Introduction
Allergy constitutes a global and aggravating health problem affecting primarily populations in developed countries. It is estimated that up to 300 million people worldwide experience allergic asthma and these estimates are expected to rise as the incidence of asthma has been steadily increasing. Etiology of the disease comprises both genetic and environmental factors, including infectious agents and air pollutants. Asthma is dependent on the presence of antigen-specific IgE antibodies [
1] and Th2 type cells [
2,
3]. Airways inflammation is thought to play a central role in the pathogenesis of asthma with mast cells [
4] and eosinophils [
5] being responsible for molecular responses via secretion of cytokines and low-molecular weight mediators.
The therapeutic approaches in allergy encompass subcutaneous (SCIT) and, more recently, sublingual (SLIT) immunotherapies (reviewed by [
6]). In the experimental models in mice a key role in protection against allergens is played by regulatory T cells and TGF-beta [
7,
8]. However, it is suggested [
9] that immune deviation of allergen-specific Th2 cells may be more effective than induction of general immune response suppression.
Among various agents capable to suppress allergy in an antigen nonspecific manner, are biologically active peptides and proteins of therapeutic value, classified as biopharmaceuticals [
10] found, among others, in whey and milk casein [
11]. For example, whey protein hydrolysate suppressed atopic dermatitis in mice [
12] and formulas containing hydrolyzed casein and whey were found as effective as breast milk to prevent allergy in infants [
13].
In this investigation we have used a mouse model of ovalbumin (OVA)-induced allergic pulmonary inflammation which has been well established to induce airways eosinophilia and extensive lung damage analogous to that seen in asthma [
14]. It is the first attempt to evaluate the effects of lactoferrin (LF), a natural immunomodulator, on amelioration of OVA-induced lung pathology.
Lactoferrin is an evolutionally old iron-binding protein, contained in body fluids and neutrophils, playing an important role in maintaining immune homeostasis [
15,
16]. LF affects both physiological and pathological immunological responses and, in the latter case, abnormal Th1/Th2 balance can be corrected by LF [
17]. Although there exists a vast literature regarding successful immune interventions in infectious, inflammatory and autoimmune disorders by LF, very few reports describe beneficial actions of LF in allergy mediated by IgE antibodies and Th2 cells [
18,
19]. Recently, LF was shown to inhibit pollen antigen-induced allergic airway inflammation in mice as assessed by histology of airways and reactive oxygen species in culture of bronchial epithelial cells [
20]. Interestingly, the robust inflammatory processes were significantly prevented by LF administration but not by a classical iron binding synthetic compound desferroxamine (DFO) [
21]. Although both LF and DFO share some characteristics of iron binding, LF is able to diminish these symptoms more efficiently than is DFO. Thus, it is clear that LF has additional activities, that may be due to its affinity to bind LPS, heparin, lysozyme, or DNA [
22], which determine its action in OVA-induced pleurisy. In a sheep model it was also shown that LF abolishes both late-phase bronchoconstriction and airway hyperresponsiveness by the means of heparin binding and inactivation of mast cell tryptase [
18]. Others demonstrated that LF decreases the release of the allergic mediator histamine when incubated with skin mast cells [
19].
The aim of this investigation was to evaluate effectiveness of buccal and other ways of administration of LF in prevention of OVA-induced pleurisy in mice.
Materials and methods
Mice
BALB/c female mice, 8 to 10 weeks old, were delivered by the Institute of Laboratory Medicine, Łódź, Poland. The mice were fed a commercial, granulated food and water ad libitum. The local ethics committee approved the study.
Reagents
Bovine LF (BLF) from milk was from Tatua Co-operative Dairy Company, New Zealand (<15 % iron saturation, <10 endotoxin U/mg) and human milk LF (HLF) from Sigma (cat. no L0520). Both LFs were low endotoxin and iron saturation less than 15 %. Dexamethasone (Dexaven) was from Jelfa, Poland, Maalox (aluminum hydroxide 3.5 g and magnesium hydroxide 4.0 g in 100 ml) from Rhone-Poulenc Rorer, France, Narcotan® from Leciva, Czech Republik. ELISA kit for determination of IL-5 was from Becton Dickinson (cat. no 555236) and ELISA kit for IFN-γ determination was from eBioscience (cat. no 88-7314-88). Endotoxin free OVA, trypan blue, Giemsa, May-Gruünwald, haematoxylin, eosin, toluidine blue and formalin were from Sigma-Aldrich Corporation, St. Louis, MO, USA.
Sensitization of mice with OVA
Mice were immunized intraperitoneally (i.p.) with 50 μg of OVA in 0.2 ml of Maalox (adjuvant) as described elsewhere [
23]. After 14 days mice were given the eliciting dose of OVA intrapleurally (12.5 μg in 50 μl of 0.9 % NaCl). This group of mice is later referred to as sensitized control mice. Mice treated i.p. only with Maalox but received OVA, intrapleurally, are referred to in the text and figure legends as the background (BG) group.
The mode of lactoferrin administration
Lactoferrin (50–800 μg) was given buccally in 25 μl of 0.9 % NaCl/dose, 24 and 3 h before administration of the eliciting dose of OVA. Alternatively, LF was administered by gavage intragastrically (500 μg/dose in 0.2 ml of 0.9 % NaCl) or by an intraperitoneal injection (500 μg/dose in 0.2 ml of 0.9 % NaCl). These mice are referred as LF groups and labeled in the figures and tables as BLF or HLF.
In vivo and ex vivo protocols
Mice were subjected to halothane anesthesia for blood collection by the retro-orbital plexus. Mice were killed by cervical dislocation and organs were collected as described below.
Assessment of the blood cell composition
Blood smears were prepared on microscope glasses, dried and stained with Giemsa and May-Grünwald reagents. The smears were subsequently reviewed under the light microscope at 1,000× magnification. The blood cell compositions were presented as a percentage of a given cell type: neutrophil precursors (band forms), mature neutrophils (segments), eosinophils, lymphocytes and monocytes.
Determination of the pleural exudates cell number, composition and cytokine level
The pleural exudates and lungs were collected from mice after the pleural cavities were washed with 0.2 ml of 0.9 % NaCl containing EDTA (10 mM) for each cavity. Fifty (50 μl) of the pleural lavage was taken for determination of cell number, the lavage then was centrifuged and the supernatant was saved for cytokine determination (frozen at −20 °C). From the cell pellet a smear was prepared, dried and stained with Giemsa and May-Grünwald reagents. Cell numbers were enumerated in a Bürker hemocytometer. The cell types composition in the pleural fluid was determined by an independent histologist at 1,000× magnification. The exudates cell compositions were presented as a percentage of a given cell type: neutrophil precursors (band forms), mature neutrophils (segments), eosinophils, basophils, lymphocytes, monocytes, macrophages and mastocytes.
The concentrations of IL-5 and IFN-γ in the pleural fluid were measured by ELISA kits according to manufacturer’s instructions.
Histological analysis
The lungs removed from each mouse were fixed in 4 % formalin solution for 48 h, washed, dehydrated, cleared in xylene and embedded in paraffin. The paraffin embedded tissues were sectioned in a Micron HM310 microtome into 6 μm sections. The sections were stained with hematoxylin and eosin (H&E) and viewed under Nikon Eclipse 801 microscope. The pathologist viewing and interpreting all histological sections was blinded to the type of experiment and treatment.
Statistics
The results of one representative experiment from three independent experiments were presented. The results are presented as mean values ± standard error (SE). Brown–Forsyth’s test was used to determine the homogeneity of variance between the groups. When the variance was homogenous, analysis of variance (one-way ANOVA) was applied, followed by post hoc comparisons with the Tukey’s test to estimate the significance of the differences between groups. Nonparametric data were evaluated with the Kruskal–Wallis’ ANOVA, as indicated in the text. Significance was determined at P < 0.05. Statistical analysis was performed using STATISTICA 7 for Windows.
Discussion
The results presented in this report demonstrated effectiveness of LF in lessening of OVA-induced pleurisy, a well-established experimental mouse model of asthma. These findings are in accordance with a generally accepted view on the immune modulatory action of LF [
17]. Our previous studies revealed that oral administration of LF to volunteers [
24] regulated cytokine production by blood cell cultures and elicited neutrophil release into circulation. On the other hand, oral application of LF before surgery attenuated postsurgical decline in immune reactivity [
25]. In mice, LF given in drinking water, reversed suppression of the immune response caused by the immobilization stress [
26]. Also LF has been shown to protect against immune-mediated tissue damage in various experimental models. For example, mice treated with BLF had increased survival and decreased gut tissue destruction after LPS injection [
27]. This also holds true for damage elicited by mycobacterial antigen trehalose 6,6″-dimycolate [
28]. Additionally, LF added together with BCG vaccine resulted in increased protection against an aerosol TB challenge, with evidence of decreased lung damage [
29]. Orally administered LF proved also to be protective in viral [
30] and fungal [
31] infections in mice.
Although the effectiveness of oral administration of LF is well established, the mechanism(s) are still under investigation. We postulate that LF may interact with cells present in the lamina propria of the oral cavity such as dendritic cells, macrophages and T lymphocytes via LF receptors (e.g. TLR, CD14, mannose receptor or sialoadhesin). Resident lingual CD4
+ T lymphocytes, bearing TLR2 and TLR4 and comprising both suppressive T cells and cells with effector function seem to be of a primary importance [
32]. These cells are able to produce main regulatory cytokines (IFN-γ, IL-4, IL-10 and IL-17) upon antigenic stimulation. It is, therefore, conceivable that these cells may proliferate and penetrate the circulation and the lymphatic system thus altering the immune reactivity upon second antigenic challenge.
Here we report significant decrease in numbers of eosinophils in the blood and pleural exudates following application of LF. That phenomenon was well correlated with the decrease of IL-5 concentration in the pleural fluid (Fig.
5a) and is in accord with generally accepted views on the interrelationship between eosinophil number and IL-5. IL-5 is a major maturation and differentiation factor for eosinophils [
33] and belongs also to Th2-type cytokines involved in development of allergic immune responses [
34]. Taking into account that LF had no effect on IFN-γ level (Fig.
5b) one can assume that the shift to Th1-type response was the major mechanism of the protective LF action in this model.
However, considering various modes of anti-inflammatory actions of LF, other phenomena may also contribute to the overall inhibitory effect of LF in OVA-induced pleurisy. For example, although administration of LF did not lower the mastocytes content in the pleural exudates (Table
3) it suppressed mastocyte de-granulation (Figs.
2,
4). That could be a consequence of inactivation of proteolytic mast cell enzymes by LF [
18,
19]. In addition, the increase in macrophage and neutrophil contents in the pleural exudates (Table
3) may also play a role in inhibition of Th2-mediated allergy [
35,
36] by mechanisms involving increased IFN-γ production.
The histological analysis revealed only an early phase of lung congestion in the LF-treated mice. Such lesions precede development of alveolar hemorrhages and pulmonary edema, which were observed in the lung of sensitized mice. On the basis of the histological examination we concluded that LF prevented the progress of the above mentioned lesions.
Of interest, the effective dose range of LF was rather narrow, i.e. 800 μg/dose was too high and 50 μg not enough to exert a strong suppressive effect. Moreover, at 800 and 400 μg doses the appearance of neutrophil precursors was noted, probably associated with the ability of LF to induce myelopoiesis at high doses [
37]. In addition, the suppression of the allergic reaction was comparable for bovine and human milk LFs. Lastly, buccal, intragastric and intraperitoneal routes of LF administrations were similarly effective suggesting involvement of the same receptor responsible for the anti-inflammatory effect. LF as an evolutionary old protein interacts with receptors associated with innate immunity, widely distributed throughout the body [
38]. Toll like receptors, in particular TLR4, are good candidates for mediation of LF protective action in this experimental model since this protein interacts with TLR4 via the carbohydrate chains [
39]. Interestingly, ligation of TLR4 on oral Langerhans cells is important to develop tolerogenic state to pathogens [
40]. Among TLRs (TLR1-8) the expression of TLR4 is significantly lower on peripheral blood cells of asthmatic patients [
41] and it was also associated with a decreased ex vivo production of Th1-type and anti-inflammatory cytokines by these cells. It is, therefore, conceivable that activation of cells by LF via TLR4 may lead to suppression of the allergic immune response described in this work. Additional studies will be necessary to determine LF’s role in maintaining immune homeostasis during allergic insults. Such studies should be grounded in the precept that the impact of LF on the regulation of physiological parameters is conditioned by the physiological state of the host.
In summary, this investigation expanded our knowledge on the suppressive effect of LF in allergy and suggests that buccal application of LF may be effective in amelioration of allergy symptoms in patients.