Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability

Abstract

Although complement activation appears to have an important role both in the early and late phases of atherosclerosis, the exact mechanism of the initiation of this activation is still unknown. Since injuries of the endothelial cells are known to result in increased stress-protein expression we tested the complement-activating ability of recombinant human 60 kDa heat-shock protein (hsp60). Human hsp60 was found to activate the complement system in normal human serum in a dose-dependent manner. Activation took place through the classical pathway. The lack of complement activation in agammaglobulinemic serum indicates that the classical pathway is triggered by anti-hsp60 antibodies. Hsp60 activated complement in the sera of 74 patients with coronary heart disease as well, and a strong positive correlation (r = 0.459, P < 0.0001) was found between the extent of complement activation and the level of anti-hsp60 IgG antibodies but there was no correlation to the level of anti-hsp65 IgG antibodies. Further distinction between anti-hsp60 and anti-hsp65 antibodies was obtained from competitive ELISA experiments: binding of anti-hsp60 antibodies to hsp60-coated plates was inhibited only by recombinant hsp60 and vice versa. Our present findings indicate that anti-hsp60 and anti-hsp65 antibodies are distinct, showing only partial cross-reactivity. Since complement activation plays an important role in the development of atherosclerosis and the levels of complement-activating anti-hsp60 antibodies are elevated in atherosclerosis-related diseases, our present findings may have important pathological implications.

Relationship of Anti-60 kDa Heat Shock Protein and Anti-Cholesterol Antibodies to Cardiovascular Events

“…Previously 11 we also measured low correlation coefficients (0.16) between anti-hsp60 and anti-hsp65 in severe CAD patients and found that these antibodies differ in their antigen specificity. 17 High serum concentration of antihsp65 and fibrinogen and to a lesser extent high concentrations of anti-hsp65 and anti-CMV had a strong joint effect in predicting cardiovascular events. High levels of anti-hsp65 were significantly associated with an increased risk of development of MI and the composite of cardiovascular events.…”

“…17,18 Serial dilutions of serum samples of healthy blood donors having high antibody levels against the tested heat shock proteins were used as standard. Data obtained as optical density values were calculated to arbitrary unit per milliliter values related to this standard.…”

Independent and Joint Effects of Antibodies to Human Heat-Shock Protein 60 and Chlamydia pneumoniae Infection in the Development of Coronary Atherosclerosis

“…The independent presence of hHSP60 and Cpn antibodies suggests that hHSP60 antibodies are predominantly induced by mechanisms other than Cpn infection. Alternatively, high levels of autoantibodies against hHSP60 may be a stable, genetically determined trait (Amarilla Veres, MD, and Tamas Szamosi, MD, unpublished observations, 2000) that predisposes to CAD by formation of abundant hHSP60 -anti-hHSP60 immune complexes 21 and may result in intense in situ complement activation and endothelial cell dysfunction. A previous study 18 concluded that high-titer antibodies to mycobacterial HSP65 correlated with the presence of antibodies to Cpn in human sera obtained from atherosclerotic patients and that the HSP65 antibodies cross-reacted with hHSP60, chlamydial HSP, and Escherichia coli Gro-EL in ELISA.…”

“…21 Serial dilutions of a control anti-hHSP60 rabbit polyclonal antiserum (StressGen SPA-840) were used as standard. OD values were calculated in arbitrary units per milliliter relative to the standard.…”

Epistatic effects of genes encoding immunoglobulin GM allotypes and interleukin-6 on the production of autoantibodies to 60- and 65-kDa heat-shock proteins

“…11 In brief, plates were coated with 0.05 mg/well human hsp60 or M. bovis hsp65. After washing and blocking (PBS, 0.5% gelatin) the wells were incubated with 50 ml of serum samples diluted 1 : 100 in PBS containing 0.5% gelatin and 0.05% Tween 20.…”