Ildikó Csuka

The effect of various inflammatory agents on the alternative metabolic pathways of arginine in mouse and rat macrophages

Abstract.Objective and design: The effects of various inflammatory stimuli on the alternative arginine metabolic pathways in mouse and rat peritoneal macrophages were investigated in vitro and compared. Treatments: Mice and rats were injected i. p. with thioglycollate, carrageenan, casein, BCG and Newcastle Disease Virus (NDV) vaccines. Methods: Peritoneal macrophages were isolated from untreated and treated animals. The activities of nitric oxide synthase (NOS) II and arginase were measured and expressions were followed by Western blotting. The uptake of arginine and nitrite formation of macrophages were also measured. Results: Inflammatory stimuli increased the NO production and the expression and activity of both NOS II and arginase in mice in vitro. On the contrary, the same treatments changed the expression and activity of NOS II only, but not those of arginase in rats. The most marked effects on NO metabolism were produced by casein and NDV treatments. Conclusions: The activity and expression of NOS II and arginase can be stimulated in peritoneal macrophages in vitro by injecting inflammatory agents into the peritoneal cavity. A markedly different response in arginine metabolism was observed in mouse and rat macrophages. Casein treatment was a potent inducer for both enzymes. NDV vaccines induced mainly NOS II, while thioglycollate induced arginase.

The effect of various inflammatory agents on the phagocytosis and cytokine profile of mouse and rat macrophages

Abstract.Objective and design:The effects of various inflammatory stimuli on the cytokine profile and phagocytic capacity of mouse and rat peritoneal macrophages were investigated in vitro. The correlations between cytokine concentrations and the expressions of NOS II and arginase were also studied.Methods:Mice and rats were injected intraperitoneally with various inflammatory agents. Peritoneal macrophages were isolated. The levels of eight cytokines were determined in macrophage cultures by ELISA test. Phagocytic capacity of macrophages was measured by the ingestion of M. Luteus.Results:The most marked changes caused by i. p. treatments were observed in the levels of IL-1 and IL-6 in mice and of IL-12 in rats. IFN-γ level were increased mainly in rat cells while TNF-α production was rather enhanced in mice. Phagocytic capacity of macrophages was higher in rat samples and it increased with all treatments, except BCG, without marked differences between different treatments.Conclusions:Each inflammatory agent caused an increase in cytokine productions in both species, with marked differences among cytokines. Correlations were found in mouse between IL-6 level and NOS II expression, and IL-10 level with arginase expression. In rat macrophages, IFN-γ, TNF-α and MIP-2 productions were in good correlation with NOS II expression.

Subphases of DNA Replication in Drosophila Cells

Exponentially growing Drosophila S2 cells in suspension culture were synchronized at low- and high-resolution centrifugal elutriation, and DNA synthesis was measured by [(3)H]-thymidine incorporation throughout the S phase. At low resolution, one repair peak at the G(1)/G(0) border and two replication peaks known as early and late S subphases were observed. At high resolution, six chronologic compartments were distinguished. The distribution of these peaks indicated one repair peak at 2.05 C value, one minor replication peak at 2.43C, and four major subphases of replication corresponding to 2.64C, 2.89C, 3.32C, and 3.60C, representing 6.7%, 3.4%, 15.3%, 20.4%, 32.1%, and 22.0% of the synthetic activity, respectively. The five major peaks of cell growth with 2.32C, 2.56C, 2.85C, 3.18C, and 3.58C values consistently preceded those of replication subphases.

Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro

1. The de novo synthesis of arginase was much higher in murine than in rat peritoneal macrophages. This process was inhibited irreversibly by protein synthesis inhibitors and reversibly by glycolysis blockers. 2. Rat macrophages produce more nitric oxide (NO) than murine cells. NO production was inhibited by the inhibitors of protein synthesis or glycolysis. 3. The loading of macrophages by exogenous arginine for 24 hr in vitro resulted in the increase of arginase and nitrite in macrophages to different extents. 4. No great differences in lysozyme production was observed. 5. The proportion of arginine taken up and incorporated is contrasted in murine and rat macrophages.

Differences in the arginase activity produced by resident and stimulated murine and rat peritoneal macrophages

1. Murine macrophages showed a considerably higher in vitro arginase production in short time cultures than rat peritoneal cells. 2. The in vivo stimulation with casein or thioglycollate resulted in an enhanced in vitro enzyme production in mice. 3. The adherence is not the condition of the enzyme production. 4. The difference between the two species cannot be explained by the lack of bivalent ions, the absence of energy supply, proteolysis, the low number of macrophages or by the different cell types of the peritoneal exudate of mouse and rat. 5. The lysozyme production of murine and rat peritoneal macrophages was also investigated and no difference was observed between the two species.

The effect of emetine on the immune response of mice

Emetine (33 mg/kg body weight) administered intraperitoneally blocked the immune response of mice to 10(9) sheep red blood cells (SRBC). The inhibition was almost complete when the drug was administered simultaneously or 24 hr after immunization, while partial inhibition was caused by treatment at 48 and 72 hr. Incorporation of 14C-leucine and 3H-thymidine by spleen cells isolated 4 hr after emetine injection of the mice was strongly decreased. Incorporation was approaching the control level in cells isolated 72-96 hr after emetine administration. However, the incorporation of labeled precursors was less than after SRBC treatment only, even after 72-96 hr. Emetine apparently blocked the development of immune response at an early stage and, in contrast to macromolecule synthesis, the inhibition of the antibody response was irreversible.

Structural changes in the mouse thymus and lymph node after emetine treatment

The effect of emetine which is a potent immunosuppresant was studied on the thymus and lymph node. The subcapsular zone of the thymus was depleted and large number of adherent cells accumulated in this thymic region. The medulla enlarged but the cortico-medullary border remained distinct. In the paracortex (T dependent area) of the lymph node many non-lymphoid pyroninophil cells appeared which is followed by an increased cell proliferation 24 hours after emetine injection. 48-60 hours after administration many macrophage-like cells appeared in the medullary sinuses. This macrophage invasion precedes the adherent cell accumulation in the subcapsular zone of the thymus suggesting a possible non-lymphoid (adherent) cell migration from the lymph nodes paracortex to the thymus.

Binding specificity of the l-arginine transport systems in mouse macrophages and human cells overexpressing the cationic amino acid transporter hCAT-1

The uptake of l-arginine into mouse peritoneal macrophages can be inhibited by numerous amino acids and derivatives. Kinetic studies showed an almost entirely competitive inhibition for both cationic and neutral amino acids and derivatives suggesting that the comparison of their binding specificity by using a quantitative structure-activity relationship (QSAR) study is reasonable. The properties of the most efficient inhibitors were the following: the length of the aliphatic side chain, a general structural similarity to l-arginine (>0.79), cationic character, l-configuration, the presence of an α-amino group (with a mean pKa of 9.41), the van der Waals volume (mean 225 Å3) and a low logP value (mean: −2.99). The significance of four other descriptors (neutral character, presence and the pKa of an α-carboxyl group, and the presence of a modified guanidino group) is much lower. Similar results were obtained for the hCAT-1 cell line, but the significance of the descriptors was slightly different. The l-configuration, van der Waals volume, the low logP value and the length of aliphatic side chain were the most significant, while the pKa value of the side chain (mean pKa = 11.6) was found to be more important than that of the α-amino group. In addition, the general similarity to l-arginine, the presence of an amino group in the terminal position of the side chain (Orn, Lys) and the basic character were significant descriptors, while the significance of the acidity is negligibly low. As a final conclusion, the following descriptors were found to be important generally for the cationic transporters: the van der Waals volume, hydrophobicity (log P); l-configuration; the size of the side chain; the general similarity to l-arginine; the presence of an α-amino group; the general basicity of the molecule; the pKa values of the α-amino group (in macrophages) or that of the side chain (in CAT-1 cells). These descriptors can be regarded as the general structurally important binding characteristics of the cationic amino transporters.

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells.

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.