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Unité INRA 914 Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, F-75231 Paris Cedex 05, France and * Department of Microbiology and Immunobiology Vaccine Center, UAB, Birmingham, AL 35294
1To whom correspondence should be addressed. E-mail: tome{at}inapg.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • lactoferrin • oral modes of administration • immunomodulation • mice
Lactoferrin (Lf),2 a member of the iron-binding glycoprotein transferrin family, is found in polymorphonuclear leukocytes and a variety of vertebrate exocrine secretions such as milk, saliva, tears, mucosal, and genital secretions (1). Many cell types, including human blood lymphocytes and intestinal epithelial cells from young adult mice bear Lf receptors (2,3). In vitro, Lf stimulates the growth of lymphocytes (4,5), natural killer activity (6), and the release of interleukin-8 (IL-8) from neutrophils (7). Lf also upregulates phagocytosis and cytotoxicity of neutrophils (8) and macrophages (9). Further, Lf downregulates granulocyte/macrophage colony-stimulating factor production by macrophages (10), the release of IL-1, IL-2, and tumor necrosis factor (TNF) from leukocytes (11), or complement activation (12). Lf is believed to be the colostral protein that enhances proliferation of human blood lymphocytes in response to T-cell mitogens (13). Of interest for regulation of mucosal immunity, Lf can pass intact through the infant gut, as evidenced by its presence in the feces of breast-fed infants (14) and can be transported intact from the apical to the basolateral side of intestinal epithelial cells in vitro (15).
The meaning and consequences of Lf in milk are still not completely understood, and its role as an exogenous oral immune modulator has often been questioned (16–20). A number of studies have addressed the potential regulatory role of milk proteins on the immune system in vivo but in most of these studies, proteins were parenterally administered i.v. or by i.p. injection (21,22). It is now well established that when a protein is administered orally, it comes in contact with specific secondary lymphoid organs such as the tonsils and the Peyer’s patches. Complex interactions that then take place in these sites are crucial for the development of a specific immune response to the antigen (23,24). Thus, although oral ingestion of a foreign protein generally leads to the induction of oral tolerance (25), oral ingestion of Lf, in heterologous species, can induce an antigen-specific response. The mechanisms underlying the regulation of adaptive immunity by Lf remain to be understood. For example, it remains unclear how the oral mode of Lf ingestion influences the regulatory effect of this milk protein.
The aim of the present study was to evaluate the consequences of chronic exposure of mice to bovine Lf as a solution or as solid diet. More specifically, we analyzed how oral modes of administration, which allow Lf to interact only with lymphoid tissues of the gastrointestinal tract (i.e., gastric intubation), differ from those that allow its interaction with other components of the mucosal-associated lymphoreticular tissues (i.e., buccal doses).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bovine Lf (Solarec-Sodelac Recogne) was 96.6% pure with a 16.2% iron saturation level. No contaminating protein was detected after analysis by HPLC and electrospray ionization-MS. All other reagents were of analytical grade.
Mice and diets.
The animal protocol complied with NIH guidelines. Female BALB/c mice (4 wk old; Charles River France) were used in the experiments and housed in a barrier room under the following conditions: temperature 22 ± 2°C; relative humidity 50 ± 20%; ventilation 10–15 changes/h; and a 12-h light:dark cycle (lights on 0700–1900h). Body weight was measured regularly and routine clinical observations were carried out throughout the experiments. The mice were fed for 2 wk a standard laboratory food (cereal, 839 g/kg; vitamins and minerals, 41 g/kg; fish proteins, 40 g/kg; and vegetable proteins, 80 g/kg; Extralabo). Mice were then divided into 6 groups of 10 mice (body weight ranged from 17.5 to 19.5 g). The control group (control) received only the standard diet and had free access to sterile water. The intramuscular (IM) group was injected i.m. with 10 µg of Lf in complete Freund’s adjuvant (Sigma), and administered a booster dose in incomplete Freund’s adjuvant 4 wk later. The intragastric gavage (IG) group was given 0.5 mL of Lf preparation at 8 g/L (4 mg/mouse/d) through a stainless steel feeding tube, for 5 consecutive days each week for 6 wk. After overnight food and water deprivation, the morning buccal dose (BD) group was allowed to swallow 0.5 mL of Lf (8 g/L) dropped into their mouth with the tip of an Eppendorf pipette [4 mg/(mouse · d)]. The continuous dose drink (CDdrink) group consumed ad libitum via the drinking water a sterile 1 or 25 g/L Lf solution at a level of 4.00 ± 0.25 mL/(mouse · d) [4 or 100 mg/(mouse · d)]. To prevent bacterial proliferation, this Lf solution was replaced twice a day. Last, the continuous dose diet (CDdiet) group consumed ad libitum 100 mg Lf/d as a powder in the diet. The food containers were refilled daily with fresh food and were fitted with bars to reduce losses.
Collection of intestinal secretions and serum.
Intestinal secretions were collected on d 0, 14, 28, and 42 as previously described (26). Briefly, mice were given 0.5 mL (48 mol/L) polyethylene glycol MW 4000 (Prolabo) every 15 min for 1 h. At 30 min after i.p. injection of pilocarpine-HCl, intestinal contents were collected in 3 mL of a cold PBS solution containing 1.5 x 10-2 mol/L diisopropylfluorophosphate (Sigma). The resulting material was clarified by two successive centrifugations (3000 x g, 4°C, 15 min) and stored at -20°C until assay. Mice were bled from the retroorbital plexus throughout the experiment on d 0, 7, 14, 28, 35, and 42. After centrifugation (3000 x g, 4°C, 15 min), individual sera were collected and stored at -20°C until assay.
Isolation of Peyer’s patch and spleen cells.
Mice were killed by i.m. injection of pentobarbital sodium (Sanofi). The spleen was aseptically removed, slit by a surgical blade, and finely minced in RPMI-1640 medium (Gibco Life Technologies) supplemented with 20 mg/L gentamicin (Gibco). Single cells were separated using a cell strainer (70 µm) (Falcon; Polylabo). RBC were removed by incubating spleen cells for 5 min with NH4Cl solution (8.4 g/L) at 4°C, and cell suspensions were washed twice with cold RPMI-1640 medium. Peyer’s patches were carefully excised from the whole length of the small intestine, removed and transferred into Petri dishes containing RPMI-1640 medium. They were slit by a surgical blade and crushed gently. Cell suspensions were passed through a cell strainer to remove cell debris and washed twice with cold medium. These isolation procedures yield 0.5–1 x 109 spleen cells and 2–3 x 106 Peyer’s patch cells per mouse with 90 and 80% viability, respectively.
Lactoferrin-specific antibody responses.
Immunoglobulin (Ig)A, IgE, IgG, IgG1, IgG2a, and IgM antibodies to Lf in intestinal secretions and sera were quantified by an ELISA (27). Flat-bottomed microtiter plates (96-well Nunc-Immuno Plate Maxisorp) were coated overnight at 4°C with 2 mg/L Lf in 0.1 mol/L NaHCO3 buffer, pH 9.6. After addition of the samples, the antibodies (Abs) were detected with biotinylated-specific antibodies (IgA, IgG and IgM from Sigma and IgE, IgG1 and IgG2
from Pharmingen) followed by extravidin peroxidase (Sigma). The plates were developed with H2O2 (0.003 g/L, Sigma) and o-phenylene-diamine (0.5 g/L, Sigma). Absorbances were read at 490 nm in an automatic ELISA reader (EL-309, Bio-Tech, OSI Paris). Results are given as the reciprocal logarithmic dilution of the last sample dilution that gave an A490 > 0.1 U. The values obtained with preimmune samples were <0.1 U.
Total immunoglobulins.
For detection of total IgA, IgG and IgM Abs microtiter plates were coated with 2 mg/L anti-mouse IgA (Sigma) or anti-mouse IgG (Sigma) or anti-mouse IgM (Sigma). The sequence described earlier for specific antibodies was used. The antibody concentration was calculated by comparison of A490 values with a reference curve obtained from purified monoclonal antibodies: IgA (Sigma), IgG (Sigma) and IgM (Sigma). The results are expressed in µg/L. The limit of detection was <2 µg/L for assays.
In vitro stimulation and proliferation assay.
Spleen or Peyer’s patch cells were cultured in 96-well flat bottom plates (Falcon) at 4 x 106 cells/mL in a final volume of 200 µL. The culture medium consisted of complete RPMI-1640 medium containing 100 mL/L fetal calf serum (FCS), 2 mmol/L L-glutamine, 105 U/L penicillin, 10 mg/L streptomycin, 10 mg/L gentamicin, and 5 x 10-5 mol/L 2-mercaptoethanol. Cultures were incubated for 48 or 96 h at 37°C in 5% CO2 atmosphere, in the presence or absence of Lf (20–100 g/L), concanavalin A (ConA; 2 g/L), or lipopolysaccharide (LPS; 4 g/L). Culture supernatants were collected and stored at -80°C until assayed for Ig synthesis (96 h) and cytokine production (48 h). For the evaluation of proliferative responses, triplicate wells were stimulated with 60 g/L of Lf. After 48 h incubation, [6-3H] desoxy-thymidine (0.5 µCi/well, specific activity: 185 GBq/mmol, Amersham) was added in a volume of 20 µL per well, and the incorporation of 3H-thymidine into DNA was measured with an Inotech counter (Automatic filter counting system INB-384, Dottikon). The results are expressed as mean disintegration per min (dpm) ± SD of triplicate cell cultures.
Th1 and Th2 cytokine responses.
IL-4 and interferon-
(IFN-) concentrations were determined by ELISA using Duoset ELISA kits (R & D Systems). Assays were performed according to the manufacturers’ guidelines. The detection threshold was 1 and 20 ng/L for IL-4 and IFN-, respectively. To determine IL-2 and IL-5 levels in culture supernatants, microtiter plates were coated with 2 g/L anti-mouse IL-2 (Pharmingen) or 2 g/L anti-mouse IL-5 (Pharmingen) in 0.1 mol/L carbonate buffer, pH 8.2, overnight at 4°C. Standards and samples diluted in RPMI-1640 containing 100 g FCS/L were tested in duplicate and placed overnight at 4°C. The next day, 50 µL of biotin-conjugated anti-IL-2 (at 1 g/L in PBS containing 10 g bovine serum albumin/L; Pharmingen) or anti-IL-5 (1.5 mg/L; Pharmingen) was added to the appropriate wells and incubated for 1 h at room temperature. After washing, 50 µL of 1:1000 extravidin-conjugated peroxidase was added to each of the wells and incubated for 1 h at room temperature. The next steps were the same as those described earlier. The sensitivity was 40 ng/L for IL-2 and 50 ng/L for IL-5, as judged by the respective standard curves. The results are expressed in ng/L.
Statistical analysis.
The results are reported as mean ± SD. Comparisons of means were made by ANOVA. When the ANOVA was significant, post-hoc testing of differences between groups was performed using Duncan’s test. All calculations were performed using SAS software (SAS Institute). A value of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All of the oral modes of exposure to Lf, except when Lf was added into the drinking water (CDdrink), induced higher levels of total IgA and IgG in the intestinal secretions, at d 28 (Table 1). In contrast with most of the oral mode of exposure, mice administered Lf by i.m. injection had no increase in total IgA in intestinal secretions. Further, mice exposed to Lf orally maintained high levels of total IgG in their intestinal secretions at d 42, whereas increased IgG levels were seen only at d 28 in mice administered Lf by i.m. injection. None of the oral or systemic modes of exposure to Lf affected the serum levels of total IgG (data not shown).
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The addition of at least 20 mg/L of Lf in culture media further increased proliferative responses of both cells from mice orally exposed to Lf, except the CDdrink groups, and cells from mice administered Lf by the i.m. route (data not shown). The stimulatory effect of Lf in vitro was not increased at higher Lf concentrations. Thus, in further investigations, cells were routinely stimulated in vitro with Lf at 60 mg/L. In the absence of further stimulation, the pattern of in vitro cytokine secretion by Peyer’s patch mononuclear cells from mice orally exposed to Lf did not differ from that of mice administered Lf by i.m. injection or control naïve mice (Table 3). However, in vitro stimulation with Lf or Con A promoted significantly higher Th2 cytokine responses by mononuclear cells from mice orally exposed to Lf compared with control naïve mice or mice administered Lf by i.m. injection. On the other hand, Th1 responses were not different between the groups that were administered Lf orally and by i.m. injection (Table 3). These results suggest that, in contrast to systemic i.m. injection, the oral mode of exposure to Lf, except the CDdrink group, biases the response of Peyer’s patch T cells toward Th2-type responses.
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In the absence of additional in vitro stimulation, the levels of cytokine secreted by cells from mice orally exposed to Lf did not differ from those of mice administered Lf by i.m. injection or control naïve mice. The addition of Lf to the culture medium stimulated elevated Th2 cytokine secretion by spleen cells from all of the groups orally exposed to Lf, except the CDdrink group. Of interest, in contrast to the Peyer’s patch cells above, spleen cells from mice administered Lf by i.m. injection also produced elevated Th2 cytokine levels after in vitro restimulation with Lf. Further, cells from mice administered Lf by i.m. injection and oral Lf i.g., but none of the other groups, secreted elevated levels of Th1 cytokines after in vitro culture in the presence of Lf (Table 5). The same pattern of cytokine responses was detected after mitogenic stimulation of spleen cells with ConA. Indeed, upon stimulation with ConA, IL-4 (Th2 cytokine) secretions were elevated in culture supernatants of spleen cells from all of the groups orally exposed to Lf, except the CDdrink group; a similar increase was measured in spleen cells from mice administered Lf by i.m. injection. Further, mitogenic stimulation with ConA induced higher and similar levels of IL-2 secretions by cells from mice administered Lf i.g. and by i.m. injection.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It is interesting that only mice administered Lf in the drinking water (CDdrink) did not have changes in mucosal and systemic immune status. Indeed, the CDdrink mode of exposure, like the BD and CDdiet modes, allows Lf to interact with the oral- and gut-associated lymphoid organs such as the nasopharyngeal tissues and the Peyer’s patches, respectively (23,24). It is now well accepted that oral tolerance can be induced by chronic administration of low antigen doses or isolated exposure to very large doses of antigen (25). Several groups have shown Lf to be quite resistant to proteolysis by various enzymes in vitro (28,29). In vivo studies (14,30,31) also showed that a small percentage of ingested Lf survives the passage through the entire intestinal tract of breast-fed infants. Moreover, Mikogami et al. (15) reported that a small fraction (
10%) of human Lf is transported across HT-29 intestinal epithelial cells. The induction of oral tolerance depends on antigen processing by both intestinal cells and classical antigen-presenting cells (macrophages, B cells, and dendritic cells) (25,32). Indeed, specific receptors for Lf have been described in intestinal epithelial cells (3), B lymphocytes (2), and macrophages (33). Thus, one could hypothesize that the addition of Lf to the drinking water leads to tolerance (34,35), possibly by persisting in body fluids and inducing a state of immune unresponsiveness in most T lymphocytes (36). This could also be related to the absence of interactions between Lf and immunocompetent organs because immunomodulation requires intact molecules or active peptides capable of reaching immunocompetent cells (37). Our findings are consistent with observations by others that the addition of ovalbumin to the drinking water suppressed immune responses in Peyer’s patch and spleen cells and induced Th2 tolerance (38,39). Madsen and Pilegaard (40) reported decreased immune response in newborn rats dosed with albumin by mouth compared with the priming by intragastric intubation. We have also observed that 
-carrageenan mixed with ovalbumin (OVA) to form a solution and given i.g. induced oral tolerance to OVA, whereas -carrageenan mixed with OVA to form a gel failed to induce oral tolerance (41). These results also agree with data indicating that the nasal route is an effective route for immunization (42), that the sublingual route is superior for desensitization (43,44), and that the oral route is the preferred route for induction of mucosal tolerance (25,45).
We also found important differences among the BD, CDdiet, and IG oral mode of exposure. All of these oral modes of exposure induced Lf-specific IgG1, a subclass associated with Th2 responses in mice (46,47). However, only the i.g. administration promoted both Lf-specific IgG2a and IgG1 Abs, a pattern seen in mice administered Lf by i.m. injection and associated with Th1 and Th2 cells, respectively. Our in vitro studies further confirmed that only Peyer’s patch cells from mice administered Lf i.g. were able to produce high levels of IgA and IgG after in vitro stimulation with Lf and LPS, respectively. Spleen cells from the same IG mice were also the only cells from the orally treated mice that secreted higher levels of both IgA and IgG after in vitro stimulation with Lf, a pattern only seen with cells from mice administered Lf by i.m. injection. It is striking that IgG subclasses more related to Th2-type responses were seen after the more physiologic modes of oral administration, i.e., the morning buccal dose (BD) and the continuous dose via the diet (CDdiet).
The analysis of T-cell responses in vitro provided some clues concerning potential mechanisms involved in the immune regulatory effect of oral Lf. Thus, it appears that oral exposures to Lf affected innate immune response because they enhanced the spontaneous proliferation of spleen cells. More importantly, the oral Lf biased both antigen-specific (i.e., Lf-specific) and nonspecific (i.e., ConA-mediated) Peyer’s patch and spleen cell responses toward Th2 cytokine responses. The less natural i.g. mode of oral exposure was the only oral treatment that promoted Th1 cytokine responses by spleen cells, as did the i.m. injection of Lf. We showed previously that administration of Lf via gastric intubation promoted strong Th2-type immune responses, which accounted for the generation of antigen-specific IgA and IgG responses (20). Our data are also in agreement with Hashizume et al. (48) who reported that Lf is able to stimulate proliferation and Ig production by murine lymphocyte in vitro. The stimulatory effect of Lf was also observed with human lymphocytes activated with phytohemagglutinin and is believed to be related to the iron-binding activity of this protein (2). This effect could also be mediated by its specific receptor on the cell surface and/or by its potential direct transcriptional function (49). Further, Wong et al. (50) demonstrated that bovine Lf can inhibit proliferation and IFN- production of ovine blood lymphocytes, adding support to the bias toward Th2 responses induced by Lf.
The results reported here show that distinct oral modes of administration lead to qualitatively different immune responses in mice. More detailed kinetic analysis of antibody isotype and subclass responses and corresponding cytokines would be required to determine the magnitude and duration of these regulatory effects. Although one cannot directly extrapolate the results obtained in BALB/c mice to humans, our findings clearly indicate that the mode of oral delivery of immunologically active food proteins should be carefully considered to more clearly establish their regulatory effects in vivo.
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FOOTNOTES |
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, interferon-; Ig, immunoglobulin; IG, intragastric gavage group; IL, interleukin; IM, intramuscular group; Lf, lactoferrin; LPS, lipopolysaccharide; OVA, ovalbumin; Th, T helper; TNF, tumor necrosis factor. 
Manuscript received 13 June 2003. Initial review completed 23 July 2003. Revision accepted 4 November 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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