Oral administration of type-II collagen peptide 250–270 suppresses specific cellular and humoral immune response in collagen-induced arthritis

doi:10.1016/j.clim.2006.08.004

Clinical Immunology

Volume 122, Issue 1, January 2007, Pages 75-84

https://doi.org/10.1016/j.clim.2006.08.004 Get rights and content

Abstract

Oral antigen is an attractive approach for the treatment of autoimmune and inflammatory diseases. Establishment of immune markers and methods in evaluating the effects of antigen-specific cellular and humoral immune responses will help the application of oral tolerance in the treatment of human diseases. The present article observed the effects of chicken collagen II (CII), the recombinant polymerized human collagen II 250–270 (rhCII 250–270) peptide and synthesized human CII 250–270 (syCII 250–270) peptide on the induction of antigen-specific autoimmune response in rheumatoid arthritis (RA) peripheral blood mononuclear cells (PBMC) and on the specific cellular and humoral immune response in collagen-induced arthritis (CIA) and mice fed with CII (250–270) prior to immunization with CII. In the study, proliferation, activation and intracellular cytokine production of antigen-specific T lymphocytes were simultaneously analyzed by bromodeoxyuridine (BrdU) incorporation and flow cytometry at the single-cell level. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively. CII (250–270) was found to have stimulated the response of specific lymphocytes in PBMC from RA patients, including the increase expression of surface activation antigen marker CD69 and CD25, and DNA synthesis. Mice, fed with CII (250–270) before CII immunization, had significantly lower arthritic scores than the mice immunized with CII alone, and the body weight of the former increased during the study period. Furthermore, the specific T cell activity, proliferation and secretion of interferon (IFN)-γ in spleen cells were actively suppressed in CII (250–270)-fed mice, and the serum anti-CII, anti-CII (250–270) antibody activities and the frequency of specific antibody-forming spleen cells were significantly lower in CII (250–270)-fed mice than in mice immunized with CII alone. These observations suggest that oral administration of CII (250–270) can suppress the cellular and humoral immune response in collagen-induced arthritis, and the simultaneous analysis of antigen-specific cellular and humoral immune responses at single-cell level will help the understanding of the oral tolerance mechanisms in CIA and the development of innovative therapeutic intervention for RA.

Introduction

Oral tolerance, classically defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen by the oral route, is an attractive approach for the treatment of autoimmune and inflammatory diseases thanks to its lack of toxicity, ease of administration over time and its antigen-specific mechanisms of action [1]. Type II collagen (CII), an orally administered autoantigen in suppressing autoimmune diseases, was first reported in 1986 [2]. Since then, a large number of studies have been conducted on the mucosal treatment of arthritis [3], [4]. Immunodominant collagen peptides also have been found effective in suppressing collagen-induced arthritis model (CIA) by oral administration [5].

CIA, an autoimmune disease mediated by CD4+ T cells, has been widely used as an animal model for rheumatoid arthritis (RA) [6], [7]. Both T and B cell activations are crucial in inducing CIA. At the onset of the disease, a Th1 cytokine profile has been reported to predominate, and the development of CIA is dependent on a T-cell-mediated activation of autoreactive B cells [8], [9]. The major role of B cells is production of arthritogenic anti-CII antibodies, which is clearly shown by the fact that antibodies reactive with CII can bind to cartilage and induce arthritis [10], [11]. But the specific cellular and humoral immune response in the process of CIA and oral tolerance is still not very clear. The results of some recent researches indicate that the successful application of oral tolerance for the treatment of human diseases depends on the dosage of the orally administrated agents, the establishment of immune markers to assess the immunologic effects and early therapy [12].

In this reported study, we used low dosage of orally administered CII (250–270) in early-stage CIA with an aim to further investigating the modulation of autoantigen (CII) and immunodominant peptides [CII (250–270)] (GPKGQTGKPGIAGFKGEQGPK), containing T cell epitope CII 260–270 (IAGFKGEQGPK) [13], [14], [15], in the specific cellular and humoral immune response in vitro and in vivo. We employed the recombinant polymerized human collagen type II polypeptide 250–270 (rhCII 250–270), synthesized (syCII 250–270) peptide and CII in assessing anti-CII and anti-CII (250–270) autoimmune responses, and simultaneously measured the lymphocyte proliferation, activation and intracellular cytokine production by bromodeoxyuridine (BrdU) incorporation and flow cytometry at the single-cell level. We also detected the antigen-specific antibody and antibody-forming cells by enzyme-linked immunospecific assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively.

Section snippets

Patients

Samples of peripheral blood were obtained from 16 patients (3 men and 13 women) with active RA and 16 healthy volunteers (5 men and 11 women) as normal control from Xijing Hospital affiliated to the Fourth Military Medical University. The mean age of the patients was 44.7 years (range 25–72 years). According to the 1987 revised diagnostic criteria of the American College of Rheumatology [16], all the patients suffered from active RA including morning stiffness > 45-min duration, > 6 swollen

CII (250–270)-specific cellular immune response in RA patients

As shown in Fig. 1, flow cytometry analysis indicated that the PBMC from RA patients stimulated by rhCII (250–270) (1 mg/ml to CD69 or 2 mg/ml to CD25) for 8 h/CD69 or 1 d/CD25 produced the optimal activation of antigen-specific T cells. The frequencies of CD69+/CD3+ and CD25+/CD3+ cells were 36.51% and 41.76% in total T cell population in PBMC from RA patients stimulated by rhCII (250–270), which were significantly higher than those cultured with PBS alone.

The proliferation of the CII or rhCII

Discussion

Previous studies on animal models show that a number of factors modulate oral tolerance. As oral tolerance has usually been defined in terms of Th1 cell responses, anything that suppresses Th1 cell activity and/or enhances Th2 or Th3 cell development will promote the induction of oral tolerance. It has been reported that CII (257–273) peptide preferentially binds to DR alleles that are associated with RA susceptibility [21], [22] and CII (250–270) can activate T cells by binding specifically to

Acknowledgment

This research was supported by grants from the National Focal Point Tackle Key Problems in science and technology, special subject “Study of Human Type II Collagen Peptide” (96-901-05-262).

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Osteoarthritis (OA) is a degenerative joint disease, affecting 32.5 million US adults or 242 million people worldwide. There is no cure for OA. Many animal and clinical trials showed that oral administration of undenatured type II collagen could significantly reduce the incidence of OA or alleviate the symptoms of articular cartilage. Type II collagen is an important component of cartilage matrix. This article reviewed research progress of undenatured type II collagen including its methods of extraction and preparation, structure and characterization, solubility, thermal stability, gastrointestinal digestive stability, its role in improving OA, and the mechanism of its action in improving OA. Type II collagen has been extensively explored for its potential in improving arthritis. Methods of extraction of type II collagen are inefficient and tedious. The method of limited enzymatic hydrolysis is mainly used to prepare soluble undenatured type II collagen (SC II). The solubility, thermal and gastrointestinal digestive stability of SC II are affected by the sources of raw material, pH, salt ions, and temperature. Oral administration of undenatured type II collagen improves OA, whereas its activity is affected by the sources, degree of denaturalization, intervention methods and doses. However, the influence of the structure of undenatured type II collagen on its activity and the mechanism are unclear. The findings in this review support that undenatured type II collagen can be used in the intervention or auxiliary intervention of patients with OA.
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Several self-antigens, such as bovine or chicken collagen II, HCgp39 and dnaJp1, have been investigated for restoration of oral tolerance in RA. Ingestion of collagen II peptides or whole collagen II proteins markedly reduced arthritis incidence and severity in collagen-induced arthritis (CIA) mice [14,17,18]. While most studies have focused on prophylactic effects of oral treatment, i.e. by starting it before or concomitantly to animal immunisation, one study showed a beneficial impact on disease severity in a therapeutic setting, paving the way for human diseases translation [18].
Rheumatoid arthritis is a chronic systemic autoimmune disease, affecting mainly the joints. It is caused by an adaptive immune reaction against self-antigens, leading to the over production of inflammatory cytokines and autoantibodies, mainly mediated by autoreactive CD4+ T cells and pathological B cell clones. The treatment options currently available rely on palliative global immunosuppression and do not restore tolerance to self-components. Here, we review antigen-specific tolerance approaches that have been developed to inhibit or delete autoreactive clones, while maintaining a potent immune system for rheumatoid arthritis. The first attempts relied on the oral ingestion of self-reactive peptides, with lukewarm results in human clinical trials. To enhance treatment efficacy, self-peptides have been engineered and combined with immunosuppressive molecules. In addition, several routes of delivery have been tested, in particular, nanoparticles carrying self-antigens and immunomodulatory molecules. More recently, transfer of immune cells, such as tolerogenic dendritic cells or regulatory T cells, has been considered to restore tolerance. Although promising results have been obtained in mouse models, the translation to humans remains highly challenging, mainly because the disease is already well developed when treatments start and because patient's specific self-antigens are often unknown. Nevertheless, these approaches hold great promises for long-term RA treatment.
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One of the most precise selections of vaccine components exist in epitope-based peptide vaccines where the peptide should mimic the naturally processed epitope to serve as tolerogens in autoimmune diseases [73]. Low dose oral administration of recombinant polymerized human rhCII250-270 or synthesized human CII250-270 suppressed the development of CIA in DBA/1 mice [74]. Altered peptide ligands (APLs) are analogs of immunogenic epitopes that have been modified by introducing one to few amino acid substitutions while retaining the MHC binding property.
Chronic inflammatory autoimmune diseases leading to target tissue destruction and disability are not only causing increase in patients’ suffering but also contribute to huge economic burden for the society. General increase in life expectancy and high prevalence of these diseases both in elderly and younger population emphasize the importance of developing safe and effective vaccines. In this review, at first the possible mechanisms and risk factors associated with chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE) and type 1 diabetes (T1D) are discussed. Current advances in the development of vaccines for such autoimmune diseases, particularly those based on DNA, altered peptide ligands and peptide loaded MHC II complexes are discussed in detail. Finally, strategies for improving the efficacy of potential vaccines are explored.
Isolation, purification and characterization of pepsin soluble collagen isolated from silvertip shark (Carcharhinus albimarginatus) skeletal and head bone
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Type II pepsin soluble collagens (PSC) were isolated from skeletal and head bone of silvertip shark; and examined for their biochemical and structural properties. Among the raw materials, the protein content (8.99%) was high in skeletal bone and the ash content (28%) was high in head bone. After the collagen extraction, the raw materials contained higher amount of ash content ranging from 82 to 88%. The hydroxyproline content of skeletal and skeletal PSC (30 and 113 mg/g) was higher than those head and head PSC. Both collagens were composed of two different α-chains (α₁- and α₂-chains) and were characterized as type II collagen. Amino acid analysis of skeletal and head PSC indicated imino acid contents of 156 and 175 amino acid residues per 1000 residues, respectively. Similar, Fourier transform infrared spectra of SCII and HCII were observed, which suggested that the isolation process did not affect the secondary structure and molecular order of collagen, particularly the triple–helical structure. Denaturation temperature of skeletal PSC (31 °C) was higher than that of head PSC. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. These results suggested that the PSC isolated from skeletal and head bone of silvertip shark were found to be suitable biomaterial in commercial applications as alternatives to mammalian collagen.
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One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2^b) mice as donors and CBA.J (H2^k) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K^b, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K^b encoding nanoparticles (K^b-NP) or Balb/c (H2^d) grafts were used instead of C57BL/6 (H2^b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K^b-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce K^b-specific tolerance and to reduce the development of transplant arteriosclerosis.
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Citation Excerpt :
Detection of anti-OVA serum antibodies was carried out in microtiter plates covered with 20 μg/mL OVA in sodium carbonate buffer, pH 9.0, as described elsewhere [15]. To detect anti-type II collagen serum antibodies, plates were covered with 20 μg/mL chicken type II collagen (Chondrex Inc, Redmond, WA, USA) in phosphate buffered saline (PBS) pH 7.2 as described elsewhere [16]. Absorbance was read at 492 nm in an ELISA reader (Labsystem MS, Finland).
Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3⁺ and IL-10⁺ cells were higher, whereas IFNγ⁺ cells and IL-17⁺ cells were lower, among gated CD4⁺ spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.

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¹: The first four authors contributed equally to this work.

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Oral administration of type-II collagen peptide 250–270 suppresses specific cellular and humoral immune response in collagen-induced arthritis

Abstract

Introduction

Section snippets

Patients

CII (250–270)-specific cellular immune response in RA patients

Discussion

Acknowledgment

J. Immunol. Methods

Cell. Immunol.

Cell Biol. Int.

J. Autoimmun.

J. Autoimmun.

Eur. J. Pharmacol.

J. Immunol. Methods

Suppression of type II collagen-induced arthritis by intragastric administration of soluble type II collagen

Proc. Natl. Acad. Sci.

Suppression of collagen-induced arthritis with pregastrically or intravenously administered type II collagen

Agents Actions

Characterization of recombinant type II collagen: arthritogenicity and tolerogenicity in DBA/1 mice

Immunology

Oral and nasal administration of chicken type II collagen suppresses adjuvant arthritis in rats with intestinal lesions induced by meloxicam

World J. Gastroenterol.

Oral administration of an immunodominant human collagen peptide modulates collagen-induced arthritis

J. Immunol.

Autoimmunity to type II collagen: an experimental model of arthritis

J. Exp. Med.

Immunization against heterologous type II collagen induces arthritis in mice

Nature

Resistance to development of collagen-induced arthritis in C57BL/6 mice is due to a defect in secondary, but not in primary, immune response

J. Clin. Immunol.

Primed B cells present type-II collagen to T cells

Scand. J. Immunol.

Collagen type II (CII)-specific antibodies induce arthritis in the absence of T or B cells but the arthritis progression is enhanced by CII-reactive T cells

Arthritis Res. Ther.

Oral tolerance

Immunol. Rev.