Antibody-mediated Complement Lysis Research Articles

Beyond Binding: The Outcomes of Antibody-Dependent Complement Activation in Human Malaria.

Antibody immunity against malaria is effective but non-sterile. In addition to antibody-mediated inhibition, neutralisation or opsonisation of malaria parasites, antibody-mediated complement activation is also important in defense against infection. Antibodies form immune complexes with parasite-derived antigens that can activate the classical complement pathway. The complement system provides efficient surveillance for infection, and its activation leads to parasite lysis or parasite opsonisation for phagocytosis. The induction of complement-fixing antibodies contributes significantly to the development of protective immunity against clinical malaria. These complement-fixing antibodies can form immune complexes that are recognised by complement receptors on innate cells of the immune system. The efficient clearance of immune complexes is accompanied by complement receptor internalisation, abrogating the detrimental consequences of excess complement activation. Here, we review the mechanisms of activation of complement by alternative, classical, and lectin pathways in human malaria at different stages of the Plasmodium life cycle with special emphasis on how complement-fixing antibodies contribute to protective immunity. We briefly touch upon the action of anaphylatoxins, the assembly of membrane attack complex, and the possible reasons underlying the resistance of infected erythrocytes towards antibody-mediated complement lysis, relevant to their prolonged survival in the blood of the human host. We make suggestions for further research on effector functions of antibody-mediated complement activation that would guide future researchers in deploying complement-fixing antibodies in preventive or therapeutic strategies against malaria.

Trypanosoma brucei gambiense-iELISA: A Promising New Test for the Post-Elimination Monitoring of Human African Trypanosomiasis.

BackgroundThe World Health Organization targeted Trypanosoma brucei gambiense human African trypanosomiasis (gHAT) for elimination as a public health problem and for elimination of transmission. To measure gHAT elimination success with prevalences close to zero, highly specific diagnostics are necessary. Such a test exists in the form of an antibody-mediated complement lysis test, the trypanolysis test, but biosafety issues and technological requirements prevent its large-scale use. We developed an inhibition ELISA with high specificity and sensitivity that is applicable in regional laboratories in gHAT endemic countries.MethodsThe T. b. gambiense inhibition ELISA (g-iELISA) is based on the principle that binding of monoclonal antibodies to specific epitopes of T. b. gambiense surface glycoproteins can be inhibited by circulating antibodies of gHAT patients directed against the same epitopes. Using trypanolysis as reference test, the diagnostic accuracy of the g-iELISA was evaluated on plasma samples from 739 gHAT patients and 619 endemic controls and on dried blood spots prepared with plasma of 95 gHAT and 37 endemic controls.ResultsOverall sensitivity and specificity on plasma were, respectively, 98.0% (95% CI 96.7–98.9) and 99.5% (95% CI 98.6–99.9). With dried blood spots, sensitivity was 92.6% (95% CI 85.4–97.0), and specificity was 100% (95% CI 90.5–100.0). The g-iELISA is stable for at least 8 months when stored at 2–8°C.ConclusionThe g-iELISA might largely replace trypanolysis for monitoring gHAT elimination and for postelimination surveillance. The g-iELISA kit is available for evaluation in reference laboratories in endemic countries.

A Promoter Polymorphism in the CD59 Complement Regulatory Protein Gene in Donor Lungs Correlates With a Higher Risk for Chronic Rejection After Lung Transplantation.

Complement activation leads primarily to membrane attack complex formation and subsequent target cell lysis. Protection against self-damage is regulated by complement regulatory proteins, including CD46, CD55, and CD59. Within their promoter regions, single-nucleotide polymorphisms (SNPs) are present that could influence transcription. We analyzed these SNPs and investigated their influence on protein expression levels. A single SNP configuration in the promoter region of CD59 was found correlating with lower CD59 expression on lung endothelial cells (p = 0.016) and monocytes (p = 0.013). Lung endothelial cells with this SNP configuration secreted more profibrotic cytokine IL-6 (p = 0.047) and fibroblast growth factor β (p = 0.036) on exposure to sublytic complement activation than cells with the opposing configuration, whereas monocytes were more susceptible to antibody-mediated complement lysis (p < 0.0001). Analysis of 137 lung transplant donors indicated that this CD59 SNP configuration correlates with impaired long-term survival (p = 0.094) and a significantly higher incidence of bronchiolitis obliterans syndrome (p = 0.046) in the recipient. These findings support a role for complement in the pathogenesis of this posttransplant complication and are the first to show a deleterious association of a donor CD59 promoter polymorphism in lung transplantation.

Platelets made HLA deficient by acid treatment aggregate normally and escape destruction by complement and phagocytes in the presence of HLA antibodies.

The presence of antibodies against HLA Class I can lead to platelet (PLT) transfusion refractoriness, that is, the repeated failure to achieve adequate posttransfusion PLT count increments. PLT refractoriness can be overcome by transfusion of HLA-matched donor PLTs. A different approach is to remove HLA from the PLT surface using low pH. Previous case studies using HLA-stripped PLTs showed encouraging but inconsistent results and lacked information on the biologic effects of acid treatment on PLT function as well as sensitivity to PLT destruction in the presence of HLA antibodies. PLTs prepared from buffy coats were stripped from HLA Class I using a brief incubation at pH 2.9. Kinetics of acid stripping, viability, phenotypic alterations, and sensitivity to complement-mediated lysis and phagocytosis were determined by flow cytometry. Functional potential was evaluated using a multiplate analyzer. Acid-treated PLTs were viable, upregulated activation markers normally and aggregated to a similar extent as untreated PLTs in response to stimulation with three natural agonists. Acid treatment removed 70% to 90% of HLA Class I complexes from the PLT surface, which led to complete protection from HLA antibody-mediated complement lysis and reduced monocyte-mediated phagocytosis in the presence of anti-HLA in vitro. Our study fills an important knowledge gap in how acid treatment affects PLT function and interactions with immune cells, paving the way for controlled clinical trials to evaluate acid-treated PLTs as an alternative to HLA-matched donors in PLT refractoriness.

Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection.

BackgroundTo explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus.Methods and FindingsSera from two groups of patients—25 with acute HIV-1 infection and 31 with chronic infection—were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env) proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies.ConclusionsCollectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.

PagPIs Required for Resistance to Antibody-Mediated Complement Lysis duringBordetella bronchisepticaRespiratory Infection

To efficiently colonize and persist in the lower respiratory tract, bacteria must survive multiple host immune mechanisms. Bordetella bronchiseptica is a gram-negative respiratory pathogen that naturally infects mice and persists in the lower respiratory tract for up to 49 days postinoculation. In this work, we examined the effect of mutation of the pagP gene on the persistence of B. bronchiseptica in the lower respiratory tract of mice. The pagP gene encodes a palmitoyl transferase that is responsible for the addition of a palmitoyl group to the lipid A region of B. bronchiseptica lipopolysaccharide. Data presented here confirm that a B. bronchiseptica deltapagP mutant demonstrates defective persistence in the lower respiratory tract of wild-type mice. We hypothesized that the defective persistence of the B. bronchiseptica deltapagP mutant was due to an increased susceptibility of this mutant to a host immune response. In vivo data indicate that both B cells and the complement component C3 are required for the reduced bacterial numbers of the deltapagP mutant on day 14 postinoculation. In addition, an in vitro complement killing assay demonstrated that B. bronchiseptica exhibits pagP-dependent resistance to antibody-mediated complement killing at low concentrations of immune serum. Taken together, these results suggest that pagP is required for B. bronchiseptica to resist antibody-mediated complement lysis during respiratory infection.

Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors.

Platelet-derived growth factor alpha-receptors (PDGFR alpha) are expressed by a subset of neuroepithelial cells in the ventral half of the embryonic day 14 (E14) rat spinal cord. The progeny of these cells subsequently proliferate and migrate into the dorsal parts of the cord after E16. Here, we show that E14 ventral cells are able to generate oligodendrocytes in culture but that dorsal cells acquire this ability only after E16, coinciding with the appearance of PDGFR alpha-immunoreactive cells in the starting population. PDGFR alpha-positive cells in optic nerve and spinal cord cultures co-labelled with antibody markers of oligodendrocyte progenitors. When PDGFR alpha-positive cells were purified from embryonic rat spinal cords by immunoselection and cultured in defined medium, they all differentiated into oligodendrocytes. Very few oligodendrocytes developed in cultures of embryonic spinal cord cells that had been depleted of PDGFR alpha-expressing cells by antibody-mediated complement lysis. These data demonstrate that all PDGFR alpha-positive cells in the embryonic rat spinal cord are oligodendrocyte progenitors and that most or all early-developing oligodendrocytes are derived from these ventrally-derived precursors.

Immunopanning and Developmental Stage-Specific Primary Culture of Oligodendrocyte Progenitors (O4+GalC−) Directly from Postnatal Rodent Cerebrum

An immunopanning technique for capturing the immediate progenitors of oligodendrocytes (OLs) that first appear In developing rat cerebrum at P4-P5 is described. Identified with two monoclonal antibodies, O4 and anti-galactocerebroside (GalC), these progenitors have the surface antigenic phenotype O4+GalC−. Their isolation from dissociated cell suspensions of tissue begins with a negative immunoselection step using antibody-mediated complement lysis to remove O4+ cells (15%) that have already differentiated to become GalC+ OLs at the time of dissociation. The remaining progenitors are labeled with monoclonal antibody 04, and 04+ cells are bound to a secondary antibody-coated polystyrene dish. After nonadherent cells are washed away, O4+GalC− progenitors are recovered and placed into primary culture. Yields of 3-4 × 105 progenitors per cerebrum are obtained with >90% viability and 95% purity. Culturing the progenitors in a defined, insulin-containing medium conditioned by type 1 astrocytes promotes extensive differentiation and long-term cell survival. Marked differences in the time course of progenitor accumulation and efficacy of immunopanning indicate that purification of O4+GalC− progenitors from regions other than cerebrum is necessary for some strains of mice.

Separation of Murine Splenic B- and T-Lymphocytes for Use in Immunotoxicological Studies

The effects of xenobiotics on the immune system are often diverse and can involve secondary effects on cell types other than those of primary immune origin. Thus, to better understand the effects of chemicals and drugs on immuno-competence, exposure of immune cell types to xenobiotics and assessment of functional activity have been conducted under in vitro culture conditions. Interpretation of data obtained using this approach is reliant on the known composition of the cell populations used in these assay systems as well as on the ability to obtain purified and functionally viable cells from mixed primary cell suspensions. The objective of this study was to develop an effective and reproducible means of separating murine splenic B- and T-cells into purified populations free of monocyte/macrophage and polymorphonuclear cell (PMN) contamination. Cell adherence and discontinuous Percoll gradients in conjunction with immunomagnetic negative selection or antibody-mediated complement lysis were used to obtain B...

Evaluation of murine splenic cell type metabolism of benzo[ a]pyrene and functionality in vitro following repeated in vivo exposure to benzo[ a]pyrene

Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benzo[ a]pyrene (B(a)P) within the spleens of untreated mice. Since repeated exposure to B(a)P results in immunosuppression and B(a)P is known to induce cytochrome P450 levels, the first objective of this study was to investigate whether exposure of mice to B(a)P could increase the amounts of immunosuppressive B(a)P metabolites generated and/or alter the pattern of B(a)P metabolites formed by several different splenic cell types. Mice were dosed with a daily sc dose of 200 mg/kg B(a)P or vehicle for 4 days. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was used to obtain different splenic cell populations. Cells were incubated with [ 3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate B(a)P metabolites. Results indicate that splenic macrophages of B(a)P-treated mice produced significantly greater amounts of some metabolites compared to those of vehicle-treated mice. The three major metabolites produced were an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10- and B(a)P-7,8-dihydrodiols. Other splenic cell types examined did not produce metabolite amounts significantly above (T-cells, PMNs, or the capsule) or just above (B-cells) background. The second objective was to investigate the splenic cell type(s) targeted by B(a)P resulting in suppression of humoral immunity. Separation-reconstitution studies along with in vitro sensitization techniques with several different antigens (sheep red blood cells (SRBC), dinitrophenyl-Ficoll (DNP-Ficoll), lipopolysaccharide (LPS)) were used to identify splenic target cells following exposure of mice to B(a)P (200 mg/kg/day, sc for 4 days). Findings indicate that in vitro plaque-forming cell (PFC) suppression was due to alterations in the adherent (macrophage) cell population. Exposure also suppressed the PFC response to the T-dependent antigen SRBC and the T-independent antigen DNP-Focoll, but did not suppress the PFC response to the polyclonal antigen, LPS. These data suggest that B(a)P is targeting macrophages.

Metabolism of benzo[ a]pyrene by murine splenic cell types

The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was capable of metabolizing B(a)P. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was utilized to obtain highly enriched populations of splenocytes for B(a)P metabolism studies. Immunofluorescent cell staining in conjunction with flow cytometry and examination of Giemsa-stained cytospin cell preparations indicated that B- or T-cell populations of greater than 95% purity and an 80–90% pure population of splenic macrophages were routinely attained. Splenic cell populations were incubated with [ 3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate the B(a)P metabolites generated by the enriched splenic cell populations. The results of these studies demonstrate that the macrophage is the cell type responsible for the metabolism of B(a)P within the spleen. The major metabolites of B(a)P produced were as follows: an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10-dihydroxy-9,10-dihydrodiol, and B(a)P-7,8-dihydroxy-7,8-dihydrodiol. Other splenic cell types examined, including B and T cells, polymorphonuclear cells, or the spleen capsule did not produce amounts of B(a)P metabolites significantly above background levels. Based on these findings, macrophages are the splenic cell types which metabolize B(a)P. As a result, macrophages may be the cell type targeted by B(a)P resulting in suppression of splenic humoral immune responses.

Trypanosome variant surface glycoprotein transfer to target membranes: a model for the pathogenesis of trypanosomiasis.

The variant surface glycoprotein (VSG) of trypanosomes is attached to the cell surface by means of a phosphatidylinositol-containing glycolipid membrane anchor. The studies presented in this paper support the hypothesis that the transfer of VSG from trypanosomes to erythrocytes could lead to one of the pathological features associated with trypanosome infection--i.e., anemia. Migration of trypanosome VSG from live trypanosomes to target cells (sheep erythrocytes) could be shown by preincubating erythrocytes with trypanosomes and subsequently testing the washed erythrocytes for insertion of VSG by their susceptibility to lysis by complement in the presence of an anti-VSG antibody. Complement-mediated lysis was found to depend on the strain-specific anti-VSG antibody used. Extent of erythrocyte lysis increased with time of cell exposure to trypanosomes and with trypanosome concentration. No erythrocyte lysis was observed when trypanosomes were preincubated with anti-VSG antibody before adding erythrocytes. Purified membrane-form VSG (which retains the glycolipid anchor), but not soluble VSG (which no longer has the terminal diacylglycerol moiety), could sensitize erythrocytes to anti-VSG antibody-mediated complement lysis. The intermembrane transfer of VSG from trypanosomes to cells of the infected host could provide a molecular mechanism for the pathogenesis of trypanosomiasis.

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin.

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.

Host and parasite factors affecting the invasion of mononuclear phagocytes by Trypanosoma cruzi.

Epimastigotes and trypomastigotes of Trypanosoma cruzi enter mouse and human macrophages by phagocytosis. Organisms are initially found in a parasitophorous vacuole, and fusion with lysosomes promptly ensues. Epimastigotes are rapidly killed and digested in phagolysosomes. Trypomastigotes, however, soon escape the confines of the vacuolar system and reach the cytoplasm, where they replicate. Metacyclic trypomastigotes are quickly taken up by mouse and human macrophages. In contrast, blood-form trypomastigotes are resistant to interiorization by mononuclear phagocytes. This effect can be overcome, without affecting parasite survival, by opsonization of the parasites with immunoglobulin G (IgG)-class antibodies or by removal of trypomastigote surface peptides with trypsin. The major surface glycoprotein (relative molecular mass 90 000) of the mammalian stages of T. cruzi has been implicated in this antiphagocytic effect. This surface glycoprotein seems to have glycosidase activity, and the antiphagocytic effect may be mediated via the removal of sugar residues necessary for parasite internalization by macrophages. This enzyme activity may explain other evasion mechanisms displayed by T. cruzi bloodstream forms. These include antibody-mediated complement lysis and Fc-receptor-mediated phagocytosis. Trypomastigotes are, however, promptly killed in mouse and human macrophages activated by either in vivo infection or in vitro treatment with sensitized, antigen-stimulated, T-cell products. Increased production of reactive oxygen intermediates by activated cells has been implicated as the mechanism of intracellular killing in mononuclear phagocytes. Opsonization by IgG-class antibodies enhances uptake of the parasites but does not affect their fate in normal mononuclear phagocytes. In contrast, it enhances their killing by activated macrophages, suggesting that antibodies may have a major protective role only in the presence of concomitant cell-mediated immunity.

Cytotoxicity of Tumour‐Bearer Serum to D23 Hepatoma Cells Derived from Solid Tumours, Ascites or in Vitro Cultures

The complement-dependent cytotoxicity of antibodies in tumour-bearer serum (TBS) from rats carrying the chemically induced D23 hepatoma was investigated. Target cells were D23 cells from solid tumours (D23sol), from ascites tumours (D23asc), or from in vitro growing cell cultures (D23cc). The D23asc and D23cc cells were not lysed when used as target cells in the assay, although they evoke cytotoxic antibodies when growing in vivo. The D23 ascites cells became susceptible to complement-dependent lysis after trypsin treatment. This was, however, not due to unmasking of target antigens, since untreated D23 ascites cells absorbed cytotoxic antibodies as efficiently as trypsinized cells. No increase in susceptibility to complement-dependent lysis was observed after trypsin treatment of D23cc cells. Absorption of cytotoxic antibodies with D23cc cells showed no or very low antigen expression on the surface of these cells. They did, however, contain the antigen(s), since 3 M KCl extracts of the D23cc cells could inhibit the complement-dependent cytotoxicity of D23sol TBS against D23sol cells. From these data it was concluded that the susceptibility of hepatoma cells for antibody-mediated complement lysis is not only correlated with antigen expression, as was the case with the in-vitro-cultivated cells, but is also dependent on increased lytic susceptibility after trypsin treatment of the cells.