Extractable polysaccharides of Panax quinquefolius L. (North American ginseng) root stimulate TNFalpha production by alveolar macrophages.

We have investigated the immunostimulatory activity of the medicinal plant Panax quinquefolius L. (North American ginseng). Rat alveolar macrophages were treated with different extracts from 4-year old roots, and tumour necrosis factor alpha (TNF) production was used as a measure of immunostimulatory activity. Aqueous extracts of P. quinquefolius root (1-100 microg/ml) were found to significantly stimulate alveolar macrophage TNF release. Both a P. quinquefolius methanol extract containing ginsenosides (but no polysaccharides), and pure ginsenoside-Rb1, the major ginsenoside present in P. quinquefolius, were found to be inactive as TNF-stimulating agents. Significant TNF-stimulating activity was found in the extractable polysaccharide fraction, which was hydrolyzed and found to contain glucose, galactose, arabinose, rhamnose, and mannose. This represents the first evidence that North American ginseng exerts cytokine-stimulating activity on macrophages.

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells.

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.

Stimulation of interleukin-1 and -6 production in alveolar macrophages by the neotropical liana, Uncaria tomentosa (uña de gato)

Two extracts of different collections of the traditional medicine uña de gato (Uncaria tomentosa) from Peru were characterized by High Pressure Liquid Chromatography as containing approximately 6 mg/g total oxindole content prior to studies with alveolar macrophages. The plant preparations greatly stimulated IL-1 and IL-6 production by rat macrophages in a dose dependent manner in the range of 0.025-0.1 mg/ml. They were also able to enhance IL-1 and -6 in lipopolysaccharide-stimulated macrophages. The results suggest a strong immunostimulant action of this plant.

Differential effects of macrophage- and granulocyte-macrophage colony-stimulating factors on cytokine gene expression during rat alveolar macrophage differentiation into multinucleated giant cells (MGC): role for IL-6 in type 2 MGC formation.

Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF stimulate the differentiation of rat alveolar macrophages (AM) into multinucleated giant cells (MGC) with distinct phenotypes (type 1 and type 2 MGC). In the present study, we analyzed the profile of cytokine gene expression induced respectively, by M-CSF and GM-CSF during rat AM differentiation using reverse transcription-PCR. Enhanced mRNA expression for IL-1alpha, TNF-alpha, and TGF-beta1 was observed 3 h after treatment with M-CSF (50 U/ml) or GM-CSF (50 U/ml). In contrast, IL-6 mRNA expression was increased by GM-CSF but not M-CSF. Kinetic analysis indicated that GM-CSF stimulated IL-6 expression early (1.5 h), with maximal effect observed at 24 h and up to 5 days thereafter. Increased mRNA levels for IL-6 were associated with higher IL-6 activity in the culture media of differentiating AM. IL-6 activity was stimulated 3 h after treatment with GM-CSF and increased with time (up to 5 days). Interestingly, addition of exogenous IL-6 (20-100 ng/ml) alone or in combination with GM-CSF to AM cultures increased slightly and selectively the formation of MGC with type 2 phenotype. Conversely, neutralization of endogenous IL-6 during AM differentiation into MGC inhibited significantly (up to 50%) the formation of type 2 MGC. These results suggest a role for IL-6 in the formation of type 2 MGC and provide some insights into the mechanisms of MGC formation and the processes that regulate it positively.

Combined chylous neck fistula, chylothorax and chyloperitoneum after transhiatal esophagectomy.

A 65-year-old man sequentially developed a chylous neck fistula, left-sided chylothorax, and chylous ascites after a transhiatal total esophagectomy for adenocarcinoma of the distal esophagus. The pathophysiology of this unusual accumulation of chyle in three separate anatomic compartments is examined.

M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes.

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25-75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-gamma (IFN-gamma) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3-16 nuclei, dense cytoplasm, and lower expression of beta3 integrin (Type 1) and (2) irregular MGC with 3-30 nuclei, thin and vacuolated cytoplasm, and higher expression of beta3 integrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to be different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.

Effects of amiodarone-induced phospholipidosis on pulmonary host defense functions in rats.

The effect of the induction of pulmonary phospholipidosis by amiodarone on selected pulmonary host defense functions was studied in male Fischer-344 rats. One week of daily amiodarone treatment resulted in a 4.5-fold increase in total phospholipid in alveolar macrophages recovered from the lungs by bronchoalveolar lavage. The presence of the phospholipidosis had no effect on the phagocytosis of heat-killed yeast cells, the induction of luminol-dependent chemiluminescence, or the spontaneous release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), or spontaneous and LPS-stimulated release of IL-1 by alveolar macrophages in vitro. In contrast, the LPS-stimulated release of IL-6 and TNF-alpha by phospholipidotic alveolar macrophages was enhanced compared with control cells. The pulmonary clearance of Listeria monocytogenes following intratracheal administration of the bacteria was not affected by the phospholipidotic condition. It appears that, in the context of the functions studied, the induction of pulmonary phospholipidosis by amiodarone does not impair pulmonary host defense processes in rats, and may actually be associated with the augmentation of some activities.

Distinctive profile of alveolar macrophage-derived cytokine release induced by fibrogenic and nonfibrogenic mineral dusts.

Groups of 7 Wistar rats each received a single intratracheal instillation of either saline (control), UICC chrysotile B asbestos (5 mg), or very short 4T30 chrysotile asbestos fibers (5 mg). Five animals in each group were killed at 1, 3, and 6 wk posttreatment and analyzed by bronchoalveolar lavage (BAL) for BAL cell populations and cytokine production in conjunction with histopathological assessment of lung tissue. Chrysotile B and short 4T30 chrysotile fibers induced chronic inflammatory reactions characterized by alveolar macrophage (AM) accumulation that resulted, respectively, in lung fibrosis and resolving granuloma. Alveolar macrophages (AM) obtained from rats treated with UICC chrysotile B and short 4T30 chrysotile produced enhanced levels of interleukin-1 (IL-1) and interleukin-6 (IL-6), both spontaneously and in response to lipopolysaccharide (LPS). A different pattern of response was observed for tumor necrosis factor-alpha (TNF-alpha). Fibrogenic chrysotile B caused biphasic changes characterized by significant inhibition of LPS-induced TNF-alpha release by AM 1 and 3 wk after treatment, followed by stimulation of spontaneous and LPS-induced TNF-alpha at 6 wk. In contrast, no significant change in spontaneous and LPS-induced TNF-alpha release was seen with AM from animals with resolving granuloma (4T30 group). Thus, modulation of AM-derived TNF-alpha was correlated under these conditions with the fibrogenic potential of asbestos dusts. These data support a role for TNF-alpha in fibrosis and suggest that TNF-alpha may represent a useful marker of lung damage induced by fibrogenic dusts.

Role of transforming growth factor-beta(1) in down-regulating TNF production by alveolar macrophages during asbestos-induced pulmonary fibrosis.

Activation of alveolar macrophages (AM) for tumour necrosis factor production is suppressed initially during the inflammatory response to fibrogenic dusts. We investigated the mechanisms involved in TNF suppression, notably the role of other AM-derived mediators including prostaglandin E(2) (PGE(2)), transforming growth factor-beta(1) (TGF-beta(1)), and interleukin 6 (IL-6). The action of PGE(2) and TGF-beta(1), on AM was different. At physiologically relevant doses (25-300 pg/ml), PGE(2) did not cause significant inhibition of Hpopolysaccharide (Lps)-induced TNF release by AM in vitro but stimulated IL-6 (up to six fold), an inhibitor of AM-derived TNT. In contrast, TGF-beta(1) (0.5-50 ng/ml) inhibited both LPS-induced TNT and IL-6 release by 50% but had no effect on PGE(2) production by AM. To determine the respective contribution of these different inhibitors in TNF suppression, AM from rats exposed to fibrogenic asbestos for weeks were treated with neutralizing antibody against TGF-beta(1) or indomethacin, an inhibitor of PGE(2) synthesis. Treatment of rat AM with anti-TGF-beta(1) but not indomethacin, abrogated the observed TNT suppression. These results suggest that an autocrine, TGF-beta(1)-dependent mechanism is involved in the down-regulation of TNF production by rat AM from animals with lung fibrosis.

Histogranin, a modified histone H4 fragment endowed with N-methyl-D-aspartate antagonist and immunostimulatory activities.

Histogranin is a naturally-occurring pentadecapeptide with a structure 80% homologous with that a fragment-(86-100) of histone H4. First isolated from bovine adrenal medulla, the peptide was also shown to be present in the pituitary, brain, adrenal glands, blood plasma, lungs and spleen. At the subcellular level, histogranin is concentrated in secretory vesicles and it is released from perfused bovine adrenal glands 15-35 min after stimulation with carbamylcholine as opposed to catecholamines and [Leu5]enkephalin which are released immediately after stimulation. Rat brain membranes possess specific binding sites for [125I][Ser1]histogranin with characteristics of a receptor, namely high affinity, saturability, reversibility and sensitivity to heat and proteolytic enzyme treatments. Intracerebroventricular injections of synthetic histogranin (10-100 nmol) in mice protect them against N-methyl-D-aspartate (NMDA)-induced convulsions without affecting convulsions induced by (R,S)-alpha-amino-3-hydroxy -5-methyl-4-isoxazole-propionate (AMPA), kainate and bicuculline. The peptide also binds to specific sites on human peripheral blood mononuclear cells and it evokes the release of tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) from isolated rat macrophages in culture. Since the structure of histone H4 is considered as one of the most conservative, it is presumed that histogranin possesses its own precursor and that its gene is distinctly expressed.

Alveolar macrophage inhibition of lung-associated NK activity: involvement of prostaglandins and transforming growth factor-beta 1.

Natural killer (NK) activity plays an important role in host defense. It is likely that this defensive role is shaped by compartmental and local environmental factors. The present study investigated the regulatory effects of alveolar macrophages (AM) on lung-associated NK activity. AM and lung lymphocytes (LL) were permitted to interact in a two-chamber system which prohibited cell contact but supported diffusion of soluble factors. AM were found to inhibit NK activity from LL in a time-dependent and reversible manner. The inhibitory event was shown to be mediated by soluble factors acting upon a post-binding event(s) in the lytic pathway of LL. AM inhibition was sensitive to indomethacin treatment (10(-5) M), which caused a decrease in prostaglandin E2 (PGE2) concentrations. Quantitation of PGE2 levels and treatment of LL with exogenous PGE2 indicated that the inhibitory effect could not be exclusively due to PGE2. It was subsequently found that exogenous transforming growth factor-beta 1 (TGF-beta 1) also inhibited LL NK activity and that treatment of inhibitory AM supernatant with a neutralizing antibody to TGF-beta 1 adsorbs up to 55% of its inhibitory activity. Moreover, the amount of TGF-beta 1 found in AM-LL co-culture media (25 pg/mL) correlated well with the level of NK inhibition observed. By contrast, platelet-derived growth factor and nitric oxide did not play a significant role in mediating AM suppression. Taken together, the data suggest that AM inhibit lung NK activity by interfering with post-binding lytic event(s) through the production of PGE2 and TGF-beta 1.

Up-regulation of cytokine production in alveolar macrophages by histogranin, a novel endogenous pentadecapeptide.

Recently, histogranin (HN), a newly found pentadecapeptide, was shown to enhance tumor necrosis factor (TNF) production by alveolar macrophages (AM). In this study, we have investigated whether HN was present in tissues rich with immune cells and further explored the effect of HN and [Ser1]HN on the production of TNF and other key cytokines. Relatively high levels of immunoreactive (ir)-HN were found in rat lung (14.9 pmol/g) and spleen (12.3 pmol/g), indicating its localization in close proximity to macrophages/monocytes and lymphocytes. Furthermore, HN and [Ser1]HN (10(-8)-10(-7) M) stimulated basal and lipopolysaccharide (LPS)-induced interleukin 1 (IL-1) mRNA expression and IL-1 release from rat AM. [Ser1]HN also stimulated basal and LPS-induced interleukin-6 (IL-6) release. Although HN did not affect the kinetics of cytokine production, the maximal enhancing effect of HN was seen at 3 h for TNF, 6 h for IL-1 and 18 h for IL-6. These data indicate that HN can up-regulate a cytokine cascade involving TNF, IL-1 and IL-6 and suggest a role for this endogenous peptide in immune regulation.

Characterization of histogranin receptors in human peripheral blood lymphocytes.

Histogranin (HN), a peptide recently isolated from bovine adrenal medulla, is also present in the spleen. In present studies, specific high affinity binding sites for HN were characterized on membrane preparations of human lymphocytes by radioligand binding. [125I]-[Ser1]HN binding was found to be dependent on time and protein concentration and to be sensitive to trypsin treatment. The binding displayed high affinity (Kd = 1.1 +/- 0.3 nM) and saturability (Bmax = 40.2 +/- 5.0 fmol/mg protein), and it was reversed upon addition of unlabelled [Ser1]HN and closely related peptides. The relative potency of various fragments in displacing [125I][Ser1]HN binding indicated that the active core of the molecule resides inside the C-terminal fragment, HN-(6-15). Interestingly, depressed patients displayed a marked decrease in the binding activity (from 15.4 to 8.55 fmol/mg protein at 0.5 nM of [125I][Ser1]HN). The presence of high affinity HN binding sites on lymphocytes provides evidence for a modulatory role for HN in the regulation of lymphocyte functions.

Bidirectional modulation of TNF-alpha production by alveolar macrophages in asbestos-induced pulmonary fibrosis.

Bronchoalveolar lavage (BAL) cells were isolated from rats 1, 3, and 6 weeks after a single intratracheal instillation of saline, UICC chrysotile asbestos (5 mg), or silica (5 mg). In asbestos-exposed rats, the pulmonary response was characterized by a significant increase in the number of alveolar macrophages (AMs) and the appearance of fibrotic lesions within 1 week. By contrast, mixed macrophage and neutrophil accumulations were observed in the silica group without evidence of fibrosis. Tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS)-stimulated BAL cells from asbestos-treated rats was significantly lower than controls 1 and 3 weeks after exposure. However, by 6 weeks higher levels of TNF-alpha production were noticeable in this group. Decreases in LPS-induced TNF-alpha production were also observed with BAL cells from silica-treated animals at all time points studied. Lower levels of TNF-alpha were not related to decreased BAL cell viability or the presence of a significant proportion of neutrophils in the silica group. Furthermore, biphasic changes in TNF-alpha production seen in the asbestos group were correlated with concomitant decreases (3 weeks) and increases (6 weeks) in levels of TNF-alpha mRNA in AMs. These data indicate that lower levels of TNF-alpha resulted from inhibition at the gene expression level and provide evidence for bidirectional modulation of TNF-alpha production by AMs during inflammatory reactions.

Effects of hyperthermia on natural killer cells: inhibition of lytic function and microtubule organization.

Cells with natural killer activity (NK) may play an important role in host defence against tumour cells. The lytic function of NK cells is very sensitive to hyperthermic inactivation. However, cells with NK activity isolated from rat spleen and exposed to 41-42.5 degrees C for 30 min could partially recover their cytotoxic activity after incubation at 37 degrees C. The recovered cytotoxicity was still NK-specific, as it only resulted in the lysis of YAC-1 sensitive targets, and could not lyse NK-resistant P815 mastocytoma cells. Conjugate formation assay using NK cells labelled with specific monoclonal antibody (mAb) 3.2.3 indicated that the binding of NK cells to targets was not significantly affected by heat treatment. Compared to controls, however, microtubule organizing centre (MTOC) reorientation towards the region of intercellular contact was reduced by 40% in heated effector cells. This was accompanied by a greater inhibition (62-77%) of NK lytic activity. Kinetic analysis indicated that MTOC reorientation capacity recovered following incubation at 37 degrees C. MTOC recovery was maximal 4 h after treatment whereas that of lytic activity peaked at 6 h. These data indicate that NK cells recover NK-specific lytic activity after heat inactivation. Moreover, our study demonstrates that hyperthermia interferes with post-binding MTOC reorientation, and further supports a role for microtubule in secretory processes involved in NK-mediated cytolysis.

Bombesin-related peptides modulate interleukin-1 production by alveolar macrophages.

We have tested the effect of bombesin (BN) and BN-related peptides on the production of interleukin-1 (IL-1) by rat alveolar macrophages. BN incubated with AM alone had no direct effect on IL-1 release. However, at concentrations ranging from 10(-11) M to 10(-6) M, BN significantly enhanced IL-1 release by AM activated with lipopolysaccharide (LPS). A typically U-shaped dose-response relationship was observed with maximal effect obtained between 10(-9) M and 10(-8) M. BN also potentiated the stimulatory effects of other IL-1 inducers including muramyl dipeptide (MDP) and granulocyte-macrophage colony stimulating factor (GM-CSF). The 2- to 3-fold enhancement in IL-1 production seen with BN was blocked by the bombesin receptor antagonist [Leu13,-psi(CH2NH)Leu14]-Bombesin. Furthermore, bombesin-related peptides, gastrin-releasing peptides, (GRP)-27 and GRP-10 also potentiated the stimulatory effects of LPS whereas Neuromedin B (NeB) had no effect. These results suggest that BN-related peptides might play an important role as local modulator(s) of cytokine production and inflammatory reactions in the lung.

Bombesin-like peptides in alveolar macrophage: increased release in pulmonary inflammation and fibrosis.

Rat bronchoalveolar cells (99% alveolar macrophages (AM] were obtained by bronchoalveolar lavage and examined for their content of bombesin-like immunoreactivity (BLI) by radioimmunoassay (RIA), immunocytochemistry and high performance liquid chromatography (HPLC) analysis. Rat AM contained and released in their culture media significant levels of BLI, the major molecular form corresponding to gastrin-releasing peptide (GRP). Release of BLI by AM was not affected by in vitro activation of AM with lipopolysaccharide and muramyl dipeptide, but was enhanced following in vivo treatment with inflammatory agents. AM from animals with inflammation and fibrosis released higher levels of BLI than controls at 3 and 6 weeks after treatment. These changes were correlated with a significant increase in the proportion of low density mature AM as determined by Percoll density gradient fractionation. Together, our data indicate that increased release of BLI by AM may be related to AM maturation and support a role for bombesin-like peptides as modulator(s) of inflammatory reactions.