Cyclosporin H Research Articles

Cyclosporin H Improves the Transduction of CD34+ Cells with an Anti-Sickling Globin Vector, a Possible Therapeutic Approach for Sickle Cell Disease.

Sickle cell disease (SCD) is a debilitating monogenic disease originating from mutations in the hemoglobin beta chain gene producing an abnormal hemoglobin HbS. The polymerization of HbS is responsible for the sickling of erythrocytes leading to anemia and vaso-occlusive events. Gene therapy is a promising treatment of SCD, and two different gene therapy drugs, using gene editing or gene transfer, have already reached the marketing stage. There is still a need to improve the efficacy of gene therapy in SCD, particularly when using anti-sickling beta-globin gene transfer strategies, which must outcompete the pathological HbS. One possibility is to increase transduction by inhibiting lentiviral restriction factors such as interferon-induced transmembrane proteins (IFITMs). This can be achieved by the addition of cyclosporin H (CsH) during the transduction process. This strategy was applied here in CD34+ hematopoietic progenitor and stem cells obtained from cord blood (CB). A first series of experiments with lentiviral vector coding for a green fluorescent protein (GFP) gene confirmed that the addition of CsH enhanced transgene expression levels and vector copy number per cell (VCN), while CD34+ cells remained viable and functional. Notably, the production of colony-forming cells (CFC) remained unaffected unless very high VCN values were reached. In a second step, CD34+ cells obtained from the CB of newborns with homozygous (n = 2) or heterozygous (n = 1) SCD mutations were transduced with the GLOBE-AS3 lentiviral vector coding for the HbAS3 anti-sickling beta globin. As with GFP, GLOBE-AS3 lentiviral transduction was clearly enhanced by CsH, leading to VCN > 2 and therapeutic levels of expression of the HbAS3. Moreover, the process did not affect the viability or functions of CFC. The combination of CB progenitors, the GLOBE-AS3 vector, and CsH is thus shown here to be a promising approach for the treatment of SCD.

Effect of Cyclosporin H on ischemic injury and neutrophil infiltration in cerebral infarct model of rats via PET imaging.

Brain ischemia-reperfusion injury is a complex process, and neuroinflammation is an important secondary contributing pathological event. Neutrophils play major roles in ischemic neuroinflammation. Once activated, neutrophils express formyl peptide receptors (FPRs), which are special receptors of a class of chemoattractants and may be potential targets to regulate the activity of neutrophils and control cerebral ischemic injury. This study was aimed to explore the ameliorating effect of Cyclosporin H (CsH), a potent FPR antagonist, on brain ischemic injury by inhibiting the activation and migration of neutrophils, and improving cerebral blood flow. We employed a middle cerebral artery occlusion (MCAO) Model on rats and performed behavioral, morphological, and microPET imaging assays to investigate the potential restoring efficacy of CsH on cerebral ischemic damages. Peptide N-cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF), an antagonist to the neutrophil FPR with a high binding affinity, was used for imaging neutrophil distribution. We found that CsH had similar effect with edaravone on improving the neurobehavioral deficient symptoms after cerebral ischemia-reperfusion, and treatment with CsH also alleviated ischemic cerebral infarction. Compared with the MCAO Model group, [18F]FDG uptake ratios of the CsH and edaravone treatment groups were significantly higher. The CsH-treated groups also showed significant increases in [18F]FDG uptake at 144h when compared with that of 24h. This result indicates that like edaravone, treatment with both doses of CsH promoted the recovery of blood supply after cerebral ischemic event. Moreover, MCAO-induced cerebral ischemia significantly increased the radiouptake of [68Ga]Ga-cFLFLF at 72h after ischemia-reperfusion operation. Compared with MCAO Model group, radiouptake values of [68Ga]-cFLFLF in both doses of CsH and edaravone groups were all decreased significantly. These results showed that both doses of CsH resulted in a similar therapeutic effect with edaravone on inhibiting neutrophil infiltration in cerebral infarction. Potent FPR antagonist CsH is promisingly beneficial in attenuating neuroinflammation and improving neurobehavioral function against cerebral infarction. Therefore, FPR may become a novel target for regulating neuroinflammation and improving prognosis for ischemic cerebrovascular disorders.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard exvivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Abstract 22: Formyl Peptide Receptor-1 Activation Is Crucial For The Cause Of Spontaneous Hypertension In Dahl Salt Sensitive Rats

Mitochondria evolved from bacteria and use N-formylated peptides (NFPs) to synthetize protein. Bacterial and mitochondrial NFPs activate formyl peptide receptor 1 (FPR-1) and lead to vascular injury. We previously observed that Dahl Salt Sensitive rats (S) fed a low-salt (LS, 0.3% NaCl) diet presented spontaneous hypertension, vascular dysfunction, and overexpression of FPR-1 in arteries when compared to Dahl Salt Resistant (R) rats. High salt (HS, 2% NaCl) diet worsened these phenotypes in S rats. Interestingly, HS diet induced leaky gut and amoxicillin (AMO) treatment decreased BP in S-HS. Due to the dual sources of NFPs (microbiota and host mitochondria), we hypothesized that cell death-derived mitochondria and/or leaky gut-derived bacterial NFPs lead to FPR-1 activation, vascular injury and elevated BP in S rats independent of HS diet. For this, we used flow cytometry to measure cell necrosis and early and late apoptosis in kidney, bone marrow-derived macrophages and mesenteric resistance arteries (MRA) from male S and R rats (8-week old) on a LS diet. Zonulin, a biomarker for leaky gut, was measured in plasma. In another group, rats were treated with FPR-1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, osmotic mini-pump, 14 days], vehicle (VEH) or received water with AMO (5 mg/kg/day) for 21 days to deplete bacteria. BP was measured by telemetry and vascular function and structure were assessed in MRA. S rats presented increased kidney cell necrosis (R: 3.8±0.3 vs. S: 5.3±0.5* %). CsH decreased spontaneous elevation of BP [Diastolic: R+VEH: 77±2.7 vs. R+CsH: 81±1.2 vs. S+VEH: 126±3.0* vs. S+CsH:115±2.7 # ] and vascular hypercontractility [KCl (120mM): R+VEH: 9.4±1 vs. R+CsH: 10.2±0.4; S+VEH: 15.5±0.9* vs. S+CsH:11.7±0.8 # mN; Phenylephrine (10μM): R+VEH: 9.3±1 vs. R+CsH: 9.7±1; S+VEH: 14.5±1*vs. S+CsH: 11.4±0.6 # mN) in S-LS rats. AMO did not change vascular contraction or BP. Leaky gut was not observed in Dahl S-LS diet. In conclusion, FPR-1 can serve as a causative agent for the spontaneous elevation of BP and kidney-derived mitochondria, but not gut-derived microbiota, are the main source for NFPs.

Formyl Peptide Receptor‐1 Activation is Crucial for Spontaneous and Salt‐Induced Hypertension in Dahl Salt Sensitive Rats: Mitochondria vs. Microbiota

AimsMitochondria evolved from bacteria and carry the same hallmarks that are an important source of inflammatogenic damage‐associated molecular patterns. One hallmark is the use of N‐formylated peptides (NFPs) as an initiator of protein synthesis. NFPs activate formyl peptide receptor (FPR‐1) and lead to vascular injury. When we first worked with Dahl Salt Sensitive (S) rats model, we observed that Dahl S fed a low‐salt (LS) diet presented signs of vascular dysfunction and spontaneous elevation of BP compared to Dahl Salt Resistant (R) rats. High‐salt (HS) diet worsened the hypertensive phenotype. Intriguingly, we observed that FPR‐1 is upregulated in arteries, and mitochondrial NFPs are elevated in the circulation of S rats (LS). We hypothesized that cell death‐derived mitochondrial NFPs and FPR‐1 activation leads to vascular dysfunction and the genesis of spontaneous elevation of BP in S rats. Because of the dual source of NFPs (from microbiota and host mitochondria), we also hypothesized that HS diet leads to gut barrier disruption, and a synergistic action of mitochondria and bacterial‐derived leaky gut NFPs are the major driving force in the maintenance of hypertension.MethodsMale S and R rats (6‐week old) were given a LS (0.3% NaCl) or HS diet (2% NaCl). Blood pressure (BP) was measured by telemetry. After 3 weeks on LS or HS diets, rats were treated (osmotic mini‐pump) with FPR‐1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, 14 days]. In another group, rats received water with amoxicillin (AMO, 5 mg/kg/day) for 3 weeks to deplete bacteria. Mesenteric resistance arteries (MRA) were collected to assess vascular function via wire myograph. Blood samples were collected to measure zonulin, a leaky gut biomarker via Elisa. t‐test p<0.05 *vs. R; #vs. absence of treatment; n=8–11 for R and S; n=6 for R and S+AMO; n=5–6 for R and S+CsH.Results and ConclusionS rats (LS) have a spontaneous elevation in BP. Treatment with FPR‐1 antagonist (CsH) decreased this response [BP (mmHg): Diastolic: R: 77±2.7 vs. R+CsH: 81±1.2 vs. S: 126±3.0* vs. S+CsH:115±2.7#]. Also, CsH decreased hypercontractility observed in arteries from S LS when compared to R rats [KCl 120mM (mN): R: 9.4±1.0 vs. R+CsH: 10.2±0.4 vs. S: 15.5±0.9* vs. S+CsH:11.7±0.8#). This suggests that FPR‐1 serves as a causative agent to the spontaneous elevation of BP. AMO did not decrease BP in S‐LS diet [BP (mmHg): Diastolic: R: 77±2.7 vs. R+AMO: 82±2.7 vs. S: 126±3.0* vs. S+CsH: 124±1.3]. In addition, zonulin is not increased in plasma in S‐LS, suggesting that leaky gut is not associated with spontaneous elevation of BP in S‐LS. However, zonulin is increased in plasma during HS diet (severe hypertension) [Zonulin (ng/mL): R‐LS: 4.09±0.1 vs. S‐LS: 4.01±0.3 vs. R‐HS: 4.31±0.1 vs. S‐HS: 6.3±0.2#]. Overall, FPR‐1 activation due to increased levels of mitochondrial NFPs is associated with the genesis of spontaneous elevations of BP in Dahl S rats. High‐salt diet leads to gut barrier disruption and, subsequently, a synergistic action of mitochondria and bacterial‐derived leaky gut NFPs are the major driving force in the maintenance of hypertension.Support or Funding InformationNSF AGEP:1432878, NIH:R00GM118885, AHA:18POST34060003

Mitochondrial peptides cause proinflammatory responses in the alveolar epithelium via FPR-1, MAPKs, and AKT: a potential mechanism involved in acute lung injury.

Acute lung injury (ALI) is characterized by alveolar epithelial damage and uncontrolled pulmonary inflammation. Mitochondrial damage-associated molecular patterns (DAMPs), including mitochondrial peptides [ N-formyl peptides (NFPs)], are released during cell injury and death and induce inflammation by unclear mechanisms. In this study, we have investigated the role of mitochondrial DAMPs (MTDs), especially NFPs, in alveolar epithelial injury and lung inflammation. In murine models of ALI, high levels of mitochondrial NADH dehydrogenase 1 in bronchoalveolar lavage fluid (BALF) were associated with lung injury scores and increased formyl peptide receptor (FPR)-1 expression in the alveolar epithelium. Cyclosporin H (CsH), a specific inhibitor of FPR1, inhibited lung inflammation in the ALI models. Both MTDs and NFPs upon intratracheal challenge caused accumulation of neutrophils into the alveolar space with elevated BALF levels of mouse chemokine KC, interleukin-1β, and nitric oxide and increased pulmonary FPR-1 levels. CsH significantly attenuated MTDs or NFP-induced inflammatory lung injury and activation of MAPK and AKT pathways. FPR1 expression was present in rat primary alveolar epithelial type II cells (AECIIs) and was increased by MTDs. CsH inhibited MTDs or NFP-induced CINC-1/IL-8 release and phosphorylation of p38, JNK, and AKT in rat AECII and human cell line A549. Inhibitors of MAPKs and AKT also suppressed MTD-induced IL-8 release and NF-κB activation. Collectively, our data indicate an important role of the alveolar epithelium in initiating immune responses to MTDs released during ALI. The potential mechanism may involve increase of IL-8 production in MTD-activated AECII through FPR-1 and its downstream MAPKs, AKT, and NF-κB pathways.

Inhibition of the AnxA1/FPR1 autocrine axis reduces MDA-MB-231 breast cancer cell growth and aggressiveness in vitro and in vivo

Breast Cancer (BC) is a highly heterogeneous disease whose most aggressive behavior is displayed by triple-negative breast cancer (TNBC), which lacks an efficient targeted therapy. Despite its controversial role, one of the proteins that having been linked with BC is Annexin A1 (AnxA1), which is a Ca+2 binding protein that acts modulating the immune system, cell membrane organization and vesicular trafficking. In this work we analyzed tissue microarrays of BC samples and observed a higher expression of AnxA1 in TNBCs and in lymph node metastasis. We also observed a positive correlation in primary tumors between expression levels of AnxA1 and its receptor, FPR1. Despite displaying a lesser strength, this correlation also exists in BC lymph node metastasis. In agreement, we have found that AnxA1 was highly expressed and secreted in the TNBC cell line MDA-MB-231 that also expressed high levels of FPR1. Furthermore, we demonstrated, by using the specific FPR1 inhibitor Cyclosporin H (CsH) and the immunosuppressive drug Cyclosporin A (CsA), the existence of an autocrine signaling of AnxA1 through the FPR1. Such signaling, elicited by AnxA1 upon its secretion, increased the aggressiveness and survival of MDA-MB-231 cells. In this manner, we demonstrated that CsA works very efficiently as an FPR1 inhibitor. Finally, by using CsA, we demonstrated that FPR1 inhibition decreased MDA-MB-231 tumor growth and metastasis formation in nude mice. These results indicate that FPR1 inhibition could be a potential intervention strategy to manage TNBCs displaying the characteristics of MDA-MB-231 cells. FPR1 inhibition can be efficiently achieved by CsA.

Abstract P286: Circulating Lipoxin A4 Causes Resolution of Inflammation via Estrogen Receptor Activation, While Mitochondria N-Formyl Peptides Induce Inflammation via Formyl Peptide Receptor in Hypertension

Recently, we observed that mitochondrial components have inflammatogenic proprieties and that their fragments are elevated in the circulation of spontaneously hypertensive rats (SHR). Also, infusion of mitochondrial N-formyl peptides (F-MITs) into rats induces inflammation and vascular dysfunction via formyl peptide receptor (FPR) activation. In contrast, Lipoxin A4 (LXA4), a mediator of resolution of inflammation, also binds FPR. However, LXA4-FPR-dependent effects seem to be opposite from F-MITs. We first hypothesized that LXA4 is decreased in SHR. We collected plasma from male SHR and Wistar Kyoto rats (WKY) (12 weeks old) and LXA4 was extracted via hydrophobic, reverse-phase, silica-based column. Our initial hypothesis was refuted, since LXA4 was increased in SHR (WKY: 0.33±0.05 vs. SHR: 0.79±0.08 ng/ml, p<0.05). As a result, we inhibited LXA4 synthesis in SHR with a 5-lipoxygenase (5-LOX) inhibitor Zileuton (10mg/kg/day i.p.) for 10 days. 5-LOX blockade did not change blood pressure (BP), but did cause a deterioration of vascular function in intrarenal and mesenteric resistance arteries in SHR. On the other hand, the blockade of FPR in SHR for 10 days with Cyclosporin H (CsH) and WRW4 (0.1 mg/kg/day) reduced BP (telemetry: SHR+control: 151±7.2 vs. SHR+CsH+WRW4: 138±0.4 mmHg, p<0.05) and left ventricle mass. FPR blockade also ameliorated acetylcholine-induced relaxation of intrarenal (Emax: SHR+control: 10±5 vs. SHR+CsH+WRW4: 28±6 %, p<0.05) and mesenteric resistance arteries (Emax: SHR+vehicle: 74±10 vs. SHR+CsH+WRW4: 99±0.6 %, p<0.05). From these data, we suggest that LXA4 does not act on FPR to promote resolution of inflammation. We believe that another factor binds FPR (e.g., F-MITs) and leads to vascular damage in SHR. In support of this, we observed that LXA4 phosphorylates estrogen receptor (ER) alpha Ser118 in aortic vascular smooth muscle cells (VSMC) and ER antagonist ICI-182 (100nM), but not FPR antagonist, abolishes these responses. On the other hand, FMITs increased phosphoERK 1/2 in VSMC and FPR antagonists blocked this increase. In conclusion, FMITs and LXA4 have opposite effects in SHR. LXA4 induces resolution of inflammation, possibly via ER activation, and F-MITs lead to damage via FPR activation.

V101L of human formyl peptide receptor 1 (FPR1) increases receptor affinity and augments the antagonism mediated by cyclosporins

Genetic variation plays a major role in drug response variability. CsA (cyclosporin A), a widely used immunosuppressive agent, is a specific antagonist for FPR1 (formyl peptide receptor 1), which is an important G-protein-coupled chemoattractant receptor in the innate immune system. In order to study the variable responses of cyclosporins to different FPR1 mutants, we investigated the distribution of human FPR1 haplotypes among 209 healthy Han Chinese subjects. The haplotype pattern in Han Chinese were characterized on the basis of five SNPs (single nucleotide polymorphisms), including rs5030878 (p.T11I), rs2070745 (p.V101L), rs5030880 (p.R190W), rs1042229 (p.N192K) and rs867228 (p.A346E). Receptor binding affinity of cyclosporins to FPR1 haplotypes was assessed using N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys–FITC in CHO-Gα16 cells stably transfected with cDNAs encoding the top 12 FPR1 haplotypes in the Han Chinese. Variants of FPR1 carrying a single amino acid substitution of leucine for valine at position 101 (p.Leu101) displayed significantly higher pKi values for CsA and CsH (cyclosporin H), indicative of an improved receptor affinity. The polymorphism of FPR1 p.Leu101 also enhanced the inhibitory effects of cyclosporins on fMLF (N-formyl-methionyl-leucyl-phenylalanine)-induced activities, including calcium mobilization, cell chemotaxis and MAPK (mitogen-activated protein kinase) phosphorylation. These results point to a possible complication for clinical use of CsA in patients carrying the p.Leu101 allele of FPR1.

The Neutron Structure of the Formyl Peptide Receptor Antagonist Cyclosporin H (CsH) Unambiguously Determines the Solvent and Hydrogen-Bonding Structure for Crystal Form II

Single-crystal neutron diffraction data were collected at 20 K to a resolution of 1.05 A on a crystal of the inverse formyl peptide receptor agonist cyclosporin H, CsH, (crystal form II, CsH-II) on the Laue diffractometer VIVALDI at the Institut Laue-Langevin (Grenoble). The solvent structure and hydrogen bonding network of CsH-II have been unambiguously determined by single-crystal neutron diffraction; the agreement factor R(F 2) is 13.5% for all 2726 reflections. All hydrogen atom positions, including methyl-group orientations, have been determined by crystallographic refinement. The neutron structure of cyclosporin provides unique and complementary insights into methyl orientation, hydrogen-bonding, and solvent interactions that are not available from X-ray analysis alone. CsH neutron structure showing the 7 waters and trace of the main chain N1---N11. All water hydrogens were determined experimentally. Thermal ellipsoids are plotted at 85% probability. Drawn with ORTEP-III/RASTER (Burnett and Johnson, Report ORNL-6895, 1996; Merritt and Bacon, Methods in Enzymology 277:505, 1997) as implemented in the program suite WinGX (Farrugia, Journal of Applied Crystallography 32(4):837, 1999) and generated by ORTEP-3 for Windows (Farrugia, Journal of Applied Crystallography 30(5):565, 1997). The main chain trace was drawn with RASMOL (Sayle, Glaxo research and development, 1994).

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

The Immunosuppressant Cyclosporin A Antagonizes Human Formyl Peptide Receptor through Inhibition of Cognate Ligand Binding

Cyclosporin A (CsA) is a fungus-derived cyclic undecapeptide with potent immunosuppressive activity. Its analog, cyclosporin H (CsH), lacks immunosuppressive function but can act as an antagonist for the human formyl peptide receptor (FPR). More recent studies have shown that CsA also inhibits fMLF-induced degranulation in differentiated HL-60 promyelocytic leukemia cells. However, it is unclear whether CsA interferes with ligand-receptor interaction, G protein activation, or other downstream signaling events. In this study we used human neutrophils, differentiated HL-60 cells, and rat basophilic leukemia (RBL)-2H3 cells expressing human FPR (RBL-FPR) to identify the action site of CsA. In functional assays, CsA inhibited fMLF-stimulated degranulation, chemotaxis, calcium mobilization, and phosphorylation of the MAPKs ERK 1/2 and the serine/threonine protein kinase Akt. CsA also blocked Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm)-induced functions in RBL-FPR cells. Concentrations for half-maximal inhibition with CsA are generally 6- to 50-fold higher than that of CsH. CsA was compared with another immunosuppressant, ascomycin, relative to the inhibitory effects on FPR-mediated chemotaxis, calcium mobilization, and degranulation. In these experiments, ascomycin produced no inhibitory effects at low micromolar concentrations (1-4 microM), whereas the inhibitory effects of CsA were prominent at comparable concentrations. Finally, CsA dose-dependently inhibited the uptake of fNle-Leu-Phe-Nle-Tyr-Lys-fluoresceine and [3H]fMLF or [125I]WKYMVm binding to FPR. However, CsA and CsH did not show any obvious inhibitory effect on FPR-like 1-mediated cellular functions. These results demonstrate that CsA is a selective antagonist of FPR and that its inhibition of fMLF-stimulated leukocyte activation is at the level of cognate ligand binding.

Differential Control of Glucocorticoid Receptor Hormone-Binding Function by Tetratricopeptide Repeat (TPR) Proteins and the Immunosuppressive Ligand FK506

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.

Cyclosporine A and FK506 induce osteoclast apoptosis in mouse bone marrow cell cultures

Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures. Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFκB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures. CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG. All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity. Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period. CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture. When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased. Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity. The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology. The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.

Suppression of Mitochondrial Permeability Transition Pore and Induction of Lymphoma P388 Cell Death by Cyclosporin A

Suppression of the mitochondrial permeability transition pore (PTP) and induction of lymphoma P388 cell death were studied in the presence of cyclosporin A (CsA) and its derivatives. In experiments with permeabilized P388 cells, CsA and its nonimmunosuppressive derivative N-methyl-Val-4-CsA, but not cyclosporin H (CsH), enhanced Ca2+ accumulation in mitochondria and suppressed PTP opening. Moreover, CsA was able either itself to induce or to enhance a prooxidant-induced P388 programmed cell death. Blebbing and formation of apoptotic bodies were among the observed CsA effects. N-Methyl-Val-4-CsA showed similar effects, but CsH had no effect on P388 cell death. These results show that initial-stage P388 tumour cell death is not related to PTP opening but can be the result of PTP closing with a corresponding increase in the formation of reactive oxygen species.

High Constitutive Activity of the Human Formyl Peptide Receptor

The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent ("basal") Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist, i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Gialpha2beta1gamma2 reconstituted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTPgammaS-sensitive high affinity [3H]FMLP binding. The FPR "antagonist" cyclosporin H (CsH) potently and efficiently reduced basal GTPgammaS binding in Sf9 membranes. Another FPR antagonist, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine did not inhibit basal GTPgammaS binding but blocked the inhibitory effect of CsH on GTPgammaS binding. Na+ reduced basal GTPgammaS binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.

Human synovial mast cells. II. Heterogeneity of the pharmacologic effects of antiinflammatory and immunosuppressive drugs.

To evaluate the in vitro effects of 4 antiinflammatory and 5 immunosuppressive agents on the release of preformed and de novo-synthesized mediators from human synovial mast cells (HSyMC) activated by immunologic and nonimmunologic stimuli. The effects of antiinflammatory and immunosuppressive agents were evaluated on the in vitro release of histamine and tryptase and the de novo synthesis of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) by HSyMC challenged with anti-IgE and substance P. Nimesulide, a sulfonanilide nonsteroidal antiinflammatory drug (NSAID) chemically unrelated to other acidic NSAIDs (such as acetylsalicylic acid [ASA], diclofenac, and piroxicam) inhibited in a concentration-dependent manner the release of preformed (histamine and tryptase) mediators from HSyMC challenged with anti-IgE. In contrast, diclofenac and piroxicam had little or no effect on HSyMC activated by anti-IgE. ASA, diclofenac, piroxicam, and nimesulide caused a concentration-dependent inhibition of IgE-mediated PGD2 release from HSyMC. Nimesulide, but not diclofenac or piroxicam, also inhibited the de novo synthesis of LTC4 by HSyMC challenged with anti-IgE. Nimesulide, diclofenac, and piroxicam had no effect on HSyMC activated by substance P. Cyclosporin A (CSA) inhibited histamine release from HSyMC challenged with anti-IgE, whereas cyclosporin H (CSH) had no effect. FK-506 also inhibited histamine release from HSyMC activated by anti-IgE, whereas rapamycin had no effect. Neither CSA, CSH, FK-506, nor rapamycin inhibited the release of histamine from HSyMC induced by substance P. Methotrexate had no effect on the release of mediators from these cells, whereas adenosine (R-phenylisopropyl adenosine and 5'-N-ethylcarboxamide adenosine) enhanced histamine release from immunologically activated HSyMC in a concentration-dependent manner. Mast cells isolated from human synovia display 4 levels of pharmacologic heterogeneity with regard to 1) the inhibitory effects of 4 antiinflammatory drugs; 2) the capacity of different immunosuppressive drugs to exert antiinflammatory activity; 3) the inhibition of the release of different mediators; and 4) the capacity of antiinflammatory and immunosuppressive drugs to modulate HSyMC activated by different stimuli. This complexity of pharmacologic modulation of HSyMC in vitro might help explain the different activity of the compounds used to treat various pathophysiologic aspects of the inflammatory arthritides.

Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N-formyl-methionyl-leucyl-phenylalanine

Background:Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, whereas cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with its D-isomer. Methods: We compared the effects of CsA and CsH on the release of preformed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C 4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). Results: CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C 4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1β (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (K d) for the CsH-FMLP receptor complex was approximately 9 × 10 -8 mol/L, more than 10-fold lower than that (≅ 1.3 × 10 -6 mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 × 10 -7 mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 × 10 -5 mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the B max (0.22 ± 0.04 nmol/L/5 × 10 6 cells vs 0.09 ± 0.01 nmol/L/5 × 10 6 cells; p < 0.05), without a significant difference in the K d (5.16 ± 1.22 nmol/L vs 6.32 ± 2.42 nmol/L; p = NS). Conclusions: CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors. (J A LLERGY C LIN I MMUNOL 1996;98:152-64.)

Anti-Inflammatory Effect of Cyclosporin A on Human Skin Mast Cells

We have examined the effects of cyclosporin A (CsA) and cyclosporin H (CsH), which bind with different affinity to cyclophilin, to evaluate the role of this protein in the release of preformed (histamine) and de novo synthesized (prostaglandin D2[PGD2]) mediators of inflammatory reactions from human skin mast cells (HSMC). CsA (2.4-800 nM)-inhibited (5-60%) histamine release from HSMC challenged with anti-IgE. CsA exerted little, if any, inhibitory effect on histamine release from HSMC challenged with compound A23187 and substance P, whereas it completely suppressed A23187-induced histamine release from human basophils. Inhibition of histamine release from HSMC challenged with anti-IgE was extremely rapid and was not abolished by washing (three times) the cells before anti-IgE challenge. CsA (2.4-800 nM) markedly inhibited (25-70%) the de novo synthesis of PGD2 from HSMC challenged with anti-IgE. CsH, which has an extremely low affinity for cyclophilin, had no effect on skin mast-cell mediator release. These data suggest that CsA is a potent anti-inflammatory agent acting on HSMC, presumably by interacting with cyclophilin.

Cyclosporin a regulates the expression of HLA-DR on human monocytes by two different mechanisms

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-γ in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-γ, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-γ receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-α also inhibited the IFN-γ-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-α is proposed.