Adherent human mesangial cells (HMC) were unable to phagocytose serum-treated zymosan (STZ), nevertheless this stimulus (1 mg/ml) induced a marked immediate increase of H2O2 and O2- release at a rate of 3.15 +/- 0.35 and 3.40 +/- 0.12 nmol/10(6) HMC/hr, respectively. Zymosan alone resulted in no release of either H2O2 or O2-. Phorbol myristate acetate (PMA, 2 X 10(-6) M) had only marginal effects on HMC leading to the generation of 0.273 +/- 0.014 nmol O2-/10(6) HMC/hr. After a lag period, human recombinant tumor necrosis factor-alpha (TNF-alpha) and human recombinant interleukin 1-alpha IL-1 alpha) both induced significant O2- production measured as SOD inhibitable reduction of cytochrome c, 5 X 10(-5) M, by adherent HMC for up to five hours, the maximum rates being 3.04 +/- 0.08 and 3.2 +/- 0.08 nmol/10(6) HMC/hr for IL-1 alpha and TNF-alpha, respectively. Significant O2- release was detectable at 0.625 ng/ml (37 pM) IL-1 alpha or 1 ng/ml (59 pM) TNF-alpha (P less than 0.05). Catalase inhibitable H2O2 production was also induced by IL-1 alpha and TNF-alpha in a dose dependent manner. Using scopoletin (40 nM) and 1 microM peroxidase we fluorimetrically measured 1.73 +/- 0.14 and 1.49 +/- 0.19 nmol H2O2/10(6) HMC/hr induced by IL-1 alpha (25 ng/ml) and TNF-alpha (20 ng/ml). Finally, we ascertained the type of radical species produced by HMC stimulated by cytokines employing ESR-spin-trapping with DMPO.2+ These results demonstrated that O2- was the primary radical species formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemokines are a family of small related proteins that play an important role in the selective recruitment of different leukocyte populations to the sites of inflammation. Human glomerular mesangial cells are potent producers of a variety of chemokines. Here we examined the kinetics of mesangial cell chemokine expression with focus on the C-C or beta chemokines monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1 alpha (MIP-1 alpha), and the C-X-C or alpha chemokine interleukin-8 (IL-8) in response to lymphocyte- or monocyte-derived cytokines and mesangial cell growth factors. It was found that interferon-gamma (IFN-gamma), a cytokine produced by TH1 lymphocytes, synergized with tumor necrosis factor-alpha (TNF-alpha) in RANTES expression and with IL-1 beta in MCP-1 synthesis. Time course studies revealed an early peak of mRNA expression of monocyte-specific MCP-1 upon activation with TNF-alpha in contrast to T cell-specific RANTES, which reached the highest mRNA level after 18 hours. This sequence of TNF-alpha-induced MCP-1 and RANTES expression was confirmed on the protein level. As another T-lymphocyte specific chemokine, MIP-1 alpha mRNA and protein was expressed only in response to TNF-alpha plus IFN-gamma with kinetics similar to those of RANTES expression. Finally, unlike other mesangial growth factors basic fibroblast growth factor (bFGF) induced MCP-1, RANTES, and IL-8 mRNA expression, suggesting an involvement of autocrine regulation mechanisms in mesangial chemokine expression.

Devices and methods for Integrated Bioprocessing have been developed for production of recombinant proteins with the yeast Pichia pastoris. In doing so cross flow filtration techniques for cell separation and product concentration are connected directly to high instrumented cultivation processes. These are equipped with on‐line measuring techniques for substrates and products, e.g., glycerol, methanol and pyruvate as well as recombinant proteins, e.g., the chemokines 1–8del MCP‐1 and vMIP‐II. Complex automation structures allow for process development at virtual plants which can be used as the basis for establishing and implementing fully automated real processes. Experiments for determination of reaction kinetics, optimization of productivity in high‐cell density cultures and Integrated Bioprocessing are outlined, along with detailed illustration of the realization of the methods at industrial pilot plant scale.

The capacity of intrinsic, glomerular mesangial cells (MC) to cause an autoreactive response of syngeneic lymphocytes in vitro are presented. Initial experiments demonstrated the MHC class II dependent capacity of MC to present exogenous antigen to sensitized lymph node lymphocytes (LN) and to activate naive, allogeneic LN in the absence of a nominal antigen. However, the most striking finding of the present investigation was that mouse MC (C57BL/6 or DBA/2) augmented a significant activation of naive, syngeneic lymphocytes. The extent of the proliferative lymphocyte response was comparable to that observed after stimulation with allogeneic MC. Moreover, during syngeneic coculture substantial amounts of interferon bioactivity were generated. Equipotent concentrations of rm IFN-gamma were sufficient to induce class II MHC expression of mouse MC. In control experiments the macrophage cell line, IC-21 (C57BL/6), or freshly prepared DBA/2 mouse peritoneal macrophages did not elicit a syngeneic LN response. Using MC, which had not been pretreated, the MC-specific LN stimulation occurred after prolonged periods of coculture. The stimulation index (S.I.) was 9.77 after 144 hours compared with LN controls (S.I. = 1). However, a 48 hour pretreatment of MC with either rm IFN-gamma alone or in combination with rh TNF-alpha and/or the continuous presence of rm IL-1 alpha during coculture periods from 72 to 144 hours substantially enhanced the proliferative LN response. Analysis of non-adherent LN by flow cytometry (FACS) after 96 or 120 hours coculture with MC revealed an increased ratio of Thy1.2+ to B220+ cells with a predominant rise of L3T4+ T-helper cells compared to Lyt2+ cytotoxic T-cells. Furthermore, immune fluorescence microscopy showed that a fraction of Thy1.2+ lymphoblasts adhered to MC. FACS analysis of these adherent LN after detachment demonstrated that in comparison to cocultures with untreated MC, cocultures of LN with IFN-gamma/TNF-alpha pre-treated MC resulted in a 24.4% increase of Thy1.2+ cells, with 89% of these being L3T4+ T-helper lymphocytes. In conclusion, autoreactivity of preferentially T-helper cells to cocultured glomerular MC was shown, which may represent a useful model of T-lymphocyte dependent glomerulonephritis.

Out of the 29 cyclosporin (Cs) metabolites defined so far, 10 representative ones were isolated from bile of liver grafted patients, purified by HPLC, and their structure specified by FAB-MS and 1H NMR. These were used to determine the growth inhibitory effects on Sprague Dawley rat glomerular mesangial cells (MC). Metabolite dilutions were added to cultured MC for 72 hours and [3H]-thymidine incorporation was measured. A 50% growth inhibition by single metabolites (M) on MC was achieved at the following concentrations (mg/liter): Cs: 1.25; M21: 6.0; M18: 9.0; M26: 10.5; M1: 10.8; M8: 10.8; M17: 12.5; M13: greater than 20.0; M25: greater than 25.0; M203-218: greater than 50.0; H355: greater than 50.0. The activity was correlated to the degree of metabolization as the group of six "active" compounds included four primary metabolites (hydroxylated or demethylated derivatives of Cs: M21, M18, M1, M17), whereas the four "inactive" compounds exclusively were secondary metabolites (demethylated, hydroxylated and/or oxidized primary metabolites: M13, M25, M203-218, H355). Combinations of active metabolites with or without Cs resulted in an additive antiproliferative effect. Although single metabolite activities are not relevant in vivo, already combinations of three (M1 + M17 + M18) or four metabolites (M17 + M18 + M21 + H355) resulted in a significant growth inhibition at concentrations of the participating metabolites measured in urine of liver transplanted patients. Moreover, significant synergistic activities were determined with combinations including secondary metabolites. A final set of experiments discharged unspecific cytotoxic effects. The inhibition of MC [3H]-thymidine incorporation was completely reversible and moreover, direct mesangiolysis was excluded for both single and combined metabolite actions. Thus, considering rat MC proliferation as an initial kidney cell model system for subsequent, more detailed studies measuring functional parameters, we have demonstrated that activities of single metabolites are related to their chemical structure. More importantly, mimicking to some extent the patients' situation, combinations of metabolites at concentrations occurring in vivo reduced MC proliferation in culture in an at least an additive fashion, suggesting that side effects of Cs treatment might be attributed to combined Cs metabolite actions.