David M. Conrad

Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines

Bovine lactoferricin (LfcinB) is a cationic, amphipathic peptide that is cytotoxic for human and rodent cancer cells. However, the mechanism by which LfcinB causes the death of cancer cells is not well understood. Here, we show that in vitro treatment with LfcinB rapidly induced apoptosis in several different human leukemia and carcinoma cell lines as determined by DNA fragmentation assays and phosphatidylserine headgroup inversion detected by Annexin V binding to the surface of cancer cells. Importantly, LfcinB treatment did not adversely affect the viability of untransformed human lymphocytes, fibroblasts, or endothelial cells. Studies with different LfcinB-derived peptide fragments revealed that the cytotoxic activity of LfcinB resided within the amino acid sequence FKCRRWQWRM. Treatment of Jurkat T leukemia cells with LfcinB resulted in the production of reactive oxygen species followed by caspase-2-induced dissipation of mitochondrial transmembrane potential and subsequent activation of caspase-9 and caspase-3. Selective inhibitors of caspase-2 (Z-VDVAD-FMK), caspase-9 (Z-LEHD-FMK), and caspase-3 (Z-DEVD-FMK) protected both leukemia and carcinoma cells from LfcinB-induced apoptosis. Conversely, a caspase-8 inhibitor (Z-IETD-FMK) had no effect, which argued against a role for caspase-8 and was consistent with the finding that death receptors were not involved in LfcinB-induced apoptosis. Furthermore, Jurkat T leukemia cells that overexpressed Bcl-2 were less sensitive to LfcinB-induced apoptosis, which was characterized by mitochondrial swelling and the release of cytochrome c from mitochondria into the cytosolic compartment. We conclude that LfcinB kills cancer cells by triggering the mitochondrial pathway of apoptosis at least in part through the generation of reactive oxygen species.

Adenosine Acts through A2 Receptors to Inhibit IL-2-Induced Tyrosine Phosphorylation of STAT5 in T Lymphocytes: Role of Cyclic Adenosine 3′,5′-Monophosphate and Phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A1, A2a, A2b, A3) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A2 receptor agonists and was blocked by selective A2a and A2b receptor antagonists, indicating that adenosine was mediating its effect through A2 receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A2 receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.

Ryanodine receptor signaling is required for anti‐CD3‐induced T cell proliferation, interleukin‐2 synthesis, and interleukin‐2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca++ mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR‐selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor‐mediated activation events and cytokine‐dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)‐2 by T lymphocytes responding to mitogenic anti‐CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation‐induced expression of the α chain (CD25) of the high affinity IL‐2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL‐2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL‐2‐dependent proliferation of CTLL‐2 T cells. These data indicate that RyR are involved in regulating IL‐2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR‐associated Ca++ signaling regulates T cell proliferation by promoting both IL‐2 synthesis and IL‐2‐dependent cell cycle progression.

Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation.

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) alpha chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy‐1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy‐1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy‐1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti‐CD3 and anti‐CD28 mAb, indicating that Thy‐1 cross‐linking enhanced T cell receptor/CD28‐driven T cell activation. Consistent with this finding, Thy‐1 blockade with a soluble nonactivating anti‐Thy‐1 mAb (clone 30‐H12) inhibited anti‐CD3‐induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose‐dependent fashion. Interleukin‐2 synthesis and CD25 expression were also impaired by Thy‐1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti‐Thy‐1‐mediated inhibition of anti‐CD3‐induced T cell activation. The CTL that were induced in the presence of blocking anti‐Thy‐1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy‐1 blockade. We conclude that Thy‐1 signalling promotes the in vitro generation of CTL that kill in a granule‐dependent fashion.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

The Ca2+ channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells

Flunarizine is a Ca2+ channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.

Pleurocidin-family cationic antimicrobial peptides mediate lysis of multiple myeloma cells and impair the growth of multiple myeloma xenografts

Abstract Multiple myeloma is a common hematological malignancy that urgently requires new approaches to treatment, since the disease is not curable using current chemotherapeutic regimens. The aim of this study was to determine whether human and mouse multiple myeloma cells are killed by the pleurocidin-like cationic antimicrobial peptides NRC-03 and NRC-07, previously shown to be active against breast cancer cells. We demonstrate here that NRC-03 and NRC-07 bound to and rapidly killed multiple myeloma cells by causing extensive membrane damage, as well as DNA cleavage. NRC-03 showed greater binding to multiple myeloma cells and a more potent cytotoxic effect than NRC-07. In addition, intratumoral injections of NRC-03 impaired the growth of multiple myeloma xenografts in immune-deficient mice. We conclude that NRC-03 warrants further investigation for its possible use in the treatment of multiple myeloma.

The hardscrabble hypothesis: A reduction in chronic tissue damage has increased the incidence of autoimmune disease

The adaptive immune system, fine tuned through millions of years of vertebrate evolution, discriminates self from foreign antigens through the deletion of auto-reactive clones and/or tolerization to self antigens. In this system, commonly encountered antigens should be recognized as self while rarely encountered antigens such as microbial or cancer derived molecules should be recognized as foreign. Throughout evolutionary history, the availability of cryptic self-antigens for presentation was enhanced though immune processing of damaged tissues due to infections, parasites and trauma - in other words, the hardscrabble conditions that prevailed throughout the vast majority of our evolutionary past. In situations where chronic tissue injury is reduced there is an increase in the incidence of autoimmune disease. A unifying hypothesis is thereby provided to explain the major epidemiological trends in autoimmune diseases, namely, (1) an historic increase in incidence, (2) increase in the incidence with increasing latitude, (3) increase in the incidence with higher socioeconomic status.

Induction of CD4 + CD25 + Foxp3 − regulatory T cells by Thy-1 stimulation of CD4 + T cells

Thy‐1 (CD90) on mouse T cells has been reported to have both T‐cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)‐mediated crosslinking of Thy‐1 on CD4+ mouse T‐cells‐induced regulatory T (Treg) cells that expressed CD25, CD39 and glucocorticoid‐induced tumor necrosis factor receptor family‐related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4+ Tresponder cells in response to anti‐CD3/anti‐CD28mAb‐coated T‐cell expander beads or syngeneic dendritic cells and soluble anti‐CD3mAb was inhibited by Thy‐1‐induced Treg cells, in spite of elevated IL‐2 levels in the co‐cultures. Interestingly, stimulation with T‐cell expander beads caused Thy‐1‐induced Treg cells to synthesize large amounts of interleukin‐2 (IL‐2). IL‐10 was also elevated in co‐cultures of activated Tresponder cells and Thy‐1‐induced Treg cells. However, mAb‐mediated neutralization of IL‐10 did not restore Tresponder‐cell proliferation to control levels, which excluded IL‐10 as a potential mediator of Thy‐1‐induced Treg‐cell suppressor function. In addition, Thy‐1‐induced Treg cells did not inhibit IL‐2‐dependent proliferation of CTLL‐2 cells, suggesting that IL‐2 receptor signaling remained intact in the presence of Thy‐1‐induced Treg cells. We suggest that Treg cells induced by Thy‐1 ligation in vivo may contribute to the maintenance of T‐cell homeostasis.