Discovery of the C5b-9 complement complex in human arterial walls with atherosclerosis.
Our initial studies began in the Laboratory of Immunology at the Medical Clinic No.1 in Cluj-Napoca, Romania, when both of us were medical students working for the MD thesis . Dr. Anca Cristea, had just finished settling down in this new laboratory with the direct help and supervision of our mentor, Prof. Dr. Roman Vlaicu. The research question that had attracted our interest was the role of immune/inflammatory mechanisms in atherogenesis.
Initially, we used direct and indirect immunofluorescence to localize immunoglobulins (IgG, IgA, IgM) and individual complement components (C1q, C3, C4, C9) in cryostat sections of various types of atherosclerotic lesions in human aortic wall samples obtained at autopsy. Very soon we extended our studies to include iliac and femoral arteries obtained at surgery, as a means of avoiding any possible artifacts induced by the use of postmortem tissues. Both studies revealed that immunoglobulins, complement components and complement inhibitory proteins were present in the atherosclerotic wall of human arteries and that their accumulation was related to the degree of atherosclerotic involvement.
This initial work was communicated at several international cardiology and immunology meetings and was met with considerable skepticism. The question we were asked was: Why should an inflammatory mechanism play a role in this process, if lipids accumulating in the arterial wall can explain the damage that we see in atherosclerosis? We were convinced at this point that what we had observed was not just an epiphenomenon as some of researchers in our clinic believed, but something important. We sent our data to the journal Atherosclerosis and, to our surprise, they agreed to publish our work (1). In this paper we reported the interesting finding that immunoglobulins and complement components are preferentially retained in the intimal thickenings and fibrous plaques of the arterial wall when compared to their plasma levels, a finding which suggested that these immunoreactive proteins may be trapped in the intima from the plasma (1).
However, we wanted to establish that complement activation occurred in situ in the area of tissue damage. At that time, Dr. Suharit Bhakdi was working in Giessen to make an antibody against the neoantigens of the C5b-9 complex. These neoantigens, normally hidden within the individual proteins, are exposed on the surface of C5b-9 complexes after complement activation (2). He generously provided us his antibody, and later, he supplied us with reagents to allow us to quantify the SC5b-9 complement complex in arterial samples by ELISA. Indeed, we were able to show that the C5b-9 neoantigens were present in the arterial atherosclerotic wall in direct relationship to the level of atherosclerotic involvement (3-5). By ELISA we found that in intimal thickenings the levels of C5b-9 were higher than those in the fibrous plaques. C5b-9 was also detected in grossly normal human aortic intima, suggesting that activation of complement begins in the prelesional stages of atherogenesis (5). The presence of C5b-9 indicated that in situ complement activation occurs and may participate in the atherosclerotic process.
The neoantigens of C5b-9 complex were shown to be present in other tissues, but it was not clear whether these complexes represented lytic complexes, or what structures were actually targeted by C5b-9. Or, as Professor Nicolae Simionescu asked us at the time: Are these complexes localized on cells or parenchyma? To find an answer to this question, we spent 2 months at the Institute of Cellular Biology and Pathology in Bucharest, which he directed. We were fortunate to work with Dr. Elena Constantinescu, an immunoelectron microscopy expert who had just returned from a year of postdoctoral studies in Dr. Marilyn Farquahar's laboratory at Yale.
Our published report of these immunoelectron microscopic studies was the first to localize the C5b-9 complex in tissues (6). We successfully demonstrated that C5b-9 deposits were associated with cell debris, lipid droplets and cholesterol clefts. Back in Cluj-Napoca, we continued this project with the help of the laboratory of electron microscopy at the University of Medicine and Pharmacy. In later studies we also showed that some of the C5b-9 deposits are present on intact cells in the arterial wall and may be co-localized with S-protein/vitronectin (7,8). The presence of C5b-9 complexes on intact cells and in cell debris associated with arterial tissue suggested that complement activation might induce cell lysis and subsequent tissue damage. These C5b-9 complexes can either be formed through direct activation and assembly on the cell surface or in the cell vicinity inducing bystander cell lysis. These studies provided some of the early and important evidences that inflammation and the complement system are involved in atherogenesis.
References:
1. Vlaicu R., Niculescu, F., Rus, H.G., and Cristea, A.: Immunoglobulins and complement components in human aortic atherosclerotic intima: Atherosclerosis 55:35-55, 1985.
2. Bhakdi, S. and Tranum-Jensen, J. Membrane damage by complement. Biochim Biophys Acta, 737: 343-372, 1983.
3. Vlaicu, R., Niculescu, F., Rus, H. G., and Cristea, A. Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous plaque. Atherosclerosis, 57: 163-177, 1985.
4. Niculescu, F., Rus, H. G., and Vlaicu, R. Immunohistochemical localization of C5b-9, S protein, C3d and apolipoprotein B in human arterial tissues with atherosclerosis. Atherosclerosis, 65: 1-11, 1987.
5. Niculescu, F., Hugo, F., Rus, H. G., Vlaicu, R., and Bhakdi, S. Quantitative evaluation of the terminal C5b-9 complement complex by ELISA in human atherosclerotic arteries. Clin Exp Immunol, 69: 477-483, 1987.
6. Rus, H. G., Niculescu, F., Constantinescu, E., Cristea, A., and Vlaicu, R. Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque. Atherosclerosis, 61: 35-42, 1986.
7. Rus, H. G., Niculescu, F., Porutiu, D., Ghiurca, V., and Vlaicu, R. Cells carrying C5b-9 complement complexes in human atherosclerotic wall. Immunol Lett, 20: 305-310, 1989.
8. Niculescu, F., Rus, H. G., Porutiu, D., Ghiurca, V., and Vlaicu, R. Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall. Atherosclerosis, 78: 197-203, 1989.
Florin Niculescu
Horea Rus
University of Maryland
School of Medicine
Baltimore MD 21201
