ARTICLE
Cutaneous inflammation is an important aspect of the psoriatic lesion.
Already in pinpoint psoriatic lesions, in the peripheral zone of the margin
of psoriatic plaques, and during early relapse following discontinuation
of treatment, accumulation of T-lymphocytes, monocytes, mast cells and
polymorphonuclear leukocytes (PMN) can be observed [1]. No substantial
inflammatory changes have been seen in the symptomless skin distant from
the psoriatic lesion, although some authors claim increased density of
T-lymphocytes [2-4]. Until now, no consensus has been reached on which
cells are first in invading the "pre-psoriatic skin".
The invasion by polymorphonuclear leukocytes (PMN) in psoriatic skin
is an early feature. The epidermal accumulation of PMN adopts the psoriasis-specific
configurations of spongiform pustules of Kogoj in the stratum Malpighi,
and micro-abcesses of Munro as intracorneal accumulations. In pustular
psoriasis, PMN accumulation is the dominating feature [5, 6].
In peripheral blood, functional and biochemical characterisation has
revealed normal, decreased and increased expression of PMN activation
features [7]. The activity of the psoriatic process whether expanding
or stable proved to be an important factor for the characteristics of
peripheral blood PMN [8].
Integrin adhesion molecules are of great importance in intercellular
and cell-matrix interactions [9]. They consist of non-covalently linked
alpha- and ß-chains and are categorised by their ß-chain [10].
The Mac-1-integrin (CD11b/CD18) which is part of the ß2-subpopulation,
is known to be of particular importance to various PMN functions [11-13].
It is pivotal to PMN adhesion to vascular endothelium, extravasation,
tissue migration, the oxidative burst, and degranulation.
The Mac-1-receptor can be up-regulated by various biochemical compounds
such as formyl-Met-Leu-Phe, platelet activating factor, interleukin 8,
and leukotriene B4 [14-17]. To the best of our knowledge, there
are no known active down-modulators of Mac-1. In particular, the up-regulation
of CD11b by leukotriene B4 (LTB4) is of interest
since LTB4 is produced in large quantities in psoriatic lesional
skin [18-21].
LTB4 which is formed in the arachidonic acid cascade, is
a potent upregulator of CD11b. It causes a rapid increase in cell-surface
presence through qualitative and quantitative up-regulation [11, 22-24].
We chose to evaluate CD11b as it is far more specific for PMN than CD18,
which is also present on other leukocyte subsets.
In psoriatic skin, up to the most peripheral zone of the psoriatic lesion,
the number of CD11b-positive cells is increased compared to normal skin
[25], which suggests that the expression of CD11b by PMN is an important
factor in the pathogenesis of psoriasis. So far, no information is available
on CD11b expression by peripheral blood PMN of psoriatic patients.
A flow cytometrical study was performed in order to investigate whether
there are any systemic changes detectable in the basal levels of PMN CD11b
expression in patients with extensive plaque psoriasis compared to healthy
volunteers. Secondly, we assessed whether PMN from psoriatics respond
abnormally to ex vivo LTB4-stimulation, with respect
to CD11b-up-regulation.
Materials and methods
Subjects
Venous blood samples were obtained from 15 patients with extensive plaque
psoriasis. All systemic antipsoriatic treatments were stopped at least
three months prior to the investigation, and local antipsoriatic therapy
was stopped for at least two weeks. Fifteen healthy volunteers, without
any history or signs of skin disease, served as the control group. Subjects
were at least 20 years of age. Both groups were matched for gender and
age. No systemic, anti-inflammatory or immunomodulating drugs were allowed.
The severity of plaque psoriasis was assessed by the psoriasis area and
severity index (PASI). Patients were judged to have extensive plaque psoriasis
when PASI was greater than 10.0.
Unstable plaque psoriasis was defined as an increasing size of the individual
lesions during the two weeks preceding the study and/or the occurrence
of pinpoint papules around chronic plaques. In stable psoriasis, these
signs were not present.
CD11b integrin up-regulation assay [26]
Blood specimens for assessment of ex vivo neutrophil CD11b surface-expression
were obtained. Peripheral blood (4 ml) was collected by venepuncture,
kept in ethylene-di-amine-tetra-acetic-acid (EDTA) at 4° C and processed
within 3 h of collection to prevent non-specific up-regulation of CD11b-expression
as a result of neutrophil activation.
Blood samples were processed in triplicate using 90 µl aliquots
which were incubated with LTB4 (10 µl 1 x 10-7
M) in Hanks' balanced salt solution (Sigma Chemical Corp., St. Louis,
USA) containing 0.1% bovine serum albumin (HBSS-BSA), or with HBSS (10
µl) alone for 30 min at 37° C. Samples were then cooled and
incubated in the dark for 30 min at 4° C with 10 µl (0.045 g/l)
anti-human CD11b-fluorescein conjugate (Mo-1-FITC, Coulter Corp., Hialeah,
USA). Erythrocytes were lysed and the remaining cells were washed with
HBSS-BSA, fixed in 1% paraformaldehyde solution and stored at 4°
C until analysis.
The analysis was always performed within one week of preparation of
the leukocyte suspensions, because previous experiments showed that assessment
within 7 days minimises storage artefacts.
Flow cytometry analysis
All specimens were analysed on an Epics Elite Flow Cytometer (Coulter,
Luton, UK). Cells were excited with an air-cooled 488 nm argon laser set
at 15 mW. Green fluorescence (FITC) was measured through a 525 nm (band
width 30 nm) band pass filter. Calibration and sensitivity were checked
by using FITC-labelled beads (Standard-Brite, Coulter Source, Hialeah,
USA). Forward and side scatter were used for gating granulocytes only.
For each sample 5,000 gated cells were analysed (Fig.
1).
Statistical analysis
For comparison between different groups, the Mann-Whitney test was used.
Correlation between disease activity and CD11b-expression, and between
CD11b-expression before and after in vitro LTB4-stimulation
was calculated using the Pearson test.
Results
Psoriatic patients (N = 15) and healthy controls (N = 15) were matched
for gender and age. Table I
summarizes the age of patients and healthy controls as well as the severity
of psoriasis indicated by the PASI-score. Among the 15 patients with psoriasis,
8 patients had unstable psoriasis, characterised by aggravation of the
lesions during the two weeks preceding the study and pinpoint papules
around the chronic plaques.
The ages of patients and healthy controls were comparable. All patients
were suffering from severe plaque psoriasis as indicated by the PASI-score.
The extent of skin involvement did not differ between patients with unstable
and stable psoriasis.
The mean PMN CD11b expression, as measured by flow cytometry in psoriatic
patients and healthy volunteers, is summarized in Figure
2. The basal CD11b expression in the group of psoriatic patients
was 4.48 ± 0.72 (mean fluorescence units per cell ± SEM) and
in healthy volunteers 6.50 ± 0.84 (mean fluorescence units per cell
± SEM). The difference between psoriatic and normal, unstimulated
CD11b expression was not significant (p = 0.06), However, there seemed
to be a tendency towards a decreased CD11b expression in psoriatics compared
to controls.
The PMN CD11b-expression after in vitro stimulation with LTB4
showed a highly significant difference between the patient and control
group (p = 0.001). In the patient group, the presence of CD11b on the
cell surface of PMN was 10.7 ± 1.77 compared to 17.6 ± 1.13
in healthy volunteers. No relation was found between the individual severity
of psoriasis as expressed by PASI, and PMN CD11b-expression.
In order to compensate for the interindividual variation of CD11b expression,
the relative "CD11b up-regulatability" for every individual (PMN CD11b-expression
after LTB4-stimulation divided by CD11b-expression present
on unstimulated PMN) was calculated. Remarkably, this relative PMN CD11b
up-regulation in psoriatic patients did not differ from that found in
healthy volunteers (2.51 ± 0.23 and 3.03 ± 0.25 respectively,
p = 0.19). Subtraction of the basal level of PMN CD11b-expression from
the level that was reached after in vitro LTB4 up-regulation
revealed that the absolute LTB4-induced increase in PMN CD11b-surface
molecules was significantly lower in psoriatic patients compared to healthy
volunteers (6.26 ± 1.31 and 11.10 ± 0.69 respectively, p = 0.001).
No differences were observed between patients with stable and unstable
psoriasis with respect to basal levels of PMN CD11b-expression, and LTB4-stimulated
PMN CD11b expression. However, when comparing patients with unstable psoriasis
to healthy volunteers, the differences were more pronounced than they
were between the overall group of patients and healthy volunteers. Both
unstimulated and in vitro LTB4-stimulated PMN CD11b-expressions
were far lower in patients with unstable psoriasis (p = 0.02 and p <
0.001 respectively).
In order to assess whether the number of CD11b-molecules present on
unstimulated blood-PMN is of relevance to the total number of these molecules
present after in vitro up-regulation with LTB4, the
correlation between these two parameters was calculated in the psoriatic
patients and healthy volunteers (Fig.
3). There is a clear correlation between unstimulated and LTB4-stimulated
CD11b-expression in both groups (p = < 0.001).
Discussion
The present study showed a tendency to decreased expression of PMN CD11b
in psoriatic patients compared to healthy volunteers. After in vitro
stimulation of PMN by LTB4 there was a clear decrease in PMN
CD11b expression in psoriatic patients compared to healthy volunteers.
No correlation was observed between the severity of psoriasis as expressed
by the PASI-score, and PMN CD11b expression. Comparison of patients with
unstable psoriasis and healthy subjects accentuated the difference between
patients and healthy controls considerably. However, in contrast to the
difference with respect to absolute values, the relative CD11b up-regulation
was virtually identical in patients and healthy subjects.
Experimental variability of flow cytometrical CD11b assessment proved
to be below 10% comparing the triplicate measurements. Therefore, the
present investigation reconfirms the high experimental reproducibility
of the methods used.
In patients and healthy volunteers, the CD11b-levels on
peripheral blood PMN following stimulation with LTB4 correlate
with the basal levels of CD11b, as illustrated in Figure
3. Therefore, unstimulated PMN CD11b expression determines LTB4-induced
PMN CD11b expression. The correlation between unstimulated and stimulated
CD11b-levels in psoriatic patients and healthy controls seems to be comparable
since there is no difference in relative CD11b up-regulation between both
groups. This provides a strong indication that the signalling from LTB4-receptor
binding up to CD11b expression on peripheral blood PMN is essentially
normal in psoriasis.
The tendency to decreased basal expression of CD11b and the significantly
decreased LTB4-induced psoriatic PMN CD11b expression compared
to normal PMN indicates a decreased number of CD11b-hemireceptors on psoriatic
PMN. The decreased expression of CD11b on peripheral blood PMN, further
decreasing in unstable psoriasis, is in sharp contrast with the in
vitro chemotaxis and protease activity which increases in unstable
psoriasis [8].
Three explanations for the decreased CD11b expression of circulating
psoriatic PMN may be hypothesized: (1) compartmentalisation of PMN subpopulations;
(2) habituation to increased levels of leukotriene B4; (3)
active down-modulation of CD11b-levels on psoriatic peripheral blood PMN.
The hypothesis of compartmentalisation of circulating
blood PMN is based on PMN subset selection. It is probable that the PMN-subpopulation
with the highest CD11b expression is most likely to be recruited to invade
the inflammed psoriatic skin, whereas PMN with a more modest density of
CD11b remain in the blood circulation. Indeed, patients with unstable
psoriasis proved to have an even lower CD11b expression on peripheral
blood PMN than the overall group of psoriatic patients. In active psoriatic
lesions, PMN-influx has been reported in 78% of patients and in chronic
plaque lesions, PMN-influx proved to occur in 41% of patients [27].
An alternative explanation is habituation of peripheral blood PMN to
increased LTB4-levels present in psoriatic skin. This hypothesis
is supported by the observation that in psoriatic uninvolved skin a decreased
accumulation of PMN occurs following a standardized stimulus with LTB4
[28, 29]. Repeated LTB4 applications resulted in a decreased
PMN accumulation as compared to the response following a single application
[29]. However, in view of the fact that the relative CD11b up-regulation
by LTB4 in psoriatics proved to be essentially normal, this
hypothesis is not supported by the observations in the present study.
Active down-modulation of CD11b is another possible mechanism that could
explain the decreased PMN CD11b expression in psoriatic patients. It may
be possible that such a defence mechanism exists in order to prevent massive
cutaneous damage due to the abundant skin presence of PMN. To the best
of our knowledge there are no known active down-modulators of CD11b. However,
integrin alpha-units, like CD11b need divalent cations (calcium or magnesium)
for their adhesive functions, and receptor function can be rapidly modulated
through phosphorylation reactions [9]. It might well be possible that
CD11b can be down-modulated on a functional level by changes in cation-concentrations
and phosphorylase activity.
Since LTB4-induced signalling in psoriatic PMN is essentially
normal, the question arises as to what extent LTB4 is relevant
to the CD11b up-regulation of psoriatic PMN in vivo. The role of
LTB4 in psoriasis has been challenged further by the modest
effects of 5-lipoxygenase inhibitors in the treatment of psoriasis [30-33].
The decreased CD11b expression on peripheral blood PMN in psoriasis
remains an intriguing finding, and further studies should be aimed at
mediators and factors involved in PMN compartmentalisation in psoriasis,
and at mechanisms involved in the in vivo regulation of CD11b in
psoriatic patients.
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