Augmentation of Transient Donor Cell Chimerism and Alloantigen-Specific Regulation of Lung Transplants in Miniature Swine.

Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major histocompatibility complex-mismatched single lung transplantation was performed in 28 minipigs followed by a 28-day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low-dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4(+) CD25(high+) T cell frequencies were detected in peripheral blood associated with decreased interferon-γ production of leukocytes. Secondary third-party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third-party splenocytes or donor splenocyte protein extracts. While animals treated with third-party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third-party, splenocyte infusions may develop long-term donor-specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.

Novel genetic tools to tackle c-di-GMP-dependent signalling in Pseudomonas aeruginosa.

AIMS: To develop new genetic tools for studying 3',5'-cyclic diguanylic acid (c-di-GMP) signalling in Pseudomonas aeruginosa. METHODS AND RESULTS: Plasmid pPcdrA::lux, carrying a transcriptional fusion between the c-di-GMP responsive promoter PcdrA and the luxCDABE reporter genes, has been generated and validated in purpose-built P. aeruginosa strains in which c-di-GMP levels can be increased or reduced upon arabinose-dependent induction of c-di-GMP synthetizing or degrading enzymes. CONCLUSIONS: The reporter systems described so far were able to detect a decrease in the c-di-GMP levels only in engineered strains overproducing c-di-GMP. Conversely, pPcdrA::lux could be used for studying any process or chemical compound expected to cause both an increase or a decrease with respect to the c-di-GMP levels produced by wild type P. aeruginosa. Another relevant aspect of this study has been the development of novel and improved genetic devices for the fine arabinose-dependent control of c-di-GMP levels in P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic tools developed and validated in this study could facilitate investigations tackling the c-di-GMP signalling process on different fields, from cellular physiology to drug-discovery research.

Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection.

Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY(+)) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY(+) intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY(+) intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.

Pharmacokinetics of ertapenem in colorectal tissue.

BACKGROUND: There are only limited data on tissue kinetics of ertapenem in colorectal tissue more than 3 h after administration of the drug. The purpose of this study was to assess the pharmacokinetics (PK) of ertapenem in colorectal tissue via population PK modeling. PATIENTS AND METHODS: Patients ≥18 years requiring surgical intervention at the colon and/or rectum were eligible (ClinicalTrials.gov identifier: NCT 00535652). Tissue and blood samples were taken during surgery after a single dose of 1 g ertapenem. Ertapenem concentration was determined by high-performance liquid chromatography/mass spectrometry. Population PK modeling was performed in S-ADAPT. RESULTS: Twenty-three patients were enrolled. The highest tissue concentration was 6.4 ± 2.3 mg/kg, the highest total plasma concentration 51.34 ± 9.4 mg/l, the highest unbound plasma concentration 7.05 ± 1.1 mg/l, and the unbound fraction in plasma was 14-15% for total ertapenem concentrations below approximately 22 mg/l, 19% at 100 mg/l, and 25% at 250 mg/l. The estimated geometric mean terminal half-life was 2.5 h for plasma and tissue. In the Monte Carlo simulation, a single dose of 1,000 mg ertapenem achieved robust (≥90%) probabilities of target attainment up to a minimum inhibitory concentration (MIC) of approximately 2 mg/l for the bacteriostasis target (free time above MIC, fT(>)(MIC) = 20%) and up to 0.25-0.5 mg/l for the near-maximal killing target (40% fT(>)(MIC)). CONCLUSION: Our data indicate an adequate penetration of ertapenem into uninfected colorectal tissue up to 8.5 h (35% of the dosing interval) after administration of 1 g intravenously.

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes.

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.

T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-alpha.

Activation and translocation of protein kinases C is a key event in the regulation of T lymphocyte activation, proliferation and function. Stimulation of human peripheral blood lymphocytes with the monoclonal antibody BMA 031 raised against the T cell antigen receptor led to a bimodal activation of protein kinases C. The immediate activation and translocation of the protein kinase C isoform PKC-alpha was followed by activation and translocation of the protein kinase C-beta isoenzyme after 90 min of stimulation. Pretreatment of the cells with cholera toxin for 90 min completely abolished activation of protein kinase C-alpha. In sharp contrast, activation and translocation of protein kinase C-beta was not influenced by the bacterial toxin, suggesting that activation and translocation of different protein kinase C isoenzymes are regulated by distinct mechanisms of transmembrane signalling coupled to the T cell antigen receptor/CD3 complex. The expression of high affinity IL-2 receptors was completely inhibited by cholera toxin, while IL-2 synthesis and secretion were not influenced in BMA 031-stimulated human lymphocytes. Extensive control experiments have shown that the effects of cholera toxin were not mediated by its B subunit, and were independent of elevation of intracellular cAMP concentration, suggesting that cholera toxin interfered with a signalling pathway leading to activation of protein kinase C-alpha, which could be responsible for the inhibition of IL-2 receptor expression. This hypothesis was substantiated by the finding that upon introduction of antibodies against protein kinase C-alpha, IL-2 receptor gene expression was completely suppressed. The results suggest, that protein kinase C-alpha might be the major protein kinase C isoenzyme of a signal transduction cascade regulating IL-2 receptor expression in stimulated human lymphocytes.

Identification of two different lysophosphatidylcholine:acyl-CoA acyltransferases (LAT) in pig spleen with putative distinct topological localization.

The lysophosphatidylcholine:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) is an integral membrane protein participating in the membrane turnover and the T-cell activation process. Here, we present data that crude membranes of pig spleen contain two different LAT enzyme activities based on topological localization studies and the enzyme specificities towards various acyl-CoAs. When crude membranes are washed with solutions of high ionic strength the supernatant contains a distinct LAT activity that we refer to as peripheral LAT (pLAT). The majority of LAT activity is found in the membrane pellet also after treatment with CHAPS. The CHAPS-insoluble LAT activity is named integral LAT (iLAT) accordingly. While pLAT prefers arachidonoyl-CoA rather than oleoyl-CoA, iLAT shows no specificity towards both unsaturated acyl-CoAs. Further investigations reveal that the CHAPS-insoluble LAT activity in the membranes can be solubilized by n-octyl glucoside and restored to original activity by reconstitution with artificial membranes. The reconstituted iLAT prefers arachidonoyl-CoA rather than oleoyl-CoA. Despite a great deal of effort by several groups little progress has been made so far in LAT purification because of the enzyme instability. We establish experimental conditions that enhance the stability of both enzyme activities and, therefore, allow further protein purification.

A novel metabolic pathway for leukotriene B4 in different cell types: primary reduction of a double bond.

Addition of leukotriene B4 together with trace amounts of tritiated leukotriene B4 to different cell types, such as bone marrow-derived macrophages, T-lymphocytes, mesangial cells or fibroblast tumor cells, led to the formation of several hitherto unknown degradation products within hours. None of them could be identified as 20-hydroxy- or 20-carboxyleukotriene B4, known to be produced by polymorphonuclear leukocytes. The primarily formed transient leukotriene B4 metabolite was less polar than leukotriene B4 and was detectable by measuring its ultraviolet absorbance at 232 nm or its radioactivity. Mass spectral analysis showed very similar fragmentation spectra of leukotriene B4 and its primary metabolite. The most abundant ion and the main fragments of the new metabolite were increased by two mass units compared to leukotriene B4. These observations suggest that, in a variety of cells, leukotriene B4 is first reduced to a 5,12-dihydroxyeicosatrienoic acid, which is further converted to secondary hydrophilic degradation products. This raises the question of the major route of leukotriene B4 metabolism in vivo.

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937.

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE2 and TxB2 secretion was induced 50-100-fold, and both PGF2 alpha and PGI2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5-10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB4 nor cysteinyl-leukotrienes were detectable in the supernatants of either undifferentiated or TPA-differentiated U937 cells.

Virus-transformed macrophage-like cell line as tool for eicosanoid research.

The ability of a virus-transformed murine macrophage like cell line HA 38 to produce different eicosanoid metabolites was examined. HA 38 cells release similar amounts of prostaglandins and leukotrienes as did murine peritoneal macrophages in response to both physiological and non-physiological stimuli. Enzyme systems known to be involved in the regulation of eicosanoid synthesis are expressed. HA 38 cells thus are a well defined macrophage model system and are well suited to study eicosanoid synthesis in macrophages and effects of drugs on the prostaglandin and leukotriene synthesis pathways.

Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP-1.

The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.

Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation.

We investigated regulation of macrophage prostaglandin production during activation by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). An in vitro model was established using the mouse macrophage-like cell line RAW 264.7. Cells were cultivated in the presence of IFN-gamma and LPS for up to 48 h and changes in the secretion of nitric oxide (NO.) and tumor necrosis factor alpha (TNF-alpha) were observed as activation markers. Under these conditions a prompt and strong increase in PGE2 production was found in the first 8 h followed by nearly constant generation of PGE2 during the next 40 h. In contrast, the activity of prostaglandin endoperoxide synthase (PGHS), measured as PGE2 production of microsomal protein fractions, was also increased, but reached a clear maximum at 24 h. Recently a second form of PGHS was cloned (PGHS-2) and specific antibodies and mRNA probes for both isoforms are available. PGHS-2 enzyme was expressed maximally after 24 h of activation whereas PGHS-1 was not influenced. In the presence of IFN-gamma and LPS, PGHS-2 mRNA expression reached a maximum at 8 h but PGHS-1 mRNA was not induced during the whole time period. These data indicate that changes in PG synthesis following macrophage activation are due to regulation of PGHS-2 expression.

Viruses transfer the antiviral second messenger cGAMP between cells.

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.

Biological properties of dihydro-leukotriene B4, an alternative leukotriene B4 metabolite.

Dihydro-leukotriene B4 (a 5,12-dihydroxy-eicosatrienoic acid) has been shown to be the primary metabolite of leukotriene B4 (LTB4) in a variety of cells other than human polymorphonuclear leukocytes (PMNLs). In this report we show that dihydro-LTB4 is significantly less active than LTB4 in different biological assay systems, i.e. leukocyte chemotaxis, chemokinesis, aggregation, adhesion to endothelium and superoxide anion production. This suggests that primary reduction constitutes a second so far unknown deactivation pathway for LTB4.

Differentiation-associated expression of prostaglandin G/H synthase in monocytic cells.

Significant progress in the investigation of the regulation of prostanoid formation has recently been made by cloning a second gene coding for prostaglandin G/H synthase (PGHS; EC 1.14.99.1). In this study we examined the expression of the two PGHS isoforms during phorbol ester induced monocytic differentiation of human myeloid leukemia cells (U937). Murine and ovine PGHS-1 probes hybridized to 2.8- and 5.5-kb mRNA species, whereas the murine PGHS-2 probe hybridized to a 5.3-kb species. Western blot analysis using antisera to mouse PGHS-1 and to a synthetic peptide derived from a mouse PGHS-2-specific region revealed a band of 70 kDa for PGHS-1 and a doublet of about 85 kDa for PGHS-2. Unlike PGHS-2, which was not expressed in U937 control cells, both PGHS-1 protein and mRNA were detected in untreated U937 cells. TPA strongly induced PGHS-2 protein and also increased the amount of PGHS-1 protein. Correspondingly, a marked induction of PGHS-2 mRNA was found, but virtually no change in the expression of the PGHS-1 2.8-kb mRNA occurred. The induction of both PGHS isoforms turned out to be dexamethasone-sensitive. The suppression of PGHS-2 induction was more pronounced. These results suggest that both PGHS-1 and to a larger extent PGHS-2 contribute to the upregulation of prostanoid synthesis during monocytic differentiation.

GCAP-II: isolation and characterization of the circulating form of human uroguanylin.

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP-II). GCAP-II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C-terminal amino acids are identical to uroguanylin from human urine. native and synthetic GCAP-II activate GC-C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP-II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP-II specific antibodies were used to localize the peptide by immunohistochemistry in entero-endocrine cells of the colonic mucosa.

Immunomodulatory effects of the alkaloid sinomenine in the high responder ACI-to-Lewis cardiac allograft model.

Extracts of the plant Sinomenium acutum have been used safely since ancient times in Chinese medicine for treatment of rheumatic diseases, and the purified alkaloid, sinomenine, was recently shown to have anti-inflammatory and antirheumatic effects. This study describes the effects of sinomenine in the high responder ACI-->Lewis cardiac transplant model in which allograft rejection occurred at 5 days posttransplant. Treatment with sinomenine (15-30 mg/kg/day i.p.) or a subtherapeutic dose of cyclosporine (CsA, 1.5 mg/kg/day, i.m.) prolonged allograft survival only marginally (mean survival of 5.4 and 7.8 days, respectively). In contrast, the combination of sinomenine and CsA had a statistically significant synergistic effect, with a mean survival of 42.2 days (P < 0.001). Allografts harvested at day 5 from recipients treated with either sinomenine or CsA showed dense mononuclear cell infiltrates with widespread subepicardial infarcts, edema, and microvascular platelet and fibrin deposition. Immunohistologic analysis showed that intragraft leukocytes consisted of >75% macrophages with approximately 10-20% T cells and <5% B or NK cells. Mononuclear cell activation was shown by expression of IL-2R (CD25, 10-20%) and labeling for IL-2 (approximately 10%), and IFN-gamma (10-20%), as well as TNF-alpha (>50%) and iNOS (>50%), but only low levels of IL-4 or IL-10 (<5%). Intragraft endothelial cells were activated, as shown by upregulation of MHC class II antigen and ICAM-1 (CD54) compared with only basal levels in normal donors hearts. Combined sinomenine/CsA therapy significantly enhanced graft morphology, resulting in only mild mononuclear cell infiltration, and an absence of infarcts, platelets, or fibrin deposition. Though residual intragraft mononuclear cells at day 5, as in control grafts, consisted primarily of macrophages plus small numbers of IL-2R+ T cells, these cells lacked expression of IL-2, had only low levels of IFN-gamma, but showed dense labeling for IL-4 and IL-10. In addition, TNF-alpha and iNOS were reduced to basal levels and no endothelial cell activation was observed, despite high titers of endothelium-bound IgM, IgG, and C3. Mitogen-induced in vitro proliferation of rat thymocytes was also more effectively decreased by the sinomenine/CsA combination than by either agent alone. These studies demonstrate the therapeutic value of sinomenine in transplantation, and indicate that this agent has novel and interesting antimacrophage, T cell, and endothelial effects that warrant further evaluation.

Cyclosporin A suppresses proliferation of renal mesangial cells in culture.

Rat glomerular mesangial cells were isolated and grown in culture for a prolonged period of time. The proliferation of these cells was suppressed by the immunosuppressivum Cyclosporin A (CSA) up to 75%, as measured by the incorporation of radiolabeled thymidine. This was dependent on the concentration of CSA used, being in the range of 125-2000 ng/ml. This effect was specific for CSA and other immunosuppressive derivatives thereof, while a non-immunosuppressive cyclosporin was ineffective. Neither the density of the cultures, nor the time of application had any influence on the susceptibility of the cells to CSA. The suppression of MC proliferation was proliferation was reversible after removal of CSA. These studies demonstrate a suppressive effect of Cyclosporin A on the proliferation of non-lymphocytic cells, the glomerular mesangial cells. This observation may help to explain the beneficial results reported with CSA in the treatment of some kidney diseases.

The ratio of macrophage prostaglandin and leukotriene synthesis is determined by the intracellular free calcium level.

The induction of eicosanoid synthesis in various cell types by different physiological stimuli is dependent on an increase in the intracellular calcium level and stimulation of the protein kinase C (PKC). In a model system this can be mimicked by using calcium ionophores and direct PKC activators. In mouse peritoneal macrophages calcium ionophores induced the formation of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4). A synergistic enhancement of both eicosanoids could be achieved by simultaneous addition of the calcium ionophore A23187 together with a suboptimal dose of the direct protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Low concentrations of the ionophore, resulting in only marginally increased intracellular calcium levels, led to a more than additive prostaglandin E2 production in combination with TPA. Higher concentrations of A23187 together with TPA favoured LTC4 synthesis, whereas PGE2 levels at the same time were even diminished. This observed shift from prostaglandin to leukotriene formation was amplified by simultaneous addition of indomethacin. Manganese as inhibitor of the A23187-induced calcium influx decreased PGE2 synthesis. On the other hand, in the presence of manganese LTC4 production was also inhibited at high concentrations of A23187 but elevated in the absence or at low doses of A23187. Our data provide evidence that in macrophages the ratio of cyclooxygenase and lipoxygenase products caused by mediators, acting via the phospholipase C or D/PKC signal transduction pathway, is regulated by the extent of the intracellular calcium increase.