Nicole Schäfer

Interrelation Between Oxidative Stress and Complement Activation in Models of Age-Related Macular Degeneration

Millions of individuals older than 50-years suffer from age-related macular degeneration (AMD). Associated with this multifactorial disease are polymorphisms of complement factor genes and a main environmental risk factor-oxidative stress. Until now the linkage between these risk factors for AMD has not been fully understood. Recent studies, integrating results on oxidative stress, complement activation, epidemiology and ocular pathology suggested the following sequence in AMD-etiology: initially, chronic oxidative stress results in modification of proteins and lipids in the posterior of the eye; these tissue alterations trigger chronic inflammation, involving the complement system; and finally, invasive immune cells facilitate pathology in the retina. Here, we summarize the results for animal studies which aim to elucidate this molecular interplay of oxidative events and tissue-specific complement activation in the eye.

Complement Regulator FHR-3 Is Elevated either Locally or Systemically in a Selection of Autoimmune Diseases

The human complement factor H-related protein-3 (FHR-3) is a soluble regulator of the complement system. Homozygous cfhr3/1 deletion is a genetic risk factor for the autoimmune form of atypical hemolytic-uremic syndrome (aHUS), while also found to be protective in age-related macular degeneration (AMD). The precise function of FHR-3 remains to be fully characterized. We generated four mouse monoclonal antibodies (mAbs) for FHR-3 (RETC) without cross-reactivity to the complement factor H (FH)-family. These antibodies detected FHR-3 from human serum with a mean concentration of 1 μg/mL. FHR-3 levels in patients were significantly increased in sera from systemic lupus erythematosus, rheumatoid arthritis, and polymyalgia rheumatica but remained almost unchanged in samples from AMD or aHUS patients. Moreover, by immunostaining of an aged human donor retina, we discovered a local FHR-3 production by microglia/macrophages. The mAb RETC-2 modulated FHR-3 binding to C3b but not the binding of FHR-3 to heparin. Interestingly, FHR-3 competed with FH for binding C3b and the mAb RETC-2 reduced the interaction of FHR-3 and C3b, resulting in increased FH binding. Our results unveil a previously unknown systemic involvement of FHR-3 in rheumatoid diseases and a putative local role of FHR-3 mediated by microglia/macrophages in the damaged retina. We conclude that the local FHR-3/FH equilibrium in AMD is a potential therapeutic target, which can be modulated by our specific mAb RETC-2.

Complement Components Showed a Time-Dependent Local Expression Pattern in Constant and Acute White Light-Induced Photoreceptor Damage

Background: Photoreceptor cell death due to extensive light exposure and induced oxidative-stress are associated with retinal degeneration. A correlated dysregulation of the complement system amplifies the damaging effects, but the local and time-dependent progression of this mechanism is not thoroughly understood. Methods: Light-induced photoreceptor damage (LD) was induced in Balb/c mice with white light illumination either for 24 h with 1000 lux (constant model) or 0.5 h with 5000 lux (acute model). Complement protein and mRNA expression levels were compared at 1 and 3 days post-LD for C1s, complement factor B (CFB), mannose binding lectin A, mannose-binding protein-associated serine protease 1 (MASP-1), C3, C4, C9, and complement factor P in retina and RPE/choroid. Histological analyses visualized apoptosis, microglia/macrophage migration, gliosis and deposition of the complement activation marker C3d. Systemic anaphylatoxin serum concentrations were determined using an ELISA. Results: Apoptosis, gliosis and microglia/macrophage migration into the outer nuclear layer showed similar patterns in both models. Local complement factor expression revealed an early upregulation of complement factor mRNA in the acute and constant light regimen at 1 day post-treatment for c1s, cfb, masp-1, c3, c4 and c9 in the RPE/choroid. However, intraretinal complement mRNA expression for c1s, cfb, c3 and c4 was increased at 1 day in the constant and at 3 days in the acute model. A corresponding regulation on protein level in the retina following both LD models was observed for C3, which was upregulated at 1 day and correlated with increased C3d staining in the ganglion cell layer and at the RPE. In the RPE/choroid C1s-complex protein detection was increased at 3 days after LD irrespectively of the light intensities used. Conclusion: LD in mouse eyes is correlated with local complement activity. The time-dependent local progression of complement regulation on mRNA and protein levels were equivalent in the acute and constant LD model, except for the intraretinal, time-dependent mRNA expression. Knowing the relative time courses of local complement expression and cellular activity can help to elucidate novel therapeutic options in retinal degeneration indicating at which time point of disease complement has to be rebalanced.

Cell type-specific complement expression from healthy and diseased retinae

Retinal degeneration is associated with complement system activation, but retinal sources of complement are unknown. Here, we describe the human and murine complement transcriptomes of Müller cells, microglia/macrophages, vascular cells, neurons and retinal pigment epithelium (RPE) in health and disease. All cell populations expressed c1s, c3, cfb, cfp, cfh and cfi. Murine Müller cells contributed the highest amount of complement activators (c1s, c3, cfb). RPE mainly expressed cfh, while cfi and cfp transcripts were most abundant in neurons. The main complement negative regulator in the human retina was cfi, while cfh dominated in the murine retina. Importantly, the expression of c1s, cfb, cfp, cfi increased and that of cfh decreased with aging. Impaired photoreceptor recycling led to an enhanced c3 expression in RPE and to a reduced cfi expression in microglia/macrophages. Expression of complement components was massively upregulated after transient retinal ischemia in murine microglia, Müller cells and RPE. The individual signature of complement expression in distinct murine and human retinal cell types indicates a local, well-orchestrated regulation of the complement system in both species.

A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common tumors within the oral cavity. Early diagnosis and prognosis tools are urgently needed. This study aimed to investigate the activation of the complement system in OSCC patients as potential biomarker. Therefore, an innovative complement activation array was developed. Characterized antibodies detecting the complement activation specific epitopes C3a, C5a and sC5b-9 along with control antibodies were implemented into a suspension bead array. Human serum from a healthy (n = 46) and OSCC patient (n = 57) cohort were used to investigate the role of complement activation in oral tumor progression. The novel multiplex assay detected C3a, C5a and sC5b-9 from a minimal sample volume of human tears, aqueous humor and blood samples. Limits of detection were 0.04 ng/mL for C3a, 0.03 ng/mL for C5a and 18.9 ng/mL for sC5b-9, respectively. Biological cut-off levels guaranteed specific detections from serum. The mean serum concentration of a healthy control cohort was 680 ng/mL C3a, 70 ng/mL C5a and 2247 ng/mL sC5b-9, respectively. The assay showed an intra-assay precision of 2.9–6.4% and an inter-assay precision of 9.2–18.2%. Increased systemic C5a (p < 0.0001) and sC5b-9 (p = 0.01) concentrations in OSCC patients were determined using the validated multiplex complement assay. Higher C5a concentrations correlated with tumor differentiation and OSCC extension state. Systemic sC5b-9 determination provided a novel biomarker for infiltrating tumor growth and C3a levels were associated with local tumor spreading. Our study suggests that systemic complement activation levels in OSCC patients may be useful to assess disease progression.