Clin. exp. Immunol. (1977) 27, 319-321.

Levamisole and human lymphocyte surface markers J. WYBRAN & A. GOVAERTS Department of Immunology, Hospital University St Pierre, Brussels (Received 13 August 1976)

SUMMARY

The action of various concentrations of levamisole on normal human lymphocytes was investigated in vitro. The action of levamisole (10 - 1 mg-10 8 mg%) upon rosette formation was studied using two rosette assays with SRBC (the active and the total T-rosette tests) to quantify T cells and the EAC assay to quantify B cells. Levamisole at only 10'- mg%/ significantly increased the percentage of both active and total T cells. In sharp contrast, levamisole between 10-2 and 10' mg% significantly decreased the EAC percentage. It is concluded that levamisole promotes a better expressivity of T-cell receptor and decreases the expressivity of C3 receptor.

INTRODUCTION Levamisole, an antihelmintic drug, has been shown to potentiate and restore both in vitro and in vivo immune responses in animals and man. Levamisole can influence the response to mitogens of normal human lymphocytes (Hadden et al., 1975; Sampson & Lui, 1976; Wybran & Govaerts, 1976). However, the augmentation in lymphocyte response is not always present, is usually not important and detectable only for a narrow concentration range of the drug. This probably explains some of the controversial results reported in the literature. In order to investigate in vitro another potential parameter, we looked at the expressivity of lymphocyte surface markers in presence of levamisole. We have used rosette assays between sheep red blood cells (SRBC) and T lymphocytes and the EAC rosetting assay for detecting B lymphocytes. The T-cell rosettes studied are the total T-cell test which investigates all human T cells and the active T-cell test detecting a subpopulation of these total T-cells (Wybran, Chantler & Fudenberg, 1973a; Wybran & Fudenberg, 1973b). MATERIALS AND METHODS Blood mononuclear cells were isolated over a Ficoll-Hypaque gradient, washed in Hanks's basic salt solution (HBSS) without magnesium and calcium. Monocytes were removed with carbonyl iron powder. The final concentration of lymphocytes was adjusted to 15-106 cells per ml in medium RPMI 1640 buffered with 25 mM HEPES (Gibco-Biocult). Pure levamisole powder was a gift of Janssen company (Beerse, Belgium). It was dissolved in RPMI 1640 with HEPES to a final concentration varying between 10-8 mg/100 ml and 1 mg/100 ml. A volume of 0-066 ml of lymphocytes (1.106 cells) was incubated for 1 hr at 370C in waterbath in presence of 0-1 ml of various concentrations of levamisole or in 0-1 ml of medium (control). Then, the cells were washed twice in HBSS and were tested for active T rosettes (TEa), total T rosettes (Tet) and EAC rosettes. The techniques for active and total T rosettes have been previously described. Briefly, for detecting the TEa (Wybran & Fudenberg, 1973c), lymphocytes were incubated for 1 hr at 37°C in 0 033 ml of foetal calf serum (FCS, Gibco-Biocult); then, SRBC in saline were added to obtain a final ratio of eight red cells for one lymphocytes. The tubes were centrifuged for 5 min at 200 g and resuspended. An aliquot of cells was taken, put in a haemocytometer and TEa were counted with a

light microscope. The TEt were determined as follows (Wybran et al., 1973a): to the lymphocytes were added 0 033 ml of FCS and 0 033 ml of SRBC (final ratio: 100 SRBC for one lymphocyte). The tubes are centrifuged for 5 min at 400 g and left vertically in a rack overnight at 4°C. The next day, after being gently resuspended, an aliquot was taken and TEt were counted.

Correspondence: Dr J. Wybran, Service d'Immunologie, H6pital Universitaire St Pierre, 322 rue Haute, 1000 Bruxelles. I

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EAC rosettes were determined as follows: SRBC were sensitized with subagglutinating doses of 19 S anti-SRBC antibody (Cordis, Miami) and with fresh human AB serum. After centrifugation, these EAC were incubated with lymphocytes (final ratio of fifty red cells for one white cell) for 30 min at 370C and vigorously resuspended with a Vortex. An aliquot of cells was taken and EAC rosettes were read. Statistical analysis was done using the Student's t-test.

RESULTS Ten normal subjects (between 19 and 62 years old) were investigated for TEa, TEt and EAC in the absence or presence of levamisole. The results are reported in Table 1. It can be seen that levamisole significantly increased the percentage of TEt only for the concentration of 10-3 mg/100 ml. The other concentrations had no effect on the percentage of TEt. The TEa percentage was also significantly increased in presence of 10-3 mg/100 ml of the drug; other concentrations were without effect. In sharp contrast, levamisole between 10' and 10- 2 mg/100 ml significantly decreased the percentage of EAC rosettes. TABLE 1. Effect of levamisole on human blood lymphocytes

Concentration (mg/100 ml)

1

10-1

10- 2

10-3

10- 4

10

10 6

10-7

10- 8

0

Total T lymphocytes (%) I = 10 Active T lymphocytes (0O) n = 10

58 0

57 0

59 9

63.4*

618

59-8

59 6

59 6

55 6

56 1

27.8

290

30 7

33.8*

32.7

32 5

29 4

28 3

25 8

26.8

EAC(00)

14.1

14.0

11.4*

11.3*

11.3*

13 9

16.1

n

9.8*

9.3*

9.6*

10 The results are expressed as the mean of duplicates results. n = Number of subjects tested. * P< 0 05 compared to controls.

DISCUSSION This study reports the in vitro influence of levamisole on B- and T-cell surface markers of blood normal human lymphocytes. It has previously been shown that levamisole can slightly enhance the lymphocyte response to phytohemagglutinin (PHA) (Hadden et al., 1975; Sampson & Lui, 1976; Wybran & Govaerts, 1976). One striking feature of these studies is the narrow range of dose in which this effect can be detected. Indeed, other authors and ourselves have reported that this action is measurable only for 10- 3 mg/100 ml which corresponds roughly to the enhancing concentration of 10'8 M reported by Hadden et al. (1975). Since the response to PHA mainly represents a T-cell function, it is not unexpected to observe an increase in the percentage of T-cell rosettes. The action was, like for PHA, restricted to the concentration of 10'- mg/100 ml. The active T-cell rosette represents a subpopulation of T-cells that closely reflects some immune functions whereas the total T test should detect all T-cells (Wybran & Fudenberg, 1973b; Felsburg, Edelman & Gilman, 1976). Since the absolute augmentation of both TEa and TEt was very similar (increment of 6%), it is suggested that levamisole has acted upon the same T-cell subpopulation. As we usually find 7000 of T-cells in the normal blood and here only about 60% of TEt, it is likely that the technical manipulations (incubations, washings) used in present experiments have slightly decreased the T-cell rosetting capacity in the TEt assay, which was thus suboptimal. TEa assay is always done in a suboptimal way (low SRBC-lymphocyte ratio and 37°C incubation) (Wybran & Fudenberg, 1973b; Chisholm & Tubergen, 1976). The interpretation of these results is highly speculative. The most likely hypothesis is that levamisole facilitates the expressivity of SRBC receptors and thus increases the percentage of T-cell rosettes when the rosette assay has been done in suboptimal conditions. The mechanism responsible for this action

Levamisole and human lymphocyte surface markers

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may be linked to cyclic nucleotides. Indeed, it is known that levamisole decreases lymphocyte cyclic AMP (Hadden et al., 1975) and that an increase in cyclic AMP diminishes the percentage of detectable T-cell rosettes (Galant & Remo, 1975). In the thymectomized mouse levamisole restores, in vivo, the azathioprine sensitivity of spontaneous rosette-forming-cells (a T-cell characteristic) suggesting, as in the present observations, an action on T-cell maturation (Van Ginckel & Hoebeke, 1976). The decrease in the percentage of EAC rosettes was unexpected. The significance and mechanisms of regulation of the C3 receptor are not known. In contrast to the T-cell rosette, the effect was observed over a wide range of concentrations of the drug. Whether this effect can be related to some loss of B-cell function is highly speculative. It is generally agreed that levamisole has no action on the humoral response. However, it has also been reported that levamisole, in some circumstances can depress the in vivo, response to SRBC in mouse (Renoux & Renoux, 1972) and that levamisole treatment of patients with rheumatoid arthritis is associated with a decrease in immunoglobulins (Szpilman et a!., 1976). Recently, levamisole has also been shown to be able to increase the receptors for IgG on human blood monocytes (Schreiber, Parson & Cooper, 1975). This observation may be relevant to the known enhancing effect of levamisole on macrophage function. In summary, levamisole can affect the surface markers of all human immune cells and it is proposed that such changes are directly related to the functional properties of the drug. The authors are very grateful to the excellent technical assistance of Miss Marie-Claire Delire. This work was supported by the Belgian Fonds de la Recherche Scientifique Medicale. REFERENCES CHISHOLM, R.L. & TUBERGEN, D.G. (1976) The significance Effect of levamisole in the human monocyte IgG receptor. of varying SRBC/lymphocyte ratio in T cell rosette Blood, 46, 1018. formation. 3. Immunol. 116, 1397. SZPILMAN, H., LUFT, S., GLINSKA-URBAN, D., FISCHER, W. FELsBURG, P.J., EDELMAN, R. & GILMAN, R.H. (1976) The & PLACHECKA, M. (1976) Levamisole and cell-mediated active E rosette test: correlation with delayed cutaneous immunity and serum-immunoglobulins on rheumatoid hypersensitivity. 3. Immunol. 116, 1110. arthritis. Lancet, ii, 208. GALANT, S.P. & REMo, R.A. (1975) fJ-Adrenergic inhibition VAN GINCKEL, R.F. & HOEBEKE (1976) Effects of levamisole of human T lymphocyte rosettes, 3. Immunol. 114, 512. on spontaneous rosette-forming cells in murine spleen. HADDEN, J.W., COFFEY, R.G., HADDEN, E.M., LOPEZEurop. J. Immunol. 6, 305. CORRALES, E. & SUNSHINE, G.H. (1975) Effects of WYBRAN, J., CHANTLER, S. & FUDENBERG, H.H. (1973a) levamisole and imidazole on lymphocyte proliferation Isolation of normal T cells in chronic lymphatic leuand cyclic nucleotide levels. Cell Immunol. 20, 98. kaemia. Lancet, i, 126. RENOuX, G. & RENoux, M. (1972) Antigenic competition WYBRAN, J. & FUDENBERG, H.H. (1973b) Thymus derived and non specific immunity after a rickettsial infection in rosette-forming cells. N. Engl. 5. Med. 288, 1072. mice: restoration of antibacterial immunity by phenyl- WYBRAN, J. & FUDENBERG, H.H. (1973c) Thymus-derived imidothiazole treatment. 3. Immunol. 109, 761. rosette forming cells in human disease state: cancer, SAMPSON, D. & Lui, A. (1976) The effect of levamisole on lymphoma, viral and bacterial infections, and other cell-mediated immunity and suppressor cell function. diseases. J. cdin. Invest. 52, 1026. Cancer Res. 36, 952. WYBRAN, J. & GOVAERTS, A. (1976) Levamisole and human SCMEIBER, A.D., PARSONS, F. & COOPER, R.A. (1975) lymphocyte cultures. Europ. J. Cancer (In press.)