Clin. exp. Immunol. (1976) 25, 493-496.
BRIEF COMMUNICATION
The effect of Levamisole on peripheral blood lymphocyte subpopulations in patients with rheumatoid arthritis and
ankylosing spondylitis M. RO S EN THAL, U. TRABERT & W. MULLER Department ofRheumatology, University of Basel, Switzerland
(Received 14 April 1976)
SUMMARY
Although Levamisole, an antihelmintic drug, has shown to have some modulatory effect on the immune response in clinical trials and experimental models, its mode of action remains obscure. In a group of fifteen patients with rheumatoid arthritis and ankylosing spondylitis receiving Levamisole on an intermittant regime, simultaneous determinations of the lymphocyte subpopulations were made prior to Levamisole administration and 3 months thereafter. No significant changes were observed either in the absolute or in the relative numbers of T- and B-cell populations, while a statistically significant reduction was found in the 'null' cells. These findings suggest that the immune potentiating effect of Levamisole may at least partially be due to a maturation process of the 'null' cells. INTRODUCTION Levamisole, an antihelmintic drug, gained increasing significance in recent months, since its modulatory effect on the immune response, preferentially the cell-mediated type, was demonstrated (Pabst & Crawford, 1975; Whitcomb, Merluzzi & Cooperband, 1976; Sampson & Lui, 1976). Its mode of action remains unknown in spite of the increasing amount of experimental work and clinical experience. Evidence was provided that the drug restors an impaired cellular immune response (Tripodi, Parks & Brugmans, 1973). Recently we reported that a possible true immunostimulation may be observed in the adjuvants disease of the rat where a marked accentuation of the disease occurs on continuous Levamisole administration (Trabert, Rosenthal & Mueller, 1976). Among other clinical conditions, where Levamisole was beneficially applied, are some rheumatic diseases like rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and Reiter's syndrome (Huskisson et al., 1976; Rosenthal, Trabert & Mueller, 1976; Ippen & Quadripur, 1975; Gordon & Keenan, 1935). In spite of the clinical improvement no reliable immunological correlation could be established. In this communication we studied simultaneously the different lymphocyte subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis receiving Levamisole in order to determine to what extent lymphocyte counts can be used as a possible immunological parameter for the therapeutic effect of this drug. Furthermore, such studies might provide some clues for the mode of action of Levamisole. MATERIALS AND METHODS Patients. Ten patients with classical or definite rheumatoid arthritis (RA) (Ropes et al., 1968) and five patients with established ankylosing spondylitis (AS) were examined. All patients showed evidence of active disease with demonstrable joint tenderness, soft tissue swellings, effusions or elevated sedimentation rate, and received only symptomatic therapy. Correspondence: Dr M. Rosenthal, Felix Platter-Spital, Rheumatologische Universitatsklinik, Burgfelderstr. 101, CH-4055 Basel, Switzerland.
493
494
M. Rosenthal, U. Trabert & W. Muller
Levamisole (Janssen Pharmaceutica, Beerse, Belgium) was administered orally in a dose of 150 mg daily for three days a week. The estimation of different lymphocyte subpopulations. This was made on peripheral blood lymphocytes obtained by FicollHypaque gradient separation (Boyum, 1968) from heparinized venous blood. The lymphocytes were fluorescinated with polyvalent goat antihuman immunoglobuline (Meloy Laboratories, Springfield, Virginia), and thereafter incubated overnight with neuraminidase-pretreated sheep red blood cells as described earlier (Papamichail et al., 1972; Weiner, Bianco & Nussenzweig, 1973). Membrane immunofluorescence (B lymphocytes) was read by Epi-illumination with a Zeiss binocular photomicroscope. Cells and rosettes (T lymphocytes) in the same field were displayed by changing to transmitted light with a dark ground cardioid condenser. Exclusion of monocytes was made by peroxidase staining (Preud'homme & Flandrin, 1974). Utilizing this procedure one could account for all lymphocytes present in the smear. The determinations of the different lymphocyte subpopulations were performed prior to the start of therapy and 3 months thereafter.
RESULTS Table 1 presents the mean values of lymphocyte counts obtained in patients with rheumatoid arthritis and ankylosing spondylitis during Levamisole therapy. T-, B- as well as double-marker lymphocyte counts did not change significantly as compared to the counts obtained prior to therapy. However, a statistically significant drop in the 'null' lymphocyte counts to about 50%° (P< 0.025) was observed during the therapy. Similar findings were obtained (Table 2) comparing the relative lymphocyte counts for the different subpopulations showing no significant difference for the T, B, and double-marker cells, while a marked reduction in the 'null' lymphocyte populations could be observed. The correlation between the clinical improvement and the change in 'null' cell counts for the individual patient (Table 3) shows that out of the eight patients who improved on Levamisole therapy only in five TABLE 1. Absolute counts of different lymphocyte subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis (n= 15)
Mean lymphocyte counts per mm3+ s.d. LevamisoleUntreated treated Statistics
1606+445 992+ 319 498+ 209 103+ 54 13+29
1655+ 598 Lymphocytes (total) T lymphocytes 1031 + 454 B lymphocytes 463+ 207 O lymphocytes 205+ 173 Double marker lymphocytes 4+7
n.s. n.s. n.s.
P< 0-025 n.s.
n.s. =Not significant.
TABLE 2. Relative lymphocyte counts of different subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis (n= 15)*
Untreated
Levamisoletreated Statistics
61-93+ 8.77 61-93+ 10-13 T lymphocytes 28-23+ 9-06 30-56+ 8-76 B lymphocytes 10-13+5-18 6-67+3-16 O lymphocytes Double marker lymphocytes 0-5+0.76 0-87+ 1-89
n.s. n.s.
P
BRIEF COMMUNICATION
The effect of Levamisole on peripheral blood lymphocyte subpopulations in patients with rheumatoid arthritis and
ankylosing spondylitis M. RO S EN THAL, U. TRABERT & W. MULLER Department ofRheumatology, University of Basel, Switzerland
(Received 14 April 1976)
SUMMARY
Although Levamisole, an antihelmintic drug, has shown to have some modulatory effect on the immune response in clinical trials and experimental models, its mode of action remains obscure. In a group of fifteen patients with rheumatoid arthritis and ankylosing spondylitis receiving Levamisole on an intermittant regime, simultaneous determinations of the lymphocyte subpopulations were made prior to Levamisole administration and 3 months thereafter. No significant changes were observed either in the absolute or in the relative numbers of T- and B-cell populations, while a statistically significant reduction was found in the 'null' cells. These findings suggest that the immune potentiating effect of Levamisole may at least partially be due to a maturation process of the 'null' cells. INTRODUCTION Levamisole, an antihelmintic drug, gained increasing significance in recent months, since its modulatory effect on the immune response, preferentially the cell-mediated type, was demonstrated (Pabst & Crawford, 1975; Whitcomb, Merluzzi & Cooperband, 1976; Sampson & Lui, 1976). Its mode of action remains unknown in spite of the increasing amount of experimental work and clinical experience. Evidence was provided that the drug restors an impaired cellular immune response (Tripodi, Parks & Brugmans, 1973). Recently we reported that a possible true immunostimulation may be observed in the adjuvants disease of the rat where a marked accentuation of the disease occurs on continuous Levamisole administration (Trabert, Rosenthal & Mueller, 1976). Among other clinical conditions, where Levamisole was beneficially applied, are some rheumatic diseases like rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and Reiter's syndrome (Huskisson et al., 1976; Rosenthal, Trabert & Mueller, 1976; Ippen & Quadripur, 1975; Gordon & Keenan, 1935). In spite of the clinical improvement no reliable immunological correlation could be established. In this communication we studied simultaneously the different lymphocyte subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis receiving Levamisole in order to determine to what extent lymphocyte counts can be used as a possible immunological parameter for the therapeutic effect of this drug. Furthermore, such studies might provide some clues for the mode of action of Levamisole. MATERIALS AND METHODS Patients. Ten patients with classical or definite rheumatoid arthritis (RA) (Ropes et al., 1968) and five patients with established ankylosing spondylitis (AS) were examined. All patients showed evidence of active disease with demonstrable joint tenderness, soft tissue swellings, effusions or elevated sedimentation rate, and received only symptomatic therapy. Correspondence: Dr M. Rosenthal, Felix Platter-Spital, Rheumatologische Universitatsklinik, Burgfelderstr. 101, CH-4055 Basel, Switzerland.
493
494
M. Rosenthal, U. Trabert & W. Muller
Levamisole (Janssen Pharmaceutica, Beerse, Belgium) was administered orally in a dose of 150 mg daily for three days a week. The estimation of different lymphocyte subpopulations. This was made on peripheral blood lymphocytes obtained by FicollHypaque gradient separation (Boyum, 1968) from heparinized venous blood. The lymphocytes were fluorescinated with polyvalent goat antihuman immunoglobuline (Meloy Laboratories, Springfield, Virginia), and thereafter incubated overnight with neuraminidase-pretreated sheep red blood cells as described earlier (Papamichail et al., 1972; Weiner, Bianco & Nussenzweig, 1973). Membrane immunofluorescence (B lymphocytes) was read by Epi-illumination with a Zeiss binocular photomicroscope. Cells and rosettes (T lymphocytes) in the same field were displayed by changing to transmitted light with a dark ground cardioid condenser. Exclusion of monocytes was made by peroxidase staining (Preud'homme & Flandrin, 1974). Utilizing this procedure one could account for all lymphocytes present in the smear. The determinations of the different lymphocyte subpopulations were performed prior to the start of therapy and 3 months thereafter.
RESULTS Table 1 presents the mean values of lymphocyte counts obtained in patients with rheumatoid arthritis and ankylosing spondylitis during Levamisole therapy. T-, B- as well as double-marker lymphocyte counts did not change significantly as compared to the counts obtained prior to therapy. However, a statistically significant drop in the 'null' lymphocyte counts to about 50%° (P< 0.025) was observed during the therapy. Similar findings were obtained (Table 2) comparing the relative lymphocyte counts for the different subpopulations showing no significant difference for the T, B, and double-marker cells, while a marked reduction in the 'null' lymphocyte populations could be observed. The correlation between the clinical improvement and the change in 'null' cell counts for the individual patient (Table 3) shows that out of the eight patients who improved on Levamisole therapy only in five TABLE 1. Absolute counts of different lymphocyte subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis (n= 15)
Mean lymphocyte counts per mm3+ s.d. LevamisoleUntreated treated Statistics
1606+445 992+ 319 498+ 209 103+ 54 13+29
1655+ 598 Lymphocytes (total) T lymphocytes 1031 + 454 B lymphocytes 463+ 207 O lymphocytes 205+ 173 Double marker lymphocytes 4+7
n.s. n.s. n.s.
P< 0-025 n.s.
n.s. =Not significant.
TABLE 2. Relative lymphocyte counts of different subpopulations in patients with rheumatoid arthritis and ankylosing spondylitis (n= 15)*
Untreated
Levamisoletreated Statistics
61-93+ 8.77 61-93+ 10-13 T lymphocytes 28-23+ 9-06 30-56+ 8-76 B lymphocytes 10-13+5-18 6-67+3-16 O lymphocytes Double marker lymphocytes 0-5+0.76 0-87+ 1-89
n.s. n.s.
P
