Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 10⁴ cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2^d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2^b B10BR; H-2^k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, N^G-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of N^G-monomethyl-L-arginine.     

Different Metabolic Effects of Ganglioside GM1 in Brain Synaptosomes and Phagocytic Cells

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe²⁺-H₂O₂ system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of ⁴⁵Ca²⁺ influx and oxidative inactivation of Na⁺,K⁺-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10^–11–10^–8 M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10^–11–10^–10 M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.     

formation of leukotriene E5 by murine peritoneal cells

Resident mouse peritoneal cells, stimulated with opsonized zymosan, produced leukotriene C₄ and E₄, with LTE₄ being the major (80–90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTC₅, LTE₅, 11-trans LTC₅ and 11-trans LTE₅, were identified. The identity of LTE₅ was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n-6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells ( ) produced higher quantities of LTE₅ (30.2 ± 5.4 ng/10⁶ cells) than LTE₄ (22.8 ± 7.3 ng/10⁶ cells). In addition, studies demonstrated a 60% reduction in LTC₄ (42.0 ± 10.8 ng/10⁶ cells to 16.7 ± 6.2 ng/10⁶ cells) and the appearance of LTC₅ (2.1 ± 0.9 ng/10⁶ cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT , in particular LTE₅, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor

Summary Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2^– and asialo-GM1⁺ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages. Abbreviations used: M-CSF, macrophage-colony-stimulating factor; NABMC, nonadherent bone marrow cells; CM, conditioned medium; NK, natural killer; N-CWS, Nocardia rubra cell-wall skeleton     

Regulation of phagocytic process of macrophages by noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenyl-glycol. Role of α- and β- adrenoreceptors

The regulatory capacity of noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenylglycol (HMPG) on the complete phagocytic process of macrophages were investigated. Either noradrenaline or HMPG did not modify adherence. However, 10^–12 M of noradrenaline stimulated the chemotaxis of macrophages, mainly mediated by -adrenergic receptors. In contrast, 10^–12 M of HMPG induced an opposed effect on this stage of the phagocytic process. To stimulate phagocytosis, it is necessary to employ a higher concentration (10^–5 M) of noradrenaline and this effect was blocked with either 10^–6 M propranolol or 10^–6 M phentolamine, and maintained by HMPG. Noradrenaline and HMPG did not modify the microbicide capacity of macrophages (measured by O₂ ^– production after phagocytosis). In conclusion, noradrenaline modulates the phagocytic process of macrophages, and this modulation is completed by HMPG, maintaining the phagocytic functions at physiologically optimal levels. Modulation of chemotaxis is mainly mediated by a-receptors and phagocytosis needs both - and -receptor-stimulation.     

Production and decomposition of eelgrass (Zostera marina L.) in saline Lake Grevelingen

Summary During five 28-hours measurements in 1981, the oxygen production and consumption in an eelgrass community in saline Lake Grevelingen were investigated using light plexiglass enclosures. Applying a conversion factor of 0.29 the amount of carbon fixed and the amount of organic carbon mineralized were estimated. Gross and net production were estimated over 24-hours periods.There appeared to be a good correlation between production and insolation on the water surface. For every measurement period the production as a function of light and aboveground eelgrass biomass in the enclosure were calculated. This showed a maximum of 5.10^–6 mg C.J.^–1 g dry weight^–1 in April and minimum of 1.4.10^–6 mg C.J.^–1 g^–1 in August.Using the calculated production coefficients, the insolation and the eelgrass biomass the gross production, net production and consumption during the growing season of 1976 were calculated. Gross production amounted to 340 gC.m^–2, and net production came to 130 g C.m^–2. Approximately 60 gC.m^–2 was respired by the eelgrass plants while the remaining 150 gC.m^–2 was consumed or mineralized by other organisms on the sampling spot. Approximately 120 g C.m^–2.yr^–1 was transported by wind and wave action towards the eastern part of the lake where it became anaerobically degraded. This resulted in the formation of sulfide and methane.Communication no. 236 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Interaction of murine colony-stimulating factor (CSF-1) with alveolar mononuclear phagocytes

Colony-stimulating factor (CSF-1) was purified from serum-free L-cell-conditioned medium (LCM) and iodinated so that we could study its interaction with murine alveolar macrophages. At 0 °C, the binding of ¹²⁵ICSF-1 to alveolar macrophages reached a stable maximum within 16 h. Under this condition, the binding of ¹²⁵ICSF-1 at various concentrations was saturated at about 3 ng/ml. The binding sites of ¹²⁵ICSF-1 were sensitive to trypsin but not to DNase or RNase treatment. At 37 °C, the trypsin-treated cells regenerated more than 90% of their original binding sites within 12 h. Whereas more than 97% of these alveolar macrophages were phagocytic and esterase-positive, autoradiographic studies showed that only 10–31 % of them were capable of binding to ¹²⁵ICSF-1. These results indicate that the frequencies of CSF-1-binding cells and alveolar macrophage colony-forming cells (AL-CFC) are closely correlated, but no causal relationship has been established.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 10⁶ cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.     

Characterization of surface alloantigens of murine neutrophils

Serological analysis of highly purified (>97%) mouse peritoneal exudate neutrophils using a protein-A rosetting technique, showed that these cells possessed the surface phenotype: Ig^–, Thy-1^–, Ly-1^–, Ly-2^–, Ly-3^–, Ly-4⁺, Ly-5⁺, Ly-6⁺, Ly-7^–, Ia^–, FcR⁺ and C3R⁺.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m² there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m² the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m² did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.     

Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 micrograms/ml, 100 micrograms/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.     

On the mechanism of action of the phagocytosis-stimulating peptide tuftsin

Summary Incubation of human polymorphonuclear leukocytes (PMNL) or thioglycollate-stimulated mouse peritoneal macrophages with the phagocytosis-stimulating peptide, tuftsin (2.5 × 10^–7 M, at 37 °C), caused an increase of 89–90% in intracellular cGMP levels, accompanied by a decrease of 20–25% in intracellular cAMP levels. Significant changes in cyclic nucleotide levels were detectable after 4 min of incubation, were maximal at 10–20 min and persisted for at least 60 min. The concentration dependences of the stimulatory effect of tuftsin on modulation of intracellular levels of cyclic nucleotides and on phagocytosis are similar, suggesting a cause and effect relationship between the two phenomena. This notion is further supported by the finding that 8-Br-cGMP and 8-Br-cAMP elicit stimulatory and inhibitory effects on macrophage phagocytosis, respectively. Measurement of ⁴⁵Ca²⁺ influx into PMNL and macrophages in the presence and absence of tuftsin did not reveal any change in ⁴⁵Ca²⁺ uptake from the media. However, tuftsin did enhance release of ⁴⁵Ca²⁺ from cells preloaded with the isotope. Results suggest that modulation of both the amount of cell-associated ⁴⁵Ca²⁺ and the intracellular levels of cyclic nucleotides are key steps in the mechanism by which tuftsin augments phagocytosis.     

Evidence for an indirect effect of radiation on mammalian chromosomes : I. Indirectly-induced chromosome loss from somatic cell hybrids

Mouse cells UV-irradiated with doses of 0–72 J/m² were fused with unirradiated Chinese hamster cells, and the chromosome constitutions of cell hybrids were examined. The number of mouse chromosomes retained by hybrids decreased with UV dose, and, unexpectedly, the number of hamster chromosomes also decreased in a dose-dependent manner. It is suggested that some component contributed by the irradiated mouse parent cell has indirectly induced damage and loss of hamster chromosomes.