Detection of β-defensins secreted by human oral epithelial cells

Journal of Immunological Methods 256 Ž2001. 65–76 www.elsevier.comrlocaterjim

Detection of b-defensins secreted by human oral epithelial cells Deborah L. Diamond a , Janet R. Kimball b, Suttichai Krisanaprakornkit b, Tomas Ganz c , Beverly A. Dale b,d,e,) a

Ciphergen Biosystems, Fremont, CA, USA Department of Oral Biology, UniÕersity of Washington, Seattle, WA, USA c Department of Medicine, UniÕersity of California, Los Angeles, CA, USA d Department of Periodontics, UniÕersity of Washington, Seattle, WA, USA Department of Mediciner Dermatology, UniÕersity of Washington, Seattle, WA, USA b

e

Received 22 January 2001; received in revised form 26 April 2001; accepted 5 June 2001

Abstract Human b-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of b-defensins in vitro and in biological fluid using ProteinChip w Array, surface enhanced laser desorptionrionization ŽSELDI. technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of b-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size. q 2001 Elsevier Science B.V. All rights reserved. Keywords: ProteinChip array; SELDI; Mass spectrometry; Oral keratinocytes; Gingiva; Innate immunity; Antimicrobial peptides; Defensins

1. Introduction AbbreÕiations: hBD, human b-defensin; SELDI, surface enhanced laser desorptionrionization; EAM, energy absorbing matrix; GEC, gingival epithelial cells; GCF, gingival crevicular fluid; HNP, human neutrophil peptide; RT-PCR, reverse transcriptasepolymerase chain reaction. ) Corresponding author. Department of Oral Biology, University of Washington, P.O. Box 357132, Seattle, WA 98195-7132, USA. Tel.: q1-206-543-4393; fax: q1-206-685-3162. E-mail address: [email protected] ŽB.A. Dale..

Defensins are small, cationic antimicrobial peptides that comprise an important component of mammalian innate immune defense. They have broadspectrum antimicrobial activity against gram-positive and gram-negative bacteria, fungi and some viruses Žreviewed by Weinberg et al., 1998; Huttner and Bevins, 1999.. In humans, six a-defensins and three b-defensins have been identified. Human b-de-

0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 4 4 2 - 2

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fensins are expressed in epithelial tissues and may be critical in host response to mucosal infection. Further, b-defensins also exhibit cross-talk between the innate and acquired immune responses ŽYang et al., 1999; Tani et al., 2000.. Human b-defensin 1 ŽhBD1. is constitutively expressed in many epithelial tissues including kidney, lung, gut, and in the stratified epithelium of the oral cavity ŽZhao et al., 1996; Mathews et al., 1999.. Human b-defensin 2 ŽhBD2. was originally isolated from epidermis ŽHarder et al., 1997.; its expression is induced in response to inflammation in all epithelia tested ŽDiamond et al., 2000; Krisanaprakornkit et al., 2000.. HBD3 is a newly reported member of this family ŽHarder et al., 2001.. Both hBD1 and hBD2 have also been identified in biological fluids including urine, bronchial fluids, nasal secretions, and saliva ŽValore et al., 1998; Cole et al., 1999.. The localization and characteristics of the b-defensins strongly support the hypothesis that these peptides have a key role in defense of mucosal and body surfaces ŽHancock and Scott, 2000.. For example, antimicrobial activity of

defensins is inhibited in respiratory epithelia in cystic fibrosis, thus contributing to increased susceptibility to chronic infection in this disorder ŽGoldman et al., 1997.. Sensitive methods of detection and assay of b-defensins are needed to better understand the expression, secretion and function of these peptides that may have critical importance in a number of medical applications. We and others have previously shown expression of hBD1 and hBD2 mRNA and peptide in oral epithelial cells derived from the gingiva ŽGECs. and in gingival tissue ŽKrisanaprakornkit et al., 1998, 2000.. In tissue, the peptide is detected near the surface of oral stratified epithelia, consistent with a role in the epithelial protective barrier function ŽDale et al., in press.. However, the physiologic role of b-defensins in the oral environment has not been definitively shown. These peptides can be envisioned to function within oral epithelial cells by protecting against cellular invasion by bacteria. However, a more general antimicrobial barrier effect would be achieved by

Fig. 1. Analysis of proteins secreted by cultured human gingival epithelial cells using ProteinChip w array SELDI time-of-flight mass spectrometry. Serum-free media and cell culture supernatants from unstimulated and F. nucleatum crude cell wall extract stimulated cells were analyzed for proteins bound to a normal phase ŽNP2. ProteinChip array. The binding was performed as described in Section 2, and the chips were washed for 5 min with 0.1 M sodium acetate, pH 4.5, then with MQ water. The molecular weight range of 3000–5500 Da is shown here. Since the media contains bovine insulin that also bound to the chip Žnot shown, MW 5733.58 Da., we used the singly and doubly charged peaks of insulin for internal calibration of peak masses. ŽA. Representative spectrum of cell culture supernatant from unstimulated cells. A peak at 5068 Da corresponding to the predicted MW of hBD-1 Ž47-amino acid form. was detected. ŽB. Protein profile of cell culture supernatant from stimulated cells. In addition to the peak corresponding to hBD1, a peak at 4328 Da corresponding to the predicted MW of hBD2 was detected in cells exposed to stimulant. Representative spectra of serum-free media in the absence ŽA. or presence ŽB. of the stimulant are shown below the corresponding cell culture supernatants.

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their secretion at mucosal sites. Both hBD1 and hBD2 have signal peptide domains. Both are expressed in gingival epithelial cells in a perinuclear distribution consistent with the localization of the Golgi apparatus and secretion via this route ŽDale et al., in press.. In this report, we describe the application of ProteinChip w Array, surface enhanced laser desorptionrionization ŽSELDI. technology ŽCiphergen Biosystems, Fremont, CA. to detect b-defensins secreted into the media of cultured human oral keratinocytes and in gingival crevicular fluid which accumulates in vivo between the epithelium and the tooth surface. The ProteinChip system combines ProteinChip technology with time-of-flight mass spectrometry and enables rapid, reproducible protein profiling directly from crude samples such as serum, urine, cerebrospinal fluid, cell and tissue extracts, and cell culture supernatants. A description of recent advances in SELDI technology and comparisons to other laser-based mass spectrometry techniques can be found in a

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recent review ŽMerchant and Weinberger, 2000.. We show, using this method, that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major forms secreted from oral epithelial cells and that hBD1 is found in several smaller forms in gingival crevicular fluid possibly as a result of extracellular processing and proteolysis. 2. Methods and materials 2.1. Oral epithelial cell culture Human gingival epithelial cells ŽGECs. were grown in serum-free medium as previously described ŽKrisanaprakornkit et al., 1998, 2000.. Some cultures were stimulated for 24 h with 10 mgrml crude cell wall extract from Fusobacterium nucleatum prepared as previously described ŽKrisanaprakornkit et al., 1998.. Cells from both subconfluent and confluent cultures were extracted with 5% acetic acid ŽValore et al., 1998. and acid-extracted proteins

Fig. 2. Analysis of proteins bound to various chemically defined ProteinChip arrays. Shown here are representative spectra of cell culture supernatants from stimulated cells. The binding was performed as described in Section 2, and the chips were washed as described here. ŽA. WCX2 Žweak cation exchange. —washed 5 min with T–PBS, pH 7.0. ŽB. H4 Žhydrophobic. —washed with 5 ml 10% acetonitrile. ŽC. NP2 Žhydrophilic. —washed for 5 min with 0.1 M sodium acetate, pH 4.5. Each array binds a different subset of proteins. However, peaks corresponding to the predicted MW of hBD1 Ž47-amino acid; 5068 Da. and hBD2 Ž4328 Da. were detected on each surface. See text for further discussion.

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examined by SDS–Tricine gel electrophoresis and Western immunoblotting or SELDI. Supernatants were collected from subconfluent and confluent cultures that were either unstimulated or stimulated for 24 h for analysis by SELDI.

consent in accordance with University of Washington Human Subjects protocols.

2.2. Biological fluid samples

Each ProteinChip Array has multiple spots that contain chemical Žionic, hydrophobic, hydrophilic, etc.. or biological Žantibody, receptor, DNA, etc.. Adocking sitesB designed for the selective capture of proteinsrpeptides from complex mixtures. After the sample is applied to the surface, unbound proteins and interfering substances Ži.e. salts, detergents. are washed away. An energy absorbing molecule ŽEAM., or matrix, is then added and allowed to dry; the laser energy absorbing matrix molecules co-crystallize with the adsorbed proteins. The captured proteins are detected using surface enhanced laser desorptionr

Gingival cervicular fluid ŽGCF. samples were collected from individuals with shallow to moderate periodontal pocket depth Ž3–6 mm. using Periopaper gingival fluid collection strips ŽOraFlow, Plainview, NY.. Strips were kept in place until saturated. This took 30 s less and the estimated volume was 5 ml. Samples were eluted with 100 ml 5% acetic acid with gentle agitation for 30 min at room temperature, then evaporated to dryness and resuspended in 10 ml 0.1% acetic acid. All samples were collected with

2.3. ProteinChip analysis of cell culture supernatants, cell extracts, and biological fluid samples

Fig. 3. Immunoaffinity capture of hBD1 and hBD2 secreted from human gingival epithelial cells. Protein G was covalently attached to a preactivated ŽPS2. ProteinChip array. The spots were then incubated with either preimmune serum or serum containing anti-hBD1 or anti-hBD2. Cell culture supernatants from unstimulated and stimulated cells were then screened for the presence of hBD1 and hBD2. Immunoaffinity capture of hBD1: the 47-amino acid form of hBD1 ŽMW 5068 Da, arrows. was secreted from both unstimulated cells Žpanel a. and stimulated cells Žpanel c.. Immunoaffinity capture of hBD2: hBD2 ŽMW 4328 Da, arrow. was secreted from stimulated cells Žpanel g..

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ionization ŽSELDI. time-of-flight mass spectrometry. Specifically, 3–10 ml cell supernatant from unstimulated and stimulated samples, control media, cell extract, or 2.5 ml of GCF samples was spotted on a variety of chemical surfaces: normal phase ŽNP2. Žhydrophilic surface: SiO 2 ., reverse phase H4 Žhydrophobic surface: C-16 Žlong chain aliphatic., weak cation exchanger ŽWCX2: carboxylate. or strong anion exchanger ŽSAX2: quaternary ammonium. ProteinChip arrays ŽCiphergen Biosystems.. After incubation in a humid chamber for 1 h at room temperature, the chips were subjected to appropriate gradient washes to remove unbound proteins and a final wash with water to remove interfering substances such as salts and detergents. After drying, a 10% saturated solution of alpha-cyano-hydroxy cinnamic acid in 50% acetonitrile Žvrv., 0.5% trifluoroacetic acid Žvrv. was added and mass analysis was performed by time-of-flight mass spectrometry in a Ciphergen Protein Biology System II ŽPBS II.. Studies of cell culture supernatants were done on three separate samples and cell lysates on two samples.

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water and allowed to dry. Alpha-cyano-hydroxy cinnamic acid was added and mass analysis was performed as described above. 2.5. Limits of detection of synthetic hBD1 and hBD2 Synthetic forms of mature hBD1 Ž3927.9 Da. and hBD2 Ž4328.2 Da. were diluted in serum-free media to concentrations of 0.0005–0.5 ngrml, and 10 ml total volume was added to the spots of normal phase ŽNP2. ProteinChip arrays. After incubation for 1 h at room temperature in a humid chamber, the unbound proteins were removed and the chips were washed

2.4. ProteinChip immunoassays The preactivated ProteinChip array ŽPS2. contains a functional group that allows covalent attachment of proteins through free amine groups. Protein G ŽSigma, St. Louis, MO. was coupled to a PS2 chip by adding 2 ml of a stock solution Ž0.5 mgrml in PBS. to 3 ml PBS already on the spots and incubating for 1 h at room temperature in a humid chamber. All subsequent incubations and washes were also done at room temperature. The unreacted sites were blocked with 1M Tris base pH 9.0 for 30 min, then washed for 15 min with PBS containing 0.5% Triton X100 and then for 5 min with PBS. Two microliters of polyclonal anti-hBD1 or anti-hBD2 rabbit serum Ž0.5 mgrml. was added to individual spots and incubated for 1 h in a humid chamber. Preimmune sera were used as controls for non-specific binding. The unbound antibodies were removed by washing with PBS containing 0.1% Triton X100 ŽT–PBS.. A final 2-min wash in PBS was performed prior to adding 5–10 ml of test sample to the spots. After incubation for 1 h in a humid chamber, unbound proteins were removed and the arrays were washed with T–PBS, then PBS. The chips were rinsed with

Fig. 4. Detection of hBD1 and hBD2 in cell lysates. ŽA. Western immunoblot of hBD1 and hBD2 from lysates of either preconfluent or postconfluent gingival epithelial cells that were unstimulated or stimulated with 10 mgrml F. nucleatum cell wall extract. hBD1 Ž44-amino acid form. and hBD2 are also shown. Migration of molecular weight standards Žlane MW. and their sizes Žright. are also shown. Reaction was with polyclonal antiserum or preimmune control as indicated. Note reaction in postconfluent cell cultures. ŽB. Immunoaffinity capture of hBD1 and hBD2 from human gingival epithelial cell lysates. Protein G was covalently attached to a preactivated ŽPS2. ProteinChip array. The spots were then incubated with either preimmune serum or serum containing anti-hBD1 or anti-hBD2. Cell lysates from unstimulated and stimulated cells were then screened for the presence of hBD1 and hBD2. Only stimulated cell lysates are shown. The 47-amino acid form of hBD1 ŽMW 5068 Da, arrow. and the 41-amino acid form of hBD2 ŽMW 4328 Da, arrow. were detected in stimulated cells.

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for 5 min with 0.1 M sodium acetate, pH 4.5 followed by a 1-min wash with MQ water. Alphacyano-hydroxy cinnamic acid was added and mass analysis was performed as described above. Duplicate or triplicate measurements were made for concentrations from 0.0005 to 0.1 ngrml. Single measurements were made for concentrations from 0.175 to 0.5 ngrml. Replicate measurements were averaged and all concentrations were used to generate standard curves for hBD1 and hBD2. 2.6. Immunoblot analysis SDS–Tricine gel electrophoresis and Western immunoblotting were conducted as previously described ŽSahasrabudhe et al., 2000.. Polyclonal antisera to hBD1 and hBD2 were used as previously described ŽValore et al., 1998; Liu et al., 1998.. Standard synthetic hBD1 and hBD2 were obtained from Peptide Institute, Osaka, Japan.

3. Results 3.1. ProteinChip analysis of cell culture supernatants Cell supernatants and control media were analyzed on a variety of ProteinChip array chemical surfaces to characterize protein expression patterns. The mass spectra of proteins bound to a normal phase ŽNP2. chip are shown in Fig. 1. A peak of the predicted mass of hBD1 Ž47-amino acid form, 5068 Da. was detected in media from both unstimulated and stimulated cells ŽFig. 1A and B, respectively.. By contrast, a peak of the predicted mass of hBD2 Ž4328 Da. was detected in the media from stimulated cells, but not unstimulated cells. Neither of these peaks is detected in the media alone Žalso shown in Fig. 1A and B., thus indicating that these proteins originate from the epithelial cells. The biochemical properties of these antimicrobial peptides make them amenable to isolation using a

Fig. 5. Limit of detection of synthetic forms of hBD1 and hBD2 bound to a normal phase ŽNP2. ProteinChip array. Synthetic forms of hBD1 Ž36 amino acids; 3927.9 Da. and hBD2 Ž41 amino acids; 4328.2 Da. were simultaneously spiked into serum-free media at concentrations from 0.0005 to 0.5 ngrml. Synthetic hBD1 with 36 amino acids was used for this purpose because the 47-amino acid form is not available commercially. Peptide solution Ž10 ml. was spotted on a normal phase ŽNP2. chip and processed as described in Fig. 1. ŽA. Representative spectra showing the increase in peak intensity with increasing concentrations of hBD1 and hBD2. The limit of detection for hBD1 was approximately 0.0025 ngrml Ž; 6 fmol. under the experimental conditions described here. The limit of detection for hBD2 was approximately 0.025 ngrml Ž; 58 fmol. under the experimental conditions described here. ŽB. hBD1 standard curve. ŽC. hBD2 standard curve.

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variety of chromatographic matrices. For example, since these peptides are cationic at neutral pH, they may be isolated by ion-exchange chromatography. Alternatively, the amphipathic nature of these peptides makes them amenable to isolation by reversephase chromatography facilitating binding to cationexchange and reverse-phase ProteinChip surfaces. Fig. 2 shows the protein profiles generated by analysis of stimulated cell culture supernatants on weak cation exchange ŽWCX2; Fig. 2A., hydrophobic ŽH4; Fig. 2B., and hydrophilic Žnormal phase ŽNP2.; Fig. 2C. ProteinChip arrays. The results demonstrate the ability to capture different groups of proteins based on the selectivity of the chip surface. Consistent with the biochemical properties of the b-defensins, peaks corresponding to the masses of hBD1 Ž5068 Da. and hBD2 Ž4328 Da. were detected on all three chip surfaces, but surprisingly, detection on the weak

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cationic exchange chip Ž2A. was very limited relative to the background. Neither peak was detected in the media alone thus confirming that these peptides are coming from the cells Ždata not shown.. Since the hydrophilic chip generated the least complex spectra with greatest enrichment of both peaks, all subsequent experiments with chemical surfaces were performed using the normal phase ŽNP2. chip as described in Fig. 1. 3.2. ProteinChip immunoassays 3.2.1. Detection of hBD1 and hBD2 in cell supernatants We confirmed that the peaks detected on the chemical surfaces are in fact hBD1 and hBD2 by specific immunoaffinity capture on ProteinChip arrays containing polyclonal antisera recognizing hBD1

Fig. 6. Analysis of proteins in gingival crevicular fluid ŽGCF.. GCF from two individuals was analyzed for proteins bound to a normal phase ŽNP2. ProteinChip array as described in Section 2. Subject 1 was sampled in two sites. The molecular weight range of 3000–4000 Da is shown in panels A–C. Three peaks corresponding to the predicted masses of the alpha defensins HNP1 Ž3442.09 Da., HNP2 Ž3371.01 Da., and HNP3 Ž3486.10 Da. were identified in each sample Žlabeled a, b and c, respectively.. The molecular weight range of 4000–6000 Da, which contains multiple peaks, is shown for the same individuals in panels D–F.

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and hBD2, respectively ŽFig. 3.. The peak at 5068 Da was specifically captured by anti-hBD1 from both unstimulated and stimulated cell supernatants ŽFig. 3a and c.. Minor hBD1 peptides Ž4751, 4639 and 4537.1 Da. were also detected with longer incubation times Ždata not shown.. hBD2 Ž4328 Da. was only detected in supernatant from stimulated cells Žcompare Fig. 3e and g.. 3.2.2. Detection of hBD1 and hBD2 in cell lysates Cell lysates from unstimulated and stimulated cells were prepared and analyzed by western blotting and immunoaffinity capture on a ProteinChip array ŽFig. 4A and B.. Consistent with mRNA expression analysis, western analysis demonstrated that hBD1 and hBD2 expression was detected in postconfluent cells;

hBD1 was constitutively expressed while hBD2 protein was detected only after stimulation ŽFig. 4A.. Similar results were obtained when the lysates were analyzed on ProteinChip arrays containing polyclonal antisera recognizing hBD1 and hBD2. 3.3. Limits of detection of synthetic hBD1 and hBD2 The limits of detection of synthetic hBD1 Ž36amino acid form. and hBD2 Ž41 amino acids. were determined under the same experimental conditions used to analyze the cell supernatants. hBD1 and hBD2 were spiked into serum-free media at concentrations from 0.0005 to 0.5 ngrml and analyzed on a normal phase ŽNP2. chip. Fig. 5A shows representative spectra demonstrating the increase in peak inten-

Fig. 7. Immunoaffinity capture of hBD1 and hBD2 from gingival crevicular fluid. Samples from two different individuals were profiled as described in Section 2. Human insulin, bovine insulin beta chain, and somatostatin ŽCiphergen Biosystems. were used for external calibration. The molecular weight range of 4200–6000 Da is shown. Several peaks Ždenoted by asterisks. were specifically captured by anti-hBD1 Žcompare panels a and b, c and d, respectively. although the species varied for each individual. Similarly, hBD2 was detected in both individuals Žcompare panels e and f, g and h.. An additional peak of 5717.8 Da was specifically captured by anti-hBD1 in individual a1 Žpanel a.. This mass does not correspond to known forms of hBD1, and the identity is unknown.

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sities with increasing concentrations of hBD1 and hBD2. Although equal amounts of both peptides were present in each case; lower signals were obtained for hBD2 than for the same amount of hBD1. This is not surprising since analyte ion yields are affected by a variety of external factors including the intrinsic properties of the peptides ŽKrause et al., 1999.. The limit of detection was approximately 0.0025 ngrml Ž; 6 fmol. for hBD1 and 0.025 ngrml Ž; 58 fmol. for hBD2. A linear standard curve could be obtained with both hBD1 ŽFig. 5B. and hBD2 ŽFig. 5C.. Above 0.1 ngrml hBD1, the curve was no longer linear. For hBD2, the curve was linear up to the highest concentration tested Ž0.5 ngrml.. 3.4. ProteinChip profiling of gingiÕal creÕicular fluid Since our ultimate goal is to understand the physiologic role of b-defensins in the oral environment, we evaluated the effectiveness of ProteinChip technology to monitor protein secretion in gingival crevicular fluid samples. Fig. 6 shows the results of our initial experiments with GCF samples. The mass spectra of proteins bound to a normal phase ŽNP2. chip showed many peaks in the mass range of hBD1 and hBD2 ŽFig. 6D–F.. Therefore, we used immunoaffinity capture with anti-hBD1 and anti-hBD2

Table 1 b-defensin peptides observed in oral cells or gingival crevicular fluid Amino acids Mass

Mass observeda

Cell lysate

Cell media

GCF

hBD1

5068; 5069.2 ) 4751; 4753.1) 4639; 4639.5 ) 4537.1 4368.9 ) 4328; 4330.1)

q

q q q q

q q q

q Žstim.

q Žstim.

hBD2 a

47 44 43 42 40 41

5074.8 4756.5 4643.3 4372.0 4334.2

q q

Observed mass is 6 Da less than predicted mass due to the presence of three disulfide bonds. The observed masses varied depending on whether an internal calibration or external calibration Žfor GCF, denoted by ) . was performed. The observed mass assignment errors are consistent with the expected performance of this analytical system.

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polyclonal antibodies ŽFig. 7.. Several hBD1 peptides Ž5069.2, 4368.9, 4639.5 and 4753.1 Da. were detected ŽFig. 7A and C, Table 1.. We also detected hBD2 ŽFig. 7E and G.. Finally, proteins corresponding to the predicted masses of the a-defensins HNP 1 Ž3442.09 Da., HNP 2 Ž3371.01 Da., and HNP 3 Ž3486.10 Da. were detected in each of the GCF samples profiled on NP2 ŽFig. 6..

4. Discussion We have used ProteinChip array, surface enhanced laser desorptionrionization ŽSELDI. technology to examine the secretion of b-defensins in an in vitro model and in gingival cervicular fluid. This methodology offers several technical advantages for detection of these peptides; first, the ease and speed of screening to profile biological samples; second, prior processing by HPLC is not required; and third, only a small sample size is required. On the other hand, identification of unknown proteins from mass peaks requires additional studies such as peptide mapping and tandem mass spectrometry, and the method is qualitative or semi quantitative rather than a quantitative one. We show that b-defensins are secreted by GECs and that secretion is detected under conditions anticipated from our previous analysis of GEC expression of b defensin mRNAs—specifically that hBD1 is detected in culture supernatant from both unstimulated and stimulated cells and that hBD2 is detected only in stimulated cells. Expression of both peptides is enhanced in postconfluent cell cultures ŽFig. 4. in agreement with results demonstrating that peptide expression accompanies differentiation in vivo and in vitro ŽDale et al., in press.. hBD1 is secreted primarily as the 5068-Da Ž47-amino acid. form; this is the size predicted by cleavage of the signal sequence ŽNielsen et al., 1997. and accounting for a 6-Da reduction in mass associated with the formation of three disulfide bonds. hBD2 is secreted primarily as the 4328-Da Ž41-amino acid. form consistent with the form originally detected in epidermis and in a cultured epithelial cell line derived from lung ŽHarder et al., 1997, 2000.. We did not detect a peak with the expected mass of the recently identified hBD3 Ž5161

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Da. under these conditions, although mRNA was detected by RT-PCR in GECs Žunpublished data.. The identity of the presumed hBD1 and hBD2 peaks was confirmed by immunoaffinity capture using ProteinChip arrays containing specific polyclonal antibodies. Unlike other techniques, such as enzyme-linked immunosorbent assays ŽELISA. that rely on indirect chemical or radioactive methods of detection, this approach offers the significant advantage that accurate and sensitive detection of captured analytes directly by native mass allows for simultaneous identification of multiple immunoreactive fragments. Our results suggest that epithelial cells secrete primarily the 47-amino acid form of hBD1, but we have also observed several minor peaks corresponding to other hBD1 peptides when longer incubation times were employed. Several forms of hBD1 ranging from 36 to 47 amino acids were also demonstrated in urine ŽValore et al., 1998; Hiratsuka et al., 2000.. These species may represent the secreted form and additional proteolytically cleaved forms that exhibit different profiles of antimicrobial activity against Escherichia coli ŽValore et al., 1998.. However, since the 47-amino acid form is the most prominent species secreted in culture, the shorter cleaved forms may result from extracellular proteolysis, as shown for the Paneth cell a-defensins ŽWilson et al., 1999.. In contrast, only one form of hBD2, the 41-amino acid form, representing the only form of hBD2 reported to date, was captured by the immunoaffinity method. This result is consistent with the single peak in this mass region in the stimulated cell supernatant on the NP2 chip ŽFig. 1.. Experiments with synthetic peptides demonstrate that hBD1 and hBD2 can be detected in femtomole amounts using chemically defined normal phase ŽNP2. arrays. Similar levels of detection were observed using ProteinChip arrays containing polyclonal antibodies recognizing hBD1 and hBD2 Ždata not shown.. The small volume and sensitivity of this technology makes it especially valuable for application to biological tissues and fluids. In this regard, recent studies aimed at investigating the processing of the amyloid precursor protein in Alzheimer’s disease have used a polyclonal antibody on the ProteinChip surface to capture and purify multiple immunoreactive amyloid beta fragments secreted into the media of human cultured cells expressing amy-

loid precursor protein ŽFrears et al., 1999; Davies et al., 1999.. The assay has been successfully extended to the analysis of brain tissue homogenates and serum to study the mechanisms of amyloid beta peptide generation ŽLi et al., 2000; Beher et al., 1999; Vehmas et al., 2000.. Taken together, these observations suggested that GCF, the physiological equivalent of the GEC culture supernatants, should be amenable to similar analyses monitoring secretion and processing of hBD1 and hBD2. The complex profile of peaks in the mass region of b-defensins was difficult to directly interpret, but use of affinity capture allowed detection of hBD2 and several forms of hBD1. We also detected several peaks corresponding to the masses of a-defensin species on the normal phase ŽNP2. surface. However, unambiguous identification of the a-defensins would require further investigation Ži.e. immunoaffinity capture or tandem mass spectrometry.. The a-defensins, derived from neutrophils which migrate into the space between the epithelium and the tooth surface in response to the chemokine IL-8 ŽTonetti et al., 1998., are normal constituents of the GCF. Immunoaffinity capture experiments revealed variation between individuals and sites in the relative amount of hBD2 and the hBD1 peptides. It is interesting to note that more hBD2 and a-defensins, with little hBD1, was seen in the case with greater inflammation Žindividual 1.. This is consistent with increased levels of both neutrophils and hBD2 in inflammation ŽMcKay et al., 1999; Singh et al., 1998.. We show using ProteinChip array technology that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major forms detected in cell lysates and correspond to the secreted forms, respectively, and that several smaller forms of hBD1 are found in gingival crevicular fluid. These studies demonstrate that this technology is a powerful tool for examining defensin secretionrprocessing in vitro and in biological fluids.

Acknowledgements This work was supported by NIH NIDCR grants R01 DE013573 and P60 DE97002.

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