British Journal of Dermatology (1979) 100, 543.

Effect of 8-methoxypsoralen plus UVA (PUVA) on lymphocyte transformation and T cells in psoriatic patients JORMA E.FRAKI, JUSSI ESKOLA* AND VAINO K.HOPSU-HAVU Departments of Dermatology and *Medical Microbiology, University of Turku, SF-20520 Turku 52, Finland Accepted for publication 13 October 1978

SUMMARY

The peripheral blood lympochytes of the psoriatic patients studied initially showed a normal response to phytohaemagglutinin (PHA) and to purified protein derivative of tuberculin (PPD) in a microculture using whole blood, but a lower than normal response to a high concentration of concanavalin A (Con A). The initial level of E-rosette-forming T cells in psoriatic patients (51 3%) was significantly lower than that in healthy controls (66 7/,,). A single exposure to 8-methoxypsoralen (8-MOP) and UVA light (PUVA) did not suppress the lymphocyte responses to PHA, Con A or PPD, rather the responses showed a tendency to be slightly increased. Sitnilarly, no changes in the mitogen responses of peripheral blood lymphocytes were recorded during or after 12 weeks PUVA therapy for psoriasis. The lymphocytes still showed a weaker than normal response to a high concentration of Con A. However, the percentage of E-rosette-forming T cells in the peripheral blood increased from 51*3% to 62-8% after 12 weeks PUVA therapy. The low initial level of E-rosette-forming T cells was found to correlate more closely with the activity than with the extent of the disease. The increase found in the E-rosette-forming cells during PUVA therapy did not correlate with the improvement of the psoriatic lesions. The low response of peripheral blood lymphocytes of psoriatic patients to a high concentration of Con A correlated with the age of the patients but not with the activity or extent of the disease.

It is now apparent that there are abnormalities in the cell-mediated immunological mechanisms in psoriatics. A definite decrease in the E-rosette-forming lymphocytes has been reported by several investigators (Clot et al, 1975, 1978; Cormane, Hunyadi & Hamerlinck, 1977; Ghnski et al, 1978). Normal levels of E-rosette-forming cells have also been claimed (Levantine & Brostoff, 1975; Heid et al, 1976; Lischka, t976). The lymphocyte response to phytohaemagglutinin (PHA) in psoriatics Correspondence: Dr Jorma E. Fraki, Department of Dermatology University of Turku, SF-20520 Turku 52, Finland. 0007-0963/79/0500-0543 J02.00 © 1979 British Association of Dermatologists E

543

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J.E.FrakiJ.Eskola

and V.K.Hopsu-Havu

has been reported to be normal (Hopsu-Havu & Helander, 1975; Clot et al, 1978) but that to concanavalin A (Con A) abnormally low (Clot et al, 1978). It has been suggested that psoriasis therapy with 8-methoxypsoralen (8-MOP) and UVA light (PUVA) also affects the immunologically active cells beneath the hyperproliferative epidermis. Ortonne et al. (i977) claimed that PUVA treatment causes a decrease in circulating T ceils in psoriasis, a finding not confirmed by Glinski et al. (1978). Kraemer & Weinstein (1977) found a reduction in thymidine incorporation of leukocytes after PUVA treatment in psoriatic patients. Inhibition of mitogen-induced lymphocyte reactions by 8-MOP plus UVA in vitro has been reported by several investigators (Bohncrt et al, 1977; Lischka et al, 1977; Scherer, Kern & Braun-Falco, 1977; Kruger, Christophers & Schlaak, 1978). A depression of lymphocyte turnover (Wuif & W'ettermark, 1977) and of leukocyte chemotaxis (Langner & Christophers, 1977) after exposure to 8-MOP and UVA light in vitro, as well as an increase in chromosome aberrations (Swanbeck et al, 1975) and sister chromatid changes (Carter, Wolf & Schnedl, 1976; Wolf-Schreiner et al, 1977) have also been reported. In this work we have studied the effect of PUVA treatment in vivo on the responses of peripheral blood lymphocytes to mitogens PHA and Con A and purified protein derivative of tuberculin (PPD) as well as on the number of circulating T cells in psoriatic patients. MATERIAL AND METHODS

Patients Patients selected for the follow-up suffered from severe psoriasis without atopic or other diseases and they usually had no medications. The intensity of psoriaris was graded from 0 to 4 and the area of psoriatic lesions was measured and it is given as percentage of total body surface (see Table i). Agematched healthy persons were selected for the control group. PUVA treatment 8-MethoxypsoraIen (Puvalen tablets. Star, Tampere, Finland) was administered (06 mg/kg) 2 h I. The clinical data of the patients and the T cell percentages of the peripheral blood lymphocytes before and after 12 weeks PUVA therapy Patients

Psoriasis

No

Age

Sex

I

23

2

66 57 38

6

63

7 8

48

M F M M M F M M

54 35

M M

52

M F

3 4 5

9 10 II 12

Mean

31 26

33 438

Years 7

Treatment

T cells (•/.,

Activity

I/cm^

Result

2O

2

107

N.D.

30 30

4

35 35

2

97 68 94 89

Area ("„)

Before

7

25

2

93

3 3 3 4 4 4 4

19 II

10

I

112

4

61

20

I

76

68

5

20

156

15

25

3 3 4

100

3 3 3 4

25

104

35

51-2

II

9 4 7

2

2

30

3 3

2O

9-8

35

26-3

N.D.= not done.

180

75

28 40

48 39 60

66

61

N.D. 42

After 65 56 42 61

43 79 59 76 78 70 61

63 62-8

^-MOP and PUVA on lymphocyte transformation

545

before black light exposure with a PUVA-22 unit (Airam, Helsinki, Finland). The intensity of treatment was 9 mW/cm^ throughout the study. The scheme for PUVA treatment was usually as follows: 3 min three times during thefirstweek, 4 min three times during the second week, 6 min 2-3 times during the third week, 9 min twice during the fourth week, 12 min twice during the fifth week and thereafter 15-30 min 1-2 times per week. Variations of the presented scheme were allowed in some patients, depending on skin sensitivity to light and on the severity of psoriatic skin lesions. Neither systemic corticosteroids nor anti-metabolites were used during this therapy. Improvement of skin lesions was graded from 0 to 4. Blood samples To study the effect of a single PUVA treatment, blood samples were collected just before and immediately after a 15 min UVA light exposure (8-methoxypsoralen was taken 2 h earlier) into sterile tissue culture tubes (Falcon Plastics, Los Angeles, Ca., U.S.A.) containing preservative-free heparin (Medica, Helsinki, Finland) 50 u/ml of blood. In the follow-up study, blood samples were collected just before UVA exposure every second week. Additional samples were collected before and after the whole follow-up study (without any treatment or 8-methoxypsoralen on the same day). Emimeration of T lymphocytes Lymphocytes were isolated from heparinized blood by the Ficoll-Isopaque technique (Beyum, 1968). T lymphocytes were estimated by an E-rosette test according to Wybran, Chantler & Fudenberg (1973)Lymphocyte transformation Our routine method for lymphocyte stimulation with mitogens (Eskola et al, 1975) and PPD (Viljanen & Eskola, 1977) were used with slight modifications. In brief, a mixture of 25 /d of heparinized blood and 150 /d of RPMI 1640 was placed into each well of a microtiter plate. Thereafter, the mitogens and PPD in 25 /il of RPMI 1640 were added into the desired wells. Control cultures received 60r

60r

40

•10

20

20

125 PHA

625

0

_J 25 125 Con A Concentralion (;Ag /ml)

5

0

0-01

I PPD

100

FIGURE I. Lymphocyte transformation (mean + s.e.) induced by different concentrations of PHA, Con A and PPD in twelve psoriatics (•) and in sixteen healthy controls (•).

546

J E.Frdki, J.Eskola and V.K.Hopsu-Havu PHA

60

40

20

Ib)

Con A

icl

PPO

40

0"-

r

2 -

FIGURE 2, Lymphocyte transformation (mean±s.e.) induced by different concentrations of PHA (a), Con A (b) and PPD (c) in twelve psoriatics during i2 weeks PUVA therapy. The corresponding values of sixteen healthy controls are presented beside the ordinate. (a) PHA (//g/ml): • , 625; • , 125; _•, 25. (b) Con A (/(g/ml): «, 125; • , 25; • , 5. (c) PPD (//g/ml): • , 100; • , i ; , o-oi.

25 (il of plain medium. The final concentrations of PHA (PHA M, Difco Laboratories, Detroit, Mich., U.S.A.) were 625, 125 and 25 fig/ml, those of Con A (Pharmacia Fine Chemicals, Uppsala, Sweden) 125, 25 and 5 /(g/ml and those of PPD (Batch 288, Ministry of Agriculture, Fisheries & Food, Central Veterinary Laboratory, Weybridge, Surrey, England) 100, i and 0 01 /ig/ml. Eighteen hours

i-MOP and PUVA on lymphocyte transformation

547

before harvesting, 20/il of'^^I-labelled 5-iodo-2'-deoxyuridine ('^^IUdR) (025 /jCi, sp. act. 90-110 mCi/mg, The Radiochemical Centre, Amersham, England) was added to each well together with 5fluoro-2'-deoxyuridine (FUdR) (Asantila & Toivanen, 1974). The total incubation time for mitogenstimulated cultures was 90 h and that for PPD-stimulated cultures 114 h. The results are expressed as counts per min (ct/min). RESULTS

Findings in psoriatics without PUVA therapy Lymphocyte transformation induced by different concentrations of PHA and PPD was of the same magnitude in psoriatics and controls (Figs i and 2). The transformation induced by Con A was, however, a little lower in psoriatics than in controls with low concentrations of Con A but significantly lower at a high Con A concentration (125 //g/ml; P < o o i ; Fig. i). The same pattern can be seen in the PUVA-treated patients (Table 2). The percentage of T cells in psoriaties (51-3%) was significantly lower than that in healthy controls (667%) (P 0 05 when compared to the corresponding value before UVA.

548

J.E.ErdkiJ.Eskola

and V.K.Hopsu-Havu

were collected before UVA exposure and thus 2 h after intake of 8-MOP. The low response of psoriatic lymphocytes to 125 //g/ml of Con A was again verified (Fig. 2). The findings on T cells in blood samples collected before and after the 12 week PUVA therapy are given in Table i. A significant increase of T cell proportion was found during PUVA therapy (513% and 62-8";;, respectively; P < o o i , /-test for matched pairs). No changes were observed in the total number of leukocytes during PUVA therapy. During the therapy a marked improvement in the clinical state of the psoriasis occurred in the patients. No correlation between the improvement of the psoriatic lesions and the increase of the E-rosette-forming T cells was found in this group of ten patients. A weak correlation between the low T-cell percentage and the activity of psoriatic lesions was found in sixteen patients including these ten and an additional six psoriatics (r = 0434, P < o i ) , but not between the area of psoriatic lesions and low T-cell percentage. No correlation between the time that the patients had suffered from psoriasis or the age of patients and the T cells were found. Also no clear correlation between the activity or the area of psoriatic lesions and lymphocyte responses to a high Con A concentration {12$ /Jg/ml) was found in twelve patients studied. In contrast, a clear correlation between the low response of lymphocytes to 125 //g/ml of Con A and the age of twelve psoriatics was recorded (r = 0720, P< o-oi). No similar correlation was found in sixteen controls (r ^ 0130, P