Granulocyte macrophage colony-stimulating factor contributes to enhanced monocyte survival in chronic atopic dermatitis.

Evidence suggesting that prolonged effector cell survival may contribute to perpetuation of inflammation prompted us to ask whether monocyte macrophages, the predominate inflammatory cell in the lesion of chronic atopic dermatitis (AD), exhibit enhanced survival in AD. Cultures of peripheral blood monocytes from patients with chronic AD, psoriasis, and from normal (NL) donors were examined for morphologic features and DNA fragmentation characteristic of cells undergoing the process of apoptosis (programmed cell death). Cultures of AD monocytes exhibited a significantly lower incidence of apoptosis than did cultures of NL monocytes (45 vs 68%, P < 0.01), or psoriatic monocytes (45 vs 80%, P < 0.01). Furthermore, AD monocytes were unresponsive to both IL-1, an inhibitor of apoptosis, and IL-4, an enhancer of apoptosis, in comparison to cultured NL monocytes. Of note, GM-CSF in a concentration-dependent fashion, decreased the incidence of apoptosis in NL monocyte cultures and rendered them unresponsive to these cytokines. These findings suggested that GM-CSF may enhance monocyte survival in AD. In support of this hypothesis, AD monocyte cultures produced fivefold more GM-CSF than did cultures of NL monocytes or psoriatic monocytes (P < 0.05). Additionally, there was a significantly greater number of GM-CSF mRNA expressing cells detected by in situ hybridization in biopsies of lesions of chronic AD than in acute AD or NL skin (P < 0.05). Finally, NL monocytes incubated with supernatants obtained from monocytes of AD patients exhibited significant inhibition of apoptosis, an effect that could be ablated by a neutralizing antibody to GM-CSF. Taken together, these data strongly suggest that increased production of GM-CSF by cells from patients with AD inhibits monocyte apoptosis and may contribute to the chronicity of this inflammatory disease.

Verapamil reversal of doxorubicin resistance in multidrug-resistant human myeloma cells and association with drug accumulation and DNA damage.

Verapamil reversed resistance to doxorubicin in a human multiple myeloma cell line selected for multiple drug resistance. The drug-resistant cell line 8226/DOX40 is known to have reduced intracellular drug accumulation associated with the overexpression of P-glycoprotein when compared to the sensitive parent cell line 8226/S. Verapamil alone was minimally cytotoxic in both cell lines, but reversed doxorubicin resistance in a dose-related manner in 8226/DOX40. A similar dose-response relationship was observed for verapamil in increasing net intracellular doxorubicin accumulation. This increased net accumulation was secondary to block of enhanced doxorubicin efflux by verapamil from resistant cells. In contrast, verapamil did not alter initial doxorubicin accumulation over the first 60 s when incubated with resistant cells. Addition of verapamil to the 8226/DOX40 cells enhanced the formation of doxorubicin-induced DNA single strand breaks, double strand breaks, and DNA-protein cross-links. Verapamil had no effect on these lesions in the drug-sensitive cells. In addition, verapamil did not affect chemotherapeutic cytotoxicity or transport in the drug-sensitive cell line. Verapamil appears to reverse doxorubicin resistance in this human myeloma cell line by blocking enhanced drug efflux, leading to increased drug accumulation and enhanced DNA damage.

A model for the extracellular release of PAF: the influence of plasma membrane phospholipid asymmetry.

Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably release, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native membrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that 'acceptor' molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular 'acceptor' molecules.

The mechanism of internalization of platelet-activating factor in activated human neutrophils. Enhanced transbilayer movement across the plasma membrane.

Recent studies suggest that cellular internalization of platelet-activating factor (PAF), a potent ether phospholipid mediator of inflammation, is modulated by, as yet undefined cellular mechanisms. Using an albumin extraction method, the internalization of PAF and several PAF analogues was studied in the resting and stimulated human neutrophil. Our data demonstrate that internalization of these analogues is largely dependent on the state of cellular activation and that the process is not specific for certain unique structural features of the PAF molecule including the 1-position ether linkage, 2-position acetyl substitution, or choline polar head group. Furthermore, the internalization process was shown not to be dependent on the PAF receptor, metabolism of the molecule, or the process of endocytosis. Data are presented to suggest that the route of internalization of PAF is enhanced transbilayer movement (flipping) across the plasma membrane occurring as a result of changes in membrane physical properties accompanying cellular activation. It is proposed that in addition to enhanced internalization of PAF, modulation of PAF biosynthesis and net release from the stimulated neutrophil may be consequences of enhanced transbilayer movement of PAF across the activated plasma membrane.

Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase.

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBD-labeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED50 of nearly 100 microM, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance.

Staphylococcal toxic shock syndrome toxin-1 inhibits monocyte apoptosis.

BACKGROUND: Chronic atopic dermatitis (AD) lesions are associated with colonization by exotoxin-producing Staphylococcus aureus. Evidence suggests that cytokine production in AD, particularly of GM-CSF, prolongs survival of both peripheral blood monocytes and dermal monocyte-macrophages, the predominate inflammatory cell in lesions caused by chronic AD. OBJECTIVE: We sought to determine whether the staphylococcal exotoxin, toxic shock syndrome toxin-1 (TSST-1), could stimulate prosurvival cytokine production in monocytes and thereby inhibit apoptosis. METHODS: Cultures of peripheral blood monocytes from normal donors and subjects with AD were incubated with various concentrations of TSST-1, and the incidence of apoptosis was assessed by examining cytospin preparations and the appearance of hypodiploid DNA in the flow cytometer. Culture supernatants were analyzed for GM-CSF, IL-1beta, and TNF-alpha by ELISA. RESULTS: TSST-1, in a concentration-dependent manner starting at 0.1 pg/mL, significantly inhibited monocyte apoptosis and resulted in the production of the prosurvival cytokines GM-CSF, IL-1beta, and TNF-alpha. In coculture conditions with conditioned media from TSST-1-stimulated monocytes, with or without neutralizing antibody to the various cytokines, the data show GM-CSF production was responsible for the inhibition of apoptosis. CONCLUSIONS: The data strongly suggest that staphylococcal exotoxins known to colonize skin lesions on patients with chronic AD may induce the production of GM-CSF, resulting in inhibition of monocyte-macrophage apoptosis, and thereby contribute to the chronicity of this inflammatory disease.

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta.

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.