Smooth Muscle Antibody in Heroin Addicts GUNNAR HUSBY, M.D., PAUL E. PIERCE, M.D., and RALPH C. WILLIAMS, Jr., M.D., F.A.C.P., Albuquerque, New Mexico
The occurrence of autoantibodies and the concentrations of serum immunoglobulins were studied in 102 heroin addicts, 20 former addicts who had abstained from heroin for 1 year or more, and 40 normal control subjects. Antibodies to smooth muscle ( 4 6 % ) and lymphocytotoxic antibodies ( 3 0 % ) were detected in the active heroin users, and there was a significantly positive correlation between these autoantibodies. Absorption experiments strongly suggested antigenic cross-reactivity between smooth muscle and lymphocyte membrane antigens. The presence of smooth muscle antibody in addicts was not clearly correlated with signs of active liver disease. The occurrence of smooth muscle antibody ( 1 0 % ) and lymphocytotoxic antibodies ( 1 5 % ) was significantly lower in the former heroin addicts than among active drug users. A significant elevation of IgM was found in the active heroin addicts. Gamma-M globulin was lower among the former heroin addicts but still elevated when compared with normal controls.
H E R O I N ADDICTS are notoriously prone to develop various
infectious complications ranging from tetanus to acute or subacute bacterial endocarditis (1-6). Previous studies of serologic or immunoglobulin-related abnormalities of this patient population have indicated a substantial increase in serum IgM (7, 8 ) , as well as a high prevalence of falsepositive serologic tests for syphilis (9, 10). In addition, opsonic antibodies for Staphylococcus aureus, as well as for various Gram-negative organisms, seem to be markedly increased in serum from heroin addicts ( 7 ) . A recent report has recorded the occurrence of cryoglobulins among a group of addicts during the course of subacute bacterial endocarditis (11). Our present study represented the results of an examination of serums from a large group of heroin addicts for other autoantibodies besides those already documented in previous reports (7, 9 ) . We thought that continuous and longstanding intravenous drug use must surely subject both the reticuloendothelial system and the immune system to a constant extraneous stimulus. Because mitochrondrial enzymes and other intracellular systems must be stimulated during long-term addiction and called on to react in an extraordinary fashion, we hypothesized that possible druginduced alteration in such intracellular machinery might be capable of stimulating autoantibody to altered cellular • From the Department of Medicine, Bernalillo County Medical Center, University of New Mexico School of Medicine, Albuquerque, New Mexico. Annals of Internal Medicine 83:801-805, 1975
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constituents. Our present report confirms the high prevalence of biologic false-positive serologies for syphilis and elevation of IgM in such patients and also indicates that a large proportion of heroin addicts show substantial amounts of smooth muscle antibody. In addition, we found lymphocytotoxic antibodies with increased frequency in these subjects. Materials and Methods SUBJECTS
Serums from 102 heroin addicts were collected during a year's interval when individuals were undergoing their initial examination for entry into a methadone maintenance program. At this time, all subjects underwent a complete history and physical examination, together with a screening battery of laboratory studies that included complete blood count, urinalysis, serologic test for syphilis (rapid plasma reagin and fluorescent treponemal antibody), serum glutamic oxalacetic transaminase, and blood glucose. Based on these examinations, the active heroin addicts were divided into two main groups, namely those with liver involvement and those without evidence of liver dysfunction (Table 1). Aliquots of serum were stored at - 7 0 °C until retested in many of the assays described below. Twelve serum samples were obtained 2 or 3 years later from persons subsequently maintained on methadone who were known on the basis of frequent urinary screens for opiates to have clearly discontinued intravenous or other parenteral routes of drug use. Eight additional serum samples were collected from persons who were residents of a drug-free therapeutic community; all of the latter were also known by urine monitoring to have been drugfree for at least 1 year. In addition, fresh serum samples were obtained from a small group of heroin addicts hospitalized with pneumonia, overdose, or concussion in order to confirm the general pattern of reactivity in the panel of tests examined. SPECIAL STUDIES
Immunoglobulin levels (IgG, IgA, IgM) were estimated in 70 active heroin addicts and in 20 former heroin addicts by means of the Oudin tube (12) or Mancini radial diffusion method (13). Gamma-D globulin was also quantitated in 50 addict serums using low level Ig quantitation plates from Meloy Laboratories, Inc., Springfield, Virginia, while quantitative determinations of IgE were done on 50 serums using the Phaedabas Kit obtained from Pharmacia Laboratories, Inc., Piscataway, New Jersey. Screening for hepatitis B associated antigen (HBAg) was done in all subjects studied using a modified gel diffusion method and the hepatitis-associated antibody AUS-tect kit purchased from Abbott Laboratories, North Chicago, Illinois. Forty serums obtained from blood donors, laboratory personnel, and medical students constituted normal controls. AUTOANTIBODIES
Antigammaglobulin antibodies were determined using the Ripley cell agglutination method (14). In addition, lympho-
801
Table 1. Serum Immunoglobulins in Active Heroin Addicts, Former Heroin Addicts, and Controls Subjects Active heroin addicts With liver involvement, 46 Without liver involvement, 56 Total, 102 Former heroin addicts, 20 f Normal controls, 40
1198 1156 1176 1263 1278
± + + ± ±
IgM
IgA
IgG 163 198 182 102 361
247 213 229 298 282
± + ± ± ±
62 65 65 80 128
291 287 298 206 135
± 40* ±90* ±88* ± 65* ± 65
IgD
IgE
7 ±5 5 ±4 5 ±4 Not done 8 ± 11
418 ± 764 178 ± 109 293 ± 541 Not done 175 ± 114
* IgM is significantly higher in both groups of active heroin addicts than in formeir heroin addicts ( P < 0 . ()1) and in normal coritrols ( P < 0.001). IgM is significantly higher in former hieroin addicts than in n ormal controls (P < 0.(M t Twelve former heroin addicts had t>een on methadone maintenance for > 1 year, ;and 8 had been completer,y drug-free for > 1 yeai
cytotoxic antibodies were determined by the method of Terasaki and McClelland (15) using a panel of 20 normal donors of varying HL-A phenotype. In absorption experiments, selected serums were absorbed three times with purified normal peripheral blood lymphocytes (16), 5 X 107 cells/ml, two times for 1 hour at 15 °C, and the third time at 4 °C overnight. Antinuclear-antibodies were determined using mouse and rat liver (17, 18), and an indirect immunofluorescence method with application of polyvalent rabbit antihuman Ig after initial overlay of sections of dilutions of test serum beginning at 1:10. Antiparietal cell and antimitochondrial antibodies were also determined by indirect immunofluorescence using mouse and rat stomach and rat kidney as test tissue substrates (19, 20). Smooth muscle antibodies were assayed using frozen sections of mouse, rat, and guinea pig ileum and stomach. Positive serums were also examined using fluoresceinated antiserums specific for IgG, IgA, and IgM to determine the immunoglobulin class of the actual autoantibodies. Specificity of antismooth muscle antibodies was determined by absorption of serums with homogenates of several preparations of mouse, rat, and human smooth muscle. The smooth muscle preparations, 30 to 60 mg protein/ml, were insolubilized using glutaraldehyde (21) to avoid the formation of immune complexes and dilution of the absorbed serums. Insolubilized normal human serums served as control. In addition, blocking experiments using absorption of anti-Ig antiserums with purified IgG, IgA, or IgM preparations were done to confirm specificity of fluorescence. In serologic tests for syphilis, we used the rapid plasma reagin card method initially, which used agglutination of cardiolipin absorbed onto charcoal particles. Since a majority of heroin addict serums studied showed weakly reactive or positive rapid plasma reagin reactions, it was initially thought that such results might be associated somehow with the other autoantibodies, such as smooth muscle antibody present in the same serums. Accordingly, absorptions of rapid plasma reagin positive addict serums with cardiolipin charcoal reagent were
done and smooth muscle and other antibodies measured before and after such treatment. In addition, the specificity of the positive rapid plasma reagin reactions were tested with the indirect immunofluorescence method (fluorescent treponemal antibody). Finally, addict serums were examined for any particular or unique reactivity that they might show with antigens present in addict liver by immunofluorescence with fresh liver biopsy frozen sections obtained from other addicts hospitalized for various diagnostic procedures. In these instances, the liver biopsy samples were obtained from patients who had been and were currently negative for antibody to hepatitis B antigen. Results
In all, some 102 serums from active heroin addicts were examined. Clearcut elevation of serum IgM (289 ± 88 mg/ml) was recorded, with no marked or uniform changes in IgG, IgA, or IgD. Gamma-E globulin seemed to be increased in the group of heroin addicts who had evidence of liver involvement, but this increase was not statistically significant (Table 1). In the 20 subjects who had abstained from heroin for more than 1 year, the serum IgM (206 ± 65 mg/ml) was significantly lower than in the active heroin users (P < 0.01) but still considerably elevated when compared with the normal controls (P < 0.001) (Table 1). Hepatitis B associated antigen was found in the serum of 13 of the 102 (12%) active heroin addicts, and 9 of these belonged to the group with liver dysfunction (Table 2 ) . A similar frequency of HBAg-positive serums (13%) was found among the former heroin addicts (Table 3). False-positive serology for syphilis was found in 69% of the active heroin addicts and in 40% of the subjects who
Table 2. Serum Antibodies and HBAg in Active Heroin Addicts
Antibody
Active Heroin Addicts Total
Rapid plasma reagin Fluorescent antitreponemal antibody False positive rapid plasma reagin Smooth muscle antibodies Lymphocytotoxic antibodies Rheumatoid factor Antinuclear antibodies Mitochondrial antibodies Parietal cell antibodies HBAg 802
no. positive/ % positive no. tested 77/102 75 7/102 7 70/102 69 47/102 46 12/40 30 10/102 10 4/102 4 1/102 1 0/102 13/102 12
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With Liver Involvement no. positive/ no. tested 34/46 3/46 31/46 23/46 6/21 3/46 3/46 1/46 0/46 9/46
% positive 74 7 67 50 29 7 7 2
... 20
Without Liver Involvement no. positive/ no. tested 43/56 4/56 39/56 24/56 6/19 7/56 1/56 0/56 0/56 4/56
% positive 11 1 70 43 32 13 2
... 7
Table 3. Serum Antibodies and HBAg in Former Heroin Addicts and Normal Controls
Antibody Rapid plasma reagin Fluorescent antitreponemal antibody False positive rapid plasma reagin Smooth muscle Lymphocytotoxic antibodies Rheumatoid factor Antinuclear antibodies Mitochondrial antibodies Parietal cell antibodies HBAg
Normal Controls
Former Heroin Addicts no. positive/no. tested 9/20 1/20 8/20 2/20 3/20 0/20 1/20 1/20 0/20 3/20
had abstained from heroin for 1 year or longer (Tables 2 and 3) (0.1 > P > 0.05). The most interesting reactions involving antibodies to tissue antigens in the addict serums were found in the high prevalence of reactivity for smooth muscle among 46% of 102 serums tested with titers varying from 10 to 256. Some variation in general pattern of staining was noted; however, typical full-fledged general myofibrillar staining was seen with most serums (Figure 1). Variations of the immunofluorescent staining pattern that also involved apical portions of gastric gland cells and to a certain extent other intracellular regions were also observed (Figure 2 ) . These patterns were clearly abolished by absorption with smooth muscle antigen extracts. No staining of skeletal muscle fibers was noted. Indirect immunofluorescence using addict serum and monospecific antibody to IgG, IgA, and IgM indicated that in all instances the addict serum smooth muscle antibody was mainly IgG, but in some instances also IgM or IgA, or both. Control experiments showed that the pepsin F ( a b / ) 2 fragments of isolated IgG from smooth muscle antibodypositive serums showed antibody activity to smooth muscle with titers comparable with those of the whole serums. Thus, the reactions observed were true antibody reactions and not, for example, binding of the Fc part of the antibody molecules to smooth muscle. No positive correlation between the occurrence of smooth muscle antibody and positive rapid plasma reagin tests was found. Absorption of serums positive in both test systems with rapid plasma reagin antigen completely abolished the rapid plasma reagin reactivity, while no difference was observed in the reactivity with smooth muscle after such absorption (Table 4 ) . The frequency of smooth muscle antibody was only slightly higher in the former heroin addicts ( 1 0 % ) than in the controls ( 5 % ) . Lymphocytotoxic antibodies were detected in 12 out of 40 active heroin users tested ( 3 0 % , Table 2 ) . Ten of these subjects also had smooth muscle antibodies, and this correlation was statistically significant (P < 0.01, FisherIrvin test), although several serums studied showed lymphocytotoxic antibody without smooth muscle antibody or the opposite situation. After absorption of several such serums with lymphocytes, neither lymphocytotoxic antibodies nor smooth muscle antibody could be seen (Table
% positive 45 5 40 10 15
. .. 5 5
13
no. positive/no. tested 0/40 Not done 0/40 2/40 1/40 0/40 1/40 0/40 0/40 0/40
% positive Not done
2.5
... . ..
4 ) . Furthermore, absorptions of three such serums with smooth muscle preparations also abolished the reactivity in the lymphocytotoxic assay, as well as the test for smooth muscle antibody, suggesting similarities between smooth muscle and lymphocytic antigens. In contrast, absorption with rapid plasma reagin antigen did not affect the lymphocytotoxic antibodies (Table 4 ) . In the former heroin users lymphocytotoxic antibodies were detected in 3 of the 20 subjects, and smooth muscle antibody was found in two of these serums, further suggesting a positive correlation between these autoantibodies. No significant increase in antimitochondrial, parietal cell, or antinuclear antibody was recorded in the active or former heroin addicts, while 10% of the active addicts had rheumatoid factor activity in serum (Tables 2 and 3 ) . No pattern showing unique reactivity for antigens present in addict hepatic tissues was observed when liver tissues from heroin addicts were used as substrate for antigen. Because it has been our experience that many of the heroin addicts seen at this institution also have a history of intermittent or concomitant alcohol abuse, interval clinical records and hospital charts on all patients were examined for indications of initial or interval liver enlargement, alcoholism, or evidence of chronic hepatic dysfunction. It was thought that in view of the high prevalence of smooth
Figure 1. Smooth muscle antibodies in the serum of a heroin addict, detected by indirect immunofluorescence method, using mouse stomach as substrate for antigen. Strongly fluorescent smooth muscle fibers are shown both in the muscularis mucosae and in the muscularis propriae. (Magnification, X 400.) Husby et al. • Smooth Muscle Antibody in Addicts
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5" 2.5
803
Figure 2. Serum from a heroin addict showing reactivity with cytoplasmic antigens of cells in mouse gastric mucosa. Note the strong staining in the apical portion of the cells. (Magnification, X 400.)
muscle antibody, this was of particular importance. However, no significant differences in the frequency of serum antibodies or level of immunoglobulins were found in addicts with or without evidence of liver involvement; however, slightly higher concentrations of IgE were recorded in the former group (Table 1). Serum HBAg was also more frequent among these subjects (Table 2 ) . Admittedly, in these persons exact definition of those with and without any vestiges of low grade hepatitis is difficult without concurrent liver biopsy. However, this was not possible in the entire group of heroin addicts studied. Discussion
The present study again documents the marked elevation of serum IgM among heroin addicts (7, 8). Previous studies of serums from ex-addicts maintained on oral methadone subsequent to their presumed discontinuation of intravenous drug use have indicated that IgM then tends to be lower and closer to normal levels (8). Our data confirm this earlier report. A clear explanation for the presence of elevated IgM during active heroin abuse is not yet available. It is conceivable that it could be used as a surveillance check on methadone-program patients who might be thought to be surreptitiously adding intravenous heroin to their daily methadone maintenance program. However, more long-term data on comparative values before and after well-documented discontinuation of intravenous drug use need to be made before a valid conclusion can be drawn. 804
The striking finding of the presence of smooth muscle antibody among 46% of all active heroin addicts studied is a phenomenon of general interest. Smooth muscle antibody itself was originally described in the serums of patients with chronic active hepatitis (22). Subsequently, the occurrence of this antibody in a wide variety of other diseases, including viral hepatitis, malignancy, and infectious mononucleosis, has been documented (23-26). At present, a clear explanation for the occurrence of smooth muscle antibody in heroin addict serums is not available. It may represent a cross-reaction between antibodies to various bacterial or fungal antigens adulterating the usual batches of illicit drugs and some of the antigenic constituents of smooth muscle antigen. The latter has recently been characterized by Trenchev, Sneyd, and Holborow (27) as a complex mixture of at least six distinct molecular species of antigens related, in many instances, to indigenous contractile proteins present in many tissues of the body. The concomitant presence of smooth muscle antibody and lymphocytotoxic antibody in a large proportion of subjects studied here is of considerable interest. A recent report by Fagraeus, Lidman, and Biberfeld (28), has documented the apparent presence of some lymphocyte membrane-related contractile proteins similar to actomyosin. This could suggest that the antismooth muscle reactivity in heroin addict serums might also react with contractile elements present in certain lymphocyte surface proteins (29). Absorption experiments documented in this study were of interest in this regard. Cross-reactions of this sort may also be involved in the genesis of strongly lymphocytotoxic antibodies in the serums of some patients with chronic active hepatitis (30) or systemic lupus erythematosus (31, 32). Recent reports by Rizetto and Doniach (33) have recorded reticulin antibodies in serums of heroin addicts. However, we were not able to find staining patterns fulfilling the authors' criteria for reticulin antibodies in the present material. Heroin addiction, unfortunately, remains a clinical cornucopia of unusual medical complications (1, 34). Abnormalities related to the immune response, serum immunoglobulins, and the occurrence of various autoantibodies are probably part and parcel of this disorder (35). The clinical condition itself provides an interesting but unfortunate model for the study of certain aspects of what could be considered an autoimmune response, since selfadministration of illicit opiate preparations seems to result Table 4. Antibody Activity in Serum After Absorptions with Rapid Plasma Reagin, Smooth Muscle, and Normal Lymphocytes
Absorption With
Rapid plasma reagin Smooth muscle Normal lymphocytes Normal human serum
Smooth Muscle Antibody
Lymphocytotoxic Antibodies
0
+
+
+
+
+ + +
* 3 serums were subjected to absorption.
December 1975 • Annals of Internal Medicine • Volume 83 • Number 6
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Antibody Activity* Rapid Plasma Reagin
0 0
0 0
in the formation of autoantibodies. Whether this is due to tissue enzymatic alterations related to drug metabolism or to drug-tissue interactions remains to be elucidated.
15. TERASAKI PI, MCCLELLAND JD: Microdroplet assay of human serum cytotoxins. Nature (Lond) 204:998-1000, 1964 16. B0YUM A: Separation of leukocytes from human blood and bone marrow. Scand J Clin Lab Invest [(Suppl)21] 97:9-88, 1968
ACKNOWLEDGMENTS: The authors thank Mrs. Celia Diaz for technical assistance and Ms. Bernadette Marquez for help with the manuscript. Grant support: in part by grants AM13690-05 and AM AI13824-04 from the U.S. Public Health Service, and by a grant from the New Mexico Arthritis Foundation. Received 9 June 1975; revision accepted 27 August 1975.
17. ROTHFIELD NF, STOLLAR BD: The relation of immunoglobulin
• Requests for reprints should be addressed to Ralph C. Williams, Jr., M.D., Department of Medicine, Bernalillo County Medical Center, 5th Floor, University of New Mexico School of Medicine, Stanford and Lomas, Albuquerque, NM 87106. References 1. LOURIA DB, HENSLE T, ROSE J: The major medical complica-
tions of heroin addiction. Ann Intern Med 67:1-22, 1967 2. BRIGGS JH, MCKERRON CG, SOUHAMI RL, et al: Severe systemic
infections complicating "mainline" heroin addiction. Lancet 2: 1227-1231, 1967 3. CHERUBIN CE, BROWN J: Systemic infections in heroin addicts. Lancet 1:298-299, 1968 4. CONWAY N: Endocarditis in heroin addicts. Br Heart J 31:543545, 1969 5. WILLIS J: Systemic infection in heroin addicts. Lancet 1:361, 1968
class, pattern of antinuclear antibody, and complement-fixing antibodies to DNA in sera from patients with systemic lupus erythematosus. J Clin Invest 46:1785-1794, 1967 18. TAN EM: Relationship of nuclear staining patterns with precipitating antibodies in systemic lupus erythematosus. / Lab Clin Med 70:800-812, 1967 19. IRVINE WJ: Gastric antibodies studied by fluorescence microscopy. Q J Exp Physiol 48:427-438, 1963 20. WALKER JG, DONUCH D, DONIACH I: Mitochondrial antibodies
and subclinical liver disease. Q J Med 39:31-48, 1970 21. AVRAMEAS S, TERNYNCK T: The cross linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. lmmunochemistry 6:53-66, 1969 22. JOHNSON GD, HOLBOROW EJ, GLYNN LE: Antibody to smooth
muscle in patients with liver disease. Lancet 2:878-879, 1965 23. DONUCH D, WALKER JG: A unified concept of autoimmune hepatitis. Lancet 1:813-815, 1969 24. WHITEHOUSE JMA, HOLBOROW EJ: Smooth muscle antibody in malignant disease. Br Med 7 4:511-513, 1971 25. HOLBOROW EJ, HEMSTED EH, MEAD SV: Smooth muscle auto-
antibodies in infectious mononucleosis. Br Med J 3:323-325, 1973 26. FARROW U , HOLBOROW EJ, JOHNSON GD, et al: Autoantibodies
and the hepatitis-associated antigen in acute infective hepatitis. Br Med J 2:693-695, 1970
6. CHERUBIN CE, BADEN M, KAVALER F, et al: Infective endo-
27. TRENCHEV P, SNEYD P, HOLBOROW EJ: Immunofluorescent trac-
carditis in narcotic addicts. Ann Intern Med 69:1091-1098, 1968
ing of smooth muscle contractile protein antigens in tissues other than smooth muscle. Clin Exp Immunol 16:125-136, 1974
7. NICKERSON DS, WILLIAMS RC JR, BOXMEYER M, et al: Increased
opsonic capacity of serum in chronic heroin addiction. Ann Internal Med 72:671-677, 1970 8. CUSHMAN P, GRIECO MH: Hyperimmunoglobulinemia associated
with narcotic addiction. Effects of methadone maintenance treatment. Am J Med 54:320-326, 1973 9. BOAK RA, CARPENTER CM, MILLER JN: Biologic false-positive
reactions for syphilis among narcotic addicts. A report on the incidence of BFP reactions as measured by TPI test. JAMA 175: 326, 1961 10. CHERUBIN CE: The medical sequelae of narcotic addiction. Ann Intern Med 67:23-33, 1967 11. HURWITZ D, QUISMORIO FP, FRIOU GH: Cryoglobulinaemia in
patients with infectious endocarditis. Clin Exp Immunol 19:131141, 1975
28. FAGRAEUS A, LIDMAN K, BIBERFELD G:
Reaction of human
smooth muscle antibodies with human blood lymphocytes and lymphoid cell lines. Nature (Lond) 252:246-247, 1974 29. PAINTER RG, SHEETZ M, SINGER SJ: Detection and ultrastruc-
tural localization of human smooth muscle myosin-like molecules in human non-muscle cells by specific antibodies. Proc Natl Acad Sci USA 72:1359-1363, 1975 30. DEHORATIUS RJ, STRICKLAND RG, WILLIAMS RC JR: T and B
lymphocytes in acute and chronic hepatitis. Clin Immunol Immunopathol 2:353-360, 1974 31. MITTAL KK, ROSSEN RD, SHARP JT, et al: Lymphocytotoxic
antibodies in systemic lupus erythematosus. Nature (Lond) 225: 1255-1256, 1970 32. TERASAKI PI, MOTTIRONI VD, BARNETT EV: Cytotoxins in dis-
quantitation of antigens by single radial diffusion, lmmunochemistry 2:235-254, 1965
ease: autocytotoxins in lupus. N Engl J Med 283:724-728, 1970 33. RIZETTO M, DONUCH D: Types of 'reticulin' antibodies detected in human sera by immunofluorescence. J Clin Pathol 26:841851, 1973 34. SAPIRA JD: The narcotic addict as a medical patient. Am J Med 45:555-588, 1968
14. WALLER M, LAWLER SD: A study of the properties of the
35. BROWN SM, STIMMEL B, TAUB RN, et al: Immunologic dys-
Rhesus antibody (Ri) diagnostic for the rheumatoid factor and its application to Gm grouping. Vox Sang 7:591-606, 1962
function in heroin addicts. Arch Intern Med 134:1001-1011, 1974
12. WILSON ID, WILLIAMS RC JR, TOBIAN L JR: Renal tubular
acidosis. Three cases with immunoglobulin abnormalities in the patients and their kindreds. Am J Med 43:356-370, 1967 13. MANCINI G, CARBONARA AD, HEREMANS JF: Immunochemical
Husby et a/. • Smooth Muscle Antibody in Addicts
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805
The occurrence of autoantibodies and the concentrations of serum immunoglobulins were studied in 102 heroin addicts, 20 former addicts who had abstained from heroin for 1 year or more, and 40 normal control subjects. Antibodies to smooth muscle ( 4 6 % ) and lymphocytotoxic antibodies ( 3 0 % ) were detected in the active heroin users, and there was a significantly positive correlation between these autoantibodies. Absorption experiments strongly suggested antigenic cross-reactivity between smooth muscle and lymphocyte membrane antigens. The presence of smooth muscle antibody in addicts was not clearly correlated with signs of active liver disease. The occurrence of smooth muscle antibody ( 1 0 % ) and lymphocytotoxic antibodies ( 1 5 % ) was significantly lower in the former heroin addicts than among active drug users. A significant elevation of IgM was found in the active heroin addicts. Gamma-M globulin was lower among the former heroin addicts but still elevated when compared with normal controls.
H E R O I N ADDICTS are notoriously prone to develop various
infectious complications ranging from tetanus to acute or subacute bacterial endocarditis (1-6). Previous studies of serologic or immunoglobulin-related abnormalities of this patient population have indicated a substantial increase in serum IgM (7, 8 ) , as well as a high prevalence of falsepositive serologic tests for syphilis (9, 10). In addition, opsonic antibodies for Staphylococcus aureus, as well as for various Gram-negative organisms, seem to be markedly increased in serum from heroin addicts ( 7 ) . A recent report has recorded the occurrence of cryoglobulins among a group of addicts during the course of subacute bacterial endocarditis (11). Our present study represented the results of an examination of serums from a large group of heroin addicts for other autoantibodies besides those already documented in previous reports (7, 9 ) . We thought that continuous and longstanding intravenous drug use must surely subject both the reticuloendothelial system and the immune system to a constant extraneous stimulus. Because mitochrondrial enzymes and other intracellular systems must be stimulated during long-term addiction and called on to react in an extraordinary fashion, we hypothesized that possible druginduced alteration in such intracellular machinery might be capable of stimulating autoantibody to altered cellular • From the Department of Medicine, Bernalillo County Medical Center, University of New Mexico School of Medicine, Albuquerque, New Mexico. Annals of Internal Medicine 83:801-805, 1975
Downloaded from https://annals.org by Tulane University user on 01/20/2019
constituents. Our present report confirms the high prevalence of biologic false-positive serologies for syphilis and elevation of IgM in such patients and also indicates that a large proportion of heroin addicts show substantial amounts of smooth muscle antibody. In addition, we found lymphocytotoxic antibodies with increased frequency in these subjects. Materials and Methods SUBJECTS
Serums from 102 heroin addicts were collected during a year's interval when individuals were undergoing their initial examination for entry into a methadone maintenance program. At this time, all subjects underwent a complete history and physical examination, together with a screening battery of laboratory studies that included complete blood count, urinalysis, serologic test for syphilis (rapid plasma reagin and fluorescent treponemal antibody), serum glutamic oxalacetic transaminase, and blood glucose. Based on these examinations, the active heroin addicts were divided into two main groups, namely those with liver involvement and those without evidence of liver dysfunction (Table 1). Aliquots of serum were stored at - 7 0 °C until retested in many of the assays described below. Twelve serum samples were obtained 2 or 3 years later from persons subsequently maintained on methadone who were known on the basis of frequent urinary screens for opiates to have clearly discontinued intravenous or other parenteral routes of drug use. Eight additional serum samples were collected from persons who were residents of a drug-free therapeutic community; all of the latter were also known by urine monitoring to have been drugfree for at least 1 year. In addition, fresh serum samples were obtained from a small group of heroin addicts hospitalized with pneumonia, overdose, or concussion in order to confirm the general pattern of reactivity in the panel of tests examined. SPECIAL STUDIES
Immunoglobulin levels (IgG, IgA, IgM) were estimated in 70 active heroin addicts and in 20 former heroin addicts by means of the Oudin tube (12) or Mancini radial diffusion method (13). Gamma-D globulin was also quantitated in 50 addict serums using low level Ig quantitation plates from Meloy Laboratories, Inc., Springfield, Virginia, while quantitative determinations of IgE were done on 50 serums using the Phaedabas Kit obtained from Pharmacia Laboratories, Inc., Piscataway, New Jersey. Screening for hepatitis B associated antigen (HBAg) was done in all subjects studied using a modified gel diffusion method and the hepatitis-associated antibody AUS-tect kit purchased from Abbott Laboratories, North Chicago, Illinois. Forty serums obtained from blood donors, laboratory personnel, and medical students constituted normal controls. AUTOANTIBODIES
Antigammaglobulin antibodies were determined using the Ripley cell agglutination method (14). In addition, lympho-
801
Table 1. Serum Immunoglobulins in Active Heroin Addicts, Former Heroin Addicts, and Controls Subjects Active heroin addicts With liver involvement, 46 Without liver involvement, 56 Total, 102 Former heroin addicts, 20 f Normal controls, 40
1198 1156 1176 1263 1278
± + + ± ±
IgM
IgA
IgG 163 198 182 102 361
247 213 229 298 282
± + ± ± ±
62 65 65 80 128
291 287 298 206 135
± 40* ±90* ±88* ± 65* ± 65
IgD
IgE
7 ±5 5 ±4 5 ±4 Not done 8 ± 11
418 ± 764 178 ± 109 293 ± 541 Not done 175 ± 114
* IgM is significantly higher in both groups of active heroin addicts than in formeir heroin addicts ( P < 0 . ()1) and in normal coritrols ( P < 0.001). IgM is significantly higher in former hieroin addicts than in n ormal controls (P < 0.(M t Twelve former heroin addicts had t>een on methadone maintenance for > 1 year, ;and 8 had been completer,y drug-free for > 1 yeai
cytotoxic antibodies were determined by the method of Terasaki and McClelland (15) using a panel of 20 normal donors of varying HL-A phenotype. In absorption experiments, selected serums were absorbed three times with purified normal peripheral blood lymphocytes (16), 5 X 107 cells/ml, two times for 1 hour at 15 °C, and the third time at 4 °C overnight. Antinuclear-antibodies were determined using mouse and rat liver (17, 18), and an indirect immunofluorescence method with application of polyvalent rabbit antihuman Ig after initial overlay of sections of dilutions of test serum beginning at 1:10. Antiparietal cell and antimitochondrial antibodies were also determined by indirect immunofluorescence using mouse and rat stomach and rat kidney as test tissue substrates (19, 20). Smooth muscle antibodies were assayed using frozen sections of mouse, rat, and guinea pig ileum and stomach. Positive serums were also examined using fluoresceinated antiserums specific for IgG, IgA, and IgM to determine the immunoglobulin class of the actual autoantibodies. Specificity of antismooth muscle antibodies was determined by absorption of serums with homogenates of several preparations of mouse, rat, and human smooth muscle. The smooth muscle preparations, 30 to 60 mg protein/ml, were insolubilized using glutaraldehyde (21) to avoid the formation of immune complexes and dilution of the absorbed serums. Insolubilized normal human serums served as control. In addition, blocking experiments using absorption of anti-Ig antiserums with purified IgG, IgA, or IgM preparations were done to confirm specificity of fluorescence. In serologic tests for syphilis, we used the rapid plasma reagin card method initially, which used agglutination of cardiolipin absorbed onto charcoal particles. Since a majority of heroin addict serums studied showed weakly reactive or positive rapid plasma reagin reactions, it was initially thought that such results might be associated somehow with the other autoantibodies, such as smooth muscle antibody present in the same serums. Accordingly, absorptions of rapid plasma reagin positive addict serums with cardiolipin charcoal reagent were
done and smooth muscle and other antibodies measured before and after such treatment. In addition, the specificity of the positive rapid plasma reagin reactions were tested with the indirect immunofluorescence method (fluorescent treponemal antibody). Finally, addict serums were examined for any particular or unique reactivity that they might show with antigens present in addict liver by immunofluorescence with fresh liver biopsy frozen sections obtained from other addicts hospitalized for various diagnostic procedures. In these instances, the liver biopsy samples were obtained from patients who had been and were currently negative for antibody to hepatitis B antigen. Results
In all, some 102 serums from active heroin addicts were examined. Clearcut elevation of serum IgM (289 ± 88 mg/ml) was recorded, with no marked or uniform changes in IgG, IgA, or IgD. Gamma-E globulin seemed to be increased in the group of heroin addicts who had evidence of liver involvement, but this increase was not statistically significant (Table 1). In the 20 subjects who had abstained from heroin for more than 1 year, the serum IgM (206 ± 65 mg/ml) was significantly lower than in the active heroin users (P < 0.01) but still considerably elevated when compared with the normal controls (P < 0.001) (Table 1). Hepatitis B associated antigen was found in the serum of 13 of the 102 (12%) active heroin addicts, and 9 of these belonged to the group with liver dysfunction (Table 2 ) . A similar frequency of HBAg-positive serums (13%) was found among the former heroin addicts (Table 3). False-positive serology for syphilis was found in 69% of the active heroin addicts and in 40% of the subjects who
Table 2. Serum Antibodies and HBAg in Active Heroin Addicts
Antibody
Active Heroin Addicts Total
Rapid plasma reagin Fluorescent antitreponemal antibody False positive rapid plasma reagin Smooth muscle antibodies Lymphocytotoxic antibodies Rheumatoid factor Antinuclear antibodies Mitochondrial antibodies Parietal cell antibodies HBAg 802
no. positive/ % positive no. tested 77/102 75 7/102 7 70/102 69 47/102 46 12/40 30 10/102 10 4/102 4 1/102 1 0/102 13/102 12
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With Liver Involvement no. positive/ no. tested 34/46 3/46 31/46 23/46 6/21 3/46 3/46 1/46 0/46 9/46
% positive 74 7 67 50 29 7 7 2
... 20
Without Liver Involvement no. positive/ no. tested 43/56 4/56 39/56 24/56 6/19 7/56 1/56 0/56 0/56 4/56
% positive 11 1 70 43 32 13 2
... 7
Table 3. Serum Antibodies and HBAg in Former Heroin Addicts and Normal Controls
Antibody Rapid plasma reagin Fluorescent antitreponemal antibody False positive rapid plasma reagin Smooth muscle Lymphocytotoxic antibodies Rheumatoid factor Antinuclear antibodies Mitochondrial antibodies Parietal cell antibodies HBAg
Normal Controls
Former Heroin Addicts no. positive/no. tested 9/20 1/20 8/20 2/20 3/20 0/20 1/20 1/20 0/20 3/20
had abstained from heroin for 1 year or longer (Tables 2 and 3) (0.1 > P > 0.05). The most interesting reactions involving antibodies to tissue antigens in the addict serums were found in the high prevalence of reactivity for smooth muscle among 46% of 102 serums tested with titers varying from 10 to 256. Some variation in general pattern of staining was noted; however, typical full-fledged general myofibrillar staining was seen with most serums (Figure 1). Variations of the immunofluorescent staining pattern that also involved apical portions of gastric gland cells and to a certain extent other intracellular regions were also observed (Figure 2 ) . These patterns were clearly abolished by absorption with smooth muscle antigen extracts. No staining of skeletal muscle fibers was noted. Indirect immunofluorescence using addict serum and monospecific antibody to IgG, IgA, and IgM indicated that in all instances the addict serum smooth muscle antibody was mainly IgG, but in some instances also IgM or IgA, or both. Control experiments showed that the pepsin F ( a b / ) 2 fragments of isolated IgG from smooth muscle antibodypositive serums showed antibody activity to smooth muscle with titers comparable with those of the whole serums. Thus, the reactions observed were true antibody reactions and not, for example, binding of the Fc part of the antibody molecules to smooth muscle. No positive correlation between the occurrence of smooth muscle antibody and positive rapid plasma reagin tests was found. Absorption of serums positive in both test systems with rapid plasma reagin antigen completely abolished the rapid plasma reagin reactivity, while no difference was observed in the reactivity with smooth muscle after such absorption (Table 4 ) . The frequency of smooth muscle antibody was only slightly higher in the former heroin addicts ( 1 0 % ) than in the controls ( 5 % ) . Lymphocytotoxic antibodies were detected in 12 out of 40 active heroin users tested ( 3 0 % , Table 2 ) . Ten of these subjects also had smooth muscle antibodies, and this correlation was statistically significant (P < 0.01, FisherIrvin test), although several serums studied showed lymphocytotoxic antibody without smooth muscle antibody or the opposite situation. After absorption of several such serums with lymphocytes, neither lymphocytotoxic antibodies nor smooth muscle antibody could be seen (Table
% positive 45 5 40 10 15
. .. 5 5
13
no. positive/no. tested 0/40 Not done 0/40 2/40 1/40 0/40 1/40 0/40 0/40 0/40
% positive Not done
2.5
... . ..
4 ) . Furthermore, absorptions of three such serums with smooth muscle preparations also abolished the reactivity in the lymphocytotoxic assay, as well as the test for smooth muscle antibody, suggesting similarities between smooth muscle and lymphocytic antigens. In contrast, absorption with rapid plasma reagin antigen did not affect the lymphocytotoxic antibodies (Table 4 ) . In the former heroin users lymphocytotoxic antibodies were detected in 3 of the 20 subjects, and smooth muscle antibody was found in two of these serums, further suggesting a positive correlation between these autoantibodies. No significant increase in antimitochondrial, parietal cell, or antinuclear antibody was recorded in the active or former heroin addicts, while 10% of the active addicts had rheumatoid factor activity in serum (Tables 2 and 3 ) . No pattern showing unique reactivity for antigens present in addict hepatic tissues was observed when liver tissues from heroin addicts were used as substrate for antigen. Because it has been our experience that many of the heroin addicts seen at this institution also have a history of intermittent or concomitant alcohol abuse, interval clinical records and hospital charts on all patients were examined for indications of initial or interval liver enlargement, alcoholism, or evidence of chronic hepatic dysfunction. It was thought that in view of the high prevalence of smooth
Figure 1. Smooth muscle antibodies in the serum of a heroin addict, detected by indirect immunofluorescence method, using mouse stomach as substrate for antigen. Strongly fluorescent smooth muscle fibers are shown both in the muscularis mucosae and in the muscularis propriae. (Magnification, X 400.) Husby et al. • Smooth Muscle Antibody in Addicts
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5" 2.5
803
Figure 2. Serum from a heroin addict showing reactivity with cytoplasmic antigens of cells in mouse gastric mucosa. Note the strong staining in the apical portion of the cells. (Magnification, X 400.)
muscle antibody, this was of particular importance. However, no significant differences in the frequency of serum antibodies or level of immunoglobulins were found in addicts with or without evidence of liver involvement; however, slightly higher concentrations of IgE were recorded in the former group (Table 1). Serum HBAg was also more frequent among these subjects (Table 2 ) . Admittedly, in these persons exact definition of those with and without any vestiges of low grade hepatitis is difficult without concurrent liver biopsy. However, this was not possible in the entire group of heroin addicts studied. Discussion
The present study again documents the marked elevation of serum IgM among heroin addicts (7, 8). Previous studies of serums from ex-addicts maintained on oral methadone subsequent to their presumed discontinuation of intravenous drug use have indicated that IgM then tends to be lower and closer to normal levels (8). Our data confirm this earlier report. A clear explanation for the presence of elevated IgM during active heroin abuse is not yet available. It is conceivable that it could be used as a surveillance check on methadone-program patients who might be thought to be surreptitiously adding intravenous heroin to their daily methadone maintenance program. However, more long-term data on comparative values before and after well-documented discontinuation of intravenous drug use need to be made before a valid conclusion can be drawn. 804
The striking finding of the presence of smooth muscle antibody among 46% of all active heroin addicts studied is a phenomenon of general interest. Smooth muscle antibody itself was originally described in the serums of patients with chronic active hepatitis (22). Subsequently, the occurrence of this antibody in a wide variety of other diseases, including viral hepatitis, malignancy, and infectious mononucleosis, has been documented (23-26). At present, a clear explanation for the occurrence of smooth muscle antibody in heroin addict serums is not available. It may represent a cross-reaction between antibodies to various bacterial or fungal antigens adulterating the usual batches of illicit drugs and some of the antigenic constituents of smooth muscle antigen. The latter has recently been characterized by Trenchev, Sneyd, and Holborow (27) as a complex mixture of at least six distinct molecular species of antigens related, in many instances, to indigenous contractile proteins present in many tissues of the body. The concomitant presence of smooth muscle antibody and lymphocytotoxic antibody in a large proportion of subjects studied here is of considerable interest. A recent report by Fagraeus, Lidman, and Biberfeld (28), has documented the apparent presence of some lymphocyte membrane-related contractile proteins similar to actomyosin. This could suggest that the antismooth muscle reactivity in heroin addict serums might also react with contractile elements present in certain lymphocyte surface proteins (29). Absorption experiments documented in this study were of interest in this regard. Cross-reactions of this sort may also be involved in the genesis of strongly lymphocytotoxic antibodies in the serums of some patients with chronic active hepatitis (30) or systemic lupus erythematosus (31, 32). Recent reports by Rizetto and Doniach (33) have recorded reticulin antibodies in serums of heroin addicts. However, we were not able to find staining patterns fulfilling the authors' criteria for reticulin antibodies in the present material. Heroin addiction, unfortunately, remains a clinical cornucopia of unusual medical complications (1, 34). Abnormalities related to the immune response, serum immunoglobulins, and the occurrence of various autoantibodies are probably part and parcel of this disorder (35). The clinical condition itself provides an interesting but unfortunate model for the study of certain aspects of what could be considered an autoimmune response, since selfadministration of illicit opiate preparations seems to result Table 4. Antibody Activity in Serum After Absorptions with Rapid Plasma Reagin, Smooth Muscle, and Normal Lymphocytes
Absorption With
Rapid plasma reagin Smooth muscle Normal lymphocytes Normal human serum
Smooth Muscle Antibody
Lymphocytotoxic Antibodies
0
+
+
+
+
+ + +
* 3 serums were subjected to absorption.
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Antibody Activity* Rapid Plasma Reagin
0 0
0 0
in the formation of autoantibodies. Whether this is due to tissue enzymatic alterations related to drug metabolism or to drug-tissue interactions remains to be elucidated.
15. TERASAKI PI, MCCLELLAND JD: Microdroplet assay of human serum cytotoxins. Nature (Lond) 204:998-1000, 1964 16. B0YUM A: Separation of leukocytes from human blood and bone marrow. Scand J Clin Lab Invest [(Suppl)21] 97:9-88, 1968
ACKNOWLEDGMENTS: The authors thank Mrs. Celia Diaz for technical assistance and Ms. Bernadette Marquez for help with the manuscript. Grant support: in part by grants AM13690-05 and AM AI13824-04 from the U.S. Public Health Service, and by a grant from the New Mexico Arthritis Foundation. Received 9 June 1975; revision accepted 27 August 1975.
17. ROTHFIELD NF, STOLLAR BD: The relation of immunoglobulin
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