INFECTION AND IMMUNITY,0019-9567/99/$04.0010

May 1999, p. 2561–2566 Vol. 67, No. 5

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

The Human Cationic Antimicrobial Protein (hCAP18), a PeptideAntibiotic, Is Widely Expressed in Human Squamous

Epithelia and Colocalizes with Interleukin-6MARGARETA FROHM NILSSON,1 BENGT SANDSTEDT,2 OLE SØRENSEN,3

GUNTHER WEBER,4 NIELS BORREGAARD,3 AND

MONA STÅHLE-BACKDAHL1*

Departments of Dermatology,1 Pathology,2 and Clinical Genetics,4 Karolinska Hospital, S-17176 Stockholm,Sweden, and Granulocyte Research Laboratory, Department of Hematology,

Rigshospitalet, Copenhagen DK-2100, Denmark3

Received 15 October 1998/Returned for modification 25 November 1998/Accepted 6 January 1999

Peptide antibiotics are widespread in nature and, by providing a rapid first line of defense, may be keyplayers in the innate immune system. Although epithelia are the main barriers shielding the internal envi-ronment from microorganisms, the role for peptide antibiotics in epithelial protection is unclear. We recentlyreported that the human cationic antimicrobial protein hCAP18, the precursor of the antimicrobial peptidecalled LL-37, is not expressed by normal human keratinocytes but is induced in various inflammatory skindisorders. In the present study we demonstrate that hCAP18 is consistently expressed at both mRNA andprotein levels in squamous epithelia of the mouth, tongue, esophagus, cervix, and vagina in humans. The genefor hCAP18 contains promoter elements that are potentially regulated by interleukin-6, and our data furthershow a colocalization between interleukin-6 and hCAP18 expression in these tissues. Our finding that hCAP18is widely produced in squamous epithelia suggests a role for this peptide in epithelial antimicrobial defense.Furthermore, colocalization with interleukin-6 indicates a potential local mechanism for the upregulation ofhCAP18 at the epithelial surfaces.

Peptide antibiotics may serve a key protective role in thehost defense. They are participants in the innate immune sys-tem (nonadaptive immune system), acting as effector mole-cules with the capacity to kill a broad spectrum of microorgan-isms (6). In contrast to highly specific adaptive immunity, theinnate immune system provides a rapid and nonspecific re-sponse and thereby contributes to the first line of defense. Thefirst peptide antibiotics were discovered in the 1980s whencecropins were isolated from insects (25) and defensins wereisolated from rabbit macrophages (24). Numerous antimicro-bial peptide antibiotics occur in nature, and over a dozen havebeen identified in humans, including several salivary histatins,lactoferricin, six a-defensins, two b-defensins, and the humancationic antimicrobial protein hCAP18.

hCAP18, the C-terminal domain of which is called LL-37,has been isolated from specific granules of human neutrophilgranulocytes and is structurally distinct from the defensins (9,15). hCAP18 belongs to the cathelicidin family of proteinscharacterized by a conserved cathelin proregion and a variableantibacterial peptide in the C-terminal domain. The gene forhCAP18, named CAMP, is the only member of the cathelicidinfamily so far identified in humans. CAMP appears to have arestricted spatial expression pattern and has thus far beenreported to be produced constitutively only in bone marrow, intestis, and in airway epithelium and to be upregulated in skinepithelium in association with inflammation (1, 5, 12). Thepromoter region of the CAMP gene contains potential bindingsites for the transcription factors, the acute-phase responsefactor, and the nuclear factor for interleukin-6 (IL-6) (15). IL-6regulates the activation of these two transcription factors, in-

dicating that this cytokine may play an important role in mod-ulating CAMP gene expression (2, 3). IL-6 is a multifunctionalcytokine involved in various inflammatory responses and isconsistently found in association with infectious processes (22).

Epithelia serve a critical role in separating the organismfrom the environment. In addition to providing fundamentalphysical and mechanical shields, epithelia function as activeimmunological organs by presenting antigens and producingcytokines (7), and epithelia are, in fact, able to produce anti-bacterial peptides (5, 16, 23). Thus, different members of thedefensin family are encountered in several human epitheliaand are induced by human keratinocytes in culture (16, 19, 30);a recent study by Bals et al. described the expression ofhCAP18 in airway epithelium (5). We have previously shownthat hCAP18 is produced in human epidermis in associationwith a variety of inflammatory disorders (12). These findingsled us to suggest that hCAP18, although not constitutivelyexpressed in the epidermis, is induced nonspecifically in re-sponse to inflammation. The present study further delineatesthe expression of hCAP18 in nonkeratinizing human squamousepithelia, and we report here that hCAP18 was consistentlyexpressed at both mRNA and peptide levels in squamous ep-ithelia of the buccal mucosa, tongue, esophagus, cervix, andvagina. In all tissues the expression of hCAP18 colocalized withthe immunoreactivity for IL-6, a finding which is noteworthy inview of the potential role for this cytokine in modulatinghCAP18 gene expression. Our findings suggest the presence ofa powerful antimicrobial defense system at the epithelial in-terfaces and a putative local mechanism for its regulation.

MATERIALS AND METHODS

Tissues. Formalin-fixed and paraffin-embedded archival human tissues, in-cluding samples from the buccal mucosa (n 5 3), tongue (n 5 2), esophagus (n 52), cervix (n 5 5), and vagina (n 5 3), were obtained from the Department of

* Corresponding author. Mailing address: Department of Derma-tology, Karolinska Hospital, S-17176 Stockholm, Sweden. Phone: 46-8-5177-3348. Fax: 46-8-5177-7851. E-mail: mba@hud.ks.se.

2561

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Pathology, Karolinska Hospital, Stockholm, Sweden. All material was obtainedat surgery (Table 1).

Preparation of RNA probes. A 435-bp hCAP18 full-length cDNA (9) wassubcloned in Bluescript KS 11 and, after linearization with BamHI and EcoRI,was used as a template for in vitro transcription to generate 35S-labeled antisenseand sense probes. After transcription, the RNA probes were ultrafiltered (Mi-cron 100; Amicon, Inc., Beverly, Mass.) before hybridization.

Northern analysis. To ascertain the specificity of the probe derived fromfull-length hCAP18 cDNA, we performed a Northern analysis by using a multi-ple-tissue Northern blot containing poly(A)1 RNA from samples from the stom-ach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow(Clontech Laboratories). The quality of the blot was confirmed through hybrid-ization with cDNA probes detecting ubiquitously expressed transcripts. Prehy-bridization and hybridization with a randomly 32P-labeled cDNA probe forhCAP18 were performed according to the manufacturer’s instructions. The blotswere washed at 0.13 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate)–0.1% sodium dodecyl sulfate at 65% and, after overnight autoradiogra-phy, were evaluated with a phosphorimager system (Fuji Bas 1000).

In situ hybridization. In situ hybridization was performed essentially as de-scribed earlier (26). Briefly, 5-mm sections were hybridized overnight with 2.5 3106 to 5.0 3 106 cpm of 35S-labeled RNA probes at 55°C. After hybridization, theslides were washed under stringent conditions that included incubation with 50mg of RNase A (Sigma) per ml for 30 min at 37°C and were processed forautoradiography. Autoradiographic exposure was for 3 to 4 weeks.

Immunohistochemistry. (i) hCAP18. Protein A-purified antibodies raised inrabbits against recombinant hCAP18 were used at a 1:500 dilution (27). Depar-affinized, rehydrated sections were treated with 1% H2O2 in methanol for 30 minat room temperature to quench endogenous peroxidase activity. After beingrinsed in phosphate-buffered saline, the sections were microwave treated twicefor 5 min. All sections were stained according to the indirect peroxidase method(17) by using a Vectastain ABC Elite Kit (Vector Laboratories) and followingthe manufacturer’s instructions. Control sections from the same tissues weresimilarly processed except that no hCAP18 antibody was added. As an additionalcontrol, we performed immunohistochemistry as described above with rabbitserum diluted 1:500 (Dako, Copenhagen, Denmark) instead of primary hCAP18antibody.

(ii) Immunoadsorption of hCAP18. To ascertain the specificity of the immu-nostaining, we performed immunoadsorption, adding 10 ml of the hCAP18 re-combinant protein (27) to 150 ml of a 1:500 dilution of anti-hCAP18 antibody.The final concentration of the hCAP18 recombinant peptide in the reactionmixture was 33 mg/ml. The mixture was preincubated at 4°C overnight with gentleshaking prior to immunohistochemical analyses according to the protocol de-scribed above. Control sections were processed according to the same protocolexcept that no peptide was added to adsorb the antibody.

(iii) IL-6. A monoclonal IL-6 antibody diluted 1:50 (Janssen, Beerse, Belgium)(8) was used for the immunostaining on serial sections from the same tissues.The sections were stained according to the indirect peroxidase method describedabove.

RESULTS

Probe specificity. Northern blot analysis confirmed the spec-ificity of the hCAP18 probe. A single prominent message ofless than 1 kb was detected specifically in bone marrow (Fig. 1).

hCAP18 mRNA and peptide are expressed in human squa-mous epithelia. All tissues were reviewed by an experienced

FIG. 1. Autoradiogram of a multiple tissue Northern blot hybridized with a0.4-kb CAP18 cDNA fragment. The sizes of the RNA markers are indicated onthe left. A single transcript of less than 1 kb was detected specifically in bonemarrow, in agreement with published data.

TABLE 1. Clinical patient data

Tissuea Histopathological evaluation Associated condition Sexb Age (yr)

Tongue Normal epithelia Sjogren’s syndrome M 74Tonguea Normal epithelia Control, 4 mo after

nonspecific ulcerM 75

Buccal mucosa Normal epithelia Oral cavity tumor F 60Buccal mucosa Normal epithelia None F 70Buccal mucosa Normal epithelia None F 55

Esophagusa Mild esophagitis Helicobacter gastritis F 20Esophagus Normal epithelia Gastric carcinoma M 60

Cervix Mild dysplasia None F 30Cervixa Severe dysplasia Inflammation F 35Cervixa Normal epithelia Uterus myoma F 47Cervix Normal epithelia None F 43Cervix Mild dysplasia Inflammation F 35

Vagina Normal epithelia None F 43Vagina Hyperplastic epithelia Inflammation F 42Vagina Normal epithelia None F 31

a Tissues visualized in figures.b M, mule; F, female.

2562 NILSSON ET AL. INFECT. IMMUN.

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

pathologist and were evaluated as listed in Table 1. In alltissues there was a positive signal upon in situ hybridization forhCAP18 mRNA in epithelial cells (Fig. 2B and 4B and D). Inmost tissues the signal for hCAP18 mRNA was more promi-nent in the basal layers of the epithelium and decreased towardthe surface (Fig. 2B and 4B), but in cervical tissues found to bedysplastic and inflammatory the strongest expression ofhCAP18 mRNA was seen in the upper part of the epitheliumtoward the surface (Fig. 4D). Sections hybridized with senseRNA probe showed a only background autoradiographic signal(Fig. 2D).

In all tissues there was immunoreactivity for hCAP18 inareas of squamous epithelium expressing hCAP18 mRNA, in-dicating the ability of these cells to produce hCAP18 peptide(Fig. 2A, 3A, and 4A, C, and E). As mentioned above, innoninflammatory tissues the signal for hCAP18 mRNA wasprimarily detected in the basal epithelial layers, althoughhCAP18 immunoreactivity was also seen in occasional supra-basal cells, suggesting that under normal conditions hCAP18 ismainly transcribed in the basal epithelial layers and is thentranslated and transported toward the surface in the process ofnormal cell turnover. However, in the two dysplastic and in-flammatory cervix samples, hCAP18 was strongly expressed inthe outermost cell layers at both mRNA and protein levels,indicating a spatially distinct induction of hCAP18 under theseconditions. In addition, in all cervix tissues there was positivesignal and immunoreactivity for hCAP18 in scattered stromalcells and in the mucous glands (Fig. 4F and G). The stromalcells had a fibroblastic appearance but were not further char-acterized. As an internal positive control, strong immunostain-

ing for hCAP18 was consistently detected in neutrophil gran-ulocytes (not shown). Control slides without hCAP18 antibodywere devoid of immunoreactivity, as were slides processed withonly rabbit serum (Fig. 3B). Immunoadsorption at 33 mg ofhCAP18 peptide per ml almost completely abolished immuno-reactivity for hCAP18 (Fig. 3C).

Expression of hCAP18 colocalizes with immunoreactivityfor IL-6 in squamous epithelia. Since the gene for hCAP18contains promoter elements regulated by IL-6, we performedimmunohistochemistry with antibodies directed against thiscytokine. In all investigated tissues, immunoreactivity for IL-6colocalized with that for hCAP18 (Fig. 2C and 3D).

DISCUSSION

In the present investigation we demonstrate that the anti-microbial peptide hCAP18 is expressed at both RNA and pro-tein levels in nonkeratinized squamous epithelia in humans.Most samples showed only minor histopathological signs ofinflammation, indicating that there may be a basal constitutiveexpression of hCAP18 in these tissues. This contrasts with whatwe previously reported in human skin, where there was nodetectable expression of hCAP18 in normal quiescent epider-mis although there was a pronounced upregulation in associ-ation with a variety of inflammatory conditions (12). Onemight argue that a continuous expression of antimicrobial ac-tivity may be more critical in epithelia lacking an outer kera-tinized cover. Still, hCAP18 is likely to be a regulated gene,and in the present investigation there was a spatially distinct

FIG. 2. hCAP18 is expressed in the squamous epithelium of the tongue and colocalizes with immunoreactivity for IL-6. (A) Section of tongue demonstrating positiveimmunostaining for hCAP18 in the epithelium. Immunoreactivity (red precipitate) is most pronounced in the basal cell layers, but scattered immunopositive cells canalso be seen in the suprabasal layers. (B) Section from the same tissue hybridized with antisense 35S-labeled cRNA probe for hCAP18 mRNA. Intense autoradiographicsignal for hCAP18 mRNA (appears as white grains under dark-field illumination) is seen in the lower portion of tongue epithelium. (C) Immunoreactivity for IL-6 isseen in tongue epithelium with the same pattern as for hCAP18 and in submucosal blood vessels. (D) Hybridization with the control sense probe for hCAP18 lacksthe autoradiographic signal for hCAP18 mRNA. The shiny appearance is due to the autofluorescence of the tissue. Bars, 100 mm.

VOL. 67, 1999 THE PEPTIDE ANTIBIOTIC hCAP18 IN HUMAN EPITHELIA 2563

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

pattern of hCAP18 production in the cervical samples exhib-iting dysplasia and inflammation compared to normal nonin-flamed tissues (Fig. 4). In the former case, the expression ofhCAP18 was clearly upregulated in the superficial layers, cre-ating a striking shield-like band (Fig. 4C to E). Interestingly, inall of the cervical samples, hCAP18 was also detected in scat-tered stromal cells and in the mucous glands (Fig. 4F and G),indicating that hCAP18 may serve a key role in the defense ofthis organ.

The complete functional repertoire of hCAP18 remains tobe fully elucidated. The C-terminal domain of hCAP18 exhib-its potent antibacterial activity in vitro against both gram-pos-itive and gram-negative bacteria, a trait which most likely re-flects a major biological role for this protein (18, 20, 28). Inaddition, several studies have demonstrated that hCAP18binds to the endotoxin bacterial lipopolysaccharide, neutraliz-ing many of its biological effects (11, 21). Thus, hCAP18 mayplay a role in the protection against septic shock as was dem-onstrated in a pig model of endotoxemia (29). Interestingly,the induction of defensins in the trachea is mediated by CD14on the epithelial cell surface, further supporting a role forepithelial cells in the local host defense (10). Besides directeffects against microbial invasion, it seems conceivable thatantibacterial peptides, including hCAP18, serve additional bi-ological roles. Thus, defensins modulate the inflammatory re-sponse through the enhancement of chemotaxis, and anotherpeptide, NK lysin, possesses antitumor activity (4). Interest-ingly, a porcine homologue of hCAP18, PR-39, can inducesyndecans in association with wound healing (14). Syndecansare cell surface proteoglycans required for cellular response togrowth factors and thereby important in tissue repair. It is

plausible that hCAP18 plays a similar role in tissue repairprocesses in humans.

In all of the tissues there was a striking colocalization be-tween hCAP18 expression and immunoreactivity for IL-6 (Fig.2 and 3). IL-6 is a proinflammatory cytokine produced by avariety of cell types, including keratinocytes. Typically, theproduction of IL-6 is induced in response to bacterial productsand tissue damage. The regulation of the hCAP18 gene ap-pears to be complex and remains to be fully clarified. However,the identification of several elements responsive to IL-6 in thegene promoter strongly suggests that this cytokine plays aprominent role in modulating the transcription of the hCAP18gene. Thus, the colocalization of IL-6 and hCAP18 reportedhere is suggestive of a mechanism through which hCAP18 maybe rapidly upregulated in epithelial cells in response to micro-bial or other challenges.

The existence of immediate and efficient antimicrobial de-fense systems in epithelial tissues seems both natural and nec-essary. Epithelia form the borders between the organism andthe frequently hostile environment and are continuously ex-posed to a large variety of potential intruders. Relying solelyon the highly evolved but slow adaptive immune system wouldnot be quick enough to stop the invasion, growth, and subse-quent takeover by pathogens capable of rapid division. Indeed,there is accumulating evidence that peptide antibiotics areprominent players in the protection of epithelial surfaces.Thus, the expression of hCAP18 was recently demonstrated inairway epithelium, and the peptides human beta defensin 1 andhuman beta defensin 2 were reported in human skin; the latterwas also found to be upregulated by contact with differentmicroorganisms (5, 13, 16). Our findings demonstrate that

FIG. 3. hCAP18 and IL-6 colocalize in esophagus epithelia. (A) Section of esophagus demonstrates hCAP18-immunoreactive epithelial cells. (B) No immunore-activity is detected in the epithelium when rabbit serum is used instead of hCAP18 antibody. (C) Immunoadsorption with hCAP18 recombinant peptide almostcompletely abolished hCAP18 immunoreactivity in esophagus epithelium. (D) Immunostaining for IL-6 demonstrates immunoreactive cells in the same areas as forhCAP18. Bars, 100 mm.

2564 NILSSON ET AL. INFECT. IMMUN.

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

hCAP18 is widely expressed in squamous epithelia and supportthe notion of an innate antimicrobial shield at epithelial inter-faces. The coexpression of hCAP18 with IL-6 might provide alocal mechanism for modulating hCAP18 gene transcription inthese tissues.

ACKNOWLEDGMENTS

We thank Kerstin Bruce, Anna Hulthen, and Anna-Lena Kastmanfor excellent technical assistance.

This work was supported by grants from the Karolinska Institute, theWelander-Finsen Foundation, the SSAC Foundation for Research inAntimicrobial Therapy, and the Swedish Psoriasis Association.

REFERENCES

1. Agerberth, B. A., H. Gunne, J. Odeberg, P. Kogner, H. G. Boman, and G. H.Gudmundsson. 1995. FALL-39, a putative human peptide antibiotic, is cys-teine-free and expressed in bone marrow and testis. Proc. Natl. Acad. Sci.USA 92:195–199.

2. Akira, S., H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T.Nakajima, T. Hirano, and T. Kishimoto. 1990. A nuclear factor for IL-6expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9:1897–1906.

3. Akira, S., Y. Nishio, M. Inoue, X.-J. Wang, S. Wei, T. Matsusaka, K. Yo-shida, T. Sudo, M. Naruto, and T. Kishimoto. 1994. Molecular cloning ofAPRF, a novel IFN-stimulated gene factor 3 p91-related transcription factorinvolved in the gp130-mediated signaling pathway. Cell 77:63–71.

4. Andersson, M., H. Gunne, B. A. Agerberth, T. Bergman, R. Sillard, H.Jornvall, V. Mutt, B. Olsson, H. Wigzell, Å. Dagerlind, H. G. Boman, andG. H. Gudmundsson. 1995. NK-lysin, a novel effector peptide of cytotoxic Tand NK cells. Structure and cDNA cloning of the porcine form, induction by

FIG. 4. In the dysplastic and inflammatory cervix, hCAP18 is expressed in a bandlike pattern in the superficial epithelial layers. (A) Noninflammatory cervixdemonstrates positive immunoreactivity for hCAP18 predominantly found in the basal epithelium. (B) In situ hybridization shows matching signal for hCAP18 mRNAin the same tissue. The shiny appearance of the surface layer is due to the autofluorescence of the tissue and does not represent an autoradiographic signal. (C) Sampleof a dysplastic and inflammatory cervix demonstrates a striking band of hCAP18 immunoreactivity in the superficial epithelium. (D) In situ hybridization shows thathCAP18 is actively expressed in the outermost layers of the epithelium. (E) High-power view of superficial hCAP18-immunoreactive epithelial cells. (F) hCAP18-immunoreactive fibroblast-like cell in the cervix stroma (same tissue sample as in panel A). (G) hCAP18-immunoreactive mucous glands in the cervix (same tissuesample as in panel C). Bars: A to D and G, 100 mm; E and F, 25 mm.

VOL. 67, 1999 THE PEPTIDE ANTIBIOTIC hCAP18 IN HUMAN EPITHELIA 2565

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

interleukin 2, antibacterial and antitumour activity. EMBO J. 14:1615–1625.5. Bals, R., X. Wang, M. Zasloff, and J. M. Wilson. 1998. The peptide antibiotic

LL-37/hCAP-18 is expressed in epithelia of the human lung where it hasbroad antimicrobial activity at the airway surface. Proc. Natl. Acad. Sci. USA95:9541–9546.

6. Boman, H. G. 1995. Peptide antibiotics and their role in innate immunity.Annu. Rev. Immunol. 13:61–92.

7. Bos, J. D., and L. Kapsenberg. 1993. The skin immune system: progress incutaneous biology. Immunol. Today 14:75–78.

8. Brakenhoff, J. P., F. D. de Hon, V. Fontaine, E. ten Boekel, H. Schooltink, S.Rose-John, P. C. Heinrich, J. Content, and L. A. Aarden. 1994. Developmentof a human interleukin-6 receptor antagonist. J. Biol. Chem. 269:86–93.

9. Cowland, J. B., A. H. Johnson, and N. Borregaard. 1995. hCAP-18, a cathe-lin/bactenecin like protein of human neutrophil specific granules. FEBS Lett.368:173–176.

10. Diamond, G., J. P. Russell, and C. L. Bevins. 1996. Inducible expression ofan antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithe-lial cells. Proc. Natl. Acad. Sci. USA 93:5156–5160.

11. Fletcher, M. A., M. A. Kloczewiak, P. M. Loiselle, M. Ogata, M. W. Ver-meulen, E. M. Zanzot, and H. S. Warren. 1997. A novel peptide-IgG con-jugate, CAP18 106-138-IgG that binds and neutralizes endotoxin and killsgram-negative bacteria. J. Infect. Dis. 175:621–632.

12. Frohm, M., B. A. Agerberth, G. Ahangari, M. Ståhle-Backdahl, S. Liden, H.Wigzell, and G. H. Gudmundsson. 1997. The expression of the gene codingfor the antibacterial peptide LL-37 is induced in human keratinocytes duringinflammatory disorders. J. Biol. Chem. 272:15258–15263.

13. Fulton, C., M. G. Anderson, M. Zasloff, R. Bull, and A. Quinn. 1997. Ex-pression of natural peptide antibiotics in human skin. Lancet 350:1750–1751.

14. Gallo, R. L., M. Ono, T. Povsic, C. Page, E. Eriksson, M. Klagsbrun, and M.Bernfield. 1994. Syndecans, cell surface heparan sulfate proteoglycans, areinduced by proline-rich antimicrobial peptide from wounds. Proc. Natl.Acad. Sci. USA 91:11035–11039.

15. Gudmundsson, G. H., B. A. Agerberth, J. Odeberg, T. Bergman, B. Olsson,and R. Salcedo. 1996. The human gene FALL39 and processing of thecathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur.J. Biochem. 238:325–332.

16. Harder, J., J. Bartels, E. Christophers, and J.-M. Schroder. 1997. A peptideantibiotic from human skin. Nature 387:861.

17. Hsu, S. M., L. Raine, and H. Fanger. 1981. Use of avidin-biotin peroxidasecomplex (ABC) in immunoperoxidase techniques: a comparison between

ABC and unlabeled antibody (PA) procedures. J. Histochem. Cytochem.29:577–580.

18. Johansson, J., G. H. Gudmundsson, M. E. Rottenberg, K. D. Berndt, and B.Agerberth. 1998. Conformation-dependent antibacterial activity of the nat-urally occurring human peptide LL-37. J. Biol. Chem. 273:3718–3724.

19. Krisanaprakornkit, S., A. Weinberg, C. N. Perez, and B. A. Dale. 1998.Expression of the peptide antibiotic human b-defensin 1 in cultured gingivalepithelial cells and gingival tissue. Infect. Immun. 66:4222–4228.

20. Larrick, J. W., M. Hirata, Y. Shimomoura, M. Yoshida, H. Zheng, J. Zhong,and S. C. Wright. 1993. Antimicrobial activity of rabbit CAP 18-derivedpeptides. Antimicrob. Agents Chemother. 37:2534–2539.

21. Larrick, J. W., M. Hirata, H. Zheng, J. Zhong, D. Bolin, J.-M. Cavaillon,S. H. Warren, and S. C. Wright. 1994. A novel granulocyte-derived peptidewith lipopolysaccharide-neutralizing activity. J. Immunol. 152:231–240.

22. Le, M., and J. Vilcek. 1989. Interleukin 6: a multifunctional cytokine regu-lating immune reactions and the acute phase protein response. Lab. Invest.61:588–602.

23. Schonwetter, B. S., E. D. Stolzenberg, and M. A. Zasloff. 1995. Epithelialantibiotics induced at sites of inflammation. Science 267:1645–1648.

24. Selsted, M. E., D. M. Brown, R. J. DeLange, and R. I. Lehrer. 1983. Primarystructures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lungmacrophages. J. Biol. Chem. 258:14485–14489.

25. Steiner, H., D. Hultmark, Å. Engstrom, H. Bennich, and H. G. Boman. 1981.Sequence and specificity of two antibacterial proteins involved in insectimmunity. Nature 292:246–248.

26. Ståhle-Backdahl, M., B. D. Sudbeck, A. Z. Eisen, H. G. Welgus, and W. C.Parks. 1992. Expression of 92 kDa type IV collagenase in eosinophils asso-ciated with basal cell carcinoma. J. Invest. Dermatol. 99:497–503.

27. Sørensen, O., J. B. Cowland, J. Askaa, and N. Borregaard. 1997. An ELISAfor hCAP-18, the cathelicidin present in human neutrophils and plasma.J. Immunol. Methods 206:53–59.

28. Turner, J., Y. Cho, N. N. Dinh, A. J. Waring, and R. Lehrer. 1998. Activitiesof LL-37, a cathelin-associated antimicrobial peptide of human neutrophils.Antimicrob. Agents Chemother. 42:2206–2214.

29. VanderMeer, T. J., M. J. Menconi, J. Zhuang, H. Wang, R. Murtaugh, C.Bouza, P. Stevens, and M. P. Fink. 1995. Protective effects of a novel32-amino acid C-terminal fragment of CAP18 in endotoxemic pigs. Surgery117:656–662.

30. Zhao, C., I. Wang, and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett.396:319–322.

Editor: J. R. McGhee

2566 NILSSON ET AL. INFECT. IMMUN.

on February 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from