Commercially Available Complement Component-Depleted Sera Are Unexpectedly Codepleted of Ficolin-2

Brady, Allison M.; Geno, K. Aaron; Dalecki, Alex G.; Cheng, Xiaogang; Nahm, Moon H.

doi:10.1128/cvi.00370-14

Cited by 9 publications

(14 citation statements)

References 30 publications

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“…In line with our observations, C1q-mediated enhancement of efferocytosis was previously shown to be independent on the quantity of C1q bound to dying cells [41]. However, as C1q depletion from serum was shown to also affect the levels of other factors involved in AC clearance such as properdin, Factor D [15] and ficolin-2 [31], we cannot exclude that lack of one or more of these other factors may have partly affected phagocytosis in depleted serum.…”

Section: Discussionsupporting

confidence: 88%

“…Physiologic clearance of apoptotic cells takes place very rapidly [28,29], and dead cell accumulation occurs only under certain pathogenic conditions [30]. While efforts have been made in vitro to dissect the relative importance of C1q from downstream complement components, artificial depletion of individual complement components from normal sera has been shown to cause reduction of other serum factors [15,31], and serum obtained from patients with complement deficiencies usually has elevated cytokines and autoantibodies that may confound interpretation of the results [32,33]. We reasoned that specific inhibition of enzymatic C1s activity would be expected to leave C1q binding to AC unaffected, while blocking classical pathway-mediated activation of C3.…”

Section: Introductionmentioning

confidence: 99%

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Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Colonna

Parry²,

Panicker³

et al. 2016

Clinical Immunology

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Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Colonna

Parry²,

Panicker³

et al. 2016

Clinical Immunology

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“…We and others have reported the specific inhibition of ficolin-2 by the coating in plastic serum collection tubes (Brady et al, 2014c; Hein et al, 2013), and others have observed the depletion of ficolin-2 by heparin-treated cardio bypass circuits (Hein et al, 2015). Ficolin-2 was observed to bind to derivatized Sepharose (Kilpatrick & Chalmers, 2012), and commercial complement component-depleted sera are deplete of ficolin-2 as well (Brady et al, 2014b). Ficolin-2 studies therefore require careful consideration of various analytical factors.…”

Section: Discussionmentioning

confidence: 99%

“…Serum samples (3 µl per lane) were assayed for ficolin-2 by SDS-12%PAGE as previously described (Brady et al, 2014b). …”

Section: Methodsmentioning

confidence: 99%

Ficolin-2 inhibitors are present in sera after prolonged storage at −80 °C

Geno

Kennedy

Sawyer

et al. 2016

PeerJ

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Ficolins can activate the lectin pathway of the complement system that provides innate immune protection against pathogens, marks host cellular debris for clearance, and promotes inflammation. Baseline inflammation increases with aging in a phenomenon known as “inflammaging.” Although IL-6 and C-reactive protein are known to increase with age, contributions of many complement factors, including ficolins, to inflammaging have been little studied. Ficolin-2 is abundant in human serum and can recognize many target structures; therefore, ficolin-2 has potential to contribute to inflammaging. We hypothesized that inflammaging would alter ficolin-2 levels among older adults and examined 360 archived sera collected from older individuals. We found that these sera had apparently reduced ficolin-2 levels and that 84.2% of archived sera exhibited ficolin-2 inhibitors, which suppressed apparent amounts of ficolin-2 detected by enzyme-linked immunosorbent assay. Fresh serum samples were obtained from donors whose archived sera showed inhibitors, but the fresh sera did not have ficolin-2 inhibitors. Ficolin-2 inhibitors were present in other long-stored sera from younger persons. Furthermore, noninhibiting samples and fresh sera from older adults had apparently normal amounts of ficolin-2. Thus, ficolin-2 inhibitors may arise as an artifact of long-term storage of serum at −80 °C.

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“…BC069825 ) in pcDNA3.1(−) was described previously (Brady et al, 2014a; Brady et al, 2014b). All cell culture was performed in a humidified 37°C incubator with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Rapid and efficient purification of ficolin-2 using a disposable CELLine bioreactor

Geno

Spencer

Nahm

2015

Journal of Immunological Methods

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The human opsonin ficolin-2 (L-ficolin) is an innate pattern-recognizing molecule that binds to acetylated moieties. Upon binding, ficolin-2 activates complement through the lectin pathway, opsonizing the target to promote phagocytic clearance. Ficolin-2 has been found to interact with a growing number of pathogenic bacteria, fungi, and viruses. Ficolin-2 also has proposed roles in host homeostasis, including the clearance of apoptotic cells. Consequently, there is an increased interest in studying ficolin-2, and access to purified ficolin-2 is necessary for these studies. Ficolin-2 purified from serum, plasma, or cell culture supernatants has been a useful tool in the characterization of ficolin-2 function; however, available protocols are laborious and inefficient, requiring additional processing of starting materials (e.g., polyethylene glycol precipitation or dialysis) and multiple steps of purification. Here, we investigated a simple solution to the problem: use of a simple, disposable bioreactor requiring only standard tissue culture equipment. Using this system, we generated cell culture supernatants containing high concentrations of recombinant ficolin-2, which permitted rapid purification of high-purity recombinant ficolin-2 without processing the supernatants. Purified recombinant ficolin-2 retained its binding capacity and supported complement activation in vitro. Bioreactor cultivation will likely be generally useful in the production of other recombinant proteins in the study of the complement system.

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Commercially Available Complement Component-Depleted Sera Are Unexpectedly Codepleted of Ficolin-2

Cited by 9 publications

References 30 publications

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Ficolin-2 inhibitors are present in sera after prolonged storage at −80 °C

Rapid and efficient purification of ficolin-2 using a disposable CELLine bioreactor

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