In vitro complement binding on cytoplasmic structures in normal human skin: immunoelectronmicroscopic studies.

We have previously provided evidence that suggests that exposure of cryostat skin sections to normal human serum (NHS) results in the antibody-independent Clq binding to cytoplasmic structures of various cell types, leading to classical complement pathway activation as evidenced by cytoplasmic C3 deposition. In the present study, we have employed immunoelectronmicroscopic methods to clarify the exact nature of cytoplasmic C3 binding structures. Incubation of cryostat skin sections with NHS followed by peroxidase-labeled rabbit anti-human C3 serum (HRP-R/Hu C3) revealed that intracytoplasmic binding of C3 occurred in suprabasal keratinocytes, melanocytes, fibroblasts, smooth muscle cells, endothelial cells, pericytes, Schwann cells, and nerve axons, but not in basal keratinocytes, Langerhans cells, and other cellular constituents of the skin. C3 binding, as revealed by the deposition of HRP reaction product, was exclusively confined to intermediate-sized filaments (ISF), which can therefore be considered to represent the subcellular site for classical complement pathway activation. Under experimental conditions that do not allow classical complement pathway activation, ISF were not decorated. Our observation that ISF of ontogenetically different cell types share the capacity of complement fixation is in accordance with the recent finding that different ISF types, despite their biochemical and antigenic heterogeneity, have common alpha-helical domains and may provide a clue to the mechanism and site of interaction between complement components and ISF.

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Activation of the Classical Pathway of Complement by Resting and Shear Stress-Stimulated Human Endothelial Cells.

Blood ◽

10.1182/blood.v106.11.2664.2664 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2664-2664

Author(s):

Wei Yin ◽

Babette Weksler ◽

David Varon ◽

Naphtali Savion ◽

Berhane Ghebrehiwet ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Complement Activation ◽

Binding Domain ◽

Complement Pathway ◽

Complement Components ◽

Classical Complement Pathway ◽

Pathway Activation ◽

Static Conditions ◽

Classical Complement

Abstract Complement activation is associated with a variety of inflammatory conditions including atherosclerosis, but the mechanism of complement activation in these settings is poorly understood. Endothelial cells (EC) play an important role in vascular pathology and express a variety of complement receptors, including gC1qR/p33, recognizing the globular domain of the complement component C1q. In preliminary studies, purified recombinant gC1qR/p33 was found to support C1q-dependent C4 activation in vitro, comprising 19.5% ± 8.3% (mean ± S.D., n=5) of that produced by aggregated IgG. In contrast, a truncated form of gC1qR/p33, lacking the C1q binding domain, failed to support C4 activation. Additional studies were performed with immortalized bone marrow microvascular EC to investigate classical complement pathway activation and deposition. EC were exposed to anticoagulated (0.32 % sodium citrate) human plasma, diluted (1/10) in 0.01 M HEPES buffered modified Tyrode’s solution, pH 7.5, containing 2 mM Mg Cl2 and 1 mM CaCl2, for 60 min, 37°C. A solid phase ELISA approach was used to detect EC-associated C1q and C4 activation (C4d). Statistically significant deposition of C4d (0.72 ± 0.3. OD units (ODU), n=4)(p=0.04) and C1q (0.57 ± 0.19. ODU, n=4) (p=0.002) was observed on EC that had been immobilized on poly-L-lysine coated microtiter wells. Consistent with classical complement pathway activation, C4d deposition remained at baseline (0.23 ± 0.13, ODU, n=4) in the presence of 10 mM EDTA, but C1q deposition was unaffected. Moreover, no significant C1q or C4d deposition occurred when endothelial cells were exposed to C1q depleted serum. Similar studies were performed using EC grown to confluence on Type I collagen to examine the effect of shear stress (12 dynes/cm2 for 1 hour in a cone-and-plate shearing device), simulating flow conditions in coronary arteries, on classical complement pathway activation and deposition. Compared to static conditions, shear stress resulted in an approximately 50% increase in C1q and C4d deposition on EC. This was accompanied by an approximately 2-fold increase in EC binding of a monoclonal antibody, 60.11, recognizing the N-terminal C1q binding domain of gC1qR/p33. Taken together, these data present evidence for a potential paradigm shift, illustrating immune complex independent classical complement pathway activation by gC1qR/p33, and deposition of activated classical complement components on EC. The generation and deposition of active complement components on EC is likely to contribute directly to vascular inflammation and atherosclerotic changes.

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Heptose I Glycan Substitutions on Neisseria gonorrhoeae Lipooligosaccharide Influence C4b-Binding Protein Binding and Serum Resistance

Infection and Immunity ◽

10.1128/iai.01109-06 ◽

2007 ◽

Vol 75 (8) ◽

pp. 4071-4081 ◽

Cited By ~ 20

Author(s):

Sanjay Ram ◽

Jutamas Ngampasutadol ◽

Andrew D. Cox ◽

Anna M. Blom ◽

Lisa A. Lewis ◽

...

Keyword(s):

Binding Protein ◽

Alternative Pathway ◽

Serum Resistance ◽

Classical Pathway ◽

Complement Pathway ◽

Classical Complement Pathway ◽

Pathway Activation ◽

Normal Human ◽

Classical Complement ◽

Strain Background

ABSTRACT Lipooligosaccharide (LOS) heptose (Hep) glycan substitutions influence gonococcal serum resistance. Several gonococcal strains bind the classical complement pathway inhibitor, C4b-binding protein (C4BP), via their porin (Por) molecule to escape complement-dependent killing by normal human serum (NHS). We show that the proximal glucose (Glc) on HepI is required for C4BP binding to Por1B-bearing gonococcal strains MS11 and 1291 but not to FA19 (Por1A). The presence of only the proximal Glc on HepI (lgtE mutant) permitted maximal C4BP binding to MS11 but not to 1291. Replacing 1291 lgtE Por with MS11 Por increased C4BP binding to levels that paralleled MS11 lgtE, suggesting that replacement of the Por1B molecule dictated the effects of HepI glycans on C4BP binding. The remainder of the strain background did not affect C4BP binding; replacing the Por of strain F62 with MS11 Por (F62 PorMS11) and truncating HepI mirrored the findings in the MS11 background. C4BP binding correlated with resistance to killing by NHS in most instances. F62 PorMS11 and its lgtE mutant were sensitive to NHS despite binding C4BP, secondary to kinetically overwhelming classical pathway activation and possibly increased alternative pathway activation (measured by factor Bb binding) by the F62 background. FA19 lgtF (HepI unsubstituted) resisted killing by only 10% NHS, not 50% NHS, despite binding levels of C4BP similar to those of FA19 and FA19 lgtE (both resistant to 50% serum), suggesting a role for the proximal Glc in serum resistance independently of C4BP binding. This study provides mechanistic insights into how HepI LOS substitutions affect the serum resistance of N. gonorrhoeae.

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Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2016.11.015 ◽

2017 ◽

Vol 140 (1) ◽

pp. 89-100.e2 ◽

Cited By ~ 11

Author(s):

Griet A. Van Roey ◽

Christopher C. Vanison ◽

Jeffanie Wu ◽

Julia H. Huang ◽

Lydia A. Suh ◽

...

Keyword(s):

Chronic Rhinosinusitis ◽

Complement Pathway ◽

Classical Complement Pathway ◽

Nasal Tissue ◽

Pathway Activation ◽

Classical Complement

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The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

Infection and Immunity ◽

10.1128/iai.73.4.2400-2410.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2400-2410 ◽

Cited By ~ 45

Author(s):

Ahmed S. Attia ◽

Eric R. Lafontaine ◽

Jo L. Latimer ◽

Christoph Aebi ◽

George A. Syrogiannopoulos ◽

...

Keyword(s):

Human Serum ◽

Resistant Strain ◽

Moraxella Catarrhalis ◽

Normal Human Serum ◽

Serum Resistance ◽

Complement Pathway ◽

Classical Complement Pathway ◽

Amino Acid Region ◽

Normal Human ◽

Classical Complement

ABSTRACT Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.

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Inefficient and abortive classical complement pathway activation by the calcium inositol hexakisphosphate component of the Echinococcus granulosus laminated layer

Immunobiology ◽

10.1016/j.imbio.2019.05.009 ◽

2019 ◽

Vol 224 (5) ◽

pp. 710-719 ◽

Cited By ~ 2

Author(s):

Anabella A. Barrios ◽

Leticia Grezzi ◽

Sebastián Miles ◽

Mara Mariconti ◽

Gustavo Mourglia-Ettlin ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Complement Pathway ◽

Inositol Hexakisphosphate ◽

Classical Complement Pathway ◽

Pathway Activation ◽

Laminated Layer ◽

Classical Complement

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Continual Low-Level Activation of the Classical Complement Pathway

Journal of Experimental Medicine ◽

10.1084/jem.194.6.747 ◽

2001 ◽

Vol 194 (6) ◽

pp. 747-756 ◽

Cited By ~ 43

Author(s):

Anthony P. Manderson ◽

Matthew C. Pickering ◽

Marina Botto ◽

Mark J. Walport ◽

Christopher R. Parish

Keyword(s):

Complement Activation ◽

Alternative Pathway ◽

Blood Stasis ◽

Classical Pathway ◽

Complement Pathway ◽

Complement Components ◽

Classical Complement Pathway ◽

Alternative Complement ◽

Classical Complement

There is evidence that the classical complement pathway may be activated via a “C1-tickover” mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.

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Influence of β-Glucan (Macrogard®) on Innate Immunity of Carp Fry

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2013-0039 ◽

2013 ◽

Vol 57 (2) ◽

pp. 219-223 ◽

Cited By ~ 7

Author(s):

Ganna Sych ◽

Patrick Frost ◽

Ilgiz Irnazarow

Keyword(s):

Protein Content ◽

Total Protein ◽

Lysozyme Activity ◽

Whole Body ◽

Total Protein Content ◽

Complement Pathway ◽

Classical Complement Pathway ◽

Pathway Activation ◽

Experimental Feeding ◽

Classical Complement

Abstract β-glucan (Macrogard®) was administrated to enhance the immunity and growth of Cyprinus carpio fry. The whole body homogenate of fish sampled with one week intervals during 2nd - 6th weeks post hatching was assayed for the total protein content, lysozyme activity, α-2-macroglobulin content, classical complement pathway activation, and weight gain. After the 3rd week of experimental feeding, the total protein content and the classical complement activity of fish fed β-glucan supplemented diet were higher than controls. Significantly higher lysozyme activity and α-2-macroglobulin levels were noted in group fed β-glucan at the 2nd and 3rd week of diet application. It was demonstrated that β-glucan enriched feeding increased the immunity of common carp at the earliest stages of their development.

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