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Circulating CLA ⁺ T cell subsets inversely correlate with disease severity and extension in acute psoriasis but not in chronic plaque psoriasis

European Journal of Dermatology. Volume 18, Number 6, 647-50, Novembre-Décembre 2008, Investigative report

DOI : 10.1684/ejd.2008.0513

Summary

Author(s) : Marta Ferran, Ana M Giménez-Arnau, Beatriz Bellosillo, Ramon M Pujol, Luis F Santamaria-Babi , Department of Dermatology, Department of Pathology, Hospital del Mar, IMAS-IMIM. Passeig Marítim 25-29, 08003 Barcelona, Spain.

Summary : Circulating CLA ⁺ T cells represent a subset of lymphocytes functionally associated to several cutaneous diseases. This population of peripheral lymphocytes is poorly characterized in acute stage psoriasis. We studied, by flow cytometry, the relationship between disease severity and extension and different subsets of circulating T cells in 31 psoriatic patients (7 guttate, 8 acute and 16 chronic psoriasis). An inverse correlation between circulating CLA ⁺ CD3 ⁺/CD4 ⁺ subsets and disease severity and extension was found in the acute form of psoriasis. Interestingly, we also observed that circulating CLA ⁺CD4 ⁺CD25 ⁺ cells inversely correlated with PASI and BSA in guttate patients, which had not been shown previously. These results may contribute to clarify the role of circulating T cells in psoriasis, especially in early stages of psoriasis.

Keywords : BSA, CLA, cutaneous lymphocyte-associated antigen, PASI, psoriasis

ARTICLE

Auteur(s) : Marta Ferran¹, Ana M Giménez-Arnau¹, Beatriz Bellosillo², Ramon M Pujol¹, Luis F Santamaria-Babi¹

¹Department of Dermatology,
²Department of Pathology, Hospital del Mar, IMAS-IMIM. Passeig Marítim 25-29, 08003 Barcelona, Spain

accepté le 23 Juillet 2008

Trafficking of circulating T lymphocytes to skin is not a random process. There is an array of molecules expressed by a subpopulation of circulating antigen-experienced T cells that allow them to home to cutaneous sites under normal and inflammatory conditions [1]. The cutaneous lymphocyte-associated antigen (CLA), which is considered a homing receptor for T cells with skin tropism, is a carbohydrate-modified P-selectin glycoprotein ligand-1 (PSGL1) which confers to memory T cells the potential of migrating to the skin. This antigen binds to the vascular selectins resulting in the initial steps of leukocyte recruitment to the skin [1]. Circulating CLA⁺ T cells represent a subset of lymphocytes functionally associated to different T cell mediated cutaneous diseases such as atopic dermatitis, contact dermatitis, vitiligo, drug allergy and viral infections [2]. Those lymphocytes respond to cutaneous antigens/allergens [3], and their presence and activation state appear to correlate with clinical symptoms [4, 5]. As in other T cell mediated skin diseases, in psoriasis, CLA⁺ T cells are present in circulating T cells and constitute most of T cells infiltrating lesions [6]. In psoriasis, early migration of circulating skin-homing T cells, together with other dendritic cell related mechanisms [7, 8] may be one of the mechanism by which psoriasis lesions develop. Since there are no studies addressing this question [9], we are interested in understanding the phenotype of those circulating lymphocytes in the acute stages of psoriasis and its relation with the severity of the lesions.

We have recently shown that, in acute psoriatic patients, in contrast to chronic psoriasis or controls, increased percentages of circulating activated (CD3⁺ and CD4⁺) T cells expressing CLA and HLA-DR markers are found. In those patients, the frequency of circulating CLA⁺ T cells directly correlated with PASI and BSA [4], suggesting a major role of those circulating lymphocytes in the acute rather than in the chronic stage of psoriasis. In addition, in chronic plaque psoriasis, a correlation between the percentage of circulating CLA⁺CD8⁺ T cells and the severity of the disease has been also detected [10].

To further characterize the role of circulating CLA⁺ T cells in acute and chronic psoriatic lesions, we have evaluated the correlation between the number of different circulating CLA⁺ T cell subsets with PASI and BSA in acute psoriasis.

Material and methods

Study population

Thirty-one adult patients with psoriasis (15 acute psoriasis vulgaris and 16 chronic psoriasis) were studied. All patients registered and included had previously signed an informed consent. Patients with erythroderma, pustular lesions or joint involvement were excluded from the study. Following a systematized protocol, two different groups of psoriasis vulgaris patients were defined: one group of patients presenting with “acute lesions” younger than six weeks, including 7 guttate psoriasis and 8 patients with a flare of a previous plaque psoriasis (15 patients); and a second group of patients presenting with “chronic lesions”, defined as chronic and stable psoriatic plaques persisting for more than six weeks (16 patients). The extension and clinical features of patients included in the study were variable. The size of a single lesion varied from few centimetres in diameter to large plaques involving extensive areas of the body. In all cases, PASI score estimation was performed, as previously reported. Mean PASI scores ranged from moderate to severe [10, 11] (PASI score from 5 to 60). The affected body surface area (BSA) was also calculated. Possible triggering factors were also recorded (streptoccocal infections, stress, etc.). Patients had been previously treated either with topical treatments (glucocorticosteroids, tars, anthralin, vitamin D3 analogues, tazarotene, bland emollients), physical treatments (photochemotherapy) or even systemic therapeutic approaches (acitretin, cyclosporine A, or methotrexate). In all patients, blood samples were obtained after a minimum blanching period (for any systemic or topical treatment) of 6 weeks.

Reagents and antibodies

Phosphate Buffered Saline (PBS) and Foetal Bovine Serum (FBS) were supplied from Cambrex (Verviers, Belgium). The HECA-452 monoclonal antibody, HECA-452 and rat IgM (HECA-452 isotype control) both conjugated to FITC, anti-rat IgM-FITC and mouse IgG2aκ PE-conjugated were from Pharmingen (San Diego, CA, USA). Antibodies as anti-CD3-PerCP, anti-CD4-PerCP, anti-CD25-PE, were supplied from Becton Dickinson (San Jose, CA). Finally, anti-CD3 PE-conjugated was obtained from Sigma (St. Louis, MO).

Cell staining and flow cytometry

Five-parameter analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson) installed with a power pack equipped with an argon laser of 488 nm wavelength. Fluorescence 1 was measured using a 530 nm band pass filter, fluorescence 2 with a 585 nm band pass filter and fluorescence 3 with a band pass filter of 650 nm. For cell staining, cold FACS buffer consisted in PBS with 2% FBS and 0.32 mg/mL sodium azide was used. Routinely, 1 × 10⁵ PBMCs were simultaneously stained in 100 μL volume of FACS buffer with HECA-452-FITC, anti-CD3-PerCP and eanti-CD25 conjugated to PE. The same staining was performed with anti-CD4-PerCP antibody instead of anti-CD3-PerCP. After an incubation period of 30 min on ice, cells were washed and fixed in PBS with 2% paraformaldehyde and stored at 4 °C until analysis. Specific staining was determined for each marker using appropriate isotype control antibodies. Cells were analyzed using CellQuest software from Becton Dickinson. During cell acquisition, peripheral blood lymphocytes were selected by their FS/SS properties and 25,000 events were acquired. Lymphocyte cell numbers were obtained by multiplying CD3 or CD4 total counts by the percentage of a given lymphocyte subset.

Statistical analysis

Correlations were calculated by the Pearson’s Correlation Coefficient.

Results

Correlation between different circulating lymphocyte subsets and PASI, in guttate psoriasis, acute plaque psoriasis and chronic stage psoriasis.

CD3⁺/CD3⁺CLA⁺: Within the circulating CD3⁺CLA⁺ subset of cells, an inverse correlation was found between the amount of those cells and the severity of the disease, in both guttate (r = –0.66 (p = 0.004)) and acute plaque psoriasis (r = –0.64 (p = 6 × 10^-4)), as shown in table 1. In contrast, no significant correlation was observed between the circulating CD3⁺ subset and PASI in any of the groups.

CD4⁺/CD4⁺CLA⁺: Similarly to former results, circulating CD4⁺CLA⁺ cells inversely correlated with PASI in guttate psoriasis (r = –0.70 (p = 0.003)) and acute plaque psoriasis (r = –0.60 (p = 0.001)), whereas no significant correlation was detected between circulating CD3⁺ cells and PASI (table 1).

Correlation between different circulating lymphocyte subsets of and BSA, in guttate psoriasis, acute plaque psoriasis and chronic stage psoriasis.

CD3⁺/CD3⁺CLA⁺: Regarding the circulating CD3⁺CLA⁺ subset of cells, an inverse correlation was found between those cells and the extent of the disease, only in the group of guttate psoriasis (r = –0.66 (p = 0.005)), which is reflected in table 2. No significant correlation was observed between the circulating CD3⁺ subset of any of the groups of psoriasis and BSA.

CD4⁺/CD4⁺CLA⁺: As in the former case, in guttate psoriasis, a significant inverse correlation was also observed between circulating CD4⁺CLA⁺ and BSA (r = –0.66 (p = 0.005)), which was not found for CD4⁺ cells (table 2).

Correlation between circulating CD4⁺/CD3⁺ CLA^+/– CD25⁺ cells and PASI/ BSA, in guttate psoriasis, acute plaque psoriasis and chronic stage psoriasis.

Guttate psoriasis showed an inverse correlation between the CD4⁺CLA⁺CD25⁺ subset and PASI and BSA (r = –0.81 (P = 0.01) and r = –0.79 (p = 0.002), respectively) (table 3). Interestingly, CD4⁺CLA^–CD25⁺ cells also correlated inversely with the PASI and BSA (r = –0.6 (P = 0.01) and r = –0.57 (p = 0.02), respectively) (data not shown). No significant correlations were found in the CD3 subset between CLA⁺ or CLA^– CD3⁺CD25⁺ cells and PASI or BSA, in any of the three groups of psoriatic patients.
Table 1 Correlation between different circulating lymphocyte subsets in various forms of psoriasis and PASIAn inverse correlation was found between counts of either CD3⁺ or CD4⁺ CLA⁺ subsets and the severity of the disease, in both guttate and acute plaque psoriasis

Psoriasis Type	PASI
CD3⁺	CD3⁺CLA⁺	CD4⁺	CD4⁺CLA⁺
Guttate	0.41 (0.04)	–0.66 (0.004)	0.42 (0.02)	–0.70 (0.003)
Acute	–0.40 (0.002)	–0.64 (0.0006)	-0.20 (0.01)	–0.60 (0.001)
Chronic	0.44 (2.6 × 10^-5)	–0.19 (0.0006)	0.26 (0.0004)	–0.21 (0.005)

Table 2 Correlation between different circulating lymphocyte subsets in various forms of psoriasis and BSAAn inverse correlation was found between counts of both CD3⁺ and CD4⁺ CLA⁺ subsets and the extent of the disease in the group of guttate psoriasis

Psoriasis Type	BSA
CD3⁺	CD3⁺CLA⁺	CD4⁺	CD4⁺CLA⁺
Guttate	0.38 (0.05)	–0.66 (0.005)	0.42 (0.002)	–0.66 (0.005)
Acute	–0.14 (0.008)	–0.45 (0.002)	–0.065 (0.03)	–0.39 (0.004)
Chronic	0.44 (0.0001)	–0.22 (0.002)	0.26 (0.001)	0.24 (0.01)

Table 3 Correlation between circulating CD4⁺CLA⁺CD25⁺ subset in various forms of psoriasis and PASI/BSA. An inverse correlation between the counts of CD4⁺CLA⁺CD25⁺ subset of cells and PASI and BSA in guttate psoriasis can be observed

Psoriasis type	CD4⁺CLA⁺CD25⁺
	PASI	BSA
Guttate	–0.81 (0.01)	–0.79 (0.002)
Acute	–0.49 (0.02)	–0.26 (0.07)
Chronic	–0.14 (0.01)	–0.11 (0.02)

Discussion

The results of this study indicate that in acute psoriasis (guttate and flares of plaque psoriasis), an inverse correlation between the numbers of circulating CLA⁺ CD3⁺/CD4⁺ and disease severity can be found. Conversely, we did not find any relevant correlation between circulating CLA⁺ T cells (CD3⁺ and CD4⁺) and PASI in chronic plaque psoriatic patients. Interestingly, a striking reduction in circulating CLA⁺ T cells has also been detected in other acute situations, for example after acute cutaneous psoriatic exacerbation under methotrexate treatment [13]. In nickel sensitive individuals developing a skin flare after oral nickel intake, a significant decrease in the percentage of circulating CD3⁺CD45R0⁺CLA⁺ cells has recently been described [14].

It is very interesting that we have not found relevant correlations between CD3⁺ and CD4⁺ regarding BSA or PASI. The reason is that CD3 and CD4 are too general cell markers and cannot reflect changes in minor subtypes of peripheral T cells with a cutaneous immune response in psoriasis. Conversely, evaluating CLA⁺ T cells, lymphocytes with cutaneous tropism are analyzed, providing relevant information on skin diseases with T-cell mediated mechanisms [2].

When analyzing circulating CLA⁺ T cells with BSA, we also found a significant inverse correlation between both CLA⁺CD3⁺ and CLA⁺CD4⁺ cells and BSA scores in guttate psoriasis. This relationship was less intense in acute plaque psoriasis cases. A possible hypothesis can be postulated to explain such a phenomenon: in guttate psoriasis, skin lesions develop synchronically and are widely distributed through the body, whereas in acute plaque psoriatic patients lesions co-exist with other with different stages, influencing the reduction in the circulating pool of skin-homing T cells. In addition, no relevant correlations were found for total CD3⁺, CD4⁺ cells and BSA or CLA⁺CD3⁺, CLA⁺CD4⁺ cells in chronic stage psoriasis.

Since CD25 identifies activated T cells, we further explored different subsets of circulating CLA⁺ T cells expressing this marker, and their correlation with PASI or BSA. Unexpectedly, we found a significant inverse correlation of circulating CLA⁺CD4⁺CD25⁺ cells with PASI and BSA in guttate patients. It has recently been shown that the skin-homing CLA⁺CD4⁺ T cell population contains a high proportion of CD4⁺CD25⁺ T reg cells [15, 16]. Although some studies have analyzed this population in chronic stage psoriasis [12, 16], this is the first data showing a correlation of the CLA⁺CD4⁺CD25⁺ subset with PASI and BSA in guttate psoriasis. Further studies are necessary to completely characterize such a population of potentially regulatory cells in guttate psoriasis.

Recently, it has been shown in some patients with guttate psoriasis that CLA⁺ T cells from tonsils and skin lesions express the same TCR, indicating a migration of those CLA⁺ from blood to skin cells to lesions [17, 18]. It is well known that psoriatic lesions express chemokines and cell adhesion molecules required for the migration of leukocytes from blood to skin [7, 8]. Based on these facts, one possible explanation for our results is that in acute forms of psoriasis, due to their migration to skin, the number of CLA⁺ T cells in the periphery decreases inversely to disease severity and extension. In fact, in patients with psoriasis induced by infection, a decrease in the percentage of circulating CD4⁺ cells expressing IL-6 receptor has been observed [19].

Our data may be in accordance with previous studies in psoriasis which show that in distant uninvolved skin, before epidermal hyperproliferation takes place, a significant infiltration of mononuclear cells [20, 21] and CLA⁺ memory T cells is observed [22, 23]. This suggests that in the initiation of the disease, when skin lesions are being induced, an extravasation of CLA⁺ T cells takes place towards cutaneous sites. This initial T cell driven phase would be followed by the involvement of innate immune system [8, 24]. Once T cells reach the skin, they interact with antigen presenting cells (i.e. dendritic cells), resulting in their activation and differentiation into Th17 lymphocytes. Th17 lymphocytes produce IL17/IL-22, cytokines which promote expansion and recruitment of innate immune response as well as affects keratinocytes function [25].

During transendothelial migration, CLA⁺ T cells require, among other molecular interactions, the LFA-1/ICAM-1 adhesion [26]. Anti-LFA-1 is one of the biological treatments for psoriasis that, besides improving psoriasis lesions, increases the number of peripheral lymphocytes [27]. A possible explanation for this fact is that by blocking migration from lymphocytes to skin their total number increases in blood. Biologicals directed to TNF-α are more effective in psoriasis, which could be explained by the fact that these new treatments are being used in patients with chronic psoriasis, where intrinsic factors in the psoriatic plaque (i.e. TNF-alpha) seem to play a more important role in the maintenance of the disease in comparison to T lymphocytes. Unfortunately, at the moment there is not much experience in the clinical benefit of using biological treatments that interfere with T cell migration in early stages of psoriasis, when migrating T cells may play a more important role. A recent work pointed out that anti-LFA-1 treatment may be beneficial for unstable psoriasis [28]. One possible explanation for the heterogeneity of response of different biological treatments in psoriasis could be that psoriasis is a multifactorial polygenic disease with variable involvement of driving forms [9].

In conclusion, our data indicate that in contrast to chronic psoriasis, in gutatte psoriasis there is an inverse relationship between PASI/BSA and circulating CLA⁺CD3⁺ and CLA⁺CD4⁺. Further observations of the immunological mechanism underlying the early stages of psoriasis are required to clarify the initial events taking place in psoriasis lesions.

Acknowledgments

Conflict of interests: none. Financial support: grant “Premio Fundación Salud 2000 (2003-2006)” from Serono, received by Ramon M. Pujol in Hospital del Mar.

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Circulating CLA + T cell subsets inversely correlate with disease severity and extension in acute psoriasis but not in chronic plaque psoriasis