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Overexpression of phosphodiesterases in experimental autoimmune myasthenia gravis: suppression of disease by a phosphodiesterase inhibitor

Revital Aricha^*, Tali Feferman^*, Miriam C. Souroujon^*, and Sara Fuchs^*,1

^* Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; and
The Open University of Israel, Raanana, Israel

¹ Correspondence: Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: sara.fuchs{at}weizmann.ac.il

SPECIFIC AIMS

Our main goal is to identify new, yet unknown molecules that are involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder characterized by muscle weakness, and to test their possible application as drug targets for treatment of the disease. Microarray analysis has indicated that phosphodiesterase (PDE) expression is up-regulated in experimental autoimmune myasthenia gravis (EAMG) in rats as compared with healthy controls. Therefore, our specific aims were to 1) analyze alterations in the expression of PDE subtypes in lymph node cells (LNC) and muscle in rat EAMG; and 2) test the effect of PDE inhibition, particularly by pentoxifylline (PTX), on the course of EAMG and on PDE expression, and to study the underlying mechanism(s) of EAMG suppression.

PRINCIPAL FINDINGS

1. Specific PDE subtypes are up-regulated in EAMG
DNA microarray analysis of rat EAMG revealed an up-regulated expression of PDEs, enzymes that are currently grouped in 11 broad families differing in their substrate specificity, inhibitor sensitivity, tissue distribution, and regulation of activity. PDEs catalyze the hydrolysis of cAMP and cGMP and are therefore critical in determining the intracellular levels of these second messengers that play a pivotal role in the regulation of a wide range of cellular functions including cellular immune responses. DNA microarray analysis revealed increased mRNA levels of PDE subtypes 1, 2, and 4 in EAMG rats as compared with healthy controls. Further analysis by quantitative real-time PCR analysis indicated that EAMG is characterized by an increase of PDE subtypes 1, 3, 4, and 7 in lymph node cells (LNC), and of PDE subtypes 2, 3, 4, and 7 in muscle - the target organ in MG (Fig. 1 A).

View larger version (39K): [in this window] [in a new window]   Figure 1. mRNA expression of PDEs in EAMG (A) and after treatment by PTX (B). A) LNC and muscle samples were harvested when rats in the AChR-immunized group reached a clinical score of 2. mRNA expression levels of PDEs in LNC (left) and muscles (right) from EAMG (n=10 and n=6 respectively) and control healthy rats (n=10 and n=4, respectively) were determined by quantitative real-time RT-PCR. *P < 0.05. B) mRNA expression levels of PDEs in LNC (left) and muscles (right) from rats treated by PTX (180 mg/kg) (n=13 and n=9, respectively) or PBS (n=15 and n=9, respectively), starting at the acute phase of EAMG were determined by quantitative real-time RT-PCR 8 wk after EAMG induction. Data are presented as relative expression value for each PDE subtype of the EAMG group compared with its control, which was assigned a value of 100. ß-Actin was used as an inner control for normalization for each PDE. Error bars indicate SEM value, *P < 0.05.

2. Inhibition of phosphodiesterases suppresses ongoing EAMG
In view of the observed up-regulation of various PDE subtypes in EAMG, we tested whether inhibition of PDE has a suppressive effect on the disease. For that purpose we chose to employ pentoxifylline (PTX), a xanthine-derived general phosphodiesterase inhibitor that is commonly used in patients suffering from peripheral vascular diseases. PTX has also been proposed as an anti-inflammatory and immunomodulating agent due to its inhibitory activity on Th1 differentiation pathways, its suppressive effect on the synthesis of several pro-inflammatory cytokines such as TNF-, IL-12, and IL-18 and its antiproliferative activity demonstrated in vitro on B and T lymphocytes. These properties render PTX a candidate therapeutic agent for Th1-mediated autoimmune disorders. However, it has not been demonstrated whether PTX may exert its suppressive effect in autoimmune diseases also by affecting the levels of PDE expression, and whether PDE expression is disregulated in these diseases.

Administration of PTX had a suppressive effect on EAMG when treatment was initiated at the acute and even at the chronic phase of disease as monitored by mean clinical score and weight changes (Fig. 2 ). This suppression was associated with down-regulation of humoral and cellular responses to the major autoantigen in myasthenia, the acetylcholine receptor (AChR), as measured respectively by ELISA with a rat AChR fragment corresponding to the extracellular domain of the AChR -subunit (R1-205) and by the in vitro response of splenocytes to AChR. Total anti-R1-205 IgG levels, as well as the levels of IgG1 were significantly lower in PTX-treated rats as compared with control PBS-treated rats. IgG2a and IgG2b subtypes were slightly decreased in PTX-treated rats. Treatment with PTX significantly suppressed T cell proliferation in response to AChR of splenocytes isolated from rats 4 to 6 wk after disease induction.

View larger version (31K): [in this window] [in a new window]   Figure 2. Suppression of EAMG by PTX. Mean clinical score and body weight of rats treated by PTX starting at the acute (A) or at the chronic phase (B) of EAMG. Treatment with PTX (120 or 180 mg/kg body weight) (n=8 each) or PBS (n=8) was initiated at 1 wk (acute phase of EAMG) or 4 wk (chronic phase of EAMG) after disease induction by injection with Torpedo AChR. Data represent 1 of 3 independent experiments. Error bars indicate SEM values. P < 0.0005 for panels A, B (left) and P < 0.05 in panel B (right). Analyzed by the 2-way ANOVA test.

3. PTX down-regulates the expression level of selected PDE subtypes
Although PTX is known to affect the activity of PDE it also down-regulated the elevated PDE levels observed in myasthenic rats. As shown in Fig. 1B , PTX treatment resulted in a significant decrease in LNC of PDE4, and in the muscle of PDE1, PDE4, and PDE7 as compared with control, PBS-treated rats.

4. PTX decreases the expression of several cytokines in LNC and of TNF- in muscles
To gain insight into the possible mechanism of EAMG suppression by PTX we analyzed the cytokine profile of treated rats. PTX treatment resulted in down-regulation of the Th1-type cytokines TNF-, IL-12, and IL-18 but had no significant effect on IFN-. The levels of the Th2-type cytokine IL-4 did not change but the level of IL-10 decreased significantly, when compared with their respective levels in PBS-treated rats. TGF-ß, a Th3-type cytokine did not change significantly.

In the muscle, which has been suggested to play an active role in producing immunomodulating factors TNF- expression was significantly reduced as a result of PTX administration. Other cytokines were either unchanged or undetectable.

5. PTX affects the function, but not the number of CD4⁺CD25⁺ regulatory T cells
To find out whether T regulatory (Treg) cells have a role in the suppressive effect of PTX, we tested rats treated with PTX or PBS for the content of Treg cells and for the expression of FoxP3, which is essential for their function. The percentage of CD4⁺CD25⁺ cells measured by FACS analysis did not reveal any difference between PTX-treated rats (mean=8.5%) and PBS-treated rats (mean=8.33%). However, the mRNA levels of FoxP3, a transcription factor essential for Treg function were 2.6-fold higher in PTX-treated rats compared with PBS-treated controls.

6. Cathepsin-l expression in muscle is increased in EAMG and decreased after PTX treatment
Multiple types of skeletal muscle pathologies involve a common program of changes in gene expression. The lysosomal cysteine endopeptidase cathepsin-l is one of the genes strongly induced upon muscle damage and loss. We therefore measured the expression of the cathepsin-l gene in EAMG and after PTX treatment. Indeed, cathepsin-l mRNA levels in muscles of EAMG rats were markedly increased (up to 12-fold) as compared with healthy rats. Treatment with PTX (180 mg/kg body weight) resulted in a 70% reduction in the cathepsin-l mRNA levels, possibly reflecting muscle recovery.

CONCLUSIONS AND SIGNIFICANCE

In this study we have demonstrated that rat EAMG is associated with increased mRNA expression of several phosphodiesterase (PDE) subtypes in lymph node cells and in muscles. To our knowledge, this is the first time that alterations in the expression levels of PDEs in an autoimmune disease have been reported. In view of these findings we tested the effect of PDE inhibition on the course of EAMG in rats and showed that PTX, a general PDE inhibitor, suppresses the progression of EAMG. This suppression is associated with down-regulation of PDE expression, as well as of humoral and cellular immune responses specific for EAMG and of certain cytokines.

In the immune system, drugs that increase cAMP levels, including PDE inhibitors, are known to prevent or attenuate inflammatory responses. Therefore, PDE inhibitors have been employed for the treatment of autoimmune disorders. However, the direct involvement of PDE in autoimmune disorders and in MG in particular, has not been demonstrated so far. As in the present study we have shown that PDEs expression is up-regulated in EAMG it was logical to test whether inhibition of PDE is capable of suppressing the disease.

Treatment by PTX led to a decrease in the humoral and cellular responses to AChR that is the autoantigen in myasthenia. The mechanism underlying this suppression seems to involve reduced levels of IL-12, IL-18, and TNF-, which are known to regulate Th1- differentiation and the Th2-type cytokine IL-10, all of which play a key role in the pathogenesis of MG. We did not find a significant difference in the number of CD4⁺CD25⁺ lymphocytes after PTX treatment nor in the gene expression of TGF-ß. However, the expression of the transcription factor FoxP3 that was shown to be necessary and sufficient for Tregs generation and function was increased (2.6-fold) after PTX treatment.

The possibility that the mechanism of action underlying the suppressive effects of PTX on EAMG is not related only to its anti-inflammatory activity cannot be excluded. PTX could also act on the muscle, as suggested by our observation that PTX treatment lead to down-regulation in the muscle of PDE1, PDE4, and PDE7 and of cathepsin-l and TNF-. As we did not observe any infiltration of lymphocytes into the muscle, it is possible that PTX acts directly on muscle cells.

Taken together, our results point to a multifaceted mechanism of action of PTX (Fig. 3 ). It acts by affecting the expression level of selected PDE subtypes as well as by modulating immune responses. PTX suppresses T cells proliferation, down-regulates Th1 cytokines and IL-10 and up regulates Foxp3 by modulating intracellular levels of cAMP in immune cells. PTX could also decrease muscle fatigue or even muscle loss in myasthenic rats by a combined effect on the muscle. The data provided in this study indicate that various PDE subtypes are involved in the pathogenesis of EAMG. Moreover, as PTX is already being used in patients with other disorders with no serious side effects, our results justify well-designed clinical trials to test the efficacy of treating MG patients by PTX, and possibly by additional, subtype-selective PDE inhibitors.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram describing changes associated with EAMG and after treatment by PTX.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4909fje;

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