Inflammatory Effect of Cleaved Bovine Lactoferrin by Elastase on Staphylococcal Mastitis

Abstract

ABSTRACT. Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFκB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

Lactoferrin: Balancing Ups and Downs of Inflammation Due to Microbial Infections

“…Thus, this approach may contribute to decrease the effects of inflammation on tissue damage and also to reduce the antibiotic dosage for the eradication of staphylococcal infection by multiresistant strains. Inflammatory response in staphylococcal mastitis seems to be correlated with the elicitation of peptides derived from bLf-elastase proteolysis that display low concanavalin A and low iron-binding affinities, as well as antibacterial properties, but induce the expression of pro-inflammatory cytokines IL-8 and TNFα leading to neutrophil infiltration [122,123]. Like staphylococcal mastitis, elicitation of low ConA affinity Lf-peptide derivatives in parotid saliva was correlated with the severity of symptoms of periodontitis patients [186].…”

Single Intramammary Infusion of Recombinant Bovine Interleukin-8 at Dry-Off Induces the Prolonged Secretion of Leukocyte Elastase, Inflammatory Lactoferrin-Derived Peptides, and Interleukin-8 in Dairy Cows

“…This recruitment of PMNL, especially neutrophils, is expected to induce further events. Neutrophils release both Lf [19] and proteases such as elastase [20, 21], resulting in the production of LDP [22]. Inflammatory LDP, especially peptides containing the GQRDLLFKDSAL sequence, such as the 22 and 23 kDa peptides, act on mammary epithelial cells to enhance IL-8 gene expression [22] and production.…”

“…The elastase activity in the whey sample was detected by zymography, in duplicate, according to the method described by Komine et al [22], with human leukocyte elastase (Sigma-Aldrich, St. Louis, MO, USA) used as a standard.…”

“…The concentrations of Lf and LDP were determined by quantitative western blot analyses using bovine Lf (Sigma-Aldrich) as a standard, in duplicate, according to the previously reported method [22, 34]. Briefly, skimmed-milk samples were separated by SDS-PAGE (13.5% polyacrylamide gel) and then blotted onto a polyvinylidene difluoride membrane.…”

“…The latter produce inflammatory Lf-derived peptides (LDP) including 22 and 23 kDa peptides [22]. Inflammatory LDP containing the GQRDLLFKDSAL sequence, such as the 22 and 23 kDa peptides, induce IL-8 gene expression in bovine mammary epithelial cells [22], which are major sources of IL-8 production in the mastitic mammary gland [23, 24]. Therefore, it is suggested that a positive amplification loop for IL-8 production, involving the sequential release of Lf and proteases from activated neutrophils by IL-8, exists in cattle.…”