Clin. exp. Immunol. (1978) 31, 321-327.
Levamisole maintains cyclophosphamide-induced remission in murine lupus erythematosus J. ZULMAN, J. MICHALSKI, CANDACE McCOMBS, J. GREENSPAN & N. TALAL Immunology and Arthritis Section, Department of Medicine, School of Medicine, and Department of Oral Medicine, School of Dentistry, University of California at San Francisco, San Francisco, California, U.S.A.
(Received 20 August 1977)
SUMMARY
Antibodies to DNA in 14 month old male NZB/W F1 mice were markedly decreased after 4 weeks of cyclophosphamide therapy. A group of these mice and untreated controls subsequently received levamisole for 12 weeks. Antibodies to DNA returned to pretreatment levels within 8 weeks in animals receiving only cyclophosphamide, whereas levamisole treatment after cyclophosphamide delayed the reappearance of these antibodies. Glomerular immunoglobulin deposition and the degree of lymphocytic tissue infiltrate were decreased in animals receiving cyclophosphamide followed by levamisole as compared with non-treated control animals or those receiving cyclophosphamide or levamisole alone. In vitro T-cell mitogen reactivity was inversely correlated with the final DNA antibody titres of individual mice, suggesting a relationship between decreased autoantibody production and augmented cellular immunity. We discuss possible mechanisms by which levamisole may delay the reappearance of autoantibodies and decrease the histological evidence of lupus nephritis when given to New Zealand mice first treated with cyclophosphamide.
INTRODUCTION New Zealand black x New Zealand white (NZB/W)F, hybrid mice develop a spontaneous autoimmune disease characterized by autoantibodies, severe immune complex glomerulonephritis and lymphocytic infiltrates in many organs (Talal & Steinberg, 1974). These mice are considered to be models for human systemic lupus erythematosus (SLE) and Sjogren's syndrome (Talal, 1976). Autoantibody production may result from the impaired regulation of B lymphocytes, possibly related to decreased suppressor Tcell activity (Talal, 1976). Cyclophosphamide is an alkylating agent effective in the therapy of human SLE (Steinberg & Decker, 1974). It also reduces serum antibody levels, retards the development of immune complex glomerulonephritis and increases longevity in young NZB/W F1 females (Morris et al., 1976; Russel & Hicks, 1968; Walker & Bole, 1975). Levamisole enhances cellular immune responses in animals (Renoux & Renoux, 1972; Spreafico et al., 1975) and restores cutaneous delayed hypersensitivity in anergic individuals with cancer and Hodgkin's disease (Tripodi, Parks & Brugman, 1973; Ramot et al., 1975). Furthermore, levamisole has been shown to maintain the remission of murine lymphoid leukaemia induced by the alkylating agent 1,3-bis(2-chlorethyl)-1-nitrosurea (BCNU) (Chirigos, Pearson & Pryor, 1973). The latter study prompted us to investigate the hypothesis that immunostimulation with levamisole might maintain cyclophosphamide-induced remission in NZB/W autoimmune disease. Correspondence: Dr J. Michalski, Immunology and Arthritis Section, 151T, VA Hospital, 4150 Clement Street, San Francisco, California 94121, U.S.A.
0099-9104/78/0200-0321$02.00 ©)1978 Blackwell Scientific Publications
321
322
J. Zulman et al. MATERIALS AND METHODS
Animals. Male NZB/W mice were obtained from our colony maintained at the vivarium of the University of California, San Francisco. The animals were 14 months old at the onset of the experiment and had established autoimmune disease, as evidenced by proteinuria and elevated levels of serum anti-DNA antibodies. Unlike females, male NZB/W mice die with renal disease in the second year of life. Individual mice were identified by numbered ear tags. Experimental and control animals were housed in the same area and were allowed food and water ad libitum. Therapeutic agents. Cyclophosphamide (Mead Johnson and Company, Evansville, Indiana), and levamisole (Janssen R & D Inc., New Brunswick, New Jersey) were freshly dissolved and diluted in sterile water. All medications were administered as 0 5 cm3 intraperitoneal injections. Experimental design. Mice were randomly assigned to four different treatment groups. Two groups were given cyclophosphamide (50 mg/kg body weight) twice weekly for 4 weeks and two groups of control mice were untreated. After 4 weeks, one of the cyclophosphamide-treated groups and one of the untreated groups were given levamisole (125 ug per mouse) on 2 consecutive days each week for a total of 16 weeks, starting on day 3 after the last dose of cyclophosphamide. All mice were bled by orbital sinus puncture at intervals of 4 weeks. At the completion of 16 weeks of levamisole therapy, animals were killed by cervical dislocation and in vitro immunological studies and histological examinations were performed. Survival at 18 months was 90% in the cyclophosphamide only group and 60% in the other three groups. One animal in each of the latter three groups died after the last bleeding, prior to being killed. In the cyclophoshamide-levamisole group, three mice died in the first week of levamisole therapy. These were all in one cage, whereas there were no deaths in two other cages of similarly treated mice. In order to investigate a possible lethal effect of sequential cyclophosphamide-levamisole therapy, twenty-eight 9 month old DBA mice were given cyclophosphamide (2-5 mg/per mouse) twice weekly for 4 weeks. Groups of seven mice were then given levamisole, 125 ,pg daily, on two consecutive days each week for a total of 5 weeks starting on day 0, 4, 7 or 11 after the last dose of cyclophosphamide. None of the mice in the four groups died. Procedures. Anti-DNA assay. Blood obtained by orbital sinus puncture at various ages was allowed to clot at room temperature for 1 hr, and left at 4VC overnight. Serum was separated by centrifugation at 205 g for 15 min. DNAbinding activity of sequential individual sera was assayed by a cellulose ester filter radioimmunoassay previously described (Talal & Pillarisetty, 1976). Radioactive double-stranded DNA (900 ct/min per 4 ng DNA) from KB cells (obtained from Electro-Nucleonics Laboratories, Bethesda, Maryland) was incubated with decomplemented 10 ul aliquots of serum for 30 min at 37°C followed by an overnight incubation at 4°C. The antigen-antibody complexes were collected onto cellulose ester filters. The filters were placed in counting vials and covered with 10 cm3 of Liquifluor toluene scintillation medium. Radioactivity was determined in a Packard liquid scintillation counter. The results are expressed as corrected ct/min retained on the filter, which is directly related to the serum-binding capacity (Attias, Sylvester & Talal, 1973). Fractionation of serum. Serum samples from each treatment group were pooled separately. 200 ,l of pooled serum samples were subjected to ultracentrifugation in a 10-35% linear sucrose density gradient (0-15 M NaCl, pH 8.0) as previously described (Talal & Pillarisetty, 1975). Bovine thyroglobulin (19S,) human gamma globulin (7S) and haemoglobin (4S) were used as sedimentation markers. Forty fractions were collected and each one was analysed for antibody to DNA. Peak fractions were tested for immunoglobulin content. In the 19S region (fractions 10-20) only p chains were detected by Ouchterlony analysis, whereas in the 7S region (fractions 20-30) y chains but not pu chains were present. Antibodies to DNA were assayed in each fraction, as described above, using 50 pl aliquots from the gradient fractions. Comparison of 7S to 19S antibody binding. The radioactive binding profiles for DNA revealed clear distribution into the 7S and 19S peaks of activity after sucrose gradient fractionation (Roubinian, Papoian & Talal, 1977a). The radioactivity representing the fractionated 7S and 19S peaks within a single gradient were added and compared for total binding activity. Results with these fractionated sera were generally comparable to results with unfractionated whole serum. Assay of in vitro proliferative response. The spleens from each mouse were minced in RPMI 1640 medium and individual single cell suspensions were prepared. Spleen cell proliferative responses to concanavalin A and phytohaemagglutinin were measured in a microtitre plate proliferative assay (McCombs, Michalski & Talal, 1976). 5 x 105 spleen cells were cultured for 48 hr in 0-2 ml RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, 1% antibiotic-antimycotic and 1% glutamine (all from Pacific Biological Company). Stimulated cultures contained a final concentration of 30 ,pg/ml of purified phytohaemagglutinin, lot No. K6951 (Wellcome Research Laboratories, Brakenham, England), or 50 ,pg/ml of concanavalin A, lot No. 4218 (Nutritional Biological Company, Cleveland, Ohio). 10 ,pCi of tritiated thymidine, sp. act. 3 0 Ci/mmole (Schwartz-Mann, Orangeburg, New York) was added 4 hr before the cells were harvested onto glass fiber filters using a multiple automated sample harvester (Otto Hiller, Madison, Wisconsin). Results are expressed as the loglo of the net counts of quadruplicate stimulated and control cultures (Gottlieb, 1974). The stimulated responses of three tumourbearing animals were less than background and were not included in the analysis of the results. Statistical analysis. The significance of mean differences were determined by the Student's two-tailed t-test. Pathology. A full autopsy was performed on all animals killed after 16 weeks and specimens of kidney, liver, spleen, pancreas, adrenals, duodenum, mesenteric lymph nodes, gonads, lung, heart, thymus, submandibular, parotid and sublingual salivary glands and cervical lymph nodes were fixed in 10% formol saline and processed for routine histopathological exam-
ination. Only three animals had abnormalities on gross post-mortem examination. One mouse in the cyclophosphamide only group and one mouse in the levamisole only group had an apparent extension of a splenic tumour into the retroperitoneal and mesen-
Levamisole in murine lupus erythematosus
323
teric tissue. The tumours proved to be poorly differentiated spindle cell malignancies. One animal in the levamisole only group had massive splenomegaly. Specimens of kidney were quick-frozen in freon, and were cooled and stored in liquid nitrogen until sectioned. Cryostat sections were mounted on formol gelatin-coated slides, air-dried for 30 min and washed for 15 min twice in PBS, pH 7-2. Each slide was rinsed in distilled water and wiped dry around the section. The slides were then placed in a moist chamber, covered with antiserum and incubated for 30 min at room temperature in the dark. Rabbit polyvalent anti-mouse immunoglobulin (Behring Diagnostic, Sommerville, New Jersey, U.S.A., Lot 656 F/protein 2-8) was used at a titre of 1:20. Slides were then rinsed quickly in PBS and washed twice for 15 min in PBS with gentle agitation, and were then rinsed in distilled water and mounted using glycerin-PBS. Coded sections were examined in a Wild fluorescence microscope using transmitted u.v. light and a dark ground condenser. Direct glomerular immunofluorescence for immunoglobulin deposition was graded on a scale of 0 to + + + + by two independent observers who read the coded slides. The grading took into consideration the extent and intensity of immunoglobulin immunofluorescence. The incidence and extent of lymphomonocytic infiltrates in a major salivary gland, lung and kidney were similarly expressed on a scale of 0 to + + + + after examination by light microscopy.
RESULTS Treatment with cyclophosphamide significantly reduced antibodies to DNA in 14 month old NZB/W male mice (Fig. 1). In animals treated with cyclophosphamide alone, anti-DNA antibody levels rose within 1 month after termination of the cyclophosphamide therapy, and surpassed pretreatment values after 2 months. Levamisole treatment initiated 3 days after the last dose of cyclophosphamide was associated with a depression of anti-DNA antibodies for an additional 2 months after cyclophosphamide was stopped. 600
C
.E u
c 0 (V
2
4
4
8 Weeks
FIG. 1. Mean DNA-binding activity of survivors of different treatment groups. The ordinate shows the mean ct/min [3H]DNA bound by 10 ,ul ofserum from the survivors, the abscissa shows time. The arrow shows when cyclophosphamide was stopped. (-) No treatment (six survivors); (0) cyclophosphamide alone (nine survivors); (A) cyclophosphamide followed by levamisole (six survivors).
324
J.
Zulman et a.
700
600 _
500-
400-
C~~~~~~~~~ E~~~~~~~~~~~~~~~ 300
200-
00~~~~
0
4
8
12
16
Weeks
FIG. 2. Sequential serum DNA-binding activity of mice receiving levamisole only. The ordinate shows the ct/ min [3H]DNA bound by 10 Al of serum, the abscissa shows time. (0) Survivors; (0) non-survivors.* Death of mouse.
To determine whether the treatment had a specific effect on either IgG or IgM anti-DNA antibodies, measured the DNA-binding activity in serum fractions after separation by sucrose gradient ultracentrifugation. A marked decrease of DNA-binding activity was noted after 4 weeks of cyclophosphamide therapy, with a slightly greater depression of IgM than IgG. Levamisole therapy following cyclophosphamide was associated with continued suppression of both IgG and IgM anti-DNA antibodies. Levamisole treatment without prior cyclophosphamide was associated with the death of the mice with the highest initial DNA-binding activity (Fig. 2). The surviving mice had an initial mean DNA-binding activity of 160 ct/min compared to 290 ct/min for mice which died during treatment (P 0 3.
Levamisole in murine lupus erythematosus
325
TABLE 2. Immunoglobulin deposition in renal glomeruli and lymphocytic infiltrate of the kidney, salivary gland and lung (scale 0 to ++++). The fraction of specimens in each treatment group rated as + + + or + + + + is given in the parentheses
Treatment group and Glomerular immunoglobulin animal number deposition
Kidney, salivary gland and lung
lymphomonocytic infiltrate (A) No treatment 18 20 22 28 29 (B) Cyclophosphamide only 14 15 16 17 23 24 31 32 33 (C) Levamisole only 38 39 40 44 53 (D) Cyclophosphamide followed by levamisole 05 06 09 11 13
++(3/5) ++ +++ +++ +++
++++-(6/9)
++ +(4/5) + +++ +++
++ ++ ++++ ++
++ +(5/9) ++ ++ ++ ++++
++++ +++ ++++ ++++
+++ +++ ++
+++(3/5)
++(1/5)
+++ +++
++ +++ ++ ++
+(1/5)
+(0/5)
+ ++ + ++++
+ + + ++
++ ++
To determine if an augmented T-lymphocyte function (as measured by mitogen stimulation) was associated with decreased autoimmunity, we performed a linear regression analysis between the mitogenstimulated [3H]TdR incorporation and the final anti-DNA antibody titre of the individual mice. There was a significant inverse correlation between mitogen reactivity and anti-DNA levels for both Con A (r = 0-604, P
Levamisole maintains cyclophosphamide-induced remission in murine lupus erythematosus J. ZULMAN, J. MICHALSKI, CANDACE McCOMBS, J. GREENSPAN & N. TALAL Immunology and Arthritis Section, Department of Medicine, School of Medicine, and Department of Oral Medicine, School of Dentistry, University of California at San Francisco, San Francisco, California, U.S.A.
(Received 20 August 1977)
SUMMARY
Antibodies to DNA in 14 month old male NZB/W F1 mice were markedly decreased after 4 weeks of cyclophosphamide therapy. A group of these mice and untreated controls subsequently received levamisole for 12 weeks. Antibodies to DNA returned to pretreatment levels within 8 weeks in animals receiving only cyclophosphamide, whereas levamisole treatment after cyclophosphamide delayed the reappearance of these antibodies. Glomerular immunoglobulin deposition and the degree of lymphocytic tissue infiltrate were decreased in animals receiving cyclophosphamide followed by levamisole as compared with non-treated control animals or those receiving cyclophosphamide or levamisole alone. In vitro T-cell mitogen reactivity was inversely correlated with the final DNA antibody titres of individual mice, suggesting a relationship between decreased autoantibody production and augmented cellular immunity. We discuss possible mechanisms by which levamisole may delay the reappearance of autoantibodies and decrease the histological evidence of lupus nephritis when given to New Zealand mice first treated with cyclophosphamide.
INTRODUCTION New Zealand black x New Zealand white (NZB/W)F, hybrid mice develop a spontaneous autoimmune disease characterized by autoantibodies, severe immune complex glomerulonephritis and lymphocytic infiltrates in many organs (Talal & Steinberg, 1974). These mice are considered to be models for human systemic lupus erythematosus (SLE) and Sjogren's syndrome (Talal, 1976). Autoantibody production may result from the impaired regulation of B lymphocytes, possibly related to decreased suppressor Tcell activity (Talal, 1976). Cyclophosphamide is an alkylating agent effective in the therapy of human SLE (Steinberg & Decker, 1974). It also reduces serum antibody levels, retards the development of immune complex glomerulonephritis and increases longevity in young NZB/W F1 females (Morris et al., 1976; Russel & Hicks, 1968; Walker & Bole, 1975). Levamisole enhances cellular immune responses in animals (Renoux & Renoux, 1972; Spreafico et al., 1975) and restores cutaneous delayed hypersensitivity in anergic individuals with cancer and Hodgkin's disease (Tripodi, Parks & Brugman, 1973; Ramot et al., 1975). Furthermore, levamisole has been shown to maintain the remission of murine lymphoid leukaemia induced by the alkylating agent 1,3-bis(2-chlorethyl)-1-nitrosurea (BCNU) (Chirigos, Pearson & Pryor, 1973). The latter study prompted us to investigate the hypothesis that immunostimulation with levamisole might maintain cyclophosphamide-induced remission in NZB/W autoimmune disease. Correspondence: Dr J. Michalski, Immunology and Arthritis Section, 151T, VA Hospital, 4150 Clement Street, San Francisco, California 94121, U.S.A.
0099-9104/78/0200-0321$02.00 ©)1978 Blackwell Scientific Publications
321
322
J. Zulman et al. MATERIALS AND METHODS
Animals. Male NZB/W mice were obtained from our colony maintained at the vivarium of the University of California, San Francisco. The animals were 14 months old at the onset of the experiment and had established autoimmune disease, as evidenced by proteinuria and elevated levels of serum anti-DNA antibodies. Unlike females, male NZB/W mice die with renal disease in the second year of life. Individual mice were identified by numbered ear tags. Experimental and control animals were housed in the same area and were allowed food and water ad libitum. Therapeutic agents. Cyclophosphamide (Mead Johnson and Company, Evansville, Indiana), and levamisole (Janssen R & D Inc., New Brunswick, New Jersey) were freshly dissolved and diluted in sterile water. All medications were administered as 0 5 cm3 intraperitoneal injections. Experimental design. Mice were randomly assigned to four different treatment groups. Two groups were given cyclophosphamide (50 mg/kg body weight) twice weekly for 4 weeks and two groups of control mice were untreated. After 4 weeks, one of the cyclophosphamide-treated groups and one of the untreated groups were given levamisole (125 ug per mouse) on 2 consecutive days each week for a total of 16 weeks, starting on day 3 after the last dose of cyclophosphamide. All mice were bled by orbital sinus puncture at intervals of 4 weeks. At the completion of 16 weeks of levamisole therapy, animals were killed by cervical dislocation and in vitro immunological studies and histological examinations were performed. Survival at 18 months was 90% in the cyclophosphamide only group and 60% in the other three groups. One animal in each of the latter three groups died after the last bleeding, prior to being killed. In the cyclophoshamide-levamisole group, three mice died in the first week of levamisole therapy. These were all in one cage, whereas there were no deaths in two other cages of similarly treated mice. In order to investigate a possible lethal effect of sequential cyclophosphamide-levamisole therapy, twenty-eight 9 month old DBA mice were given cyclophosphamide (2-5 mg/per mouse) twice weekly for 4 weeks. Groups of seven mice were then given levamisole, 125 ,pg daily, on two consecutive days each week for a total of 5 weeks starting on day 0, 4, 7 or 11 after the last dose of cyclophosphamide. None of the mice in the four groups died. Procedures. Anti-DNA assay. Blood obtained by orbital sinus puncture at various ages was allowed to clot at room temperature for 1 hr, and left at 4VC overnight. Serum was separated by centrifugation at 205 g for 15 min. DNAbinding activity of sequential individual sera was assayed by a cellulose ester filter radioimmunoassay previously described (Talal & Pillarisetty, 1976). Radioactive double-stranded DNA (900 ct/min per 4 ng DNA) from KB cells (obtained from Electro-Nucleonics Laboratories, Bethesda, Maryland) was incubated with decomplemented 10 ul aliquots of serum for 30 min at 37°C followed by an overnight incubation at 4°C. The antigen-antibody complexes were collected onto cellulose ester filters. The filters were placed in counting vials and covered with 10 cm3 of Liquifluor toluene scintillation medium. Radioactivity was determined in a Packard liquid scintillation counter. The results are expressed as corrected ct/min retained on the filter, which is directly related to the serum-binding capacity (Attias, Sylvester & Talal, 1973). Fractionation of serum. Serum samples from each treatment group were pooled separately. 200 ,l of pooled serum samples were subjected to ultracentrifugation in a 10-35% linear sucrose density gradient (0-15 M NaCl, pH 8.0) as previously described (Talal & Pillarisetty, 1975). Bovine thyroglobulin (19S,) human gamma globulin (7S) and haemoglobin (4S) were used as sedimentation markers. Forty fractions were collected and each one was analysed for antibody to DNA. Peak fractions were tested for immunoglobulin content. In the 19S region (fractions 10-20) only p chains were detected by Ouchterlony analysis, whereas in the 7S region (fractions 20-30) y chains but not pu chains were present. Antibodies to DNA were assayed in each fraction, as described above, using 50 pl aliquots from the gradient fractions. Comparison of 7S to 19S antibody binding. The radioactive binding profiles for DNA revealed clear distribution into the 7S and 19S peaks of activity after sucrose gradient fractionation (Roubinian, Papoian & Talal, 1977a). The radioactivity representing the fractionated 7S and 19S peaks within a single gradient were added and compared for total binding activity. Results with these fractionated sera were generally comparable to results with unfractionated whole serum. Assay of in vitro proliferative response. The spleens from each mouse were minced in RPMI 1640 medium and individual single cell suspensions were prepared. Spleen cell proliferative responses to concanavalin A and phytohaemagglutinin were measured in a microtitre plate proliferative assay (McCombs, Michalski & Talal, 1976). 5 x 105 spleen cells were cultured for 48 hr in 0-2 ml RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, 1% antibiotic-antimycotic and 1% glutamine (all from Pacific Biological Company). Stimulated cultures contained a final concentration of 30 ,pg/ml of purified phytohaemagglutinin, lot No. K6951 (Wellcome Research Laboratories, Brakenham, England), or 50 ,pg/ml of concanavalin A, lot No. 4218 (Nutritional Biological Company, Cleveland, Ohio). 10 ,pCi of tritiated thymidine, sp. act. 3 0 Ci/mmole (Schwartz-Mann, Orangeburg, New York) was added 4 hr before the cells were harvested onto glass fiber filters using a multiple automated sample harvester (Otto Hiller, Madison, Wisconsin). Results are expressed as the loglo of the net counts of quadruplicate stimulated and control cultures (Gottlieb, 1974). The stimulated responses of three tumourbearing animals were less than background and were not included in the analysis of the results. Statistical analysis. The significance of mean differences were determined by the Student's two-tailed t-test. Pathology. A full autopsy was performed on all animals killed after 16 weeks and specimens of kidney, liver, spleen, pancreas, adrenals, duodenum, mesenteric lymph nodes, gonads, lung, heart, thymus, submandibular, parotid and sublingual salivary glands and cervical lymph nodes were fixed in 10% formol saline and processed for routine histopathological exam-
ination. Only three animals had abnormalities on gross post-mortem examination. One mouse in the cyclophosphamide only group and one mouse in the levamisole only group had an apparent extension of a splenic tumour into the retroperitoneal and mesen-
Levamisole in murine lupus erythematosus
323
teric tissue. The tumours proved to be poorly differentiated spindle cell malignancies. One animal in the levamisole only group had massive splenomegaly. Specimens of kidney were quick-frozen in freon, and were cooled and stored in liquid nitrogen until sectioned. Cryostat sections were mounted on formol gelatin-coated slides, air-dried for 30 min and washed for 15 min twice in PBS, pH 7-2. Each slide was rinsed in distilled water and wiped dry around the section. The slides were then placed in a moist chamber, covered with antiserum and incubated for 30 min at room temperature in the dark. Rabbit polyvalent anti-mouse immunoglobulin (Behring Diagnostic, Sommerville, New Jersey, U.S.A., Lot 656 F/protein 2-8) was used at a titre of 1:20. Slides were then rinsed quickly in PBS and washed twice for 15 min in PBS with gentle agitation, and were then rinsed in distilled water and mounted using glycerin-PBS. Coded sections were examined in a Wild fluorescence microscope using transmitted u.v. light and a dark ground condenser. Direct glomerular immunofluorescence for immunoglobulin deposition was graded on a scale of 0 to + + + + by two independent observers who read the coded slides. The grading took into consideration the extent and intensity of immunoglobulin immunofluorescence. The incidence and extent of lymphomonocytic infiltrates in a major salivary gland, lung and kidney were similarly expressed on a scale of 0 to + + + + after examination by light microscopy.
RESULTS Treatment with cyclophosphamide significantly reduced antibodies to DNA in 14 month old NZB/W male mice (Fig. 1). In animals treated with cyclophosphamide alone, anti-DNA antibody levels rose within 1 month after termination of the cyclophosphamide therapy, and surpassed pretreatment values after 2 months. Levamisole treatment initiated 3 days after the last dose of cyclophosphamide was associated with a depression of anti-DNA antibodies for an additional 2 months after cyclophosphamide was stopped. 600
C
.E u
c 0 (V
2
4
4
8 Weeks
FIG. 1. Mean DNA-binding activity of survivors of different treatment groups. The ordinate shows the mean ct/min [3H]DNA bound by 10 ,ul ofserum from the survivors, the abscissa shows time. The arrow shows when cyclophosphamide was stopped. (-) No treatment (six survivors); (0) cyclophosphamide alone (nine survivors); (A) cyclophosphamide followed by levamisole (six survivors).
324
J.
Zulman et a.
700
600 _
500-
400-
C~~~~~~~~~ E~~~~~~~~~~~~~~~ 300
200-
00~~~~
0
4
8
12
16
Weeks
FIG. 2. Sequential serum DNA-binding activity of mice receiving levamisole only. The ordinate shows the ct/ min [3H]DNA bound by 10 Al of serum, the abscissa shows time. (0) Survivors; (0) non-survivors.* Death of mouse.
To determine whether the treatment had a specific effect on either IgG or IgM anti-DNA antibodies, measured the DNA-binding activity in serum fractions after separation by sucrose gradient ultracentrifugation. A marked decrease of DNA-binding activity was noted after 4 weeks of cyclophosphamide therapy, with a slightly greater depression of IgM than IgG. Levamisole therapy following cyclophosphamide was associated with continued suppression of both IgG and IgM anti-DNA antibodies. Levamisole treatment without prior cyclophosphamide was associated with the death of the mice with the highest initial DNA-binding activity (Fig. 2). The surviving mice had an initial mean DNA-binding activity of 160 ct/min compared to 290 ct/min for mice which died during treatment (P 0 3.
Levamisole in murine lupus erythematosus
325
TABLE 2. Immunoglobulin deposition in renal glomeruli and lymphocytic infiltrate of the kidney, salivary gland and lung (scale 0 to ++++). The fraction of specimens in each treatment group rated as + + + or + + + + is given in the parentheses
Treatment group and Glomerular immunoglobulin animal number deposition
Kidney, salivary gland and lung
lymphomonocytic infiltrate (A) No treatment 18 20 22 28 29 (B) Cyclophosphamide only 14 15 16 17 23 24 31 32 33 (C) Levamisole only 38 39 40 44 53 (D) Cyclophosphamide followed by levamisole 05 06 09 11 13
++(3/5) ++ +++ +++ +++
++++-(6/9)
++ +(4/5) + +++ +++
++ ++ ++++ ++
++ +(5/9) ++ ++ ++ ++++
++++ +++ ++++ ++++
+++ +++ ++
+++(3/5)
++(1/5)
+++ +++
++ +++ ++ ++
+(1/5)
+(0/5)
+ ++ + ++++
+ + + ++
++ ++
To determine if an augmented T-lymphocyte function (as measured by mitogen stimulation) was associated with decreased autoimmunity, we performed a linear regression analysis between the mitogenstimulated [3H]TdR incorporation and the final anti-DNA antibody titre of the individual mice. There was a significant inverse correlation between mitogen reactivity and anti-DNA levels for both Con A (r = 0-604, P
