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Keywords:

  • autoimmunity;
  • dendritic cells;
  • spontaneous polychondritis;
  • type II collagen;
  • vaccination

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Immature dendritic cells (iDCs) have a tolerogenic potential due to low expression of important co-stimulatory cell surface molecules required for antigen presentation and induction of an effective immune response. We report here that injection of iDCs pulsed with chick type II collagen (CII) delayed the onset significantly and suppressed the severity of spontaneous polychondritis (SP) in the human leucocyte antigen (HLA)-DQ6αβ8αβ transgenic mouse model. Bone marrow-derived iDCs were pulsed in vitro with CII and transferred into 6-week-old HLA-DQ6αβ8αβ transgenic mice. Mice receiving CII-pulsed iDCs did not display any clinical signs of disease until 5·5 months of age, indicating the ability of the DC vaccine to delay significantly the onset of SP. Control groups receiving unpulsed iDCs or phosphate-buffered saline (PBS) developed polyarthritis at 3·5 months, as we have reported previously. The severity and incidence of disease was reduced in mice injected with CII-pulsed iDCs. Proinflammatory cytokines were in low to undetectable levels in the serum and tissue in the CII-pulsed iDC mice, correlating with the protection. This is the first evidence of iDC therapy controlling SP and suggests that iDC vaccination may provide a tool to reducing clinical manifestations in human inflammatory autoimmune disease such as relapsing polychondritis and rheumatoid arthritis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Relapsing polychondritis (RP) is an autoimmune disorder in which the cartilaginous tissues are the primary targets of destruction [1]. The clinical diagnosis is determined by inclusion of various clinical criteria that include chondritis of auricles, inflammatory polyarthritis, nasal chondritis, ocular inflammation, respiratory tract chondritis and cochlear and/or vestibular damage [2]. RP is relatively rare, affecting 3·5–4/1 000 000 people per year, in which cartilaginous sites are destroyed by cyclic inflammatory episodes beginning most commonly during the fourth or fifth decade of life [3].

Autoimmune diseases such as RP are characterized by the loss of tolerance to self-determinants, activation of autoreactive lymphocytes and subsequent damage to single or multiple organs. Accumulating evidence suggests that the process of determining the outcome of immune responses (immunity or tolerance) may be initiated by antigen-presenting cells (APC) [4,5]. Dendritic cells (DC), being the most potent professional APC, not only activate lymphocytes, but can tolerize T cells to specific antigens, thereby minimizing autoimmune reactions [6]. Currently the selective enhancement of the tolerogenicity of DCs has been achieved by the use of immature DCs (iDCs), by the pharmacological inhibition of DC maturation or by the use of genetically engineered DCs expressing immunosuppressive molecules [7]. DCs differentiated in the presence of exogenously added cytokines, e.g. transforming growth factor (TGF)-β, interleukin (IL)-10, granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4, possess the immunophenotypic and functional features of iDCs. Lutz et al.[8] showed that iDCs generated in the presence of GM-CSF but absence of IL-4 are maturation resistant and prolong haplotypes specific cardiac allograft survival when administered 7 days before transplantation. In vitro cultured iDCs induce CD4+CD25+ regulatory T cells which suppress the response of antigen-primed CD4+ T cells to allogeneic normal mature DCs [9,10].

The tolerogenic properties of in-vitro antigen-pulsed iDCs have been demonstrated in various autoimmune conditions. Papaccio et al.[11] demonstrated that injection of syngeneic DCs pulsed in-vitro with human gammaglobulin (HGG) could prevent spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice. Bone marrow-derived DCs pulsed in-vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86) and injected subcutaneously back into Lewis rats have been shown to transfer immune tolerance to induced experimental allergic encephalomyelitis [12]. Most recently, Popov et al.[13] demonstrated the effectiveness of in-vitro generated, chick type II collagen (CII) antigen-pulsed tolerogenic iDCs in suppressing collagen-induced arthritis (CIA). Antigen-pulsed iDCs also have tolerogenic properties in humans. Dhodapkar et al.[14] have demonstrated that injection of iDCs pulsed with influenza matrix protein (MP) and keyhole limpet haemocyanin (KLH), in two healthy subjects, led to the specific inhibition of the MP-specific CD8+ T cell effector function and the appearance of MP-specific IL-10 producing cells demonstrating antigen-specific inhibition of effector T cell function in-vivo in humans. These results provide direct evidence that DCs can mediate tolerance in experimental autoimmune diseases and have potential in human disease as well.

In this study we report that vaccination with CII-pulsed iDCs into human leucocyte antigen (HLA)-DQ6αβ8αβ transgenic mice susceptible for spontaneous polychondritis (SP) reduced the severity and delayed initiation of disease in these mice. This is the first evidence showing that DC vaccination can prevent spontaneous polychondritis in this HLA-DQ double transgenic mouse model, which has clinical applications in human RP and rheumatoid arthritis.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Mice

Mice expressing both HLA-DQ6αβ (DQA1*0103/DQB*0601) and HLA-DQ8αβ (DQA*0301/DQB*0302) transgenes in the absence of endogenous major histocompatibility complex (MHC) class II expression (Aβ0) were generated as described previously [15]. Briefly, the transgenes were introduced into the 2f background crossed with Aβ0 mice, resulting in transgenic mice expressing the DQ transgene and lacking both 2-A and 2-E expression. All breeding occurred in laminar flow isolation hoods, and all mice were bred and housed in a clean conventional area in accordance with the institutional animal care and usage guidelines in the Center for Biological Research at the University of North Dakota.

Spontaneous model of polychondritis

Middle-aged mice (between 4 and 6 months of age) expressing the DQ6αβ8αβ transgene and lacking endogenous 2 expression develop SP, with inflammatory events occurring at the ears, the peripheral joints and the nose [3]. All mice in these studies were allowed to age in the conventional mouse colony. All mice were monitored and scored two times a week for erythema, inflammation and deformity or ankylosis of the peripheral joints. The grading criterion for polychondritis used for scoring was as follows. 0: no clinical signs of disease, 1: swelling of less than three digits, 2: swelling of three or more digits or swelling of ankle, 3: ankylosis of the joint. Each limb was scored with a possible combined score of 0–12 for each animal. The mice used in our study were scored for development of polychondritis but we focused our research on joint inflammation.

Preparation and antigen-loading of dendritic cell cultures

Bone marrow cells were flushed from the femur and tibia bones of 4- to 5-week-old DQ6αβ8αβ double transgenic mice. Cells were washed, viability determined in a haemocytometer using 0·2% of trypan blue dye and cultured in 24-well plates (Costar, Cambridge, MA, USA) at 37°C in 5% humidified CO2, at a concentration of 2 × 106 cells/well in 2 ml of complete medium [RPMI-1640 supplemented with 2 mM L-glutamine, 100 mM sodium pyruvate (Cellgro; Mediatech Inc., Manassas, VA, USA), 5000 U/ml penicillin G, 5000 µg/ml streptomycin sulphate, 10% fetal calf serum (FCS) (Gibco BRL, Grand Island, NY, USA) and 50 µM 2-ME (Sigma, St Louis, MO, USA)] supplemented with recombinant GM-CSF (20 ng/ml) (eBioscience, San Diego, CA, USA) as described earlier [16,17].

Non-adherent aggregates were removed on day 2 and fresh medium was added after every 2 days. On day 4, adherent cells were displaced by gentle pipetting and collected. These cells were purified further based on expression of CD11c, a specific DC cell surface marker using magnetic cell separation (MACS). Cells (2 × 108 cells/ml) were incubated with 100 µl of anti-CD11c-coated magnetic microbeads (MACS®, Auburn, CA, USA) for 15 min at 4°C. The cells were then washed in MACS® buffer and passed through a MACS® magnetic column for selection. Purity and phenotypic analysis of the cells was performed using flow cytometry on a fluorescence activated cell sorter (FACS)Calibur (Becton Dickinson, San Jose, CA, USA). The cells were stained using monoclonal antibodies (mAbs) against DC-specific cell surface markers such as fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11c and CD86, phycoerythrin (PE)-conjugated anti-mouse CD80 (eBiosciences) and MHCII (HB144 hybridoma) secondary-stained using FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Appropriate antibody isotype controls were used.

The purified iDCs were pulsed with chick CII (50 µg/ml) for 6–8 h at 37°C in 5% CO2. Cells were washed; viable cells were enumerated and were used for subsequent immunization experiments. Three consecutive injections of 100 ul of CII-pulsed iDCs (2 × 106 cells/ml) were given in hind footpads of 6-week-old DQ6αβ8αβ transgenic mice. Age-matched control mice were injected with 100 ul of unpulsed iDCs or phosphate-buffered saline (PBS), respectively.

Enzyme-linked immunosorbent assay (ELISA) for anti-mouse type II collagen IgG levels

One hundred µl of collagen solution (Arthrogen-CIA® mouse type II collagen, 5 mg/ml) were diluted in 1 × collagen dilution buffer as per the manufacturer's instructions (Chondrex, Redmond, WA, USA), added to Nunc Maxisorb microtitre plates (NNI, Rochester, NY, USA) and incubated overnight at 4°C. Plates washed three times with PBS/0·5% Tween 20 (PBST) were blocked by adding 1% bovine serum albumin (BSA) in PBS (blocking buffer) for 2 h at room temperature (RT) and washed again three times with PBST. Sera were diluted fourfold, 1/1000–1/64 000, in PBST, and 200 µl was added in duplicate. High-titre CII-specific IgG-positive and negative controls were run with each plate. Sera were incubated for 2 h at 37°C and were washed three times with PBST. CII-specific mouse IgG was detected with peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich) for 1 h at 37°C. The plates were washed three times with PBST and the reaction visualized with para-nitrophenylphosphate (pNPP) substrate (Sigma-Aldrich). The colorimetric change of each well was determined at an optical density of 405 nm on a Thermomax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and the data were analysed using Microsoft Excel.

Total IgG levels

Total serum IgG levels were determined by ELISA as described previously [18]. Briefly, sera were diluted fourfold, 1/1000–1/640 000, in coating buffer (1 M Na2CO3 + 1 M NaHCO3 + 1 M NaN3), and 200 µl/well was added to Nunc Maxisorb microtitre plates in duplicate and incubated for 2 h at RT. Plates were washed three times with PBST, 100 µl/well goat anti-mouse immunoglobulin (Sigma-Aldrich) was added and plates were incubated for 2 h at 37°C. Plates were washed three times with PBST, 200 µl/well pNPP substrate (Sigma-Aldrich) was added and incubated for 1 h at 37°C to visualize the reaction. The colorimetric change of each well was determined as above.

Serum and tissue cytokine analysis

Cytokine levels were measured in serum samples obtained from the CII-pulsed iDC, unpulsed iDC and PBS injected mice by Proteoplex murine cytokine array (Novagen, Madison, WI, USA). Briefly, serum samples were diluted 1 : 3 along with the cytokine standards. One hundred µl of samples and standards were loaded into wells and incubated for 1 h at RT. After washing four times with PBST buffer, 80 µl of murine detection antibody cocktail was added and incubated for another hour at RT. PBXL-3 Fluorophore was added after washing off the cocktail and incubated for 1·5 h at RT. The slide was removed from the assembly, rinsed in 1× rinse solution for 10 s and air-dried for 1 min at 200 g at RT. The slide was scanned and analysed using the Scanning Service Packet. Serum was obtained at different days post-immunization with CII-pulsed iDCs, unpulsed iDCs and PBS. Days 5, 30, 70 and 90 serum samples were analysed for the measurement of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, GM-CSF, IFN-γ and TNF-α. Novadigm discontinued production of the mouse cytokine array kit we used earlier; therefore, we duplicated our cytokine data by using a similar kit by Quansys, the Q-Plex® mouse cytokine array. The principle and methodology used in this kit was similar to the one we used before and was as mentioned above.

For immunohistochemistry analysis, paws were fixed in 4% paraformaldehyde for 1 h at 4°C and later in 30% sucrose overnight at 4°C. Fixed and frozen tissues were sectioned and mounted on slides for staining. Cytokines were detected in tissue sections by immunohistochemical staining using the diamino benzidine (DAB) staining method.

Statistical analysis

Statistical analysis was carried out using the two-way analysis of variance (anova) and t-test. P-value <0·05 was considered statistically significant unless noted otherwise. Mean for samples was calculated and data were plotted as standard deviation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

CII-pulsed iDCs prevent induction of SP

Our study focused on the abrogation of joint inflammation by vaccination with CII-pulsed iDCs. The control groups of DQ6αβ8αβ transgenic mice injected with unpulsed iDCs (n = 8) or PBS (n = 8) developed SP after day 30 (∼ 3·5 months of age), as has been shown previously [3]. Mice (n = 18) injected with 2 × 106 cells/ml of purified iDCs pulsed with CII showed delayed development of SP (Fig. 1). Significant reduction in disease severity was seen in mice injected with CII-pulsed iDCs compared to the control group. The onset of inflammation was delayed by approximately 90 days post-immunization.

image

Figure 1. Mean severity scores after immunization with chick type II collagen (CII)-pulsed immature dendritic cells (iDCs), unpulsed iDCs and phosphate-buffered saline (PBS); 6-week-old mice each received three consecutive footpad injections of in-vitro CII-pulsed iDCs (n = 18), unpulsed iDCs (n = 8) or PBS (n = 8). Mice were scored twice weekly until day 210 (∼ 9 months of age). Two-way analysis of variance variance test showed statistically significant difference between groups (P < 0·001).

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One hundred per cent disease incidence was observed in control mice injected with PBS beginning approximately around day 60; 32% of control mice injected with unpulsed iDCs started developing disease by day 60, eventually reaching 100% incidence thereafter. In mice injected with CII-pulsed iDCs, disease did not develop till day 120, and showed reduced disease incidence compared to the controls (Fig. 2). Therefore, immunization with CII-pulsed iDCs delayed the onset of SP in the HLA-DQ6αβ8αβ transgenic mice and caused significant reduction in clinical severity.

image

Figure 2. Percentage incidence of polyarthritis after immunization with chick type II collagen (CII)-pulsed immature dendritic cells (iDCs), unpulsed iDCs and phosphate-buffered saline (PBS). Disease was monitored in the 6-week-old mice vaccinated with CII-pulsed iDCs, unpulsed iDCs and PBS until 9 months of age. Percentage incidence of disease was determined in the three groups of mice.

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CII-pulsed iDC immunization induced antibody production

Significantly low levels of anti CII-IgG were detected in the mice immunized with CII-pulsed iDCs (Fig. 3). Similar results were reported previously, showing that DQ6αβ8αβ transgenic mice developing SP display undetectable levels of CII-specific antibodies [3]. Therefore, immunization with CII-pulsed iDCs did not lead to a CII-specific immune response in these mice. Non-significant (P > 0·05) levels of serum CII antibody levels were detected in CII-pulsed iDC vaccinated mice when compared to the control groups of mice immunized with unpulsed iDCs (Fig. 3) or PBS (data not shown) and also serum from non-immunized young naive mice. Serum from CII immunized mice (CIA) served as a positive control and showed high levels of anti-CII antibody, as seen previously [3].

image

Figure 3. Anti-mouse chick type II collagen (CII)-specific antibody response in mice immunized with CII-pulsed immature dendritic cells (iDCs) compared to controls. Anti-CII-specific enzyme-linked immunosorbent assay was performed for serum samples collected on day 40 from mice immunized with CII-pulsed iDCs or controls immunized with unpulsed iDCs. CII immunized mice (collagen-induced arthritis) serum was used as a positive control and serum from young naive mice was used as negative control. Readings were taken at an optical density of 405 nm. Statistical analysis was performed by one-way analysis of variance and Newman–Keuls multiple comparison test with P-value less than 0·05 considered significant.

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Total IgG levels were measured in the mice injected with CII-pulsed iDCs, control mice injected with unpulsed iDCs or PBS. High levels of total IgG were seen in the mice immunized with CII-pulsed iDCs compared to the controls (data not shown). Isotype ELISA results showed that IgG1 was the most predominant isotype produced in all the groups, compared to IgG2a, suggesting a T helper type 2 (Th2) immune response. A high level of IgG1 antibody was produced in mice immunized with CII-pulsed iDCs compared to the controls injected with unpulsed iDCs or PBS (Fig. 4). Serum from CII immunized mice also showed comparable levels of IgG1 antibody production, as has been shown previously [3].

image

Figure 4. Antibody isotype profiles in sera from different groups of mice as determined by enzyme-linked immunosorbent assay (ELISA). Serum samples were collected on day 40 from mice injected with chick type II collagen (CII)-pulsed immature dendritic cells (iDCs), unpulsed iDCs and phosphate-buffered saline (PBS). Serum from aged diseased CII immunized (collagen-induced arthritis) mice was used for comparison. The serum samples were used to measure levels of immunoglobulin (Ig)G1 and IgG2a antibody isotype by ELISA at an optical density of 405 nm. Data are plotted as mean ± standard deviation.

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CII-pulsed iDCs modulate cytokine response

Serum was collected from mice immunized with CII-pulsed iDCs on days 5, 30, 70 and 90 and control mice injected with unpulsed iDCs or PBS were bled on days 5 and 30 post-immunization and serum cytokine levels were measured. Low levels of IFN-γ were detected in the CII-pulsed iDCs immunized mice compared to the non-immunized young naive mice. Manifestation of the clinical disease in mice immunized with CII-pulsed iDCs towards day 90 was associated with an increase in the levels of IFN-γ. Sera collected on day 30 from mice injected with PBS or unpulsed iDCs showed high levels of IFN-γ, which correlated with the initiation of clinical disease (Fig. 5a). Serum IFN-γ levels seen in mice injected with CII-pulsed iDCs and controls were consistent with the inflammation time-line. Histological sections of mouse paws were obtained on days 60, 90 and 120 post-immunization for immunodetection of IFN-γ at the tissue level. Immunohistochemistry results correlated with serum samples, with undetectable levels of IFN-γ seen in the day 60 sections compared to days 90 and 120 (Fig. 5b).

imageimageimageimage

Figure 5. Chick type II collagen (CII)-pulsed immature dendritic cells (iDCs) modulate cytokine secretion. (a) Serum interferon (IFN)-γ and tumour necrosis factor (TNF)-α level were analysed in mice immunized with CII-pulsed iDCs, unpulsed iDCs and phosphate-buffered saline (PBS). Serum samples were separated from mice bled retro-orbitally at days 5, 30 and 70 post-immunization with CII-pulsed iDCs. Mice injected with unpulsed iDCs and PBS were bled on days 5, 30 and 180 post-immunizations. Days 5 and 30 serum levels of IFN-γ and TNF-α from mice injected with unpulsed iDCs and PBS are shown for comparison with CII-pulsed iDC immunized mice. Representative experimental results are shown here as obtained by the Proteoplex murine cytokine array. (b) Immunohistochemistry analysis for the detection of IFN-γ in paw tissue sections taken from mice immunized with CII-pulsed iDCs. Paws were taken from the CII-pulsed iDC vaccinated mice on days 60, 90 and 120 post-immunization. Day 60 paw sections were also taken from mice injected with unpulsed iDCs and PBS and used for comparison with CII-pulsed iDC vaccinated day 60 sections (data not shown). Paraformaldehyde-fixed, frozen paws sections were immunostained for IFN-γ by the 3, 3′-diaminobenzidine (DAB) staining method. Sections were visualized in a blinded fashion under a light microscope at 10× magnification. (c) Immunodetection of TNF-α in paw tissue sections taken from mice immunized with CII-pulsed iDCs. Paw sections (10–15 um) were immunostained and analysed for in-situ production of TNF-α in mice after vaccination with CII-pulsed iDCs on days 60, 90 and 120 post-immunization. Paws taken and immunostained on day 60 from mice vaccinated with unpulsed iDCs and PBS were used for comparison (data not shown). The immunoreaction was visualized on sections as brown/black spots under a light microscope at 10× magnification. (d) Serum interleukin (IL)-10 and IL-12 levels were measured in mice immunized with CII-pulsed iDCs, unpulsed iDCs and PBS. Days 5, 30 and 70 post-immunization serum samples were collected from mice immunized with CII-pulsed iDCs. Days 5 and 30 serum levels of IL-10 and IL-12 from mice injected with unpulsed iDCs and PBS were used for comparison with CII-pulsed iDC vaccinated mice. Representative data are shown here as obtained by the Proteoplex murine cytokine array.

TNF-α was undetectable on day 5 post-immunization in mice injected with CII-pulsed iDCs, but increased steadily by day 90. Control mice injected with PBS showed high levels of TNF-α production on day 30 compared to the CII-pulsed iDC immunized group (Fig. 5a). Immunostained tissue sections showed less or undetectable levels of TNF-α in the day 60 section compared to days 90 and 120, which increased gradually (Fig. 5c). The results indicate a decrease in production of proinflammatory cytokines after vaccination with CII-pulsed iDCs.

Levels of IL-12 decreased with age irrespective of the treatment. Low levels of IL-10 were detected in day 5 serum samples taken from mice injected with CII-pulsed iDCs. The IL-10 levels remained low by day 90 compared to the controls injected with unpulsed iDCs, PBS or as seen in the non-immunized young mice (Fig. 5d). Although increased IL-10 production has been thought traditionally to indicate a predominantly Th2 type of immune response, IL-10 may be playing an immunostimulatory role in the disease process. Injection of CII-pulsed iDCs led to a decrease in the IL-10 serum levels, which correlates with absence or decrease in clinical induction of SP in the transgenic mouse model.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

As has been demonstrated previously [19], we show in this study that bone marrow-derived iDCs generated in the presence of GM-CSF are potent immunomodulatory cells, able to induce protection against SP. In this study, we show for the first time that bone marrow-derived syngeneic iDCs pulsed with CII delay the development, reduce severity and lead to decreased disease incidence of SP in the transgenic mouse model. Joint inflammation was reduced significantly in mice injected with CII-pulsed iDCs. This is in agreement with studies conducted in different experimental models of autoimmunity that provide evidence of the tolerogenic potential of antigen-pulsed iDCs [11–13].

The putative autoantigen involved in the disease process in SP is still unknown. The possibility that autoimmunity to types II and XI collagen might contribute to the pathogenesis of human rheumatic disease has been suggested by studies demonstrating antibodies to these collagens in the sera of patients with rheumatoid arthritis and RP [20,21]. Of the three cartilage-specific collagens isolated, type II is the most abundant, constituting 80–90% of the total collagen content of the cartilage compared to types XI and IX collagen, which each constitute 5–10% of cartilage collagen [22,23].

In the present study we have shown that consecutive injections of CII-pulsed iDCs prevented the induction of clinical disease in the HLA-DQ transgenic mice. In order to determine if the vaccination induced a CII-specific immune response, serum CII antibodies were measured. It has been shown in a previous study [3] that transgenic mice developing SP do not produce CII-specific IgG antibodies. In our study non-significant levels of CII specific antibody were seen in the mice injected with CII-pulsed iDCs compared to the controls. This indicates that the protection induced by the DC vaccine is antigen non-specific. It has been shown previously that α3 (XI) and α1 (II) are both arthritogenic in DBA/1 mice and they share significant sequence homology [24]. Because CII shares homology with other cartilage collagen antigens, cross-reactivity might be involved and thus responsible for protection or disease prevention in the mice injected with CII-pulsed iDCs.

Low levels of Th1 cytokines, IFN-γ and TNF-α, respectively, were produced in the CII-pulsed iDC injected mice compared to controls. IFN-γ was produced in the mice injected with CII-pulsed iDCs after clinical disease was initiated (post-day 90), therefore showing correlation of production of the proinflammatory cytokine with inflammation. Similarly, TNF-α was not detected in serum on day 5 post-immunization but production increased steadily until day 90. The protection seen in the CII-pulsed iDCs injected mice can be associated with the absence or reduced production of IFN-γ and TNF-α cytokine levels in the serum and at tissue level.

IL-10 levels were high in the control mice compared to the mice injected with CII-pulsed iDCs. We hypothesize that IL-10 might be playing an immunostimulatory role instead of being immunosuppressive in the pathogenesis of SP. Various in-vivo and in-vitro models have demonstrated the immunostimulatory property of IL-10. In human autoimmune conditions such as systemic lupus erythematosus and rheumatoid arthritis, increased levels of IL-10 have been detected in serum and joints, respectively [25,26]. It has been shown that in the experimental autoimmune encephalomyelitis animal models, autoantibody production and immune complex pathology can be reduced substantially by treatment with anti-IL-10 mAb [27]. Neutralization of endogenous IL-10 with anti-IL-10 mAb inhibits the development of insulin-dependent diabetes mellitus when performed early in mice life [28]. IL-10 is known to exert immunostimulatory effects on certain cells types such as mouse mast cells and thymocytes [29]. Blocking mast cell degranulation reduces the severity of clinical arthritis occurring in SP in the transgenic model (data not shown). Therefore, mast cells may be one of the important cells to be affected as a result of reduced IL-10 production in the CII-pulsed iDCs mice. IL-10 also enhances immunoglobulin production by naive and committed B cells and acts as a switch factor for IgG1 and IgG3 production [30].

The functional activities of DCs are mainly dependent on their state of activation and differentiation [31]. Immature, semimature and mature antigen-pulsed dendritic cell vaccinations have been shown to be effective due to induction of either one or all of the following mechanisms: generation of regulatory forkhead box P3 (FoxP3)+ T cells, anergic T cells or production of immunoregulatory cytokines [32–34]. The mechanisms by which CII pulsed iDCs suppress disease in the transgenic mouse model are under study. Recent experiments have shown that the numbers of CD4+25+FoxP3+ regulatory T cells increased after injections with CII-pulsed mature dendritic cells. Generation of regulatory T cells in-vivo may be one of the potential mechanisms in our model and is currently under further study.

Antigen-specific iDCs are attractive tools for therapy in human autoimmune conditions. The study demonstrates that unique non-pathogenic epitopes can be used for loading antigen-presenting cells which can help to modulate the immune response. Future studies will clarify if the ability of non-pathogenic tissue-specific loaded iDCs have an application in other diseases. This study has shown the preventative effects of immunization with CII-pulsed iDCs in the SP transgenic mouse model and also suggests the potential application of this form of therapy in human relapsing polychondritis cases.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We thank Dr Chella S. David, Mayo Clinic College of Medicine, Rochester, MN for the HLA-DQ6αβ8αβ transgenic breeding stock, Dr Thomas J. Santoro, Associate Dean for Graduate Medical Education, University of Illinois College of Medicine at Peoria, IL for his scientific input and Dr Patrick Carr, University of North Dakota, SMHS, Grand Forks, ND and his laboratory members for their help with the immunostaining of tissue sections. This study was supported by research grant from NIH-NIAMS (AR30752).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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