Downregulation of CD43 in RAEB and RAEB-T patients. Report of 3 cases

CD43 (leukosialin, sialophorin) is a cell surface mucin expressed at high levels on most leukocytes and is reported to be involved in adhesion, anti-adhesion, and signal transduction prodders. Regulation of its expression is thought to take place through methylation of the DNA in the nonproducing cells, and the methylation inhibitor 5-azacytidine induces expression of the sialophorin gene. Here we report three cases of patients with myelodysplastic syndromes in which acquired severe deficiency of the CD43 antigen on the surface of most hemopoietic cells was observed. Peripheral blood mononuclear (PBMC) cells from 32 MDS patients and 20 healthy individuals were analyzed by flow cytometry after labeling with an anti-CD43 (DF-T1) monoclonal antibody. In 1 patient with refractory anemia with excess of blasts (RAEB) and 2 patients with refractory anemia with excess of blasts in transformation (RAEB-t), the percentages of CD43(+) PBMC were 3.8%, 6%, and 9.9%, respectively. The deficiency was observed at protein and RNA level as confirmed by western and southern blot, while analysis of the DNA by single-strand conformation polymorphism and sequencing did not reveal any difference in the gene sequence between the CD43(+) and CD43(-) cells of these patients. It is known that patients with MDS may have normal and dysplastic population of hemopoietic cells. Further studies are needed to reveal the mechanism of downregulation of the gene in these 3 patients and whether the phenomenon is related to the dysplastic population only or not.     

Quantitative assessment of human neutrophil chemiluminescence induced by opsonized Escherichia coli K-12

The interaction of opsonized E. coli K-12 bacteria and polymorphonuclear leukocytes (PMN) was quantified, using luminol-enhanced chemiluminescence (CL) as a parameter of PMN stimulation. On a double-logarithmic scale light emission depended on the opsonin concentration used during pre-opsonisation. The most potent CL-inducing agent was fresh human serum, and its stimulatory activity depended on an intact complement (C) system. Both inactivation of C by heating or blocking the classical C pathway with EGTD decreased the CL-inducing potency by a factor of 8 to 16. Hypogammaglobulinemic heated serum mediated little CL. IgG for intravenous use mediated CL generation, but reduction/alkylation and sulphitolysis reduced the stimulatory power. Evidence is presented that the anti-K-12 antibodies within commercial IgG and IgM used for substitution do not improve the stimulatory power of IgG-deficient, IgM- and C-sufficient serum, unless very high Ig concentrations are substituted.     

Specific sensitivity of CD43 to neutrophil elastase [see comments]

CD43 (sialophorin, leukosialin), an O-glycosylated and sialylated membrane protein (surface sialomucin) with antiadhesive properties, is thought to protect circulating leukocytes by preventing cell surface interactions. Although it is resistant to several proteases, the granule enzyme elastase was recently implicated in loss of extracellular CD43 regions from incubated neutrophils. Flow cytometry showed that neutrophil CD43 is cleaved by low levels of neutrophil elastase with half-maximal cleavage at 5 micrograms/mL; pancreatic elastase, in contrast, did not cleave CD43. Related neutrophil granule proteases proteinase-3 and cathepsin-G did not cleave CD43 or required greater than 10-fold higher enzyme levels, respectively. The 115-kD CD43 isoform on T-lymphoid cells, which differs in glycosylation from 135-kD neutrophil CD43, was equally sensitive to neutrophil elastase, suggesting that cleavage susceptibility extends to various leukocytes. Enzymatic removal of sialic acid did not facilitate CD43 cleavage by neutrophil elastase, a feature that distinguishes the action of neutrophil elastase from other proteases. Western blots of elastase- treated neutrophils detected an 83-kD CD43 fragment that, together with the released 52-kD fragment and 40-kD subfragment, accounts for the entire molecule and indicates that CD43 is cleaved at two sites only, releasing the distal approximately 40% of the sialomucin region. The specificity of the CD43 cleaving reaction was shown by the insensitivity of other neutrophil and lymphoid surface proteins to elastase levels that deplete CD43. Exceptions were P-selectin glycoprotein ligand-1 on neutrophils, also a surface mucin, and CD16 (Fc gamma RIII), which was previously characterized as elastase sensitive. The sensitivity and specificity of CD43 cleavage by neutrophil elastase, the very high levels of elastase in human neutrophils and its ready release by stimulating conditions suggest important physiologic/pathologic roles for this CD43 cleaving reaction.     

Inhibition of zymosan activation of human neutrophil oxidative metabolism by a mouse monoclonal antibody

We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ- induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.     

Patterns of guanine nucleotide exchange reflecting disparate neutrophil activation pathways by opsonized and unopsonized Candida albicans hyphae

Through guanosine triphosphate (GTP) regulatory proteins are crucial components in signal transduction by most soluble and opsonized particulate stimuli, previous data suggest that neutrophil (PMNL) activation by unopsonized hyphae differs. Most of the PMNL superoxide response evoked by unopsonized hyphae was independent of both Ca++ ions and pertussis toxin-sensitive guanine nucleotide regulatory proteins. To determine whether related regulatory proteins were involved in PMNL activation by unopsonized hyphae, separated PMNL plasma membranes were incubated with GTP and a poorly hydrolyzed, radiolabeled GTP analogue, 5'-guanylylimido-diphosphate, then stimulated. Particulate Candida albicans hyphae and soluble chemotactic peptide induced comparable guanine nucleotide release. In contrast, while unopsonized hyphae caused release, it was considerably delayed, though opsonization discernibly affected neither PMNL attachment nor spreading over hyphal surfaces. This paralleled earlier observations of other delayed responses by intact PMNL to unopsonized hyphae: phospholipase C activation, the rise in cytosolic free Ca++ ions, and actin polymerization.     

Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins

Recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary immunological response to invading pathogens and has also emerged as a hallmark of vascular inflammation. One of the principal enzymes released upon PMN activation is myeloperoxidase (MPO), a heme protein that not only generates cytotoxic oxidants but also impacts deleteriously on nitric oxide-dependent signaling cascades within the vasculature. Because MPO also associates with the membrane of PMN, we evaluated whether MPO could also function as an autocrine modulator of PMN activation. The extent of PMN membrane-associated MPO was elevated in patients with acute inflammatory vascular disease compared with healthy individuals. Isolated PMNs bound free MPO by a CD11b/CD18 integrin-dependent mechanism. PMNs exposed to MPO were characterized by increased tyrosine phosphorylation and p38 mitogen-activated protein kinase activation. Also, nuclear translocation of NFkappaBin PMN was enhanced after incubation with MPO, as was surface expression of CD11b. Binding of PMN to MPO-coated fibronectin surfaces amplified PMN degranulation, as evidenced by increased release of MPO and elastase. MPO also augmented PMN-dependent superoxide (O(2)(*-)) production, which was prevented by anti-CD11b antibodies, but not MPO inhibitors. Collectively, these results reveal that binding of MPO to CD11b/CD18 integrins stimulates PMN signaling pathways to induce PMN activation in a mechanism independent of MPO catalytic activity. These cytokine-like properties of MPO thus represent an additional dimension of the proinflammatory actions of MPO in vascular disease.     

Signaling through CD43 regulates CD4 T-cell trafficking

The mucin-like protein CD43 is excluded from the immune synapse, and regulates T-cell proliferation as well as T-cell migration. While the CD43 cytoplasmic domain is necessary for regulation of T-cell activation and proliferation, the mechanism via which CD43 regulates trafficking is not well defined. To investigate whether CD43 phosphorylation regulates its function in T cells, we used tandem mass spectrometry and identified Ser76 in murine CD43 as a previously unidentified site of basal phosphorylation. Interestingly, mutation of this single serine to alanine greatly diminishes T-cell trafficking to the lymph node, while CD43 exclusion and CD43-mediated regulation of T-cell proliferation remain intact. Furthermore, the CD43 extracellular domain was also required for T-cell trafficking, providing a hitherto unknown function for the extracellular domain, and suggesting that the extracellular domain may be required to transduce signals via the cytoplasmic domain. These data reveal a novel mechanism by which CD43 regulates T-cell function, and suggest that CD43 functions as a signaling molecule, sensing extracellular cues and transducing intracellular signals that modulate T-cell function.     

Inhibition of cellular immune reactions by zymosan

Mice treated intraperitoneally with zymosan showed a strong inhibition of the DTHR (delayed-type hypersensitivity reaction) against sheep erythrocytes (SE), ovalbumin (OA), and alloantigen. Furthermore, the rejection time of skin transplants was nearly doubled while antibody formation against SE was significantly enhanced. When a DTHR against OA and an antibody formation against SE were induced at the same time in the same animals, than the suppressive and stimulating effects cancelled each other. These results are discussed with regard to the sensitivity of lymphocyte subpopulations, which may be different if exposed to phagocytosis-induced oxygen radicals.     

抑制细胞间通讯功能可加速人成纤维细胞的衰老

目的研究间隙连接蛋白connexin43在人成纤维细胞衰老过程中的调节作用。方法设计并合成针对人connexin43的双链RNA(siRNA),抑制人二倍体成纤维细胞WI-38细胞的connexin43表达和细胞间通讯功能,观察对其衰老相关β-半乳糖苷酶(SA-β-gal)染色阳性率、细胞增殖能力、细胞周期调节蛋白P27、P21表达水平等衰老表型的影响。结果我们合成的siRNA-cx43可高效、特异的抑制connexin43 mRNA和蛋白质表达及细胞间通讯功能。转染WI-38细胞后,细胞增殖能力降低、细胞SA-β-gal染色阳性率(75.32±5.17)较对照组(32.48±3.94)和转染siRNA-con组(37.81±4.1 2)细胞升高(P<0.05),P27、P21表达增加,转染siRNA-cx43的WI-38细胞增殖能力显著降低,细胞变扁平、肥大,细胞SA-β-gal染色阳性率达100%,细胞提前出现衰老。结论间隙连接蛋白connexin43对WI-38细胞的衰老进程有重要的调节作用,其表达减少和细胞间通讯功能降低,可以促进WI-38细胞衰老进程。     

Exocytosis of zymosan particles by human phagocytes

The kinetics of human leucocyte phagocytosis and exocytosis of fluorescein-isothiocyanate (FITC)-labelled zymosan particles were studied by flow cytometry (FCM). The leucocytes rapidly associated with zymosan particles, and internalization was confirmed by a fluorescence quenching technique. For incubation periods longer than about 60 min, exocytosis of ingested particles was observed. All human phagocytes ejected zymosan particles. The rate of exocytosis was about 1 particle per phagocyte per h, and was independent of the number of internalized particles. Exocytosis was dependent on temperature and glucose, but did not require Ca++ and Mg++ ions. Phagocytosis and exocytosis occurred concurrently, and phagocytosis accelerated ejection of previously internalized particles. The exocytosed particles were partially degraded by the phagocytes, and rephagocytosis of ejected zymosan particles was slower than the uptake of control particles. Phagocytes undergoing exocytosis remained intact during the 210 min examined. The findings indicate a heretofore neglected vector in the interaction of prey and phagocytosing cells.     

Downregulation of CD38 activation markers by atorvastatin in HIV patients with undetectable viral load

Immune activation and chronic inflammation are recognized as major component of HIV disease even in patients with undetectable viral load. We evaluated the effect of atorvastatin on CD38 activation in such patients, in a case-control study (133 cases - 266 controls). At week 48, CD38 activation was significantly lower in cases vs. controls, with no difference in high-sensitivity C-reactive protein (hsCRP) and CD4. These results suggest that atorvastatin reduces the level of immune activation in patients with undetectable viral load.     

CD43 interferes with T-lymphocyte adhesion.

CD43 is a cell-surface sialoglycoprotein of uncertain physiologic function expressed to various degrees by most leukocytes. We tested whether or not CD43 participates in intercellular adhesion by comparing the binding of human T lymphocytes to transfected HeLa cells stably expressing CD43 and sham-transfected HeLa cells (CD43-negative). Significantly fewer T lymphocytes adhered to the CD43-positive HeLa cells than to the CD43-negative HeLa cells. Diminished T-cell adherence to the CD43-positive HeLa cells was seen for all T lymphocytes tested, irrespective of their source or derivation. Antibody-blocking experiments revealed that CD43 interference with T-cell adhesion largely represented interference with T-cell leukocyte function-associated antigen 1 binding to HeLa cell intercellular adhesion molecule 1. The CD43 anti-adhesion effect was not overcome by treating cells with phorbol 12-myristate 13-acetate, a chemical that increases the binding avidity of leukocyte function-associated antigen 1 for intercellular adhesion molecule 1. However, neuraminidase treatment of the HeLa cell transfectants diminished the CD43 antiadhesion effect. These data indicate that CD43 expression by opposing cells can interfere with cell-cell adhesion. The data also suggest that CD43 might regulate T-cell adhesion by interfering with leukocyte function-associated 1 binding to intercellular adhesion molecule 1, a major activation-induced adhesion pathway among lymphocytes.     

CD43 in B-cell chronic lymphocytic leukemia

CD43 (other names: sialophorin, leukosialin, sialoglycoprotein of white blood cells) is an integral cell membrane mucin. In population of peripheral B cells CD43 occurs only on activated B cells and CD5 positive B cells. These last cells create neoplasm population in patients with B-cell chronic lymphocytic leukemia (B-CLL). Anti-CD43 monoclonal antibodies are used routinely in investigations of tissue fragments in cases of non-Hodgkin's lymphoma, whereas we did not find publication on theme of CD43 expression on peripheral blood B cells in patients with B-cell chronic lymphocytic leukemia. Wherefore advisable appeared estimation CD43 expression on B-CLL cells and comparison it with expression of typical B-CLL markers--such as CD5 and CD6. Immunological phenotype of peripheral blood and bone marrow lymphocytes has been evaluated using flow cytometry (Cytoron Absolute Ortho-Diagnostic Systems) and two-color staining. Twenty six untreated patients with B-CLL were studied. Because on well-known correlations between CD43 expression and metastasis potential of tumor, patients were divided on two groups differing score of total tumor mass (score TTM). Score TTM was evaluated according to criterion of Jaksic and Vitale. Twelve patients whose TTM score was equal or lower than 9 and median lymphocytosis was 24.6 x 10(9) in microliter were included in group I. 14 patients whose TTM score was higher than 9 were included in group II. Median lymphocytosis in these patients was 152.6 x 10(9) in microliter. The median percentage of CD43+/CD19+ cells in peripheral blood was 62.6% in the group I, and 75% in the group II (p < 0.05). Median fluorescence intensity (MFI) of CD43 antigen was 87.7 in the I group comparing to 77.4 in the group II. So one observed tendency to lowering MFI during tumor growing but the difference was not significant (p = 0.25). In peripheral blood during progression of disease more clearly than CD43+ cells increased percentage of CD5+ and CD6+ cells. The median percentage of CD19+/CD5+ cells was 62.7% in the group I, 82.4% in the group II and the difference was significant (p < 0.002). The difference in the median percentages CD6+/CD19+ cell 71.8% in group I and 84.3% in the II one were also significant (p < 0.03). MFI of CD5 and also CD6 antigens did not change in course of disease. Moreover, examination of CD43 and CD5 expression in marrow additionally to blood study were performed in 12 cases (6 from group I, 2 from group II and 4 new not included). The median percentage of CD43+/CD19+ cell was 35.1% in blood and 43.7% In marrow, in contrast to these results was the median percentage of CD19+/CD5+ cell, which was higher in peripheral blood (70.4%) than in bone marrow (60.9%). The results of this study indicate that CD43 is present on peripheral blood B-CLL cells. Moreover, percentage of these cell increases during progression of disease however more weakly than percentage of CD5 and CD6 positive cells. Expression of CD43 is independent from expression CD5 and CD6 and diminishes during tumor mass increasing, what can depended from releases exocellular domains of CD43. CD43+ cell from B-CLL patients have a tendency to accumulation in tissues what is illustrated by higher percentage of CD43+ cell in bone marrow than in peripheral blood.     

Activation of phagocyte oxidative metabolism by opsonized Plasmodium falciparum merozoites

A luminol-dependent chemiluminescence (CL) method was used to measure the oxidative burst of phagocytes triggered by antimalarial antibodies and Plasmodium falciparum merozoites. A specific antibody-dependent increase in chemiluminescence response was obtained using both polymorphonuclear leukocytes and monocytes as effector cells. Using various sera from malaria infected subjects it was observed that antibodies involved in the increased chemiluminescence responses were encountered at higher levels in sera from protected subjects than in those susceptible to clinical manifestations. In the conditions used the assay shows substantial intertest variation but appears of interest to assess the protective status of groups of exposed individuals.     

Graded responses of human neutrophils induced by serum-treated zymosan

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3'- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.     

CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6.

Regulated adherence of polymorphonuclear leukocytes (PMNs) to endothelium and subendothelial matrix is a critical event for PMN localization at and migration into inflammatory sites. We previously reported that human PMNs stimulated in vitro adhere to laminin, the major glycoprotein of mammalian basement membrane, by both CD11/CD18 (beta 2 integrin)-dependent and CD11/CD18-independent mechanisms. This CD11/CD18-independent adherence is inhibited by monoclonal antibodies (MoAbs) directed against the beta 1 subunit of integrins (very late antigens [VLA]). The specific PMN VLA receptor responsible for stimulated CD11/CD18-independent PMN adherence to laminin was not elucidated. We show here that this CD11/CD18-independent adherence is mediated by a member of the beta 1 integrins, VLA-6. MoAbs GoH3 and 450-30, which bind the alpha 6 subunit of VLA-6, significantly reduced adherence of phorbol myristate acetate-stimulated PMNs to laminin-coated surfaces when CD11/CD18-independent adherence was blocked with anti-CD11/CD18 MoAbs. Furthermore, GoH3 completely inhibited stimulated adherence of CD11/CD18-deficient PMNs to laminin. Analysis by flow cytometry showed that human PMNs express VLA-6. The PMN alpha 6 is identical in size and pl to the platelet alpha 6, but the PMN beta 1 exhibits considerable heterogeneity in molecular weight compared with the platelet beta 1. This activation-dependent adherence receptor for laminin may play a role in PMN interaction with basement membrane laminin during PMN movement through vascular walls.     

Alterations of CD43 expression in transfusion-dependent myelodysplastic syndromes

CD43 is a sialylated glycoprotein expressed on the surface of most haemopoietic cells and has been implicated in cell adhesion and signaling. It has previously been shown that CD43 expression is altered in patients with myelodysplastic syndrome (MDS). This raised the question of whether the alteration is associated with transfusions in these patients. We studied the expression of this antigen on peripheral blood leucocytes in two groups of patients with refractory anaemia, 22 transfused and 20 non-transfused. We found decreased expression of CD43 on the monocytes and neutrophils of patients receiving transfusions. Other activation molecules were studied (CD11b, CD18) and were found up-regulated suggesting the existence of activated leucocytes in these patients. The increased levels of soluble vascular cellular endothelial molecule after transfusions in these patients suggested vascular endothelial activation in the absence of infection. Given together, these results show that decreased CD43 in the transfused group of MDS patients is associated with an activated endothelial phenotype.     

Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation

Human neutrophils are primed in the presence of complexes of lipopolysaccharide (LPS) with its serum binding protein (LBP) in a manner dependent on CD14. Cellular consequences of priming include increased responsiveness, the upregulation of surface proteins including the adhesive integrin CD11b/CD18 (Mac-1), the increased binding of certain ligands to CD11b/CD18, and the concurrent shedding of the L-selectin homing receptor. Because expression of both CD11b/CD18 and L-selectin is obligatory for formyl peptide-stimulated neutrophil aggregation in vitro (Simon et al, Blood 82:1097, 1993), we have examined the consequences of bacterial endotoxin on the expression of neutrophil adhesive molecules. We observed that the exposure of neutrophils to LPS/LBP, while enhancing the surface numbers and adhesive function of CD11b/CD18 for latex particles, did not induce aggregation. In contrast, as the LPS/LBP concentration increased (ED50 = 30 ng/mL LPS/LBP), the ability of neutrophils to aggregate decreased in parallel with the shedding of L-selectin. Moreover, when L-selectin adhesive activity was blocked by treatment with Fab fragments of Dreg- 200, aggregation was inhibited to an extent roughly proportional to the available L-selection. Blocking of LPS/LBP with CD14-specific monoclonal antibodies suppressed L-selectin shedding and preserved formyl peptide-stimulated aggregation. Taken together, the data suggest that inhibition of neutrophil aggregation by LPS/LBP is related to the expression of L-selectin via CD14 rather than LPS inhibition of CD11b/CD18 function during cellular stimulation.     

Downregulation of the CD95 receptor and defect CD40-mediated signal transduction in B-chronic lymphocytic leukemia cells

Cross-linking of the CD40 receptors has been shown to induce protein tyrosine kinases (PTK) phosphorylation and prevent apoptosis in Bcl-2 negative germinal center B cells. The expression of CD40 on B chronic lymphocytic leukemia (B-CLL) cells was found to be similar to that of normal B cells. Activation of normal B cells with soluble anti-CD40 monoclonal antibody (mAb) induced tyrosine phosphorylation, prolonged survival and prevented apoptosis. However, activation of CD40 on B-CLL cells using soluble anti-CD40 mAb does not influence survival or apoptosis. Normal B cells entered apoptosis when cultured in the presence of soluble anti-CD95 mAb. This process was independent of PTK activity. On B-CLL cells, the CD95 molecules were downregulated and a transient PTK signal was observed when cross-linking of the receptor by soluble anti-CD95 mAb occurred. Interestingly, B-CLL cells did not enter apoptosis in the presence of anti-CD95 mAb. Our study indicates that survival signals mediated through the CD40 molecule and death signals mediated through the CD95 molecule used different intracellular pathways in control donor B cells. In contrast, B-CLL cells do not respond to these signals. The leukemic B cells showed a defective CD40-mediated signal transduction and downregulated CD95 receptor expression. As a consequence, no apoptosis could be induced in B-CLL cells by a soluble anti-CD95 mAb. The abnormalities of these receptors may contribute to the long-lived status of B-CLL cells.     

Macrophage recognition of zymosan particles

Zymosan particles have served as a model for recognition of microbes by the innate immune system for over 50 years. Zymosan induces inflammatory signals in macrophages through Toll-like receptors TLR2 and TLR6. In addition, phagocytic receptors on macrophages bind zymosan and stimulate particle engulfment. We have further examined the requirements for induction of inflammatory responses such as TNF-alpha production and NF-kappaB activation by zymosan in mouse macrophages. We have observed that direct particle contact is required (excluding a role for soluble components of zymosan preparations) and that contact with a single particle is sufficient to trigger cytokine production. Further, ablation of the Toll-like receptor-stimulating activity of zymosan does not affect the ability of phagocytic receptors to internalise the particle.