Clearance of circulating DNA-anti-DNA immune complexes in mice.

Abstract

Immune complexes composed of DNA and antibodies to DNA contribute to tissue injury in systemic lupus erythematosus (SLE) (1, 2). High titers of antibodies to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are associated with the disease (2, 3), and antibodies to DNA have been eluted from the kidneys of SLE patients (4, 5). Direct demonstration of circulating DNA-anti-DNA immune complexes has been reported (6, 7), but not confirmed (8).Whereas the fate of circulating immun e complexes such as HSA-anti-HSA has been well characterized (9), the behavior of immune complexes containing DNA has not been studied. DNA is cleared from the circulation extremely rapidly, faster than large-latticed complexes (10). Furthermore, in vitro DNA binds to glomerular and skin basement membranes (11). These unique features of DNA might also influence the fate of immune complexes containing DNA as the antigen. In this study, immune complexes were prepared in vitro with ssDNA and antibodies to DNA from a patient with SLE. The clearance and organ localization of these immune complexes was studied in normal mice and compared with the clearance of heat aggregated immunoglobulin (IgG), serving as a model for immune complexes. Our results demonstrate that the clearance of ssDNA-anti-DNA immune complexes parallels the clearance of DNA alone and is much more rapid than the clearance of aggregated IgG. Materials and MethodsAntibodies to DNA. Serum from a patient with active SLE, kindly provided by Dr. J. V. Jones of the University of Chicago, was precipitated with 50% saturated ammonium sulphate and resuspended in borate buffer (0.2 M borate, 0.15 M NaCI; pH 8.0). This material was radiolabeled with 125I by the iodine monochloride method (12), and free 125I was removed by dialysis (final sp act 30,000 epm//~g). This preparation, containing IgM and IgG, was gel filtered over Sepharose 4B (Pharmacia Fine Chemicals, Div. of Pharmaeia Inc., Piscataway, N J) and the included IgG peak was pooled (see Figure 1 A). Reehromatography of this peak showed no excluded protein.ssDNA. Calf thymus DNA (Worthington Biochemical Corp., Freehold, N J) was heat denatured and labeled with laxI according to the method of Commerford (13). After dialysis to remove free xa'I, the DNA was passed over Sepharose 4B, and the excluded material was pooled (Fig. 1 B). Analysis of this DNA on 2% agarose gel electrophoresis revealed a mean mol wt of 640,000 with a range of 400,000-800,000.Preparation of Immune Complexes. I0 mg of 125I IgG was mixed with 20/~g of lalI ssDNA and incubated at 37°C for 30 rain. This mixture (2 ml) was gel filtered on a 50-ml Sepharose 4B column, and the 125I and 131I counts per 0.5 ml fraction were determined (Fig. 1C).

DNA-anti-DNA immune complexes. Antibody protection of a discrete DNA fragment from DNase digestion in vitro.

“…DNA itself was cleared rapidly from the circulation by digestion with circulating nucleases and by hepatic binding (5,6,7). Immune complexes containing large DNA were cleared from circulation at the same rate and with the same distribution as DNA alone (8). Studies by Harbeck et al (9) suggested that DNase is able to digest DNA away from the DNA-anti-DNA complex, and can thereby release free antibody.…”

The double edged sword of the immune response: mutational analysis of a murine anti-pneumococcal, anti-DNA antibody.

“…However, it has not been possible to demonstrate routinely the presence of DNA, either free in the circulation or bound in immune complexes, in patients with SLE (37). DNA-anti-DNA complexes prepared ex vivo and injected into animals showed no affinity for glomerular basement membrane (GBM), and were rapidly cleared from the circulation by the reticuloendothelial system (38). In addition, it seems unlikely that the negatively charged DNA in the immune complex could bind easily to the negatively charged GBM.…”

Endothelial cell binding by human polyclonal anti-DNA antibodies: relationship to disease activity and endothelial functional alterations

“…Tissue deposition of DNA-anti-DNA immune complexes has been regarded as a prerequisite in disease pathogenesis [3,4]. However, it remains doubtful whether this represents the predominant pathogenic mechanism, in view ofthe inconsistent detection of circulating immune complexes, and the failure to produce similar pathologies upon injection of pre-formed immune complexes in animal experiments [5,6]. Cross-reactivity of anti-DNA antibodies towards a heterogeneous group of cellular and extracellular components has been demonstrated Correspondence: Dr T. M. Chan, Department of Medicine, 4th floor.…”