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Gene Therapy (1998) 5, 1584–1592 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt Prevention of collagen-induced arthritis by gene delivery of soluble p75 tumour necrosis factor receptor RA Mageed1, G Adams1, D Woodrow2, OL Podhajcer3 and Y Chernajovsky1 1 Kennedy Institute of Rheumatology and 2Department of Histopathology, Charing Cross Hospital, Hammersmith, London, UK; and Fundación Campomar, Universidad de Buenos Aires, Argentina 3 Collagen type II-induced arthritis (CIA) in DBA/1 mice can be passively transferred to SCID mice with spleen B- and T-lymphocytes. In the present study, we show that infection ex vivo of splenocytes from arthritic DBA/1 mice with a retroviral vector, containing cDNA for the soluble form of human p75 receptor of tumour necrosis factor (TNF-R) before transfer, prevents the development of arthritis, bone erosion and joint inflammation in the SCID recipients. Assessment of IgG subclass levels and studies of synovial histology suggest that down-regulating the effector functions of T helper-type 1 (Th1) cells may, at least in part, explain the inhibition of arthritis in the SCID recipients. In contrast, the transfer of splenocytes infected with mouse TNF-␣ gene construct resulted in exacerbated arthritis and enhancement of IgG2a antibody levels. Intriguingly, infection of splenocytes from arthritic DBA/1 mice with a construct for mouse IL-10 had no modulating effect on the transfer of arthritis. The data suggest that manipulation of the immune system with cytokines, or cytokine inhibitors using gene transfer protocols can be an effective approach to ameliorate arthritis. Keywords: collagen-induced arthritis; gene therapy; tumour necrosis factor p75 receptor Introduction The properties and outcome of T cell-dependent (TD) immune responses can be predicted by the cytokine profile and phenotype of the responding T helper (Th) lymphocytes.1 This tenet is based primarily on the paradigm that Th cells comprise functionally distinct populations characterised by the pattern of cytokines they produce and their effector functions.2 Furthermore, there is emerging evidence that the inappropriate induction of functionally distinct T cell populations may contribute to many autoimmune, allergic and chronic infectious diseases.3 In inflammatory joint diseases, such as rheumatoid arthritis (RA) and CIA, there is increasing evidence for the involvement of pro-inflammatory cytokines induced, or produced by Th1-type cells in disease pathogenesis. For example, TNF-␣ and IL-1 have been identified at sites of disease and treatment with antibodies to these cytokines ameliorates arthritis.4–6 Furthermore, IFN-␥ has been detected in culture supernatants of draining lymph node cells taken during the early phases of CIA in DBA/1 mice suggesting a dominant role for Th1-type cells at the onset of arthritis.7 In addition to the stimulatory effect of cytokines produced or induced by Th1-type cells on the inflammatory arm of the immune system, these cytokines can directly cause cartilage and bone resorption, inhibit the synthesis of proteoglycan and collagen in cartilage and cause mild arthritis when injected directly into the Correspondence: RA Mageed, Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK Received 18 December 1997; accepted 12 August 1998 joint space.8,9 Furthermore, TNF-␣ induces the release of prostaglandin E2 and collagenase by synovial cells, contributes to fibrosis and facilitates inflammatory cell infiltration by promoting adhesion of neutrophils and lymphocytes to endothelial cells. This has led to the hypothesis that excessive production of TNF-␣ may be important in the pathogenesis of arthritis.10 One of the early studies to validate this concept was performed using in vitro cultures of mononuclear cells from RA synovial membranes.11 This study revealed that anti-TNF antibodies diminished the production of other proinflammatory cytokines such as IL-1 and GM-CSF. Consequently, this was followed by treatment of mice with CIA and RA patients with antibodies to TNF. These studies confirmed TNF-␣ as a useful target for therapeutic intervention.6 However, the precise way by which blocking TNF-␣ ameliorated arthritis is not clear. Furthermore, there can be problems associated with antibodybased therapies, such as repeated intravenous administration to overcome the problem of absorption and clearance and immunogenicity of the antibody itself. In the present study, we use a transfer model of CIA to define how TNF-␣ influences arthritis, and assess the value of a gene delivery system to block its actions. CIA has been well characterised and the model is regarded as useful to elucidate pathogenic mechanisms relevant to human RA and identify potential targets for therapeutic intervention.4 To examine the processes that underlie CIA, a previous study showed that the disease can be transferred from arthritic DBA/1 mice to SCID recipients with spleen cells.12 However, the nature of the cellular interactions and molecules that regulate disease remained largely undefined. Furthermore, the effect of manipulating the cytokine network by cytokine, or cyto- Gene therapy of murine arthritis RA Mageed et al kine inhibitor administration have yet to be adequately assessed. This has primarily been because of the need to administer large amounts of recombinant cytokines or cytokine inhibitors which have a short half-life, in repeated doses. Gene delivery obviates the need to express, purify and administer multiple doses of cytokine or cytokine inhibitory proteins. Using IgG2a and IgG1 antibodies as serological markers of Th1- and Th2-type effector functions, data from the present study suggest that TNF-␣ may be involved in CIA by directly inducing and/or enhancing Th1-type cell effector functions. These findings imply that knowledge of the immune response mode may be an important first step in designing new immunotherapeutic strategies for human diseases. Results Transfer of collagen-induced arthritis from DBA/1 to SCID mice All SCID recipients of unmodified splenocytes from arthritic DBA/1 mice developed arthritis within 11–13 days after intraperitoneal injection. The arthritis was characterised by variable synovitis mainly in the distal interphalangeal joints (Figure 1). Splenocytes from naı̈ve non-arthritic DBA/1 mice with, or without preincubation with CII in vitro, did not cause arthritis nor produce anti-CII antibodies in the SCID recipients. Furthermore, infection of splenocytes from arthritic DBA/1 mice with a retroviral vector expressing the herpes simplex virus thymidine kinase (HSVtk) did not affect the transfer of arthritis, paw thickness or the level of antiCII antibodies (data published in detail in Ref. 13). SCID recipients that developed arthritis appeared otherwise healthy and without clinical signs of graft-versus-host disease (eg ruffled fur, hunched posture or diarrhoea). Figure 1 Photomicrograph of a synovial section from a SCID recipient of unmodified spleen cells from arthritic DBA/1 mice 15 days after cell transfer. The section was stained with haematoxylin and eosin and photographed at an original magnification of ×80. The Figure depicts a joint showing synovial hyperplasia (some indicated by an arrow) and an underlying lymphocytic infiltrate with changes mainly in the distal interphalangeal joint (indicated by arrow heads). The effect of ex vivo infection of splenocytes from arthritic DBA/1 mice with retroviral constructs expressing soluble human p75 TNF-R, mTNF-␣ or mIL-10 on the transfer of arthritis to SCID recipients Seventeen of the 20 SCID recipients of arthritic DBA/1 splenocytes infected with the retroviral construct for the soluble TNF-R failed to develop clinical features of arthritis although on histological examination they showed a mild and acute form of synovitis (Table 1, pathology in Figure 2). These SCID mice had measurable levels of TNF-R in the serum until termination of the experiments (15 days after transfer). However, serum levels of the p75 TNF-R did not correlate with paw swelling and there was no consequent correlation with the clinical response seen (Spearman’s non-parametric correlation, r = 0.4362, P = 0.0545, Table 2). The decrease in paw swelling, however, correlated with lower levels of serum antiCII antibodies. In contrast, SCID recipients of DBA/1 splenocytes infected with the mTNF-␣ or the mIL-10 construct had more severe disease affecting many joints and higher levels of anti-CII antibodies compared with SCID recipients of unmodified splenocytes from arthritic DBA/1 mice (Tables 1 and 3). Pathological indices of arthritis in the SCID recipients Results of histological examination of joints from arthritic SCID recipients of unmodified splenocytes, or SCID recipients of splenocytes infected with the soluble p75 TNF-R, mTNF-␣, or mIL-10 construct are summarised in Table 1. Arthritic SCID recipients of unmodified DBA/1 splenocytes showed variable synovitis, which was mainly present in the distal interphalangeal joints (Figure 1). This synovitis was characterised by an increase in the number of synovial lining layer cells and an intraluminal accumulation of fibrin and neutrophils. In contrast, mice receiving splenocytes infected with the soluble p75 TNF-R construct had a mild synovitis that consisted of few focal increases in the number of synovial lining layer cells (Figure 2). Mice given arthritic DBA/1 splenocytes infected with mTNF-␣ or mIL-10 construct had severe arthritis characterised by chondrocyte necrosis with focal fragmentation of the articular cartilage together with pannus formation, which particularly eroded through the bone beneath the lateral edge of the cartilage (Figures 3 and 4, respectively). The synovial lining layer cells were increased in numbers and fibrin and neutrophils accumulated both in the lining layer and the joint space. The surrounding tissue contained a prominent infiltrate of lymphocytes, macrophages, neutrophils and fibroblasts with many dilated blood vessels engorged with red blood cells. The number of macrophages was more evident in the SCID recipients of splenocytes infected with mTNF␣ or mIL-10 construct than recipients of unmodified splenocytes. In contrast, these changes were not seen in the SCID recipients of splenocytes infected with the soluble p75 TNF-R construct. Levels of total IgG1 and IgG2a subclasses and IgG antibody subclasses to CII in the SCID recipients Levels of total IgG1 and IgG2a isotypes, and bovine CIIspecific IgG, IgG1 and IgG2a antibody subclasses were measured in all SCID recipients. Levels of total IgG2a were consistently higher than those of IgG1 in the arthritic SCID recipients of unmodified DBA/1 splenocytes, or splenocytes infected with the mTNF-␣ or mIL- 1585 Gene therapy of murine arthritis RA Mageed et al 1586 Table 1 Comparison of the pathological indices of arthritis in SCID recipients of splenocytes from arthritic DBA/1 mice without infection with retroviral vectors or after infection with retroviral vector constructs for the soluble human p75 TNF-R, mTNF-␣ or mIL-10 Disease indices Synovial lining cell hyperplasia Fibrin deposition Chondrocyte necrosis Fragmented cartilage Neutrophils Lymphocytes Macrophages Fibroblasts Pannus Location of observed changes SCID recipients of unmodified splenocytes SCID recipients of SCID recipients of SCID recipients of splenocytes infected with splenocytes infected with splenocytes infected with mIL-10 construct the soluble p75 TNF-R mTNF-␣ construct construct ++ + − − + ++ ++ ++ − Mainly in the distal interphalangeal joints + +++ +++ − − − − − − − − +++ +++ +++ +++ +++ +++ +++ +++ Most joints +++ +++ +++ +++ +++ +++ +++ +++ Most joints Paws from arthritic mice (one or two paws from each of 20 mice) were removed and fixed in buffered formalin. For the purpose of the microscopic examination, the paws were decalcified with EDTA in buffered formalin and embedded in paraffin. Six micrometrethick sections were stained with haematoxylin and eosin and disease parameters assessed microscopically in comparison with sections obtained from control untreated SCID mice. No histopathological changes are indicated by the − sign. Occasional changes are indicated as +; changes seen frequently are indicated as ++; widespread changes are indicated as +++. Figure 2 Photomicrograph of a synovial section from a SCID recipient of arthritic DBA/1 mice splenocytes infected with the soluble human p75 TNF-R construct. The section, from a SCID mouse killed 15 days after cell transfer, shows mild synovial lining layer hyperplasia (indicated by arrows). The section was stained with haematoxylin-eosin and photographed at a magnification of ×80. 10 constructs (Table 3), with two exceptions. In the experiments presented in this manuscript, two SCID recipients of non-infected splenocytes had very high levels of IgG1 and to a lesser extent, IgG2a. The increase in the level of total IgG in these two SCID recipients was also reflected in the level of specific anti-CII antibodies. However, the increase in the level of anti-CII antibodies did not strictly reflect the change in the ratio of total IgG2a to IgG1. The reason for the disparity in the level of IgG1 and IgG2a in these two SCID recipients compared with the rest is unclear, but is not likely to be due to the mice being ‘leaky’. All possible leaky SCID mice were excluded from the transfer experiments before initiation of the study. The levels of IgG2a anti-bovine CII antibodies were significantly higher than IgG1 in all the arthritic SCID recipients, with one exception (one of the aforementioned two mice). When the data were expressed as ratios of IgG2a to IgG1 (total and CIIspecific) the values were always disproportionately high in the three arthritic groups. These values and ratios were reversed in favour of IgG1 (total and CII-specific) in the SCID recipients of splenocytes infected with the soluble p75 TNF-R construct (Table 3). This coincided with the prevention of arthritis transfer and amelioration of histopathological features of the disease. The experiments were repeated on two other occasions confirming the reversal in the ratio of IgG2a:IgG1 in favour of IgG1 in SCID recipients of DBA/1 splenocytes infected with the soluble TNF-R construct. The data suggested that there was a dominance of a Th1-driven IgG2a antibody production in SCID recipients of unmodified arthritic DBA/1 splenocytes, or splenocytes infected with mTNF␣ or mIL-10. The latter observation was somewhat surprising in view of the effect of IL-10 on down-regulating macrophage functions, including T cell activation and Th1-type cytokine production (eg TNF-␣).14 The explanation for this finding may lie in the relatively low quantity of IL-10 produced by cells infected with the construct compared with levels required for an effective action on arthritis.15 Alternatively, it is possible that the exacerbated arthritis may be due to the effect of mIL-10 on B cell differentiation and antibody production. It is noteworthy in this respect that levels of both total IgG1 and IgG2a, and of anti-CII antibodies were generally higher in the SCID recipients of splenocytes infected with the mIL-10 construct compared with the other groups. Discussion Collagen-induced arthritis in DBA/1 mice has been extensively used to study clinical and therapeutic aspects of human RA.4,16 Clinically, both diseases involve sustained proliferative synovitis with cartilage and bone destruction.17 Immunologically, CIA has a number of Gene therapy of murine arthritis RA Mageed et al Table 2 Lack of correlation between serum levels of the soluble human p75 TNF-R, IgG anti-CII antibodies and paw thickness in SCID recipients of splenocytes from DBA/1 mice infected with the retroviral construct for soluble p75 TNF-R Serum concentration of soluble p75 TNF-R (pg/ml) 3832 3344 4042 564 2409 1203 7701 4400 5450 ⬍20 6650 565 300 515 590 ⬍20 5200 ⬍20 5610 1675 Mean ± s.d. (median) Level of anti-bovine CII in SCID recipients (mg/ml) Paw thickness of SCID recipients (mm) 0.04 0 0 0.01 0 0 0 7.7 18.6 1.8 13.3 2.0 1.6 1.7 1.8 0.5 0.9 0 0.6 0.3 2.54 ± 4.98 (0.550) 2.0 2.1 2.2 2.1 1.9 2.0 1.9 2.7a 2.9a 2.0 3.2a 2.2 1.9 1.7 1.9 2.0 2.1 1.9 2.0 1.9 2.13 ± 0.37 (2.0) Each of the 20 SCID recipients included in the Table (from three separate experiments) received 5 × 107 splenocytes, in single cell suspension, from arthritic DBA/1 mice intraperitoneally after 48 h incubation in vitro with 100 g/ml bovine CII. Four hours before culture termination, the cells were centrifuged and resuspended in culture medium containing supernatants from the packaging cell line. The mean ± standard deviation of paw thickness in control SCID mice receiving non-infected splenocytes and mice receiving splenocytes infected with retroviral constructs containing HSVtk, mTNF-␣ or mIL-10 genes were: eight mice receiving non-infected splenocytes, 2.69 ± 0.2 (median 2.7; range 2.3–3.0); four mice receiving splenocytes infected with a retroviral gene construct for HSVtk, 2.76 ± 0.2 (median 2.75; range 2.6–3.0); four mice receiving splenocytes infected with the retroviral construct for mTNF-␣, 2.63 ± 0.16 (median 2.6; range 2.4–2.8); 10 mice receiving the retroviral construct containing mIL-10 gene, 2.75 ± 0.21 (median 2.7; range 2.3–3.1). Paw thickness was significantly different between control mice receiving non-infected splenocytes from arthritic DBA/1 mice and those receiving similar splenocytes infected with the soluble p75 TNF-R (Mann– Whitney test; P ⬍ 0.001). Spearman’s non-parametric correlation coefficient for serum levels of soluble p75 TNF-R with anti-CII antibodies and paw thickness were: r = 0.0702 (P = 0.769) and r = 0.4362 (P = 0.0545), respectively. a These three mice did not show beneficial therapeutic effect and developed arthritis. similarities with RA, eg the requirement for genetic predisposition factors involving both MHC- and non-MHCrelated gene loci. Furthermore, attainment of reactivity with self antigens, native CII in CIA, is important to the development of both diseases.18 More recently, passive transfer of CIA from DBA/1 to SCID recipients has shown that interactions between antigen presenting cells, B and T cells are crucial for establishing disease.12 In the present study, we demonstrate that the transfer of arthritis from arthritic DBA/1 mice to SCID recipients can be blocked by modifying some of the effector functions of Th1- and/or Th2-type cells.1,2 This apparent modification of the immune response was achieved by infecting lymphocytes with a retroviral construct carrying cDNA for the soluble form of human p75 TNF-R. Alteration in the ratio of total IgG subclasses and of antiCII antibodies in favour of IgG1 compared with IgG2a levels and histological changes within the synovium suggest that the prevention of arthritis was associated with down-regulating the effector function of Th1-type cells. A link between TNF-␣ and Th1-type responses was also observed in TNF-␣ knock-out mice, which were shown to be incapable of mounting IgG2a antibody responses when challenged with antigens.19 However, it is not yet clear whether down-regulation of IgG2a production shown in our study resulted from a direct inhibitory effect on Th1 cells or indirectly through the induction of Th2 cells. In immune responses to exogenous antigens, Th lymphocytes mature along two alternative pathways into Th1-type cells, which play the major role in cell-mediated immunity by producing IL-2, TNF-␣ and IFN-␥, or Th2type cells which are involved in antibody mediated responses by producing IL-4, IL-5 and IL-10.1,2 Both Th subpopulations cross-regulate each other through the production of cytokines. For example, IFN-␥ induces Th1-type cells and inhibits the differentiation and functions of Th2-type cells.1 Conversely, IL-4 induces Th2type cells and inhibits Th1-type cell proliferation and opposes the effects of IFN-␥.19 Therefore, reciprocal regulation occurs between Th1/Th2-type cells. The extent of the reciprocal action of Th1- and Th2-type cells in the CIA on each other and influence on the disease process is only emerging. A recent study of in vitro cultured lymph node cells from immunised DBA/1 mice showed increased IFN-␥ production during the first 6 days of immunisation.7 However, a role for imbalances in the cytokine network in CIA in favour of cytokines induced, or produced by Th1-type cells has long been suggested.20,21 For example, it was shown that mice transgenic for human TNF-␣ develop arthritis.20 Furthermore, natural levels of IL-1 are chronically increased in mice with CIA and lymphocytes from these mice produce significantly increased amounts of IL-2.21 The contribution of TNF-␣ and IL-1 (induced by Th1-type cells) to cartilage and bone destruction and the recruitment of immune cells into the synovium are also well known.4,8 Most of these studies stressed that the role of TNF-␣ and IL-1 in arthritis was due to the pro-inflammatory nature of these two cytokines, their ability to induce prostaglandin E2 in culture and enhancement of bone and cartilage destruction. The experiments described here suggest that TNF-␣ also contributes to the immune-mediated arthritis through the induction of IgG2a production, an isotype known to be induced by Th1-type cells. The data also showed that systemic levels of soluble p75 TNF-R did not reflect a positive therapeutic effect. Studies over the last few years have shown that two distinct forms of TNF exist, a membrane-expressed form (mTNF) and a soluble form which is derived from proteolytic cleavage of the mTNF.22 Despite this relationship, however, it has been established that mTNF is significantly more bioactive than soluble TNF and that engagement of mTNF confers intracellular signalling typical of TNF responses such as 1587 Gene therapy of murine arthritis RA Mageed et al 1588 Table 3 Levels of total serum IgG1 and IgG2a subclasses and IgG1 and IgG2a antibodies to bovine CII in the serum of SCID recipients of arthritic DBA/1 splenocytes SCID recipient of A. Unmodified splenocytes Mouse 1 2 3 4 5 mean ± s.e.m. B. Splenocytes with mTNF-␣ construct Mouse 1 2 3 4 5 mean ± s.e.m. C. Splenocytes with mIL-10 construct Mouse 1 2 3 4 5 mean ± s.e.m. D. Splenocytes with p75 TNF-R construct Mouse 1 2 3 4 5 mean ± s.e.m. Total IgG1 (g/ml) Total IgG2a (g/ml) IgG1 anti-CII (g/ml) IgG2a antiCII (g/ml) Ratio of total IgG2a:IgG1 Ratio of anti-CII IgG2a:IgG1 16 17 19 8318 8260 928 771 876 1882 2854 0.4 2.2 0.2 182 380 14 3 45 210 236 58.0 45.4 46.1 0.2 0.4 30.0 ± 27.6 35.0 1.4 225.0 1.2 0.6 52.6 ± 97.5 35 49 31 166 135 786 992 1613 2214 926 11 2 2 98 51 35 23 39 472 312 22.4 20.4 52.4 13.3 6.9 23.0 ± 17.3 3.4 15.5 19.4 4.8 6.1 9.2 ± 6.6 38 112 258 179 56 1130 2278 1894 1997 1164 11 24 92 62 2 57 223 417 287 23 30.0 20.3 7.3 11.1 21.0 18.0 ± 8.9 5.1 9.5 4.5 4.7 15.1 7.02 ± 3.2 886 732 585 870 1032 241 316 127 174 357 110 209 48 64 95 5 14 11 23 29 0.3 0.4 0.2 0.2 0.4 0.3 ± 0.1 0.05 0.06 0.22 0.35 0.30 0.2 ± 0.14 The level of total IgG1 and IgG2a subclasses and bovine CII-specific IgG1 and IgG2a were determined in ELISA. For total IgG1 and IgG2a, the plates were sensitised with anti-mouse ␥-chain subclass-specific antibodies while for CII-specific antibodies the plates were sensitised with bovine CII. Concentration of bound IgG isotypes was determined by extrapolation from standard curves constructed from known inputs of purified IgG1, IgG2a or bovine CII-specific antibodies. The ratios of IgG2a anti-CII to IgG1 anti-CII antibodies in SCID recipients of unmodified (non-infected) splenocytes (group A) or splenocytes infected with a construct for mTNF-␣ or mIL-10 gene were significantly higher than those obtained for SCID recipients of splenocytes infected with the soluble p75 TNF-R gene construct (group D) (Mann–Whitney test; P ⬍ 0.05). The ratios of total IgG2a to IgG1 were different in the different groups but the difference between group A and D was not significant in the Mann–Whitney test (P = 0.13). However, multivariate analysis using Pillai’s trace showed that the four groups were significantly different in the level of total and anti-CII antibodies (F = 2.652, P = 0.009). The mean and standard error of the mean (mean ± s.e.m.) for the ratio of IgG2a:IgG1 for each group is given. This experiment was repeated on two other occasions with similar outcome (data not shown). cytotoxicity and B cell activation.23 The lack of a direct relationship between serum levels of soluble p75 TNF-R and the therapeutic effects seen may suggest that the soluble receptor functions by controlling the bioavailability of mTNF in the microenvironment of activated T cells. This implies that manipulation of local TNF production may be required to achieve a therapeutic effect. It is, however, unclear at this stage whether the soluble form of the p75 TNF-R is modulating the biological effects of mTNF (and lymphotoxin) by blocking signaling through p75, p55 or both receptors. Earlier studies have suggested that the contribution of the soluble form of p75 TNF-R to cellular responses was to act as a buffer for excessive soluble TNF, while the membrane form of the p75 TNF-R enhanced p55 TNF-R signaling after binding to TNF (ligand passing).24 However, recent data suggest that mTNF can directly induce cellular responses through the membrane p75 receptor and independent of the p55 TNFR.25 The precise pathway through which the soluble p75 TNF-R inhibits Th1-type cells, therefore, requires further investigation. The data, nevertheless, suggest that the observed effect of soluble p75 TNF-R may possibly be through blocking cognate immune cell interactions. This suggestion is in line with previous findings that antibodies to the CD40 ligand (gp39) and anti-CD4 antibodies, which down-regulate Th1 cells, are effective in treating arthritic DBA/1 mice.26,27 Histological examination of joints from arthritic and treated mice revealed a reduction in cellularity of the synovium of mice receiving splenocytes infected with the soluble p75 TNF-R construct suggesting that a factor in regulating arthritis may be the prevention of extrava- Gene therapy of murine arthritis RA Mageed et al Figure 3 Photomicrograph of a synovial section from a SCID recipient of arthritic DBA/1 mice splenocytes infected with the mTNF-␣ construct. The section is from a SCID mouse killed 15 days after cell transfer. The articular cartilage contains necrotic chondrocytes (indicated by arrow heads) and there is pannus eroding the cartilage edge (indicated by arrows). The joint space contained fibrin and neutrophils with a surrounding chronic synovitis. The section was stained with haematoxylin-eosin (×80 magnification). Figure 4 Photomicrograph of a synovial section from a SCID recipient of arthritic DBA/1 mice splenocytes infected with the mIL-10 construct 15 days after cell transfer. The fragmented articular cartilage contains many necrotic chondrocytes (indicated by arrow heads). There is also pannus eroding on both sides of the joint (indicated by arrows). The section was stained and photographed as for the other Figures. sation of immune cells to the synovium. It was not clear from previous studies whether leucocyte migration was inhibited through manipulation of the cytokine network. It was, however, suggested that TNF-␣ can directly influence the migration of leucocytes through its effect on endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin and indirectly through the induction of chemokines such as IL-8.28 Furthermore, studies of human RA patients treated with a chimeric monoclonal anti-TNF antibody decreased serum levels of E-selectin and ICAM-1 levels suggesting that this may reflect diminished activation of endothelial cells in the synovial microvasculature leading to reduced migration of leucocytes into the synovial joints.29 These studies all point to the possibility that TNF-␣ is a potent cytokine in inducing chemotactic activity and leucocyte recruitment into the joint. Our study provides some evidence for these suggestions and suggests that migration of immune cells into the joint and cartilage and bone destruction may have been prevented by suppressing immune activation through the TNF-␣ pathway. It is important, however, to point out that the study can not exclude the possibility that the reduction in synovial cellularity may be due to an increase in apoptosis. This is clearly an area that needs further investigation. In view of these data and of previous studies, we hypothesise that the pathogenesis of CIA in SCID recipients may be dependent, at least during the initial phases, on a Th1-type response in which TNF-␣ is involved. Furthermore, it appears that the transfer of arthritis can be inhibited when the immune response is deviated to a Th2-type response. It remains, however, to be conclusively determined whether the biological effect of the p75 TNF-R was secondary to its proposed inhibitory effect on Th1 cells and cytokine production, or to the blocking of other TNF-␣ pathways. Although manipulation of the cytokine network appears critical in disease prevention, it is unlikely that cytokines alone can precipitate diseases. It is known, for example, that cytokines are incapable on their own of overcoming genetic resistance to CIA in mouse strains that do not express H-2q or H-2r MHC haplotypes, and that cytokines failed to facilitate the development of arthritis in CD4-deficient mice.30 This is, however, contrasted by the recent finding that mice transgenic for human TNF-␣ develop arthritis, although this arthritis does not appear to be mediated by the immune system.20 Thus it is possible that in CIA in the DBA/1 mouse deregulated Th1-type cytokine production may lead to inappropriate induction of autoreactive Th1type cells in susceptible mice. The induction of autoimmune diseases by these cells is likely to be contingent on excessive IFN-␥ production, which can lead to changes in the cleavage pattern of self-antigens, upregulation of MHC antigens and increase presentation of self-peptides.3 Th2 cells, on the other hand, can act as regulatory T cells by limiting the response to self-antigens. It is interesting in this respect to note that administration of IFN-␥ at the time of immunisation with CII has been shown to augment the incidence of arthritis and accelerates its onset.31 Furthermore, there is evidence for an association between the early stages of CIA in DBA/1 mice and IFN-␥ production by lymph node cells.7 These findings suggest that the role of IFN-␥ in enhancing Th1 responses to self-peptides is perhaps an important aspect of arthritis and that other Th1-associated effector functions, such as extravasation of inflammatory cells, are also crucial for the inflammation and tissue destruction. Materials and methods Animals Eight- to 12-week-old DBA/1 and CB-17 scid/scid (SCID) mice used in the study were bred and maintained under germ-free conditions at the Kennedy Institute of Rheumatology. ‘Leaky’ SCID mice, producing detectable levels of immunoglobulin (⬎5 ng/ml), were identified before the experiments using ELISA and excluded from the study. 1589 Gene therapy of murine arthritis RA Mageed et al 1590 Induction and transfer of arthritis Type II collagen (CII) was purified from bovine articular cartilage as previously described.4 Arthritis was induced in male DBA/1 mice by intradermal injection of 100 g of the purified CII emulsified in Freund’s complete adjuvant (Difco Laboratories, East Mosley, UK) at the base of the tail. Onset of arthritis was macroscopically visible as paw swelling 3 weeks after immunisation. Spleens from arthritic DBA/1 mice were aseptically removed and used to transfer arthritis to SCID mice. Separated splenocytes were washed with Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Paisley, UK) containing 5% heat inactivated foetal calf serum and a mixture of penicillin (100 units/ml) and streptomycin (100 g/ml) before incubation with bovine CII for 48 h in culture before use for the transfer of arthritis. Supernatants from the packaging cell line CRIP transfected with the retroviral gene constructs were added to the culture 4 h before their inoculation into the SCID recipients. Clinical features of arthritis were monitored by assessing paw swelling and the number of swollen paws using callipers once every 2 days. Onset of arthritis in the SCID recipients was generally observed at day 11. Severity of arthritis was assessed according to established clinical scores.4,12 The results were expressed as paw thickness in millimetres (mm) and compared with paw thickness before disease onset. Histopathology Paws (one to two per mouse) were removed after death, fixed in 10% (wt/vol) buffered formalin, and decalcified with EDTA in buffered formalin (5.5%). The paws were embedded in paraffin, sectioned and stained with haematoxylin and eosin for microscopic evaluation of cellular and architectural changes in a blinded fashion. The severity of arthritis in each joint was classified as mild: mild, minimal synovitis, cartilage loss and bone erosions limited to discrete foci; moderate, synovitis and erosions present but normal joint architecture intact; severe, synovitis, extensive erosions and joint architecture disrupted. Antibody detection Anti-CII antibody levels were determined by ELISA.4,12,16 Microtitre ELISA plates (ICN Biomedicals, Thame, UK) were sensitised with native bovine CII (at 2 g/ml) in Tris-buffered saline (TBS) pH 7.4 by incubation at 4°C overnight. Non-occupied sites were quenched with a solution of 2% (wt/vol) casein in TBS for 1 h at room temperature and then serial dilutions of test sera in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS/Tween) were added to the wells and incubated for 1 h at 37°C. Bound total IgG, or IgG subclasses 1 and 2a, were revealed with horseradish peroxidase (HRP) conjugated sheep anti-mouse IgG-Fc, IgG1 or IgG2a antibodies, respectively (Binding Site, Birmingham, UK). IgG or IgG subclass antibodies were quantified by extrapolation using standard curves constructed from optical density values obtained for known inputs of purified total IgG, IgG1 or IgG2a anti-collagen antibodies. Total IgG1 and IgG2a levels were determined using a capture ELISA with sheep anti-mouse IgG1 or IgG2a (at 2 g/ml in PBS; Binding Site) sensitised plates. Doublefolding dilutions of serum samples starting from 1:200 were added to each well and incubated for 2 h at 37°C. A standard control containing known inputs of affinity- purified mouse IgG1 or IgG2a proteins (Binding Site) were added and titrated to give known concentration of IgG content to construct a standard curve. Bound IgG1 or IgG2a were detected using HRP-conjugated antimouse IgG1 or IgG2a. Quantification was achieved by extrapolation from standard curves constructed as above. The sensitivity limits of the assays used in the study were about 5 ng/ml of IgG1 or IgG2a. Retroviral vectors and constructs Construction of the retroviral vector pBabe-pAGO-Neo, expressing the Herpes simplex virus thymidine kinase gene has been described before.13 The soluble human p75 TNF-R construct was prepared using cDNA for the extracellular domain of human p75 TNF-R obtained from a baculovirus clone (pVL139 hp75).13 The fragment was digested with XbaI, blunted with Klenow fragment of DNA polymerase, ligated to BamHI linkers and then digested with NcoI and BamHI and cloned into the NcoIBamHI sites of the retroviral vector MFG. The packaging cell line CRIP was transfected with this retroviral vector and the selectable marker pSV2neo at a molar ratio of 20:1. Clones resistant to geneticin (G418; Gibco-BRL) at 1 mg/ml were isolated and a high producer clone obtained after multiple infections with transient ecotropic virus obtained from infection of BOSC23 cells with this virus. This construct expressed 260 ng/ml p75-TNF-R per 106 cells in 24 h, as determined by a previously described ELISA.32 Subsequent measurement of serum levels of human TNF-R in the SCID recipient of splenocytes infected with the p75 TNF-R were determined using the same ELISA protocol. The mIL-10 construct was prepared using mouse IL-10 (mIL-10) cDNA (kindly provided by Dr K Moore, DNAX, Palo Alto, CA, USA) obtained from a 1.2 kb XhoI fragment containing the whole of the open reading frame of the IL-10 cDNA, and cloned into the SalI site of the retroviral vector pBabe puro.33 The resulting retroviral construct (named mIL-10 Babe puro) was packaged after calcium phosphate transfection into GPenvAM12 cells34 and cell clones resistant to 1.5 g/ml puromycin (Sigma) in culture medium were pooled and used as a source of viral supernatant. These cells produced 2–4 ng/ml mouse IL-10 per 106 cells in 24 h, as determined by ELISA using monoclonal antibodies with specificity for mIL-10 (Pharmingen, San Diego, CA, USA). Mouse TNF-␣ (mTNF-␣) construct was prepared using a 2.8 kb genomic clone of mouse TNF-␣, kindly donated by Drs N Sarvetnick (Scripps Research Institute, La Jolla, CA, USA) and S Thompson (Cantab Pharmaceuticals, Cambridge, UK), isolated from Bluescript (IIKS) phagemid as an XhoI to BamHI segment. The fragment also contained a hepatitis B virus polyadenylation signal. After filling the ends with Klenow, this fragment was cloned into the ‘filled-in’ EcoRI site of pBabe bleo. The hepatitis polyadenylation signal was removed by digestion with EcoRI. The vector was named mTNF-Babe bleo. Packaged retrovirus was obtained as above after selection in 25 g/ml pleomycin (Sigma). Tissue culture and infection of spleen cells The packaging cell line GPenvAM1234 was maintained in DMEM, 10% new-born calf serum with glutamine, penicillin and streptomycin. One day before transfection with the retroviral constructs, cells were trypsinised and Gene therapy of murine arthritis RA Mageed et al plated out at 5 × 105 cells per 9-cm diameter dish. Four hours before DNA was added as a calcium phosphate precipitate, the medium was changed and fresh medium added. The transfection was carried out as previously described.13 Transfected cells were selected in 1 mg/ml G418 and more than 30 clones were pooled as a population and maintained in G418 at 0.5 mg/ml. Viral supernatants were collected overnight from confluent 15-cm plates each containing 12 ml medium with 10% foetal bovine serum (FBS) or 10% new-born calf serum without the selective drug. The supernatants were centrifuged at 400 g for 10 min to remove cellular debris, transferred to sterile tubes and snap frozen on dry ice. These supernatants were stored at −70°C before use. Spleens were removed and dissected aseptically and put into DMEM medium containing 10% FBS. The spleens were sieved through a cell strainer and washed with Hank’s solution containing 2% FBS. The cells were centrifuged and the pellet resuspended in 2 ml of Tris buffer containing 0.15 m ammonium chloride to lyse red blood cells. The cells were centrifuged and washed once in the same medium and passed through a sieve to remove cell clumps. The cells were plated at 5 × 106 cells/ml in DMEM with 10% FBS and CII at 100 g/ml and incubated for 48 h at 37°C in a humidified incubator with 5% CO2 in the air. When necessary, cells were infected with retroviruses as follows: at the start of the last 4 h of incubation with CII, the cells were centrifuged at 500 g for 5 min, resuspended at 2 × 106 in viral supernatant supplemented with DEAE dextran at a final concentration of 70 g/ml. After 4 h, the cells were centrifuged, washed twice and resuspended in DMEM without serum at 5 × 107/ml and 1 ml injected intraperitoneally into each SCID recipient. Statistical analyses Differences between control mice receiving non-infected splenocytes and the treated group receiving soluble p75 TNF-R gene construct in paw thickness and ratio of IgG2a:IgG1 were determined using the two-sided nonparametric Mann–Whitney test for non-paired samples. Correlation coefficients were determined using Spearman’s non-parametric correlation. Both analyses were performed using the GraphPad Instat software (GraphPad Software, San Diego, CA, USA). 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