Early events associated to the oral co-administration of type II collagen and chitosan: induction of anti-inflammatory cytokines
Carina Porporatto1,
Ismael D. Bianco2,
Ana M. Cabanillas1 and
Silvia G. Correa1
1 Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina 2 Centro de Excelencia en Productos y Procesos de la Provincia de Córdoba, 5000 Córdoba, Argentina
Correspondence to: S. G. Correa; E-mail: scorrea{at}bioclin.fcq.unc.edu.ar
Transmitting editor: S. Hedrick
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Abstract
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Oral administration of an antigen can result in local and systemic priming or tolerance and the basis of this dichotomy is poorly understood. The intestinal microenvironment, and factors such as nature of the antigen, dose, genetic background, uptake and concentration of the antigen that gain access to the internal milieu via the mucosa influence these active immunologic processes. Chitosan is a biocompatible natural polysaccharide able to promote the transmucosal absorption of peptides and proteins. The aim of our work was to study the effect of the co-administration of type II collagen (CII) and chitosan during the initial contact of the antigen with the immune system. Sixteen hours after feeding we evaluated several molecular events in mucosal and in systemic lymphoid tissues. We determined in Peyers patches (PP) and spleen cells the number and activation of T cells, the arrival of the antigens, and the cytokine profile. In PP we found a reduction in the cell number without changes in CD3+ cells. In spleen, instead, we observed an increase in CD3+ cells as well as the internalization of the CD3 complex. CII:chitosan-fed animals exhibited a reduced secretion of IL-2 with an increase of IL-10 in PP and spleen respectively. In addition, in PP, CII:chitosan-fed rats showed increased levels of mRNA for transforming growth factor-ß, IL-4 and IL-10. Together, our data suggest that the co-administration with chitosan modifies the uptake and/or the distribution of the relevant antigen, and promotes an anti-inflammatory environment early after feeding.
Keywords: collagen, chitosan, cytokine, oral tolerance, transforming growth factor-ß
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Introduction
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Chitosan is a biocompatible natural polysaccharide built by units of glucosamine and N-acetylglucosamine (1). This mucoadhesive polymer is able to promote the transmucosal absorption of peptides and proteins. Chitosan solutions at 0.5% (w/v) concentration are highly effective at increasing the absorption of insulin across the nasal mucosa in rats and sheep (2). Significantly higher amounts of macromolecular drugs can be transported after co-administration with chitosan (3), and solution formulations seem to be potent carrier systems and absorption enhancers for mucosal immunointervention (4). The mechanism of action of chitosan combines bioadhesion and a transient widening of the tight junctions in the membrane (5), and the polysaccharide induces a redistribution of cytoskeleton F-actin and the tight junction protein ZO-1, thereby modifying the integrity of the tight junctions (6,7).
Oral administration of an antigen can result in local and systemic priming or tolerance, and the basis of this dichotomy is poorly understood (8,9). These active immunologic processes are well established within 17 days of antigen administration (9). The effectiveness of mucosal immune responses is influenced by factors such as nature of the antigen, dose, genetic background, antigen uptake and concentration of the immunologically relevant antigen that gains access to the internal milieu via the mucosa (911). The intestinal microenvironment influences an immune response in several ways and the cytokine milieu might determine the outcome of the immune response (12).
Type II collagen (CII), the major protein in cartilage, is an interesting antigen to study because it is potentially relevant to common human diseases. Even when CII is frequently administered via the nasal and oral mucosa to promote tolerance in patients or experimental models of arthritis (1315), the events that govern the induction of mucosal immune responses to CII remain poorly defined. Interestingly, under different conditions, CII and chitosan form complexes by electrostatic interactions or hydrogen bonding that are resistant to digestion by enzymes like collagenase, suggesting a protective effect of this polysaccharide (1618). Considering that this polysaccharide promotes the mucosal uptake of native antigens and protects CII from digestion (1618), the aim of our work was to study the effect of the co-administration of CII and chitosan during the initial contact of the antigen with the immune system. Early after feeding CII and chitosan, we analyzed the molecular events both in mucosa and in systemic lymphoid tissues: in Peyers patches (PP), mesenteric lymph node (MLN) and spleen cells, we determined the activation of T cells, the arrival of the antigens and the cytokine profile. The results shown herein demonstrate that chitosan modifies the uptake and/or the distribution of the relevant antigen as well as the cytokine microenvironment at the time of the initial contact with the immune system, promoting an anti-inflammatory milieu in PP.
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Methods
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Animals
Female 8- to 10 week-old Wistar rats (weighing 180230 g) were used in this study. Animals were housed and cared for at the Animal Resource Facilities, Departamento de Bioquimica Clinica, Universidad Nacional de Córdoba, in accordance with the institutional guidelines.
Preparation of CII and chitosan
Native atelocollagen was isolated from the septum cartilage of young animals (
1 year old) using essentially the same procedure as previously described (19,20). Briefly, bovine cartilage was washed, defatted, ground, and digested with pepsin in 0.5 M acetic acid to remove the telopeptides and release collagen from the tissue. The collagen was treated via a series of salt fractions and dissolution in acid to reduce the content of other types of collagen (i.e. types IV, IX and XI). The purity of the preparation was analyzed by SDSPAGE as described elsewhere (21) and Western blotting developed with anti-bovine CII. The purified CII was finally dissolved in 0.1 M acetic acid to a final concentration of 0.25 mg/ml.
Low-mol.-wt chitosan (mol. wt 50 kDa), 85% deacetylated, used throughout this study (Sigma, St Louis, MO) was prepared as described previously (22).
Study of the resistance to pepsin degradation
The study was performed as described previously (18,23). Briefly, a stock solution of pepsin was prepared at 2 mg/ml in a 20 mM acetate buffer at pH 5; CII (5 mg/ml) or CII:chitosan (at 10 or 100% of the CII weight) was incubated at 37°C in 10 mM HClKCl, pH 2, at a pepsin/CII ratio of 1/40 in a dialysis bag immersed in 200 ml 10 mM HClKCl. At different times, aliquots were collected both from the dialysis bag and in the external liquid for electrophoresis and absorbance at 230 nm assessment respectively. Samples were separated electrophoretically in 10% polyacrylamide minigels (Bio-Rad, Richmond, CA) under reducing conditions (21).
Feeding protocols and cell preparation
Animals were fed in the afternoon a final volume of 200 µl. According to the fed agent, rats were assigned to one of four groups: diluent (0.1 M acetic acid), chitosan (1 mg chitosan in 0.1 M acetic acid), CII (1 mg CII in 0.1 M acetic acid) and CII:chitosan (1 mg CII + 1 mg chitosan in 0.1 M acetic acid). Sixteen hours later rats were killed and 10 randomly selected PP/rat, MLN and spleens were removed. Single-cell suspensions were prepared by mechanical dispersion according to standard procedures in RPMI medium supplemented with gentamicin, heparin and 5% FCS (24). In some experiments rats received 1 mg of chitosan from days 1 to 5 and 1 mg CII + 1 mg chitosan on day 6 (group chitosan:CII:chitosan).
Determination of cell viability
Cell damage or death after exposure to chitosan was measured by assessing the release of lactate dehydrogenase (LDH) in cell-free supernatants (25). A UV optimized method for the determination of LDH was used (Wiener, Rosario, Argentina).
Flow cytometry
Analysis of intracellular CD3 expression was performed as described (26). Briefly, 1 x 106 cells were incubated with FITC-labeled mouse anti-CD3 (PharMingen, San Diego, CA). After washing, cells were incubated with a 3- to 5-fold excess of purified anti-CD3 (PharMingen) to saturate extracellular CD3 sites. All staining steps were performed at 4°C in RPMI/EDTA/FCS. Isotype controls (Sigma) were run with each sample and matched for fluorochrome. After incubation cells were washed, fixed, permeabilized with 0.1% saponine and incubated with phycoerythrin (PE)-labeled mouse anti-CD3 (PharMingen) for intracellular protein detection. After incubation cells were washed, resuspended in 2% formaldehyde and at least 10,000 events were analyzed using a Cytoron Absolute (Ortho Diagnostic Systems, Raritan, NJ). Monocytes/macrophages, granulocytes and dead cells were gated out on the basis of forward and right angle light scatter.
Antigen-presentation assay
Splenocytes from rats fed 16 h earlier with the different treatments were incubated for 1 h at 37°C with medium or 40 µg/ml CII, washed exhaustively and irradiated (3000 cGy for 15 min). Antigen-presentation experiments were performed in 96-well microtiter plates in a total volume of 200 µl of RPMI/gentamicin/10% FCS containing irradiated 1 x 105 splenocytes as antigen-presenting cells and 1 x 105 inguinal lymph node cells from rats immunized 15 days before with CII/complete Freunds adjuvant (27). Cell cultures were maintained at 37°C in 5% humidified CO2 for 72 h. Eighteen hours before ending the culture, 1 µCi/well of [3H]thymidine was added. Cells were harvested onto glass fiber filters and [3H]thymidine incorporation was evaluated by liquid scintillation counting.
Cytokine measurement
To evaluate cytokine production, 4 x 106/ml PP or spleen cells were cultured with medium or 40 µg/ml CII. Supernatants were harvested after 24 or 48 h, and assayed for IL-2 or IL-10 respectively by ELISA using reagents and protocols obtained from PharMingen.
Intracellular cytokine detection was performed by flow cytometry stimulating 4 x 106/ml PP or spleen cells with 40 µg/ml CII for 48 h at 37°C in 5% CO2 (28). Then, cells were stained with PE- or FITC-labeled anti-CD3 antibodies, washed once, fixed in PBS/2% formaldehyde for 5 min, permeabilized by washing twice in PBS/1% FCS containing 0.1% saponine and then stained with PE-conjugated anti-IL-10 or FITC-conjugated anti-IFN-
mAb (PharMingen). After extensive washing, cells were treated with 2% formaldehyde/PBS and 10,000 events per sample were analyzed.
Evaluation of IL-10, IL-4 and transforming growth factor (TGF)-ß mRNA content in PP cells was performed as described (29). Briefly, total RNA was prepared by the TRIzol reagent method (Life Technologies, Gibco). Two micrograms of total RNA was incubated with 0.5 µg of 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 Ribonuclease inhibitor (RNasin) (Promega, Madison, WI), 1.25 mM deoxynucleoside triphosphates (Invitrogen, Life Technologies, CA), 200 U MMLV reverse transcriptase (Promega) in MMLV 5 x reaction buffer (Promega) in a final volume of 25 µl. In a total volume of 50 µl PCR buffer (Invitrogen, Life Technologies, Brazil) 2 µl (for IL-10 and ß-actin) or 5 µl (for TGF-ß and IL-4) of cDNA was incubated with 1.25 U of Taq DNA polymerase (Invitrogen), 1.5 mM (ß-actin, lL-10 and IL-4) or 2 mM (TGF-ß) MgCl2 (Invitrogen), 1 mM deoxynucleotide triphosphates and 0.2 µM (IL-10 and ß-actin) or 1 µM (TGF-ß and IL-4) sense and anti-sense primers (29). Each sample was incubated in a thermal cycler (PTC-100 thermal cycler; M. J. Research) using 1 cycle at 94°C for 5 min; this was followed by 25 cycles for ß-actin, 30 cycles for IL-10 and IL-4 or 35 cycles for TGF-ß. Each cycle consisted of 1 min at 94°C, 1 min at 55°C (IL-10 and ß-actin) or 1 min at 58°C (TGF-ß and IL-4) and 1 min at 72°C. To measure PCR products semiquantitatively, 2 µl of cDNA product was serially diluted 2-fold, and amplified by using 20, 25, 30 and 35 cycles for cytokines and 20, 22, 24 and 26 cycles for ß-actin genes under the same conditions described above. The linear range of amplification for each primer pair was established in independent preliminary studies. The PCR products were analyzed by 2% agarose gel electrophoresis in the presence of 0.5 mg/ml ethidium bromide. The immunoreactive protein bands were analyzed with the Scion Image program. Results were expressed as relative densitometric units.
Statistical analysis
All data are shown as mean values ± SD. Statistical significance and differences among groups were determined by analysis of variance and StudentNewmanKeuls tests. P < 0.05 values were considered statistically significant.
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Results
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Study of the protective effect of chitosan on the enzymatic digestion of CII
Orally administered proteins are exposed to the activity of enzymes like pepsin or trypsin, yielding peptides with different immunogenic activity. We evaluated the ability of chitosan to protect CII during the digestion with pepsin in the absence or in the presence of 10 or 100% chitosan. The reaction was made in a dialysis bag immersed in KClHCl solution; in such conditions, short chains of CII resulting from the digestion by the enzyme were able to cross the membrane bag. Aliquots from internal and external liquids were sampled at different times for absorbance at 230 nm and electrophoresis assessment respectively. Figure 1(A) shows OD values at different times of the digestion. Compared with the absorbance plot of CII, when 10 or 100% chitosan was included in the mixture, lower OD values were obtained through the hydrolysis period studied, suggesting a reduced number of short fragments in the external fluid. Figure 1(B) shows electrophoresis of the CII and CII plus 100% chitosan at the initial and final times of the digestion assay respectively. As can be seen, CII exhibits thinner and less defined bands after enzymatic treatment, according to a more pronounced digestion. For CII plus 100% chitosan, instead, more intense bands were observed at the final time. Protein bands at the initial and final times were semiquantified by densitometry, and the percentage of CII present at the final time was 74.41% for CII and 87.26% for CII plus 100% chitosan of the initial sample. These results show that chitosan is able to protect CII, suggesting that a co-administration protocol could contribute to preserve the native antigen after feeding. Similar results were obtained with trypsin (data not shown).

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Fig. 1. Protective effect of chitosan on the enzymatic digestion of CII. A digestion assay with pepsin was performed in a dialysis bag immersed in HClKCl, pH 2, solution. At different times, aliquots from internal and external fluids were collected for absorbance at 230 nm (A) and electrophoresis assessment (B) respectively. Equal volumes were resolved on SDSPAGE; a representative experiment for the digestion assay in the presence of 100% chitosan is shown: (diamonds) CII; (squares) CII in the presence of 10% chitosan; (triangles) CII in the presence of 100% chitosan. IT: initial time, FT: final time.
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Cellularity in PP 16 h after feeding
Evidence supports the notion that early events are crucial in the priming or tolerance at mucosal sites (9). Within 24 h of feeding, the antigen traverses the gut epithelium, is presented by antigen-presenting cells, and triggers activation, anergy and deletion of T cells (26). To assess the effect of the co-administration of CII and chitosan during the initial contact with the immune system, we evaluated several parameters 16 h after feeding a single dose of diluent, CII, chitosan and CII:chitosan. Single-cell suspensions were prepared by mechanical dispersion of 10 randomly selected PP/rat according to standard procedures and cellularity was expressed as cell number/PP. As shown in Fig. 2, the cellularity in PP is reduced in animals receiving CII, chitosan and CII:chitosan (P < 0.01 versus diluent), suggesting the traffic of cells between compartments or cell loss by damage/apoptosis.

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Fig. 2. Cellularity in PP 16 h after feeding. Diluent-, 1 mg CII-, 1 mg chitosan- or 1 mg CII plus 1 mg chitosan-fed rats were killed, and 10 randomly selected PP/rat were obtained and single-cell suspensions were prepared and counted according to standard procedures. Results are means ± SD of at least four rats/group; data from three experiments were pooled. ** P < 0.01 versus diluent.
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To monitor cell damage/death, supernatants of PP treated in vitro with chitosan were assayed for LDH activity (25). Levels of LDH did not increase significantly after 6, 12 or 24 h of culture (data not shown), excluding the possibility of cell damage and suggesting that traffic between lymphoid compartments was taking place. In addition, at the time of our study we found no microscopic evidence of apoptosis (data not shown).
Changes in percentages of CD3+ cells after feeding
Peripheral T cells are immediately activated in vivo after oral administration of antigens like myelin basic protein and activation is associated with the antigen-induced TCRCD3 complex down-modulation (26). To examine this possibility, PP, MLN and spleen cells were labeled with FITC-conjugated anti-CD3 antibody, permeabilized and labeled with PE-conjugated anti-CD3; positive FITCCD3 cells were gated and analyzed for internal CD3 expression. Table 1 shows percentage and MFI of surface and internal CD3 expression of different groups 16 h after feeding. The percentage of surface CD3+ cells is reduced in PP of CII- or chitosan-fed rats (versus diluent P < 0.01), but remains unchanged in the CII:chitosan group (versus diluent P > 0.05; versus CII and chitosan P < 0.01). On the other hand, the percentage of CD3+ cells in spleen is increased in both groups receiving chitosan (versus diluent P < 0.01; versus CII P < 0.01 or P < 0.05). We believe that this finding could represent the recruitment of naive T cells by the polysaccharide. We did not assess the contribution of recruitment versus proliferation in spleen. However, chitosan itself is not able to induce or to expand T cell proliferation (1,2). In additional experiments, rats fed FITC-labeled chitosan showed positive cells in the spleen 16 h after feeding, supporting the arrival of the polysaccharide to the spleen (data not shown). No changes in MLN CD3+ cells were observed.
Results of the assessment of internal CD3 expression show that only a significant increase in the percentage of spleen positive cells was observed in rats receiving CII:chitosan (versus diluent, CII and chitosan P < 0.05) with no differences in PP or MLN cells. The density of CD3 expression evaluated as the MFI was similar in all groups for both surface and internal CD3. In agreement, it has been shown that a single dose of antigen induced immediate TCR down-modulation and T cell activation in vivo (26).
Uptake of the antigen after a single feeding
Orally administered proteins either intact or degraded are rapidly distributed to and recognized in the spleen (25,27). Then, we tested whether spleen cells have the capacity to stimulate CII-specific T cell responses in vitro by an antigen-presentation assay. Spleen mononuclear cells obtained 16 h after feeding were incubated with or without 40 µg/ml CII for 1 h, irradiated and co-cultured with inguinal lymph node cells from rats immunized 15 days before with CII in adjuvant. Figure 3 shows that ex vivo, splenocytes from CII-fed rats stimulated the proliferation of CII-specific T cells (P < 0.01 versus diluent) with no effect when cells from the CII:chitosan group were used. When splenocytes were incubated with CII before the assay, a similar proliferation was observed with cells from the different groups (P = NS). Together, these findings suggest that a reduced amount of CII is reaching the spleen in CII:chitosan-fed animals, possibly due to a local retention of the antigen in PP.

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Fig. 3. Spleen cells from CII:chitosan-fed rats are unable to stimulate CII-specific T cells in vitro. Splenocytes from rats killed 16 h after a single feeding (n = 4/group) were incubated for 1 h with medium or 40 µg/ml CII, irradiated and cultured with 1 x 105 inguinal lymph node cells from rats immunized 15 days before with CII with complete Freunds adjuvant. White bars: splenocytes cultured with medium before irradiation. Black bars: splenocytes cultured with 40 µg/ml CII before irradiation. Proliferation is expressed as c.p.m. and results are means ± SD of a representative of three independent experiments. **P < 0.01 versus diluent.
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Cytokine analysis
Supernatants from in vitro cultures of PP and spleen cells obtained 16 h after feeding were tested for the production of IL-2 and IL-10 by ELISA. When PP or spleen cell cultures were examined without stimulation of any kind, levels of cytokines secreted were below our level of detection. As shown in Fig. 4(A), following 24 h stimulation with CII, the amount of IL-2 was significantly reduced in cultures of PP from CII:chitosan-fed rats, achieving about half the values from the diluent group (P < 0.05). In cultures of spleen cells from chitosan- and CII:chitosan-fed rats, the IL-2 production was significantly less (P < 0.05 versus diluent). For IL-10 production instead (Fig. 4B), following a 48-h stimulation with CII, the amount of IL-10 was significantly increased in cultures of PP from chitosan- and CII:chitosan-fed rats (P < 0.05 versus diluent). In cultures of spleen cells, only CII:chitosan-fed rats produced increased levels of IL-10 (P < 0.05 versus diluent).

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Fig. 4. Production of IL-2 and IL-10 in PP and spleen after feeding. Rats fed a single dose of diluent, 1 mg CII or 1 mg CII plus 1 mg chitosan were killed 16 h later and single suspensions of cells were prepared as described in Methods; 4 x 106/ml cells were cultured with 40 µg/ml CII. Supernatants were harvested after 24 or 48 h and assayed for IL-2 or IL-10 by ELISA respectively. Results are means ± SD of at least four rats/group; data from three experiments were pooled. *P < 0.05 versus diluent.
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Moreover, IL-10 and IFN-
production was assessed by flow cytometry; histograms depicted in Fig. 5 show the percentage of IL-10+ CD3 lymphocytes in PP or spleen of different groups. This is a representative experiment out of three. As can be seen, the percentage of IL-10+ cells in PP of chitosan- and CII:chitosan-fed rats is higher compared with diluent. On the other hand, the proportion of IL-10+ cells increases in spleens of chitosan-, and in a minor proportion of CII- and CII:chitosan-fed rats. For IFN-
, instead, levels matched the isotype control in all groups, showing that this cytokine was not re-stimulated after feeding (data not shown).

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Fig. 5. Production of IL-10 in CD3+ cells of PP and spleen after feeding. Rats fed a single dose of diluent, 1 mg CII or 1 mg CII plus 1 mg chitosan were killed 16 h later and single suspensions of cells were prepared as described in Methods. Intracellular cytokine detection was performed by flow cytometry stimulating 4 x 10 6 cells/ml with 40 µg/ml CII for 48 h at 37°C in 5% CO2. Cells (1 x 106) were stained with FITC-labeled anti-CD3 antibodies, fixed in PBS/2% formaldehyde, permeabilized and stained with PE-conjugated anti-IL10 mAb; 10,000 events per sample were analyzed. Positive CD3 cells were gated and analyzed for IL-10 staining. Results are presented as histograms; the percentage of IL-10+ CD3 cells of each group is indicated. A representative experiment out of three is shown.
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Together, the decreased production of IL-2, the activation of T cells evaluated as the down-modulation of CD3 and the production of IL-10 are very suggestive of cells rendered anergic in vivo that may become regulatory cells as described previously (30).
To better characterize at the local level the cytokine microenvironment that could participate in this early step of the induction of the immune response, PP cells from different groups were analyzed for TGF-ß, IL-4 and IL-10 mRNA expression using RT-PCR 16 h after feeding. Figure 6 shows that TGF-ß, IL-4 and IL-10 mRNA were up-regulated in CII:chitosan-fed rats, in contrast to animals receiving diluent, CII or chitosan (Fig. 6A and C). In agreement, low doses of antigens can induce expansion and/or activation of cells secreting regulatory cytokines and single or multiple feeds can modify the expression level of these cytokines in PP (31).

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Fig. 6. Suppressive/regulatory cytokine mRNA expression is up-regulated in PP after chitosan feeding. Cytokine mRNA content in PP was evaluated in rats fed a single dose of CII, chitosan or CII:chitosan; additionally, a group received 1 mg chitosan during 5 days and CII:chitosan the day later (group Ch:CII:Ch). Sixteen hours after the last feeding, rats were killed, and total RNA was prepared and subjected to RT-PCR with specific primers as described in Methods. PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. Relative intensity was normalized with ß-actin and expressed as relative densitometric units. Results are representative of two experiments, each performed using RNA obtained from a pool of three rats/group.
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In view of the fact that in chitosan-fed animals we observed systemic and local effects indicating some modulatory activity of this polysaccharide, five doses of 1 mg chitosan were administered to naive rats on alternate days plus a single dose of CII:chitosan on day 6 (group chitosan:CII:chitosan) to assess whether chitosan was able to condition the mucosal site before the arrival of the antigen. As can be seen (Fig. 6B and C), mRNA expression levels of the suppressive/regulatory cytokines TGF-ß, IL-4 and IL-10 are enhanced in PP after treatment with a marked increase in TGF-ß and IL-4 transcripts.
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Discussion
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The biocompatible and biodegradable polysaccharide chitosan is able to increase the paracellular transport of macromolecules, accelerate wound healing and stimulate innate immune cells (1,32,33). Our hypothesis was that the initial contact of the fed antigen CII with the immune system could be modified by the co-administration of chitosan considering that this polysaccharide promotes the mucosal uptake of native antigens. Our work demonstrates that chitosan reduced the enzymatic digestion of CII, suggesting the protective effect of the polysaccharide. In addition, when co-administered with CII, chitosan modified the availability of the relevant antigen, induced changes in T cell activation in mucosal and systemic compartments, and stimulated the expression of anti-inflammatory cytokines, particularly in PP. These events occurring early after feeding of the antigen could be relevant in the settling of a microenvironment to promote the induction of tolerance to CII.
CII and chitosan can form polyanionpolycation complexes by means of electrostatic interactions or by hydrogen bonding (1618). Depending on the conditions, chitosan induces a strong protection toward CII digestion by collagenase. In contrast to other polymers such as polyacrylates, chitosans do not exhibit any enzymatic inhibitory properties (34), and it is accepted that interactions between chitosan and the enzymes could modify the activity (17,18). In fact, the building of CII:chitosan complexes implies a denaturation of CII triple helices which become less sensitive to specific collagenases (18). A similar mechanism could explain the milder enzymatic digestion of CII observed in our experimental condition, suggesting that the co-administration with chitosan may have a protective effect against proteases found in gastric and intestinal fluids.
To characterize early events associated with the oral administration of CII:chitosan, we started evaluating the input of cells into the mucosal compartment. The diminished PP cellularity 16 h after feeding in CII, chitosan and CII:chitosan groups was indicative that loss of cells or emigration occurred. Cell damage assessed by LDH levels was discarded and we found no evidence that apoptosis was taking place at the time of our study. In addition, apoptosis is not considered an early event after oral administration of antigens. In T cell transgenic models apoptosis is induced in the intestine only 4872 h after feeding (35). Our data suggest instead that in normal naive rats, a pronounced emigration was taking place after feeding chitosan, CII or both. Accordingly, evidence shows that antigen-primed cells migrate to effector sites in the gut lamina propria and epithelium as well as to other mucosal tissues after feeding (36). However, the kinetics of the migration from the gut to the peripheral lymphoid organs has not been established (26).
Despite the reduced cellularity observed in PP, after surface CD3 labeling we observed that whereas the percentage of CD3+ cells diminished in CII and chitosan groups, in CII:chitosan-fed rats it remained unchanged. Studies with transgenic TCR mice show that antigen-specific T cells appear rapidly in PP after a feed of antigen, suggesting that tolerance may be initiated in these organs (3539). Rapid T cell activation localized particularly in mucosal tissues is observed within hours after feeding soluble protein antigens such as ovalbumin (39). On the other hand, the internalization of the CD3 complex was observed only in the spleen of CII:chitosan-fed rats. Remarkably, after oral exposure to immunogenic or tolerogenic antigens T cell activation and division occurs simultaneously at the local and systemic level, suggesting that fed antigen is presented throughout the animal during priming and tolerance induction (40). We consider that our findings are suggestive of differences in the amount and the nature of CII that is reaching the immune system in CII- or CII:chitosan-fed animals. The form, site and dose of the antigen dictate the fine microenvironmental influence of T cell migration and sensitization (41). T cells are sequestered at the site of antigen presentation (42) and, in animal models after oral delivery of the relevant antigen, a small accumulation of transgenic T cells in PP is observed (40). Interestingly, feeding immunogenic antigen, i.e. associated with cholera toxin, could result in increased persistence of T cells when compared with the soluble form of the antigen (40).
Consistently, our results of antigen uptake showed that CII is not reaching the spleen, at least in an immunogenic way, when co-administered with chitosan. Several possibilities may explain these data. First, it is possible that properties of chitosan operate to retain the antigen in PP avoiding the pronounced emigration of T cells observed in CII- or chitosan-fed rats. We have no data yet on the effective dose of CII that is gaining access to mucosal and systemic lymphoid tissues in our system. It is well accepted that oral administered proteins are partially, if not completely, digested before crossing the mucosal lining (43). However, the protective effect of chitosan on the enzymatic digestion combined with the absorption enhancement (1,3,32) could determine in our model a higher and sustained supply of less degraded CII at inductive mucosal sites that strongly influence the outcome of the immune response. On the other hand, it is important to point out here that CII, being a matrix protein, differs from classical immunogens: collagens in general are notoriously bad immunogens, and dendritic cells are unable to process and present CII (44). This is currently under study, but it is tempting to speculate that very particular events are taking place in PP after the uptake of CII:chitosan.
Secondly, soluble factors released locally by chitosan-stimulated innate cells may affect the activation state of T lymphocytes even in the presence of adequate levels of the antigen. Accordingly, chitosan enhances the functions of inflammatory cells such as macrophages after binding N-acetyl-D-glucosamine residues to mannose receptors, which mediate the internalization of the chitosan particles (33,45). In that way, chitosan promotes the production of TGF-ß-1 and platelet-derived growth factor (7,33), and enhances the alternative metabolic pathway of L-arginine in rat macrophages, which is associated with an anti-inflammatory profile (45).
The increase of internal positive CD3 spleen cells observed in CII:chitosan-fed rats could represent recently emigrated lymphocytes that, exposed to the antigen in PP, experience in the spleen the activated state that precedes anergy. This process, associated with a down-regulation of TCR expression, is usually coupled with the inability of a mature T cell to proliferate and secrete IL-2 (26,41). It has been shown that TCR is internalized in vivo in response to oral administration of a high dose of myelin basic protein (26) and the level of TCR down-modulation in vivo is related directly to the dose of tolerogen (26).
In the present study, we found that CII:chitosan-fed animals exhibited a reduced secretion of IL-2 in PP with an increase in IL-10 both locally and systemically. Interestingly, chitosan per se stimulated IL-10 production in PP as well as reducing the levels of IL-2 in spleen. An important question from this study involves the mode of action of chitosan: RT-PCR analysis revealed that IL-10, IL-4 and TGF-ß mRNA expression in PP was higher in CII:chitosan-fed rats and much higher in rats receiving multiple doses of chitosan before the antigen feed.
The immune response to fed antigens has both local and systemic components (8,9,40) and the cytokine milieu drives the differentiation to priming or tolerance (30,46). The cytokines elicited after CII:chitosan feeding are frequently found in different models of oral tolerance (12,26,27,31) and participate actively in the induction of the tolerance state. Our data suggest that the co-administration of CII with the polysaccharide chitosan modifies the uptake and or the distribution of the relevant antigen as well as the microenvironment at the time of the initial contact with the immune system. Although we did not address the point in the present report, the local and systemic effects could result in an enhancement of the tolerance induction to CII. Considering that CII is a frequent target of tolerance studies aimed at developing alternative arthritis treatments, further studies will help to determine the impact of chitosan effects in the tolerance to this particular antigen.
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Acknowledgements
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This work was supported by grants of CONICET (PEI 0307/98), Agencia Cordoba Ciencia, Agencia Nacional de Promoción Científica y Tecnológica (FONCYT) and Fundación ANTORCHAS. We would like to thank Paula Icely for excellent technical assistance. C. P. is a recipient of a fellow from Agencia Cordoba Ciencia. I. B., A. M. C. and S. C. are Career Members from CONICET.
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Abbreviations
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CIItype II collagen
LDHlactate dehydrogenase
MLNmesenteric lymph nodes
PEphycoerythrin
PPPeyers patches
TGFtransforming growth factor
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References
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