Confocal microscopy abnormalities preceding antibody positivity and manifestations of Sjogren’s syndrome

Abstract

An asymptomatic 26-year-old woman underwent confocal microscopy as part of a control population for a research study. Images revealed reduced sub-basal corneal nerve density and multiple activated dendritic cells. Three years later, she presented with a self-limited cutaneous vasculitis in her lower extremities which prompted an evaluation for autoimmune diseases. Laboratory testing revealed positive antinuclear antibodies (1:320, thick granular pattern), and anti-SSA/SSB (SSA, 53.6 U/mL, moderately positive; SSB, 142.7U/mL, strongly positive). Two weeks later, she presented with ocular pain and an ophthalmologic examination revealed ocular surface staining. An ocular ultrasound was consistent with posterior scleritis. Based on this picture, a diagnosis of Sjögren syndrome (SS) was made. SS is a chronic autoimmune disease that can present with symptoms that diminish the patient’s quality of life. Confocal microscopy might be a valuable tool for the early diagnosis of disease.

Introduction

The human cornea is one of the most densely-innervated surfaces of the body; with approximately 7000 nerves per square millimeter and ~400x increased sensitivity compared to skin.¹ An active topic of interest in ophthalmology is to understand how corneal nerves relate to various ocular pathologies. Since the creation of the two-sided mirror confocal microscope in 1969 by Svishchev, neural tissue within the cornea can be imaged in-vivo.² In recent years, advances in In-vivo laser scanning confocal microscopy (IVCM) have allowed for high resolution images of corneal nerves to be obtained and these images have been examined in reference to various eye conditions including dry eye (DE), contact lens wear, keratoconus, vernal keratoconjunctivitis, corneal dystrophies, and keratitis.³

DE has been most robustly examined with confocal microscopy. DE is a heterogeneous, commonly encountered disease that can significantly diminish a patient’s quality of life.⁴ DE is most frequently divided into sub-types including aqueous tear deficiency (ATD) , which can occur as an isolated condition or in the setting of a systemic immune disorder (most commonly Sjögren syndrome (SS) and graft versus host disease (GVHD)), and evaporative DE.⁵ With regards to confocal microscopy, studies have reported different confocal findings in different DE sub-types.⁶ Specifically, a confocal signature of immune mediated ATD is a decrease in corneal nerve density and an increase in dendritic cells as compared to isolated ATD, evaporative DE, and controls.^6–9 However, the utility of confocal microscopy in predicting the development of immune mediated ATD has not been examined. We present a case of an asymptomatic woman with incidental findings on IVCM consistent with immune mediated DE who years later developed ocular and extraocular symptoms and signs of SS.

Case Report

A 26-year-old Hispanic female was scanned with IVCM imaging (HRT III, Heidelberg Engineering, Heidelberg, Germany) as a control for a research study that aimed to examine confocal findings in individuals with DE. Imaging revealed few detectable corneal nerve fibers and numerous activated dendritic cells in multiple areas of the cornea in both eyes (OU) (Figure 1). The clinical examination was unremarkable for symptoms or signs of DE and for other ocular abnormalities. Specifically, her tear film was stable, and she had no ocular surface staining; a Schirmer’s test was not performed. She had no past ocular history or surgeries; past medical history was significant for Hashimoto thyroiditis with positive anti-thyroperoxidase and anti-thyroglobulin antibodies five years prior. At that time, antinuclear antibodies and rheumatoid factor testing was performed with negative results. At age 29, she presented with three separate episodes of a self-limited petechial and purpuric rash over her lower extremities (Figure 2a–b). An autoimmune panel was performed with positive antinuclear antibodies (1:320, thick granular pattern), anti-Ro (SSA, 53.6 U/mL, moderately positive), and anti-La (SSB, 142.7U/mL, strongly positive). At the time of presentation, the patient was adherent with levothyroxine treatment and thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) levels were within normal limits. Two weeks after the first episode of skin rash, the patient reported ocular pain in both eyes (OU) exacerbated with ocular movements. A complete ophthalmologic examination was performed, and slit-lamp examination revealed prominent corneal nerves, and limbal and conjunctival staining with lissamine green (Figure 3a–b). Tear production was normal (anesthetized Schirmer’s of 15mm and 20mm in the right and left eye, respectively) and tear stability was intact (tear breakup time of 10 seconds in both eyes). Corneal sensation with Cochet Bonnet aesthesiometer was also intact at 6.0cm OU. An ocular ultrasound demonstrated thickened extraocular muscles and inflammation of the posterior sclera, consistent with posterior scleritis OU. (Figure 3c–d) Based on the clinical picture and laboratory data, a diagnosis of SS was made.

Figure 1.

Representative 400×400 μm confocal microscopy images of the subbasal nerve plexus shows a paucity of corneal nerves and the presence of numerous activated dendritic cells.

Figure 2.

Photographs of lower extremities showing purpuric and petechial rash consistent with cutaneous vasculitis.

Figure 3.

Slit-lamp photograph of the right eye revealing a) prominent corneal nerves in meridians 7,8,10, and 11, and b) nasal corneal, limbal, and conjunctival staining with lissamine green. Ocular ultrasound of the right eye c) hyperechogenic scleral lesion (yellow arrow) consistent with scarring and d) hypoechogenic linear lesion (yellow arrows) characteristic of active posterior scleritis.

Discussion

SS is a prevalent autoimmune disease characterized by mucosal dryness due to reduced exocrine gland secretion. There are two widely known criteria used to diagnose SS: the American College of Rheumatology/European league against rheumatism (ACR/EULAR) criteria and the Clinical Practice Guideline proposed by the Research Team for Autoimmune Diseases, Research Program for Intractable Disease of the Ministry of Health, Labor and Welfare (MHLW), in Japan.^{10, 11} ACR-EULAR criteria is based on the sum of 5 items: anti SSA/Ro antibody positivity, abnormal ocular staining score, abnormal Schirmer’s test result, unstimulated salivary flow and focal lymphocytic sialadenitis. MHLW practice guidelines propose a series of clinical questions with evidence-based recommendations that range from very weak to strong and divide primary Sjogren into glandular and extra glandular forms. Our patient fulfilled criteria for SS using both definitions even though the guidelines have different approaches. The former focuses on manifestations related to exocrine gland dysfunction while the latter examines the sum of multiorgan manifestations.

In our patient, the strongest indicator of SS was autoantibody positivity which occurs in 50–70% of patients who are diagnosed with primary SS.¹² According to MHLW, even without symptoms of dryness, a positive anti-SSA/SSB has a sensitivity and specificity of 83.7 and 91.5, respectively, for SS.¹¹ Various methods can be used to detect SSA/SSB, with the RNA precipitation assay having the highest sensitivity and specificity, as compared to other techniques such as immunodiffusion, electrophoresis, and enzyme-linked immunosorbent assay (ELISA).¹³ In our case, SSA/SSB was detected using ELISA methodology.

Ocular staining is also a defining feature of SS. The ACR-EULAR grades staining using the van Bijsterveld score, which ranges from 0 to 9. To arrive at the score, the ocular surface is separated into 3 zones (cornea, nasal and temporal conjunctiva) and the intensity of staining in each zone is graded on a scale of 0 to 3. For SS, a score of ≥4 is needed. In our case, the patient scored a 6 based on confluent corneal and conjunctival staining. Interestingly, in our case, the staining was found in the setting of normal tear stability and production. Many factors such as ocular surface inflammation (histocompatibility antigen (HLA-DR) expression and T cell infiltration) and hyperosmolarity may underlie desquamation of corneal wing and basal epithelial cells, in the absence of aqueous tear deficiency.^14–16

Immune-complex depositions have also been described as a manifestation of SS^{17, 18}, and such deposits may underlie the non-palpable purpura noted in 5 to 10% of individuals with SS. As in our patient, the lower limbs are preferentially involved in SS associated cutaneous vasculitis.^{19, 20} The presence of cutaneous vasculitis also increases the risk of other extraglandular manifestations.²⁰ A study of 89 individuals with primary SS found a higher frequency of peripheral neuropathy (31% vs. 4%, p < 0.001), antinuclear antibodies (88% vs. 60%, p = 0.002), and anti-Ro/SS-A antibodies (70% vs. 43%, p = 0.011) in individuals with (n=52, 58%) vs without (n=37, 42%) cutaneous vasculitis.²¹

In addition to peripheral neuropathy, corneal nerve abnormalities have also been described in SS, however with variable findings across studies. With regards to function, studies using a non-contact aesthesiometer (Belmonte aesthesiometry) have demonstrated conflicting results, some reporting hypo-aesthesia^{22, 23} and others hyper-aesthesia^{24, 25} in SS vs controls. With contact aesthesiometry (Cochet-Bonnet), one study reported hypo-aesthesia in SS vs controls.¹⁴ With regards to structure, many studies have reported decreased nerve density and increased dendritic cell numbers in SS vs controls.^{14, 26, 27} In addition, increased nerve beading and tortuosity, thought to be indicative of high metabolic activity, has been reported in individuals with SS.^26–29 Similar findings have been reported in diseases associated with SS. For example, one study examined function and structure in individuals with systemic lupus erythematosus (SLE) as compared to controls. In this study, sensation (measured via contact aesthesiometer) was similar between groups but individuals with SLE had lower nerve fiber density and higher numbers of activated dendritic cells compared to controls.³⁰ Our patient had intact sensation on Cochet-Bonnet but a reduced sub-basal nerve density on IVCM. This is not unexpected as some studies noted an association between corneal sensitivity and nerve density^{8, 14} while others found the two metrics to be independent.^{24, 30} Sub-basal nerve fibers are an important contributor to epithelial cell trophism, normal blinking, and lachrymal reflex. Thus, alterations in innervation may contribute to tear hyposecretion, inflammation, and epithelial cell damage, the latter of which was noted in our patient.¹⁴

A natural extension of these data is to question whether IVCM metrics can be used to differentiate between DE subtypes, which can include immune mediated, meibomian gland dysfunction associated, and neuropathic, to name a few. As note above, individuals with SS have decreased nerve fiber density, and increased beading and tortuosity compared to healthy populations, but these differences become less distinct when examining across DE sub-types.^{26, 28, 29} On the other hand, increased numbers of dendritic cells in the central cornea are more common in SS compared to other DE sub-types.⁸ In fact, a retrospective study of individuals with DE symptoms found that the presence of ≥2 activated dendritic cells (defined as hyperreflective cells with at least 3 processes emanating from the cell trunk that were of the same size or longer than the cell body) in the central cornea had a sensitivity of 60% and specificity of 77% for the presence of a diagnosed systemic immune disorder, a finding mostly driven by individuals with secondary SS.⁹ Other studies have reported increased numbers of activated dendritic cells in RA,^{31, 32} active and inactive thyroid eye disease,³³ ankylosing spondylitis,³⁴ and systemic lupus erythematosus³⁵ as compared to controls. However, this finding has not been described prior to the onset of a systemic disease. Of note, our patient had a history of Hashimoto thyroiditis, a pathology that has not been explored with respect to confocal microscopy, and thus we cannot comment on the contribution of this disorder to our patient’s findings.

The unique aspect of our case is that our patient showed these confocal findings three years before her SS diagnosis and, most importantly, without symptoms or signs of DE. This case raises the possibility that findings on confocal microscopy can be used as a prognostic test in SS, a disease where current markers often arise late in the course of disease.³⁶ While interesting, further studies with a larger number of patients are needed to evaluate the utility of IVCM in predicting the onset of SS in various populations.

Availability of data and material

All data generated are included in this published article and available from the corresponding author on reasonable request.