Eugene V. Barnett

Circulating Immune Complexes: Their Immunochemistry, Detection, and Importance

The size and molecular composition of circulating immune complexes depend on various factors, including the concentrations and valences of antigens and antibodies and the antigen-antibody ratio. The composition and biological properties of circulating immune complexes, in turn, influence their fate in vivo as well as the likelihood of their detection by various assays. Several assays clearly detect circulating immune complexes, but no single assay has yet been shown to be the most sensitive and the most specific for the entire spectrum of circulating immune complexes. Assays correlate poorly with each other, but this may be desirable if we are to determine which circulating immune complexes have diagnostic, prognostic, or pathogenic importance. Circulating immune complexes are found in numerous rheumatologic disorders and infectious diseases. Their presence in the circulation statistically correlates with disease activity; however, the assays currently used have limited value for diagnosing or aiding in therapeutic decisions. Nevertheless, the future holds promise for such uses.

Characterization of anti-F(ab′)2 antibodies in SLE patients evidence for cross-reacting auto-anti-idiotypic antibodies☆

Abstract In this study we have characterized anti-F(ab′)2 antibodies from SLE patients sera which inhibit binding of antigen (125I-ss-DNA) to anti-ss-DNA antibodies from autologous patient serum, other SLE patients, mice, and ss-DNA-immunized rabbit sera. The SLE anti-F(ab′)2 antibodies did not block other antigen-antibody reactions, i.e., hepatitis surface antigen (HBsAg) with anti-HBsAg and phospholipase A with its antibody. Anti-F(ab′)2 antibodies isolated from rheumatoid arthritis (RA) sera did not block 125I-ss-DNA binding with anti-ss-DNA antibodies. Anti-F(ab′)2 antibodies in SLE appeared to contain cross-reacting, auto-anti-idiotypic antibodies directed against anti-DNA antibodies.

The occurrence and nature of precipitating antibodies in anti-DNA sera

Abstract The binding technique of Farr and counterimmunoelectrophoresis were compared as methods for measuring the occurrence of anti-DNA antibodies. Ninety-one anti-nuclear antibody positive sera were examined. Twenty-nine were positive by binding and counterimmunoelectrophoresis, 18 were positive by binding alone, 15 were positive by counterimmunoelectrophoresis alone, and 29 were negative in both test systems. No consistent differences in quantity of antibody, inhibition with native or single-strand DNA or DNA digest, average equilibrium constants, or antigen content could be demonstrated between sera with high and with low precipitin titers to DNA. It is suggested that one explanation for large amounts of precipitating antibody to DNA is the presence of antibodies in a patients serum directed against multiple antigenic determinants on the DNA molecule; this results in a functional multivalency for the DNA and enhanced ability to engage in lattice formation and precipitation.

Substrate competition between DNase I and anti-DNA antibody

Sera from rabbits immunized with DNA and patients known to have SLE were shown to inhibit the digestion of 125I-labeled ss-DNA by pancreatic DNase I. Similarly, DNase present in the serum of a patient with SLE was found to digest the radio-labeled DNA at one-sixth the rate of an equal quantity of DNase present in NHS. The inhibition was shown to be due to isolated anti-DNA antibody in both the immunized rabbit sera and the SLE sera. The type of inhibition involved was demonstrated to be of the competitive variety in which the binding of antibody to DNA presumably blocks enzyme access to the substrate. The possible pathogenetic implications of these findings are discussed.

Characterization and measurement of anti-IgG antibodies in human sera by radioimmunoassay (RIA)

A sensitive direct binding radioimmunoassay (RIA) was developed which detected low avidity anti-IgG antibodies in sera negative in the latex fixation test (LFT). IgG class antibodies could be detected and were commonly found along with IgM class antibodies. Additionally, the RIA was more reproducible than the LFT, was easily adapted to measure relative avidities of anti-IgG antibodies, and had other technical advantages over the LFT.

The role of immunological tests in routine synovial fluid analysis.

Reports describing the analysis of synovial fluid were few until the work of Kling 17, in 1 938, and the classic monograph of Ropes and Bauer 24, in 1953. On the basis of these studies, synovial fluid analysis focused on certain laboratory tests which attempted to determine the degree of inflammatory change manifest in the synovial fluid at the time of aspiration. This analysis was done by determining the color and appearance of the fluid and the number and type of cells present. Qualitative estimates of hyaluronate were also made by measuring viscosity and the quality of the mucin clot (Rope’s test). Values for these observations have been grouped into four classes (I to IV) 7,24 in an attempt to correlate the degree of synovial inflammation with the disease producing the synovitis. However, the degree of synovial inflammation may vary considerably in any arthritic joint. Thus, the synovial fluid present at the time of arthrocentesis reflects both the existing activity of the synovitis which may be acute, chronic, or in remission, and the patient’s previous or current systemic or local therapy. In addition to what can be learned from these laboratory tests, further diagnostic information may be obtained by the identification and quantitation of specific immunologic factors in synovial fluid, factors which may be characteristic of certain diseases that produce clinical synovitis. These same factors have been implicated in the pathogenic mechanisms which may initiate or perpetuate synovial inflammation ‘ ‘ ‘ 13, 16, 19, 20, 22, 27, 31, 33, In an attempt to improve the diagnostic specificity of synovial fluid analysis, a group of serologic tests were incorporated into our standard synovial fluid analysis. These tests are based in part on the three major classes of immunoglobulins which are found in the globulin fraction of protein. The term itninunoglobulin (Ig) is a functional description ofcertain protein classes that are known to have antibody activity 15#{149} These three major classes have been given the arbitrary designations IgG, IgA, and 1gM. We therefore quantitated each of the major immunoglobulin classes present in both the serum and the synovial fluid of our patients. Other tests included the determination of rheumatoid factors, antinuclear antibodies, and the identification of intracytoplasmic inclusions within synovial fluid leukocytes. The total cornplement and the third component of the complement system were also measured. These tests were selected because they identify certain immune factors and interactions thought to be involved in the pathogenesis of chronic inflammatory synovitis. Rheumatoid factors are primarily found in the 1gM immunoglobulin class. They are now considered to be antibodies to gamma globulins and are also called anti-

DIETARY RESTRICTION IN MICE BEGINNING AT 1 YEAR OF AGE: EFFECTS ON SERUM IMMUNE COMPLEX LEVELS

One year old mice from a long-lived strain were gradually subjected to life-prolonging dietary restriction. Serum immune complex and immunoglobulin G (abbreviated IgG) levels were measured at 13 and 23 months of age. Longitudinal and cross-sectional analyses showed reduced serum immune complex and IgG levels for mice underfed in adulthood.

Immunologic Changes in Synovial Fluid Following Synovectomy of the Knee for Rheumatoid Arthritis

The composition of synovial fluid changes in some systemic diseases that affect joints. Traditional methods of synovial-fluid analysis focused on appearance, mucin clot, vicosity, cells, sugar, protein, and presence of bacteria l1,13,48#{149} Recently specific crystals arid their relationship to disease have been identified 36#{149} Rheumatoid arthritis arid related diseases produce immunologic changes in synovial fluid as well as in serum, and they can now be measured by sensitive tests. Immunoglobulins, complement, and antinuclear antibodies constitute the three groups of factors involved which are the subject of this report. At least three groups of gamma globulins have been identified that appear to be involved in resistance to infectious diseases 18 and are missing from the serum of patients with agammaglobiriemia 27#{149} They are called immunoglobulins (Ig), and the three major components are nowclassified IgG, IgA, and 1gM. The only quantitative determination of immunoglobulin in synovial fluid was reported by Brown, Cooper, and Bluestone 8#{149} They measured only 1gM in rheumatoid synovial fluid and serum and reported

Role of nuclear antigens and antinuclear antibodies in inflammation

Abstract The sera of patients with lupus erythematosus and other “autosensitivity” diseases, such as rheumatoid arthritis, frequently contain not only the LE cell factor but antinuclear antibodies directed against denatured and native DNA, Sm antigen of the nucleus, protein associated with RNA, and probably many other nuclear antigens. The patients rarely have antibody only to a single nuclear antigen and, furthermore, these antinuclear antibodies are found to be heterogeneous immunoglobulins of the γG, γA, and γM immunoglobulin classes. Data are presented which suggest that at least in rheumatoid arthritis the antibodies may be the result rather than the cause of the disease. Similar antibodies have been elicited by immunization of animals with killed bacteria, human serum, and DNA components conjugated to carrier proteins. Animals with such experimentally induced antibodies do not acquire “human” diseases and it is of further interest that such antinuclear antibodies are found in low concentrations in humans without disease. The above data, taken together with the observations that antinuclear antibodies are generally incapable of injuring intact cells, initially suggest that these antibodies have limited pathogenetic significance. This may not be the case, however, because there is increasing evidence that nuclear antigens and antinuclear antibodies may be one of a series of antigen-antibody combinations capable of eliciting inflammation of the synovium within the joint and along the basement membrane of the renal glomerulus. One animal disease which may bear on this question is Aleutian disease of mink. We have recently had the opportunity to examine 16 pairs of mink sera taken before and after the onset of the disease. Nuclear antigen (DNA) was detected in all these sera both before and after onset of the disease. Only in the diseased animals was antinuclear antibody detectable. It has been suggested by others that the vasculitis and other immunopathologic features of Aleutian disease are due to circulating antigen-antibody complexes even though the disease can be transmitted from mink to mink by filterable material.

THE ROLE OF RHEUMATOID (ANTIGLOBULIN) FACTORS IN HEMOLYTIC ANEMIA

Autoimmune hemolytic anemias are characterized by anti-erythrocyte antibodies that usually belong to one class of immunoglobulins. Warm-antibody hemolytic anemia is associated with yG-type autoantibodies, whereas antibodies of the yM class are responsible for chronic cold-agglutinin disease. The transient cold-agglutinin hemolytic anemia present in patients with infectious mononucleosis, however, is frequently caused by a mixed y G y M cold agglutinin. In the mixed cold agglutinin, neither the yG or yM component alone is capable of destroying the red blood cell; the presence of both is required for cold-,agglutinin activity. The hemolytic mechanism appears to depend on the sensitization of erythrocytes with y G at 4 O C, and the subsequent interaction of the sensitized red cells with a cold-reactive yM-antiglobulin (FIGURE 1 ) .