In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin.

The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.     

Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.     

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain¹. The discovery in 2004 of mutations in LRRK2 that cause Parkinson''s disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state^2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities^4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations^7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool^9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson''s is open to debate.A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads^4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK2¹². Recently, Daniëls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available¹³. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP^14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Use of liposomes in probing the uptake of liposomal phosphatidylcholine by rabbit lung in vitro.

The purpose of this study was to characterize the uptake of liposomal phosphatidylcholine by lung tissue and its subcellular organelles. Multilamellar liposomes were prepared from egg yolk phosphatidylcholine, dicetyl phosphate, and cholesterol (molar ratio 7 : 2 : 1). Liposomal phosphatidylcholine labeled with [1-14C]dipalmitoyl phosphatidylcholine was taken up by lung slices and incorporated into subcellular organelles including lamellar bodies, mitochondria, and microsomes. In addition, when liposomes were incubated with lamellar bodies, mitochondria, or microsomes, the transfer of liposomal phosphatidylcholine to these subcellular fractions was facilitated by the cytosolic fraction. In tissue slice experiments after 1 h of incubation, about 86% of the total radioactivity absorbed by lung slices and subcellular organelles was recovered in phosphatidylcholine. The ratio of the radioactivity of fatty acids at 1- and 2-positions of dipalmitoyl phosphatidylcholine recovered from all fractions was nearly 1 : 1. This suggests that most phosphatidylcholine molecules were taken up intact. In conclusion, this study provides a method using liposomes as a tool for probing the phosphatidylcholine transfer mechanism in lung.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Modulation of adhesion molecules on human large granular lymphocytes by interleukin-2 in vivo and in vitro.

The modulation of adhesion molecules on human large granular lymphocytes (LGL) by interleukin (IL)-2 was investigated both in vivo and in vitro. Intercellular adhesion molecule-1 (ICAM-1; CD54) expression increased on LGL of cancer patients receiving IL-2 adoptive immunotherapy. ICAM-1 expression on LGL isolated by Percoll gradient centrifugation, LGL purified, and expanded by adherence to plastic surfaces and LGL identified by Leu 19 (CD56) monoclonal antibody were increased significantly in response to IL-2 in vitro. Exposure of LGL to IL-1, interferon (IFN)-gamma, and tumor necrosis factor (TNF) in vitro did not induce ICAM-1. The expression of LFA-1 (CD11a/CD18), a receptor for ICAM-1, and other leukocyte adhesion molecules, including Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), was only maintained by IL-2. IL-2 induction of ICAM-1 and the maintenance of CD18 complex expression on small lymphocytes separated by Percoll gradients were similar to that on LGL. We conclude that IL-2 enhances the expression of ICAM-1 on multiple human lymphocyte populations including LGL effectors. Expression of the CD18 complex on LGL does not appear to be highly regulated by IL-2. These findings may have implications relevant to the role of these adhesion molecules in the activities of LGL modulated by IL-2.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, -26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4 °C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, ?26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4°C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.     

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro

We investigated the intracellular metabolic fate of [Me-14C]choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5:4:1). With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of water-soluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine. With [Me-14C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents. It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.     

The antitumor activity of liposomal aclarubicin in vitro and in vivo]

Study on antitumor activity of free and liposomal anthracycline antibiotic aclarubicin in vitro and in vivo showed that liposomal aclarubicin was characterised by activity against ascitic Ehrlich carcinoma comparable to that of free aclarubicin when used in a dose of 25 mg/kg. Liposomal antibiotic had a more pronounced antimetastatic action and showed no toxicity (in a dose of 30 mg/kg). Liposomal aclarubicin had a higher activating capacity with respect to the macrophage tumoricidal properties.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Expression of murine interleukin-2 cDNA in E. coli and biological activities of recombinant murine interleukin-2

Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E. coli. Recombinant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation.

Sialoglycolipids shed by tumor cells have been implicated in tumor-induced inhibition of T-lymphocyte responses to interleukin-2 (IL-2). In the present study, we have used glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, to investigate whether shedding of glycoproteins might also contribute to immunosuppression. Glycophorin A inhibited IL-2-stimulated proliferation of the IL-2-dependent cell lines HT-2 and CTLL-2 in a dose-dependent manner. Time course studies on synchronized cell populations indicated that the glycoprotein acted early in the activation process. On the other hand, glycophorin A had essentially no effect on IL-1-mediated stimulation of the IL-1-sensitive thymocyte cell line EL-4 NOB-1. Gel filtration FPLC demonstrated that IL-2 was able to bind to glycophorin aggregates under physiological conditions. Reconstituted vesicles containing glycophorin were also shown to bind IL-2. In addition, both soluble glycophorin aggregates and lipid vesicles containing glycophorin blocked binding of IL-2 to high-affinity cellular IL-2 receptors. Taken together, these results suggest that shedding of tumor sialoglycoproteins with oligosaccharide chains similar to glycophorin A might contribute to negative modulation of IL-2-mediated immune responses.     

Phenytoin induces interleukin-1 production in vitro

Human adherent mononuclear cells and subcloned cell lines established from the human histiocytic cell line U-937 were cultured with phenytoin (PHT) and/or lipopolysaccharide (LPS) purified from Bacteroides fragilis. After the cultivation period, the cell-free supernatants were tested for interleukin-1 (Il-1) activity. The results showed that PHT induces Il-1 activity and potentiates LPS-induced Il-1 production. In the monocytic cell line U-937, the induced Il-1 production was found to be clonally distributed indicating that the response to PHT may be exerted by a subpopulation of monocytes. The PHT-induced Il-1 activity may be of importance in the development of gingival overgrowth. The induced Il-1 could also contribute to other known side effects such as dermatologic reactions accompanied by transient fever seen in patients medicating the drug.     

Recombinant interleukin-2-induced polyclonal proliferation of in vitro unstimulated human peripheral blood lymphocytes

Human peripheral blood mononuclear cells as well as T-cell-enriched or T-cell-depleted populations were found to proliferate in response to recombinant interleukin-2 (IL-2) in vitro in the absence of a lectin preactivation signal. This proliferative response was detected at Day 2, peaked at Day 5, and was dependent on the concentration of IL-2 used. At the initiation of culture, these cells did not appear to be activated as determined by the expression of Tac antigens. In cultures of unfractionated T-cell-enriched suspensions, high concentrations of IL-2 resulted in preferential expansion of the OKT8+ population, although both OKT4+ and OKT8+ cells proliferated in response to IL-2 when cultured alone. These studies demonstrate that human lymphocytes obtained by standard fractionation procedures from peripheral blood are capable of proliferation in response to IL-2 without in vitro preactivation signals given by the addition of mitogens or antigens to cultures. These findings suggest that in vivo IL-2, in the absence of other exogenous stimuli, may directly influence immune responses and thus may have a potential role as a clinical immunopharmacologic agent.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4.

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

Biopharmaceutics of liposomal interleukin 2, oncolipin

Oncolipin is a multilamellar liposomal (dimyristoyl phosphatidylcholine) formulation of interleukin 2 (IL-2) and human serum albumin (HSA) with distinct surface characteristics which may influence its biological activities. IL-2 and HSA were detected on the surface of the liposomes using specific antibody staining. Surface expression of IL-2 was also demonstrated by the observation that Oncolipin bound to cells expressing IL-2 receptors (IL-2R) containing alphabetagamma or betagamma subunits. Binding and internalization of Oncolipin by cells expressing alphabetagamma or betagamma receptor subunits was blocked by excess free IL-2 or a neutralizing antibody against the beta chain. The display of surface IL-2 on Oncolipin's liposomes was maintained in vivo after intravenous injection into mice. IL-2 was also present between the lipid bilayers of the multilamellar liposomes based on the unique physical characteristics detected by freeze fracture electron microscopy. The bulk of the liposome-associated IL-2 was released from the liposomes upon incubation at 37 degrees C in medium containing serum, indicating that the IL-2 was not irreversibly entrapped on or in the liposome structure. Thus, Oncolipin is receptor-targeted to activated T and NK cells by virtue of its surface expression of IL-2 and has the potential to release IL-2 following deposition within lymphoid organs. These properties may confer distinct advantages over soluble IL-2 for immunotherapy of cancer and viral diseases.