DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K125 adjacent to K126, K127 at the junction of CCPs2-3 and spatially near R96, and R100, all identified as being critical for DAF's function. Functional analyses of glucose or ribose treated DAF protein showed profound loss of its regulatory activity. The data argue that de-regulated activation of systemic complement and de-regulated activation of T cells and leukocytes could result from non-enzymatic glycation of DAF.

DAF as a therapeutic target for steroid hormones: implications for host-pathogen interactions.

In this chapter, we present a concise historic prospective and a summary of accumulated knowledge on steroid hormones, DAF expression, and therapeutic implication of steroid hormone treatment on multiple pathologies, including infection and the host-pathogen interactions. DAF/CD55 plays multiple physiologic functions including tissue protection from the cytotoxic complement injury, an anti-inflammatory function due to its anti-adherence properties which enhance transmigration of monocytes and macrophages and reduce tissue injury. DAF physiologic functions are essential in many organ systems including pregnancy for protection of the semiallogeneic fetus or for preventing uncontrolled infiltration by white cells in their pro- and/or anti-inflammatory functions. DAF expression appears to have multiple regulatory tissue-specific and/or menstrual cycle-specific mechanisms, which involve complex signaling mechanisms. Regulation of DAF expression may involve a direct or an indirect effect of at least the estrogen, progesterone, and corticosteroid regulatory pathways. DAF is exploited in multiple pathologic conditions by pathogens and viruses in chronic tissue infection processes. The binding of Escherichia coli bearing Dr adhesins to the DAF/CD55 receptor is DAF density dependent and triggers internalization of E. coli via an endocytic pathway involving CD55, lipid rafts, and microtubules. Dr+ E. coli or Dr antigen may persist in vivo in the interstitium for several months. Further understanding of such processes should be instrumental in designing therapeutic strategies for multiple conditions involving DAF's protective or pathologic functions and tailoring host expression of DAF.

Complement and cellular cytotoxicity in antibody therapy of cancer.

The effective and practical use of mAbs in cancer therapy became a reality with the development of the chimeric anti-CD20 mAb, rituximab. Several additional mAbs have since been approved for clinical use. Despite these successes, the mechanisms by which mAbs mediate antitumor activity are still unclear. Preclinical studies indicate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) both can contribute to mAb-induced tumor cell lysis. However, evidence related to the relative clinical importance of each mechanism, and whether they are synergistic or antagonistic, is conflicting. New ways to enhance both CDC and ADCC are being developed in attempt to develop a more effective anticancer mAb. Continued research on the mechanisms of mAb therapy will be necessary if we are to take optimal advantage of the current mAbs and develop more effective mAbs in the future.

Neurosteroids reduce inflammation after TBI through CD55 induction.

The inflammatory cascade that follows traumatic brain injury may lead to secondary cell death and can impede recovery of function. Complement factors and their convertases are increased in glia after brain injury and lead to the production of inflammatory products that kill vulnerable neurons. Progesterone and its metabolite allopregnanolone (5alpha-pregnan-3beta-ol-20-one) have been shown to reduce the expression of inflammatory cytokines in the acute stages of brain injury, although how they do this is not completely understood. In this study we show that both progesterone and allopregnanolone treatments enhance the production of CD55 following contusion injuries of the cerebral cortex in rats. CD55, a single-chain type 1 cell surface protein, is a potent inhibitor of the complement convertases which are activators of the inflammatory cascade. The increased expression of CD55 could be an important mechanism by which steroids help to reduce the cerebral damage caused by inflammation.

Down-regulation of human complement factor H sensitizes non-small cell lung cancer cells to complement attack and reduces in vivo tumor growth.

Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.

Minimal residual disease in ovarian cancer as a target for complement-mediated mAb immunotherapy.

Ovarian cancer is potentially well suited for local monoclonal antibody (mAb) immunotherapy, because it remains within the peritoneal cavity for a long period of time before giving rise to distant metastases. At the stage of minimal residual disease, the cells appear to be in a state of dormancy (G(0)) or at least have lower rates of tumour cell proliferation. They should be a promising target for immunotherapy. Here we first examined the cell-cycle expression of CD59 and decay-accelerating factor (DAF; CD55) on four different ovarian carcinoma cell lines, using simultaneous flow cytometric analysis of DNA content or the cell-cycle-specific nuclear proliferation protein Ki67 and CD59 or DAF surface expression. We found that CD59 and DAF are stably expressed throughout the cell cycle. The polyvalent approach to target-independent antigens to improve the efficiency of mAb complement (C)-mediated damages was promising, and tumour cells become sensitive to C damage, when incubated with cross-linked mAb against different tumour-associated antigens. Although, such immune complex-mediated C activation was rather ineffective in killing the cells, it could be potentiated by the addition of blocking mAb against CD59 and DAF. Our results suggest that the activities of intrinsic C regulators must be neutralized to make minimal residual disease a promising target for antibody therapy.

Polymorphic expression of decay-accelerating factor in human colorectal cancer.

BACKGROUND: We have previously shown that expression of decay-accelerating factor (DAF), a complement regulatory protein, is enhanced immunohistochemically on the luminal surface of cancer glands in human colorectal cancer and is detected in stool specimens of patients with colorectal cancer. The amount of DAF present in the stools might be influenced by the stability of DAF on the cell surface which is regulated by biochemical properties such as glycosylation of the protein. In the present study, to help elucidate the mechanism for the release of DAF from human colorectal cancers, we biochemically analyzed DAF expression by western and northern blotting by using surgically resected specimens of colorectal cancers. METHODS: Surgically resected colorectal cancer tissues were obtained from 10 patients. Expression of DAF was determined by western and northern blotting, and glycosylation of DAF protein was analyzed with glycosidase digestion. RESULTS: Northern blot analysis demonstrated that the expression of DAF mRNA in colorectal cancer was enhanced two- to threefold compared with normal tissues. In western blotting, expression of DAF protein in the cancer tissue was increased, and heterogeneity in the apparent molecular weight of DAF was observed among patients. When o-linked sugars were removed, this heterogeneity of DAF size diminished. CONCLUSIONS: The polymorphic expression of DAF in colorectal cancer is likely to reflect variability in the o-glycosylation of the protein. We speculate that this variability could affect the stability of DAF on the surfaces of cancer cells and, in turn, the amount of DAF shed into the stools of colorectal cancer patients.

Glucose-induced loss of glycosyl-phosphatidylinositol-anchored membrane regulators of complement activation (CD59, CD55) by in vitro cultured human umbilical vein endothelial cells.

AIMS/HYPOTHESIS: This study examines whether increased glucose concentrations are responsible for a decreased expression of membrane regulators of complement activation molecules. The effect of high glucose in determining an increase in membrane attack complex deposition on endothelial cells was also investigated. METHODS: Endothelial cells were isolated from umbilical cord tissue, cultured in the presence of increased concentrations of glucose, and the expression of CD46, CD55, and CD59 was detected by ELISA (enzyme-linked immunosorbent assay) and by flow cytometry. Glucose-treated endothelial cells were also incubated with antiendothelial cell antibodies and fresh complement to assess the amount of membrane attack complex formation. RESULTS: High concentrations of glucose decreased the expression of CD59 and CD55 by endothelial cells in a time-dependent and glucose concentration-dependent manner without affecting CD46 expression. High concentrations of soluble CD59 were found in the supernatants of cells treated with high glucose. The decrease in CD59 expression induced by high glucose concentrations was reversed by coincubation of cells with a calcium channel blocking agent (Verapamil). All of these effects were not reproduced by osmotic control media. Cells treated with concentrations of high glucose were more susceptible to complement activation and membrane attack complex formation after exposure to antiendothelial cell antibodies. CONCLUSION/INTERPRETATION: We speculate that hyperglycaemia could directly contribute to a loss of CD59 and CD55 molecules through a calcium-dependent phosphoinositol-specific phospholipase C activation and subsequent regulation of cell wall expression of GPI-anchored proteins. This phenomenon could facilitate the activation of a complement pathway and could play a part in the aetiology of endothelial dysfunction in diabetes.

Therapeutic inhibition of the complement system.

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.

Decreased expression of endometrial decay accelerating factor (DAF), a complement regulatory protein, in patients with luteal phase defect.

PROBLEM: We investigated the level of decay accelerating factor (DAF) in the endometrium of luteal phase defect (LPD) patients, before and after treatment with progesterone. METHODS: Endometrial samples from fourteen normal-cycling controls and six samples from infertility patients with LPD before and four samples after progesterone treatment were stained by anti-DAF IgG using immunohistochemistry. RESULTS: Mean DAF OD in LPD patients was 15% compared to 60% in the control group. The mean DAF OD was 88% after treatment with progesterone. CONCLUSION: Results support interpretation that progesterone upregulates DAF while decreased progesterone may be associated with reduced expression of the DAF in LPD patients.