Thyroid follicular cell function after non-lethal complement membrane attack

Terminal complement complexes have been identified around thyroid follicles in Graves' disease and Hashimoto's thyroiditis, and the concentrations of such complexes are increased in the sera of these patients, suggesting a role for complement activation and membrane attack complexes (MAC) in autoimmune thyroiditis. This has been investigated further using cultured human and rat thyroid cells. Thyrocytes were resistant to lysis by homologous complement, in contrast to the effects of heterologous (rabbit) complement. The formation of non-lethal amounts of MAC, using reactive lysis or classical pathway activation, significantly reduced cAMP production by these cells in response to thyroid-stimulating hormone (TSH) (P less than 0.01); similar effects were seen with thyroid-stimulating antibodies. Thyroid cells were able to recover rapidly from complement attack after washing and incubation for 30 min. Non-lethal MAC formation also resulted in reactive oxygen metabolite production, detected by luminol-dependent chemiluminescence in three out of five thyroid cell preparations tested. Ionomycin, but not TSH, also stimulated reactive oxygen metabolite production. These results suggest that repeated or continuous sub-lethal complement attack on thyroid cells may exacerbate hypothyroidism in Hashimoto's thyroiditis, or partially counter the effects of thyroid-stimulating antibodies in Graves' disease. Furthermore, the production of reactive oxygen metabolites in these circumstances could increase the intra-thyroidal inflammatory response; oxygen radical scavenging by anti-thyroid drugs (which are concentrated by thyrocytes) may account in part for the amelioration of thyroiditis observed with such treatment.     

Mechanisms of complement resistance induced by non-lethal complement attack and by growth arrest.

Non-lethal complement (C) attack on K562 cells has been shown to induce a transient resistance to lethal amounts of C. We have previously shown that incubation of K562 with phorbol 12-myristate 13-acetate (PMA) caused an increase in both CD59 expression and resistance to C killing and we were interested to examine whether non-lethal C attack caused a similar effect. We here demonstrate that expression of the C inhibitors decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 was unaltered on K562 after non-lethal C attack and that neutralization of these inhibitors with specific blocking antibodies did not reverse the induced resistance. In an effort to understand the mechanisms of resistance we searched for other conditions that might induce C resistance in K562 cells. Growth-arrested cells showed a similar degree of resistance to C killing. The levels of DAF and MCP on these cells were unaltered whereas expression of CD59 was markedly reduced. Non-lethal C attack on these growth-arrested cells induced a further increase in resistance to C killing, suggesting that the mechanisms of resistance were not identical. Indeed, resistance of non-lethally attacked cells was completely lost within 8 hr of attack whereas resistance of growth-arrested cells was detectable for up to 48 hr after returning to cell cycle. These data demonstrate that C resistance induced by two distinct strategies is not mediated by the known membrane C inhibitors. Resistance may be a result of the expression of a novel inhibitor or due to metabolic depletion, a likely common consequence of non-lethal C attack and induction of growth arrest, implying that cells take an active role in C-mediated killing.     

补体攻膜复合物细胞非致死性效应及其信号转导机制

有核细胞能通过自身机制抵抗同源补体溶破,同时亚溶破MAC在细胞膜上沉积后,能刺激多种细胞的多种生物学活性改变,包括产生活性氧、释放花生四烯酸及其代谢产物、产生多种细胞因子、诱导膜蛋白表达和诱导细胞进入细胞周期等,此称MAC的细胞非致死效应。这些效应是由MAC活化细胞跨膜信号转导机制介导的。     

Increased biosynthesis of complement components by cultured monocytes, synovial fluid macrophages and skynovial membrane cells from patients with rheumatoid arthritis.

Monocytes, synovial fluid (SF) and synovial membrane (SM) macrophages from patients with rheumatoid arthritis (RA) were maintained in short-term tissue culture for up to 10 days, and the synthesis of C4, C2, C3, C5, factor B(B), D, properdin (P), C3b inactivator (C3bINA) and beta 1H globulin studied. Functionally active C2, B, D, P, C3bINA and beta 1H were synthesized by the cells in each type of culture. C4, C3 and C5 could be detected, but were functionally inactive. RA monocytes synthesized more C2 than monocytes from patients with degenerative joint disease (DJD) (P < 0.001). Similar studies revealed that SF macrophages synthesized more C3 than SM macrophages (P < 0.001) which in turn produced more C2 than monocytes (P < 0.001). Other experiments showed that SF macrophages synthesized more of each component than the other cell types. SM macrophages made more C2 than B than RA and DJD monocytes, but synthesized only small quantities of P, D and beta 1H. RA monocytes synthesized more of each component than DJD monocytes. The results of these studies show that (1) in RA, complement components can be synthesized locally in the inflamed joints, and (2) local factors in the joints probably stimulate complement synthesis.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.     

A novel membrane glycoprotein capable of inhibiting membrane attack by homologous complement

Neuraminidase-treated human erythrocytes become sensitive to haemolysis by heterologous serum via activation of the alternative complement pathway (ACP), while remaining insensitive to homologous serum because of the presence of inhibitors on the cell membrane. We obtained a monoclonal antibody which renders the neuraminidase-treated erythrocytes sensitive to haemolysis by homologous human serum via the ACP. This antibody reacts with a 20 KDa membrane glycoprotein which interferes with the terminal stage of complement action on cell membranes. The 20 KDa protein is anchored to the membrane via phosphatidylinositol.     

Comparative proliferation of non-rheumatoid and rheumatoid human synovial cells

While normal synovial membrane cells have a very long doubling time, rheumatoid arthritis increases cell turn-over leading to the formation of a pannus. We studied the comparative proliferative behaviour in culture of synoviocytes of rheumatoid (RA) and non-rheumatoid (NR) origin in order to evaluate the usefulness of this model to investigate the drugs used in the treatment of inflammatory diseases. First-passage cultures of cells from patients with clinically defined non-inflammatory joint disease or rheumatoid arthritis were observed for 8 days. In the presence of various combinations of supplemented media, 3H-thymidine incorporation, protein content and cell density were assessed. In addition to a relationship between the fetal calf serum (FCS) concentration and cell growth, it was found that RA cells proliferated more rapidly than NR cells. In 1% FCS, protein content and cell density increased in RA cultures whereas NR synoviocytes accumulated in the quiescent phase. In 5% and 10% FCS, RA cells responded more strongly than NR in terms of protein and DNA synthesis and cell division. After 48 hours of relative FCS deprivation, NR cells abruptly started to proliferate; the response of RA cultures was delayed, but the synoviocytes quickly reached preconfluence.     

In vitro activation of human complement by polyacrilonitrile dialyzer membrane assayed by complement binding to bystander red cells.

Quantitation of complement activation by polyacrilonitrile (PAN) dialyzer membrane is complicated by the high adsorptive capacity of the membrane for fluid phase anaphylotoxins. Assays for these anaphylotoxins, therefore, underestimate the degree of complement activation produced by this membrane. Alternative methods of measuring in vitro complement activation by the PAN and Cuprophan membranes were explored by incubating normal human erythrocytes with the membranes in the presence of serum. This led to deposition of C3d on these "innocent bystander" red cells, and provided an independent parameter for measuring complement activation. The PAN membrane caused significantly more C3d deposition on red cells, and thus more complement activation than Cuprophan. The possible significance of complement activation by PAN membrane, in consideration of its property of binding the resultant anaphylotoxins, is discussed.     

The membrane attack complex of complement induces permeability changes via thresholds in individual cells.

Flow cytometry was used to quantify the fluorescence of propidium iodide in rat polymorphonuclear leucocytes (PMN) attacked by the membrane attack complex (MAC) in order to establish the existence of permeability and lytic thresholds in individual cells, a 'threshold' being defined as a cellular event involving the rapid transition of cells from one state to another under physiological conditions. Activation of the complement pathway resulted in PMN being attacked by MAC within 5 min. Approximately 30-40% of the cell population subsequently became permeable to small molecules and macromolecules. Individual PMN passed through 'thresholds' of cell permeability and cell lysis, or recovered from complement attack at different times. In the flow cytometer, three distinct populations of PMN were identified: cells that had recovered before the permeability 'threshold', cells that had recovered after the permeability 'threshold' but before the lytic 'threshold', and cells that failed to recover from complement attack. Individual PMN attacked by MAC passed through permeability and lytic thresholds at different times after an initial lag of 7.5 +/- 2.5 min and 11.5 +/- 1.0 min, respectively. Adenosine, an activator of adenylate cyclase, inhibited removal of MAC from the cell surface. Consequently, more cells passed through the permeability and lytic 'thresholds', resulting in an increased percentage of lysed cells.     

Mononuclear phagocytes of normal and rheumatoid synovial membrane identified by monoclonal antibodies.

The presence of cells bearing epitopes of the mononuclear phagocyte series was studied immunohistochemically in synovium removed from joints involved by trauma (T), osteoarthritis (OA) and rheumatoid arthritis (RA). Mononuclear phagocytes were the most consistent feature of the inflamed rheumatoid synovium. They shared at least five epitopes expressed by mononuclear phagocytes in other tissues. In OA/T samples, cells bearing markers of the less mature monocyte were present at the surface of the synovial membrane, namely the intimal layer, while those bearing macrophage epitopes were apparent throughout the intimal layer and subintimally. This suggested that maturation of the monocyte population takes place after the monocytes have entered the synovial tissues, settled at the surface, then moved downward into the subintimal layer. The synovial monocytes accounted for all the HLA-D region positive cells in the lining layer.     

Regulation of CD59 expression on K562 cells: effects of phorbol myristate acetate, cross-linking antibody and non-lethal complement attack.

CD59 is the major membrane attack complex of complement (MAC) inhibiting protein on human cells. Its regulation is therefore an important factor in determining the fate of cells at sites of complement activation. We have chosen the K562 erythroleukaemia cell line as a model for studies of the regulation of CD59 expression, because it has previously been reported that phorbol 12-myristate 13-acetate (PMA) caused a 15-fold up-regulation of CD59 mRNA in these cells, implying a substantial capacity for CD59 synthesis. However, no assessment of CD59 protein expression was made in these studies. We show here that surface expression of CD59, as assessed by flow cytometry, was increased four-fold over a 16-hr incubation with PMA, whereas surface expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) was not altered. The newly expressed CD59 was functionally active and anchored through glycosyl-phosphatidylinositol (GPI). Increased expression was dependent upon de novo protein synthesis. CD59 released into cell supernatant was also increased seven-fold by PMA, this 'secreted' CD59 retained its GPI anchor. Non-lethal complement attack did not alter CD59 expression but antibody cross-linking of CD59 caused a rapid loss of the CD59-antibody complexes. However, CD59 was quickly restored to pre-attack levels. This rapid restoration was not dependent upon protein synthesis, suggesting release from preformed stores.     

Type III collagen: A major constituent of rheumatoid and normal human synovial membrane.

The findings establish that type III collagen is a major constituent of grossly proliferated rheumatoid and normal synovium. Unlike the collagen of normal synovium most of that in rheumatoid tissue could be solubilised by pepsin at 4 degrees C. Moore than half the pepsin-solubilised collage was identified as type III, the remainder being type I, by CM-cellulose chromatography; SDS-polyacrylamide electrophoresis with and without reduction of disulphide bonds; and amino acid analysis. Moreover, at least half the total collagen in several samples of normal as well as rheumatoid tissue was clearly type III when cyanogen bromide-derived peptides were run on SDS-polyacrylamide electrophoresis and compared with peptides prepared from purified types I and III collagens. This conclusion was supported by the isolation on phosphocellulose and quantitation by amino acid analysis of the collagen peptides alpha(1)CB2 and alpha(III)CB2 from a cyanogen bromide digest of rheumatoid synovium.     

Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin

Larvae and adults of the parasitic blood fluke Schistosoma mansoni are resistant to killing by human complement. An earlier search by Parizade et al. for a schistosome complement inhibitor identified a 94-kDa surface protein which was named SCIP-1 (M. Parizade, R. Arnon, P. J. Lachmann, and Z. Fishelson, J. Exp. Med. 179:1625-1636, 1994). Following partial purification and analysis by mass spectrometry, we have determined SCIP-1 to be a surface-exposed form of the muscle protein paramyosin. As shown by immunofluorescence, anti-paramyosin antibodies label the surface of live schistosomula and adult worms. Like SCIP-1, purified native paramyosin reacts with a polyclonal rabbit anti-human CD59 antiserum, as shown by Western blot analysis. Also, the human complement components C8 and C9 bind to recombinant and native paramyosin. Analysis of paramyosin binding to fragments of C9 generated by thrombin or trypsin has demonstrated that paramyosin binds to C9 at a position located between Gly245 and Arg391. Paramyosin inhibited Zn(2+)-induced C9 polymerization and poly-C9 deposition onto rabbit erythrocytes (E(R)). In addition, paramyosin inhibited lysis of E(R) and of sensitized sheep erythrocytes by human complement. Finally, anti-paramyosin antibodies enhanced in vitro killing of schistosomula by normal and C4-depleted human complement. Taken together, these findings suggest that an exogenous form of S. mansoni paramyosin inhibits activation of the terminal pathway of complement and thus has an important immunomodulatory role in schistosomiasis.     

人脐静脉内皮细胞清除补体攻膜复合物的机制

目的：探讨内皮细胞清除补体攻膜复合物（MAC）的途径及其清除动力学，方法：原代培养的人脐静脉内皮细胞以RH414荧光标记质膜双层，0℃组装亚溶剂量的MAC，37℃复苏后，LSCM实时监MAC沉积诱导的质膜囊泡化形成和胞吞，胞吐情况,流式细胞仪定量检测内皮细胞表面MAC抗原的清除情况，结果：MAC沉积后，内皮细胞有的质膜囊泡化形成，囊泡以胞吞和胞吐2种方式离开细胞，并以前者占优，37度条件下，内皮细胞清除表面MAC的半衰期约为5min。结论：内皮细胞可通过胞吞和胞吐2种机制清除细胞表面沉积的MAC，并以胞吞方式为主。     

Regulation of complement membrane attack complex formation in myocardial infarction.

Recent studies have suggested that the complement (C) system is involved in the development of tissue injury of myocardial infarction. As it is not known why the strictly controlled C system starts to react against autologous heart tissue, we have analyzed the expression of various membrane regulators of C (CR1, DAF, MCP, CD59, C8 binding protein) and the pattern of deposition of C components and plasma C regulators (C4b binding protein and vitronectin) in normal (n = 7) and infarcted (n = 13) human myocardium. In the infarcted myocardium deposits of the C membrane attack complex (MAC) were observed by immunofluorescence microscopy, and lesions resembling the transmembrane channels of MAC were detected by transmission electron microscopy. CD59 and C8 binding protein were strongly expressed by muscle cells of normal myocardial tissue. Little or no CR1, MCP, and DAF was observed on these cells. The assembly of MAC was accompanied by the deposition of vitronectin (S-protein) and C4b binding protein in the infarcted areas of myocardium. In accordance with our earlier results the expression of CD59 but not of C8 binding protein was clearly diminished in the lesions. The results show that C8 binding protein, vitronectin, and C4b binding protein do not prevent complement attack against the infarcted myocardium but rather become codeposited with the MAC. Ischemia-induced transformation of nonviable cells into complement activators, acquired loss of resistance to the MAC by shedding of CD59, and recruitment of multifunctional serum proteins by MAC could thus constitute a general process aimed at the clearance of injured tissue.     

Targeted neutralization of the complement membrane attack complex inhibitor CD59 on the surface of human melanoma cells

Major problems in the immunotherapy of human tumors with complement-activating monoclonal antibodies (mAb) are (i) inherent resistance of tumor cells to complement cytolysis and (ii) a possible undiscriminatory attack against normal cells. In the present study we have developed a procedure to simultaneously direct the complement membrane attack complex and neutralize its inhibitor CD59 (protectin) on human melanoma cells in vitro. G361 melanoma cells were selectively recognized in heterogenous cell mixtures by a complement-fixing mAb (R24) against the tumor cell GD3-ganglioside. Biotinylated anti-CD59 mAb (YTH53.1) was directed to the tumor cells with a high-affinity biotin-avidin bridge using a proportion of R24 as a biotinylated targeting mAb and avidin as a linker. Biotinylated anti-CD59 mAb lost its ability to activate complement, but retained its CD59-neutralizing activity. Thus, it was possible to avoid nonspecific lysis of surrounding erythrocytes and endothelial cells and direct the CD59-neutralizing effect to the tumor cells. As a result the tumor cells were efficiently killed by R24 plus complement while the bystander cells remained viable. These results suggest that it is possible to target an unrestricted complement membrane attack against GD3- and CD59-positive melanoma cells.     

Membrane attack by complement

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.     

Lymphocyte migration in the rheumatoid synovial membrane

There are impressive similarities between the lymphocyte populations in lymph node and synovial membrane: similar amounts and classes of immunoglobulins are synthesized; lymphoid aggregates, when present in the rheumatoid synovial membrane, resemble those of the lymph node paracortex in being composed predominantly of helper-inducer T4 cells; B cells which leave tall endothelial PCV together with the T cells, tend to segregate themselves from the T cell aggregates and migrate to areas rich in macrophages where they proliferate and differentiate. In the lymph node, the B cells migrate to the primary follicles of the outer cortex and to the medullary region, while in the synovial membrane the corresponding region is the transitional area, which contains lymphocytes (predominantly T8 cells), macrophages and plasma cells.     

Structural correlations of phorbol-ester-induced stimulation of PGE2 production by human rheumatoid synovial cells

Eight phorbol esters were studied for their ability to stimulate prostaglandin production in human rheumatoid synovial cells over the dose range 0.1 ng to 1.0 g. These derivatives were based upon phorbol, 4-deoxyphorbol, and 12-deoxyphorbol nuclei. This activity was structurally dependent and, although it did not correlate with the actions of the same compounds to induce erythemain vivo, it did correlate with their ability to stimulate human lymphocyte mitogenesis. Stimulation of PGE₂ production by a phorbol and a 12-deoxyphorbol analog was inhibited in this system by both indomethacin and dexamethasone.