J Vet Sci.  2012 Mar;13(1):33-38. 10.4142/jvs.2012.13.1.33.

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes

• Dec M
• Wernicki A
• Puchalski
• Urban-Chmiel R
• Wasko A

• KMID: 1365000
• DOI: http://doi.org/10.4142/jvs.2012.13.1.33

Abstract

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca(2+)-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H2O2 in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.

Keyword

conglutinin; reactive oxygen species

MeSH Terms

Animals
Cattle/*immunology
Collectins/*pharmacology
Enzyme-Linked Immunosorbent Assay/veterinary
Female
Granulocytes/*drug effects/immunology
Hydrogen Peroxide/immunology
Immunity, Innate/drug effects/immunology
Phagocytosis/immunology
Reactive Oxygen Species/*immunology
Respiratory Burst/*drug effects/immunology
Serum Globulins/*pharmacology
Statistics, Nonparametric
Superoxides/immunology

Figure

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  Fig. 1 Conglutinin purified from bovine serum using a two-step chromatographic method; (A) SDS-PAGE analysis of non-reduced conglutinin in 3~20% polyacrylamide gel stained with Coomassie brilliant blue R-250, (B) Western blotting and (S) molecular weight standard.

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  Fig. 2 The effect of mannan-bound conglutinin (2.5 µg/well) coated onto microplate on superoxide production by bovine leukocytes as measured by a nitroblue tetrazdium (NBT) assay. Control wells contained neither mannan nor conglutinin, and positive control wells contained PMA (200 ng/mL). The results are presented as mean ± SD for four independent experiments, each done in sextuplicate. PMA: phorbol myristate acetate. *p < 0.05 vs. the control.

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  Fig. 3 The effect of conglutinin on superoxide production by bovine leukocytes as measured by an NBT assay. Different concentrations of bovine conglutinin were added to a cell suspension in plate wells. Control wells received HBSS instead of conglutinin, and positive control wells contained PMA (200 ng/mL). The results are presented as mean ± SD for four independent experiments, each done in sextuplicate. *p < 0.05 vs. the control.

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  Fig. 4 The effect of mannan-bound conglutinin (2.5 µg/well) coated onto microplate wells on H2O2 production by bovine leukocytes determined by the peroxidase-dependent oxidation of phenol red. Control wells contained neither mannan nor conglutinin, and positive control wells contained PMA (200 ng/mL). The results are presented as mean ± SD for four independent experiments, each done in quintuplicate. *p < 0.05 vs. the control.

• 

  Fig. 5 The effect of conglutinin on H2O2 production by bovine peripheral polymorphonuclear leukocytes measured by the peroxidase-dependent oxidation of phenol red. Different concentrations of bovine conglutinin were added to a cell suspension in plate wells. Control wells received HBSS instead of conglutinin, and positive control wells contained PMA (200 ng/mL). The results are presented as mean ± SD for four independent experiments, each done in quintuplicate. *p < 0.05 vs. the control.

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