Clin:. exp. Immunol. (1979) 35, 478-483.

The in vitro evaluation of certain neutrophil and lymphocyte functions following the ingestion of 150 mg oral dose of levamisole: assessment of the extent and duration of stimulation of neutrophil chemotaxis, protein iodination and lymphocyte transformation R. ANDERSON, ROBYN OOSTHUIZEN, ANNETTE THERON & A. J. VAN RENSBURG Immunology Section, Department of Microbiology, Institute of Pathology, University ofPretoria, South Africa

(Received 4 September 1978)

SUMMARY

Certain functions of human blood neutrophils and lymphocytes were investigated at varying time intervals after the ingestion of a single 150 mg dose of levamisole. The functions tested were neutrophil chemotaxis and post-phagocytic metabolic activity and mitogen-induced DNA and protein synthesis of lymphocytes. It was found that levamisole causes a stimulation of neutrophil motility (cell- and serum-associated) and post-phagocytic hexose monophosphate shunt activity and protein iodination. Increased lymphocyte DNA synthesis, but not protein synthesis, to the mitogen phytohaemagglutinin was observed. The stimulation which was detected almost immediately of these neutrophil and lymphocyte functions was still evident 24 hr later but not at 48 hr, indicating that a single oral dose of levamisole can cause the alteration (stimulation) of leucocyte functions which persists until 24-48 hr after intake of the drug.

INTRODUCTION Various functions of normal and abnormal phagocytic cells and lymphocytes have been reported to be enhanced by the anthelminthic drug levamisole (L-2,3,5,6-tetrahydro-6-phenylimidazo-(2, Ib)-thiazolehydrochloride) in vivo and in vitro. Stimulation of phagocytosis in vitro by normal (Molin & Stendahl, 1977) and abnormal (De Cree et al., 1974) neutrophils has been reported. Likewise, stimulation of macrophage phagocytosis in vivo (Hoebeke & Franchi, 1973) and in vitro (Lima et al., 1974) has also been described. Furthermore, stimulation of neutrophil post-phagocytic metabolism by levamisole has been reported by Wright, Kirkpatrick & Gallin (1977). Levamisole has also been reported to increase the directional and random locomotion of normal blood neutrophils (Anderson et al., 1976; Wright et al., 1977) and of neutrophils with abnormal motility from patients with hyperimmunoglobulinaemia E and recurrent pyogenic infections (Wright et al., 1977), recurrent Herpes simplex infections (Rabson et al., 1977) and rheumatoid arthritis (Mowat, 1978). The motility of normal blood monocytes can also be improved in vitro by levamisole (Pike & Snyderman, 1976). In vivo improvement of abnormal neutrophil motility following levamisole therapy has been reported in patients with Crohn's disease (Segal, Levi & Loewi, 1977) and in patients with rheumatoid arthritis (Mowat, 1978). Lymphocyte functions which can be enhanced by levamisole include E-rosetting capacity in vitro (Ramot et al., 1976) and in vivo (Ramot et al., 1976; De Cree et al., 1978) and mitogen- and antigeninduced transformation in vitro (Lichtenfeld et al., 1974; Pabst & Crawford, 1975; Hadden et al., 1975; Correspondence: Dr R. Anderson, Department of Microbiology, P.O. Box 2034, Pretoria, South Africa. 0099-9104J79/0030-0478502.00 ©0 1979 Blackwell Scientific Publications

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Levanisole effects on1 leucocytefunctions 479 Copeland, Steward & Harris, 1974; Wachi et al., 1974; Woods, Seigel & Chirigos, 1974; Wybrand & Govaerts, 1977). The present study was undertaken to determine the effects of a single oral dose (150 mg) of levamisole on the functions of neutrophils and lymphocytes from normal healthy adult volunteers. The neutrophil functions assessed were chemotaxis and post-phagocytic hexose monophosphate shunt activity (HMS) and protein iodination. Lymphocyte functions assessed were mitogen-induced lymphocyte transformation (DNA synthesis) and protein synthesis. Each function was assessed prior to the ingestion of levamisole and 2 hr post-levamisole. To assess the duration of the levamisole effects the cell functions were evaluated at 0, 2, 4, 6, 24, 48 and 72 hr. To exclude effects of repeated bleedings over a short time interval on the cell functions tested (possibly due to factors generated during the taking of the blood), a second group of volunteers who received no levamisole underwent testing of the same leucocyte functions at 0, 2 and 4 hr.

MATERIALS AND METHODS Neutrophil studies. Studies of neutrophil function were performed on ten individuals prior to and following levamisole intake. Pre-levamisole blood specimens correspond to time 0 hr in the Results section. Subsequent bleeding and testing of cells from each individual was carried out at 2, 4, 6, 24, 48 and 72 hr following the ingestion of a single 150 mg oral dose of levamisole. The protocol for the assessment of lymphocyte function was almost identical, the only difference being that six individuals were included in this component of the study. Neutrophil chemotaxis. Blood for neutrophil chemotactic studies was taken into heparin (5 units/ml), centrifuged briefly at 300 g for 10 min and the plasma and upper red cell and leucocyte-rich layers removed and sedimented at 37 C for 30 min following which the buffy layer was removed and lightly centrifuged (250 g/ 10 min). The supernatant was removed and the cell pellet washed twice with Hanks' balanced salt solution (HBSS, Grand Island Biological Co., Grand Island, New York) and cells resuspended to 3 x 106 neutrophils/ml. Only one leucoattractant was used in this study, viz autologous serum activated with 500 ug/ml bacterial endotoxin (Escherichia coli 0127: B8 Fifco, Detroit, Michigan). Two techniques were used to evaluate chemotactic responsiveness. (a) The method of Boyden (1962) using modified chemotactic chambers (Wilkinson, 1971) which were incubated at 37 C for 3 hr after which filters were removed, processed, inverted and the number of cells which achieved complete transfilter passage enumerated microscopically. Using this technique results were expressed as cells per microscope high power field (cells/HPF). (b) The method of Zigmond & Hirsch (1973) which utilizes a shortened incubation time (30 min) during which the cells do not usually attain complete transfilter passage. Using this technique the migratory response was expressed in terms of the distance of migration into the filter from the top of the 'fastest moving cell front', and results were expressed as microns migrated per unit time (,um/30 min). Using these techniques the pre- and post-levamisole chemotactic responses of neutrophils to EAS derived from autologous sera were assessed. Neutrophils and EAS were matched for each time interval tested. In another series of experiments to determine if levamisole effects were related to cells or serum alone or to both, pre-levamisole neutrophils from three volunteers were tested against pre-levamisole and 2 hr post-levamisole EAS derived from autologous serum to test for serum-mediated effects. In the corresponding series of experiments pre-levamisole and 2 hr post-levamisole neutrophils from the same subjects were tested for chemotactic responsiveness against autologous pre-levamisole sera to investigate cell-associated effects. Each assay was always run in triplicate. Studies of post-phagocytic metabolic activity. Hexose moiiophosphate shunt activity. The extent of HMS activity wras assessed according to the method of Wood, Katz & Landau (1963) with minor modifications. Neutrophils from heparinized venous blood were separated from mononuclear cells by density gradient centrifugation (Ficoll, sodium metrizoate gradients) at 400 g for 15 min. The resultant erythrocyte/neutrophil fraction was sedimented with 3% gelatin for 30 min at 370C to remove most of the red cells. The neutrophil-rich supernatant was centrifuged at 250 g for 10 min and the erythrocytes in the cell pellet lysed with hypotonic (0-85%) ammonium chloride. The neutrophil rich supernatant was centrifuged and washed once with 015 m phosphate buffered saline (PBS) and resuspended to 107 PMNIN/ml. The assay system employed 0-2 ml PMN suspension (2x 106 PMN), 0 1 ml autologous serum 0 1 ml of a Candida albicans suspension containing 108 organisms per ml, (cell: C. albicauis ratio 1: 5) and 0-6 (0 06 ,uCi) ml of glucose radiolabelled in the C' position (New England Nuclear, Boston, Massachusetts, as o-Glucose 1-"4C). In control systems, 01 ml of PBS substituted for C. albicans and the isotope control contained 0 4 ml PBS and 0-6 ml radiolabelled glucose. The apparatus used to test HMS activity is a double-chambered stoppered system in which the outer chamber contains the neutrophils and C. albicans and the inner chamber contains 0-6 ml of 1 N KOH. Each chamber was incubated at 37"C for 1 hr, after which the reaction was terminated by the addition of 2-0 ml of 1 N HCl to the outer chamber. The chambers were allowed to stand for a further hour at room temperature to permit absorption of released '4C02 by the KOH. After this time 0 2 ml of KOH was removed and added to 3 0 ml of scintillation fluid (Insta-gel acidified with 5 5 ml of 17 N HCl/litre). HMS activity was expressed as corrected radioactive counts per minute (ct/min) after deduction of the isotope control and unstimulated controls.

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Protein iodination. Determination of post-phagocytic iodination of ingested C. albicans was performed according to the method of Root & Stossel (1974). Pure neutrophil suspensions were used and adjusted to a final count of 107 PMN/ml. Each experimental tube contained 0-1 cell suspension (106 PMN), 0-1 ml C. albicans (107 organisms, to give a PMN:C. albicans ratio of 1:10), 0-1 ml autologous serum, 0-1 ml 125I (New England Nuclear, sodium iodate) solution in PBS (0.6 U/ml) and 0-6 ml PBS. Controls were of two types: (a) cell and C. albicans control in which the serum was omitted, and (b) corresponding serum control in which the cells and C. albicans were omitted. Tubes were rotated on a turntable for 1 hr at 370C, after which protein was precipitated by the addition of 2-0 ml of 10% perchloric acid (PCA). The protein precipitate was centrifuged down and twice washed with 2-0 ml of 10% PCA. The amount of protein-associated 125I was determined by solid scintillation counting. Results were expressed as corrected ct/min for experimental tubes by summing the average counts from the two controls and deducting this value from the corresponding experimental value. For both HMS and protein iodination determination assay systems were always run in duplicate for each time interval tested. As for cell motility studies, corresponding neutrophils and autologous sera were matched for each time interval. Lymphocyte studies. Mitogen-induced DNA synthesis. Blood for studies of lymphocyte function was defibrinated and fractionated by density gradient centrifugation (Ficoll sodium metrizoate gradients) at 400 g for 25 min. The mononuclear cell layer was removed and twice washed in medium RPMI 1640 pH 7-2 (Grand Island Biological Co., New York) supplemented with HEPES (Sigma, St. Louis, Missouri) 2-0 g/litre and 10% heat-inactivated autologous serum. The cell suspension was adjusted to 4 x 106 mononuclear cells/ml. Aliquots of 50 ul (2 x 105 cells) of this suspension were placed in wells of 6-0 mm Linbro tissue culture plates (Flow Laboratories, Inglewood, California), together with 100 ul of serum supplemented RPMI. The mitogens used in this study were phytohaemaglutinin (PHA, Wellcome Reagents, Beckenham, UK) and concanavalin A (Sigma) at concentrations of 25 pg/ml and 50 pg/ml. Mitogens were added in 20 pl volumes to triplicate wells and unstimulated controls received 20 p1 of RPMI. The plates were mixed and incubated for 48 hr in a humidified atmosphere of 5% CO2 in air, after which 20ul of tritiated thymidine (3H-T) (Thymidine-methyl-3H, New England Nuclear) containing 0-2 pCi was added to each well and the plates incubated for a further 18 h. Harvesting was performed using a multiple automated sample harvester (MASH-II, Microbiological Associates, Bethesda, Maryland). Incorporation of 3H-T was assessed in a liquid scintillation spectrophotometer. Mitogen-induced protein synthesis. In these studies 0 5 ml aliquots of cell suspensions (2 x 106 mononuclear cells) in serum-supplemented RPMI were added to 17 x 100 mm round-bottom polypropylene culture tubes (Falcon, Division of Becton, Dickinson and Co., Oxnard, California). The final volume in each tube was brought to 1-0 ml by the addition of 0 3 ml of medium, 0-1 ml of medium (controls) or 0-1 ml ofPHA (experimentals) and 0 1 ml of a tritiated amino acid mixture (3H-aa) containing 5 uCi (L-amino acid mixture. New England, Nuclear) which was added at the outset. Two PHA concentrations were used, 25 jug/ml and 50 pg/ml, since these were previously found to induce maximal protein synthesis. The tubes were incubated for 72 hr at 37°C in a humidified atmosphere of 5% CO2 and air. Following incubation, 1-0 ml of 10% tricarboxylic acid (TCA) was added to each tube. The resultant protein precipitate was twice washed with TCA and redissolved in 1-0 ml of 1 N KOH. Aliquots of 0-2 ml were transferred to 3 0 ml of acidified Insta-Gel (5 5 ml of 17 N HCI/ litre) and the extent of incorporation of 3H-aa into protein was assessed in a liquid scintillation spectrophotometer.

RESULTS

Calculation and expression of results Results for each post-levamisole time were expressed as the percentage increase of control (pre-levamisole) values. Corresponding values for each post-levamisole time for each individual (ten individuals tested) were averaged and the results expressed as mean (percentage) stimulation with standard error. This mode of expression of results, although unnecessary for cell migratory values since considerable stimulation post-levamisole was observed, was considered important for studies of neutrophil postphagocytic metabolic activity and lymphocyte transformation. In these latter studies stimulation, although consistent and significant, was of lower magnitude.

Neutrophil studies Significant stimulation of neutrophil chemotactic responsiveness was observed following ingestion of levamisole (Table 1). Stimulation was detected using both the intra-filter penetration technique of Zigmond & Hirsch (1973) and the method of Boyden (1962) and was present at 2, 4, 6 and 24 hr. Thereafter no stimulation was evident. Results shown in Table 2 indicate that the stimulation of cell locomotion is a property both of cells and serum, since enhanced motility was observed when pre-levamisole neutrophils were tested against EAS derived from 2 hr post-levamisole autologous serum and when 2 hr

Levanzisole effects on leucocyte functions

481

TABLE 1. Neutrophil chemotaxis, post-phagocytic hexose monophosphate shunt activity and protein iodination at varying time intervals following ingestion of 150 mg levamisole Mean increase (%) of chemotaxis

Time (hr)

Zigmond method

2 4 6 24 48 72

105+ 10-73* 113+7-02 91+ 13-46 105+ 8.74 -6+ 812 3+9-12

Mean increase (%) in hexose monophosphate Mean increase (%) in shunt activity protein iodination Boyden method

67+ 10-36 69+11-20 59+ 14-97 90+ 23-20 2+ 13-12 -9+ 10-17

17± 1-96t 18+1-47 n.d. n.d. n.d. n.d.

22+4.19* 21+5 34 n.d. 23+ 5-62 2+4-31

-4+6-10

* Results are expressed as the mean stimulation (percentage) ± s.d. (ten separate experiments) for each time interval tested. t Mean stimulation (percentage) ± s.e. (six separate experiments).

TABLE 2. Comparison of the chemotactic responses of pre- and postlevamisole neutrophils to EAS derived from pre- and post-(2 hr) levamisole sera Chemotactic responsiveness

Neutrophils Pre-levamisole Post-levamisole* Pre-levamisole Post-levamisole

EAS

(,um/30 min)

Pre-levamisole Pre-levamisole Post-levamisole Post-levamisole

64+ 5-55t 116+ 6-57 105+ 5-17 122+ 8-50

Post-levamisole results all from 2 hr testing. t Results as mean+ s.e. of three separate experiments.

*

post-levamisole neutrophils were tested against pre-levamisole EAS. No effects on cell motility were observed in control subjects who received no levamisole, indicating that repeated bleeding has no effects on cell motility (results not shown). Likewise, stimulation (although not of the same magnitude) of neutrophil post-phagocytic hexose monophosphate shunt activity and protein iodination was observed following ingestion of levamisole (Table 1). This stimulation was not evident at 48 hr. As for chemotactic studies no effects, either stimulatory or inhibitory, of repeated bleeding on these activities were detected. The mean (with standard error) pre-levamisole values for cell migration, HMS activity and protein iodination were 63 pm+ 3 2/30 min, 127+22 cells/HPF, 20, 291 ct/min+ 950 and 27,014 ct/min+ 1888, respectively. Lymphocyte studies Stimulation of lymphocyte transformation to PHA at both concentrations was detected in five out of six individuals up to 24 hr. However, less consistent stimulation (in three out of six) was evident when con A was used as the mitogen (Table 3). No alteration in basal and PHA-stimulated lymphocyte protein synthesis was detected following ingestion of levamisole (results not shown). The mean (with standard error) pre-levamisole values for lymphocyte transformation to 25 ,g/ml PHA, 50 ug/ml PHA, 25 ,ug/ml con A and 50 yug/ml con A were 39,914 ct/min+ 593, 34,382+ 999, 6446+ 920 and 7620+ 948, respectively. L

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TABLE 3. Lymphocyte transformation to phytohaemagglutinin and concanavalin A at varying intervals following ingestion of 150 mg of levamisole Mean increase (%) in lymphocyte transformation to the mitogens:

Phytohaemagglutinin Time 2 4 6 24 48 72

Concanavalin A

25 ,ug/ml

50 ,g/ml

25 ,g/ml

50 ,g/ml

18-30+7-29*

40.43+ 16-75

10*21+8-31

27A40+ 13-68

36-25+29-60 48-15+24-07 11-75+ 5*39 -6-12+ 12-31 2-41+ 11-21

14-21+9-21 15-10+ 10-12 6-20+ 9-12 5 10+ 10-12 -4-15+7-85

17-81+8-20 15-21+9-12 16-12+8*45 20-12+ 9-12 -8-13+ 10-1 -5-12+8-12

19-49+9-75 15 90+ 9-75 2-30+ 10-12 0 45+9 62

* Results expressed as mean increase percentage (of six experiments) and s.e. of the mean normal (pre-levamisole) value for each time interval.

DISCUSSION Levamisole causes stimulation of many leucocyte functions. In vitro, the drug promotes stimulation of random migration, chemotaxis and directional motility of normal and abnormal human neutrophils (Anderson et al., 1976; Wright et al., 1977) and can also cause reversal of deactivation of chemotaxis by EAS (Anderson et al., 1976). However, the effects of levamisole on neutrophil post-phagocytic metabolic activity in vitro are somewhat controversial. Wright et al. (1977) have reported stimulation of hexose monophosphate shunt activity, whereas Repine & Douglas (1977) and Anderson, Glover & Rabson (1978) found no detectable stimulation. The results of the present study show that a single 150 mg oral dose of levamisole can cause stimulation of neutrophil chemotactic responsiveness to EAS derived from autologous serum. The stimulation is a property of both cells and serum and persists for at least 24 hr. Enhanced neutrophil post-phagocytic metabolic activity of a similar duration, but lesser magnitude was also observed. We have also investigated (results not shown) the effects of a single 75 mg oral dose of levamisole on the post-phagocytic activities of neutrophils from two related children with chronic granulomatous disease. The precise enzyme defect has not yet been identified. There was no detectable pre-levamisole post-phagocytic nitro-blue tetrazolium reduction, HMS activity or protein iodination. These functions were tested 2 hr post-levamisole, but no stimulation was observed. Both children had markedly defective neutrophil chemotaxis in the absence of any apparent infection. However both had elevated serum IgE levels (8000 iu/ml and 450 iu/ml) which may be related to the low chemotaxis (Hill et al., 1976). The chemotactic responsiveness of the neutrophils from patients to EAS derived from autologous serum improved considerably after levamisole (approximately three-fold stimulation). This finding is in agreement with the reports of Wright et al. (1977) and De Cree et al. (1978). Stimulation of lymphocyte transformation to PHA was evident in five out of six individuals, with no detectable alteration in resting levels. The one individual who showed no stimulation had a particularly high response to PHA and further stimulation may not have been possible. Variable stimulation was found with con A. It is possible that a longer incubation time may be required to detect effects on con A stimulation of lymphocytes or that the differences may be too slight to be significant. The effects on PHA transformation are in agreement with our in vitro findings of stimulation of lymphocyte transformation by 105 M levamisole (unpublished observations) and with those of others (Hadden et al., 1975; Wybrand & Govaerts, 1977). No effects of levamisole on PHA-induced protein synthesis were observed. This study has indicated that a 150 mg oral dose of levamisole can potentiate certain functions of leucocvtes from normal individuals. The stimulation was still evident 24 hr following levamisole intake.

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De Cree et a!. (1978) have recently reported in vivo correction of depressed neutrophil (chemotaxis, phagocytosis and antimicrobial activity) and T lymphocyte (E-rosetting and transformation) functions in a child with recurrent infection by levamisole. When levamisole was administered for 3 consecutive days each week, no clinical improvement was evident. However, when the dose schedule was changed to 3 alternate days each week there was significant clinical improvement. This finding would appear to confirm the results of the present study in that one 150 mg oral dose of levamisole can promote stimulation of certain cell functions until at least 24 hr after intake, suggesting that a more lasting protection may ensue if the drug is administered on alternate days. REFERENCES

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