Transcutaneous IL-2 uptake mediated by Transfersomes depends on concentration and fractionated application

INTRODUCTION: Transfersomes (TF) are new, ultradeformable carriers with characteristics that enable them to penetrate the skin spontaneously. TFs are able to transport noninvasively both low- and high-molecular-weight molecules into the body. MATERIALS AND METHODS: TFs contain phosphatidylcholine and sodium cholate. Recombinant human interleukin-2 (Proleukin, Chiron) was added to the TFs and incubated for 24 h at 4 degrees C. The immunotransfersomes (ITF) were isolated from free interleukin-2 (IL-2) by filtration (Centrisart, Sartorius). Twenty-five thousand, 50,000 and 150,000 IU pure IL-2 and ITFs, which had been incubated with the same concentrations of IL-2, were applied subcutaneously (s.c.) (n = 8) and epicutaneously (e.c.) (n = 8) to mice. The IL-2 serum concentrations in the mice were then measured by ELISA after 2, 4, 6, 8, 10 and 24 h. Fractionation of the transdermal IL-2 application was also examined as a means of improving uptake. RESULTS: In concentrations of 25,000 and 50,000 IU IL-2, the subcutaneous application of ITFs resulted in a longer lasting IL-2 serum concentration than did the subcutaneous application of pure IL-2. While at 25,000 IU, the epicutaneous application of ITFs resulted in serum concentrations comparable to those resulting from s.c. application, at 50,000 and 150,000 IU, only 50% and 22.6% of the maximum serum concentration resulting from the s.c. application of pure IL-2 was obtained. Fractionating the transdermal IL-2 application improved uptake. CONCLUSION: We were able to show that biologically active IL-2 can be bonded to TFs up to 75%. It is possible to transport IL-2 through the skin using TFs. Both the concentration-dependent saturation of the TFs with IL-2 and fractionation of the application resulted in differing degrees of transcutaneous IL-2 uptake.     

Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines

We have investigated the effects of IL-2 and IL-4 on different parameters of T cell activation using three T cell lines. The Th cell line L14 and the cytotoxic T cell line C30.1, both grown in IL-2-containing medium, and a line derived from C30.1 cells (line 1) cultured in IL-4 for a prolonged period were studied. All three cell lines could be activated with IL-2 or IL-4. T cell stimulation by either IL-2- or IL-4-induced identical patterns of cell size enlargement and transferrin receptor expression. However, only IL-2 up-regulated cell-surface expression of the p55 subunit of the IL-2R (p55 IL-2R) as measured by flow cytometry and RIA. This difference was also reflected by the accumulation of soluble p55 IL-2R in the culture medium. No significant increase in expression of membrane or soluble forms of p55 IL-2R was detected after IL-4 stimulation. mAb specific for p55 IL-2R which block IL-2-induced T cell growth did not affect IL-4-mediated T cell proliferation indicating that p55 IL-2R is not involved in IL-4-mediated T cell growth. Analysis of IL-4R expression performed on line 1 using biotinylated IL-4 revealed that IL-4, but not IL-2, is capable of increasing IL-4R expression. Together these results suggest that during IL-2- or IL-4-induced T cell proliferation, each lymphokine specifically up-regulates its own receptor.     

Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.     

IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15, and IL-21

Cytokines that use the common receptor gamma-chain for regulating CD8(+) T cell responses to Ag include IL-2, IL-15, and the recently identified IL-21. The ability of these cytokines to regulate antitumor activity in mice has generated considerable interest in understanding their mode of action. In this study we compare the abilities of IL-2, IL-15, and IL-21 to stimulate immunity against tumors in a syngeneic thymoma model. Durable cures were only achieved in IL-21-treated mice. By monitoring both endogenous and adoptively transferred tumor Ag-specific CD8(+) T cells, it was determined that IL-21 activities overlap with those of IL-2 and IL-15. Similar to IL-2, IL-21 enhanced Ag activation and clonal expansion. However, unlike IL-2 treatment, which induces activation-induced cell death, IL-21 sustained CD8(+) T cell numbers long term as a result of increased survival, an effect often attributed to IL-15. These findings indicate that the mechanisms used by IL-21 to promote CD8(+) T cell responses offer unique opportunities for its use in malignant diseases and infections.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

The role of IL-12, IL-23 and IFN-gamma in immunity to viruses

IL-12, IL-23 and IFN-gamma form a loop and have been thought to play a crucial role against infectious viruses, which are the prototype of "intracellular" pathogens. In the last 10 years, the generation of knock-out (KO) mice for genes that control IL-12/IL-23-dependent IFN-gamma-dependent mediated immunity (STAT1, IFN-gammaR1, IFNgammaR2, IL-12p40 and IL-12Rbeta1) and the identification of patients with spontaneous germline mutations in these genes has led to a re-examination of the role of these cytokines in anti-viral immunity. We here review viral infections in mice and humans with genetic defects in the IL-12/IL-23-IFN-gamma axis. A comparison of the phenotypes observed in KO mice and deficient patients suggests that the human IL-12/IL-23-IFN-gamma axis plays a redundant role in immunity to most viruses, whereas its mouse counterparts play a more important role against several viruses.     

Tumor-specific Tc1, but not Tc2, cells deliver protective antitumor immunity.

We investigated whether secretion of multiple cytokines by CD8+ T cells is associated with improved protection against tumor challenge. We show that antitumor immunity induced by immunization with dendritic cells and a MHC class I-binding tumor peptide are dependent on secretion of IFN-gamma but not IL-4 or IL-5 by host cells. To further address the role of IL-4 and IL-5 in antitumor immunity, tumor-specific TCR-transgenic CD8+ T cells were activated in vitro to generate cytotoxic T (Tc) 1 cells that secrete high IFN-gamma and no IL-4 or IL-5 or Tc2 cells that secrete IL-4, IL-5, and some IFN-gamma. Both cell types killed target cells in vitro. Tc1 and Tc2 cells were adoptively transferred into syngeneic hosts, and their ability to protect against tumor challenge was compared. Tc1 cells were able to significantly delay tumor growth, whereas Tc2 cells or Tc2 cells from IFN-gamma(-/-) donors had no effect. This was due to neither the inability of Tc2 cells to survive in vivo or to migrate to the tumor site nor their inability to secrete IL-4 and/or IL-5 in the presence of limiting amounts of anti-CD3. However, IFN-gamma secretion by Tc2 cells was triggered inefficiently by restimulation with Ag compared with anti-CD3. We conclude that the ability to secrete "type 2" cytokines, and cytotoxic ability, have a limited role in antitumor immune responses mediated by CD8+ T cells, whereas the capacity to secrete high amounts of IFN-gamma remains the most critical antitumor effector mechanism in vivo.     

Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4-/- Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4-/- Th2 cells are transferred into IL-4Ralpha-/- mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5-/- Th2 cells were transferred into IL-5-/- recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Ralpha is critically important in Th2 cell stimulation of mucus production.     

The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation

Although host immunity offers the obvious benefit of reducing parasite infection, it is often traded-off with other fitness components. We investigated whether the cost of an immune response in the yellow fever mosquito, Aedes aegypti, is modulated by the antigen that activates the melanization immune response. Thus, one of three different novel antigens were injected into the mosquito's thorax--either a glass bead, a negatively charged (C-25) Sephadex bead, or a neutral (G-25) Sephadex bead--and fecundity and bead melanization were observed. Glass beads are immunologically inert and were therefore used as an inoculation control. The fecundity of mosquitoes inoculated with these beads did not differ from the fecundity of mosquitoes that did not melanize negatively charged or neutral beads. The ability of A. aegypti to melanize negatively charged Sephadex beads was associated with reduced fecundity, showing a clear cost of immunity. In contrast, melanization of the neutral beads was quite strong but had no effect on fecundity. Thus, the cost of what appeared to be the same immune response--melanization of a bead--depended on the type of bead that stimulated the immune system. Such differences might help to explain variation of immune efficacy against different parasites in natural populations.     

IL-4 inhibits IL-2 synthesis and IL-2-induced up-regulation of IL-2R alpha but not IL-2R beta chain in CD4+ human T cells.

There is growing evidence to suggest a regulatory role of IL-4 in the immune system affecting both proliferation and lymphokine production. In the present work we have analyzed the effect of IL-4 on IL-2 and IFN-gamma synthesis by stimulating CD4+ human T cells (+10% accessory cells) with Con A in the presence of several doses (1 to 100 U/ml) of human rIL-4. The results showed an impaired IL-2 and IFN-gamma synthesis in the presence of IL-4. This inhibition was dose dependent and was evident only when IL-4 was added in the first 2 h of culture. Moreover, the external addition of IL-2 did not revert the inhibitory effect of IL-4 on IL-2 and IFN-gamma synthesis induced by Con A. We have also analyzed the effect of IL-4 on the expression of both alpha- and beta-chains of the IL-2R. Although the expression of IL-2R alpha mRNA was not modified after 6 h in culture in the presence of IL-4, a decrease was observed at 24 and 48 h. The addition of rIL-2 showed that the inhibition in IL-2R alpha expression could be explained by an impairment in the up-regulatory signal transmitted through the IL-2R. In addition to this, IL-4 did not modify the IL-2R beta mRNA expression at 6 and 24 h although a decreased expression was observed at 48 h which could be explained by the defective IL-2 production. The differential effect of IL-4 on the up-regulatory effect of IL-2 in the expression of IL-2R alpha and IL-2R beta suggest the existence of different regulatory mechanisms acting on the expression of both chains.     

Efficient internalization of IL-2 depends on the distal portion of the cytoplasmic tail of the IL-2R common gamma-chain and a lymphoid cell environment

The common gamma-chain (gammac), a subunit of the IL-2R, is essential for high affinity ligand binding and signal transduction due to Jak3 association to gammac. Another consequence of IL-2/IL-2R interaction is rapid receptor-mediated endocytosis of the receptor-ligand complex. In the present study, we establish that this rapid endocytosis of IL-2 in a T cell tumor line is dependent upon the cytoplasmic tail of gammac. Deletion mutants of the cytoplasmic tail mapped this activity to 9 aa of gammac, 45-54 aa distal to the transmembrane region. In contrast, ligand-independent constitutive endocytosis of gammac occurred more slowly and was dependent upon a PEST sequence in a more membrane-proximal region of the cytoplasmic tail of gammac. Thus, this receptor subunit may use distinct sorting signals for its constitutive regulation and ligand-induced endocytosis. Rapid endocytosis of IL-2 was inhibited by the tyrosine kinase inhibitor genistein, implicating a role for a signal transduction pathway in IL-2 internalization. However, one T cell line bearing a mutant gammac exhibited impaired endocytosis of IL-2, despite normal IL-2-induced Jak/STAT activation. Furthermore, inefficient endocytosis of IL-2 was noted after transfection of the COS7 epithelial cell line with the IL-2R, and further reconstitution of these cells with Jak/STAT proteins did not enhance this internalization. Collectively, these latter findings indicate that rapid endocytosis of IL-2 is dependent upon cellular signaling in lymphoid cell environment that is not solely a consequence of the presence of the Jak/STAT pathway.     

IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1alpha in the murine model. The production and activity of IL-1alpha is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10(-/-) mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4(-/-) exhibited no increased resorption. IL-10(-/-) had markedly elevated IL-1alpha production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4(-/-) exhibited decreased IL-1alpha production (p < 0.05). IL-10 also suppressed IL-1alpha production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1alpha expression.     

Delta 9-tetrahydrocannabinol treatment suppresses immunity and early IFN-gamma, IL-12, and IL-12 receptor beta 2 responses to Legionella pneumophila infection

The marijuana cannabinoid, delta 9-tetrahydrocannabinol (THC), suppresses immunity to Legionella pneumophila and development of Th1 activity and cell-mediated immunity. In the current study, THC effects on cytokines regulating the development of Th1 cells were examined. BALB/c mice showed significant increases in serum IL-12 and IFN-gamma within hours of infection; however, the levels of these Th1-promoting cytokines as well as resistance to a challenge infection were suppressed by THC (8 mg/kg) injected 18 h before priming. The Th2-promoting cytokine, IL-4, was increased within hours of a Legionella infection and was further increased by THC treatment. These results suggested that THC injection suppressed the cytokine environment promoting Th1 immunity. In additional experiments, THC pretreatment and infection of IL-4 knockout mice showed that serum IL-12 and IFN-gamma were suppressed equally in both knockout and normal mice. This suggested that the drug-induced increase in IL-4 was not responsible for the decreases in serum IL-12 and IFN-gamma. However, THC treatment was shown to suppress the expression of IL-12 receptor beta 2 mRNA, indicating that, in addition to suppression of IL-12, THC injection suppressed the expression of IL-12 receptors. Finally, the role of cannabinoid receptors in Th1-promoting cytokine suppression was examined, and results with receptor antagonists showed that both cannabinoid receptors 1 and 2 were involved. It is suggested that suppression of Th1 immunity to Legionella is not due to an increase in IL-4 production but to a decrease in IFN-gamma and IL-12. Furthermore, both types of cannabinoid receptors are involved.     

Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation

Transforming growth factor-beta1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-beta1 signaling. Despite the physiological importance, the mechanisms that TGF-beta1 uses to regulate T cell expansion remain poorly understood. TGF-beta1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3(-/-) mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-beta1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-beta1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3(-/-) T cells. In contrast, disruption of Smad3 did not affect TGF-beta1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-beta1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-beta1 to inhibit TCR-induced proliferation of Smad3(-/-) T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-beta1 signals through multiple pathways to suppress T cell proliferation.     

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.     

Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status

Mouse spermatozoa from the caudae epididymides could be freeze-dried without losing their ability to support normal development. Immature spermatozoa from the testes, in contrast, were damaged by freeze-drying. However, immature spermatozoa became resistant to freeze-drying after their treatment with diamide, which oxidizes free -SH groups. Conversely, epididymal spermatozoa were damaged by freeze-drying if first treated with dithiothreitol (DTT), which reduces -SS- bonds. The potential for freeze-drying damage seems likely to relate to the -SS- status of sperm proteins, in particular its protamines.     

Heat production by sacred lotus flowers depends on ambient temperature, not light cycle

Flowers of the sacred lotus, Nelumbo nucifera, maintain receptacle temperature (Tr) between about 30C and 36C during their 2-4 d sequence of anthesis by increasing the rate of heat production, measured as oxygen consumption (VO2), at low ambient temperature (Ta) at night and reducing it at high Ta during the day. The effects of Ta and photoperiod on VO2 were separated by manipulating Ta around outdoor flowers under natural photoperiod or constant darkness. There was no effect of time of day on either Tr or VO2, but both depended on Ta. Rising Ta resulted in an increase in Tr, a brief upward spike in VO2, followed by a long-term decrease in VO2. Decreasing Ta had the opposite effects: a decrease in Tr and a brief depression, followed by a slow rise, in VO2. The two-phase responses to sudden Ta change indicate a rapid, mass-action effect and a slower, regulatory adjustment. Temperature regulation continued in constant darkness for over 3 d.Keywords: Thermoregulation, lotus, photoperiod, temperature, heat production, oxygen consumption, Nelumbonaceae.