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) RT-PCR product using specific primer for cathelicidin. The PCR product was extracted given the selected primer. Lanes 1 through 5 represent the normal salivary gland tissues; lanes 6-10, the tissues from the glands with chronic sialadenitis. The plus sign indicates positive control from normal lung tissue showing cathelicidin; the minus sign, negative control of RT-PCR amplification without cathelicidin primer of RT; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Comparison of the cathelicidin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio between the tissues from normal salivary glands and the glands with chronic sialadenitis. The expression was significantly increased in the glands with chronic sialadenitis compared with that in the normal salivary glands.
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 ×400.)
Woo J, Jeong JY, Hwang YJ, Chae SW, Hwang SJ, Lee H. Expression of Cathelicidin in Human Salivary Glands. Arch Otolaryngol Head Neck Surg. 2003;129(2):211-214. doi:10.1001/archotol.129.2.211
Salivary secretions play a critical role in maintaining oral health via innate host defense mechanisms and secretion of secretory IgA. One of the antimicrobial peptides, LL-37, is the only cathelicidin protein that has yet been identified in humans. Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens.
To examine the expression of cathelicidin in human salivary glands and to investigate up-regulation of cathelicidin in inflammatory conditions.
Reverse transcriptase–polymerase chain reaction and immunohistochemical staining were performed on 20 salivary gland tissues, 10 from normal salivary glands and 10 from glands with chronic sialadenitis.
Cathelicidin messenger RNA transcripts were detected in the tissues from the normal salivary glands and the glands with chronic sialadenitis. The level of cathelicidin messenger RNA in glands with chronic sialadenitis was significantly increased compared with that in normal salivary glands. Cathelicidin protein was expressed in the glandular epithelium of the normal salivary glands and the glands with chronic sialadenitis.
The results indicate that cathelicidin might play an important role in innate host defense of human salivary glands.
THE ORAL CAVITY and the ducts of the salivary glands are open to the oral environment. They are exposed to a variety of biological, chemical, and mechanical insults. Oral mucosal and salivary glandular defenses range from simple mechanical rinsing by salivary flow to complex mechanisms of host defense and adaptive immunity. The interplay of these defenses keeps the oral environment and salivary gland relatively disease free in spite of constant challenges.
Antimicrobial peptides or proteins have a significant role in innate host defenses in many human organs. Their role is killing bacteria, which invade or reside as natural microbial flora.1 The antimicrobial peptides that have been identified in humans are salivary histatin, lactoferricin, defensins, and the human antimicrobial protein hCAP182 (also called LL-37), which is the only cathelicidin family protein that has yet been identified in humans. It was cloned from complementary DNA isolated from human bone marrow, and it is primarily expressed in neutrophil granulocytes.3- 5 Their expressions were reported in testis, bone marrow, and lung and the squamous epithelia of the mouth, tongue, esophagus, cervix, and vagina.2,3 However, to our knowledge, there have been no reports about the expression of cathelicidin in the human salivary glands. We postulated that the salivary gland is likely to be protected by cathelicidin and tested the hypothesis that cathelicidin is expressed in salivary gland tissue by using reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical staining.
Tissues were obtained from surgical biopsy procedures performed at the Department of Otorhinolaryngology–Head and Neck Surgery, Korea University College of Medicine, Seoul. Salivary tissues were recovered from excision of parotid glands from 10 patients with chronic sialadenitis, and distant normal salivary tissues were obtained from 10 patients with benign parotid gland tumors. One portion was immediately flash frozen in liquid nitrogen and stored at −70°C for subsequent RNA studies. For RT-PCR–positive control of cathelicidin, lung epithelium was prepared in the same manner. Another portion was fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline (pH 7.4), stored overnight at 4°C, and then embedded in paraffin for immunohistochemical staining. Informed consent had been given by all patients. The tissue procurement procedures were approved by the institutional review board at Korea University.
Tissue was homogenized in 1 mL of Trizol reagent (Gibco BRL, Tucson, Ariz), and RNA was extracted according to the manufacturer's instructions. Samples were air-dried and resuspended in water treated with diethyl pyrocarbonate and were kept on ice for immediate use or stored at −70°C. Aliquots of RNA were treated with RQ1 RNAase-free Dnase (Promega, Madison, Wis) according to the manufacturer's instructions. Concentrations of RNA were determined spectrophotometrically, and the integrity was checked by electrophoresis in agarose gels containing formaldehyde.
The total RNA from each sample was reverse-transcribed in 20 µL of reaction mixture containing 2.5 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL) and 50 pmol of random hexanucleotides at 42°C for 60 minutes. Based on the published sequences, oligonucleotide primers were synthesized commercially at Bioneer Co (Daejon, South Korea) for PCR as follows: 5′-GAA GAC CCA AAG GAA TGG CC-3′ and 5′-CAG AGC CCA GAA GCC TGA GC-3′ for LL-37 and 5′-GTG GAT ATT GTT GCC ATC AAT GAC C-3′ and 5′-GCC CCA GCC TTC ATG GTG GT-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Amplification of the complementary DNA was carried out using 35 cycles at 94°C 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. Specificity of the 570–base pair (bp) PCR product was verified by the predicted size, restriction digestion, and DNA sequencing. To establish the specificity of responses, negative controls were used in which input RNA was omitted or in which RNA was used but reverse transcriptase was omitted. As a positive control, messenger RNA (mRNA) was extracted from lung tissues known to express cathelicidin. To ensure RNA quality, all preparations were subjected to analysis of GAPDH expression. To analyze semiquantitatively the result of RT-PCR, we scanned the gel images and measured the intensity of the PCR product through use of NIH Image software (National Institutes of Health, Bethesda, Md). We determined the relative intensity of individual bands on a gel image as the ratio of the intensity of cathelicidin to the intensity of GAPDH.
The paraffin blocks were sliced into 5-µm-thick sections. Deparaffinization with xylene and rehydration with 100% and then 75% alcohol were done serially. Paraffin sections from the paraformaldehyde-fixed salivary tissues were treated with 3% hydrogen peroxide methanol to block endogenous peroxidase and were incubated with a rabbit polyclonal antibody to cathelicidin (a generous gift of Dr Ole Sorenson, Granulocyte Research Laboratory, National University Hospital, Copenhagen Denmark) was used at a dilution of 1:100 and incubated overnight at 4°C in a humidified chamber. Immunoreactive cathelicidin was visualized with a Vectastatin Elite ABC Kit (Vector Lab Inc, Burlingame, Calif). Controls included the substitution of primary or secondary antibody with phosphate-buffered saline.
Data were expressed as the mean ± SEM. Comparisons of quantitative data between 2 groups were analyzed with the Mann-Whitney test. Differences were considered significant for P values less than .05.
The RT-PCR studies showed that salivary tissue contained mRNA encoding for cathelicidin. The PCR products from the salivary tissue had the size (570 bp) that was expected from the selected primers. The same-sized product was expressed in the positive control (Figure 1). There was significant difference in the amount of cathelicidin mRNA expression between the tissues of the chronic sialadenitis and normal salivary glands. The ratio was 2.69 ± 0.28 for the chronic sialadenitis gland group and 0.36 ± 0.14 for the normal salivary gland group. The difference between the 2 groups was statistically significant (P = .009) (Figure 2).
Immunostaining showed that cathelicidin expression was localized to the ductal cells and to the inflammatory cells of chronic sialoadenitis (Figure 3A) and normal salivary glands (Figure 3B); acinar cells were uniformly negative for cathelicidin staining (Figure 3D). There was no specific localization with negative control, confirming the specificity of the cathelicidin antibody (Figure 3C).
Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. In contrast to highly specific adaptive immunity, the innate immune system provides a rapid and nonspecific response and thereby contributes to the first line of defense. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system (nonadaptive immune system) and serve a key protective role in the host defense. They are acting as effector molecules with the capacity to kill a broad spectrum of microorganisms.1 The peptides are strategically located at sites exposed to microorganisms such as the airway and gastrointestinal tract and in phagocytes. The first peptide antibiotics were discovered in the 1980s when cecropins were isolated from insects6 and defensins were isolated from rabbit macrophages.7 Numerous antimicrobial peptide antibiotics occur in nature, and over a dozen have been identified in humans, including several salivary histatins, lactoferricin, 6 α-defensins, 2 β-defensins, and the human cationic antimicrobial protein hCAP18.2
The hCAP18 protein (human cationic antimicrobial protein of 18 kd) is a newly described protein of human neutrophilic granulocytes, which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The family of antimicrobial peptides, whose members contain this conserved N-terminus, are provisionally called the cathelicidins.8 Four cathelicidins have been identified in bovine neutrophils and 9 in porcine neutrophils. However, only 1 cathelicidin, hCAP18, is found in human neutrophilic granulocytes.3- 5
The hCAP18 protein is synthesized in neutrophil progenitor, myelocytes, and metamyelocytes in the bone marrow and stored in the peroxidase-negative granules of mature neutrophils.9 It is synthesized as an 18-kd proprotein from which a 5-kd C-terminal fragment, LL-37, bearing all of the hitherto known biological activity, is cleaved.10 The plasma level of cathelicidin is 1.18 µg/mL, which is severalfold higher than that for other specific granule proteins of neutrophils. The cathelicidin is present in plasma as high-molecular-weight complexes.11 The cathelicidin is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.12 The cysteine-free peptide LL-37 can adopt an amphipathic α-helical conformation, has lipopolysaccharide-binding properties, and manifests antibacterial effect against a wide range of bacterial species through C-terminal domain.13,14 The bacterial-killing properties of cathelicidin are synergistic with lactoferrin and lysozyme.14 The promotor region of the cathelicidin gene contains potential binding site for transcription factors, the acute-phase response factor, and the nuclear factor for interleukin 6.10 Interleukin 6 may play an important role in modulating cathelicidin gene expression.15,16
In the present study, we demonstrated the antimicrobial peptide cathelicidin mRNA and protein in human salivary glands. Cathelicidin is primarily detected in the duct cells of the gland, so cathelicidin can be one of the protection materials in salivary gland itself. Because ducts of the salivary glands are open to the oral environment, they are open to environmental insults from bacteria, viruses, and fungi. There is opportunity for retrograde infection into the gland, which is essentially a cul de sac. Cathelicidin could be active at the point of entry for oral bacteria, thus acting as a first-line host defense for this vulnerable space as well as having a functional role as a secreted protein in saliva. By immunohistochemical staining, the cathelicidin protein was found to be localized predominantly in the duct cells of salivary gland. This indicates that cathelicidin, which was previously found in the lung, squamous epithelia of the mouth, tongue, esophagus, and vagina, and nasopharynx,2,4 is constitutively expressed in the salivary gland, a site of constant microorganism challenge. By RT-PCR, the cathelicidin mRNA was detected in all cases of chronic sialadenitis and had an increased expression compared with that in normal salivary glands. This indicates that the expression of the cathelicidin gene seems to be not only constitutive but also induced during inflammation.17
Recently, human cathelicidin has been shown to have chemotactic effects on host cells. It is chemotactic for neutrophils, monocytes, and CD4 T cells.18 The chemotactic activity of cathelicidin is mediated by formyl peptide receptor-like 1.18 The capacity of cathelicidin to mobilize various types of phagocytic cells with other effects provide evidence for their participation in alerting, mobilizing, and amplifying innate and adaptive antimicrobial immunity of the host.
In the present study, we showed the localizing expression of the human cathelicidin in salivary glands and up-regulation in the inflammatory conditions. This finding suggested the physiologic importance of cathelicidin in the defense of retrograde infection. Other functions for this broadly expressed peptide and possible expression of cathelicidin in saliva need to be evaluated.
Corresponding author and reprints: Heung-Man Lee, MD, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul 152-703, South Korea (e-mail: firstname.lastname@example.org).
Accepted for publication July 9, 2002.