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Toll‐like receptors (TLRs) play an important role in inducing innate and acquired immune responses against infection. However, the effect of Toll‐like receptor 7 (TLR7) on follicular helper T (Tfh) cells in mice infected with Plasmodium is still not clear. The results showed that the splenic CD4+CXCR5+PD‐1+ Tfh cells were accumulated after Plasmodium yoelii NSM infection, the content of splenic Tfh cells was correlated to parasitemia and/or the red blood cells (RBCs) counts in the blood. Moreover, the expression of TLR7 was found higher than TLR2, TLR3 and TLR4 in splenic Tfh cells of the WT mice. TLR7 agonist R848 and the lysate of red blood cells of infected mice (iRBCs) could induce the activation and differentiation of splenic Tfh cells. Knockout of TLR7 leads to a decrease in the proportion of Tfh cells, down‐regulated expression of functional molecules CD40L, IFN‐γ, IL‐21 and IL‐10 in Tfh cells; decreased the proportion of plasma cells and antibody production and reduces the expression of STAT3 and Ikzf2 in Tfh cells. Administration of R848 could inhibit parasitemia, enhance splenic Tfh cell activation and increase STAT3 and Ikzf2 expression in Tfh cells. In summary, this study shows that TLR7 could regulate the function of Tfh cells, affecting the immune response in the spleen of Plasmodium yoelii NSM‐infected mice. [ABSTRACT FROM AUTHOR]

Non‐immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B‐cell expression of HLA‐DR/DP/DQ, whilst reconstitution reduced the levels of B‐cell activation markers HLA‐DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream. [ABSTRACT FROM AUTHOR]

Summary: Protein 4.1R, an 80 000 MW membrane skeleton protein, is a vital component of the red blood cell membrane cytoskeleton that stabilizes the spectrin–actin network and regulates membrane properties of deformability and mechanical stability. It has been shown that 4.1R is expressed in T cells, including CD8+ T cells, but its role in CD8+ T cells remains unclear. Here, we have explored the role of 4.1R in CD8+ T cells using 4.1R−/− mice. Our results showed that cell activation, proliferation and secretion levels of interleukin‐2 and interferon‐γ were significantly increased in 4.1R−/− CD8+ T cells. Furthermore, the phosphorylation levels of linker for activation of T cells (LAT) and its downstream signaling molecule extracellular signal‐regulated kinase were enhanced in the absence of 4.1R. In vitro co‐immunoprecipitation experiments showed a direct interaction between 4.1R and LAT. Moreover, 4.1R−/− CD8+ T cells and mice exhibited an enhanced T‐cell‐dependent immune response. These data enabled the identification of a negative regulation function for 4.1R in CD8+ T cells by a direct association between 4.1R and LAT, possibly through inhibiting phosphorylation of LAT and then modulating intracellular signal transduction. [ABSTRACT FROM AUTHOR]

It is widely accepted that antibodies and CD4 T cells play critical roles in the immune response during the blood stage of malaria, whereas the role of CD8 T cells remains controversial. Here, we show that both CD8 and CD4 T cells robustly responded to an acute self-limiting blood-stage infection with Plasmodium yoelii. Similar to antigen-specific T cells, both CD8 and CD4 T cells showed dynamic expression of the surface proteins interleukin (IL)-7R and programmed death-1 (PD-1). Additionally, activated CD8 T cells showed differences in the expression of Killer cell lectin-like receptor G1, L-selectin and B cell lymphoma-2 and produced granzyme B, indicating cytotoxic activity, and the initially high expression of T-box transcription factor TBX21 in malaria-activated CD4 T cells indicated an early T helper type 1 (Th1)-skewed immune response. Our data demonstrate that blood-stage malaria infection results in a striking T-cell response and that activated CD8 and CD4 T cells have phenotypic and functional characteristics that are consistent with conventional antigen-specific effector and memory T cells. Therefore, a better understanding of the CD8 and CD4 T-cell response induced by blood-stage infection may prove to be essential in the development of a vaccine that targets the erythrocytic stage of the malarial parasite. [ABSTRACT FROM AUTHOR]

CD46 is a complement regulatory molecule expressed on every cell type, except for erythrocytes. While initially described as a regulator of complement activity, it later became a ‘magnet for pathogens’, binding to several viruses and bacteria. More recently, an alternative role for such complement molecules has emerged: they do regulate T-cell immunity, affecting T-cell proliferation and differentiation. In particular, CD46 stimulation induces Tr1 cells, regulatory T cells characterized by massive production of interleukin-10 (IL-10), a potent anti-inflammatory cytokine. Hence, CD46 is likely to control inflammation. Indeed, data from CD46 transgenic mice highlight a role for CD46 in inflammation, with antagonist roles depending on the cytoplasmic tail being expressed. Furthermore, recent data have shown that CD46 is defective in multiple sclerosis, IL-10 production being severely impaired in these patients. This lack of IL-10 production probably participates in the inflammation observed in patients with multiple sclerosis. This review will summarize the data on CD46 and T cells, and how CD46 is likely involved in multiple sclerosis. [ABSTRACT FROM AUTHOR]

Anti-D has been widely and effectively used in Rhesus blood group D negative mothers for the prevention of haemolytic disease of the fetus and newborn; its mechanism of action however, often referred to as antibody-mediated immune suppression (AMIS), remains largely unresolved. We investigated, in a murine model, whether active immune suppression or clonal deletion mediated by anti-red blood cell (RBC) immunoglobulin G (IgG) could explain the phenomenon of AMIS. Transfusion of IgG-opsonized foreign RBCs (i.e. AMIS) strongly attenuated antibody responses compared to transfusion of untreated foreign RBCs. When the AMIS-mice were subsequently transfused with untreated RBCs, no immune suppression was observed at 5 and 35 days after AMIS induction; in fact, the mice responded to retransfusion with untreated RBCs in a manner that was characteristic of a secondary immune response. When IgG-opsonized RBCs were transfused concurrently with untreated RBCs, a dose-dependent reduction of the antibody response was observed. This work suggests that the attenuation of the antibody responsiveness by anti-RBC IgG is not associated with active immune suppression or clonal deletion at either the T-cell or B-cell level; rather, the effect appears more characteristic of B-cell unresponsiveness to IgG-opsonized RBCs. These results may have implications for the understanding of the mechanism of action of anti-D in haemolytic disease of the fetus and newborn. [ABSTRACT FROM AUTHOR]

It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in β2-microglobulin-deficient (β2m(–/–)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8+ T cells and natural killer T (NKT) cells. When C57BL/6 and β2m(–/–) mice were injected with parasitized ( Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1– αβΤ-cell receptorint cells in both mice. The difference was a compensatory expansion of NK and γδT cells in β2m(–/–) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8+ T and NKT cells may be partially related to this protection. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is associated with anaemia. Although the underlying mechanisms remain unclear, the accompanying reticulocytosis implies that erythrocyte lifespan in the circulation is shortened. Among the factors that permit prolonged survival of erythrocytes are the membrane complement regulators. In conditions such as paroxysmal nocturnal haemoglobinuria, where erythrocyte expression of these regulators is reduced, erythrocyte survival is compromised and anaemia follows. Recentin vitroevidence indicates that one of the key membrane complement regulators, CD59, is inactivated by glycation in the presence of high concentrations of glucose or other glycating sugars. To ascertain whether glycation-induced inactivation of CD59 occurrsin vivowe examined CD59 surface expression and function on erythrocytes from a cohort with poorly controlled type 1 diabetes (hyperglycaemic) and from matched normoglycaemic controls. Although expression of CD59, assessed using polyclonal anti-CD59 antiserum, was similar in the two groups, erythrocytes from hyperglycaemic individuals were more susceptible to lysis by complement, entirely as a result of the loss of functional CD59. These data implicate glycation-induced inactivation of CD59 as a factor contributing to anaemia in type 1 diabetes. [ABSTRACT FROM AUTHOR]

Summary A cDNA encoding a feline homologue of CD2 (fCD2) was identified. Several amino acids (aa) important for ligand interaction, molecular folding or signal transduction, found in other mammalian CD2, were found to be highly conserved in the predicted fCD2 aa sequence. fCD2-expressing cells were able to form rosettes with human red blood cells (probably via human CD58), and the rosette formation was inhibited by an anti-fCD2 monoclonal antibody. These results are indicative of the similarity of feline and human CD2 structures. fCD2 was found to be expressed in feline peripheral blood T lymphocytes, monocytes and cultured lymphoid cells. [ABSTRACT FROM AUTHOR]

Summary This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D‐74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C‐terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly‐histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18–20 000 as compared with 20–28 000 for GPI‐anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)‐triggered ‘reactive‐lysis’ system and a C5b‐7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b‐7 site assay, using purified terminal components to develop lysis, was 10‐fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement‐mediated diseases. [ABSTRACT FROM AUTHOR]

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythocytes sensitized with specific immunoglobulin G(IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fcγ receptor (FcγR)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of FcγR functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify FcγR expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to FcγR activity. We conclude that LPS enhance FcγR dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules. [ABSTRACT FROM AUTHOR]

The rosetting of T cells by sheep erythrocytes is mediated through the interaction of the CD2 molecule on T cells with T11TS, a molecule on sheep erythrocytes homologous to lymphocyte function-associated antigen-3 (LFA-3, CD58). We cloned a TI ITS cDNA from sheep leucocyte mRNA which encodes a soluble molecule comprising the distal D1 and the D2 extracellular domains, but not the transmembrane domain, cDNA for this soluble D1+D2 form of sheep LFA-3 (sLFA-3) was expressed in Escherlchia coli and the properties of the purified recombinant protein were assessed by inhibition of T-cell rosette formation, sLFA-3 inhibited rosette formation, but its activity was low, 50% inhibition occurring at 25 micro;g/ml, consistent with the observed Iow binding avidity of fluorescein isothiocyanate (FITC)-labelled sLFA-3, sLFA-3 was made multimeric to increase its affinity, by crosslinking biotinylated sLFA-3 to streptavidin-biotinylated dextran complexes. The binding of crosslinked sLFA-3 multimers, tested by fluorescence-activated cell sorting (FACS) analysis, was significantly increased compared to sLFA-3 monomers. Competition with monoclonal antibodies demonstrated that multimeric sLFA-3 bound to the T111 epitope on CD2. The multimeric form of sLFA-3 was significantly more potent than the monomer in inhibiting proliferation of human T cells in response to purified protein derivative (PPD), tetanus toxoid (TT) or allogeneic cells. Multimeric sLFA-3 might, therefore, have potential as an immunotherapeutic agent to inhibit and/or anergize antigen-specific T-cell responses. [ABSTRACT FROM AUTHOR]

Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients' B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI-deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo-deposited C3 fragments and in vitro complement-activating ability. CR1 levels on B cells were significantly lower in FI-deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI-deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo-bound C3b fragments were degraded to C3d,g when the patients' PBL were incubated in homologous serum containing EDTA. Finally, the patients' erythrocytes failed to exert any inhibition on AP activation in homologous serum. [ABSTRACT FROM AUTHOR]

With age, New Zealand black (NZB) mice spontaneously develop anti-mouse red blood cell (RBC) autoantibodies resulting in the development of autoimmune haemolytic anemia (AIHA). Previously, we characterized a panel of monoclonal autoantibodies derived from unimmunized, adult NZB mice. One of these antibodies (G8) was shown to be pathogenic, inducing AIHA in a non-autoimmune-prone mouse strain (BALB/c). Using G8, and two other antibodies from our panel, we have characterized two distinct autoantigens on the surface of mouse RBCs. The autoantigen, historically referred to as antigen X (AgX), was found to be partially hidden on the surface of the mouse RBC because glycosidase treatment or mild digestion with proteinase K resulted in increased reactivity with autoantibodies. One of the monoclonal antibodies (3H5G1) was found to immunoprecipitate a 110000 MW protein identified as the erythrocyte anion transporter (band 3) whereas the pathogenic antibody (G8) as well as a third monoclonal antibody (2E6m) were shown to immunoprecipitate a 60000 MW protein that was not reactive with the anti-band 3 serum. Finally, we show that the autoantigen recognized by G8 is expressed on differentiated mouse erythroleukaemia (MEL) cells. The results suggest that a protein distinct from band 3 can serve as a target for AIHA in NZB mice. [ABSTRACT FROM AUTHOR]

IgG and IgM antibodies were detected on non-parasitized as well as parasitized erythrocytes (E) from mice surviving over 15 days after infection with rodent malaria, Plasmodium berghei, whereas C3 was detected exclusively on parasitized E. Parasitized E, however, were quite resistant to the haemolytic activity of guinea pig complement and effectively inactivated human C3b to iC3b on their surface. Similarly, parasitized E were extremely resistant to homologous complement as assessed by haemolysis and C3 binding even when regulatory proteins (decay-accelerating factor, DAF; complement receptor related gene y, Crry; heat-stable antigen, HSA) were blocked with specific antibodies. DAF and Crry were equally expressed on both normal E and parasitized E from mice within a week post-infection; therefore, molecules that inhibit the haemolysis or C3 binding of parasitized E appear to be independent of DAF and Crry. Unexpectedly, the molecular forms of HSA and DAF in parasitized erythrocyte membranes were found to be different from those of normal erythrocyte membranes: DAF was detected as three bands (85 000, 64 000 and 30 000 MW) by immunoblotting. HSA was detected as more hishly glyocsylated forms than normal HSA. These alterations of DAF and HSA could be explained by the modification of membrane proteins and polysaccharides induced by parasitization, and we hypothesize that these changes of membranes or membrane protons are involved in the resistance of parasitized E against homologous complement. [ABSTRACT FROM AUTHOR]

Band 3, the red blood cell (RBC) anion channel protein, is the target autoantigen for the pathogenic RBC autoantibodies and T-helper (Th) cells in New Zealand Black (NZB) mice with autoimmune haemolytic anaemia (AIHA). To determine the subpopulation of these Th cells, they were stimulated with Band 3 and the profile of the cytokines elaborated by the responding cells was measured. NZB T cells stimulated with Band 3 produced high levels of the Th1 cytokine, interferon-γ (IFN-γ), but little or no interleukin-4 (IL-4), IL-5 or IL-10. Similar patterns were produced by NZB T cells responding to a spectrin preparation from the RBC membrane skeleton, or to mycobacterial heat-shock protein (hsp) 65 following immunization of mice with hsp 65 in incomplete adjuvant. By contrast, T cells from CBA mice similarly immunized with hsp 65 produced high levels of IL-4 and IL-5 in response to hsp 65. Examination of the isotype of the RBC-bound immunoglobulins in NZB mice revealed that immunoglobulin G2a (IgG2a) autoantibodies were the first to be detected in most mice and that later in the disease, IgG3 autoantibodies were often prominent. It is concluded that, contrary to expectation, the development of RBC autoantibodies in NZB mice is associated with Thl cytokine-dominated responses. [ABSTRACT FROM AUTHOR]

Crude extracts of Plasmodium falciparum schizont-infected erythrocytes (PfSE) induce polyclonal activation of peripheral blood T lymphoeytes from naive (malaria unexposed) humans. We demonstrate that the active component of PfSE is membrane bound, soluble in sodium dodecyl sulphate (SDS) and partially heat stable, but distinct from the turnout necrosis factor (TNF)inducing, exoantigen-like activity of schizont extracts. Malaria pigment induces little or no T-cell activation. The responding cells arc predominately CD4+, CD45RO+, T-cell receptor (TCR)αβ+. Contrary to previous reports, expansion of the TCRγδ+ subset was observed in cells from only one of eight donors. Proliferating cells secrete interferon-γ (IFN-γ) and release large amounts of soluble interleukin-2R (sIL-2R) into the culture supernatant but produce no detectable interleukin-4 (IL-4), a phenotype typical of the T-helper (Th)1 subset of CD4+ T cells. We propose that these activated T cells may initiate the inflammatory response to malaria infection in non-immunes and may contribute to the pathology of the disease. [ABSTRACT FROM AUTHOR]

The excessive production of tumour necrosis factor (TNF) is associated with the pathology of blood-stage malaria and phosphatidylinositol-containing phospholipid antigens from parasitized erythrocytes stimulate its secretion by macrophages, thus acting as toxins. This brief report describes some properties of an inhibitor present in lysates from erythrocytes infected with malarial parasites that blocked the detection of recombinant TNF in an enzyme-linked immunosorbent assay and diminished or abolished the cytotoxicity of TNF. It was not found in control lysates of normal erythrocytes. Its addition to macrophage cultures stimulated by toxic malarial preparations or by bacterial lipopolysaccharide also blocked the detection of TNF. These findings may explain the contradictory results obtained from different assays for TNF, and emphasize the need for caution when interpreting the results of a single assay system. If released when parasitized erythrocytes rupture in vivo, the inhibitor could help protect both parasite and host from the damaging effects of TNF. [ABSTRACT FROM AUTHOR]

The MIC 11 antigen is expressed on human cells and is characterized by reaction with a monoclonal antibody (mAb), 16.3A5. The gene controlling MIC 11 was recently mapped to the p13 region of chromosome 11 within 500 kb of the gene encoding CD59, a complement regulatory protein. The present report investigates the antigenic relationship between these cell-membrane determinants and sets out evidence that MIC 11 and CD59 are encoded by the same gene. Western blotting of human erythrocyte membrane proteins and purified membrane CD59 showed that 16.3A5 anti-MIC 11 antibody bound to a 19-24 000 MW band with the characteristic appearance of CD59 protein, and gave staining patterns identical to those obtained with the CD59 antibody, BRIC 229. The binding of 16.3A5 monoclonal lgG to purified urine-derived CD59 in enzyme-linked immunosorbent assay (ELISA) was inhibited by YTH 53.1 rat CD59 antibody, indicating that the MIC 11 epitope is the same as, or close to, that recognized by CD59 antibodies such as YTH 53.1, BRIC 229 and 2/24. Prior exposure of erythrocytes to 16.3A5 anti-MIC 11 also reduced the ability of the CD59 antibodies, BRIC 229 and YTH 53.1, to block the complement-inhibiting function of membrane CD59. Anti-MIC 11 antibody alone, however, had no inhibitory effect on CD59 function. This may be due to its relatively low binding affinity or to some slight difference in epitope specificity. Further studies using immunofluorescence showed that the MIC 11 epitope, like CD59, is absent from EBV-B cells lacking GP1-anchored proteins and from a B cell line specifically deficient in CD59 protein. Overall, the results provide strong evidence that MIC 11 is a determinant on the CD59 molecule. [ABSTRACT FROM AUTHOR]

The adhesion of erythrocytes infected with Plasmodium falciparum (P. falciparum) is one of the major pathological features of severe malaria. Several potential receptors to endothelium for falciparum-infected erythrocyte on endothelium have been described. Recently, the malaria binding site on ICAM-1(CD54) has been mapped to a site distinct but overlapping with the LFA-1 (CD11a/CD18) site. We detected by flow cytometry, confocal laser microscopy and immunoprecipitation, a molecule expressed at the surface of erythrocytes infected with mature stages of the M96 strain of P. falciparum that was recognized by a monoclonal antibody (mAb) (TS1/22) directed against an LFA-1 epitope. However, this molecule was not recognized by mAbs directed against other epitopes of LFA-1 or against other integrins. Furthermore, the mAb TS1/22 partially inhibited cytoadherence of parasitized red blood cells to human-brain rnicrovascular endothelial cells. The expression of a molecule sharing an epitope with human LFA-1 integrin on the parasitized erythrocyte surface could be involved in the sequestration of these cells and thus in the pathogenesis of severe disease. [ABSTRACT FROM AUTHOR]

The four subclasses of IgG have different biological activities associated with their Fc regions. Fcγ receptors on leucocytes (FcγR) mediate binding and phagocytosis of opsonized particles. Two structurally and functionally distinct allelic polymorphisms of the FcγR have been defined: the H/R131 forms of FcγRIIa (CD32), and the neutrophil antigen 1 (NA1)/NA2 forms of FcγRIIIb (CD16). In this study the activities of allotypes of CD16 are analysed with antibacterial IgG subclass antibodies and with IgG 1 and IgG3 anti-Rhesus D, and the activities of CD32 with IgG1 and IgG3 anti-Rhesus D. With respect to the allotypes of CD16, polymorphonuclear leucocytes (PMN) homozygous for FcγRIIIb-NA2 exhibited a lower (21-25%) IgG1-mediated phagocytosis of Staphylococcus aureus strain Wood (STAW), Haemophilus influenzae type b (Hib), and Neisseria meningitidis group B (NMen) than IIIb-NA1 PMN. The difference was apparent only when the micro-organisms were opsonized in the absence of complement, and was furthermore enhanced (34-52%) upon blockade of FcγRIIa. In addition, monoclonal IgG3 anti-D-mediated rosette formation and phagocytosis was consistently found to be lower (16%) with FcγRIIIb-NA2 than with IIIb-NA1 PMN. For the allotypes of CD32 we now show that IgG3 anti-D sensitized erythrocytes formed more (50%) rosettes and were phagocytosed at a higher rate with PMN carrying FcγRIIa-H131 than with PMN carrying IIa-R131. Heterozygous FcγRIIa-H/R131 PMN exhibited intermediate phagocytic activity in this respect. This study illustrates a critical role of FcγR allotypes in functional interactions with biologically relevant IgG subclass antibodies. [ABSTRACT FROM AUTHOR]

The role of FcγRI in the immune destruction of blood cells is uncertain as serum IgG levels are sufficient to competitively inhibit interactions between this high-affinity receptor and sensitized red cells. In the current study, it is proposed that, rather than functioning as a receptor for opsonized red cells, FcγRI might, under appropriate conditions, mediate the passive sensitization (or 'arming') of human macrophages with IgG antibodies resulting in the in vivo destruction of unsensitized cells expressing the corresponding antigen. To examine this hypothesis, FcγRI-bearing human monocytes and U937 cells were first passively sensitized by incubation in vitro with human monoclonal anti-D, and then incubated with D-positive red cells. The uptake of monoclonal anti-D by U937 cells was rapid and, in the presence of 2·5 µg/ml IgG1 or IgG3 anti-D was almost complete after 5 min at 37°. Subsequent incubation of passively sensitized U937 cells in an IgG-free medium for 1 hr at 37° resulted in the loss from the cell surface of approximately 50% cell-bound IgG; the remaining cell-bound IgG was lost more slowly despite repeated washing. In functional assays, passively sensitized monocytes (M-IgG) mediated adherent, phagocytic and chemiluminescent (CL) responses to D-positive red cells. After incubation of M-IgG in 50% v/v fresh normal human serum (FNHS) for 2 hr, sufficient anti-D remained bound to monocytes to promote the adherence of red cells. The adherence and phagocytosis of red cells by M-IgG was enhanced by the simultaneous addition of 50% FNHS, probably owing to the binding of low levels of C3bi to red cells. In contrast, phagocytic and CL responses of unsensitized monocytes to anti-D-sensitized red cells (E-IgG) were abrogated in the presence of 0·25% v/v FNHS, presumably owing to blocking of FcγRI by IgG. It is considered that in vivo, FcγRI may mediate the passive sensitization of macrophages in close proximity with antibody-secreting cells in the reticular network of the splenic cords. Once 'armed' in this way, macrophages may destroy cells expressing the appropriate antigen. [ABSTRACT FROM AUTHOR]

We sought to characterize factors released by sonicated human erythrocytes that stimulate peripheral blood mononuclear cells (PBMNC) to release tumour necrosis factor-α (TNF). This response is not inhibited by polymyxin B, indicating that contaminating lipopolysaccharide (LPS) is not responsible. When erythrocyte lysates are fractionated by reverse-phase chromatography using a gradient of n-propanol on Sep-Pak® C18 cartridges, the TNF-inducing activity elutes as a single peak. The erythrocyte-derived TNF-inducing activity is unaffected by digestion with proteases but is destroyed by mild base hydrolysis or digestion by lipases, indicating that compounds containing ester-linked acyl chains may be essential. These properties are similar to those of TNF stimulators that we have previously identified in erythrocytes infected with malaria parasites, except that the TNF-inducing activity per cell is about 200 times higher in parasitized erythrocytes than in uninfected erythrocytes. Lipase-digested erythrocyte lysates inhibit the TNF- inducing factors of both normal and malaria-infected erythrocytes, suggesting that lipase digestion creates partial structures which compete with active components for macrophage receptors. Such receptors may recognize a common structure that contains an inositol monophosphate (IMP)-like component, as IMP also inhibits the TNF response to erythrocyte-derived factors and to parasite lysates whereas it does not affect the response to LPS. We conclude that lysed erythrocytes release specific cytokine-inducing factors that may contribute to the fever response to non-infectious tissue injury. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the treatment of neutrophils with [roteolytic enzymes markedly reduces the expression of receptor III for the Fc portion of IgG (FcγRIII), but it does not affect the number of FcγRII on the cell surface. In the present study, we analysed the effect of proteolytic enzymes on functional responses of neutrophils induced by immune complexes (IC). Our results showed that treatment with pronase or chymotrypsin markedly increased the binding of IgG-coated erythrocytes (IgG-E) to neutrophils, as well as their capability to display IgGmediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and chemiluminescence (CL) induced by IgG-E, responses that have been shown to be completely dependent on FcγRII. A similar enhancing effect was observed, in all cases, after neutrophil treatment with neuroaminidase. We also studied the effect of proteolysis on neutrophil activation induced by other types of IC. It was found that pronase and chymotrypsin significantly enhanced CL responses induced by soluble IC (sIC) but did not modify the responses induced by either precipitating IC (pIC) or soluble IC prepared with cationized antibodies (catIC). On the other hand, neuroaminidase significantly enhanced CL induced by either sIC, pIC or catIC. Taken together, our data suggest that the activity of FcγRII can be up-regulated by proteolysis. However, this effect appears to be strongly dependent on the characteristics of the IC employed as stimulus. [ABSTRACT FROM AUTHOR]

A cell-ELISA was developed using monolayers of glutaraldehyde-fixed normal as well as Plasmodium berghei-infected mouse erythrocytes for quantification and characterization of anti-erythrocytic autoantibodies in routine malaria. Testing normal (NMS) and peak parasitaemic sera (PPS) on erythrocyte monolayers treated with trypsin, sodium meta periodate, neuraminidase or heat, and competitive inhibition of antibodies with soluble sialic acid, revealed that some anti-erythrocytic antibodies (which increase during the parasitaemic phase of infection) recognize N-acetyl neuraminic acid (NANA) residues on host erythrocytes. High levels of antibodies to NANA covalently conjugated to bovine serum albumin (BSA) were detectable in PPS. Such antibodies could be significantly absorbed out by preincubation of PPS with mouse erythrocytes (MRBC). Antibodies in PPS, when affinity-purified on a column of Fetuin- Agarose, were found to be reactive to normal as well as parasitized erythrocyte monolayers. Immunoglobulin isotyping and IgG subgroup typing revealed that most of the anti-erythrocytic autoantibodies in NMS were IgM and IgA, while in PPS there was an appreciable increase in IgG2a and IgG3. Affinity-purified anti-NANA antibodies reacted with DNA when tested in an ELISA. There was a significant positive correlation between anti-erythrocytic antibody and DNA-binding levels in NMS as well as PPS. The DNA-binding antibodies in PPS could be effectively absorbed out by preincubation of sera with fresh MRBC. Affinity determination of anti-erythrocytic antibodies eluted from MRBC revealed binding characteristics in the following order: MRBC > single-stranded DNA > double-stranded DNA. [ABSTRACT FROM AUTHOR]

This study focused on the mode in which maternal T cells induce suppression of plaque-forming cell (PFC) response in offspring. The maternal T cells of C57BL/6J pregnant mice, which had been intraperitoneally injected with 2 × 108 of sheep red blood cells (SRBC) on day 12 of gestation, were transferred, 5 days after immunization, into (C3H/HeJ × C57BL/6J)F1 normal pregnant mice on day 12 of gestation. The (C3H/HeJ × C57BL/6J)F1 × C3H/HeJ offspring of (C3H/HeJ × C57BL/6J)F1 recipient pregnant mice were reared to more than 6 weeks of age, and their anti-SRBC PFC responses were examined. Suppression of anti-SRBC PFC response was observed in H-2bxk but not H-2k offspring. Thus, maternal T cells of SRBC-immunized pregnant mice induce suppression of antiSRBC PFC in offspring with restriction to major histocompatibility complex (MHC) haplotype utilized in maternal T-cell responses during pregnancy. Maternal CD4+ T cells are responsible for the MHC-restricted induction of PFC suppression in offspring. Furthermore we demonstrated, in this report, using adoptive transfer of maternal T cells from SRBC-immunized pregnant mice and in vitro secondary PFC assay in the offspring, that maternal T-cell-mediated suppression results from the development of CD4+ suppressor T cells in offspring. Moreover, the activation of suppressor T cells in offspring depends on the recognition of SRBC antigens presented in association with the same MHC haplotype as that utilized in the maternal T-cell response during pregnancy. Thus, the maternal T cells of SRBC-immunized pregnant mice generate a repertoire of suppressor T cells in their offspring. [ABSTRACT FROM AUTHOR]

Naturally occurring anti-band 3 antibodies appear to mediate opsonization of oxidativcly stressed and in vivo aged red cells. Their low concentration in plasma (< 100 ng/ml) and weak affinity (estimated association constant, 5-7 x l06 l/mol) contrasted with their biological efficiency. In compensating for their inadequate properties they have an affinity for C3 at a site independent of the antigen binding domain, with an estimated association constant of 2-3 x l05 l/mol. Though weak, their bindin8 to C3 was about 100 times higher than that of whole IgG, which is known to have an affinity for C3. The affinity for C3 may render these antibodies preferred targets of the short-lived nascent C3b and result in a preferential C3b-anti-band 3 complex formation. C3b-IgG complexes represent the best opsonins and can nucleate alternative complement pathway C3 convertases by which opsonization is further enhanced. [ABSTRACT FROM AUTHOR]

Freshly recovered pulmonary alveolar macrophages (AM) and the AM-derived established cell line, MH-S, have previously been shown to be highly suppressive of in vitro IgM anti-sheep erythrocyte (SRBC) responses. Supernatants obtained from cultures of AM incubated with antigen-stimulated lymphocytes or from the MH-S cell line alone have also been shown to be suppressive when added to the in vitro antibody-forming system. In order to determine if AM and MH-S cells, owing to their mucosal location, could differentially regulate antibody responses including immunoglobulin isotypes other than IgM, an in vitro system for the detection of cells producing IGG, IgA and IgE anti2,4 dinitrophenol (DNP) antibody was developed. These studies demonstrate that AM, MH-S cells, and MH-S culture supernatants suppress the in vitro generation of IgM, IgG, IgA and IgE anti-DNP spot-forming cells (SFC). No apparent differential regulation of any of the four murine IgG anti DNP antibody subclasses was observed. Time-course experiments suggested that optimal AM- and MH-S-mediated suppression occurred 18 hr after culture initiation. Both AM and MH-S cells suppressed IgM and IgG anti-DNP antibody responses in a dose-related manner, suggesting that MH-S is a good model for the study of AM-mediated immunoregulation. [ABSTRACT FROM AUTHOR]

The capacity to trap immune complexes (IC) was examined using purified peroxidase-antiperoxidase IgG on frozen sections of mouse spleen. In the absence of serum, binding of IC was confined to macrophages in the red pulp. However, when IC were applied in the presence of fresh mouse serum as a source of complement, trapping occurred on follicular dendritic cells (FDC) in the primary follicles. Trapping was specifically inhibited with monoclonal antibodies against complement receptor type 1 (CRI) (8C12) and CR½ (7G6), but not with anti-CR3 (Mac-1). The binding mechanism of IC in the secondary follicles of animals immunized with sheep red blood cells differed from that observed in unimmunized mice. Here, trapping occurred both in the absence and presence of fresh mouse serum. In the absence of fresh mouse serum, trapping on FDC was mediated by FcRγII, as demonstrated by blocking with monoclonal antibody 2.4G2. FcRγII-mediated trapping was only observed on a relatively small proportion of FDC that was located in the light zone of the secondary follicles, while complement receptor-mediated IC trapping was observed in more expanding areas, including dark zone and corona. These results show that both complement receptor as well as Fc receptor can function in the binding of IC to FDC. They also provide evidence for the existence of functionally different FDC populations in the secondary lymphoid follicles of mouse spleen. [ABSTRACT FROM AUTHOR]

Repeated immunization of mice with rat red blood cells (RRBC) results in the production of both erythrocyte autoantibodies and anti-RRBC antibodies. The manner in which erythrocyte selftolerance is broken has been little studied. It has been assumed that help for autoreactive B cells is provided by The cells specific for the foreign RRBC. We show here that autoreactive The cells can be recovered from RRBC-immunized mice. The cells proliferate in vitro whether stimulated by self or rat erythrocytes. Analysis of the specificity of the proliferating cells revealed extensive cross-reactivity for the two types of erythrocytes, it is therefore surprising that, in initial cultures, a slower response is evident when mouse erythrocytes are used as the antigen. From cytotoxic depletion of T-cell subsets, the phenotype of the proliferating cells was identified as Thy-1.2+, Lyt-1.1+, Lyt-2.1-, L3T4+. During in vitro stimulation of the T cells, growth factors characteristic of Tho cells are secreted. Finally, we demonstrate that the responding T cells are able to help primary in vitro antibody responses to self and rat erythrocytes. We conclude that autoreactive Tho cells are likely to be involved in the experimental induction of autoimmune haemolytic disease in mice. [ABSTRACT FROM AUTHOR]

The NZB mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by the production of pathogenic anti-mouse red blood cell (MRBC) autoantibodies. Although it is believed that the predisposition to autoimmune anaemia is multigenic in nature, the main pathogenic mechanism is attributed to antiMRBC autoantibodies. We have generated eight anti-MRBC monoclonal antibody (mAb)producing hybridomas derived from splenocytes of 9- and 12-month-old NZB mice with spontaneous autoimmune anaemia to dissect the molecular and cellular mechanisms resulting in the production of these pathogenic antibodies. The predominant immunoglobulin isotype was IgG2a, produced by five out of eight hybridomas (63%), while IgM, IgG1 and IgG2b were each produced by one hybridoma cell line (12%). Antigen specificity analysis of all eight hybridomas revealed that antibodies from seven out of eight hybridomas were monospecific for MRBC antigen(s). Only one hybridoma (clone 4-16-1) cross-reacted with rat RBC. None of the hybridomas produced antibodies reactive with single- or double-stranded DNA (ss- or dsDNA). Surface and cytoplasmic staining for the CD5 antigen revealed that none of the hybridomas was derived from CD5+ B lymphocytes. All hybridomas cause anaemia when implanted intraperitoneally into normal BALB/c mice. Molecular studies of five of the eight anti-MRBC mAb reveal that all use functionally rearranged genes from the VH J558 gene family. Three of these five mAb used FL16.1 DH genes while one had a CDR3 that resulted from a fusion between two DH genes (SP2.3 and SP2.2) from the SP family. [ABSTRACT FROM AUTHOR]

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of murine RBC autoantigens and cross-reactive rat antigens. Sera and RBC eluates from the AIHA-positive mice immunoprecipitated a murine RBC autoantigen of 42,000 MW that comigrates with a zone of glycophorin in PAS-stained polyacrylamide gels. In addition, the eluates immunoprecipitated a 105,000 MW protein corresponding to band 3, the erythrocyte anion channel, and two further components of 34,000 and 29,000 MW within the glycophorin zone. The 42,000 MW band was not detected by immunoblotting, indicating that it bears autoantigenic epitope(s) that are denatured during electrophoresis. Cross-reactive autoantibody in the eluates immunoprecipitated a rat RBC protein that comigrated with band 3, together with two bands of 36,000 MW and 34,000 MW that may represent minor rat glycophorins. In contrast, rat-specific serum IgG from mice with AIHA reacted predominantly with major rat glycophorins of 75,000 MW and 38,000 MW. Immunoblotting revealed that normal murine sera contain IgG that binds autologous spectrin from the RBC membrane skeleton, and that this activity is increased in mice with AIHA. Sera from AIHA-positive mice also reacted with other internal murine RBC components that are not exposed on the surface of intact cells. It is evident from the data that immunization of mice with rat RBC results in the generation of multiple autoantibodies with a complex range of specificities. [ABSTRACT FROM AUTHOR]

An inhibitor of the membrane attack complex of complement was isolated from the membranes of sheep erythrocytes. Fast protein liquid chromatography (FPLC) and affinity purification procedures for this sheep complement-inhibiting protein (SCIP) both yielded a pure protein with an apparent Mr of 19,000 under reducing and non-reducing conditions. Incubation of the denatured protein with neuraminidase and Endo-F reduced the apparent Mr to 18,000 and 15,000 respectively, while treatment with O-deglycosidase or phosphatidylinositol-specific phospholipase C (PIPLC) did not affect the apparent MrSCIP was detectable on erythrocytes and lymphocytes but not on platelets and could partially be removed by PIPLC treatment. Deglycosylation of the pure protein markedly reduced and PIPLC treatment abolished its activity. A monoclonal antibody (mAb) raised against sheep complement-inhibiting protein (SCIP) enhanced the susceptibility of sheep erythrocytes to lysis by homologous complement. SCIP inhibited complement after the stage of C5b-7 formation. Amino terminal protein sequence was obtained and was shown to be similar to that human CD59. All these features suggest that SCIP is the sheep equivalent of human CD59. Human CD59 has been reported to be species in that it inhibits complement from relatively few species. However, SCIP efficiently inhibited lysis of guinea-pig erythrocytes by complement from a wide range of species tested indicating that it is a potent and non-selective inhibitor of the membrane attack complex of complement (MAC). [ABSTRACT FROM AUTHOR]

The primary IgM response of murine B lymphocytes against red blood cell-bound antigens can be induced by incubating antigen-reactive B cells either with the lymphokines interleukin-1 (IL-1) and IL-2 together with the nucleoside cAMP, or by the addition of antigen-specific helper T cells. The reactivity of B cells is strongly influenced by the T-cell lymphokine IL-2. IL-2 inhibits the cyclic adenosine 3',5'-phosphate (cAMP)-dependent B-cell response when it is allowed to act on the cells prior to cAMP. On the other hand, if IL-2 acts on B cells together with or after cAMP, it synergizes with the nucleoside and enhances the immune response. A similar effect of IL-2 is observed in the Tcell-mediated activation of B cells. If IL-2 is present before helper T cells interacted with B cells, it inhibits antibody production. The inhibitory IL-2 effect is reversed by the simultaneous addition of exogenous cAMP. The finding supports the hypothesis that Ia ligation by T cells results in B cells in the elevation of cAMP which acts as an important second messenger in B cells. The antagonism between cAMP and IL-2 was also examined in the pre-B-cell line 70Z/3. The nucleoside is highly toxic to 70Z/3 pre-B cells and a majority disintegrates within hours of exposure to the nucleoside. The surviving cells undergo phenotypic differentiation expressing surface Igκ chains and major histocompatibility complex (MHC) class II molecules, and increase the expression of IL-2 receptor (R). The phenotypic differentiation requires the presence of IL-1. IL-2 inhibits both of these B-cell responses to cAMP, the IL-1-independent cell death, and the IL-1-dependent phenotypic differentiation. [ABSTRACT FROM AUTHOR]

The expression of both high-affinity E receptor (EhR) to sheep erythrocytes and CD2-11.3 epitope on activated human T lymphocytes suggests that these two structures may be functionally identical or closely associated. Therefore the aim of the present work was to determine if the CD2-11.3 epitope is involved in the early E rosette formation. The ability of normal and phytohaemagglutin (PHA)activated human T lymphocytes to express the CD2-11.3 epitope and to form early E rosettes (T cells with EhR) was studied simultaneously. The partial divergence of CD2-11.3 expression on T lymphocytes from the ability of these cells to form early E (Ee) rosettes was found. The results indicated that the expression of CD2-11.3 epitope alone is insufficient to form the Ee rosettes by activated T lymphocytes, yet it may facilitate this phenomenon in the presence of EhR. The above data clearly show that the CD2-11.3 epitope is functionally closely associated although not identical to EhR. Accordingly, it seems that these two structures may co-adhere to the appropriate ligand. Thus it is possible that the CD2-11.3 epitope, as well as its established role in activation signalling, may also act as a co-adhesion molecule. [ABSTRACT FROM AUTHOR]

The present study focused on the influence of maternal immunity during pregnancy on the responsiveness of the immune system(s) in offspring. Maternal immunization of pregnant mice with T-dependent foreign antigen sheep red blood cells (SRBC) induced suppression of anti-SRBC plaque-forming cell (PFC) responses in their offspring. We attempted to identify the cell species among the maternal lymphoid cells of the immunized pregnant mice that induced this suppression in their offspring, by separating the maternal cells into T cells, B cells and macrophages, or T-cell subsets, and then adoptively transferring them into other normal pregnant mice. The results demonstrated the following: first, maternal CD4+ T cells of immunized pregnant mice induced immune suppression in their offspring. Second, maternal T cells could be activated during pregnancy in the same fashion as in non-pregnant mice. The T-cell factor(s) for the immune suppression in offspring is produced not only by maternal T cells of immunized pregnant mice but also by T cells activated in non-pregnant mice. Third, cellular organization was required for maternal T cells to induce this immune suppression in their offspring. [ABSTRACT FROM AUTHOR]

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8 -CD19 or -CD56 antibodies to yield T4, T8. B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fcγ receptor II (FcγRII) and Fcγ receptor III (FcγRIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by FcγRIII) in antibody- dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these `ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with `ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind FcγRIII via CH2 and CH3 domains whereas ADCC antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of FcγRII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of 18 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1. [ABSTRACT FROM AUTHOR]

The notion that bile-dedicated antibody is made within the liver by migratory antibody-forming cells (AFC) was examined further in rats. Livers from immunized animals were removed to perfusion in isolation so that plasma influences on bile antibody would be obviated. Antibody was secreted for at least 5 hr by the livers of rats that had received intravenous (i.v.) or intra-Peyer's patch (IPP) immunization with horse erythrocytes. After initially declining, the titres stabilized at 5-8% of the starting value for IPP-immunized rats and at 0·8% for i.v.-immunized animals, levels that were then sustained. In other experiments, the biliary antibody output was measured in immunized rats in the period immediately following splenectomy, an expedient that would deny the liver any newly formed AFC. Splenectomy during spleen-based. IgM antibody responses led to bile titres falling, over about 12 hr, to 21% of initial values. This level was then maintained for at least another 12 hr. Serum titres over this period remained static. Lastly, the bile ducts of immunized rats were ligated to test whether locally made antibody that was destined for bile could be forced instead to reflux to blood. Biliary obstruction during IgM responses to horse erythrocytes and pneumococcal polysaccharide, type 3, was found to raise significantly serum antibody titres. For pneumococcal polysaccharide, the serum response was also noticeably prolonged. These findings are consistent with the biliary antibody of immunized rats being constituted, in part, from local sources and not from plasma alone. [ABSTRACT FROM AUTHOR]

Rat oligodendrocytes, which activate the classical pathway of complement in the absence of antibody, are highly sensitive in a reactive lysis assay using human C566 and EDTA serum. Oligodendrocytes may be relatively deficient in glycolipid-linked complement regulatory protein(s), since digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) failed to increase their sensitivity to serum, whereas complement-insensitive astrocytes, when treated with PI-PLC, became strikingly sensitive. To test the hypothesis that oligodendrocytes lack terminal complement regulatory molecule(s), human erythrocyte CD59, a recently described complement regulatory protein, was purified to homogeneity. The biological activity of the preparation was confirmed by reincorporating the protein into guinea-pig erythrocytes through its glycolipid anchor, which resulted in dose-dependent protection against human C566 and EDTA serum. Incorporation of 105 molecules of human CD59 into rat oligodendrocytes resulted in good protection against homologous human complement (76%), and significant protection against rat complement homologous to the cell (36%). Protection could be reversed using an antibody to CD59. [ABSTRACT FROM AUTHOR]

The influence of the In(Lu) gene on human red blood cell (RBC) expression of CDw75 antigens was examined. CDw75 antigens were increased in expression on Lu(a-b-) cells of the dominant inhibitor type in comparison with red cells from donors of other Lutheran (Lu) phenotypes. In contrast, CD44 epitopes detected with F10-44-2, A3D8 and BU52 monoclonal antibodies (mAb) were decreased in expression on Lu(a-b&-) red cells. Among normal blood donors of the common phenotype Lu(a-b+) there was a wide range of expression of CDw75 antigens on red cells. The results show that CDw75 is a quantitative polymorphism of human red cells and, among antigens influenced by the In(Lu) gene, is unique in being up-regulated in expression. [ABSTRACT FROM AUTHOR]

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59. [ABSTRACT FROM AUTHOR]

In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgAl-E), and the effect of mAb binding valency on FcγRI-mediated ADCC. All hybrid monospecific (ms) anti-HulgAl and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgAl-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mlgG2a H chains was predominantly mediated via FcγRI, as shown by inhibition studies on monocytes with FcγRI-blocking mAb TB-3 (&ap;80% inhibition). However, partial inhibition of mtgGl-2a and mlgG2a-2b-induced cytotoxicity (20-50%) was observed by using FcγRll-blocking mAb IV.3 or CIKM5. For hybrid mIgGl-l mAb the opposite was true; the cytotoxicity was predominantly mediated via FcαRlI (70-80%) and less via FcγRl (20-30%). Comparing the hybrid ms anti-HulgA 1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgAl/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgAl-E sensitized with univalently bound bs anti-HuIgAl/HRP mAb. This decrease was only found for FcγRI-mediated ADCC (mlgG2ae2a, mlgGl -2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgC by FcγRI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on FcγRI-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (l0.l) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37° to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1–1.5 × l03 anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory, In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it. [ABSTRACT FROM AUTHOR]

In this study we show a spontaneous mobilization at 37° of the complement receptor for C3b (CR1) of granulocytes prepared by a method in which erythrocytes were removed by specific lysis, as well as a method where granulocytes were prepared by dextran sedimentation at low temperature without using centrifugation. This increase of CR1-expression was not obtained when erythrocytes were present during the incubation. This inhibitory effect of erythrocytes was maximal at an erythrocyte: granulocyte ratio of 600:1 or more and was not caused by interference with the fluorescence of the immunoassay. EDTA plasma had no inhibitory effect on CR1 mobilization, indicating that the phenomenon was not due to plasma proteins, nor the used anti-coagulant. An increased CR1 mobilization was, however, obtained in the presence of erythrocytes if the granulocytes were simultaneously exposed to the chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (FMLP) at the concentration 10-9 M or more. However, to obtain a CR1 expression comparable to systems without erythrocytes, a 10-fold higher concentration of FMLP was needed. These results suggest that the inhibitory effect of erythrocytes on the spontaneous receptor mobilization of granulocytes could be a mechanism to keep the complement receptors and other surface structures within the cells while circulating in the blood, to be expressed at the cellular surface only by the appropriate signal, such as propagated by soluble mediators from an inflammatory focus. [ABSTRACT FROM AUTHOR]

Using monoclonal antibodies (mAb) characterized at the last International Workshop on Human Leucocyte Antigens, we examined the expression of CDw75 antigens (β-galactoside α 2,6- sialyltransferase) on normal peripheral blood cells and on cells from patients with B-cell chronic lymphocytic leukaemia (CLL). The mAb used (HH2, EBU.65, EBU.141 and OKB4) detect different epitopes of CDw75. Normal peripheral blood B cells expressed high levels of CDw75 detectable with HH2, EBU.65 and EBU.141 but did not react with OKB4. Cells from patients with B-cell CLL closely resembled normal B cells. All CDw75 epitopes, including OKB4, were strongly expressed on some Namalwa variant Burkitt lymphoma cell lines. The OKB4 epitope was also present on red cells from all normal donors. The other COw75 mAb were unreactive with red cells from some normal donors. The CDw75 epitope detected with EBU.65 was present on most CO4+ T cells and on a minority of CD8+ cells. HH2 and EBU.141 stained only small numbers of T lymphocytes. OKB4 did not react with T cells. EBU.65+, CD4+ T cells had low levels of expression of CD45R0, CD29, CD54 and CD58, and had high levels of CD45RA antigen. Phylohaemagglutinin (PHA)activation of cells led to the loss of EBU.65 binding. These results suggest that the CDw75 epitope recognized by the EBU.65 mAb is a marker of naive T lymphocytes. On B CLL cells the epitopes detected with HH2, EBU.65 and EBU.141 were destroyed by treatment with neuraminidase. Treatment of B-CLL cells and red cells with neuraminidase increased the binding of OKB4, suggesting that this epitope is masked by sialic acid. The results suggest that CDw75 is a sialylated cell-surface antigen expressed in a number of tissue-specific isoforms. [ABSTRACT FROM AUTHOR]