The frequency of CLA+ CD8+ T cells in the blood of psoriasis patients correlates closely with the severity of their disease

Authors


Prof. Helgi Valdimarsson, Department of Immunology, Landspítali University Hospital, 101 Reykjavik, Iceland.  E-mail: helgiv@landspitali.is

Abstract

Psoriasis is thought to be a T cell-mediated skin disease and the cutaneous lymphocyte antigen (CLA) is an important skin homing epitope for T cells. We have studied the relationship between disease severity (PASI) and phenotypic analysis of T cells in the blood of 36 patients with psoriasis focusing on the expression of CLA, VLA-4 and CD25 on CD4+ and CD8+ T cells. The patients had a higher frequency of circulating CLA+ CD8+ cells than healthy controls. Furthermore, a much stronger correlation was observed between PASI and the frequency of CLA+ CD8+ than CLA+ CD4+ T cells. The frequency of CLA+D8+ T cells correlated more strongly with redness, thickness and scaling of the skin lesions than the total affected body surface area. In contrast to CLA the T cell expression of VLA-4 did not demonstrate any such correlation. Finally, the expression of the activation marker CD25 on CD8+ T cells showed a strong correlation with disease severity in patients with moderate to severe psoriasis (PASI > 10) but such correlation was not observed for CD4+ T cells. These findings support the notion that circulating CLA+ CD8+ T cells may play an important role in the pathogenesis of psoriasis.

Introduction

Psoriasis is a common chronic skin disease in which T cells are thought to play a key pathogenic role [1–3]. Cutaneous lymphocyte antigen (CLA) is a carbohydrate epitope that interacts with E-selectin [4] and is a product of fucosyl transferase VII dependent post-translational modification of the P-selectin glycoprotein ligand 1 (PSGL-1) [5,6]. In humans CLA is normally detected on less than 20% of peripheral blood T cells [7–9]. CLA positive cells are found in healthy skin [7] and most of skin-infiltrating T cells express this epitope [10] while T cells infiltrating other tissues are predominantly CLA negative [11]. It has therefore been postulated that CLA controls the influx of memory T cells to cutaneous sites [10,11]. Accordingly, it has been reported that T cells in psoriatic lesions express CLA, whereas T cells infiltrating arthritic joints did not [12]. This suggests that CLA may play a pathogenic role in psoriasis. It has recently been reported that injection of superantigen activated human T cells can induce psoriatic lesions in uninvolved skin transplanted on to SCID mice [13,14]. CLA expression has also been reported in this model [15]. Increased expression of the tissue non-specific adhesion molecule very late antigen VLA-4 has also been observed in psoriasis [16,17].

It has been reported that patients with active psoriasis have increased frequency of T cells expressing the alpha chain of the IL-2 receptor (CD25), a marker of activated T cells [18,19] and the level of serum CD25 has shown a moderate correlation with disease severity in psoriasis [20]. Other activation markers and immunological mediators in serum that have shown correlation with PASI in psoriasis include E-selectin [21–23], TNF-alpha [24] and neopterin [25].

It has been suggested that CD8+ T cells may play an important pathogenic role in psoriasis [13]. In this study we demonstrate that the frequency of CLA+ CD8+ T cells in the peripheral blood of psoriatic patients correlates strongly with disease severity as measured by the PASI score, and that this also applies for CD25+ CD8+ T cells and disease severity in patients with moderate to severe psoriasis (PASI > 10).

Materials and methods

Study population

Thirty-six patients with chronic plaque psoriasis were evaluated by the same physician for the Psoriatic Area and Severity Index (PASI) and the individual components of this score were also recorded separately. None of these patients had guttate type lesions at the time of the study. In the PASI scoring system [26], the area of psoriatic involvement of the head (H), trunk (T), upper limbs (U) and lower limbs (L) is given a numerical value from 0 to 6, and in each area erythema (E), infiltration (I) and desquamation (D) are assessed on a scale of 0–4. The PASI is calculated from the following formula: PASI = 0·1(EH + IH + DH)AH + 0·3(ET + IT + DT)AT + 0·2(EU + IU + DU)AU + 0·4(EL + IL*DL)AL. The total affected body surface area (TBS) was also calculated. The patients had not been on any treatment for at least 1 month prior to their recruitment to the study and none had any evidence of psoriatic arthritis. Blood samples were obtained but clinical assessment was done without knowledge of the results of the T cell analysis. Thirteen individuals with no history of psoriasis were included in the study as healthy controls. The study was approved by the Ethics Committee of the Landspítali University Hospital in Reykjavík, Iceland.

Isolation of peripheral mononuclear blood cells

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density gradient. The cells were washed twice and resuspended in RPMI 1640 (Gibco BRL, Life Technologies, Paisley, UK) supplemented with 2 mm glutamin (Gibco), 100 U/ml penicillin/100 µg streptomycin (Gibco) with 10% human AB serum (The Blood Bank, Landspítali University Hospital, Reykjavík, Iceland).

Antibodies

Monoclonal antibodies (MoAbs) to the following surface antigens conjugated with fluorescein isothyocyanate (FITC) or phycoerythrin (PE) were used: anti-CD25-PE, anti-CD4-FITC, anti-CD4-PE, anti-CD8-FITC, anti-CD8-PE anti-CD49d/VLA-4α-PE (Becton Dickinson, Oxford, UK) and anti-CLA-(HECA-452)-FITC (Pharmingen, Oxford, UK). FITC and PE-conjugated mouse IgG1 were used as isotype controls.

Double immunofluorescence staining and flow cytometry analysis

The PMBC (1 × 105) were double-stained with the FITC- and PE-conjugated monoclonal antibodies for 30 min on ice and washed twice in phosphate buffer saline (PBS) before fixation in 0·5% formalin. Stained cells were analysed by flow cytometry using a FACScan (Becton Dickinson). The data were analysed by Cell Quest software™ (Becton Dickinson).

Statistical analysis

Statistical significance was determined by Student's t-test or Mann–Whitney rank sum test. When equal variance was not observed correlations were determined by the Spearman's rank order correlation (r) using the SigmaStat 2·01 (Jandel Scientific, Erkrath, Germany). P-values below 0·05 were considered significant.

Results

Frequency of CLA+ CD8+ T-cells correlated strongly with PASI

There was no overall difference in the frequencies of CD4+ and CD8+ T cells in the peripheral blood of the psoriatic patients and the healthy controls. However, the patients had a significantly higher frequency of CLA+ CD8+ T cells compared to the healthy controls (P = 0·029), while the frequency of CLA+ CD4+ T cells was similar (Fig. 1). Furthermore, the PASI score correlated with the frequency of T cells expressing CLA (r = 459, P = 0·005) (Table 1, Fig. 2a) and this was due mainly to a strong correlation between PASI and CLA+ CD8+ T cells (r = 0·635, P < 0·001). This applied equally when the frequency of CLA+ T cells was expressed as a percentage of the total T cells or a percentage of CD8+ T cells (data not shown). As shown in Table 1 all the individual components that the PASI score is based upon showed a correlation with the frequency of CLA+ CD8+ T cells. However, it should be noted that this correlation was not as striking for the total affected body surface area as for the other components of the PASI score that reflect disease activity, namely the redness, thickness and scaling (Table 1). Again, these associations were much stronger for the CD8+ T cells. In contrast, no significant correlation was observed between any of the disease severity parameters and the adhesion molecule VLA-4, neither for CD4+ nor CD8+ T cells (Table 1).

Figure 1.

The frequency of CLA+ CD4+ (a) and CLA+ CD8+ (b) T cells in the blood of healthy controls and psoriatic patients. The frequencies are expressed as a percentage of the total T cells. Each box plot represents the mean/median and the 25th and 75th percentiles. The error bars represent the lowest and the highest values that are not outliers (outside the 5th/95th percentiles.

Table 1.  Spearman's correlation between T cell subsets in the blood and the total and individuals component of the PASI score and total affected body surface area (TBS)
 Total PASITBSRednessThicknessScaling
T cell subsetsrPrPrPrPrP
Total CLA+ T cells0·4590·0050·4000·0160·4120·0140·4570·0060·4790·004
CLA+ CD4+0·4020·0150·3820·0220·3300·0530·4020·0170·4080·015
CLA+ CD8+0·635< 0·0010·4710·0040·614< 0·0010·607< 0·0010·716< 0·001
Total VLA-4+ T cells0·4380·1430·4620·1230·2910·3690·0460·8810·1050·734
VLA-4+ CD4+0·4480·1360·4830·1050·3090·3390·0560·8600·0730·818
VLA-4+ CD8+0·1230·6830·3080·3170·3870·2210·3690·2450·3820·233
Figure 2.

Figure 2.

Correlation between disease severity and the expression of CLA+ T cells; (a) PASI versus total CLA+ T cells, (b) PASI versus CLA+ CD4+ T cells and (c) PASI versus CLA+ CD8+ T cells. The frequencies are expressed as a percentage of the total T cells.

The frequency of CD25+ CD8+ T-cells correlated with PASI in patients with moderate to severe psoriasis.

The T cells activation marker CD25 was also evaluated separately for the CD4+ and CD8+ T cell populations but there was no overall difference in the frequency of these subpopulations between the patients and the healthy controls (data not shown). Furthermore, the frequencies of the CD25+ subpopulations did not show a significant correlation with the PASI score when all the patients were analysed (data not shown). However, when those patients who had a relatively severe psoriasis (PASI > 10) were analysed separately, the frequency of circulating CD25+ CD8+ T cells correlated strongly (P = 0·007) with PASI, while such correlation was not observed for CD25+ CD4+ T cells (P = 0·178) (Fig. 3).

Figure 3.

Figure 3.

Correlations between disease severity and the expression of CD25+ CD4+ (filled circels) and CD25+ CD8+ (open circles) T cells in patients with moderate to severe psoriasis (PASI > 10). The frequencies are expressed as a percentage of the total T cells.

Discussion

In this study we demonstrate for the first time a strong correlation between the extent and severity of psoriasis (PASI) and the frequency of circulating CD8+ T cells expressing the skin homing epitope CLA. Furthermore, in patients with severe disease, the frequency of circulating CD25+ CD8+ T cells correlated with the PASI score while this did not apply to CD25+ CD4+ T cells. In contrast, the expression of the tissue non-specific adhesion molecule VLA-4 did not correlate with PASI. Increased frequency of CLA+ T cells in peripheral blood of psoriatic patients has been reported before [27], but in another study such an increase for CD4+ or CD8+ T cells was not detected [28]. However, that study involved only 10 patients and the findings were not analysed in relation to disease severity nor treatment. We have found that treatment can have a marked influence on CLA expression (manuscript in preparation).

CD8+ T-cells have been implicated in the pathogenesis of psoriasis because of the strong association with the HLA class I antigen Cw6 [29,30]. Furthermore, it has recently been suggested that CD8+ positive NK T cells may play an important role in the generation of psoriatic lesions [31,32]. Our findings are consistent with this possibility, although we have not analysed the CLA+ CD8+ T cells for NK markers. Thus, although CD4+ T-cells may be necessary for initiating psoriatic skin lesions [13], CD8+ T-cells could play an important role in relation to the disease activity in terms of redness, thickness and scaling of the skin lesions [33]. It should be noted in this context that the frequency of the CLA+ CD8+ T cells showed a stronger correlation with these parameters of disease activity than with the size of the area of affected skin (Table 1). Thus, interactions of relatively few CD4+ T cells with antigen-presenting cells could create the microenvironment required for recruiting CD8+ T cells to psoriatic lesions. This might explain why psoriasis can worsen dramatically in HIV infected individuals with relatively few circulating CD4+ T cells [34].

The frequency of CLA+ CD8+ T cells in the blood seems to reflect very closely the extent and severity of skin lesions in psoriatic patients and we are not aware of any laboratory parameter that is superior in this respect. It remains to be determined whether changes in the numbers of circulating CLA+ CD8+ T cells is a useful parameter for monitoring therapeutic trials for patients with psoriasis. Although the observed correlation could be an epiphenomenon, it is possible that the activity and severity of psoriasis is dependent upon constant recruitment to the skin of CD8+ T cells from the blood. This is also supported by the correlation observed between the PASI score and the frequency of CD25+ CD8+ T cells in the blood of patients with moderate to severe disease. Our findings clearly call for a simultaneous comparison of CLA+ T cells in the blood and psoriatic lesions.

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