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ORAL PATHOLOGY - EXPRESSION OF iNOS MESSAGE IN HUMAN BUCCAL MUCOSAL (KERATINOCYTES) CELL LINE TR146 USING RT-PCR AND IMMUNOCYTOCHEMISTRY.

Byline: MUHAMMAD RIZWAN and SADIA RIZWAN

ABSTRACT

This study was carried out at the department of Oral Pathology, Queen Mary College of Medicine and Dentistry, Barts and The London to determine whether human oral buccal mucosal (keratinocytes) cell line, TR 146, expressed iNOS message and whether the expression of iNOS is varied when TR146 cells were exposed to different cytokines such as IL-15, IL-8, IL-1 ss, or TNF- a using RT-PCR and Immuno-cytochemistry and to determine the effect of the above mentioned cytokines on NO function by measuring nitrite production in TR146 cells. Immuno-cytochemistry analysis revealed that TR146 cells expressed iNOS proteins when incubated with IL-15 and IL-8 and a modest increase was seen with IL-1 ss /TNF-a. RT-PCR for iNOS indicated a marked increase in expression when the cells were exposed to IL-8, IL-15 or IL-1ss/ TNF-a. NOS activity was assessed by measuring nitrite activity.

It was observed that treating the cells with cytokines caused significant increase in nitrite levels, except in the case of IL-8. These results sugest that TR146 cells expressed iNOS , the levels of which varied with various cytokines. It is clear that IL-15, IL-1ss /TNF-a and IL-8 are regulators of iNOS expression in oral keratinocytes and affect NOS activity.

Key words: NOS, LPS, Cytokines, TR146, Immunocytochemistry, RT-PCR. Keratinocytes

INTRODUCTION

Nitric oxide (NO) is a dissolved gas, produced from the amino acid L-arginine by NO synthase (NOS) and is thought to have a physiological and patho-physi-ological roles in maintaining blood pressure. NO causes smooth muscle cells in the vessel walls to relax, so that the vessels dilate and blood flows more freely through them.1 Lipooxygenase products regulate NO produc-tion in IL-1a stimulated vascular smooth muscle cells (VSMCs).2

NO is also used by many nerve cells to signal neighboring cells. Dissolved NO diffuses readily out of the cells that makes it and into neighboring cells.1 NO stimulates soluble guanylyl cyclase (sgc) to produce cGMP in endothelial cells.3 NO prevents 6-hydroxy-dopamine-induced apoptosis in PC12 cells through cGMP-dependent p13 kinase/ AKT activation.4

Endothelial cells, the flattened cells that line every blood vessel, release NO in response to stimulation by nerve endings, and this NO causes smooth muscle cells in vessel walls to relax. Dendritic cells and macroph-ages produce NO in response to LPS stimulation.5 NO is also produced by intestinal epithelial cells in response to challenge from gut microorganisms and may be involved in host defense against inner pathogens.6

NO is important in an early stage of the innate immune response.5 NO is shown to have physiological and pathophysiological roles in neurotransmission, blood pressure homeostasis, and immunological re-sponses.7

Nitric oxide synthase is an enzyme that produces nitric oxide from L-arginine. Three isoforms of NOS have been identified and cloned. Endothelial NOS is expressed in endothelial cells, neural NOS is expressed constitutively in the neural cells and inducible nitric oxide synthase (iNOS) is seen a variety of cells types.6,8,9,10 Nitric oxide synthase governs the production of NO during nervous system development as well as in certain disease states such as stroke, multiple sclerosis Parkinson's disease, and HIV demen-tia.11

INOS is an isoform of NOS and its expression is seen in a wide variety of cells such as macrophages, hepatocytes, and keratinocytes and is induced by sev-eral agents as LPS and cytokines and has the ability to synthesize large amounts of NO.6, 8,9,10 iNOS expression has also been observed in the basal layers of gingival epithelia of non-inflammaed and inflamed periodontal tissues.12 Pulmonary epithe-lial cells express iNOS 6 as do human intestinal epithe-lial cells.13 Lipooxygenase productsregulate iNOS pro-duction in IL-1a stimulated vascular smooth muscle cells (VSMCs).2 Agents such as LPS and cytokines induced expression of iNOS.6, 8,9,10 Recent evidence suggests the possible involvement of iNOS in the development and maintainence of hypertension in certain animal models.1

PKC may be involved in the post-translational modification of iNOS and in the regulation of the availability of iNOS substrates.14 Studies suggest that the MAP kinase pathway is partly involved in cytokine-induced iNOS expression in rat vascular smooth muscle cells (VSMCs).15

Cytokine or LPS-induced inos is involved in the abrogation of intracellular pathogens and inflamma-tory responses.13, 16, 17

METHODOLOGY

Present study was carried out at The depart-ment of Oral Pathology, Queen Mary College of Medicine and Dentistry, Barts and The London, UK, The human oral keratinocytes cell line, TR146 was obtained from Dr Alan Cruchley, Deptt of Oral Pathology. Cell line was cultured in T75 cm2 plastic tissue culture flasks containing 10ml of Dulbecco's Modified Eagle growth Medium (DMEM), as growth medium, supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin/streptomycin (PS). Cells were maintained in a humidified atmosphere of 95% air / 5% CO2.

The experiments of this project were carried out in three phases. Firstly, Immunocytochemistry was per-formed to determine iNOS expression in TR146 cells. Secondly, Reverse transcription followed by polymerase chain reaction (RT-PCR) was carried out to determine whether the cells expressed iNOS mRNA. Thirdly, nitrite levels in the cells were measured in response to various cytokines to determine if iNOS is active in TR146 cells.

Immunocytochemistry

It was carried out to determine whether human oral keratinocytes express iNOS message. Oral keratinocytes (TR146) cells were cultured in a growth medium, Dulbecco's Modified Eagle Medium (DMEM by invitrogen, Paisley, Scotland), supplemented with 10% fetal Bovine Serum (FBS) and 0.1% Penicillin/ Streptomycin (PS). Cells were maintained in a humidi-fied atmosphere of 95% air / 5% CO2. After growth, cells from the flasks cultured in DMEM growth medium were obtained and separated by separating solution. Immunocytochemistry was performed in 24-wells plate using, Anti-human IL-15R-a antibody (R& D Systems, Human IL-15R- a extra-cellular domain specific goat IgG type) and Sf 21-derived rhIL-15R-a extra-cellular domain Immunogen) (Table 1). The experiment was repeated 3 times.

TABLE 1: TREATMENTS USED FOR VARIOUS CYTOKINES IN 24 WELLS PLATE

Treatment###Control###IL-15###IL-1 ss/###IL-8

Materials###TNF- a

PBSul###200ul###200ul###200ul###200ul

iNOSul###200ul###200ul###200ul###200ul

2- RT-PCR (Reverse Transcription-Polymerase

Chain Reaction)

PCR is a technique for the in-vitro amplification of specific DNA sequences by the simultaneous primer extension of complimentary strands of DNA. In RT-PCR, single stranded RNA is used as a technique to template to synthesize double stranded DNA. The purpose of this experiment was to determine whether oral epithelial cells express mRNA encoding the gene for iNOS.

Oral keratinocytes were cultured in a growth medium DMEM. Cells were separated for PCR and following steps were followed

RNA extraction

Extraction of RNA from the cells. The concentra-tion of the RNA was measured by using UV-VIS Spec-trophotometer (UV-mini 1240 Spectrophotometr by Shamzadu Deutschland, GmbH) at A260 and A280 nm. And RNA concentration was calculated using the fol-lowing formula: A260 x dilution factor = ug RNA / ml

DNA Synthesis

After RNA extraction, cDNA synthesis was per-formed using following materials and protocol (Table 2).

Dctp, Aatp, dgtp, dttp (100Mm), Promega ltd. UK.

DTT (0.1M), Oligo Dt Subscript. Primer (0.5ug / ul), RNase H (3U /ul), Superscript RT, 5 x 1st strand buffer all from Invitrogen.

RNase inhibitor (40U/ul), Helena Biosciences, UK. RNase A (10mg/ml), Sigma, Poole, UK.

TABLE 2: MATERIALS USED FOR cDNA SYNTHESIS

Materials###Control IL-15 IL-8###IL-1ss/

###TNF

Oligo Dt###1ul###1ul###1ul###1ul

RNA (2ug)###5ul###5ul###5ul###5ul

DEPC Water###4ul###4ul###4ul###4ul

Total volume###10ul###10ul###10ul###10ul

Polymerase Chain Reaction (PCR)

PCR reagents 10x PCR Buffer 5.0ul, Distilled water 42.8ul, dNTPs (200uM) 0.5ul, sensenprimer 0.2ul, antisense primer 0.2ul, Cdna 1.0ul, Taq polymerase 0.3ul and total of 50.0ul were set up in 0.5ml microfuge tubes and mixed gently.

DNA was substituted for water as a negative control for PCR. And then PCR reactions were put through a cycling program in a hybaid thermocycler. The program was run as mentioned in table 3.

Then about 10ul of the PCR products were mixed with 2ul of loading dye and ran in a 1% agarose gel and then viewed by UV illumination and pictures taken.

Nitrite Assay

The accumulation levels of nitrate and nitrite resulting from NO produced by IL-15 stimulated hu-man buccal epithelial cells were measured, as NO is rapidly converted into these two stable end products.18

The accumulation level of nitrite resulting from NO activity by IL-15 stimulated TR146 cells, was measured using as in-house 96 well assay. These assays were performed by the help of Dr Justin Doel, Department of Oral Microbiology, Queen Mary College Of Medicine and Dentistry, London, UK.

Cells cultured in growth medium, were removed from the culture substrate by treatment with trypsin.

TABLE 3

Process###Tempe-###Time###Cycles

###rature###

DNA denaturation###94 c###5 minutes###1 cycle

DNA denaturation###94 c###1 minute###35 cycles

Primer melting/###60c###I minute###35 cycles

annealing###

Primer extension###72c###2 minutes###35 cycles

Primer extension###72c###7 minutes###1 cycle

Cells were stimulated with by IL-15 100ng/ml, IL-8 100ng/ml, or IL-1 ss /TNF-a 100ng/ml overnight and were incubated (in a 500ul volume) overnight. Medium was then harvested and stored at-20 degree C until assayed for nitrite. Nitrite concentrations were deter-mined in 96-well plates, in duplicate using 50ul sample volume of each treatment used. Each sample was treated with 50ul sulphanilamide solution and incu-bated for 5 minutes at room temperature protecting from light. Then 50ul NED sol. (N-1-naphthylethy-lenediamine dihydrochloride in water) was added and incubated for 5 minutes at room temp. protecting from light.

Plates were then read using a plate reader at 540nm (Table 4). Values of nitrite were estimated by comparing with a standard curve of nitrite concentra-tions including 250um, 125um, 62.2 um, 31.1um, and 0um nitrite (Fig 3).

RESULTS

Immunostaining of the treated and untreated cells was carried out to see the expression of iNOS protein in TR146 cells. Figure 1 showed that cells with no primary antibody and cells with no treatment showed no expression of iNOS. (Fig 1A and 1B). Cells treated

TABLE 4: NITRITE ASSAY

Nitrite (um)###At 540 nm

0###0.04

31.1###0.45

62.2###1.1

125###2.3

250###4.1

The effect of IL-15, IL-8, and IL-1 ss /TNF-gmma on NO activity in TR146 cells was estimated by measuring nitrite levels. TR146 cells were incubated with or without 100ng/ml of cytokines. Nitrite levels in the culture supernatants were determined. Untreated cells demonstrated some nitrite production where as IL-15 clearly showed increases (more than double the con-trol) production of nitrite. IL8 and IL-1ss /TNF- a also showed increased production of nitrite than the con-trolled cells. Data indicates that these cells have NOS activity which increased when treated with various cytokines.

DISCUSSION

Prior to the present study, it had been reported that IL-15 protein was expressed in a constitutive manner in basal epithelial keratinocytes in healthy human gingivae, and that IL-15 and IL15-R a chain mRNA could be detected in human gingival epithelial cells (HGEC) by RT-PCR analysis.

Previous studies have reported higher levels of iNOS mRNA expression in the periotoneal macroph-ages in IL-15 transgenic mice after stimulation of Mycobact-erium bovis bacillus, compared with those of wild mice.19 Another study showed both iNOS and IL-15R mRNA expression were up-regulated in mouse dendritic cells after stimulation of LPS or propione-bacterium acnes.20 These studies suggested that IL-15 may be involved in iNOS expression in other cell types. This study directly demonstrated that human oral keratinocytes cells express IL-15R and IL-15 has the ability to induce iNOS expression and nitrite produc-tion.

Studies have also found that gingival keratinocytes can be induced to express iNOS by stimulation with IL-1ss, IL-8, TNF-a, or LPS as well as their combina-tions.6,8,22 It has been reported that epithelial cells highly express iNOS at both mRNA and protein levels in inflammatory lesions, including those of Periodontitis.12 Although the physiological and pathophysiological roles of iNOS expression in periodontal lesion are not yet fully defined, it is expected that hyperproduction of NO in inflamed periodontal lesions may be involved in epithelial bar-rier dysfunction and alveolar bone resorption. IL-15 receptors are known to be expressed in a variety of tissues, including epithelial cells 25, ss cells and NK cells.24

It was shown that IL-15 was also able to induce iNOS and NO production by epithelial cells.13,23,26 Our present findings provide new evidence that an oral keratinocytes cell line express IL-15R and iNOS and that IL-15 is also a potent regulatory cytokine for the stimulation of iNOS and nitrite production in these cells. Immunostaining to see the presence of iNOS in TR146 cells (Fig 4) show that cells treated with IL-15 and other inflammatory cytokines IL-8, and IL-1ss/ TNF-a caused an increase in IL-15R and iNOS expres-sion in these cells.

IL-15 acts via IL-15R, which shares ss and Gamma subunit with IL-2R. however, unlike IL-2R, IL-15R expression is widely distributed throughout a variety of tissues and cells. It remains uncertain what kind of intracellular signal transduction is involved in IL-15R mediated iNOS expression in TR146, though several signaling pathways have been implicated in the up-regulation of iNOS mRNA in HGEC. Among these factors, NF-kappaB is a pivotal transcription inducer of iNOS expression in various cells.

It is possible that endothelial cells and fibro-blasts underlying buccal epithelial cells are also influ-enced by IL-15 generated from these cells. A recent report has revealed that stimulation with IL-15 in-creases hyaluronan synthesis by endothelial cells,27 and gingival fibroblasts. Since hyaluronan is known to function as a scavenger of nitric oxide (NO) derivatives such as peroxynitrite,21 which exerts deleterious ef-fects on host cells, the IL-15 induced hyaluronan generation by endothelial cells and fibroblasts may contribute to the prevention of NO associated tissue damage.

In summary, findings of the present study demon-strate that TR146 cells express IL-15R and iNOS message, and IL-15 is a potent inducer and regulator of iNOS expression and nitrite production in these cells.

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Author:Rizwan, Muhammad; Rizwan, Sadia
Publication:Pakistan Oral and Dental Journal
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2010
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