Expression of Cathelicidin in Human Salivary Glands

ORIGINAL ARTICLE Expression of Cathelicidin in Human Salivary Glands

Jeong-Su Woo, MD; Ji Yong Jeong, MD; You Jin Hwang, PhD; Sung Won Chae, MD; Soon Jae Hwang, MD; Heung-Man Lee, MD, PhD

Background: Salivary secretions play a critical role in salivary glands and 10 from glands with chronic sialad- maintaining oral health via innate host defense mecha- enitis. nisms and secretion of secretory IgA. One of the antimicrobial peptides, LL-37, is the only cathelicidin protein Results: Cathelicidin messenger RNA transcripts were that has yet been identified in humans. Cathelicidins detected in the tissues from the normal salivary glands are a family of peptides thought to provide an innate de- and the glands with chronic sialadenitis. The level of ca- fensive barrier against a variety of potential microbial thelicidin messenger RNA in glands with chronic sialad- pathogens. enitis was significantly increased compared with that in normal salivary glands. Cathelicidin protein was ex- Objectives: To examine the expression of cathelicidin pressed in the glandular epithelium of the normal sali- in human salivary glands and to investigate vary glands and the glands with chronic sialadenitis. up-regulation of cathelicidin in inflammatory conditions. Conclusion: The results indicate that cathelicidin might play an important role in innate host defense of human Design: Reverse transcriptase–polymerase chain reac- salivary glands. tion and immunohistochemical staining were performed on 20 salivary gland tissues, 10 from normal Arch Otolaryngol Head Neck Surg. 2003;129:211-214

HE ORAL CAVITY and the row, and it is primarily expressed in ducts of the salivary glands neutrophil granulocytes.3-5 Their expres- are open to the oral envi- sions were reported in testis, bone mar- ronment. They are ex- row, and lung and the squamous epithelia posed to a variety of bio- of the mouth, tongue, esophagus, cervix, logical,T chemical, and mechanical insults. and vagina.2,3 However, to our knowl- Oral mucosal and salivary glandular de- edge, there have been no reports about the fenses range from simple mechanical rins- expression of cathelicidin in the human sali- ing by salivary flow to complex mecha- vary glands. We postulated that the sali- nisms of host defense and adaptive vary gland is likely to be protected by cat- immunity. The interplay of these de- helicidin and tested the hypothesis that fenses keeps the oral environment and sali- cathelicidin is expressed in salivary gland vary gland relatively disease free in spite tissue by using reverse transcriptase– of constant challenges. polymerase chain reaction (RT-PCR) and Antimicrobial peptides or proteins immunohistochemical staining. From the Department of have a significant role in innate host de- Otorhinolaryngology–Head and fenses in many human organs. Their role Neck Surgery, Communication is killing bacteria, which invade or reside METHODS Disorder Institute of Medical as natural microbial flora.1 The antimi- Research Center, Korea crobial peptides that have been identified TISSUE SAMPLES University College of Medicine, in humans are salivary histatin, lactoferri- Seoul (Drs Woo, Jeong, Chae, cin, defensins, and the human antimicro- Tissues were obtained from surgical biopsy pro- Soon Jae Hwang, and Lee), and 2 cedures performed at the Department of Oto- Department of Molecular bial protein hCAP18 (also called LL- rhinolaryngology–Head and Neck Surgery, Ko- Biology, Gil Medical Center, 37), which is the only cathelicidin family rea University College of Medicine, Seoul. Gachon Medical School, Inchon protein that has yet been identified in hu- Salivary tissues were recovered from excision City, Korea (Dr You Jin mans. It was cloned from complemen- of parotid glands from 10 patients with chronic Hwang). tary DNA isolated from human bone mar- sialadenitis, and distant normal salivary tis-

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Cathelicidin 530 bp 3.0

GAPDH 248 bp

+ – 2.0 Normal Chronic Sialadenitis

Figure 1. Expression of cathelicidin in human salivary gland by reverse transcriptase–polymerase chain reaction (RT-PCR). Ethidium bromide–stained agarose gel showing the presence of 570–base pair (bp) 1.0 RT-PCR product using specific primer for cathelicidin. The PCR product was extracted given the selected primer. Lanes 1 through 5 represent the normal Cathelicidin/GAPDH mRNA Ratio salivary gland tissues; lanes 6-10, the tissues from the glands with chronic sialadenitis. The plus sign indicates positive control from normal lung tissue 0 showing cathelicidin; the minus sign, negative control of RT-PCR Normal Chronic amplification without cathelicidin primer of RT; and GAPDH, Sialadenitis glyceraldehyde-3-phosphate dehydrogenase. Figure 2. Comparison of the cathelicidin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio between the tissues sues were obtained from 10 patients with benign parotid from normal salivary glands and the glands with chronic sialadenitis. The gland tumors. One portion was immediately flash frozen in expression was significantly increased in the glands with chronic sialadenitis liquid nitrogen and stored at −70°C for subsequent RNA stud- compared with that in the normal salivary glands. ies. For RT-PCR–positive control of cathelicidin, lung epithelium was prepared in the same manner. Another portion was the gel images and measured the intensity of the PCR product fixed with 4% paraformaldehyde in 0.1M phosphate-buffered through use of NIH Image software (National Institutes of saline (pH 7.4), stored overnight at 4°C, and then embedded Health, Bethesda, Md). We determined the relative intensity in paraffin for immunohistochemical staining. Informed con- of individual bands on a gel image as the ratio of the intensity sent had been given by all patients. The tissue procurement of cathelicidin to the intensity of GAPDH. procedures were approved by the institutional review board at Korea University. IMMUNOHISTOCHEMICAL STAINING FOR CATHELICIDIN PROTEIN EXTRACTION OF RNA The paraffin blocks were sliced into 5-µm-thick sections. De- Tissue was homogenized in 1 mL of Trizol reagent (Gibco BRL, paraffinization with xylene and rehydration with 100% and then Tucson, Ariz), and RNA was extracted according to the manu- 75% alcohol were done serially. Paraffin sections from the para- facturer’s instructions. Samples were air-dried and resus- formaldehyde-fixed salivary tissues were treated with 3% hy- pended in water treated with diethyl pyrocarbonate and were drogen peroxide methanol to block endogenous peroxidase and kept on ice for immediate use or stored at −70°C. Aliquots of were incubated with a rabbit polyclonal antibody to cathelici- RNA were treated with RQ1 RNAase-free Dnase (Promega, Madi- din (a generous gift of Dr Ole Sorenson, Granulocyte Re- son, Wis) according to the manufacturer’s instructions. Con- search Laboratory, National University Hospital, Copenhagen centrations of RNA were determined spectrophotometrically, Denmark) was used at a dilution of 1:100 and incubated over- and the integrity was checked by electrophoresis in agarose gels night at 4°C in a humidified chamber. Immunoreactive cathe- containing formaldehyde. licidin was visualized with a Vectastatin Elite ABC Kit (Vector Lab Inc, Burlingame, Calif). Controls included the substitu- REVERSE TRANSCRIPTASE–POLYMERASE tion of primary or secondary antibody with phosphate- CHAIN REACTION buffered saline. The total RNA from each sample was reverse-transcribed in 20 µL of reaction mixture containing 2.5 U of Moloney murine STATISTICAL ANALYSIS leukemia virus reverse transcriptase (Gibco BRL) and 50 pmol Data were expressed as the mean±SEM. Comparisons of quan- of random hexanucleotides at 42°C for 60 minutes. Based on titative data between 2 groups were analyzed with the Mann- the published sequences, oligonucleotide primers were syn- Whitney test. Differences were considered significant for P val- thesized commercially at Bioneer Co (Daejon, South Korea) for ues less than .05. PCR as follows: 5Ј-GAA GAC CCA AAG GAA TGG CC-3Ј and 5Ј-CAG AGC CCA GAA GCC TGA GC-3Ј for LL-37 and 5Ј- GTG GAT ATT GTT GCC ATC AAT GAC C-3Ј and 5Ј-GCC RESULTS CCA GCC TTC ATG GTG GT-3Ј for glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Amplification of the REVERSE TRANSCRIPTASE–POLYMERASE complementary DNA was carried out using 35 cycles at 94°C CHAIN REACTION for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a final extension cycle of 72°C for 7 minutes. Speci- The RT-PCR studies showed that salivary tissue con- ficity of the 570–base pair (bp) PCR product was verified by tained mRNA encoding for cathelicidin. The PCR prod- the predicted size, restriction digestion, and DNA sequencing. ucts from the salivary tissue had the size (570 bp) that To establish the specificity of responses, negative controls were was expected from the selected primers. The same-sized used in which input RNA was omitted or in which RNA was used but reverse transcriptase was omitted. As a positive con- product was expressed in the positive control (Figure 1). trol, messenger RNA (mRNA) was extracted from lung tissues There was significant difference in the amount of cathe- known to express cathelicidin. To ensure RNA quality, all prepa- licidin mRNA expression between the tissues of the rations were subjected to analysis of GAPDH expression. To chronic sialadenitis and normal salivary glands. The ra- analyze semiquantitatively the result of RT-PCR, we scanned tio was 2.69±0.28 for the chronic sialadenitis gland group

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C D

∗

Figure 3. The expression of cathelicidin in human salivary glands. Immunohistochemical staining using polyclonal antibody against cathelicidin reveals strong immunoreactivity for cathelicidin in the ductal epithelia of a gland with chronic sialadenitis (A) and normal salivary gland (B). There is no immunoreactivity for cathelicidin in negative control (C) and acinus (asterisk) (D). (Original magnification ϫ400.)

and 0.36±0.14 for the normal salivary gland group. The microorganisms.1 The peptides are strategically located difference between the 2 groups was statistically signifi- at sites exposed to microorganisms such as the airway cant (P=.009) (Figure 2). and gastrointestinal tract and in phagocytes. The first peptide antibiotics were discovered in the 1980s when ce- IMMUNOHISCTOCHEMICAL STAINING cropins were isolated from insects6 and defensins were OF CATHELICIDIN isolated from rabbit macrophages.7 Numerous antimicrobial peptide antibiotics occur in nature, and over a Immunostaining showed that cathelicidin expression was dozen have been identified in humans, including sev- localized to the ductal cells and to the inflammatory cells eral salivary histatins, lactoferricin, 6 ␣-defensins, 2 ␤-de- of chronic sialoadenitis (Figure 3A) and normal sali- fensins, and the human cationic antimicrobial protein vary glands (Figure 3B); acinar cells were uniformly nega- hCAP18.2 tive for cathelicidin staining (Figure 3D). There was no The hCAP18 protein (human cationic antimicro- specific localization with negative control, confirming the bial protein of 18 kd) is a newly described protein of hu- specificity of the cathelicidin antibody (Figure 3C). man neutrophilic granulocytes, which belongs to the cathelicidin family of antimicrobial proteins. Members of COMMENT this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cat- Innate immunity is important for the integrity of the host ionic C-terminus. The family of antimicrobial peptides, against potentially invasive pathogenic microorganisms whose members contain this conserved N-terminus, are in the environment. In contrast to highly specific adap- provisionally called the cathelicidins.8 Four cathelici- tive immunity, the innate immune system provides a rapid dins have been identified in bovine neutrophils and 9 in and nonspecific response and thereby contributes to the porcine neutrophils. However, only 1 cathelicidin, first line of defense. Antibiotic peptides with broad an- hCAP18, is found in human neutrophilic granulo- timicrobial activity are part of the innate immune sys- cytes.3-5 tem (nonadaptive immune system) and serve a key pro- The hCAP18 protein is synthesized in neutrophil tective role in the host defense. They are acting as effector progenitor, myelocytes, and metamyelocytes in the bone molecules with the capacity to kill a broad spectrum of marrow and stored in the peroxidase-negative granules

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 of mature neutrophils.9 It is synthesized as an 18-kd pro- din in the defense of retrograde infection. Other func- protein from which a 5-kd C-terminal fragment, LL-37, tions for this broadly expressed peptide and possible bearing all of the hitherto known biological activity, is expression of cathelicidin in saliva need to be evaluated. cleaved.10 The plasma level of cathelicidin is 1.18 µg/ mL, which is severalfold higher than that for other spe- Accepted for publication July 9, 2002. cific granule proteins of neutrophils. The cathelicidin is Corresponding author and reprints: Heung-Man Lee, present in plasma as high-molecular-weight com- 11 MD, PhD, Department of Otorhinolaryngology–Head and plexes. The cathelicidin is processed to the antimicro- Neck Surgery, Guro Hospital, Korea University College of bial peptide LL-37 by extracellular cleavage with pro- 12 Medicine, 80 Guro-dong, Guro-gu, Seoul 152-703, South teinase 3. The cysteine-free peptide LL-37 can adopt an Korea (e-mail: [email protected]). amphipathic ␣-helical conformation, has lipopolysaccharide-binding properties, and manifests antibacterial effect against a wide range of bacterial species through REFERENCES C-terminal domain.13,14 The bacterial-killing properties

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