Local and systemic activity of the polysaccharide chitosan ... - CiteSeerX

Uncorrected Version. Published on April 4, 2005 as DOI:10.1189/jlb.0904541

Local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration Carina Porporatto,* Ismael D. Bianco,† and Silvia G. Correa*,1 *Inmunologıá, CIBICI (CONICET), Departamento de Bioquı´mica Clıńica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Ciudad Universitaria, Argentina; and †Centro de Excelencia en Productos y Procesos de la Provincia de Co´rdoba (CEPROCOR), Agencia Co´rdoba Ciencia S.E. and CONICET, Argentina

Abstract: Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin, which exhibits particular properties: interacts with negatively charged sites on the cell surface; changes the permeability of intestinal epithelium, enhancing the uptake of peptides and proteins; and activates leukocytes. Antigens coadministered or encapsulated with the polysaccharide show improved mucosal and systemic humoral immune responses, although the mechanism is poorly understood. Herein, we characterized in Peyer’s patches mesenteric lymph nodes and spleen molecular events triggered after oral administration of chitosan in the absence of protein antigen. Sixteen hours after feeding, we studied the uptake and distribution of the polysaccharide, the phenotype of recruited antigen-presenting cells (APC), the induction of cytokines such as tumor necrosis factor ␣, interleukin (IL)-12, IL-4, IL-10, and transforming growth factor-␤ (TGF-␤), and the activation of T lymphocytes. We show here that the uptake of chitosan at inductive mucosal sites involves CD11b/cⴙ APC and that chitosan feeding increases the percentage of OX62ⴙ dendritic cells, which up-regulate the major histocompatibility complex class II antigens without changing the expression of costimulatory CD80 or CD86 molecules. The polysaccharide elicits the release of IL-10 as well as the expression of IL-4 and TGF-␤ in mucosa, and in spleen, the activation of CD3ⴙ T cells occurs. Our results demonstrate that chitosan acts by enhancing the T helper cell type 2 (Th2)/ Th3 microenvironment in the mucosa. A single dose of this polysaccharide exhibits local and systemic effects, and its activity could be relevant in the maintenance of the intestinal homeostasis. J. Leukoc. Biol. 78: 000 – 000; 2005. Key Words: mucosa 䡠 immature dendritic cell 䡠 cytokines

INTRODUCTION

nucleotide-binding oligomerization domain proteins, the cytokine milieu, and the genetic background of the host [1– 4]. Mucosal immunity can be modulated by the coadministration of antigens with adjuvants, bacterial components, or delivery systems that improve the uptake [5–7]. Currently, the only widely tested mucosal adjuvants are cholera toxin, the Escherichia coli heat-labile toxin, and their derivatives [8, 9]. As their suitability for use in humans is not possible as a result of their toxicity, evaluation of additional mucosal adjuvants is needed [10]. Potential candidates are the bacterial-derived immunostimulant monophosphoryl lipid A, which retains some of the toxicity of the lipopolysaccharide (LPS) [11], and the family of the aminoalkylglucosamine phosphates, which are chemically synthesized [12, 13]. Chitosan is a cationic biocompatible polysaccharide built by repeated units of N-acetyl-D-glucosamine and D-glucosamine, derived from the partial deacetylation of chitin, a natural polysaccharide extracted from the crustacean shells. Chitosan is also found in some microorganisms in yeasts and fungi [14]. It has been administered to humans by several routes without toxic effects [15]; it also has been used as dietary fiber [16, 17] and represents a new generation system for antigen delivery [18 –21]. At the mucosal level, this mucoadhesive polysaccharide interacts through positively charged amino groups with negatively charged sites on the cell surface. The interaction causes a redistribution of protein zonula occludens-1 and F-actin without affecting the viability of epithelial cells, although reversible perturbation of plasma membrane may occur [18]. As a consequence, changes in the permeability of epithelia take place, enhancing the uptake of peptides and proteins and increasing the contact with the immune system [18, 19]. Given its ability to deliver drugs across the mucosal lining and its potential adjuvant activity, chitosan is considered an ideal candidate for mucosal immunization [22, 23]. Coadministered with protein antigens, chitosan modifies the uptake and/or the distribution of the relevant antigen and enhances the release of regulatory cytokines associated to the antigen-specific stimulation early after feeding [24]. Antigens coadminis-

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The outcome of the immune response to orally delivered antigens strongly depends on the nature, degradation, and uptake of the antigen, the signaling through Toll-like receptors and 0741-5400/05/0078-0001 © Society for Leukocyte Biology

Correspondence: Inmunologıá, CIBICI (CONICET), Departamento de Bioquı´mica Clıńica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Co´rdoba, Argentina. E-mail: [email protected] Received September 21, 2004; revised February 24, 2005; accepted March 4, 2005; doi: 10.1189/jlb.0904541

Journal of Leukocyte Biology Volume 78, July 2005

Copyright 2005 by The Society for Leukocyte Biology.

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tered or encapsulated with the polysaccharide show improved mucosal and systemic humoral immune responses, although the mechanism is poorly understood [22, 23, 25]. Moreover, this polysaccharide is able to activate leukocytes in vitro and in vivo [24, 26] with different effects depending on the metabolic status of the cell [27]. Its mucoadhesive properties and the adjuvant, activity reported previously encouraged us to study early events associated with the coadministration of chitosan with antigens frequently evaluated in oral tolerance protocols, such as type II collagen [24]. The effects observed in rats fed single or repeated doses of the polysaccharide prompted us to study the intrinsic ability of chitosan to promote a T helper cell type 2 (Th2)/Th3-biased environment at the mucosal level. In this work, we characterized molecular events triggered after a single oral administration of chitosan. Sixteen hours after feeding, we studied in Peyer’s patches (PP), mesenteric lymph nodes (MLN), and spleen the uptake and distribution of the polysaccharide, the phenotype of recruited antigen-presenting cells (APC), the induction of cytokines tumor necrosis factor ␣ (TNF-␣), interleukin (IL)-12, IL-4, IL-10, and transforming growth factor-␤ (TGF-␤), and the activation of T lymphocytes. We show here that at inductive mucosal sites, the uptake of chitosan involves CD11b/c⫹ APC. Early after chitosan feeding, the percentage of OX62⫹ dendritic cells (DC) increases, which up-regulates major histocompatibility complex (MHC) class II antigens without changing the expression of costimulatory CD80 or CD86 molecules. At mucosal tissues, the polysaccharide elicits the release of IL-10 as well as the expression of IL-4 and TGF-␤ mRNA, and in the spleen, the activation of CD3⫹ T cells occurs. To the best of our knowledge, our work provides the first evidence in vivo of the activation of mucosal immune cells after feeding a low dose of this cationic polysaccharide in the absence of the protein antigen.

MATERIALS AND METHODS Animals In this study, we used female 8- to 10-week-old Wistar rats weighing 180 – 230 g. Animals were housed and cared for at the Animal Resource Facilities, Departamento de Bioquı´mica Clıńica, Universidad Nacional de Co´rdoba (Argentina), in accordance with institutional guidelines.

Feeding and cell preparation We used 85% deacetylated, low molecular weight chitosan (⬃80 kDa; Aldrich Chemical Co., Milwaukee, WI) prepared as described [24, 28] and fluorescein

TABLE 1.

isothiocyanate (FITC)-chitosan (CarboMer, San Diego, CA), as we found previously that low molecular weight chitosan has a higher ability to stimulate APC than other chitosans [27]. In the afternoon, we fed rats a final volume of 200 ␮l 0.1 M acetic acid (diluent group) or acetic acid containing 1 or 3 mg chitosan. Sixteen hours later, we removed PP, MLN, and spleens. We prepared single-cell suspensions by mechanical dispersion (PP and MLN) or spleen mononuclear cells using high-density gradient Ficoll-Paque according to standard procedures in RPMI medium supplemented with gentamicin, heparin, and 5% fetal calf serum (FCS) [24]. Table 1 shows representative numbers of cells recovered from each tissue. For uptake studies, we fed rats 1 or 3 mg of the 1:3 FITC-chitosan:chitosan mixture, and 16 h later, we removed the entire length of the small intestine. For comparative purposes, five ⬃20-cm sections (I–V) from each animal obtained from identical anatomical positions of the small intestine were obtained. We isolated two randomly selected PP per section, and we prepared cell suspensions as above. Cells were washed, fixed in 2% formaldehyde, and resuspended in phosphate-buffered saline (PBS); 50,000 events were analyzed using Cytoron Absolute (Ortho Diagnostic Systems, Raritan, NJ). In each experiment, we included PP cell suspensions from diluent-treated rats to control for intrinsic autofluorescence [29]. On the basis of forward- and side light-scatter, mononuclear cells were gated in, and dead cells were gated out. Positivity was defined as fluorescence that exceeded 98% of controls [30, 31].

Flow cytometry Analysis of phenotype and activation marker expression was performed as described [24, 31]. Briefly, 1 ⫻ 106 cells were incubated with FITC- or phycoerythrin (PE)-conjugated antibodies. Cells were stained for CD54 (1A29), CD71 (OX-26), CD3 (G4.18), CD45RA (OX-33), macrophage subset (HIS36), CD11b/c (OX-42), CD80 (3H5), and CD86 (24F; all from BD PharMingen, San Diego, CA) and MHC class II (OX-6; MCA46G) and OX62 (1029B; Serotec, Oxford, UK). All staining steps were performed at 4°C in PBS-EDTA-FCS. After incubation, cells were washed, fixed in 1% formaldehyde, and resuspended; 10,000 events were analyzed using Cytoron Absolute. Isotype controls (Sigma Chemical Co., St. Louis, MO) were run with each experiment and matched for fluorochrome. In each experiment, we included PP, MLN, and spleen cells from diluent-treated rats to control for intrinsic autofluorescence. On the basis of forward- and side light-scatter, mononuclear cells were gated in, and dead cells were gated out. Isotype-matched control histograms were included. Positivity for each probe was defined as fluorescence that exceeded 98% of controls [30].

Cytokine assessment by enzyme-linked immunosorbent assay (ELISA) and flow cytometry We determined IL-10 by ELISA using reagents and protocols obtained from BD PharMingen in supernatants of PP, MLN, or spleen cell suspensions, which were cultured with medium or restimulated with 10 ␮g/ml chitosan for 48 h at 37°C, 5% CO2. Levels of cytokines in cultures of cells from different groups without stimulation of any kind were below our level of detection. The in vitro restimulation was adapted from protocols used to assess cytokine production after oral administration of protein antigens [32–35].

Absolute Number of Mononuclear and CD3⫹ Cells of Different Experimental Groups 16 h after Feeding Mean ⫾

Mononuclear cells (⫻106) Diluent 1 mg Chitosan 3 mg Chitosan CD3⫹ cells (⫻106) Diluent 1 mg Chitosan 3 mg Chitosan

SD

Peyer’s patches

Mesenteric lymph nodes

Spleen

6.4 ⫾ 1.6 7.2 ⫾ 1.6 5.1 ⫾ 0.8

102.4 ⫾ 37.9 130.1 ⫾ 30.9 131.0 ⫾ 62.8

138.3 ⫾ 38.7 263.6 ⫾ 44.4 160.0 ⫾ 50.4

1.5 ⫾ 0.3 1.14 ⫾ 0.05 0.9 ⫾ 0.1

77.5 ⫾ 3.0 71.5 ⫾ 5.5 64.0 ⫾ 13

74.5 ⫾ 24.6 258.7 ⫾ 8.5 90.0 ⫾ 11.8

Female Wistar rats (n ⫽ 5– 6 per group) were fed diluent, 1 or 3 mg chitosan. Single-cell suspensions from PP, MLN, and spleens were prepared 16 h after feeding.

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We evaluated intracellular IL-10 and interferon-␥ (IFN-␥) content ex vivo in T lymphocytes of PP, MLN, and spleen cells prepared as described above and cultured for at least 6 h in RPMI–10 ␮g/ml brefeldin A. Then, cells were treated with FITC- or PE-labeled anti-CD3 antibodies, fixed in 4% formaldehyde for 15 min, permeabilized with PBS–1% FCS– 0.1% saponine, and stained with PE-conjugated anti-IL-10 or FITC-conjugated anti-IFN-␥ antibodies (BD PharMingen) as described [24, 36]. Isotype controls (Sigma Chemical Co.) were run with each experiment and matched for fluorochrome. After extensive washing with PBS–1% FCS– 0.1% saponine, cells were resuspended in PBS, and 10,000 events per sample were analyzed.

Expression of mRNA content for TNF-␣, IL-4, IL-12, and TGF-␤ Evaluation of TNF-␣, IL-4, and IL-12, in PP and TGF-␤ mRNA in PP, MLN, and mononuclear spleen cells was performed as described [24, 37]. Briefly, total RNA was extracted by the TRIzol威 reagent method (Life Technologies, Gibco, Grand Island, NY). TNF-␣, IL-4, and IL-12 (4 ␮g) and TGF-␤ (2 ␮g) of total RNA were incubated with 0.5 ␮g oligo(dT; Biodynamics, Buenos Aires, Argentina) for 5 min at 70°C and allowed to stand on ice for 5 min. The sample was incubated for 1 h at 42°C with 25 U RNase inhibitor (RNasin, Promega, Madison, WI), 1.25 mM deoxynucleoside triphosphate (Invitrogen, Life Technologies, Carlsbad, CA), and 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase (RT; Promega) in MMLV 5⫻ reaction buffer (Promega) in a final volume of 25 ␮l. In a total volume of 25 ␮l polymerase chain reaction (PCR) buffer (Invitrogen, Life Technologies, Brazil), 1 ␮l (for ␤-actin), 2.5 ␮l (for TNF-␣), or 5 ␮l (for TGF-␤, IL-12, and IL-4) cDNA was incubated with 1.25 U Taq DNA polymerase (Invitrogen), 1.5 mM (␤-actin, IL-12, TNF-␣, and IL-4) or 2 mM (TGF-␤) MgCl2 (Invitrogen), 1 mM deoxynucleotide triphosphate, and 0.2 ␮M (␤-actin, TNF-␣, and IL-12) or 1 ␮M (TGF-␤ and IL-4) sense and antisense primers [24]. Each sample was incubated in a thermal cycler (PTC-100 thermal cycler, M.J. Research, Watertown, MA) using one cycle at 94°C for 5 min; this was followed by 25 cycles for ␤-actin, 30 cycles for IL-4, or 35 cycles for TGF-␤, TNF-␣, and IL-12; each cycle consisted of 1 min at 94°C, 1 min at 55°C (␤-actin, TNF-␣, and IL-12) or 58°C (TGF-␤ and IL-4), and 1 min at 72°C, with a final extension at 72°C for 5 min. The linear range of amplification for each primer pair was established in independent preliminary studies. PCR products were analyzed by 2% agarose gel electrophoresis in the presence of 0.5 mg/ml ethidium bromide. Bands were analyzed with the Scion Image program and expressed as densitometric units. Results were depicted as the ratio of mRNA levels for each cytokine relative to ␤-actin mRNA levels.

To evaluate the uptake in different intestine sections, we fed rats a single dose of 1 or 3 mg FITC-chitosan:chitosan mixture. Sixteen hours later, the complete small intestine (up to the ileocaecal junction) was removed and divided in five sections (see Materials and Methods). Single-cell suspensions from two PP per section were prepared and analyzed by flow cytometry to determine the percentage and mean fluorescence intensity (MFI) of total FITC-positive cells or FITC-positive cells gated on R1, where on the basis of forward- and side light-scattering, APC are found (Fig. 1A). We used PP cell suspensions from diluent-treated rats as negative controls. As shown in Figure 1, the percentage of FITC-positive cells (Fig. 1, B and D) and the MFI (Fig. 1, C and E) for total cells (Fig. 1, B and C) and cells from the R1 region (Fig. 1, D and E) decreased down the gut,

Statistical analysis Data are shown as mean values ⫾ SD. Statistical significance and differences among groups were determined by ANOVA and Student-Newman-Keuls tests. P ⬍ 0.05 values were considered statistically significant.

RESULTS Uptake and phenotypic characterization of cells loaded with chitosan at local and systemic lymphoid tissues To determine the uptake and distribution of the polysaccharide chitosan, we used a mixture of 1 or 3 mg FITC-chitosan: chitosan. The stability of FITC-chitosan was assessed by digestion assays as described [38]. FITC-chitosan (10 mg/ml) was incubated with pepsin (0.1 mg/ml in 50 mM HCl-KCl, pH 2.0) or pancreatin (0.1 mg/ml in 50 mM phosphate, pH 8.0) for at least 4 h. Following the incubation, the pH was adjusted to 8.5 with phosphate buffer to precipitate all chitosans present. After the centrifugation at 10,000 g during 5 min, the chitosancontaining pellets were solubilized in 0.1 M acetic acid. Under all those conditions, at least 85% of the fluorescence remained associated to the polysaccharide (data not shown).

Fig. 1. Analysis of chitosan uptake at different sections of the small gut. Rats were fed 1 mg (␱) or 3 mg (•) FITC-chitosan:chitosan, and 16 h after polysaccharide administration, the gut was divided in five 20-cm sections (I–V), and cell suspensions were prepared out of two randomly selected PP per section. Cells were analyzed by flow cytometry, defining total and R1 cells on the basis of scatter (A). Data are shown as percentage and MFI of total positive cells (B and C) or positive cells gated on R1 (D and E). The correlation analysis between fluorescence (percentage or MFI) and gut segment is shown (F). A representative experiment of two similar ones is shown.

Porporatto et al. Activity of chitosan after feeding

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with the lowest values in the distal sections (V section). Compared with total cells, R1-gated cells showed higher percentages (P⬍0.05) and a lower MFI (P⬍0.05). The reduced MFI values in this subset could be a result of a more efficient processing of the polysaccharide by enzymes such as chitinases present in APC [39]. No differences were observed with 1 and 3 mg chitosan on the basis of per-section analysis. The correlation between fluorescence recovery (percentage or MFI) and gut segment showed a significant negative trend (Fig. 1F). Together, these results demonstrate that the uptake of chitosan takes place mostly at the upper portion of the small gut and is mediated mainly by cells of the region, where on the basis of forward- and side light-scattering, APC are found. To identify the subpopulations loaded with FITC-chitosan following oral administration of the polysaccharide, we used PE-labeled antibodies against CD3 (T cells), CD45RA (B cells), the macrophage subset, and CD11b/c (monocyte/DC) molecules. Figure 2A shows the percentage of CD3-, CD45-, macrophage-, and CD11b/c-positive cells in PP, MLN, and the spleen as well as the percentage of FITC-positive cells obtained after gating on PE-positive cells. Representative histograms of PP, MLN, and spleen FITC⫹ cells gated on PEpositive cells from diluent and 1 mg FITC-chitosan:chitosanfed rats are also shown (Fig. 2B). At mucosal and systemic tissues, the fluorescence associated with the polysaccharide was located mainly in the APC subset, including DC and macrophages. However, a clear difference in the frequency of CD11b/c⫹ FITC⫹ cells was observed in PP, MLN, and spleen. Although PP and MLN showed a similar frequency of CD11b/c⫹ cells (⬃7%), the highest percentage of CD11b/c⫹ FITC⫹ cells (⬃33%) was observed in PP 16 h after feeding the polysaccharide. Approximately 15% of CD11b/c⫹ cells in MLN was FITC⫹, and in spleen, only 7% of CD11b/c⫹ cells were also FITC⫹. These results show that 16 h after feeding, chitosan-loaded cells are present in PP, MLN, and spleen. However, the polysaccharide is mainly distributed in the DC/ macrophage subpopulation at the inductive sites of the mucosal immune system.

Cytokine analysis after chitosan feeding Cytokines such as IL-10 and TGF-␤ participate in the mucosal response to food antigens. However, little is known about the expression pattern of cytokines induced by the oral administration of polysaccharides in the absence of protein antigen. For this purpose, we assessed ex vivo the expression of TNF-␣, IL-12, and IL-4 mRNA in PP 16 h after the administration of a single dose of chitosan. As shown in Figure 3A, the polysaccharide did not modify the IL-12 mRNA expression or even decreased the TNF-␣ mRNA expression at the 3 mg dose (P⬍0.05 vs. diluent). However, a significant increment in the IL-4 mRNA expression was observed with both doses of chitosan (P⬍0.05 vs. diluent). As a result of the pivotal role of TGF-␤ and IL-10 in mucosal responses, we also assessed ex vivo the expression of TGF-␤ in cell suspensions obtained after feeding. As shown in Figure 3B, the polysaccharide up-regulated the TGF-␤ mRNA expression in PP and MLN (P⬍0.05 vs. diluent). No differences were observed in the spleen (data not shown). In addition, we studied the production of IL-10 in mononuclear cells of rats fed 4


Fig. 2. Phenotype of chitosan-loaded cells in PP, MLN, and spleen. Rats were fed diluent or 1 mg FITC-chitosan:chitosan, and 16 h later, PP, MLN, and spleen cell suspensions were stained with PE-conjugated anti-CD3, antiCD45RA, antimacrophage subset (mac), and anti-CD11b/c. For the analysis, PE-positive cells corresponding to the different subpopulations were gated on and evaluated for their levels of FITC fluorescence. (A) Data are the mean ⫾ SD of the percentage of PE-positive cells (open bars) and FITC-positive cells (solid bars) present in each lymphoid tissue. The results are representative of two separate experiments using three animals per group. (B) Representative histograms of PP, MLN, and spleen FITC⫹ cells gated on PE-positive cells. The thick, bold line represents the staining of cells from diluent group with isotype control. The thin line with shading below represents the FITC fluorescence of gated PE⫹ cells.

chitosan and restimulated in vitro with the polysaccharide for 48 h as described for protein antigens [32–35] (Fig. 3C). For unstimulated cells of different groups, the amount of IL-10 secreted was below the level of detection. The 3 mg dose stimulated the increment in levels of IL-10 in PP, MLN, and spleen (P⬍0.05 vs. diluent) with no significant effect after feeding 1 mg.

Assessment of T cell phenotype and cytokine production It is well accepted that immediately after oral administration of protein antigens, peripheral T cells become activated in vivo [34]. We evaluated the expression of CD71 (early activation marker) and CD54 (activation marker) molecules in CD3⫹ cells after feeding a single 1-mg dose of chitosan (Fig. 4A). The percentage of CD3⫹ CD71⫹ cells remained unchanged in PP, MLN, and spleen, although a significant increment of http://www.jleukbio.org

Fig. 3. Cytokine production early after chitosan feading. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later PP, MLN, and the spleen were removed. (A and B) Total RNA was prepared out of cell suspensions and subjected to RT-PCR to determine mRNA expression for TNF-␣, IL-12, and IL-4 in PP, and TGF-␤ and ␤-actin in PP and MLN with specific primers as described in Materials and Methods. PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. Bands were analyzed with the Scion Image program and expressed as densitometric units. Results are depicted as the ratio of mRNA levels for each cytokine relative to ␤-actin mRNA levels. Data are the mean ⫾ SD of two experiments, each performed using total RNA obtained from two to four animals per group (*, P⬍0.05 vs. diluent). (C) PP, MLN, and spleen cell suspensions were restimulated with 10 ␮g/ml chitosan for 48 h to determine IL-10 in supernatants by ELISA. Shown is IL-10 concentration in picograms per milliliter and the SD of triplicate determinations. (*, P⬍0.05, vs. diluent and 1 mg dose; N.D., not detected).

CD3⫹ CD54⫹ cells (P⬍0.05 vs. diluent) was observed 16 h after feeding the polysaccharide. We also evaluated ex vivo in CD3⫹ cells of PP, MLN, or spleen the production of IL-10 and IFN-␥. Representative histograms show an increment of 41% in the percentage of CD3⫹ IL-10⫹ cells in PP of 3 mg chitosan-fed rats compared with the diluent group (Fig. 4B). Similar results were obtained with 1 mg chitosan (data not shown). A slight but significant (P⬍0.05 vs. diluent) increase in the percentage of CD3⫹ IFN-␥⫹ cells was observed in PP at both doses (Fig. 4C).

Assessment of costimulatory molecules in OX62⫹ cells in mucosal tissues APC from inductive mucosal sites include immature DC, which show high expression of MHC class II antigens, display low stimulatory activity, and secrete TGF-␤ and IL-10 [40 – 42]. In rat, a subpopulation of DC expresses the ␣ε2 integrin recognized by the OX62 mAb [43, 44]. In our experimental condition, FITC-chitosan⫹ cells are mainly located in R1 (Fig. 1) and CD11b/c⫹ cell macrophages and DC uptake chitosan (Fig. 2). In consequence, we examined PP and MLN cell suspensions gated in a similar region (R1) for OX62, MHC class II antigens, and stimulatory CD80 and CD86 molecule expression. After the administration of the 3 mg dose, the percentage of OX62⫹ cells increased in PP (P⬍0.05 vs. diluent) without changes or even diminution in MLN (Fig. 5). OX62⫹-gated cells showed a significant increment on the expression of MHC class II antigens after 3-mg chitosan administration (P⬍0.05 vs. diluent), with no changes in the levels of CD80 and CD86 molecules.

DISCUSSION The intrinsic processes that operate in mucosal tissues appear distinct from conventional mechanisms of innate and specific immunity [45]. Although mucosal surfaces are exposed to the external environment and taxed with antigenic loads consisting of commensal bacteria, dietary antigens, and viruses, the bias to anti-inflammatory responses is a normal attribute of the mucosal immune system [1, 4]. Herein, we studied in vivo events that occur in mucosal and systemic lymphoid tissues after oral administration of low doses of the polysaccharide chitosan in the absence of protein antigen. Early after feeding, FITC-chitosan⫹ cells are present in PP, but the percentage decreases down the small gut, which may be a result of the insolubility of the polysaccharide at higher pH in the intestinal lumen. Beneath the epithelial barrier, the polysaccharide is taken up mainly by CD11b/c⫹ cells of PP and MLN. Chitosan administration increases the percentage of OX62⫹ cells in PP. However, the contact does not fully mature DC that up-regulate MHC class II antigens but maintain a low expression of costimulatory CD80 and CD86 molecules. Unique features of immune responses in the gut are related to the microenvironment and to the presence of specialized APC populations. Freshly isolated PP DC seem phenotypically and functionally immature, express comparably low levels of costimulatory CD80 and CD86 molecules, and release IL-10 and TGF-␤ after stimulation [46, 47]. Mucosal resident DC have a propensity to induce responses rich in IL-10 and TGF-␤ cytokines, although Th1 responses can be induced following intestinal infections. We demonstrate here that a single dose of chitosan, in the absence of protein antigen, triggers at the local microenvironment the release of IL-10 as well as the expresPorporatto et al. Activity of chitosan after feeding

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Fig. 4. Phenotypic and functional changes of CD3⫹-positive cells after chitosan administration. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later, PP, MLN, and spleen cells (1⫻106) were stained with FITC-labeled anti-CD3 antibodies and PE-labeled anti-CD71 or anti-CD54 antibodies (A), PE-conjugated anti-IL-10 (B), or PE-conjugated anti-IFN-␥ (C). For intracellular cytokines, after CD3 staining, cells were fixed in PBS– 4% formaldehyde, permeabilized, and treated with the appropriate monoclonal antibody (mAb); 10,000 events per sample were analyzed. (B) Shown are representative histograms for intracellular IL-10 levels, and the percentage of CD3⫹ IL-10⫹ cells of each group is indicated. The thick, bold line represents the staining of cells with isotype control. The thin line with shading below represents the intracellular staining of CD3⫹ cells with anti-IL-10 antibody. A representative experiment out of three is shown. (C) Data are the mean ⫾ SD of the percentage of intracellular, IFN-␥-positive cells of three animals per group. The results are representative of two separate experiments (*, P⬍0.05, vs. diluent).

sion of IL-4 and TGF-␤ mRNA. Cytokines such as IL-10, TGF-␤, and IL-4 are known to suppress the function of APC. For instance, IL-10 selectively inhibits the expression of B7 on murine macrophages without effect on the up-regulation of MHC class II expression [48]. In agreement, in our experimental condition, the minor release of IFN-␥ in PP and the upregulation of MHC class II molecules seem to be counterbal6


anced by the huge production of IL-10 triggered by the polysaccharide. We show here that chitosan has biological activity at mucosal and systemic levels. The uptake and distribution of the polysaccharide as well as the activation of spleen T cells that we found are similar to previous results with peptides, proteins, or food antigens [32, 34, 49]. The transient T cell activation induced by food antigens has been related to the maintenance of the peripheral T cell population and to the shape of the T cell repertoire [32]. Considering that chitosan is a weak base that precipitates at neutral pH or higher [18], it is tempting to speculate that free chitosan is not reaching systemic lymphoid tissues as described for soluble antigens. In agreement, soluble antigens crossing the epithelial-cell barrier through the epithelium converge in the liver, and those transported by M cells or DC that reach lymphatics converge in the spleen [4]. Substances that can adhere to the epithelium or M cells are transported in much larger amounts. It is interesting that the route by which antigens cross the intestinal epithelial-cell barrier is likely to dictate the type of response that is generated [45]. Chitosan could exhibit natural properties to modulate the mucosal immune response: As a dietary fiber, it can influence local immune function by changing the intestinal flora and mucosal microenvironment [16, 17]. As a delivery agent, chitosan can decrease the clearance rate and stimulate the uptake of antigens by M cells [21–23]. As a component of fungal cell walls and parasite sheaths, chitosan could provide “danger signals” acting as an adjuvant [50, 51], possibly through the activation of components of the innate immune system such as macrophages [26, 27]. Immunizations with several immunogens and chitosan administered at the mucosal interface increase antibody responses [15, 20 –23], particularly the immunoglobulin A production, a TGF-␤- and IL-10-driven process [2, 4, 47]. When fed with a single low dose of protein, chitosan enhances the antigen-specific release of IL-10 and TGF-␤ [24]. Besides, the IL-10, IL-4, and TGF-␤ mRNA expression in PP is noticeably higher in rats fed multiple doses of chitosan before the antigen administration [24]. In the same way, the intranasal administration of the immunodominant epitope of the Dermatophagoides pteronyssinus 1 adsorbed to chitosan suppresses airway inflammation and triggers the IL-10 production by antigen-specific T cells [52]. In that experimental condition, an increment of IFN-␥ is observed and is associated to the induction of a tolerogenic state [52]. The strong stimulatory activity of chitinous derivatives has been mainly attributed to the N-acetyl-D-glucosamine residues [22, 27], and the effect could be mediated by a macrophage lectin receptor, as with mannose specificity [53]. The activity of the polysaccharide seems to depend on the activation status of the cell: Chitosan inhibits the production of inflammatory mediators by proteose-peptone-elicited peritoneal macrophages [27] or LPS/IFN-␥-activated RAW 264.7 cells [54, 55], but it stimulates the release of proinflammatory mediators by resident peritoneal macrophages [27] or unstimulated RAW 264.7 cells [54, 55]. The apparent conflicting results obtained in vitro and in vivo are not necessarily opposite effects but could reflect differences in experimental conditions or parameters evaluated. It is important to point out here that previous reports http://www.jleukbio.org

Fig. 5. Phenotype of DC of PP and MLN after chitosan feeding. Rats were fed diluent (hatched bars) or 1 mg (open bars) or 3 mg (solid bars) chitosan, and 16 h later, PP and MLN (1⫻106) were stained with FITC-labeled anti-OX62 and PE-labeled anti-MHC II anti-CD80 or anti-CD86 antibodies. The upper-left graph is a representative granularity versus size-density plot, where the R1 region was defined. Results are expressed as percentage of OX62⫹ cells in R1 in cell suspensions of PP and MLN or MFI of OX62⫹-gated cells for MHC class II, CD80, and CD86 markers. Average data of three rats per group are depicted (*, P⬍0.05, vs. diluent). RT-SC, Right-scatter; FW-SC, forward-scatter.

using chitosan as an adjuvant, as well as this study where the intrinsic activity of chitosan was evaluated, show that in vivo, this polysaccharide consistently enhances the natural phenomena that occur at the mucosal level. Together, our results demonstrate that orally administered chitosan, in the absence of protein antigen, enhances a naturally Th2/Th3-biased microenvironment at the mucosal level by stimulating the production of regulatory cytokines. Its activity could be relevant in the maintenance of the intestinal homeostasis. Our results open the possibility that in a near future, this or other polysaccharides could be used to modulate the immune response to orally administered antigens.

ACKNOWLEDGMENTS This work was supported by grants from CONICET (PEI 6163/04), Agencia Cordoba Ciencia, Agencia Nacional de Promocioń Cientı´fica y Tecnolo´gica (FONCYT), and Fundacioń ANTORCHAS. C. P. is a recipient of a fellow from CONICET. I. D. B. and S. G. C. are career members of CONICET.

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