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Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on 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

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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 -binding 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 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. 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, and dendritic cell IL-12 production (3–7); 2) en- 24) by a process that is mediated via a Toll-like -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 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 ( (pI), 8.4–9.0) The costs of publication of this article were defrayed in part by the payment of page monomeric (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 with 18 U.S.C. Section 1734 solely to indicate this fact. (29–31). It is also a major constituent of 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 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 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. The tip of the centrifuge tube containing the cell pellet was cut

from the tube using a clipper and placed into a tube which allowed deter- Downloaded from the interaction of the protein with eukaryotic DNA. Recently, there mination of 125I-labeled human lactoferrin content by gamma counter. has been the suggestion that there may be a sequence specificity for Each experiment included conditions in which 125I-labeled human apo- binding of eukaryotic DNA to lactoferrin and that lactoferrin may lactoferrin was added to HBSS which lacked cells and was processed in an play a role in transcriptional activation (61). analogous manner. This was done to control for nonspecific sticking of Given previous evidence that 1) lactoferrin can bind ssDNA; 2) lactoferrin to the tubes (background). These background counts were sub- tracted from the cell-containing values to arrive at cell-specific lactoferrin interaction with another immunostimulatory molecule, LPS, is binding. modified upon its binding to lactoferrin; and 3) lactoferrin is http://www.jimmunol.org/ present in high concentrations at both mucosal surfaces and sites of Statistical analyses acute bacterial infection, we hypothesized that lactoferrin will bind CpG-containing ODN and that this will lead to alterations in the Results obtained under different experimental conditions were com- pared by Student’s paired t test when independent variables were being biological activity of these ODN toward the immune system. Data assessed or by ANOVA when analyses of trends were being deter- reported herein support that hypothesis. mined. For both types of analyses, results were considered to be sig- nificant at p Յ 0.05. Materials and Methods 32P-labeled ODN Results by guest on September 23, 2021 Lactoferrin binds CpG ODN The following ODN, which contained nondetectable levels of endotoxin, were provided by the Coley Pharmaceutical Group (Wellesley, MA): 2006, Given that lactoferrin had previously been shown to bind to both TCG TCG TTT TGT CGT TTT GTC GTT; 1818, GAG AAC GCT GGA ssDNA and dsDNA (47, 58–60), we examined the ability of lac- CCT TCC AT; 1906, TCC ATG ACG TTC CTC TCC ATG ACG TTC toferrin to bind synthetic ODN with or without CpG motifs. Using CTC TCC ATG ACG TTC TTC CTC; and 1858, TCC AGT CTA CGC 32 CTA GTT CT; 1888: TCC ATG ACG TTC CTA GTT CT. P-labeled ODN in conjunction with EMSA analysis, human lac- The ODN of interest (200 ng) was incubated with T4 polynucleotide kinase toferrin was found to bind such ODN in a concentration-dependent (10 U) and [32P]ATP (30 ␮Ci) in a volume of 40 ␮l for 30–60 min at 37°C. manner following a brief (2- to 5-min) exposure (Fig. 1). The The solution was then centrifuged through a G-25 Sepharose column for 4 min binding was saturable, as it could be inhibited by the presence of at 2000 rpm at room temperature. The radiolabeled ODN was then harvested from the eluate, counted in a gamma counter, and then stored at Ϫ20°C until a 100-fold concentration excess of unlabeled ODN (Fig. 1A). No needed. For cellular uptake experiments, ODN with a 5Ј fluorescein modifi- difference in binding to lactoferrin was distinguishable among the cation (Operon Technology, Alameda, CA) were used. CpG-and non-CpG-containing ODN examined (Fig. 1B), indicat- ing that lactoferrin binding did not require the presence of the CpG EMSA motif. Neither was binding unique to human lactoferrin, as CpG Binding of ODN to lactoferrin, transferrin, and lactoferricin B was assessed ODN binding was also readily detectable using bovine lactoferrin by EMSA. Lactoferrin (bovine and human) as well as transferrin were (Fig. 1C). purchased from Sigma (St. Louis, MO). Lactoferricin B was kindly pro- Binding of iron to lactoferrin results in conformational changes in vided by the Morinaga Milk Industry (Zama City, Japan). 32P-labeled ODN the molecule (64–66). However, comparison of ODN binding to apo- was incubated alone or with the protein of interest for 2–5 min in H Oat 2 lactoferrin and iron-loaded forms of human lactoferrin revealed no room temperature. Samples were then subjected to native PAGE followed by overnight autoradiography (Ϫ80°C). differences in their ability to bind the CpG-containing ODN (Fig. 1D). In contrast, the related iron-binding protein transferrin was unable to Ramos cell CD86 expression bind the ODN under the same conditions (Fig. 2). Interestingly, in studies of lactoferrin binding to ODN 1906 at The Ramos human B cell line (Burkitt lymphoma B cell line, ATCC relatively low ratios of lactoferrin to ODN, we observed a single CRL-1923) was maintained in RPMI 1640 supplemented with L-glu- tamine, penicillin, streptomycin, and HEPES. Cells were harvested and band that migrated well into the gel. As the concentration of lac- incubated in RPMI 1640 containing 10% FCS at a concentration of toferrin increased, additional slower migrating bands appeared, 1–2 ϫ 106/ml. To these cell suspensions was then added ODN alone or and with further increases a single band was eventually seen with ODN that had been preincubated with lactoferrin, lactoferricin B, or much slower migration into the gel (Fig. 1, A–C). Interestingly, transferrin in H2Ofor1hat37°C. After overnight incubation in 96-well flat-bottom plates, the cells were removed and assessed for CD86 ex- this was not seen with the other ODN examined (Fig. 1B). ODN pression by surface Ag staining using FITC-conjugated Ab to CD86 1906 is nearly twice as long as the other ODN studied, and we (2331 (FUN-1); purchased from BD PharMingen, San Diego, CA) in hypothesize that this banding pattern reflects the ability of the 1906 The Journal of Immunology 2923 Downloaded from http://www.jimmunol.org/

32 FIGURE 1. Lactoferrin binds ODN. Human lactoferrin, at the indicated amounts, was incubated with 1 ng of P-labeled ODN in H2O for 2–5 min and then subjected to EMSA. Results are representative of three separate experiments. A, Lactoferrin binding to ODN; 1906 was readily detectable and was containing 100 ng of unlabeled ODN). B, No difference in binding to lactoferrin was ,ء inhibited by a 100-fold excess of unlabeled ODN (far right lane distinguishable between the CpG (1906)- and non-CpG (1858)-containing ODN. C, Both human and bovine lactoferrin were able to bind the ODN 1906. D, Both lactoferrin and iron-loaded (di-ferric) lactoferrin were able to bind the ODN 1906. Results shown are representative of at least three separate experiments.

ODN to bind several molecules of lactoferrin, resulting in the step- interaction that likely involved the N-terminal portion of the lac-

wise banding pattern on the gel. toferrin molecule. To test this hypothesis, the effect of increasing by guest on September 23, 2021 salt concentration on the ability of lactoferrin to bind to CpG ODN Mechanism of CpG oligonucleotide binding to lactoferrin was assessed. Consistent with a charge-charge-mediated interac- The failure of the less cationic protein transferrin to bind the ODN, tion, high salt conditions (50 mM NaCl) resulted in a progressive in conjunction with previous work by others (47, 58–60) exam- decline in association of the ODN with lactoferrin (Fig. 3). ining the nature of lactoferrin-DNA interactions suggested that Previous work by others (47, 58) suggested that lactoferrin bind- binding of CpG ODN occurred on the basis of a charge-charge ing of eukaryotic DNA occurs at the same N-terminal portion of

FIGURE 2. Transferrin does not bind ODN. Five micrograms of human FIGURE 3. Lactoferrin binding of ODN is inhibited by high salt. Hu- lactoferrin and transferrin were incubated with 1 ng of 32P-labeled ODN man lactoferrin (400 ng) was incubated with 0.5 ng of 32P-labeled ODN

1906 in H2O for 2–5 min and then subjected to EMSA to determine their 1906 in either H2O or solutions containing various amounts of NaCl and relative ability to bind the ODN. Results are representative of three sepa- subjected to EMSA. Results are representative of three separate rate experiments. experiments. 2924 LACTOFERRIN INHIBITS CpG ODN IMMUNOSTIMULATORY EFFECTS

the lactoferrin molecule at which binding of other negatively charged biological molecules (e.g., LPS, heparin, and heparan sul- fates) occurs (47, 48). In the present work, we found that both heparin and LPS inhibit binding of 32P-labeled ODN in a concen- tration-dependent manner (Figs. 4 and 5). These data are consistent with the primary binding site for the ODN being on the N-terminal portion of lactoferrin. To confirm this, we examined the ability of purified lactoferricin B, which is comprised of a short portion of the N-terminal sequence of bovine lactoferrin (42, 67), to bind 32P-labeled ODN. As shown in Fig. 6, lactoferricin B exhibited, on a molar basis, a similar ability to bind ODN. Lactoferricin B contains a disulfide bond (67). This bond does not appear to be necessary for the ability of lactoferricin B to bind ODN as pretreatment of the molecule with the reducing agent DTT to break those bonds had no effect on subsequent binding of 32P- labeled ODN (data not shown). Downloaded from Binding to lactoferrin alters the biological activity of CpG ODN FIGURE 5. Lactoferrin binding of ODN is inhibited by LPS. 32P-la- Previous work has shown that the binding of LPS to lactoferrin alters beled ODN 1906 (1 ng) was incubated with 1 ␮g of lactoferrin in the LPS biological activity (49, 50, 52, 53). Therefore, we hypothesized presence of increasing concentrations of Escherichia coli LPS (0.1–10 ␮g) that binding of CpG ODN to lactoferrin or lactoferricin B would in- and subjected to EMSA. The presence of LPS interfered with the ability of hibit the immunomodulatory effects of that molecule. Alternatively, it lactoferrin to bind 32P-labeled ODN 1906 in a concentration-dependent manner. Results are representative of three separate experiments.

was also possible that lactoferrin would enhance the CpG activity by http://www.jimmunol.org/ increasing cellular uptake of the DNA. To distinguish between these two possibilities, we examined the effect of lactoferrin/lactoferricin B binding on the ability of CpG ODN to increase CD86 expression on enhance Ramos cell CD86 expression was examined. No effect was the human B lymphocyte-derived Ramos cell line. This parameter observed (Fig. 7), indicating that the effect of lactoferrin/lactoferricin was chosen because our previous experience had indicated that CD86 B does not result from some general toxicity, but rather is specific for expression in this cell line is not modulated by LPS exposure (A. M. the ODN-induced response. Krieg, unpublished observation), a response which if present could potentially confound data interpretation, given the ability of lactofer- Lactoferrin inhibits binding and internalization of CpG ODN

rin to bind LPS as well. As shown in Fig. 7, binding of the ODN to by guest on September 23, 2021 The biological effect of CpG ODN on cellular systems has been either lactoferrin or lactoferricin B significantly decreased the mag- shown to require their binding and internalization by a mechanism nitude of resulting expression of CD86 on the cell surface. This in- that remains ill-defined (23, 24). Accordingly, we assessed the hibition could be overcome by increasing the concentration of the effect of binding of CpG ODN to lactoferrin on its uptake by ODN in the assay (data not shown). In contrast, the related iron- Ramos cells by measuring the ability of the cells to take up FITC- binding protein transferrin was without effect. As a control for any conjugated ODN (expressed as mean fluorescence index (MFI) by possible generalized cytotoxic effects of lactoferrin, the effect of lac- FACS). Lactoferrin (0.5 ␮g/ml) significantly inhibited ODN up- toferrin and lactoferricin B on the ability of polyclonal anti-IgM to take over3hat37°Cto35Ϯ 6% of control (mean MFI Ϯ SEM, n ϭ 3, p Ͻ 0.05). In contrast, transferrin (0.5 ␮g/ml) was without effect: MFI was 83 Ϯ 12% of control (n ϭ 3, p Ͼ 0.05). The same concentration of lactoferrin also decreased ODN binding at 4°Cby 50 Ϯ 28% (n ϭ 3, p Ͻ 0.05), reflecting an effect on surface binding as well as uptake over time. Transferrin again was without effect: MFI was 107 Ϯ 32% of control (n ϭ 3, p Ͼ 0.05).

FIGURE 4. Lactoferrin binding of ODN is inhibited by heparin. 32P- ␮ labeled ODN 1906 (1 ng) was incubated with 1 g of lactoferrin in H2O FIGURE 6. Lactoferricin B binds ODN. Lactoferricin B, at the indi- in the presence of varying concentrations of and subjected cated concentrations, was incubated with 0.5–1ngof32P-labeled ODN

to EMSA. The presence of heparin interfered with the ability of lactoferrin 1906 in H2O for 2–5 min and then subjected to EMSA. Lactoferricin B to bind 32P-labeled ODN 1906 in a concentration-dependent manner, re- binding to ODN 1906 was readily detectable and was inhibited by a 100- containing 100 ng of ,ء flected as serial dilutions of heparin. Results are representative of three fold excess of unlabeled ODN (far right lane separate experiments. unlabeled ODN). Results are representative of three separate experiments. The Journal of Immunology 2925

ecules to inhibit ODN binding indicates that this most likely occurs on the basis of charge-charge interactions. The fact that lactoferrin, but not the related iron-binding protein transferrin, was able to bind the ODN suggests that the high pI of lactoferrin relative to transferrin may be an important determinant of this activity. This is further supported by that fact that a small portion of the N- terminal sequence of lactoferrin that is responsible for much of the cationic nature of the molecule (lactoferricin) (42, 67) exhibited the same ability to bind ODN as the entire lactoferrin molecule. This observation also has important implications for the poten- tial for this interaction to occur in vivo where lactoferrin can be cleaved through protease activity to generate lactoferricin (42, 69, 70). Thus, even at sites of high protease activity, the ability of lactoferrin to bind DNA should remain intact. Furthermore, since the oxidation state of the sulfhydryl group of lactoferricin B does not appear to dramatically alter the ability of lactoferricin to bind ODN, this process should function both in highly oxidizing and FIGURE 7. Lactoferrin (LF) binding of CpG ODN decreases bioactiv- ity. The Ramos human B cell line was incubated overnight with the CpG- reducing environments. Downloaded from containing ODN 1906 (1 ␮g/ml) alone or with the same amount of ODN Previous work by others has shown that lactoferrin is capable of that had been preincubated with human lactoferrin (0.5 mg/ml), transferrin binding ssDNA and dsDNA (47, 58–60). Furmanski and col- (0.5 mg/ml), or lactoferricin B (30 ␮g/ml) for 1 h before it was added to the league (61) indicated that there is a preferred sequence specificity cell suspension. Following this incubation, the ability of the ODN to induce for the binding of ODN by lactoferrin. These authors also reported CD86 expression on the cell surface was assessed by FACS. Also shown that lactoferrin could be internalized and trafficked to cell nuclei are results in which the cells were exposed to an alternative stimulus of where it could potentially serve as a regulator of gene transcription ␮ CD86 expression, anti-IgM (2 g/ml), alone or in the presence of lacto- (61). Our data indicate that, at least in the case of DNA fragments http://www.jimmunol.org/ ferrin (0.5 mg/ml), as a control for possible nonspecific effects of lacto- that lactoferrin would initially encounter extracellularly, this pro- ferrin on CD86 expression. Results are reported as mean Ϯ SEM of the cess serves to inhibit rather than enhance intracellular localization. percentage of cells expressing CD86, representing three or more separate experiments. Results with the addition of lactoferrin or lactoferricin B to tk;4Binding of LPS to the N-terminal sequence of lactoferrin the ODN were significantly less than ODN alone at p Ͻ 0.05. No other has been shown to alter many of the proinflammatory effects of results were significantly different from ODN alone. LPS (49, 50, 52, 53). We find that binding of CpG ODN to the same site of lactoferrin has a similar negative effect on the proin- flammatory action of CpG sequences on human B cells. CpG- These latter data suggested that the binding of the ODN to lac- induced up-regulation of CD86 was blocked by lactoferrin as well by guest on September 23, 2021 toferrin did not direct the ODN to a different surface receptor sys- as lactoferricin B. Since lactoferrin also decreased uptake of the tem, as has been described previously with monocytes and LPS ODN by these cells and since that uptake appears to be required for (52). ODN also altered the interaction of lactoferrin with the cellular activation (23, 24), it is likely that lactoferrin binding in- Ramos cells. Binding of lactoferrin to Ramos cells was decreased hibits many additional effects of the CpG ODN as well. This effect by 71% when lactoferrin was preincubated with the ODN, decreas- was specific for lactoferrin relative to transferrin. Furthermore, as ing from 150 Ϯ 18 fmol lactoferrin/106 cells to 43 Ϯ 8 fmol evidenced by the lack of effect of lactoferrin on the cellular re- lactoferrin/106 cells (mean Ϯ SEM, n ϭ 4) when the lactoferrin sponse to polymeric anti-IgM, the inhibition appears to be specific was preincubated with ODN before addition to the cells ( p Ͻ for the ODN-mediated response and is not a generalized cytotoxic 0.02). Thus, it appears that the interaction of ODN and lactoferrin effect. decreases the ability of both agents to bind to the Ramos cell Lactoferrin binds to cells by at least two mechanisms: 1) high surface. capacity low-affinity binding that may occur via charge-charge in- teractions with surface glycolipids or other molecules such as DNA (71–76) and 2) lower capacity/higher affinity binding to one Discussion or more proteinaceous plasma membrane receptors (77–83). Lac- The unmethylated CpG sequences that distinguish bacterial from toferrin has been reported to bind to B cells via interaction with eukaryotic DNA have been identified as potentially important sig- DNA on the cell surface (74, 75) and it appears that B cell binding naling molecules whose recognition allows various aspects of the is mediated through the highly charged N-terminal sequence of host immune system to be activated to respond to invading bac- lactoferrin (84). To our knowledge, no specific protein receptors terial pathogens (1, 2). Both beneficial and deleterious conse- for lactoferrin on the B cell surface have been reported. Thus, the quences of such activation have been described (1, 2). Although biological response to CpG ODN bound to lactoferrin could be much attention has been paid to the mechanism whereby CpG- different in those cells possessing one or more high-affinity lacto- containing ODN activate cellular immune responses in vitro and in ferrin-binding mechanisms such as , hepatocytes, vivo, there has been relatively little focus on the means whereby PHA-activated lymphocytes, and gastrointestinal epithelial cells these responses are negatively modulated. (77–83). This requires further investigation. In the present work we show that lactoferrin, a protein present Nevertheless, these data suggest that binding of bacterial DNA in high concentrations at mucosal surfaces, milk, and neutrophil- or DNA fragments could be a mechanism to modulate the proin- specific granules (29–34, 68), readily binds CpG-containing DNA. flammatory effects of these compounds. Lactoferrin would seem to Although lactoferrin plays a major role as an iron-binding protein, be ideally suited for this role as it is present at high concentrations our work and results from other investigators (47) indicate that the at mucosal surfaces (1 mg/ml) and in human milk (10 mg/ml) iron status of the protein does not influence its DNA-binding ac- (29–31). At these concentrations, which are far higher than those tivity. The ability of high salt and other negatively charged mol- used in our experiments, it would be readily available to deal with 2926 LACTOFERRIN INHIBITS CpG ODN IMMUNOSTIMULATORY EFFECTS bacterial DNA routinely present from those microorganisms col- References onizing these locations. This could serve to limit excessive immu- 1. Bird, A. P. 1999. CpG-rich islands and the function of DNA methylation. Nature nostimulatory activity at mucosal surfaces colonized by large num- 321:209. 2. Krieg, A. M., A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, bers of microbes, e.g., gut, without losing the ability to detect G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger pathogens that have invaded into tissues past the anti-inflammatory direct B-cell activation. Nature 374:546. layer of lactoferrin. Interestingly, CpG ODN are being explored as 3. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y. Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson, and E. Raz. 1997. an immunoadjuvant for a variety of vaccines and as immunother- Immunostimulatory DNA sequences function as T-helper-1 promoting adjuvants. apeutic agents for cancer and allergic diseases (3, 22). The poten- Nat. Med. 3:849. tial impact of lactoferrin in oral vaccine strategies using CpG ODN 4. Chu, R. S., O. S. Targoni, A. M. Krieg, P. V. Lehmann, and C. V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper (Th1) im- may need to be explored, particularly if they involve administra- munity. J. Exp. Med. 186:1623. tion of CpG-containing vaccines to infants who are being breast- 5. Hartmann, G., G. J. Weiner, and A. M. Krieg. 1999. CpG DNA: a potent signal fed. Likewise, the delivery of CpG ODN to other mucosal surfaces for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96:9305. such as the nasal cavity or lungs for allergy therapy (15) may 6. Halpern, M. D., R. J. 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