Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Host factors in metastasis: Direct lysis of bloodborne metastatic tumor cells by murine vascular endothelial cells

During the process of cancer metastasis, the majority of circulating tumor cells arrest in microcapillary beds and then rapidly die. To study whether vascular endothelial cells can directly lyse tumor cells, we isolated vascular endothelial cells by perfusion of lungs from immunocompetent or nude mice. The cells were grown in culture, and then cloned and characterized. Cloned endothelial cells were incubated with several lymphokines and cytokines. Cells incubated with IFN-$\gamma$ and TNF lysed a variety of tumor cells with different metastatic potential. Mouse skin and lung fibroblasts treated with the same cytokines did not. Endothelial cell mediated tumor cell lysis was not due to different binding ability of tumor cells to cytokine treated and untreated endothelial monolayers. Kinetic studies demonstrated that the continuous presence of cytokines in the tumor-endothelial cocultures was necessary to produce maximal lysis of tumor cells. Target cell lysis was not due to the direct effects of IFN-$\gamma$ or TNF, since vascular endothelial cells isolated from the lung of nude mice lysed human melanoma cells that are sensitive or resistant to TNF. Cytokine treated endothelial cells produced a high level of nitric oxide, which is known to be cytotoxic to a variety of target cells. The level of nitric oxide production was directly correlated with the degree of tumor cell lysis. A specific inhibitor of nitric oxide synthesis(N$\sp{\rm G}$-monomethyl-L-arginine), completely inhibited production of nitric oxide and tumor cell lysis. Treatment of cytokine activated endothelial cells with dexamethasone also inhibited tumor cell lysis. This inhibition was independent of tumor-endothelial adhesion but correlated with inhibition of nitric oxide production. Collectively, these results suggest that vascular endothelial cells can directly destory tumor emboli and thus play an active role in the pathogenesis of cancer metastasis. ^

The role of nitric oxide in apoptosis and cancer metastasis

The purpose of these studies was to investigate the role of nitric oxide (NO) in tumor metastasis. K-1735 Metastatic cells survived in blood circulation to produce experimental lung metastases, whereas nonmetastatic cells did not. After incubation with combination cytokines or lipopolysaccharide (LPS), nonmetastatic cells exhibited high levels of inducible nitric oxide synthase (iNOS) activity and NO production, whereas metastatic cells did not. The production of NO directly correlated with cytotoxic effects of cytokines or LPS. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases, highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.^ Furthermore, multiple systemic administrations of multilamellar vesicle-liposomes (MLV) containing the lipopeptide CGP 31362 (MLV-31362) or MLV-31362 combined with murine interferon-gamma (IFN-$\gamma$) eradicated the metastases by M5076 reticular cell sarcoma. Tumor regression correlated with iNOS expression within the tumor lesions and with increased NO production. The administration of NMA significantly decreased NO production and diminished the antitumor activities. These data imply that the activation of iNOS can serve as a target for immunotherapeutic agents for treatment of murine reticulum cell sarcoma metastases. ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Differentiative requirements for human CD8$\sp+$ suppressor lymphocytes

The aim of this research was to characterize the differentiative requirements of human CD8$\sp{+}$ suppressor lymphocytes. We investigated the role of monocytes in cellular interactions required for generation of T suppressor cells (Ts) in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMC). We observed that the functional activity of CD8$\sp{+}$ T cells was dependent on the concentration of monocytes in the inductive cultures; at concentrations normally present in peripheral blood, PWM stimulation induced potent suppressor activity, whereas under conditions of moderate monocyte depletion the same phenotypic subset of CD8$\sp{+}$ cells enhanced responses. We also demonstrated that differentiation of CD8$\sp{+}$CD28$\sp{-}$ suppressor cells could be mediated by soluble products elaborated by monocytes and CD4$\sp{+}$ cells, identified as PGE$\sb2$ and IFN$\gamma$ respectively. These two signals were required sequentially to cause Ts induction. That is PGE$\sb2$ was required initially, followed by an IFN$\gamma$-dependent differentiative step. We also explored the possibility that PGE$\sb2$ caused modulation of the IFN$\gamma$ receptor number and/or affinity on CD8$\sp{+}$ cells, which might render these cells responsive to the differentiative effect of the IFN$\gamma$-signal. Using radiolabelled $\sp{125}$I-IFN$\gamma$, direct binding assays demonstrated that 10$\sp{-8}$M PGE$\sb2$ selectively increased the number of receptors on the CD8$\sp{+}$ cells. In contrast CD4$\sp{+}$ cells treated similarly exhibited no significant change in their number of IFN$\gamma$ receptors. These results, thus, suggest a relationship between PGE$\sb2$ induced expression of IFN$\gamma$ receptor and the initial requirement for PGE$\sb2$ in IFN$\gamma$-dependent differentiation of Ts cells. Together, our results suggest a crucial role for PGE$\sb2$ and IFN$\gamma$ in regulation of the immune response. Furthermore, such detailed definition of the differentiative requirements for CD8$\sp{+}$ suppressor cells should provide new insight into fundamental mechanisms of immunoregulation. ^

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^

Suppression of T lymphocyte activation in simulated microgravity

Immune dysfunction is encountered during spaceflight. Various aspects of spaceflight, including microgravity, cosmic radiation, and both physiological and psychological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. Clinostatic RWV bioreactors that simulate aspects of microgravity were used to analyze the response of human PBMC to polyclonal and oligoclonal activation. PHA responsiveness in the RWV bioreactor was almost completely diminished. IL-2 and IFN-$\gamma$ secretion was reduced whereas IL-1$\beta$ and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Antigen specific T cell activation, including the mixed-lymphocyte reaction, tetanus toxoid responsiveness, and Borrelia activation of a specific T cell line, was also suppressed in the RWV bioreactor.^ The role of altered culture conditions in the suppression of T cell activation were considered. Potential reduced cell-cell and cell-substratum interactions in the RWV bioreactor may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions was not affected. Furthermore, increasing cell-population density, and therefore cell-cell interactions, in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Finally, activation of purified T cells with crosslinked CD2/CD28 or CD3/CD28 antibody pairs, which does not require costimulation through cell-cell contact, was completely suppressed in the RWV bioreactor suggesting a defect internal to the T cell.^ Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation in simulated microgravity, there is a specific dysfunction within the T cell involving the signaling pathways upstream of PKC activation. ^

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^

IMMUNOLOGICAL AND CYTOLOGICAL INVESTIGATION OF GAMMA INTERFERON PRODUCTION IN HUMAN T-LYMPHOCYTES INDUCED BY PMA AND PHA (IMMUNOGOLD, CELL CYCLE ANALYSIS)

The present study examined cellular mechanisms involved in the production and secretion of human (gamma)IFN. The hypothesis of this investigation was that (gamma)IFN is an export glycoprotein whose synthesis in human T lymphocytes is dependent on membrane stimulation, polypeptide synthesis in the rough endoplasmic reticulum, packaging in the Golgi complex, and release from the cell by exocytosis.^ The model system for this examination utilized T lymphocytes from normal donors and patients with chronic lymphocytic leukemia (CLL) induced in vitro with the tumor promoter, phorbol 12-myristate 13-acetate (PMA) and the lectin, phytohemagglutinin (PHA) to produce (gamma)IFN. This study reconfirmed the ability of PMA and PHA to synergistically induce (gamma)IFN production in normal T lymphocytes, as measured by viral inhibition assays and radio-immunoassays for (gamma)IFN. The leukemic T cells were demonstrated to produce (gamma)IFN in response to treatment with PHA. PMA treatment also induced (gamma)IFN production in the leukemic T cells, which was much greater than that observed in similarly treated normal T cells. In these same cells, however, combined treatment of the agents was shown to be ineffective at inducing (gamma)IFN production beyond the levels stimulated by the individual agents. In addition, the present study reiterated the synergistic effect of PMA/PHA on the stimulation of growth kinetics in normal T cells. The cell cycle of the leukemic T cells was also responsive to treatment with the agents, particularly with PMA treatment. A number of morphological alterations were attributed to PMA treatment including the acquisition of an elongated configuration, nuclear folds, and large cytoplasmic vacuoles. Many of the effects were observed to be reversible with dilution of the agents, and reversion to this state occurred more rapidly in the leukemic T cells. Most importantly, utilization of a thin section immuno-colloidal gold labelling technique for electron microscopy provided, for the first time, direct evidence of the cellular mechanism of (gamma)IFN production and secretion. The results of this latter study support the idea that (gamma)IFN is produced in the rough endoplasmic reticulum, transferred to the Golgi complex for accumulation and packaging, and released from the T cells by exocytosis. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

The cyclotron production and nuclear imaging of bromine-77

In this investigation, bromine-77 was produced with a medical cyclotron and imaged with gamma cameras. Br-77 emits a 240 kev photon with a half life of 56 hours. The C-Br bond is stronger than the C-I bond and bromine is not collected in the thyroid. Bromine can be used to label many organic molecules by methods analogous to radioiodination. The only North American source of Br-77 in the 70's and 80's was Los Alamos National Laboratory, but it discontinued production in 1989. In this method, a p,3n reaction on Br-77 produces Kr-77 which decays with a 1.2 hour half life to Br-77. A cyclotron generated 40 MeV proton beam is incident on a nearly saturated NaBr or LiBr solution contained in a copper or titanium target. A cooling chamber through which helium gas is flowed separates the solution from the cyclotron beam line. Helium gas is also flowed through the solution to extract Kr-77 gas. The mixture flows through a nitrogen trap where Kr-77 freezes and is allowed to decay to Br-77. Eight production runs were performed, three with a copper target and five with a titanium target with yields of 40, 104, 180, 679, 1080, 685, 762 and 118 uCi respectively. Gamma ray spectroscopy has shown the product to be very pure, however corrosion has been a major obstacle, causing the premature retirement of the copper target. Phantom and in-vivo rat nuclear images, and an autoradiograph in a rat are presented. The quality of the nuclear scans is reasonable and the autoradiograph reveals high isotope uptake in the renal parenchyma, a more moderate but uniform uptake in pulmonary and hepatic tissue, and low soft tissue uptake. There is no isotope uptake in the brain or the gastric mucosa. ^

The role of endogenous IFN-beta in cutaneous melanoma: Consequences of epidermal hyperplasia

The purpose of this study was to characterize epidermal hyperplasia overlying malignant melanoma, to determine the mitogenic factor responsible for the induction of this hyperplasia and to investigate its biological consequence. Whether increased keratinocyte proliferation overlying melanoma is due to production of growth factors by the tumor cells or to other mechanisms is unknown. Epidermal hyperplasia overlying human melanoma was found overlying thick (>4.0mm), but not thin (<1.0mm) tumors. Immunostaining of the sections for growth factors related to angiogenesis revealed that epidermal hyperplasia was associated with loss of IFN-β production by the keratinocytes directly overlying the tumors. Since previous studies from our laboratory have demonstrated that exogenous administration of IFN-β negatively regulates angiogenesis, we hypothesize that tumors are able to produce growth factors which stimulate the proliferation of cells in the surrounding tissues. This hyperplasia leads to a decrease in the endogenous negative regulator of angiogenesis, IFN-β. ^ The human melanoma cell line, DM-4 and several of its clones were studied to identify the mitogenic factor for keratinocytes. The expression of TGF-α directly correlated with epidermal hyperplasia in the DM-4 clones. A375SM, a human melanoma cell line that produces high levels of TGF-α, was transfected with a plasmid encoding full-length antisense TGF-α. The parental and transfected cells were implanted intradermally into nude mice. The extent of epidermal hyperplasia directly correlated with expression of TGF-α and decreased production of IFN-β, hence, increased angiogenesis. ^ In the next set of experiments, we determined the role of IFN-β on angiogenesis, tumor growth and metastasis of skin tumors. Transgenic mice containing a functional mutation in the receptor for IFN α/β were obtained. A375SM melanoma cells were implanted both s.c. and i.v. into IFN α/βR −/− mice. Tumors in the IFN α/β R −/− mice exhibited increased angiogenesis and metastasis. IFN α/βR −/− mice were exposed to chronic UV irradiation. Autochthonous tumors developed earlier in the transgenic mice than the wild-type mice. ^ Collectively, the data show that TGF-α produced by tumor cells induces proliferation of keratinocytes, leading to epidermal hyperplasia overlying malignant melanoma associated with loss of IFN-β and enhanced angiogenesis, tumorigenicity and metastasis. ^

The role of MEKK3 in T cell homeostasis and IFNgamma production

MEKK3, a member of the MAP3K family, is involved in regulating multiple MAPK and NF-κB pathways. The MAPK and NF-κB signaling pathways are important in regulating T cell functions. MEKK3 is expressed through the development of T cell and also in subsets of T cell in the peripheral. However, the specific role of MEKK3 in T cell function is unknown. To reveal the in vivo function of MEKK3 in T cells, I have generated MEKK3 T cell conditional knock-out mice. Despite a normal thymus development in the conditional knock-out mice, I observed a decrease in the number of peripheral T-cells and impaired T-cell function in response to antigen stimulation. T cells undergo homeostatic proliferation under lymphopenia condition, a process called lymphopenia-induced proliferation (LIP). Using a LIP model, I demonstrated that the reduction of peripheral T cell number is largely due to a severe impairment of the self-antigen/MHC mediated T cell homeostasis. Upon anti-CD3 stimulation, the proliferation of MEKK3-deficient T cell is not significantly affected, but the production of IFNγ by naïve and effector CD4 T cells are markedly decreased. Interestingly, the IL-12/IL-18 driven IFNγ production and MAPK activation in MEKK3-deficient T cells is not affected, suggesting that MEKK3 selectively mediates the TCR induced MAPK signaling. Furthermore, I found that MEKK3 is activated by TCR stimulation in a RAC1/2 dependent manner, but not by IL-12/IL-18 stimulation. Finally, I showed that basal level of ERK and JNK activation is defective under LIP condition. I showed that the TCR induced ERK, JNK and p38 MAPK activation is also defective in MEKK3 deficient CD4 T cells. Taken together, my data demonstrate a crucial role of MEKK3 in T cell homeostasis and IFNγ production through regulating the TCR mediated MAPK pathway. ^