transcription initiation and the putative translation start codons are shown on the diagonal by arrows. Regions 1, 2, and 3 highlighted by arrows are highly conserved between HD-5 and HD-6, as shown in B. B, nucleotide similarity of 5`-flanking regions of HD-5 (top sequence) and HD-6 (bottom sequence). The sequences were aligned for maximal sequence identity. Regions of particularly striking identity are underlined. The major site of transcription initiation defined by 5` RACE analysis for HD-6 and HD-5(18) is indicated with an arrow. The TATA and CAT box sequences are boxed.
Figure 4:
RT-PCR analysis of HD-5 and HD-6 expression in fetal intestine. RT-PCR products from human
intestinal RNA samples were analyzed by agarose gel electrophoresis and
ethidium bromide detection. A, HD-5 expression
detected as a 304-bp cDNA fragment after RT-PCR using amplification of
a 572-bp cDNA fragment from -glucocerebrosidase as a control. B, HD-6 expression detected as a 325-bp cDNA fragment
after RT-PCR with the same
-glucocerebrosidase control. Each lane in both gels contains 20% of a RT-PCR product using 1
µg of total RNA as template from: 13.5-week gestational age small
intestine (lane 1), 13.5-week colon (lane 2),
14.5-week distal small intestine (lane 3), 15-week colon (lane 4), 16-week distal small intestine (lane 5),
16-week colon (lane 6), 17-week distal small intestine (lane 7), 17-week colon (lane 8), 6-month neonatal
jejunum (top, lane 9), no RNA (control, top, lane 10), no reverse transcriptase (control, top, lane 11), no RNA (control, bottom, lane 9),
no reverse transcriptase (control, bottom, lane 10),
Life Technologies, Inc./BRL 1-kilobase ladder size standards (top, lane 12, and bottom, lane
11).
Total RNA isolated from fetal specimens of distal small intestine ranging in gestational age from 19 to 24 weeks and from adult small intestine was analyzed by Northern blot hybridization (Fig. 5). The blot was probed sequentially with antisense oligonucleotide probes specific for HD-5 (HSIA-309a) and HD-6 (HSIB-309a). To control for possible cross-hybridization of the defensin probes under the experimental conditions, a slot blot containing HD-5 and HD-6 clones was hybridized and washed under the same conditions as the Northern blot. Specific hybridization was observed with both of the defensin probes. PhosphorImager analysis of the Northern blot indicated that approximately 40 times more HD-5 mRNA is detectable in the adult than in the 24-week fetal sample shown in Fig. 5. 3-6-fold less mRNA is detectable in the other specimens on this blot. RNA from a second specimen at 24 weeks of gestation showed lower levels than those in the first sample, comparable with the 21-week sample (data not shown). The relative ratio of HD-5 to HD-6 was estimated to be approximately 3:1, similar to that found in the 3` RACE analysis with DEF15sI. Defensin mRNA from specimens at earlier gestational ages was not detectable by Northern blot analysis under these conditions (data not shown).
Figure 5:
Northern blot analysis of small intestinal
RNA from fetal and adult specimens. Total RNA (20 µg) from the
distal half of fetal small intestine obtained at indicated gestational
ages and RNA (0.8, 4.0, and 20 µg) from adult small intestine were
fractionated by formaldehyde-agarose gel electrophoresis, blotted to a
nylon filter, and hybridized sequentially to indicated probes. Ethidium
bromide staining indicated essentially equivalent RNA in each of the
fetal samples (not shown). A, hybridization with the HD-6 probe HSIB309a. A slot blot containing plasmid DNA with HD-5 and HD-6 cDNA inserts was simultaneously hybridized and
washed to control for conditions of stringency (see ``Materials
and Methods''). Autoradiographic exposure at -70 °C with
an intensifying screen was 2 weeks for the Northern blots and 2 days
for the slot blot controls. The Northern blot was then stripped of
probe under high stringency and re-exposed prior to use in subsequent
experiments. B, hybridization of the same filter with the HD-5 probe HSIA309a under the same conditions as in A. C, hybridization of the same filter with a
glyceraldehyde-3-phosphate dehydrogenase cDNA probe under similar
conditions as above except that the hybridization temperature was 42
°C and the final wash was at in 0.1 SSC/0.1% SDS at 50
°C.
Figure 6:
Detection of HD-5 and HD-6 mRNA in the Paneth cells of the small intestine by in situ hybridization. Paraffin-embedded sections of fetal (24-weeks
gestational age; A, D, and G), newborn (B, E, and H), and adult (C, F, and I) ileum were hybridized with HD-5 and HD-6 riboprobes labeled with
[S]UTP, washed under high stringency, coated
with photographic emulsion, exposed, developed, and then stained with
hematoxylin and eosin as described
previously(18, 37) . A-C, low power
view of sections of ileum hybridized with a HD-5 antisense
riboprobe. D-F, low power view of parallel sections
hybridized with a HD-6 antisense riboprobe. G and H, low power view of parallel sections treated with 20
µg/ml of RNase A for 15 min at room temperature prior to
hybridization as in A and E, respectively. I, low power view of a parallel section hybridized with an HD-5 sense riboprobe. The dense silver grains at the base of
the intestinal crypts represent positive signal (A-F).
All sections are shown at the same magnification. The bar equals 100 microns. The arrows indicate the locations of
representative Paneth cells in each
section.
Comparison of the
nucleotide sequence of HD-5 and HD-6 shows an unusual
and very striking pattern of similarity, with two testable hypotheses
emerging from this observation. First, the high similarity across 850
nucleotides of flanking region suggests that cis-elements important in
tissue specific and developmentally regulated expression of these genes
might be found in this region. Transgenic studies using 6.5 kilobases
of 5`-flanking DNA from a mouse enteric defensin gene ligated to a
reporter gene showed expression largely restricted to Paneth cells in
mature intestinal crypts(47) . It is possible that the
information necessary for tissue-specific expression is located in the
proximal region where we observe high nucleotide identity. Second, the
presence of several nuclear factor interleukin-6 recognition sequences (39) throughout the 5`-flanking region offers a rationale to
test if constitutive levels of defensin gene expression in the bowel
are up-regulated in response to inflammation. Certain members of
another group of mammalian antibiotic peptides, the -defensins,
are highly inducible with their expression in differentiated epithelial
cells responsive to inflammatory stimuli (48, 49, 50) .
Northern analysis detected enteric defensin mRNA in the second trimester of gestation at levels approximately 40-250-fold less than those observed in the adult (Fig. 5). Because of the difficulty in establishing gestational ages with precision at this stage of development, we interpret the variability in our Northern blot analysis with some caution. We conclude conservatively that readily quantifiable enteric defensin mRNA accumulates in the latter part of the mid-trimester but at levels much lower than found in adults. In situ hybridization analysis localized the expression of enteric defensin mRNA to Paneth cells (Fig. 6) and suggests that fewer numbers of Paneth cells in the fetal crypts, as compared with the newborn and adult, account for part of the lower level of enteric mRNA observed (Fig. 6, A versus B and C and D versus E and F). Our data are consistent with anatomic data showing lower numbers of Paneth cells in crypts of the mouse at early gestational ages(47, 53) .
In the mouse, there is evidence for expression of 16
defensin-encoding mRNAs(26) . The six characterized murine
enteric defensin genes (25) all have very high overall
nucleotide similarity (85%), suggesting gene duplication events that
occurred relatively recently in evolution. The data reported here (Fig. 3), consistent with previous data from screening of a
phage cDNA library(18) , suggest that only two defensin genes, HD-5 and HD-6, are expressed in human small
intestine. These two genes are not as closely related (Fig. 2A) as those in the mouse(25) ,
consistent with duplication and subsequent divergence much earlier in
evolution. The striking species difference in enteric
defensin gene numbers remains an enigma and may reflect selective
pressures resulting from complex interactions between host and
microbial environment.
An immaturity of specific and nonspecific effectors of the immune response is thought to predispose premature infants to infection. For example, necrotizing enterocolitis is an illness that causes substantial morbidity and mortality among premature infants yet is uncommon in term newborns(28, 41, 43) . Histologically, necrotizing enterocolitis is characterized by inflammation and necrosis at affected sites. The etiology appears to be multifactorial, and a likely central feature is clinical infection caused by microbes colonizing the intestinal tract. It has been proposed that characteristics of an immature gastrointestinal tract lead to the development of necrotizing enterocolitis(28, 43) . Our results demonstrate very low level expression of defensin by the fetal intestine through 24 weeks of gestation, the lower limit of extrauterine viability. Limited expression of intestinal defensins by the fetus might, therefore, place a preterm infant at risk for bacterial invasion of the intestine and possibly the development of necrotizing enterocolitis. Future studies will be needed to define the biological activities of the human enteric defensins, to determine if levels of defensin expression are altered following premature birth, and to define the possible role of enteric defensins in the pathophysiology of necrotizing enterocolitis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33317[GenBank].