Edited by: Sofie Struyf, KU Leuven, Belgium
Reviewed by: Ahmed M. Abu El-Asrar, King Saud University College of Medicine, Saudi Arabia; Undurti Narasimha Das, UND Life Sciences LLC, United States
†Shared first authorship.
Specialty section: This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Patients with juvenile idiopathic arthritis often develop chronic anterior uveitis (JIAU). JIAU patients possess a particularly high risk for developing secondary glaucoma when inflammatory inactivity has been achieved. By using multiplex bead assay analysis, we assessed levels of pro- and anti-inflammatory cytokines, chemokines, or metalloproteinases in the aqueous humor (AH) of patients with clinically inactive JIAU with (JIAUwG) or without secondary glaucoma (JIAUwoG), or from patients with senile cataract as controls. Laser-flare photometry analysis prior to surgery showed no significant differences between JIAUwG or JIAUwoG. Compared with the control group, levels of interleukin-8, matrix metalloproteinase-2, -3, -9, serum amyloid A (SAA), transforming growth factor beta-1, -2, -3 (TGFβ-1, -2, -3), and tumor necrosis factor-alpha in the AH were significantly higher in patients with clinically inactive JIAUwG or JIAUwoG. Samples from JIAwoG patients displayed significantly higher levels of SAA (
Approximately 11–13% of patients with juvenile idiopathic arthritis eventually develop uveitis (JIAU), too, typically as chronic anterior uveitis with insidious onset, in the absence of redness or pain. Risk factors for developing uveitis include the presence of anti-nuclear antibodies, oligoarthritis subtype, and early onset of the disease (
Topical treatment with corticosteroids is the first-line therapy, but it may promote elevated intraocular pressure (IOP) and cataract development. Systemic medication with methotrexate, adalimumab, and other disease-modifying anti-rheumatic drugs (DMARDs) can be used if the topical treatment is ineffective or for chronic disease. Uveitis patients have an increased risk for elevated IOP and uveitic glaucoma (UG), which might be induced by the inflammatory disease, but may also result from corticosteroids given as the first treatment approach to reduce ocular inflammation (
Such local immune responses may be controlled by proinflammatory and anti-inflammatory cytokines, which can be secreted by immune cells and certain other cell types to regulate inflammation. Proinflammatory cytokines are predominantly produced by helper T cell and macrophages to upregulate inflammatory reactions (
We hypothesized that altered outflow rates might be reflected by alterations in the MMP, interleukin (IL)-8, and TGFβ pathways. Therefore, measuring cytokine concentrations in the AH of JIAU patients with and without glaucoma could increase our knowledge of the local pathogenetic processes and may help to explain why some patients with JIAU develop glaucoma and others do not. The multiplex bead immune assay technique allows the simultaneous detection of multiple cytokines even from small clinical samples, as shown previously in patients with uveitis (
The study design complies with the standards put forth by the Declaration of Helsinki. The study was approved by the local ethics committee. All subjects provided written informed consent for AH collection during independently planned surgery. For underaged JIAU patients, written informed consent was obtained from the patients’ parents.
Uveitis was classified according to the recommendations put forth by the international uveitis study group (IUSG), under consideration of the recent modifications (
Diagnosis and treatment of JIAU followed the German national recommendations for management of disease (
For all groups, suspicion of steroid-induced ocular hypertension constituted an exclusion criterion. None of the JIAU eyes had active inflammation, as defined by anterior chamber (AC) cells ≥0 during the 3 months prior to AH collection, and all had been treated 5× daily with prednisolone acetate 1% for one week prior to surgery. In the control group, any history of systemic or eye inflammation represented an exclusion criterion. Only one eye of an individual patient was included in the study in all groups.
Aqueous humor samples (100 to 200 µl) were collected at the beginning of the surgery through limbal paracentesis using a 0.5-ml syringe with a 30-gauge needle (BD Hamburg, Germany). Care was taken to prevent contamination with blood or intraocular tissue. All samples were immediately frozen and stored at −80°C until analysis.
Concentrations of IL-8, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases-1, -2, -3, -9 (MMP-1, -2, -3, -9), serum amyloid A (SAA), TGFβ-1, -2, -3, and TNF-α were measured by multiplex bead assay analysis (Luminex® Performance Assay TGFβ Multiplex Kit R&D Systems Abingdon UK; Procarta® Immunoassay Affymetrix, Santa Clara US) according to the manufacturer’s instructions. Samples were analyzed with LUMINEX® 100/200™ using the Luminex xPonent software (Luminex, USA).
Statistical analysis was performed using MedCalc statistical software 10.0.1.0 (Marikerke, Belgium). For analysis of normally distributed data, Student’s
Overall, 30 JIAU patients, 15 JIAU subjects without glaucoma (JIAUwoG) (average 15.1 years at AH collection) and another 15 JIAU subjects with glaucoma (JIAUwG) (average 13.1 years at AH collection), were included. Additionally, 24 subjects with senile cataract (average 75.5 years at AH collection) were used as controls. The demographic data of the subjects, including number of patients, age, duration of uveitis, lens status, and most recent laser-flare values, are summarized in Table
Demographic data of patients, treatment, and non-invasive laser-flare measurements.
C | JIAUwoG | JIAUwG | ||
---|---|---|---|---|
Number of eyes | 24 | 15 | 15 | |
Age (years; mean ± SD) | 75.5 ± 9.1 | 15.1 ± 11.5 | 13.1 ± 3.4 | <0.001 |
Duration of uveitis (years, mean ± SD) | n.a. | 5.6 ± 4.5 | 6.9 ± 3.1 | 0.37# |
Lens status | n.a. | 15 phakic | 9 phakic, 3 PC IOL, 3 aphakic | 0.023+ |
Posterior synechiae (eyes) | n.a. | 13 | 6 | 0.03+ |
Systemic corticosteroids | n.a. | 6 | 2 | 0.21+ |
csDMARD | n.a. | 13 | 13 | 1+ |
bDMARD | n.a. | 3 | 2 | 1+ |
Non-invasive laser-flare photometric analysis (KOWA LF-500) (photons/ms, mean ± SD) | n.a. | 110.3 ± 40.8 | 65.5 ± 15.3 | 0.26# |
Age of JIAUwG and JIAUwoG patients did not significantly differ (
Six JIAUwG patients had received topical prostaglandins (Latanoprost or Bimatoprost) until 2 weeks before glaucoma surgery. Six of 15 JIAUwoG subjects had received steroids systemically, while this was the case in only 2 of 15 JIAUwG patients (Table
The lens status of the 15 eyes with JIAUwoG were phakic, whereas only 9 of 15 eyes in JIAUwG were phakic, 3 were pseudophakic, and another 3 were aphakic.
Non-invasive laser-flare measurements did not differ between JIAUwoG (110.3 ± 40.8 photons/ms) and JIAUwG (65.5 ± 15.3 photons/ms) patients (
Laser-flare photometric measurement of the six JIAUwG eyes treated with prostaglandins prior to surgery (62.9 ± 19.8 photons/ms) compared with the nine JIAUwoG eyes (68.93 ± 26.2 photons/ms) revealed no differences (
Compared with the cataract group (control), in whom SAA expression could not be detected, JIAUwoG patients showed an extensive level of SAA (
Box plots of matrix metalloproteinases (MMP)-9, serum amyloid A (SAA), and transforming growth factor beta (TGFβ)-2 in patients with juvenile arthritis with (JIAUwG) or without glaucoma (JIAUwoG). Specimens collected from patients with senile cataract served as control. Dots represent individual values. ANOVA with Bonferroni correction (MMP-9, SAA, TGFβ-2:
The control group showed a slight constitutive expression of TGFβ-2. The levels of TGFβ-2 in the JIAUwG patients were significantly higher than in the JIAUwoG patients (
Compared with the cataract group, the concentration of IL-8, MMP-2, MMP-3, MMP-9, TGFβ-1, TGFβ-3, and TNF-α was significantly increased in subjects with JIAUwG or JIAUwoG. However, no significant differences in MCP-1 and MMP-1 were observed between the control group and both JIAU groups (Figure
Box plots of interleukin-8 (IL-8), matrix metalloproteinases (MMP)-2, MMP-3, transforming growth factor beta (TGFβ)-1, TGFβ-3, and tumor necrosis factor-alpha (TNF-α) in patients with juvenile idiopathic arthritis-associated uveitis with or without glaucoma. Specimens collected from patients with senile cataract served as a control. Dots represent individual values. ANOVA with Bonferroni correction (IL-8:
The correlation among cytokine levels are shown in Table
Correlation between cytokines.
ρ/ |
IL-8 | MCP-1 | MMP-1 | MMP-2 | MMP-3 | MMP-9 | SAA | TGFβ-1 | TGFβ-2 | TGFβ-3 | TNF-α |
---|---|---|---|---|---|---|---|---|---|---|---|
IL-8 | 0.199 | 0.828 | 0.731 | 0.827 | 0.771 | 0.725 | 0.489 | 0.307 | 0.616 | 0.695 | |
MCP-1 | 0.146 | 0.181 | 0.187 | 0.24 | 0.159 | 0.086 | 0.079 | 0.043 | 0.042 | 0.078 | |
MMP-1 | <0.0001 |
0.1874 | 0.694 | 0.802 | 0.736 | 0.815 | 0.536 | 0.254 | 0.610 | 0.679 | |
MMP-2 | <0.0001 |
0.1728 | <0.0001 |
0.846 | 0.574 | 0.697 | 0.485 | 0.33 | 0.579 | 0.733 | |
MMP-3 | <0.0001 |
0.0807 | <0.0001 |
<0.0001 |
0.673 | 0.781 | 0.513 | 0.338 | 0.59 | 0.75 | |
MMP-9 | <0.0001 |
0.2468 | <0.0001 |
<0.0001 |
<0.0001 |
0.621 | 0.499 | 0.275 | 0.544 | 0.656 | |
SAA | <0.0001 |
0.53 | <0.0001 |
<0.0001 |
<0.0001 |
<0.0001 |
0.655 | 0.298 | 0.760 | 0.652 | |
TGFβ-1 | 0.009 |
0.5923 | 0.003 |
0.001 |
0.0005 |
0.0007 |
<0.0001 |
0.79 | 0.846 | 0.351 | |
TGFβ-2 | 0.376 | 0.7714 | 0.085 | 0.0251 | 0.0218 | 0.0624 | 0.0433 | <0.0001 |
0.593 | 0.261 | |
TGFβ-3 | <0.0001 |
0.7781 | <0.0001 |
0.0001 |
0.0001 |
0.0002 |
<0.0001 |
<0.0001 |
0.0001 |
0.423 | |
TNF-α | <0.0001 |
0.57 | <0.0001 |
<0.0001 |
<0.0001 |
<0.0001 |
<0.0001 |
0.0172 | 0.0763 | 0.0042 |
Correlation of cytokines in the two JIAU groups showed no significant differences with regard to age, uveitis duration, or laser-flare values. Only MMP-2 was significantly elevated in patients with increased laser-flare photometric values (
Correlation of cytokines with age, duration of uveitis, or laser-flare values.
Age (years) |
Duration of uveitis (years) |
Laser-flare photometric values (photons/ms) |
||||
---|---|---|---|---|---|---|
Correlation coefficient | Correlation coefficient | Correlation coefficient | ||||
IL-8 | −0.21 | 0.25 | −0.078 | 0.69 | 0.171 | 0.41 |
MCP-1 | −0.364 | 0.05 | −0.167 | 0.36 | 0.35 | 0.09 |
MMP-1 | 0.033 | 0.85 | −0.042 | 0.81 | 0.256 | 0.22 |
MMP-2 | −0.227 | 0.221 | −0.3 | 0.16 | 0.603 | 0.0038 |
MMP-3 | 0.006 | 0.97 | −0.021 | 0.90 | 0.512 | 0.014 |
MMP-9 | −0.298 | 0.10 | −0.131 | 0.48 | 0.227 | 0.27 |
SAA | −0.063 | 0.73 | −0,254 | 0.17 | 0.399 | 0.056 |
TGFβ-1 | 0.128 | 0.48 | 0.107 | 0.56 | 0.0038 | 0.98 |
TGFβ-2 | 0.147 | 0.43 | 0.185 | 0.31 | 0.039 | 0.85 |
TGFβ-3 | 0.161 | 0.93 | −0.122 | 0.51 | 0.115 | 0.58 |
TNF-α | −0.181 | 0.32 | −0.239 | 0.19 | 0.36 | 0.084 |
No difference in cytokine levels were observed in JIAU eyes whether patients were treated with csDMARD or prostaglandins. However, in patients treated with bDMARD, we detected significantly higher levels of MMP-1 (
Influence of csDMARD, bDMARD, or prostaglandin medication on the expression of cytokines in the anterior chamber.
csDMARD |
bDMARD |
Prostaglandins |
|||||||
---|---|---|---|---|---|---|---|---|---|
Neg | Pos | Neg | Pos | Neg | Pos | ||||
Number | 4 | 26 | 25 | 5 | 9 | 6 | |||
IL-8 | 4.2 ± 3.2 | 7.1 ± 9.5 | 0.56 | 7.3 ± 9.6 | 3.3 ± 2.7 | 0.37 | 7.4 ± 12.7 | 4.7 ± 2.3 | 0.61 |
MCP-1 | 110.1 ± 35.5 | 160 ± 100 | 0.33 | 158 ± 97 | 130 ± 91 | 0.56 | 141 ± 72 | 151 ± 65 | 0.79 |
MMP-1 | 11.2 ± 8.5 | 196 ± 807 | 0.65 | 38.4 ± 58.9 | 838.45 | 0.027 | 12.7 ± 6.3 | 30.7 ± 41.3 | 0.21 |
MMP-2 | 821 ± 363 | 984 ± 380 | 0.42 | 1,014 ± 376 | 704 ± 280 | 0.09 | 1,099 ± 428 | 875 ± 192 | 0.2567 |
MMP-3 | 6,211 ± 4,074 | 7,924 ± 4,526 | 0.48 | 8,210 ± 4,525 | 5,124 ± 3,200 | 0.16 | 7,779 ± 3,522 | 8,574 ± 3,794 | 0.68 |
MMP-9 | 2.3 ± 2.1 | 5.5 ± 5.3 | 0.25 | 5.5 ± 5.3 | 2.8 ± 3 | 0.28 | 4.1 ± 3.9 | 2.9 ± 1.9 | 0.54 |
SAA | 2,074 ± 2,034 | 7,166 ± 7,433 | 0.18 | 6,648 ± 7,002 | 5,686 ± 8,705 | 0.78 | 3,708 ± 4,723 | 2,663 ± 2,240 | 0.62 |
TGFβ-1 | 311 ± 250 | 1,844 ± 2,053 | 0.15 | 1,213 ± 1,261 | 3,775 ± 3,471 | 0.0059 | 1,026 ± 8,48 | 1,511 ± 1,087 | 0.35 |
TGFβ-2 | 1,188 ± 1,243 | 2,643 ± 1,877 | 0.14 | 2,448 ± 1,987 | 2,456 ± 1,147 | 0.99 | 3,047 ± 1,751 | 4,180 ± 1,074 | 0.18 |
TGFβ-3 | 76 ± 32 | 289 ± 291 | 0.16 | 252 ± 299 | 301 ± 171 | 0.73 | 195 ± 244 | 201 ± 154 | 0.96 |
TNF-α | 19 ± 32 | 22 ± 32 | 0.87 | 25 ± 33 | 5.3 ± 3.2 | 0.2 | 28 ± 41 | 21 ± 24 | 0.71 |
Statistical calculation for clinical factors associated with a higher risk for glaucoma manifestation by using univariate regression analysis of age of patients, duration of uveitis, systemic treatment with conventional immunosuppressant (methotrexate, azathioprine, or cyclosporine A) or treatment with adalimumab revealed no significant influence. The presence of posterior synechiae at the time of surgery was associated with a decreased risk for secondary glaucoma development (OR 0.1025, 95% CI 0.0168–0.6280;
The present study investigated the expression of pro- and anti-inflammatory cytokines, chemokines, or metalloproteinases in the AH of patients with clinically inactive uveitis associated with JIA, both with or without glaucoma. Quiescence of uveitis disease was confirmed by clinical observation and ensured by treatment with individually adjusted anti-inflammatory medication before surgery.
We found that the concentrations of IL-8, MMP-2, MMP-3, MMP-9, TGFβ-1, TGFβ-2, TGFβ-3, SAA, and TNF-α were significantly elevated in JIAU samples compared with the control group (senile cataract patients). These results show that even patients with clinically inactive JIAU receiving intensive anti-inflammatory treatment may not achieve an entirely inactive state from an immunological point of view. Instead, the AH in JIAU eyes displayed a constitutive expression of the proinflammatory cytokines IL-8 and TNF-α. We also found an increased expression of MMP-2, MMP-3, MMP-9, TGFβ-1, and TGFβ-3 in eyes with JIAU, independent of the presence of glaucoma. Eyes with JIAUwG displayed increased levels of TGFβ-2, while eyes with JIAUwoG showed significantly increased levels of SAA.
Elevated levels of TGFβ-2 have previously been strongly correlated with increased outflow resistance and IOP elevation in POAG. TGFβ-2 is an anti-proliferative and anti-inflammatory cytokine, which has been discussed in the context of various disorders, including glaucoma (
Increased SAA concentrations in the AC of eyes with glaucoma have been found previously, too. Here, the expression of SAA2 was increased in TM tissues from donors with glaucoma. Furthermore, treatment of cultured human TM cells with recombinant SAA affected upregulation of IL-8 and increased IOP in the human ocular perfusion organ culture (
In a recent study, various inflammatory cytokines in the AH of patients with POAG, exfoliation glaucoma (EXG) and senile cataract were determined. Herein, increased TGFβ-1, IL-8, and SAA levels were found in POAG and EXG when compared with the cataract control group. IL-6 was decreased in the POAG group. The levels of TGFβ-1, IL-8, and SAA correlated positively with each other, with IOP, and the number of glaucoma medications. The authors concluded that TGFβ-1, IL-8, and SAA have critical roles in IOP elevations in patients with POAG (
Moreover, previous studies of rheumatoid arthritis have shown that SAA in the serum was strongly associated with disease activity, but also with the risk of cardiovascular and renal involvement and could, therefore, serve as a marker to help identify patients with persistent inflammation and at risk of extra-articular complications (
It has been shown previously that SAA may attract migrating phagocytes to the site of inflammation, serve as an agonist for toll-like receptor 4, activate nuclear factor NF-κB, and induce expression of both inflammatory cytokines and M2 macrophage markers and to influence polymorphonuclear neutrophils (PMN) by inhibiting apoptosis and enhancing efferocytosis (
However, inflammation responses in the eye must be downregulated, for example, by anti-inflammatory cytokines, to minimize tissue damage in the eye. It has been shown previously that TGFβ-2 may influence the levels of SAA. A previous study has shown that IL-10−/− mice that develop a form of inflammatory bowel disease (a model for Crohn’s disease) gained more weight and developed lower pathological scores, lower TNF-α concentrations, and lower SAA content when mice were fed a TGFβ-2-containing diet (
Some of the patients with JIAUwG received medication with Latanoprost or Bimatoprost to treat elevated IOP. Previous studies have shown that topical treatment with Latanoprost decreased levels of TGFβ-1, MMP-2, and tissue inhibitor of metalloproteinases-2 in patients suffering from PEXG (
No information has been published about the impact of csDMARD on intraocular cytokine concentrations. However, our data show that treatment with csDMARD did not significantly alter cytokine concentrations in the AH. In contrast, our data demonstrate that there was a significant difference in the expression of MMP-1 or TGFβ-1 after treatment with bDMARD. An explanation could be that bDMARD are favored in patients with a particularly severe JIAU disease course with or without glaucoma. However, our results imply that the use of bDMARD modulated the local immune response of the patients.
Tumor necrosis factor-alpha is a proinflammatory cytokine with pleiotrophic effects on various cell types. Recent results suggest that the TNF-α neutralizing function dictates not only the success of anti-inflammatory treatment, but also other biological effects. Anti-TNF-α medications are increasingly being used to treat JIAU (
In some diseases, e.g., rheumatoid arthritis, TGFβ signaling is enhanced and seems to play a proinflammatory rather than an anti-inflammatory role. TGFβ-1 promoted the expression of proinflammatory cytokines and metalloproteinases (
Recent studies have shown that pharmacological blockade of IL-6 by a humanized anti-IL-6 receptor antibody is beneficial in patients with rheumatoid arthritis and JIA, suggesting that IL-6 might be an essential key cytokine in these diseases. IL-6 was shown to be involved in the induction of Th17 cell stimulation with TGFβ. Together with TNF-α and IL-1, IL-6 induces CRP or SAA signaling. Indeed, IL-6 was among those cytokines previously detected in the AH of JIAU patients (
In conclusion, the results of the current study show that pro- and anti-inflammatory cytokine, chemokine, or metalloproteinase levels are increased in clinically inactive JIAU eyes, suggesting that in these eyes disease is seemingly not inactive from an immunological point of view and that the eyes show a cytokine profile typical of chronic inflammation. The pathogenetic process for the development of glaucoma in some of the JIAU patients may be related to the severity of ocular inflammation, the use of corticosteroids, and complications such as posterior synechiae, damage of anterior chamber angle or to the TM. We therefore conclude that the etiologic mechanisms involved are multifactorial. However, the significantly increased levels of SAA in JIAUwoG and of TGFβ-2 in JIAUwG suggest that the cytokines could play important roles in modulating IOP. Previous studies have found that SAA may induce IL-8 production of TM. TGFβ-2 has been shown to decrease SAA and TNF-α and to regulate ECM synthesis and remodeling (
The study design complies with the standards put forth by the Declaration of Helsinki. The study was approved by the local ethics committee. All subjects provided written informed consent for AH collection during independently planned surgery. For underaged JIAU patients, written informed consent was obtained from the patients’ parents.
DB, MK, and CH designed the study and wrote the manuscript. DB, MK, PM, BK, KW, and CH performed the experiments and analyzed the data. KW, JK, AH, and CH performed the eye surgery, collected the aqueous humor, and provided clinical data. All authors approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.