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PubMed1988

Immunopathologic studies of cutaneous lupus erythematosus.

The studies, as outlined above, strongly suggest that there may be several pathophysiologic mechanisms resulting in the development of cutaneous lupus lesions. It appears that all lupus lesions are associated predominantly with a T-cell infiltrate. Based upon the studies of the neonatal lupus infants, it has been hypothesized that the U1RNP and Ro(SS-A) autoantibodies of maternal origin play a direct pathologic role in the genesis of the annular polycyclic SCLE lesions and this may be mediated by antibody-dependent cellular cytotoxicity mechanisms in which the antibody binds to the respective antigen present on the keratinocyte plasma membrane and the effector cells are T cells derived from the infants. Other studies, using direct immunofluorescence techniques, have demonstrated an association of cutaneous lupus lesions occurring in the presence of immunoglobulin and complement at the dermal/epidermal junction (positive lupus band test) in which the neoantigen of the complement membrane attack complex (C5b-C9) is detected. These data have been interpreted as indicating that immunoglobulin and complement, perhaps in the form of immune complexes, may play a role in the pathogenesis of some cutaneous lupus lesions. Additional studies have determined that there is a substantial number of lupus patients with cutaneous disease, without antinuclear antibodies, who fail to demonstrate the deposition of immunoglobulin and complement at the dermal/epidermal junction. Furthermore, other studies have indicated that ultraviolet light is capable of inducing lesions in lupus patients that histologically are identical to those of cutaneous lupus erythematosus but that failed to demonstrate the deposition of the immunoglobulin and complement components. Since discoid lupus lesions demonstrate a preponderance of T cells, it has been proposed that some of these lesions are the direct result of a T-cell cytotoxic event. However, the nature of the autoantigens responsible for this putative T cell-mediated cytotoxic response is unknown at the present time. The role of ultraviolet light in the genesis of the cutaneous lupus lesions appears to involve, within the epidermis, the generation of autoantigen macromolecules which then react with autoantibodies or specific T cells of the lupus host.

Humans↗
PubMed2000

Complement-induced phospholipase A2 activation in experimental membranous nephropathy.

BACKGROUND: In the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. By analogy, in cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). This study addresses the mechanisms of PLA2 activation. METHODS: PLA2 expression was assessed with the polymerase chain reaction or immunoblotting, and activity was determined using an in vitro assay or by measurement of free AA. RESULTS: Under basal conditions, GEC in culture expressed a relatively low level of cytosolic PLA2 (cPLA2) protein, while mRNAs of groups IB, IIA and V secretory PLA2s (sPLA2) were not detectable. Incubation of GEC with sublytic C5b-9 induced 1.5- to 2.0-fold increases in free [3H]AA at 40 minutes, and three and 24 hours. C5b-9 did not increase cPLA2 protein, and did not induce group IB, IIA or V sPLA2 mRNAs. Stable overexpression of cPLA2 in GEC amplified the C5b-9-induced increases in free [3H]AA, while analogous overexpression of group IIA sPLA2 had no effect. PLA2 activity was increased in glomeruli of rats with PHN, and this enhanced activity was characterized as cPLA2. There were no differences in cPLA2 protein expression between PHN and control glomeruli. CONCLUSIONS: Release of AA by C5b-9 in GEC in culture and in vivo is mediated by cPLA2, and the mechanism is consistent with post-translational regulation of cPLA2 activity. C5b-9 does not induce expression or stimulate activity of sPLA2 isoforms in GEC.

Animals↗
PubMed1987

Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes exhibit abnormalities in decay accelerating factor (DAF), acetylcholinesterase, and resistance to autologous C5b-9 attack. To investigate the nature of the lesion underlying PNH cells, we examined the relationship of these abnormalities to one another. Analyses of DAF in acetylcholinesterase-negative erythrocytes revealed that these two abnormalities involve functionally independent molecules, coincide precisely in the same cell populations, and are similarly expressed in PNH II and more complement-sensitive PNH III erythrocytes. The DAF and acetylcholinesterase deficiencies contrast with the C3b/C4b receptor (CR1) deficit, which is less profound and similarly distributed in complement-insensitive cell populations. Hemolytic studies showed that defective resistance to autologous C5b-9 attack is mediated by another mechanism. Whereas reconstitution of PNH II erythrocytes with DAF completely corrected their complement sensitivity, DAF reconstitution of PNH III erythrocytes restored their ability to circumvent C3b uptake but had no effect on their heightened susceptibility to reactive lysis. Assays of complement-insensitive (PNH I) erythrocytes surviving after reactive lysis disclosed partial DAF and acetylcholinesterase deficits. These findings indicate that the PNH lesion involves multiple membrane components and that PNH I erythrocytes are also abnormal.

Acetylcholinesterase↗
PubMed1992

Inhibition of the alternative pathway of complement by glomerular chondroitin sulphate proteoglycan.

Cultured rat glomerular epithelial cells (GEC) synthesize and secrete a complement inhibitory chondroitin sulphate B proteoglycan (termed GCRF). As proteoglycans and their component glycosaminoglycans may affect several different steps of complement activation, the functional properties of GCRF were investigated in this study. GCRF inhibited preformed alternative pathway convertases (C3bBbP), but did not substantially accelerate their decay. In contrast to other polyanions, GCRF did not affect the activity of human or rat factor H, nor did it inhibit the terminal complement proteins. GCRF inhibited the effect of decay-accelerating factor (DAF) on C3bBbP when the two were incubated together and DAF was in excess, but it did not affect the decay of DAF of classical pathway convertases. However, when DAF was first incorporated into the erythrocyte membrane, the effect of GCRF on C3bBbP was additive to that of DAF. Thus, GCRF inhibits the activity of C3bBbP and blocks the action of DAF, but not that of factor H, perhaps by binding to factor B in the alternative pathway convertase.

Animals↗
PubMed1988

Molecular mechanisms of lymphocyte-mediated killing.

Lymphocytes kill tumor cells and other target cells by a contact-dependent mechanism. Killer lymphocytes acquire cytoplasmic granules upon lymphokine stimulation and these granules in turn contain potent cytotoxic mediators, some of which have been characterized recently. These include (i) a pore-forming protein (perforin) of 70 kDa that polymerizes into transmembrane tubules in the presence of calcium and that is structurally related to the terminal components (C5b-6, C7, C8 and C9) of the complement cascade, (ii) a cytokine related to tumor necrosis factor and lymphotoxin that causes DNA fragmentation in target cells, and (iii) a family of serine esterases presently without any known function. The relevance of these mediators in lymphocyte-mediated killing is currently being investigated. The identification and characterization of additional putative mediators of cytotoxicity in the future will undoubtedly provide us with a better understanding of the molecular basis of cell-mediated killing.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

Susceptibility of human trophoblast to killing by human complement and the role of the complement regulatory proteins.

The susceptibility of trophoblast to cytolysis by human complement was investigated using cells purified to over 90% from first trimester placentae. Two assay systems were employed to measure the killing of trophoblasts, an antibody-dependent complement-mediated cytolysis and the reactive lysis. The antibody obtained from a patient with Addison's disease reacted specifically with syncytiotrophoblasts and induced a dose-dependent killing of the cells not exceeding 50% even in the presence of excess antibody and complement. The percentage of cells killed by the terminal complement complex in the reactive lysis system was somewhat higher, reaching values of 60%. Immunofluorescence analysis revealed the presence of CD46 and CD59 on all syncytiotrophoblasts, whereas CD55 was only detected on approximately 30% of the cells. Inhibition of CD46 and CD59 resulted in increased susceptibility of syncytiotrophoblasts to complement lysis. The protective function of CD55 could not be evaluated because of its reduced expression on isolated trophoblasts. These results suggest that syncytiotrophoblasts may be killed by complement and that membrane regulators to some extent protect these cells from complement damage.

Antigens, CD↗
PubMed1984

Membrane attack by complement.

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.

Animals↗
PubMed1983

Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex.

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.

Animals↗
PubMed1994

Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.

Animals↗
PubMed1985

The relationship between channel size and the number of C9 molecules in the C5b-9 complex.

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.

Animals↗
PubMed2001

Transgenic porcine valves show no signs of delayed cardiac xenograft rejection.

BACKGROUND: Glutaraldehyde fixation stiffens the structural integrity of porcine valves, although the solution also destroys tissue viability and accelerates calcification. Recently, we demonstrated that fresh cardiac valves from domestic pigs do not express the galactose alpha1, 3 galactose (alpha-Gal) antigen and may be immunologically unique. The absence of alpha-Gal explained why the valves remained pristine while the rest of the porcine heart was destroyed by primate immunoglobulin M (IgM) and complement membrane attack complex (MAC) within 60 minutes. We sought to clarify whether fresh porcine valves from transgenic pigs bearing human complement regulatory proteins (CD59/DAF) can survive longer in primates and whether porcine cardiac valves remained immunologically privileged after prolonged exposure. METHODS: Tissue sections from wild-type untransplanted (n = 6), wild-type transplanted (n = 3), and transgenic pigs expressing human CD59/DAF proteins transplanted (n = 3) porcine-to-primate cardiac grafts were examined by hematoxylin and eosin, and by immunohistochemistry for the porcine endothelial marker (GalNac), alpha-Gal, primate IgM and MAC. RESULTS: alpha-Gal antigens were highly expressed on the vascular, but not valvular, endothelium of transgenic pigs. Hearts from CD59/DAF transgenic pigs survived 5, 7, and 11 days, but showed increasing IgM and MAC deposition until failure. Valves remained morphologically intact at explant, and strong GalNac staining suggested an intact endothelial surface. However, the valves showed no signs of IgM- or MAC-mediated damage. CONCLUSIONS: Although hearts from transgenic pigs expressing human complement regulatory proteins can survive for days in the primate recipient, the xenografts eventually fail because of escalating attacks of primate IgM and MAC. The absence of the alpha-Gal antigens protects unfixed porcine valves from rejection.

Animals↗
PubMed1985

Possible involvement of terminal complement complex in active Heymann nephritis.

We investigated whether the appearance of various complement components in renal deposits of immune complexes correlated with the development of proteinuria in rats with active Heymann nephritis. Sequential kidney biopsy specimens and serum samples were obtained from Lewis rats immunized with Fx1A in complete Freund's adjuvant. Circulating antibodies against purified auto-antigen renal tubular epithelial glycoprotein, as measured by ELISA, were found in the circulation together with a diffuse granular deposition of IgG1, IgG2a, and IgG2b in the glomeruli within 2 weeks after immunization. Biopsy specimens taken 4 weeks after immunization showed diffuse deposition of C4 and C3, which indicated that activation of complement by the classical pathway had occurred. The detection of the C5b-9 complex of complement in glomerular deposits coincided with the development of abnormal proteinuria indicating that the glomerular damage in this autoimmune disease may be caused by complement-mediated lesions in the glomerular capillary walls.

Animals↗
PubMed1988

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa.

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.

Adenosine Diphosphate↗