Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells.

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Schistosoma japonicum reveals distinct reactivity with antisera directed to proteases mediating host infection and invasion by cercariae of S. mansoni or S. haematobium.

Serine proteases released from the acetabular glands of cercariae, also known as cercarial elastases, are key enzymes in the penetration process of schistosomes through the skin of the final host. Antisera against these enzymes secreted from Schistosoma mansoni or S. haematobium reveal differences in the patterns of elastase expression among schistosome species and among different developmental stages of the larvae. Immunolocalization studies showed that antisera raised against the enzyme s28 protease react with S. mansoni, S. haematobium and also S. japonicum, in developing as well as mature cercariae and in both pre- and post-acetabular glands. Antisera against the enzyme SmCE detect the respective antigen solely in the pre-acetabular glands. Remarkably, the SmCE-1a isoform is detectable with DNA-vaccinated mouse sera in S. mansoni and S. haematobium only, but is apparently absent from the acetabular glands of S. japonicum. These differences in immunoreactivity of cercarial enzymes may be related to the distinct infection process of S. japonicum.

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Complement resistance of human carcinoma cells depends on membrane regulatory proteins, protein kinases and sialic acid.

Nucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC-3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti-mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC-3> SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC-dependent process. Combined treatment of CA with anti-CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement-mediated lysis. Therefore, the effectiveness of antibody- and complement-based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.

Complement and apoptosis.

Apoptosis and necrosis are two forms of cell death characterized by distinct morphologies. Until recently, complement-mediated cell lysis has been presented as a classical example of necrotic cell death. However, recent reports on apoptogenic effects of complement have shaken this dogma. The field has become even more confusing with descriptions of anti-apoptotic effects of complement. Necrosis has been associated traditionally with inflammation, whereas apoptosis has been regarded as noninflammatory. Therefore, first descriptions of the capacity of the complement system to identify apoptotic cells and to be activated by them, led to the development of the concept that complement opsonizes apoptotic cells for fast clearance by phagocytic cells. In the absence of such opsonization, (eg in C1q or C4 deficiency), apoptotic cells may remain longer in the body and may stimulate autoantibody production or undergo pro-inflammatory secondary necrosis. This has been associated in man and mouse with the development of an autoimmune disease like systemic lupus erythematosus. However, complement may also interfere with the programmed intention of apoptosis to avoid triggering of inflammation. Our recent results show that, under specific conditions, early apoptotic cells are not only opsonized by complement but may also be lysed, raising the possibility that under certain conditions apoptosis will be associated with an inflammatory reaction. The review describes and discusses the reports covering the various aspects of the interface between complement and apoptosis and its possible relevance to autoimmune diseases and inflammation and raises the following questions: 1. Can activated complement proteins induce apoptotic cell death? 2. Can complement protect cells from apoptosis? 3. Do apoptotic cells activate complement? and 4. What role is complement playing in clearance of apoptotic cells and which complement receptors are involved? Clearly, this research field is highly complex and still at its formation stage, yet interesting and important mechanisms are bound to emerge from these studies.

K562 erythroleukemic cells are equipped with multiple mechanisms of resistance to lysis by complement.

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.

Contribution of heat shock proteins to cell protection from complement-mediated lysis.

The possible participation of hsc70 and hsp70 in cellular protection from complement damage was studied. Human erythroleukemia K562 cells were pretreated with reagents affecting hsc70 or hsp70, and cell sensitivity to lysis by antibody and human complement was examined. Treatment with deoxyspergualin, an hsc70 inhibitor, sensitized K562 cells to complement lysis, whereas treatment with ethanol, butanol or hemin, inducers of hsc70 synthesis, protected the cells from complement-mediated lysis. Incubation of K562 at either 42 degrees C or with the amino acid analogue L-azetidine-2-carboxylic acid induced synthesis of hsp70, but not of hsc70. The latter treatment also conferred elevated resistance to complement lysis on K562 cells. Pretreatment of K562 cells with sub-lethal doses of complement desensitizes them to lethal complement doses. No effect of sublytic complement on synthesis of hsc70 and hsp70 was found. However, the results demonstrated that complement stress causes translocation of hsc70 from the cytoplasm to the K562 cell surface. Two monoclonal and two polyclonal antibodies identified hsc70 on the surface of intact, viable complement-stressed cells, while antibodies directed to hsp70 did not bind to these cells. Altogether, the results suggest that the heat shock proteins hsc70 and hsp70 play a role in cell defense against complement.

Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis.

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.

Immunolocalization of the Toxin Latrunculin B within the Red Sea Sponge Negombata magnifica (Demospongiae, Latrunculiidae).

The location of latrunculin B, the major toxin of the Red Sea sponge Negombata magnifica, was revealed using specific antibodies. Antibodies from rabbits immunized with a conjugate of latrunculin B with keyhole limpet hemocyanin (KLH) were purified over a latrunculin B-Sepharose affinity column. Analysis of immunohistochemical and immunogold-stained sponge sections, using light and transmission electron microscopy, revealed latrunculin B labeling mostly beneath the sponge cortex at the border between the external (ectosome) and internal (endosome) layers (ectosome-endosome border). The endosome was less labeled than the border. Immunogold localization revealed latrunculin B in the sponge cells but not in its prokaryotic symbionts. Archeocytes and choanocytes were significantly more labeled than other cells. The antibodies primarily labeled membrane-limited vacuoles within archeocytes and choanocytes that are perhaps latrunculin B secretory or storage vesicles. Peripheral latrunculin B may have a role in defense against external epibionts, predators, and competitors.

Cell desensitization by sublytic C5b-9 complexes and calcium ionophores depends on activation of protein kinase C.

Basal cell resistance to lysis by complement C5b-9 complexes depends on extracellular and intracellular protection. Cell membrane regulatory proteins and enzymes interfere with complement activation and intracellular processes of protein phosphorylation and synthesis support cell resistance and damage repair. K562 human erythroleukemic cells treated with sublytic complement doses become protected from lytic doses of complement within 50 min. The early signaling processes leading to cell desensitization to complement-mediated lysis were studied. Treatment with calcium ionophores or phorbol 12-myristate 13-acetate rapidly induced in K562 cells protection from complement as well as synthesis of a large protein complex similar to the large complement-induced protein complex L-CIP induced by sublytic complement. Both ionophore- and complement-induced protection were blocked by treatment with protein kinase C (PKC) inhibitors. Calphostin C, sphingosine and GF109203X abrogated complement-induced protection almost completely, whereas Go6976 inhibited it only partially. Since Go6976 is a selective inhibitor of the conventional PKC type, it is proposed that sublytic complement doses activate both conventional and non-conventional PKC types. Immunofluorescence analysis of K562 cells demonstrated sublytic complement-induced translocation of the conventional PKCalpha and PKCbetaII from the cytoplasm to the plasma membrane. These results indicate that PKC activation is an early obligatory signal in cell desensitization by sublytic C5b-9 or calcium ionophore.

Phosphorylation of the complement component, C9, by an ecto-protein kinase of human leukemic cells.

Ecto-protein kinases (ecto-PK) are surface constituents of many, if not all, animal cell types; normal, transformed or malignant. The occurrence of ecto-PK on the surface of human leukemia cell lines was described [Paas, Y., Fishelson, Z., 1995. Shedding of tyrosine and serine/threonine ecto-PK from human leukemic cells. Arch. Biochem. Biophys. 316 780-788.]. These ecto-PKs have been shown to phosphorylate several exogenous substrates, including the complement C9 protein, an essential component of the terminal complement system. C9 is phosphorylated by ecto-PK of K562 cells on serine residue(s). Phosphorylation occurs in the N-terminal C9a portion produced by cleavage of phosphorylated C9 with human alpha-thrombin. C9 polymers generated upon incubation of C9 with ZnCl2 do not serve as substrates for the K562 ecto-PK. In contrast, unfolded C9, obtained by reduction and alkylation, serves as a superior substrate for the K562 ecto-PK. Native C9 phosphorylation produced a rather low stoichiometry of incorporated phosphate (around 3%) per C9. Despite that, the phosphorylated C9 expressed reduced hemolytic activity. The complement-sensitive variant of K562 (K562/S) did not express the C9 phosphorylating activity. Various PK inhibitors tested failed to block C9 phosphorylation. Only heparin and 2,3-diphosphoglycerate (dpGA) prevented C9 phosphorylation, indicating that the ecto-PK is related to the casein kinase CK2. C9 can be phosphorylated by ecto-PK from other tumor cells, including Jurkat, SK-OV-3 and BT-474. These results suggest that extracellular phosphorylation of C9 may serve as a protective mechanism against complement in tumor cells.

Neutralization of complement regulatory proteins augments lysis of breast carcinoma cells targeted with rhumAb anti-HER2.

The capacity of recombinant human monoclonal anti-p185HER2 IgG (rhumAb anti-HER2) to activate human complement was investigated. Complement activation by rhumAb anti-HER2 on various human breast carcinoma cell lines resulted in deposition of complement proteins on these cells. Complement activation was also observed in a solid-phase binding assay, in which purified p185HER2 was immobilized onto a microtiter plate. rhumAb anti-HER2 induced some complement-mediated tumor cell lysis by rabbit complement, but not by human complement. Analysis of membrane complement regulatory proteins (mCRP) on breast carcinoma cells revealed a heterogenous expression of CD46, CD55 and CD59. After blocking the mCRP activity with specific antibodies, rhumAb anti-HER2 induced about 15% lysis of p185HER2-expressing tumor cells. Tumor cell sensitization with rabbit polyclonal anti-tumor antiserum following mCRP neutralization, augmented cell lysis from 10 to 80%. Expression of mCRP was upregulated by treatment with PMA, and correlated with increased protection of the tumor cells from complement lysis. These results suggest that humanized antibodies like rhumAb anti-HER2 promote complement activation leading to tumor cell phagocytosis and cell-mediated cytotoxicity. They further demonstrate that a successful tumor immunotherapeutical approach, based on antibody and complement treatment, requires mCRP neutralization.

Distinction between processing of normal and mutant complement C3 within human skin fibroblasts.

Inherited C3 deficiency may result from mutations in the C3 gene affecting transcription or translation (type I deficiency). We described a type II C3 deficiency caused by a mutation yielding an abnormal non-secreted C3. The post-translational processing of mutant and normal C3 was analyzed in fibroblasts grown from skin biopsies. Mutant C3 is located mainly in the endoplasmic reticulum (ER), whereas normal C3 is seen evenly distributed throughout the cytoplasm. Most of the mutant C3 is degraded within the cell, and only a small fraction (around 8%) is secreted after 20 h chase. Processing of C3 at 19 degrees C was reduced in normal fibroblasts but completely blocked in mutant fibroblasts. ATP depletion blocked processing of normal proC3 to C3. In contrast, the mutant proC3 was partly degraded in ATP-depleted cells, yet its complete degradation and secretion were blocked. Intracellular degradation of the mutant C3 was not inhibited by NH4Cl, thus excluding cleavage within lysosomes. These results demonstrate that the type II mutant C3 studied here is retained in the ER probably by a quality contol machinery that identifies abnormal protein folding. Consequently, it is destined to undergo a two-step intracellular degradation; an initial ATP-independent step followed by an ATP-dependent step.

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis.

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3-1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3-1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3-1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3-1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3-1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3-1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.

Protection of human nasal respiratory epithelium from complement-mediated lysis by cell-membrane regulators of complement activation.

Complement in the respiratory tract protects the host from invading micoorganisms and other inhaled insults, but may damage normal tissue. Recently we reported that human respiratory epithelium from the nose to the alveoli expresses three cell-membrane regulators of complement activation: membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF; CD55), and CD59. In this study we investigated whether two of these complement-regulatory proteins, DAF and CD59, protect human nasal epithelial cells from complement-mediated lysis. Treatment of nasal epithelial cells in suspension with 50% or 100% normal human serum (NHS) lysed small percentages of cells (8% and 16%, respectively). Addition of complement activators, rabbit serum antinasal epithelial cells (anti-NEC), or lipopolysaccharide (LPS) increased cell lysis in the presence of 50% NHS in a dose-dependent manner up to 50% and 35% lysis, respectively. Human serum deficient in C3 or C7 did not lyse nasal epithelial cells even in the presence of anti-NEC. To assay the contribution of DAF and CD59 to cell protection against lysis, nasal epithelial cells in suspension were treated with appropriate blocking antibodies. Both anti-DAF and anti-CD59 markedly increased the susceptibility of human nasal epithelial cells to lysis by complement. At 50% NHS, anti-DAF and anti-CD59 antibodies increased epithelial cell lysis from 8% to 24% and 67%, respectively. A similar pattern of response to complement was demonstrated by monolayers of substrate-anchored cultured cells. These results indicate that DAF and CD59 protect human nasal epithelial cells from complement-mediated lysis; however, intense activation of complement may overcome this protection, leading to cell death and tissue injury. We speculate that imbalance between complement regulation and complement activation in the human respiratory tract in disease may result in tissue injury and impaired tissue function.

Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula.

Transformation of cercariae of Schistosoma mansoni into schistosomula is accompanied by release of a soluble 28-kDa serine protease (s28) from the acetabular glands. The postulated activities of s28 include cleavage of skin connective tissue proteins (elastin, etc.), release of the cercarial glycocalyx, and cleavage of complement proteins. Our previous results demonstrated the presence of an antigenically cross-reactive protein on the surface of mechanically transformed schistosomula. As shown here, schistosomula express on their surface a 28-kDa serine protease (m28) which can be immunoprecipitated with anti-s28 antibodies. m28 eluted from the schistosomular tegumental membrane with NP-40 was purified to homogeneity in one step by adsorption on a chymotrypsin inhibitor column: 6-aminocaproyl-D-tryptophan methyl ester-Sepharose. Proteolytic activity of m28 was completely inhibited by the chymotrypsin inhibitor N-succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone. Efficient removal of m28 from schistosomula was achieved with NP-40, deoxycholate, cholate, Tween 20, and phospholipases A2 and C, but not with papain, trypsin, pronase, or proteinase K. Furthermore, treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) followed by hydroxylamine also released m28. Anti-cross-reactive determinant antibodies which recognize a neo epitope exposed in glycosyl phosphatidyl inositol-containing molecules cleaved by PI-PLC bind to purified m28. The latter results suggest that m28 is anchored to the tegumental membrane of schistosomula by a lipid anchor and that perhaps some of the m28 molecules are bound via glycosylphosphatidyl inositol. Based on inhibitor sensitivity and antigenic cross-reactivity, it is conceivable that s28 and m28 are related, if not identical, proteins. Finally, m28 was detected antigenically also on lung-stage and adult worms of S. mansoni.

Complement membrane attack complex, perforin, and bacterial exotoxins induce in K562 cells calcium-dependent cross-protection from lysis.

The complement membrane attack complex (MAC), the cytolytic granule protein of cytotoxic lymphocytes perforin, the streptococcal exotoxin streptolysin O (SLO), and the bee venom polypeptide melittin utilize a similar mechanism to incorporate into cell membranes, induce a Ca2+ influx and a rise in intracellular Ca2+ concentration, and produce cell lysis. At sublytic concentrations, these proteins trigger several cellular activities, including protein phosphorylation and synthesis. We have recently demonstrated that human leukemic cells treated with sublytic doses of human complement become more resistant to lytic complement doses. The study has now been extended to include three other pore-formers: murine perforin, SLO and melittin. As shown here, sublytic MAC induces in the K562 human erythroleukemic cells protection from lytic perforin, and vice versa, sublytic perforin induces protection from complement. Also, sublytic SLO and melittin increase resistance of K562 cells to lytic complement and perforin doses. The capacity of Ca2+ ionophores to induce resistance to the lytic proteins has been examined. Exposure of K562 cells to sublytic concentrations of ionomycin or A23187 for 1 h at 37 degrees C confers on them resistance to complement- and perforin-mediated lysis. The protective effects of the ionophores can be abrogated by chelation of extracellular Ca2+ and by inhibition of RNA or protein synthesis in the cells. These results indicate the following: 1) nucleated cells exposed to sublytic complement MAC, perforin, SLO, or melittin may become resistant to the four pore-formers. Physiologically, this may be regarded as an immunologic tachyphylaxis. 2) Ca2+ influx induced by these pore-formers is an essential and sufficient factor to produce this tachyphylaxis.

Novel mechanisms of immune evasion by Schistosoma mansoni.

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28--A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56--A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70--A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1--A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.