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PubMed1998

Temporal induction of clusterin in the peri-infarct zone after experimental myocardial infarction in the rat.

Clusterin, a glycoprotein with potent cellular cohesive properties, is induced in many organs at times of tissue injury or remodeling. After renal infarction, for example, clusterin is localized to tubular epithelial cells in the peri-infarct zone. The purpose of this study was to examine the spatial and temporal expression of cardiac clusterin after myocardial infarction. Sprague-Dawley rats underwent permanent coronary ligation or sham operation. Hearts were harvested at 6 hours and at 2, 14, and 28 days after infarction. Cardiac clusterin expression was examined by immunohistochemistry and in situ hybridization. Left ventricular clusterin staining was evident at 6 hours and 2 days after myocardial infarction, although not at later time periods. Clusterin was localized to peri-infarct zone myocytes and endothelial cells of this region, and local synthesis of clusterin by myocytes was confirmed by in situ hybridization. Clusterin was not present in inflammatory cells or in left ventricular tissue distant from the infarct. The distribution of clusterin was different from the membrane attack complex of complement (C5b-9), with the latter being present diffusely throughout the infarct zone. Although the role of cardiac clusterin is not known, we speculate that clusterin's cohesive properties serve to promote myocyte interactions that are perturbed in the peri-infarct zone after myocardial infarction.

Animals↗
PubMed1989

Local complement activation, thromboxane-mediated vasoconstriction, and vascular leakage in isolated lungs. Role of the terminal complement sequence.

In situ complement activation was induced in isolated, ventilated, and blood-free perfused rabbit lungs. Six to 11% human serum (vol/vol) was repeatedly admixed to the recirculating Krebs Henseleit buffer during 10-min periods, interrupted by rinsing phases with serum-free medium. This caused dose-dependent, reversible, and reproducible pulmonary artery pressor responses with pressure peaks or plateaus of 8 to 22 mm Hg. Pressor responses were paralleled by a marked release of thromboxane (TxA2) and prostaglandin I2 into the medium. Heat-inactivated serum, purified fluid-phase SC5b-9, and serum that had been activated with inulin in the absence or presence of a carboxypeptidase inhibitor all failed to elicit pressor responses. Both pressor response and prostanoid release were strictly dependent on complement factor C8, and evidence for C8 binding was obtained with the use of 125I-C8. Inhibitor studies collectively indicated that TxA2 was the predominant vasoconstrictive agent. Although repetitive short-term application of human serum induced no major lung weight gain, prolonged exposure to serum (40 to 80 min) without intermittent rinsing phases caused a delayed increase in the capillary filtration coefficient to maximally 3- to 4-fold values, with marked lung edema formation. We conclude that in situ activation of complement in the rabbit lung vasculature causes immediate intrapulmonary prostanoid generation and thromboxane-mediated vasoconstriction, independent of circulating leukocytes, but dependent on the formation of terminal membrane-complement complexes. Continuous complement activation results in a delayed increase in lung vascular permeability.

Animals↗
PubMed1983

Interaction of human beta-endorphin with nonopiate binding sites on the terminal SC5b-9 complex of human complement. Significance of COOH-terminal beta H-endorphin fragments.

We have characterized the binding of 125I-labeled human beta-endorphin (125I-beta H-endorphin) to sites present on the terminal fluid-phase complex of human complement, consisting of complement components C5b, C6, C7, C8, C9, and the S-protein (SC5b-9 complex). Specific binding exhibited saturability, reversibility, structural specificity, temperature dependence, and absence of negative cooperative effects. Binding was maximal at 4 degrees C and pH 7.0; it was diminished by monovalent and divalent cations as well as by increasing concentrations of urea and Triton X-100 and apparently required intact disulfide groups. Binding was not inhibited by a number of opioid peptides sharing common sequences with the NH2 terminus of beta H-endorphin. In contrast, binding was inhibited by beta H-endorphin, N-acetyl-beta H-endorphin, and a series of COOH-terminal beta H-endorphin fragments, where of the COOH-terminal dipeptide Gly-Glu represented the minimal effective structure. Stepwise extension towards the NH2 terminus led to an increased binding affinity of the respective fragment. Computer resolution of competition curves yielded one binding component for several shorter COOH-terminal beta H-endorphin fragments and for beta H-endorphin (1-5) + (16-31), whereas two distinct binding components were obtained when beta H-endorphin (27-31), beta H-endorphin (6-31), N-acetyl-beta H-endorphin or beta H-endorphin were used as inhibitors. This study presents detailed data on the binding of COOH-terminal beta H-endorphin fragments to specific nonopiate binding sites present on the terminal SC5b-9 complex of human complement. We suggest that through this interaction, beta H-endorphin may modulate certain functions within the immune system.

Binding Sites↗
PubMed2004

Potent antileukemic interactions between flavopiridol and TRAIL/Apo2L involve flavopiridol-mediated XIAP downregulation.

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L), were examined in human leukemia cells (U937 and Jurkat). Coexposure of cells to marginally toxic concentrations of TRAIL and FP (24 h) synergistically increased mitochondrial injury (eg, cytochrome c, AIF, Smac/DIABLO release), cytoplasmic depletion of Bax, activation of Bid as well as caspase-8 and -3, PARP cleavage, and apoptosis. Coadministration of TRAIL markedly increased FP-induced apoptosis in leukemic cells ectopically expressing Bcl-2, Bcl-x(L), or a phosphorylation loop-deleted form of Bcl-2 (DeltaBcl-2), whereas lethality was substantially attenuated in cells ectopically expressing CrmA, dominant-negative-FADD, or dominant-negative-caspase-8. TRAIL/FP induced no discernible changes in FLIP, DR4, DR5, Mcl-1, or survivin expression, modest declines in levels of DcR2 and c-IAP, but resulted in the marked transcriptional downregulation of XIAP. Moreover, cells stably expressing an XIAP-antisense construct exhibited a pronounced increase in TRAIL sensitivity comparable to degrees of apoptosis achieved with TRAIL/FP. Conversely, enforced XIAP expression significantly attenuated caspase activation and TRAIL/FP lethality. Together, these findings suggest that simultaneous activation of the intrinsic and extrinsic apoptotic pathways by TRAIL and FP synergistically induces apoptosis in human leukemia cells through a mechanism that involves FP-mediated XIAP downregulation.

Apoptosis↗
PubMed1989

Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor.

When whole serum C is activated by cobra venom factor complexes (CoFBb), paroxysmal nocturnal hemoglobinuria (PNH) III E (the most C-sensitive type) are hemolyzed, but normal and PNH II E (the intermediately sensitive type) are not. Previous studies have shown that after exposure to CoFBb and serum, PNH III E bind relatively large amounts of the trimolecular C complex, C5b67, whereas normal and PNH II E bind virtually none. In the studies reported herein, we have observed that when normal and PNH III E are incubated with isolated C5, C6, and 125I-C7 in the presence CoFBb, the normal E bind more C5b-7 than the PNH cells. When C7-deficient serum is included in the reaction mixture, however, the PNH E are once again observed to bind much greater amounts of C5b-7. These observations suggest that plasma and membrane factors act in concert to restrict the assembly of the trimolecular C5b-7 complex on human E. PNH III E appear to be deficient in the membrane component of this inhibitory system.

Animals↗
PubMed1997

Mixed connective tissue disease. A clinical, histologic, and immunofluorescence study of eight cases.

A study of the cutaneous eruptions of eight patients with mixed connective tissue disease (MCTD) was performed to better characterize its dermatopathology and to explore a role for the membrane attack complex of complement C5b-9 in lesional pathogenesis. Nine lesional skin biopsies were obtained from eight patients with MCTD and analyzed by conventional light microscopy. Direct immunofluorescence (IF) and indirect IF using a monoclonal antibody to C5b-9 were applied in six and five cases respectively. The biopsied cutaneous eruptions were characterized clinically as photo-distributed erythematosus annular and/or papulosquamous lesions mimnicking subacute cutaneous lupus erythematosus (SCLE) in five of eight patients as an ill-defined, telangiectatic, scaly patch on the face in one patient, palpable purpura in one patient, and dorsal hand blisters resembling porphyria cutanea tarda (PCT) in another. With the exception of the latter two patients, the histology appeared similar, comprising a cell poor and/or lichenoid interface dermatitis with suprabasilar exocytosis around necrotic keratinocytes in the absence of deep periadnexal or perivascular extension or conspicuous follicular plugging, a pattern similar to that of SCLE. However, the lesions differed from SCLE by virtue of vasculopathic alterations comprising vascular ectasia, hypovascularity, and/or luminal thrombosis confined to the superficial vascular plexus and a sclerodermoid tissue reaction, the latter seen in two cases. One biopsy showed a pustular leukocytoclastic vasculitis (LCV). In another case, a biopsied hand blister demonstrated a PCT-like appearance histologically, namely, pauci-inflammatory subepithelial blister formation with hyalinization of dermal papillae capillaries accompanied by an LCV. There was nuclear keratinocyte decoration with IgG and C5b-9 in all cases studied, accompanied by a positive lupus band test in two cases and homogenous deposition of immunoreactants along the dermoepidermal junction and within vessels in the PCT-like eruption. Granular vascular decoration with immunoreactants including C5b-9 was seen in two LCV cases and in two biopsies from rashes clinically mimicking SCLE. Although the epidermal pathology of MCTD mimicks that of SCLE, a concomitant vasculopathy paralleling that seen in skin lesions of dermatomyositis distinquishes the dermatopathology of MCTD from that of SCLE. Corroborating the role of microangiopathy in the pathogenesis of the skin lesions of MCTD was the demonstration of C5b-9 in blood vessels. The deposition of C5b-9 in keratinocytes may explain the pattern Of IgG decoration of keratinocytes; the formation of plasmalemmal pores may permit binding of immunoglobulin to antigens in the nucleus and/or cytosol. The C 5b-9 complex may be the effector mechanism of epithelial and/or endothelial cell injury in MCTD or may serve to augment the effects of antibody-dependent cellular cytotoxicity.

Adult↗
PubMed2001

Interaction between host complement and mosquito-midgut-stage Plasmodium berghei.

After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.

Animals↗
PubMed1985

Early- and late-phase activation of complement evaluated by plasma levels of C3d,g and the terminal complement complex.

Activation of the initial part (early phase) and terminal part (late phase) of the complement cascade was examined. C3d,g and the fluid-phase terminal complement complex were quantified and compared after spontaneous in vitro activation and after acute in vivo activation caused by extracorporeal circulation during coronary artery surgery. The results suggest that there is a close but not complete correlation between early- and late-phase activation of complement, that C3d,g and the terminal complement complex have different elimination rates in vivo, and that these two indicators are valuable for evaluation of early- and late-phase activation, respectively.

Complement Activation↗
PubMed2000

Complement activation in nephropathia epidemica caused by Puumala hantavirus.

BACKGROUND AND AIM: Nephropathia epidemica (NE) is a hemorrhagic fever with renal syndrome caused by Puumala hantavirus. There is considerable variability in the clinical severity of NE. Its pathogenesis is largely unknown and data on complement activation in NE are scant. We sought here to establish how the complement system behaves during NE and whether the variation in the clinical outcome is related to the activation of complement. PATIENTS AND METHODS: The complement component levels and complement activation products and the clinical course of 25 hospital-treated acutely ill NE patients were studied. RESULTS: Complement activation was observed in 23 patients. In 10 patients the complement system was activated mainly through the alternative route, in 6 mainly through the classical route and in 5 through both the alternative and the classical route. An elevated soluble terminal complex SC5b-9 was the most sensitive indicator of complement activation (17 out of 25 patients). Two patients had only an elevated SC5b-9 value and 2 had no complement activation. The C4d/C4 maximal ratio was significantly higher in patients with clinically severe NE compared to the mild or moderate disease groups. CONCLUSION: Our results show that complement activation is common in NE. Although both pathways are usually activated, the classical pathway activation by immune complexes or directly by viral components is associated with a severe clinical course of NE.

Adolescent↗
PubMed1984

On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum.

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.

Animals↗
PubMed1988

Terminal complement complexes and anaphylatoxins in septic and ischemic patients.

Terminal complement complex (TCC) and anaphylatoxin formation in 18 patients with sepsis and 20 patients with acute limb ischemia were studied before the start of treatment and seven days later. The septic or ischemic patients had elevated levels of plasma TCC before start of therapy. In successfully treated patients these concentrations were within the normal range one week later. Similarly, the plasma anaphylatoxin level was increased before therapy and returned to the normal range within seven days. Escherichia coli incubated in vitro in fresh human serum at body temperature started formation of TCC in a dose-related manner. As complement will induce cellular lysis via TCC and edema via anaphylatoxins, anemia and impaired respiration in these patients may be influenced by increased concentrations of terminal complement complexes and of C3a and C5a.

Aged↗
PubMed1987

Complement dependence of antibody-induced mesangial cell injury in the rat.

Intravenous administration of rabbit anti-rat thymocyte serum (ATS) reactive with Thy-1-like antigens present on rat mesangial cells induces almost immediate (1-hr) mesangial cell injury in rats followed by sequential mesangiolytic and mesangial-proliferative/infiltrative lesions. To determine the role of complement in these ATS-induced glomerular lesions, ATS was given to Lewis rats that had been depleted of C3 by cobra venom factor (CVF). CVF treatment prevented the degenerative changes in mesangial cells and accumulation of even the few polymorphonuclear leukocytes (PMN) seen in the glomeruli (2.67 PMN/glomerulus) 1 hr after ATS-treatment in rats not given CVF. In addition, CVF prevented the mesangiolysis and mesangial hypercellularity seen at day 4. Rat C3 and late complement components identified in the mesangial of ATS-treated rats in close association with the deposition of rabbit immunoglobulin G was also absent as a result of CVF treatment. CVF treatment did not affect binding of ATS to glomeruli as studied by immunofluorescence or paired label radioisotope techniques. The depletion of leukocytes and/or PMN by irradiation or treatment with anti-I-MN serum had no effect on the induction of the acute mesangial cell damage or the mesangiolytic lesion. Irradiation did diminish the 4-day proliferative/infiltrative lesion. Complement depletion normalized the ATS-induced increase in mesangial uptake of heat-aggregated human gamma-globulin (655.0 +/- 35.2 micrograms in ATS-treated vs 20.3 +/- 2.9 micrograms/5 X 10(4) glomeruli in ATS plus CVF-treated rats; mean +/- SEM). Small immune deposits present in the mesangial areas of kidneys 4 to 5 days after CVF treatment represented CVF-anti-CVF antibody-C3 complexes. The model of mesangial cell damage induced by ATS in the rat is complement-dependent and may relate, at least in part, to complement-mediated mesangial cell lysis.

Animals↗
PubMed1984

Killing of Leishmania tropica amastigotes by factors in normal human serum.

Amastigotes of Leishmania tropica and L. donovani were incubated with fresh or heat-inactivated normal human serum. Viability was estimated by amastigote conversion to promastigote forms and by the ability of serum-treated amastigotes to infect human monocytes. L. tropica, a parasite that causes local skin infection, was killed by fresh but not by heat-inactivated serum. The serum cytotoxic effect on L. tropica was inhibited by EDTA but not by Mg-EGTA. C2-deficient serum killed normally; C6-deficient serum was ineffective. These data indicate that L. tropica is killed by the complement membrane attack complex, in a sequence of reactions initiated by components of the alternate pathway. In contrast, L. donovani, a parasite that causes systemic visceral leishmaniasis, was 10-fold less susceptible to the cytotoxic effects of normal serum. Thus, a profound difference exists in the susceptibility of amastigotes of two species of Leishmania to a defense mechanism present in human serum. Serum complement factors may play an important role in limiting L. tropica to the skin. The resistance of L. donovani to such factors may be the primary reason for its ability to escape from the site of inoculation and cause catastrophic, disseminated disease.

Animals↗
PubMed1990

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.

Animals↗
PubMed2004

Ectopic expression of Bcl-XL or Ku70 protects human colon cancer cells (SW480) against curcumin-induced apoptosis while their down-regulation potentiates it.

Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin.

Antigens, Nuclear↗
PubMed1984

Mechanism of complement cytolysis and the concept of channel-forming proteins.

Complement damages membranes via the terminal reaction sequence that leads to the formation of membrane-bound, macromolecular C5b-9(m) protein complexes. These complexes represent C5b-8 monomers to which varying numbers of C9 molecules can be bound. Complexes carrying high numbers of C9 (ca. 6/8-12/16?) exhibit the morphology of hollow protein channels. Because they are embedded within the lipid bilayer, aqueous transmembrane pores are generated that represent the primary lesions caused by complement in the target cell membrane. Many other proteins damage membranes by forming channels in a manner analogous to the C5b-9(m) complex. Two prototypes of bacterial exotoxins, Staphylococcus aureus alpha-toxin and streptolysin-O, are discussed in this context, and attention is drawn to the numerous analogies existing among these protein systems. Common to all is the process of self-association of the native proteins to form supramolecular complexes. This event is in turn accompanied by a unique transition of the molecules from a hydrophilic to an amphiphilic state.

Animals↗
PubMed1987

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes.

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal polymorphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4-6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation of C9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.

Animals↗