Department of Medicine, University of New South Wales, Liverpool Hospital, Lock Mail Bag 7103, Liverpool BC, NSW 1871, Australia
1 Department of Medicine, University of Colorado, Denver, CO 80262 USA
Correspondence to: S. J. Hodgkinson
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Abstract |
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Keywords: anti-CD3, autoimmunity, experimental allergic encephalomyelitis, immunotherapy, multiple sclerosis, neuroimmunology, regulatory T cells, Th1, Th2
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Introduction |
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The inflammation in EAE is thought to be mediated by Th1 cells, that are activated by IL-12 (19) and produce IL-2, IFN- and TNF-
, but not Th2 cytokines (20). Induction of Th2 cells is promoted by IL-4, and these cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, but not Th1 cytokines (21). Th1 cytokines, such as IFN-
, inhibit the generation of Th2 cells (22), and the Th2 cytokines IL-4 and IL-10 inhibit Th1 cells (23). The role of Th2 cells in EAE is less clear, but these cells may be active in both the mediation and regulation of EAE (24). Recombinant IL-4 therapy has been shown to delay and limit the development of passive EAE induced by the transfer of encephalitogenic Th1 clones (25). They enhance Th2 responses, and inhibit Th1 responses through production of IL-4 and IL-10 (24,26). Although earlier studies had shown that clones producing Th1 cytokines, especially TNF-
, were best able to mediate EAE (27), other studies suggest that Th2 effector clones can also mediate EAE (28). Th3 cells, which produce transforming growth factor (TGF)-ß, are regulatory cells that can transfer tolerance to EAE and are induced in oral tolerance to EAE by feeding with MBP (29). In Th1-mediated autoimmune colitis (30,31) and NOD mice (32), tolerance may be mediated by Tr1 regulatory cells that produce IL-5, IL-10 and TGF-ß, but not IL-4 or other Th2 cytokines. The role of Tr1 cells in the regulation of EAE is unknown.
In multiple sclerosis, anti-CD3 mAb therapy was found to exacerbate disease symptoms, which was postulated to be due to the release of pro-inflammatory cytokines, IFN- and TNF-
(33). Anti-CD3 mAb therapy is widely used in organ transplantation models to prevent and reverse rejection, which is also predominantly Th1 mediated (34). Most anti-CD3 mAb activate Th1 cells to release cytokines including IL-2, IFN-
and TNF-
(35,36). This effect is mediated by the Fc of the mAb binding to antigen-presenting cells (APC), and the facilitation of T cell activation by mAb binding to CD3 and inducing proliferation (37,38). Anti-CD3 mAb which do not bind Fc receptors because of their isotype, or through the Fc portion being altered by enzymatic digestion (F(ab)2 fragments) or genetic modification, do not induce activation or proliferation of T cells (3739). These non-mitogenic anti-CD3 mAb selectively block Th1 cells, but not Th2 cells, both in vivo (40) and in vitro (41,42). In vivo treatment of rodents with these mAb can induce transplantation tolerance (43,44) and re-establish tolerance to insulin-dependent diabetes in NOD mice (45).
In this study, we examined if G4.18, a non-mitogenic mouse anti-rat CD3 mAb of the IgG3 isotype (44), could prevent induction or reverse established EAE in Lewis rats. In vivo, G4.18 induces tolerance to fully allogeneic cardiac allografts in rats, but only causes a minor depletion of circulating T cells (<20%) and transient modulation of the TCRCD3 complex from the cell surface (44). G4.18 therapy is not associated with T cell activation or release of cytokines, but preferentially inhibits Th1 cytokines and spares Th2 cytokine induction (40). We examined if G4.18 also inhibits Th1 cells in EAE.
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Methods |
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Disease induction and monitoring
Active EAE was induced in 12-week-old female Lewis rats immunized with 50 µg of guinea pig MBP emulsified with incomplete Freund's adjuvant (Sigma-Aldrich, St Louis, MO) and 5 mg/ml of heat killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Groups of at least six animals were immunized and treated with G4.18, MRC OX-81 mAb or rIL-4 alone or in combination. Controls included isotype-matched mAb without reactivity to rats, supernatant from non-transfected CHO-K1 cell lines or rIL-5.
All rats were clinically assessed and weighed daily. The animals were graded for disease severity as: 0, normal; 0.5, partial loss of tail tone; 1, complete loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis and front limb weakness; 4, hind and front limb paralysis; 5, moribund or dead. Onset of active EAE was normally at about day 11 post-immunization when there was an abrupt onset of paralysis and marked weight loss. Clinical recovery occurred between days 18 and 30, with gradual weight gain. Due to a variation in the time of onset of disease and the day of peak activity, disease activity was assessed as the mean of the daily results for clinical score and percent weight loss of each animal over days 1218. Group data was expressed both as the mean ± SD of the daily mean values for days 1218 inclusive and the mean of the maximal mean clinical score for each animal. At least two animals in each group were sacrificed at day 14 and 18 post-immunization, when brain stems and popliteal lymph nodes (PLN) were collected for immunohistology and analysis of cytokine mRNA by RT-PCR.
Production and administration of mAb
mAb used included G4.18 (mouse IgG3) (44) and MRC OX-81 (mouse IgG1), a mAb that blocks rat IL-4 function (46) (a kind gift of Dr Don Mason, MRC Cellular Immunology Unit, Oxford, UK). Isotype control mAb with no reactivity to rat included BLCA8, a mouse IgG3 mAb reactive to human bladder cancer (a kind gift of Dr N. Pearce, Centenary Institute, Sydney University, Sydney, Australia), and A6, a mouse IgG1 mAb reactive only to human CD45RO (47). mAb were grown in ascites of BALB/c mice primed by i.p. injection of incomplete Freund's adjuvant (Difco, Detroit, MI) 3 days prior to injection of clones. Ascites were purified on a DEAESepharose column (Pharmacia, Uppsala, Sweden) and antibody concentration was determined by radial immunodiffusion. All mAb preparations had <0.06 U/ml of endotoxin as determined by a Limulus amebocyte lysate assay (Coatest Gel-LAL; Chromogenix, Moindal, Sweden).
Animals were treated with G4.18, BCLA8, MRC OX-81 or A6 mAb at a dose of 7 mg/kg daily by i.p. injection for 14 days or control BCLA8 mAb were given for 14 days, starting at day 0, 7 or 12 after immunization. With this dosage, there is sufficient MRC OX-81 mAb in serum 8 days after a single injection to block 10,000 U of rIL-4 function in vitro (48). This dose of MRC OX-81 also inhibits IL-4 function in vivo; it reduces inflammation in experimental uveoretinitis (49,50) and blocks IgG isotype switching in alloimmune responses (48).
Rat rIL-4 production
rIL-4 was obtained from the supernatant of cultures of CHO-K1 cell lines stably transfected with rat IL-4 cDNA sequence subcloned into the pEE6. MCMV-GS expression vector (kindly provided by N. Barclay, MRC Cellular Immunology Unit, Oxford, UK). DMEM-F12 medium (Gibco) was supplemented with 10% FCS and 0.05 mM L-methionine sulfoximine (Sigma) as a selection marker. Final cultures were serum free, and supernatant was concentrated 25 times using a Sartocon micro-cross flow filtration unit (Sartorius, Gottingen, Germany) and stored at 70°C. rIL-4 activity was assayed for its ability to induce class II MHC on B cells, as described (51). One unit of rIL-4 was that which induces 50% of maximal class II MHC expression on 5x105 B cells. rIL-4 preparations were assayed for endotoxin and only those with <0.06 U/ml were used. rIL-4 was given i.p. daily at 30 µg (104 U)/kg for 14 days, either from the day of immunization or the day of onset of disease, usually day 12 post-immunization. Control animals were treated with either supernatant from non-transfected CHO-K1 cell lines or rat rIL-5 produced by transfected CHO-K1 cells in a manner similar to rIL-4 (51). rIL-5 activity was assessed by its ability to support an IL-5 dependent line, as described (52); 5 µg/day of rIL-5, equivalent to 3000 U, was given daily for 14 days.
Staining of mononuclear cells in blood and tissues with mAb to identify subsets
Anti-rat mAb used included G4.18 (CD3), MRC OX-35 (CD4), MRC OX-12 ( chain of Ig), MRC OX-6 (RT-1B, MHC II), MRC OX-8 (CD8), MRC OX-33 (CD45RA, B cell), MRC OX-22 (CD45RC), MRC OX-39 (IL-2 receptor,
chain), MRC OX-34 (CD2) and R7.3 (TCR
ß) (PharMingen, San Diego, CA), and ED1 (macrophage) was obtained from Serotec (Oxford, UK). Tissue specimens were stained with an indirect immunoperoxidase technique as described (53). Single-cell suspensions of mononuclear cells were stained with a direct immunoflourescent method and analyzed on a FACScan, as described (53).
Semi-quantitative PCR
Methods for RNA extraction, reverse transcription and PCR for cytokines have been described (53). cDNA samples were serially diluted in diethyl pyrocarbonate-treated water as neat (N), 1/10, 1/20, 1/40 and 1/80. PCR used 1 µl of cDNA, 0.5 U of Taq Polymerase (Biotech International, Perth, Australia), 1.5 mM of MgCl2 and 125 µM dNTP (Promega, Madison, WI) using specific primers for rat IL-2, IL-10, IL-4, IFN-, TNF-
and TGF-ß, as described (54). IL-12Rß2 primers were designed from regions of mouse and human genes with high similarity, and were 5'-cct ata tct gtt atg aaa tca gg-3' and 5'-ctg tca cag ctg tca tcc ata-3' (55,56). Each cDNA sample was analyzed in duplicate, with a positive (cDNA from concanavalin A-activated lymphocytes) and negative (no reverse transcriptase) control included.
All samples were assayed using GAPDH primers, a housekeeping gene, to confirm that the concentration of template cDNA was uniform in all samples. Standard PCR conditions consisted of an initial 3 min denaturation (94°C) followed by 30 s each of denaturation (94°C) and primer annealing (60°C), and 50 s extension (72°C) cycles, then a final extension at 72°C for 4 min, performed on a Corbett thermal cycler (Corbett Research, Sydney, Australia). The number of cycles for each primer set was between 23 and 35 cycles, and was determined to ensure the dilutions were tested on the linear phase of amplification. A quarter of total PCR products were run on 6% polyacrylamide gels with pUC19/HpaII-digested mol. wt marker, stained with ethidium bromide, and photographed under UV light using a Kodak DC40 camera and Digital Science software (Rochester, NY).
ELISA assay of antibody to MBP
Serum from all animals collected at day 0, 3, 7, 10, 14, 18 and 21 was used in ELISA assays to detect antibody responses against MBP. Ninety-six-well plates were coated overnight at 4°C with MBP (25 µg/ml) in coating buffer (0.15% Na2CO3, 0.29% NaHCO3, pH 9.5). Plates were washed twice before triplicate samples of serum at 1/200 dilution were added, incubated for 2 h, and then washed and reacted with horseradish peroxidase-conjugated rabbit anti-rat Ig (Dako) diluted at 1/2000. The plates were again washed twice then reacted with phosphatase substrate (Sigma) and read on a microplate reader (BioRad, Hercules, CA) at 450 nm. All data were expressed as a mean of triplicate values and SE < 20% of the mean. All assays included positive controls of hyperimmunized sera from rats repeatedly immunized with complete Freund's adjuvant (CFA)/MBP after recovery and were collected at day 50 post-immunization. Results were expressed as a percentage of the positive control.
Passive EAE
To examine the effects of G4.18 on EAE induced by passive transfer of activated cells, spleens from Lewis rats immunized 10 days prior with MBP/CFA were cultured with concanavalin A (2 µg/ml) for 4 days in DMEM (Difco) and 10% FCS. Then, 3.6x107 cultured cells were injected i.v. into three groups of normal Lewis rats. One group was treated with G4.18 7 mg/kg/day from day of transfer for 14 days. Controls were treated with BLCA8 (Ig isotype control) from day of transfer for 14 days. A third group was treated with G4.18 from day of onset of clinical disease (day 5) until 19 days.
Statistics
Significant differences in maximal disease and weight loss between different groups of animals were assessed using the MannWhitney U-test and P < 0.05 was considered a significant difference. Data was expressed as mean ± SD.
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Results |
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To examine what effect of G4.18 given at the time of immunization on the response against MBP, the PLN draining the site of immunization was examined. At day 14 post-immunization, G4.18-treated nodes were enlarged (56 ± 22mg, n = 8) compared to normal lymph nodes (4.2 ± 0.84 mg, n = 6), but of similar size to those from rats immunized with CFA without MBP (57 ± 6 mg, n = 3). MBP/CFA-immunized untreated control PLN were significantly larger then all other groups (87.8 ± 14 mg, n = 6, P 0.009 for normal, 0.001 for CFA and 0.01 for G4.18 treated). G4.18-treated PLN had less CD4+ (20 ± 7 versus 58 ± 10%) and CD8+ (10.5 ± 3.5 versus 33.5 ± 10%) T cell infiltration compared to untreated EAE controls.
In RT-PCR of mRNA extracted from G4.18-treated PLN at day 3 post-immunization there was less Th1 cytokines IL-2 and IFN-, as well as the marker of Th1 cells IL-12Rß2 than in control (Fig. 3A
). Th2 cytokines IL-4 and IL-5 mRNA were more abundant in G4.18-treated than untreated controls. The levels of macrophage cytokines IL-10 and TNF-
as well as the Th3 cytokine TGF-ß were comparable in all groups, as were control GAPDH levels. At day 8 there was no difference in Th1 marker mRNA in G4.18-treated compared to untreated controls. However, increased IL-4 and a stronger IL-5 band in the neat dilution was observed in G4.18-treated PLN (Fig. 3B
). Again, there was no difference in IL-10, TNF-
and TGF-ß levels. Thus, G4.18 therapy given from the time of immunization was associated with an early reduction of Th1 cytokines and enhanced induction of Th2 cytokines.
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Treatment with MRC OX-81 delayed the onset of clinical EAE by 2 days, but there was no difference in the severity of disease, its clinical course or the weight loss observed when compared to untreated control animals (Fig. 1D). The isotype control A6 mAb-treated group had an identical course to CHOK1 supernatant and untreated controls.
Effect of G4.18 commenced at time of onset of clinical symptoms in active EAE
To examine if G4.18 could reverse EAE at the time of onset of clinical symptoms, treatment with G4.18 was started on day 12 post-immunization, when the majority of rats had signs of clinical disease. All groups were stratified to match for clinical score at the time of treatment. The combined results of four experiments are summarized in Table 1. All but two of the 103 rats immunized developed clinical signs of EAE, indicating that the immunization schedule was reliable and effective. Once treated with G4.18, rats that had developed EAE did not progress clinically; their disease stabilized the following day and they started to recover within 2 days (Fig. 4
). The number of days of illness when rats had clinical symptoms was significantly reduced with G4.18 therapy compared to untreated controls. In the peak disease activity period from 12 to 18 days, clinical score and weight loss for each days were also significantly less in G4.18-treated rats compared to controls. This was due to the earlier recovery of G4.18-treated rats. The mean of the maximal disease activity in all G4.18-treated groups was also significantly less than all control groups (P < 0.01). Nearly all G4.18-treated rats had recovered by day 18, but control rats had a mild disease that persisted throughout the period of observation and up to day 30 post-immunization (Fig. 4
). Thus, G4.18 therapy reduced the peak severity of EAE and led to recovery 23 days sooner than in untreated controls. Isotype control mAb (BCLA8)-treated rats had similar disease severity, clinical score and weight loss to that of untreated controls, which indicated that G4.18's effects are related to its T cell specificity.
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Combining rIL-4 and G4.18 therapy significantly shortened the days of illness and reduced the mean of daily disease activity compared to G4.18 or rIL-4 treatment alone (Table 1 and Fig. 4
). There was no difference in mean maximal score in the G4.18/rIL-4- and G4.18-treated groups. Controls given G4.18 therapy combined with control CHO-K1 supernatant had a similar clinical course to rats treated with G4.18 alone (Fig. 4
). Combining MRC OX-81 with G4.18 therapy had no effect on EAE remission induced by G4.18. There was no significant difference in days of illness, mean of the mean daily disease activity or mean of the mean daily weight loss. Controls treated with G4.18 and A6, an isotype-matched control for MRC OX-81, were also comparable to G4.18 treatment alone. Taken together, these studies showed that a combination of rIL-4 with G4.18 was more effective than G4.18 therapy alone. The effect of G4.18 on recovery from active EAE was not dependent upon IL-4 as blocking this cytokine with MRC OX-81 had no impact on the effect of G4.18.
Examination of the effects of G4.18 alone and with rIL-4 on mononuclear cell infiltrates in brainstem
Mononuclear cell infiltrates in brain stem were examined at day 14, when there was first evidence of recovery, and day 18, when there was complete recovery, this corresponded to 2 and 6 days after commencement of therapy (Table 2). Untreated controls showed a large infiltration of TCR
ß+, CD4+, CD8+, B cells, dendritic cells and macrophages at both day 14 and 18. This infiltration persisted for up to 30 days (data not shown). Normal brain stem showed no positive staining for any lymphocyte markers used, thus all cells stained in other groups are an inflammatory infiltrate. The number of CD4+ cells exceeded that of TCR
ß+ cells in all samples, as rat macrophages also express CD4.
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Examination of the effects of G4.18 and G4.18/rIL-4 on the cytokines expressed by the mononuclear cell infiltrates in brainstem
RT-PCR was used to examine cytokine mRNA levels for the brain stem at day 14 and 18 of rats treated from day 12. GAPDH levels were similar in all samples (Fig. 5). Th1 cytokine mRNA (IL-2, IFN-
and IL-12Rß2) was detected in brain stems of controls but not in G4.18- and G4.18/rIL-4-treated rats at day 14 (Fig. 5
) and 18 (data not shown). This was consistent with the marked reduction in brain stem cellular infiltration found with G4.18 and combined G4.18/rIL-4 therapy. Th2 cytokines, IL-4 and IL-10, were detected in untreated controls, but in G4.18 or rIL-4/G4.18-treated rats no IL-4 and only low levels of IL-10 were detected. IL-5 was different in that at day 14 there were higher levels of IL-5 in both G4.18 and G4.18/IL-4-treated rats compared to controls (P = 0.035 and 0.015 respectively). At day 18, the IL-5 mRNA levels were similar in G4.18-treated and control animals. As the T cell infiltrate in rIL-4/G4.18-treated brain stems was trivial, these results suggested that the residual cells predominantly produced IL-5. A marked reduction in the macrophage cytokines TNF-
and IL-10 was also found with G4.18 and rIL-4/G4.18 treatment compared to controls. TGF-ß mRNA was comparable at both days 14 and 18 for all groups. These results show that there was sparing of IL-5 and TGF-ß with G4.18 and G4.18/IL-4 treatment, while Th1, other Th2 and macrophage cytokines were reduced or not detected, consistent with depletion of T cells and macrophages.
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The effect of G4.18 on passive EAE
As the G4.18 effect appeared to be on activated T cells, we tested its effect on passive EAE that was induced by transfer of activated spleen cells from Lewis rats immunized with MBP/CFA. The adoptive hosts developed clinical signs of EAE by day 5 and maximal disease by day 6 with recovery by day 8. G4.18 given from time of transfer of cells markedly reduced severity of EAE with only one out of six rat developing any clinical disease with mean maximal disease of 1.5 compared to control treated with isotype-matched non-functional mAb (BCLA8) which developed severe clinical disease (3 ± 0.35). G4.18-treated rats at onset of clinical disease has reduced severity (1.9 ± 0.6, P = 0.02) and accelerated recovery compared to the control group (Fig. 7).
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Discussion |
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Early G4.18 treatment from the time of immunization had no effect on the clinical development of active EAE. There was an early inhibition of Th1 cell cytokine mRNA expression at day 3; however, this appears to have only delayed induction, as Th1 cytokine expression was comparable to untreated controls by day 8. At both time points (day 3 and 8), mRNA for the Th2 cytokines IL-4 and IL-5, but not IL-10, was higher in G4.18-treated compared to untreated controls. This is consistent with the reported inability of anti-CD3 mAb to block Th2 cell function, both with Th2 clones in vitro (41,42) and by the sparing of Th2 cells in allograft tolerance induction in vivo (40). The failure of the mAb to maintain this blocking function or to effect onset of disease when given from the time of immunization was found to be due to the development of anti-idiotypic antibodies. These antibodies neutralized the function of G4.18, preventing the mAb binding to CD3 and enhancing clearance of the mAb from the circulation. Delaying treatment by commencing 7 days after immunization only delayed the course of EAE until anti-G4.18 antibodies developed to G4.18. These results suggest G4.18 mAb may have a limited ability to block activation of naive T cells, but works by blocking Th1 effector cell function (40). The findings that G4.18 blocks induction of passive EAE, as well as reversing clinical disease with late treatment, is also consistent with a dominant effect of anti-CD3 mAb being on activated T cell function.
The most dramatic effects with G4.18 were found when treatment was commenced after the onset of active or on passive EAE, when there are activated T cells. This effect was shown to be specific for G4.18, in that isotype-matched mouse IgG3 mAb (BCLA8) which is non-reactive to rat antigen had no effect on the clinical course of either active or passive EAE. G4.18 therapy was associated with less cellular infiltrate into the brain stem, which paralleled earlier clinical recovery of active EAE. There was a persistent infiltrate of CD4+ T cells and macrophages for >1 week after spontaneous recovery of EAE in untreated control (data not shown). A major infiltrate of T cells and macrophages precedes onset of clinically active EAE (data not shown), thus the G4.18 leads to a reduction of central nervous system infiltrate. The remaining cells after G4.18 treatment did not express mRNA for Th1 cell markers including IL-2, IFN- and IL-12Rß2, but there was continued expression of IL-5 and TGF-ß. The macrophage cytokines TNF-
and IL-10 were also markedly reduced compared to untreated controls, consistent with reduction of macrophages from the brain stem. The absolute readings did not compensate for the marked loss in all mononuclear cells in the infiltrate, which makes the persistent expression of IL-5 and TGF-ß a much more significant finding.
This data is consistent with the relative sparing of Tr1 cells but not Th1 or Th2 cells in the brainstem. Tr1 cells are a recently described subset of T cells that have been shown to have regulatory function in autoimmune colitis (30,31) and immune-mediated non-obese diabetes (32). These cells have been cultured in vitro by repeated antigenic stimulation with IL-10 and antigen, characterized by high IL-5, IL-10 and TGF-ß levels with low IL-2 and no IL-4. These cells proliferate slowly compared to other T cell phenotypes. In our experiments, both IL-5 and TGF-ß were preserved, but IL-10 was markedly reduced. This was most likely related to the high expression of IL-10 by macrophages in the rat (37,58). The key Th2 cytokine, IL-4, was not preserved in the brainstem but was in the PLN. This is consistent with G4.18's previously described selective ability to inhibit activated Th1 cells but spare Th2 cells in vivo and in vitro (40). In this study, the effect of G4.18 was examined in vivo, and thus determined the overall trends in cytokine profiles not the number of Th1 and Th2 cells. Studies of individual cell cytokines would require in vitro stimulation in the absence of G4.18 that may have changed the profile, thus these studies were not performed.
Studies with rIL-4 failed to demonstrate that treatment with this cytokine alone could affect the course of EAE, irrespective of whether treatment was instituted at induction or at the onset of clinical EAE. This is consistent with some reports (23), but at variance with others in the mouse and rat, that showed delayed onset and reduced severity of EAE with IL-4 therapy (25,29). Likewise, treatment with rIL-5, or supernatant from non-transfected CHO-K1 cell lines, had no effect on EAE. The rIL-4-treated rats developed an enhanced IgG1 response to MBP and mRNA for IL-4 was increased in lymph nodes, consistent with induction of Th2 responses. rIL-4 prepared and given in a similar manner in our laboratory has been shown to delay organ allograft rejection in DA rats (51) and inhibit induction of Heymann nephritis in Lewis rats (manuscript in preparation), both associated with enhanced Th2 cytokine expression and increased IgG1 with reduced IgG2a and IgG2b responses. Thus, our protocol is able to significantly modify Th1/Th2 responses in Lewis rats. There is no ready explanation for the variation in reports on the efficacy of rIL-4 in modifying EAE (23,24,26). A similar protocol of administration of rIL-4 used in our study failed to inhibit Th1-mediated uveitis in Lewis rats (49). Differences in dose, time of administration and the models used may account for the conflicting reports of rIL-4 treatment on EAE. Whilst, Th2 have been considered to regulate EAE, this is not always the case as Th2 clones can mediate EAE (28). The dosage of rIL-4 was close to the maximum tolerated by rats and caused ascites with an inflammatory infiltrate of eosinophils. Higher doses of rIL-4 caused a high mortality in Lewis rats (data not included). rIL-4 combined with G4.18 enhanced the effect of the mAb in that a further reduction in the severity and duration of EAE was observed. The combined treatment was associated with a lesser mononuclear cell infiltrate in the brain stem than in G4.18-treated rats. There was also enhanced up-regulation of IL-4 mRNA in the draining lymph nodes, but not in the persisting mononuclear cell infiltrate. The modest delay in onset of active EAE with blocking anti-IL-4 mAb therapy suggests Th2 responses may have contributed to part of the early injury in active EAE in this model.
The efficacy of G4.18 was not dependent upon IL-4, as treatment with MRC OX-81 which blocks IL-4 function in vivo (46,41) did not affect the recovery of EAE induced by G4.18. Thus, G4.18's effects were most likely due to inhibition of activated Th1 cells. Secondary effects were the sparing of cells expressing Th2 and Tr1 cytokines, which may have promoted the re-establishment of tolerance. Such regulatory cell-mediated tolerance may have prevented relapse of EAE once the anti-CD3 mAb was blocked by the development of anti-idiotypic antibodies. Late G4.18 treatment groups were observed for up to 50 days and never developed major relapses, unlike groups treated from day 7, which relapsed when anti-idiotype antibodies to G4.18 developed.
It is possible that with late treatment, regulatory cells had time to develop and these could prevent relapse. The specific effects of anti-CD3 mAb on activated Th1 cells have been identified in clones grown in vitro (42). Anti-CD3 mAb affect specific signaling pathways required for activation of Th1 but not Th2 cells (38,59). Our results suggested that anti-CD3 mAb may also spare Tr1 cells. Thus, there are two potential regulatory cells subsets that could have promoted recovery from EAE induced by anti-CD3 mAb. The effect of rIL-4 in enhancing recovery suggested that Th2 cells can contribute to recovery in this model. The precise role of Tr1 cells or TGF-ß-producing Th3 cells in the anti-CD3 mAb effect requires further investigation. These studies suggest that a non-mitogenic, non-activating anti-CD3 mAb may have potential in treating Th1-mediated autoimmune diseases in man, including demyelinating diseases. The effects observed are similar to those observed in NOD mice, where onset of diabetes is stopped by non-mitogenic anti-CD3 mAb (60). Furthermore, anti-CD3 therapy may be enhanced by co-administration of rIL-4.
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Acknowledgments |
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Abbreviations |
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CFA complete Freund's adjuvant |
EAE experimental autoimmune encephalomyelitis |
MBP myelin basic protein |
PLN popliteal lymph node |
TGF transforming growth factor |
TNF tumor necrosis factor |
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Notes |
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Received 6 November 2000, accepted 30 May 2001.
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References |
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