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Review

Methotrexate in Psoriasis: From A to Z


Abdulla Saleh, Mohn’d Abuhilal, Bernard Cheung,* MBChB, MRCP (UK), MSc Derm

Address: Research Trainee, Division of Dermatology, Leeds General Infirmary, Leeds, UK; Senior Lecturer, Division
of Dermatology, Leeds General Infirmary, Leeds, UK
E-mail: bernard.cheung76@yahoo.com
* Corresponding Author: B. Cheung, Division of Dermatology, Leeds General Infirmary, Great George Street, Leeds,
UK, West Yorkshire, LS1 3EX

Published:
J Turk Acad Dermatol 2010; 4 (1): 04101r
This article is available from: http://www.jotad.org/2009/4/jtad04101r.pdf
Key Words: psoriasis, methotrexate, mechanism

Abstract

Background: For decades methotrexate has been used in the treatment of psoriasis with well-
established efficacy. Although many theories regarding methotrexate’s mechanism of action in
psoriasis were suggested, the exact the mechanism of action is still not very well understood. This
paper reviews the published literature on methotrexate mechanism of action in psoriasis. Articles
published with English abstracts between January 1970 and December 2008 identified in MEDLINE
were reviewed. It is likely that methotrexate interferes with the inflammatory pathways critical to
psoriasis pathogenesis by multiple mechanisms. Current evidence suggests that methotrexate, as
an immunomodulatory agent and anti-metabolite, decreases T-cell-mediated inflammation at
multiple steps and that explains its efficacy in treating various inflammatory diseases including
psoriasis.

Introduction effects have been ascribed to methotrexate in


both in vitro and in vivo studies, and it is be-
For more than three decades, methotrexate
coming more apparent that methotrexate
has been used in the treatment of psoriasis
may intercept several points in the inflamma-
with well-established record of efficacy. Even
with the era of novel biological agents, met- tory pathways that lead to psoriasis. This re-
hotrexate remains commonly used for the view was performed by searching MEDLINE
treatment of moderate to severe psoriasis. for articles published between 1970 and
Despite the long history, methotrexate’s mec- 2008 containing the keywords methotrexate,
hanism of action in psoriasis remains not en- psoriasis and mechanism. Studies investiga-
tirely clear. Many theories for methotrexate’s ting the mechanism of action of methotrexate
mechanism of action in psoriasis were sug- in rheumatoid and psoriatic arthritis were in-
gested. When the chemotherapeutic agent cluded. We therefore provide a review of cur-
methotrexate was found to be effective in rent knowledge on methotrexate mechanism
treating psoriasis, it was assumed that its ef- of action in the treatment of psoriasis.
ficacy lay in its cytotoxic effects on hyperp-
roliferating keratinocytes [1, 2]. However, as
Pathogenesis of Psoriasis
appreciation of psoriasis as a T-cell mediated
inflammatory process grew, interest in met- Psoriasis is a chronic inflammatory disease
hotrexate’s potential role as an immuno- characterized by T-cell-driven keratinocyte
suppressant has also increased. Several hyperproliferation and hypervascularity,
immunomodulatory and immunosuppressive which presents clinically as scaly, erythema-
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tous plaques.[3, 4] The current model of pso- Healthy cell membranes are impermeable to
riasis pathogenesis is that antigen-presenting trypan blue, while dying cell membranes are
cells (APCs) from the skin migrate to regional permeable, resulting in positive staining. Ne-
lymph nodes and activate T cells with an, as utral red will positively stain the nuclei of vi-
yet, unidentified antigen. This APC and T-cell able cells. The study reported similar
interaction depends on cell surface molecules amounts of staining between the two groups,
such as intercellular adhesion molecule-3 suggesting that methotrexate did not reduce
(ICAM-3). T-cell activation induces expression the viability of the human keratinocytes.
of surface molecules such as cutaneous Three years later, Jeffes et al provided evi-
lymphocyte associated antigen (CLA-1) and dence suggesting that lymphocytes in psoria-
leukocyte function-associated antigen-1 (LFA- tic lesions were significantly more vulnerable
1). CLA-1 binds endothelial leukocyte adhe- to methotrexate cytotoxic effects than kerati-
sion molecule 1 (E-selectin), a surface molecule nocyte and epithelial cells [6]. Proliferating
on activated endothelial cells that facilitates the lymphoid cells that included macrophages
migration of T cells to the inflammatory site via and T cells, normal human keratinocytes and
blood vessels. Once in close proximity to the epithelial cells, were cultured for 24 hours
inflammatory site, the T cells’ LFA-1 binds to with methotrexate and the number of killed
intercellular adhesion molecule-1 (ICAM-1), cells was counted. Methotrexate was emplo-
which is found on endothelial cells. This bin- yed at concentrations that would be achieved
ding facilitates T-cell adhesion to endothelial with once weekly low-dose methotrexate the-
cells and allows for subsequent T-cell diape- rapy. They reported that over 95% of the
desis from the intravascular space into the lymphoid cells were killed while less than
dermis. In the dermis, activated T cells me- 10% of the proliferating epidermal cells were
diate inflammation characterized by the pre- affected by this methotrexate exposure. In
dominant involvement of TH-1 cytokines. 1998, Heenen et al investigated the hypothe-
This inflammation is believed to drive kerati- sis that low-dose methotrexate triggers kera-
nocyte hyperproliferation. Each of these tinocyte apoptosis [7]. The keratinocyte model
steps represents potential sites of methotre- used consisted of normal skin epidermal exp-
xate action. lants cultivated for 10 days on dead de-epi-
dermized dermis. This model was incubated
with methotrexate 10-7 M to 10-6 M for 5
Effects on Keratinocytes
days. Cells exhibiting chromatin aggregation,
Methotrexate’s anti-metabolite activity initi- separation from neighboring cells, and apop-
ally led to theories that it worked by disrup- totic bodies under light microscopy were dee-
ting keratinocyte reproduction. Keratinocyte med apoptotic. The TUNEL assay was used to
hyperproliferation can be disrupted by indu- detect fragmented DNA and the presence of
cing cell death, inhibiting proliferation, or in- p53, a transcription factor that can activate
ducing maturation. Methotrexate’s role in the apoptosis pathway in the presence of se-
each of these processes has been investiga- vere cellular stress resulting from DNA da-
ted. Although keratinocyte hyperproliferation mage. Light microscopy revealed an increased
is a key feature of psoriasis, few studies have percentage of apoptotic cells in the basal
been published that explore methotrexate ef- layer of the methotrexate-treated skin versus
fects on keratinocytes. the control (1% ± 0.4 apoptotic in methotre-
xate treated versus 0.02% ± 0.01 apoptotic in
the control). In the methotrexate-treated cul-
Cell Death tures, TUNEL was positive in some basal and
In 1992, Schwartz et al explored methotre- para-basal cells. No controls were described
xate effects on keratinocyte cell viability [5]. for the TUNEL assay. Forty percent of the
The authors compared the levels of cell viabi- basal layer stained positive for p53 but no
lity between human neonatal foreskin kerati- controls were conducted for the p53 assay.
nocytes cultured with methotrexate 10-5 M Heenen et al also used TUNEL and histology
to 10-6 M for 72 hours and control kerati- to examine punch biopsies from four psoria-
nocytes not exposed to methotrexate. Neutral sis patients 24 hours after they completed an
red vital staining and the trypan blue exclu- eight-week course of 12 mg to 15 mg/week
sion assay were used to assess cell viability. methotrexate therapy. They reported three

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apoptotic cells per 1,000 germinative cells, ded that methotrexate did not inhibit the
but did not describe a control group. Based growth of normal human keratinocytes at
on the findings from both the model and bi- concentrations achieved with once-weekly
opsies, the authors of the study concluded methotrexate therapy. In a 2005 study pub-
that low-dose methotrexate induces apopto- lished by Yazici et al, 10 psoriasis patients
sis in human keratinocytes. provided lesional skin biopsies before and
after a six-week course of methotrexate 10 mg
to 25 mg/ week [9]. The biopsy specimens
Hyperproliferation were assessed by immunohistochemistry for
The most intuitive approach for discouraging expression of surface molecule proliferating
keratinocyte hyperproliferation would be to cell nuclear antigen (PCNA) and Ki-67. PCNA
inhibit keratinocyte reproduction. This app- and Ki-67 are nuclear proteins associated
roach was employed in several studies. In with proliferation and have been found to be
1992, Schwartz et al incubated healthy increased in psoriasis [10, 11]. They found
human keratinocytes with methotrexate 10- that lesions expressed lower levels of both Ki-
5 M to 10-7 M for 72 hours in thymidine-free 67 (p<0.01) and PCNA (p<0.01) following met-
media [5]. An electronic cell count of kerati- hotrexate treatment. This suggests that
nocytes revealed a 75% inhibition of growth methotrexate therapy may result in decrea-
in the methotrexate exposed cells compared sed hyperplasia within lesional skin.
to the controls, suggesting that methotrexate
had a growth inhibitory effect on the kerati-
Keratinocyte Maturation and
nocytes. The authors, however, note that
Differentiation
methotrexate’s effects on proliferation were
completely prevented by the addition of Inducing keratinocyte maturation and diffe-
thymidine, the nucleotide whose synthesis is rentiation may also potentially slow kerati-
prevented when methotrexate acts as a folate nocyte turnover. An in vitro study published
anti-metabolite. Based on these observations, by Schwartz et al in 1992 examined methot-
the authors concluded that methotrexate is rexate effects on keratinocyte proliferation
capable of inhibiting keratinocyte growth. and differentiation [5]. Human neonatal fo-
Furthermore, the mechanism by which kera- reskin keratinocytes were cultured with met-
tinocyte growth is inhibited appears to be hotrexate 10-5 M to 10-7 M for three to six
based upon methotrexate’s actions as an days. After 72 hours of exposure, an electro-
anti-metabolite. In 2003, Pol et al used a 96- nic cell counter detected a mean cell volume
wellplate assay system to investigate the an- increase of 225%, which suggested an in-
tiproliferative effects of several anti-psoriatic crease in cell size associated with kerati-
drugs, including methotrexate, on human ke- nocyte differentiation. Immunohistochemical
ratinocytes [8] Keratinocytes were exposed to staining for involucrin, a marker of terminal
methotrexate 10-5 M to 10-7 M and their pro- keratinocyte differentiation, was increased in
liferation over four days was assessed using the methotrexate-treated cells (1×105 met-
a sensitive DNA binding dye. Increased stai- hotrexate versus 5.5×104 control). Microsco-
ning is directly correlated to increased cell pic evaluation of these keratinocytes revealed
mass and indicates cell proliferation. Cytoto- larger, flatter cells with decreased nuclear-to-
xic effects were controlled for with lactate cytoplasm ratios, consistent with a more ma-
dehydrogenase surveillance. Lactate dehyd- ture stage of differentiation. At five days of
rogenase is normally an intracellular enzyme; methotrexate 10-6 M exposure, scintillation
detection of an increased release of lactate counts and fluorography showed a 2.0 to 2.3
dehydrogenase suggests a loss of cell viabi- increase in radioactive amino acid incorpora-
lity. Methotrexate exposure resulted in a 20% tion, suggesting an increase in protein
inhibition of keratinocyte cell growth on day synthesis. After six days of methotrexate 10-6
2, which was not statistically significant. The M exposure, a several-fold increase of inso-
study concluded that methotrexate did not luble protein staining, thought to represent
significantly affect proliferation at therapeu- cornified envelope protein, was observed. No
tically relevant concentrations. These results significant findings were reported for cultures
were similar to the earlier findings of the exposed to methotrexate concentrations less
1995 study by Jeffes et al, who also conclu- than 10-6 M. The authors concluded that,

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based on the morphological and biochemical methotrexate decreases endothelial expres-


findings, methotrexate induces maturation sion of E-selectin. Similar findings were re-
and terminal differentiation in keratinocytes. ported in the study of methotrexate effects on
endothelial activation markers in patients
with bullous pemphigoid. A decrease in
Methotrexate’s Effects on Endothelial ICAM-1 and E-selectin expression was found
Cells in patients with bullous pemphigoid treated
Downregulation of Adhesion Molecules with methotrexate [15].

T-cell migration from the intravascular space


into the dermis is a crucial step in the patho- Angiogenesis Inhibition
genesis of psoriasis, and this process is de- Histologically, hypervascularity is noted in
pendent on interactions between endothelial psoriatic skin, which contributes to the
cells and T cells. Endothelial expression of grossly observed erythema. When used at the
appropriate adhesion ligands such as E-se- high dosages necessary for chemotherapy,
lectin and ICAM-1, are necessary for success- methotrexate is capable of inhibiting angioge-
ful T-cell adhesion and migration [12, 13]. In nesis. When hypervascularity became appre-
2003, Yamasaki et al investigated how thera- ciated as a feature of psoriasis, it was natural
peutic concentrations of methotrexate (10-6 to to ask whether methotrexate exerted thera-
10-7 M) affected the expression of ICAM-1 on peutic effects in psoriasis by inhibiting angio-
human umbilical vein endothelial cells [14]. genesis. In 1989 Hirata et al performed in vivo
Immunohistochemistry revealed a decrease studies to examine whether methotrexate can
in ICAM-1 expression after exposure to 10-6 inhibit angiogenesis [16]. Methotrexate 5 ×
M of methotrexate. Furthermore, they found 10-9 M was injected intramuscularly into rab-
that endothelial cells exposed to methotrexate bits and the degree of corneal neovasculari-
expressed lower levels of ICAM-1 mRNA as zation was assessed. This early in vivo study
measured by PCR. Based on these results, it concluded that low-dose methotrexate inhi-
was concluded that methotrexate downregu- bits angiogenesis. In 2003 Yamasakai et al
lates ICAM-1 on endothelial cells, and may do examined methotrexate effects on endothelial
so by downregulating gene expression. The growth [14]. Human umbilical vein endothe-
following year, Sigmundsdottir et al studied lial cells were incubated with 10-8 M to 10-7 M
the effects of methotrexate on endothelial of methotrexate for three, six and eight days.
expression of E-selectin [13]. A psoriasis pa- Cells were stained with Alamar blue dye and
tient was treated with low-dose methotrexate the number of cells was determined by mea-
for five weeks. At days 4, 11 and 16 post-the- suring absorbance of cell culture media at
rapy, punch biopsies of the skin lesions and 590 nm. They found that methotrexate had a
immunohistochemistry were performed to de- dose-dependent inhibitory effect on human
tect E-selectin. Methotrexate was restarted, umbilical vein endothelial cell growth (culture
and two additional punch biopsies performed treated with methotrexate 10-7 M: 2 × 102 OD
on days 21 and 32 were also stained for E-se- at 590 nm versus control culture with no
lectin. Two observers blinded to the timing of methotrexate exposure: 5 × 102 OD at 590
the biopsies evaluated the immunohistoche- nm, p < 0.001). The authors concluded that
mistry independently. They found that clini- these findings indicated that methotrexate
cal exacerbation coincided with rising had an inhibitory effect on endothelial cell
E-selectin levels and CLA+ influx into the der- growth. Two years later, in 2005, Yazici et al
mis. Conversely, the resumption of methotre- employed immunohistochemistry on lesional
xate was followed by decreased E-selectin skin biopsies to study the effects of methot-
expression and decreased CLA+ T-cell infilt- rexate on angiogenesis [9]. CD31 is a com-
rate in the dermis. This chronologically cor- monly used endothelial marker and increased
related relationship between the level of levels suggest newly formed blood and
E-selectin and the subsequent number of in- lymphatic vessels [17, 18]. Ten psoriasis pa-
filtrating CLA+ lymphocytes demonstrated tients were treated with methotrexate 10 mg
the potential significance of E-selectin expres- to 25 mg per week. Biopsies were performed
sion in the psoriasis pathogenesis pathway. before and six weeks after treatment. All bi-
Overall, Sigmundsdottir et al concluded that opsies were stained with CD31 antibodies.

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The degree of CD31 staining was evaluated ability to stimulate lymphocyte proliferation
using an arbitrary four-point scale: (0) no after LC is pre-incubated with the agent. Pre-
staining, (1) weak staining limited to the pa- incubation for 24 hours with methotrexate
pillary endothelium, (2) moderate diffuse en- did not affect the LC’s capacity to stimulate
dothelial staining, and (3) severe diffuse lymphocytes; however, co-incubation of LC
endothelial staining. A statistically significant and peripheral lymphocytes with methotre-
reduction in CD31 staining (p < 0.05) was ob- xate completely inhibited the lymphocytes
served in the post-methotrexate skin biopsies. ability to respond to stimuli. In 1997, Liu et
The authors concluded that methotrexate dec- al tested methotrexate effects on LC immu-
reased the expression of the endothelial mar- nostimulatory effects and LC viability [20].
ker CD31, which suggests that methotrexate Mixed LC-lymphocyte reaction (MLCLR) was
inhibited the formation of new blood vessels. used to assess methotrexate effect on LC sti-
In the same year, Fiehn et al reported conflic- mulation. LC was incubated with methotre-
ting findings [18]. They studied methotrexate xate, then washed and re-incubated with
effects on angiogenesis using a human pla- peripheral blood lymphocytes. To assess LC
cental angiogenesis assay and a murine mat- viability, LC was incubated with methotrexate
rigel model. The human placental assay and subsequently stained with trypan blue.
exposed fresh placental tissue to 10 μg to 100 Methotrexate had a modest effect on the
μg of methotrexate and measured angiogene- MLCLR and no effect was seen above 1
sis by counting the number of new microves- μg/mL. The authors concluded that it was
sels. They found that 100 μg of methotrexate unlikely that methotrexate exerted its immu-
failed to inhibit angiogenesis in the human nomodulatory effects via LC suppression.
placental assay. The murine matrigel model Methotrexate showed no effect on LC viability,
is used to study anti-angiogenic drugs. A even at very high pharmacological levels (1
matrigel containing basic fibroblast growth mg/mL).
factor and heparin was injected intracutaneo-
usly into 24 mice. Twice weekly, 14 mice recei-
ved methotrexate 35 mg/kg intraperitoneally; Methotrexate’s Effects on T Cells
12 control mice received the placebo. The mice
Evidence supports that activated T cells are
were killed on day 10 and the matrigel matrix
key players in the immunopathogenesis of
removed for analysis. The matrigel matrix was
psoriasis. The following components involving
homo-genized and the hemoglobin content,
T cells are considered crucial in the pathoge-
which parallels the matrigel vessel content,
nesis of psoriasis: T-cell migration into lesio-
was measured. No significant difference in
nal tissue, the number of activated CLA+ T
matrigel hemoglobin content was found (1.61
cells in lesional tissue, and T-cell cytokine
± 1.59 g/L). Based on the findings from both
production. How methotrexate affects each of
models, Fiehn et al concluded that methotre-
these steps has been the focus of the studies
xate did not inhibit angiogenesis. They sug-
listed below.
gested that Hirata et al findings [16] may
have differed because the aqueous humor
may act as a compartment where methotre-
Downregulation of T-Cell Adhesion
xate can accumulate in high concentrations
Molecules
for prolonged periods.
In 2004, Sigmundsdottir et al studied methot-
rexate effects on peripheral T-cell CLA expres-
Antigen-Presenting Cells sion in 16 psoriasis patients who received
The stimulation of antigen-presenting cells in methotrexate 5 mg to 25 mg per week [13].
the skin, also known as dendritic cells or Blood samples were collected three to four
Langerhans cells (LC) may be a key upstream days after dosing; and isolated peripheral T
event in psoriasis pathogenesis. Modulation cells were stained with CLA-specific monoc-
of this process may significantly influence lonal antibodies (mAbs) and analyzed by flow
whether psoriatic lesions develop. A 1983 cytometry. A negative correlation (r = -0.505,
study by Morhenn et al at Stanford examined p = 0.046) between methotrexate dosage and
the inhibition of LC with various anti-psoria- the percentage of cells staining positive for
tic agents using the skin cell lymphocyte re- CLA among peripheral T cells was observed,
action [19]. This assay measures an agent suggesting that methotrexate reduced CLA
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expression in a dose-dependent manner. In T-Cell Cytolysis


another branch of this study, one psoriasis
Since the discovery of a positive correlation
patient treated with methotrexate 25 mg per
between the number of CLA+ T cells and di-
week underwent similar evaluation during
sease severity in psoriasis, interest has esca-
which blood was collected for five weeks. Pe- lated in methotrexate’s cytolytic effect on
ripheral T cells were isolated, stained and activated T cells. As previously discussed,
analyzed with flow cytometry for surface CLA. Jeffes et al suggested that methotrexate pos-
The percentage of peripheral T cells staining sessed substantial cytotoxic effects on
positive for CLA decreased in the first three lymphocytes, and that lymphocytes are 1,000
to four days following methotrexate adminis- times more sensitive to methotrexate than
tration, and then increased steadily until the epithelial cell lines. [6] Other studies have
next methotrexate dose. Methotrexate also confirmed T-cell sensitivity to methotrexate
decreased the amount of CLA expressed per [22], including a recent study by Herman et
T cell. Sigmundsdottir et al concluded that a al [23]. In addition to verifying methotrexate
dose-dependent, inverse relationship exists ability to induce T-cell death, Herman et al in-
between methotrexate administration and the vestigated the mechanism by which cell death
frequency and intensity of CLA expression on occurs. T cells were incubated with methot-
T cells in psoriasis patients. Methotrexate tre- rexate 10-5 M to 10-9 M for 24 hours prior to
atment resulted in fewer peripheral T cells assessment. They found that T cells stained
expressing CLA and fewer CLA molecules per positive for apoptotic cell markers at a rate
cell on the T cells that continued to express three times greater than controls (22.5 ± 1.5%
CLA. Johnston et al in 2005 investigated the versus 9.1 ± 0.7% respectively, p ≤ 0.05).
effects of low-dose methotrexate on the These findings suggest that methotrexate can
lymphocyte expression of several adhesion induce apoptosis in T cells. Methotrexate may
molecules, including CLA and ICAM-1 [21]. also induce cell death via free radical oxygen
Peripheral T cells were stimulated with strep- species. Phillips et al inhibited methotrexate
tococcal antigen and then incubated with induced T-cell death with the addition of the
methotrexate 10-9 M to 10-5 M for five days. antioxidant glutathione and its precursor, N-
Adhesion molecule expression was assessed acetylcysteine [22].
with immunohistochemical staining and flow
cytometry. They concluded that fewer T cells
expressed CLA or ICAM-1 following methot- T-Cell TNF-α Production
rexate incubation at concentrations greater Accumulating evidence suggests that methot-
than 10-7 M. Follow-up experiments revealed rexate’s anti-inflammatory qualities arise
that methotrexate suppression of CLA exp- from its effects on T-cell cytokine production,
ression could be reversed by folinic acid (leu- specifically, by reducing inflammatory cyto-
covorin) supplementation. Folinic acid is kine and increasing inhibitory cytokine pro-
commonly used to rescue cells from the anti- duction [24, 25, 26, 27]. As a key element in
metabolite effects of methotrexate by provi- psoriasis pathogenesis, the cytokine TNF-α is
ding an alternate route of thymidylate found at higher levels in psoriasis plaques
synthesis. Folinic acid supplementation sug- and the synovial fluid of patients with psoria-
gests that CLA suppression by methotrexate tic arthritis [27, 28, 29]. The clinical efficacy
occurs via a folate-dependent pathway. In a of newer anti-psoriatic drugs that target TNF-
2005 study published by Yazıcı et al, 10 pso- α, for example, etanercept, infliximab and
riasis patients provided lesional skin biopsies adalimumab, underscore the importance of
before and after a six-week regimen of met- TNF-α in psoriasis pathogenesis. Associations
hotrexate 10 mg to 25 mg per week [9.] The between methotrexate and TNF-α levels have
biopsy specimens were assessed for expres- been observed since the 1990s. A 1995 study
sion of surface molecule ICAM-3 by immuno- by Seitz et al found that psoriasis patients
histochemistry. ICAM-3 is associated with who clinically improved while on methotre-
T-cell and APC interactions. ICAM-3 expres- xate had reduced TNF-α production among
sion was evaluated using an arbitrary four- their peripheral blood mononuclear cells
point scale. The authors concluded that (PBMCs), comprised of monocytes, T cells and
methotrexate decreased the expression of B cells [27]. Two studies involving patients
ICAM-3 (p < 0.01). with rheumatoid arthritis reported reduced
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TNF-α levels in the synovial fluids of patients rexate required for inhibition varied between
undergoing methotrexate therapy [30, 31]. In donors: in the control group not exposed to
1999, two studies investigated the relations- methotrexate, TNF-α concentrations ranged
hip between methotrexate and TNF-α pro- from 470 pg/mL to 11,000 pg/mL. Due to
duction by activated T cells [26, 33]. In one the wide inter-individual variability, data
study, Neurath et al used mice to study met- from each donor was analyzed using his or
hotrexate effects on cytokine production by her own control trials. A statistically signifi-
splenic T cells [26]. Splenic T cells were iso- cant reduction in TNF-α production was ob-
lated from healthy mice and cultured. These
served at methotrexate concentrations
T cells were activated by stimulation with va-
greater than 8 ng/mL. At a methotrexate
rious commonly used antigens. The cultures
concentration of 1 μg/mL, TNF-α levels were
were exposed to methotrexate 10-7 M to 10-5
M. Serial ELISAs of the supernatant tracked suppressed by greater than 80% compared to
the production of TNF-α for 50 hours. They control culture concentrations. Interestingly,
found that methotrexate significantly redu- the addition of folinic acid or thymidine ab-
ced TNF-α production by activated T cells rogates methotrexate inhibitory effects on
(500 pg/mL TNF-α, methotrexatetreated TNF-α production. A more recent study by
group, versus 1,800 pg/mL TNF-α, control Lange et al echoed the findings of earlier stu-
group). The authors concluded that in T cells, dies, concluding that methotrexate does in-
methotrexate reduces TNF-α production in deed reduce TNF-α production by activated T
murine models, and suggested that TNF-α cells [34]. This study compared the levels of
modulation contributes to methotrexate’s ef- TNF-α, among others, in methotrexate-trea-
ficacy in rheumatoid arthritis. In another ted and untreated mice. Findings included a
study, Hildner et al used peripheral CD4+ T significant reduction in the amount of TNF-α
cells to study the relationship between met- produced.
hotrexate and TNF-α production [33]. They
harvested CD4+ T cells from the peripheral
blood of healthy volunteers. Primed T cells Discussion
were stimulated and cultured with IL-2 for
nine days in the presence or absence of met- Methotrexate is a folate anti-metabolite origi-
hotrexate (0.1 to 10.0 µg/mL). These primed nally introduced as a chemotherapeutic
T cells were then restimulated with two addi- agent; thus, early research regarding methot-
tional days of anti-CD3 mAb and IL-2 stimu- rexate mechanism of action focused on its
lation, along with methotrexate (0.1 to 10.0 cytotoxic capabilities. However, since its in-
µg/mL). The concentration of TNF-α in the troduction, methotrexate has been found ef-
supernatant was analyzed by ELISA. A sta- fective in the treatment of a variety of
tistically significant reduction of TNF-α pro- inflammatory diseases such as psoriasis,
duction was observed at all methotrexate rheumatoid arthritis and inflammatory bowel
dosages. At 0.1 µg/mL, TNF-α was reduced disease [35]. Recognition of methotrexate’s
to < 10% (p < 0.01) of the levels observed in therapeutic efficacy in inflammatory disor-
the control group. They concluded that met- ders redirected research efforts toward its po-
hotrexate can inhibit TNF-α production by tential role in immunomodulation. This
primed human T cells. In a follow-up study, review of available literature on methotrexate
Gerards et al compared the production of se-
mechanism of action in psoriasis therapy re-
veral cytokines by peripheral blood mono-
vealed that methotrexate does possess im-
nuclear cells collected from 20 healthy
munomodulating capabilities. Moreover, we
human volunteers [32]. Blood monocytes
were collected from healthy volunteers and found a lack of evidence for earlier but still
rheumatoid arthritis patients and stimulated commonly held beliefs that methotrexate pri-
with various antigens. The cells were cultu- marily acts by interfering with keratinocyte
red for four days with methotrexate concen- reproduction. The following is a discussion of
trations between 2 μg/mL and 2 ng/mL. what is known, what has been shown to be
ELISA was used to measure the amount of unlikely, and what remains to be substantia-
cytokine present in the culture supernatant. ted regarding the components underlying
They found that the concentration of methot- methotrexate efficacy in psoriasis treatment.
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ICAM-1 and E-Selectin Downregulation CLA and ICAM-3 Downregulation


Endothelium expression of adhesion molecu- Several studies provide clear evidence that
les on dermal vessels comprises a critical step methotrexate therapy reduces T-cell expres-
in the pathogenesis of psoriasis, as it facilita- sion of the adhesion molecules CLA and
tes T-cell localization to the inflammation ICAM-3. These molecules play pivotal roles in
site. Yamasaki et al provided in vitro evidence T-cell localization to inflammation sites, T-cell
of ICAM-1 downregulation in endothelial cells diapedesis to inflamed tissue, and interacti-
following methotrexate exposure [14]. Their ons with APCs, respectively. The 2004 study
research suggested that methotrexate did this by Sigmundsdottir et al offers clear in vivo
by suppressing gene expression. Concurrent evidence that methotrexate reduces the per-
immunohistochemical experiments demons- centage of peripheral T cells expressing CLA,
trated a significant reduction in the number and the intensity of CLA expression per cell.
of cell surface ICAM-1 molecules on endothe- Frequent blood draws on psoriasis patients
lial cells exposed to therapeutic concentrati- receiving methotrexate therapy provided data
ons of methotrexate. Taken together, these sufficient to reveal a clear pattern of decrea-
findings suggest that methotrexate effectively sing CLA+ T-cell counts in response to each
reduces endothelial cell surface expression of methotrexate administration. Furthermore,
this group demonstrated a statistically signi-
ICAM-1 by suppressing ICAM-1 gene expres-
ficant negative correlation between methotre-
sion.
xate dosage and CLA+ T-cell frequency, which
Another cell adhesion molecule elevated in provides further support of methotrexate ef-
psoriasis and correlated with disease severity fect on T-cell expression of CLA. Two other
is E-selectin. As previously mentioned, E-se- studies reflect these findings, also reporting
lectin allows T cells to enter the skin. Inhibi- a decrease in CLA+ T cells following methot-
tion of this step inhibits psoriasis [36, 37, rexate exposure. [9,40] There is also some evi-
38, 39]. Sigmundsdottir et al reported that dence of reduced ICAM-3 expression in
successive skin biopsies on a psoriatic pati- lesional skin following methotrexate therapy.
ent on low-dose methotrexate therapy revea- While the evidence presented by these studies
led a progressive decrease in E-selectin provide interesting and rather promising gro-
expression with continued methotrexate ad- und work in the topic of T-cell adhesion mo-
ministration [13]. This was followed by a mar- lecules, further studies are necessary for
ked reduction in the number of CLA+ corroboration.
leukocytes in the skin lesions. The reduction
in E-selectin and CLA+ leukocyte infiltrate co-
T-Cell Death
incided with clinical improvement. This fin-
ding reiterates the importance of E-selectin in In the 1990s, it was initially assumed that
the disease process. Although these results methotrexate achieved its therapeutic benefit
were derived from a single subject, they are by targeting keratinocytes; therefore, the dis-
compelling nonetheless due to the distinct covery that T cells were more vulnerable to
patterns and correlations between methotre- methotrexate cytotoxic effects were surpri-
xate administration, E-selectin levels, CLA+ sing. In a breakthrough study, Jeffes et al de-
leukocyte levels and clinical disease. Similar monstrated that T cells were more than 1,000
results of decreased adhesion molecules were times more sensitive to methotrexate cytoto-
seen in other disease models such as bullous xicity than epithelial cell lines [6]. Since then,
pemphigoid [15]. many studies have yielded similar results,
confirming that methotrexate can induce ac-
These studies provide promising preliminary tivated T-cell death [41, 42, 43, 44]. While it
evidence that methotrexate is capable of has been argued that methotrexate is unli-
downregulating endothelial expression of the kely to function via its cytotoxic effects since
cell adhesion molecules ICAM-1 and E-selec- methotrexate is used at much lower doses in
tin. Given the significance of ICAM-1 and E- psoriasis than in chemotherapy, these stu-
selectin to the pathogenesis of psoriasis, this dies employed methotrexate concentrations
likely represents a major mechanism by corresponding to the exposure achieved du-
which methotrexate exerts its clinical efficacy ring low-dose methotrexate therapy. It is li-
in psoriasis. kely that the activated T cells vulnerability to
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methotrexate toxicity allows for cytolysis at dual variability, the clinical significance of
lower doses than necessary for other cell this mechanism in the average patient rema-
types. Together with the T cells demonstrated ins unknown.
enhanced susceptibility to methotrexate, it is
probable that T-cell cytolysis contributes in
part to methotrexate efficacy in psoriasis. Keratinocyte Maturation and
Current studies are now focused on elucida- Differentiation
ting the mechanism of cytotoxicity. Work by Methotrexate might have a role in inducing
Herman et al suggested that apoptosis is the keratinocyte differentiation and maturation.
mechanism of cytotoxicity [23], while Phillips In Schartz et al, keratinocytes exposed to
et al concluded that the generation of radical methotrexate exhibited histological changes
oxygen species plays a role [22]. Regardless consistent with a more mature stage of deve-
of the mechanism by which methotrexate in- lopment. Furthermore, contrary to intuition,
duces T-cell death, T cells are undoubtedly protein synthesis increased after methotre-
key players in psoriasis pathogenesis, and xate exposure, producing insoluble protein,
the induction of T-cell death likely contribu- possibly representing cornified envelope pro-
tes significantly to methotrexate clinical effi- tein. The most compelling evidence, however,
cacy in psoriasis. was the increased intracellular staining of in-
volucrin, a marker of terminal keratinocyte
differentiation [5].
TNF−α Production by Activated T Cells
Accumulating evidence suggests that methot-
rexate alters T-cell production of several cyto- Keratinocyte Death
kines, including IL-1, IL-2, IL-4, IL-8, INF-γ Methotrexate is an anti-metabolite that binds
and TNF-α [25, 28, 32, 42]. Although these irreversibly to dihydrofolate reductase with a
cytokines have well-established clinical signi- greater affinity than folic acid. This binding
ficance in psoriasis, the discussion of each is prevents the de novo synthesis of the precur-
beyond the scope of this review, which focu- sor for the DNA nucleotide thymidine. Cells
ses on methotrexate effects on TNF-α. Over a are less likely to enter the synthesis or S-
decade ago, it was observed that psoriasis phase with a reduced availability of precur-
and rheumatoid arthritis patients improving sors; cells already in the S-phase die.
on methotrexate therapy had reduced con- Researchers initially studied methotrexate ef-
centrations of serum and synovial TNF-α [24, fects on keratinocytes because it was thought
30, 31]. This association was further elucida- that methotrexate exerted its greatest effect
ted by Gerard et al, who demonstrated that a there. A search for evidence of methotrexate
single oral administration of low-dose met- antiproliferative effect on keratinocytes revea-
hotrexate induced a significant drop in serum led few studies investigating this topic. Avai-
TNF-α levels in 2 hours [32]. Initial in vitro lable literature is inconclusive owing to the
studies, however, failed to demonstrate a signi- small number of studies and/or their poor
ficant effect of methotrexate on TNF-α produc- design. Results diverged widely, reporting no
tion, foreshadowing the complex relationship increased cell death, some effect, and signifi-
between TNF-α and psoriasis [24, 45, 46, 47, cant apoptosis. One study employing viability
48, 49]. The relationship between methotre- stains reported no difference in cell viability
xate and TNF-α depends on several factors in- between methotrexate-treated and untreated
cluding the stage and route of T-cell keratinocytes [5]. Another reported that less
activation, the presence of folinic acid and/or than 10% of proliferating keratinocytes were
thymidine, the duration of contact while in killed at concentrations achieved with once-
culture, and intrinsic differences between in- weekly low-dose methotrexate therapy [6]. A
dividuals [32, 33]. Recent studies considering study by Heenen et al concluded that low-dose
these factors have presented evidence that methotrexate induces significant apoptosis in
methotrexate inhibits T-cell TNF-α produc- keratinocytes [7]. This study, however, is limi-
tion [28, 32, 33]. Accumulating evidence sug- ted by its design: The TUNEL assay was em-
gests that methotrexate can reduce TNF-α ployed, which may not be a valid assay to
production by activated T cells; however, be- assess keratinocyte apoptosis because positive
cause TNF-α production exhibits interindivi- results also could be attributed to other cell
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states, such as increased cell turnover (i.e. limit keratinocyte hyperproliferation by inhi-
keratinocyte proliferation) [50, 51]. The study biting de novo nucleotide synthesis. Schwartz
was further limited by an absence of controls. et al concluded in 1992 that methotrexate in-
Together these factors limit the interpretabi- hibited proliferation of keratinocytes, but also
lity of the study results. It should be noted noted that these effects were completely pre-
that all of these studies used healthy kerati- vented by thymidine [5]. It can be argued that
nocytes. Keratinocytes within psoriatic pla- thymidine supplementation rescues these
ques have been shown to possess increased keratinocytes from cell cycle suspension by
resistance to apoptosis induction compared providing the desired DNA precursors. Re-
to healthy keratinocytes [51]. Because these gardless of the molecular explanation for this
studies used healthy keratinocytes, it rema- observation, a mechanism dependent on the
ins to be seen whether these findings are app- complete absence of thymidine is unlikely to
licable to psoriasis. Little conclusive data exert much clinical effect because thymidine
exists to support the common conception is present in vivo. Furthermore, the human
that methotrexate acts in psoriasis by indu- body can circumvent the de novo pathway
cing keratinocyte death. However, the idea via a salvage pathway to supply DNA precur-
that methotrexate acts in psoriasis by slowing sors. A 2003 in vitro study reported no sta-
skin turnover remains pervasive in lay litera- tistically significant difference in keratinocyte
ture. Additional studies are necessary to de- growth following methotrexate exposure [8].
finitively identify methotrexate effects on The study did not address the presence of
keratinocytes. thymidine or folate in the culture medium;
therefore, the findings cannot be compared to
Angiogenesis those obtained by Schwartz et al. Finally, a
Angiogenesis inhibition by methotrexate is recent ex vivo study reported a decrease in
still a relatively new area of research. Very few the markers associated with proliferation in
studies discussed this issue. Among available the skin biopsies of psoriasis patients who
studies, no agreement exists regarding met- had undergone methotrexate therapy [9]. The
hotrexate inhibitory effects on angiogenesis presence of these markers was measured by
during low-dose therapy. A 2003 in vitro immunohistochemistry and graded using an
study reported significant methotrexate inhi- arbitrary four-point scale. Each patient de-
bition of endothelial cell proliferation, sugges- monstrated marked disease improvement as
ting that methotrexate may prevent measured by the psoriasis area and severity
angiogenesis in vivo [14]. Two years later, an index (PASI) score. The finding of reduced
ex vivo study substantiated the earlier in vitro markers of proliferation may suggest that the
studies, reporting a statistically significant resolution of psoriasis is associated with re-
decrease in the endothelial marker CD31 duced proliferation, though not necessarily a
after treatment with methotrexate [9]. Unfor- direct result of methotrexate exposure. Re-
tunately, the authors did not state whether ports of clinical efficacy achieved with refor-
blinding was employed during the quantifica- mulated versions of topical methotrexate are
tion of CD31 staining. Conversely, that same emerging [52, 53]. The clinical efficacy of to-
year, a study employing two different angio- pical methotrexate may stimulate renewed in-
genesis assay systems reported that methot- terest in the effects of methotrexate on
rexate had no effect on angiogenesis [16]. keratinocytes. However, based on evidence
Results from both an in vitro human placen- available today, the inhibition of keratinocyte
tal model and an in vivo murine matrigel an- hyperproliferation has not proven to be met-
giogenesis assay showed no increase in blood hotrexate primary mechanism of action in
vessel formation following methotrexate expo- psoriasis.
sure. Based on currently available evidence,
it is still not known whether methotrexate in-
Antigen-Presenting Cell Inhibition
hibition of angiogenesis translates to clinical
efficacy during psoriasis therapy. The inhibition of a key upstream event in pso-
riasis pathogenesis would be an elegant mec-
Inhibiting Keratinocyte Proliferation
hanism of action. The published studies
Methotrexate beneficial effect on psoriasis is addressing methotrexate effect on Langer-
frequently ascribed to its presumed ability to hans cells (LCs) confirm that methotrexate
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has no effect on LC viability [19, 20]. Furt- rexate. J Invest Dermatol 1995; 104:183-188. PMID:
hermore, the studies implicate peripheral 7829873
lymphocytes as methotrexate target. Liu et al 7. Heenen M, Laporte M, Noel JC , de Graef C. Methot-
found only a modest reduction in LC’s immu- rexate induces apoptotic cell death in human kerati-
nocytes. Arch Dermatol Res 1998; 290:240-245.
nostimulatory capabilities after incubation PMID: 9681674
with methotrexate [20]. They concluded, ho-
8. Pol A, Bergers M, Schalkwijk J. Comparison of antip-
wever, that it was unlikely for methotrexate roliferative effects of experimental and established
to exert its efficacy via LC suppression due to antipsoriatic drugs on human keratinocytes, using a
the high levels of methotrexate required for simple 96-well-plate assay. In Vitro Cell Dev Biol
the modest response. 2003; 39:36-42. PMID: 12892525
9. Yazıcı AC , Türsen U, Apa DD, et al. The changes in
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Conclusion
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Methotrexate decreases T-cell-mediated in- 16215763
flammation at multiple steps. In the treat- 10. Hannuksela-Svahn A, Paakko P, Autio P, Reunala T,
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tein before and after PUVA treatment in psoriasis.
exert its effects by acting as both an immu-
Acta Derm Venereol 1999; 79:195-199. PMID:
nomodulatory agent and anti-metabolite [6, 10384915
23, 35, 37, 54]. Methotrexate’s multiple mec-
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