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H H Radeke

Publications and source records attributed to H H Radeke.

At least 19 recordsLinked to original sources
PubMed2006

Upregulation of group IB secreted phospholipase A(2) and its M-type receptor in rat ANTI-THY-1 glomerulonephritis.

Treatment of rat glomerular mesangial cell (GMC) cultures with pancreatic secreted phospholipase A(2) (sPLA(2)-IB) results in an enhanced expression of sPLA(2)-IIA and COX-2, possibly via binding to its specific M-type sPLA(2) receptor. In the current study, we have investigated the expression and regulation of sPLA(2)-IB and its receptor during glomerulonephritis (GN). In vivo we used the well-established rat model of anti-Thy 1.1 GN (anti-Thy 1.1-GN) to study the expression of sPLA(2)-IB and the M-type sPLA(2) receptor by immunohistochemistry. In addition, in vitro we determined the interkeukin (IL)-1beta-regulated mRNA and protein expression in primary rat glomerular mesangial and endothelial cells as well as in rat peripheral blood leukocytes (PBLs). Shortly after induction of anti-Thy 1.1-GN, sPLA(2)-IB expression was markedly upregulated in the kidney at 6-24 h. Within glomeruli, the strongest sPLA(2)-IB protein expression was detected on infiltrated granulocytes and monocytes. However, at the same time, the M-type receptor was also markedly upregulated on resident glomerular cells. In vitro, the most prominent cytokine-stimulated secretion of sPLA(2)-IB was observed in monocytes isolated from rat PBLs. Treating glomerular endothelial cells (GECs) with cytokines elicited only weak sPLA(2)-IB expression, but treatment of these cells with exogenous sPLA(2)-IB resulted in a marked expression of the endogenous sPLA(2)-IB. Mesangial cells did not express sPLA(2)-IB at all. The M-type sPLA(2) receptor protein was markedly upregulated on cytokine-stimulated mesangial and endothelial cells as well as on lymphocytes and granulocytes. During anti-Thy 1.1 rat GN, sPLA(2)-IB and the M-type sPLA(2) receptor are induced as primary downstream genes stimulated by inflammatory cytokines. Subsequently, both sPLA(2)-IB and the M-type sPLA(2) receptor are involved in the autocrine and paracrine amplification of the inflammatory process in different resident and infiltrating cells.

Animals↗
PubMed2005

Interference with MCP-1 gene expression by vector generated triple helix-forming RNA oligonucleotides.

Triple helix-forming oligonucleotides (TFOs) that specifically bind to double-stranded DNA sequences can be rationally designed, while intracellular delivery of single stranded RNA TFOs has not yet been studied in detail. In this report, we demonstrate gene and sequence-specific inhibition of MCP-1 gene expression due to interference of intracellular-generated single-stranded RNA (CU-TFO) with an overlapping SP-1/AP-1 target. Binding of synthetic 19-nucleotide (19-nt) CU-TFO to the MCP-1 promoter duplex was verified by triplex blotting. Furthermore, we confirmed binding of a 1.1-kb fusion transcript containing the 19-nt pyrimidine CU sequence to a plasmid-encoded MCP-1 promoter target duplex at pH 7.0. In tumour necrosis factor-alpha-stimulated HEK cells, CU-TFOs inhibited MCP-1 protein release by 76 +/- 10.2% compared to intracellular-generated control oligonucleotides. Interleukin-8 as a control target gene was not affected by CU-TFO, confirming both highly specific and effective chemokine gene repression by transfectable TFO-shuttle vectors.

Base Sequence↗
PubMed2005

22-ene-25-oxa-vitamin D: a new vitamin D analogue with profound immunosuppressive capacities.

BACKGROUND: The biologic role of 1,25-dihydroxyvitamin D(3), such as anti-inflammatory functions, reduction of cytokine production by T cells and immunoglobulin production by B cells, is well established. However, its clinical use as an immunosuppressive agent is limited because of the hypercalcemic toxicity occurring after systemic application. The purpose of this study was to investigate the immunmodulatory effects of 22-ene-25-oxa-vitamin D (ZK156979), a novel low calcemic vitamin D analogue. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using the Ficoll Hypaque technique, cultured for 24 h and treated with different concentrations of ZK156979 ranging from 10(-5) to 10(-10) mol L(-1) compared with 1,25-dihydroxyvitamin D(3)[10(-5)-10(-10) mol L(-1)] following phytohaemagglutinin (PHA) stimulation. Interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha), interleukin 1 beta (IL-1beta), interleukin 10 (IL-10) and interleukin 4 (IL-4) secretion in supernatants were measured by ELISA. RESULTS: ZK156979 inhibited the PHA-induced Th1-response (IFNgamma and TNFalpha levels) and the macrophage-product IL-1beta in a concentration-dependent manner (10(-10)-10(-5) mol L(-1)) with the efficiency on cytokine expression compared with 1,25-dihydroxyvitamin D(3) being slightly reduced. In contrast, ZK156979 and 1,25-dihydroxyvitamin D(3) both affected the Th2 response, leading to significantly increased IL-10- and IL-4 secretion. CONCLUSIONS: ZK156979 is a member of novel vitamin D analogues revealing prominent immunomodulatory and suppressive characteristics with distinctive inhibition of Th1-cytokines whereas the Th2 compartment is augmented, thus providing a considerable therapeutic potential in T-cell -mediated diseases.

Calcitriol↗
PubMed2002

IFNgamma induces functional chemokine receptor expression in human mesangial cells.

Infiltration of leucocyte populations into sites of inflammation is a common feature in renal diseases. Glomerular mesangial cells are potent producers of a variety of chemokines, leading to specific attraction of distinct types of inflammatory leucocytes into the glomerulus, but so far there is limited knowledge about the responsiveness of mesangial cells to chemokines. We investigated the expression of chemokine receptors and the responsiveness of primary human mesangial cells (HMC) to the chemokines which they produce, namely monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8. We found that mRNAs of the chemokine receptors CCR1, which has been shown before, CCR2 and CXCR2 were induced by T-helper cytokine interferon-gamma (IFNgamma). In IFNgamma-stimulated cells, CCR2 and CXCR2 were detectable by flow cytometry. Following treatment with IFNgamma, HMC responded to MCP-1 and IL-8 with an increase of IL-6 mRNA and protein expression, which was in part blocked by pertussis toxin. Moreover, chemokine stimulation of transfected HMC led to an activation of the immunoregulatory transcription factors NFkappaB and AP-1. Additionally, we found that MCP-1 enhanced the expression of its own mRNA in cells activated to express CCR2, suggesting autocrine feedback mechanisms in MCP-1 regulation. Finally, IFNgamma-activated cells migrated towards an MCP-1 gradient in a chemotaxis assay. These results strengthen the assumption that chemokines are not only involved in the recruitment of immune cells to inflamed tissues, but also seem to play a central role in the autocrine regulation of local tissue cells, leading to proceeding inflammation and possibly contributing to healing by mediating cell growth and migration.

Autocrine Communication↗
PubMed2000

Selective inhibition of monocyte chemoattractant protein-1 gene expression in human embryonal kidney cells by specific triple helix-forming oligonucleotides.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed by a variety of tissue cells in response to inflammatory stimuli, such as IL-1beta, TNF-alpha, and IFN-gamma. A major function of MCP-1 is the recruitment and activation of monocytes and T lymphocytes. Overexpression of MCP-1 has been implicated in a number of diseases, including glomerulonephritis and rheumatoid arthritis, indicating that the modulation of MCP-1 activity and/or expression is a desired therapeutic strategy. In the present study, our aim was to test whether the MCP-1 expression could be inhibited at the transcriptional level using triple helix-forming oligonucleotides (TFOs). We designed a TFO targeted to the SP-1 binding site in the human MCP-1 gene promoter. Gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequence-specific triplex with its dsDNA target with an EC50 of approximately 1.9 x 10(-7) M. The corresponding phosphorothioated oligonucleotide was also effective in this assay with an 8-fold higher EC50 value. Binding of the TFO to the target DNA prevented the binding of rSP-1 and of nuclear proteins in vitro. The TFO could also partially inhibit endogenous MCP-1 gene expression in cultured human embryonic kidney cells. Treatment of TNF-alpha-stimulated human embryonic kidney 293 cells with the TFO inhibited the secretion of MCP-1 in a dose-dependent manner (up to 45% at 5 microM oligonucleotide). The inhibition of MCP secretion was caused at the level of gene transcription, because MCP-1 mRNA levels in oligonucleotide-treated cells were also decreased by approximately 40%.

Binding, Competitive↗
PubMed1999

Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers.

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

Animals↗
PubMed1999

Mycophenolate mofetil inhibits rat and human mesangial cell proliferation by guanosine depletion.

BACKGROUND: Mycophenolate mofetil (MMF) is used for immunosuppression after renal transplantation because it reduces lymphocyte proliferation by inhibiting inosine monophosphate dehydrogenase (IMPDH) in lymphocytes and GTP biosynthesis. In the present study we asked if therapeutic concentrations of MMF might interfere with mesangial cell (MC) proliferation which is involved in inflammatory proliferative glomerular diseases. METHODS: Rat and human MCs were growth-arrested by withdrawal of fetal calf serum (FCS) and stimulated by addition of FCS, platelet-derived growth factor (PDGF) or lysophosphatidic acid (LPA). Different concentrations of MMF (0.019-10 microM) were added concomitantly in the presence or absence of guanosine. MC proliferation was determined by [3H]thymidine incorporation. Cell viability was assessed by trypan blue exclusion. Apoptotic nuclei were stained using the Hoechst dye H33258. Cytosolic free Ca2+ concentrations were determined with the fluorescent calcium chelator fura-2-AM. RESULTS: MMF inhibited mitogen-induced rat MC proliferation with an IC50 of 0.45 +/- 0.13 microM. Human MCs proved to be even more sensitive (IC50 0.19 +/- 0.06 microM). Inhibition of MC proliferation was reversible and not accompanied by cellular necrosis or apoptosis. Addition of guanosine prevented the antiproliferative effect of MMF, indicating that inhibition of IMPDH is responsible for decreased MC proliferation. Early signalling events of GTP-binding-protein-coupled receptors, such as changes in intracellular Ca2+ levels were not affected by MMF. CONCLUSIONS: The results show that MMF has a concentration-dependent antiproliferative effect on cultured MCs in the therapeutic range, which might be a rationale for the use of this drug in the treatment of mesangial proliferative glomerulonephritis.

Animals↗
PubMed1998

IFN-gamma induces the high-affinity Fc receptor I for IgG (CD64) on human glomerular mesangial cells.

The deposition of immune complexes, followed by activation of complement and/or Fc receptors and generation of chemoattractants, is the most common feature of human glomerulonephritis. Recently we have shown that primary cultured human glomerular mesangial cells (HMC), which are normally negative for IgG Fc receptors, can be stimulated to express the low-affinity FcgammaRIII-A receptor isoform. In this study we further demonstrate that activation of HMC through IFN-gamma resulted in the functional expression of the high-affinity Fc receptor for IgG (FcgammaRI, CD64). IFN-gamma-dependent induction of classical FcgammaRIa1 mRNA as well as a2, b2 splice variants were evident after 24 h in proliferating HMC and after 48 h in resting HMC. Transcription of FcgammaRI mRNA was also induced by IL-10 in proliferating HMC, whereas other cytokines such as IL-3, transforming growth factor-beta1 and granulocyte-macrophage colony-stimulating factor were not effective. Cell surface expression of FcgammaRI could be detected by flow cytometric analysis after IFN-gamma stimulation and was accompanied by the augmentation of MHC class II and the up-regulation of intercellular adhesion molecule-1 expression. Triggering of HMC by cross-linking FcgammaRI with F(ab')2 fragments of the anti-CD64 monoclonal antibody 22 led to enhanced synthesis of mRNA for the chemokines IL-8 and monocyte chemoattractant protein-1, indicating that the FcgammaRI of HMC is functionally active. These in vitro data suggest that engagement of both FcgammaRI and FcgammaRIII-A on activated HMC through IgG immune complexes may result in an increased chemoattraction of leukocytes into the glomerulus, contributing to the development of glomerulonephritis.

Cells, Cultured↗
PubMed1997

Lymphocyte-derived cytokines induce sequential expression of monocyte- and T cell-specific chemokines in human mesangial cells.

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.

Autocrine Communication↗
PubMed1996

Mesangial cell DNA synthesis induced by hydrogen peroxide, interleukin-6, and platelet-derived growth factor: effects of indomethacin and dazmegrel.

This study indirectly examined the role of prostanoids (PG) in mediating rat mesangial cell (MC) DNA synthesis induced by hydrogen peroxide (H2O2), interleukin 6 (IL-6), and platelet-derived growth factor (PDGF). MC were exposed to three daily pulses of 10(-6) mol/l H2O2 alone or in combination with IL-6 (5 ng/ml) or PDGF (10 ng/ml). In order to examine (indirectly) the role of PG in mediating changes in MC DNA synthesis, indomethacin (1.5 x 10(-5) mol/l) or the thromboxane A2 synthetase inhibitor Dazmegrel (10(-5) mol/l) was added to the medium and DNA synthesis assessed after 72 h using 3H-thymidine incorporation (3H-TdR). Stimulation of MC by H2O2 alone resulted in an increase in 3H-TdR of 34.7 +/- 5.5% (p < 0.01). H2O2 enhanced the mitogenic effects of IL-6 and PDGF, 3H-TdR increasing by 52 +/- 12.1% (p < 0.01) and 100 +/- 21% (p < 0.001), respectively. Indomethacin suppressed the DNA synthesis induced by H2O2 alone, 3H-TdR decreasing by 33 +/- 12% (p < 0.05). Indomethacin also reduced the mitogenic response to H2O2 plus IL-6 and H2O2 plus PDGF by 91 +/- 17 and 97 +/- 12%, respectively (p < 0.05). Dazmegrel reduced 3H-TdR when MC were exposed to H2O2 alone by 31.8 +/- 16% (p < 0.05) and when combined with IL-6 or PDGF by 80 +/- 26 and 120 +/- 13%, respectively (p < 0.05). These data suggest that the pathways through which H2O2-induced growth of MC is mediated appear, at least in part, to involve PG, particularly thromboxane A2.

Animals↗
PubMed1995

Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor.

For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine growth-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic fibroblast growth factor (bFGF). The basal growth-inducing capacity of supernatants collected from serum-free cultured HMC and concentrated 100-fold above a cut-off size of 10 kd was significantly increased by interleukin (IL)-1 beta, platelet-derived growth factor (PDGF), and LPS up to 15-fold, but not by IL-1 alpha, IL-6, or bFGF. An anti-human bFGF antibody blocked the majority of IL-1 or LPS-induced proliferative effect of supernatants; complete inhibition was achieved by a combination of anti-human bFGF- and anti-human platelet-derived growth factor antibodies. HMCs express different isoforms of bFGF (18, 21.5, and 24 kd) in membrane, cytosolic, and nuclear fractions. All isoforms of bFGF were found in the nuclear fraction of HMC, whether stimulated or not. Immunoblots for bFGF protein of HMC demonstrated that only a approximate to 16 kd bFGF protein was released into HMC supernatants after stimulation with IL-1 beta, platelet-derived growth factor-BB, and LPS. The 18 kd isoform of bFGF accumulated in the membranes but was not released after stimulation with IL-1 alpha, IL-6, and bFGF, suggesting that its release was a prerequisite for autocrine growth stimulation. By means of reverse transcription polymerase chain reaction controlled by Southern blots, bFGF-mRNA expression of HMC was enhanced by IL-1 alpha, IL-1 beta, and LPS. Finally, we were able to show that HMCs are expressing bFGF receptors. In summary, our data demonstrate for the first time that the autocrine proliferative response of HMC to major inflammatory factors may primarily be mediated by bFGF.

Base Sequence↗
PubMed1995

[Activation-dependent expression of Fc gamma-receptors on glomerular mesangial cells].

Resting, non-cycling mesangial cells (MCs) can be induced by IFN gamma to express the Fc gamma-RIIIa and Fc epsilon RI-gamma subunits of the CD16TM receptor complex. After cell surface expression of CD16TM by IFN gamma induction the binding of immune complexes to MCs induces IL-6 secretion. A Fc epsilon RI-gamma gene knockout has recently been described. It is now possible to study the function of Fc gamma RIIIa and other gamma chain associated Fc receptors in the initiation and progression of chronic glomerular inflammation in the context of the intact immune system in vivo.

Animals↗
PubMed1994

Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFc gamma RIII-A) and the associated Fc epsilon RI gamma-chain.

Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.

Amino Acid Sequence↗
PubMed1994

Activation of autoreactive T-lymphocytes by cultured syngeneic glomerular mesangial cells.

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.

Animals↗
PubMed1993

Effects of cyclosporin and FK-506 on glomerular mesangial cells. Evidence for direct inhibition of thromboxane synthase by low cyclosporin concentrations.

The cellular sources or molecular mechanisms responsible for the derangement of vasoactive prostanoid levels during immunosuppressive cyclosporin (CSA) therapy have not been defined. Using cultured rat glomerular mesangial cells (MC), the cytostatic, cytotoxic and prostanoid synthesis modulating effects of CSA and FK-506 have been measured and compared with the immunosuppressive action of these drugs. Both, CSA and FK-506 inhibited proliferation of MC at similar doses (IC50 approximately 1 microgram.ml-1). Lymphoproliferation was suppressed with IC50s of 50 ng.ml-1 and < 1 ng.ml-1, respectively. In contrast, and unlike FK-506, CSA caused mesangiolysis (IC50 = 4.5 micrograms.ml-1) and concentration dependently inhibited the interleukin-1 beta (IL-1 beta) stimulated mesangial cell release of TXB2 at nanomolar doses (IC50 = 50 ng.ml-1). In kinetic experiments (6-48 h), CSA 1 ng.ml-1 partially and 1 microgram.ml-1 completely abolished the IL-1 beta augmented mesangial secretion TXB2 at all the time points tested. Both, low and high doses of CSA reduced PGE2 release by only 20-40% and then not until at least 24 h of incubation. Measuring enzymatic capacity of membrane fractions of MC to generate TXB2 or PGE2 from added arachidonic acid (10(-5) M), CSA (0.1-1000 ng.ml-1) caused a dose dependent reduction in cyclooxygenase (COX)/thromboxane synthase activity up to 76%, while PGE2 synthesis (COX/prostaglandin synthase) was decreased by 34%. Immunoblots with a specific COX-1 antiserum revealed that COX-1 protein expression of MC was not affected by CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The inflammatory function of renal glomerular mesangial cells and their interaction with the cellular immune system.

The autoimmune nature of chronic progredient glomerular diseases has been well established. Like in other chronic inflammatory diseases, the active role of organ-borne cells has become increasingly apparent--both for the inflammatory process and for the initiation and perpetuation of the immune reaction. In most forms of glomerulonephritis, intrinsic glomerular mesangial cells are likely candidates to come into intimate contact with immune cells such as monocytes or lymphocytes. On the basis of cell culture studies we would like to integrate the current knowledge available about the responsiveness of mesangial cells to inflammatory agents and the resulting secretory capacity and, moreover, their possible role in sustaining chronic inflammatory injury and autoimmune reactions through a direct interaction with lymphocytes. Apart from being responsive to physiological stimuli such as angiotensin II, glomerular mesangial cells are predominantly activated by agents related to inflammation. This includes exogenous substances such as the components of gram-negative bacteria and an array of highly potent immunological stimuli like antigen-antibody complexes, activated complement, or various cytokines. The transformation of resting mesangial cells to proliferating cells with an accompanying expansion of their secretory profile and responsiveness is due to mediators like platelet-derived growth factor, transforming growth factor, and others. Numerous low-molecular-weight substances (O2-, H2O2, NO, platelet-activating factor, eicosanoids), proteins (proteinases, matrix components, interleukins 1 and 6, colony-stimulating factors, growth factors), and cell-surface molecules released or expressed by mesangial cells participate in the inflammatory process. Among these products interleukin 1 and/or 6, class II major histocompatibility antigen and integrins also support an interaction with the cellular immune system. It has been well documented that mesangial cells induced in vitro by recombinant T-cell lymphokines, such as interferon-gamma, do express MHC II and ICAM-1 and could function as antigen-presenting cells. However, and perhaps more interestingly, our own recent experiments with cocultures of syngeneic mouse lymphocytes and mesangial cells have demonstrated that T-cells are directly activated by cultured mesangial cells, thus resembling a mesangial cell-specific autoimmune reaction. In parallel to clinical studies searching for a mesangial autoantigen these experiments might help to elucidate the mechanisms of initiation and perpetuation of mesangial cell-dependent autoimmune glomerulonephritis.

Autoimmune Diseases↗
PubMed1992

The synergistic immunosuppressive potential of cyclosporin metabolite combinations.

Out of the 29 cyclosporin (CS) metabolites defined so far seven representatives were isolated from the bile of liver grafted patients, purified by HPLC and characterized by FAB-MS and/or 1H-NMR. These were used to determine the growth inhibitory effects on concanavalin A stimulated rat lymphocytes (LN). Metabolites diluted in culture medium at concentrations re-checked by HPLC at the respective assay time were added and proliferation determined by [3H]-thymidine incorporation after 48 h. A 50% growth inhibition of LN by single metabolites (AM) was achieved at the following concentrations (mg/l): CS: 0.023; primary metabolites AM1: 0.11; AM1c: 0.65; AM9: 1.05; secondary metabolites AM19: 1.02; AM4N9: 1.02; H355: 1.85; AM1A: 4.5. Although all metabolites were immunosuppressive at higher concentrations in vitro on a single metabolite level, only AM1 with 20% of the activity of native CS seemed to play a role in vivo. However, when we tested the antiproliferative effects of double or triple metabolite combinations, we found a strong synergism not only of primary metabolites, but even with combinations including secondary metabolites. The concentration of the participating metabolites necessary to decrease LN growth by 50% was far below the trough levels observed in vivo. Finally, to mimic to some extent the in vivo situation we determined the interaction of native CS with single metabolites or double combinations. In contrast to the clear synergism in the absence of CS the combinations of metabolites with native CS resulted in an additive growth inhibition. These results indicate an immunosuppressive potential of all metabolites tested and a clear synergism of metabolites in the absence of CS. Although up to double metabolite combinations did only additively enhance CS induced immunosuppression, the combination of 29 metabolites occurring in vivo might have significant immunosuppressive effects in situations where CS levels drop below active concentrations.

Bile↗