International Journal of Pediatric Otorhinolaryngology (2006) 70, 487—492
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Expression of cathelicidin in recurrent throat infection Jae-Jun Song a, Kyu Sung Hwang b, Jeong Soo Woo a, Sung Won Chae a, Jae Gu Cho a, Hee Joon Kang a, Soon Jae Hwang a, Heung-Man Lee a,* a
Department of Otorhinolaryngology - Head and Neck Surgery and Communication Disorder, Institute of Medical Science Research Center, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, 152-703 Seoul, South Korea b Gachon Medical School Gil Hospital, Inchon, South Korea Received 30 May 2005; accepted 30 July 2005
KEYWORDS Cathelicidin; Tonsil; Antimicrobial peptide
Summary Background: Epithelial cells can be called the first line of a defense barrier to microorganisms by the innate immune system. The antimicrobial peptides are the major participants of this system. Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens. Objectives: To evaluate the expression of the cathelicidin in recurrent throat infection. Patients and methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed for 10 palatine tonsil tissues with hypertrophy and 10 palatine tonsil tissues with recurrent throat infection. Results: Cathelicidin mRNA transcripts were detected in recurrent throat infection. The expression levels of cathelicidin mRNA in recurrent throat infection was significantly higher compared with those in hypertrophic tonsils. Cathelicidin protein was localized on the tonsillar surface epithelium and inflammatory cells in the tonsillar crypt of recurrent throat infection patients. Conclusion: These results suggest that cathelicidin is one of antimicrobial peptides in the human palatine tonsils, and that cathelicidin may also play an important role in innate host defense of human tonsils. # 2005 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
* Corresponding author. Tel.: +82 2 818 6750; fax: +82 2 868 0475. E-mail address:
[email protected] (H.-M. Lee).
The tonsils and adenoids are situated at the entrance of the respiratory and alimentary tracts and represent the first site of contact with a variety of microorganisms and other antigenic substances
0165-5876/$ — see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2005.07.025
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present in food and inhaled air. Therefore, human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles [1]. Tonsillar epithelia protect against microbial invasion. This defense function of the epithelial layer is conducted by the innate immune system and provides a rapid and early response in the host defense system. The antimicrobial peptides are the major participants of this innate immune system and are produced in the various respiratory epithelial cell layers. Their role is to kill the bacteria which invade the respiratory mucosa. Antimicrobial peptides which have been identified in humans are: salivary histatins, lactoferricin, defensins, and the human cationic antimicrobial protein hCAP18 [2,3]. hCAP18, which is also called LL-37, is the only cathelicidin family protein so far identified in humans. Its expression has been reported in testis, bone marrow, lung, and the squamous epithelia of the mouth, tongue, esophagus, cervix, and vagina [4]. However, there have been no reports about the localization of cathelicidin in the human palatine tonsil. The purpose of this study was to evaluate the differential expression of cathelicidin in recurrent throat infection and in non-inflamed tonsils through reverse transcriptase-polymerase chain reaction (RT-PCR), and to localize the cathelicidin protein by immunohistochemistry.
2. Patients and methods 2.1. Patients The study group was composed of 10 children (five males and five females, age range 9—13 years) who underwent tonsillectomy for recurrent throat infection. The children were severely affected with recurrent throat infection, i.e. seven or more throat infections in the preceding year, five or more in each of the previous 2 years, or three or more in each of the previous 3 years (Paradise criteria) [5]. The control group comprised 10 children (five males and five females, age range 7—12 years) who underwent surgery for obstructive sleep apnea due to tonsillar hypertrophy. The patients undergoing tonsillectomy for tonsillar hypertrophy had 3+ or 4+ hypertrophy on a scale described by Brodsky et al [6]. None of the children in the control group fulfilled the criteria for recurrent throat infections,
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Table 1
Clinical characteristics of patients
Characteristics
Recurrent throat infection (n = 10)
Tonsillar hypertrophy (n = 10)
Mean (S.D.) age (months)
134 (18.1)
112 (15.3)
5 5
5 5
6 (4—8)
1 (0—3)
7 (4—10)
5 (0—8)
Sex Male Female Median number of counting episodes in each of past 3 years (range) a Median duration (days) of each counting episode (range)
a Counting episodes are those characterized by one or more of the following features: oral temperature of at least 38.3 8C; cervical lymphadenopathy (enlarged > 2 cm) or tender cervical lymph nodes; tonsillar or pharyngeal exudates; or a positive culture for Group A beta-hemolytic streptococcus.
had evidence of tonsillar infections pre-operatively, and none had an upper respiratory infection in the 6 weeks preceding tonsillectomy. None of the patients in the study group and the control group had any history of allergy, and none were receiving any ongoing drug treatment. Clinical characteristics of the patients in the study group and the control group are summarized in Table 1.
2.2. Tissue preparations Palatine tonsils were obtained from 20 patients who underwent tonsillectomy. Tissues were cut into two portions. One portion was immediately frozen in liquid nitrogen and stored at 70 8C for subsequent RNA process. For immunohistochemical staining, the second portion was fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer saline (PBS, pH 7.4) overnight at 4 8C and then embedded in paraffin. Informed consents had been given by all patients. The tissue procurement procedures were approved by the Committee of Ethics, Korea University College of Medicine.
2.3. Cathelicidin mRNA isolation and reverse transcriptase-polymerase chain reaction The tonsil epithelium was selectively collected for cell suspension. Total RNA was extracted from palatine tonsil specimens using TRIzol1 (Gibco BRL, Grand Island, NY) according to the manufacturer’s suggestion. Total RNA from each sample was
Cathelicidin in tonsil
reverse-transcribed in 20 ml of reaction mixture containing 2.5 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Gibco BRL, Grand Island, NY) and 50 pM random hexanucleotides at 42 8C for 60 min. Based on the published sequences, oligonucleotide primers for PCR were synthesized commercially (Bioneer Co., Daejon, South Korea): cathelicidin sense primer 50 -GAA GAC CCA AAG GAA TGG CC-30 and cathelicidin antisense primer 50 -CAG AGC CCA GAA GCC TGA GC-30 . Amplification of the cDNA was carried out using 35 cycles at 94 8C for 45 s, 55 8C for 30 s, and 72 8C for 1 min followed by a final extension cycle of 72 8C for 7 min. Specificity of the 570 bp PCR product was verified by predicted size, restriction digestion and DNA sequencing. As negative controls, cathelicidin primer or reverse transcriptase were omitted from some RT-PCR reactions. For the positive control, messenger RNA (mRNA) was extracted from lung tissues known to express cathelicidin. The primers 50 -GTG GAT ATT GTT GCC ATC AAT GAC C-30 and 50 -GCC CCA GCC TTC ATG GTG GT-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were run simultaneously as an internal control (the expected size of this PCR product was 270 bp). The PCR product was analyzed by gel electrophoresis. To analyze semiquantitatively the result of RTPCR, we scanned the gel images and measured the intensity of the PCR product through use of NIH Image software (National Institute 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.
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block endogenous peroxide and were incubated with a polyclonal antibody to cathelicidin (a generous gift of Dr. Ole Sorensen, Granulocyte Research Laboratory, National University Hospital, Denmark) for 1 h at a dilution of 1:500. Immunoreactive cathelicidin was visualized with the Vectastain Elite ABC kit (Vector Lab Inc., Burlingame, CA) by using 3-amino-9-ethylcarbazole as a chromogen. Negative control included the substitution of primary antibody with PBS or non-immune serum IgG.
2.5. Statistical analysis Data were expressed as the mean S.E.M. Comparisons of quantitative data between two groups were analyzed with the Mann—Whitney test. Differences were considered significant for P values less than 0.05.
3. Results 3.1. Reverse transcriptase-polymerase chain reaction RT-PCR showed prominent amplification products of the predicted size (570 bp) representing cathelicidin transcripts in all recurrent throat infection cases (Fig. 1). There was a statistically significant difference in the amount of cathelicidin mRNA expression between the tissue from recurrent throat infection and hypertrophied tonsils (P < 0.05) (Fig. 2).
2.4. Immunolocalization of cathelicidin
3.2. Immunohistochemical localization of cathelicidin
The paraffin blocks were sliced into 5 mm thickness. Deparaffinization with xylene and rehydration with 100%, and then 75% alcohol was done serially. Paraffin sections from the paraformaldehyde-fixed tonsil tissues were treated with 0.3% H2O2/methanol to
The tissue distribution of cathelicidin was analyzed by immunohistochemical staining. Positively immunostained cells appeared red. The non-keratinizing squamous epithelium of tonsil was immunostained positively for cathelicidin in the recurrent throat
Fig. 1 Expression of cathelicidin in human palatine tonsils by reverse transcriptase-polymerase chain reaction (RTPCR). Ethidium bromide-stained agarose gel showing the presence of 570 base pair (bp) RT-PCR product using a specific primer for cathelicidin. The PCR product was extracted given the selected primer. The (+) sign indicates positive control from normal lung tissue showing cathelicidin; the () sign, negative control of RT-PCR amplification without cathelicidin primer of RT; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 2 Comparison of the cathelicidin/glyceraldehyde3-phosphate dehydrogenase (GAPDH) messenger RNA ratio between the tonsillar tissues from hypertrophied tonsil and recurrent throat infection. The expression was significantly increased in the tonsils with recurrent throat infection compared with that in the hypertrophied tonsil.
infection group (Fig. 3). The inflammatory cells in crypt of tonsil were stained more strongly stained in tissues with recurrent throat infection. There was no specific localization of immunostained cells in the negative control, confirming the specificity of the cathelicidin antibody.
4. Discussion In this study, we investigated whether LL-37 is expressed in human tonsils under inflammatory
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and noninflammatory conditions. We detected expression of cathelicidin mRNA by RT-PCR in all the specimens studied. There was a significant increase in cathelicidin expression in the recurrent throat infection group compared with the tonsillar hypertrophy group. In addition, cathelicidin is localized in the surface epithelium and inflammatory cells in crypts. This suggests that cathelicidin is not only constitutively expressed in the human palatine tonsil, but is also upregulated during chronic inflammation, and may play a role in the host defense. The tonsil epithelia are consistently exposed to environmental pathogens and are particularly vulnerable to damage and infection. The epithelium of the palatine tonsils is stratified and is characterized as lymphoepithelium, which consists not only of epithelial cells but also of non-epithelial cells, lymphocytes, macrophages, and dendritic cells [7]. Until now, immunoglobulins (Ig), and secretory neutrophils in the surface of tonsil were thought to be the main factors in the defense of tonsilar surface from frequently encountered microorganisms. Secretory IgA is an important first-line defense system that protects the body against invasion by microorganisms and the entrance of foreign molecules. Unfortunately, palatine tonsils have no secretory IgA for lack of a secretory component. The only surface protection of the tonsil is mediated by antibodies that passively diffuse into the lumen by intercellular epithelial diffusion [8]. Because these antibodies are induced by antigenic stimulation, it is not feasible
Fig. 3 Expression of cathelicidin in human palatine tonsils. Immunohistochemical staining reveals strong immunoreactivity for cathelicidin in the surface epithelium, in inflammatory cells and in secreted cathelicidin in crypt of tonsils with recurrent throat infection (Original magnification 400).
Cathelicidin in tonsil
to use these antibodies as an immediate protection mechanism of the tonsillar surface. For the prevention of microorganism’s entrance into the parenchyme of tonsil, another protection mechanism is needed in the surface epithelium. And the antimicrobial peptides are the major participants of this innate immune system and are produced in the tonsillar epithelial cell layer. The hCAP18 protein is a newly described protein of human neutrophilic granulocytes, which belongs to the cathelicidin family of antimicrobial proteins. Four kinds of cathelicidins have been identified in bovine neutrophils and nine in porcine neutrophils. However, only one cathelicidin, hCAP18, is found in human neutrophilic granulocytes [9—11]. Cathelicidin exhibits strong antibiotic activity against a number of gram-positive and gram-negative organisms including group A streptococcus; the bactericidal effect of cathelicidin is synergistic with lactoferrin and lysozyme [12]. Cathelicidin is upregulated in chronic inflammation of human keratinocyte. Frohm et al. demonstrated that the expression levels of cathelicidin mRNA and protein were increased in keratinocytes from inflamed skin in psoriasis patients and psoriatic scales were shown to contain cathelicidin [13]. Cathelicidin is also upregulated in inflammations of respiratory mucosa. Tracheal aspirates from newborns with pulmonary or systemic infections were demonstrated to contain increased levels of cathelicidin compared with non-infected newborns [14]. In addition, cathelicidin is known to be induced by H. influenza infection in pharyngeal cells and upregulated in chronic nasal inflammatory disease [15,16]. In contrast, Islam et al. demonstrated that Shigella infection decreased cathelicidin expression in colon epithelial cells both in vitro and in vivo. These bacterial effects on LL-37 expression were suggested to be mediated via bacterial DNA [17]. Nell et al. demonstrated the increased expression of cathelicidin in nasal sinus epithelium in inflammation and the relationship with inflammation via stimulation of IL-8 released by epithelial cells [18]. In this study, we demonstrated that the chronic tonsillar inflammation upregulates the expression of cathelicidin in tonsillar epithelium. Thus, cathelicidin may play an important role in infection and inflammation in tonsil. By immunohistochemistry, the cathelicidin protein was localized predominantly in the surface epithelium and inflammatory cells in the crypt of the palatine tonsil. This indicates that cathelicidin, which was previously found in the lung, squamous epithelium of the mouth, tongue, esophagus, and vagina, and nasopharynx is expressed in the palatine tonsil, a site of constant microorganism challenge
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[15,19]. In addition, cathelicidin may provide a basal antimicrobial activity at the tonsillar surface to guard against infection. Furthermore, many inflammatory cells were in crypt area and were positively stained for cathelicidin in recurrent throat infection. In a study of tight junctions of tonsillar epithelium, the crypt epithelium was shown to have an epithelial barrier different from that of the surface epithelium [20]. Many inflammatory cells and increased expression of cathelicidin in the tonsillar crypts in our study suggest that the tonsillar crypt would be the site of frequent infection and play an important role in the tonsillar defense mechanism. In summary, we detected the antimicrobial peptide cathelicidin in the inflamed and non-inflamed human palatine tonsil, and demonstrated that the cathelicidin mRNA and protein were upregulated in recurrent throat infection. From these results, it is feasible that cathelicidin is secreted by tonsils and thus may play a role in the tonsillar host defense mechanism.
Acknowledgment This work was supported by the Brain Korea 21 Project of the Ministry of Education & Human Resources Development, Republic of Korea in 2004.
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