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JEADV ISSN 1468-3083 334 JEADV 2007, 21, 334– 339 © 2006 European Academy of Dermatology and Venereology Blackwell Publishing Ltd ORIGINAL ARTICLE The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis B Torres-Álvarez,* JP Castanedo-Cazares, C Fuentes-Ahumada, B Moncada Department of Dermatology, Hospital Central, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico Keywords CD69, cell adhesion molecules, methotrexate, psoriasis *Corresponding author, Bertha Torres-Álvarez, Dermatology Department, Hospital Central ‘Dr Ignacio Morones Prieto’, Av. Venustiano Carranza 2395, CP 78240, San Luis Potosí, S.L.P. Mexico, tel./fax +52 444 8 34 27 95; E-mail: [email protected] Received: 3 November 2005, accepted 20 February 2006 DOI: 10.1111/j.1468-3083.2006.01916.x Abstract Background The mechanism of the action of methotrexate (MTX) in the treatment of psoriasis has not been completely elucidated. Objective To assess the effect of MTX on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, activation molecule CD69 and T-cell phenotype in skin specimens from patients with psoriasis. Methods We performed an immunohistochemical analysis of the expression of T-cell phenotype and cell adhesion/activation molecules in skin biopsies from patients with psoriasis treated with a fixed dose of MTX (12.5 mg/week). To determine data on the epidermal/dermal T-cell infiltration we carried out a manual quantification. Results Skin samples prior to therapy showed a moderate to severe inflammatory infiltrate, mainly due to T lymphocytes with a helper/inducer (CD4) phenotype. Most of these cells also expressed ICAM-1 and VCAM-1. Blood vessels showed expression of E-selectin and VCAM-1, and keratinocytes were positive for ICAM-1 staining. The cell infiltrate was reduced after therapy, as well as the expression of cell adhesion molecules. However, we also noted the persistence of the T lymphocyte phenotype CD8 + , expressing the CD69 activation molecule, after the MTX treatment. Conclusions MTX downregulates the expression of some adhesion molecules, a phenomenon that may contribute to its anti-inflammatory therapeutic effect in psoriasis. The infiltrating T cells post-treatment have an activated cytotoxic phenotype, which may suggest a pathogenic role in the continuation and/or recurrence of psoriasis. Introduction The pathogenic mechanisms of psoriasis are still unclear. Current evidence strongly suggests that psoriasis is an immunologically mediated disease of the T-helper 1 (Th1)-type cell-mediated response. This is supported by recent evidence that reveals an early influx into lesions of activated T lymphocytes, and by the production of cytokines and growth factors in early lesions. 1,2 The importance of the T cells has been recognized because of the proven efficacy of immunosuppressive therapy such as anti-CD4 monoclonal antibodies, 3 diphtheria toxin-related interleukin-2 fusion protein (DAB389 IL-2), 4 cyclosporin A 5 and tacrolimus, 6 as well as the new biological agents. 7 The presence of T cells in skin with psoriasis is mediated by an upregulated expression of leucocyte adhesion mole- cules, such as endothelial leucocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), on the vascular endothelium and epidermal keratinocytes and by the production of inflammatory cytokines. 8,9 Whereas VCAM-1 and E-selectin are important in continuous traf- ficking, ICAM-1 strongly enhances the infiltration of T cells. 9 Methotrexate (MTX) is an effective treatment for

The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis

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Page 1: The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis

JEADV ISSN 1468-3083

334

JEADV

2007,

21

, 334–339 © 2006 European Academy of Dermatology and Venereology

Blackwell Publishing Ltd

ORIGINAL ARTICLE

The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis

B Torres-Álvarez,* JP Castanedo-Cazares, C Fuentes-Ahumada, B Moncada

Department of Dermatology, Hospital Central, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Mexico

Keywords

CD69, cell adhesion molecules, methotrexate,

psoriasis

*Corresponding author, Bertha Torres-Álvarez,

Dermatology Department, Hospital Central

‘Dr Ignacio Morones Prieto’, Av. Venustiano

Carranza 2395, CP 78240, San Luis Potosí,

S.L.P. Mexico, tel./fax +52 444 8 34 27 95;

E-mail: [email protected]

Received: 3 November 2005,

accepted 20 February 2006

DOI: 10.1111/j.1468-3083.2006.01916.x

Abstract

Background

The mechanism of the action of methotrexate (MTX) in thetreatment of psoriasis has not been completely elucidated.

Objective

To assess the effect of MTX on the expression of intercellular adhesionmolecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin,activation molecule CD69 and T-cell phenotype in skin specimens from patientswith psoriasis.

Methods

We performed an immunohistochemical analysis of the expressionof T-cell phenotype and cell adhesion/activation molecules in skin biopsies frompatients with psoriasis treated with a fixed dose of MTX (12.5 mg/week). Todetermine data on the epidermal/dermal T-cell infiltration we carried out amanual quantification.

Results

Skin samples prior to therapy showed a moderate to severe inflammatoryinfiltrate, mainly due to T lymphocytes with a helper/inducer (CD4) phenotype.Most of these cells also expressed ICAM-1 and VCAM-1. Blood vessels showedexpression of E-selectin and VCAM-1, and keratinocytes were positive forICAM-1 staining. The cell infiltrate was reduced after therapy, as well as theexpression of cell adhesion molecules. However, we also noted the persistenceof the T lymphocyte phenotype CD8

+

, expressing the CD69 activation molecule,after the MTX treatment.

Conclusions

MTX downregulates the expression of some adhesion molecules,a phenomenon that may contribute to its anti-inflammatory therapeutic effectin psoriasis. The infiltrating T cells post-treatment have an activated cytotoxicphenotype, which may suggest a pathogenic role in the continuation and/orrecurrence of psoriasis.

Introduction

The pathogenic mechanisms of psoriasis are still unclear.Current evidence strongly suggests that psoriasis is animmunologically mediated disease of the T-helper 1(Th1)-type cell-mediated response. This is supported byrecent evidence that reveals an early influx into lesions ofactivated T lymphocytes, and by the production of cytokinesand growth factors in early lesions.

1,2

The importance ofthe T cells has been recognized because of the provenefficacy of immunosuppressive therapy such as anti-CD4monoclonal antibodies,

3

diphtheria toxin-related interleukin-2

fusion protein (DAB389 IL-2),

4

cyclosporin A

5

and tacrolimus,

6

as well as the new biological agents.

7

The presence of T cells in skin with psoriasis is mediatedby an upregulated expression of leucocyte adhesion mole-cules, such as endothelial leucocyte adhesion molecule-1(ELAM-1), vascular cell adhesion molecule-1 (VCAM-1)and intercellular adhesion molecule-1 (ICAM-1), on thevascular endothelium and epidermal keratinocytes and bythe production of inflammatory cytokines.

8,9

WhereasVCAM-1 and E-selectin are important in continuous traf-ficking, ICAM-1 strongly enhances the infiltration ofT cells.

9

Methotrexate (MTX) is an effective treatment for

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psoriasis.

10

The precise mechanism of action of MTX onthis disease is unclear but it is known to inhibit DNAsynthesis by competing as a substrate for dihydrofolatereductase and acting on dividing epidermal cells.

11

Itseffect on proliferating lymphoid cells, as well as on theexpression of cutaneous lymphocyte antigen (CLA) andE-selectin, ICAM-3 and CD31, has been described.

12,13

There is also evidence that MTX affects T-cell activationand adhesion molecule expression

in vitro.

14

The CD69 molecule is an activation-inducer molecule,expressed by activated lymphocytes, that performs animportant role in activation, proliferation and differenti-ation of T cells as well as in the synthesis of cytokines suchas tumour necrosis factor-

α

(TNF-

α

).

15

CD69 is detected insmall subsets of T and B cells in peripheral lymphoidtissues from healthy subjects.

16

This molecule is persistentlyexpressed by CD8

+

infiltrating cells found in several chronicinflammatory diseases, such as chronic viral hepatitis,

17

rheumatoid arthritis

18

and halus naevus.

19

The functionalrole of CD69 expressed by T cells at inflammatory focushas not been described, but it has been suggested thatthese cells participate in the tissue damage observed inthese conditions.

17,19

Thus, the aim of this study was to assess the effect ofMTX on the expression of intercellular adhesion molecules,such as ICAM-1, VCAM-1, E-selectin and the activationmolecule CD69, and also the T-cell phenotype on skinspecimens from patients affected by psoriasis.

Subjects and methods

Patients

Sixteen consecutive patients (12 males and four females)diagnosed with psoriasis were included after informedconsent was obtained. Subjects with a history of alcoholism,hepatitis or renal disease were excluded. None of thepatients had been receiving treatment for 3 months beforeenrolment. Severity of psoriasis was evaluated usingthe psoriasis area and severity index (PASI) before andafter MTX treatment. A fixed dose of MTX 12.5 mg perweek (over a 36-h period) was administered for 8 weeks.Individuals were seen on a monthly basis throughout thestudy. Skin biopsies were taken from psoriasis lesions,prior to therapy and 4 days after the last MTX doseadministration.

Detection of cell adhesion molecules in tissues

The monoclonal antibodies (mAb) assessed were:anti-VCAM-1 (CD106) and ICAM-1 (CD54), CD4, CD8(BioSource International, Camarillo, CA, USA), activa-tion inducer molecule (AIM) (CD69; Ortho Diagnostics,

Raritan, NJ, USA) and anti-E-selectin (CD62E, 68-5H11,BD-Pharmingen, Woerden, the Netherlands).

Acetone-fixed frozen skin sections (4

µ

m thick) wereincubated with a blocking reagent (normal horse serum)diluted in phosphate-buffered saline solution (PBS) for20 min, washed and then incubated with the mAb for60 min in a humidity chamber at room temperature. Theskin sections were washed with PBS, incubated for 30 minwith a biotinylated secondary antibody and subsequentlytreated with the avidin-biotin-peroxidase complex (Vec-tostain, Vector Laboratories, Burlingame, CA, USA). Theslides were developed with 3-amino-ethyl-carbazole for5 min, washed with water, and counterstained with Mayer’shaematoxylin. The slides were evaluated by three separateinvestigators, and the results are expressed as positive ornegative for each mAb; when positive staining was found,the intensity of the expression of that molecule wasscored as weak (+), moderate (++) or strong (+++).

Quantification of the cellular infiltrate

Stained sections were counted manually by two independentblinded observers. Using a 0.5

×

0.5 mm ocular grid andat

×

200 magnification, the positive cells were counted inthe entire section. The epidermal and dermal regionswere counted separately. The results are expressed as thenumber of positive or double positive cells per millimetre(epidermis) or per millimetre squared (dermis).

Results

All of the patients completed the study and MTX was welltolerated without adverse effects. All patients had moderatepsoriasis with an average PASI score before treatmentof 26 (range 24–28), and 13 (range 5–19) at the end of the8 weeks of therapy.

We observed that clinical improvement was related tothe expression of the intercellular adhesion molecules;the results are summarized in Table 1.

In the pretreatment skin samples we found an expressionof ICAM-1 by keratinocytes (fig. 1a); a moderate to severeinflammatory infiltrate mainly due to T lymphocytes witha helper/inducer (CD4) phenotype, a few of them with aweak expression of CD69 and an upregulated expressionof VCAM-1 and ICAM-1. Endothelial cells showed increasedexpression of ICAM-1, VCAM-1 and E-selectin.

After 8 weeks of MTX therapy, the cell infiltrate and theexpression of the cell adhesion molecules were signific-antly reduced (fig. 1b and Table 2).

A gradual reduction in the number of CD8

+

T cells inthe epidermis and dermis was observed; however, in thedermis CD69

+

cells were also observed with a moderateexpression in almost all biopsies (fig. 2).

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69 in p

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Table 1

Expression of intercellular adhesion molecules before and after MTX treatment, correlated with PASI

Patient no.

Age/Gender

Weight (kg)

Onset

PASI

ICAM-1 VCAM-1 E-selectin CD8/69 CD4/69

Keratinocytes EndotheliumDermal lymphocytes

Dermal lymphocytes

Endothelial cells

Endothelial cells Epidermal Dermal Epidermal Dermal

1 45/M 71 28 +++ +++ +++ ++ ++ ++ – – – +2 40/F 68 28 +++ +++ +++ ++ ++ ++ – – + +3 46/M 78 27 +++ +++ +++ ++ ++ ++ – – – +4 39/F 55 24 +++ +++ +++ ++ ++ ++ – – – +5 53/M 67 24 +++ +++ +++ ++ ++ ++ – – + ++6 50/M 70 27 +++ +++ +++ ++ ++ ++ – – + +7 52/M 65 28 +++ +++ +++ ++ ++ ++ – – + ++8 50/M 68 27 +++ +++ +++ ++ ++ ++ – – + ++9 40/F 66 24 +++ +++ +++ ++ ++ ++ – – + ++10 38/M 67 26 +++ +++ +++ ++ ++ ++ – – + ++11 30/F 54 28 +++ +++ +++ ++ ++ ++ – – + ++12 60/M 63 24 +++ +++ +++ ++ ++ ++ – – + ++13 42/F 64 24 +++ +++ +++ ++ ++ ++ – – + ++14 38/M 70 28 +++ +++ +++ ++ ++ ++ – – + ++15 32/M 68 24 +++ +++ +++ ++ ++ ++ – – + ++16 39/F 67 24 +++ +++ +++ ++ ++ ++ – – + +

Patient no.

Age/Gender

Weight (kg)

After 8 weeks of MTX treatment

PASI

ICAM-1 VCAM-1 E-selectin CD8/69 CD4/69

Keratinocytes EndotheliumDermal lymphocytes

Dermal lymphocytes

Endothelial cells

Endothelial cells Epidermal Dermal Epidermal Dermal

1 45/M 71 15 + + + + + + ++ ++ – –2 40/F 68 13 + + + + + + ++ ++ – –3 46/M 78 17.5 + + + + + + ++ ++ – –4 39/F 55 7.6 + + + + + + + + – –5 53/M 67 12 + + + + + + ++ ++ – –6 50/M 70 12 + + + + + + ++ ++ – –7 52/M 65 19 + + + + + + ++ ++ – –8 50/M 68 12 + + + + + + ++ ++ – –9 40/F 66 12 + + + + + + + + – –10 38/M 67 15 + + + + + + ++ ++ – –11 30/F 54 16 + + + + + + ++ ++ – –12 60/M 63 12 + + + + + + ++ ++ – –13 42/F 64 16 + + + + + + ++ ++ – –14 38/M 70 15 + + + + + + ++ ++ – –15 32/M 68 12 + + + + + + ++ ++ – –16 39/F 67 5 + + + + + + ++ ++ – –

Results were graded as follows: –, undetectable; +, weak expression; ++, moderate expression; +++, strong expression.MTX, methotrexate; PASI, psoriasis activity and severity index.

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Discussion

The pathogenesis of psoriasis is currently thought toinvolve T lymphocytes, antigen-presenting cells, cytokinesand adhesion molecules.

1

MTX is an effective antipsoriaticagent that has been widely used to treat severe psoriasis.

11

The action of MTX in psoriasis has not been clarified, buta reduction in the expression of CLA and E-selectin

12

andalso the suppression of T-cell activation and T-cell adhesionmolecule expression

in vitro

have been described recently.

14

In this paper we report our study of the effect of MTXon the expression of adhesion and activation intercellularmolecules as well as T-cell phenotype on skin biopsies

from patients with psoriasis. In all pretreatment skinbiopsies we found a strong expression of ICAM-1 onkeratinocytes, a moderate to severe inflammatory infil-trate indicated by the presence of CD4

+

T cells, a few ofthem with a weak expression of the early activation mol-ecule CD69, and with a strong expression for ICAM-1,moderate for VCAM-1, and the expression of VCAM-1and E-selectin in blood vessels.

After therapy, we observed a marked clinical improve-ment, accompanied by a reduction in cell infiltrate as wellas in the expression of cell adhesion molecules. Keratino-cyte ICAM-1 expression tended to disappear accordingto the clinical response. However, despite the clinical

Table 2 Results of immunoperoxidase single and double staining of skin

biopsies at baseline and at the end of treatment. The data are expressed

as average numbers of positive cells per millimetre (epidermal cells) or per

millimetre squared (dermal cells)

Baseline Post-treatment biopsy

Epidermis

CD4+ 5 ± 3 3 ± 2CD8+ 18 ± 5 3 ± 1

CD4+CD69+ 2 ± 3 –

CD8+CD69+ – 4 ± 2Dermis

CD4+ 100 ± 40 58 ± 20

CD8+ 40 ± 25 25 ± 18

CD4+CD69+ 10 ± 20 –

CD8+CD69+ – 15 ± 8

fig. 1 Representative images of ICAM-1 immuno-

peroxidase staining in (a) lesional psoriatic skin at

baseline (original magnification × 200) and (b) a

residual lesion of psoriasis 4 days after the last

MTX dose administration (original magnification

× 200).

fig. 2 Double immunoperoxidase staining of biopsy 4 days after the last

MTX dose administration, showing the persistence of CD8+ CD69+ T cells in

the cellular infiltrate of the dermis (original magnification × 200).

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improvement and the reduction in inflammatory cellswith MTX treatment, we found the persistence of CD8

+

T cells expressing the CD69 molecule. The persistence ofCD8

+

cells in lesional plaque psoriasis after clobetasol-17propionate treatment has been described recently.

20

Ourfindings are in accordance with this observation and sup-port the hypothesis that CD8

+

T cells play a key role in thepathogenesis of psoriasis. The feature of T cells withthe phenotypes CD69

+

and CD8

+

, despite resolution of theskin lesions, may explain the rapid recurrence of psoriasison cessation of treatment or could suggest an importantrole for this molecule in immune reactivity and theinflammatory reaction. Thus, the role of CD69 expressedby T cells at inflammatory sites has not been elucidated,but in our case, they identified the persistently activatedT cells in psoriasis, as has been described by Ferenczi

et al

.

21

These CD69

+

T cells could signify a reactivation eventoccurring within the local microenvironment. Ishikawa

et al

. suggest that this expression could be due to an aner-gic state, characteristic of CD69 expressing lymphocytesin chronic inflammatory diseases.

22,23

More studies arenecessary to identify specific T-cell clones that possess apathogenic phenotype, as well as the specific interactionswith the CD8

+

T cells during treatment for psoriasis.To summarize, we can confirm that MTX has an anti-

inflammatory action in psoriasis through reduction of theactivation and adhesion molecules, and the expression ofCD69 suggests a role for this molecule in the inflamma-tory chronic reaction in psoriasis.

MTX has been considered as a cytotoxic drug but itsuse in a diverse range of inflammatory diseases, alongwith the findings shown here, as well as other author’sfindings,

14

indicates that MTX has an important anti-inflammatory effect, similar to that found in other drugsnot primarily developed as anti-inflammatories, such thalid-omide,

24

pentoxifylline

25

or rolipram.

26

It would be inter-esting to clarify in what proportion this anti-inflammatoryeffect is responsible for the positive response observed inpsoriasis considering the cytotoxic effect, keeping in mindthat other anti-inflammatory drugs without antiprolifer-ative effects may improve psoriasis, as is the case withbenoxaprofen

27

and meclofenamate.

28

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