ARTICLE
Auteur(s) : Xinhua Li1, Xing Fan2,
Kaiming
Zhang2, Guohua Yin2, Yufeng
Liu1
1Institute of Dermatology of Chinese PLA, Xijing
Hospital, Fourth Military Medical University, Xi’an 710032,
China
2Institute of Dermatology, Taiyuan City Centre Hospital,
Affiliated to Shanxi Medical University, Taiyuan City, Shanxi
Province 030009, P.R. China
accepté le 27 Avril 2007
Psoriasis is a common and enigmatic inflammatory skin disease of
multifactorial etiology. Changes in the epidermis are the most
striking feature of psoriasis. However, basic research in the last
decade has strongly promoted the understanding of the pathogenesis
of the disease [1]. T Lymphocytes and not keratinocytes are the
relevant driving force in psoriasis [2]. Pathogenic T lymphocytes
play a critical role by triggering the chain reaction of the
cellular and molecular networks in the formation of psoriatic
lesions [3]. The importance of T cells in the immunopathogenesis of
psoriasis was established with clinical therapy observation,
including a wide range of immunosuppressive agents,
immunomodulating drugs, fusion proteins blocking T cell activation
or anergizing T cells, relevant cytokine therapies, and biologics
inhibiting T cell migration [4-8]. In vivo, psoriatic T cells
injected intradermally into skin from normal persons or into
non-lesional skin from psoriatic patients transplanted onto severe
combined immunodeficient (SCID) mice, induced psoriatic lesion
conversion [9, 10]. In vitro, lesional and circulating T-cell
clones of psoriatic patients promote the proliferation of
keratinocytes through secretion of soluble factors, in particular
gamma-interferon (IFN-γ) and interleukin-8 (IL-8) [11-13].Evidence
accumulated that intraepidermal T cells, including CD4+ T
cells and CD8+ T cells, both of which are involved in the
immune reaction, were important in the formation of psoriatic
lesions. Immunohistochemical staining of biopsy specimens obtained
from psoriatic lesions show that CD4+ T cells are prevalent in
the dermis and CD8+ T cells are prevalent in the epidermis
[14]. The evolution of a psoriatic plaque appears to be dependent
initially on an influx of CD4+ T cells into the dermis and
epidermis, while the maintenance of plaque is dependent in part on
the presence of CD8+ T cells [15]. How do T cells play a role
in the hyperproliferation of keratinocytes and which T-cell subset
is central? Direct evidence on this line of thought is still
lacking. The aim of this study was therefore to investigate
keratinocytes C-Myc,Bcl-xL and Ki67 gene expression in responsible
to psoriatic CD4+ T and CD8+ T lymphocytes.
Materials and methods
Recruitment and clinical evaluation of patients
The fifteen patients with chronic plaque type psoriasis who were
enrolled in this study were undergoing in-patient treatment for
psoriasis and had not received systemic antipsoriatic treatment for
at least 8 weeks prior to biopsy (9 males and 6 females, age range
22-49 y). The patients were all in the progressive phase and their
mean psoriasis area and severity index (PASI) score at the time of
blood collection was 15.7 ± 7.26 (Mean ± SD). The normal control
group consisted of 20 normal human volunteers (13 males and 7
females, age range 25-52y). Five milliliters of heparinized
peripheral blood was obtained from each individual respectively
after written informed consent according to the guidelines of the
Medical Ethics Committee of Taiyuan City Centre Hospital.
Isolation of PBMC, T cells and T subsets
Peripheral blood mononuclear cells (PBMC) were purified from
peripheral blood from patients with psoriasis and normal volunteers
by density gradient centrifugation. Then total T cells, CD4+ T
cells and CD8+ T cells were isolated respectively from these
cells, using magnetic anti-human CD3 beads, magnetic anti-human CD4
beads, magnetic anti-human CD8 beads and an anti-idiotype antibody
(Dynal Biotech) according to the manufacturer’s instructions.
Purified cells were incubated on ice with anti-CD3-FITC,
anti-CD4-FITC, anti-CD8-FITC and an isotype negative control
antibody (BD Pharmingen) and evaluated by flow cytometry. Total
cells, (CD3+ cells), CD4+ cells and CD8+ cells isolated by
immunomagnetic bead methods, were all with > 95% purity.
Keratinocyte culture models
Normal human keratinocytes were isolated from skin obtained from
adult foreskin excision. Specimens were washed thoroughly with
ice-cold calcium-free minimal essential medium containing
penicillin-streptomycin 100 U, 100 μg per mL), then
washed thoroughly using sterile physiological saline buffer.
Subcutaneous fat and deep dermis were removed, and the remaining
tissue was cut to about 5 × 5 mm2 and incubated at
0.25% trypsin above two layers of gauze for 16 h at 4 °C.
Then the epidermis was separated from the dermis by forceps and
digested into single-keratinocytes by incubating in Hank’s liquid
with 0.25% trypsin at 37 °C for 10 min with frequent
agitation. Keratinocytes were cultured on glass coverslips in
24-well plates at 1 × 106/mL concentration with
2 mL RPMI-1640 media supplemented with 10% FBS, 100 IU/mL
penicillin and 100 μg/mL streptomycin. T cells, CD4+ T
cells and CD8+ T cells isolated from PBMC of psoriatic
patients or normal controls were added to the keratinocyte culture
system at 1 × 106/mL per well. After 72 h of
incubation, the supernatants in the co-culture system were
collected for IL-4, IL-8 and IFN-γ level assays by respective
ELISA. Then, T cells, which were nonadherent on the glass
coverslips or plastic culture plates, were removed by washing three
times with culture media. The remaining adherent keratinocytes
grown on glass coverslips were fixed in methanol: acetone (1:1)
solution.
Immunohistochemistry
The cells were rehydrated, blocked with 1% bovine serum albumin/5%
bovine serum, rinsed and incubated with mAbs against human C-myc,
Bcl-xL and Ki67 proteins (Santa Cruz, CA, USA). After washing, the
cells were incubated with first a biotinylated secondary antibody,
then HRP-conjugated streptavidin (Santa Cruz, CA, USA), and finally
diaminobenzidine substrate. The percentages of keratinocytes
positively stained for C-myc, Bcl-xL and Ki67 protein expression
were respectively evaluated with bright field microscopy
observations.
Statistical analysis
Data of assay were expressed as mean ± SD. Statistical analysis was
performed using Statistical Package for Social Sciences (SPSS 10.0
Inc, Chicago, IL, USA). Values of p < 0.05 were considered
statistically significant.
Results
Increased C-myc and Ki67 protein expression on keratinocytes
was observed in response to psoriatic peripheral T cells
An increase in the positive percentage of C-myc and Ki67 proteins,
but not Bcl-xL protein was present in keratinocytes in response to
psoriatic T cells compared with medium alone (figure 1A). This increase
was variable and ranged from 10% to 20%. The difference was
statistically significant for the group as a whole (P < 0.05).
In contrast, in the normal control group no increase of C-myc and
Ki67 protein expression on keratinocytes was observed. This
difference in C-myc and Ki67 protein expression, influenced by
psoriatic versus normal control T cells, was statistically
significant (P < 0.05).
An increase in the positive percentage of C-myc and Ki67 protein
was also detected in keratinocytes in response to both CD4+ T
cells and CD8+ T cells from psoriatic patients in comparison
to medium alone and CD4+ T cells or CD8+ T cells from
normal controls (figures
1B and C). Nevertheless, it should be noted that the
positive percentage of C-myc and Ki67 protein in response to
psoriatic CD4+ T cells significantly exceed that to
CD8+ T cells (figure 1D).
T cells, CD4+ T cells and CD8+ T cells overproduce
IL-8 and IFN-γ
Another hallmark was the mechanism of psoriatic T cells to
stimulate keratinocytes to overexpress C-myc and Ki67. Therefore,
IL-4, IL-8 and IFN-γ levels in the culture supernatant of the
keratinocytes co-cultured with T cells were assayed by ELISA. IL-8.
IFN-γ levels in the supernatant of the keratinocyte culture system
in the presence of psoriatic T cells were substantially higher (p
< 0.01) than of those in the presence of medium alone or normal
T cells. Interestingly, much more IL-8 and IFN-γ could be detected
in the supernatant of the culture system where keratinocytes were
cultured with psoriatic CD4+ T cells, compared with that with
CD8+ T cells. The difference was not significant for IL-4
production in the culture supernatant for either psoriatic T cells
or normal control T cells (figure 2).
Discussion
Recent research efforts have focused on the primary
T-lymphocyte-based immunopathogenesis of the disease, where the
fundamental phenomenon is the recruitment and activation of
preferentially type 1 T cells secreting type 1 (Th1) cytokines [12,
16]. Regardless of the presence of activation signals, circulating
blood T cells from psoriasis patients showed a type 1
differentiation bias which indicated an imbalance within the T cell
population [17]. In addition, intradermal injection of periphery
blood immunocytes (mainly T lymphocytes) from psoriasis patients,
but not normal persons, induced psoriasis in normal human skins
grafted on SCID mice under appropriate conditions [18].
Collectively, T cells are both functionally and numerically
impaired with a peculiar activation immunology in psoriasis. As a
result, they can not restrain a starting inflammation, and in fact
actively perpetuate the process. Once inflammation is initiated in
potentially psoriatic skin, these pathogenic T cells become
activate, enter the skin, and release cytokines and chemokines to
attract other immune cells to perpetuate the inflammatory cascade
in the epidermis, which is psoriasis.
A majority of studies show that psoriatic T lymphocytes play an
important role in the pathogenesis of psoriasis epidermal
hyperplasia, but the concrete mechanism is lacking. Keratinocyte
proliferation is a characteristic of psoriatic lesions and could be
marked by overexpression of growth-regulating proteins including
C-myc, Bcl-xL and Ki67 [19-21]. High expression of C-myc
accelerates hyperproliferation of keratinocytes and in some
circumstances induces cell apoptosis. Bcl-xL protein is a member of
the anti-apoptotic Bcl-2 family and is responsible for blocking
cell apoptosis and for keratinocyte survival. Ki67 is a sensitive
index reflecting the dynamic of epidermis hyperplasia and is
expressed in all cell cycle phases other than G0 in parallel with
proliferating cell nuclear antigen (PCNA) [22]. Ki67 can resist
apoptosis caused by C-myc but does not influence the hyperplasia
function of C-myc [20, 21]. In our experimental system of coculture
of foreskin keratinocytes with T cells, the T cells of psoriatic
origin induced keratinocytes to overexpress C-myc and Ki67
proteins, but not Bcl-xL,while the T cells of normal origin did
not. The study demonstrated that psoriatic peripheral blood T cells
could induce keratinocytes to proliferation by inducing
pro-proliferation proteins to over-express.
In psoriatic lesions, there is a complex cytokine network
consisting of elevated levels of IFN-γ, TNF-α, and several
interleukins including IL-1, IL-2, IL-6, IL-8, IL-12, IL-17, and
IL-19 [23]. The Th1-cytokine microenvironment is probably essential
for over-proliferation of the keratinocytes. A distinguishing
feature of circulating and lesional T cells of psoriatic patients
is their high capability for secreting TH1 cytokines in vitro [24,
25], whereas T cells usually function mainly via
cytokine-independent fashions. In the study, we detected
significantly higher levels of IFN-γ and IL-8, but not IL-4 in the
keratinocyte-psoriatic T cell culture supernatants. It seemed that
peripheral blood T cells from psoriatic patients provide a growth
stimulatory environment for keratinocytes via a
cytokine-independent fashion, induce overexpression of epidermal
proliferation regulation proteins and further fully promote
keratinocyte proliferation. T cell activation as a mediator of
psoriatic epidermis hyperplasia, plays an extremely important role
in the formation and maintenance of psoriatic lesions.
Previous studies have shown that CD4+ T cells preponderate
(64%-85%) in the early lesions of psoriasis, while CD8+ T
cells account for only 10%-32%. CD4+ T cells but not
CD8+ T cells derived from peripheral blood were capable of
converting PN skin engrafted onto SCID mice to PP skin after
intradermal injections [9, 10]. But recent evidence has shown
CD8+ T cells have even more important pathophysiological
functions in psoriasis, since a remarkable increase of CD8+ T
cells was detected in psoriatic lesions [26]. Using human skin-SCID
mouse, only T cells isolated from psoriatic lesions, mainly
CD8+ T cells by immunohistochemical staining, could keep the
pathology characteristics [27]. It indicated that the CD8+ T
population plays a key role in the maintenance of chronic psoriatic
lesions. The above mentioned results suggest the main function of
CD4+ T cells may lie in triggering the lesion, while
CD8+ T cells are essential to maintaining the pathological
changes of psoriasis. Furthermore, in this study, we demonstrated a
difference in keratinocytes in response to CD4+ T cells and
CD8+ T cells from psoriatic patients. CD4+ T cells can
secrete much higher levels of IL-8 and IFN-γ and induced much
stronger C-myc and Ki67 expression in keratinocytes, compared with
CD8+ T cells. Thus we can infer that both CD4+ T cells
and CD8+ T cells in psoriasis can induce keratinocyte
hyperproliferation, but CD4+ T cells might play a major role
in epidermis dysfunction and early lesion formation.
In conclusion, these findings demonstrate that psoriatic
peripheral blood T lymphocytes are remarkably different from T
lymphocytes of normal volunteers in their functioning. T cells and
the subpopulation of CD4+ T and CD8+ T cells in psoriasis
induce pro-proliferation proteins to express dysfunctionally,
probably in a cytokine-independent fashion, while CD4+ T cell
subpopulations have a more noticable influence on keratinocyte
hyperplasia. It further illustrates that T cells, especially
CD4+ T cells, play a vital role in the immunopathogenesis of
lesion formation in psoriasis, thereby contributing to a better
understanding of the pathogenesis of psoriasis. Furthermore, no
specificity for psoriasis has been shown in this study, as other
inflammatory skin diseases may also show similar effects of T
cells, e.g. modifying the phenotype, function and proliferation of
normal keratinocytes [28]. Once the molecular mechanisms of T cell
dysfunction are better delineated, immunomodulatory strategies by
manipulating T cells may eventually allow for improvement of the
disease in general.
Acknowledgments
The authors would like to acknowledge the support of Natural
Science Foundation of Shanxi Province of P.R China and all the
patients concerned for their voluntary participation in the present
study. There is no conflict of interest.
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