Weiguo Hu

Complement Regulator CD59 Protects Against Atherosclerosis by Restricting the Formation of Complement Membrane Attack Complex

Complement is a central effector system within the immune system and is implicated in a range of inflammatory disorders. CD59 is a key regulator of complement membrane attack complex (MAC) assembly. The atherogenic role of terminal complement has long been suspected but is still unclear. Here, we demonstrate that among mice deficient in apolipoprotein (Apo)E, the additional loss of murine CD59 (mCd59ab−/−/ApoE−/−) accelerated advanced atherosclerosis featuring occlusive coronary atherosclerosis, vulnerable plaque, and premature death and that these effect could be attenuated by overexpression of human CD59 in the endothelium. Complement inhibition using a neutralizing anti-mouse C5 antibody attenuated atherosclerosis in mCd59ab−/−/ApoE−/− mice. Furthermore, MAC mediated endothelial damage and promoted foam cell formation. These combined results highlight the atherogenic role of MAC and the atheroprotective role of CD59 and suggest that inhibition of MAC formation may provide a therapeutic approach for the treatment of atherosclerosis.

The critical roles of platelet activation and reduced NO bioavailability in fatal pulmonary arterial hypertension in a murine hemolysis model

Pulmonary arterial hypertension (PAH) is suspected to be a strong mortality determinant of hemolytic disorders. However, direct contribution of acute intravascular hemolysis to fatal PAH has not been investigated. The roles of nitric oxide (NO) insufficiency and platelet activation in hemolysis-associated fatal PAH have been suspected but not been experimentally studied. We recently generated a unique intravascular hemolysis mouse model in which the membrane toxin, intermedilysin (ILY), exclusively lyses the erythrocytes of transgenically expressing human CD59 mice (ThCD59(RBC)), thereby inducing ILY-dose-dependent massive hemolysis. Using this murine hemolysis model, we found that the acute increase in pulmonary arterial pressure leading to right ventricle failure caused sudden death. Reduced NO bioavailability and massive platelet activation/aggregation leading to the formation of massive thrombosis specifically in the pulmonary microvasculature played the critical roles in pathogenesis of acute hemolysis-associated fatal PAH. Therapeutic interventions enhancing NO bioactivity or inhibiting platelet activation prevented sudden death or prolonged survival time via the suppression of the acute increase in pulmonary arterial pressure and improvement of right ventricle function. These findings further highlight the importance of the inhibition of platelet activation and the enhancement of NO bioavailability for the treatment and prevention of hemolysis-associated (fatal) PAH.

Human CD59 Inhibitor Sensitizes Rituximab-Resistant Lymphoma Cells to Complement-Mediated Cytolysis

Rituximab efficacy in cancer therapy depends in part on induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex, thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell non-Hodgkins lymphoma (NHL), and upregulation of hCD59 is an important determinant of the sensitivity of NHL cells to rituximab treatment. Here, we report that the potent hCD59 inhibitor rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing rituximab-resistant lymphoma cells and primary chronic lymphocytic leukemia cells (CLL) to rituximab treatment. By defining pharmcokinetic/pharmacodynamic profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. Increasing expression levels of the complement regulators CD59 and CD55 in rituximab-resistant cells occur due to selection of preexisting clones rather than de novo induction of these proteins. Moreover, lymphoma cells overexpressing CD59 were directly responsible for the resistance to rituximab-mediated CDC therapy. Our results rationalize the use of rILYd4 as a therapeutic adjuvant for rituximab treatment of rituximab-resistant lymphoma and CLL. Furthermore, they suggest that preemptive elimination of CD59-overexpressing subpopulations along with rituximab treatment may be a useful approach to ablate or conquer rituximab resistance.

rILYd4, a Human CD59 Inhibitor, Enhances Complement-Dependent Cytotoxicity of Ofatumumab against Rituximab-Resistant B-cell Lymphoma Cells and Chronic Lymphocytic Leukemia

Purpose: Ofatumumab is an anti-CD20 antibody recently approved for treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL); it mediates much stronger complement-dependent cytotoxicity (CDC) than rituximab. Human CD59, a key membrane complement regulator that inhibits CDC, is highly expressed in B-cell malignancies and its upregulation is an important determinant of the sensitivity of B-cell malignancies to rituximab treatment. Previously, we have shown that the potent CD59 inhibitor rILYd4 sensitizes rituximab-resistant lymphoma cells to rituximab-mediated CDC. Here, we further investigated whether rILYd4 can sensitize B-cell malignancies to ofatumumab-mediated CDC and whether either ofatumumab-mediated CDC or rILYd4-enhanced ofatumumab-mediated CDC correlates with CD20 or CD59 expression, known biomarkers involved in rituximab activity. Experimental Design: Rituximab-resistant cell lines and primary CLL cells were used to investigate the antitumor efficacy of the combination of rILYd4 with ofatumumab or rituximab. Propidium iodide staining or alamarBlue assay were used to evaluate the CDC effect. The levels of CD20 and CD59 on the cell membrane were analyzed by flow cytometry. Results: rILYd4 enhanced CDC effects mediated by ofatumumab or rituximab on rituximab-resistant lymphoma cells and primary CLL cells in vitro. The sensitivity to CDC effects mediated by ofatumumab positively correlated with the ratio of CD20/CD59 and negatively correlated with CD59 levels on CLL cells. The degree to which rILYd4 enhanced CDC correlated positively with the CD59 levels on CLL cells. Conclusions: These data suggest that rILYd4 may enhance the anticancer activity of ofatumumab and rituximab in B-cell malignancies that have relapsed after prior antibody-based therapies. Clin Cancer Res; 17(21); 6702–11. ©2011 AACR.

A high-affinity inhibitor of human CD59 enhances complement-mediated virolysis of HIV-1: implications for treatment of HIV-1/AIDS.

Many pathogenic enveloped viruses, including HIV-1, escape complement-mediated virolysis by incorporating host cell regulators of complement activation into their own viral envelope. The presence of complement regulators including CD59 on the external surface of the viral envelope confers resistance to complement-mediated virolysis, which may explain why human pathogenic viruses such as HIV-1 are not neutralized by complement in human fluids, even in the presence of high Ab titers against the viral surface proteins. In this study, we report the development of a recombinant form of the fourth domain of the bacterial toxin intermedilysin (the recombinant domain 4 of intermedilysin [rILYd4]), a 114 aa protein that inhibits human CD59 function with high affinity and specificity. In the presence of rILYd4, HIV-1 virions derived from either cell lines or peripheral blood mononuclear cells of HIV-1–infected patients became highly sensitive to complement-mediated lysis activated by either anti–HIV-1 gp120 Abs or by viral infection-induced Abs present in the plasma of HIV-1–infected individuals. We also demonstrated that rILYd4 together with serum or plasma from HIV-1–infected patients as a source of anti–HIV-1 Abs and complement did not mediate complement-mediated lysis of either erythrocytes or peripheral blood mononuclear cells. These results indicate that rILYd4 may represent a novel therapeutic agent against HIV-1/AIDS

Balancing role of nitric oxide in complement-mediated activation of platelets from mCd59a and mCd59b double-knockout mice

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. mCd59 knockout mice reportedly exhibit hemolytic anemia and platelet activation. This phenotype is comparable to the human hemolytic anemia known as paroxysmal nocturnal hemoglobinuria (PNH), in which platelet activation and thrombosis play a critical pathogenic role. It has long been suspected but not formally demonstrated that both complement and nitric oxide (NO) contribute to PNH thrombosis. Using mCd59a and mCd59b double knockout mice (mCd59ab−/− mice) in complement sufficient (C3+/+) and deficient (C3−/−) backgrounds, we document that mCd59ab−/− platelets are sensitive to complement‐mediated activation and provide evidence for possible in vivo platelet activation in mCd59ab−/− mice. Using a combination of L‐NAME (a NO‐synthase inhibitor) and NOC‐18 or SNAP (NO‐donors), we further demonstrate that NO regulates complement‐mediated activation of platelets. These results indicate that the thrombotic diathesis of PNH patients could be due to a combination of increased complement‐mediated platelet activation and reduced NO‐bioavailability as a consequence of hemolysis. Am. J. Hematol. 2009.

Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59RBC) or on endothelia (strain ThCD59END). Intravenous injection of ILY in ThCD59RBC mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59END mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.

Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells

Many monoclonal antibodies (mAbs) have been extensively used in the clinic, such as rituximab to treat lymphoma. However, resistance and non-responsiveness to mAb treatment have been challenging for this line of therapy. Complement is one of the main mediators of antibody-based cancer therapy via the complement-dependent cytolysis (CDC) effect. CD59 plays a critical role in resistance to mAbs through the CDC effect. In this paper, we attempted to investigate whether the novel CD59 inhibitor, recombinant ILYd4, was effective in enhancing the rituximab-mediated CDC effect on rituximab-sensitive RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells. Meanwhile, the CDC effects, which were mediated by rituximab and anti-CD24 mAb, on the refractory multiple myeloma (MM) cell line ARH-77 and the solid tumor osteosarcoma cell line Saos-2, were respectively investigated. We found that rILYd4 rendered the refractory cells sensitive to the mAb-mediated CDC effect and that rILYd4 exhibited a synergistic effect with the mAb that resulted in tumor cells lysis. This effect on tumor cell lysis was apparent on both hematological tumors and solid tumors. Therefore, rILYd4 may serve as an adjuvant for mAb mediated-tumor immunotherapy.

Generation and phenotyping of mCd59a and mCd59b double-knockout mice.

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. Humans express only one, whereas mice express two CD59 genes. We previously reported the targeted deletion of the mCd59b gene in which absence of mCd59b together with an unintended down regulation of mCd59a caused hemolytic anemia with spontaneous platelet activation. To confirm the complement role in the hemolytic anemia caused by abrogation of mCd59 function, we have developed a mCd59a and mCd59b double knock out mice and analyzed its phenotype in complement sufficient and deficient (C3−/−). We report here that total abrogation of mCd59 function in mCd59ab−/− mice results in complement‐mediated hemolytic anemia that is rescued by the deficiency of C3 in compound mCd59ab−/−/C3−/− mice. Am. J. Hematol., 2009.

Analysis of the promoters and 5′-UTR of mouse Cd59 genes, and of their functional activity in erythrocytes

The complement regulatory protein CD59 inhibits formation of the membrane attack complex (MAC), the terminal effector of the complement system. There are two mouse Cd59 genes in mice but only one in humans. In the work reported here we (a) mapped the promoter regions of both mCd59a and mCd59b genes, (b) identified two different promoters for each mCd59 gene, (c) defined a previously unrecognized additional exon 1 in each mCd59 gene, (d) identified that each mCd59 gene expresses two different tissue-specific transcripts that differ in their 5′-UTR, and (e) confirmed the presence of mCd59b mRNA in multiple tissues. At the functional level, comparison of the sensitivity of mCd59ab−/− and mCd59a−/− red blood cells to MAC-mediated lysis revealed that mCd59b protects RBC from MAC-mediated lysis, at least in the setting of mCd59a deficiency. Together these findings indicate that the mCd59 genes may have complex and perhaps different regulatory mechanisms in different tissues.