Proteomics Differentiate Between Thyroid-Associated Orbitopathy and Dry Eye Syndrome

Abstract

Purpose.: In patients with thyroid-associated orbitopathy (TAO), the dry eye syndrome occurs frequently, and symptoms and signs of both disorders overlap making early and accurate differential diagnosis difficult. A differentiation via specific markers is warranted.

Methods.: Tear fluid samples of 120 subjects with TAO, TAO + dry eye, dry eye, and controls were collected. The samples were measured using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were tested with antibody microarrays.

Results.: Proteomics identified deregulated proteins in TAO and dry eye. Compared with dry eye, proline-rich protein 1 (PROL1, P = 0.002); uridine diphosphate (UDP)–glucose-dehydrogenase (UGDH, P = 0.017); calgranulin A (S10A8, P < 0.0001); transcription-activator BRG1 (SMCA4, P < 0.0001); annexin A1 (P = 0.007); cystatin (P = 0.009); heat shock protein 27 (P = 0.03); and galectin (P = 0.04) were markedly downregulated in TAO. Compared with healthy controls, PROL1 (P < 0.05.); proline-rich protein 4 (PRP4, P < 0.05), S10A8 (P = 0.004) and SMCA4 (P = 0.002) were downregulated in TAO. In contrast, the proteins midasin and POTE-ankyrin–domain family-member I were upregulated in TAO versus healthy controls (P < 0.05). Protein dysregulation was associated with inflammatory response and cell death. Antibody microarray confirmed significant changes of PRP4, PROL1, and UGDH between TAO and dry eye or healthy controls (P < 0.01). The presence of these three proteins was negatively correlated with smoking (P < 0.05).

Conclusions.: Proteomics of tear fluid demonstrated an upregulation of inflammatory proteins versus a downregulation of protective proteins in TAO, and a significantly different protein panel in TAO versus dry eye and/or controls. The spectrum of inflammatory and protective proteins might be a useful indicator for disease activity and ocular surface disease in patients with TAO.

Thyroid-associated orbitopathy (TAO) is the most frequent extrathyroidal manifestation in patients with autoimmune thyroid disease.¹ In patients with TAO, involvement of the lacrimal gland and a reduced tear production has been reported.^2,3 Eye lid retraction, an abnormally wide palpebral fissure, insufficient eyelid closure, proptosis, reduced blinking, and impaired Bell's phenomenon are factors regarded to contribute to an increased tear film evaporation.⁴ Also, the composition of tear fluid is altered in TAO.^5–7 Thus, patients with TAO often exhibit symptoms and signs of the dry eye syndrome⁸ and the combination of both makes an early and accurate differential diagnosis difficult. The quality of life and visual functioning of these patients is markedly impaired.⁹ Dry eye is defined by an abnormal tear film that results in changes of the ocular surface often accompanied by ocular discomfort. Its symptoms may include blurred vision, burning, itchiness, redness or grittiness in the eye, and sensitivity to light.¹⁰ Dry eye is a common disease and has an increased prevalence in people with autoimmune disease and thyroid disorders.^11–12 

In thyroid-associated orbitopathy, circulating pro-inflammatory cytokines (e.g., TNF alpha) increase the expression of the Fas molecule on the surface of the lacrimal cells.¹³ This leads to apoptosis of the target cells and to the release of a membrane bound 264 KD protein, fodrin, which is known as the autoantigen in patients with Sjögren syndrome.¹⁴ Sjögren syndrome, frequently observed in patients with various autoimmune diseases, encompasses symptoms and signs of dry eye. 

In previous studies, we have shown that protein profiles of TAO patients differ from those in healthy controls using the surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) technology.¹⁵ These proteins were identified with the help of the matrix assisted laser desorption ionization time of flight (MALDI-TOF) technology.⁵ In the present prospective and controlled study, we aimed to identify specific protein patterns in the tear fluid of patients with TAO. To gain more insight into the relation of TAO and dry eye on tear composition, we compared the protein expression in the tear fluid of TAO patients with and without concomitant dry eye syndrome and in healthy controls. Thus, we analyzed tear samples of patients with TAO, dry eye, combined TAO + dry eye, and controls with the MALDI-TOF MS to detect and identify disease specific altering proteins. To verify the MALDI results, we performed antibody microarray studies. 

Methods

Subjects

A total of 120 subjects were included in the study. Of those, 60 patients had various degrees of clinical activity and severity of TAO with and without concomitant dry eye syndrome, 30 patients had dry eye syndrome only, and 30 were healthy, euthyroid control persons. All patients and controls gave their written informed consent. The protocol was approved by the local Ethics Committee of the state Rhineland Palatinate, Germany, in accordance with the tenets of the Declaration of Helsinki. At the joint thyroid eye clinic of the JGU Medical Center, complete endocrine and ophthalmic investigations were performed prior to tear sampling. Diagnosis and definition of clinical activity and severity of TAO followed the criteria recommended by the Consensus Statement of the European Group on Graves' orbitopathy.¹⁶ Further information was obtained using a questionnaire retrieving patients' clinical history and previous treatment. All participants underwent the following ophthalmic investigations: Schirmer's test with anaesthesia (STA), measurement of tear film breakup time, staining of the conjunctiva (Oxford system), and slit lamp examination of lid parallel conjunctival folds. 

Tear Fluid Collection

To obtain samples of tear fluid from all 120 subjects, STA was performed and the Schirmer strips were stored −80°C until use. The Schirmer strips were eluted with 0.1% dodecyl maltoside. Subsequently, the samples were precipitated with 100% ice cold acetone. Per study collective, 30 samples were pooled in five samples per pool resulting in six pool groups. A total of 10 μg protein of each sample was pooled, allowing at least 50 μg protein per pool group to be applied on a SDS Gel. 

MALDI Mass Spectrometry and Data Acquisition

The samples were separated on a 1D SDS–PAGE. Per lane and pool group, 50 μg (5 × 10 μg) were applied on the gel. An overnight in gel digestion and an elution of the peptides followed. The samples were evaporated and adjusted to pH ≤ 4 with 0.5% trifluoroacetic acid for fractionation with zip tips (Zip Tip Pipette Tips; Merck Millipore, Darmstadt, Germany) according to the manufacturer's protocol. The samples were eluted in three steps with 15%, 35%, and 50% acetonitrile solution and then directly spotted to a steel target. The sample was cocrystallized with an energy-absorbing matrix (cinnamic acid). Data acquisition was accomplished using a MALDI-TOF/TOF mass spectrometer (Ultraflex II TOF/TOF; Bruker Daltonics, Billerica, MA, USA) with a nitrogen laser. After acquiring the digest spectra with 100 laser shots averaged from five sample positions in the linear mode, peptides below m/z 4000 Da with good peak intensity were selected for fragmentation analysis using a reflector mode. Peptide fragmentation was performed using collision-induced dissociation and 50 laser shots from five sample positions were summed up for each parent ion. All spectra were externally calibrated by using the peptide calibration standard (Angiotensin II 1047, 19 Angiotensin I 1297.49, Substance P 1348.64, Bombesin 1620.86, ACTH clip 1-17 2093.08, ACTH clip 18-39 2465.19, Somatostatin 28 3147.47; Bruker Daltonics). Data processing of raw spectra, peak detection, and protein identification were performed using commercial software (Flex analysis 2.4 and BioTools 3.1; Bruker Daltonics; and MASCOT (Matrix Science, Boston, MA, USA). The MALDI spectra obtained were used for database searches with MASCOT using SwissProt release 11_1 (Swiss Institute of Bioinformatics, Geneva, Switzerland) database. MASCOT compares the peptide and lift spectra against peptide patterns in the database and searches for homologies. In this case the BioTools Software which is linked with the MASCOT-server was used. If the data of the spectra are matching the data in the database, the probability is measured if this was a contingency. The protein queries were run under MudPIT scoring conditions with a significance threshold of P < 0.05 for protein identification. 

Antibody Microarrays

The proteins with significant associations in mass spectrometry and differing between TAO, dry eye, and controls were investigated with antibody microarrays. Antibodies against the detected proteins were spotted on nitrocellulose slides (OnCyte; Grace Bio-Labs, Bend, OR, USA) with a spotter (Scienion AG, Berlin, Germany) with three drops per spot. The arrays were blocked with blocking substance (Super G blocking buffer; Grace Bio-Labs) for 1 hour. The samples were labeled with fluorescent dye cy5 at RT for 1 hour in the dark. The slides were incubated with the labeled samples for 2 hours at 4°C shaking. After washing with 0.5% Tween PBS, the slides were scanned with 10 db. We used commercial software (Imagene; BioDiscovery, Inc., Hawthorne, CA, USA) for spot analysis. 

Data Analysis

With the help of the P²M (Proteomics Pipeline Mainz, Experimental Ophthalmology, Mainz, Germany), software intensities of identified proteins were normalized and clustered.¹⁷ Corresponding cluster lists containing the normalized peak intensity values and the identifications for each sample were exported to a statistical analysis program (Statistica, version 6.2; StatSoft, Tulsa, OK, USA). After testing for normal distribution, we used the Kruskal-Wallis test based on combinations of multiple peaks for analysis. This analysis calculates the most significant proteins differing over all groups. Further, a post hoc test was run to perform a pairwise testing between the groups. For analysis of the microarray data, an ANOVA and subsequently a post hoc analysis were performed. 

Results

Demographic, Clinical, and Serological Data

The demographic data of all subjects included in the study are summarized in Table 1. Baseline serum thyroid stimulating hormone (TSH) levels were within the normal range (median TSH: 1.23 mU/L; range, 0.45–3.3 mU/L) in patients with dry eye and in healthy controls, respectively. Table 2 also shows the dry eye related test results in all subjects. Clinical and serological data of the 60 patients with TAO are summarized in Table 3 showing no significant differences between the two groups. 

Table 1

 

Demographic Data

 

Table 2

 

Clinical Parameters

 

Table 3

 

Clinical and Serological Data of the 60 Patients With TAO Without and With Concomitant Dry Eye Syndrome (TAO + dry eye)

 

Mass Spectrometry

A total of 69 proteins with over 400 peptides were identified by the Mascot method with a peptide mass tolerance of ±100 ppm and a fragment mass tolerance ±0.6 Dalton (Da). A total of 28 proteins identified in the tear fluid were significantly different over all four study groups (Table 4). Eighteen proteins (64%) significantly differed between TAO and dry eye, eight (28%) between TAO and controls and 11 (39%) between dry eye and controls. 

Table 4

 

Deregulated Proteins and Their Regulation in the Study Groups

 

Compared with dry eye, proline-rich protein 1 (PROL1, P = 0.002); uridine diphosphate (UDP)–glucose-dehydrogenase (UGDH, P = 0.017); calgranulin A (S10A8, P < 0.0001); transcription activator BRG1 (SMCA4, P < 0.0001); annexin (P = 0.006); cystatin (P = 0.008); heat shock protein 27 (P = 0.032); and galectin (LEG3; P = 0.039) were markedly downregulated in TAO (Figs. 11552–3). The highest downregulations in TAO were noted for S10A8 (5-fold) and SMCA4 (4-fold) compared with dry eye. Also compared with controls, PROL1 was 5-fold (P < 0.05); proline-rich protein 4 (PRP4, P < 0.05) 2-fold (Fig. 4); SMCA4 2-fold; and S10A8 1.8-fold downregulated in TAO. 

Figure 1

 

Box-and-whisker plots of PROL 1. (A) Mass spectrometry data. (B) Microarray data. The x-axis represents the groups and the y-axis the measured intensity values. (C) Scatterplot of PROL 1 versus the number of pack years in patients with TAO (r = −0.301; P = 0.027).

Figure 2

 

Box-and-whisker plots of UGDH. (A) Mass spectrometry data. (B) Antibody microarray data. The x-axis represents the groups and the y-axis the measured intensity values. (C) Scatterplot of UGDH versus the number of pack years in patients with TAO (r = −0.287; P = 0.035).

Figure 3

 

Box-and-whisker plots of proteins that are deregulated in TAO compared with dry eye obtained with mass spectrometry. (A) Calgranulin A. (B) Transcription activator BRG1. (C) Annexin A1. (D) Cystatin N. The x-axis represents the groups and the y-axis the measured intensity values.

Figure 4

 

Box-and-whisker plots of PRP4. (A) Mass spectrometry data. (B) Microarray data. The x-axis represents the groups and the y-axis the measured intensity values. (C) Scatterplot of PRP4 versus the number of pack years in patients with TAO (r = −0.31; P = 0.023).

In contrast, the following proteins were upregulated in TAO versus dry eye and/or controls. Lysozyme C was 1.9-fold upregulated in TAO versus TAO + dry eye (P = 0.02) and Midasin and POTE ankyrin domain family member I (POTEI) were 1.7- and 3.9-fold upregulated in TAO versus controls (P < 0.05), respectively. Also significantly upregulated in dry eye versus controls (Table 4) were the proteins LEG3 (P = 0.033) and S100A8 (P < 0.001, 2.5-fold); BRG1 (P < 0.001); and HSP27 (P = 0.042, 2-fold), as well as ANXA1 (P = 0.004, 1.8-fold). 

Using the IPA pathway software Ingenuity Systems (Ingenuity Systems, Inc., Redwood City, CA, USA) the identified proteins above were found to be involved in inflammatory response, cell-to-cell signaling and interaction, cellular movement, and cell death. 

Antibody Microarray

Antibody microarray confirmed significant changes of PRP4, PROL1, and UGDH between TAO with and without dry eye, dry eye, or controls (P < 0.01). These three proteins negatively correlated with smoking, P < 0.05 (Figs. 1–4). The higher the number of pack years, the lower the protein intensity was. 

Discussion

This prospective and controlled study is the first that investigates the tear fluid composition of TAO patients with and without concomitant dry eye syndrome in comparison to dry eye only and healthy controls. Proteome analysis of tear fluid can be used to identify specific proteins in patients with TAO and to differentiate between TAO and the dry eye syndrome as well as between TAO and normal controls. Protein dysregulation was associated with inflammatory response, cell-to-cell signaling and interaction, cellular movement, and cell death. In our hands, sampling of tear fluid was well-accepted by both patients and controls and proteome analysis of tear samples was technically easy to perform. It thus represents an ideal tool to investigate lacrimal gland involvement in patients with TAO and dry eye. Further characterization of these proteins as well as their demonstration in the sera of the corresponding patients may help to identify protein regulations of the disease, especially in the various stages of TAO. 

In this study, protective proteins (i.e., PROL1/PRP4) were markedly downregulated in patients with TAO. Proline-rich proteins are highly expressed and secreted by lacrimal acinar cells which have a large share of the cellular mass of the lacrimal gland.¹⁸ Lacrimal PRPs have protective functions similar to those of salivary acidic PRPs by modulating the microflora (e.g., through agglutination and clearance of pathogens on the ocular surface).^19,20 Downregulation might be explained by the FAS ligand mediated apoptosis of lacrimal cells during the pathogenesis of TAO.¹⁴ This downregulation was further verified and confirmed with the antibody microarray experiments. The results are also compliant with the previously reported downregulation of PRP4 in tears of patients with dry eye compared to controls.¹¹ In line with the above data was the downregulation of the “protective” cystatin in TAO. Cystatins are cysteine proteinase inhibitors belonging to the cystatin superfamily.²¹ High concentrations of cystatins are reported in saliva and tears and have a protective function in case of inhibition of cysteine proteases from microorganisms.²² Hence, the downregulation is a result of the destruction of the lacrimal cells in the late phase of TAO. 

Further, the anti-inflammatory protein annexin A1 (ANXA1) was downregulated in TAO versus dry eye. Annexin A1 acts as an endogenous downregulator of inflammation in cells of the innate immune system as it blocks the interaction of activated neutrophils to endothelial cells. There is evidence that it also plays a role in the adaptive immune system as a tuner of T cell receptor signaling and a subsequent differentiation of Th1/Th2 cells over the activation of both the extracellular signal-regulated kinases/mitogen-activated protein kinases pathway and protein kinase B pathway via formyl peptide receptor like 1(FPRL-1).²³ Also downregulated in TAO was UDP-α-d-glucose (UGDH) which catalyzes the reaction of UDP-α-d-glucuronic acid to UDP-α-d-glucose through a 2-fold oxidation. Uridine diphosphate–α-d-glucose is a precursor of glycosaminoglycans which are produced in activated fibroblasts and released in the florid and active phase of TAO.^24–26 Hydrophilicity of these mucopolysaccharides attracts water and leads to the clinical phenotype of proptosis. Since over 60% of the TAO patients included in this study were in the late and hence inactive phase of the disease, down-regulation of this enzyme may be explained by the late stage of TAO.²⁷ 

In contrast, proteins that are involved in inflammatory processes were upregulated in TAO (e.g., the POTE isoform POTE-2α-actin), which is elevated in Hela cells treated with proapoptotic factors (e.g., the FAS ligand, antibodies to FAS Ligand, and TAIL). Also, overexpression of POTEI was demonstrated to induce apoptosis. In the present study, upregulation of POTEI in TAO points to an inflammatory process in the orbit. Furthermore, Midasin, which is required for maturation and nuclear export of pre-60S ribosome subunits involved in the posttranslational modification of cellular proteins that regulate various cellular inflammatory responses was upregulated, probably related to the increased cellular processes during inflammatory progression in the orbit. Patients with TAO have an abnormally high tear film osmolarity.⁴ Hyperosmolarity stimulates proinflammatory cytokines including interleukin 1B, tumor necrosis factor α, and matrix metalloproteinase 9 (MMP-9) in mice.²⁸ These cytokines activate mitogen-activated protein kinases cascades that stimulate further inflammatory cytokines.²⁹ This cycle can lead to a high amount of ocular inflammation. Evidence suggests that ocular inflammation mediated by T lymphocytes is also important in the pathogenesis of dry eye.³⁰ Hyperosmolarity may also cause pathological changes to the corneal epithelium cells, where MMP-9 can lyse substrates such as the corneal epithelial basement membrane and tight junction proteins that normally have a corneal epithelial barrier function.³¹ 

Scarce data and only a few comparable studies are available in the literature. One study recently showed in tear samples of TAO subjects an essential reduction of the protein fractions of inflammation-related protein immunoglobulin kappa chain C region (IgKC) and serum albumin, whereas a novel isoform of complement component 3 was completely absent in the tears of TAO patients.³² These reduced protein concentrations in the tears of TAO patients may contribute to changes in their ocular surfaces via diminished reactive oxygen species depletion and adaptive immune responses. In another study,³³ comparison of orbital fat protein from TAO with age-matched controls showed significant differences in the proteome, and upregulation of specific proteins in orbital tissue from TAO was associated with biochemical mechanisms or capacities against endoplasmic reticulum stress, mitochondria dysfunction, and cell proliferation as well as apoptosis in TAO orbital tissues. Also a correlation between the decreased protein concentrations of proline-rich proteins and UGDH with smoking was shown. This is in line with findings that show that smoking is a risk factor for developing TAO and has an impact on tear composition.⁷ In our present study, a limitation lies in the small smoker amount in the control group and a subsequent study with groups matched for smoking is foreseen in our lab. 

In conclusion, proteome analysis of tear fluids demonstrated an upregulation of inflammatory proteins and a downregulation of protective proteins in TAO as well as a significantly different protein panel in TAO versus dry eye and/or controls. Microarray investigation confirmed the proteomics results in several characterized proteins. In our lab, similar studies are currently being performed analyzing orbital tissue of patients with various clinical stages of TAO versus orbital control tissue looking for specific protein regulations of the complex autoimmune disease. Subsequent to their identification in the serum of patients with TAO and/or dry eye, proteomics may offer a useful tool to identify specific markers of this disease. 

Acknowledgments

The authors thank Tanja Diana, MSc, Molecular Thyroid Research Lab, JGU Medical Center, for manuscript evaluation. 

Disclosure: N. Matheis, None; F.H. Grus, None; M. Breitenfeld, None; I. Knych, None; S. Funke, None; S. Pitz, None; K.A. Ponto, None; N. Pfeiffer, None; G.J. Kahaly, None