Uveitis is one of the main causes of blindness worldwide (). Behçet's disease (BD) and acute anterior uveitis associated with ankylosing spondylitis (AS) are two commonly seen uveitis entities in Asia ([2-4]). However, the pathogenesis and clinical features of these diseases are quite different. BD is a chronic systemic inflammatory disorder characterized by nongranulomatous uveitis, recurrent oral ulcers, genital ulcers, and skin lesions (). AS is a chronic autoimmune arthritis that primarily affects the spine and is characterized by inflammatory back pain, asymmetrical peripheral oligoarthritis, and enthesitis (). Acute anterior uveitis is considered to be the most frequent extraarticular manifestation of AS (). Although the exact etiology of uveitis remains unclear, host genetic factors as well as immune imbalance are potential cofactors implicated in the initiation and development of the disease.
The complement system is seen as a major element of innate immunity and is regarded as the first line of defense against intrinsic and extrinsic antigens. It has been shown to be involved in the development of several ocular inflammatory diseases ([8-10]). Furthermore, a variety of genetic variants of the complement system are associated with ocular inflammatory diseases, such as age-related macular degeneration ([11-13]) and anterior uveitis ([14, 15]). Wakefield et al () were the first to address a possible role for complement C4 allotypes in uveitis and found a significantly increased frequency of the C4B2 allotype in patients with anterior uveitis. C4, which is located on chromosome 6q21.3, encodes 2 functionally distinct C4A (MIM 120810) and C4B (MIM 120820) isoforms. Previous studies indicated that a low copy number of C4A or C4B confers risk for multiple autoimmune disorders, including systemic lupus erythematosus (SLE) ([16-18]), rheumatoid arthritis (RA) (), and Graves' disease (). A positive correlation between gene copy number variations of C4 and the quantity of C4 protein has been demonstrated (). Additionally, specific C4 deficiency has been associated with multiple autoimmune disorders, including SLE (), RA (), and juvenile idiopathic arthritis ().
The aforementioned data suggest that copy number variations of C4 are associated with multiple immune diseases and may contribute to C4 deficiency in patients with SLE or RA. Whether C4 levels are aberrant in uveitis patients, including patients with BD and patients with AS-associated acute anterior uveitis, and whether copy number variations of C4 are responsible for an altered level in uveitis has not yet been reported. We therefore analyzed the level of C4 and C4 copy number variations in two frequently encountered clinical uveitis entities in China and investigated associations between the variants and a number of immune response parameters. Our findings provide further support for the notion that C4 plays an important role in the pathogenesis of intraocular inflammation.
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In the present study, we showed that the protein level of C4 in serum was increased in patients with active BD as compared to healthy controls, whereas no difference was observed between controls and the patients with AS and acute anterior uveitis. We furthermore found that having 2 copies or more than 2 copies of the C4A gene significantly increased the risk of BD. Functional studies showed significantly increased expression of C4A messenger RNA by PBMCs in the high copy number group. Increased production of IL-6 was found in the high C4A copy number group. We did not detect an effect of C4 copy number variations on the activation of CD4+ T cells.
Complement C4 plays several effector roles in the immune system, including involvement in the activation of the classical and the lectin complement pathways, which leads to cytolysis or neutralization of invading microbes, clearance of immune complexes, removal of apoptotic cells, and regulation of the activation of T and B lymphocytes ([35, 37]). Consistent with the findings of a previous study showing an increased level of C4 in patients with active BD (), the present study showed increased expression of C4 in patients with active BD, but not in patients with AS-associated acute anterior uveitis.
C4 is cleaved into C4A and C4B by a subunit of the first complement component, C1s. Activated C4A will usually form an amide bond with amino groups on antigens, and it thereby plays a role in immune complex clearance, whereas activated C4B is strongly reactive with hydroxyl groups on glycerols or glycosylated antigens and is more relevant in the defense against microbes ([18, 38]). Furthermore, C4A may mediate an inflammatory response by stimulating smooth muscle contraction, histamine release from mast cells, vasodilation, and increased vascular permeability (), leading to disease manifestations. The involvement of C4A in BD demonstrated in the present study, combined with data on vascular permeability changes (), may provide another view to strengthen the hypothesis that the pathogenesis of BD includes a systemic vascular inflammation caused by the generation of immune complexes (). Interestingly, we also found that the frequency of high copy numbers of C4A was increased in BD and that a higher copy number of C4A was associated with a higher expression of C4A. Our findings are consistent with those of an earlier study that showed a positive relationship between gene copy number variations of C4 and C4 protein quantity (). These findings suggest that C4A copy number variations may contribute to the increased C4 level in BD.
The role of C4 in an autoimmune disease such as SLE seems to differ from the role of C4 in BD indicated by our findings. Hauptmann et al () were the first to report a link between C4 deficiency and SLE. Animal studies with mice also confirmed that C4 deficiency can lead to SLE (). Immune complexes are considered to play an important role in the pathogenesis of SLE, and C4 deficiency may impair the clearance of these complexes, leading to tissue deposition. Low copy numbers of C4A or C4B have been shown to be more frequent in patients with SLE ([16-18]), RA (), and Graves' disease (). How C4 copy number affects RA or Graves' disease is unknown. Taken together, these data suggest that the role of C4 copy number variations may vary among immune-mediated diseases in humans.
We performed a number of experiments to assess a possible role for C4 copy number in cytokine expression and T cell activation. We performed these studies using samples from healthy controls, since the patients included in the present study represented a heterogeneous group of individuals with marked differences in the degree of inflammation and immunosuppressive therapy received. Our results showed that high copy numbers of C4A enhanced the production of IL-6. Despite the lack of a direct association between C4A and IL-6 expression, previous studies have found simultaneously elevated levels of C4 and IL-6 in patients undergoing cardiopulmonary bypass or with major depression ([44, 45]). Animal studies showed that C4 did not affect the expression of IL-6 in mice (). One possible explanation for this discrepancy could be species differences. IL-6 has recently been shown to be a critical regulator of Th17 differentiation and is seen as a marker of inflammation. Increased production of IL-6 in BD patients has been reported previously ([32, 47]). Not surprisingly, IL-6–knockout mice were shown to be resistant to the induction of experimental autoimmune uveoretinitis (EAU), an animal model of human endogenous uveitis (). Additionally, IL-6 blockade showed a therapeutic effect on EAU ([49, 50]), suggesting that it plays an important role in the pathogenesis of EAU. Taken together, these data suggest that the high copy number of C4A may play a role in BD pathogenesis via enhanced expression of IL-6. We did not find an effect of C4 copy number on the activation of PBMCs and the production of other proinflammatory cytokines.
It should be noted that the present study has certain limitations that need to be taken into account when considering its contributions. The patients were recruited from our ophthalmic center and may represent a selected population of patients. Confirmation of our data is therefore needed by studies of patients recruited from other medical specialties, such as rheumatology, to confirm our data for the BD and AS patient groups. The results presented here also need to be validated in other ethnic cohorts.
In conclusion, our results indicate that a high copy number of C4A confers risk for BD, but not AS-associated acute anterior uveitis, in a Han Chinese population. A higher C4A copy number may be associated with an effect on the regulation of C4A expression and control of a proinflammatory cytokine such as IL-6. These findings highlight the important role of C4 in the pathogenesis of BD.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Yang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Hou, Yang.
Acquisition of data. Hou, Qi, Liao, Fang.
Analysis and interpretation of data. Hou, Qi, Liao, Q. Zhang, Fang, Zhou, Liu, Bai, M. Zhang, Kijlstra, Yang.