A novel label-free cell-based assay technology using biolayer interferometry.

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the ß2-adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization.

Immunoglobulin M-enriched human intravenous immunoglobulin prevents complement activation in vitro and in vivo in a rat model of acute inflammation.

An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is their ability to block complement activation. The purpose of this study was to compare the complement-inhibitory activity of four IVIG preparations differing in isotype composition. The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany. Their complement inhibitory activity was assessed in vitro by measurement of the blocking of C1q-, C4-, and C3 deposition on solid-phase aggregated rabbit IgG by enzyme-linked immunosorbent assay (ELISA). Complement inhibition in this ELISA was best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not exhibit an inhibitory effect. Control experiments with excess concentrations of C1q as well as with C1q-depleted serum showed that the inhibitory effects of IVIG were not caused by complement activation and thus, consumption, but that C4 and C3 were scavenged by IgM and to a lesser extent by IgG. These results were confirmed in vivo in the rat anti-Thy 1 nephritis model, in which a single dose of 500 mg/kg of IVIgM prevented C3-, C6-, and C5b-9 deposition in the rat glomeruli, whereas the effect of IVIgG was much less pronounced. Reduction of complement deposition was paralleled by a diminished albuminuria, which was completely absent in the IVIgM-treated rats. IVIgM and to a lesser extent IVIgG also prevented rat C3 deposition on cultured rat glomerular mesangial cells in vitro, but did not influence anti-Thy 1 binding. Neither IVIgM nor Pentaglobin nor IVIgG negatively affected in vitro phagocytosis of Escherichia coli (E coli) by human granulocytes. In conclusion, we have shown that IgM enrichment of IVIG preparations enhances their effect to prevent the inflammatory effects of complement activation.

Production of inflammatory mediators and cytokine responsiveness of an SV40-transformed human proximal tubular epithelial cell line.

Proximal tubular epithelial cells (PTEC) play a central role in the physiology of the renal tubulointerstitium. To be able to study the relationship between tubular cells and inflammatory renal diseases the availability of cultured cells is of importance. This study describes an immortalized proximal tubular epithelial cell line which was generated using SV40 DNA. To determine whether the transformation altered the cell line, the transformed cell line was characterized phenotypically using different monoclonal antibodies directed against peptidases, which are characteristic of PTEC, such as adenosine deaminase binding protein (CD26), leucine amino peptidase and carboxy peptidase M by immunofluorescent staining and FACS analysis. All peptidases were clearly present on the parental cell line and the transformed cell line. However, the level of expression of the peptidases was lower on the transformed cell line as compared to the parental nontransfected cells. The morphology of the transformed cell line, determined using a transwell culture system and electron microscopy, showed a polarized morphology of the tubular cells, tight junctions and microvilli. The transformed cell line was compared with the parental proximal tubular epithelial cells in its ability to respond to inflammatory cytokines such as IL- 1alpha TNF-alpha, IFN-gamma. Stimulation with these cytokines resulted in enhanced production of complement components C2, C3, C4 and factor H, IL-6 and the chemokines IL-8 and MCP-1. The transformed cell line responded in a similar fashion as the parental cell line, although the amount of the different proteins produced was significantly higher in the transformed cell line. Overall, the transformed tubular cell line seems to be a suitable model to study different effects on tubular cells in relation to inflammatory kidney diseases.

Transforming growth factor-beta 1 regulates chemokine and complement production by human proximal tubular epithelial cells.

Previously it has been demonstrated that human proximal tubular epithelial cells (PTEC) are able to produce chemokines (such as IL-8 and MCP-1) and complement components (such as C2, C3, C4 and factor H), and that production of these proteins is regulated by pro-inflammatory cytokines such as interleukin-1 alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Since TGF-beta is also expressed in the renal interstitium during inflammation, we investigated the effect of TGF-beta on the production of chemokines and complement components by PTEC in culture. Transforming growth factor-beta 1 up-regulated IL-8 production by an average of 4.17 +/- 1.0 fold. macrophage chemoattractant phagocyte (MCP-1) production, on the other hand, was down-regulated by TGF-beta 1 by an average of 2.2 +/- 0.7 fold. The production of C3 and C4 was also down-regulated after incubation with TGF-beta 1 (1.9 +/- 0.3- and 3.0 +/- 1.2-fold, respectively). All effects were dose- and time-dependent and were found to be specific for TGF-beta 1, as assessed by inhibition of the effect with a neutralizing antibody against TGF-beta 1. These data, together with the knowledge that TGF-beta, chemokines and complement components play a role in several types of renal disease, suggest that TGF-beta is involved in the regulation of local expression of chemokines and complement components by tubular cells.

Expression of the IL-2 receptor on human renal proximal tubular epithelial cells.

The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the alpha, beta, and gamma chains. Previous studies have shown that human proximal tubular epithelial cells (PTEC) express IL-2R alpha during inflammation, such as in the kidneys after renal transplantation or in crescentic glomerulonephritis. Furthermore, in this study it is shown that PTEC in culture produce more complement C3 after stimulation with IL-2, suggesting the presence of a functional IL-2R on PTEC. In these experiments, the binding of IL-2 to PTEC was analyzed. PTEC stimulated with IL-2 exhibited very low binding of digoxigenin-labeled IL-2; however, stimulation of PTEC with IL-2, in combination with interferon (IFN)-gamma, resulted in increased binding of the digoxigenin-labeled IL-2. In addition, it was found that IL-2, together with IFN-gamma, enhanced the production of C3 by PTEC from baseline levels of 69.6 +/- 3.4 to 198.6 +/- 3.3 ng of C3 per 10(6) cells. The surface expression of IL-2R alpha and IL-2Rbeta was analyzed using monoclonal antibodies. Although unstimulated PTEC hardly showed detectable expression of these chains, stimulation of PTEC with the combination of IL-2 and IFN-gamma resulted in increases of the index of mean fluorescence for the alpha- and beta-chains of 5.5 +/- 0.5 and 9.7 +/- 0.7, respectively. Reverse transcription-PCR analysis revealed mRNA expression not only for the alpha- and beta-chains, but also for the gamma-chain, after stimulation with the combination of IL-2 and IFN-gamma. This study demonstrates that PTEC express the high-affinity IL-2R and that the IL-2R might be involved in the regulation of production of complement component C3 found during renal inflammation.

Interferon-gamma induces biosynthesis of complement components C2, C4 and factor H by human proximal tubular epithelial cells.

In a previous study the authors demonstrated that the production of complement component C4 by human kidney proximal tubular epithelial cells (PTEC) is upregulated by interferon gamma (IFN-gamma). In the present study the authors describe that PTEC in culture express both mRNA and protein of the IFN-gamma receptor complex, and that culture of PTEC with 1000 U/ml IFN-gamma for 72 h results in enhanced production not only of C4 (36.1 ng/10(6) cells), but also of C2 (10.8 ng/10(6) cells) and Factor H (17.5 ng/10(6) cells). Unstimulated PTEC produced 0.5 ng/10(6) cells, 0.5 ng/10(6) cells and 0.4 ng/10(6) cells of C2, C4 and Factor H, respectively. The upregulation of the three complement components was dose- and time-dependent and specific for IFN-gamma because the effect of IFN-gamma was abolished by a monoclonal antibody directed against IFN-gamma. Furthermore no effect of other cytokines was observed. The regulation of synthesis of C2, C4 and Factor H occurred at the transcriptional level as shown by semi-quantitative RT-PCR and dot-blot analysis. Taken together with the observation that in normal kidney tissue the tubuli express IFN-gamma receptor alpha-chain and a signal transducing protein, the present study implies that enhanced production of complement by PTEC may occur during a local immune response by in situ generation of IFN-gamma by infiltrating T-cells in the interstitium of the kidney.

Interleukin-1 alpha enhances the biosynthesis of complement C3 and factor B by human kidney proximal tubular epithelial cells in vitro.

Local production in tubular cells of complement has been shown to occur in several kidney diseases by in situ hybridization, but the regulation at the local site during an inflammation is still unknown. In the present study, we demonstrate that human proximal tubular epithelial cells (PTEC) are able to produce complement components C3 and Factor B under non-stimulated conditions in vitro. The basal production of both was increased by 0.5 ng/ml interleukin-1 alpha (IL-1 alpha) for C3: from 95.5 +/- 4.0 ng/10(6) cells to 416.5 +/- 4.9 ng/10(6), and for Factor B: from 271 +/- 7.0 ng/10(6) cells to 457.5 +/- 7.0 ng/10(6) cells. In contrast cytokines such as TNF-alpha, IFN-gamma, IL-10 and IL-15 had no detectable effect. The upregulation by IL-1 alpha was dose- and time-dependent. The response to IL-1 alpha was shown to be mediated via the IL-1 receptor, as the addition of recombinant interleukin-1 receptor antagonist inhibited the IL-1 alpha induced complement production by more than 80%. IL-1 alpha enhanced mRNA expression of both C3 and Factor B as demonstrated by RT-PCR and dot-blot analysis. This indicated that IL-1 alpha upregulated the expression of the C3 and Factor B at the transcriptional level. We hypothesize that in vivo the production of C3 and Factor B at the local site during an inflammatory response in the kidney may be regulated by IL-1 alpha produced by inflammatory cells.

Regulation and production of IL-8 by human proximal tubular epithelial cells in vitro.

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL-8. In the present study we have analysed the production of IL-8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum-free conditions, were found to produce IL-8 to different degrees from not detectable levels up to 10.8 +/- 1.5 ng IL-8 per 1 x 10(5) cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL-8 of PTEC is 15.1 and 8.1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0.5 ng/ml rIL-1 alpha or 1000 U/ml recombinant tumour necrosis factor-alpha (rTNF-alpha) to the culture media of PTEC induced an up-regulation of IL-8 production up to 6.3-fold and 3.0-fold, respectively. The up-regulation by IL-1 alpha and TNF-alpha was dose- and time-dependent. In contrast, 500 U/ml recombinant interferon-gamma (rIFN-gamma) down-regulated the production of IL-8 3.4-fold. Northern blot analysis showed that IL-1 alpha and TNF-alpha increased the expression of IL-8 mRNA, whereas IFN-gamma reduced IL-8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL-8 in the kidney, and that IL-8 produced in the proximal tubule can be induced by various proinflammatory cytokines.

Production and cytokine-mediated regulation of monocyte chemoattractant protein-1 by human proximal tubular epithelial cells.

Impairment of renal function in various types of glomerular disease is associated with tubulointerstitial changes. The mechanism of mononuclear cell infiltration in the interstitium is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a monocyte-specific chemotactic factor. We analyzed the presence of MCP-1 in renal biopsies from patients with various forms of glomerular disease and demonstrated that MCP-1 expression is increased in renal tubular epithelial cells during disease. Further analysis showed that various cell lines of human proximal tubular epithelial cells (PTEC) produce MCP-1 in culture under serum-free conditions and that the production is inhibited by cycloheximide. IL-1 alpha and TNF-alpha enhanced the production by each cell line in a dose- and time-dependent manner as measured by radioimmunoassay. Northern blot analysis demonstrated that IL-1 alpha and TNF-alpha markedly enhanced the expression of MCP-1 mRNA. Taken together these observations support the notion that MCP-1 is synthesized de novo by PTEC. MCP-1 produced by PTEC is found to be 13 kD by gel filtration chromatography. It is chemotactically active for monocytes. We conclude that in various types of glomerular disease, MCP-1 expression in tubular epithelial cells is associated with up-regulation of MCP-1 production by PTEC. These findings raise the possibility that macrophages may accumulate in renal interstitium as a consequence of MCP-1 production by PTEC.

Differences and similarities in adjuvant chemotherapy in mouse and man.

The effectiveness of adjuvant chemotherapy is easily demonstrated in many mouse models, whereas in man the response is good but far less spectacular. With the aid of a mathematical model the following variables in which differences may exist between mouse and man were tested: 1. Uniformity of tumour load in population 2. Cell kinetic factors in drug sensitivity 3. Variation of drug sensitivity among tumours 4. Drug penetration into tumour 5. Tumour antigenicity 6. Variation in growth rate among tumours It is concluded that the factors 1 and 3 are responsible for the difference. Mouse models are more sensitive by selection of an effective drug and an optimal tumour load. Other factors seem not to affect the results.

Adjuvant chemotherapy: theoretical considerations and model studies.

Comparison of response of experimental tumors to adjuvant chemotherapy for minimal disease with the response of solid large tumors to similar drug regimens has indicated that for many tumors the small microscopic foci are more responsive. There are, however, a number of model tumors that respond far less to chemotherapy when applied to minimal disease than when applied to large tumors. The response of these tumors to adjuvant treatment shows similarities with the clinically observed response. The data do not support the hypothesis that the success of a drug combination given as adjuvant chemotherapy may be predicted from the success of the same treatment when applied for manifest disease.