Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.

Specific alterations in keratin biosynthesis in mouse epidermis in vivo and in explant culture following a single exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

Treatment of mouse skin with a single dose of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) causes dramatic alterations in the biosynthesis of specific epidermal keratins. TPA was applied either topically to the skin of mice or added to the culture medium of skin explants maintained in vitro. Twenty-four h after exposure, skin samples were pulse labeled with [35S]methionine, and epidermal proteins were extracted and resolved by two-dimensional gel electrophoresis. In whole animals, TPA caused a marked decrease in the biosynthesis of the Mr 67,000 (basic) and 59,000 (acidic) keratins, specific markers for suprabasal differentiation in the epidermis. In addition, the synthesis of an acidic keratin of Mr 48,000 and a basic keratin of Mr 62,000 was also decreased. Concomitantly, TPA caused an increase in the synthesis of a basic keratin of Mr 60,000 and acidic keratins of Mr 52,000 and 49,000, markers for proliferating cells and primary epidermal cell cultures. The normal pattern of keratin synthesis observed in explants from normal skin was similar to that observed in skin samples from animals treated with TPA, except that synthesis of the Mr 67,000 basic keratin subunit was maintained. The addition of TPA to culture medium containing the skin explants resulted in a dose-dependent decrease in the synthesis of this keratin. Furthermore, resulting patterns of keratinization were identical to epidermis treated with TPA in vivo. These results suggest that a single acute exposure to TPA alters normal differentiation of mouse epidermal cells in vivo while causing a pronounced basal cell hyperproliferation. This response can be reproduced following TPA exposure to skin explants in culture, suggesting that the changes in keratinization observed are independent of the animal. Specific keratins modulated during TPA-induced hyperplasia may serve as marker proteins for aberrant epidermal cell growth and differentiation leading to the development of neoplasia.

Effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on neurite outgrowth from chick embryo sensory ganglia.

The addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to defined growth medium stimulated an intense neurite outgrowth from chick sensory ganglia explants. The development of radial neurites was concentration dependent. At low concentrations of TPA (1.6 to 16 nM), neurites were long but moderate in numbers. At higher concentrations (160 to 480 nM), TPA produced dense, short neurites that formed thick fascicles. This reduced outgrowth in length was not related to cytotoxicity and was found to be reversible when the tumor-promoting agent was removed. Neurite outgrowth was also accompanied by an increase in proliferation of nonneuronal cells. Blocking the proliferation of nonneuronal components by the mitotic inhibitor 1-beta-D-arabinofuranosylcytosine did not inhibit neurite outgrowth, suggesting that the TPA-induced neurite differentiation was independent of nonneuronal cells. When TPA was tested in the presence of nerve growth factor, the induction of neurite differentiation was not affected. Other active tumor promoters including phorbol-12,13-didecanoate and mezerein also elicited neurite outgrowth, while nonpromoting agents such as phorbol and 4 alpha-phorbol-12,13-didecanoate were ineffective.

Effect of tumor promoters on sarc gene expression in normal and transformed chick embryo fibroblasts.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the sarc protein kinase of normal chick embryo fibroblasts (CEF) and of the src kinase of cells transformed by a temperature-sensitive mutant of avian sarcoma virus (CEF-tsASV) were studied and compared with the known effects of TPA on cell morphology and plasminogen activator (PA) activity. One hr after the addition of TPA to normal CEF, there was a 3- to 8-fold stimulation of kinase activity when compared to control cultures; during the subsequent 24 hr, TPA produced less than a 2-fold stimulation. Although TPA induced levels of PA in CEF which were equivalent to those produced by cEF-tsASV grown at 36 degrees, the latter cells contained much higher levels of kinase activity than those of CEF plus TPA. In addition, TPA failed to enhance the kinase activity of CEF-tsASV at either 36 degrees or 40 degrees, even though at both temperatures TPA induced morphological changes and markedly enhanced PA activity. These results suggest that the effects of TPA on morphology and PA are not due to an effect on these protein kinases.

Induction of chemotaxis in mouse peritoneal macrophages by phorbol ester tumor promoters.

The ability of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), to induce chemotaxis in three different populations of mouse peritoneal macrophages was studied. TPA in the range of 10(-9) to 10(-7) M produced a dose- and time-related increase in chemotaxis in resident, thioglycollate-elicited, and divinyl ether maleic anhydride copolymer-activated macrophages. A maximal response was obtained after 4 hr incubation with 10(-7) M TPA, and this concentration of TPA was as effective as inducing chemotaxis as was endotoxin-activated mouse serum. Orientation of macrophages towards TPA was also observed by microscopy. Within 2 hr, cells exposed to TPA sent out cytoplasmic processes along the TPA gradient. Parallel arrays of cells oriented towards the TPA were observed after 4 hr incubation. Two other diterpene tumor promoters, phorbol-12,13-didecanoate and mezerein, were also chemotactic for the macrophages, as was the peptide epidermal growth factor, which shares a number of effects with TPA on cells in culture. On the other hand, two phorbol esters inactive as tumor promoters, 4-alpha-phorbol-12,13-didecanoate and phorbol, were not chemotactic for macrophages. Retinoic acid, which inhibits tumor promotion, inhibited TPA-induced, but not endotoxin-activated mouse serum-induced chemotaxis. These findings, taken together with previous studies, indicate that phorbol ester tumor promoters are potent modulators of macrophage function.

5-Fluorouracil suppresses nitric oxide biosynthesis in colon carcinoma cells.

Nitric oxide is an important cellular mediator that plays a role in regulating cellular proliferation of both normal and tumor cells. In the present study, we characterized nitric oxide production by the human colon adenocarcinoma cell line DLD-1 and examined the effects of 5-fluorouracil (5-FUra), an antimetabolite effective against colon tumors, on nitric oxide production. IFN-gamma was found to be a potent inducer of nitric oxide production in DLD-1 cells. This effect was dependent on L-arginine and blocked by the nitric oxide synthase inhibitors NG-monomethyl-L-arginine, nitroarginine, and aminoguanidine. Production of nitric oxide by DLD-1 cells was due to the expression of the inducible (type II) form of nitric oxide synthase. mRNA for the nitric oxide synthase was present in both untreated and IFN-gamma-stimulated cells, as determined by RT-PCR, suggesting that expression of enzyme is regulated posttranscriptionally. Treatment of DLD-1 cells with concentrations of 5-FUra that are not growth inhibitory or cytotoxic strongly inhibited their ability to express nitric oxide synthase and produce nitric oxide in response to IFN-gamma. This effect was not reversed with thymidine, indicating that inhibition of nitric oxide production was due to incorporation of 5-FUra into RNA. However, pretreatment of DLD-1 cells with 5-FUra before stimulation with IFN-gamma also suppressed nitric oxide production. Thus, inhibition of nitric oxide production was not due directly to incorporation of 5-FUra into the mRNA for nitric oxide synthase. Taken together, these data suggest that inhibition of nitric oxide biosynthesis in colon tumor cells by 5-FUra may underlie, at least in part, the efficacy of this antitumor agent.

Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis.

Topical application of curcumin, the yellow pigment in turmeric and curry, strongly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase activity, DNA synthesis, and tumor promotion in mouse skin (Huang et al., Cancer Res., 48: 5941-5946, 1988). Chlorogenic acid, caffeic acid, and ferulic acid (structurally related dietary compounds) were considerably less active. In the present study, topical application of curcumin markedly inhibited TPA- and arachidonic acid-induced epidermal inflammation (ear edema) in mice, but chlorogenic acid, caffeic acid, and ferulic acid were only weakly active or inactive. The in vitro addition of 3, 10, 30, or 100 microM curcumin to cytosol from homogenates of mouse epidermis inhibited the metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) by 40, 60, 66, or 83%, respectively, and the metabolism of arachidonic acid to 8-HETE was inhibited by 40, 51, 77, or 85%, respectively [IC50 (concentration needed for 50% inhibition) = 5-10 microM]. Chlorogenic acid, caffeic acid, or ferulic acid (100 microM) inhibited the metabolism of arachidonic acid to 5-HETE by 36, 10, or 16%, respectively, and these hydroxylated cinnamic acid derivatives inhibited the metabolism of arachidonic acid to 8-HETE by 37, 20, or 10%, respectively (IC50 greater than 100 microM). The metabolism of arachidonic acid to prostaglandin E2, prostaglandin F2 alpha, and prostaglandin D2 by epidermal microsomes was inhibited approximately 50% by the in vitro addition of 5-10 microM curcumin. Chlorogenic acid, caffeic acid, and ferulic acid (100 microM) were inactive. In vitro rat brain protein kinase C activity was not affected by 50-200 microM curcumin, chlorogenic acid, caffeic acid, or ferulic acid. The inhibitory effects of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on TPA-induced tumor promotion in mouse epidermis parallel their inhibitory effects on TPA-induced epidermal inflammation and epidermal lipoxygenase and cyclooxygenase activities.

Enhancement of macrophage-induced cytotoxicity by phorbol ester tumor promoters.

The potent promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), markedly enhanced the ability of mouse peritoneal macrophages to inhibit the growth of L5178Y tumor cells as measured by growth in agar. Three populations of macrophages, resident, divinylether maleic anhydride copolymer, and thioglycollate-recruited, were used. In general, TPA reduced both the cocultivation time and the number of macrophages required to induce cytotoxicity in all three macrophage types. With divinylether maleic anhydride copolymer macrophages, TPA enhanced cytotoxicity in a dose-dependent manner in the concentration range of 1.7 to 170 nM at macrophage: tumor cell ratios of 10:1 and 1:1. For reasons that were not apparent, inhibition of cytotoxicity was found at higher cell ratios. With both thioglycollate-elicited and resident macrophages, TPA (170 nM) enhanced cytotoxicity at all ratios tested. Even 1:1 ratios of macrophages:tumor cells, which were not cytotoxic alone, inhibited cell viability by 50% to 60% in the presence of TPA. A correlation was found between the biological activity of related macrocyclic diterpenes and their ability to enhance macrophage-mediated cytotoxicity. Thus, mezerein and phorbol didecanoate enhanced macrophage cytotoxicity, while the biologically inactive analogs, phorbol, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, and 4-alpha-phorbol-12, 13-didecanoate were without effect in this assay. Cytotoxicity towards untransformed BALB/c/3T3 cells was also demonstrated using a liquid cloning assay. These target cells were much less sensitive to growth inhibition by the macrophages than were the L5178Y cells. A 50% decrease in survival occurred only after 48 hr incubation and required macrophage: target cell ratios of 100:1. The addition of 170 nM TPA led to a dramatic enhancement of cytotoxicity in these cells at macrophage:target cell ratios of 10:1 and 1:1. The results observed with the phorbol esters in the present studies are compatible with other evidence that these compounds can modulate a variety of macrophage functions.

Differentiation of U-937 histiocytic lymphoma cells towards mature neutrophilic granulocytes by dibutyryl cyclic adenosine-3',5'-monophosphate.

Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The ability of murine epidermal cells to produce intracellular hydrogen peroxide was analyzed by flow cytometry and the measurement of 2',7'- dichlorofluorescin (DCFH) oxidation. Epidermal cells isolated from acetone-treated CD-1 mice for 24 h were relatively homogeneous in cell size and density and oxidized low levels of DCFH. However, following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 micrograms; 24 h), two cytokeratin-positive populations of cells were identified that were heterogeneous with respect to size and density. These two TPA-derived cell populations oxidized levels of DCFH that were time and dose dependent and were between 2- and 10-fold higher than levels of DCFH oxidized by cells isolated from acetone-treated mice. The ability of catalase, the enzyme that detoxifies hydrogen peroxide, to suppress DCFH oxidation to control levels suggested that intracellular hydrogen peroxide was responsible for the enhanced rate of DCFH oxidation in epidermal cells isolated from TPA-treated mice. The ability of mouse epidermal keratinocytes to oxidize DCFH in response to TPA treatment was confirmed using a cloned keratinocyte cell line. These results suggest that specific subpopulations of keratinocytes produce elevated levels of intracellular peroxides following treatment with TPA either in vivo or in culture.

Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid.

A methanol extract of the leaves of the plant Rosmarinus officinalis L. (rosemary) was evaluated for its effects on tumor initiation and promotion in mouse skin. Application of rosemary to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited tumor initiation by B(a)P and 7,12-dimethylbenz[a]anthracene (DMBA). Topical application of 20 nmol B(a)P to the backs of mice once weekly for 10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in the formation of 7.1 tumors per mouse. In a parallel group of animals that were treated topically with 1.2 or 3.6 mg of rosemary 5 min prior to each application of B(a)P, the number of tumors per mouse was decreased by 54 or 64%, respectively. Application of rosemary to mouse skin also inhibited TPA-induced ornithine decarboxylase activity, TPA-induced inflammation, arachidonic acid-induced inflammation, TPA-induced hyperplasia, and TPA-induced tumor promotion. Mice initiated with 200 nmol DMBA and promoted with 5 nmol TPA twice weekly for 19 weeks developed an average of 17.2 skin tumors per mouse. Treatment of the DMBA-initiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol TPA twice weekly for 19 weeks inhibited the number of TPA-induced skin tumors per mouse by 40, 68, or 99%, respectively. Topical application of carnosol or ursolic acid isolated from rosemary inhibited TPA-induced ear inflammation, ornithine decarboxylase activity, and tumor promotion. Topical application of 1, 3, or 10 mumol carnosol together with 5 nmol TPA twice weekly for 20 weeks to the backs of mice previously initiated with DMBA inhibited the number of skin tumors per mouse by 38, 63, or 78%, respectively. Topical application of 0.1, 0.3, 1, or 2 mumol ursolic acid together with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiated mice inhibited the number of tumors per mouse by 45-61%.

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C.

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.

Nitric oxide in the lung: therapeutic and cellular mechanisms of action.

Nitric oxide is produced by many cell types in the lung and plays an important physiologic role in the regulation of pulmonary vasomotor tone by several known mechanisms. Nitric oxide stimulates soluble guanylyl cyclase, resulting in increased levels of cyclic GMP in lung smooth muscle cells. The gating of K+ and Ca2+ channels by cyclic GMP binding is thought to play a role in nitric oxide-mediated vasodilation. Nitric oxide may also regulate pulmonary vasodilation by direct activation of K+ channels or by modulating the expression and activity of angiotensin II receptors. Administration of nitric oxide by inhalation has been shown to acutely improve hypoxemia associated with pulmonary hypertension in humans and animals. This is presumably due to its ability to induce pulmonary vasodilation. Inhaled nitric oxide improves oxygenation and reduces the need for extracorporeal membrane oxygenation in term and near-term infants with persistent pulmonary hypertension. However, long-term benefits to these infants have been difficult to demonstrate. In other pathologic conditions, such as prematurity and acute respiratory distress syndrome, short-term benefits have not been shown conclusively to outweigh potential toxicities. For example, high-dose inhaled nitric oxide decreases surfactant function in the lung. Inhaled nitric oxide also acts as a pulmonary irritant, causing priming of lung macrophages and oxidative damage to lung epithelial cells. Conversely, protective effects of nitric oxide have been described in a number of pathological states, including hyperoxic and ischemia/reperfusion injury. Nitric oxide has also been reported to protect against oxidative damage induced by other reactive intermediates, including superoxide anion and hydroxyl radical. The dose and timing of nitric oxide administration needs to be ascertained in clinical trials before recommendations can be made regarding its optimal use in patients.

Pharmacologic therapy of persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is a potentially life-threatening condition characterized by a failure of pulmonary vascular resistance to decrease adequately during the transition to extrauterine life. Inhaled nitric oxide, a vasodilator that acts selectively on the pulmonary circulation, has revolutionized the treatment of this condition. However, inhaled nitric oxide has not proven effective in all patients, particularly those with congenital diaphragmatic hernias or meconium aspiration syndrome. Furthermore, large clinical trials of inhaled nitric oxide have failed to demonstrate significant differences in mortality between nitric oxide-treated and control infants with PPHN. Other therapeutic approaches to PPHN have been limited by a relative lack of specificity for the pulmonary circulation, and have received much less attention. Pharmacologic approaches, including pulmonary surfactants, prostacyclin, endothelin antagonists, Ca(2+)-channel blockers, magnesium sulfate, and tolazoline, have exhibited varying degrees of efficacy in lowering pulmonary vascular pressures in humans and/or animals. A number of these agents are also effective when used in combination. For example, phosphodiesterase inhibitors have been reported to act synergistically with inhaled nitric oxide. Surfactants also appear to be useful in PPHN, particularly in patients with congenital diaphragmatic hernia, when used in combination with other therapies. Surfactant lavage and other novel therapies may also be effective in combination therapy of meconium aspiration syndrome. Further studies should be directed at defining the optimal therapies in specific clinical settings. Validation of multiple therapeutic modalities for PPHN, including inhaled nitric oxide, will allow for rational, combined vasodilator strategies that are specific for the underlying pathophysiology in each patient.

Multifunctional role of nitric oxide in inflammation.

In response to infection or tissue damage, an array of soluble and lipid mediators as well as cytokines and growth factors cause both immune and nonimmune cells to produce rather large amounts of nitric oxide. Nitric oxide and its oxidation products are toxic and can cause tissue injury. The endocrine system can protect against nitric-oxide-mediated tissue damage by producing corticosteroids, growth factors, and cytokines that are potent inhibitors of nitric oxide production. This review focuses on our current understanding of the role of nitric oxide in the inflammatory response. An emphasis has been placed on the potential for nitric oxide in tissue damage.

Functional heterogeneity in liver and lung macrophages.

Although initially considered merely "scavenger cells" that participate in immunologic responses only after B and T lymphocytes have performed their biological tasks, more recent evidence suggests that macrophages play a key role in host defense as well as in the maintenance of normal tissue structure and function. For macrophages to perform their biological functions, they must be activated. This involves up-regulation of an array of signaling pathways resulting in altered gene expression and increased biochemical and functional activity. Macrophages have been identified in almost all tissues of the body. However, the basal activity of these cells, as well as their ability to respond to inflammatory mediators, varies considerably with their location. In addition, even within a particular tissue, there is evidence of macrophage heterogeneity. The largest populations of macrophages in the body are located in the liver and lung. Because of the unique attributes of these tissues, hepatic and pulmonary macrophages play essential roles not only in nonspecific host defense but also in the homeostatic responses of these tissues. In this review, the functional and biochemical activities of macrophages localized in the liver and lungs are compared. Evidence suggests that these represent distinct cell populations with unique functions and responsiveness to inflammatory agents.

Distinct biochemical responses of hepatic macrophages and endothelial cells to platelet-activating factor during endotoxemia.

Acute endotoxemia is associated with activation of hepatic macrophages and endothelial cells. These cells release a variety of inflammatory mediators that have been implicated in tissue injury. In the present studies, we analyzed the biochemical responses of these cells to platelet-activating factor (PAF), a lipid autacoid released during hepatic inflammatory responses. To induce acute endotoxemia, rats were injected intravenously with lipopolysaccharide (LPS). Using the calcium sensitive fluorescent indicator dye Indo-1, we found that PAF induced a rapid and transient increase in intracellular calcium in both hepatic macrophages and endothelial cells. Induction of acute endotoxemia resulted in an increase in the amount of calcium mobilized by both cell types. Although endothelial cells from control rats were less responsive to PAF than macrophages, these cells were more sensitive to in vivo endotoxin. PAF was also found to cause a rapid decrease in intracellular pH in hepatic macrophages that was quantified by fluorescence image analysis using the pH sensitive dye SNAFL-calcein. This decrease occurred more rapidly in macrophages from endotoxemic rats. In cells from both control and endotoxemic rats, the effects of PAF on intracellular pH were inhibited by the specific PAF antagonist triazolam. In contrast to hepatic macrophages, PAF had no effect on intracellular pH in endothelial cells from either control or endotoxemic rats. Ligand binding studies demonstrated that both hepatic macrophages and endothelial cells possess high affinity binding sites for PAF. Macrophages expressed 6- to 7-fold more binding sites/cell than endothelial cells and exhibited a higher Kd. Whereas treatment of rats with LPS had no effect on the Kd for PAF binding to macrophages or on the number of binding sites, a significant increase in both of these receptor characteristics was observed in endothelial cells. Taken together, the present data suggest that the biochemical responses of endothelial cells and macrophages to PAF are distinct. Furthermore, cellular activation induced by PAF in endothelial cells appears to be independent of changes in intracellular pH.

Platelet-activating factor-induced calcium mobilization and oxidative metabolism in hepatic macrophages and endothelial cells.

Fluorescence image analysis of the calcium sensitive dye Indo-1 was used to characterize platelet-activating factor (PAF)-induced calcium mobilization in hepatic macrophages and endothelial cells. PAF, but not lyso-PAF, an inactive analog, induced a rapid and transient increase in intracellular levels of calcium that appeared to depend on the presence of extracellular calcium. In both macrophages and endothelial cells, these effects were dose dependent, reaching maximal levels with 10 nM PAF. However, the kinetics of the calcium response to PAF in macrophages and endothelial cells was distinct. The endothelial cells responded more rapidly to PAF than the macrophages (within 1 min vs. 2-3 min, respectively) and displayed longer recovery periods (4 min vs. > 10 min, respectively). In contrast, the magnitude of the response to PAF was greater in the macrophages. In both cell types, triazolam and alprazolam, two PAF antagonists, and the calcium channel blocker verapamil inhibited PAF-induced calcium mobilization. PAF also stimulated superoxide anion production alone and in combination with the macrophage activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in both cell types. Macrophages produced more of this reactive oxygen intermediate in response to PAF and TPA than endothelial cells. Interestingly, in the absence of extracellular calcium or when verapamil was added to the cultures, superoxide anion production by the macrophages, but not the endothelial cells, was significantly reduced. These results demonstrate that, although hepatic macrophages and endothelial cells mobilize calcium in response to PAF, the characteristics of the response in each cell type are different. These differences may underlie, at least in part, distinct functional responses of the cells to PAF.

Selective inactivation of lymphocytes after psoralen/ultraviolet light (PUVA) treatment without affecting systemic immune responses.

Psoralens, together with ultraviolet light A (PUVA), are used for the treatment of several proliferative diseases. It has been suggested that the effectiveness of this treatment is due to induction of a specific immune response by PUVA-treated lymphocytes that down-regulates untreated cells of the same specificity. The present studies show that the clinically used psoralen analogue 8-methoxypsoralen (8-MOP), as well as 4,8-dimethyl-5'-(pyridiniummethyl)psoralen bromide (4NQ), a derivative that does not cross the cell membrane, when activated by UVA light, render lymphocytes unresponsive to mitogenic, antigenic, or phorbol myristate acetate + ionomycin-induced stimulation. This state of unresponsiveness was not reversed by exogenous recombinant interleukin-2. Treatment of lymphocytes with 4NQ and UVA light had no effect on the viability or the homing pattern of the cells to spleen or liver, whereas 8-MOP induced toxicity in about 50% of cells after 3 days in culture. Using an adoptive transfer mouse model, we found that antigen-specific lymphocytes treated with PUVA (8-MOP or 4NQ) had no effect on the ability of these mice to respond to a subsequent challenge with the same or an irrelevant antigen. This was tested by measuring the T cell proliferative responses of lymph node cells from mice primed with keyhole limpet hemocyanin (KLH) or fowl gamma globulin (FGG) before or after adoptive transfer of large numbers of KLH- or FGG-specific PUVA-treated lymph node cells. No effects of antigen-primed PUVA-treated cells on antigen-primed untreated cells were observed in vitro in mixed lymphocyte cultures responding to the relevant antigen. These results suggest that, in our model system, PUVA induces cell inactivation but does not affect systemic T cell responses.