Transferrin-derived synthetic peptide induces highly conserved pro-inflammatory responses of macrophages
Introduction
Transferrins are a family of 75–80 kDa iron-binding proteins that are widely spread across many phyla (Taboy et al., 2001). The transferrin molecule is composed of two relatively homologous lobes (C-and N-lobe; ∼40% sequence identity), with a single iron-binding site in each lobe. Due to the highly conserved biological function of transferrin, it is not surprising that transferrins from diverse taxa are highly homologous. For example, the percent amino acid identity between mouse transferrin and that of goldfish, bovine and human molecules is 41, 63 and 72%, respectively, and that of transferrin homologue lactoferrin is 60% (Baker et al., 2001). Transferrin is synthesized mainly in the liver and also by macrophages (Djeha et al., 1992) suggesting a possible role for transferrin in host defense.
The central role of transferrin as an iron transporting protein has been extended by observations that modified versions of this protein also participate in the regulation of innate immunity. For example it has been reported that transferrin acts as an acute phase protein (Gabay and Kushner, 1999) and that it can create a bacteriostatic environment by sequestering free iron from invading pathogens (Ong et al., 2006). Transferrin fragments are frequently observed in bronchoalveolar lavage (BAL) of cystic fibrosis patients (Britigan et al., 1993), and in the supernatant of mitogen-activated macrophage cultures of lower vertebrates (Stafford and Belosevic, 2003) suggesting that transferrin fragments may act as danger signals, “warning” the immune system of the presence of pathogens or of tissue injury.
Recent evidence suggests that modified transferrin was involved in the induction of antimicrobial function of macrophages, since immunopurified transferrin fragments present in the supernatants of mitogen-stimulated goldfish leukocytes, induced significant production of reactive nitrogen intermediates in goldfish in vitro-derived macrophages (Stafford and Belosevic, 2003). Similarly, lactoferrin fragments, found in parotid saliva of periodontitis patients, induced the production of interleukin-6 (IL-6), monocytes chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) (Komine et al., 2007).
To exert the observed pro-inflammatory activity transferrin must be present at the inflammatory sites and it must be modified (cleaved) into fragments that are recognized by immune cells. Since transferrin is abundant in serum, (2–5 mg/mL of serum) (Regoeczi and Hatton, 1980) it is probable that during an ongoing inflammatory response and increased vascular permeability (Bryniarski et al., 2003), transferrin could reach and be subjected to proteolytic cleavage by both host and pathogens proteases released at inflammatory sites (Britigan and Edeker, 1991). Furthermore, transferrin and lactoferrin fragments are normal constituents of airway secretions (Thompson et al., 1990). For example, elevated levels of neutrophil-derived elastase are frequently observed in cystic fibrosis patients (Goldstein and Doring, 1986), suggesting that this enzyme may be involved in the modification of transferrin into immunostimulatory fragments.
We report that elastase can cleave transferrin of distantly related species (mouse, bovine, human N-lobe and fish) into immunostimulatory fragments. Mass spectrometry analysis of elastase-treated mouse apo-transferrin revealed the presence of a 31 amino acid long fragment containing six-cysteine residues resembling antimicrobial defensin proteins. This peptide was located in the N2 sub-domain of N-lobe, which corresponds to amino acids 174–204 of the full murine transferrin. The peptide was synthetically produced and shown to induce activation of different macrophage populations. Consequently, the synthetic peptide was named TMAP (transferrin macrophage-activating peptide).
TMAP induced a strong nitric oxide response in P388D1 murine macrophage cell line, mouse peritoneal macrophages, mouse bone marrow-derived macrophages (BMDM) and in vitro-derived goldfish macrophages. A cytokine microarray analysis revealed that TMAP also induced increased production of several pro-inflammatory cytokines and chemokines by macrophages, including TNF-α, IL-6, MCP-5, MIP-1α, MIP-1γ, MIP-2, G-CSF, KC, VEGF, and RANTES), suggesting an important regulatory role for this peptide in inflammation of lower and higher vertebrates.
Section snippets
Animals
Four-to-six week old C57BL/6 female mice were purchased from Charles River (Wilmington, MA) and maintained according to Canadian Council for Animal Care (CCAC) guidelines in filter-top cages in the Biological Sciences Animal Facility, University of Alberta.
The goldfish were obtained from Mt. Parnell Fisheries Inc. (Mercersburg, PA). The fish were maintained in the aquatic facility of the Department of Biological Sciences, University of Alberta. The goldfish were kept at 20 °C using a
Induction of macrophage activation using elastase-cleaved transferrin
The FPLC fractions generated using Superdex 75 size-exclusion chromatography of elastase-digested murine transferrin were tested for their ability to induce nitric oxide response in macrophage-like P388D1 cells. Fig. 1A shows an activity profile of fractions B8-C3 that induced a strong nitric oxide response in P388D1 cells, indicating the presence of transferrin fragments with immunostimulatory activity. Similarly, elastase-generated transferrin fragments from other species (bovine, human
Discussion
At the inflammatory site different enzymes are released by the immune cells including metalloproteases (macrophages) and serine proteases (neutrophils) that have been shown to act on both pathogens as well as host proteins (Bryniarski et al., 2003). Elastase was one enzyme characterized at the inflammatory site and was shown to be secreted by neutrophils early (first 24 h) of the inflammatory response (Britigan et al., 1993). Previous studies have shown that host elastase can cleave transferrin
Conclusion
The elastase-generated murine, bovine, human N-lobe and goldfish transferrin fragments were reported in this study to induce pro-inflammatory functions of macrophages suggesting that this may be a novel mechanism for control of inflammatory responses of macrophages. That transferrin-derived synthetic peptide TMAP characterized in this study, induced potent pro-inflammatory responses of different macrophage populations obtained from different host species, suggests that this macrophage
Acknowledgements
This work was supported by a grant from the Natural Sciences and Engineering Council of Canada (NSERC) to M.B. G.H. received Teaching Assistantship Award from the Department of Biological Sciences, University of Alberta. We wish to thank Dr. Ross MacGillivray University of British Columbia for providing human N-lobe of transferrin, and Dr. James Stafford University of Alberta, for helpful advice and critical review of the manuscript.
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