CHARACTERISTICS OFl' IMMUNE CONIPLEXES IN CONNECTIVE TISSUE DISEASES JOIIN S. DAVIS, IV, M.D. AND (by invitation) FIIEDRICA E. SMITH, M.l). CHARLOTTESVILLE
Immune complexes have been shown to be associated with vasculitis in experimental animals in numerous studies performed during the last 30 years.-13 i\Iany investigators are now directing attention to the detection of immune complexes in humans w-ith connective tissue diseases, especially systemic lupus erythematosus (SLE), where immune complex deposition has been demonstrated in the kidney4 as well as other sites.5' 6 In this study we have evaluated substances w-hich may represent facets of immune complexes: cryoprecipitates (cryos), substances precipitated by the first compo)nent of complement (Clq precipitins), and antiDNA antibody (wshich may bind with DNA to form circulating complexes in some patients with SLE). Because these three serologic parameters have been identified in many of our patients with connective tissue diseases, we attempted to correlate their presence or absence with clinical disease activity. HISTORICAL BACKGROUND In the 1940's Arnold Rtichl injected rabbits repeatedly wN-ith horse serum and wtas able to produce lesions resembling human polyarteritis. In 19.153, Germuth2 showed that the vasculitis produced in rabbits occurred when antigen-antibody complexes were formed and that serum complement levels fell simultaneously. Subsequently, Dixon3 studied experimental serum sickness by injecting rabbits -ith bovine serum albumin. He noted that antigen excess was followed by a period of antigen-antibody equivalence and that it was during this period of equivalence that heart disease, arthritis, nephritis, and other manifestations of serum sickness occurred. Once antigen-antibody complexes were cleared and free antibody was present in excess, clinical disease disappeared. In 1963 Christian and co-workers described cryoprecipitates consisting of IgG, IgiAl, and the Clq component of complement in patients with SLE.7 Cryos have also been described in the sera of patients with a variety of other diseases such as glomerulonephritis, SBE and mixed cryoglobuliFrom the I)ivision of Rheumatology D)epartment of Medicine, University of Virginia School of MIedicine, Charlottesville, Virginia. Supported in part by a grant from the John A. Hartford Founldation and USPHS Grant AM-11766. 86
IMMilUNE COMIPLEXES IN CONNECTIVE TISSUE DISEASES
nemia. In at least one report a correlation has been noted between cryos, low serunm complement levels, and nephritis in SLE.8 Unique antigens anid corresponding antibodies (such as DNKA and anitiDNA) have not been consistently detected, but many cryos may be immune complexes. Factors in human sera wthich react with isolated Clq were first described by Agnello and co-workers.9 They found an association between Clq precipitins and low complement levels, especially in patients w-ith SLE. Recently Clq precipitins have been reported in sera of patients with ulcerative colitis, regional enteritis, dermatitis herpetiformis, and other conditions.10' 11 Some of these precipitins might also be immune complexes and miglht be (ryoprecipitable. In our study data were gathered over an 18-month period and were analyzed to determine 1) if there were distinctive and independent properties of the cryoprecipitates and Clq precipitins, 2) if free serum antiDNA activity was associated with cryoprecipitates and Clq precipitins and 3) if clinical correlations with their presence or absence in serum might suggest a diagnostic or prognostic usefulness for them in the management of patients with connective tissue diseases. MIETHODS Sera from patients with SLE and other connective tissue diseases were examined for cryoprecipitates, Clq precipitins and free antiDNA; serum complement levels were also measured. Cryos, when present, were precipitated from fresh serum at 40 C for 48 hours and were evaluated as a cryocrit. For evaluation of cryos in double diffusion, solubilized cryos were conicentrated 10 fold over original serum concentrations. Clq reagent was isolated by standard techniques9 and precipitin reactions were read after 48 hours. Hemolytic complement activity was measured by a modification of the method of Kabat and 'Mayer."2 AntiDNA and DNA activity was measured by counterimmunoelectrophoresis (CIE).13, 14 CHARACTERISTICS OF CRYOPRECIPITATES AND Clq PRECIPITINS We first attempted to define some of the characteristics of cryos and Clq precipitins in SLE sera containing both cryos and Clq precipitins. Several lupus sera, resolubilized cryoprecipitates, and cryo-free supernatants (from the same sera) were placed in agarose wells opposite wells containing Clq reagent. A precipitin line was found between Clq and serum as well as between Clq and cryo-free serum supernatant, but not between Clq and cryoprecipitates. Since Clq could be detected in the cryoprecipitates (by precipitation with anti-Clq in agarose), it appeared likely that the failure of Clq to react w-ith resolubilized cryoprecipitates was due to the fact that binding sites were already saturated. The finding that Clq continued to
88
JOHN S. DAVIS AND FREDRICA E.
SM1ITH
react with the cryo-free sera suggested that the precipitin reacting with Clq was distinct from the cryoprecipitate. Clq reactions were then determined in 156 SLE sera, their cryoprecipitates and cryo-free supernatants. Only 53 sera actually contained cyroprecipitates, but all sera were handled as if they did indeed have cryoprecipitable material. In no case did a cryoprecipitate react further Nith Clq in double diffusion. However, all of the supernatants from Clq precipitinpositive sera continued to react with Clq after removal of the cryo. In addition, 10 serum Clq precipitins appeared in the supernatant material for the first time after the cryoprecipitate had been removed, a phenomenon w-hich remains to be elucidated. Thus, this study suggested that, at least in this assay system, cryoprecipitates and Clq precipitins were distinctive complexes which appeared to be essentially independent of each other. As a further step in evaluation of the relationship between Clq precipitins and cryos, patient sera containing both, ervoprecipitates and Clq precipitins were passed through Sepharose columns which were linked to Clq by activated eyanogen bromide. By this method Clq precipitins could be quantitatively removed for further analysis. Sera were first passed through the column to eff ect adsorption of complexes to Clq and the material bound to Clq was subsequently eluted Nith 1 molar sodium chloride. Table 1 shows results of one study with the first column indicating the amount of various components which had been adsorbed to the column from an SLE serum (E.F.). The second column show-s the amount of material adsorbed to Clq fromn this same serum from which a cryoprecipitate had previously been TABLE 1 Eluates Cotnponients of Clq-Bonncd (Clq Precipitinis) Obtained front Whole Serum and Cryo-Free Serum of Otte SLE Patientt (E.F.)* Clq-bound eluates
Cryoprecipitatel
Component
W'hole Serum Cryo-Free Serum
Protein (mg)......................... IgG (mg)............................. ,gM (mg)........................... IgA (mg)............................ Clq ................................... I)NA ................................ Anti-DNA ............................ Clq precipitin......................... *
2.62 2.12 0.333
2.81 2.28
+
+
I1_
_T_
0.312
0.784 0.535 0.089 0.043
+
2 ml serum.
t Direct analysis for similar components in the cryo of the same patient is shown for comparison.
IMMUNE COMPLEXES IN CONNECTIVE TISSUE DISEASES
89
removed. It can be seen that the amount of IgG and IgM in both eluates was roughly the same; no IgA was seen in either eluate. DNA and antiDNA Nere not found, and the re-concentrated material was shown to react with Clq in double diffusion as expected. The third column shows a direct analysis of the isolated, unwashed cryoprecipitate for comparison. The cryoprecipitate also contained IgMi and IgG and a small amount of IgA which may have been a nonspecific contaminant. No antiDNA or DNA activity -as detected, and as previously stated, there was no reactivity with Clq in double diffusion. This study suggested that the components of the Clq precipitin were not affected by prior removal of a cryo from the starting serum; they were thus apparently independent complexes. In order to determine whether there might be unique antigens in either the cryo of Clq precipitin of a single lupus patient with large amounts of both complexes, rabbits were immunized with isolated complexes and the antiserum subsequently absorbed with pooled normal human serum. No antigens specific for these complexes were noted when the antiscra were reacted with the original precipitins in double diffusion. Subsequently, all sera from 99 patients with SLE which had been screened for both Clq precipitins and cryoprecipitates were evaluated in the light of a varietv of parameters. Table 2 shows the results of this assessment. In this study, patients were grouped according to the tvpe of complex which wsas most frequently positive in their sera. Of the 99 patients, 50 were fouind to have one or both complexes on at least one occasion. Of those positive, 50'7c had cryos only, 32 had Clq precipitins only, and 18 NO had both factors positive. AntiDNA activity in serum as not exclusively associated writh either type of complex but was most frequently found when both cryos and Clq precipitins were present. When serum complement levels were evaluated at the time complexes were found, or within one month of the complex determinations, significantly low levels were TABLE 2 SLE Patients Grouped Accordinig to Type of Most Frequently Recurring Serum Complex Serum
Patients
I Cryo only II Clq pptn. only III Cryo and Clq pptn.
25 (50%) 16 (32%) 9 (18%)
Anti-DNA*
Complement Levelt
Renal Involvement
8/25 (32%) 3/16 (19%) 5/9 (56%c)
35 42 28
10/25 (40/0c) 9/16 (56%)
4/9 (44%)
50 total *
Inicidence of free, serum anti-DNA activity in each group. mean values given. 34-48 is normal range for
t CH50 U/ml;
our
laboratory.
90
JOHN S. DAVIS AND FREDRICA E. SMITH
found only in sera containing both cryos and Clq precipitins. Low normal levels were found in sera of patients wvith cryos only, while patients who had only Clq precipitins appeared to have complement levels in the midnormal range. The frequency of renal involvement in these patients was also examined (as one parameter of serious clinical disease), but those patients with renal involvement were not distinguished by this grouping of patients according to the type of complex found in their sera. Table 3 depicts the incidence of these complexes in SLE patients who had three or more determinations positive for one or both complexes. It can be seen that there is a distinct mimetic pattern in this lupus patient population. Patienits tended to have recurring episodes during which either Clq precipitins or cryos or both were noted, but, except for the latter group, very little overlap was seen. Thus, there did appear to be a recurring pattern in the type of complexes that were seen. As shown in Table 4 sera were also screened from 268 patients with diseases other than lupus. Sera from 33 patients diagnosed as having connective tissue diseases were positive for either cryos, Clq precipitins or both. This table shows the distribution of cryos and Clq precipitins in these TABLE 3 Mimetic Pattern of Serum Complexes in 18 SLE Patientts on Serial Determinations Patient %
I Cryos Only*
1........................................
3/5
2 ........................................ 3 ........................................ 4 ........................................ 5 ........................................
8/8 6/6
d)........................................
7/7 2/3 13/13 3/3
7 ........................................ 8 ........................................
9........................................
III II Both* Cryos Clq pptn. Only* and Clq pptn.
2/5
4/4
1/6
5/6 1/3
10 ........................................ 11 ........................................
4/4 3/3
12 ........................................ 13 ........................................ 14 ........................................
2/3
1/3
4/4 3/4
1/4
2/4 1/5 4/15 1/4
2/5 7/15 2/4
15 ........................................ 16 .......... ............................. 17 ........................................ 18 ........................................ *
1/4 2/5 4/15 1/4
Number of times positive/total positive determinations.
1/4
91
I1MMUNE CO'MPLEXES IN CONNECTIVE TISSUE DISEASES
TABLE 4
Conintective Tissue Disease Patienlts (Exclutdinig Those W/rith SLE) Grolnped According to Typc of Mlost Frequenttly Recutrrinig Serutm Comlplex Group
I Cryo Only II Clq pptn. only III Cryo and Clq pptn.
S Patients
Anti-DNA*
Serum Complement
Renal Involvement
7 (21%) 21 (64%) 5 (15%)
0/7 (0%) 1/19 (5%) 0/3 (0%)
47 50 49
2/7 (29%o) 1/21 (5%) 0/5 (0%o)
Levell
33 Total Incidence of free, serum anti-DNA activity in each group. t CH50 U/ml; mean values given. 34-48 is normal range for our laboratory.
*
TABLE 5 Serologic Abnormallties* int Patients IWith Active SLE
Active Renal Disease Active CNS Disease Other Active Diseaset
Crvo
Clq Precipitin
Anti-DNA
Low Complement
2/9 22% 0/2
3/9 33% 2/2
0/9 0C/o
0%
100%
0%
3/9 33% 0/2
16/43 37%
9/43
12/43
21%
28%
0/2
Levelt
0% 7/39 18%
* Positive on over half the determinations. CH50 < 30 units/ml (normal range 34-48). t Arthritis, pneumonitis, pericarditis, etc.
33 patients. As expected, antiDNA activity was rarely found in the sera of this non-SLE group of patients. Furthermore low complement levels were not associated with either of these complexes. Renal involvement was found in 2 of 7 patients with recurrent cryos in their sera, but the number of patients is too small to draw any definite conclusions. CORRELATION OF CO.MPLEXES AND ACTIVE DISEASE IN SLE In evaluating the occurrence of cryos, Clq precipitins, antiDNA, and complement in relation to disease activity, patients were again grouped into those with SLE and those with other connective tissue diseases. As shown in patients with SLE in Table 5, attempts were first made to correlate the presence of cryos, Clq precipitins, antiDNA, and low complement levels with active renal disease. For the purpose of this evaluation, the criteria for determining active renal disease were 2 or more of the following: pro-
92
JOHN S. DAVIS AND FREDRICA E. SMITH
teinuria greater than 1.5. gin, 94 hours, hematuria and/or cellular casts, rising serum creatinine (associated in one patient with acute renal failure), anid positive renial biopsy (whein biopsy was prompted by one or more of the preceding findings). Surprisingly, when one examines these serologic abnormalities in patients with active renal disease, one notes that antiDNA activity, as detected by CIE, is not regularly present in the nine patients Imeeting the criteria. In fact, of 57 determinations of antiDNA in these 9 patients, only 4 w-ere positive. This agrees with a recent observation by Johnson and associates,"5 in which their 6 patients with nephropathy had no antiDNA detectable by CIE, but our findings are at variance with other reports showing free serum antiDNA activity by hemagglutination and binding techniques in patients with active lupus nephropathy.'6' 17 It is possible that antiDNA has combined with DNA to form immune complexes during this period of active nephritis, under which circumstances free, precipitable antiDNA would not be detected. Low complement levels were not uniformly present in these patients, but they were seen more frequently than in patients with other facets of active disease. In patients with active CNS disease, the number of patients is too small for any conclusions to be drawn. Patients with disease activity other than renal or CNS disease were grouped together. In these patients, Clq precipitins and cryos were seen with about the same frequency as in patients with active renal disease. AntiDNA was usually present in 28 %/ of these patients, in contrast to its absence in patients with active renal disease. The role of drug therapy in modifying these observations is currently being evaluated, since many of these patients were receiving steroids during these episodes of disease activity. In patients with connective tissuc diseases other than SLE, the number of patienrts wsitlh active renal involvemeint was too small to draw any con(lusiOis (only one hiad rensal disease). Vlasculitis, however, w-as found in 40 %X of all these complex positive patients -ith active disease (13/33); furthermore, in all 5 patients w-ith both cryoprecipitates and Clq precipitins in their sera, vasculitis wN-as present. CONCLUSIONS In conclusion, it appears that cryoprecipitates and Clq precipitins may be distinctive immune complexes which are in some respects independent of each other. In SLE, although not in the other connective tissue disCaWes, the presence of both cryos and Clq precipitins was associated with low complement levels. Also in SLE patients, there was a strong mimetic pattern in the recurrence of either cryos, Clq precipitins, or both. In examining antiDNA activity by CIE in patients with SLE, an ab-
IMMUNE COMPLEXES IN CONNECTIVE TISSUE DISEA SES
93
sea ce of this antibody was found in those individuals with active nephritis. This failure to find free antiDNA may mean that DNA and antiDNA have combined to form complexes during epidodes of renal disease activity. It is hoped that further study of circulating complexes in patients with connective tissue diseases who have specific organ injury may make it possible to predict subsequent tissue damage in patients with early disease. Careful analysis of components of these complexes may also reveal specific antigens (and antibodies) associated with specific diseases and disease patterns. ACKNOWLEDGMENTS The authors wish to thank Mrs. Stephania M. Godfrey and :\Ir. Timothy Nealon for their helpful assistance with this project. REFERENCES 1. ltiCH, A. R.: Hypersensitivity in I)isease, with especial reference to periarteritis nodosa, rheumatic fever, disseminated lupus erythematostus and rheumatoid arthritis. The Harvey Lechtres. Series 42: 106, 1946-47. 2. GERMUTH, F. G.: A comparative histologic and immunologic study in rabbits of inducted hypersensitivity of the serum sickness type. J. Exp. Med. 97: 257, 1953. 3. DixON, F. J.: The role of antigen-antibody complexes in diseases. The Harvey Lectures. Series 58: 21, 1962-63. 4. PARONETTO, F. AND KOFFLER, D.: Immunofluorescent localization of immunoglobulins, complement and fibrinogen in human diseases. I. Systemic lupus erythematosus. J. Clin. Invest. 44: 1657, 1967. 5. SVIEC, K. H. AND ALLEN, S. T.: Antibody to nuclear material eluted from isolated spleen vessels in systemic lupus erythematosus. Science 170: 550, 1970. 6. ATKINS, C. M., KONDON, J. J., QUISMORIO, F. P. AND FRIOU, G. J.: The choroid plexus in systemic lupus erythematosus. Ann. IJt. Med. 76: 65, 1972. 7. CHRISTIAN, C. L., HATFIELD, W. B., AND CHASE, P. H.: Systemic lupus erythematosus: cryoprecipitation of sera. J. Clin. Invest. 42: 823, 1963. 8. AGNELLO, V., KOFFLER, 1)., EISENBERG, J. W., WINCHESTER, R. J. AND KUNKEL, H. G.: Clq precipitins in the sera of patients with systemic lupus erythematosus and other hypocomplementemic states: characterization of high and low molecular weight types. J. Exp. Med. 134: 228, 1971. 9. AGNELLO, V., WINCHESTER, R. J., AND KUNKEL, H. G.: Precipitin reactions of the Clq components of complement with aggregated gamma globulin and immune complexes in gel diffusion. Immunol. 19: 909, 1970. 10. DOE, W. F., BOOTH, C. C. AND BROWN, D. L.: Evidence for complement-binding immune complexes in adult coeliac disease, Crohn's disease and ulcerative colitis. Lancet 1: 402, 1973. 11. MOWBRAY, J. F., HOLBOROW, E. J., HOFFBRAND, A. V., SEAH, P. P., AND FRY, L.: Circulating immune complexes in dermatitis herpetiformis. Lancet 1: 400, 1973. 12. DAVIS, J. S.: Complement and its role in disease in Vistas in Connective Tissue Diseases. Ed. J. C. Bennett, C. C. Thomas, Springfield, 1968, p. 129. 13. DAVIS, G. L. AND DAVIS, J. S.: Detection of circulating DNA by counterimmunoelectrophoresis. Arth. Rheum. 16: 52, 1973.
94
JOHN S. DAVIS AND FREDRICA E. SMITH
14. DAVIS, J. S. AND WINFIELD, J. G.: Serum antibodies to DNA by counterimmunoelectrophoresis. Clin. Immun. Immutnopath. 2: 510, 1974. 15. JOHNSON, G. 1)., EDMONDS, J. P., AND HOLBoLROW, E. J.: Precipitating antibody to DNA detected by two-stage electroimmunodiffusion. Laticet 2: 883, 1973. 16. SHARP, G. C., IRVIN, W. S., LAROQUE, 11. L., VEWc, C., DALY, V., KAISER, A. D., AND HOLMAN, H. R.: Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J. Clin. Invest. 50: 350, 1971. 17. PINCUS, T., SCHUR, P. H., ROSE, J. A., DECKER, J. L., TALAL, N.: Measurement of serum DNA-binding activity in systemic lupus erythematosus. N. Eng. J. Med. 281: 701, 1969.
Immune complexes have been shown to be associated with vasculitis in experimental animals in numerous studies performed during the last 30 years.-13 i\Iany investigators are now directing attention to the detection of immune complexes in humans w-ith connective tissue diseases, especially systemic lupus erythematosus (SLE), where immune complex deposition has been demonstrated in the kidney4 as well as other sites.5' 6 In this study we have evaluated substances w-hich may represent facets of immune complexes: cryoprecipitates (cryos), substances precipitated by the first compo)nent of complement (Clq precipitins), and antiDNA antibody (wshich may bind with DNA to form circulating complexes in some patients with SLE). Because these three serologic parameters have been identified in many of our patients with connective tissue diseases, we attempted to correlate their presence or absence with clinical disease activity. HISTORICAL BACKGROUND In the 1940's Arnold Rtichl injected rabbits repeatedly wN-ith horse serum and wtas able to produce lesions resembling human polyarteritis. In 19.153, Germuth2 showed that the vasculitis produced in rabbits occurred when antigen-antibody complexes were formed and that serum complement levels fell simultaneously. Subsequently, Dixon3 studied experimental serum sickness by injecting rabbits -ith bovine serum albumin. He noted that antigen excess was followed by a period of antigen-antibody equivalence and that it was during this period of equivalence that heart disease, arthritis, nephritis, and other manifestations of serum sickness occurred. Once antigen-antibody complexes were cleared and free antibody was present in excess, clinical disease disappeared. In 1963 Christian and co-workers described cryoprecipitates consisting of IgG, IgiAl, and the Clq component of complement in patients with SLE.7 Cryos have also been described in the sera of patients with a variety of other diseases such as glomerulonephritis, SBE and mixed cryoglobuliFrom the I)ivision of Rheumatology D)epartment of Medicine, University of Virginia School of MIedicine, Charlottesville, Virginia. Supported in part by a grant from the John A. Hartford Founldation and USPHS Grant AM-11766. 86
IMMilUNE COMIPLEXES IN CONNECTIVE TISSUE DISEASES
nemia. In at least one report a correlation has been noted between cryos, low serunm complement levels, and nephritis in SLE.8 Unique antigens anid corresponding antibodies (such as DNKA and anitiDNA) have not been consistently detected, but many cryos may be immune complexes. Factors in human sera wthich react with isolated Clq were first described by Agnello and co-workers.9 They found an association between Clq precipitins and low complement levels, especially in patients w-ith SLE. Recently Clq precipitins have been reported in sera of patients with ulcerative colitis, regional enteritis, dermatitis herpetiformis, and other conditions.10' 11 Some of these precipitins might also be immune complexes and miglht be (ryoprecipitable. In our study data were gathered over an 18-month period and were analyzed to determine 1) if there were distinctive and independent properties of the cryoprecipitates and Clq precipitins, 2) if free serum antiDNA activity was associated with cryoprecipitates and Clq precipitins and 3) if clinical correlations with their presence or absence in serum might suggest a diagnostic or prognostic usefulness for them in the management of patients with connective tissue diseases. MIETHODS Sera from patients with SLE and other connective tissue diseases were examined for cryoprecipitates, Clq precipitins and free antiDNA; serum complement levels were also measured. Cryos, when present, were precipitated from fresh serum at 40 C for 48 hours and were evaluated as a cryocrit. For evaluation of cryos in double diffusion, solubilized cryos were conicentrated 10 fold over original serum concentrations. Clq reagent was isolated by standard techniques9 and precipitin reactions were read after 48 hours. Hemolytic complement activity was measured by a modification of the method of Kabat and 'Mayer."2 AntiDNA and DNA activity was measured by counterimmunoelectrophoresis (CIE).13, 14 CHARACTERISTICS OF CRYOPRECIPITATES AND Clq PRECIPITINS We first attempted to define some of the characteristics of cryos and Clq precipitins in SLE sera containing both cryos and Clq precipitins. Several lupus sera, resolubilized cryoprecipitates, and cryo-free supernatants (from the same sera) were placed in agarose wells opposite wells containing Clq reagent. A precipitin line was found between Clq and serum as well as between Clq and cryo-free serum supernatant, but not between Clq and cryoprecipitates. Since Clq could be detected in the cryoprecipitates (by precipitation with anti-Clq in agarose), it appeared likely that the failure of Clq to react w-ith resolubilized cryoprecipitates was due to the fact that binding sites were already saturated. The finding that Clq continued to
88
JOHN S. DAVIS AND FREDRICA E.
SM1ITH
react with the cryo-free sera suggested that the precipitin reacting with Clq was distinct from the cryoprecipitate. Clq reactions were then determined in 156 SLE sera, their cryoprecipitates and cryo-free supernatants. Only 53 sera actually contained cyroprecipitates, but all sera were handled as if they did indeed have cryoprecipitable material. In no case did a cryoprecipitate react further Nith Clq in double diffusion. However, all of the supernatants from Clq precipitinpositive sera continued to react with Clq after removal of the cryo. In addition, 10 serum Clq precipitins appeared in the supernatant material for the first time after the cryoprecipitate had been removed, a phenomenon w-hich remains to be elucidated. Thus, this study suggested that, at least in this assay system, cryoprecipitates and Clq precipitins were distinctive complexes which appeared to be essentially independent of each other. As a further step in evaluation of the relationship between Clq precipitins and cryos, patient sera containing both, ervoprecipitates and Clq precipitins were passed through Sepharose columns which were linked to Clq by activated eyanogen bromide. By this method Clq precipitins could be quantitatively removed for further analysis. Sera were first passed through the column to eff ect adsorption of complexes to Clq and the material bound to Clq was subsequently eluted Nith 1 molar sodium chloride. Table 1 shows results of one study with the first column indicating the amount of various components which had been adsorbed to the column from an SLE serum (E.F.). The second column show-s the amount of material adsorbed to Clq fromn this same serum from which a cryoprecipitate had previously been TABLE 1 Eluates Cotnponients of Clq-Bonncd (Clq Precipitinis) Obtained front Whole Serum and Cryo-Free Serum of Otte SLE Patientt (E.F.)* Clq-bound eluates
Cryoprecipitatel
Component
W'hole Serum Cryo-Free Serum
Protein (mg)......................... IgG (mg)............................. ,gM (mg)........................... IgA (mg)............................ Clq ................................... I)NA ................................ Anti-DNA ............................ Clq precipitin......................... *
2.62 2.12 0.333
2.81 2.28
+
+
I1_
_T_
0.312
0.784 0.535 0.089 0.043
+
2 ml serum.
t Direct analysis for similar components in the cryo of the same patient is shown for comparison.
IMMUNE COMPLEXES IN CONNECTIVE TISSUE DISEASES
89
removed. It can be seen that the amount of IgG and IgM in both eluates was roughly the same; no IgA was seen in either eluate. DNA and antiDNA Nere not found, and the re-concentrated material was shown to react with Clq in double diffusion as expected. The third column shows a direct analysis of the isolated, unwashed cryoprecipitate for comparison. The cryoprecipitate also contained IgMi and IgG and a small amount of IgA which may have been a nonspecific contaminant. No antiDNA or DNA activity -as detected, and as previously stated, there was no reactivity with Clq in double diffusion. This study suggested that the components of the Clq precipitin were not affected by prior removal of a cryo from the starting serum; they were thus apparently independent complexes. In order to determine whether there might be unique antigens in either the cryo of Clq precipitin of a single lupus patient with large amounts of both complexes, rabbits were immunized with isolated complexes and the antiserum subsequently absorbed with pooled normal human serum. No antigens specific for these complexes were noted when the antiscra were reacted with the original precipitins in double diffusion. Subsequently, all sera from 99 patients with SLE which had been screened for both Clq precipitins and cryoprecipitates were evaluated in the light of a varietv of parameters. Table 2 shows the results of this assessment. In this study, patients were grouped according to the tvpe of complex which wsas most frequently positive in their sera. Of the 99 patients, 50 were fouind to have one or both complexes on at least one occasion. Of those positive, 50'7c had cryos only, 32 had Clq precipitins only, and 18 NO had both factors positive. AntiDNA activity in serum as not exclusively associated writh either type of complex but was most frequently found when both cryos and Clq precipitins were present. When serum complement levels were evaluated at the time complexes were found, or within one month of the complex determinations, significantly low levels were TABLE 2 SLE Patients Grouped Accordinig to Type of Most Frequently Recurring Serum Complex Serum
Patients
I Cryo only II Clq pptn. only III Cryo and Clq pptn.
25 (50%) 16 (32%) 9 (18%)
Anti-DNA*
Complement Levelt
Renal Involvement
8/25 (32%) 3/16 (19%) 5/9 (56%c)
35 42 28
10/25 (40/0c) 9/16 (56%)
4/9 (44%)
50 total *
Inicidence of free, serum anti-DNA activity in each group. mean values given. 34-48 is normal range for
t CH50 U/ml;
our
laboratory.
90
JOHN S. DAVIS AND FREDRICA E. SMITH
found only in sera containing both cryos and Clq precipitins. Low normal levels were found in sera of patients wvith cryos only, while patients who had only Clq precipitins appeared to have complement levels in the midnormal range. The frequency of renal involvement in these patients was also examined (as one parameter of serious clinical disease), but those patients with renal involvement were not distinguished by this grouping of patients according to the type of complex found in their sera. Table 3 depicts the incidence of these complexes in SLE patients who had three or more determinations positive for one or both complexes. It can be seen that there is a distinct mimetic pattern in this lupus patient population. Patienits tended to have recurring episodes during which either Clq precipitins or cryos or both were noted, but, except for the latter group, very little overlap was seen. Thus, there did appear to be a recurring pattern in the type of complexes that were seen. As shown in Table 4 sera were also screened from 268 patients with diseases other than lupus. Sera from 33 patients diagnosed as having connective tissue diseases were positive for either cryos, Clq precipitins or both. This table shows the distribution of cryos and Clq precipitins in these TABLE 3 Mimetic Pattern of Serum Complexes in 18 SLE Patientts on Serial Determinations Patient %
I Cryos Only*
1........................................
3/5
2 ........................................ 3 ........................................ 4 ........................................ 5 ........................................
8/8 6/6
d)........................................
7/7 2/3 13/13 3/3
7 ........................................ 8 ........................................
9........................................
III II Both* Cryos Clq pptn. Only* and Clq pptn.
2/5
4/4
1/6
5/6 1/3
10 ........................................ 11 ........................................
4/4 3/3
12 ........................................ 13 ........................................ 14 ........................................
2/3
1/3
4/4 3/4
1/4
2/4 1/5 4/15 1/4
2/5 7/15 2/4
15 ........................................ 16 .......... ............................. 17 ........................................ 18 ........................................ *
1/4 2/5 4/15 1/4
Number of times positive/total positive determinations.
1/4
91
I1MMUNE CO'MPLEXES IN CONNECTIVE TISSUE DISEASES
TABLE 4
Conintective Tissue Disease Patienlts (Exclutdinig Those W/rith SLE) Grolnped According to Typc of Mlost Frequenttly Recutrrinig Serutm Comlplex Group
I Cryo Only II Clq pptn. only III Cryo and Clq pptn.
S Patients
Anti-DNA*
Serum Complement
Renal Involvement
7 (21%) 21 (64%) 5 (15%)
0/7 (0%) 1/19 (5%) 0/3 (0%)
47 50 49
2/7 (29%o) 1/21 (5%) 0/5 (0%o)
Levell
33 Total Incidence of free, serum anti-DNA activity in each group. t CH50 U/ml; mean values given. 34-48 is normal range for our laboratory.
*
TABLE 5 Serologic Abnormallties* int Patients IWith Active SLE
Active Renal Disease Active CNS Disease Other Active Diseaset
Crvo
Clq Precipitin
Anti-DNA
Low Complement
2/9 22% 0/2
3/9 33% 2/2
0/9 0C/o
0%
100%
0%
3/9 33% 0/2
16/43 37%
9/43
12/43
21%
28%
0/2
Levelt
0% 7/39 18%
* Positive on over half the determinations. CH50 < 30 units/ml (normal range 34-48). t Arthritis, pneumonitis, pericarditis, etc.
33 patients. As expected, antiDNA activity was rarely found in the sera of this non-SLE group of patients. Furthermore low complement levels were not associated with either of these complexes. Renal involvement was found in 2 of 7 patients with recurrent cryos in their sera, but the number of patients is too small to draw any definite conclusions. CORRELATION OF CO.MPLEXES AND ACTIVE DISEASE IN SLE In evaluating the occurrence of cryos, Clq precipitins, antiDNA, and complement in relation to disease activity, patients were again grouped into those with SLE and those with other connective tissue diseases. As shown in patients with SLE in Table 5, attempts were first made to correlate the presence of cryos, Clq precipitins, antiDNA, and low complement levels with active renal disease. For the purpose of this evaluation, the criteria for determining active renal disease were 2 or more of the following: pro-
92
JOHN S. DAVIS AND FREDRICA E. SMITH
teinuria greater than 1.5. gin, 94 hours, hematuria and/or cellular casts, rising serum creatinine (associated in one patient with acute renal failure), anid positive renial biopsy (whein biopsy was prompted by one or more of the preceding findings). Surprisingly, when one examines these serologic abnormalities in patients with active renal disease, one notes that antiDNA activity, as detected by CIE, is not regularly present in the nine patients Imeeting the criteria. In fact, of 57 determinations of antiDNA in these 9 patients, only 4 w-ere positive. This agrees with a recent observation by Johnson and associates,"5 in which their 6 patients with nephropathy had no antiDNA detectable by CIE, but our findings are at variance with other reports showing free serum antiDNA activity by hemagglutination and binding techniques in patients with active lupus nephropathy.'6' 17 It is possible that antiDNA has combined with DNA to form immune complexes during this period of active nephritis, under which circumstances free, precipitable antiDNA would not be detected. Low complement levels were not uniformly present in these patients, but they were seen more frequently than in patients with other facets of active disease. In patients with active CNS disease, the number of patients is too small for any conclusions to be drawn. Patients with disease activity other than renal or CNS disease were grouped together. In these patients, Clq precipitins and cryos were seen with about the same frequency as in patients with active renal disease. AntiDNA was usually present in 28 %/ of these patients, in contrast to its absence in patients with active renal disease. The role of drug therapy in modifying these observations is currently being evaluated, since many of these patients were receiving steroids during these episodes of disease activity. In patients with connective tissuc diseases other than SLE, the number of patienrts wsitlh active renal involvemeint was too small to draw any con(lusiOis (only one hiad rensal disease). Vlasculitis, however, w-as found in 40 %X of all these complex positive patients -ith active disease (13/33); furthermore, in all 5 patients w-ith both cryoprecipitates and Clq precipitins in their sera, vasculitis wN-as present. CONCLUSIONS In conclusion, it appears that cryoprecipitates and Clq precipitins may be distinctive immune complexes which are in some respects independent of each other. In SLE, although not in the other connective tissue disCaWes, the presence of both cryos and Clq precipitins was associated with low complement levels. Also in SLE patients, there was a strong mimetic pattern in the recurrence of either cryos, Clq precipitins, or both. In examining antiDNA activity by CIE in patients with SLE, an ab-
IMMUNE COMPLEXES IN CONNECTIVE TISSUE DISEA SES
93
sea ce of this antibody was found in those individuals with active nephritis. This failure to find free antiDNA may mean that DNA and antiDNA have combined to form complexes during epidodes of renal disease activity. It is hoped that further study of circulating complexes in patients with connective tissue diseases who have specific organ injury may make it possible to predict subsequent tissue damage in patients with early disease. Careful analysis of components of these complexes may also reveal specific antigens (and antibodies) associated with specific diseases and disease patterns. ACKNOWLEDGMENTS The authors wish to thank Mrs. Stephania M. Godfrey and :\Ir. Timothy Nealon for their helpful assistance with this project. REFERENCES 1. ltiCH, A. R.: Hypersensitivity in I)isease, with especial reference to periarteritis nodosa, rheumatic fever, disseminated lupus erythematostus and rheumatoid arthritis. The Harvey Lechtres. Series 42: 106, 1946-47. 2. GERMUTH, F. G.: A comparative histologic and immunologic study in rabbits of inducted hypersensitivity of the serum sickness type. J. Exp. Med. 97: 257, 1953. 3. DixON, F. J.: The role of antigen-antibody complexes in diseases. The Harvey Lectures. Series 58: 21, 1962-63. 4. PARONETTO, F. AND KOFFLER, D.: Immunofluorescent localization of immunoglobulins, complement and fibrinogen in human diseases. I. Systemic lupus erythematosus. J. Clin. Invest. 44: 1657, 1967. 5. SVIEC, K. H. AND ALLEN, S. T.: Antibody to nuclear material eluted from isolated spleen vessels in systemic lupus erythematosus. Science 170: 550, 1970. 6. ATKINS, C. M., KONDON, J. J., QUISMORIO, F. P. AND FRIOU, G. J.: The choroid plexus in systemic lupus erythematosus. Ann. IJt. Med. 76: 65, 1972. 7. CHRISTIAN, C. L., HATFIELD, W. B., AND CHASE, P. H.: Systemic lupus erythematosus: cryoprecipitation of sera. J. Clin. Invest. 42: 823, 1963. 8. AGNELLO, V., KOFFLER, 1)., EISENBERG, J. W., WINCHESTER, R. J. AND KUNKEL, H. G.: Clq precipitins in the sera of patients with systemic lupus erythematosus and other hypocomplementemic states: characterization of high and low molecular weight types. J. Exp. Med. 134: 228, 1971. 9. AGNELLO, V., WINCHESTER, R. J., AND KUNKEL, H. G.: Precipitin reactions of the Clq components of complement with aggregated gamma globulin and immune complexes in gel diffusion. Immunol. 19: 909, 1970. 10. DOE, W. F., BOOTH, C. C. AND BROWN, D. L.: Evidence for complement-binding immune complexes in adult coeliac disease, Crohn's disease and ulcerative colitis. Lancet 1: 402, 1973. 11. MOWBRAY, J. F., HOLBOROW, E. J., HOFFBRAND, A. V., SEAH, P. P., AND FRY, L.: Circulating immune complexes in dermatitis herpetiformis. Lancet 1: 400, 1973. 12. DAVIS, J. S.: Complement and its role in disease in Vistas in Connective Tissue Diseases. Ed. J. C. Bennett, C. C. Thomas, Springfield, 1968, p. 129. 13. DAVIS, G. L. AND DAVIS, J. S.: Detection of circulating DNA by counterimmunoelectrophoresis. Arth. Rheum. 16: 52, 1973.
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JOHN S. DAVIS AND FREDRICA E. SMITH
14. DAVIS, J. S. AND WINFIELD, J. G.: Serum antibodies to DNA by counterimmunoelectrophoresis. Clin. Immun. Immutnopath. 2: 510, 1974. 15. JOHNSON, G. 1)., EDMONDS, J. P., AND HOLBoLROW, E. J.: Precipitating antibody to DNA detected by two-stage electroimmunodiffusion. Laticet 2: 883, 1973. 16. SHARP, G. C., IRVIN, W. S., LAROQUE, 11. L., VEWc, C., DALY, V., KAISER, A. D., AND HOLMAN, H. R.: Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J. Clin. Invest. 50: 350, 1971. 17. PINCUS, T., SCHUR, P. H., ROSE, J. A., DECKER, J. L., TALAL, N.: Measurement of serum DNA-binding activity in systemic lupus erythematosus. N. Eng. J. Med. 281: 701, 1969.
