Purpose
To assess clinical outcomes and tear cytokine levels in patients with moderate and severe meibomian gland dysfunction (MGD) after treatment with oral minocycline and artificial tears versus artificial tears only.
Design
Prospective, randomized clinical trial.
Methods
Sixty eyes of 60 patients with stage 3 or 4 meibomian gland dysfunction were enrolled. We evaluated the tear film break-up time, Schirmer test results, corneal and conjunctival fluorescein staining results, biomicroscopic examination results of lid margins and meibomian glands, and tear cytokine levels before and after 1 month and 2 months of oral minocycline and artificial tears (group 1) or artificial tears only (group 2). Tear samples were collected and analyzed using a BD Cytometric Bead Array (BD Bioscience, San Jose, California, USA) for detection of interleukin (IL)-1β, IL-6, IL-7, IL-8, IL-12p70, IL-17α, interferon-γ, tumor necrosis factor-α, and monocyte chemotactic protein-1. The Wilcoxon signed-rank test, Mann–Whitney U test, generalized linear model, and linear mixed model were performed.
Results
Patients in group 1 showed statistically significant improvement in all clinical signs and symptoms after 1 month and 2 months of treatment. Patients of group 1 showed more significant improvement compared with those in group 2. Patients in group 1 also showed statistically significant reductions in IL-6, IL-1β, IL-17α, tumor necrosis factor-α, and IL-12p70 after 2 months of treatment.
Conclusions
Oral minocycline can provide clinical benefits in treating moderate and severe meibomian gland dysfunction by reducing inflammatory cytokine levels.
Blepharitis is a common ocular surface disorder with a complex cause, affecting approximately 39% to 50% of the adult population. It may be associated with several systemic diseases, particularly rosacea and seborrheic dermatitis, and is related to other ocular conditions like dry eye, chalazion, conjunctivitis, and keratitis. Common symptoms are burning sensation, irritation, tearing, photophobia, blurred vision, and red eyes. Objective signs, including eyelid margin redness, conjunctival redness, hyperkeratinization of eyelid, telangiectasia of the lid margin, increased discharge of the meibomian gland, irregular thickening of the dirty lipid layer, and eyelid crusting and/or loss of eyelashes, often accompany these symptoms.
Meibomian gland dysfunction (MGD), a specific type of posterior blepharitis, is a very prevalent condition and a major cause of dry eye. MGD usually is secondary to dysfunction or structural changes on the meibomian glands. Under normal conditions, lipids in these glands are expressed through meibomian openings and form the lipid layer of the tear film. The tear film lipid layer prevents tear evaporation, tear outflow, and contamination, acting as a barrier against microorganisms or skin sebum. However, meibum lipids are modified in patients with MGD, resulting in tear instability, evaporative dry eye, and eyelid inflammation. These changes add to corneal damages and exacerbate ocular symptoms, which are all associated with the constant release of inflammatory mediators.
In one study evaluating the possible association of tear proteins with severity of MGD in dry eye, increasing levels of distinct tear proteins, S100A8 (calgranulin A) and S100A9 (calgranulin B), were correlated with MGD severity. Tear cytokines also play an important role in the chronic inflammation of MGD. Interleukin (IL)-1β, IL-7, IL-12, IL-17, and macrophage inflammatory protein-1β levels in MGD patients were higher than those in a normal group when comparing the inflammatory cytokine levels in tears of MGD patients and those in a normal group. IL-6, IL-8, IL-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels were higher in dysfunctional tear syndrome with MGD than in the control group. The gelatinases (matrix metalloproteinase [MMP]-9) were elevated significantly and were correlated with IL-1α in MGD associated with ocular rosacea. In one study measuring inflammatory cytokine levels in tears of a normal group and those of MGD patients, IL-6 and pro-MMP-9 were elevated significantly in MGD patients. Moreover, there is a strong interaction between MMP and inflammatory cytokines, each one activating the other type of mediator from its respective inactive precursor. Although there was no study describing the association between monocyte chemotactic protein-1 (MCP-1) and MGD, MCP-1 has been known as a major proinflammatory cytokine and has been described in development of T helper cell 1 and T helper cell 2. Thus, measuring tear cytokine levels can be used as objective criteria in diagnosing MGD and analyzing the efficacy of treatment.
Although the pathogenesis of MGD was unclear, clinical improvement perhaps is obtained with a continuous regimen of eyelid scrubs and warm compresses. When, in case of more severe MGD, this treatment is insufficient, it is helpful to use topical and systemic antibiotics with anti-inflammatory properties such as azithromycin, tetracycline, doxycycline, and minocycline. Azithromycin, a macrolide antibiotic with anti-inflammatory properties, has been used to treat ocular surface infections and MGD. In a recent study, azithromycin suppresses inflammatory cytokines (TNF-α, IL-1β), chemokines (IL-8, RANTES [Regulated on Activation, Normal T Cell Expressed and Secreted]), and MMP (MMP-1, MMP-3, and MMP-9) by blocking nuclear factor-κB activation in human corneal epithelial cells. Tetracycline, doxycycline, and minocycline also were effective in treating MGD through anti-inflammatory, antimetalloproteinase, and antiapoptotic properties. Compared with tetracycline, minocycline and doxycycline have fewer side effects. Because minocycline is well tolerated with excellent bioavailability, absorption, a prolonged half-life, and a highly lipophilic nature, it may be a therapeutic option in MGD. To our knowledge, there has been no study on tear cytokine levels in MGD patients treated with oral minocycline. Thus, we evaluated both inflammatory tear cytokine levels and corresponding clinical outcomes for analyzing the efficacy of minocycline in moderate and severe MGD. The aim of this research was to determine the concentration of inflammatory tear cytokines in patients with MGD and to compare the changes in tear cytokine levels between a group treated with minocycline and artificial tears and a group treated with artificial tears only.
Methods
A total of 60 eyes of 60 patients who met inclusion and exclusion criteria were enrolled in this study. Inclusion criteria included patients with stage 3 or 4 MGD. MGD was diagnosed by evidence of lid margin or tarsal conjunctival erythema, bulbar conjunctival hyperemia, telangiectasia, thickening and irregularity of the eyelid margins, or meibomian gland orifice inclusions. Stages of MGD were assessed using MGD staging criteria. Exclusion criteria included a history of previous ocular or intraocular surgery, ocular infection, non–dry eye ocular inflammation, ocular allergy, autoimmune disease, history of intolerance or hypersensitivity to any component of the study medications, wearing contact lenses during the study period, presence of current punctal occlusion, pregnancy, lactating women, and children. Additionally, patients were excluded if they were using any topical ocular or systemic medication that could be used for the treatment MGD or dry eye, including topical or oral antibiotics, topical cyclosporine A, topical or oral steroids, topical nonsteroidal anti-inflammatory drugs, topical ocular allergy medications, or artificial tears. If patients were using any other topical or systemic medication for the treatment of MGD and dry eye, they were instructed to discontinue them 2 weeks before study initiation (wash-out period). Sixty patients were allocated randomly into 2 groups. Odd-numbered patients were included in group 1 and the even-numbered were included in group 2. Thirty eyes of 30 patients received 50 mg minocycline orally (Minocin; SK Chemical, Seoul, Korea) twice daily and 0.1% sodium hyaluronate (Kynex; Alcon Laboratory, Seoul, Korea) 4 times daily (group 1). Thirty eyes of 30 patients received 0.1% sodium hyaluronate 4 times daily (group 2) for 2 months treatment. After the appropriate 2-week wash-out period, patients were examined for baseline measurements. At study initiation, all patients completed an Ocular Surface Disease Index questionnaire and underwent an ocular surface, tear, and meibomian gland evaluation that consisted of fluorescein tear film break-up time (TBUT), Schirmer test, corneal and conjunctival fluorescein staining, biomicroscopic examination of lid margins and meibomian glands, and tear cytokine levels. The study eye was chosen to be the eye having a higher stage of MGD. If the total scores for each eye were equal, the right eye was chosen as the eye for tear collection. All measurements except tear cytokine levels were conducted in the same manner before treatment, after 1 month, and after 2 months of treatment. Tear cytokine levels were evaluated before treatment and after 2 months of treatment. Patients were instructed not to wipe their eyelid margins and to use artificial tears 4 hours before tear sampling. All measurements and grading were performed by 1 physician (T.-I.K.). To minimize the extent to which one test influences the results of tests that follow, each test was performed in the same order. Tear collection was performed first, followed by TBUT, corneal and conjunctival fluorescein staining, Schirmer basic secretion tear test with topical anesthesia, and biomicroscopic examination of lid margins and meibomian glands. At least 10 minutes were allowed between tests.
Tear Film Break-Up Time
To measure TBUT, a drop of nonpreserved saline solution was added to a fluorescein strip (Haag-Streit, Koeniz, Switzerland), which was applied to the inferior palpebral conjunctiva. The patients were instructed to blink 3 or 4 times for a few seconds to ensure adequate mixing of the dye, then were examined using a slit lamp with maximum cobalt blue light. The patients were asked to open the eye widely and to look straight ahead, and the physician measured the time it took for a single black dot or line to appear on the cornea using a stop watch. The test was performed 3 times and the results were averaged.
Schirmer Test
The patients underwent the Schirmer basic secretion tear test with topical anesthesia. A 5 × 35-mm Whatman filter paper strip (Haag-Streit, Koeniz, Switzerland) was inserted in the lateral one third of the inferior fornix. After 5 minutes, the length that had become wet by the tear film was measured.
Tear Collection and Multiplex Bead Analysis
Thirty microliters of phosphate-buffered saline were injected into the inferior conjunctival sac using a micropipette. Approximately 20 μL tear fluid and buffer were collected with a micropipette. To minimize irritation of the ocular surface or lid margin, unstimulated tear fluid was collected from the marginal tear strip of the lower lid near the lateral canthus. Anesthetic drops were not instilled. Tear samples immediately were transferred into 0.5-mL Eppendorf tubes (Eppendorf, Fremont, California, USA), were placed on dry ice, and were kept in a −70 C freezer until used for immunoassay. Cytokines were measured using the BD Cytometric Bead Array (BD Bioscience, San Jose, California, USA). The cytokines analyzed were IL-6, IL-7, IL-8, IL-1β, IL-17α, MCP-1, TNF-α, IL-12p70, and IFN-γ. The measurements were performed essentially as previously described. Briefly, 20 μL tear fluid was thawed and added to a 50-μL mixture containing each capture antibody-bead reagent and 50 μL detector antibody-phycoerythrin reagent. The mixture subsequently was incubated for 3 hours at room temperature and was washed to remove unbound detector antibody-phycoerythrin reagent before flow cytometry. Data were acquired and analyzed using BD Cytometric Bead Array software that calculates the cytokine concentration based on the standard curves and a 4-parameter logistic curve-fitting model. Flow cytometry was performed using the BD LSRII system (BD Bioscience). The lower limits of detection were the following: IL-1β, 2.3 pg/mL; IL-6, 1.6 pg/mL; IL-7, 0.5 pg/mL; IL-8, 1.2 pg/mL; IL-12p70, 0.6 pg/mL; IL-17α, 0.3 pg/mL; IFN-γ, 1.8 pg/mL; TNF-α, 0.7 pg/mL; and MCP-1, 1.3 pg/mL. The lowest cytokine concentration in the linear portion of the standard curve was used for statistical comparison of tear samples with concentrations of less than this level.
Meibomian Gland Dysfunction Staging
Microscopic examination of the meibomian glands was performed last because examination of the gland can affect both TBUT and Schirmer test results. The operator scored for the presence or absence of lid margin abnormalities: lid margin irregularity, plugging of the meibomian orifices, lid margin vascular engorgement, and anterior or posterior replacement of mucocutaneous junction, giving a score of 0 to 4. The degree of expressibility using firm digital pressure applied over 5 lower lid glands was based on the following: grade 0, all 5 glands expressible; grade 1, 3 to 4 glands expressible; grade 2, 1 to 2 glands expressible; grade 3, 0 glands expressible. The degree of meibum quality using firm digital pressure applied over 8 lower lid glands was based on the following: grade 0, clear; grade 1, cloudy; grade 2, cloudy with granular debris; grade 3, thick, like toothpaste. Each of the 8 glands of the lower eyelid was graded on a scale from 0 to 3. The scores of the 8 glands were summed to obtain a total score (maximum score for each eye, 24). Staining scores were obtained by summing the scores of the exposed cornea and conjunctiva (Oxford staining score range, 1 to 15; Dry Eye Workshop staining (DEWS) score range, 0 to 33). The patients were asked to rate subjective symptoms (ocular discomfort, itching, and photophobia with limitations of activities) on a scale of 0 (no symptoms) to 3 (severe symptoms). The stage of MGD was assessed using all of these clinical parameters and the symptom questionnaire.
Assessment of Safety
Safety was assessed by monitoring any adverse events during the course of the study. The patients also were instructed to report any unfavorable symptoms or signs, such as gastrointestinal trouble, itching, irritation, and hyperemia.
Statistical Analysis
The Wilcoxon signed-rank test was used to compare clinical outcomes and cytokine levels before and after treatment. The Mann–Whitney U test was used to compare the clinical outcomes and cytokine levels between the 2 groups.
A linear mixed model with post hoc analysis was used to evaluate possible differences between groups and time courses in the measurement of clinical outcomes and cytokine levels, all with the unstructured covariance matrix. In the presentation of noncontinuous scale values, such as MGD staging, expressibility, and ocular irritation symptom, we performed the generalized linear model analysis. The statistical analysis was performed with SAS software version 9.2 (SAS Institute, Cary, North Carolina, USA). Differences were considered statistically significant when P values were less than .05.
Results
Patients’ characteristics are summarized in Table 1 , and the results of clinical signs and symptoms are shown in Table 2 . In the linear mixed analysis, considering the interaction effect between the 2 groups and the 3 time courses, there were statistically significant differences in the measurement of clinical outcomes ( P = .034, P = .029, P < .001, P < .001, P = .011, and P = .006 for TBUT, Schirmer test, lid margin abnormality, meibum quality, ODSI, and DEWS, respectively). There was no statistically significant difference between the 2 groups in baseline clinical signs and symptoms ( Table 3 ). Group 1 patients showed statistically significant improvement in all clinical signs and symptoms after 1 month and 2 months of treatment, compared with values before treatment. Group 2 patients showed statistically significant improvement in all clinical signs and symptoms except Schirmer test after 1 month and 2 months of treatment, compared with values before treatment. When comparing clinical signs and symptoms of the 2 treatment groups after 2 months of treatment, there were statistically significant differences in TBUT, corneal fluorescein staining score, lid margin abnormality, and meibum quality, with better clinical outcomes in group 1. There was no significant difference in Schirmer test results, conjunctival fluorescein staining score, Ocular Surface Disease Index, DEWS score, and Oxford scale. In general linear model analysis, oral minocycline treatment was associated with a significant improvement in MGD staging and expressibility compared with artificial tears alone (L’Beta estimate, 0.001; P < .001 for MGD staging; and L’Beta estimate, 0.038; P < .001 for expressibility). In terms of ocular irritation symptom, patients may not take significant advantages from oral minocycline (L’Beta estimate, 0.536; P = .220). We also performed the Wilcoxon signed-rank test and Mann–-Whitney U test. The results from these statistical analyses were similar to those from linear mixed analysis.
| Group 1 a | Group 2 b | |
|---|---|---|
| Patients (eyes) | 28 (28) | 30 (30) |
| Location | ||
| Right | 18 | 17 |
| Left | 10 | 13 |
| Sex | ||
| Female | 14 | 18 |
| Male | 14 | 12 |
| Age (years) | ||
| Mean (SD) | 63.19 (9.25) | 63.79 (13.51) |
| Median | 62 | 65 |
| Range | 46 to 78 | 45 to 79 |
a Oral minocycline and artificial tears treatment group.
| Group 1 (n = 28) a | Group 2 (n = 30) b | |||||
|---|---|---|---|---|---|---|
| Before | 1 Month | 2 Months | Before | 1 Month | 2 Months | |
| TBUT (sec) c | 3.17 (0.45) | 7.19 (0.51) | 11.81 (0.78) | 3.13 (0.48) | 5.61 (0.53) | 8.89 (0.82) |
| Schirmer test results (mm) c | 6.71 (1.32) | 9.05 (1.16) | 10.14 (1.09) | 7.05 (1.39) | 7.16 (1.22) | 7.84 (1.15) |
| Corneal stain score c | 2.14 (0.29) | 0.76 (0.20) | 0.14 (0.13) | 2.00 (0.30) | 0.89 (0.21) | 0.68 (0.13) |
| Conjunctival stain score c | 3.81 (0.22) | 1.90 (0.16) | 0.62 (0.14) | 3.37 (0.23) | 2.21 (0.17) | 0.89 (0.14) |
| Lid margin abnormality c | 3.62 (0.12) | 1.95 (0.14) | 0.67 (0.12) | 3.42 (0.13) | 2.58 (0.15) | 1.63 (0.12) |
| Meibum quality c | 12.90 (0.70) | 8.86 (0.55) | 4.62 (0.45) | 11.42 (0.74) | 9.26 (0.58) | 8.42 (0.47) |
| OSDI c | 23.38 (1.71) | 9.10 (1.31) | 4.33 (0.87) | 20.68 (1.80) | 12.68 (1.38) | 6.11 (0.91) |
| DEWS c | 8.14 (0.75) | 3.00 (0.33) | 1.43 (0.27) | 6.05 (0.79) | 3.89 (0.35) | 1.32 (0.28) |
| Oxford c | 4.81 (0.25) | 2.52 (0.20) | 1.29 (0.23) | 4.42 (0.26) | 2.89 (0.21) | 1.47 (0.24) |
| MGD staging, n (%, proportion of ≥ stage 3) d | 28 (100) | 13 (46.4) | 0 (0) | 30 (100) | 17 (56.7) | 14 (46.7) |
| Ocular irritation symptom, n (%, proportion of ≥ grade 2) d | 28 (100) | 11 (39.3) | 0 (0) | 30 (100) | 19 (63.3) | 2 (6.7) |
| Expressibility, n (%, proportion of ≥ grade 1) d | 28 (100) | 13 (46.4) | 2 (7.1) | 30 (100) | 30 (100) | 27 (90) |
a Oral minocycline and artificial tears treatment group.
b Artificial tears alone treatment group.
c Results are presented as least square mean (standard error) in linear mixed model with post hoc analysis.
d Generalized linear model analysis for noncontinuous scale values.
| P Value | |||||
|---|---|---|---|---|---|
| Group 1 (n = 28) a | Group 2 (n = 30) b | Group 1 vs Group 2 after 2 Months c | |||
| Before Treatment vs 1 Month after Treatment c | Before Treatment vs 2 Months after Treatment c | Before Treatment vs 1 Month after Treatment c | Before Treatment vs 2 Months after Treatment c | ||
| TBUT (sec) | <.001 | <.001 | <.001 | <.001 | .014 |
| Schirmer test results (mm) | .034 | <.001 | .925 | .272 | .154 |
| Corneal stain score | <.001 | <.001 | <.001 | <.001 | .005 |
| Conjunctival stain score | <.001 | <.001 | <.001 | <.001 | .170 |
| Lid margin abnormality | <.001 | <.001 | <.001 | <.001 | <.001 |
| Meibum quality | <.001 | <.001 | .004 | <.001 | <.001 |
| OSDI | <.001 | <.001 | <.001 | <.001 | .167 |
| DEWS | <.001 | <.001 | .009 | <.001 | .773 |
| Oxford | <.001 | <.001 | <.001 | <.001 | .580 |
a Oral minocycline and artificial tears treatment group.
b Artificial tears alone treatment group.
c P values from linear mixed model with post hoc analysis were adjusted by the Bonferroni correction method.
Table 4 summarizes tear cytokine levels. The total spread of IL-6, IL-7, IL-8, IL-1β, IL-17α, MCP-1, TNF-α, IL-12p70, and IFN-γ was 1.6 to 88.29 pg/mL, 0.5 to 19.29 pg/mL, 1.2 to 757.5 pg/mL, 2.3 to 23.35 pg/mL, 0.3 to 36.64 pg/mL, 1.3 to 365.4 pg/mL, 0.7 to 36.4 pg/mL, 0.6 to 33.27 pg/mL, and 1.32 to 27.5 pg/mL, respectively. In group 1, there was a statistically significant decrease in cytokine IL-6, IL-1β, IL-17α, TNF-α, and IL-12p70 levels after 2 months of treatment. There was a similar trend in the decreases of cytokines IL-7 and IL-8 after 2 months of treatment, although it was not statistically significant. However, in group 2, no significant differences were found in the levels of all cytokines after 2 months of treatment. When comparing the cytokine levels of 2 groups after 2 months of treatment, there were statistically significant differences in IL-1β and MCP-1 levels. We also performed a linear mixed model analysis to evaluate possible differences between groups and time courses in the measurement of cytokine levels ( Table 5 ). There was no statistically significant difference between the 2 groups in baseline tear cytokine concentrations. In group 1, there was a statistically significant decrease in cytokine IL-6 after 2 months of treatment with minocycline. In group 2, no significant differences were found in the levels of all cytokines after 2 months of treatment. When considering the interaction effect between the 2 groups and the 2 time courses, there was no statistically significant difference in the measurement of cytokine levels ( P = .311, P = .546, P = .398, P = .909, P = .254, P = .063, P = .311, P = .555, and P = .552 for IL-6, IL-7, IL-8, IL-1β, IL-17α, MCP-1, TNF-α, IL-12p70, and IFN-γ, respectively). However, there were statistically significant differences in IL-1β, MCP-1, and TNF-α between the 2 groups after 2 months of treatment.
| Group 1 (pg/mL; n = 28) a | Group 2 (pg/mL; n = 30) b | P Value d | |||||
|---|---|---|---|---|---|---|---|
| Before | After 2 Months | P Value c | Before | After 2 Months | P Value c | ||
| IL-6 | 15.73 (20.64) | 3.89 (4.93) | <.001 | 14.78 (13.24) | 9.09 (11.01) | .340 | .184 |
| IL-7 | 4.89 (3.74) | 3.75 (2.22) | .050 | 5.16 (2.25) | 4.95 (4.75) | >.999 | >.999 |
| IL-8 | 88.32 (168.29) | 72.51 (160.99) | .050 | 86.62 (53.76) | 101.53 (81.36) | .398 | .334 |
| IL-1β | 4.28 (3.37) | 2.74 (1.11) | .024 | 6.65 (6.04) | 4.91 (2.49) | >.999 | <.001 |
| IL-17α | 4.52 (7.92) | 1.76 (1.73) | .018 | 5.12 (4.85) | 4.83 (6.55) | >.999 | .662 |
| MCP-1 | 56.07 (101.28) | 24.49 (34.36) | .254 | 64.92 (52.58) | 72.16 (27.89) | .110 | .002 |
| TNF-α | 4.66 (6.73) | 2.08 (2.49) | .036 | 5.38 (8.88) | 5.92 (7.00) | >.999 | .716 |
| IL-12p70 | 7.59 (2.66) | 5.58 (3.85) | .028 | 7.59 (9.62) | 7.69 (9.94) | >.999 | .420 |
| IFN-γ | 4.13 (2.48) | 3.37 (2.88) | .289 | 6.15 (6.70) | 6.88 (7.31) | >.999 | .386 |
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