Adriamycin sensitizes the adriamycin-resistant 8226/Dox40 human multiple myeloma cells to Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-mediated (TRAIL) apoptosis.

The newly discovered member of the tumor necrosis factor superfamily, Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been identified as an apoptosis-inducing agent in sensitive tumor cells but not in the majority of normal cells, and hence it is of potential therapeutic application. However, many tumor cells are resistant to Apo2L/TRAIL-mediated apoptosis. Various chemotherapeutic drugs have been shown to sensitize tumor cells to members of the tumor necrosis factor family. However, it is not clear whether sensitization by drugs and sensitivity to drugs are related or distinct events. This study examined whether an Adriamycin-resistant multiple myeloma (MM) cell line (8226/Dox40) can be sensitized by Adriamycin (ADR) to Apo2L/TRAIL-mediated apoptosis. Treatment with the combination of Apo2L/TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental 8226/S and the 8226/Dox40 tumor cells. Adriamycin treatment modestly up-regulated Apo2L/TRAIL-R2 (DR5) and had no effect on the expression of Fas-associated death domain, c-FLIP, Bcl-2, Bcl(xL), Bax, and IAP family members (cIAP-1, cIAP-2, XIAP, and survivin). The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR, whereas pro-caspase-9 and Apaf-1 were up-regulated. Combination treatment with Apo2L/TRAIL and ADR resulted in significant mitochondrial membrane depolarization and activation of caspase-9 and caspase-3 and apoptosis. Because ADR is shown to sensitize ADR-resistant tumor cells to Apo2L/TRAIL, these findings reveal that ADR can still signal ADR-resistant tumor cells, resulting in the modification of the Apo2L/TRAIL-mediated signaling pathway and apoptosis. These in vitro findings suggest the potential application of combination therapy of Apo2L/TRAIL and subtoxic concentrations of sensitizing chemotherapeutic drugs in the clinical treatment of drug-resistant/Apo2L/TRAIL-resistant multiple myeloma.

Preferential induction of TNF-alpha and IL-1beta and inhibition of IL-10 secretion by human peripheral blood monocytes by synthetic aza-alkyl lysophospholipids.

Several newly synthesized aza-alkyl lysophospholipids (AALP) have been shown to exert a potent antitumor cytotoxicity in vitro. Their potential use in vivo prompted us to study their effects on the immune system. The present study investigated the effect of AALP on the secretion of cytokines (TNF-alpha, IL-1beta, IL-6, and IL-10) by normal and activated human peripheral blood-derived monocytes (PBM). Five AALP compounds (BN52205, BN52207, BN52211, BN52218, and BN52227) were tested. Human peripheral blood monocytes were cultured for 18 h at 37 degrees C in the presence of AALP and/or LPS (1 microg/ml) or IFN-gamma (1000 U/ml) and the supernatants tested for the presence of cytokines by ELISA. All five AALP compounds stimulated TNF-alpha and IL-1beta secretion but not IL-6 secretion from nonstimulated PBM. There were no significant differences among the five AALP compounds tested and BN52207 was selected for further studies. Secretion of TNF-alpha was significantly potentiated by BN52207 when the PBM were activated by either IFN-gamma or LPS. There was also an upregulation of TNF-alpha mRNA transcription as detected by RT-PCR. The induction of TNF-alpha secretion by BN52207 was dependent on de novo protein synthesis as the specific TNF-alpha inhibitor, pentoxifylline, and the protein synthesis inhibitors, cyclohexamide and emetine, abolished TNF-alpha secretion. BN52207 also stimulated IL-1beta secretion by resting and activated PBM in a concentration-dependent manner. Unlike TNF-alpha and IL-1beta, however, BN52207 had no effect on IL-6 secretion. Noteworthy, unlike the induction of TNF-alpha and IL-1beta secretion, BN52207 inhibited completely the secretion of IL-10 by resting and LPS-activated PBM. Further, BN52207 enhanced the macrophage killing activity of tumor target cells. Overall, this study demonstrates that AALP are endowed with a selective regulation of cytokine synthesis and secretion by resting and activated PBM. This regulation is manifested by upregulating TNF-alpha and IL-1beta secretion and abolishing IL-10 secretion. The selective regulation of cytokine synthesis and secretion by AALP suggest that AALP may have potential therapeutic uses in vivo in clinical disease manifestations that are regulated by cytokines.

Cocaine enhances monocyte migration across the blood-brain barrier. Cocaine's connection to AIDS dementia and vasculitis?

Cocaine has wide-ranging effects on the immune and neuroendocrine systems (Fiala et al., 1996) resembling an inflammatory "stress" response with upregulation of pro-inflammatory cytokines and stimulation of the HPA axis (Gan et al., 1997). Cocaine abuse has also been associated with vascular pathology, including vasculitis, vasospasm and hemorrhage. These effects suggest that cocaine could perturb the function of endothelial cells, including the blood-brain barrier, and influence the progression to AIDS in HIV-infected individuals (Shapshak et al., 1997; Goodkin et al., 1997). In order to understand clinical consequences of cocaine abuse, it is important to gain insight into molecular and cellular basis of cocaine's effects on immune and endothelial cells. Cocaine's in vitro effects on (a) permeability, (b) immune cell migration, (c) adhesion molecules, and (d) cytokine expression were investigated in a blood-brain barrier model constructed with brain microvascular endothelial cells and fetal astrocytes with the following results: (a) cocaine and tumor necrosis factor-alpha (TNF-alpha) increased the model's permeability to inulin similarly in a dose-responsive fashion; (b) cocaine (10(-4) to 10(-8_ M) enhanced monocyte migration across the barrier with the maximum increase, approximately 100%, by 10(-5) M cocaine; (c) cocaine treatment also increased the expression of endothelial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecules-1 (VCAM-1) and platelet/endothelial cell adhesion molecule-1 (PECAM-1); (d) although the cocaine in vitro effects on cytokine production by mononuclear cells have been difficult to assess due to a heterogeneity in the degree of responsiveness between individuals, the data suggest that mononuclear cells from cocaine addicts are sensitized to in vitro cocaine challenge with hypersecretion of inflammatory cytokines. Cocaine's in vivo manifestations are compatible with these in vitro effects: (A) chronic cocaine treatment of rats significantly increased rolling white blood cell flux, leukocyte-endothelium adhesion, and ICAM-1 expression in the mesentery (House et al., 1996); (B) cocaine injection to cocaine-dependent subjects tipped the balance of cytokine secretion by mononuclear cells to Th1-type (Gan et al., 1997), and (C) cocaine injection stimulated the hypothalamic-pituitary axis (HPA) to increase both anti- and pro-inflammatory hormonal secretion. Collectively, these results suggest that the immune effects of cocaine on endothelial, immune and neuroendocrine cells impair the function of the blood-brain, barrier, increase cell emigration from the blood vessels, in particular into the brain, and may cause vasculitis. These effects could also increase importation of HIV-1 into the brain.

Differential secretion of TNF-alpha and IFN-gamma by human peripheral blood-derived NK subsets and association with functional maturation.

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF-alpha and IFN-gamma by both the binder and the killer subsets but not by the free subset. IFN-alpha activated the secretion of IFN-gamma only, whereas IL-2 activated the secretion of both TNF-alpha and IFN-gamma by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF-alpha and IFN-gamma secretion in both the binder and the killer subsets, though IFN-gamma secretion was more pronounced in the binder subset. Activation of TNF-alpha and IFN-gamma secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-gamma. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF-alpha and IFN-gamma, whereas the free NK subset secretes little or no TNF-alpha and IFN-gamma following activation. These data suggest that the ability of NK cells to secrete TNF-alpha and IFN-gamma following activation correlates with the functional stage of maturation of NK cells.

Natural killer cell anergy to cytokine stimulants in a subgroup of patients with heart failure: relationship to norepinephrine.

Heart failure is a disease characterized by chronically high levels of plasma norepinephrine and anergy in the cytotoxicity of circulating natural killer (NK) lymphocytes. This study shows that NK anergy extends to a significantly reduced cytotoxicity in response to the powerful NK stimulants, interleukin (IL)-2 and interferon (IFN)-alpha. Fifteen patients with heart failure, New York Heart Association stage III or IV, were studied for NK-cell-mediated cytotoxicity. The patients were divided into two groups based upon their NK cytotoxicity function: (1) those who had minimal baseline cytotoxicity and failed to respond following stimulation by IL-2 and IFN-alpha (n = 6), and (2) those who were about at the level of normal controls, and were responsive to IL-2 and IFN-alpha (n = 9). There was no relationship between the anergy and the etiology of the heart failure, laboratory indicators of heart failure, serum albumin or sodium, state anxiety, age or sex of the subjects. There was a statistically significant negative correlation between the response of NK cells to the stimulators IL-2 and IFN-alpha and the level of plasma norepinephrine in the heart failure patients. This was corroborated by in vitro testing of direct effects of norepinephrine on normal NK cells, which indicated that baseline cytotoxicity and the ability of these cells to respond to IL-2 were inhibited in a dose-dependent manner. The findings indicate that the NK cell anergy seen in heart failure patients extends to the response to the stimulators IL-2 and IFN-alpha in a subgroup of patients.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion but not IL-6 from activated human peripheral blood monocytes by a new synthetic demethylpodophyllotoxin derivative.

A newly synthesized demethylpodophyllotoxin derivative, 4-O-butanoyl-4'-demethylpodophyllotoxin (BDPT) or BN58705, has recently been shown to exert a potent cytotoxic activity in vitro against a variety of drug-resistant human tumor cell lines. The effect of this agent on effector cells of the immune system, however, has not been examined. The present study investigated the effect of BDPT on the response of activated human peripheral blood derived monocytes (PBM) to secrete cytokines. Activation of PBM overnight with LPS, IFN-gamma, or PMA resulted in secretion into the supernatant of TNF-alpha, IL-1 beta, IL-6, and IL-8 as assessed by ELISA. The addition of BDPT to the stimulated cultures resulted in significant inhibition of TNF-alpha and IL-1 beta secretion, whereas the secretion of IL-6 and IL-8 was not affected. The selective inhibition of TNF-alpha and IL-1 beta secretion by BDPT-treated PBM was observed with all three stimuli tested. The inhibitory effect mediated by BDPT was concentration dependent and was optimal at 6-20 microM. Time kinetic analysis indicated that the inhibition of secretion was rapid and detected as soon as 2 hr following stimulation of the PBM and lasted for as long as 24 hr. A comparison was made between BDPT and pentoxyfilline, a xanthine-derived phosphodisterase inhibitor that was reported to inhibit TNF-alpha and IL-1 beta secretion by PBM. Both BDPT and PTX showed similar time kinetics and patterns of inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of human peripheral-blood-derived monocytes by cis-diamminedichloroplatinum: enhanced tumoricidal activity and secretion of tumor necrosis factor-alpha.

The anticancer agent cis-diamminedichloroplatinum (CDDP) has been shown to have immunopotentiating and immuno-suppressive properties depending on the CDDP concentration used. Treatment of human peripheral-blood-derived monocytes (PBM) in vitro with low concentrations of CDDP resulted in a significant potentiation of antitumor cytotoxicity as assessed in an 18-hour 51Cr release assay. The potentiation of cytotoxicity by CDDP was also observed with PBM treated with recombinant interferon-gamma (rIFN-gamma). The monocyte-mediated cytotoxicity was significantly inhibited when antitumor necrosis factor (TNF) antibody was added to the assay culture. The role of TNF in the cytotoxic mechanism was further corroborated by demonstrating that significant levels of immunoreactive TNF-alpha in the supernatants were detected by ELISA. Further, supernatants derived from CDDP-treated monocytes were cytotoxic to the TNF-sensitive tumor cells and the cytotoxicity was neutralized by the addition of anti-TNF antibody. The secretion of TNF-alpha by CDDP-treated monocytes was readily detected as early as 4 h after culture and was dependent on de novo protein synthesis as inhibitors of RNA and protein synthesis abolished TNF-alpha secretion. Altogether, these results demonstrate that CDDP can potentiate monocyte-mediated antitumor cytotoxicity and stimulates TNF-alpha synthesis and secretion.