Reinhold Deppisch

Induction of mediator release from human glomerular mesangial cells by the terminal complement components C5b-9

Exposure of cultured human glomerular mesangial cells (GMC) to normal human serum and an activator of the complement system results in rapid uptake of the terminal complement proteins C5b-9 by the cells. This innocent bystander complement attack, however, does not result in cell killing, but in the stimulation of the GMC to release prostaglandin E (PGE), interleukin 1 (Il-1) and tumor necrosis factor (TNF). Endogenously synthesized Il-1 in turn activates PGE release, indicating that the C5b-9 attack initiates an autocrine feedback stimulation. Together with the fact that C5b-9 is found in many forms of glomerulonephritis, the data point to a role of the terminal complement proteins in the initiation and perpetuation of an inflammatory response.

Stimulation of Mononuclear Cells by Contact With Cuprophan Membranes: Further Increase of β2-Microglobulin Synthesis by Activated Late Complement Components

Contact of mononuclear cells (MNC) with cuprophan membranes in vitro causes an increase in beta 2-microglobulin (beta 2m) synthesis. Since in vivo the dialyzer membrane is rapidly coated with plasma proteins, contact activation of MNC was tested in the presence of normal human serum (NHS). After contact with cuprophan, deposition of C5b-9 on the cells was seen, followed by an increase in beta 2m synthesis and cytokine release, exceeding that seen after contact activation in the absence of serum. Inactivated serum or serum deficient in C8 did not increase beta 2m production, indicating that the additional activation was due to complement C5b-9. The results suggest that there are two cuprophan-related mechanisms of cell activation: one by contact of cells with the membrane, the other by the complement activation products. Both might synergistically contribute to an increased beta 2m synthesis in hemodialysis patients.

Activation of human neutrophils after contact with cellulose-based haemodialysis membranes: intracellular calcium signalling in single cells

PURPOSE OF STUDYnIn vitro contact of human leukocytes with cellulose-based dialysis membranes under complement-independent conditions results in activation of various leukocyte functions. To analyse signals involved in the mechanism of cell activation, we measured changes in cytosolic free calcium ([Ca2+]i) in individual human blood neutrophils (PMN) upon contact with flat sheet haemodialysis membranes.nnnRESULTSnBy confocal laser-scanning microscopy (CLSM), changes in [Ca2+]i were monitored in Fluo-3-labelled cells up to 10 min after contact with a regenerated cellulose (RC) membrane. Multiple [Ca2+]i transients were observed for cells in contact with RC; biostochastic analysis showed that up to 67% of the PMN responded with a high increase in [Ca2+]i, the rest were low- or non-responding cells. After contact with the new synthetic polycarbonate-polyether (PC-PE) membrane only non-responding cells were seen, indicating reduced cellular contact activation. The increase in [Ca2+]i of cells on RC could be inhibited by 5mM L-fucose. This monosaccharide was recently found to be present in cellulose-based polymers in picomolar concentrations.nnnCONCLUSIONSnThe data supports the hypothesis that dialysis-membrane-associated L-fucose residues participate in complement-independent leukocyte activation during haemodialysis therapy.

Complement Activation and C3 Allotype Distribution in Patients with Bronchial Asthma

61 patients suffering from intrinsic (idiotypic) or extrinsic (allergic) asthma were investigated for signs of complement activation and for C3 phenotype distribution. Activation of both the classical and alternative pathway of the complement system and generation of the membrane attack complex could be assessed by ELISAs for the activation-specific protein-protein complexes C1rsC1 inhibitor, C3b(Bb)P and SC5b-9, respectively. A possible deficiency of the complement regulatory proteins C1 inhibitor, factor H and factor I was excluded. In contrast to earlier studies, C3 allele frequencies did not differ from those found in the healthy population. Our results support the role of complement activation during bronchial asthma and, thereby, provide further evidence for the inflammatory nature of the disease.

25-Hydroxyvitamin D3 Metabolism in vitro by Mononuclear Cells from Hemodialysis Patients

Hemodialysis patients have a capacity for extrarenal production of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]; however, the source of the hormone is unknown in these patients. Since 1,25(OH)2D3 synthesis by cultured hematopoietic cells has been demonstrated previously, we assessed hormone production by mononuclear cells from peripheral blood obtained from normal subjects (n = 6), uremic patients not yet requiring dialysis (n = 4) and hemodialysis patients (n = 14). 1,25(OH)2D3 production was analyzed by sequential straight phase and reverse phase HPLC. In the hemodialysis group, the mean specific production of a metabolite co-eluting with 1,25(OH)2D3 (in fmol/100,000 cells/h) both by monocyte-enriched adherent cells (Mo) and lymphocyte-enriched non-adherent cells (Ly) was increased as compared to non-dialyzed subjects (119 vs. 22 for Mo, not significant, 65 vs. 14 for Ly, p < 0.05). Taken together, Mo and Ly from hemodialysis patients synthesized significantly more 1,25(OH)2D3 (p < 0.02) than non-dialyzed subjects (184 vs. 36, means). No differences were found between cells from normal subjects and patients with preterminal renal failure. Exposure of cultured normal Mo (n = 6) to cuprophane (CU), polyacrylonitrile (AN69) or polycarbonate-polyether (PC) membrane devices resulted in increased 1,25(OH)2D3 production as compared to control incubations without membrane. The rank order of increase was PC > AN69 > CU, whereby only PC (p < 0.05) was significantly different from control. Our results suggest that blood mononuclear cells contribute to extrarenal 1,25(OH)2D3 synthesis in hemodialysis patients, and that this synthetic activity may be related to the hemodialysis procedures.

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in β2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm−2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal α-l-fucopyranoses with α-l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm2 for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm2 was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.