CD8alpha+beta(low) effector T cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by the loss of self-tolerance to nuclear antigens. Aberrant T-cell function plays a central role in lupus pathogenesis. We and others previously demonstrated that peripheral TCRalphabeta+CD3+ T cells express CD8beta either at a high (CD8beta(high)) or low density (CD8beta(low)), thereby defining two functionally distinct subsets. CD8beta(low) T cells express predominantly CD8alphaalpha and less CD8alphabeta as a coreceptor, display a differentiated phenotype and exert effector function. CD8beta(high) T cells appear to be the precursors expressing predominantly the heterodimeric efficient CD8alphabeta coreceptor, exhibiting a naïve phenotype and high proliferative capacity. In the present study, the distribution and functional properties of CD8beta(high) and CD8beta(low) T cells of SLE patients were compared (n = 20) with those of healthy subjects (n = 16). It was found that expansion of CD8beta(low) T-cell subset correlated with disease activity indicating chronic antigenic stimulation leading to a major lack of naïve CD8beta(high) precursor T cells in SLE. Functional characteristics of CD8beta(low) T cells including production of cytokines and cytotoxic granules were not significantly different between patients with SLE and healthy individuals. We speculate that unbalanced CD8beta(high)/CD8beta(low) T-cell relation reflects a skewed homeostasis within the CD8+ T-cell compartment towards fully differentiated effector T cells possibly due to persistent antigen stimulation in SLE.

Lack of specific receptors for C-reactive protein on white blood cells.

The classic acute-phase reactant C-reactive protein (CRP) plays an important role in innate immunity. Specific CRP receptors have been described on white blood cells and were further characterized as Fcgamma receptors I and II. Here, we used biotinylated, highly purified natural CRP and recombinant human CRP from E. coli to investigate binding to white blood cells. The structural integrity of recombinant CRP was demonstrated by proof of pentamer assembly using non-denaturing gel electrophoresis. Furthermore, the functional capability was confirmed by calcium-dependent ligand binding (phosphorylcholine-coupled BSA and nuclear constituents), and by complement activation (C3 deposition). The monocytic cell line U937 expresses FcgammaRI and FcgammaRII--the proposed CRP receptors--in high density. Binding of biotinylated CRP was only detected by flow cytometry using a partially purified CRP preparation, that contained additional proteins, e.g. IgG as demonstrated by immunoblotting. Highly purified and recombinant CRP, free of IgG, were not bound. To exclude blocking of binding epitopes by labeling on recombinant CRP, biotinylation was performed at various biotin to protein ratios. In addition, competition assays demonstrated that binding of biotinylated, partially purified CRP was only inhibited by partially purified CRP and IgG, but not by highly purified and recombinant CRP. Recombinant CRP bound to U937 cells only after contamination with 0.5 microg IgG per 100 microg CRP before biotinylation. Therefore, we conclude that CRP itself is not bound to white blood cells and strongly suggest a reassessment of previous data.

Heterogeneous PIG-A mutations in different cell lineages in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells in which affected cells are characterized by the lack of glycosylphosphatidylinositol (GPI)-anchored proteins. The lesion in PNH lies in the defective synthesis of N-acetyl-D-glucosaminyl-phosphatidylinositol (GlcNAc-Pl), the first intermediate in GPI biosynthesis. Reintroduction of the PIG-A gene into GPI(-) patient cells reportedly complements this defect. We have analyzed here PIG-A transcripts of six PNH patients. GPI+ and GPI- cell lines from each individual were used, ie, Epstein-Barr virus-transformed B-lymphoblastoid cell lines, T-cell lines, and natural killer cell clones. Reverse transcriptase polymerase chain reaction and sequencing showed three different PIG-A splicing variants in GPI+ cell lines, in which the largest transcript contained the wild-type PIG-A coding region sequence. GPI-deficient cell lines showed abnormal splicing variants. Sequencing of PIG-A complementary DNA and genomic DNA showed heterogeneous mutations ranging from different point mutations to small deletions. Two lymphocyte cell lines (T- and B-cell lines) of one patient presented with the same mutation. For another patient, two different mutations were detected in one natural killer cell line. Therefore, different cell lineages have somatic mutations in PIG-A that lead to PNH.

The loss of Fc gamma RIIIb in paroxysmal nocturnal hemoglobinuria is functionally replaced by Fc gamma RII.

Neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH) show a deficiency for the glycosylphosphatidylinositol- (GPI) linked Fc gamma receptor IIIb (Fc gamma RIIIb, CD16). The functional consequences of this defect are not clear. Here, we examined Fc gamma RIIIb-deficient neutrophils for their activation via Fc gamma receptors. Hydrogen peroxide (H2O2) production and change of intracellular free calcium [Ca2+]i were used as parameters for cell activation. Fc gamma RII and Fc gamma RIIIb stimulation was reached by cross-linking using fragments of monoclonal antibodies or incubation with monoclonal IgG cryoglobulin complexes. In parallel to the deficiency of Fc gamma RIIIb expression, H2O2 production and [Ca2+]i influx were decreased after cross-linking of Fc gamma RIIIb in PNH neutrophils compared with that for normal neutrophils. Stimulation via Fc gamma RII was not affected. Cryoglobulin complexes previously shown to activate normal neutrophils predominantly via Fc gamma RIIIb stimulated PNH neutrophils at a level not significantly weaker than controls. But this activation was mediated only via Fc gamma RII as shown by blocking studies. The results suggest that the loss of GPI-anchored Fc gamma RIIIb is functionally replaced by Fc gamma RII during the immune complex stimulation of PNH neutrophils. Therefore, the equipment of neutrophils with pleomorphic Fc gamma receptors prevents an immunodeficiency in PNH.

Development of the glycosylphosphatitylinositol-anchoring defect characteristic for paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia.

The introduction of immunosuppressive therapy for treatment of aplastic anemia has led to a considerable improvement in the prognosis of this disease. However, long-term follow-up of these patients showed a high incidence of "late" hematologic complications such as myelodysplasia and paroxysmal nocturnal hemoglobinuria (PNH). The detection of the glycosylphosphatitylinositol (GPI)-anchoring defect on peripheral blood cells of patients with aplastic anemia is now available as a new tool for early specific detection of PNH and is more sensitive than the Ham-test. Granulocytes appear to be the first cells affected in 11 patients with a GPI-anchoring defect of 29 suffering from aplastic anemia investigated in the present study. The later involvement of erythrocytes and a positive Ham test was observed in 1 patient. From our data it can be concluded that the rate of PNH resulting from aplastic anemia might be higher than reported in the literature when the Ham test alone was used for follow-up. Furthermore, our results suggest the clinical response to immunosuppressive therapy appears to be worse in the group developing the GPI-anchoring defect than in the group without this deficiency.

Fc gamma receptor activation of neutrophils in cryoglobulin-induced leukocytoclastic vasculitis.

OBJECTIVE: The role of Fc gamma receptors (Fc gamma R) in type I cryoglobulinemia was investigated to characterize novel mechanisms of neutrophil activation in the pathogenesis of leukocytoclastic vasculitis. METHODS: Neutrophils from healthy donors were incubated with purified monoclonal IgG1 kappa cryoglobulin complexes in vitro. Changes in surface antigen expression and mechanisms of intracellular hydrogen peroxide production and calcium release were measured by flow cytometry. RESULTS: After incubation for 2 hours, surface expression of Fc gamma RI (CD64), CD66, and CD67 was up-regulated; Fc gamma RII (CDw32), Fc gamma RIII (CD16), and LAM-1 were down-regulated. Using solubilized and complexed cryoglobulins, it was demonstrated that complex formation is necessary to induce intracellular H2O2 production and calcium release from intracellular stores. Both H2O2 generation and calcium mobilization could be inhibited by pretreatment with F(ab')2 fragments of monoclonal antibodies (MAb) against Fc gamma RIII. In contrast, Fab fragments of anti-Fc gamma RII MAb failed to block these activations. Neither the cryoglobulin complex-induced production of H2O2 nor the increase in cytoplasmic calcium was affected by treatment with pertussis toxin, which suggests that pertussis toxin-sensitive G proteins are not involved in signal transduction. CONCLUSION: These results indicate that Fc gamma RIII plays a major role in the pathogenesis of leukocytoclastic vasculitis.

Differences in activation of normal and glycosylphosphatidylinositol-negative lymphocytes derived from patients with paroxysmal nocturnal hemoglobinuria.

In these studies, the role of glycosylphosphatidylinositol (GPI)-anchored surface molecules during T cell activation was investigated in fresh T cells and T cell lines obtained from patients with paroxysmal nocturnal hemoglobinuria. For control, GPI-expressing T cells of the same patients were used. Unstimulated GPI- T cells exhibited significantly reduced surface expression of the activation Ag CD45R0, compared with GPI+ T cells. In addition, in measurements of proliferation, IFN-gamma production, and induction of second messengers such as cytoplasmic Ca2+, CD48- lymphocytes showed a similar response to TCR-specific stimulation, compared with CD48+ lymphocytes. In contrast, stimulation with the lectin PHA produced a decreased response of CD48- lymphocytes in these functions. In addition, stimulation with cross-linked CD59 mAb increased the proliferation of GPI-molecule expressing CD48+ T cell lines only. From these data, it can be concluded that GPI-anchored surface molecules play an important role in T lymphocyte activation.

Diagnosis of paroxysmal nocturnal haemoglobinuria using immunophenotyping of peripheral blood cells.

Paroxysmal nocturnal haemoglobinuria (PNH) is now generally accepted as a disease in which bone marrow derived cells are deficient in phosphatidylinositolglycan (PIG)-anchored surface molecules. A series of new monoclonal antibodies detecting PIG-anchored surface structures on human leucocytes (CD48, CD55, CD59) has recently been described. In the present study 12 patients with the diagnosis PNH and a positive Ham test were examined for PIG-anchored surface antigen expression on various cell lineages using immunofluorescence. In all patients deficient cells were detected in erythrocyte, granulocyte and monocyte analysis. A deficient lymphocyte subset was also observed in all but one of these patients. Using two-colour analysis, all lymphocyte subpopulations such as T, B and NK cells were found to be affected. In addition, peripheral blood cells of 22 patients with severe aplastic anaemia (SAA) were tested for the PIG-anchoring defect. In five of these patients the defect was detected, and in four of the five the lack of PIG-anchored molecules was confined to the granulocyte and monocyte lineages apparently without affecting the erythrocytes. The results of these studies demonstrate that cytofluorographic testing of peripheral blood cells provides a simple and reliable method for establishing the diagnosis of PNH. Furthermore, especially in the case of aplastic anaemia patients, the sensitivity of immunophenotyping might be superior to conventional laboratory tests.

[Severe vasculitis in cryoglobulinemia--mechanisms of Fc gamma receptor activation of granulocytes]. / Schwere Vaskulitis bei Kryoglobulinämie--Mechanismen der Fc gamma-Rezeptor-Aktivierung von Granulozyten.

Monoclonal IgG-cryoglobulins activate PMN through Fc gamma-receptors. The expression of Fc gamma RII and Fc gamma RIII is decreased, the expression of Fc gamma RI, CD66 and CD67 is increased. During this activation substances like superoxide anions and lysosomal enzymes subsequently damaging the endothelium are released.