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2921.Full.Pdf Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells This information is current as of September 23, 2021. Bradley E. Britigan, Troy S. Lewis, Mari Waldschmidt, Michael L. McCormick and Arthur M. Krieg J Immunol 2001; 167:2921-2928; ; doi: 10.4049/jimmunol.167.5.2921 http://www.jimmunol.org/content/167/5/2921 Downloaded from References This article cites 90 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/167/5/2921.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells1 Bradley E. Britigan,2*†‡ Troy S. Lewis,* Mari Waldschmidt,† Michael L. McCormick,*†‡ and Arthur M. Krieg*†§¶ Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. Downloaded from The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge- charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve http://www.jimmunol.org/ to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection. The Journal of Immunology, 2001, 167: 2921–2928. acterial, but not eukaryotic, DNA contains a large num- These and other experimental data have suggested that immune ber of unmethylated CpG dinucleotides (1). When these stimulation by bacterial DNA may serve as an important signaling B unmethylated CpGs are in a particular base context mechanism to activate protective immune responses to invasion by (“CpG motifs”) they stimulate various innate and acquired immu- pathogenic bacteria (22). Given the potency of the response, the nological responses in murine and human systems (2). Among the role of bacterial DNA and CpG ODN as potential vaccine adju- effects of unmethylated CpG oligodeoxynucleotides (ODN)3 are: vants is being explored (3, 22). The biological effect of CpG ODN by guest on September 23, 2021 1) enhancement of Ag-specific Th1 responses including enhanced appears to involve binding and internalization of the molecule (23, macrophage and dendritic cell IL-12 production (3–7); 2) en- 24) by a process that is mediated via a Toll-like receptor-depen- hanced NK cell production of IFN-␥ (8); 3) activation of B cell dent pathway (25–27) and perhaps formation of reactive oxygen proliferation and IL-6 and Ig secretion (7); 4) stimulation of pro- species (28). tective immunity against intracellular pathogens (9–14); 5) inhi- Although there has been much interest in the immunostimu- bition of allergen-and LPS-induced airway inflammation (15–17); latory effects of bacterial DNA, there has been relatively little 6) induction of direct airway inflammation (18); and 7) stimulation attention paid to mechanisms whereby the host may limit and of immune responses to tumor Ags (19, 20). CpG-containing ODN thereby help to regulate such responses. At many mucosal sur- have also been shown to be able to induce a systemic inflammatory faces there is a normal resident microflora. Continued stimula- response syndrome in animals that is analogous to that resulting tion of the local immune system in response to the DNA of this from i.v. challenge with LPS (21). microflora could be deleterious and it would be expected that a mechanism would have evolved to limit such events. Similarly, *Research Service and Department of Internal Medicine, Veterans Affairs Medical at a local site of bacterial infection, it would likely be beneficial Center, Iowa City, IA 52246; †Department of Internal Medicine, ‡Free Radical Re- to the host to have a means of terminating CpG-mediated stim- search Program, Department of Radiology, and §Immunology Program, University of Iowa College of Medicine, Iowa City, IA 52242; and ¶Coley Pharmaceutical Group, ulation of the immune system as the infection comes under Wellesley, MA 02481 control. Received for publication April 2, 2001. Accepted for publication July 13, 2001. Lactoferrin is a highly cationic (isoelectric point (pI), 8.4–9.0) The costs of publication of this article were defrayed in part by the payment of page monomeric glycoprotein (76–80 kDa) that is found in high con- charges. This article must therefore be hereby marked advertisement in accordance centrations (1–10 mg/ml) at many mucosal surfaces and in milk with 18 U.S.C. Section 1734 solely to indicate this fact. (29–31). It is also a major constituent of neutrophil secondary 1 This work was supported by research grants from the Research Service of the De- (specific) granules (32, 33). It is secreted by local neutrophils and partment of Veterans Affairs (to B.E.B., M.L.M., and A.M.K.), National Institutes of Health Awards RO1AI34954 (to B.E.B.) and RO1CA66570 (to A.M.K.), and a grant is present at high levels at sites of bacterial infection (34, 35). from the Coley Pharmaceutical Group (to A.M.K.). Lactoferrin contains two high-affinity ferric iron binding sites and 2 Address correspondence and reprint requests to Dr. Bradley E. Britigan, Department is thought to function as host defense in part by sequestering iron of Internal Medicine, Division of Infectious Diseases, University of Iowa Hospitals from pathogenic microbes (36, 37). Lactoferrin is also thought to and Clinics, SW54, GH, Iowa City, IA 52242. E-mail address: bradley-britigan@ uiowa.edu serve as an antioxidant since iron bound to the protein is unable to 3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; pI, isoelectric point; participate as a catalyst for the generation of the hydroxyl radical MFI, mean fluorescence index. (38–40). Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2922 LACTOFERRIN INHIBITS CpG ODN IMMUNOSTIMULATORY EFFECTS Lactoferrin has antimicrobial activity that is independent of its conjunction with FACS analysis (62). In some experiments, polyclonal iron-binding activity (41–43). Lactoferrin binds to the outer mem- antihuman IgM (Sigma) was substituted for the ODN as an alternative brane of Gram-negative bacteria, leading to alterations in bacteria stimulus for Ramos cell CD86 expression. membrane permeability barriers, which in turn causes microbial cell injury and death (41, 44). This latter activity has been linked Cellular uptake of CpG ODN to the proximal N terminus of the molecule, termed lactoferricin, Ramos cells were incubated at 37°C with FITC-conjugated ODN in PBS, which contains a large number of arginine residues. These are after initial incubation of the ODN for 30 min in H2O alone or H2O that responsible for much of the cationic nature of the protein (42, 45). contained desired concentrations of lactoferrin, transferrin, or lactoferricin B. After 3 h, the cells were washed and cell-associated FITC was deter- The related iron-binding protein transferrin, which lacks these ar- mined by FACS (63). To correct for cell membrane binding, parallel cell ginines, has a pI of 5–5.5 (46). samples were also incubated with ODN on ice for 30 min to prevent The cationic N-terminal component of lactoferrin has also been uptake. shown to result in the ability of the molecule to bind a variety of biologically important, negatively charged molecules, via charge- Binding of lactoferrin to Ramos cells charge interactions. These include LPS (47–54), heparin, and Ramos cells were washed twice in HBSS and suspended at a concentration heparan sulfates (47, 55–57) and DNA (47, 58–60). Binding of of 106/ml in 0.5 ml of HBSS at 4°C. To the cell suspension was added 5 LPS to lactoferrin has been shown to modulate the bioactivity of LPS ␮Ci of 125I-labeled human apolactoferrin. After 30 min at 4°C, the cells by altering how it interacts with target cells (49, 50, 52, 53). Most of were washed three times with cold HBSS and finally pelleted at 200 ϫ g the interest in the DNA-binding properties of lactoferrin has been on for 10 min.
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