Fusion of the Fc part of human IgG1 to CD14 enhances its binding to Gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils

Vida, András; Bardoel, Bart W.; Milder, Fin J.; Majoros, László; Sümegi, Andrea; Bácsi, Attila; Vereb, György; Kessel, Kok P. M. van; Strijp, Jos A. G. van; Antal‐Szalmás, Péter

doi:10.1016/j.imlet.2012.04.008

Cited by 2 publications

(3 citation statements)

References 34 publications

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“…Additionally, the mechanisms involved in delivery of these ligands to multiple Toll-like receptor complexes are also unresolved. To address this gap in knowledge, many groups have purified various recombinant forms of soluble CD14 using bacteria, yeast, insect, and human cellular expression systems (12, 41-51), often with the goal of structure determination (49, 50). Currently, only the unliganded crystal structure of mouse CD14, purified from SF9 insect cells, is known (52).…”

Section: Introductionmentioning

confidence: 99%

The Crystal Structure of Human Soluble CD14 Reveals a Bent Solenoid with a Hydrophobic Amino-Terminal Pocket

Kelley

Lukk

Nair

et al. 2013

The Journal of Immunology

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Human monocyte differentiation antigen CD14 is a pattern recognition receptor that enhances innate immune responses to infection by sensitizing host cells to bacterial lipopolysaccharide (LPS; endotoxin), lipoproteins, lipoteichoic acid and other acylated microbial products. CD14 physically delivers these lipidated microbial products to various Toll-like receptor signaling complexes that subsequently induce intracellular proinflammatory signaling cascades upon ligand binding. The ensuing cellular responses are usually protective to the host, but can also result in host fatality through sepsis. In this work, we have determined the X-ray crystal structure of human CD14. The structure reveals a bent solenoid typical of leucine rich repeat proteins with an amino terminal pocket that presumably binds acylated ligands including LPS. Comparison of human and mouse CD14 structures show great similarity in overall protein fold. However, compared to mouse CD14, human CD14 contains an expanded pocket and alternative rim residues that are likely to be important for LPS binding and cell activation. The X-ray crystal structure of human CD14 presented herein may foster additional ligand bound structural studies, virtual docking studies, and drug design efforts to mitigate LPS induced sepsis and other inflammatory diseases.

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Section: Introductionmentioning

confidence: 99%

The Crystal Structure of Human Soluble CD14 Reveals a Bent Solenoid with a Hydrophobic Amino-Terminal Pocket

Kelley

Lukk

Nair

et al. 2013

The Journal of Immunology

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“…The use of flow cytometry to assess granulocyte, phagocytosis, and oxidative burst was first described in 1994 as a two‐color experiment involving isolated polymorphonuclear (PMN) cells and labeled bacteria . Since this initial description, the use of flow cytometry to assess granulocyte function has become routine . While flow cytometry techniques are commonly used in a variety of disease models, the information that can be obtained is limited by current flow cytometer technology.…”

Section: Statement Of Purposementioning

confidence: 99%

“…Studies that have used a series of assay incubations are able to better describe the time course of observed changes in granulocyte function . A final limitation of existing methods is the reliance on forward scatter (FSC) and side scatter (SSC) to identify granulocytes , rather than using definitive cell‐surface markers like CD66b and/or CD45. The one drawback associated with the use of immunophenotypic markers is that their expression may be altered during granulocyte activation; however, definitive markers allow more accurate ID of cell types, ensuring that the granulocyte population is free of monocytes or other phagocytic leukocytes.…”

Section: Statement Of Purposementioning

confidence: 99%

Image‐based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst

et al. 2013

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Granulocytes play a key role in innate immunity and the most common functional assays are phagocytosis and oxidative burst. The purpose of this technical note is to use imagebased flow cytometry to divide activated granulocytes into unique subsets based on their degree of phagocytosis and oxidative burst in response to different experimental incubations. Prior to the experiments, all reagents were titered to determine the lowest dose that resulted in an acceptable signal to noise ratio. Heparinized, whole blood (100 ml) was mixed with one of two bioparticles (E. coli and S. aureus) and DHE (10 mg/ml) and incubated for 5, 10, 20, 40, 60, 80, 100, 120, and 140 min in a 37 C water bath. An additional tube kept on ice was used as a negative control. All subsequent processing steps were completed on ice in the dark to minimize additional activation of cells. After the 37 C incubation, N-ethylmaleimide (15 mM) was added to halt phagocytosis, preventing the uptake of additional microparticles. Suspensions were labeled with CD66b-APC and CD45-APCeFluor780 for 60 min and a fix/lyse solution was added. Prior to acquisition, 7AAD was added to stain nuclear DNA. A minimum of 5,000 granulocyte (CD66b1) events were acquired using a Millipore-Amnis FlowSight equipped with blue (488 nm, 60 mW), red (642 nm, 100 mW), and side scatter (785 nm, 12 mW) lasers. Samples were compensated and analyzed using Amnis IDEAS software (v.5.0.983.0). Image-based analysis allowed us to divide activated granulocytes into three distinct subsets, whose relative abundance changed as a function of both bioparticle type and incubation length. The method described in this technical note represents a potential novel adaptation to common methods of assessing granulocyte function. More research is needed to test and validate our image-based method in clinical conditions that impair granulocyte function. V C 2013 International Society for Advancement of Cytometry

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Fusion of the Fc part of human IgG1 to CD14 enhances its binding to Gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils

Cited by 2 publications

References 34 publications

The Crystal Structure of Human Soluble CD14 Reveals a Bent Solenoid with a Hydrophobic Amino-Terminal Pocket

The Crystal Structure of Human Soluble CD14 Reveals a Bent Solenoid with a Hydrophobic Amino-Terminal Pocket

Image‐based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst

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