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Suppression of Collagen-induced Arthritis by Oral or Nasal Administration of Type II Collagen
Article No. jaut.1999.0320, available online at http://www.idealibrary.com on
Journal of Autoimmunity (1999) 13, 315–324
Suppression of Collagen-induced Arthritis by Oral or Nasal Administration of Type II Collagen Gabriela Garcia1,2, Yoshinori Komagata1, Anthony J. Slavin1, Ruth Maron1 and Howard L. Weiner1 1
Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA 2 Astra Research Center Boston, 128 Sidney St., Cambridge, MA 02139, USA
Received 26 April 1999 Accepted 20 May 1999 Key words: arthritis, collagen, mucosa, NALT, oral tolerance
We directly compared the effects of oral and nasal administration of collagen type II (CII) on disease progression, cytokine production and T cell responses in DBA/1 mice. Lymphocytes were assayed for proliferation and cytokine production and cell lines established. T cells from fed or nasally treated groups proliferated significantly less and produced markedly less IFN- than the non-fed immunized group 10 days after immunization and prior to onset of arthritis. T cell lines established from fed or nasally treated mice showed a pattern of cytokine production involving IL-4, IL-10 and TGF-, whereas T cell lines from the control group produced more IFN- and IL-2. Suppression of clinical measures of arthritis was equivalent in the nasal and orally treated groups. Animals were then tested for IFN- production 70 days after a booster immunization at a time when disease was apparent. Mucosally treated animals secreted less IFN- as compared to controls, even at this late time point. Suppression of collagen induced arthritis (CIA) by nasal treatment of mice with CII was associated with diminished levels of TNF- and IL-6 mRNA expression in the joints of tolerized mice, two cytokines known to be involved in the inflammatory and pathological process of CIA. These results demonstrate the induction of antigen specific Th2 and TGF- secreting regulatory cells following both oral and nasal treatment, which is associated with suppression of local inflammation in the joints and decreased Th1 type responses in the periphery throughout the course of the illness. © 1999 Academic Press
Introduction
activity of matrix metaloproteinases (MMPs), produced by activated macrophages and fibroblasts in response to IL-1 and TNF- [4]. These proteinases can be inhibited by two immunoregulatory cytokines, TGF- and IL-10, which not only inhibit the production of proinflammatory cytokines that induce MMPs but also induce the production of their native inhibitors (TIMPs) [6]. The first studies to demonstrate that an orally administered autoantigen can suppress an autoimmune disease were the use of type II collagen (CII) in CIA [7, 8]. Since then, a large number of studies have investigated mucosal treatment of arthritis [9–13]. The primary mechanisms by which oral tolerance is mediated include deletion, anergy and active suppression; the determining factor in this process is the dose of antigen fed [14]. High dose antigen appears to induce systemic anergy or deletion; however, a regime of multiple feedings with low dose antigen favors the induction of regulatory T cells whose suppressive activities are mediated through the production of anti-inflammatory cytokines such as IL-4, IL-10 and TGF- [15, 16]. TGF--secreting cells appear to be a unique subset that has been termed Th3 cells [17, 18].
Murine type II collagen-induced arthritis (CIA) is an autoimmune disease mediated by CD4 + T cells [1–3] and is widely used as an animal model for rheumatoid arthritis. It is characterized by chronic inflammation within the joint, associated with synovitis and erosion of cartilage and bone [1]. Following T cell activation, an inflammatory cascade involving T cells, macrophages, neutrophils, synoviocytes, cytokines, chemokines and antibodies is triggered, which leads to the characteristic joint destruction of arthritis [4, 5]. The pivotal role of cytokines and chemokines in the pathogenesis of arthritis suggests that the source of these molecules and their target cells may provide potential sites for interrupting the network of cellular and molecular interactions that leads to cartilage and bone destruction. The destruction of the cartilage is now widely considered in most part to be due to the Correspondence to: Dr Howard L. Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Fax: 617 525–5252. E-mail: [email protected] 315 0896–8411/99/070315+10 $30.00/0
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Results from experimental autoimmune models suggest that cytokines associated with the Th1 phenotype of lymphocytes, IFN-, TNF- and IL-12, promote inflammation in the target organ whereas cytokines associated with Th2 responses (IL-4, IL-10) and Th3 responses (TGF-) have a role in suppressing disease [19, 20]. The finding that Th1 and Th2 subsets of CD4 + T cell can cross-regulate each other via production of distinct cytokine patterns supports the idea that oral tolerance induced by low doses of antigen reflects the downregulation of Th1 CD4 + cells by Th2 cells [16]. Moreover, IL-4, IL-10 and TGF- have been implicated not only with the inhibition of proinflammatory cytokines and chemokines present in the arthritic joints but also the suppression of PGE2, collagenase, and NO production [6, 21, 22]. The studies reported here show that both oral and nasal administration of collagen type II induce the generation of Th2/Th3 responses and suppress arthritis.
Materials and Methods Mice Male, 6–8-week-old, DBA/1 mice were purchased from The Jackson Laboratory. Mice were housed in Harvard Medical School Animal Care Facilities.
Induction of oral or nasal tolerance Oral tolerance was induced by a multiple low-dose feeding regimen. Each mouse was fed for 5 consecutive days with 30 g of chicken collagen type II (Sigma St. Louis, MO, USA) dissolved in 0.2 ml of 0.01 N acetic acid by gastric intubation with an 18-gauge stainless steel feeding needle (Thomas Scientific). Nasal tolerance was induced by nasal administration for 3 consecutive days of 10 g of chicken collagen type II dissolved in 10 l of 0.01 N acetic acid. Control groups were fed or nasally treated with PBS.
Induction and evaluation of CIA Two days after the last oral or nasal treatment each mouse received 100 g (total dose) of chicken collagen type II emulsified in an equal volume of complete Freund’s adjuvant (CFA) containing 1 mg of Mycobacterium tuberculosis (H37RA) per ml (Difco). The intradermal injections were divided between five sites on the back and at the base of the tail. Twenty-one days later the animals received a booster of 100 g of collagen type II dissolved in 0.2 ml of 0.01 N acetic acid by intraperitoneal (ip) injection. Mice were observed twice a week for the presence of distal joint swelling and erythema. Each limb was scored on a scale from 0 to 4 (0, absence of arthritis; 1, erythema and mild swelling of the tarsus; 2, moderate erythema
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and swelling of tarsus and ankles; 3, severe swelling of tarsus and ankles; 4, ankylosis and bony deformity). A maximum arthritic index (MAI) was obtained for each mouse by summing the greatest score recorded for each limb (0, no disease; 16, highest possible score). The MAI for each group was calculated according to the formula: mean MAI ×number of arthritic mice number of mice in the group
Isolation and propagation of antigen-specific T cell lines Two days after the last feeding or nasal treatment, mice from each group were immunized in the footpad. Each mouse received a subcutaneous (sc) injection in the footpad of 100 g of collagen type II emulsified in an equal volume of CFA (as described above). Ten days later, a single cell suspension was prepared from pooled lymph nodes or spleens removed from mice. The cells were suspended in culture medium (DMEM) supplemented with 10 mM HEPES, 4 mM glutamine, 10 mM non-essential amino acid, 1 mM sodium pyruvate, 5×105 M 2-mercaptoethanol, 50 U/ml penicillin, 50 U/ml streptomycin, and either 1% syngeneic serum or 10% fetal calf serum. Lines were propagated by alternate cycles of stimulation with 50 g/ml chicken collagen type II for three days with irradiated syngeneic splenocytes as antigen presenting cells. After three days of antigen specific stimulation, cells were maintained for 11 days in complete medium supplemented with 0.6% (v/v) T-Stim culture supplement (Collaborative Biomedical Products, Bedford, MA, USA). All other cell culture reagents were purchased from Bio Whittaker.
Proliferation and cytokine assays For proliferation assay, 5×105 cells/well were cultured in 96-well plates with 5–50 g/ml antigen for 84 h 3 H-thymidine (1 Ci/well) was added for the last 14 h of culture. For cell-line proliferation 5×104 cells/well were used in the presence of 5×105 syngeneic irradiated spleen cells, serving as antigen presenting cells. Incorporation of 3H-thymidine was measured using a LKB betaplate liquid scintillation counter following harvest on a Tomtec harvester. The proliferation response was measured as CPM (cpm of wells with antigen minus cpm of wells without antigen). Cytokine concentration was determined in the supernatant of 10×106 cells per/ml cultured with 5–50 g/ ml of antigen. Supernatant for IL-2 and IL-4 was collected after 24 h of culture, 40 h for IFN- and IL-10 and 72 h for TGF-. Quantitative ELISA for IL-2, IL-4, IL-10 and IFN- was performed using paired mAbs specific for corresponding cytokines according to manufacturer’s recommendations (Pharmingen, San Diego, CA, USA) as previously described [18]. Briefly,
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active TGF- (without acid treatment) was determined by a sandwich ELISA. Chicken anti-TGF- (R&D Systems, Minneapolis MN, USA) was used as a coating antibody and murine anti-TGF- (Celtrix and Genzyme) and peroxidase labelled goat anti-mouse IgG (Kirkergard and Perry) were used to capture and develop the assay.
Table 1. Oral or nasal administration of CII suppresses IFN-
RT-PCR for detection of cytokine mRNAs in joints
Proliferation and cytokine secretion from DBA/1 mice fed or nasally treated and immunized as described in Materials and Methods. Popliteal lymph nodes were taken 10 days after immunization and the cells stimulated in vitro with 50 g/ml CII and proliferation and cytokine secretion measured. The values represent the proliferation or cytokine secretion (pg/ml in test culture with antigen −pg/ml in control culture without antigen).
Sixty days after the booster, joints were taken from each mouse. The joints were dissected free of skin and bones above the paw, and frozen in liquid nitrogen. After homogenization with Trizol solution (Gibco BRL, Gaithersburg, MD, USA), debris of soft tissue was separated by centrifugation. Total RNA was extracted from the supernatant with isopropanol following the manufacturer’s instructions. Ten micrograms of total RNA, quantitated by (260 nm) spectrophotometer, was reverse transcribed using oligo d(T)12–18 (Gibco BRL), RNaseH-reverse transcriptase (Superscript II, Gibco BRL) at 42°C for 2 h. The reverse transcriptase was inactivated at 95°C. To adjust the amounts of cDNA of each sample, semi-quantitative PCR was performed using -actin specific primers and a competitive fragment (CF) that contains -actin cDNA sequence yielding different size PCRamplification products from endogenous cDNA product [23]. The -actin cDNA concentration of each sample was determined using -actin primers by keeping the cDNA concentration constant and adding serial dilutions of CF. PCR reaction for -actin was 95°C for 1 min, 57°C for 2 min, and 72°C for 2 min, for 36 cycles. Amplified products were stained with ethidium bromide to determine the amount of CF that gave amplified products from the CF and cDNA of equal intensity. After the adjustment of concentration of each cDNA, another housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA concentration of each sample was checked using HPRT specific primers for validation. cDNA samples were then amplified with IL-4, IL-6, IL-10, IFN-, TNF- and TGF-1 specific primers, and products were separated on agarose gels and stained with ethidium bromide. The number of cycles in these PCR reactions was 36 cycles for IL-6, TNF-, TGF-1, and 40 cycles for IL-4, IL-10, IFN-. The annealing temperature for IL-4, IL-10 was 57°C, and for the others was 55°C. The sequences of primers for cytokine genes are as follows: -actin forward, 5′-TGGAATCCTGT GGCATCCATGAAAC-3′; -actin reverse, 5′-TAAAACGCAGC TCAGTAACAGTCCG-3′; IL-4 forward, 5′-CGAAGAACACCA CAGAGAGTGAGCT-3′; IL-4 reverse, 5′-GACTCATTCATGG TGCAGCTTATCG-3′; IL-6 forward, 5′-TTCCTCTCTGCAAGAGACT-3′; IL-6 reverse, 5′-TGTATCTCTCTGAAGGACT-3′; IL-10 forward, 5′-GGACAACATA CTGCTAACCGA CTC-3′;
CII fed PBS fed CII nasal PBS nasal
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IL-10 reverse, 5′-AAAATCACTC TTCACCTGCTCCAC-3′; IFN- forward, 5′-CACACTGCATCTTGGCTT-3′; IFN- reverse, 5′-ACTCCTTTTCCGCTTCCT-3′; TNF- forward, 5′-GCGACGTGGA ACTGGCAGAAG-3′; TNF- reverse, 5′-GGTACAACCCA TCGGCTGGCA-3′; TGF-1 forward, 5′-CTTTAGGAAG GACCTGGGTT-3′; TGF-1 reverse, 5′-CAGGAGCGCACAATCATGTT-3′; HPRT forward, 5′-CTCGAAGTGTTGGATACAGG-3′; HPRT reverse, 5′-TGGCCTATAGGCTCATAGTG-3′;
Results Oral and nasal administration of CII inhibits activation of IFN- production To study the effect of oral and nasal administration of CII we tested proliferative responses and cytokine production of lymph node and spleen cells from DBA/1 mice pretreated by the oral or nasal route with CII. Mice were subsequently immunized in the footpad and popliteal lymph node and spleen cells were taken 10 days after immunization and cultured in vitro with CII. Suppression of proliferative responses and IFN- was seen in lymph nodes of both nasal and oral treated groups (Table 1). Furthermore, oral administration of CII induced TGF- in the spleen. No IL-10 or IL-4 was observed in primary culture. A similar suppression of IFN- was observed in spleen cell culture (data not shown). T cell lines derived from mice treated by the oral or nasal route produce Th2-type cytokines The cytokine production of long term T cell lines from animals treated by the oral or nasal route was investigated upon restimulation of the lines in vitro with CII. T cell lines derived from orally treated animals produced less IFN- than PBS fed controls (Table 2), consistent with the suppression of IFN- observed in primary cultures (Table 1). In addition, these lines
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Table 2. Long-term T cell lines from orally and nasally treated mice produce IL-4 and IL-10
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Cytokine secretion from long-term T cell lines derived from lymph nodes of CII fed or PBS fed animals immunized with CII in CFA as described in Materials and Methods. Lines represent stable cytokine production after being maintained for 2–3 months stimulated every 14 days in vitro with antigen presenting cells pulsed with 50 g/ml CII. Supernatants were measured for cytokines by ELISA. The value represents the cytokine secretion (pg/ml in test culture with antigen −pg/ml in control culture with no antigen).
10
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Figure 1. Oral and nasal administration of collagen suppresses onset and severity of CIA. Prior to immunization mice were treated 5 consecutive days with either oral PBS ( ), 30 g oral CII ( ) or three consecutive days nasally with 10 g CII ( ). Mice were immunized with 100 g CII in CFA and boosted 21 days later. Beginning 5 days after the booster, mice were monitored for CIA and scored as described in Materials and Methods.
produced IL-10 and IL-4. T cell lines derived from animals treated by the nasal route were also found to have a decreased production of Th1 type cytokines and an increase in IL-4 and IL-10 (Table 2).
treated (n=16] was also significantly reduced at 40 days after the booster (PBS vs. oral, P=0.02) and at 80 days after the booster in those mice receiving CII nasally [100% in PBS treated (n =12) vs. 53% in nasally treated (n=17); PBS vs. nasal, P=0.009]. In order to investigate the immune responses associated with this effect, we analysed the cytokine production and proliferative responses of spleen cells at different times throughout the disease course. To induce arthritis in DBA/1 mice two immunizations are required, the first with CII emulsified in adjuvant and the second 21 days later with soluble CII. We first tested animals 10 days after the first immunization. Although immune responses were examined before the booster and before the animals started to show the first symptoms of arthritis, a clear distinction was observed between the treated and control groups. As shown in Figure 2, animals treated by either the oral or nasal route showed a suppression of proliferation and INF- production in comparison to the control group. These results suggest that IFN- responses were suppressed prior to clinical evidence of joint involvement. The second time point studied was 70 days after the booster. At this time all animals from the control group were sick and the spleen cells showed proliferative responses greater than 5000 CPM (Figure 3). By contrast, the orally and nasally treated groups which had a decrease in both disease severity and incidence, had lower proliferative responses. A comparison of the proliferative response between the groups indicates that sick animals from the treated groups generally showed lower levels of proliferation than the control group (Figure 3). Similar results were observed for IFN- production (Figure 4). All of the sick animals from the PBS treated group produced measurable IFN-; however, only 1/3 nasally treated, and 1/4 orally treated mice produced IFN-. None of the non-sick mice produced measurable amounts of IFN-.
Nasal administration of CII suppresses inflammatory cytokines in the joints To further examine the mechanisms involved in the suppression of arthritis by nasal administration of CII we investigated the cytokine pattern in the joints of treated and control animals. Joints of treated animals showed lower levels of TNF- and IL-6, two inflammatory cytokines, in comparison to arthritic joints of control animals (Figure 5). Both groups showed high expression of TGF- mRNA.
Oral and nasal administration of CII suppresses CIA
Discussion
We then directly compared the effect of oral and nasal administration of CII on the incidence and severity of CIA. As shown in Figure 1, both oral and nasal CII lessened disease severity (P<0.01). The incidence of disease [100% in PBS treated (n=12) vs. 62% in orally
The gastrointestinal tract is the major site of antigenic contact in the body. The gut associated lymphoid tissue (GALT) provides for absorption of nutrients, avoiding hypersensitivity to food antigens and exclusion of pathogens [24, 25].
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Figure 2. Oral or nasal administration of collagen suppresses (A) proliferative responses and (B) IFN in vitro. Spleen and lymph node cells were collected from DBA/1 mice treated as in Figure 1, 10 days after immunization and cultured with CII in vitro. Values represent the mean ±SEM of triplicate cultures.
The uptake of macromolecules, particulate antigens and microorganisms across the intestinal epithelia is restricted by multiple mechanisms including local secretions containing mucins and secretory IgA, rigid and packed microvilli, and the glycocalyx: a thick layer of membrane anchored proteins and several enzymes [26]. An organized lymphoid tissue in the form of Peyer’s patches (PP) has a specialized epithelium, known as M cells, capable of transporting antigens across the mucosal barrier. In contrast to most epithelial cells, M cells endocytose material via a transepithelial vesicular transport system. T cells can interact with antigen presenting cells in the PP and then migrate to mesenteric lymph nodes and reach the peripheral immune system as well as other mucosal sites of the body, including the lamina propria in the gut [16, 25]. Although both Th1 and Th2 cells are present in the GALT, the overall environment is predominantly Th2 and affords IgA class switch and secretion [27, 28]. These results indicate the uniqueness of the GALT since it favors the induction of Th2 cells and TGF- secreting regulatory cells (Th3-type cells). The cytokine milieu present in the GALT strongly influences the generation of T cell subsets that provide help to induce and differentiate IgA + B cells [29]. TGF- derived from T cells and non-lymphoid cells and IL-5 derived from Th2 cells favor the switch from IgM to IgA and together with IL-6 help the terminal differentiation of IgA-secreting plasma cells in the gut [30, 31]. It has been shown that after oral administration of a number of antigens, including sheep red blood cells, ovalbumin, and myelin basic protein, T cells are generated in Peyer’s patches which migrate to the spleen and lymph node, generating peripheral tolerance [32–34]. One of the primary mechanisms of active cellular suppression seen following oral administration of antigens is via the generation of T cells which secrete anti-inflammatory cytokines such as
TGF-, IL-4 and IL-10 [17, 18, 35]. In recent years, the induction of oral tolerance to self-antigens has been extensively studied as a tool to regulate the immune system in a manner that inhibits inflammatory events that lead to autoimmunity [16]. Recent analysis of cytokine expression in rheumatoid arthritis (RA) tissues and in experimental models of RA has provided a better understanding of the role of specific cytokines involved in the inflammatory process. Thus, it has been shown that proinflammatory cytokines such as TNF-, IL-1, IL-6, GM-CSF and chemokines such as IL-8 are abundant in RA tissues [4]. Furthermore, there appears to be an attempt to regulate this inflammatory milieu through the increased production of cytokines such as IL-10 and TGF-, and cytokine inhibitors such as IL1ra and soluble TNF-R. However, these regulatory mechanisms are not sufficient to prevent tissue damage. The morphological characteristics of Peyer’s patches (PP) and nasal associated lymphoid tissue (NALT) and the adhesion patterns connected with them indicate that PP and NALT may play equally important roles in the mucosal immune response [36, 37]. Both oral and nasal administration of collagen has been reported to suppress CIA but the mechanisms associated with this suppression are not completely defined [7, 10, 11]. In the present study we show that oral and nasal administration of low doses of CII inhibit the generation and activation of antigenic specific Th1 cells in the spleen and draining lymph nodes. Furthermore, we show that cell lines producing anti-inflammatory cytokines such as IL-4 and IL-10 can be generated from mice treated by the oral or nasal route with CII. The suppression of inflammatory events in the joints of tolerized mice was further demonstrated by the diminished expression of mRNA for TNF- and IL-6, two cytokines involved with the inflammatory and pathological process of CIA. Of note, both groups
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Figure 3. Proliferative responses from spleen cells of sick and non-sick mice. Proliferative responses of spleen cells 79 days after booster. Mice were treated as in Figure 1 and spleen cells cultured in 10 ug/ml CII. The mice were pretreated with (A) PBS orally, (B) 10 g CII nasally or (C) 30 g CII orally. Solid bars represent sick and open bars non-sick mice. Each bar represents an individual mouse.
of mice (tolerized and control) expressed high amounts of TGF-, which is concordant with the cytokine pattern observed in joints of arthritic patients. In contrast to other animal models of autoimmune diseases such as EAE [19], TGF- is highly expressed in the target organ of arthritis, the inflamed joints [4]. A vast array of functions attributed to TGF-
can be demonstrated in arthritic joints, including inflammatory and non-inflammatory activities and tissue repair [38–40]. TGF- contributes to the tissue repair and inhibits the destruction of the cartilage and bone by inhibiting the production of metaloproteinases such as collagenase and inducing the production of TIMP, type I and type XI collagen [6, 41].
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Figure 4. IFN- production from spleen cells from sick mice 79 days booster. Mice were treated as in Figure 1 and spleen cells cultured in 10 ug/ml CII 79 days after booster. IFN- was determined from spleen cells of sick mice treated with (A) PBS orally, (B) 10 g CII nasally or (C) 30 g CII orally. Each bar represents an individual mouse.
Several studies have demonstrated the ability of IL-4 and IL-10 to suppress the inflammatory process and tissue damage in experimental models of arthritis. Intraperitoneal administration of recombinant IL-4 inhibits an experimental model of arthritis by decreasing the influx of inflammatory cells to the tissue, decreasing TNF- production and reactive oxygen
intermediate metabolism [21]. It also increased the production of IL-1ra, which can modulate inflammation and tissue degradation by blocking IL-1 produced PGE2 and collagenase, and neutralizing IL-1 induction of IL-6. More recently, Horsfall et al. showed that upon continuous administration of IL-4 in CIA DBA/1 mice, anti-collagen antibodies
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Figure 5. Nasal CII suppresses production of inflammatory cytokines in the joints. RT-PCR for TNF-, IL-6 and TGF- was performed on homogenates of mouse joints from mice treated with PBS or 10 g CII nasally. Joints were collected 60 days after the booster. HPRT was used as an internal control.
were reduced, histologic score improved and there was a 1000-fold decrease in TNF- secretion by synovial cells [42]. There is evidence for the involvement of endogenous IL-4 in the suppression of arthritis. It was shown in comparing the cytokine pattern of three strains of rats injected with CII [43] that the susceptible strain (DA) produced a strong humoral response and Th1 cytokine profile, whereas the resistant strain (F344) produced a limited humoral response with a Th2 profile (IL-4). Furthermore, the involvement of nonMHC genes in determining the direction of the response to CII was demonstrated by the analysis of a strain MHC congenic to the DA rat (PVG.1AV1). This strain was resistant to arthritis and produced a marked humoral response of isotype IgG1 and also IL-4. Mauri et al. demonstrated that during CIA in mice, the cellular immune response is characterized by the activation and proliferation of CD4 + T cells producing IFN- and during the spontaneous remission phase, there is a shift in favor to antagonistic cytokines such as IL-4 and IL-10 [44]. IL-10 is well known to inhibit IFN- production by Th1 cells but also can inhibit the production of a variety of cytokines from other leukocyte populations [45]. IL-10 inhibits the production of IL-1, IL-6, TNF-, IL-8, G-CSF, from macrophages and IL-1, TNF, IL-8, MIP1, and MIP1 from polymorphonuclear cells. Most of these cytokines and chemokines have been implicated in the pathological process of arthritis. The therapeutic effect of IL-10 was demonstrated by treatment of DBA/1 mice with injections of IL-10 starting before or after disease onset [46]. Both studies showed a decrease in the severity of arthritis. Interestingly, in a comparative study on the therapeutic effect of Th2 cytokines in arthritis, injection of IL-4 alone did not suppress the disease, and IL-10 suppressed it slightly, whereas combined therapy with IL-4 and IL-10 after
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disease onset reduced cellular infiltrates and provided protection from cartilage destruction [47]. Our results demonstrate the ability of oral or nasal tolerance to CII to inhibit the incidence and severity of CIA in DBA/1 mice and induce similar immune responses. The finding that spleen cells from tolerized mice proliferate less and produce less IFN- in vitro than controls is direct evidence of the suppression of a Th1 inflammatory response. Of note is the observation that the mice treated by the oral or nasal route with CII became sick, but nonetheless showed diminished proliferative responses in vitro compared to control and did not produce IFN-. This immunological response is most probably related to the mild outcome of the disease seen in these mice. Our results also demonstrate that low doses of orally or nasally administered CII preferentially induce cells which produce anti-inflammatory cytokines such as IL-4, IL-10 and this is associated with a decrease of TNF- and IL-6 in the joints, something that has not been demonstrated previously in studies of CIA treated with mucosal antigen. Thus, the administration of collagen by either the oral or nasal route is an effective immunotherapeutic intervention designed to shift the balance of the immune response from Th1 to Th2 and to suppress the inflammation and the tissue destruction of arthritic joints.
Acknowledgements This study was supported by NIH grant AI43458. GG received a fellowship from CAPES, Brazil.
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intravenously administered type II collagen. Agents Actions 19: 318–319 Thompson H.S.G., Harper N., Bevan D.J. Staines N.A. 1993. Suppression of collagen induced arthritis by oral administration of type II collagen: changes in immune and arthritic responses mediated by active peripheral suppression. Autoimmunity 16: 189–199 Khare S.D., Krco C.J., Griffiths M.M., Luthra H.S., David C.S. 1995. Oral administration of an immunodominant human collagen peptide modulates collagen-induced arthritis. J. Immunol. 155: 3653–3659 Staines N.A., Harper N., Ward F.J., Malmstro¨ m V., Holmdahl R., Bansal S. 1996. Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184–198 of bovine type II collagen (CII) expressing a dominant T-cell epitope. Clin. Exp. Immunol. 103: 368–375 Myers L.K., Seyer J.M., Stuart J.M., Kang A.H. 1997. Suppression of murine collagen-induced arthritis by nasal administration of collagen. Immunology 90: 161–164 Weiner H.L., Komagata Y. 1998. Oral tolerance and the treatment of rheumatoid arthritis. Springer Semin. Immunopathol. 20: 289–308 Friedman A., Weiner H. 1994. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl. Acad. Sci. USA 91: 6688–6692 Chen Y., Inobe J., Marks R., Gonnella P., Kuchroo V.K., Weiner H.L. 1995. Peripheral deletion of antigen-reactive T-cells in oral tolerance. Nature 376: 177–180 Faria A.M.C., Weiner H.L. 1999. Oral tolerance: mechanisms & therapeutic applications. Adv. Immunol. 73: 153–264 Chen Y., Kuchroo V.K., Inobe J.-I., Hafler D.A., Weiner H.L. 1994. Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265: 1237–1240 Inobe J., Slavin A.J., Komagata Y., Chen Y., Liu L., Weiner H.L. 1998. IL-4 is a differentiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur. J. Immunol. 28: 2780–2790 Khoury S.J., Hancock W.W., Weiner H.L. 1992. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis as associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor , interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176: 1355–1364 Hancock W.W., Khoury S.J., Carpenter C.B., Sayegh M.H. 1994. Differential effects of oral versus intrathymic administration of polymorphic major histocompatibility complex class II peptides on mononuclear and endothelial cell activation and cytokine expression during a delayed-type hypersensitivity response. Am. J. Pathol. 144: 1149–1158 Allen J.B., Wong H.L., Costa G.L., Bienkowski M.J., Wahl S.M. 1993. Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis. J. Immunol. 151: 4344–4351
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22. Kasama T, Strieter R.M., Lukacs N.W., Lincoln P.M., Burdick M.D., Kunkel S.L. 1995. IL-10 expression and chemokine regulation during the evolution of murine type II CIA. J. Clin. Invest. 95: 2868–2876 23. Platzer C., Richter G., Uberla K., et al. 1992. Analysis of cytokine mRNA levels in interleukin-4-transgenic mice by quantitative polymerase chain reaction. Eur. J. Immunol. 22: 1179–1184 24. Strobel S. 1990. Mechanisms of gastrointestinal immunoregulation and food induced injury to the gut. Eur. J. Clin. Nutr. 45 (Suppl. 1): 1–9 25. Abreu-Martin M.T., Targan S.R. 1997. Regulation of immune responses of the intestinal mucosa. Crit. Rev. Immunol. 16: 277–309 26. Neutra R.M., Kraehenbuhl J.P. 1996. M cells as a pathway for antigen uptake and processing. In Essentials of mucosal immunology. Kagnoff M.F. and Kiyono H., eds. Academic Press, San Diego, pp. 29–36 27. James S.P., Fiocchi C., Graeff A.S. Strober W. 1986. Phenotypic analysis of lamina propria lymphocytes. Gastroenterology 91: 1483–1489 28. Kramer D.R., Cebra J.J. 1995. Early appearance of ‘natural’ mucosal IgA responses and germinal centers in suckling mice developing in the absence of maternal antibodies. J. Immunol. 154: 2051–2062 29. MacDonald T.T. 1983. Immunosuppression caused by antigen feeding. II. Suppressor T-cells mask Peyer’s patches B cell priming to orally administered antigen. Eur. J. Immunol. 13: 138–142 30. Coffman R., Lebman D., Shrader B. 1989. Transforming growth factor beta (TGF-) specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes. J. Exp. Med. 144: 3411–3416 31. Deem R.L., Targan S.R. 1995. The role of TGFb in molding the acivation state of lamina propria T-cells. Gastroenterology 108: 32. Mattingly J.A., Waksman B.H. 1978. Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer’s patches after oral administration of sheep erythrocytes and their systemic migration. J. Immunol. 121: 1878–1882 33. Richman L.K., Chiller J.M., Brown W.R., Hanson D.G., Vaz N.M. 1978. Enterically induced immunological tolerance. I. Induction of suppressor T lymphocytes by intragastric administration of soluble proteins. J. Immunol. 121: 2429–2433 34. Higgins P., Weiner H.L. 1988. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J. Immunol. 140: 440–445 35. Miller A., Lider O., Roberts A.B., Sporn M.B., Weiner H.L. 1992. Suppressor T-cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of TGF- following antigen specific triggering. Proc. Natl. Acad. Sci. USA 89: 421–425 36. Kuper C.F., Koornstra P.J., Hameleers D.M. et al. 1992. The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219–224 37. Wu H.Y., Nguyen H.H., Russel M.W. 1997. Nasal lymphoid tissue (NALT) as a mucosal immune inductive site. Scand. J. Immunol. 46: 506–513 38. Allen J.B., Manthey C.L., Hand A.R., Ohura K., Ellingsworth L., Wahl S.M. 1990. Rapid onset synovial
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inflammation and hyperplasia induced by transforming growth factor b. J. Exp. Med. 171: 231–247 Thorbecke G.J., Shah R., Leu C.H., Kuruvilla A.P., Hardison A.M., Palladino M.A. 1992. Involvement of endogenous TNFa and TGFb during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89: 7375–7379 Wahl S.M., Allen J.B., Costa G.L., Wong H.L., Dasch J.R. 1993. Reversal of acute and chronic synovial inflammation by anti-TGFb. J. Exp. Med. 177: 225–230 Khalil N., Bereznay O., Sporn M., Greenberg A.H. 1989. Macropahge production of TGFb and fibroblast collagen synthesis in the chronic pulmonary inflammation. J. Exp. Med. 170: 727–737 Horsfall A.C., Butler M.D., Marinova L., et al. 1997. Suppression of collagen induced arthritis by continuous administration of IL-4. J. Immunol. 159: 5687–5696 Mussener A., Lorentzen J.C., Kleinau S., Klareskog L. 1997. Altered Th1/Th2 balance associated with
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Journal of Autoimmunity (1999) 13, 315–324
Suppression of Collagen-induced Arthritis by Oral or Nasal Administration of Type II Collagen Gabriela Garcia1,2, Yoshinori Komagata1, Anthony J. Slavin1, Ruth Maron1 and Howard L. Weiner1 1
Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA 2 Astra Research Center Boston, 128 Sidney St., Cambridge, MA 02139, USA
Received 26 April 1999 Accepted 20 May 1999 Key words: arthritis, collagen, mucosa, NALT, oral tolerance
We directly compared the effects of oral and nasal administration of collagen type II (CII) on disease progression, cytokine production and T cell responses in DBA/1 mice. Lymphocytes were assayed for proliferation and cytokine production and cell lines established. T cells from fed or nasally treated groups proliferated significantly less and produced markedly less IFN- than the non-fed immunized group 10 days after immunization and prior to onset of arthritis. T cell lines established from fed or nasally treated mice showed a pattern of cytokine production involving IL-4, IL-10 and TGF-, whereas T cell lines from the control group produced more IFN- and IL-2. Suppression of clinical measures of arthritis was equivalent in the nasal and orally treated groups. Animals were then tested for IFN- production 70 days after a booster immunization at a time when disease was apparent. Mucosally treated animals secreted less IFN- as compared to controls, even at this late time point. Suppression of collagen induced arthritis (CIA) by nasal treatment of mice with CII was associated with diminished levels of TNF- and IL-6 mRNA expression in the joints of tolerized mice, two cytokines known to be involved in the inflammatory and pathological process of CIA. These results demonstrate the induction of antigen specific Th2 and TGF- secreting regulatory cells following both oral and nasal treatment, which is associated with suppression of local inflammation in the joints and decreased Th1 type responses in the periphery throughout the course of the illness. © 1999 Academic Press
Introduction
activity of matrix metaloproteinases (MMPs), produced by activated macrophages and fibroblasts in response to IL-1 and TNF- [4]. These proteinases can be inhibited by two immunoregulatory cytokines, TGF- and IL-10, which not only inhibit the production of proinflammatory cytokines that induce MMPs but also induce the production of their native inhibitors (TIMPs) [6]. The first studies to demonstrate that an orally administered autoantigen can suppress an autoimmune disease were the use of type II collagen (CII) in CIA [7, 8]. Since then, a large number of studies have investigated mucosal treatment of arthritis [9–13]. The primary mechanisms by which oral tolerance is mediated include deletion, anergy and active suppression; the determining factor in this process is the dose of antigen fed [14]. High dose antigen appears to induce systemic anergy or deletion; however, a regime of multiple feedings with low dose antigen favors the induction of regulatory T cells whose suppressive activities are mediated through the production of anti-inflammatory cytokines such as IL-4, IL-10 and TGF- [15, 16]. TGF--secreting cells appear to be a unique subset that has been termed Th3 cells [17, 18].
Murine type II collagen-induced arthritis (CIA) is an autoimmune disease mediated by CD4 + T cells [1–3] and is widely used as an animal model for rheumatoid arthritis. It is characterized by chronic inflammation within the joint, associated with synovitis and erosion of cartilage and bone [1]. Following T cell activation, an inflammatory cascade involving T cells, macrophages, neutrophils, synoviocytes, cytokines, chemokines and antibodies is triggered, which leads to the characteristic joint destruction of arthritis [4, 5]. The pivotal role of cytokines and chemokines in the pathogenesis of arthritis suggests that the source of these molecules and their target cells may provide potential sites for interrupting the network of cellular and molecular interactions that leads to cartilage and bone destruction. The destruction of the cartilage is now widely considered in most part to be due to the Correspondence to: Dr Howard L. Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Fax: 617 525–5252. E-mail: [email protected] 315 0896–8411/99/070315+10 $30.00/0
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Results from experimental autoimmune models suggest that cytokines associated with the Th1 phenotype of lymphocytes, IFN-, TNF- and IL-12, promote inflammation in the target organ whereas cytokines associated with Th2 responses (IL-4, IL-10) and Th3 responses (TGF-) have a role in suppressing disease [19, 20]. The finding that Th1 and Th2 subsets of CD4 + T cell can cross-regulate each other via production of distinct cytokine patterns supports the idea that oral tolerance induced by low doses of antigen reflects the downregulation of Th1 CD4 + cells by Th2 cells [16]. Moreover, IL-4, IL-10 and TGF- have been implicated not only with the inhibition of proinflammatory cytokines and chemokines present in the arthritic joints but also the suppression of PGE2, collagenase, and NO production [6, 21, 22]. The studies reported here show that both oral and nasal administration of collagen type II induce the generation of Th2/Th3 responses and suppress arthritis.
Materials and Methods Mice Male, 6–8-week-old, DBA/1 mice were purchased from The Jackson Laboratory. Mice were housed in Harvard Medical School Animal Care Facilities.
Induction of oral or nasal tolerance Oral tolerance was induced by a multiple low-dose feeding regimen. Each mouse was fed for 5 consecutive days with 30 g of chicken collagen type II (Sigma St. Louis, MO, USA) dissolved in 0.2 ml of 0.01 N acetic acid by gastric intubation with an 18-gauge stainless steel feeding needle (Thomas Scientific). Nasal tolerance was induced by nasal administration for 3 consecutive days of 10 g of chicken collagen type II dissolved in 10 l of 0.01 N acetic acid. Control groups were fed or nasally treated with PBS.
Induction and evaluation of CIA Two days after the last oral or nasal treatment each mouse received 100 g (total dose) of chicken collagen type II emulsified in an equal volume of complete Freund’s adjuvant (CFA) containing 1 mg of Mycobacterium tuberculosis (H37RA) per ml (Difco). The intradermal injections were divided between five sites on the back and at the base of the tail. Twenty-one days later the animals received a booster of 100 g of collagen type II dissolved in 0.2 ml of 0.01 N acetic acid by intraperitoneal (ip) injection. Mice were observed twice a week for the presence of distal joint swelling and erythema. Each limb was scored on a scale from 0 to 4 (0, absence of arthritis; 1, erythema and mild swelling of the tarsus; 2, moderate erythema
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and swelling of tarsus and ankles; 3, severe swelling of tarsus and ankles; 4, ankylosis and bony deformity). A maximum arthritic index (MAI) was obtained for each mouse by summing the greatest score recorded for each limb (0, no disease; 16, highest possible score). The MAI for each group was calculated according to the formula: mean MAI ×number of arthritic mice number of mice in the group
Isolation and propagation of antigen-specific T cell lines Two days after the last feeding or nasal treatment, mice from each group were immunized in the footpad. Each mouse received a subcutaneous (sc) injection in the footpad of 100 g of collagen type II emulsified in an equal volume of CFA (as described above). Ten days later, a single cell suspension was prepared from pooled lymph nodes or spleens removed from mice. The cells were suspended in culture medium (DMEM) supplemented with 10 mM HEPES, 4 mM glutamine, 10 mM non-essential amino acid, 1 mM sodium pyruvate, 5×105 M 2-mercaptoethanol, 50 U/ml penicillin, 50 U/ml streptomycin, and either 1% syngeneic serum or 10% fetal calf serum. Lines were propagated by alternate cycles of stimulation with 50 g/ml chicken collagen type II for three days with irradiated syngeneic splenocytes as antigen presenting cells. After three days of antigen specific stimulation, cells were maintained for 11 days in complete medium supplemented with 0.6% (v/v) T-Stim culture supplement (Collaborative Biomedical Products, Bedford, MA, USA). All other cell culture reagents were purchased from Bio Whittaker.
Proliferation and cytokine assays For proliferation assay, 5×105 cells/well were cultured in 96-well plates with 5–50 g/ml antigen for 84 h 3 H-thymidine (1 Ci/well) was added for the last 14 h of culture. For cell-line proliferation 5×104 cells/well were used in the presence of 5×105 syngeneic irradiated spleen cells, serving as antigen presenting cells. Incorporation of 3H-thymidine was measured using a LKB betaplate liquid scintillation counter following harvest on a Tomtec harvester. The proliferation response was measured as CPM (cpm of wells with antigen minus cpm of wells without antigen). Cytokine concentration was determined in the supernatant of 10×106 cells per/ml cultured with 5–50 g/ ml of antigen. Supernatant for IL-2 and IL-4 was collected after 24 h of culture, 40 h for IFN- and IL-10 and 72 h for TGF-. Quantitative ELISA for IL-2, IL-4, IL-10 and IFN- was performed using paired mAbs specific for corresponding cytokines according to manufacturer’s recommendations (Pharmingen, San Diego, CA, USA) as previously described [18]. Briefly,
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active TGF- (without acid treatment) was determined by a sandwich ELISA. Chicken anti-TGF- (R&D Systems, Minneapolis MN, USA) was used as a coating antibody and murine anti-TGF- (Celtrix and Genzyme) and peroxidase labelled goat anti-mouse IgG (Kirkergard and Perry) were used to capture and develop the assay.
Table 1. Oral or nasal administration of CII suppresses IFN-
RT-PCR for detection of cytokine mRNAs in joints
Proliferation and cytokine secretion from DBA/1 mice fed or nasally treated and immunized as described in Materials and Methods. Popliteal lymph nodes were taken 10 days after immunization and the cells stimulated in vitro with 50 g/ml CII and proliferation and cytokine secretion measured. The values represent the proliferation or cytokine secretion (pg/ml in test culture with antigen −pg/ml in control culture without antigen).
Sixty days after the booster, joints were taken from each mouse. The joints were dissected free of skin and bones above the paw, and frozen in liquid nitrogen. After homogenization with Trizol solution (Gibco BRL, Gaithersburg, MD, USA), debris of soft tissue was separated by centrifugation. Total RNA was extracted from the supernatant with isopropanol following the manufacturer’s instructions. Ten micrograms of total RNA, quantitated by (260 nm) spectrophotometer, was reverse transcribed using oligo d(T)12–18 (Gibco BRL), RNaseH-reverse transcriptase (Superscript II, Gibco BRL) at 42°C for 2 h. The reverse transcriptase was inactivated at 95°C. To adjust the amounts of cDNA of each sample, semi-quantitative PCR was performed using -actin specific primers and a competitive fragment (CF) that contains -actin cDNA sequence yielding different size PCRamplification products from endogenous cDNA product [23]. The -actin cDNA concentration of each sample was determined using -actin primers by keeping the cDNA concentration constant and adding serial dilutions of CF. PCR reaction for -actin was 95°C for 1 min, 57°C for 2 min, and 72°C for 2 min, for 36 cycles. Amplified products were stained with ethidium bromide to determine the amount of CF that gave amplified products from the CF and cDNA of equal intensity. After the adjustment of concentration of each cDNA, another housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA concentration of each sample was checked using HPRT specific primers for validation. cDNA samples were then amplified with IL-4, IL-6, IL-10, IFN-, TNF- and TGF-1 specific primers, and products were separated on agarose gels and stained with ethidium bromide. The number of cycles in these PCR reactions was 36 cycles for IL-6, TNF-, TGF-1, and 40 cycles for IL-4, IL-10, IFN-. The annealing temperature for IL-4, IL-10 was 57°C, and for the others was 55°C. The sequences of primers for cytokine genes are as follows: -actin forward, 5′-TGGAATCCTGT GGCATCCATGAAAC-3′; -actin reverse, 5′-TAAAACGCAGC TCAGTAACAGTCCG-3′; IL-4 forward, 5′-CGAAGAACACCA CAGAGAGTGAGCT-3′; IL-4 reverse, 5′-GACTCATTCATGG TGCAGCTTATCG-3′; IL-6 forward, 5′-TTCCTCTCTGCAAGAGACT-3′; IL-6 reverse, 5′-TGTATCTCTCTGAAGGACT-3′; IL-10 forward, 5′-GGACAACATA CTGCTAACCGA CTC-3′;
CII fed PBS fed CII nasal PBS nasal
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4964 15109 587 8266
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IL-10 reverse, 5′-AAAATCACTC TTCACCTGCTCCAC-3′; IFN- forward, 5′-CACACTGCATCTTGGCTT-3′; IFN- reverse, 5′-ACTCCTTTTCCGCTTCCT-3′; TNF- forward, 5′-GCGACGTGGA ACTGGCAGAAG-3′; TNF- reverse, 5′-GGTACAACCCA TCGGCTGGCA-3′; TGF-1 forward, 5′-CTTTAGGAAG GACCTGGGTT-3′; TGF-1 reverse, 5′-CAGGAGCGCACAATCATGTT-3′; HPRT forward, 5′-CTCGAAGTGTTGGATACAGG-3′; HPRT reverse, 5′-TGGCCTATAGGCTCATAGTG-3′;
Results Oral and nasal administration of CII inhibits activation of IFN- production To study the effect of oral and nasal administration of CII we tested proliferative responses and cytokine production of lymph node and spleen cells from DBA/1 mice pretreated by the oral or nasal route with CII. Mice were subsequently immunized in the footpad and popliteal lymph node and spleen cells were taken 10 days after immunization and cultured in vitro with CII. Suppression of proliferative responses and IFN- was seen in lymph nodes of both nasal and oral treated groups (Table 1). Furthermore, oral administration of CII induced TGF- in the spleen. No IL-10 or IL-4 was observed in primary culture. A similar suppression of IFN- was observed in spleen cell culture (data not shown). T cell lines derived from mice treated by the oral or nasal route produce Th2-type cytokines The cytokine production of long term T cell lines from animals treated by the oral or nasal route was investigated upon restimulation of the lines in vitro with CII. T cell lines derived from orally treated animals produced less IFN- than PBS fed controls (Table 2), consistent with the suppression of IFN- observed in primary cultures (Table 1). In addition, these lines
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Table 2. Long-term T cell lines from orally and nasally treated mice produce IL-4 and IL-10
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Cytokine secretion from long-term T cell lines derived from lymph nodes of CII fed or PBS fed animals immunized with CII in CFA as described in Materials and Methods. Lines represent stable cytokine production after being maintained for 2–3 months stimulated every 14 days in vitro with antigen presenting cells pulsed with 50 g/ml CII. Supernatants were measured for cytokines by ELISA. The value represents the cytokine secretion (pg/ml in test culture with antigen −pg/ml in control culture with no antigen).
10
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Figure 1. Oral and nasal administration of collagen suppresses onset and severity of CIA. Prior to immunization mice were treated 5 consecutive days with either oral PBS ( ), 30 g oral CII ( ) or three consecutive days nasally with 10 g CII ( ). Mice were immunized with 100 g CII in CFA and boosted 21 days later. Beginning 5 days after the booster, mice were monitored for CIA and scored as described in Materials and Methods.
produced IL-10 and IL-4. T cell lines derived from animals treated by the nasal route were also found to have a decreased production of Th1 type cytokines and an increase in IL-4 and IL-10 (Table 2).
treated (n=16] was also significantly reduced at 40 days after the booster (PBS vs. oral, P=0.02) and at 80 days after the booster in those mice receiving CII nasally [100% in PBS treated (n =12) vs. 53% in nasally treated (n=17); PBS vs. nasal, P=0.009]. In order to investigate the immune responses associated with this effect, we analysed the cytokine production and proliferative responses of spleen cells at different times throughout the disease course. To induce arthritis in DBA/1 mice two immunizations are required, the first with CII emulsified in adjuvant and the second 21 days later with soluble CII. We first tested animals 10 days after the first immunization. Although immune responses were examined before the booster and before the animals started to show the first symptoms of arthritis, a clear distinction was observed between the treated and control groups. As shown in Figure 2, animals treated by either the oral or nasal route showed a suppression of proliferation and INF- production in comparison to the control group. These results suggest that IFN- responses were suppressed prior to clinical evidence of joint involvement. The second time point studied was 70 days after the booster. At this time all animals from the control group were sick and the spleen cells showed proliferative responses greater than 5000 CPM (Figure 3). By contrast, the orally and nasally treated groups which had a decrease in both disease severity and incidence, had lower proliferative responses. A comparison of the proliferative response between the groups indicates that sick animals from the treated groups generally showed lower levels of proliferation than the control group (Figure 3). Similar results were observed for IFN- production (Figure 4). All of the sick animals from the PBS treated group produced measurable IFN-; however, only 1/3 nasally treated, and 1/4 orally treated mice produced IFN-. None of the non-sick mice produced measurable amounts of IFN-.
Nasal administration of CII suppresses inflammatory cytokines in the joints To further examine the mechanisms involved in the suppression of arthritis by nasal administration of CII we investigated the cytokine pattern in the joints of treated and control animals. Joints of treated animals showed lower levels of TNF- and IL-6, two inflammatory cytokines, in comparison to arthritic joints of control animals (Figure 5). Both groups showed high expression of TGF- mRNA.
Oral and nasal administration of CII suppresses CIA
Discussion
We then directly compared the effect of oral and nasal administration of CII on the incidence and severity of CIA. As shown in Figure 1, both oral and nasal CII lessened disease severity (P<0.01). The incidence of disease [100% in PBS treated (n=12) vs. 62% in orally
The gastrointestinal tract is the major site of antigenic contact in the body. The gut associated lymphoid tissue (GALT) provides for absorption of nutrients, avoiding hypersensitivity to food antigens and exclusion of pathogens [24, 25].
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Figure 2. Oral or nasal administration of collagen suppresses (A) proliferative responses and (B) IFN in vitro. Spleen and lymph node cells were collected from DBA/1 mice treated as in Figure 1, 10 days after immunization and cultured with CII in vitro. Values represent the mean ±SEM of triplicate cultures.
The uptake of macromolecules, particulate antigens and microorganisms across the intestinal epithelia is restricted by multiple mechanisms including local secretions containing mucins and secretory IgA, rigid and packed microvilli, and the glycocalyx: a thick layer of membrane anchored proteins and several enzymes [26]. An organized lymphoid tissue in the form of Peyer’s patches (PP) has a specialized epithelium, known as M cells, capable of transporting antigens across the mucosal barrier. In contrast to most epithelial cells, M cells endocytose material via a transepithelial vesicular transport system. T cells can interact with antigen presenting cells in the PP and then migrate to mesenteric lymph nodes and reach the peripheral immune system as well as other mucosal sites of the body, including the lamina propria in the gut [16, 25]. Although both Th1 and Th2 cells are present in the GALT, the overall environment is predominantly Th2 and affords IgA class switch and secretion [27, 28]. These results indicate the uniqueness of the GALT since it favors the induction of Th2 cells and TGF- secreting regulatory cells (Th3-type cells). The cytokine milieu present in the GALT strongly influences the generation of T cell subsets that provide help to induce and differentiate IgA + B cells [29]. TGF- derived from T cells and non-lymphoid cells and IL-5 derived from Th2 cells favor the switch from IgM to IgA and together with IL-6 help the terminal differentiation of IgA-secreting plasma cells in the gut [30, 31]. It has been shown that after oral administration of a number of antigens, including sheep red blood cells, ovalbumin, and myelin basic protein, T cells are generated in Peyer’s patches which migrate to the spleen and lymph node, generating peripheral tolerance [32–34]. One of the primary mechanisms of active cellular suppression seen following oral administration of antigens is via the generation of T cells which secrete anti-inflammatory cytokines such as
TGF-, IL-4 and IL-10 [17, 18, 35]. In recent years, the induction of oral tolerance to self-antigens has been extensively studied as a tool to regulate the immune system in a manner that inhibits inflammatory events that lead to autoimmunity [16]. Recent analysis of cytokine expression in rheumatoid arthritis (RA) tissues and in experimental models of RA has provided a better understanding of the role of specific cytokines involved in the inflammatory process. Thus, it has been shown that proinflammatory cytokines such as TNF-, IL-1, IL-6, GM-CSF and chemokines such as IL-8 are abundant in RA tissues [4]. Furthermore, there appears to be an attempt to regulate this inflammatory milieu through the increased production of cytokines such as IL-10 and TGF-, and cytokine inhibitors such as IL1ra and soluble TNF-R. However, these regulatory mechanisms are not sufficient to prevent tissue damage. The morphological characteristics of Peyer’s patches (PP) and nasal associated lymphoid tissue (NALT) and the adhesion patterns connected with them indicate that PP and NALT may play equally important roles in the mucosal immune response [36, 37]. Both oral and nasal administration of collagen has been reported to suppress CIA but the mechanisms associated with this suppression are not completely defined [7, 10, 11]. In the present study we show that oral and nasal administration of low doses of CII inhibit the generation and activation of antigenic specific Th1 cells in the spleen and draining lymph nodes. Furthermore, we show that cell lines producing anti-inflammatory cytokines such as IL-4 and IL-10 can be generated from mice treated by the oral or nasal route with CII. The suppression of inflammatory events in the joints of tolerized mice was further demonstrated by the diminished expression of mRNA for TNF- and IL-6, two cytokines involved with the inflammatory and pathological process of CIA. Of note, both groups
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Figure 3. Proliferative responses from spleen cells of sick and non-sick mice. Proliferative responses of spleen cells 79 days after booster. Mice were treated as in Figure 1 and spleen cells cultured in 10 ug/ml CII. The mice were pretreated with (A) PBS orally, (B) 10 g CII nasally or (C) 30 g CII orally. Solid bars represent sick and open bars non-sick mice. Each bar represents an individual mouse.
of mice (tolerized and control) expressed high amounts of TGF-, which is concordant with the cytokine pattern observed in joints of arthritic patients. In contrast to other animal models of autoimmune diseases such as EAE [19], TGF- is highly expressed in the target organ of arthritis, the inflamed joints [4]. A vast array of functions attributed to TGF-
can be demonstrated in arthritic joints, including inflammatory and non-inflammatory activities and tissue repair [38–40]. TGF- contributes to the tissue repair and inhibits the destruction of the cartilage and bone by inhibiting the production of metaloproteinases such as collagenase and inducing the production of TIMP, type I and type XI collagen [6, 41].
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Figure 4. IFN- production from spleen cells from sick mice 79 days booster. Mice were treated as in Figure 1 and spleen cells cultured in 10 ug/ml CII 79 days after booster. IFN- was determined from spleen cells of sick mice treated with (A) PBS orally, (B) 10 g CII nasally or (C) 30 g CII orally. Each bar represents an individual mouse.
Several studies have demonstrated the ability of IL-4 and IL-10 to suppress the inflammatory process and tissue damage in experimental models of arthritis. Intraperitoneal administration of recombinant IL-4 inhibits an experimental model of arthritis by decreasing the influx of inflammatory cells to the tissue, decreasing TNF- production and reactive oxygen
intermediate metabolism [21]. It also increased the production of IL-1ra, which can modulate inflammation and tissue degradation by blocking IL-1 produced PGE2 and collagenase, and neutralizing IL-1 induction of IL-6. More recently, Horsfall et al. showed that upon continuous administration of IL-4 in CIA DBA/1 mice, anti-collagen antibodies
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Figure 5. Nasal CII suppresses production of inflammatory cytokines in the joints. RT-PCR for TNF-, IL-6 and TGF- was performed on homogenates of mouse joints from mice treated with PBS or 10 g CII nasally. Joints were collected 60 days after the booster. HPRT was used as an internal control.
were reduced, histologic score improved and there was a 1000-fold decrease in TNF- secretion by synovial cells [42]. There is evidence for the involvement of endogenous IL-4 in the suppression of arthritis. It was shown in comparing the cytokine pattern of three strains of rats injected with CII [43] that the susceptible strain (DA) produced a strong humoral response and Th1 cytokine profile, whereas the resistant strain (F344) produced a limited humoral response with a Th2 profile (IL-4). Furthermore, the involvement of nonMHC genes in determining the direction of the response to CII was demonstrated by the analysis of a strain MHC congenic to the DA rat (PVG.1AV1). This strain was resistant to arthritis and produced a marked humoral response of isotype IgG1 and also IL-4. Mauri et al. demonstrated that during CIA in mice, the cellular immune response is characterized by the activation and proliferation of CD4 + T cells producing IFN- and during the spontaneous remission phase, there is a shift in favor to antagonistic cytokines such as IL-4 and IL-10 [44]. IL-10 is well known to inhibit IFN- production by Th1 cells but also can inhibit the production of a variety of cytokines from other leukocyte populations [45]. IL-10 inhibits the production of IL-1, IL-6, TNF-, IL-8, G-CSF, from macrophages and IL-1, TNF, IL-8, MIP1, and MIP1 from polymorphonuclear cells. Most of these cytokines and chemokines have been implicated in the pathological process of arthritis. The therapeutic effect of IL-10 was demonstrated by treatment of DBA/1 mice with injections of IL-10 starting before or after disease onset [46]. Both studies showed a decrease in the severity of arthritis. Interestingly, in a comparative study on the therapeutic effect of Th2 cytokines in arthritis, injection of IL-4 alone did not suppress the disease, and IL-10 suppressed it slightly, whereas combined therapy with IL-4 and IL-10 after
G. Garcia et al.
disease onset reduced cellular infiltrates and provided protection from cartilage destruction [47]. Our results demonstrate the ability of oral or nasal tolerance to CII to inhibit the incidence and severity of CIA in DBA/1 mice and induce similar immune responses. The finding that spleen cells from tolerized mice proliferate less and produce less IFN- in vitro than controls is direct evidence of the suppression of a Th1 inflammatory response. Of note is the observation that the mice treated by the oral or nasal route with CII became sick, but nonetheless showed diminished proliferative responses in vitro compared to control and did not produce IFN-. This immunological response is most probably related to the mild outcome of the disease seen in these mice. Our results also demonstrate that low doses of orally or nasally administered CII preferentially induce cells which produce anti-inflammatory cytokines such as IL-4, IL-10 and this is associated with a decrease of TNF- and IL-6 in the joints, something that has not been demonstrated previously in studies of CIA treated with mucosal antigen. Thus, the administration of collagen by either the oral or nasal route is an effective immunotherapeutic intervention designed to shift the balance of the immune response from Th1 to Th2 and to suppress the inflammation and the tissue destruction of arthritic joints.
Acknowledgements This study was supported by NIH grant AI43458. GG received a fellowship from CAPES, Brazil.
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