Cryoglobulin studies in systemic lupus erythematosus.FF Sikander, DS Salgaonkar, VR Joshi
Twenty-two patients with systemic lupus erythematosus (SLE) were investigated to study the incidence and immunochemical nature of circulating immune complexes. In 68.18% of patients, the immune complexes were detected as cryoglobulins. A significantly high value of serum IgG and IgA (1980.64 +/- 1031.53 and 283.81 +/- 131.02 mg% respectively) were observed in these patients compared to those in normal (1097.04 +/- 298.10 and 200.93 +/- 89.96 mg% respectively). The patients also had significantly low levels of serum complement fraction C3 (61.35 +/- 22.64 mg%). Correlation of these parameters could help in understanding of the role of immunochemical factors in the pathogenesis of the disease.
Keywords: Adolescent, Adult, Aged, Cryoglobulins, analysis,immunology,Human, Immunoglobulin A, immunology,Immunoglobulin G, immunology,Immunoglobulin M, immunology,Lupus Erythematosus, Systemic, etiology,immunology,Middle Age,
Circulating immune complexes have been implicated in the pathology of various diseases including multiple myeloma, macroglobulinaemia, malignant lymphomas, as also diseases of autoimmune origin.
A large number of methods designed to detect immune complexes in biological fluids, based on their physical, chemical and biological properties have been reported. A commonly used method for assessing circulating immune complexes is their behaviour as "cryoglobulins"-proteins that precipitate reversibly in cold.,,
A study of these cryoglobulins was undertaken in patients of SLE in order to determine their significance in the pathogenesis of the disease.
Twenty-two patients (age range 15-65 years) with a confirmed diagnosis of SLE attending the rheumatology clinic of B.Y.L. Nair Charitable Hospital were studied. Twenty-seven healthy volunteers (age range 18-60 years) were included as controls.
Detection of cryoprecipitates: A modification of the method used by Zvaifler was used. A brief summary of the technique utilised is shown in Fig. 1. Sera negative for cryoprecipitates were diluted in a 1 : 1 proportion with distilled water for the detection of hypotonic cold precipitates.
Analysis of cryoprecipitates: The cold precipitates were tested for protein content by the method of Lowry. Ouchterlony's double diffusion technique in agar was utilised for determining the type of immunoglobulin present (IgG, IgA and/or IgM). The latex-agglutination test of Singer and Plotz was carried out for detecting the presence of IgM-rheumatoid factor (RF) in the precipitates.
Serum parameters: The serum immunoglobulins (IgG, IgA, IBM) and the third component of complement, C3 were estimated using the single radial immunodiffusion technique of Mancini. A semiquantitative assay for serum IgM-RF was carried out using the latex agglutination test.
The results of the studies on cryoprecipitates are shown in [Table - 1]and [Table - 2]. An incidence of 68.18% of cold precipitation was found in SLE patients; of these 27.27% cases showed precipitation only after the sera were rendered hypotonic. No cryoprecipitation could be detected in any of the normal sera, either before or after dilution.
The protein content of cryoprecipitates in SLE patients was found to be 0.074 ± 0.053 mg/ml. The immunoglobulin content of these precipitates is depicted in[Table - 2]. Based on the classification of Brouet et al, the cryoprecipitates were divided into "simple" (those containing only one class of immunoglobulins) and "mixed" cryoglobulins (those composed of more than one class of immunoglobulins). A distribution of simple and mixed cryoglobulins in the ratio of 47 : 53 was seen in these SLE cryoprecipitates. Of the mixed cryoglobulins IgG and IgM immunoglobulins were detected in 26.66% and IgG and IgA in 20% of the precipitates; only one precipitate (6.66%) showed all the three: IgG, IgA and IgM immunoglobulins.
The complement component C3 could not be detected in any of the precipitates.
Forty per cent of the cryoprecipitates were observed to show a positive latex agglutination test[Table - 1]. A much greater protein content (0. 115 ± 0.06 mg/ ml) was observed in RF-positive precipitates as compared to RF-negative precipitates (0.0471 ± 0.024 mg/ml). A significantly lower percentage (33.33%) of mixed cryoglobulins was noted in the RF-negative precipitates (Z > 1.96).
The serum immunochemical parameters of SLE patients are depicted in[Table - 2]. Significantly higher mean values of serum IgG and IgA were observed as compared to those in normal subjects. Also, a highly significant decrease in the serum C3 values was observed; (p < 0.01).
A highly significant close association between seropositivity and presence of circulating immune complexes in SLE sera was revealed by the Chi-square test (p < 0.01) [Table - 2].
The role of circulating immune complexes in the pathogenesis of rheumatoid diseases is being widely investigated; indeed, SLE has now been regarded as an autoimmune disease by an increasingly large number of workers. The study of the seropathology and the presence and nature of the circulating immune complexes in SLE was, therefore, undertaken in the present study.
As in the present study, a high incidence of cryoprecipitates has been reported by many workers.,, No cold precipitates could be detected in any of the 27 normal control sera in the present study. This finding is consistent. with the earlier reports., A few workers have found limited quantities of cryoglobulins in the normal sera e.g. Cream found less than 80 µg/ml, Weisman and Zvaifler less than 30 µg/ml of serum, and Erhardt et al, less than 10 µg/ml.
It has been suggested by Grey and Kohler that cryoglobulins may be more appropriately termed as "Cryoimmunoglobulins" since the vast majority contain either monoclonal immunoglobulins or mixtures of immunoglobulins. Predominance of IgG class of immunoglobulins with lesser amounts of IgM and IgA have been reported in rheumatoid sera by Erhardt et al as was also seen in the present study.
The observation that cryoprecipitates representing circulating immune complexes are composed of immunoglobulins, as also the detection of antiglobulin IgM RFs in several precipitates points towards the immunochemical nature of SLE. The IgM RFs probably play an important role in both, the formation and the pathogenic influences of immune complexes in this disease. This contention is supported by the highly significant association obtained between seropositivity and cryopositivity in the present study.
The highly significant decrease in serum complement values obtained in the present study is supported by the results of several other workers in the field;,, reduced levels have been found to be indicative of renal involvement in many cases. Depressions of serum complement may be probably due to localisation on glomerular lesions or a consequence of binding to the widespread vascular lesions, after being fixed by circulating antigen antibody complexes. The present study may thus help in the understanding of immunochemical parameters of SLE which are involved in systemic immuno-inflammation.
[Table - 1], [Table - 2]