Suppression of experimental autoimmune neuritis by phosphodiesterase inhibitor pentoxifylline

JOURNAL OF THE

NEUROLOGICAL SCIENCES ELSEVIER

Journal of the Neurological Sciences 143 (1996) 14-18

Suppression of experimental autoimmune neuritis by phosphodiesterase inhibitor pentoxifylline Cris S, Constantinescu

‘, Brendan

Hilliard “, Ehud Lavi b, Elvira Ventura ‘, Vijay Venkatesh a, Abdolmohamad

Rostami ‘“

“ Deprzrrment~f’Neurolo,qy,IInit:er.riqqf’fr,?tr,~>l(on;[z Srhml of Medii;ne, .7400SI,n,cc Slrr>el,Philudelphin, PA 1!)104-4283, USA Un;(wmil), (!fPt,ri)7.\}l/){{nili School oj’Medicine, 3400 bpruce Street, h .Deparment qf’f’atltdo,qy and Laboratot> Medicine, Difkirm of~(,,.,,]l?(zt)?,]l(]~>, Phikulrlphirr,PA 19104-428.?,USA Received 14 Jwrrarry 1996; revised 26 April 1996; acccptcd 20 May 1996

Abstract Phosphodiesterase inhibitor pentoxit’ylline (POX) has been shownto have multipleilll]llull{>lllt]dulat[~ry effects in vilruand in vivo.It inhibits T cell proliferation,‘1’helper l-type cytokines,and tumor necrosis factor. We postulatedthat POX might have an in vivo immunomodtdatory effect on a I’-cell-mediated autoimmune peripheral nervous system disease, expel-imental mttoimmtrnc ncurilis (EAN).We investigatedthe effectof POXon EANin 1-atsimmunizedwithperipheralnervemyelincontainingncurimgenicpepticieSP26. At Q()() ,ng/kg/day,

there was significant

suppression

of clinical EAN, weight loss, and T cell proliferation

to SP26 compared to

SP26 was suppressedby POX in vitro. These studiesdemonstratea beneficial role for POX in EAN, with potentialapplicabilityto humanautoimmunedemyelination. controls. Proliferation

of T cells from immunized rats to

Autoinmmnity; Cyclic AMP; Cytokine; Experimental autoimnarne neuritis; GuilluimBwk syndrome; Pentoxifylline; Plios[)l][)dicstcrasc KrJwot-&:

1. Introduction Experimental autoimmune neuritis (EAN) is a T cellmediated demyelinating disease of the peripheral nerve which represents an animal model for the Guillain-Barr6 syndrome (GBS) (Rostami, 1992; Hartung et al., 1995; Rostami, 1995). EAN can be induced in rodents by immunization, combined with adjuvants, with peripheral nerve myelin, its component, P2 protein, or P2 peptide fragment SP26, containing aminoacids 53-78 (Rostami et al., 1990; Rostami, 1992; Rostami, 1995). Passive EAN is induced by adaptively transferring P2-reactive CD4 + T cells (Liningtm et al., 1984; Rostami et al., 1985). The resulting inflammatory demyelination causes an acute illness characterized by weight loss and paralysis, followed by recovery. EAN represents the peripheral nervous system counterpart of experimental autoimmune encephalomyelitis (EAE), a T cell-mediated demyelinating disease of the central nervous system (Zamvil and Steinman, 1990). EAE and EAN are characterized by nervous system inflammatory infiltrates consisting largely of T cells and macrophages.

* Corresponding author. Tel: + 1 (215) 662-6557. Fax: + 1 (215) 573-2107.

Immunopathological studies in EAN suggest that Thl type cytokines, including interleukin (IL)-2, interferon (IFN)-Y, and tumor necrosis factor (TNF) may mediate pathology in EAN (Hartung et al., 1992; Hartung et al., 1995). Inhibition of these cytokines by counterregtrlatory cytokines (Jung et al., 1994) or by neutralization (Stoll et al., 1993) has suppressed EAN. Phosphodiesterase (PDE) inhibitor pentoxifylline (POX), a methylxanthine derivative which enhances cyclic AMP, has been shown to possess multiple immunoregulatory effects, including inhibition of T cell proliferation and of Thl -type cytokines in vitro and in vivo (Rieneck et al., 1993; Rott et al., 1993; Hecht et al., 1995). POX suppressed or prevented EAE (Nataf et al., 1993; Rott et al., 1993). We postulated that POX is of potential benefit in EAN. In the present study, we provide evidence for the ability of POX to ameliorate EAN in rats. 2. Materials and methods 2.1. Immunization and treatment EAN was induced as previously described (Rostami et 10– lz-week-old female Lewis rats al., 1990). Briefly,

0022-510X/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII S0022- 5 10X(96)O0 195-5

C.S. Cmrsturrtirrercuet u1./Joumul (fth~ Neurologicu[ Sciew~s 143 (1996,) 14-18

15

(Charles River Laboratories), weighing 135-160 g, under anesthesia were immunized in the hind footpad with 600 pg bovine peripheral nerve myelin in-complete Freund’s adjuvant. POX (a gift from Hoechst-Roussel Pharmaceuticals), or phosphate-buffered saline (PBS) as control, were given daily, intramuscularly at 30, 60, and 200 mg/lcg/ day, at final volume 300 @/animal, fropl immunization until day 21 post-immunization. Paralyzed rats had easy access k} food and water.

formalin, and embedded in paraffin. Five-pm sections were cut, stained with hematoxylin and eosin, or Luxol fast blue, and analyzed for endoneurial inflammation and for demyelination, each of which were independently characterized as absent, mild, moderate, or severe. The tissues were assessed by an investigator who was unaware of the treatmcut groups to which Lhertits belonged.

2.2. clinical

Studcnl’s t kst wits used to ussess the significance of differences between treated and untreated groups with respect to weight changes and proliferation. Differences between clinical scores were analyzed using Wilcuxon’s sign rank test. Spearrmm’s rank correlation coefficient was calculated to asses the dcgrcc of correlation between the two clinical scoring systems. X2 test was used to assess the differences between the proportion of rats with severe disease in each group. A value of p s 0.05 was considered statistically significant.

a,rsessmenr

Animals were weighed daily and scored blindly by two invcstigatols using two previously described scales, The first is a scale of O–3 with 0.5 points for intermediate clinical signs (Rostami ct al., 1990): normal, O; mild (> 5% weight loss, flaccid tail), 1; moderate (previous signs plus ataxia and inability to spread the toes), 2; severe (paraplegia or tetraplegia), 3. Parallel assessment at the peak of disease was performed using aO–10 scale (Jung et al., 1994): normal, O; decreased tone of tail, hanging tip rsf tail, 1; flaccid tail, 2; impaired righting, 3; ataxia, abnormal position, 4; mild hind limb paraparesis, 5; moderate paraparesis, 6; severe partiparesis or paraplegia, 7; tetraplcgia, 8; molibund, 9; death due to EAN, 10.

2.5. Sldisti~’s

2.3. Prvl(ferution assays Lymphocyte prolifel”ation assays were done as described (Rostami et al., 1985). Briefly, popliteal lymph node cells were incubated at 1 X IOb/well (total volume, 200 @/well) in 96-well microtiter plates with appropriate concentrations of SP26, concanavalin A (con A), PPD, or ovalbumin (OVA). Incorporation of [3H]thymidine (0.5 #Ci/well), added for the last 16 h of a 72-h incubation, was measured in a liquid scintillation counter (Beckman). Results represent mean counts per minute (cpm) of triplicates. 2.4. Histological assessment For neuropathological assessment, the cauda equina and sciatic and posterior tibial nerves were removed, fixed in

3. Results 3.1, Clinicul sign,~ Inlerobserver variability between clinical scores given blindly by the two investigators was consistently <490. The clinical scores obtained using the two scoring systems correlated well with correlation coefficient r consistently >0.95. Therefore, results are given for the O–10 scale only. Clinical signs of EAN were first recognized at day 12 after immunization. There were no significant differences in severity or duration of EAN between low- or intermediate-dose (30 or 60 mg/kg/day, n = 5 per group) POX-treated rats and controls (n = 5) (data not shown). In the high-dose (200 mg/kg/day) POX-treated group (n = 16), there was suppression of the clinical signs of EAN (Fig. 1). In the three days of peak disease activity (day 13–15), and additionally at day 19, the differences between treated (n = 16) and control (n = 18) rats were statistically significant ( p < 0.05). The maximum clinical scores were 9 in the PBS-treated group and 8 in the

Table 1 Effect of in vitro POX on proliferation of T cells from myelin-immunized rats ‘ In vitro treatment

Medium alone:

Stimulation condition and proliferation profile SP 26 No stimulus 100 ~g/ml 842+386

14274+ 1298

10 p,g\ml

Con A 5 ~g/ml

8072+ 537

105370~ 5627

Pox: 300 WM P

544*79 NS

7118 +753

4989+790

72261 + 3321

0.03 2237 + 145

NS 121879 ~6300

<0.0001

NS

30PM

776*241

0.009 7791 *442

P

NS

0.008

‘ Values are mean+ S.D. radioactive counts per minute. NS = not

wereobtainedin an additional, independent experiment.

significant (p> 0.05).ConA= concanavalin A; Pox= pentoxifylline. Similarresults

16

C.S. Constantinescu et al./Journal oj”theNeurological Sciences 143 (1996) 14-18

10 r

0

3

6

d~~s

9

12 15 18 21 24 post-immunization, ,,

Fig, 1, Effect of POX on the clinical course of EAN. Results are shown as mean clinical scores+ SEM (one-sided for graphical clarity). Asterisks indicate a significant difference (ps 0.05) between POX (200 mg/kg/ day)-treated rats and PBS-treated controls.

POX-treated group. The number of animals with severe disease (maximum score > 6) were 5 and 1 in the PBS and POX-treated groups, respectively ( p < 0.05). Comparison of weights between animals assigned to the POX or PBS treatment groups showed no pre-experiment sample bias of weight. Initial weights for the two groups had a meant S.D. = 145.23 ~ 4.65, and 146.11 +-6.11, respectively ( p > 0.05). Weight loss was first noted in immunized, untreated animals at day 11, and it reached a peak at day 15 post-immunization. This weight loss, typically associated with peak EAN (days 13–15 post-immunization), was prevented, and even reversed, compared to controls ( p < 0.02) (Fig. 2). 3.2. Prol$eration assays Six animals in each group were sacrificed at day 14 post-immunization for proliferation assays and histology.

10 S5

1 T

A

-i-

do -5 -10

Fig. 3, Effect of POX on the histological appearance of EAN. (A,B). Representative sections of proximal segments of sciatic nerves of rats sacrificed at the peak of disease (day 14 post-immunization), revealing moderate-to-severe endoneurial inflammatory cell infiltration (A,B), and perivascular infiltration (B). (C) In contrast, inflammation is absent or minimal in this representative example of a corresponding section from a POX (200 mg/kg/day)-treated animal. Hematoxylin-eosin stain.

-15 -20 I day 13

day 14

day 15

Fig. 2. Effect of POX on the weight loss associated with peak EAN. POX significantly inhibited and even reversed the EAN-associated weight loss (p< 0,02), The mean weight changes at peak disease (days 13-15 post-immunization) are shown in POX-treated animals (open bars), and PBS-treated control animals (solid bars). Error bars indicate the SEM. A wt = mean weight change in grams expressed as the difference from the weight measured on the day of immunization (day 0).

The effect of POX on proliferation of peripheral nerve myelin-reactive T cells was assessed by adding POX 300 p,M or 30 KM to the cells incubated with SP26. There was a significant reduction in proliferation at either concentration of POX added in vitro. No significant inhibition of proliferation to conA was seen (Table 1), although a trend toward inhibition was noted in the presence of 300 WM POX added in vitro.

C.S. Constantinescu et al, /Journal of th~ Neurological Sciences 143 (1996) 14-18

Lymphocytes from rats that had been treated in vivo with POX exhibited decreased proliferation to SP26 (p < 0.05), but not to the control, irrelevant antigen OVA, or to conA ( p > 0.05). With respect to the latter stimulus, the stimulation index (ratio of radioactive counts per minute beween unstimulated and stimulated cells), was 2.9 in the PBS-treated animals and 4.1 in the POX-treated animals. Although there was a trend toward suppression of proliferation to PPD, a component of the immunization adjuvant, this did not achieve statistical significance ( p > 0.05) (data not shown). 3.3. Histological assessment Histological evaluation of peripheral nerve and cauda equina sections in the animals sacrificed at day 14 post-immunization revealed significant endoneurial inflammation in controls and minimal, if any, in the POX-treated animals (Fig. 3). Demyelination was mild to moderate in PBS-treated rats and minimal or absent in POX-treated rats. These differences in the degree of demyelination in the selected sections of EAN did not reach statistical significance.

4. Discussion These studies demonstrate the ability of POX to supress the autoimmune response in EAN. POX significantly decreased the severity of paralysis — the clinical hallmark of EAN. The mean clinical scores at peak disease in the untreated group were approximately 1.5 in the O–3 scoring system and approximately 4.5 in the 0–10 scoring system, indicating significant severity. In addition, POX markedly prevented the EAN-associated weight loss — an integral part of the clinical syndrome of EAN, which reaches the highest level at the peak of neurological deficit. POX suppressed significantly the endoneurial inflammation at the peak of disease, which is consistent with the evidence for its in vivo immunomodulatory anf anti-inflammatory properties. Although demyelination was decreased in POX-treated rats, the degree of demyelination was generally mild in both groups, resulting in no statistically significant differences. Therefore the effect of POX on demyelination in EAN (which, as in EAE, does not always correlate linearly with inflammation) cannot be definitively characterized in this study. The dose of POX necessary to achieve the protective effects was slightly lower than that required to suppress EAE (Rott et al., 1993). We observed no side effects associated with this treatment in rats. Therefore, higher doses in rats may potentially be more effective. POX inhibited the SP26-dependent T cell proliferation, without significantly affecting conA-induced proliferation, in both in vivo and in vitro conditions. The significance of

17

this finding is unclear. In our study, the proliferation induced by conA in lymphocytes from rats that had been treated in vivo was lower than that induced by antigen, possibly because samples were harvested at a time point designed to ensure high antigen-specific proliferation, when conA induced proliferation probably was already declining. It is possible that, had the stimulation indices been higher, this differential effect of POX may not have been observed. On the other hand, a similar dissociation in the presence of POX between conA- and antigen-induced proliferation, while of yet unclear mechanism, has been observed with another mitogenic stimulus, phytohemagglutinin (Rieneck et al., 1993). At 10 kg/ml SP26, antigen specific proliferation of T cells appeared to be more efficiently inhibited by a lower dose of POX (30 KM) than by the 300 LM dose (although the percent inhibition by the two doses was not statistically significant). This may be explained by a nonlinear dose-response curve of POX inhibition at the selected concentrations or by the fact that POX being a nonspecific PDE inhibitor may, at higher concentrations, inhibit additional PDEs such as the cGMP-specific ones with opposite activities. The mechanisms of the POX-induced inhibition are probably multiple. PDE inhibition increases the intracellular cAMP, with several consequences. First, cAMP elevation uncouples the T cell receptor/CD3 complex from downstream signaling mediators (Lerner et al., 1988). Secondly, it interferes selectively with the activity of transcription factors for cytokines such as IL-2 and TNF (Chen and Rothenberg, 1994). Also, POX stimulates prostaglandin production, thus further suppressing proinflammatory cytokines. POX has provided benefit in several experimental and clinical situations, including endotoxic shock (Zabel et al., 1989). This study shows POX to be beneficial in EAN, a T cell mediated autoimmune disease. These findings raise the possibility of a therapeutic role for PDE inhibitors in GBS, and human autoimmune demyelinating disease in general. This is supported by recent evidence of successful prevention of autoimmune demyelination by a PDE inhibitor, rolipram, not only in the Lewis rat (Sommer et al., 1995), but also in a non-human primate rnoclel (Genainet al., ]9s)5). In addition, rolipram inhibits the production of TNF and IFN-y by human myelin basic protein-reactive T cells (Sommer et al., 1995). PDE inhibitors, including POX have been well characterized pharmacologically. Moreover, recently POX demonstrated therapeutic properties in rheumatoid arthritis, a human T cell mediated autoimmune disease (Maksymowych et al., 1995). POX causes rare significant side effects at the usual hemorrheological doses used in humans, and it was well tolerated in clinical trials employing higher dosing regimens (Dezube et al., 1995; Maksymowych et al., 1995). In addition, the relationship between equivalent therapeutic dosages in rats and humans may not be linear. The fact that suppression of EAN in our study requires a high dose

18

C.S. Constantinescu et uL/Jaarnal oj’the Neurological Sciences

regimen suggests that optimal effects are possible using POX in conjunction with other approaches. Inhibition of cytokine production may be achieved by lower doses of synergistic agents which block the production pathways at distinct points. For example, POX synergies in this way with dexamethasone for suppression of TNF production (Han et al., 1990). Also, recently, the combination of PDE inhibitors with prostanoids demonstrated synergy in the enhancing cAMP and suppressing TNF (Sinha et al., 1995). Thus, PDE inhibitors such as POX, possibly combined with synergistic interventions may successfully modulate autoimmune demyelination. Acknowledgements This study was supported by grants IK11 HD-O1O49, NS-08075 and NS-1 1037 from the NIH. We thank A. Rathblott for technical assistance, Drs. M. Bocamea and E. Bradlow for suggestions regarding statistical analysis, Dr. K. Frei for reviewing the manuscript, and the HoechstRoussel Pharmaceuticals, Somerville, New Jersey, for the gift of pentoxifylline. References Chen, D. and E.V. Rothenberg (1994) Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J. Exp. Med. 179: 93 1–942. Dezube, B..f. et al, (1995) High-dose pentoxifylline in patients with AIDS: inhibition of tumor necrosis factor production, J. Infect. Dis. 171: 1628-1632. Genain, C.P. et al. (1995) Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor. Proc. Natl. Acad. Sci. USA. 92: 3601–3605. Han, J, et al. (1990) Dexamethasone and pentoxifylline inhibit endotoxin-induced cachectin\tumor necrosis factor synthesis at separate points in the signaling pathway. J. Exp. Med. 172: 391–394. Hartung, HP. et al. (1992) Inflammatory mediators in demyelinating disorders of the CNS and PNS. J. Neuroimmunol. 39: 197-210. Hartung, H.P. et al. (1995) Immunopathogenesis and treatment of the Guiilain Barr6 syndrome. Muscle Nerve. 10: 137-153; 154-184.

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