Bovine milk fat globules do not inhibit C5a chemotactic activity

Abstract

-The C5a complement fragment is a potent inflammatory molecule but its contribution to the inflammatory response of the mammary gland remains uncertain. One of the unresolved questions is the possible interference of whole milk with C5a. In this study, the chemotactic activity of purified bovine C5a was tested in the presence of whole or skimmed milk. Milk from healthy glands acted as a chemoattractant, which could mask any inhibitory effect on C5a activity. To circumvent milk activity, washed milk fat globules were incubated with optimal (1 nM) or suboptimal (0.1 nM) concentrations of C5a, and the eventual chemoattractant activity was measured. There was no reduction in C5a-induced migration through a polycarbonate filter or shape-change of neutrophils. The concentrations of C5a determined by ELISA in the fluid phase were not reduced after incubation with the fat globules. It can be concluded that bovine milk fat globules do not trap or degrade C5a. Although these results do not explain the inhibitory effect of whole milk in the C5a-induced recruitment of neutrophils in milk, they suggest that milk does not inhibit the second major activity of C5a (apart from chemotaxis), i.e. the stimulation of phagocytic and bactericidal activities of neutrophils. C5a / chemotactism / milk / fat globuleRésumé -Les globules gras du lait de vache n'inhibent pas l'activité chimiotactique du C5a. Le fragment du complément C5a est un puissant agent inflammatoire mais sa contribution à la réponse inflammatoire de la glande mammaire reste incertaine. Une des questions non résolues est l'interfé-rence possible du lait entier avec le C5a. Dans cette étude, l'activité chimiotactique du C5a bovin purifié a été évaluée en présence de lait entier ou écrémé. Le lait de glandes saines s'est révélé être un chimioattractant, ce qui pourrait masquer un éventuel effet inhibiteur sur le C5a. En vue de contourner l'activité du lait, des globules gras lavés ont été incubés avec des concentrations optimales (1 nM) ou suboptimales (0,1 nM) de C5a, et l'activité résultante a été mesurée. La migration à travers une membrane filtrante de polycarbonate ou la déformation des polynucléaires neutrophiles n'ont pas été diminuées. Les concentrations de C5a, mesurées par ELISA, n'ont pas été réduites après incubation 413 Vet. Res. 33 (2002) (33) 2 47 42 77 79; e-mail: rainard@tours.inra.fr avec les globules gras. Il peut en être conclu que les globules gras du lait ne capturent pas ou ne dégra-dent pas le C5a. Bien que ces résultats n'offrent pas d'explication à l'effet inhibiteur du lait entier sur le recrutement C5a-dépendant des neutrophiles dans le lait, ils suggèrent que le lait n'inhiberait pas l'autre activité majeure du C5a (en plus du chimiotactisme), à savoir la stimulation des fonctions phagocytaires et bactéricides des neutrophiles. C5a / chimiotactisme / lait / globules gras

The complement in milk and defense of the bovine mammary gland against infections

“…This result indicates that the components of whole milk inhibit the inflammatory activity of ZAP. One simple assumption is that the milk fat globules interfered with the inflammatory activity of C5a, but the investigations with bovine milk fat globules and purified bovine C5a showed that fat globules do not trap or inactivate C5a [84]. Whatever the underlying mechanism, the inhibition of the C5a recruiting activity by whole milk casts doubt on the contribution of the intrinsic milk complement in the initiation of the inflammatory reaction of the lactating mammary gland.…”

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection

“…They include increased mammary blood flow facilitating delivery of neutrophils, complement factors and chemotactic factors to the gland stabilization and attenuation of bacterial toxin-related apoptosis, counter-regulation of adverse proinflammatory cytokine release, and facilitated augmentation of growth factor production including IGF-1 [38,43,[45][46][47][48]. Exactly how AM affects the biological activity of AM-BP towards processing of complement C3 and the progression through the complement cascade towards complement C5-mediated chemotactic attraction of monocytes, granulocytes and neutrophils [49] to sites of mammary infection remains unknown.…”

“…Pio et al [17] demonstrated that the minimal inhibitory concentration of AM effective against E. coli shifted from 10 to more than 35 g/ml when AM was complexed with AM-BP. Interestingly, the activity of AM-BP as complement factor-H (responsible cofactor for the factor I-dependant cleaving of complement C3B) is increased when AM is bound [17] suggesting that aspects of the innate complement cascade within the mammary gland [49] are also modulated by AM. Furthermore, increased beneficial clinical outcome (decreased morbidity and mortality) recently has been attributed to the use of an AM/AM-BP complex as an intervention strategy to limit the complicating effects of sepsis and hypovolumic shock on progression into multiple organ failure [19,[42][43][44].…”

Molecular Aspects of Neutrophils as Pivotal Circulating Cellular Innate Immune Systems to Protect Mammary Gland from Pathogens

“…The issue of blood and postdiapedetic PMN functions and concentrations in different physiological and pathological conditions remains the focus of most concern. Although the presence of strong chemotactic factors in non-inflamed udder is the subject of debate, their presence in inflammatory environment of udder and milk is indisputable (Manlongat et al, 1998;Rainard, 2002;Stevens et al, 2011a;2011b;Pezeshki et al, 2011). Most inflammatory chemoattractants are only induced and released during acute infection.…”

“…Neutrophils engage microorganisms through extracellular membrane receptors like PRRs, e.g., TLRs, C-type lectins (mannose receptor), scavenger receptors like CD36, Nod-like receptors (NLRs), and N-formyl Met-Leu-Phe (f-MLP) receptors (Nathan., 2006;Diez-Fraille et al, 2004;Bellocchio et al, 2004;Sohn et al, 2007a;2007b ;Zarbock A & Ley, 2008;Rainard, 2002;Stevens et al, 2011a;2011b). Neutrophils also bind to pathogens coated with various opsonins like IgG, complement components (e.g.…”