Stimulation of the synthesis and release of lipids in tumor cells under attack by antibody and C

Antibody-sensitized line-1 or line-10 tumor cells treated with GPC (TAC) incorporated fatty acids into complex cellular lipids and released increased amounts of fatty acids within 5 to 10 min after the addition of GPC as compared to control cells. This effect was dependent on the concentration of GPC used; however, under conditions where the cells were not killed, the enhanced synthesis and release of lipids were not dependent on the antibody concentration used to sensitize the cells. Treatment of the cells with antibody alone, GPC alone, or antibody plus heat-inactivated GPC did not result in enhanced synthesis or release of lipids. No enhancement in DNA, RNA, or protein synthesis in TAC was noted. Line-1 cells, which can be killed by GPC when sensitized with excess anti-Forssman IgM antibody, demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to the antibody-sensitized cells, before measurable killing of the cells had occurred. This effect persisted in the surviving cells when tested 5 and 10 min after the formation of TAC. Addition of GPC deficient in C4 to antibody-sensitized cells did not result in enhanced lipid synthesis or release. These data suggest that the synthesis of macromolecules of which lipids are a major component is of central importance for the ability of the cells to resist antibody-GPC mediated attack.     

Lysis of tumor cells by antibody and complement. II. Lack of correlation between amount of C4 and C3 fixed and cell lysis.

Two antigenically distinct diethylnitrosamine-induced guinea pig hepatoma cell lines, line-1 and line-10, sensitized with rabbit anti-Forssman or with tumor-specific antibody, were more susceptible to killing by human complement (HuC) than by guinea pig complement (GPC). This difference could not be ascribed to differences in the amount of C1, C4, and C3 fixed: millions of C4 and hundreds of thousands of C3 were detected on cells whether they were killed or not killed by the C sources. Tumor cells sensitized with anti-Forssman IgM antibody generally had more GP C4 and C3 than Hu C4 and C3 bound to their surfaces. Cells sensitized with anti-tumor antibody generally had more Hu C4 and C3 than GP C4 and C3 bound to their surfaces. The resistance to killing of nucleated cells by antibody and C may be due in part to intrinisic properties of the cell.     

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Interaction of concanavalin A with herpes simplex virus infected cells

Incubation of herpes simplex virus (HSV) infected cells with concanavalin A (Con A) interferes with binding of the Fc portion of antibody. In addition, the lectin inhibits complement mediated cytolysis, probably by interference with antibody binding. These results suggest that binding sites of both Fc and HSV antibody contain residues which attract Con A.     

The different roles of two distinct Fc gamma receptors on guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes

The functional roles of two distinct types of Fc gamma receptors (Fc gamma 1/gamma 2R specific for both IgG1 and IgG2, and Fc gamma 2R specific for IgG2 alone) on the surface of guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes (EA) were investigated by the use of two Fab's of monoclonal anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibodies. The binding and subsequent ingestion of IgG1 antibody-sensitized erythrocytes (EA gamma 1) by macrophages were completely inhibited by anti-Fc gamma 1/gamma 2R Fab', indicating that the reactions are mediated only by Fc gamma 1/gamma 2R. On the other hand, the binding and subsequent ingestion of IgG2 antibody-sensitized erythrocytes (EA gamma 2) were substantially inhibited by anti-Fc gamma 2R Fab', but not by anti-Fc gamma 1/gamma 2R Fab'. The inhibitory activities of anti-Fc gamma 2R Fab' were dependent upon the amount of IgG2 antibody bound on erythrocytes; increasing the amount of bound IgG2 antibody from 0.15 to 0.91 micrograms/2 X 10(8) erythrocytes resulted in a decrease in the inhibition of binding of EA gamma 2 by anti-Fc gamma 2R Fab' from 50 to 0%, and also a decrease in the inhibition of ingestion of EA gamma 2 from 100 to 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of carbohydrate in the interaction of Tamm-Horsfall protein with complement 1q and inhibition of classical complement activation

Tamm-Horsfall protein (THP) binds strongly to complement 1q (C1q), a key component of the classical complement pathway. The goals of this study were to determine whether THP altered the activation of the classical complement pathway and whether the carbohydrate portion of THP was involved in this glycoprotein's binding to C1q and alteration of complement activation. The ability of THP to prevent complement activation in diluted serum or plasma incubated at 37 degrees C was assessed using both a haemolytic assay with antibody-sensitized sheep RBC and a C4d ELISA. Both these methods showed that THP inhibited activation of the classical complement pathway in a dose-dependent manner. Glycosidases were used to remove most of the carbohydrate from THP. This partially deglycosylated THP bound human IgG with a higher affinity (KD1 = 1.4 nmol/L; KD2 = 0.31 micromol/L) than did intact THP (KD1 = 33.4 nmol/L; KD2 = 31.0 micromol/L). An ELISA showed that removal of carbohydrate from THP reduced, but did not eliminate, the ability of this protein to inhibit binding of C1q to intact THP. Haemolysis assays using antibody-sensitized sheep RBC showed that removal of THP carbohydrate eliminated the ability of THP to protect against complement activation. In conclusion, THP inhibited the activation of the classical complement pathway that occurred in diluted serum or plasma. The carbohydrate moieties of THP appeared to be important in this inhibitory activity.     

Receptors for immune complexes on cells within a non-lymphoid murine tumor.

In this paper we present evidence that antibodies unrelated to the tumor cell can comprise part of the in vivo Ig surface coat of cells derived from the non-lymphoid murine tumor, TA3/St. This was shown by incubating TA3 cells originating from other OA or BSA preimmunized mice with radioiodinated 125I-OA and 131I BSA. Radioiodine-labeled 125I-OA specifically fixed to cells derived from TA3/St tumors originating from OA-preimmunized mice. On the other hand 131I-BSA was specifically fixed by cell populations from BSA-preimmunized mice. Incubation of these cells in vitro at 37 degrees C abolished the specific binding and antibody could subsequently be detected in the tissue culture medium. Radioiodine labeled purified soluble antibody-antigen complexes could also be bound to cells derived from freshly harvested TA3/St tumors but not to their in vitro propagated counterparts. Removal of phagocytic or adherent cells from these cell populations decreased the binding of the complexed antibody on freshly harvested TA3/St populations, but did not eliminate it. Inhibition of complexed antibody binding was obtained when TA3/St cells (an H-2a tumor) were pre-incubated with anti-H2a antiserum. Propagation of the tumor in an F1 hybrid (A X C57BL) in which host cells could be distinguished from tumor cells by using an anti H-2b antiserum showed that binding of the immune complex was mostly limited to host cells infiltrating into the tumor population.     

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.     

Mouse skin graft prolongation with donor strain bone marrow and anti-lymphocyte serum: surface markers of the active bone marrow cells

The survival of C3H/HeJ skin grafts on B6AF1 mice treated with anti-lymphocyte serum (ALS) can be significantly prolonged by the injection of the host with C3H/HeJ bone marrow. Although the prolongation is apparently due at least in part to the ultimate presence in the host of specific suppressor cells of donor origin, little is known about the nature of the cells in the marrow inoculum that are responsible for this effect. The present investigation was undertaken to characterize surface markers of the active bone marrow cells. Marker-positive populations were either depleted and enriched by panning techniques or depleted by killing with specific antibody and complement, and then were assayed for their ability to prolong graft survival. Cells that were adherent to anti-Ia-coated plates extended graft survival only slightly better than did treatment with ALS alone, whereas nonadherent (Ia-depleted) cells, as well as cells treated with anti Ia and complement, retained good prolonging activity. Similarly, panning on anti-immunoglobulin (Ig)-coated plates produced an active, Ig+-depleted population and an inactive adherent population, and killing of Thy-1+ cells with antibody and complement did not compromise the ability of the bone marrow inoculum to prolong graft survival. Complement receptor-positive (EAC+) and Fc gamma receptor-positive cells (EA+) were separated by panning on plates coated with sheep erythrocytes/antibody/complement and erythrocytes/7S antibody respectively. Adherent, EAC+-enriched cells were only slightly active, whereas the nonadherent, EAC-depleted population was fully active in graft prolongation. However, both Fc gamma R+ (EA+)-enriched and depleted populations were active, with the enriched fraction producing significantly better prolongation than the depleted population. Thus, the bone marrow cells that can prolong skin graft survival in ALS-treated mice appear to be Ia-, Thy-1+, largely complement receptor negative, and Ig-, but are largely positive for Fc gamma receptors.     

The mechanism of inhibition of the activation of the complement first component by polyanions and polycations

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

Cytolytic activity of Ia-restricted T cell clones and hybridomas: evidence for a cytolytic mechanism independent of interferon-gamma, lymphotoxin, and tumor necrosis factor-alpha

Ten different helper T cell (Th) hybridomas that are specific to Ia or antigen plus Ia were found to express nonspecific cytolytic activity toward the cytotoxin (CT)-resistant P815 cells upon activation with either Con A or a monoclonal anti-T3 antibody (T3-mAb). In contrast to cytolytic Th1 clones which secrete high levels of interferon-gamma (IFN-gamma) and cytotoxin (CT) (lymphotoxin (LT, also known as TNF-beta) or tumor necrosis factor-alpha (TNF-alpha], these Th hybridomas produce low or undetectable levels of IFN-gamma and CT. No inhibitory activity of IFN-gamma and CT was observed in culture supernatants of activated Th hybridomas. Double-chamber experiments demonstrated that CT-sensitive L929 cells when physically separated from activated Th1 clones were killed by membrane-permeable CT. Under identical experimental conditions, lysis of P815 cells did not occur. Moreover, activation of Th hybridomas directly in wells containing the CT-sensitive L929 cells failed to induce target cell lysis. This confirms that these Th hybridomas produce little CT and argues against high local concentrations of CT being responsible for Th hybridoma-mediated killing of P815 cells. Finally, a polyclonal rabbit antiserum to rTNF-alpha, which strongly and specifically inhibited CT-mediated and Th1 clone-mediated killing of L929 cells, failed to inhibit P815 lysis by activated Th1 clones and Th hybridomas. These observations establish that a cytolytic mechanism independent of IFN-gamma, LT, and TNF-alpha is responsible for lysis of CT-resistant target cells.     

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.     

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab)₂ fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2AG2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.Abbreviations used in this paper Ig immunoglobulin - FcR receptor for the Fc portion of Ig - TNP trinitrophenyl - Fab antigen-binding fragment - pA Protein A - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - PBS phosphate-buffered saline - BSA bovine serum albumin - SAMIg sheep antimouse Ig - SRBC sheep red blood cells - C complement - FITC fluorescein isothiocyanate - CNBr cyanogen bromide - EA antibody-sensitized erythrocytes     

The Mechanism of Inhibitory Effect of Polyanions and Polycations on the Activation of the Complement First Component

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 g/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 ± 6 and 8.1 ± 0.1 g/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 ± 3 g/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q–antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

Stability of C3 Convertase in the Rat Classical Complement Pathway

It has been reported that rat serum complement causes efficient hemolysis of antibody-sensitized sheep erythrocytes (EA) at 20 C but not at 37 C. In connection with this, we demonstrated that C3 convertase of rat complement was significantly unstable at 37 C using purified components of rat complement.     

电剌大鼠的血清中淋巴细胞转化抑制因子的作用机制分析

Previous reports showed that EA stimulation (3V, 2Hz, 30 min/d, 5 d) induced the production of one or more lymphocyte proliferation-inhibitory factor(s) in the rat serum. In this paper, the mechanisms of the action for the inhibitory factor(s) to suppress lymphocyte proliferation were studied. (1) the lymphocytes from different immune organs of the mice were prepared and cultured with the rat serum stimulated by EA. The results show that the serum not only inhibited the mouse lymph node T cell proliferation induced by Con A, but also inhibited the mouse thymocyte and spleen T cell proliferation induced by Con A. When B cells were stimulated by LPS, the proliferative effect can also be inhibited significantly by the rat serum stimulated by EA. This implies that the effect of the lymphocyte proliferation-inhibitory factor(s) has no specificity. (2) Incubation of the mouse lymph node cell with serum for one hour is enough to cause an inhibitory effect on Con A stimulated lymphocyte proliferation. However, no inhibitory effect was observed if the mouse lymph node cells were incubated with Con A for 15 min or 30 min before the addition of rat serum. The results demonstrate that the lymphocyte proliferation-inhibitory factor(s) act on the early events of T lymphocyte activation induced by Con A. (3) Protein kinase C (PKC) is a key link in the activation of T and B lymphocyte proliferation by Con A and LPS respectively. So it would be interesting to learn whether the inhibitory effect of the lymphocyte proliferation-inhibitory factor(s) is caused by the inhibition of PKC activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the mechanism by which gangliosides inhibit the proliferative response of murine splenocytes to concanavalin A

Gangliosides are known to inhibit the proliferative response of murine and human lymphocytes to antigens and mitogens in vitro. In this study the response of murine spleen cells to concanavalin A (Con A) was used as a model system. Analysis of the cellular events by flow cytometry revealed that during the first 24 hr of culture the effect of gangliosides on Con A-treated cells was minimal. At 48 hr, however, more of the ganglioside-treated cells were in G0/G1, the cells contained more RNA, and fewer cells were in S phase. These data indicate that gangliosides inhibit the transition of the cells from G0/G1 into the S phase of the cell cycle. Expression of the interleukin 2 (IL-2) receptor, as measured by the binding of a monoclonal antibody to the receptor, was not inhibited by the gangliosides. Binding of 125I-labeled recombinant IL-2 to cells cultured for 48 hr with Con A was inhibited by ganglioside GD1a but not by asialo GM1. Inhibition was much more effective if the gangliosides were preincubated with IL-2 before addition of cells, but no inhibition was observed if the cells were preincubated with gangliosides and the unbound gangliosides were washed out prior to addition of the IL-2. These data suggest that interference with the binding of IL-2 to the high-affinity IL-2 receptor of activated T lymphocytes plays an important role in the inhibition of Con A-induced proliferation.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.