The effects of immunosuppressive agents on the function of human hepatocytes in vitro

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.     

In vitro immunosuppressive effects of cytotoxic agents conjugated to antihuman lymphocyte globulin.

Antihuman lymphocyte globulin (ALG) was either coupled to the lymphocytoxic drug chlorambucil or covalently bound to the cytotoxic alkalating agent melphalan via a polyglutamic acid carrier. Both types of complexes strongly inhibited the proliferative response in human mixed lymphocyte cultures and the ability of mixed lymphocyte culture-activated T effector cells to lyse 51Cr-labelled lymphoblast target cells, and were more potent than ALG or drug alone. These experiments indicate that it is possible to bind cytotoxic agents to ALG without destroying either the properties of the drug or the ability of the antibody to bind to lymphoid cells.     

A comparison of the inhibitory effects of immunosuppressive agents cyclosporine, tetranactin, and didemnin B on human T cell responses in vitro.

The agents cyclosporine, tetranactin (TN), and didemnin B (DB) were compared for their ability to inhibit proliferative human T cell responses in vitro, using anti-CD3, PHA, alloantigen, or tetanus toxoid as stimuli and using monocytes or Langerhans cells as antigen-presenting cells/accessory cells (APC/AC). We found that all three agents suppressed T cell activation in a dose-dependent fashion, irrespective of the stimulus of APC/AC type used. Both T cells and APC/AC were affected by the drugs. DB appeared to be the most potent suppressive drug (IC50 = 1-4 ng/ml), whereas CsA and TN exerted approximately similar potency (IC50 = 50-60 ng/ml). Remarkably however, DB was toxic at a concentration of 10 ng/ml, which is quite close to the inhibition-inducing dose. No toxicity was observed with CsA and TN at doses up to 5000 ng/ml. The agents TN and DB could interrupt ongoing T cell responses and could block responsiveness to exogenous recombinant IL-2. Expression of IL-2 receptors was slightly inhibited by all three drugs. Expression of MHC class II molecule HLA-D and of adhesion molecules LFA-1, LFA-3, and ICAM-1 was clearly reduced by DB, giving an explanation for the observed inhibition of cluster formation between T cells and APC/AC. Except for a slight reduction of LFA-3 by TN, CsA and TN did not affect the expression of any of these cell surface markers or the formation of clusters. Differences in the effects of CsA, TN, and DB on immune responses in vitro and on the phenotype of T cells and APC/AC suggest that these immunosuppressive drugs have different inhibitory mechanisms.     

Influence of immunosuppressive agents on tryptophan degradation and neopterin production in human peripheral blood mononuclear cells

The anti-proliferative and immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan via the kynurenine pathway. IDO is stimulated during cellular immune responses preferentially by Th1-type cytokine interferon-γ (IFN-γ). IDO activity is estimated by calculating the kynurenine to tryptophan ratio (Kyn/Trp). In human monocyte-derived macrophages and dendritic cells, GTP-cyclohydrolase I is induced in parallel to IDO and produces neopterin. This study investigated the effects of common immunosuppressants on freshly isolated human peripheral blood mononuclear cells (PBMC) in vitro. PBMC were incubated with compounds for 30 min and then either left unstimulated or stimulated with mitogen phytohaemagglutinin (PHA). Concentrations of tryptophan, kynurenine and neopterin were measured in supernatants after 48 h. Kyn/Trp, neopterin and IFN-γ concentrations were significantly higher in PHA-stimulated vs. unstimulated PBMC. Tacrolimus (FK506), cyclosporine A (CsA), sirolimus and methylprednisolone dose-dependently inhibited tryptophan degradation and neopterin production. FK506, CsA and sirolimus showed significant inhibition at concentrations as low as 0.1 μg/ml, whereas prednisolone and methylprednisolone required higher doses to suppress tryptophan degradation. Mycophenolate-mofetil suppressed neopterin formation more efficiently than Kyn/Trp. All tested drugs also strongly decreased mitogen-induced IFN-γ concentrations. Overall the investigated immunosuppressants are effective to inhibit IDO activity and neopterin production in a similar and dose-dependent manner, however with some differences in IC50s when comparing individual compounds. The corresponding changes of IFN-γ concentrations are in line with its role as a trigger of both biochemical changes.     

In vitro effects of chemotherapeutic agents on human osteoblast-like cells

Osteopenia is a complicating problem that may occur during and after treatment for childhood malignancy. Clinical studies suggest that chemotherapeutic agents directly affect osteoblasts in vivo. Since combinations of agents are used for treatment, we individually investigated the chemosensitivity of human osteoblast-like cells to 11 of the chemotherapeutic agents used. The relative chemosensitivity of osteoblast-like cells representing different stages of cell differentiation was also examined. Cell numbers were evaluated following culture of an established human osteoblast-like cell line (MG63) for 3 days with clinically relevant concentrations of the chemotherapeutic agents. The chemosensitivity of MG63 cells was compared to that of a human osteoprogenitor cell line (HCC1) and primary osteoblast-like (HOB) cells derived from pediatric bone. Cell numbers were reduced by all agents in all cell types, although there was a varied response between agents at equimolar concentrations. In MG63 cells the lowest concentration of agent significantly reducing cell numbers varied between agents, for example, methotrexate (10(-7) M), vincristine (10(-9) M), and etoposide (10(-7) M) (all P <0.01). The less differentiated osteoblast phenotypes were significantly more chemosensitive at equimolar concentrations of methotrexate, vincristine, asparaginase, and dexamethasone than more differentiated phenotypes (all P <0.01). Furthermore, four agents significantly increased alkaline phosphatase (AP) activity in HOB cells. We conclude that individual chemotherapeutic agents added to osteoblast cell cultures reduce cell numbers, with osteoblast precursor cells being preferentially depleted. These results suggest that most of the agents may contribute to osteopenia in childhood malignancy by direct effects on cell numbers.     

The emerging matrix of immunosuppressive agents

In recent years, significant milestones have been reached in the field of transplantation through the development of immunosuppressive drugs that inhibit lymphocyte activation, cytokine signal transduction, and cellular proliferation. However, the widespread tissue distribution of the molecular targets exploited to date-calcineurin, mammalian target of rapamycin (mTOR), and inosine monophosphate dehydrogenase-produces an array of collateral toxicities. Avoiding these side effects requires new strategies that selectively block destructive immune responses: a fifth generation of immunosuppressants. These agents must target molecules that are critical for and specific to the adaptive immune response.     

Naturally occurring immunosuppressive agents. I. The presence in normal pig liver of a factor possessing immunosuppressive properties with respect to pig lymphoid cells in vitro.

Saline cell-free extracts of normal pig liver, but not normal pig spleen, contain a noncytotoxic factor (or factors) capable of suppressing the blastogenic response of pig lymphocytes to stimulation with a number of plant mitogens: phytohemagglutinin, pokeweed, and concanavalin A. This reaction is generally considered to be a reflection of the capacity of the cell to participate in an immune reaction. Normal pig serum does not display inhibitory activity. The liver extract must be in contact with the lymphocytes for at least the final 48 hr of the 72-hr culture period in order to suppress the blastogenic response. Whether this active constituent in normal pig liver extract is an immunosuppressive agent in vivo remains to be determined.     

Differential effects of modern immunosuppressive agents on the development of intimal hyperplasia

Modern immunosuppressive agents such as tacrolimus and rapamycin are claimed to be associated with a reduction in vascular narrowing, a central feature of chronic rejection. This study assesses the effect of cyclosporine, tacrolimus and rapamycin on the development of intimal thickening, fibrosis-associated genes and deposition of extracellular matrix (ECM) proteins in a model of intimal hyperplasia. Male Sprague-Dawley rats received either no treatment or 5 mg/kg cyclosporine, 0.1 mg/kg tacrolimus or 0.05 mg/kg rapamycin. Animals underwent left common carotid balloon angioplasty, and intima medial ratios, pro-fibrotic gene expression and ECM accumulation were calculated at 14 and 28 days. Cyclosporine was associated with increased intimal thickening compared to controls (P<0.004). Tacrolimus had no effect on intimal thickening, whilst rapamycin significantly inhibited intimal thickening at both 14 and 28 days (P<0.004 and P<0.026, respectively). All groups significantly inhibited matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, transforming growth factor (TGF)- and collagen III expression at 14 days (P<0.001), but increased ECM deposition. However, rapamycin marginally reduced ECM deposition compared to cyclosporine (P<0.06). Treatment with cyclosporine was associated with worsening of vascular narrowing, whilst rapamycin showed a beneficial reduction in intimal thickening. Treatment with all immunosuppressive agents resulted in increased ECM deposition. Rapamycin may halt the progression of vascular narrowing compared to both cyclosporine and tacrolimus.     

The effects of pretreatment with donor antigen and immunosuppressive agents on fully allogenic tracheal graft

BACKGROUND: Obliterative bronchiolitis is a major clinical problem in cases involving a transplanted lung. We examined drug-induced tolerance to a fully allogenic tracheal graft in a murine heterotopic transplantation model. MATERIALS AND METHODS: Recipient mice (C57BL/6) were primed iv with 1 x 10(8) splenocytes of donor mice (BALB/c). Day 0 was the day of the splenocyte injection. Cyclophosphamide and Busulfan were injected intraperitoneally on day 2. On day 3, 1 x 10(7) donor bone marrow cells were intravenously injected. On day 28, a donor tracheal graft was implanted into a subcutaneous pocket. Grafts were harvested at 3-week intervals, and the degree of obstruction of the inner cavity, the condition of epithelium, and the viability of chondrocytes were examined. RESULTS: All of the isograft controls (BALB/c) and grafts implanted in the T cell-free recipients (BALB/c-nu) showed patent, lined epithelium and viable chondrocytes. All allografts tested showed total luminal occlusion by granulative tissue and inflammatory cells, and the epithelium was totally absent. Five of 11 drug-treated grafts were completely patent, although the epithelium was almost absent and the chondrocytes were substantially destroyed. However, when the chimerism was analyzed by flow cytometry analysis of the recipient T cells, approximately 90% of the donor cells were recognized. CONCLUSIONS: Even by this pre-treatment-induced chimerism, a transplanted allogenic trachea was not completely preserved. The present results suggest that a non-allogenic response might have contributed to the rejection.     

Pro- and anti-cancer effects of immunosuppressive agents used in organ transplantation

Development of cancer is a feared, and increasingly apparent, complication of long-term immunosuppressive therapy in transplant recipients. In addition to the need to reduce cancer occurrence in these patients, therapeutic protocols are lacking to simultaneously attack the malignancy and protect the allograft when neoplasms do occur. In this overview, we present the current literature regarding the pro- and anti-neoplastic effects of immunosuppressive agents on cancer growth and development. Recent experimental findings are paving the way for new therapeutic strategies aimed at both protecting an allograft from immunologic rejection and addressing the problem of cancer in this high-risk population.     

The immunosuppressive action of FK506. In vitro induction of allogeneic unresponsiveness in human CTL precursors.

FK506 is an unusually potent new immunosuppressive agent that inhibits T cell-mediated immunity in vivo and in vitro. In these studies we sought to further elucidate the immunosuppressive mechanism of action of FK506 on human allogeneic MLR-induced CTL activation. FK506 induced suppression of cell-mediated lympholysis by PBMC was optimal at 1-2-nM concentrations, if added at the initiation of 6 day CML cultures. The sensitivity to suppression decreased with time, and fully differentiated effectors were resistant to inhibition by FK506. Suppression of CML was not reversed by washing the cultures, adding exogenous IL-2, or restimulating with fresh cells. Pretreatment of unfractionated or adherent allogeneic PBMC with FK506 blocked the stimulating activity of these cells. Furthermore, addition of FK506-treated stimulator cells to cocultures containing untreated responder and stimulator cells resulted in suppression of CML. The inhibition in the cocultures was greatest if the FK506-pretreated cells were autologous to the original stimulator, suggesting a relative specificity in the suppression obtained under these conditions. These studies suggest that, in addition to suppressing the response of alloreactive CTL precursors, FK506 reduces the ability of irradiated allogeneic PBMC to induce CTL generation.     

The effects of clindamycin on human osteoblasts in vitro

Introduction Clindamycin is an antibiotic frequently used in different local application forms for the treatment of prosthetic joint infections, chronic osteomyelitis or as infection prophylaxis in bone cement. No information is available regarding its direct effects on bone cells, although very high local effective antibiotic concentrations can be achieved. Materials and methods We cultured pooled osteoblasts, previously derived from human trabecular bone specimens of four healthy donors, with different concentrations of clindamycin (0–500 μg/ml) for 24, 48 and 72 h. Cell proliferation (MTT), cytotoxicity [lactate dehydrogenase (LDH)-activity], cell metabolism [alkaline phosphatase (ALP)-activity] and extracellular matrix calcification (Alizarin staining) were assessed after antibiotic treatment. Results Proliferation significantly decreased in a dose-dependent manner and reached 3.5% of control samples at 500 μg/ml at 72 h. LDH-activity was unaffected at lower concentrations but significantly increased at 500 μg/ml at 48 and 72 h. ALP-activity significantly increased at 10 μg/ml at 24 and 48 h and then decreased in a time- and dose-dependent manner. Calcification increased at 10 and 25 μg/ml, while it decreased or no calcification was found at concentrations of 50 μg/ml and above. Conclusion We could demonstrate that clindamycin at lower concentrations stimulated the cell metabolism of human osteoblasts and that higher clindamycin levels of 500 μg/ml had cytotoxic effects. The observed effects of high clindamycin levels on human osteoblasts highlight a potential alteration of bone metabolism in vivo and have to be taken into account in local antibiotic administration, e.g., in clindamycin-impregnated bone cement, where such high antibiotic concentrations can be achieved.     

New immunosuppressive agents in transplantation

Immunosuppressive agents have enabled the development of allogenic transplantation during the last 40 years, allowing considerable improvement in graft survival. However, several issues remain such as the nephrotoxicity of calcineurin inhibitors, the cornerstone of immunosuppressive regimens and/or the higher risk of opportunistic infections and cancers. Most immunosuppressive agents target T cell activation and may not be efficient enough to prevent allo-immunization in the long term. Finally, antibody mediated rejection due to donor specific antibodies strongly affects allograft survival.Many drugs have been tested in the last decades, but very few have come to clinical use. The most recent one is CTLA4-Ig (belatacept), a costimulation blockade molecule that targets the second signal of T cell activation and is associated with a better long term kidney function than calcineurin inhibitors, despite an increased risk of acute cellular rejection.The research of new maintenance long-term immunosuppressive agents focuses on costimulation blockade. Agents inhibiting CD40-CD40 ligand interaction may enable a good control of both T cells and B cells responses. Anti-CD28 antibodies may promote regulatory T cells. Agents targeting this costimulation pathways are currently evaluated in clinical trials.Immunosuppressive agents for ABMR treatment are scarce since anti-CD20 agent rituximab and proteasome inhibitor bortezomib have failed to demonstrate an interest in ABMR. New drugs focusing on antibodies removal (imlifidase), B cell and plasmablasts (anti-IL-6/IL-6R, anti-CD38…) and complement inhibition are in the pipeline, with the challenge of their evaluation in such a heterogeneous pathology.     

The effects of intravenous anaesthetic agents on human neutrophil chemiluminescence

Intact neutrophil function is essential for the defence against infection. Any alteration in neutrophil function, which decreases their ability to phagocytose and kill bacteria, might contribute to mortality and morbidity. We investigated the effects of clinical concentrations of thiopentone, Alfathesin, methohexitone, morphine, lidocaine and diazepam on the microbicidal oxidative function of human neutrophils. The oxidative activity was assessed utilizing the technique of chemiluminescence, which is a measure of free radical generation. Thiopentone and Alfathesin produced a significant dose dependent depression in chemiluminescence. There was a 27 per cent reduction in activity with thiopentone 5 micrograms X ml-1, a concentration equivalent to the free plasma concentration achieved following an anaesthetizing dose of thiopentone. There was a 55 per cent reduction in chemiluminescence at an alphaxolone concentration of 1.25 micrograms X ml-1, a concentration equivalent to the free plasma level obtained after induction of Alfathesin anaesthesia. The effect of thiopentone and Alfathesin was reversed by cell washing. Methohexitone, morphine, diazepam, and lidocaine caused no significant reduction in chemiluminescence over the dose ranges studied. These observations indicate that thiopentone and Alfathesin can adversely affect leucocyte function in vitro and, therefore, may contribute to impaired host resistance in the perioperative period and in the intensive care unit.