hypothesis that antimicrobial peptides contribute to host de- fense of the respiratory tract. Antimicrobial peptides comprise a prevalent mechanism of.
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4596-4600, May 1993 Biochemistry
Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene (respiratory tract/host defense/defensin-like)
GILL DIAMOND, DOUGLAS E. JONES, AND CHARLES L. BEVINS* Division of Human Genetics and Molecular Biology, The Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Communicated by John J. Burns, February 25, 1993
tively labeled as described (5). PCR primers for amplifying the TAP gene from bovine genomic DNA were as follows: TAPG9s, 5 '-ATAAAGCGGCAATCGCAGTG-3'; TAPG1Oa, 5'-ACAACCTCAGTGACCAGTGG-3'. Oligonucleotide probes for hybridizations were as follows: TAP48A, 5' -CCAAGCAGACAGGACCAGGAAGAGGAGCGCGAGGAGCAGGTGATGGAGCCTCAT-3'; TAP54S, 5'-
We previously reported the isolation and ABSTRACT characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we cafled tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratifled epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5' exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.
ATGAGGCTCCATCACCTGCTCCTCGCGCTCCTCTTCCTGGTCCTGTCTGCTTGG-3'; TAP160A, 5'-CCTGA-
TCGGCACACAGATGCCTTTATTCCAAACACAGCTTACAGGATT-3'; TAP113S, 5'-AATCCTGTAAGCTGTGTTAGGAATAAAGGCATCTGTGTGCCGATCAGG-3'; TAP118S, 5'-CACCTGTGTTGGGCGGGCAGTAAA-3'. Genomic DNAs were obtained commercially from Clonetech or were isolated from bovine blood by standard methods (6). The bovine genomic library in EMBL3 was obtained from Clontech. Northern Blot Analysis. Radioactively labeled DNA probes were hybridized to the immobilized RNA in 20% (vol/vol) formamide/5 x standard saline citrate (SSC)/5 x Denhardt's solution/0.1% (wt/vol) SDS at 42°C, and washed in 2x SSC/0.1% SDS at 650C (3, 7). In Situ Hybridization. Tissue was hybridized with the indicated oligonucleotide probes as described (5, 8). Slides were analyzed by dark-field photomicroscopy and then stained with hematoxylin and eosin for bright-field analysis. Primer Extension. Total mRNA from bovine trachea was annealed with TAP48A (5'-end-labeled with [32P]ATP and T4 polynucleotide kinase) and extended with Moloney murine leukemia virus reverse transcriptase as described (7). Extension products were analyzed by electrophoresis on 7% polyacrylamide gels in the presence of 7 M urea using a DNA dideoxynucleotide chain-termination DNA sequence ladder as a size standard.
Antimicrobial peptides comprise a prevalent mechanism of host defense found throughout the animal kingdom (1, 2). Significant effort is currently focused on characterizing this defense system at the molecular level. In a series of experiments designed to determine whether peptide-based host defenses are intrinsic to mammalian epithelial tissue, we recently isolated an antimicrobial peptide from bovine tracheal mucosa called tracheal antimicrobial peptide (TAP) (3). The sequence of the mature TAP molecule is NPVSCVRNKGICVPIRCPGSMKQIGTCVGRAVKCCRKK. The molecule is basic in charge and contains 6 cysteines, all of which participate in intramolecular disulfide bonds. When assayed in vitro, TAP is bactericidal against both Grampositive and Gram-negative bacteria and also exhibits potent antimicrobial activity against Candida albicans. Characterization of TAP cDNA indicated that it encodes a 63-amino acid putative propeptide with the mature peptide at the C terminus (3). While TAP and the mRNA that encodes it were found to be abundant in extracts from the tracheal mucosa, the initial studies did not further localize the site of TAP expression. We now show that the TAP gene is expressed primarily, if not exclusively, in the bovine airway mucosa, specifically in the columnar epithelial cells lining the airway. We extend our analysis of TAP to the level of genomic organization and present evidence that TAP is a member of a large family of antimicrobial peptides. The findings support the hypothesis that antimicrobial peptides are important components of host defense of the mammalian respiratory epithelium.t
RESULTS Tissue-Specific Expression of TAP. While TAP was originally isolated from the tracheal mucosa (3), we were interested to determine whether it was also expressed in other epithelial tissues. RNA isolated from several tissues was studied by Northern blbt analysis. The results in Fig. 1A show that TAP expression is restricted to airway tissue, with the majority of expression in the trachea. No signal was seen in RNA from bovine fallopian tube, which like the airway is ciliated, nor was there any signal in small intestine. Furthermore, we did not observe TAP expression prenatally, as there was no signal in tracheal RNA from fetal cows of two different ages. Histological examination of sections from
MATERIALS AND METHODS General Methodology. General methods were as described (3) unless otherwise noted. Bovine tissues were obtained from local meat-processing facilities or from Pel-Freez Biologicals. Estimates of bovine fetal gestational age were based on crown-rump length (4). Oligonucleotides were radioac-
*To whom reprint requests should be addressed at: Division of Genetics, Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L13373).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4596
Biochemistry: Diamond et al. these fetal trachea showed an immature pseudostratified ciliated epithelium but similar to that seen in the adult.J As a control, the blot was also hybridized with a bovine a-tubulin probe, which indicated that relatively similar amounts of RNA were in each sample (Fig. 1A). An RNase protection assay was also performed on the RNAs described above. The results were similar to those seen by Northern blot analysis, with no signal seen in fallopian tube or fetal trachea (data not shown). Northern blot analysis of other tissues, including testes, salivary glands, kidney, and brain, also failed to elicit any signal with the TAP probe (data not shown). To further analyze the expression of TAP throughout the bovine conducting airway, and to test for the possible variation in expression between individual cows, we probed a separate Northern blot containing RNA obtained from the proximal tracheal mucosa, distal tracheal mucosa (near the first division), and bronchiolar tissue (at the third division) for each of three different cows (from the same cull). The experiment showed that there was some difference (2- to 4-fold) between the three animals examined (Fig. 1B). Some variation of TAP expression was also seen along the mucosal portion of the airway (Fig. 1B). However, this latter variation may reflect the relative proportion of ciliated epithelium obtained in the dissection. In Situ Hybridization of TAP. To further characterize the expression of TAP mRNA in the bovine airway, we performed in situ hybridization. When hybridized with the antisense TAP oligonucleotide probe, a strong signal was observed in the epithelial cells lining the airway (Fig. 2 A and E). Essentially similar results were obtained in cross-sections of proximal and distal tracheal sections (data not shown). No signal was seen in the basal layer of the epithelium (Fig. 2 A and E), in alveoli (Fig. 2 D and H), in connective tissue (Fig. 2 A and E), or in glandular cells (data not shown). The complementary sense probe did not result in any specific signal (Fig. 2 B and F), consistent with hybridization specific for the TAP message. Also, no signal was observed when the tissue was pretreated with RNase A prior to hybridization with the antisense probe (Fig. 2 C and G). Cloning of the TAP Gene. A bovine genomic library was screened with a TAP oligonucleotide probe (TAP48A) and numerous hybridizing plaques were identified. The plaques with the strongest signals were purified and then screened with a second TAP oligonucleotide probe (TAP160A). One phage (TAPG-3) strongly hybridized with both probes. Preliminary sequence analysis indicated that several of the clones hybridizing to TAP48A but not TAP160A, encode TAP-like family members (unpublished observations). Southern blot hybridization of the TAPG-3 phage DNA with other probes from the 5' and 3' regions of the TAP cDNA sequence indicated that the entire transcribed region of the gene was contained on a single 1.9-kb EcoRI fragment (data not shown). A restriction map is shown in Fig. 3A and the sequence of the EcoRI fragment plus an additional 1.4 kb of 5' flanking nucleotides was determined (Fig. 3C). Comparison of this sequence with the TAP cDNA indicated that the bovine TAP gene has two exons surrounding a 1.6-kb intron. Primer-extension analysis of mRNA from bovine tracheal mucosa exhibits two bands of comparable intensity (Fig. 3B). The size of the shorter product is consistent with transcription initiation from the adenosine, which we have assigned as nucleotide +1 (see Fig. 3C); transcription may thus also initiate from nucleotide -1. Consensus sites for eukaryotic *The fetal columnar epithelial cells were shorter, -25 ,m at 4 months gestation (tracheal diameter, 0.5 cm) and -35 ,um at 6 months gestation (tracheal diameter, 1 cm), compared with adult tissue (-50 ,um). No clear basal cell layer was observed in the fetal airway sections. These histological findings are consistent with published observations of respiratory tract morphology in the bovine fetus (9).
Proc. Natl. Acad. Sci. USA 90 (1993)
4597
B cowi A
1
cow2 COW3 fDBPDBpDr P D B P D B p D B
2 3 4 5 6
-0.4 Kb..
TAP::
:
TUBULIN
FIG. 1. Northern blot analysis of TAP RNA. Total RNA (10 pg) from various tissues was fractionated by agarose gel electrophoresis in the presence of formaldehyde and then blotted to a nylon membrane (3, 7). The membranes were hybridized with the 32P-endlabeled oligonucleotide TAP160A (TAP), or a-tubulin cDNA as a control (Tubulin). (A) Tissue distribution of TAP mRNA. Lanes: 1, nasal septum; 2, tracheal mucosa; 3, fallopian tube; 4, small intestine; 5, fetal trachea (-4 months gestation); 6, fetal trachea (-6 months gestation). (B) Distribution of TAP mRNA along the airway from three different cows (cows 1, 2, and 3). P, proximal tracheal mucosa; D, distal tracheal mucosa; B, bronchiolar tissue. Autoradiographic exposures were 1-3 days.
transcriptional regulation are observed upstream from this site. Of particular interest is a TATA box at -24; a CAAT box is at -84, and an NF-KB consensus binding sequence is at -181. Sequence comparison analysis with GenBank identified regions upstream of exon 1 with high nucleotide identity to known sequences. The sequence of highest similarity (identity 90% over 542 bases, shown underlined in Fig. 3C) is the bovine Alu type art2 sequence, a highly repetitive element found upstream from a number of bovine genes (10). This comparison defines a flanking segment containing ruminantspecific, repetitive sequences upstream from the TAP transcriptional start site. Genomic Analysis of TAP-Related Sequences. Many families of antimicrobial peptides maintain high nucleotide identity in the 5' region of the mRNA (5, 11-14). To determine whether this might be true for TAP, a Southern blot containing bovine genomic DNA was hybridized with an oligonucleotide probe (TAP48A), corresponding to a 5' sequence of TAP mRNA. Fig. 4A shows that within the bovine genome there are numerous bands that hybridize to this probe under the same stringency as used for our cloning experiments. The multiple bands are consistent with the cloning results described above, where we detected the presence of TAP-related sequences in the bovine genomic library. The same blot was subsequently hybridized with two different oligonucleotide probes from the mature peptide-coding region of TAP (TAP160A, Fig. 4B; TAP118S, Fig. 4C). TAP160A, which encodes the first 16 amino acids of the mature peptide, recognizes fewer bands under the same conditions of stringency. TAP118S, a shorter oligonucleotide that corresponds to a more C-terminal region of the peptide, yielded single bands in both lanes under comparable stringency. This latter result is consistent with the presence of the TAP gene in a single copy. The multiple bands obtained with the probes TAP48A and TAP160A are consistent with the presence of a group of TAP-related sequences in the bovine genome. This notion was validated upon sequence analysis of several genomic clones as noted above. One unexpected finding was that the single EcoRI band in Fig. 4C corresponded to -4.0 kb, while the sequence of the genomic clone indicated that the fragment size containing the probes was 1.9 kb. This discrepancy was reconciled when it was discovered that there is a polymorphism, at one of the EcoRl sites flanking the TAP gene, yielding in some
4598
Biochemistry: Diamond et al.
Proc. Natl. Acad. Sci. USA 90
(1993)
FIG. 2. In situ hybridization of TAP mRNA expression in airway tissue. Cross-sections of bovine bronchiolar tissue (diameter, 3 mm) (A-C and E;G), and lung tissue (D and H) were hybridized
with 35S-labeled TAP oligonucleo-
L
cases either a 4.0- or a 1.9-kb band (data not shown). To determine whether there were any breed-associated polymorphisms within the TAP exons, genomic DNA isolated from blood samples of Holstein, Jersey, and Simmenthal cows was subjected to PCR amplification with oligonucleotide primers flanking the TAP gene. In each case, these primers amplified a single segment of DNA, which was subcloned and partially sequenced. Sequence analysis indicated that the exon portions of the TAP gene in the three breeds were identical, although there were some single base substitutions within the intron sequence (data not shown).
DISCUSSION We previously reported that TAP is an abundant peptide with broad spectrum antimicrobial activity found in the airway of the cow (3). This work characterizes the TAP gene and the site of its tissue expression. RNA analysis indicates that TAP is expressed along the entire length of the conducting airway, from nasal to bronchiolar tissue (Fig. 1). In situ data further localize the mRNA to the more apical aspect of the pseudostratified epithelium (Fig. 2A), most consistent with ex-
tides as described (8). (A and C-E). Hybridization with antisense probe, TAP160A. (B and F) Hybridization with sense probe, TAP113S. (E and G) Hybridization with antisense probe after pretreatment of tissue with RNase at 10 ug/ml for 15 min at room temperature. (A-D) Lightfield photomicrographs of tissue stained with hematoxylin and eosin. (E-H) Dark-field photomicrographs of same sections. Ar, arteriole (A-C) or small artery (D); a, alveolus; L, lumen. Exposure to photographic emulsion was 7 days. (Bar = 25 Am.)
pression in columnar epithelial cells. No signal was observed in the more basal layer, indicating that basal cells are not major sites of expression. The data suggest expression in the ciliated cells, which make up the vast majority of this epithelium (15). At this level of resolution, it is not possible to exclude the possibility that the other less-abundant epithelial cell types might also express the message.§ No signal is observed in alveolar sections (Fig. 2D). Finally, no signal was observed in submucosal glands (data not shown), the site of expression of lysozyme, another component of the airway host defense (16). Northern blot analysis failed to detect TAP mRNA in fetal trachea or in tissues other than the airway (Fig. 1A); however, we cannot rule out the possibility that very low level TAP expression may not be detectable under our experimental conditions. We have evidence that TAP-related molecules
§Mucus-producing cells such as goblet cells and serous cells are also
found along the epithelium, but at a much lower frequency (especially serous cells) (16). Since Clara cells tend to be found more distally in the airway (17), they would not significantly contribute to the signal seen in the proximal airway.
EXON 2
EXON I
A
4599
Proc. Natl. Acad. Sci. USA 90 (1993)
Biochemistry: Diamond et al.
_..
TR
TAPG-10a
f AAAAAAAAAA
TAP 48a TAP 54s
TAP 160a TAP 113s
s _
-_
TAP 1 OS
C agcccaccaacccctcagtccatgggactcaccaggcaagaacactggagcgggttgcca
-ctcattggtttc 1800
60
tttccttcttcaaaaggaaccatcagtccagttcagttcagttcagttcagtcgctcagt 120
:ctttttcataaa 1860
ggtacccaactctttgcgaccccatgaattgcagcatgcc
180
:tataaatcttta 1920
aactcccagagttcactcaaactcatgtccatcgagtcgs
240
.caggccaagcct 1980
atcctctgtcgcccccttcttctcctgccccaaatceCtcccagcaccagggtcttttcc 300
:aggaaagtcctc 2040
aatgagtcaactcttcgcatgaggtggccaaggtattggagcttcagcttcagcatgagt 360
caggacacgtcgctgatgatgagaccctcgctccacagctgggaggcagcacatccaaag 2200
ccttccaaagaacacccaggactgatctcctttaggatggactggttggatctccttgca 420
cagtgtgcaggatcggctgtctgattttcatcttacccttgatatttccggaaataggat 2160
gtccaaaagactatcaagagtcttctccaacaccacagttcaaaagCatcaattctttgg 480
gaaaatatgtaggaaggaaggagggagggagggagagagaggctgcagagactgagacct 2220
cgctcagcttcttcacagtccaaCtctcaCatccatacatgaccactggaaaaaaaccat
540
gagacaactgattagatgtcaaaatcaagttgaaattcccttgtctgatcagtgttgttt
taggcggacctttgttggtaaagtaatatctctgcttttcaaatgctatc
600
ttccaaggagtaatcgtgttttaatttcatggctgcaatc
660
2400
720
2460
1780
2520
Hp,
agctttaagccaactttttcactctcatctttcactttcatcaagaggctttttagttcc :840
2580
Also shown is the structure of the
:2340
2280
2340
FIG. 3. Analysis of bovine TAP gene. (A) Restriction map of bovine TAP gene, including 1.4 kb of upstream sequence. A, Apa I; H, HindIII; E, EcoRI; Hc, HincII; Hpa II; P, Pst
I; X, Xho
I.
predicted precur-
tcttcactttctgccatacgggtggtgtcatctgcatatttgaggttattgacatttctc I900
2640
cDNA and the
ccggcaatcttgattccagcttgtgcttcttccagccaataaggaactatagaaataaag 960
2700
from the cDNA
11020
2760
1080
2820
indicate oligonucleotides used for PCR and hybridizations. (B)
sor
structure of TAP deduced sequence.
Arrows
Primer extension of TAP mRNA 1140
rtbg
ttttc
lc
using TAP48A as an oligonucleo-
1200
2940
1260
tcttgttgtcacagaggceatgtccgcatctcatcacataaagccccagggccttgctca 3000
t1320
gaggggacacagctggecttctcgagatgctgctttcctgctgacacgtttttccctctc
3060
:1380
tgttctcttttta
3120
t ccaetaataataaaaatccacagccagcccaggaattcctcggaacCtgggac 1440 +1 CCA I1 500 t C A ct gcggcaa tcgcagtgtcttctc
AGGCATC=TGCCGATG AGGAGICIAIACAGATI GCACISTIG sGlyIleCysValProIleArgCysProGlySerMetLysGlInIleGlyThrCysvalGl
;1560 CACrCSC-TCGCTCCTC MetArgLeuHisHisLeuLeuLeuAlaLeuLeuPheLeuValLeuSerAlaTrpSerG 1620
by
comparison with
TAP
Sequences homologous to bovine GCCCAGI
tgtcctgctacccccatc tacaegtggtcagactaaacccaccatatttgatgctcctgak 1680
GTITAAAGTra
gaagcctgcgctgagtccttagtagcgagaggggtctgagaagggccctgacaaattcct
aaatgaattc
1740
mined
cDNA. Transcriptional start site (+ 1) was assigned from primerextension studies (see text and B).
GCAA
gtgagctcttgggagccetggagggagccgtgggctctctctcctgtttctacctccttc
primer. Bands correspond to 84 and 85 nucleotides. (C) Nucleotide sequence of TAP gene. Exons (capital letters) were deter-
tide
exist and are expressed in some other tissues, indicating different tissue specificity for other members of a large family (G.D. and C.L.B., unpublished observations). There is a consensus sequence for an NF-KB recognition site 181 bases upstream from the transcriptional start site. NF-KB has been shown to enhance the expression of a wide variety of genes involved in immune and inflammatory responses and can be modulated by lipopolysaccharides (LPS) and phorbol esters (18). This is of interest, considering the recent studies on the defense peptides in Hyalophora cecropia. In this insect, expression of the genes encoding several antimicrobial peptides is strongly induced in response to injury, LPS, phorbol esters, and bacterial infection (19). LPS and phorbol esters were recently shown to induce activity of a regulatory protein similar to NF-KB, which binds to the single NF-KB-like sites located 45-200 bp upstream from each of these insect genes (20). Furthermore, NF-KB motifs were found at a similar site upstream from the inducible promoter of an antimicrobial peptide gene in Drosophila (21). In light of these results, the presence of an NF-KB site in the
ClTCGCAGAATAAAT1 C
3300
repetitive elements are underlined
3360
and NF-KB
(see text). TATA box, CAAT box, sequences are
boxed
(see text).
regulatory region of the TAP gene suggests that LPS, via airborne bacteria, may be among the factors that modulate TAP expression. Consistent with this hypothesis, preliminary experiments indicate that TAP gene expression is dramatically up-regulated in primary cultures of bovine airway epithelial cells upon incubation with LPS (100 ng/ml) (G.D. and C.L.B., unpublished observations). TAP Is a Member of a Large Family of Related Sequences. Numerous antimicrobial peptides have been identified in both plants and animals, indicating evolutionary conservation of this mechanism of host defense (for reviews, see refs. 1, 2, 19, 22, and 23). A family of structurally related antimicrobial peptides named defensins form a major component of phagocyte-mediated host defense in mammals (for review, see ref. 24). Until recently, mammalian defensins were known to exist only in hematopoietic cells. This distribution was expanded when a defensin mRNA detected in mouse small intestine (25) was localized to the Paneth cell (26). A human defensin gene was later discovered that is also expressed in these epithelial cells of the small intestine (5, 27).
4600
Proc. Natl. Acad Sci. USA 90 (1993)
Biochemistry: Diamond et al.
A
1
2
B
1
2
C
~~9.4 6:6
-
4.4
4.4
-
4.4
2.32.0-
2.3 2.0
-
2.32.0
86. 9.4-
.pM W
1
2
8:64 -
FIG. 4. Southern blot analysis of TAP-related sequences. Bovine genomic DNA (10 ,ug) was digested with restriction endonucleases and size fractionated by agarose gel electrophoresis. DNA was transferred to a nylon membrane and hybridized with 32P-end-labeled oligonucleotide TAP48A (from the signal sequence) (A); oligonucleotide TAP160A (from the N-terminal portion of the mature peptide sequence) (B); or oligonucleotide TAP118S (from the C-terminal portion of the mature peptide) (C). Hybridization was carried out in Sx SSC/1% SDS/Sx Denhardt's solution/40 ,ug of RNA per ml at 42°C in the presence of 20% formamide for A and B, and 25% formamide for C. Blots were washed four times in 2x SSC/0.1% SDS at room temperature and then in 2x SSC/0.1% SDS at 65°C. Autoradiographic exposure was -10 days for all experiments. Lanes: 1, EcoRI; 2, Taq I. Numbers on left are kb.
Together these studies demonstrate that defensin expression is not limited to cells of hematopoietic origin. TAP can be considered a defensin-like molecule in that it shares general characteristics of defensins at the gene structure and peptide levels. The defensins have a basic charge and a broad spectrum of in vitro antimicrobial activity. They are characterized by a highly conserved 6-cysteine motif, along with 4 other conserved residues, forming a partial consensus sequence found across species lines (24). In addition, the putative signal sequences of preprodefensins are remarkably well conserved on both the amino acid and the nucleotide levels (5, 24). Like defensins, TAP is a basic molecule, with a broad spectrum of antimicrobial activity and contains 6 cysteines, all involved in disulfide bonds. The TAP gene has 2 exons, similar to a human epithelial defensin gene (5). The similarities between TAP and defensins in both structure and activity may suggest a common ancestral gene. However, the conserved cysteine motif is different for TAP and the defensins, and the highly conserved putative signal sequences are dissimilar for these two classes of prepropeptides. Thus, the evolutionary relationship between these two classes of peptides remains uncertain. Our Southern blot data (Fig. 4A) and genomic sequence analysis (unpublished data) support the hypothesis that, similar to defensins, TAP is a member of a large family of antimicrobial peptide genes. Indeed, numerous antimicrobial peptides named ,B-defensins, which share an identical 6-cysteine residue array with TAP, have been recently identified in bovine neutrophils (28). DNA sequence analysis indicates that the probe that we used to identify TAP-related sequences, TAP48A, is highly conserved among the cDNAs encoding these peptides (M. Selsted, personal communication). The presence of bovine repetitive elements in the flanking region of the TAP gene suggests a possible mechanism by which this family may have evolved through unequal homologous recombination. The fact that the ,B-defensins, found in circulating phagocytes, and TAP, from the tracheal epithelium, are members of the same family of antimicrobial peptide offers strong support of the hypothesis that TAP contributes
to host defense
of the
airway.
Conclusions. We present evidence that an antimicrobial peptide gene is highly expressed in respiratory epithelial cells. The expression of this gene is localized to the ciliated epithelium of the respiratory mucosa. These results support the hypothesis that this peptide is important in host defense at this epithelial surface. This gene may be useful for studies on gene regulation in the mammalian airway and in directing expression in transgenic animals. We thank members of the Division of Human Genetics for useful discussions, especially Dr. Michael Zasloff. We value the informative discussions concerning this work with Dr. Michael Selsted. We thank Drs. Sheldon Feinstein and Phillip Ballard for helpful comments on the manuscript. This work was supported in part by National Institutes of Health Grants R01AI32234 and 5T32GM07170, The G. Harold and Leila Y. Mathers Charitable Foundation, Magainin Pharmaceuticals, Inc., and The Ben Franklin Partnership. 1. Boman, H. G. (1991) Cell 65, 205-207. 2. Zasloff, M. (1992) Curr. Opin. Immunol. 4, 3-7. 3. Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W. L. & Bevins, C. L. (1991) Proc. Natl. Acad. Sci. USA 88, 3952-3956. 4. Salisbury, G. W. & VanDemark, N. L. (1961) Physiology of Reproduction and Artificial Insemination of Cattle (Freeman, San Francisco), pp. 121-125. 5. Jones, D. E. & Bevins, C. L. (1992) J. Biol. Chem. 267, 23216-23225. 6. Aldridge, J., Kunkel, L., Bruns, G., Tantravahi, U., LaLande, M., Brewster, T., Moreau, E., Wilson, M., Bromley, W., Roderick, T. & Latt, S. A. (1984) Am. J. Hum. Genet. 36, 546-564. 7. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Plainview, NY), 2nd Ed. 8. Young, W. S., III, Mezey, E. & Siegel, R. E. (1986) Neurosci. Lett. 70, 198-203. 9. de Zabala, L. E. & Weinman, D. E. (1984) Anat. Histol. Embryol. 13, 1-14. 10. Duncan, C. H. (1987) Nucleic Acids Res. 15, 1340. 11. Lidholm, D.-A., Gudmundsson, G. H., Xantholoulos, K. G. & Boman, H. G. (1987) FEBS Lett. 226, 8-12. 12. Ganz, T., Rayner, J. R., Valore, E. V., Tumolo, A., Talmadge, K. & Fuller, F. (1989) J. Immunol. 143, 1358-1365. 13. Ouellette, A. J. & Lualdi, J. C. (1990) J. Biol. Chem. 265, 9831-9837. 14. Kylsten, P., Samakovlis, C. & Hultmark, D. (1990) EMBO J. 9, 217-224. 15. Sturgess, M. J. (1989) in Lung Cell Biology, ed. Massaro, D. (Dekker, New York), Vol. 41, pp. 115-152. 16. Basbaum, C. B. & Finkbeiner, W. E. (1989) in Lung Cell Biology, ed. Massaro, D. (Dekker, New York), Vol. 41, pp. 37-79. 17. Massaro, G. D. (1989) in Lung Cell Biology, ed. Massaro, D. (Dekker, New York), Vol. 41, pp. 81-114. 18. Lenardo, M. J. & Baltimore, D. (1990) Cell 58, 227-229. 19. Boman, H. G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126. 20. Sun, S.-C. & Faye, I. (1992) Eur. J. Biochem. 204, 885-892. 21. Reichhart, J.-M., Meister, M., Dimarcq, J.-L., Zachary, D., Hoffmann, D., Ruiz, C., Richards, G. & Hoffmann, J. A. (1992) EMBO J. 11, 1469-1477. 22. Bevins, C. L. & Zasloff, M. A. (1990) Annu. Rev. Biochem. 59, 395-414. 23. Lehrer, R. I., Ganz, T. & Sested, M. E. (1991) Cell 64, 229230. 24. Ganz, T., Selsted, M. E. & Lehrer, R. I. (1990) Eur. J. Haematol 44, 1-8. 25. Ouellette, A. J. & Cordell, B. (1988) Gastroenterology 94, 114-121. 26. Ouellette, A. J., Greco, R. M., James, M., Frederick, D., Naftilan, J. & Fallon, J. T. (1989) J. Cell Biol. 108, 1687-1695. 27. Jones, D. E. & Bevins, C. L. (1993) FEBS Lett. 315, 187-192. 28. Selsted, M. E., Tang, Y.-Q., Morris, W. L., McGuire, P. A., Novotny, M. J., Smith, W., Henschen, A. H. & Cullor, J. S. (1993) J. Biol. Chem. 268, 6641-6648.
