use of protein drugs conjugated with PEG [19,20]. In thesestudies, they purified the polymer until the polymer had asingle molecular weight. It is necessary to use the purifiedpolymer to evaluate the tissue distribution of the polymerscientifically and quantitatively. Many experiments andphysical analyses of these polymer chains in solution arenecessary to determine whether Tsutsumi and colleagues’method of preparing polymeric drug carriers can be usedgenerally for site-specific drug delivery. However, thestudy by Tsutsumi and colleagues has paved the way fordeveloping such a technique.

References

1 Brich, Z. et al. (1992) Preparation and characterization of a watersoluble dextran immunoconjugate of doxorubicin and the monoclonalantibody. J. Control. Release 19, 245–258

2 Igarashi, R. et al. (1992) A stable PGE1 prodrug for targeting therapy.J. Control. Release 20, 37–46

3 Kopecek, J. (1991) Targetable polymeric anticancer drugs. Temporalcontrol of drug activity. Ann. New York Acad. Sci. 618, 335–344

4 Nishikawa, M. et al. (1992) Pharmacokinetics of receptor-mediatedhepatic uptake of glycosylated albumin in mice. Int. J. Pharm. 85,75–85

5 Yamaoka, T. et al. (1995) Fate of water-soluble polymers administeredvia different routes. J. Pharm. Sci. 84, 349–354

6 Yamaoka, T. et al. (1994) Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenousadministration to mice. J. Pharm. Sci. 83, 601–606

7 Kamada, H. et al. (2003) Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renaldrug targeting. Nat. Biotechnol. 21, 399–404

8 Brenner, B.M. et al. (1978) Glomerular permselectivity: barrier

function based on discrimination of molecular size and charge. Am.J. Physiol. 234, F455–F460

9 Takakura, Y. et al. (1990) Disposition characteristics of macromol-ecules in tumor-bearing mice. Pharm. Res. 7, 339–346

10 Veronese, F.M. et al. (2002) Polyethylene glycol-superoxide dismutase,a conjugate in search of exploitation. Adv. Drug Deliv. Rev. 54,587–606

11 Hinds, K.D. and Kim, S.W. (2002) Effects of PEG conjugation on insulinproperties. Adv. Drug Deliv. Rev. 54, 505–530

12 Kozlowski, A. et al. (2001) Development of pegylated interferons forthe treatment of chronic hepatitis C. BioDrugs 15, 419–429

13 Duncan, R. and Spreafico, F. (1994) Polymer conjugates. Pharmaco-kinetic considerations for design and development. Clin. Pharmaco-kinet. 27, 290–306

14 Delgado, C. et al. (1992) The uses and properties of PEG-linkedproteins. Crit. Rev. Ther. Drug Carrier Syst. 9, 249–304

15 Auzely-Velty, C. et al. (2002) Galactosylated N-vinylpyrrolidone-maleic acid copolymers: synthesis, characterization, and interactionwith lectins. Biomacromolecules 3, 998–1005

16 Murthy, N. et al. (2003) Design and synthesis of pH-responsivepolymeric carriers that target uptake and enhance the intracellulardelivery of oligonucleotides. J. Control. Release 89, 365–374

17 Pedder, S.C. (2003) Pegylation of interferon alfa: structural andpharmacokinetic properties. Semin. Liver Dis. 23 (Suppl. 1), 19–22

18 Milas, L. et al. (2003) Poly(L-glutamic acid)-pacritaxel conjugate is apotent enhancer of tumor radiocurability. Int. J. Radiat. Oncol. Biol.Phys. 55, 707–712

19 Tsutsumi, Y. et al. (1994) Chemical modification of natural humantumor necrosis factor-alpha with polyethylene glycol increases its anti-tumor potency. Jpn. J. Cancer Res. 85, 9–12

20 Yamamoto, Y. et al. (2003) Site-specific PEGylation of a lysine-deficientTNF-alpha with full bioactivity. Nat. Biotechnol. 21, 546–552

0165-6147/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tips.2003.10.009

Interleukin 4 treatment of psoriasis: are pleiotropiccytokines suitable therapies for autoimmune diseases?

Roland Martin

Cellular Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke,

National Institutes of Health, 10 Center DR MSC 1400, Bethesda, MD 20892-1400, USA

Many human autoimmune diseases are still treated

by a combination of corticosteroids and general

immunosuppression. A better understanding of the

pathogenesis of these diseases has led to therapies

that are more specific than current therapies of some

of these disorders. In psoriasis, T helper (Th) cells with

a pro-inflammatory phenotype (Th1) are considered

essential to the disease process. A recent clinical trial of

interleukin 4 in psoriasis has demonstrated that such

pleiotropic cytokines can be used safely and effectively

to correct imbalances in immune function.

In many respects, psoriasis is a typical autoimmunedisease [1]. Psoriasis affects a specific organ, the skin,and, to a lesser extent, joints, tendons and ligaments.

Inflammatory infiltrates, tissue destruction, clinicalheterogeneity and a complex genetic background are allcharacteristics of psoriasis, type 1 diabetes, rheumatoidarthritis (RA), multiple sclerosis (MS) and other auto-immune diseases. Approximately 2% of the populationsuffer from papules, nodules and thickened, scaly plaquesthat appear in distinct areas of the body, such as the elbowsand knees, or affect large areas of the skin. When thedisease is more widespread and joints become involved,psoriasis can severely compromise the quality of life of thepatient and has a substantial socioeconomic impact [2].

The cause of psoriasis is unknown but a combination ofgenetic predisposition and environmental factors probablytriggers the autoimmune process. Pathogenetically, psori-asis is characterized by oligoclonal T-cell infiltrations,which accompany other immune cells such as monocytes,and a chronic change in tissue turnover and architectureCorresponding author: Roland Martin (martinr@ninds.nih.gov).

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that might be caused by the secretion of inflammatorymediators [1,3,4]. T-helper cells (CD4þ) that expressinterferon g (IFN-g), other pro-inflammatory mediatorsand surface receptors (e.g. the chemokine receptor CCR5)appear to drive the immune process [1,4,5]. These cells arereferred to as T-helper 1 (Th1) cells [6] and are crucial fororchestrating adaptive cellular and humoral immuneresponses against intracellular pathogens. CD4þ T cellsthat secrete immune mediators such as interleukin 4(IL-4), IL-5 and IL-13 are also involved in the immunedefense response (e.g. against certain parasites) and arereferred to as Th2 cells [6]. Although the separation intoTh1- and Th2-dependent immune responses is at timessimplistic, such a separation has provided a very helpfulframework for understanding the nature of antigenicstimuli that activate either Th1 or Th2 cells and for deter-mining which transcription factors, immune cells or medi-ators play a role in the immune response. The samedichotomy applies to the abovementioned human auto-immune diseases and allergies (including asthma). Thus,Th1 cells are involved in the pathogenesis of MS and RA,whereas Th2 cells are important in asthma and otherallergic disorders. Th1 cells can be activated during infec-tions via activation of the innate immune system andlocal release of tissue antigens (i.e. bystander activation)or via similarities between an infectious organism andtissue autoantigens (i.e. molecular mimicry). For unknownreasons, in disorders of the immune system, these cells arethen either periodically or chronically activated, continueto infiltrate a specific tissue (i.e. the skin and joints inpsoriasis) and lead to tissue destruction and clinical dis-ability. In recent years, the molecular pathways that leadto Th1 and Th2 differentiation have been studied in greatdetail. IFN-g and IL-4 are the major T-cell-derived medi-ators in Th1 and Th2 pathways, respectively, but numer-ous other cytokines, chemokines and cellular componentsare involved in effector T-cell differentiation in vivo [6]. Tounderstand the role of IL-4 in the treatment of psoriasis,it is important to note that many of the factors thatare vital for Th1 differentiation antagonize Th2 functionand vice versa [6].

Treatments of psoriasis and other autoimmune diseases

Current treatments of psoriasis are shown in Table 1.Methotrexate, cyclosporine, retinoids or fumarate isadministered to provide general immunosuppression totreat severe forms of psoriasis [7]. Fumarate, whosemechanism of action includes a Th1 to Th2 switch, isused in Europe [7]. The lack of specificity and side-effects of these drugs in addition to improved understanding

of the immunopathogenesis of psoriasis have led to newtreatments, such as IL-10, IL-11, cytokine inhibitors orfusion molecules of cytokine receptors, which are alldesigned to block the cytokine cascade or induce T-cellanergy (Table 1) [8].

A recent report focused on the ‘master mediator’ in Th2responses, IL-4 [9], as a therapy for psoriasis, rather thanIL-10, IL-11 or any of several other cytokines that areinvolved in some aspects of Th2 immune function. The useof IL-4 as a treatment was based on knowledge of themolecular role of IL-4 in Th2 T-cell development [6]: (i) IL-4is the only cytokine that induces a Th2 bias in vitro; and(ii) IL-4 alleviates experimental Th1-mediated autoimmunediseases [10]. The treatment was very well tolerated inpatients and doses of 0.2 and 0.5 mg kg21 three times a dayappeared to be as efficacious or better than currently usedtreatments of the disorder. Within six weeks of treatmentall patients exhibited improvement based on the psoriasisarea and severity index (PASI) [9], with 95% of patientsimproving by .50% and 75% of patients improving by.68%. Six weeks following administration of IL-4 at dosesof 0.2 and 0.5 mg kg21 the expression of two cytokines thatare directly involved in psoriasis, IL-8 and IL-19, decreasedin psoriatic plaques, as did the number of CCR5-expressingTh1 cells and the ratio of IFN-g:IL-4, an important indicatorof the local expression of pro-inflammatory (Th1) versusanti-inflammatory (Th2) cytokines. Although it was expectedthat less profound changes would be observed in theperipheral blood, the study documented subtle increases inthe number of IL-4-, IL-10- and IFN-g-secreting T cells atdifferent points of the study. The elevated peripheralexpression of CD23, the low-affinity IgE receptor, on B cellsand monocytes not only served as a control for the patients’compliance with the treatment, but also provided an in vivoindicator for the biological effect of IL-4 (Table 2).

Potential problems and advantages of using pleiotropic

cytokines to treat autoimmune diseases

Cytokines are pleiotropic (i.e. they act on different celltypes and tissues) and are often redundant [i.e. theirfunction can be completely or partially replaced by othercytokines (e.g. IL-4) (Table 2)]. Although these character-istics are important for safeguarding the vital function ofthe immune system against failure, they also presentproblems, particularly when employing cytokines for thetherapy of infectious diseases, cancer or autoimmunediseases. In the case of infections it is easy to understandthat boosting Th2 responses might be beneficial for copingwith some parasites; however, boosting Th2 responsesmight also compromise our defense against intracellular

Table 1. Current and future treatments of psoriasisa,b

Current topical therapiesc Current systemic therapiesd Therapies in development

Steroids, dithranol, Tar, salicylic acid,

vitamin A (retinoids) and vitamin D

derivatives (calcipotriol, tacalcitol and

calcitriol), phototherapy

Retinoids, fumarate [16], PUVA,

methotrexate, cyclosporine

IL-10, IL-11 and IL-4 [17,18]; TNF-a

blocking agents, monoclonal

antibodies or fusion proteins

targeting CD2, CD11a, CD80, CD86,

IL-2 receptor or CD4

aData from [2,8].bAbbreviations: IL-4, interleukin 4; PUVA, psoralens combined with ultraviolet A irradiation; TNF-a, tumor necrosis factor a.cUsed to treat mild and localized forms of psoriasis.dUsed to treat generalized and severe forms of psoriasis (e.g. psoriatic arthritis).

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organisms such as Mycobacterium tuberculosis. In auto-immune diseases, the situation is usually more compli-cated. Even in so-called Th1 autoimmune diseases such asRA or MS, antibodies play an important role and areprobably crucial in the effector phase of the disease, atleast in MS. In experimental models of Th1 autoimmunedisease it is often difficult, if not impossible, to discern theroles of different cytokines because of their overlappingand pleiotropic functions. Thus, constitutive eliminationvia gene-targeted knockout is often not informative.Furthermore, the genetic ‘autoimmune background’ inthe outbred human population is very complex (i.e. mul-tiple genes contribute weakly and additively to disease)and results in substantial pathogenetic and clinicalheterogeneity. Finally, many of these autoimmune sus-ceptibility alleles are probably shared by several auto-immune diseases [11], and modulating the molecularpathway that appears most crucial in one autoimmunedisease might lead to the appearance of another, pre-viously silent, autoimmune condition. An interestingexample of the latter hypothesis comes from antagonizingthe pro-inflammatory mediator tumor necrosis factor a

(TNF-a) in MS. Although these approaches are highlyeffective in RA and psoriasis, attempts to block TNF-a inMS have not only been unsuccessful, but have appearedto worsen the disease [12]. Furthermore, cases of opticneuritis and MS have been observed following thetreatment of RA with TNF-a blocking agents. Althoughit is not clear whether these cases can be explained by theprevalence of RA and MS in the population and thelikelihood of simultaneous development of these disordersin some individuals, the experiences of using antagonistsof TNF to treat MS raise a serious note of caution.

It is clear that treating an autoimmune disease with acytokine or cytokine inhibitor is not as straightforward asone might anticipate. A sound understanding of the patho-genesis of the disease under study, extensive knowledge ofthe molecular and in vivo mechanism of action of therespective cytokine and, ideally, knowledge of the level ofheterogeneity of the human disease are all prerequisitesfor successful cytokine-based therapy. These prerequisitesare clearly not always met. For example, based on itsantiviral properties and the assumption that viruses playan important role in the etiology of MS, interferon b

(IFN-b) was originally tested in MS and became the firstapproved therapy for MS [13]. However, further

elucidation of the pathogenesis of MS now suggests thatIFN-b acts primarily at the blood–brain barrier andinhibits the migration of Th1 cells into the brain. Thedevelopment of therapies that antagonize TNF-a in RAhas been more straightforward. After the central role ofTNF-a in joint inflammation and destruction was estab-lished, the administration of agents that antagonize TNF-a became an effective therapy for RA [14]. Because TNF-aalso appeared to be an excellent target in MS, we do notunderstand why the abovementioned problems occurredwith TNF blocking agents in MS. It is possible that TNF-ais involved in oligodendrocyte differentiation and conse-quently these approaches interfere with tissue repair.

Conclusions and remaining questions

Although many aspects of psoriasis (e.g. the antigen-specificity of Th1 cells) need further research, the assump-tion of a pathogenetically relevant Th1 bias of the auto-reactive T cells in this disorder is well supported, and therecent clinical trial of IL-4 in psoriasis appears promising.The reported trial was very well designed because it notonly incorporated a dose-finding component and clinicalsafety and efficacy measures, but also monitored biologicalmarkers in both tissue and peripheral blood. The measure-ments of the biological markers supported the in vivomechanism of action of IL-4 and the suggested pathogen-esis of psoriasis. The absence of allergic reactions or signsof general immunosuppression or compromise, combinedwith the mechanistic findings, following administration ofIL-4 suggest that IL-4 should also be tested in several Th1autoimmune diseases (e.g. MS, RA and inflammatorybowel diseases). Clearly, longer treatment regimens andfollow-up will be needed to assess the safety and efficacyof such treatments conclusively and to better understandwhy IFN-g-secreting cells are at least transiently increasedin the periphery [15]. In addition, unexpected adverseevents might occur, as highlighted by the use of TNF-aantagonizing therapies in MS (compared with RA). Theperformance of well-designed clinical trials that examinethe mechanism of action and safety of an immunomodu-latory treatment in a specific autoimmune disease is theonly way to approach these questions.

References

1 Austin, L.M. et al. (1999) The majority of epidermal T cells in psoriasisvulgaris lesions can produce type 1 cytokines, interferon-g, inter-leukin-2, and tumor necrosis factor-a, defining TC1 (cytotoxic

Table 2. Biological effects of interleukin 4a

Actions on immune cellsb Actions on non-immune cells and tissues

Activation and growth of B cells Pro-fibrotic role in lung fibrosis [19]

IgG and IgE production Analgesic (anti-nociceptive) effects via inhibition of TNF-a and IL-1 [20]

MHC class II induction Increase of GABA-ergic phenotype in retinal ganglial cells [21]

Growth and survival of T cells; Th2 differentiation Enhancement of intestinal transepithelial permeability [22]

Inhibition of macrophage activation Fusion of myotubes with myoblasts [23]

Enhancement of mast cell growth –

Enhancement of IL-2- and IL-12-induced IFN-g secretion in NK cells –

Downregulation of C5a and C3a in monocytes and Mo-derived dendritic

cells

–

aAbbreviations: IFN-g, interferon g; Ig, immunoglobulin; IL-2, interleukin 2; MHC, major histocompatability complex; Mo, macrophage; NK cells, natural killer cells;

Th2, T-helper 2 cell; TNF-a, tumor necrosis factor a.bData from [5,6].

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T lymphocyte) and TH1 effector populations: A type 1 differentiationbias is also measured in circulating blood T cells in psoriatic patients.J. Invest. Dermatol. 113, 752–759

2 Christophers, E. and Mrowietz, U. (1999) Psoriasis. In Dermatologyin General Medicine (Fitzpatrick, T.B. et al., eds), pp. 495–521,McGraw-Hill

3 Nickoloff, B.J. and Wrone-Smith, T. (1999) Injection of pre-psoriaticskin with CD4 þ T cells induces psoriasis. Am. J. Pathol. 155,145–158

4 Walters, I.B. et al. (2003) Narrowband (312-nm) UV-B suppressesinterferon gamma and interleukin (IL) 12 and increases IL-4transcripts: differential regulation of cytokines at the single-celllevel. Arch. Dermatol. 139, 155–161

5 Szegedi, A. et al. (2003) Elevated rate of T helper 1 (T(H)1) lymphocytesand serum IFN-gamma levels in psoriatic patients. Immunol. Lett. 86,277–280

6 Agnello, D. et al. (2003) Cytokines and transcription factors thatregulate T helper cell differentiation: new players and new insights.J. Clin. Immunol. 23, 147–161

7 Mrowietz, U. et al. (1999) Treatment of severe psoriasis with fumaricacid esters: scientific background and guidelines for therapeutic use.Br. J. Dermatol. 141, 424–429

8 Asadullah, K. et al. (2002) Novel immunotherapies for psoriasis.Trends Immunol. 23, 47–53

9 Ghoreschi, K. et al. (2003) Interleukin-4 therapy of psoriasis inducesTh2 responses and improves human autoimmune disease. Nat. Med. 9,40–46

10 Rocken, M. et al. (1994) Antigen-specific activation, tolerization, andreactivation of the interleukin 4 pathway in vivo. J. Exp. Med. 179,1885–1893

11 Becker, K.G. et al. (1998) Clustering of non-major histocompatibilitycomplex susceptibility candidate loci in human autoimmune diseases.Proc. Natl. Acad. Sci. U.S.A. 95, 9979–9984

12 Lenercept Multiple Sclerosis Study Group and The University ofBritish Columbia MS/MRI Analysis Group, (1999) TNF neutralization

in MS: results of a randomized, placebo-controlled multicenter study.Neurology 53, 457–465

13 IFNB MultipleSclerosis Study Group, (1993) Interferon beta-1b iseffective in relapsing-remitting multiple sclerosis. I Clinical results ofa multicenter, randomized, double-blind, placebo-controlled trial.Neurology 43, 655–661

14 Feldmann, M. et al. (2001) Cytokine blockade in rheumatoid arthritis.Adv. Exp. Med. Biol. 490, 119–127

15 Hochrein, H. et al. (2000) Interleukin (IL)-4 is a major regulatorycytokine governing bioactive IL-12 production by mouse and humandendritic cells. J. Exp. Med. 192, 823–833

16 Litjens, N.H. et al. (2003) Beneficial effects of fumarate therapy inpsoriasis vulgaris patients coincide with downregulation of type 1cytokines. Br. J. Dermatol. 148, 444–451

17 Gotlieb, A.B. and Bos, J.D. (2002) Recombinantly engineered humanproteins: transforming the treatment of psoriasis. Clin. Immunol. 105,105–116

18 Weinberg, J.M. et al. (2002) Biologic therapy for psoriasis-the firstwave: infliximab, etanercept, efalizumab, and alefacept. J. DrugsDermatol. 1, 303–310

19 Roberts, E.M. et al. (2003) IL-4 expression delays eosinophil-independent vasculopathy and fibrosis during allograft rejection inthe mouse. J. Clin. Immunol. 23, 119–131

20 Schafer, M. (2003) Cytokines and peripheral analgesia. Adv. Exp. Med.Biol. 521, 40–50

21 Sholl-Franco, A. et al. (2002) IL-4 increases GABAergic phenotype inrat retinal cell cultures: involvement of muscarinic receptors andprotein kinase C. J. Neuroimmunol. 133, 20–29

22 Di Leo, V. et al. (2002) Factors regulating the effect of IL-4 on intestinalepithelial barrier function. Int. Arch. Allergy Immunol. 129, 219–227

23 Horsley, V. et al. (2003) IL-4 acts as a myoblast recruitment factorduring mammalian muscle growth. Cell 113, 483–494

0165-6147/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tips.2003.10.006

|Letters

Sparing animals and progressing in science

Amalia Guaitani, Stefania Filippeschi and Silvio Garattini

Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157 Milano, Italy

In previous reports [1–4], we have described the constantdecrease, beginning in 1978, of the use of laboratoryanimals at the Mario Negri Institute for PharmacologicalResearch of Milan (IRFMN), a non-profit organization thatofficially started its activity in 1963 with 22 researchers.

After reaching a peak number of 188 906 animals (rats,mice and rabbits) used in experiments by the IRFMN in1978, this number decreased to 24 669 in 1997 and hassince remained essentially unchanged (22 333 animals in2002) (Figure 1a). During 1988–1998 IRFMN staffnumbers increased by 27% and the number of publishedscientific articles increased by 52%, whereas the number ofanimals used decreased by 53%. In addition, the percen-tage of published articles that reported the results ofin vivo and/or ex vivo animal experiments decreased from32% (1988; total number of articles ¼ 269) to 16% (1998;total number of articles ¼ 409) of the total number ofarticles published by the IRFMN. These results can be

explained by various factors, including: (i) advancedtechnologies in all experimental procedures, which allowmeasurements in the same animal; (ii) improved appli-cation of statistics to calculate the minimum numberof animals necessary to obtain the required information;(iii) improved protocol planning; and (iv) increased use oftissue cultures from animals and humans. Furthermore,the IRFMN has expanded its research in areas that do notrequire the use of animals, such as clinical trials, studies ofepidemiology and disease prevention. However, it shouldbe emphasized that clinical trials cannot be performedunless other organizations have carried out the necessarypreclinical and toxicological studies, which require the useof several animal species.

During the past two decades, the voluntary introductionby the IRFMN of self-regulation policies via the AnimalCare and Use Committee has made a pivotal contributionto the reduction of animal use. The plateau reached in theuse of animals largely coincides with a stabilization of thescientific personnel, beginning in 1995, with ,500 peopleCorresponding author: Silvio Garattini (garattini@marionegri.it).

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