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W. Li, Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai Jiao Tong University Medical School, 280 South Chongqing road, Shanghai 200025, China. E-mail: firstname.lastname@example.org
Vasoactive intestinal peptide (VIP) has been found to act as a potent anti-inflammatory factor through regulating the production of both anti- and pro-inflammatory mediators and promoting Th2-type responses. In this study, we used Chicken collagen II-induced experimental arthritis (CIA) model in Wistar rats to investigate the potential effects of VIP on rheumatoid arthritis. Our results showed that in vivo treatment of CIA-induced rats with VIP had great protective benefit at both clinical and histological levels. Disease suppression was associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and expanded CD4+CD25+ Treg in the periphery, which inhibited autoreactive T cell activation/expansion. In conclusion, the study provides evidence that VIP had great protective effect on CIA through its inhibition actions on pathogenic T cells.
Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology that leads to chronic inflammation in the joints and subsequent destruction of the cartilage and bone. The drugs and agents currently used to treat RA have multiple effects, some of which are undesirable, and in the long term these treatments do not prevent joint damage. To find therapeutic alternatives, several strategies have targeted the hallmark characteristics of RA-inflammation and autoimmunity.
Vasoactive intestinal peptide (VIP) is a potent Th2-produced immunosuppressive agent that has proved to be protective in several models of autoimmune diseases such as collagen-induced arthritis, inflammatory bowel disease, uveoretinitis [1–3] and experimental autoimmune encephalomyelitis . Collagen-induced arthritis (CIA) is a murine experimental disease model induced by immunization with type II collagen (CII). Because it shares a number of clinical, histologic and immunologic features with RA , we used the CIA model to study the potential effect of VIP on the pathogenesis of arthritis. In this study, we investigated whether VIP suppress disease by inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response.
Because CD4+CD25+ Treg have been found to play a pivotal role in the regulation of rheumatoid arthritis (RA) and CIA [6–9], in the present study we also investigated whether VIP might exert its protective effect against CIA by increasing the number or enhancing the suppressive function of the Treg cells.
Materials and methods
Animals and peptides. Female Wistar Rat, 4–6 weeks of age, average weight (190 ± 15) g, were purchased from Animal Center of Fudan University (Shanghai, China) and housed in the animal care facilities of Shanghai Jiao Tong University Medical School under pathogen-free conditions according to the Institutional Animal Care and Use Committee guidelines. Chicken type II collagen(CCII) was purchased from Shanghai Caoben Co. VIP was purchased from GL Biochem, Shanghai.
Introduction and assessment of CIA. Chicken type II collagen was dissolved in 0.01 m acetic acid at 4 °C overnight. The incomplete Freund’s adjuvant (IFA, Sigma, St Louis, MO, USA) was mixed with Mycobacterium tuberculosis (strain H37RA, Difco, Detroit, MI, USA), then the dissolved CCII was emulsified with an equal volume of the complete Freund’s adjuvant. At day 0, the rats were immunized subcutaneously (s.c.) at the base of the tail and the back with 0.1 ml of emulsion containing 250 μg CCII and 500 μg Mycobacterium tuberculosis respectively. At day 11, rats were boosted intraperitoneal with 0.04 mol/l emulsion of CCII and IFA at the base of the tail containing 100 μg CCII. Rats were analyzed by two independent, blinded examiners every day and monitored for signs of arthritis onset using clinical score assessment of the following system: grade 0, no evidence of erythema and swelling; grade 1, Erythema and slight swelling confined to the mid-foot (tarsals) or ankle joint; grade 2, Erythema and mild swelling extending from the ankle to the mid-foot; grade 3, Erthema and moderate swelling extending from the ankle to the metatarsal joints; grade 4, Erythema and severe swelling encompass the ankle, foot, and digits. The animal protocol was approved by the institutional review board of Shanghai Jiaotong University, Medical School.
Treatment protocol. There are four experimental groups: control, model (CIA rats), VIP-prevented group and VIP-treated group (six rats per group). 1 week before immunization, VIP-prevented group was administrated intraperitoneally (i.p.) 15 nmol VIP per injection every other day. From day 0 to the end of the experiment, VIP-treated group was administered intraperitoneally 15 nmol VIP per injection every other day. The other six rats received PBS as control.
Histological analysis. For hematoxilin/eosin(H&E) staining, the hind paws from rats transcardially perfused with 4% paraformaldehyde were postfixed overnight, decalcified in 5% formic acid, embedded in paraffin. Paraffin-embedded 5- to 10-μm sections of joints were stained with H&E and then examined by light microscopy.
Mononuclear cells preparation. Spleen mononuclear cells (MNC) suspensions from rats were prepared by grinding through a nylon mesh. Erythrocytes were osmotically lysed. MNC were suspended in RPMI 1640 (Gibco, Life technologies, Gibco, Paisley, UK) supplemented with 200 U/ml penicillin, 200 U/ml streptomycin, 55 μM β2-mercaptoethanol and 10% heat-inactivated fetal bovine serum.
Proliferation assay. A radiometric proliferation assay based on [3H]-thymidine incorporation was used. Briefly, 200 μl aliquots of MNC (2.5 × 106/ml) suspensions were applied into 96-well flat-bottomed microtiter plates. MNC suspensions were stimulated with type II collagen for 72 h. After 56 h of incubation at 37 °C with 5% CO2 and humified atmosphere, the cells were labeled for an additional 16 h with 10 μl aliquots containing 1 μCi of [3H]-thymidine. Cells were harvested and thymidine incorporation was measured in a liquid β-scintillation counter. Cultures were run in triplicate and the results were expressed as cpm.
Cytokine measurement. The levels of cytokines were determined with ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, 200 μl aliquots of MNC (1 × 107/ml) suspensions were applied into 96-well round-bottomed microtiter plates and were stimulated with type II collagen. After 48 h of incubation at 37 °C with 5% CO2 and humified atmosphere, the supernatant was harvested and cytokines were detected.
Flow cytometric analysis. Splenocytes cells incubated with various mAb: phycoerythrin (PE)–conjugated anti-CD25, PE-cy5-conjugated anti-CD4 (BD PharMingen, San Diego, CA, USA). And PE-conjugated IgG1, PE-cy5-conjugated IgG2a (BD PharMingen) were used as isotype control. After incubation for 30 min on ice, the resulting cells were washed three times in PBS-FBS, and fixed in 1% paraformaldehyde. Intracellular staining of Foxp3 was performed using an anti-rat Foxp3 staining set (eBioscience, San Diego, CA, USA), rat IgG2a (eBioscience) was used as isotype control, according to manufacturer’s instruction. Percentage of positive stained cells was analyzed using a FACS Aria instrument (BD Biosciences, San Jose, CA, USA).
RNA isolation. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA) from CD4+ T cells (5 × 105). Genomic DNA was removed from total RNA prior to cDNA synthesis using RNase-free DNase Set (Qiagen). First-strand cDNA synthesis was performed for each RNA sample using Sensiscript RT Kit (Qiagen). Random hexamers were used to prime cDNA synthesis.
Real-time RT-PCR analysis of Foxp3 gene expression. Primer Express software (ABI, Foster City, CA, USA) was used to design primers from published cDNA sequences. BLASTN searches were conducted on the primer nucleotide sequences to ensure gene specificity. The primer sequences for the Foxp3 gene were as follows: sense primer 5′-GCTTGTTTGCTGTGCGGAGAC-3′, anti-sense primer 5′-GTTTCTGAAGTAGGCGAACAT-3′. Initial PCR amplification of cDNA with the primers yielded gene products of the expected size on 2% agarose gel. This showed that primers were specific and therefore useful for further real time PCR analysis. Relative quantitation of gene expression was performed using the Applied Biosystems ABI Prism 7900 sequence detection system. SYBR Green master mix (ABI) was used for real-time RT-PCR to detect the abundance of PCR products among samples. Thermocycler conditions comprised an initial holding at 50 °C for 2 min, then 95 °C for 10 min. This was followed by a 2-step PCR program consisting of 95 °C for 15 s, and 60 °C for 60 s for 40 cycles. Data were collected and quantitatively analyzed on an ABI Prism 7900 HT Sequence Detection System (ABI). The β-actin gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. All quantities were expressed as n-fold relative to a calibrator.
Cell isolation and suppressive capacity of regulatory CD4+ T cell. CD4+ T cells were prepared from freshly isolated splenocytes by using biotinlated CD4 antibody and an additional step with biotin binding Dynabeads and subsequently with Detachbeads (Dynal Biotech, New Hyde Park, NY, USA). CD4+CD25+ T cells were subsequently isolated by positive selection using phycoerythrin (PE)–conjugated anti-CD25 and anti-PE microbeads (Miltenyi Biotec, Sunnyvale, CA, USA). The purity of the resulting CD4+CD25+ T cells was 95–98%, as determined by flow cytometry using specific antibodies. Purified CD4+CD25+ T cells were used as responder in functional analysis. CD25- T cells (10,000 cells/well) were cultured in the presence of anti-CD3 (1 μg/ml, eBioscience) and irradiated autologous APCs (50,000 cells per well) for 72 h in RPMI 1640 medium containing 10% FBS and 100 IU/ml IL-2. The ability of CD4+CD25+ T cells to suppress the proliferation of CD25− T cells was determined by [3H]-thymidine incorporation. During the final 16–18 h of the functional assays, 1 μCi [3H]-thymidine was added and cell proliferation was measured by scintillation counting. The results were calculated as % inhibition as follows : [1-(experimental CPM/control CPM)] × 100%.
VIP decreases incidence and severity of CIA
As we know that VIP could inhibit the production of pro-inflammatory molecules and lead to Th2 cell predominance, we investigated the in vivo effect of VIP on CIA, a Th1-mediated inflammatory disease model. As shown in Fig. 1A, the severity of CIA was much milder in rats treated with VIP compared with the control rats received PBS only. The incidence of the disease was 83% in control rats with a mean severity of 5.6 at its peak. In contrast, only 67% VIP-prevented rats with a mean severity of 1.33% and 50% VIP-treated rats with a mean severity of 0.67. Delayed onset day in VIP-prevented and VIP-treated rats were observed. While in VIP-treated group, we saw no remission in therapeutic effects 10 days after cessation of VIP administration indicating that no additional neuropeptide is necessary after a short period of VIP treatment to maintain protection from the disease. Histological evaluation of joints showed that rats protected from CIA by VIP had less lymphocytic infiltration, synovial hyperplasia, pannus formation, cartilage destruction and bone erosion (Fig. 1B). Thus, VIP protected rats from CIA development at both clinical and histological levels.
VIP regulates Th1/Th2 balance in CIA
We next investigated the mechanisms underlying the decrease in incidence and severity of CIA following VIP intervention. VIP has several immunomodulatory effects including inhibition of T-cell proliferation and regulation of Th1/Th2 balance [8, 11–13]. To test whether impaired T-cell functions in VIP-treated rats lead to CIA inhibition, we first tested the effect of VIP treatment on CII-specific, proliferative responses of spleen MNC suspensions from CIA rats. Whereas MNC from control rats proliferated in response to CII, T cells from rats receiving VIP responded to CII to a much lesser extent and VIP-treated group had more significant reduction with P < 0.01 (Fig. 2A). These data indicate that VIP administration during CIA development at least partially inhibits T-cell clonal expansion in response to CII challenge.
It has been well proved that pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-2, IL-6 and IL-12, play central roles in CIA and RA pathogenesis. And IL-4 and IL-10 have anti-inflammatory effect in the disease. So, we next tried to find out whether VIP administration in vivo altered cytokine profiles of MNC. As shown in Fig. 2B, C, VIP administration in vivo significantly inhibited the production of IFN-γ, while expression of IL-4 was increased. Therefore, VIP protected rats from CIA through the inhibition of Th1 response and promoting Th2 deviation.
VIP prevents the downregulation of CD4+CD25+ Treg cells during CIA
Several studies have indicated that Treg confer significant protection against CIA by decreasing the activation and joint homing of autoreactive Th1 cells [6, 7, 14]. Because the VIP also inhibited events in the inflammatory phase of CIA following the activation of antigen-specific CD4+ Th1 cells, the possibility exists that VIP induces Treg with suppressive activity during the progression of the disease. Because the former experiments showed that VIP treatment had better effect than VIP prevention, further experiment used only VIP-treated group to campare with model group.
We further addressed whether VIP could induce CD4+CD25+ Treg responses as part of the regulatory mechanism. Although the phenotype analysis indicated no changes of percentages of CD4+CD25+ Tcells in spleen after VIP treatment (Fig. 3A), it was not possible to distinguish CD4+CD25+ Tregs from in vivo–activated non-regulatory T cells based on their cell surface characteristics. Thus, experiments were carried out to determine the expression level of transcription factor Foxp3 in CD4+CD25+ T cells, as an indicator of the regulatory properties [15–17]. The results demonstrated that Foxp3 expression in CD4+CD25+ T cells was upregulated in both the model and VIP-treated group at 2 week, then there was downregulation of Foxp3 expression in the model group, whereas only minial downregulation is seen in the VIP-treated group in 3 week (Fig. 3B, C).
VIP prevents Foxp3 mRNA down expression and induces functional Treg in CIA
High expression of Foxp3 characterizes CD4+CD25+ Treg cells [15–17]. We determined whether VIP induces or contributes to the levels of Foxp3 in CD4+ Tcells. Spleen CD4+ T cells from both Model and VIP-treated group were subjected to RT-PCR for Foxp3 expression. CD4+ T cells from Model group express low level of Foxp3 in 3 week, gradually decreased from 1 to 3 week after immunization. In contrast, Foxp3 was kept strongly by VIP at 3 week (Fig. 4A). To determine whether CD4+CD25+ T cells isolated from VIP-treated CIA rats function as suppressive Treg, autoreactive CD4+ Tcells of untreated or VIP-treated CIA rats were stimulated with APCs and anti-CD3 in the absence or presence of CD4+CD25+ T cells of each group. In each group, there is also a pool with CD4+CD25+ T cells and APCs. Functional analyses with purified CD4+CD25+ T cells showed significantly increased inhibitory activity on the proliferation of T cells compared to Model group (P < 0.01, Fig. 4B), indicating that VIP induced CD4+CD25+ regulatory T cell response.
Rheumatoid arthritis is a systemic inflammatory disease, presumably of autoimmune origin. Due to its pathologic, immunologic, and clinical similarities to human RA, CIA is a commonly used model for studying RA and testing potential therapeutic agents. In this study we show that the neuropeptide VIP provides a highly effective therapy for CIA. Treatment of arthritic rats with VIP decreased the frequency of arthritis, delayed onset and prevented joint damage. VIP is effective in the induction of regulatory mechanisms and immunological properties.
The balance of Th1/Th2-type cytokines might have a substantial role in the regulation of autoimmune diseases. CIA has been identified as a Th1-mediated autoimmune disease. In contrast, Th2-mediated responses have beneficial effects on the severity and progression of the disease [18–21]. Here we demonstrate that administration of VIP to arthritic rats results in a decreased CII-specific T-cell response by specifically inducing a shift in the Th phenotype from a Th1-toward a Th2-type response. Prevention or treatment with VIP led to the inhibition of IFN-γ, and significant increase in IL-4 production. Similar VIP effects on Th1/Th2 balance following antigenic stimulation have been previously described[1–4, 12]; such effects are associated with the VIP-mediated preferential upregulation of the costimulatory molecule B7.2 against B7.1 on antigen-presenting cells, in particular macrophages, as well as the downregulation of macrophage IL-12 production and subsequent IFN-γ secretion by Th1 cells . VIP has a profound therapeutic effect on CIA through a specific effect on the Th1 response. We did not observe any adverse effects of the neuropeptide in this murine system.
The autoimmune protective actions of VIP have been demonstrated in a variety of contexts, by reducing pathologic Th1 responses and deactivating dendritic cells and macrophages [1–3, 14]. The present study demonstrated that VIP prevents the downregulation of CD4+CD25+ Tregs during CIA. CD4+CD25+ Tregs are critical for maintaining self-tolerance and preventing autoimmunity. In CIA model or patients with rheumatoid arthritis, impairment in CD4+CD25+ Tregs was proposed [9, 23]. Treg play a critical role in the therapeutic effect of VIP on CIA. CD4+CD25+ Treg have been reported to have an important function in the regulation of autoimmune diseases, including CIA and RA [6–9, 24–27]. CD4+CD25+ Treg have been characterized by high expression of the transcription repressor Foxp3 [15–17]. In CIA rats, we found that the percentage of Foxp3+ cells in CD4+CD25+ T cells was significantly decreased in 3 week. But after VIP treatment, Foxp3+ cells in CD4+CD25+ Tcells VIP-treated CIA rats from spleen was higher than those from spleen of control CIA rats. Also in mRNA level, CD4+ T cells from Model group express lower level of Foxp3 in 3 week, the level of Foxp3 was gradually decreased from 1 to 3 week after immunization. In contrast, Foxp3 was induced strongly by VIP at 3 week. Our results demonstrate that VIP prevents the downregulation CD4+CD25+ Treg during CIA.
At last, we intended to determine whether CD4+CD25+ Treg cells isolated from VIP-treated CIA rats function as suppressive Treg. Our results showed in VIP treated group that purified CD4+CD25+ T cells showed significantly increased inhibitory activity on the proliferation of T cells compared to Model group, the inhibition rate was nearly 91%, indicating that VIP induced CD4+CD25+ regulatory T cell response.
Another important aspect of the study is related to the effect of VIP on the clinical score of CIA. The observed regulatory immune responses appear to correlate with improvements in clinical observations. Suggesting that VIP downregulates systemic inflammatory processes potentially through regulatory T cell responses and altered cytokine profile.
When we were preparing our manuscript, Delgado and colleagues published their work about therapeutic effects of VIP on CIA . Our study was in agreement with their report in the clinical effect of VIP on CIA. By using collagen-induced CIA in DBA mice, they described the effect of VIP was mediated by downregulating both inflammatory and Th1-type autoreactive response. VIP is able to convert in vitro activated antigen-primed CD4+CD25− cells to very efficient CD4+CD25+Foxp3+CTLA-4+ cells. The VIP induced presence of Treg in the draining lymph nodes and joints of arthritic mice could simply be a consequence of recruitment of Treg to these sites through the effect on the production of certain synovial/APC/T cell–related chemokines.
In summary, the therapeutic effect of VIP on arthritis is associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and expanded CD4+CD25+ Treg in the periphery, which inhibited autoreactive T cell activation/expansion. These observations provide a powerful rationale for the assessment of the efficacy of VIP as a novel therapeutic approach to the treatment of RA.
The work was supported by grants from Shanghai commission of Science and Technology (Shanghai Nature Science Foundation 07ZR14066, 06ZR14058), Renji hospital and Shanghai Jiaotong University, Basic medical college collaboration Foundation (PY07005), Shanghai Board of Health Foundation 2007126 and National Natural Science Foundation of China NSF-30600517.