March 2009
Volume 50, Issue 3
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Cornea  |   March 2009
Detection and Regulation of Cationic Amino Acid Transporters in Healthy and Diseased Ocular Surface
Author Affiliations
  • Kristin Jäger
    From the Departments of Anatomy and Cell Biology and
  • Ulrike Bönisch
    From the Departments of Anatomy and Cell Biology and
  • Michaela Risch
    From the Departments of Anatomy and Cell Biology and
  • Dieter Worlitzsch
    Hygiene, Martin Luther University of Halle-Wittenberg, Halle, Germany.
  • Friedrich Paulsen
    From the Departments of Anatomy and Cell Biology and
Investigative Ophthalmology & Visual Science March 2009, Vol.50, 1112-1121. doi:10.1167/iovs.08-2368
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      Kristin Jäger, Ulrike Bönisch, Michaela Risch, Dieter Worlitzsch, Friedrich Paulsen; Detection and Regulation of Cationic Amino Acid Transporters in Healthy and Diseased Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2009;50(3):1112-1121. doi: 10.1167/iovs.08-2368.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions.

methods. Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR.

results. Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-α and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only.

conclusions. These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.

One of the prominent effects proinflammatory cytokines can exert on cells is to induce the expression of the inducible isoform of the nitric oxide synthase (NOS) family, the iNOS, resulting in high-output nitric oxide (NO) synthesis. 1 High-output NO production may serve for local defense against bacteria and viruses. 2 At the ocular surface this has been demonstrated in resistance to Pseudomonas aeruginosa ocular infection. 3 High-output NO production is also involved in toxic effects on neighboring cells 4 and in immune-mediated tissue destruction, as has been shown, for instance, for lacrimal gland acinar cells and conjunctival epithelium in autoimmune Sjögren syndrome. 5 6  
A precursor for the synthesis of NO is the semiessential amino acid L-arginine in humans, which serves also as a component for protein synthesis. In inflammation, L-arginine may become limited because of the substrate competition between abundantly expressed iNOS and arginase (needed for protein synthesis), 7 which might affect the expression pattern of genes regulating the inflammatory process. This has been shown, for example, in inflammatory bowl disease 8 and could play a role during inflammation at the ocular surface. The transport of L-arginine into the cell is enabled primarily by cationic amino acid transporters (CATs), y+ transporters. They constitute a subfamily of the solute carrier family 7 (SLC7). There are at least four different human genes 9 consisting of four confirmed transport proteins for cationic acids—CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3) 10 —exhibiting differential expression patterns and tissue localization. 11 SLC7A4 and SLC7A14 are two related proteins with yet unknown function. 10 Mammalian cells ubiquitously and constitutively express CAT-1. 12 By contrast, de novo expression of CAT-2B is observed in many different cell types under inflammatory conditions only, providing increased L-arginine availability, particularly for iNOS. 13 Notable exceptions to this known so far are hepatocytes and smooth muscle cells with constitutive expression of the splice variant CAT-2A. CAT2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate on the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. 14  
The aims of the present study were to detect and localize human CATs in ocular surface epithelial cells and tissues of the lacrimal apparatus, to investigate potential differences in the distribution pattern in the presence of corneal diseases (i.e., herpetic keratitis, keratoconus, and Fuchs dystrophy), and to analyze the regulation of hCATs by proinflammatory cytokines (IFN-γ, IL-1β, and TNF-α) and supernatants of P. aeruginosa and Staphylococcus aureus in cultured corneal epithelial cells (cell line). 
Materials and Methods
Tissues and Cell Lines
The study was conducted in compliance with institutional review board regulations, informed consent regulations, and the provisions of the Declaration of Helsinki. Lacrimal glands, upper eyelids, conjunctivas, corneas, and nasolacrimal systems (consisting of lacrimal sac and nasolacrimal duct) were obtained from cadavers (five male, eight female; age range, 49–88 years) donated to the Department of Anatomy and Cell Biology at Martin Luther University (Halle-Wittenberg, Germany). All used tissues were dissected from the cadavers within 4 to 12 hours of death. Donors were free of recent trauma, eye and nasal infections, and diseases involving or affecting lacrimal apparatus or ocular surface function. 
Twenty-one samples of the central cornea of patients with keratoconus (10 samples), Fuchs dystrophy (five samples), and herpetic keratitis (six samples) were obtained during surgical procedures at the Department of Ophthalmology, Christian Albrecht University of Kiel (Kiel, Germany). The medical histories of the patients concerning other possible eye-affecting diseases were obtained. Two patients with keratoconus had neurodermatitis. 
After dissection, tissue from the right eye of each cadaver was prepared for paraffin embedding and was fixed in 4% paraformaldehyde; tissue from each left eye was used for molecular biological investigations and were immediately frozen at −80°C. After surgery, all corneas from patients were fixed in 4% formalin, dehydrated in graded concentrations of ethanol, and embedded in paraffin. 
SV40-transformed human corneal epithelial (HCE) cells (a kind gift from Kaoru Araki-Sasaki, Tane Memorial Eye Hospital, Osaka, Japan) 15 were cultured as monolayers and used for stimulation experiments. 
For experiments, 500,000 cells each were seeded into 25-cm2 flasks and cultivated in Dulbecco modified Eagle medium (DMEM F12; PAA Laboratories GmbH, Pasching, Austria) containing 10% FCS (Biochrom AG, Berlin, Germany). At 100% confluence, the cells were exposed to IL-1β (400 U/mL; Chemicon/Millipore, Schwalbach, Germany), IFN-γ (400 U/mL; ImmunoTools, Friesoythe, Germany), TNF-α (400 U/mL; ImmunoTools), or supernatants of P. aeruginosa (1:100 dilution) or S. aureus (1:15 dilution) in serum-free DMEM with 0.05% bovine serum albumin for 24 hours. Concentrations of stimulants were chosen on the basis of previously published experiments. 16 17 18 19 20 21 Given that TNF-α alone was shown to be prosurvival, 22 a presensitization step with preincubation of the cells with IFN-γ (400 U/mL) for 24 hours was performed before the 24-hour TNF-α stimulation in addition to the stimulation experiments. Cells were used for reverse transcriptase–polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis. 
Production of Bacterial Supernatants
P. aeruginosa strain PAO1 (ATCC 15692) and S. aureus strain SA 113 (ATCC 35556) were grown overnight at 37°C during shaking in tryptone soy broth (Oxoid, Basingstoke, UK). Thereafter, bacterial dilutions were plated on Columbia agar supplemented with 10% sheep blood (Heipha, Eppelheim, Germany) and incubated overnight at 37°C, after which plate counts were performed. Bacteria were adjusted to 5 × 107 CFU/mL and centrifuged twice at 6000 rpm for 30 minutes. Supernatants were filtered twice using filters impermeable to bacteria (0.22-μm pore size; Millipore, Eschborn, Germany). Aliquots of the supernatants were confirmed sterile by overnight incubation on agar. 
RNA Preparation and Complementary DNA Synthesis
For conventional RT-PCR, tissue biopsy samples from 13 lacrimal glands, upper eyelids, conjunctivas, corneas, and nasolacrimal systems were crushed in an agate mortar under liquid nitrogen, then homogenized in 5 mL RNA pure solution (peqgold; peqLab Biotechnologie, Erlangen, Germany) with a homogenizer (Polytron, Paterson, NJ). Insoluble material was removed by centrifugation (12,000g, 5 minutes, 4°C). Total RNA was isolated (RNeasy-Kit; Qiagen, Hilden, Germany). Crude RNA was purified with isopropanol and repeated ethanol precipitation, and contaminating DNA was destroyed by digestion with RNase-free DNase I (20 minutes, 25°C; Boehringer, Mannheim, Germany). The DNase was heat-denatured for 15 minutes at 65°C. RNA (500 ng) was used for each reaction. cDNA was generated with 50 ng/μL (20 pmol) oligo (dT)15 primer (Amersham Pharmacia Biotech, Uppsala, Sweden) and 0.8 μL superscript RNase H reverse transcriptase (100 U; Gibco, Paisley, UK) for 60 minutes at 37°C. Ubiquitously expressed β-actin, which was amplifiable in each case with the specific primer pair, served as the internal control for the integrity of the translated cDNA. 
Polymerase Chain Reaction
For conventional PCR, 1 μL cDNA (from each sample) was incubated with 13.7 μL H2O, 1 μL 50 mM MgCl2, 0.5 μL dNTP, 2 μL 10× PCR buffer, 0.3 μL (5 U) Taq DNA polymerase (Invitrogen, Karlsruhe, Germany), and 0.5 μL (100 pmol) of each of the following primers: Product/GenBank accession no. human CAT1/NM/003045 sense 5′-GATTTAGGTGACACTATAGAATACATCTGCTTCATCGCCTACTT-3′, antisense 5′-TAGCAGTCCATCCTCAGCCATG-3′ (228 bp); human CAT2B/U76369 sense 5′-GATTTAGGTGACACTATAGAATACCCCAATGCCTCGTGTAATCT-3′, antisense 5′-TGCCACTGC-ACCCGATGATAAAGT-3′ (120 bp); and human CAT3/U76369 sense 5′-GATTTAGGTGACACTATAGAATACCCCAATGCCTCGTGTAATCT-3′, antisense 5′-TGCCACTGC-ACCCGATGATAAAGT-3′ (120 bp). After 5 minutes of heat denaturation at 95°C, the PCR cycle consisted of 95°C for 15 seconds; 64°C for 30 seconds; 72°C for 30 seconds and 80°C for 10 seconds for CAT1 and 95°C for 20 seconds; 55°C for 30 seconds; and 72°C for 30 seconds and 80°C for 15 seconds for CAT2b. Thirty cycles were performed with each primer pair. The final elongation cycle consisted of 80°C for 10 seconds (CAT1) and 80°C for 15 seconds (CAT2b). Primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). PCR (10 μL) was loaded on an agarose gel. After electrophoresis, the amplified products were visualized by fluorescence. Base pair values were compared with GenBank data. PCR products were also confirmed by sequencing (BigDye; Applied Biosystems, Foster City, CA). Tissue from human prostate was used as a positive control for CAT3. 
Real-Time RT-PCR
We used real-time RT-PCR to quantify levels of hCAT1 and hCAT2b mRNA in HCE cells with and without stimulation. Relative quantification of the signals was performed by normalizing the signals of the hCAT1 and hCAT2b genes with the β-actin signal. Forward and reverse primers used to generate a 228-bp hCAT1 and a 120-bp hCAT2b amplicon were as described for RT-PCR. Forward and reverse primers used to generate a 275-bp actin amplicon were 5′-CAAGAGATGGCCACGGCTGCT-3′ and 5′-TCCTTCTGCATCCTGTCGGCA-3′. Primers were designed to span intron/exon boundaries to verify PCR product amplification from cDNA. 
Real-time PCR was performed on a DNA engine (Opticon 2 System; MJ Research, Waltham, MA). All 25-μL reactions contained cDNA (4 μL), super mix (10 μL; Platinum SYBR Green qPCR SuperMix-UDG; Invitrogen), specific primers (0.8 μL), and PCR-H2O (5.2 μL). Reaction components were assembled in strip tubes with ultraclear strip caps (MJ Research). The cycle profile was 50° for 2 minutes, followed by 95°C for 2 minutes, 40 cycles at 95°C for 15 seconds, and 70°C for 30 seconds (for hCAT1, hCAT2b, and β-actin). Standard curves for hCAT1, hCAT2b, and β-actin were generated with the use of a serially diluted template of known copy number. To verify the specificity of the amplification reaction, we performed melting curve analysis by increasing the temperature from 46°C to 96°C at a transition rate of 1°C/s. In addition, reaction products were examined on agarose gels. We normalized data from triplicate reactions based on the ratio of target hCAT1 and hCAT2b cDNA, respectively, to that of β-actin. 
Sequencing of PCR Fragments
PCR fragments were collected after a real-time RT-PCR run and were purified (QIAquick purification kit; Qiagen). Purified DNA was used forsequencing with the same primer used for the PCR reaction. The sequencing reaction was performed at the Centre for Basic Medical Research at Martin Luther University Halle-Wittenberg. Sequences obtained were compared with the published sequences in National Center for Biotechnology Information. 
Antibody Generation and Testing
Antibody generation and testing has been described previously. 23 In brief, to produce antibodies to the individual hCAT proteins 1 and 2, appropriate peptides were first designed and produced by immunization of rabbits (anti-hCAT1 and anti-hCAT2 IgG [it is not possible to differentiate between hCAT2a and hCAT2b]; DPC Biermann GmbH [Bad Nauheim, Germany]; Biocarta [Hamburg, Germany]). Before treatment, the antibodies were tested for their specificity. For this purpose, the peptides of antibodies were divided into different concentrations in the incubation solution with the antibodies. It was shown by Western blot analysis that a specific 70-kDa band reduction was equivalent to the increase in peptide concentration. 23  
Immunohistochemistry
For analysis by immunohistochemistry, lacrimal glands and upper eyelids with conjunctiva, corneas, nasolacrimal systems, and corneal abnormalities were sectioned and dewaxed. Immunohistochemical staining was performed with polyclonal rabbit anti-hCAT-1 and anti-hCAT-2 IgG (both 1:100). Sections were pretreated with trypsin for 5 minutes, and nonspecific binding was inhibited by incubation with goat normal serum (Dako, Glostrup, Denmark) 1:5 in Tris-buffered saline. The primary antibody was applied overnight at room temperature. Secondary antibody anti-rabbit IgG (1:300 biotinylated; Calbiochem, La Jolla, CA) was incubated at room temperature for at least 4 hours. Visualization was achieved with peroxidase-labeled streptavidin-biotin and diaminobenzidine for at least 5 minutes. After counterstaining with hemalum, the sections were mounted (Aquatex; Boehringer, Mannheim, Germany). Two negative control sections were used in each case: one was incubated with the secondary antibody only and the other with the primary antibody only. Sections of human skin (hCAT1) and liver (hCAT2) were used for positive control. The slides were examined with a Zeiss microscope (Axiophot; Carl Zeiss, Jena, Germany). Liver served as an additional negative control for hCAT1. 
Statistical Analysis
Results of the experiments are reported as mean ± SD. Statistical analysis was made (SigmaStat software for Windows, v 3.00; SPSS Inc., Chicago, IL). Variance analysis was applied for determination of three and more samples (treatment groups) of normally distributed populations with equal variance. Because only one factor was examined in the various mean groups, the simple variance analysis (one-way ANOVA) was used. Random samples with unknown common variance were examined. The Bonferroni t-test was performed to compare three or more groups with reference to a control. All data represent a minimum of three separate experiments performed in triplicate. A standardized t-test was used, and P values were generated to establish the significance level of the data and comparisons between groups. 
Results
Expression of hCATs in Tissues of the Ocular Surface and Lacrimal Apparatus
hCAT1-specific cDNA amplification products were detected in nasolacrimal ducts, conjunctivas, and corneas, but not in lacrimal glands (n = 13 for each tissue; Fig. 1 ). hCAT2b amplification products were visible in all tissues investigated (lacrimal glands, conjunctivas, corneas, nasolacrimal ducts); n = 13 for each tissue; Fig. 1 ). mRNA of HCAT3 could not be detected in any of the investigated samples (n = 13 for each tissue; Fig. 1 ). The β-actin control PCR was positive and of similar amounts for all investigated tissues, as expected (Fig. 1) . Controls in nontranscribed RNA revealed a lack of amplification (data not shown). 
Distribution of Human Cationic Amino Acid Transporters 1 and 2 in Tissues of the Lacrimal Apparatus, Ocular Surface, and Corneal Abnormalities
Paraffin-embedded 7-μm sections from 13 lacrimal glands, conjunctivas, corneas, and nasolacrimal ducts and 21 samples of the central cornea of patients with keratoconus (10 samples), Fuchs dystrophy (five samples), and herpetic keratitis (six samples) were analyzed (Figs. 2 and 3)
hCAT1
No hCAT1 could be detected in any of the analyzed sections of the lacrimal gland. All layers of corneal epithelial cells revealed perinuclear and cytoplasmic reactivity, whereas the Bowman membrane, corneal stroma, Descemet membrane, and corneal endothelium showed no reactivity. All layers of conjunctival epithelial cells demonstrated positive reactivity with strong staining in the upper part of the cells. The secretory product of goblet cells was not reactive. In addition to epithelial cells, some subepithelial mononuclear cells also showed cytoplasmic reactivity. High columnar epithelial cells of the nasolacrimal duct epithelium (lacrimal sac and nasolacrimal duct) revealed positive reactivity, especially in the upper part of the cells. In addition, the basal cell layer demonstrated strong reactivity, whereas the secretory product of goblet cells was negative. Positive reactivity to herpetic keratitis was visible, especially in all layers of the corneal epithelium and subepithelially in the stroma surrounding herpetic lesions. Other areas of the stroma,Descemet membrane, and endothelium revealed no reactivity. Reactivity was visible in all epithelial layers of the keratoconus, especially supranuclearly. Other corneal structures showed no reactivity. Reactivity to Fuchs dystrophy was visible in all epithelial layers, especially supranuclearly. Other corneal structures showed no reactivity. As expected, human skin revealed positive reactivity in all epithelial cell layers with the exception of the stratum corneum. 
hCAT2
Positive immunoreactivity was visible in all acinar epithelial cells of the lacrimal gland. All corneal epithelial cell layers revealed weak perinuclear and cytoplasmic reactivity, whereas all other corneal layers (Bowman membrane, stroma, Descemet membrane, endothelium) were negative. Basal cells of the conjunctival epithelial cell layer revealed strong positive reactivity, whereas the other conjunctival epithelial cell layers showed no or only weak (superficial epithelial cells) reactivity. No reactivity was visible in the secretory product of goblet cells or subepithelially. Basal epithelial cells of the double-layered epithelium of the nasolacrimal duct epithelium (lacrimal sac or nasolacrimal duct) demonstrated strong reactivity, whereas weak reactivity was seen in high columnar epithelial cells. No reactivity was visible in the secretory product of goblet cells or subepithelially. Strong positive reactivity was visible in all epithelial layers of the corneal epithelium in patients with herpetic keratitis, whereas all other corneal layers (Bowman membrane, stroma, Descemet membrane, endothelium) and areas surrounding herpetic lesions were negative. Strong positive reactivity to keratoconus was visible in the superficial layer of the corneal epithelium, whereas all other epithelial layers revealed only weak reactivity. All other corneal structures showed no reactivity. Positive reactivity to Fuchs dystrophy was visible in all layers of the corneal epithelium. Other structures of the cornea were negative. In the liver (positive control), hepatocytes revealed positive staining, as expected. 
Regulation of hCAT1 and hCAT2b mRNA Expression in HCE after Stimulation with Cytokines
Stimulation of cultured HCEs with different proinflammatory cytokines was analyzed by real-time RT-PCR (Fig. 4) . Relative mRNA expression of hCAT1 and hCAT2b was significantly upregulated by TNF-α (P < 0.05), whereas IFN-γ and IL-1β had no significant effect. 
Regulation of hCAT1 and hCAT2b mRNA Expression in HCE after Stimulation with Bacterial Supernatants
Stimulation of cultured HCEs with supernatants of S. aureus and P. aeruginosa were analyzed by real-time RT-PCR (Fig. 5) . Relative mRNA expression of hCAT1 is significantly upregulated by the supernatant of S. aureus (P < 0.05), whereas the supernatant of P. aeruginosa had no significant effect. Relative mRNA expression of hCAT2b was significantly upregulated by supernatants of both S. aureus and P. aeruginosa (P < 0.05). 
Discussion
NO serves as a mediator in diverse and complex cellular processes throughout the eye, such as the regulation of aqueous humor dynamics, retinal neurotransmission, and phototransduction. 24 25 Moreover, NO has been shown to have a role in endotoxin-induced uveitis in rats, 26 27 28 29 in experimental guinea pig models of allergic conjunctivitis, 30 31 and in increased conjunctival blood flow and hyperpermeability of sensitized guinea pigs after exposure to the antigen or histamine. In the cornea, NO spontaneously produced in the corneal endothelium is highly involved in the maintenance of corneal thickness. 32 Moreover, increased levels of NO were detected in lavage fluid from the ocular surface 30 minutes after challenge, along with conjunctivitis, edema formation, and hyperpermeability. Finally, NO plays an important role in inflammatory and infectious processes. 33  
NO production depends on the action of NOS. It is well known that the induction of nitric oxide synthase (iNOS) in many cell types occurs by stimulation with a complex milieu of proinflammatory cytokines and bacterial endotoxins during periods of inflammation or infection. 34 35 In addition, at the ocular surface and the lacrimal apparatus, it has been observed that the enzymes iNOS and arginase 1 are significantly increased during inflammatory processes. 3 5 36 37 38 39 Both arginase 1 and iNOS metabolize the cationic amino acid L-arginine. Diminished L-arginine availability has been shown to create a NO deficiency associated with several diseases and pathophysiological conditions, such as induction of hyperreactive airways, contributions to epidermal hyperproliferation in patients with psoriasis, and impaired wound healing after trauma or shock. 40 41 42 On the other hand, supplementation of L-arginine has a beneficial effect on cell proliferation and improving wound healing, diminishing epidermal hyperproliferation in psoriasis and reducing asthmatic conditions. 40 41 42 Given that both enzymes use L-arginine as a substrate and are dependent on its availability, transport of L-arginine through the cell membrane is of great relevance. Among the transport systems that mediate L-arginine uptake, cationic amino acid transporters (CAT1, CAT2, and CAT3) are considered the major arginine transporters in most cells and tissues. 43  
In the present study, therefore, we investigated the distribution and regulation of the y+ transporters hCAT1 (SLC7A1), hCAT2 (SLC7A2), and hCAT3 (SLC7A3) in the tissues of the ocular surface and lacrimal apparatus in mRNA and protein levels. We found the expression of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. This finding is in accordance with differential expression patterns and tissue localizations of hCAT1 and hCAT2. 12 Interestingly, we found that acinar cells of the lacrimal gland express only hCAT2 but not hCAT1. With the sole exception of liver, hCAT1 is ubiquitously and constitutively expressed in mammalian cells. 9 Lack of hCAT1 in the lacrimal gland was unexpected because salivary glands and mammary glands have been shown to express hCAT1. 11 12 Because we were unable to detect hCAT1 at the mRNA and protein levels in all lacrimal glands analyzed, we do not think the finding is an artifact. Thus, hCAT1 is absent not only in liver but also in lacrimal gland. Moreover, our studies revealed an absence of hCAT3 in all tissues under investigation. This is in accordance with findings by Vekony et al., 11 who showed that hCAT3 is expressed in some but not all brain tissues and in some peripheral tissues such as thymus, uterus, prostate, testis, mammary gland, and some tumors. 
Some subepithelial cells with mononuclear appearance revealed positive cytoplasmic reactivity for hCAT-1 but not for hCAT2. Interestingly, a similar finding was made by Closs et al. 44 in J774A.1 macrophages (a macrophage cell line). These macrophages express CAT-1 constitutively, whereas CAT-2B is expressed in activated macrophages only. 44  
Immunohistochemical comparisons of the distribution patterns of hCAT1 and hCAT2 proteins were carried out between healthy cornea and different corneal diseases (keratoconus, Fuchs dystrophy, herpetic keratitis). No difference in staining intensity was observed for hCAT1, whereas a strong subjective increase in staining intensity was visible for hCAT2 in the epithelium of all diseased corneae (keratoconus, Fuchs dystrophy, herpetic keratitis). 
Keratoconus is a noninflammatory disease that resembles a corneal thinning disorder with development of a localized conical protrusion. 45 Histologically, fine anterior scars are visible, caused by idiopathic breaks in Bowman membrane. Descemet membrane also shows signs of disruption that may lead to a destruction of the integrity of the corneal endothelial barrier and stromal edema. 45 All processes lead to a destruction of the corneal structure. It has been observed that keratoconus is associated with the induction of iNOS in the diseased cornea. 46 Therefore, we speculated that the subjective increased staining of hCAT2 in keratoconus corneas in comparison with normal corneas accompanies the induction of iNOS. 
Fuchs dystrophy is based on a pump failure of endothelial cells 47 that leads to stromal and epithelial edema caused by aqueous humor penetrating the corneal layers. Development of microcysts or bullae within the epithelium may follow. These vesicles can rupture, followed by a scarring process. 47 Like keratoconus, Fuchs dystrophy is associated with the induction iNOS in the diseased cornea. 46 Moreover, it has been demonstrated that the anterior chamber concentrations of arginine are significantly increased in patients with Fuchs dystrophy. 48 It may also be hypothesized that the observed subjective increase in the staining intensity of hCAT2 in the corneal epithelium of patients with Fuchs dystrophy is associated with an increase of iNOS in the cornea and arginine in the aqueous humor. 
In contrast to keratoconus and Fuchs dystrophy, corneal infection with herpes simplex virus resembles a disease that can affect not only the cornea but other ocular tissues as well. The virus colonizes cervical and trigeminal ganglia and reaches peripheral tissues through the axons. In the cornea, the virus can cause epithelial and stromal keratitis with ulceration, edema, and stromal scarring. 49 Our results show hCAT1 expression in all corneal epithelial layers and in the stroma surrounding herpetic lesions. hCAT2 is expressed only in the epithelium, revealing clear, subjectively increased staining of epithelial cells in comparison with healthy corneal epithelium. De novo expression of CAT2 has been observed in many different cell types under inflammatory conditions, providing increased L-arginine availability, in particular for iNOS. 13 23 50 51 Patients with herpetic keratitis experienced a strong increased expression of iNOS and arginase 1 and as an induction of chemokines and proinflammatory cytokines. 36 52 53 Just as proinflammatory mediators have been shown to enhance the expression of CAT proteins and the influx of L-arginine in various cell models, 54 55 56 our immunohistochemical findings support the impression that this is also the case in the corneal epithelium in patients with herpetic keratitis. However, our cell culture experiments support this only in part. A significant increase in the relative hCAT2b expression was observed with TNF-α after 6 hours of stimulation but not with IL-1β or IFN-γ, though all three cytokines are known to be involved in the pathogenesis of recurrent herpetic keratitis. Moreover, in addition to hCAT2b, TNF-α significantly upregulated the relative expression of hCAT1 at 6 hours. These experimental findings are limited because only one time point (6 hours) and use of a cell line were analyzed. However, upregulation of CAT1 has been demonstrated 57 58 and may occur in corneal epithelial cells, perhaps without consequences at the protein level. 
Most causative organisms inducing bacterial keratitis are the Gram-positive pathogen S. aureus and the Gram-negative bacterium P. aeruginosa. 59 60 Both bacteria and their bacterial products and toxins lead to the induction of an immune response triggered by the production of proinflammatory cytokines such as TNF-α, IL-1β, and IFN-γ. 61 As a consequence, iNOS and eNOS expression and NO levels in the cornea are increased compared with levels in healthy, uninfected cornea. 3 39 46 62 In our study, hCAT2b mRNA expression was markedly increased in cultured HCE cells after challenge with TNF-α and bacterial supernatants of S. aureus and P. aeruginosa. Both bacteria and TNF-α are known to activate the transcription factor NF-κB, 63 64 65 and NF-κB is an essential transcription factor for the up-regulation of CAT2b. 66 On the other hand, it has been shown that inhibitors of the NF-κB pathway hinder the increase in CAT2b mRNA and the stimulation of arginine uptake. 67 It seems to be of no consequence whether P. aeruginosa is acting under aerobic or anaerobic conditions because it has been observed that the bacterium is also able to metabolize arginine under anaerobic conditions. 68 69 Further investigations addressing this issue will be of interest. TNF-α and supernatant of S. aureus also increased the expression of hCAT1 in our experiments. Similar regulation has been observed in human endothelial cells 70 in which TNF-α (10 ng/mL) upregulated hCAT1 expression until 6 hours of stimulation. However, later time points until 24 hours of stimulation led to the downregulation of hCAT1. 70 Whether such downregulation also occurs in HCE cells was not determined in the present experiments. Because TNF-α alone has been shown to be prosurvival, 22 a presensitization step involving preincubation of the cells with IFN-γ for 12 hours was performed, revealing that presensitization had no significant effect compared with TNF-α alone. 
CAT2b induction during bacterial inflammation and, thus, L-arginine transport into the epithelial cells of the ocular surface is necessary not only for high throughput NO production during infection but also for production of antimicrobial peptides. Specific bacteria such as S. aureus and P. aeruginosa lead to a strong induction of the antimicrobial peptides β-defensin 2 and β-defensin 3 in ocular surface epithelia if they come into contact with the epithelial cells. 71 The most distinct molecular feature of such defensins is cationicity, manifested by abundant arginine (with or without lysine). For the production of β-defensins, therefore, high levels of arginine and, thus, high transport capacity of the amino acid into the epithelial cell that produces β-defensins are needed. 72  
In conclusion, these studies demonstrate for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. They suggest that hCAT2 is induced during herpetic keratitis and by TNF-α and bacterial supernatants in corneal keratinocytes. Further studies are needed to analyze whether hCAT2 functions as a possible target that regulates immunity, as recently been demonstrated by Thompson et al. 73 In corneal diseases such as keratoconus or Fuchs dystrophy, hCAT2 is also upregulated. However, the reason for this is only speculative and needs further elucidation. Observed changes in the expression of hCATs lead to differences in the distribution of L-arginine in cellular subcompartments. Therefore, consumption of L-arginine by NOS increases and withdraws arginase, its substrate. We recommend further study to determine whether L-arginine could be adopted as a possible topical therapeutic treatment in patients with ocular surface inflammation, such as bacterial infection of the ocular surface and efferent tear ducts or dry eye syndrome. 
 
Figure 1.
 
Expression of human cationic amino acid transporters in tissues of the lacrimal apparatus and ocular surface. RT-PCR analysis for HCAT1 (a), 2b (a), and 3 (b) expression was performed in the nasolacrimal duct (nd), lacrimal gland (lg), conjunctiva (cj), and cornea (co). The integrity of the cDNAs was tested by amplification of the β-actin transcript. M, base pair standard; neg, negative control; pc, positive control (prostate).
Figure 1.
 
Expression of human cationic amino acid transporters in tissues of the lacrimal apparatus and ocular surface. RT-PCR analysis for HCAT1 (a), 2b (a), and 3 (b) expression was performed in the nasolacrimal duct (nd), lacrimal gland (lg), conjunctiva (cj), and cornea (co). The integrity of the cDNAs was tested by amplification of the β-actin transcript. M, base pair standard; neg, negative control; pc, positive control (prostate).
Figure 2.
 
Distribution of hCAT1 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) No hCAT1 is produced by the lacrimal gland. (b) Corneal epithelial cells reveal perinuclear and cytoplasmic reactivity. Bowman’s membrane and the corneal stroma show no reactivity with the antibody. (c) Conjunctival epithelial cells reveal positive reactivity with strong staining in the upper part of the cells. The secretory product of goblet cells is not reactive. In addition to epithelial cells, some subepithelial cells with the appearance of mononuclear cells demonstrated cytoplasmic reactivity. (d) High columnar epithelial cells of the nasolacrimal ducts reacted positively with the antibody, especially the upper part of these cells and the basal cell layer, whereas the secretory product of goblet cells did not react. (e) Immunohistochemical detection of hCAT1 in a patient with herpetic keratitis. Positive reactivity is especially visible in the epithelium and subepithelially in the stroma in the area of herpetic lesions. (f) Immunohistochemical detection of hCAT1 in a patient with keratoconus. HCAT1positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (g) Immunohistochemical detection of hCAT1 in a patient with Fuchs dystrophy. HCAT1 positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (h) Positive control section from human skin (cornifying). Positive red staining occurs in all epithelial cell layers except stratum corneum. (ah) Hemalum. Scale bars: (a, d) 82.5 μm; (b, c, eg) 176 μm; (h) 32 μm.
Figure 2.
 
Distribution of hCAT1 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) No hCAT1 is produced by the lacrimal gland. (b) Corneal epithelial cells reveal perinuclear and cytoplasmic reactivity. Bowman’s membrane and the corneal stroma show no reactivity with the antibody. (c) Conjunctival epithelial cells reveal positive reactivity with strong staining in the upper part of the cells. The secretory product of goblet cells is not reactive. In addition to epithelial cells, some subepithelial cells with the appearance of mononuclear cells demonstrated cytoplasmic reactivity. (d) High columnar epithelial cells of the nasolacrimal ducts reacted positively with the antibody, especially the upper part of these cells and the basal cell layer, whereas the secretory product of goblet cells did not react. (e) Immunohistochemical detection of hCAT1 in a patient with herpetic keratitis. Positive reactivity is especially visible in the epithelium and subepithelially in the stroma in the area of herpetic lesions. (f) Immunohistochemical detection of hCAT1 in a patient with keratoconus. HCAT1positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (g) Immunohistochemical detection of hCAT1 in a patient with Fuchs dystrophy. HCAT1 positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (h) Positive control section from human skin (cornifying). Positive red staining occurs in all epithelial cell layers except stratum corneum. (ah) Hemalum. Scale bars: (a, d) 82.5 μm; (b, c, eg) 176 μm; (h) 32 μm.
Figure 3.
 
Distribution of hCAT2 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) Positive immunoreactivity is visible in acinar epithelial cells. (b) Corneal epithelial cells reveal weak perinuclear and cytoplasmic reactivity. Bowman membrane and corneal stroma show no reactivity with the antibody. (c) Basal cells of the conjunctival epithelium reveal strong positive reactivity with the antibody, whereas other conjunctival epithelial cell layers show no or only weak (superficial epithelial cells) reactivity. No reactivity is visible subepithelially. (d) Basal epithelial cells of the double-layered epithelium of the nasolacrimal ducts demonstrate strong reactivity. Weak reactivity is seen in high columnar epithelial cells. No reactivity is visible in goblet cells. (e) Strong positive reactivity is visible in all layers of the corneal epithelium in a patient with herpetic keratitis. (f) Strong positive reactivity is visible in the superficial layer of the corneal epithelium in a patient with keratoconus. Deeper corneal epithelial layers reveal only weak reactivity. The corneal stroma is not reactive. (g) Positive reactivity is visible in all layers of the corneal epithelium in a patient with Fuchs dystrophy. (h) Positive control section from a human liver revealing positively stained hepatocytes. (ah) Hemalum. Scale bars: (a) 82.5 μm; (b, fh) 176 μm; (c, d) 42.5 μm; (e) 32 μm.
Figure 3.
 
Distribution of hCAT2 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) Positive immunoreactivity is visible in acinar epithelial cells. (b) Corneal epithelial cells reveal weak perinuclear and cytoplasmic reactivity. Bowman membrane and corneal stroma show no reactivity with the antibody. (c) Basal cells of the conjunctival epithelium reveal strong positive reactivity with the antibody, whereas other conjunctival epithelial cell layers show no or only weak (superficial epithelial cells) reactivity. No reactivity is visible subepithelially. (d) Basal epithelial cells of the double-layered epithelium of the nasolacrimal ducts demonstrate strong reactivity. Weak reactivity is seen in high columnar epithelial cells. No reactivity is visible in goblet cells. (e) Strong positive reactivity is visible in all layers of the corneal epithelium in a patient with herpetic keratitis. (f) Strong positive reactivity is visible in the superficial layer of the corneal epithelium in a patient with keratoconus. Deeper corneal epithelial layers reveal only weak reactivity. The corneal stroma is not reactive. (g) Positive reactivity is visible in all layers of the corneal epithelium in a patient with Fuchs dystrophy. (h) Positive control section from a human liver revealing positively stained hepatocytes. (ah) Hemalum. Scale bars: (a) 82.5 μm; (b, fh) 176 μm; (c, d) 42.5 μm; (e) 32 μm.
Figure 4.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with cytokines. Bars represent the mean value of cDNA from at least three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) and hCAT2b (b) were significantly upregulated by TNF-α, whereas IFN-γ and IL-1β had no significant effect. Because TNF-α alone has been shown to be prosurvival, a presensitization step involving preincubation of the cells with IFN-γ for 12 hours was performed, revealing that this has no significant effect compared with TNF-α alone.
Figure 4.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with cytokines. Bars represent the mean value of cDNA from at least three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) and hCAT2b (b) were significantly upregulated by TNF-α, whereas IFN-γ and IL-1β had no significant effect. Because TNF-α alone has been shown to be prosurvival, a presensitization step involving preincubation of the cells with IFN-γ for 12 hours was performed, revealing that this has no significant effect compared with TNF-α alone.
Figure 5.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with bacterial supernatants. Bars represent the mean value of cDNA from three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) was significantly upregulated by supernatant of S. aureus, whereas supernatant of P. aeruginosa had no significant effect. Expression of hCAT2b (b) was significantly upregulated by supernatants of both S. aureus and P. aeruginosa.
Figure 5.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with bacterial supernatants. Bars represent the mean value of cDNA from three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) was significantly upregulated by supernatant of S. aureus, whereas supernatant of P. aeruginosa had no significant effect. Expression of hCAT2b (b) was significantly upregulated by supernatants of both S. aureus and P. aeruginosa.
The authors thank Ute Beyer and Susann Möschter for expert technical assistance. 
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Figure 1.
 
Expression of human cationic amino acid transporters in tissues of the lacrimal apparatus and ocular surface. RT-PCR analysis for HCAT1 (a), 2b (a), and 3 (b) expression was performed in the nasolacrimal duct (nd), lacrimal gland (lg), conjunctiva (cj), and cornea (co). The integrity of the cDNAs was tested by amplification of the β-actin transcript. M, base pair standard; neg, negative control; pc, positive control (prostate).
Figure 1.
 
Expression of human cationic amino acid transporters in tissues of the lacrimal apparatus and ocular surface. RT-PCR analysis for HCAT1 (a), 2b (a), and 3 (b) expression was performed in the nasolacrimal duct (nd), lacrimal gland (lg), conjunctiva (cj), and cornea (co). The integrity of the cDNAs was tested by amplification of the β-actin transcript. M, base pair standard; neg, negative control; pc, positive control (prostate).
Figure 2.
 
Distribution of hCAT1 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) No hCAT1 is produced by the lacrimal gland. (b) Corneal epithelial cells reveal perinuclear and cytoplasmic reactivity. Bowman’s membrane and the corneal stroma show no reactivity with the antibody. (c) Conjunctival epithelial cells reveal positive reactivity with strong staining in the upper part of the cells. The secretory product of goblet cells is not reactive. In addition to epithelial cells, some subepithelial cells with the appearance of mononuclear cells demonstrated cytoplasmic reactivity. (d) High columnar epithelial cells of the nasolacrimal ducts reacted positively with the antibody, especially the upper part of these cells and the basal cell layer, whereas the secretory product of goblet cells did not react. (e) Immunohistochemical detection of hCAT1 in a patient with herpetic keratitis. Positive reactivity is especially visible in the epithelium and subepithelially in the stroma in the area of herpetic lesions. (f) Immunohistochemical detection of hCAT1 in a patient with keratoconus. HCAT1positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (g) Immunohistochemical detection of hCAT1 in a patient with Fuchs dystrophy. HCAT1 positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (h) Positive control section from human skin (cornifying). Positive red staining occurs in all epithelial cell layers except stratum corneum. (ah) Hemalum. Scale bars: (a, d) 82.5 μm; (b, c, eg) 176 μm; (h) 32 μm.
Figure 2.
 
Distribution of hCAT1 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) No hCAT1 is produced by the lacrimal gland. (b) Corneal epithelial cells reveal perinuclear and cytoplasmic reactivity. Bowman’s membrane and the corneal stroma show no reactivity with the antibody. (c) Conjunctival epithelial cells reveal positive reactivity with strong staining in the upper part of the cells. The secretory product of goblet cells is not reactive. In addition to epithelial cells, some subepithelial cells with the appearance of mononuclear cells demonstrated cytoplasmic reactivity. (d) High columnar epithelial cells of the nasolacrimal ducts reacted positively with the antibody, especially the upper part of these cells and the basal cell layer, whereas the secretory product of goblet cells did not react. (e) Immunohistochemical detection of hCAT1 in a patient with herpetic keratitis. Positive reactivity is especially visible in the epithelium and subepithelially in the stroma in the area of herpetic lesions. (f) Immunohistochemical detection of hCAT1 in a patient with keratoconus. HCAT1positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (g) Immunohistochemical detection of hCAT1 in a patient with Fuchs dystrophy. HCAT1 positivity is visible in the epithelium, especially supranucleally, but is absent in the stroma. (h) Positive control section from human skin (cornifying). Positive red staining occurs in all epithelial cell layers except stratum corneum. (ah) Hemalum. Scale bars: (a, d) 82.5 μm; (b, c, eg) 176 μm; (h) 32 μm.
Figure 3.
 
Distribution of hCAT2 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) Positive immunoreactivity is visible in acinar epithelial cells. (b) Corneal epithelial cells reveal weak perinuclear and cytoplasmic reactivity. Bowman membrane and corneal stroma show no reactivity with the antibody. (c) Basal cells of the conjunctival epithelium reveal strong positive reactivity with the antibody, whereas other conjunctival epithelial cell layers show no or only weak (superficial epithelial cells) reactivity. No reactivity is visible subepithelially. (d) Basal epithelial cells of the double-layered epithelium of the nasolacrimal ducts demonstrate strong reactivity. Weak reactivity is seen in high columnar epithelial cells. No reactivity is visible in goblet cells. (e) Strong positive reactivity is visible in all layers of the corneal epithelium in a patient with herpetic keratitis. (f) Strong positive reactivity is visible in the superficial layer of the corneal epithelium in a patient with keratoconus. Deeper corneal epithelial layers reveal only weak reactivity. The corneal stroma is not reactive. (g) Positive reactivity is visible in all layers of the corneal epithelium in a patient with Fuchs dystrophy. (h) Positive control section from a human liver revealing positively stained hepatocytes. (ah) Hemalum. Scale bars: (a) 82.5 μm; (b, fh) 176 μm; (c, d) 42.5 μm; (e) 32 μm.
Figure 3.
 
Distribution of hCAT2 in tissues of the lacrimal apparatus, ocular surface, and corneal abnormalities. Positive antibody (red). (a) Positive immunoreactivity is visible in acinar epithelial cells. (b) Corneal epithelial cells reveal weak perinuclear and cytoplasmic reactivity. Bowman membrane and corneal stroma show no reactivity with the antibody. (c) Basal cells of the conjunctival epithelium reveal strong positive reactivity with the antibody, whereas other conjunctival epithelial cell layers show no or only weak (superficial epithelial cells) reactivity. No reactivity is visible subepithelially. (d) Basal epithelial cells of the double-layered epithelium of the nasolacrimal ducts demonstrate strong reactivity. Weak reactivity is seen in high columnar epithelial cells. No reactivity is visible in goblet cells. (e) Strong positive reactivity is visible in all layers of the corneal epithelium in a patient with herpetic keratitis. (f) Strong positive reactivity is visible in the superficial layer of the corneal epithelium in a patient with keratoconus. Deeper corneal epithelial layers reveal only weak reactivity. The corneal stroma is not reactive. (g) Positive reactivity is visible in all layers of the corneal epithelium in a patient with Fuchs dystrophy. (h) Positive control section from a human liver revealing positively stained hepatocytes. (ah) Hemalum. Scale bars: (a) 82.5 μm; (b, fh) 176 μm; (c, d) 42.5 μm; (e) 32 μm.
Figure 4.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with cytokines. Bars represent the mean value of cDNA from at least three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) and hCAT2b (b) were significantly upregulated by TNF-α, whereas IFN-γ and IL-1β had no significant effect. Because TNF-α alone has been shown to be prosurvival, a presensitization step involving preincubation of the cells with IFN-γ for 12 hours was performed, revealing that this has no significant effect compared with TNF-α alone.
Figure 4.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with cytokines. Bars represent the mean value of cDNA from at least three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) and hCAT2b (b) were significantly upregulated by TNF-α, whereas IFN-γ and IL-1β had no significant effect. Because TNF-α alone has been shown to be prosurvival, a presensitization step involving preincubation of the cells with IFN-γ for 12 hours was performed, revealing that this has no significant effect compared with TNF-α alone.
Figure 5.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with bacterial supernatants. Bars represent the mean value of cDNA from three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) was significantly upregulated by supernatant of S. aureus, whereas supernatant of P. aeruginosa had no significant effect. Expression of hCAT2b (b) was significantly upregulated by supernatants of both S. aureus and P. aeruginosa.
Figure 5.
 
Regulation of hCAT1 (a) and hCAT2b (b) mRNA expression in HCE after stimulation with bacterial supernatants. Bars represent the mean value of cDNA from three different experiments. The fold increase in transcript levels over controls is expressed as mean ± SEM. *P < 0.05. Expression of hCAT1 (a) was significantly upregulated by supernatant of S. aureus, whereas supernatant of P. aeruginosa had no significant effect. Expression of hCAT2b (b) was significantly upregulated by supernatants of both S. aureus and P. aeruginosa.
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