Autocrine IL-6 Research Articles

Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression.

Introducing avidin-biotin complex ELISA for anti-DNA antibody, the mechanism of in vitro production of anti-ssDNA antibody as well as of polyclonal immunoglobulin mediated by an IL-6-IL-6R loop was studied in patients with systemic lupus erythematosus (SLE). Regardless of the presence or absence of T cells, B cells from SLE patients could produce IgG anti-ssDNA antibody as well as total IgG without any stimulation. Low density B cells obtained by Percoll gradient density centrifugation responded to rIL-6 to produce IgG and IgG anti-ssDNA antibody. rIL-2 and rIL-4 had lesser effects on the differentiation of low density B cells. In fact, IL-6R was preferentially expressed on low density B cells from active SLE patients, as detected by anti-IL-6R MoAb, MT18, which did not inhibit IL-6 binding. SLE B cells, especially high density B cells, produced greater amounts of IL-6 in culture supernatants than did T cells, regardless of whether disease was active or inactive. Normal T cells and B cells did not produce significant amounts of IL-6. Thus, endogenous IL-6 produced by high density B cells bound to the IL-6R preferentially expressed on the low density B cells, and drove them into terminal differentiation, especially in active SLE patients. Further, addition of polyclonal anti-IL-6 or anti-IL-6R MoAb (PM1), which inhibited IL-6 binding, both inhibited IgG anti-ssDNA antibody as well as total IgG production by SLE B cells in a dose-dependent manner. These results suggest that interruption of the autocrine IL-6 loop would be of therapeutic value in SLE.

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.

Bruton's Tyrosine Kinase Separately Regulates NFκB p65RelA Activation and Cytokine Interleukin (IL)-10/IL-12 Production in TLR9-stimulated B Cells

B lymphocytes express both B cell receptor and Toll-like receptors (TLR). We show here that Bruton's tyrosine kinase (Btk), a critical component in B cell receptor signaling, is also involved in TLR9 signaling in B cells. Stimulation of B cells with TLR9 ligand CpG oligodeoxynucleotide (ODN) leads to transient phosphorylation of Btk, and in the absence of Btk, TLR9-induced proliferation of B cells is impaired. Interestingly, Btk(-/-) B cells secrete significantly more interleukin (IL)-12 but much less IL-10 compared with wild type B cells upon TLR9 stimulation. Immunization of Btk(-/-) mice with CpG ODN also leads to elevated levels of IL-12 in vivo and consequently, a greater -fold increment in the production of Th1 type IgG2b and IgG3 antibodies in these mice compared with wild type controls. The addition of exogenous recombinant IL-10 could suppress IL-12 production by TLR9-activated Btk(-/-) B cells, suggesting that in B cells, Btk negatively regulates IL-12 through the induction of autocrine IL-10 production. TLR9 signaling also leads to the activation of NFkappaB, including the p65RelA subunit in wild type B cells. The lack of Btk signaling affects the activation of NFkappaB and impairs the translocation of the p65RelA subunit to the nucleus of B cells upon TLR9 stimulation. However, p65RelA(-/-) B cells could respond similarly to wild type B cells in terms of IL-10 and IL-12 secretion when stimulated with CpG ODN, suggesting that the defect in NFkappaB p65RelA activation is additional to the impairment in cytokine production in TLR9-activated Btk(-/-) B cells. Thus, Btk plays an important role in TLR9 signaling and acts separately to regulate NFkappaB RelA activation as well as IL-10 and IL-12 production in B cells.

Adiponectin blocks multiple signaling cascades associated with leptin‐induced cell proliferation in ApcMin/+ colon epithelial cells

We previously demonstrated that leptin, an adipose-derived hormone, induces cell proliferation in a model of preneoplastic (IMCE (Apc(Min/+)), but not normal (YAMC (Apc(+/+)), colon epithelial cells by inducing autocrine IL-6 production and trans-IL-6 signaling. Low serum adiponectin is associated with colon, prostate and breast cancer. Adiponectin is secreted by white adipose tissue; the levels of adiponectin in the blood decrease as body mass index (and leptin) increases. In our study, we tested whether murine recombinant globular adiponectin (gArcp30) could modulate leptin-induced cell proliferation, autocrine IL-6 production, trans-IL-6 signaling and other leptin-induced cell signaling events previously observed in IMCE cells but not YAMC cells. Under serum-free conditions, adiponectin (1 mug/ml) inhibited leptin-induced autocrine IL-6 production, soluble IL-6 receptor shedding, trans-IL-6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin-induced cell proliferation in the IMCE cells and this inhibition was associated with I kappa B-alpha phosphorylation, I kappa B-alpha degradation and decreased NF-kappaB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-kappaB-dependent autocrine IL-6 production and trans-IL-6 signaling. We hypothesize that adiponectin may be an important regulator of colon epithelial cell homeostasis by linking the observed reduced risk for cancer in populations with high serum adiponectin concentrations to specific mechanisms of cell number homeostasis in a model of preneoplastic colon epithelial cells. These data may have broad implications for diet and lifestyle strategies for the prevention and treatment of obesity-associated cancers.

IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland

High serum levels of IL-6 correlate with poor outcome in breast cancer patients. However, no data are available on the relationship between IL-6 and mammary stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in the MCF-7 breast cancer cell line and in primary human mammospheres (MS), multicellular structures enriched in stem/progenitor cells of the mammary gland. MS from node invasive breast carcinoma tissues expressed IL-6 mRNA at higher levels than did MS from matched non-neoplastic mammary glands. In addition, IL-6 mRNA was detected only in basal-like breast carcinoma tissues, an aggressive breast carcinoma variant showing stem cell features. IL-6 treatment triggered Notch-3-dependent upregulation of the Notch ligand Jagged-1 and promotion of MS and MCF-7-derived spheroid growth. Moreover, IL-6 induced Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promoted a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, autocrine IL-6 signaling relied upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, these data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

IL-6 Enhances C-myc Translation in Myeloma Cells Via Activation of the Myc Internal Ribosomal Entry Site (IRES) and an IRES-Binding Trans Factor, hnRNP A1.

Dysregulated c-myc expression in multiple myeloma (MM) can be due to a translocation with juxtaposition of the myc gene to the Ig enhancer resulting in constitutive transcription. However, myc protein levels can also be increased in MM cells when it is due to heightened translation. Activated translation can be mediated via an mTOR-mediated, cap-dependent mechanism, thus named because the translation initiation complex utilizes the cap structure on mRNA to recruit the ribosomal subunit. However, an alternative mechanism is cap-independent where the ribosomal subunit binds a transcript via an IRES in the RNA's 5′UTR. IRES function is regulated by IRES trans activating factors (ITAFs) which bind to the 5′UTR. We studied myc expression in ANBL-6 wild type and activated K-RAS-transfected MM cells which proliferatively respond to IL-6 but do not contain myc translocations. IL-6 increased myc protein expression in both cell lines in a concentration and time-dependent fashion but RNA expression was only enhanced in the wild type cells. The increased protein expression in K-RAS mutant cells was unaffected by rapamycin, indicating independence from mTOR and cap-dependent mechanisms. Using an IRES-dependent reporter vector, reporter expression was shown to be increased by IL-6, confirming IL-6 activates myc IRES activity. This was also demonstrated in the IL-6 autocrine U266 MM cell line where myc IRES-dependent reporter expression was ablated following exposure to anti-IL-6 antibodies. In addition, primary MM cells treated with IL-6 also demonstrated an increase in myc IRES activity. Since a yeast three-hybrid screen for proteins specifically binding to the myc IRES identified several clones encoding hnRNP A1, we evaluated hnRNP A1 as a mediator of myc IRES activity. IL-6 increased the cytoplasmic localization of hnRNP A1 in ANBL-6 MM cells. Furthermore, in MM cells where hnRNP A1 expression was knocked down, myc IRES activity was greatly diminished and there was no enhancement with addition of IL-6. These results indicate that IL-6 can enhance c-myc translation in MM cells via activation of the myc IRES and that hnRNP A1 is a critical myc ITAF involved in the IL-6 response. hnRNP A1 may serve as a new therapeutic target in myeloma.

Generation of T cell regulatory activity by plasmacytoid dendritic cells and tryptophan catabolism

Plasmacytoid dendritic cells (pDCs) represent a specialized cell population that produces large amounts of type I interferons. Recently, murine and human pDCs have been credited with a unique ability to express the inducible, tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO) as an effector means of regulatory T cell function and thus mediate immunosuppression in specific settings. Here we review and further clarify specific features of the relative efficacy and mechanisms of IDO induction in pDCs after ‘ligand’ engagement by the soluble forms of molecules present on regulatory T cell surface (‘reverse signaling’). We found that splenic pDCs do not express IDO and are not tolerogenic under steady-state conditions. B7 engagement by the two different ligands CTLA-4-Ig and CD28-Ig resulted in opposing outcomes on IDO function—i.e., activation by the former (through autocrine IFN-γ) and inhibition by the latter (via autocrine IL-6 and SOCS3). In addition, engagement of GITR ligand by GITR-Ig in pDCs also resulted in IDO activity via type I IFNs and noncanonical NF-κB signaling. Therefore, although pDCs do not express IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by those cells.

Interleukin‐6 is a potent growth factor for ER‐α‐positive human breast cancer

Bone is the primary anatomical site of breast cancer metastasis, and bone metastasis is associated with increased morbidity and mortality. Mesenchymal stem cells (MSC) are a predominant fibroblast cell population within the bone marrow, and metastatic breast cancer cells that seed within bone would predictably encounter MSC or their soluble factors. Therefore, we examined the impact of primary human MSC on a panel of estrogen receptor-alpha (ERalpha)-positive (MCF-7, T47D, BT474, and ZR-75-1) and ERalpha-negative (MDA-MB-231 and MDA-MB-468) human breast tumor cell lines. All ERalpha-positive breast tumor cell lines displayed low basal activation of signal transducer and activator of transcription 3 (STAT3) until exposed to MSC, which induced chronic phosphorylation of STAT3 on tyrosine-705. Paracrine IL-6 was found to be the principal mediator of STAT3 phosphorylation in coculture studies, and MSC induction of STAT3 phosphorylation was lost when IL-6 was depleted from MSC conditioned media or the IL-6 receptor was blocked on tumor cells. Enhanced tumor cell growth rates were observed in the ERalpha-positive mammary tumor cell line MCF-7 after paracrine and autocrine IL-6 exposure, where MCF-7 growth rates were enhanced by >2-fold when cocultured with MSC in vitro and even more pronounced in vivo with autocrine IL-6 production.

Exposure of Myeloid Dendritic Cells to Exogenous or Endogenous IL-10 during Maturation Determines Their Longevity

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-x(L) expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-x(L) in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.

AKT Kinase Addiction in Multiple Myeloma.

Cancer cells with activating kinase mutations are singularly hypersensitive to drugs that target these kinases, a phenomenon referred to as kinase or oncogene addiction. Although the AKT kinase is not known to be mutated in multiple myeloma (MM), it is frequently hyperactivated due to mutations in upstream signaling proteins. Previous work indicated that MM cells with heightened AKT activity were hypersensitive to drugs targeted to mTOR, a downstream substrate of AKT, suggesting an addiction to this pathway. “Kinase addiction” would theortically render the cell dependent on the kinase for growth. This dependency implies that other cascades of viability and/or proliferation may become downregulated subsequent to kinase activation. To test for AKT kinase addiction in MM we stably transfected the U266 MM cell line that is known to be protected against apoptosis by an autocrine Il-6/IL6R pathway (Caitlett-Falcone et al., Immunity 10, 105–115, 1999) with a constitutively active AKT construct (U266-AKT). AKT-transfected MM cells were protected against fas-induced apoptosis even when the autocrine IL-6 protective pathway was paralyzed by anti-IL6 and anti-IL6 receptor antibodies. However, as expected for an AKT addicted cell line, the U266-AKT cell line was more sensitive to agents that target the PI3/AKT/mTOR pathway, including two AKT inhibitors and the PI3K inhibitors LY294002 and wortmanin in terms of increased level of apoptosis and/or growth inhibition in comparison to the control U266 cell line. This “kinase-addicted” state was associated with down regulation of several components of the autocrine anti-apoptotic pathway of U266 cells. Expression of IL6, the IL6 receptor subunits gp80 and gp130 and STAT3 were all markedly silenced following AKT transfection. This down regulation of the survival pathway was relatively specific as expression of the fas receptor and VEGF receptors were relatively unaffected. The decrease in protein expression was also reflected in the decrease of mRNA for the receptor subunits, suggesting a transcriptional mechanism for down regulation. As expected from the decrease in autocrine IL-6 signaling, AKT-transfected cells also demonstrated a marked inhibition of Jak1 and MAPK 42/44 phosphorylation and enzymatic activity. In summary, over expression of activated AKT results in an AKT-addicted state whereby the MM cell becomes dependent on AKT for survival as other growth-promoting and anti-apoptotic cascades become paralyzed. These data support AKT as an important target for therapeutic intervention of MM and suggest such therapy would be most successful in patients whose MM cells contain the highest amount of activated AKT.

Azacitidine Down-Regulates Both IL-6 Signalling and NFkB Activity in Human Myeloma Cells.

We have previously demonstrated that Azacitidine (AZA) at clinically achievable concentrations (<10μM) induces apoptosis in a range of genetically heterogeneous human myeloma cell lines (HMCL) and prolongs survival in the murine 5T33 model of systemic multiple myeloma (MM). HMCL U266 demonstrates autocrine IL-6 production, constitutive Stat3 phosphorylation and is resistant to conventional chemotherapy and glucocorticoids. Using U266 we have evaluated further the effect of AZA on human MM cells. AZA treatment (5mM daily) of U266 induced apoptosis at 24, 48 and 72 hours of 28%, 43% and 52%, respectively. Immunoblot analyses showed rapid falls in IL-6R and p-Stat3 (both within 4 hours) prior to evidence of proteolysis (PARP cleavage). ELISA of AZA-treated U266 conditioned media demonstrated a reduction in IL-6 and an immediate and sustained reduction in soluble IL-6R levels, consistent with reduced IL-6 synthesis and IL-6 receptor shedding, respectively. RT-PCR confirmed that the rapid falls in IL-6 and IL6-R were not secondary to reduced transcription with relative increases of 1.7 and 2.4-fold, respectively, by 8 hours post-AZA. Flow cytometry showed an initial rise and subsequent modest fall in both membrane and cytosolic gp130 while RT-PCR and immunoblot analyses showed a rapid fall in both SOCS3 transcriptional activity and protein levels, respectively. Time-course experiments with AZA 5μM and the addition of either physiological (100pg/ml) or supra-physiological (3ng/ml) concentrations of exogenous IL-6 over a 96-hour period demonstrated that 3ng/ml but not 100pg/ml IL-6 was capable of partially restoring Stat3 phosphorylation but that neither concentration of exogenous IL-6 prevented AZA-induced cell death. NFkB activity was evaluated with a luciferase reporter enzyme system following treatment for 24 hours with AZA 1μM, 5μM and 10μM. Marked suppression of NFkB activity was seen at both the 5μM and 10μM dose levels. Flow cytometric evaluation of phospho-IkB and IkB showed a modest decline in the pIkB:IkB ratio over the same period. Finally, combined treatment of U266 with AZA and dexamethasone demonstrated synergistic killing (synergism quotient = 1.2) when AZA was administered prior to dexamethasone but not in the reverse order. We conclude that AZA induces down-regulation of both IL-6 signalling and NFkB activity. Furthermore, our data suggest that the former effect is most likely mediated by rapid AZA-induced inhibition of protein synthesis. The mechanism of NFkB inhibition requires further clarification. AZA represents a potentially novel therapeutic approach to MM.

Differential Effects of Immunosuppressive Drugs on Chemokine Receptor CCR7 in Human Monocyte-Derived Dendritic Cells: Selective Upregulation by Rapamycin

Appropriate recruitment of dendritic cells (DC) at sites of inflammation and migration to secondary lymphoid organs is of critical importance for the initiation of Ag-specific immune responses. The proper localization of DC in selected tissues is guided primarily by the coordinated expression of chemokine receptors (CKR). Here we show that immunosuppressive drugs have divergent effects on the modulation of CKR in maturing DC. Dexamethazone (DEX) and IL-10 inhibited human DC migration to CCL19 in vitro and mouse DC migration to lymph nodes (LN) in vivo, by impairing CCR7 expression. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) were characterized by the inability to modulate CKR expression and migratory activity. Rapamycin (RAPA) increased DC migration to CCL19 in vitro and to LN in vivo by enhancing CCR7 expression. This effect could be mediated, in LPS-maturing DC, by the inhibition of autocrine IL-10 production. The in vivo data obtained with ex vivo RAPA treated DC were confirmed in a model of in vivo drug administration in mice, suggesting a potential clinical relevance. These findings demonstrate that immunosuppressive agents differently modulate the CKR switch associated with maturing DC; in particular, RAPA selectively up-regulates CCR7 and enhances the migration of differentiated DC to regional LN. This study contributes to a better understanding of the role of immunosuppressive therapy on DC migration, a potentially relevant check point of immunosuppressive treatment.

High dexamethasone concentration prevents stimulatory effects of TNF-α and LPS on IL-6 secretion from the precursors of human muscle regeneration

A frequent finding in patients surviving critical illness myopathy is chronic muscle dysfunction. Its pathogenesis is mostly unknown; one explanation could be that muscle regeneration, which normally follows myopathy, is insufficient in these patients because of a high glucocorticoid level in their blood. Glucocorticoids can prevent stimulatory effects of proinflammatory factors on the interleukin (IL)-6 secretion, diminishing in this way the autocrine and paracrine IL-6 actions known to stimulate proliferation at the earliest, myoblast stage of muscle formation. To test this hypothesis, we compared the effects of major proinflammatory agents [tumor necrosis factor (TNF)-alpha and endotoxin lipopolysaccharide (LPS)] on the IL-6 secretion from the muscle precursors and then studied the influence of dexamethasone (Dex) on these effects. Mononuclear myoblasts, which still proliferate, were compared with myotubes in which this capacity is already lost. For correct interpretation of results, cultures were examined for putative apoptosis and necrosis. We found that constitutive secretion of IL-6 did not differ significantly between myoblasts and myotubes; however, the TNF-alpha- and LPS-stimulated IL-6 release was more pronounced (P < 0.001) in myoblasts. Dex, applied at the 0.1-100 nM concentration range, prevented constitutive and TNF-alpha- and LPS-stimulated IL-6 release at both developmental stages but only at high concentration (P < 0.01). Although there are still missing links to it, our results support the concept that high concentrations of glucocorticoids, met in critically ill patients, prevent TNF-alpha- and LPS-stimulated IL-6 secretion. This results in reduced IL-6-mediated myoblast proliferation, leading to the reduced final mass of the regenerated muscle.

Duration of CTL activation regulates IL‐2 production required for autonomous clonal expansion

The extent and magnitude of CD8(+) T lymphocyte (CTL) clonal expansion in vivo depends on the duration of stimulation provided during primary activation, whereas expansion under conventional in vitro conditions fails to reveal this. The molecular details of this differential programming are unclear. We developed a low-density culture system that recapitulates in vivo conditions and enables analysis of both proliferation and survival/apoptosis during primary expansion. We found that CTL given a prolonged stimulus produced more IL-2 and for a longer period than briefly activated CTL, and the IL-2 produced during the expansion phase was essential for cell proliferation and accumulation at low densities. CTL given a short stimulation and plated at low density followed the fate of briefly stimulated cells in vivo and underwent an abortive expansion and apoptosis, unless IL-2 was exogenously provided. Inhibition of apoptosis did not replace the requirement for IL-2 to drive clonal expansion. CTL primed for prolonged periods but in the absence of costimulation similarly behaved as though "partially programmed", undergoing abortive expansion at low densities. These results indicate that the nature and duration of antigenic stimulation regulate the autonomy of CTL expansion via the production of autocrine IL-2.

LACK‐reactive CD4+ T cells require autocrine IL‐2 to mediate susceptibility to Leishmania major

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC). The generation of MPE is largely regulated by vascular endothelial growth factor (VEGF), and upregulation of VEGF by Stat3 has been observed in several types of tumor cells. In this study, we demonstrate constitutively activated Stat3 in several human lung cancer cell lines and in tumor cells infiltrated in the pleurae of patients with adenocarcinoma cell lung cancer (ADCLC) and MPE. The observations suggest that activated Stat3 plays a role in the pathogenesis of ADCLC. In PC14PE6/AS2 cells, a Stat3-positive human ADCLC cell line, autocrine IL-6 activated Stat3 via JAKs, not via Src kinase. PC14PE6/AS2 cells express higher VEGF mRNA and protein than do Stat3-negative PC14PE6/AS2/dnStat3 cells. In an animal model, PC14P6/AS2/dnStat3 cells produced no MPE and less lung metastasis than did PC14P6/AS2 cells. PC14PE6/AS2 cells also expressed higher VEGF protein, microvessel density, and vascular permeability in tumors than did PC14P6/AS2/dnStat3 cells. Therefore, we hypothesize that autocrine IL-6 activation of Stat3 in ADCLC may be involved in the formation of malignant pleural effusion by upregulating VEGF. Higher levels of IL-6 and VEGF were also found in the pleural fluids of patients with ADCLC than in patients with congestive heart failure. The autocrine IL-6/Stat3/VEGF signaling pathway may also be activated in patients with ADCLC and MPE. These findings provide novel targets for the management of MPE.

Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin

Proliferation of Ag-specific T cells is central to the development of protective immunity. The concomitant stimulation of the TCR and CD28 programs resting T cells to IL-2-driven clonal expansion. We report that a prolonged occupancy of the TCR and CD28 bypasses the need for autocrine IL-2 secretion and sustains IL-2-independent lymphocyte proliferation. In contrast, a short engagement of the TCR and CD28 only drives the expansion of cells capable of IL-2 production. TCR/CD28- and IL-2-driven proliferation revealed a different requirement for PI3K and for the mammalian target of rapamycin (mTOR). Thus, both PI3K and mTOR activities were needed for T cells to proliferate to TCR/CD28-initiated stimuli and for optimal cyclin E expression. In contrast, either PI3K or mTOR were sufficient for IL-2-driven cell proliferation as they independently mediated cyclin E induction. Interestingly, rapamycin delayed cell cycle entry of IL-2-sufficient T cells, but did not prevent their expansion. Together, our findings indicate that the TCR, CD28, and IL-2 independently control T cell proliferation via distinct signaling pathways involving PI3K and mTOR. These data suggest that Ag persistence and the availability of costimulatory signals and of autocrine and paracrine growth factors individually shape T lymphocyte expansion in vivo.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

The Humanized Anti-CD40 Monoclonal Antibody SGN-40 Targets Hodgkin's Disease Cells through Multiple Mechanisms.

CD40 is a member of the TNF receptor superfamily expressed by multiple lineages within the hematopoietic system that carries out essential functions in the maturation of antigen-presenting cells, cross-priming of cytotoxic T cells, initiation of T cell-dependent antibody responses, and Ig class-switching. CD40 is widely expressed by lymphoid malignancies including B-cell precursor acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). SGN-40, a humanized version of the murine anti-CD40 monoclonal antibody S2C6, mediates antibody-dependent cellular cytotoxicity (ADCC) against both NHL and MM cells, directly induces apoptosis in NHL lines, and inhibits the proliferative response of MM cells toward autocrine IL-6 through down-modulation of the IL-6 receptor. Based on these activities, SGN-40 is currently in phase I clinical trials for relapsed and/or refractory MM and NHL. In this study, we examined the expression of CD40 on primary tumors isolated from 93 independent patients with HD. Seventy nine of these 93 cases (85%) demonstrated expression of CD40 by both the Reed-Sternberg cells as well as the infiltrating mononuclear cells. In a 1+ to 3+ scoring system, 35% of cases showed intense 3+ staining, 24% were positive (2+), and 26% were weakly positive (1+). Flow cytometry revealed that 8 of 9 HD cell lines expressed surface CD40, with receptor numbers ranging from 2,000 to > 370,000. SGN-40 induced CD40-positive HD cell lysis through ADCC in a dose-dependent manner, with IC50 values < 10 ng/mL. Fc-dependent effector function of SGN-40 was further exemplified by its ability to enhance macrophage mediated antibody-dependent cellular phagocytosis (ADCP). Specific ADCP against CD40-positive target cells was detected when SGN-40 was present at concentrations > 10 ng/mL. Furthermore, direct anti-proliferation signaling by SGN-40 in HD cells was also observed. In the L-1236 cell line, SGN-40 at doses ≥ 100 ng/ml reduced DNA synthesis to 25% of the untreated cells as measured by 3H-thymidine incorporation assays. Taken together, these data suggest that HD may be a suitable therapeutic target for SGN-40.

Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.