Anti-inflammatory effects of orally ingested lactoferrin and glycine in different zymosan-induced inflammation models: Evidence for synergistic activity☆
Introduction
There is a growing awareness of the interaction of food constituents with the immune system [1]. The present study aims to evaluate the anti-inflammatory effects of two of these nutritional components, i.e. the amino acid glycine and the iron-binding protein lactoferrin [2], [3].
The simple nonessential amino acid glycine is an inhibitory neurotransmitter in the central nervous system that acts via a glycine-gated chloride channel (GlyR) [4]. Apart from the nervous tissue, glycine has been presumed to be biological neutral for a long time. In the past years however, evidence accumulated indicating that glycine comprises anti-inflammatory and immunomodulatory activities, at least in part, via activation of the GlyR. The existence of a GlyR has been demonstrated on a wide variety of cells including different cell types involved in immune responses, such as macrophages, monocytes, neutrophils and T lymphocytes [3], [5], [6]. Activation of the GlyR blunts calcium ion influxes in these cells via a chloride induced hyperpolarization of the membrane [7]. Counteraction of the calcium ion influxes, which could be induced by many different stimuli, sabotages various downstream events including the production of cytokines and other inflammatory mediators. It has been demonstrated that glycine largely prevents the endotoxin-induced TNF-α production by Kupffer cells and alveolar macrophages [5], [8]. In addition, glycine reduces the lipopolysaccharide (LPS)-induced TNF-α and IL-1β expression while it stimulates the IL-10 expression in monocytes [9].
Lactoferrin (LF) is a widespread iron-binding protein and member of the transferrin family. It is produced by exocrine glands and might be released by degranulating neutrophils at the side of infection and inflammation. Iron binding is, without any doubt, a key property of LF that accounts for many of its biological roles in host defense such as bacteriostasis and protection against oxygen radicals catalyzed by free iron. Other direct effects of LF on host defense include the binding to bacteria, fungi and parasites. In addition, it has been demonstrated that LF plays a role in modulating immune responses by its ability to interact with target molecules and cells. Anti-inflammatory effects of LF have been shown by the inhibition of pro-inflammatory cytokine production [10], [11], [12] and the up regulation of anti-inflammatory cytokines [13]. On the other hand, LF may enhance directly or indirectly the immune response (in vitro and in vivo) by regulating the proliferation, differentiation and activation of both T and B cells [14], [15].
Both glycine as well as LF can modulate innate immune reactions which might offer a new opportunity for the treatment of chronic inflammatory diseases. The present study was designed to evaluate the anti-inflammatory properties in more detail. The objective was bipartite. 1) Literature describes an immune stimulatory as well as an immune inhibitory potential for LF. Therefore, the first goal of the study was to evaluate the immunomodulatory activities of orally ingested glycine and b-LF in different models of inflammation. The models used are the zymosan-induced ear–skin inflammation model and the zymosan-induced acute arthritis model. 2) The interaction between different pharmaceutical immunomodulatory medications has been studied extensively. However, the possibility that different food components might boost or counteract their individual effect on the immune system is rarely examined. The second part of the study addresses the question whether the immunomodulatory effects of glycine and LF interact with each other. For this latter goal the ear–skin inflammation model was used. The results indicated that both glycine as well as b-LF were able to significantly inhibit inflammatory responses. Furthermore, the combination of the two nutritional components showed a synergistic anti-inflammatory effect which opens new avenues for the treatment of chronic inflammatory diseases.
Section snippets
Materials and methods
All experimental procedures using laboratory animals were approved by an independent animal experiments committee (DEC Consult, Bilthoven, The Netherlands). The b-LF, with an iron saturation of 16%, was obtained from DMV (DMV International, Veghel, The Netherlands).
Ear(skin)-inflammation
To investigate the effect of glycine and b-LF on the zymosan-induced ear swelling in mice, both components were administered orally for three days. At the second day zymosan (or PBS) was injected into both ears intradermally. Ear thickness was measured before and at different time-points after injection. The detected ear swelling peaked around 6 h after injection and then slowly declined. The swelling of the ears of the zymosan injected, vehicle treated animals at 6 h (generally the maximal
Discussion
The present study demonstrated that a combination of two, orally administered, nutritional components; glycine and bovine-lactoferrin, displayed a synergistic anti-inflammatory activity. The effects of glycine on the zymosan-induced ear swelling were concentration dependent whereas only the low doses of b-LF were able to inhibit the swelling. Furthermore, both components diminished: 1) the zymosan-induced increase of inflammatory cytokines in the ear and 2) the zymosan-induced increase of TNF-α
Acknowledgments
The authors wish to thank Joyce Faber, Karen Knipping, Marije Kleinjan and Johanne Groothuismink for their technical assistance, as well as Dr. L.R. Verdooren for his advice concerning the statistical data analysis.
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This study was supported by a SenterNovem grant, TSIN1055, the Dutch ministry of economic affairs.