Osmotic and hypoxic induction of the complement factor C9 in cultured human retinal pigment epithelial cells: Regulation of VEGF and NLRP3 expression.

Purpose: Variants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells. Methods: Hyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA. Results: Extracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1ß. Conclusions: The expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.

Hepatitis C virus suppresses C9 complement synthesis and impairs membrane attack complex function.

Hepatitis C virus (HCV) proteins inhibit complement component expression, which may attenuate immunity against infection. In this study, we examined whether HCV regulates the membrane attack complex (MAC) via complement component C9. MAC is composed of C5b to C9 (C5b-9) and mediates cell lysis of invaded pathogens. Liver biopsy specimens from chronically HCV-infected patients exhibited a lower level of C9 mRNA expression than liver biopsy specimens from unrelated disease or healthy control human liver RNA. Hepatocytes infected with cell culture-grown HCV or expressing HCV core protein also displayed significant repression of C9 mRNA and protein levels. Promoter analysis suggested that the T cell factor-4 (TCF-4E) transcription factor is responsible for HCV core-mediated C9 promoter regulation. Sera from chronically HCV-infected patients displayed a lower level of C5b-9 and a reduced antimicrobial effect on model organisms compared to unrelated patient sera or sera from healthy volunteers. Together, these results for C9 regulation by HCV core protein coupled with functional impairment of the membrane attack complex underscore HCV-mediated attenuation of immune mechanisms.

Potassium cyanide protects Escherichia coli from complement killing by the inhibition of C3 convertase activity.

The exact mechanism by which deposited C5b-9 complexes kill Gram-negative bacteria is unclear. It has been proposed that during complement activation the membrane attack complex triggers an energy dependent process in Gram-negative bacteria that mediates destruction of the inner membrane. This observation in part resulted from the survival of Gram-negative bacteria that were incubated with an uncoupler (DNP) or an inhibitor (KCN) of oxidative phosphorylation during complement activation. In a reexamination of this issue we employed potassium cyanide (KCN) to block energy dependent pathways and observed a dose dependent inhibition of C9 uptake on E. coli J5 during serum incubation, suggesting that cyanide was interfering with complement activation. To verify the effect on complement activation we chose specifically to study the effects of KCN on the C3 convertase of the classical pathway. Sensitized sheep erythrocytes were employed as our model system. This system allowed us to construct a series of stable intermediates that were used to test the effect of cyanide on the formation and activity of precursors of the classical pathway C3 convertase. The data illustrate that the concentrations of potassium cyanide that inhibit complement killing of J5 also inhibit C3 convertase activity on sensitized sheep erythrocytes. The results of this study refute the principal observation made by other investigators, that potassium cyanide protects bacteria from complement killing by inhibiting bacterial energy dependent pathways that spark inner membrane destruction. A better scenario is that the organisms survive because cyanide inhibits complement activation.

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis.

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.