December 2016
Volume 57, Issue 15
Open Access
Immunology and Microbiology  |   December 2016
The Effect of Ambient Titanium Dioxide Microparticle Exposure to the Ocular Surface on the Expression of Inflammatory Cytokines in the Eye and Cervical Lymph Nodes
Author Affiliations & Notes
  • Youngsub Eom
    Department of Ophthalmology, Korea University College of Medicine, Seoul, South Korea
  • Jong Suk Song
    Department of Ophthalmology, Korea University College of Medicine, Seoul, South Korea
  • Hyun Kyu Lee
    Department of Ophthalmology, Korea University College of Medicine, Seoul, South Korea
  • Boram Kang
    Department of Ophthalmology, Korea University College of Medicine, Seoul, South Korea
  • Hyeon Chang Kim
    Department of Preventive Medicine, Yonsei University College of Medicine, Seoul, South Korea
  • Hyung Keun Lee
    Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea
  • Hyo Myung Kim
    Department of Ophthalmology, Korea University College of Medicine, Seoul, South Korea
  • Correspondence: Jong Suk Song, Department of Ophthalmology, Guro Hospital, Korea University College of Medicine, 80, Guro-dong, Guro-gu, Seoul, 152–703, South Korea; crisim@korea.ac.kr
Investigative Ophthalmology & Visual Science December 2016, Vol.57, 6580-6590. doi:10.1167/iovs.16-19944
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      Youngsub Eom, Jong Suk Song, Hyun Kyu Lee, Boram Kang, Hyeon Chang Kim, Hyung Keun Lee, Hyo Myung Kim; The Effect of Ambient Titanium Dioxide Microparticle Exposure to the Ocular Surface on the Expression of Inflammatory Cytokines in the Eye and Cervical Lymph Nodes. Invest. Ophthalmol. Vis. Sci. 2016;57(15):6580-6590. doi: 10.1167/iovs.16-19944.

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

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Abstract

Purpose: To investigate the ocular immune response following exposure to airborne titanium dioxide (TiO2) microparticles.

Methods: Rats in the TiO2-exposed group (n = 10) were exposed to TiO2 particles for 2 hours twice daily for 5 days, while the controls (n = 10) were not. Corneal staining score and tear lactic dehydrogenase (LDH) activity were measured to evaluate ocular surface damage, serum immunoglobulin (Ig) G and E were assayed by using enzyme-linked immunosorbent assay, and the size of cervical lymph nodes was measured. In addition, the expression of interleukin (IL)-4, IL-17, and interferon (IFN)-γ in the anterior segment of the eyeball and cervical lymph nodes was measured by immunohistochemistry, real-time reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis.

Results: Median corneal staining score (3.0), tear LDH activity (0.24 optical density [OD]), and cervical lymph node size (36.9 mm2) were significantly higher in the TiO2-exposed group than in the control group (1.0, 0.13 OD, and 26.7 mm2, respectively). Serum IgG and IgE levels were found to be significantly elevated in the TiO2-exposed group (P = 0.021 and P = 0.021, respectively). Interleukin 4 expression was increased in the anterior segment of the eyeball and lymph nodes following TiO2 exposure, as measured by immunostaining, real-time RT-PCR, and Western blot. In addition, IL-17 and IFN-γ levels were also increased following TiO2 exposure compared to controls as measured by immunostaining.

Conclusions: Exposure to airborne TiO2 induced ocular surface damage. The Type 2 helper T-cell pathway seems to play a dominant role in the ocular immune response following airborne TiO2 exposure.

Air pollution exposure has been studied mainly in the context of respiratory and cardiovascular diseases and has been identified as a risk factor for cardiopulmonary pathology.1,2 However, the effect of air pollution exposure on the ocular surface has not been extensively studied despite the fact that air pollution exposure has been reported to be associated with ocular surface diseases including dry eye syndrome, conjunctivitis, and blepharitis.35 
Airborne particulate pollution is composed of variously sized particles and various chemical components.6,7 In ambient particulate matter (PM), fine particles that have an aerodynamic diameter less than 2.5 μm (PM2.5) are more likely to have adverse health effects than large particles.8,9 Titanium dioxide (TiO2) is a chemical that is used in the production of cosmetics, paints, and plastics and has also been used to evaluate the effects of fine ambient PM exposure on health.10 
Recently, there has been an effort to evaluate the effect of TiO2 nanoparticle exposure on the ocular surface.11 This study demonstrates that exposure of the ocular surface to TiO2 nanoparticles induces ocular surface damage and shows the role of mucus in ocular surface defense against nanoparticle exposure.11 However, the effect of TiO2 exposure on the ocular immune system was not investigated. Previous work12 has shown that ocular exposure to ozone increases the tear levels of interleukin (IL)-1β, IL-6, IL-17, and interferon (IFN)-γ in mice. On the other hand, a study which has evaluated the effect of ambient air pollution on tear cytokine levels in healthy outdoor workers shows that high levels of PM2.5 exposure are associated with decreased IL-5 and IL-10 in tears.13 The reporting group thus wanted to evaluate the effect of PM2.5 TiO2 exposure on the ocular surface immune system through the use of a whole-body exposure chamber for rats. Corneal staining, tear lactic dehydrogenase (LDH) activity (a measure of cellular damage14), tear mucin 5 subtypes A and C (MUC5AC) concentration, serum immunoglobulin (Ig) G and E, the size of superficial cervical lymph nodes, immunohistochemistry, and the expression of IL-4, IL-17, and IFN-γ in the anterior segment of the eyeball and cervical lymph nodes were assessed and compared after 5 days of TiO2 microparticle exposure. 
Materials and Methods
Thirty-six inbred male Lewis rats, each weighing 150 to 250 g (6–8 weeks old), were used in this study. Rats were randomly divided into four groups: airflow-exposed (n = 8), control 1 (n = 8), TiO2-exposed (n = 10), and control 2 (n = 10) groups (see below). All procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.15 Approval for this study was obtained from the Korea University Guro Hospital Institutional Review Board, Seoul, South Korea. 
Microparticle Exposure and Study Groups
An exposure chamber with six fans was constructed for the exposure of TiO2 particles on the ocular surface of rats in this study. Titanium (IV) oxide, rutile, 99.5% (metal basis) with a mean diameter of 1.0 to 2.0 μm (Alfa Aesar; Johnson Matthey GmbH, Karlsruhe, Germany) was put in the exposure chamber, and airborne particle concentration in the exposure chambers was adjusted for particulate matter less than 10 μm (PM10) as measured by a MetOne 831 Aerosol Mass Monitor (Met One, Grants Pass, OR, USA). The mean ± standard deviation of airborne TiO2 particle concentration during the study period was 331 ± 83 μg/m3. Rats in the TiO2-exposed group (n = 10) were exposed to airborne TiO2 in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 2 group (n = 10) were not exposed to airborne TiO2 particles. The daily average TiO2 concentration of exposure to 331 μg/m3 TiO2 for 2 hours twice daily is approximately 55 μg/m3, which is similar to the short-term (24-hour) limit value (50 μg/m3) of the Air Quality Guidelines set forth by the World Health Organization for PM10.16 This limit value for short-term PM10 concentrations is often exceeded in various cities across Europe, Asia, and the United States.1719 In terms of exposure duration, a previous study11 has shown distinct differences in results between rabbits that underwent TiO2 instillation in the eye for 4 days and rabbits that underwent a single instillation of TiO2 in the eye. 
To investigate the effect of airflow on the results, corneal staining (n = 4 for each group), tear LDH activity (n = 4), tear MUC5AC concentration (n = 4), and serum IgG (n = 4) and IgE (n = 4) levels were compared between the airflow-exposed and control 1 groups. Rats in the airflow-exposed group (n = 8) were exposed only to airflow without airborne TiO2 in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 1 group (n = 8) were not exposed to airflow or airborne TiO2 particles. 
General anesthesia was induced via intramuscular injection of xylazine hydrochloride (Rompun 2%, 1 mg/100 g body weight; Bayer, Leverkusen, Germany) and Zoletil (8 mg/100 g body weight; Virbac Laboratories, Carros, France) before corneal staining, tear sample collection, and euthanasia. 
Corneal Staining
The bilateral corneal staining score of six TiO2-exposed and six control 2 rats (n = 6 for each group) was graded 24 hours after the last exposure of TiO2 with a slit lamp examination under general anesthesia by a single experienced ophthalmologist (YE) according to the National Eye Institute scoring scheme.20 The mean corneal staining score of both eyes of individual rats was used for statistical analysis. Briefly, a fluorescein sodium–impregnated paper strip (Haag-Streit, Bern, Switzerland) was wet with 5 μL sterile normal saline and diluted dye was dropped onto the upper bulbar surface after retracting the upper lid. After the dye was placed, the rat's eye was gently closed five times and the excessive tear fluid and dye were wiped away. Corneal staining was observed with slit lamp examination under cobalt blue illumination. In the same way, the bilateral corneal staining score of four airflow-exposed and four control 1 rats was compared 24 hours after the last exposure to airflow only. Rats used for ocular surface staining were not used for tear sample collection, but were used in the assessment of the size of superficial cervical lymph nodes, immunohistochemistry, and the expression of IL-4, IL-17, and IFN-γ. 
Tear Sample Collection
Tear samples from the bilateral eyes of four airflow-exposed, four control 1, four TiO2-exposed, and four control 2 rats (n = 8 for each group) were collected under general anesthesia 24 hours after the last exposure of TiO2 or airflow only between 10:00 and 11:00 AM. Tear samples (106 μL) were obtained from each eye by using the flush tear collection method, as previously described.21,22 To obtain eye-flush tears, 60 μL sterile normal saline was introduced into the space between the eye and the lateral canthus, and fluid was aspirated from the inferior meniscus by using a micropipette tip.21,22 In each collection of eye-flush tears, approximately 50 to 60 μL tear volume was collected. Sterile normal saline was instilled twice and the tear sampling was performed within 1 minute. For enzyme-linked immunosorbent assay (ELISA), the tear sample on ice was immediately sent to the laboratory and stored at −20°C until analysis. Lactic dehydrogenase activity was measured in 100 μL diluted tear samples (n = 8 for each group) containing 6 μL eye-flush tears and 94 μL phosphate-buffered saline (PBS; method described below). The remaining 100 μL undiluted eye-flush tears (n = 8 for each group) were used to measure MUC5AC level (method described below). The mean values of the right and left eyes in each rat were used to compare tear LDH activity (n = 4 for each group) and tear MUC5AC concentration (n = 4) between the two groups. 
Enzyme-Linked Immunosorbent Assay
Lactic dehydrogenase activity was measured in eight tear samples from each group,14 and another eight samples from each group were used to measure MUC5AC levels. Lactic dehydrogenase activity was measured with the LDH ELISA kit (CytoTox96 nonradioactive cytotoxicity assay; Promega, Madison, WI, USA), and the level of MUC5AC was measured with an ELISA kit for Mucin 5 subtype AC (MyBiosource, San Diego, CA, USA).11 
The blood samples for measuring the IgG and IgE levels in serum were collected from the abdominal aorta of four rats in each group under general anesthesia. The IgG level in serum (1:200,000 dilution) was measured with a Rat IgG ELISA kit (ab189578; Abcam, Cambridge, MA, USA), and the IgE level in serum (9:10 dilution) was measured with a Rat IgE ELISA kit (ab157736; Abcam). 
All measurements were conducted according to the manufacturer's protocol by using a microplate spectrophotometer (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA, USA). 
Size of Cervical Lymph Nodes
Three TiO2-exposed and three control 2 rats were anesthetized and humanely euthanized by using a CO2 chamber. After euthanasia, superficial cervical lymph nodes were exposed by bilateral neck dissection, and the largest node on each side was collected from three TiO2-exposed (n = 6) and three control 2 (n = 6) rats. Dissected lymph nodes were placed beside a graduated ruler, and digital images were taken. The digital images were quantified by a blinded examiner (HKyL) using ImageJ (1.43u; http://rsb.info.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). First, the unit of measurement of the still pictures was changed from distance in pixels to millimeters, based on the graduated ruler beside the lymph nodes, as previously described.23 Next, the area of the lymph node was selected and measured by using the “Freehand Selections” and “Selection Brush Tool” of ImageJ (Fig. 1).11 
Figure 1
 
Measurement of cervical lymph node size from digital images, using ImageJ.
Figure 1
 
Measurement of cervical lymph node size from digital images, using ImageJ.
Immunohistochemistry
One eye per one euthanized rat in the TiO2-exposed and control 2 groups was enucleated 24 hours after the last TiO2 exposure. One superficial cervical lymph node from each group was collected. Eyes and lymph nodes were fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin-embedded tissues were cut into 4-μm sections with a microtome (Leica RM 2255; Leica, Bannockburn, IL, USA), and tissue sections were placed on microscope slides. After deparaffinization of the tissue sections with xylene, tissue sections were immersed in a graded series of ethanol and PBS. Serially cut sections were used for immunohistochemistry of IL-4, IL-17, and IFN-γ. Primary antibodies were commercially obtained for IL-4 and IL-17 (1:500 for ab9811 and 1:500 for ab79056, respectively; Abcam), and IFN-γ (1:500, orb 48034; Biorbyt, Carrickfergus, UK). A rabbit-specific HRP/DAB (ABC) Detection IHC Kit (ab64261; Abcam) was used as a secondary antibody according to the manufacturer's instructions. The tissue sections were observed under light microscopy at ×400 magnification, and digital images were taken with an Olympus BX51 microscope and a DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). The cells positive for each stain were determined by analyzing four visual fields on conjunctival biopsy and reported as cells per square millimeter. The median IL-4–, IL-17–, and IFN-γ–positive cells per square millimeter of four fields on conjunctival biopsy were compared between the TiO2-exposed and control 2 groups. 
Quantification of Cytokines
The expression of IL-4, IL-17, and IFN-γ in the anterior segment of the eyeball (full-thickness corneoscleral button with iris) and cervical lymph nodes was evaluated in the TiO2-exposed and control 2 groups by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR). The anterior segments of both eyes from each rat were obtained from five euthanized rats in each group by using a circular scleral incision at 2 to 3 mm behind the limbus. The largest superficial cervical lymph node on each side of the neck was obtained by neck dissection as described above. 
Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis.
For the extraction of RNA, the anterior segments of both eyes from each rat were homogenized together in TRIzol solvent (Takara Bio, Inc., Shiga, Japan), and total RNA was isolated from the homogenized sample (n = 1 for each group) according to the product protocol. Bilateral cervical lymph nodes from individual rats were also homogenized together in TRIzol solvent, and total RNA was isolated from the homogenized sample (n = 1 for each group). Complementary DNA was generated by using 2 μg RNA from the anterior segment of the eyeball and 0.1 μg RNA from lymph nodes by a one-cycle reverse transcriptase reaction with a High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Real-time RT-PCR was performed with the cDNA to quantify the expression of IL-4, IL-17, and IFN-γ using the 7300 Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). Primer sequences were as follows: IL-4 (5′-TGATGGGTCTCAGCCCCCACCTTGC-3′ and 5′-CTTTCAGTGTTGTGAGCGTGGACTC-3′); IL-17 (5′-GACCCAAACCACAAGTCCAA-3′ and 5′-GTCATCTTCATCTCCGTGTCC-3′); and IFN-γ (5′-AAAGACAACCAGGCCATCAG-3′ and 5′-CTTTTCCGCTTCCTTAGGCT-3′). Messenger RNA fold change was quantified with an internal control gene (GAPDH mRNA) by using the comparative threshold (2-ΔΔCT) method.24 
Western Blot Analysis.
The anterior segments of both eyes of each rat were homogenized together in T-per tissue protein extraction reagent containing a protease inhibitor mixture (Thermo Scientific, Rockford, IL, USA), and the tissue cell extracts (n = 4 for each group) were subjected to Western blot analysis for measuring the protein levels of IL-4, IL-17, IFN-γ, and β-actin, as previously described.25 Tissue homogenates from bilateral cervical lymph nodes from individual rats were also subjected to Western blot analysis for measuring protein levels of IL-4, IL-17, IFN-γ, and β-actin (n = 4 for each group). Protein concentration per lane in the Western blot was 50 μg for the anterior segment samples and 10 μg for the cervical lymph node samples. Primary antibodies were commercially obtained for IL-4, IL-17 (1:1000 for ab9811 and 1:2000 for ab79056, respectively; Abcam), IFN-γ (1:1000, orb 48034; Biorbyt), and β-actin (1:10,000, No. 5125; Cell Signaling Technology, Danvers, MA, USA). The secondary antibody was an anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (1:10,000 for IL-4 and IFN-γ, and 1:20,000 for IL-17, No. 7074; Cell Signaling Technology). 
Statistical Analyses
Statistical analyses were performed by using the Mann-Whitney U test in SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA). In our previous study that compared ocular surface staining between the control and TiO2-4D group 24 hours after the last TiO2 instillation, rabbits in the TiO2-4D group received a 4 μL TiO2 instillation in the right eye once a day for 4 days and the untreated left eyes were allocated into the control group.11 The mean (±SD) grading of ocular surface staining in the TiO2-4D group (n = 5) is 1.40 ± 0.55 and that in the control group (n = 5) is 0.20 ± 0.45.11 Thus, analysis suggested that four measurements were needed to achieve a power of 80% and 5% significance (two tailed) to identify the difference in means. Thus, four rats were chosen for ELISA and Western blot analysis in each group. Values were expressed as the median and interquartile range (IQR). Results of Western blot analysis were expressed as a ratio to β-actin. Results were considered statistically significant if the P value was less than 0.05. 
Results
The Effect of Airflow
There were no significant differences in corneal staining score, tear LDH activity, tear MUC5AC concentration, or serum IgG and IgE levels between the airflow-exposed and control 1 groups (Table 1). 
Table 1
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 1 and Airflow-Exposed Groups
Table 1
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 1 and Airflow-Exposed Groups
Corneal Staining
The median (IQR) corneal staining score in the TiO2-exposed group (3.0 [1.9–4.1]; Fig. 2A, Table 2) was significantly higher than that of the control 2 group (1.0 [0.8–1.8]; P = 0.034; Fig. 2B, Table 2). 
Figure 2
 
Representative photographs of rat eyes after fluorescein staining. Rats in the TiO2-exposed group were exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 2 group were not exposed to TiO2. (A) TiO2-exposed group. (B) Control 2 group.
Figure 2
 
Representative photographs of rat eyes after fluorescein staining. Rats in the TiO2-exposed group were exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 2 group were not exposed to TiO2. (A) TiO2-exposed group. (B) Control 2 group.
Table 2
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 2 and TiO2-Exposed Groups
Table 2
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 2 and TiO2-Exposed Groups
Enzyme-Linked Immunosorbent Assay
The median (IQR) tear LDH activity in the TiO2-exposed group (0.2 optical density [OD] [0.2–0.4]) was significantly higher than that of the control 2 group (0.1 OD [0.1–0.2]; P = 0.021; Fig. 3A, Table 2). However, there was no significant difference in the tear MUC5AC concentration between the TiO2-exposed and control 2 groups (Fig. 3B; Table 2). There were no significant differences in tear LDH activity or tear MUC5AC concentration between eyes in each group. 
Figure 3
 
A comparison of LDH activity and MUC5AC levels in tear samples between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) A comparison of LDH activity in tears between the two groups. (B) A comparison of MUC5AC levels in tears between the two groups. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 3
 
A comparison of LDH activity and MUC5AC levels in tear samples between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) A comparison of LDH activity in tears between the two groups. (B) A comparison of MUC5AC levels in tears between the two groups. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
The median (IQR) serum IgG level (4.4 ng/mL [3.1–7.6]) and IgE level (12.1 ng/mL [5.6–16.9]) in the TiO2-exposed group were significantly higher than those of the control 2 group, respectively (P = 0.021 and P = 0.021, respectively; Table 2). 
Size of Cervical Lymph Nodes
The median (IQR) size of lymph nodes in the TiO2-exposed group (36.9 mm2 [32.4–42.4]) was significantly larger than that of the control 2 group (26.7 mm2 [20.7–30.9]; P = 0.010; Fig. 4). 
Figure 4
 
A comparison of cervical lymph node size between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 4
 
A comparison of cervical lymph node size between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Immunohistochemistry
Interleukin 4–, IL-17–, and IFN-γ–positive cells were infrequently detected in both conjunctival and cervical lymph node biopsies of the control 2 group (Figs. 5, 6). In comparison, both conjunctival and lymph node biopsies from the TiO2-exposed group had more IL-4, IL-17, and IFN-γ immunostained cells than those from the control 2 group (Figs. 7, 8). The median (IQR) IL-4–, IL-17–, and IFN-γ–positive cells per square millimeter on conjunctival biopsy in the TiO2-exposed group (72 cells/mm2 [39–105], 90 cells/mm2 [57–96], and 96 cells/mm2 [60–142], respectively) was significantly higher than in the control 2 group (12 cells/mm2 [3–21], 18 cells/mm2 [12–42], and 30 cells/mm2 [6–36] , respectively; P = 0.020, P = 0.027, and P = 0.019, respectively). 
Figure 5
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 5
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 6
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 6
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Quantification of Cytokines
Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis.
Titanium dioxide exposure on the ocular surface increased the expression level of IL-4, IL-17, and IFN-γ in the anterior segment of the eyeball from each rat by 13.3-fold, 6.0-fold, and 7.4-fold, respectively, when compared to controls. Interleukin 4, IL-17, and IFN-γ expression in the cervical lymph nodes increased by 10.7-fold, 5.1-fold, and 13.1-fold, respectively, following TiO2 exposure compared to controls. 
Western Blot Analysis.
The levels of IL-4 in both the anterior segment of the eyeball and cervical lymph nodes and the levels of IL-17 in the cervical lymph nodes were significantly increased following TiO2 exposure compared to the control 2 group (Figs. 9A, 9B; Table 3). However, the increases in the levels of IL-17 in the anterior segment of the eyeball and the levels of IFN-γ in both the anterior segment of the eyeball and cervical lymph node following TiO2 exposure were not significantly different as compared to the control 2 group (Figs. 9B, 9C; Table 3). 
Figure 7
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 5.
Figure 7
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 5.
Table 3
 
Comparison of Expression of Inflammatory Cytokines in the Anterior Segment of the Eyeball and Cervical Lymph Node of Rats Between the Control 2 and TiO2-Exposed Groups
Table 3
 
Comparison of Expression of Inflammatory Cytokines in the Anterior Segment of the Eyeball and Cervical Lymph Node of Rats Between the Control 2 and TiO2-Exposed Groups
Discussion
This study evaluated the effects of airborne TiO2 microparticle exposure on the ocular surface immune system by assaying the serum IgG and IgE levels and by measuring the expression of inflammatory cytokines in the anterior segment of the eyeball and cervical lymph nodes following TiO2 exposure. This study found a significant increase in the expression of IL-4 in both the anterior segment of the eyeball and cervical lymph nodes following TiO2 exposure. Interleukin 4 is a T helper 2 (Th2) cytokine that induces differentiation of Th0 cells to Th2 cells and stimulates B cells to produce IgE.26,27 In addition, serum IgG and IgE levels, the production of which are enhanced by Th2 cytokines, were found to be significantly elevated following TiO2 exposure in this study. Thus these results indicate that the Th2 pathway is involved in the immune response following TiO2 exposure on the ocular surface. In line with this study, intratracheal exposure of mice to TiO2 for 90 days increases the levels of several cytokines, including IL-4.28 The mRNA expression of the transcription factor GATA-3, which has been shown to play a relevant role in the Th2 pathway, is upregulated in the lung tissue of rats with pulmonary injury induced by TiO2 microparticles, although there is no significant change in IL-4.29 Previous work that has investigated the hepatotoxicity of TiO2 nanoparticles has shown that increased levels of IL-4, IL-5, and GATA-3 are accompanied by hepatic inflammation and that alteration of the Th2 pathway may be involved in the TiO2 nanoparticle–induced liver injury.30 
The results of the immunohistochemistry and real-time RT-PCR experiments in the current study indicated that airborne TiO2 exposure increases expression of IL-4, IL-17, and IFN-γ in both the anterior segment of the eyeball and cervical lymph nodes when compared to controls, which are representative cytokines for Th2, Th17, and Th1 cytokines, respectively. This result is similar to that of a previous study that has evaluated the effect of ozone exposure on the ocular surface.12 In that study, Lee et al.12 demonstrate that ozone exposure increases levels of IL-1β, IL-6, IL-17, and IFN-γ in tears of mice. Conversely, Matsuda et al.13 have shown that PM2.5 exposure does not increase levels of IL-2, IL-4, IL-5, IL-10, and IFN-γ in tears of healthy workers, but instead they have found decreased IL-5 and IL-10 in tears in groups exposed to high levels of PM2.5. Differences between long-term environmentally exposed human subjects and short-term exposed laboratory animals may explain the discrepancy between the results of the work done here and these studies.12,13 
Airborne TiO2 particle–exposed rats showed enlarged cervical lymph nodes in this study. Lymph nodes are distributed along the course of the lymphatic vessels throughout the body. Lymph node enlargement is a phenomenon that occurs as part of the body's natural immune response. A previous study31 has shown that rats have enlarged tracheobronchial lymph nodes following inhalation of TiO2. Wang et al.32 have demonstrated that intragastric administration of TiO2 in mice causes lymph nodule proliferation in the spleen. Superficial cervical lymph nodes act as the lymphatic drainage pathway in the immunopathogenesis of dry eye disease.3335 In the immune cycle of the dry eye, antigen-presenting cells from the ocular surface migrate to the superficial cervical lymph nodes where the antigen is presented to CD4+ T cells to produce cytokines and to generate immune responses.33,34 The results obtained here suggest that cervical lymph nodes play a role in the activated immune response against airborne TiO2 particle exposure. 
To investigate mucin secretion in tears by conjunctival goblet cells, tear MUC5AC concentration was measured in this study instead of performing impression cytology. Impression cytology identifies goblet cells via periodic acid–Schiff (PAS) staining of granules within the superficial layers of the conjunctival epithelium. Because all mucin granules are released in an apocrine fashion when secreted from goblet cells,36 PAS staining on impression cytology cannot identify goblet cells after all mucin granules are released. In addition, an increase in the number of goblet cells on impression cytology could indicate not only an increase in mucin synthesis, but also a decrease in mucin secretion.37 On the other hand, an increase in tear MUC5AC concentration indicates an increase in mucin secretion. 
Titanium dioxide nanoparticles can directly induce mucin secretion in human bronchial epithelial cells via a calcium signaling–mediated pathway. However, 5 days of repeated PM2.5 TiO2 exposure on the ocular surface increased tear LDH activity but not the tear MUC5AC level in this study. Similarly, a previous study11 has shown that the tear MUC5AC level increases after a single exposure but then returns to normal levels following repeated exposure to TiO2 nanoparticles. This reduction of mucin secretion may be due to exhaustion of conjunctival goblet cells following release of their secretory granules.11,38 Furthermore, 7 days of repeated allergen exposure on the ocular surface causes a reduction of MUC5AC in a mouse allergic conjunctivitis model.38 Thus, repeated TiO2 exposure could decrease ocular surface protection provided by the gel-forming mucin secreted by conjunctival goblet cells.11 On the other hand, increases in MUC5AC mRNA levels and conjunctival goblet cell hyperplasia were observed in human conjunctiva following long-term exposure to air pollution.39,40 There may be differences in the response of the ocular surface to short- and long-term exposures to air pollution. Thus, a direct comparison between short- and long-term exposure to air pollution is warranted. 
Airflow itself could cause evaporative conditions, leading to desiccation stress on the ocular surface. Thus, to investigate the effect of airflow on the ocular surface, this study compared corneal staining, tear LDH activity, tear MUC5AC concentration, and serum IgG and IgE levels between rats that were and were not exposed to this airflow. The results indicated that airflow without TiO2 particles does not influence the ocular surface and immune system. It thus seems that the effects of airflow on the ocular surface are negligible as compared to the effects of airborne TiO2 particles. In line with this study, a previous study that has evaluated the effect of airflow exposure for 1 hour, 3 times a day for 4 days on tear production and corneal staining has shown that airflow does not influence tear production and corneal staining when compared to the control group.41 
There are several limitations in the present study. First, the sample size was relatively small. Second, TiO2 exposure was not limited to the ocular surface but also included nasal exposure and inhalation. Thus, the possibility of the effect of inhalation and nasal exposure to TiO2 on cervical lymph nodes cannot be ruled out. Indeed, cervical lymph nodes act as the draining lymph nodes of intranasally induced immunologic tolerance.42 In addition, deposition of brown particles in cervical lymph nodes was observed in rats with repeated intratracheal exposure of TiO2.43 These effects are unavoidable in this study design evaluating the effect of airborne TiO2 exposure on the ocular surface. 
In conclusion, exposure of the ocular surface to airborne TiO2 particles induced ocular surface damage and increased serum IgG and IgE levels and the expression of inflammatory cytokines, especially type 2 cytokines, in both the anterior segment of the eyeball and cervical lymph nodes. These findings suggest the possibility that the Th2 pathway plays a dominant role in the immune response following airborne TiO2 particle exposure on the ocular surface. 
Figure 8
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 6.
Figure 8
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 6.
Figure 9
 
A comparison of the expression of inflammatory cytokines in the anterior segment of the eyeball and cervical lymph nodes between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) Expression of IL-4. (B) Expression of IL-17. (C) Expression of IFN-γ. A representative example of a Western blot gel image is shown at the top left of each figure. β-Actin was used as an internal loading control for the Western blot. The bars represent means ± SD of four independent experiments. The asterisk indicates a significant difference (P value < 0.05) in the expression of proinflammatory cytokines in the TiO2-exposed group compared with the control by the Mann-Whitney U test.
Figure 9
 
A comparison of the expression of inflammatory cytokines in the anterior segment of the eyeball and cervical lymph nodes between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) Expression of IL-4. (B) Expression of IL-17. (C) Expression of IFN-γ. A representative example of a Western blot gel image is shown at the top left of each figure. β-Actin was used as an internal loading control for the Western blot. The bars represent means ± SD of four independent experiments. The asterisk indicates a significant difference (P value < 0.05) in the expression of proinflammatory cytokines in the TiO2-exposed group compared with the control by the Mann-Whitney U test.
Acknowledgments
Supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant HI13C0055), by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1A02937003), and by the Busan Sungmo Eye Hospital Sodam Scholarship Committee, Busan, South Korea. The funding organization had no role in the design or conduct of this research. 
Disclosure: Y. Eom, None; J.S. Song, None; H.Ky. Lee, None; B. Kang, None; H.C. Kim, None; H.Ke. Lee, None; H.M. Kim, None 
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Figure 1
 
Measurement of cervical lymph node size from digital images, using ImageJ.
Figure 1
 
Measurement of cervical lymph node size from digital images, using ImageJ.
Figure 2
 
Representative photographs of rat eyes after fluorescein staining. Rats in the TiO2-exposed group were exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 2 group were not exposed to TiO2. (A) TiO2-exposed group. (B) Control 2 group.
Figure 2
 
Representative photographs of rat eyes after fluorescein staining. Rats in the TiO2-exposed group were exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days. Rats in the control 2 group were not exposed to TiO2. (A) TiO2-exposed group. (B) Control 2 group.
Figure 3
 
A comparison of LDH activity and MUC5AC levels in tear samples between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) A comparison of LDH activity in tears between the two groups. (B) A comparison of MUC5AC levels in tears between the two groups. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 3
 
A comparison of LDH activity and MUC5AC levels in tear samples between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) A comparison of LDH activity in tears between the two groups. (B) A comparison of MUC5AC levels in tears between the two groups. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 4
 
A comparison of cervical lymph node size between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 4
 
A comparison of cervical lymph node size between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. An asterisk indicates a P value < 0.05 by the Mann-Whitney U test.
Figure 5
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 5
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 6
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 6
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the control 2 group (not exposed to TiO2). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ.
Figure 7
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 5.
Figure 7
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of conjunctiva from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 5.
Figure 8
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 6.
Figure 8
 
Immunohistochemistry of IL-4, IL-17, and IFN-γ in serial sections of cervical lymph nodes from rats in the TiO2-exposed group (exposed to airborne TiO2 microparticles in the exposure chamber for 2 hours twice daily for 5 days). (A) Immunohistochemistry of IL-4. (B) Immunohistochemistry of IL-17. (C) Immunohistochemistry of IFN-γ. More IL-4, IL-17, and IFN-γ immunostained cells (arrows) are observed in the TiO2-exposed group than in the control 2 group in Figure 6.
Figure 9
 
A comparison of the expression of inflammatory cytokines in the anterior segment of the eyeball and cervical lymph nodes between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) Expression of IL-4. (B) Expression of IL-17. (C) Expression of IFN-γ. A representative example of a Western blot gel image is shown at the top left of each figure. β-Actin was used as an internal loading control for the Western blot. The bars represent means ± SD of four independent experiments. The asterisk indicates a significant difference (P value < 0.05) in the expression of proinflammatory cytokines in the TiO2-exposed group compared with the control by the Mann-Whitney U test.
Figure 9
 
A comparison of the expression of inflammatory cytokines in the anterior segment of the eyeball and cervical lymph nodes between the control 2 and TiO2-exposed groups. Experimental groups are the same as in Figure 2. (A) Expression of IL-4. (B) Expression of IL-17. (C) Expression of IFN-γ. A representative example of a Western blot gel image is shown at the top left of each figure. β-Actin was used as an internal loading control for the Western blot. The bars represent means ± SD of four independent experiments. The asterisk indicates a significant difference (P value < 0.05) in the expression of proinflammatory cytokines in the TiO2-exposed group compared with the control by the Mann-Whitney U test.
Table 1
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 1 and Airflow-Exposed Groups
Table 1
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 1 and Airflow-Exposed Groups
Table 2
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 2 and TiO2-Exposed Groups
Table 2
 
Comparison of LDH Activity and MUC5AC Levels in Rat Tear Samples and Serum Immunoglobulin (IgG and IgE) Levels Between the Control 2 and TiO2-Exposed Groups
Table 3
 
Comparison of Expression of Inflammatory Cytokines in the Anterior Segment of the Eyeball and Cervical Lymph Node of Rats Between the Control 2 and TiO2-Exposed Groups
Table 3
 
Comparison of Expression of Inflammatory Cytokines in the Anterior Segment of the Eyeball and Cervical Lymph Node of Rats Between the Control 2 and TiO2-Exposed Groups
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