C3 Glomerulopathy and Related Disorders in Children: Etiology-Phenotype Correlation and Outcomes.

BACKGROUND AND OBJECTIVES: Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan-Meier method. RESULTS: Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2-15 (median, 9; interquartile range, 7-11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13-8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on the initial biopsy specimen. CONCLUSIONS: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Dendritic Cells Promote the Spread of Human T-Cell Leukemia Virus Type 1 via Bidirectional Interactions with CD4+ T Cells.

Human T-cell leukemia virus type 1 (HTLV-1) propagates within and between individuals via cell-to-cell transmission, and primary infection typically occurs across juxtaposed mucosal surfaces during breastfeeding or sexual intercourse. It is therefore likely that dendritic cells (DCs) are among the first potential targets for HTLV-1. However, it remains unclear how DCs contribute to virus transmission and dissemination in the early stages of infection. We show that an HTLV-1-infected cell line (MT-2) and naturally infected CD4+ T cells transfer p19+ viral particles to the surface of allogeneic DCs via cell-to-cell contacts. Similarly organized cell-to-cell contacts also facilitate DC-mediated transfer of HTLV-1 to autologous CD4+ T cells. These findings shed light on the cellular structures involved in anterograde and retrograde transmission and suggest a key role for DCs in the natural history and pathogenesis of HTLV-1 infection.

Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis.

The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. Here we describe the use of Tissue MicroArray (TMA) methodology in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH, complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. We found that numbers of cells immunolabelled for HLA-DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. We conclude that TMA methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.

Plasma complement biomarkers distinguish multiple sclerosis and neuromyelitis optica spectrum disorder.

BACKGROUND: Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are autoimmune inflammatory demyelinating diseases of the central nervous system. Although distinguished by clinicoradiological and demographic features, early manifestations can be similar complicating management. Antibodies against aquaporin-4 support the diagnosis of NMOSD but are negative in some patients. Therefore, there is unmet need for biomarkers that enable early diagnosis and disease-specific intervention. OBJECTIVE: We investigated whether plasma complement proteins are altered in MS and NMOSD and provide biomarkers that distinguish these diseases. METHODS: Plasma from 54 NMOSD, 40 MS and 69 control donors was tested in multiplex assays measuring complement activation products and proteins. Using logistic regression, we tested whether combinations of complement analytes distinguished NMOSD from controls and MS. RESULTS: All activation products were elevated in NMOSD compared to either control or MS. Four complement proteins (C1inh, C1s, C5 and FH) were higher in NMOSD compared to MS or controls. A model comprising C1inh and terminal complement complex (TCC) distinguished NMOSD from MS (area under the curve (AUC): 0.98), while C1inh and C5 distinguished NMOSD from controls (AUC: 0.94). CONCLUSION: NMOSD is distinguished from MS by plasma complement biomarkers. Selected complement analytes enable differential diagnosis. Findings support trials of anti-complement therapies in NMOSD.

Complement Biomarkers as Predictors of Disease Progression in Alzheimer's Disease.

There is a critical unmet need for reliable markers of disease and disease course in mild cognitive impairment (MCI) and early Alzheimer's disease (AD). The growing appreciation of the importance of inflammation in early AD has focused attention on inflammatory biomarkers in cerebrospinal fluid or plasma; however, non-specific inflammation markers have disappointed to date. We have adopted a targeted approach, centered on an inflammatory pathway already implicated in the disease. Complement, a core system in innate immune defense and potent driver of inflammation, has been implicated in pathogenesis of AD based on a confluence of genetic, histochemical, and model data. Numerous studies have suggested that measurement of individual complement proteins or activation products in cerebrospinal fluid or plasma is useful in diagnosis, prediction, or stratification, but few have been replicated. Here we apply a novel multiplex assay to measure five complement proteins and four activation products in plasma from donors with MCI, AD, and controls. Only one complement analyte, clusterin, differed significantly between control and AD plasma (controls, 295 mg/l; AD, 388 mg/l: p < 10- 5). A model combining clusterin with relevant co-variables was highly predictive of disease. Three analytes (clusterin, factor I, terminal complement complex) were significantly different between MCI individuals who had converted to dementia one year later compared to non-converters; a model combining these three analytes with informative co-variables was highly predictive of conversion. The data confirm the relevance of complement biomarkers in MCI and AD and build the case for using multi-parameter models for disease prediction and stratification.

Polypropylene Sulfide Nanoparticle p24 Vaccine Promotes Dendritic Cell-Mediated Specific Immune Responses against HIV-1.

Delivery of vaccine formulations into the dermis using antigen-coated microneedle patches is a promising and safe approach because of efficient antigen delivery and safety. We evaluated an intradermal vaccine using HIV-1 p24 Gag peptide-conjugated polypropylene sulfide nanoparticles to induce immunity against HIV-1. This peptide-conjugated polypropylene sulfide nanoparticle formulation did not accelerate the maturation of blood- or skin-derived subsets of dendritic cells, either generated in vitro or purified ex vivo, despite efficient uptake in the absence of adjuvant. Moreover, dendritic cell-mediated capture of particulate antigen in this form induced potent HIV-1-specific CD4(+) T-cell responses, as well as B-cell-mediated antibody production. Nanoparticle-based intradermal antigen delivery may therefore provide a new option in the global effort to develop an effective vaccine against HIV-1.

Identification of factor H-like protein 1 as the predominant complement regulator in Bruch's membrane: implications for age-related macular degeneration.

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch's membrane. Bruch's membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch's membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch's membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.

Complement activation in multiple sclerosis plaques: an immunohistochemical analysis.

INTRODUCTION: Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain. This study aims to bring clarity to these questions. RESULTS: We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease. Active, chronic active and chronic inactive multiple sclerosis plaques (35 in total) and non-plaque areas were examined.Multiple sclerosis plaques were consistently positive for complement proteins (C3, factor B, C1q), activation products (C3b, iC3b, C4d, terminal complement complex) and regulators (factor H, C1-inhibitor, clusterin), suggesting continuing local complement synthesis, activation and regulation despite the absence of other evidence of ongoing inflammation. Complement staining was most apparent in plaque and peri-plaque but also present in normal appearing white matter and cortical areas to a greater extent than in control tissue. C1q staining was present in all plaques suggesting a dominant role for the classical pathway. Cellular staining for complement components was largely restricted to reactive astrocytes, often adjacent to clusters of microglia in close apposition to complement opsonised myelin and damaged axons. CONCLUSIONS: The findings demonstrate the ubiquity of complement involvement in multiple sclerosis, suggest a pathogenic role for complement contributing to cell, axon and myelin damage and make the case for targeting complement for multiple sclerosis monitoring and therapy.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PURPOSE: To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). DESIGN: Multicenter, cross-sectional study. PARTICIPANTS: We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. METHODS: We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). MAIN OUTCOME MEASURES: We evaluated AMD status and recipient and donor CFH Y402H genotype. RESULTS: In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). CONCLUSIONS: Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Dimerization of complement factor H-related proteins modulates complement activation in vivo.

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Tissue-specific host recognition by complement factor H is mediated by differential activities of its glycosaminoglycan-binding regions.

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6-8 and CCP19-20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6-8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19-20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6-8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19-20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay.

BACKGROUND: Research on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH. METHODS: Reagents from a commercial radial immunodiffusion (RID) assay (The Binding Site) were adapted for use on the Siemens BNII high throughput nephelometric instrument. The assay was calibrated with a highly purified human fH preparation with rigorously determined concentration, and assay performance was comprehensively evaluated using samples from healthy human volunteers, with the commercial RID assay as a comparator. The distribution and determinants of circulating fH concentration in humans were then investigated in a large representative population sample. RESULTS: The nephelometric assay had recovery close to 100%, was reproducible with intra- and inter-assay CV's of 11% and 5-15% respectively, and had a wider operating range than the RID assay. fH values were unaffected after multiple freeze-thaw cycles demonstrating that it is evidently a stable analyte for immunoassay. fH concentration was unaltered by an acute inflammatory stimulus. The population study showed that plasma fH concentration is associated with circulating lipids and indices of body fat. CONCLUSION: We present the first high throughput assay for circulating fH; the assay is accurate and reliable with reproducible measures from stored samples. It has established the distribution of fH values at a population level and demonstrated important associations with circulating lipids and indices of body fat, thus providing an important reference for future clinical and epidemiological investigations.

Epidermis instructs skin homing receptor expression in human T cells.

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. Although the mechanisms controlling memory T-cell migration to peripheral tissues are poorly understood, the current paradigm includes the localized secretion of "imprinting" signals from tissue-resident dendritic cells in the draining lymph nodes. Here we show that CCR8 expression by newly activated naive T cells is regulated by skin-specific factor(s) derived primarily from epidermal keratinocytes, thereby providing a mechanism for the preferential expression of CCR8 by skin-resident memory T cells. Importantly, no such effects were observed after coculture with primary cells from skin-unrelated epithelia, including mesothelium and small intestine. The keratinocyte-derived CCR8-inducing factor(s) were soluble, and independent of vitamins A and D. Furthermore, the induction of CCR8 under these conditions correlated with an increase in cutaneous lymphocyte-associated antigen expression. Our findings challenge current tissue homing paradigms, especially those involving CCR10, and emphasize the importance of steady-state epidermis rather than tissue-resident dendritic cells in controlling the localization of memory T cells within human skin.

Assembly and regulation of the membrane attack complex based on structures of C5b6 and sC5b9.

Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8ß-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.

Complement regulator factor H in multiple sclerosis.

A recent proteomic study published in this journal demonstrated lower cerebrospinal fluid (CSF) expression of factor H (fH), an important complement regulator, along with two other complement proteins, in active multiple sclerosis (MS) patients. We have previously demonstrated raised serum fH levels in MS and here, an extended analysis, quantifying fH in CSF, demonstrates no change in fH levels in active disease, but significantly raised levels in progressive disease. These findings support our previous work showing raised serum fH in patients with progressive MS, and our results predict that CSF fH levels will be raised rather than reduced in active disease.

Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is associated with complement alternative pathway defects in over half the cases. Point mutations that affect complement surface regulation are common in factor H (CFH); however, sometimes individuals have null mutations in heterozygosis. The latter are difficult to identify, although a consistently low plasma factor H (fH) concentration is suggestive; definitive proof requires demonstration that the mutant sequence is not expressed in vitro. Here, novel reagents and assays that distinguish and individually quantify the common factor H-Y402H polymorphic variants were used to identify alleles of the CFH gene, resulting in low or null expression of full-length fH and also normal or increased expression of the alternative splice product factor H-like-1 (FHL-1). Our assay identified three Y402H heterozygotes with low or absent fH-H402 but normal or increased FHL-1-H402 levels in a cohort of affected patients. Novel mutations explained the null phenotype in two cases, which was confirmed by family studies in one. In the third case, family studies showed that a known mutation was present on the Y allele. The cause of reduced expression of the H allele was not found, although the data suggested altered splicing. In each family, inheritance of low expression or null alleles for fH strongly associated with aHUS. Thus, our assays provide a rapid means to identify fH expression defects without resorting to gene sequencing or expression analysis.

Impaired binding of the age-related macular degeneration-associated complement factor H 402H allotype to Bruch's membrane in human retina.

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

Elevated plasma C4a levels in multiple sclerosis correlate with disease activity.

Complement plays a pivotal role in the pathogenesis of multiple sclerosis. C4a, an activated fragment of complement component C4, has been linked to disease activity. We correlated plasma C4 and plasma and CSF C4a with clinical disease in a well-characterised cohort of patients and controls. Plasma C4 was non-significantly and CSF C4a was significantly elevated overall in patients compared to controls. Plasma C4a was raised only in acute relapse, decreasing over 2 months. Results demonstrate intrathecal and systemic activation of complement, reflected in changes in CSF and plasma C4a. The data support a role for complement activation in pathogenesis and suggest a systemic component to the disease.

Complement regulator factor H as a serum biomarker of multiple sclerosis disease state.

Multiple sclerosis has a variable phenotypic presentation and subsequent disease course that, although unpredictable at disease onset, is of crucial importance in guiding interventions. Effective and accessible biomarkers are required in order to stratify patients and inform treatment. We examined whether the complement regulator factor H and its Tyr402His polymorphism, recently implicated as biomarkers in other chronic inflammatory central nervous system conditions, might identify or predict specific pathological processes and outcomes in multiple sclerosis. Employing novel assays, we measured factor H and its His402 variant in serum from 350 patients with multiple sclerosis classified according to disease course and relapse status. Serum factor H levels were significantly higher in progressive disease (P < 0.001) compared to controls and relapsing patients, after controlling for variables including disease duration, age, gender, disability and treatment. Serum factor H levels were capable of distinguishing secondary progressive from relapsing remitting disease (excluding patients in clinical relapse) with a sensitivity of 89.41%, specificity of 69.47% and a positive predictive value of 72.38%. Acute relapse was also associated with transiently increased factor H levels (P = 0.009) compared to stable relapsing disease. In clinically stable patients, factor H levels remained constant over 1 year (coefficient of variation percentage = 6.8), however, in patients in transition from relapsing to progressive disease, factor H levels significantly increased over a period of 2 years (P = 0.007). Concentration of the His402 variant in heterozytgotes was significantly higher in secondary progressive (P < 0.01) and primary progressive (P < 0.05) disease, suggesting altered expression or consumption of variants when factor H is upregulated. Serum factor H may be an effective indicator of progression and a practical and accessible biomarker and stratifying tool in determining disease course, providing objective evidence to help guide therapeutic decisions.

Complement factor H binds to denatured rather than to native pentameric C-reactive protein.

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.