Effects of serum on calcium mobilization in the submandibular cell line A253.

The effects of serum on inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization in the human submandibular cell line A253 were studied. Exposure of A253 cells to fetal bovine serum (FBS) elicited a 3.3-fold increase in IP3 formation and a concentration-dependent transient increase in cytosolic free Ca2+ concentration ([Ca2+]i), which was similar in Ca2+-containing and Ca2+-free media. Newborn bovine serum (NBS), but not bovine serum albumin (BSA), induced a similar response. The Ca2+ release triggered by FBS was significantly (88%) reduced by the phospholipase C inhibitor U73122, indicating that Ca2+ release induced by FBS is through the PLC pathway. Pretreatment with the tyrosine kinase inhibitor genistein abolished the FBS- and NBS-induced Ca2+ release, suggesting that tyrosine kinase plays an important role in mediating the Ca2+ release. Pre-exposure to ATP or thapsigargin (TG) significantly reduced the FBS-induced [Ca2+]i increase, indicating that Ca2+ release caused by FBS is from the TG- or ATP-sensitive Ca2+ store. While FBS exposure elicited a large Ca2+ release, it reduced Ca2+ influx. Furthermore, FBS significantly inhibited the Ca2+ influx activated by the depletion of intracellular stores by ATP or TG. These results suggest that (1) serum elicits Ca2+ release from ATP- and TG-sensitive stores, which is mediated by IP3; (2) the serum-induced Ca2+ release may be modulated by a tyrosine kinase-associated process; and (3) serum strongly inhibits Ca2+ influxes including the store depletion-activated Ca2+ influx.

Alterations in Ca2+ storage and mobilization in submandibular acinar cells of reserpine-treated rats.

The present study investigated the effects of the catecholamine-depleting drug reserpine on cellular Ca2+ storage and mobilization in rat submandibular acinar cells. Adult rats received seven daily injections of reserpine (0.5 mg/kg) and inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization were measured in isolated submandibular acinar cells. Ultrastructural analysis demonstrated a significant reduction in rough endoplasmic reticulum (ER) and a dramatic accumulation of secretory granules in the cells of treated animals. Reserpine reduced acetylcholine (ACh)-stimulated IP3 formation by 46% and the initial increase in cytosolic Ca2+ concentration ([Ca2+]i) in response to ACh or thapsigargin was reduced by 21 and 56%, respectively. While norepinephrine (NE) did not induce significant IP3 formation, the [Ca2+]i response to NE was increased 360% by reserpine treatment. Reserpine treatment also enhanced the sustained [Ca2+]i increase following these stimuli. After stimulation with ACh or NE, exposure to ionomycin caused a further elevation in [Ca2+]i which was significantly larger in the cells of treated animals. After exposure to agonist + ionomycin, addition of monensin induced a third increase in [Ca2+]i which was significantly larger in cells of reserpine-treated animals. While capacitative Ca2+ entry was not altered, NE-activated Ca2+ influx was abolished after reserpine treatment. Reserpine treatment therefore alters IP3-sensitive and insensitive Ca2+ stores, non-capacitative Ca2+ influx and active Ca2+ transport in submandibular acinar cells of rats.

Effects of particulate and soluble (1-3)-beta-glucans on Ca2+ influx in NR8383 alveolar macrophages.

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.

Characterization of Ca2+ mobilization in the human submandibular duct cell line A253.

The regulation of Ca2+ mobilization in the human submandibular duct cell line A253 was investigated by monitoring cytosolic free Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-triphosphate (IP3) formation. An increase in [Ca2+]i was elicited by ATP, isoproterenol (IPR), or vasoactive intestinal polypeptide (VIP), but not by acetylcholine, norepinephrine, or substance P, suggesting that Ca2+ mobilization is regulated by P2-purinergic, beta 2-adrenergic, and VIP receptors. 1,4,5-IP3 formation was significantly increased by ATP but not by the other agonists. Exposure of the cells to a membrane permeable cAMP analog, dibutyryl-cAMP, or to the adenylate cyclase activator forskolin induced a smaller increase in [Ca2+]i, indicating that the IPR-induced Ca2+ release is not mediated by cyclic AMP. Inhibition of the endoplasmic Ca(2+)-ATPase with thapsigargin (TG) in Ca(2+)-free medium induced a 207% increase in [Ca2+]i, and a subsequent exposure to ATP caused a further increase in [Ca2+]i of 104%. Similarly, TG exposure after ATP induced a further Ca2+ release, suggesting that the TG-sensitive store and the IP3-sensitive store do not overlap. Similar results were observed by sequential exposure to TG and IPR or to ATP and IPR. Ca2+ influx across the plasma membrane was enhanced after ATP or TG, but not after IPR. Our findings show a unique pattern of Ca2+ mobilization in the A253 cell line: (i) Ca2+ mobilization is regulated by P2-purinergic, beta 2-adrenergic, and VIP receptors; (ii) Ca2+ release is mediated by 1,4,5-IP3 and probably by an unknown mediator; (iii) TG, P2-, and beta 2-agonists discharge separate Ca2+ stores; and (iv) ATP and TG, but not IPR, regulate Ca2+ influx.

Regulation of cytosolic free Ca2+ in cultured rat alveolar macrophages (NR8383).

Ca2+ mobilization in the rat alveolar macrophage cell line NR8383 was examined with the Ca2+-sensitive fluorescent probe Fura-2. ATP and norepinephrine elicited a 108 and 46% increase, respectively, in cytosolic free Ca2+ concentration ([Ca2+]i). Acetylcholine, nicotine, isoproterenol, substance P, and vasoactive intestinal polypeptide did not alter [Ca2+]i. Inositol 1,4,5-trisphosphate (IP3) formation was also activated by ATP. The carbohydrate-rich cell wall preparation, zymosan, induced a gradual [Ca2+]i, increase only in the presence of external Ca2+, but did not activate IP3 formation. This increase was abolished by laminarin and by removal of extracellular Ca2+, suggesting that the [Ca2+]i increase was activated by beta-glucan receptors and mediated by Ca2+ influx. This influx was significantly reduced by SKF96365, but not by nifedipine, (omega-conotoxin GVIA, (omega-agatoxin IVA, or flunarizine. These results suggest that release of intracellular Ca2+ in NR8383 cells is regulated by P2-purinoceptors and that zymosan causes Ca2+ influx via a receptor-operated pathway.

The effects of cyclosporine and dexamethasone on an alveolar macrophage cell line (NR8383).

Lung infections are a major source of morbidity and mortality in recipients of lung transplants. Prominent among the pathogens that cause pneumonias in these subjects are gram-negative bacilli, particularly Pseudomonas strains. One important reason that bacteria infect the lungs of these patients is that pulmonary defenses are impaired by the drugs used to prevent transplant rejection. Using a rat alveolar macrophage cell line (NR8383), we measured the effects of exposure (24 hr) to cyclosporine and dexamethasone (DEX) on the ability of these cells to (1) kill Pseudomonas aeruginosa (Pa); (2) produce H2O2; and (3) release tumor necrosis factor. We found that the bactericidal activity against unopsonized or opsonized Pa of NR8383 cells was unaltered by CsA (0.1, 0.5, or 1 micrograms/ml), DEX (10(-6) M), or CsA + DEX (0.5 micrograms/ml + 10(-6) M, respectively). Likewise, LPS-induced TNF release, and zymosan A and Pa-induced H2O2 production were unaltered by CsA (0.1 or 1 microgram/ml). In contrast, H2O2 production and TNF release were decreased by about 50% and 90%, respectively, by DEX exposure (10(-6) M). Thus, while DEX but not CsA decreased TNF release and H2O2 production in NR8383 cells, bactericidal activity against Pa was unaffected. One explanation for these results is that decreases in TNF or H2O2 of the magnitude we observed do not impair bactericidal activity against Pa; however, an alternative explanation is that Pa are killed by NR8383 cells through other mechanisms. Interpretation of these results must take into consideration the fact that macrophages from different species and tissues may respond differently to various stimuli.

Pneumocystis carinii induces an oxidative burst in alveolar macrophages.

There is evidence that alveolar macrophages (AM) play a role in the clearing of Pneumocystis carinii from the lungs. To investigate the mechanisms involved in this process, we studied in vitro the induction of an oxidative burst by P. carinii in a cell line of macrophages (NR8383) and AM from normal rats. P. carinii was added to macrophage monolayers (10(6) cells), and the H2O2 produced after 4 h of incubation was measured. Both NR8383 macrophages and normal rat AM produced H2O2 in response to P. carinii cysts and trophozoites isolated from dexamethasone-treated rats, although the amount of H2O2 induced in AM from normal rats was larger. NR8383 macrophages bound and phagocytized both P. carinii cysts and trophozoites and produced increasing amounts of H2O2 as a dose-related response to cysts and trophozoites. Opsonization of P. carinii with normal rat serum increased H2O2 production by both types of macrophages; this enhancement was decreased, but not abolished, when the serum was first depleted of complement by heat treatment. These findings demonstrate that NR8383 macrophages and normal rat AM produce an oxidative burst in response to P. carinii and that this response is enhanced by complement.

Role of the zymolyase-sensitive cyst wall of Pneumocystis carinii in the oxidative burst of macrophages.

Alveolar macrophages are thought to participate in clearing Pneumocystis carinii (Pc) from the lungs. We have recently demonstrated that Pc cysts and trophozoites induce an oxidative burst in a cell line of rat alveolar macrophages (NR8383). In order to investigate the mechanism of this response, we examined the effect that disruption of the Pc cyst wall with zymolyase had on the cyst's ability to elicit H2O2 from NR8383 macrophages and correlated these results with the electron microscopic appearance of the cyst wall.

A continuous alveolar macrophage cell line: comparisons with freshly derived alveolar macrophages.

Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonized Pseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H2O2 and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations.

Resistance of mucoid Pseudomonas aeruginosa to nonopsonic phagocytosis by alveolar macrophages in vitro.

A unique, recently described rat alveolar macrophage cell line (NR8383) was used to study the interaction of the pulmonary immune system with a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa (SRM-3), its nonmucoid revertant (SRM-3R), and a non-cystic fibrosis isolate (PAO-1). Strain SRM-3 was cultivated in a chemostat system to allow maintenance of an entirely mucoid population. The alveolar macrophage response to the mucoid and nonmucoid strains of P. aeruginosa was determined by visually quantitating phagocytosis in acridine orange-stained monolayers and measuring the induction of an oxidative burst as indicated by chemiluminescence and H2O2 production. In all experiments, fewer than 2% of the NR8383 cells engulfed the mucoid SRM-3 isolate, while SRM-3R and PAO-1 were phagocytized by 15 and 41%, respectively. Opsonization by normal serum (complement) provided minimal phagocytic enhancement of these strains, whereas specific anti-P. aeruginosa antibody slightly elevated phagocytic responses to strains with nonmucoid phenotypes while providing a sevenfold increase in uptake of SRM-3. Chemiluminescent and H2O2 responses were comparable with the levels of phagocytosis observed, with very little or no response to the mucoid strain SRM-3. The data indicate that the strains with mucoid phenotypes are refractile to ingestion and that studies which describe ingestion of mucoid strains were likely measuring ingestion of revertants. Alginic acid (2 mg/ml) was found to inhibit stimulation of macrophage response to the opsonized and unopsonized nonmucoid strain PAO-1.

From growth factor dependence to growth factor responsiveness: the genesis of an alveolar macrophage cell line.

A rat pulmonary alveolar macrophage (PAM) cell line (NR8383) was initiated in culture in the presence of a gerbil lung cell conditioned medium (GLCM), and has been propagated continuously for over 36 mo. When examined at different times throughout this in vitro period, NR8383 exhibited characteristics typical of macrophages: (a) Zymosan ingestion was seen in 90 to 98% of the cells examined; (b) Pseudomonas aeruginosa phagocytosis in 50 to 80%; (c) Nonspecific esterase activity in greater than 95%. During the first 6 mo., the PAM replicated with doubling times approximating 15 to 20 d. Throughout this period, GLCM dependence was evident. After 27 wk in vitro, NR8383 replication increased markedly, and within 2 wk, the doubling time was less than 48 h. NR8383 was readily monitored by [3H]thymidine (TdR) blastogenesis assay. In the presence of GLCM uptake of [3H]TdR was fivefold greater than in control cultures. Adherence and growth kinetics were effectively controlled by modulation of GLCM or serum content in culture medium. It was demonstrated that PAM growth factor(s) is ubiquitous, not species-specific, and under certain conditions may be derived from "endogenous" sources of persisting non-PAM populations within the parent, uncloned line NR8383. Cloned progeny remain devoid of non-PAM "feeder" cells, but retain macrophage properties, including interleukin-1 secretion, Fc receptors, and H2O2 production.

Distribution of Legionella pneumophilia antibody among primate species.

Sera representing 16 different primate species were surveyed by indirect immunofluorescence for evidence of antibody to Legionella pneumophila. The presence of antibody in Old and New World monkeys and in apes supports previous observations of the ubiquity of Legionella pneumophila.

Oncornavirus-like particles in squirrel monkey (Saimiri sciureus) placenta and placenta culture.

Oncornavirus-like particles similar in morphology to type D particles were observed in 1 of 2 squirrel monkey (Saimiri sciureus) placentas. Intracytoplasmic type A particles, immature virus particles, and mature viruses with eccentric or occasionally centric nucleoids were associated with placental syncytiotrophoblasts. A spike layer typical of type B viruses was not detected in viral envelopes. Onvornaviruses, identical to those previously isolated from squirrel monkey tissues and similar to Mason-Pfizer monkey virus, were seen in cultures derived from the virus-positive squirrel monkey placenta cocultivated with a mink lung culture. The major morphologic difference between the in vivo and the in vitro squirrel monkey virus was in the nucleoid position of mature virus particles.

Viral infections of the captive Kenya baboon (Papio cynocephalus): a five-year epidemiologic study of an outdoor colony.

Rectal swabs, throat swabs, fecal samples, tissues, and sera were collected from 334 adult and infant Kenya baboons (Papio cynocephalus) in captivity at this institution over a 5-year period. A total of 4,893 specimens were collected, resulting in the isolation of 582 viral isolates (11.9%). The month of November yielded the lowest isolation rate, while the month of January produced the highest rate. The most commonly isolated viruses in adults and infants were SV6 and SV23, followed by N125, SV15, and SV17 in that order in adults, and SA7, N125, SV15, V340, and SV17 in that order in infants. Nine serotypes, namely enteroviruses SV19, SV42, SA5, A13, and N203, as well as adenoviruses SV15, SV20, SV31, and SV37, were isolated only from adults. Two adenovirus serotypes, SA7 and V340, were recovered predominantly from infants.

Oncornavirus: isolation from a squirrel monkey (Saimiri sciureus) lung culture.

An oncornavirus isolated from a squirrel monkey (Saimiri sciureus) lung culture has a density of 1.16 to 1.17 grams per milliliter, contains 70S RNA, and has an RNA-directed DNA polymerase that prefers Mg2+ over Mn2+ in an assay in which polyribocytidylate - oligodeoxyguanylate (12-18) is used as a synthetic template. Morphologically, the virus resembles Mason-Pfizer monkey virus but is antigenically distinct from this virus. The virus grows in cells of human, chimpanzee, rhesus monkey, canine, and mink origin, but not cells of squirrel monkey origin. On the basis of its properties, the newly isolated virus can be classified as a retravirus.

Oncornavirus-like particles in baboon type C virus-infected chimpanzee lung cells (SFRE:CL-1).

Intracytoplasmic type A particles were observed in a fetal chimpanzee lung culture (SFRE:CL-1) inoculated with type C virus-containing supernatants from a coculture of baboon placenta and SFRE:CL-1 cells. Budding, immature, and mature type C particles were also noted. In thin section, spike-like structures were rarely detected on budding intracytoplasmic type A particles but were occasionally observed on some immature and mature virus particles. Unlike mouse mammary tumor virus or Mason-Pfizer monkey virus, infected SFRE:CL-1 cells contained no eccentric or rodshaped nucleoids.

C-type viruses in chimpanzee (Pan sp.) placentas.

C-type virus particles were observed by electron microscopy in placentas from 7 of 9 chimpanzees (Pan sp.). These viruses were morphologically similar to those observed in placentas of other primates.

Vertical transmission of C-type viruses: their presence in baboon follicular oocytes and tubal ova.

C-type viruses were found in baboon follicular oocytes and tubal ova adjacent to the plasma membrane in the perivitelline space or along the inner margin of the zona pellucida. Their presence support the concept of vertical transmission of C-type viruses.