Chondrocyte antigen expression, immune response and susceptibility to arthritis

Vera S. F. Chan1,3, E. Suzanne Cohen1, Thomas Weissensteiner1, Kathryn S. E. Cheah2 and Helen C. Bodmer1

1 The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire RG20 7NN, UK
2 Department Biochemistry, The University of Hong Kong, Sassoon Road, Hong Kong, China

Correspondence to: H. C. Bodmer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The association of HLA-B27 with certain forms of arthritis implies a role for MHC class I-restricted T cells in the arthritic process. Our aim was to study CD8+ T cell responses towards specific antigens localized in joint tissue. Known determinants were introduced into chondrocytes of transgenic (TG) mice, under the control of the cis-regulatory sequences of the human type II collagen gene (COL2A1). Two Escherichia coli ß-galactosidase (ß-gal)-expressing lines were derived (CIIL73 and CIIL64) as well as two lines (CIINP) expressing influenza A virus nucleoprotein (NP). Expression of the antigens could be demonstrated in cartilaginous tissues. The TG lines showed variable degrees of responsiveness towards the transgene-introduced antigens; whilst 75% of CIIL73 mice had an impaired cytotoxic T lymphocyte (CTL) response towards ß-gal, the response in CIIL64 mice was essentially normal. However, both lines displayed normal proliferative and antibody responses to ß-gal. A reduced CTL response was seen to NP in the CIINP lines in ~65% of the animals. In spite of the persistence of T cell responses to the transgene antigens in these lines, induction of CTL responses alone has so far failed to induce clinical signs of arthritis. Interestingly, some animals expressing ß-gal were susceptible to arthritis following challenge with type II collagen alone, whilst their non-TG littermates and TG mice from other lines remained unaffected. As ß-gal is expressed by E. coli, a component of the normal gut flora, this suggests a possible role for gut-derived immune responses. We believe these lines could form the basis of a model for studying links between intestinal inflammation and arthritis.

Keywords: ß-galactosidase, ankylosing spondylitis, autoimmunity, BALB/c, collagen, cytotoxic T lymphocyte, influenza A virus, mice, nucleoprotein, rodent, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The human MHC allele, HLA-B27, is strongly associated with the seronegative spondyloarthropathies, including ankylosing spondylitis and reactive arthritis. One hypothesis to explain this association is that the MHC class I molecules present potentially arthritogenic peptides to cytotoxic T lymphocytes (CTL), which may contribute to the arthritic process when activated. However, the demonstration of CD8+ T cell responses to self-antigen has been hampered by the failure to identify potential arthritogenic antigens. Proteins expressed more specifically in joints, such as type II and type XI collagen, have been suggested as target antigens for autoreactive T cells (1). However, it has proved difficult to isolate collagen-specific CD8+ T cell clones from peripheral blood of patients suffering spondyloarthropathies (2).

Numerous transgenic (TG) models have been used to investigate the CD8+ T cell responses to `neo-self' antigens in the periphery (35). In a few cases, the TG antigen itself can cause spontaneous development of disease. The over-expression of MHC antigens in the pancreas (6,7) or in the central nervous system (8) can lead to spontaneous diabetes or dysmyelination respectively. However, in most cases, autoimmune disease does not occur spontaneously, either because of T cell `ignorance' (9,10) or tolerance (11,12). Challenging these TG animals with the relevant antigen can, however, yield very different results depending on the model. In the RIP-LCMV (9,10) and MBP-LCMV (13) models, LCMV infection can initiate an autoimmune response. On the other hand, in the RIP-HA system (11), influenza virus infection could not break the tolerance and pancreatic infiltration has not been observed. The human type II collagen gene (COL2A1) has been used to express an MHC class II-restricted T cell epitope involved in the development of collagen-induced arthritis (CIA) in TG mice (14,15). However, there are no reports of this approach applied to MHC class I-restricted T cell responses in joint-related disease.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Gene constructs and the generation of TG mice
The transgene construct, pKL80.3, has been described previously (16). It contains the 6.1 kb 5' flanking DNA sequence of the COL2A1 gene, the 3.5 kb Escherichia coli ß-gal gene (ß-gal; LacZ) cassette including the SV-40 poly(A) tail and the 0.3 kb enhancer element essential for tissue-specific expression (CSE) cloned into the vector pPolyIII. The second construct, pANP, contained the 4.8 kb COL2A1 promoter fragment from a second plasmid pAA2 (17). cDNA encoding nucleoprotein (NP) from the influenza A virus, A/NT/60/68 (obtained from the plasmid NP28, a gift of A. Townsend), followed by a stop codon and the poly(A) tail, replaced the majority of the LacZ gene, being cloned into an EcoRI site at position 3016 (45 bases upstream of the LacZ stop codon). This construct also has the 0.3 kb CSE from pKL80.3. In arthritis induction experiments, a line, CIIB7.1, expressing human CD80 in place of NP was used.

Vector sequence was removed from the transgene constructs using the restriction enzyme NotI. TG mice were generated by pronuclear injection of DNA into (C57BL6x BALB/c) F2 oocytes. Lines of mice were produced from five independent TG founders, two for each of the constructs: LacZ (lines CIL64 and CIIL73), NP (lines CIINP14 and CIINP45) and one for CD80 (line CIIB7.1). TG mice were genotyped by Southern hybridization of tail DNA using a 1.6 kb EcoRI fragment of the COL2A1 proximal sequence as a probe. Copy numbers were estimated by relative intensity of the transgene band to that produced by hybridization of the probe to the mouse COL2A1 promoter. Founders were backcrossed onto the BALB/c background and were kept in the specific pathogen-free unit at the Institute for Animal Health, UK.

X-gal staining of the neonatal joints
The X-gal staining procedure was modified from a previously described method (18). Briefly, ribs and limb joints were dissected out from 1- to 3-day-old neonatal mice, and were fixed in 0.2% glutaraldehyde, 2% formaldehyde, 5 mM EGTA, pH 7.3 and 2 mM MgCl2 in PBS at room temperature for 30 min. This was followed by three 5 min washes with rinse solution (0.1% deoxycholic acid, 0.2% Nonidet P-40 and 2 mM MgCl2 in PBS) at room temperature. The tissues were incubated at 30°C for 24 h in the dark in substrate solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal), 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide in rinse solution. After colour development, the tissues were washed with rinse solution and processed for paraffin wax embedding. Sections (3 µm) were cut and counter-stained with eosin for microscopic examination.

Immunohistochemistry
Ribs and limb joints were carefully dissected out from neonatal mice and were fixed in 4% paraformaldehyde in PBS at 4°C for 20 min. After two PBS washes, the tissues were embedded in Tissue-Tek. The NP transgene expression was detected using the IMAGEN influenza virus A and B detection kit (code K6105; Dako, High Wycombe, UK). Frozen sections (7 µm) were re-hydrated in PBS. Then 25 µl of reagent A (containing FITC-conjugated mAb against NP and matrix protein, and Evans Blue as a counter-stain) was added to each section and incubation was carried out in darkness in a humidified chamber at 37°C for 1 h. It was followed by two washes of PBS for 5 min. The sections were mounted and examined under a confocal fluorescence microscope (DM RBE; Leica, Heidelberg, Germany) and the images were captured using the True Confocal Scanner with TCS-NT software (Leica).

Immunization
Adult mice from the ß-gal TG lines were immunized i.p. with 107 p.f.u. vaccinia recombinant for NP. Mice from CIINP lines were infected intranasally with 50 µl PBS containing 20 HAU influenza virus A strain A/X31. For protein immunization, mice were injected s.c. in two sites with 100 µg E. coli ß-gal (Boehringer Mannheim, Germany) emulsified with an equal volume of CFA containing 0.5 mg H37RA Mycobacterium tuberculosis (Difco, Detroit MI).

In vitro re-stimulation and 51Cr-release assay
Spleens were obtained 2 weeks after viral infection for in vitro re-stimulation. Autologous splenocytes were incubated with 1 µM ß-gal876–884 (TPHPARIGL) or NP147–155 (TYQRTRALV) (Research Genetics, Huntsville, AL) peptide in RPMI at 37°C for 1 h and used as stimulators. In vitro re-stimulation cultures were set up with 1.5x107 splenocytes and 0.3x107 peptide-pulsed stimulators in 15 ml RPMI supplemented with 10% FCS, 50 IU/ml penicillin and streptomycin, 0.3 g/l L-glutamine, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, and 5 U/ml Lymphocult-T as a source of IL-2 (Biotest, Dreirich, Germany). The culture was maintained at 37°C, 5% CO2 for 5 days, at which time a standard 51Cr-release assay was performed. P815 target cells were pulsed with 1 µM peptide or unpulsed as indicated.

Proliferation assay
Two weeks after immunization, draining lymph nodes were removed and disrupted to form single-cell suspensions. Lymph node cells (3–5x105) were co-cultured, in triplicate wells, with E. coli ß-gal at various concentrations in 200 µl RPMI supplemented with 10% FCS in a 96-well flat-bottomed plate. At least four control wells without the antigen were included to represent the background counts. The culture was incubated at 37°C, 5% CO2 for 4 days and 1 µCi of [3H]thymidine (sp. act. = 25Ci/mmol; Amersham Life Science, Little Chalfont, UK) was added to each well for the last 18 h. [3H]Thymidine uptake was measured using the 1450 MicroBeta TRILUX ß-plate counter (Wallac, Turku, Finland).

ELISA
ELISA plates (Maxisorp; Nunc, Life Technologies Ltd, Paisley, UK) were coated with 50 µl ß-gal solution at 5 µg/ml in PBS containing 0.05% sodium azide at 4°C overnight. The solution was flicked off and the plates were washed 5 times with PBS-T (0.1% Tween 20 in PBS). This washing procedure was repeated between the subsequent incubation steps. The plates were then pre-treated with 100 µl blocking buffer (1% casein in PBS-T) at room temperature for 30 min followed by 100 µl diluted serum samples at 37°C for 1 h and 50 µl of 1µg/ml peroxidase-conjugated anti-mouse IgG (Vector, Laboratories, Peterborough, UK) for another 1 h. This was followed by incubation with 100 µl peroxidase substrate, ABTS (Vector), at room temperature for 20 min in darkness. Substrate colour change was measured as OD at 405 nm using the Biomek plate reader (Beckman Instruments, High Wycombe, UK ).

Induction of arthritis with collagen
Bovine type II collagen (CII) (cat. no. C-1188; Sigma) was dissolved in 0.1 M acetic acid and emulsified with equal volume of complete Freund's adjuvant (CFA; Difco, Detroit, MI) to a final concentration of 1 mg/ml. Mycobacterium tuberculosis H37 RA (Difco) was added to a final concentration of 5 mg/ml. Mice were immunized s.c. with 100 µg CII/CFA mixture. After 4 weeks, animals were boosted s.c. with 100 µg CII emulsified in incomplete Freund's Adjuvant (IFA; Difco). When mice were also challenged with vaccinia, 10 days after CII priming, they were boosted with 107 NPVV (vaccinia recombinant for NP and ß-gal). Then, 40 days after the initial challenge, the mice were injected s.c. with 100 µg CII/IFA mixture. The genetic status of the mice was not known during the monitoring period of 3–4 months. Arthritis scoring: grade 1 = redness or slight swelling of the paw, grade 2 = obvious increase in paw thickness and grade 3 = visible joint distortion. Only those mice that had an arthritis score of >=1 for 5 consecutive days were considered as showing clinical signs of arthritis. Onset was then defined as the first day the signs had appeared.

At the end of the monitoring period, joints were taken for histological assessment. Feet or knees were dissected out, fixed and decalcified in Decalcifier I solution (Surgipath Europe Ltd, Cambridge, UK). Normal samples were taken at the same time for comparison. Paraffin sections (3 µm) were cut along a longitudinal axis and stained with haematoxylin & eosin. In some experiments, only joints taken from animals having shown signs of arthritis were taken for histology. In experiments where all mice provided samples for histology, no abnormalities were detected in the absence of clinical signs.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tissue-specific expression of the transgene
The COL2A1 regulatory elements were used to direct expression of the ß-gal, NP and CD80 to tissues that synthesize CII, principally chondrocytes in the developed animal (17,19). The two ß-gal lines differed in the transgene copy number, CIIL73 having >100 copies while CIIL64 had ~20. Figure 1Go(B and C) shows X-gal-stained paraffin sections from the limb joints of ß-gal TG 1-day-old neonates. Intracellular X-gal staining was observed in isolated patches of cells in cartilaginous tissues in the knee joint (Fig. 1BGo) and in the metatarsal joint (Fig. 1CGo) of TG animals. No detectable signal was seen in the corresponding area of non-TG animals (Fig. 1AGo). Although both ß-gal-expressing lines showed similar patterns of expression in cartilage, there were differences in the non-chondrocyte expression patterns of the two TG lines. During embryonic development (day 15–16 gestation), CIIL73 was expressed strongly in the lungs, whilst CIIL64 had no lung expression but patchy expression in the heart (data not shown). Young adults, however, did not generally express the transgene antigen in tissues other than ribs, joints or, occasionally, brain (RT-PCR, data not shown).



View larger version (103K):
[in this window]
[in a new window]
 
Fig. 1. Expression of ß-gal or NP transgenes in neonatal chondrocytes. (A–C) ß-Gal expression revealed by X-gal staining, counter-stained with eosin, in chondrocytes of CIIL73 1-day-old neonates. (A) Non-TG littermate, magnification x20. (B) TG knee, magnification x200. (C) TG metatarsal, magnification x400. (D–F) confocal image of expression of NP in frozen sections of neonatal joints from the CIINP14 line. Sections are stained with FITC-conjugated anti-NP antibody and counter-stained with Evans Blue; positive staining for NP is shown in yellow. (D) Non-TG littermate. (E) TG at the same magnification. (F) Higher magnification of E.

 
NP expression in neonatal joints and ribs of the CIINP14 line was detected using immunofluorescence staining on frozen sections. Figure 1Go(E and F) shows positive staining for NP in neonatal ribs. Minimal background staining was observed in the corresponding area in non-TG mice (Fig. 1DGo). Positive staining for NP was also seen in scattered chondrocytes in the cartilaginous area in the knee joint of the TG animals (data not shown). Transgenes were not expressed in the thymus of CIINP14, CIIL64 or CIIL73 lines as assessed by RT-PCR assays or X-gal staining of 16.5-day embryos of the CIIL73 and CIIL64 lines (data not shown).

CD80 expression in the CIIB7.1 line was demonstrated in the ribs and joints of TG animals by RT-PCR, and on the cell surface of a proportion of chondrocytes isolated from neonatal cartilage (data not shown).

Transgene-dependent reduction of CTL, but not proliferative, T cell responses
To determine the effect of transgene expression on CTL responses, TG and non-TG littermates from CIIL73 and CIIL64 lines were immunized i.p. with vaccinia recombinant for NP (which also expresses E. coli ß-gal as a selection marker). Spleen cells were assayed for transgene-specific cytotoxicity and results for the CIIL73 line are represented in percent lysis of different targets at different E:T ratios (Fig. 2Go). Both the TG and non-TG mice mounted good CTL responses to the control antigen, as shown by the specific lysis of the NP147–155-pulsed targets (Fig. 2Go B). In contrast, most, but not all, of the TG animals had an impaired CTL response to the transgene antigen, ß-gal (Fig. 2Go A). However, among those CIIL73 TG mice that could mount a ß-gal CTL response, the magnitude of the response was comparable to that observed in the non-TG mice. Unlike the CIIL73 line, CIIL64 TG mice could mount a transgene-specific CTL response that was comparable to that of their non-TG littermates (Table 1Go and data not shown). In order to assess the frequency of mice with an impaired ß-gal CTL response, we counted those animals that showed <10% specific lysis at the maximum E:T ratio as non-responders. The results are summarized in Table 1Go, showing 72% of CIIL73 TG mice to be non-responders.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2. Impaired CD8+ T cell response to the transgene antigen in the CIIL73 line. Individual animals are shown and are representative of the data summarized in Table 1Go. Panel (B) shows the NP response from the same mice shown on the top row of (A). Animals were immunized with vaccinia virus recombinant for NP, then 2 weeks later spleen cells were re-stimulated with either ß-gal876–884 (A) or NP147–155 (B) and then assayed in a standard 51Cr-release assay. Open diamonds, untreated P815 targets cells; closed diamonds, ß-gal876–884-pulsed P815 target cells; closed circles, NP147–155-pulsed cells; closed squares, P815 cells transfected with ß-gal.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Summary of ß-gal CTL response in the CIIL73 and CIIL64 lines
 
In order to examine CTL responses to the transgene antigen in the CIINP14 line (expressing NP), mice were challenged by intranasal infection with influenza A virus. Figure 3Go(A) shows results of individual mice from a representative experiment. All the TG animals retained the ability to respond to NP; however, the response from the majority of these titrated out more quickly, in terms of effector cell numbers, than their non-TG littermates. Interestingly, although the reduction in CTL response to the transgene was not complete, as in the CIIL73 line, a similar proportion of TG animals was affected. Mice were grouped as low responders if the specific lysis was <50% of the maximal killing after 1 log10 titration of the highest E:T, if >50%, as high responders. Figure 3Go(A) shows an example of one high- and one low-responder TG mouse. Figure 3Go(B) shows a box and whisker plot of responses from a total of 25 TG and 25 non-TG animals to NP147–155 at an intermediate E:T ratio of 8. The difference between the two groups is highly significant (P < 0.0001). Table 2Go summarizes the results of five independent experiments, showing 64% of CIINP14 TG mice to be low responders. The difference in responder status between TG animals was unlikely to be due to a gross reduction of the affinity or avidity of the responding T cells, as a titration of peptide, at a constant E:T of 10, was equivalent for both TG and non-TG mice. Figure 4Go shows a titration from six representative animals, including three low-responder and one high-responder TG mice. Although the total lysis for the low-responder mice was lower than that for the others, peptide sensitivity remains the same for all the animals. In all, a total of 12 mice tested, irrespective of whether they were high or low responders, had very similar dose–response curves.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Reduced CD8+ T cell response to the transgene antigen in the CIINP14 line. (A) The responses of representative individual animals of the CIINP14 line are shown following in vivo Immunization with influenza A virus followed by in vitro re-stimulation of spleen cells with NP147–155 peptide. Closed symbols, P815 cells pulsed with peptide NP147–155; open symbols, unpulsed P815 cells. Data are representative of results summarized in Table 2Go. Lo, low responder; Hi, high responder. (B) Box and whisker plot representing specific lysis of NP147–155-pulsed P815 target cells at an E:T ratio of 8. Pooled data from five experiments with 25 TG and 25 non-TG animals are shown. The box represents ~50% of the values, the whiskers show the range, the filled circle is the mean response and the line bisecting the box is the median response; asterisk indicate outliers.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Summary of NP CTL response in the CIINP14 line
 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. CTL from TG and non-TG mice of the CIINP14 line have equal avidity for peptide. Specific lysis of 51Cr-labelled P815 target cells in the presence of a titration of NP147–155 peptide as shown, at a constant E:T ration of 10. Results are for individual mice. TG-lo, low responder as defined in Table 2Go; TG-hi, high responder as defined in Table 2Go. Both non-TG littermates shown are high responders. Results are representative from 12 individual mice tested.

 
CD4+ T cell response to the transgene antigen in CIIL73 TG mice
To analyse whether CD4+ autoreactive T cells are still present in the TG animals, mice from the CIIL73 line were tested for proliferative and antibody responses towards the transgene antigen. Mice were immunized with ß-gal in CFA s.c. Figure 5Go(A) shows proliferation of draining lymph node cells at various antigen concentrations. Both groups of mice responded equally well to the transgene antigen with no significant difference between the TG and non-TG mice. To measure the antibody response to the transgene antigen, blood samples were taken before immunization and at the time of sacrifice. Individual serum samples were assayed for anti-ß-gal IgG by ELISA (Fig. 5BGo). Again, no significant difference was seen between TG and non-TG animals. Thus, although there was often a profound effect on the CTL response to the transgene antigen in the CIIL73 line, the CD4+ T cell and antibody responses were unaffected.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. TG and non-TG animals from the CIIL73 line make equivalent proliferative T cell and antibody responses to ß-gal. (A) Proliferative response to ß-gal of draining lymph nodes cells following immunization with ß-gal/CFA. Mean response for each group (n = 5). Representative of two experiments. Closed squares, CIIL73 TG mice; open squares, non-TG littermates. (B) Whole IgG antibody response in the serum of CIIL73 TG mice (black bars) or non-TG littermates (white bars) from the same animals as in (A).

 
Arthritis induction in ß-gal TG mice
No mice of any of the TG lines has been observed to develop spontaneous arthritis or joint disease over and above that of normal or non-TG animals up to at least 18 months of age. Therefore expression of the transgenes per se does not appear to be detrimental to development or have any deleterious effects on the mice. In addition, some mice were left for several months following priming as for CTL responses (above). During this time animals were monitored for possible clinical signs of arthritis. None were seen during a period of at least 3 months nor were any histological signs of arthritis seen in a selection of mice tested (data not shown). Therefore, it appeared that simple induction of immune responses against the transgene antigen was insufficient to induce a significant incidence of arthritis.

In other animal models of arthritis, such as CIA, transfer of disease required both autoantibodies and self-reactive T cells (20). In our system, the transgene antigens were expressed intracellularly, either in the cytoplasm or the nucleus. This cellular location could render antigen less available as a target for antibody recognition in healthy, intact cartilage. Thus, we set out to induce an antibody response to a cartilage matrix protein, which would be antibody accessible, prior to transgene antigen immunization. We chose CII because it could elicit an antibody response without inducing arthritis in BALB/c mice (21). In the first instance mice were challenged with CII in CFA on day 0, infected with vaccinia on day 10 and boosted with CII in IFA on day 40. In two independent experiments two mice of the CIIL73 line developed arthritis, assessed by both clinical and histological analysis (Fig. 6Go and Table 3Go: CII–Vaccinia–CII). However, surprisingly, when mice were challenged and boosted with CII alone (without specific induction of responses against ß-gal) some animals from both ß-gal-expressing lines developed arthritis (Table 3Go). Clinical and histological features were similar irrespective of induction protocol, although the two CIIL64 mice had less severe disease, histologically and of later onset. Arthritic signs have only been observed in the hind limbs, predominantly involving the knee, ankle and forefoot. Onset was relatively late and was asymmetrical (generally in only one limb), very much in contrast to classical CIA. Only one of the mice had overt clinical disease in both hind limbs, though with unequal severity. Although clinical signs were seen in a number of non-TG mice (Table 3Go), there was a statistically significant difference in the number of TG animals from either of the ß-gal lines compared to their non-TG littermates (13 of 66 TG versus five of 63 non-TG P = 0.046). The histology, seen only in some of the TG mice, is particularly characterized by polymorph infiltration and synovial proliferation, with evidence of bone erosion and remodelling, cartilage damage, and chondrocyte loss (Fig. 6Go). There is a possibility that antigen expression using the COL2A1 promoter could render the chondrocytes, and thus the cartilage of TG animals, more `fragile' and therefore more susceptible to arthritis than their non-TG littermates. A structural weakness of the cartilage is highly unlikely as the NP and ß-gal transgenes are only expressed intracellularly and therefore would have no influence on the cartilage matrix. In addition, we did not see any signs of arthritis in a large number of animals expressing human CD80 (cell surface expression) under the same promoter nor did we see anything in the very high copy CIINP14 line (Table 3Go). Furthermore, histological analysis of TG, compared to non-TG, animals that remained clinically healthy did not show any deficit in cartilage (data not shown).



View larger version (143K):
[in this window]
[in a new window]
 
Fig. 6. Histological appearances of arthritis in CIIL73 mice. H & E-stained paraffin sections of decalcified samples from CIIL73 TG animals. (A and B) An animal challenged with both CII and recombinant vaccinia virus. (A) Normal tarsal joint of the left hind limb. (B) Tarsal joint from the right hind limb of the same animal, showing extensive soft tissue infiltration of inflammatory cells, peri-articular bone erosion and enthesitis. (C and D) A TG animal which was only challenged with CII. (C) Low-power view of a tarsal joint. (D) High-power view showing fibrillation of the articular cartilage surface of the same tarsal joint.

 

View this table:
[in this window]
[in a new window]
 
Table 3. Summary of arthritis induction experiments
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The association of HLA-B27 with certain forms of arthritis implies a role for MHC class I-restricted T cells in their pathogenesis. However, this does not necessarily imply that these T cells could, by themselves, precipitate the disease. In this study, we have deliberately chosen antigens that have the potential to be efficiently presented in the MHC class I pathway of antigen presentation, being cytoplasmic or nuclear, but are less likely to be efficiently presented to MHC class II-restricted T cells (22,23). We have shown that intracellular expression of TG antigens in chondrocytes can have a variable, but at times profound, effect on the CTL response to that antigen, whilst leaving the CD4+ T cell and antibody responses intact. The lack of an effect on the CD4+ T cell response may be due partially to the cellular localization of the transgene protein. In addition, although chondrocytes can express MHC class II antigens on activation, they are normally class II in mouse and man (24,25). It is of note that the CD8+ T cell response differed in the two lines expressing ß-gal. Although they differed in expression in other tissues during embryonic development, most notably with expression in the heart in the CIIL64 line and in the lung in the CIIL73 line, both lines had similar patterns of expression in cartilage. Nonetheless, a difference between the embryonic expression in the two lines may indicate other subtle differences in expression that could account for the differences seen in the CTL responses.

The reduction of the CTL response is unlikely to be the result of deletion or inactivation of the autoreactive T cells in the thymus, as the COL2A1 regulatory elements do not direct expression in lymphoid tissues (16,19). In addition, transgene expression was not detectable in the thymus by RT-PCR or X-gal staining. Failure to detect transgene expression does not completely rule out the possibility of thymic tolerance and if this were at a critical threshold, then it could explain some of the difference seen between littermates. These questions would be best addressed using TCR TG mice of the appropriate specificity.

Transgene dose is, however, a factor that has the potential to affect the CTL response. The CIIL73 line has ~5 times greater copy number than the non-tolerant CIIL64 line (data not shown) and, although this does not necessarily relate directly to expression level, it indicates a likely difference.

A more intriguing finding is the difference between littermates of the CIINP and CIIL73 lines. Approximately 30% of TG animals are able to make a near-normal CTL response to their transgene antigen, whilst their TG littermates may be profoundly tolerant. The variation between littermates could be related to genetic differences in some of the earlier backcrosses, or to chimeric or variable expression of the transgene. However, this pattern of response appears to be maintained (data not shown) and is seen in three lines of TG mice with independent insertions; therefore, we feel this is unlikely as an explanation. TG TCR animals will be required to precisely define the mechanisms of tolerance. However, the variable nature of the reduction of CTL response between littermates does imply that presentation leading to inactivation or deletion of potentially autoreactive T cells is at or near a threshold, either in level of expression or timing. Both these elements have been found to be important in the development of peripheral tolerance in models with co-expression of antigen and a specific TCR transgene (2628).

No evidence of arthritis was observed in any of the TG lines tested, even in instances when an immune response was efficiently induced against the transgene. This result is in contrast to many other TG models of autoimmunity, including diabetes and central nervous system disease (reviewed in 3,29). Indeed, expression of ß-gal under a retinal promoter did not lead to ß-gal-specific tolerance, but an autoimmune retinitis was precipitated by induction of ß-gal-specific CTL responses in these animals (30). There are a number of potential reasons for the lack disease in our mice expressing similar antigens, but under the COL2A1 promoter. Access of potentially autoreactive T cells to the TG chondrocytes is likely to be restricted in normal healthy animals due to the relatively impermeable matrix of the cartilage, together with a lack of vascularization. Even if CTL had access to the chondrocytes in the absence of any inflammatory signals, the low level of MHC class I on cells would make them essentially invisible to the T cells (E. Cohen et al., manuscript in preparation). Access might be improved by inflammation following damage or stress of the cartilage or enthesis. Although there is speculation that this mechanism could be important in humans, it may be a relatively rare event in a specific pathogen-free mouse colony and therefore not detected in our experiments.

Other induced models of arthritis, in particular CIA, generally require both a specific T cell as well as an antibody response. Transferring either arthritic serum or spleen cells from arthritic mice alone cannot induce complete CIA, which probably requires the synergetic effect from both types of response (20,31). Autoantibodies against CII can be generated in many different strains of mice, although there is a qualitative difference in terms of epitope specificity and IgG subclass from the arthritic serum (32). BALB/c mice are, however, highly resistant to CIA, although there have been reports of a low incidence of disease in some studies (21,33). In our experiments we have not seen histological evidence of arthritis in either BALB/c mouse controls (data not shown) or in non-TG littermates (Table 3Go). Nevertheless, two mice from the CIIL73 line developed a severe inflammatory arthritis as a result of challenge with bovine CII and induction of an anti-ß-gal response by infection with recombinant vaccinia virus.

Surprisingly, a few mice from both lines expressing ß-gal also developed arthritis in the absence of any other antigen-specific challenge. That this was not seen in seven CIINP14 mice or in 36 mice expressing human CD80 under the same COL2A1 promoter indicates that the arthritis may be linked to the transgene antigen, rather than be caused by potential toxic effects of transgene expression on the chondrocytes. Although we have, as yet, no definitive explanation for the low level of susceptibility of the CIIL64 and CIIL73 animals to arthritis induced by CII, the clinical picture seen is distinct from classical CIA. In addition, the nature of the antigen could be important. As ß-gal is an E. coli protein, animals are potentially exposed to this antigen in the resident bacteria of the intestine. We could not see spontaneous ß-gal responses in the spleens of these mice; however, normal mice do show a low level of spontaneous ß-gal-specific response detectable in the mesenteric lymph nodes, but not the spleen (unpublished results). There is a strong link between some forms of arthritis and gastrointestinal or genitourinary infection or inflammation (34,35). Indeed, a number of animal models also depend on the presence of gut bacteria (36,37). Therefore the TG lines described in this report could provide a useful tool for the investigation of an antigen-specific link between gut immune responses and arthritis susceptibility.


    Acknowledgments
 
We would like to thank Dr Keith Leung for providing reagents and helpful discussion and advice on the COL2A1 vector constructions, and Matthew Roddis for technical assistance. Some of this work was supported by a Senior Fellowship for H. C. B. from the Wellcome Trust at Oxford, grant no. 035643. V. S. F. C. was in receipt of a scholarship from the Croucher Foundation.


    Abbreviations
 
ß-gal ß-galactosidase
CFA Freund's complete adjuvant
CIA collagen-induced arthritis
CII type II collagen
COL2A1 human type II collagen gene
CSE cartilage-specific element
CTL cytotoxic T lymphocyte
IFA Freund's incomplete adjuvant
LacZ ß-galactosidase gene
NP nucleoprotein
TG transgenic

    Notes
 
3 Present address: Department of Biophysics, University of Toronto, Ontario Cancer Institute, 610, University Avenue, Toronto, Ontario M5G 2M9, Canada Back

Transmitting editor: E. Simpson

Received 21 July 2000, accepted 13 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Chiocchia, G., Boissier, M. C., Ronziere, M. C., Herbage, D. and Fournier, C. 1990. T cell regulation of collagen-induced arthritis in mice. I. Isolation of Type II collagen-reactive T cell hybridomas with specific cytotoxic function. J. Immunol. 145:519.[Abstract/Free Full Text]
  2. Gao, X. M., Wordsworth, P. and McMichael, A. 1994. Collagen-specific cytotoxic T lymphocyte responses in patients with ankylosing spondylitis and reactive arthritis. Eur. J. Immunol. 24:1665.[ISI][Medline]
  3. Arnold, B., Goodnow, C., Hengartner, H. and Hammerling, G. 1990. The coming of transgenic mice: tolerance and immune reactivity. Immunol. Today 11:69.[ISI][Medline]
  4. Adams, T. E., Alpert, S. and Hanahan, D. 1987. Non-tolerance and autoantibodies to a transgenic telf antigen expressed in pancreatic beta-cells. Nature 325:223.[ISI][Medline]
  5. Kruisbeek, A. M. and Amsen, D. 1996. Mechanisms underlying T-cell tolerance. Curr. Opin. Immunol. 8:233.[ISI][Medline]
  6. Allison, J., Campbell, I. L., Morahan, G., Mandel, T. E., Harrison, L. C. and Miller, J. F. 1988. Diabetes in transgenic mice resulting from over-expression of class I histocompatibility molecules in pancreatic beta cells. Nature 333:529.[ISI][Medline]
  7. Lo, D., Burkly, L. C., Widera, G., Cowing, C., Flavell, R. A., Palmiter, R. D. and Brinster, R. L. 1988. Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells. Cell 53:159.[ISI][Medline]
  8. Turnley, A. M., Morahan, G., Okano, H., Bernard, O., Mikoshiba, K., Allison, J., Bartlett, P. F. and Miller, J. F. 1991. Dysmyelination in transgenic mice resulting from expression of class I histo- compatibility molecules in oligodendrocytes [see Comments]. Nature 353:566.[ISI][Medline]
  9. Ohashi, P. S., Oehen, S., Buerki, K., Pircher, H., Ohashi, C. T., Odermatt, B., Malissen, B., Zinkernagel, R. M. and Hengartner, H. 1991. Ablation of `tolerance' and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 65:305.[ISI][Medline]
  10. Oldstone, M. B., Nerenberg, M., Southern, P., Price, J. and Lewicki, H. 1991. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response. Cell 65:319.[ISI][Medline]
  11. Lo, D., Freedman, J., Hesse, S., Palmiter, R. D., Brinster, R. L. and Sherman, L. A. 1992. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects both CD4+ and CD8+ T cells. Eur. J. Immunol. 22:1013.[ISI][Medline]
  12. Schonrich, G., Kalinke, U., Momburg, F., Malissen, M., Schmitt-Verhulst, A. M., Malissen, B., Hammerling, G. J. and Arnold, B. 1991. Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 65:293.[ISI][Medline]
  13. Evans, C. F., Horwitz, M. S., Hobbs, M. V. and Oldstone, M. B. A. 1996. Viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease. J. Exp. Med. 184:2371.[Abstract/Free Full Text]
  14. Malmstrom, V., Ho, K. K. Y., Lun, J., Tam, P. P. L., Cheah, K. S. E. and Holmdahl, R. 1997. Arthritis susceptibility in mice expressing human type II collagen in cartilage. Scand. J. Immunol. 45:670.[ISI][Medline]
  15. Malmstrom, V., Kjellen, P. and Holmdahl, R. 1998. Type II collagen in cartilage evokes peptide-specific tolerance and skews the immune response. J. Autoimmun. 11:213.[ISI][Medline]
  16. Leung, K. K., Ng, L. J., Ho, K. K., Tam, P. P. and Cheah, K. S. 1998. Different cis-regulatory DNA elements mediate developmental stage- and tissue-specific expression of the human COL2A1 gene in transgenic mice. J. Cell Biol. 141:1291.[Abstract/Free Full Text]
  17. Cheah, K. S. E., Levy, A., Trainor, P. A., Wai, A. W. K., Kuffner, T., So, C. L., Leung, K. K. H., Lovellbadge, R. H. and Tam, P. P. L. 1995. Human Col2a1-directed SV40 T-antigen expression in transgenic and chimeric mice results in abnormal skeletal development. J. Cell Biol. 128:223.[Abstract]
  18. Sanes, J. R., Rubenstein, J. L. and Nicolas, J. F. 1986. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J 5:3133.[Abstract]
  19. Lovell-Badge, R. H., Bygrave, A., Bradley, A., Robertson, E., Tilly, R. and Cheah, K. S. 1987. Tissue-specific expression of the human type II collagen gene in mice. Proc. Natl Acad. Sci. USA 84:2803.[Abstract]
  20. Seki, N., Sudo, Y., Yoshioka, T., Sugihara, S., Fujitsu, T., Sakuma, S., Ogawa, T., Hamaoka, T., Senoh, H. and Fujiwara, H. 1988. Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity. J. Immunol. 140:1477.[Abstract/Free Full Text]
  21. Anderson, G. D., Banerjee, S., Luthra, H. S. and David, C. S. 1991. Role of Mls-1 locus and clonal deletion of T-cells in susceptibility to collagen-induced arthritis in mice. J. Immunol. 147:1189.[Abstract/Free Full Text]
  22. Calin-Laurens, V., Forquet, F., Mottez, E., Kanellopoulos, J., Godeau, F., Kourilsky, P., Gerlier, D. and Rabourdin-Combe, C. 1991. Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules. Eur. J. Immunol. 21:761.[ISI][Medline]
  23. Weiss, S. and Bogen, B. 1991. MHC class II-restricted presentation of intracellular antigen. Cell 64:767.[ISI][Medline]
  24. Brennan, F. R., Mikecz, K., Buzas, E. I. and Glant, T. T. 1995. Interferon-{gamma} but not granulocyte/macrophage colony-stimulating factor augments proteoglycan presentation by synovial cells and chondrocytes to an autopathogenic T cell hybridoma. Immunol. Lett. 45:87.[ISI][Medline]
  25. Alsalameh, S., Jahn, B., Krause, A., Kalden, J. R. and Burmester, G. R. 1991. Antigenicity and accessory cell function of human articular chondrocytes. J. Rheumatol. 18:414.[ISI][Medline]
  26. Kurts, C., Sutherland, R. M., Davey, G., Li, M., Lew, A. M., Blanas, E., Carbone, F. R., Miller, J. F. and Heath, W. R. 1999. CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose. Proc. Natl Acad. Sci. USA 96:12703.[Abstract/Free Full Text]
  27. Morgan, D. J., Kreuwel, H. T. and Sherman, L. A. 1999. Antigen concentration and precursor frequency determine the rate of CD8+ T cell tolerance to peripherally expressed antigens. J. Immunol. 163:723.[Abstract/Free Full Text]
  28. Alferink, J., Tafuri, A., Vestweber, D., Hallmann, R., Hammerling, G. J. and Arnold, B. 1998. Control of neonatal tolerance to tissue antigens by peripheral T cell trafficking. Science 282:1338.[Abstract/Free Full Text]
  29. Vonherrath, M. G., Evans, C. F., Horwitz, M. S. and Oldstone, M. B. A. 1996. Using transgenic mouse models to dissect the pathogenesis of virus-induced autoimmune disorders of the Islets of Langerhans and the central nervous system. Immunol. Rev. 152:111.[ISI][Medline]
  30. Gregerson, D. S., Torseth, J. W., McPherson, S. W., Roberts, J. P., Shinohara, T. and Zack, D. J. 1999. Retinal expression of a neo-self antigen, ß-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis. J. Immunol. 163:1073.[Abstract/Free Full Text]
  31. Taylor, P. C., Plater-Zyberk, C. and Maini, R. N. 1995. The role of the B cells in the adoptive transfer of collagen-induced arthritis from DBA/1 (H-2q) to SCID (H-2d) mice. Eur. J. Immunol. 25:763.[ISI][Medline]
  32. Brand, D. D., Marion, T. N., Myers, L. K., Rosloniec, E. F., Watson, W. C., Stuart, J. M. and Kang, A. H. 1996. Autoantibodies to murine type-II collagen in collagen-induced arthritis—a comparison of susceptible and nonsusceptible strains. J. Immunol. 157:5178.[Abstract]
  33. Courtenay, J. S., Dallman, M. J., Dayan, A. D., Martin, A. and Mosedale, B. 1980. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283:666.[ISI][Medline]
  34. Mielants, H., Devos, M., Cuvelier, C. and Veys, E. M. 1996. The role of gut inflammation in the pathogenesis of spondyloarthropathies. Acta Clinica Belg. 51:340.[ISI][Medline]
  35. Leinsalo-Repo, M., Turunen, U., Stenman, S., Helenius, P. and Seppala, K. 1994. High Leirisalorepo frequency of silent inflammatory bowel-disease in spondylarthropathy. Arthritis Rheum. 37:23.[ISI][Medline]
  36. Thompson, S. J. and Elson, C. J. 1993. Susceptibility to pristane-induced arthritis is altered with changes in bowel flora. Immunol. Lett. 36:227.[ISI][Medline]
  37. Taurog, J. D., Maika, S. D., Satumtira, N., Dorris, M. L., McLean, I. L., Yanagisawa, H., Sayad, A., Stagg, A. J., Fox, G. M., Le, O. B. A., Rehman, M., Zhou, M., Weiner, A. L., Splawski, J. B., Richardson, J. A. and Hammer, R. E. 1999. Inflammatory disease in HLA-B27 transgenic rats. Immunol. Rev. 169:209.[ISI][Medline]