Phagocytosis Of Colloidal Carbon Research Articles

Colloidal carbon stimulation of Kupffer cells triggers Nrf2 activation in the isolated perfused rat liver

Activation of transcription factor Nrf2 was investigated in the isolated perfused rat liver infused with 0.5 mg of colloidal carbon (CC)/ml for 5-15 min to stimulated Kupffer cell function. Infusion of CC enhanced liver O(2) consumption over basal levels, with a time-dependent increase in CC-induced O(2) uptake, at constant rates of CC phagocytosis by Kupffer cells, as assessed histologically, and adequate viability conditions of the livers, as shown by the marginal (0.34 %) total sinusoidal lactate dehydrogenase (LDH) efflux over intrahepatic LDH activity. Under these conditions, cytosolic protein levels of Nrf2 (Western blot) and inhibitor of Nrf2 Keap1 progressively declined by CC infusion, those of nuclear Nrf2 increased, leading to enhancement in the nuclear/cytosolic Nrf2 ratios. It is concluded that the respiratory burst of CC-stimulated Kupffer cells triggers Nrf2 activation in the perfused liver, a response that may afford cellular protection under pro-oxidant conditions underlying Kupffer cell stimulation.

Thyroid Hormone-Induced Cytosol-to-Nuclear Translocation of Rat Liver Nrf2 Is Dependent on Kupffer Cell Functioning

L-3,3′,5-triiodothyronine (T3) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl3; 10 mg/kg i.v. 72 h before T3 [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T3), and determinations were performed 2 h after T3. T3 increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl3 treatment prior to T3, an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T3-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl3 administration. Similar to GdCl3, apocynin given before T3 significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T3. This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl3 or apocynin given prior to T3, thus hindering Nrf2 activation.

Modulation of Lymphocyte Migration to the Murine Spleen after Marginal Zone Macrophage Phagocytosis of Blood-Borne Particulate Material

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocye Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.

Influence of C-phycocyanin on hepatocellular parameters related to liver oxidative stress and Kupffer cell functioning.

Kupffer cells, liver macrophages involved in immunomodulation, phagocytosis, and biochemical attack, can induce cytotoxicity and inflammation when their activity is exacerbated. The aim of this study was to evaluate the effects of C-phycocyanin on Kupffer cell functioning considering its antioxidant and anti-inflammatory properties. Actions of C-phycocyanin on colloidal carbon phagocytosis, carbon-induced respiratory burst activity, and sinusoidal lactate dehydrogenase (LDH) release were studied in isolated perfused mouse liver. The influence of C-phycocyanin on tumor necrosis factor-a (TNF-alpha) and nitrite levels in serum and liver nitric oxide synthase (NOS) activity was assessed in rats subjected to thyroid hormone (T3) administration, a condition known to underlie hepatic oxidative stress comprising an increased Kupffer cell activity. C-phycocyanin elicited a concentration-dependent inhibition of carbon phagocytosis and carbon-induced O2 uptake (IC50 = 0.2 mg/ml) by perfused livers, with a 52% diminution in the carbon-induced sinusoidal release of LDH being found at a concentration of 0.25 mg/ml. Thyroid calorigenesis induced an 82-fold increase in serum TNF-alpha levels, an effect that was suppressed by pretreatment with C-phycocyanin, the antioxidant alpha-tocopherol, and by the Kupffer cell inactivator gadolinium chloride. C-phycocyanin also suppressed the T3-induced increases in serum nitrite levels (234%) and in the activity of hepatic NOS (75%). C-phycocyanin significantly decreases Kupffer cell phagocytosis and the associated respiratory burst activity, effects that may contribute to the abolition of oxidative stress-induced TNF-alpha response and NO production by hyperthyroid state.

Influence of aging on Kupffer cell respiratory activity in relation to particle phagocytosis and oxidative stress parameters in mouse liver

The influence of aging on the respiratory activity of stimulated Kupffer cells was investigated in the isolated perfused mouse liver in relation to colloidal carbon phagocytosis, and the content of glutathione (GSH) and protein carbonyls as parameters related to oxidative stress. Livers from aged (22 months) mice exhibited significant 35% and 65% decreases in the carbon uptake and in the carbon-induced O2 consumption compared to young (3 months) animals, respectively, with a concomitant 46% diminution in the carbon-induced O2 consumption/carbon uptake ratio. Hepatic GSH depletion was observed in aged mice compared to young animals, whereas protein oxidation was enhanced. It is concluded that aging leads to an impairment in the functional capacity of Kupffer cells reflected by a substantial reduction in their respiratory burst activity, lessened endocytic capacity and enhanced oxidative stress, that may contribute to increased susceptibility of the liver to noxious challenges.

Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow.

Recent studies have shown that an increased concentration of ambient particulate matter (PM10) is related to decreased pulmonary function and respiratory and cardiovascular mortality. The mechanisms responsible for this excess mortality are unknown and the relationship between the level of PM10 and the circulating leukocyte counts has not been previously investigated. We postulated that the deposition of PM10 in the peripheral lung stimulates alveolar macrophages (AM), which results in polymorphonuclear leukocyte (PMN) release from bone marrow (BM). To test this hypothesis, either colloidal carbon (CC) (n = 3) or saline (n = 4) was instilled into the lungs of rabbits and PMN release from BM was evaluated by using 5'-bromo-2'-deoxyuridine (BrdU). CC instillation in the lung shortened the transit time of PMN through the BM to 71.0 +/- 6.9 h compared with the saline controls (85.5 +/- 2.8 h, p < 0.01). The role of AM in this response was further investigated by incubating isolated AM in tissue culture medium either with or without the presence of CC, and measuring the effect of the supernatants on the release of PMN from the BM. The supernatant of AM incubated with CC shortened the PMN transit time through the BM to 74.9 +/- 3.7 h (p < 0.05) compared with the supernatant from the unstimulated AM (98.6 +/- 1.9 h) and medium alone (94.3 +/- 3.7 h). We conclude that the phagocytosis of CC by AM releases mediators (cytokines) that stimulate the BM to release PMN. We speculate that these newly released PMN may play an important role in the decline in lung function and high mortality seen in populations exposed to high concentrations of atmospheric PM10.

Dose-dependent effects of acute lindane treatment on Kupffer cell function assessed in the isolated perfused rat liver

1. Twenty-four hours after lindane exposure (5-60 mg/kg) a dose-dependent increase in the serum and hepatic levels of the insecticide was observed. Both the basal rate of O2 consumption and the sinusoidal e ux of lactate dehydrogenase (LDH) by the perfused rat liver was enhanced after the administration of 20-60 mg lindane/kg. 2. The administration of low doses of lindane (5-20 mg/kg) increased carbon uptake and the carbon-induced O2 consumption by the perfused liver, effects that were abolished by pretreatment with the Kupffer cell inactivator gadolinium chloride (GdCl3). These parameters were not modified at the higher doses of lindane used (40-60 mg/kg). 3. In the dose range of 20-60 mg/lindane kg, carbon infusion led to a further increase inliver LDHe uxover values foundinits absence,aneffect that was markedly diminished by GdCl3 in rat treated with 20 mg lindane/kg, being unaltered by GdCl3 in animals given 60 mg/kg. 4. It is concluded that lindane induces a dose-dependent biphasic effect on Kupffer cell function, which could be conditioned by differential membrane perturbation actions of the insecticide that progressively accumulates in the liver, thus altering receptor-mediated and enzymatic processes related to colloidal carbon phagocytosis. Increased Kupffer cell function at low doses of lindane leads to enhanced liver injury. However, this feature of lindane intoxication at higher doses (60 mg/kg) is independent of Kupffer cell activity and seems to be determined by an oxidative stress mechanism induced at the parenchymal cell level.

Donor nutritional status--a determinant of liver preservation injury.

In liver transplantation, the quality of the liver is determined by a number of factors including donor nutritional status. Livers from fasted donors appear to tolerate long-term preservation better than livers from fed donors. In this study we repeated earlier results and obtained 31% (4/13) survival after 40-hr preservation of livers from fed donor Brown Norway rats and 67% (8/12) survivors with donor livers from 4-day-fasted rats (P = 0.154). The explanation for this improvement is not known but may be due to inactivation of Kupffer cells due to nutritional depletion of the liver. Kupffer cell activation has been one explanation advanced to explain how cold storage injuries livers during reperfusion (transplantation). In this study, we have measured how donor fasting affects Kupffer cell function (phagocytosis of colloidal carbon) after preservation of the rat liver. In addition, we measured how enhancing liver glycogen by feeding glucose to the rat donors affected outcome and liver functions tested by isolated perfusion after 24- and 40-hr cold storage of the liver. Preservation did not cause inactivation or activation of Kupffer cell phagocytosis of colloidal carbon. In livers with 0-hr preservation, colloidal carbon uptake was 3.1 +/- 0.2 mg/g/hr, after 40-hr preservation uptake was 3.8 mg/g/hr (P < 0.05 vs. 0 hr) (fed) and 2.7 +/- 0.3 mg/g/hr (fasted, P, 0.05 vs. 0-hr and 40-hr-fed). Thus, the improved survival obtained with livers from fasted donors does not appear related to inactivation of Kupffer cell phagocytosis. Although livers from fasted donors showed improved survival, there was extensive hepatocellular injury as indicated by large LDH release from the livers after 40-hr cold storage as tested by isolated perfusion. LDH released into the perfusate increased from 35.8 +/- 10.1 U/L (fed, 40-hr CS) to 301 +/- 65 U/L (fasted, 40-hr CS) after 1-hr reperfusion. AST release showed a similar pattern and bile production was suppressed more in livers from fasted donors than fed donors. Feeding rats glucose elevated liver glycogen and significantly reduced hepatocellular injury as measured by LDH release and AST release in the isolated perfused liver after 40-hr cold storage. Feeding rats glucose (40% in drinking water for 4 days) also improved survival: fed+glucose = 85% survival versus 31% survival with no glucose and fasted+glucose = 92% survival versus 67% survival with no glucose. These results show that both extensive donor fasting and glucose feeding enhanced outcome in orthotopic liver transplantation. This dilemma (both fasting and feeding improved survival) are discussed in terms of how the interactions between Kupffer cells and hepatocytes affect liver viability. Donor fasting is probably impractical clinically as a method to improve the donor liver, but elevating liver glycogen by glucose supplementation is possible and may lead to improved preservation and outcome in liver transplantation.

Pharmacological activities of flavonoids (III) structure-activity relationships of flavonoids in immunosuppression

Effects of twenty-one different flavonoids and their related compounds on the phagocytosis of colloidal carbon by macrophages in liver and spleen, humoral immune responses against bacterial α-amylase and cellular immune responses against oxazolone and dinitrofluorobenzene were studiedin vivo andin vitro. It was shown that most of the flavonoids accelerated significantly the phagocytosis, and they suppressed significantly not only humoral and cellular immune responses but also the development of immunological memory after the antigenic stimulation. Especially, malvin was the most active in phagocysis, and disodium cromoglycate and morin were the most active in humoral and cellular immunosuppression, respectively. Daidzein had the most potent inhibitory activity in the development of memory cells. The structure-activity relationships of the flavonoids in immunosuppression became apparant from these results: 1. The presence of C2–3 double bond and C4 ketone group in C-ring was important for their immunosuppressive activity. 2. Flavonoids with benzene ring at 2 or 3 position in C-ring showed the almost same activities. 3. The opening of C-ring did not affect their immunosuppressive activity. 4. The glycosylated flavonoids at 3 position in C-ring were less potent than their aglycones. 5. Di- or tri-hydroxylated flavonoids in B-ring were more potent than mono-hydroxylated. 6. Chromanochromanone also had the immunosuppressive activity.

Influence of colloidal carbon phagocytosis as a metabolic load on the hepatic energy metabolism in rats

The influence of phagocytosis on hepatic energy metabolism was investigated in rats injected with colloidal carbon. Following injection of 6 mg colloidal carbon per 100 g body wt (BW) the hepatic energy charge potential (ECP) remained unchanged. However, oxidative phosphorylation of the isolated mitochondria (PR) increased significantly to 120 and 124% of the control at 1 and 3 hr, respectively, and returned to the normal range at 6 hr. The pyruvate level rose to 195 and 150% at 3 and 6 hr. The lactate level was elevated to 195 and 143% at 3 and 6 hr. Following injection of 20 mg colloidal carbon per 100 g BW, the ECP decreased from 0.865 to 0.783 at 1 hr, but recovered to the normal range thereafter. The PR increased more markedly to 135% at 1 hr, concomitant with an elevation of the ketone body ratio. At 3 hr it remained elevated at 118%, but returned to the normal level at 6 hr. The pyruvate and lactate levels increased to more than 250% at 1 and 3 hr. At 6 hr, the lactate level returned to the control level, but the pyruvate level showed still higher level than the control. These results indicate that phagocytosis exerts an acute metabolic load on the total liver. It was tentatively proposed that parenchymal and nonparenchymal cells in the liver have interrelation in the energy metabolism, and that an enhancement of hepatocellular mitochondrial phosphorylative activity occurs to assist the accelerated metabolism due to phagocytosis.

The ontogenetic development of the reticulo‐endothelial system in the rainbow trout, Salmo gairdneri Richardson

Abstract The phagocytosis of colloidal carbon in the adults and fry of Salmo gairdneri Richardson was investigated. Adult fish accumulated trapped carbon in the spleen, kidney and heart. Fry, as early as 4 days post‐hatch, had an efficient phagocytic system, the carbon being engulfed by free macrophages which accumulated in the connective tissue, under the skin, in the gut and in the gills. By 18 days post‐hatch, the situation was similar to that found in the adult. The trapping of carbon in the gills of young fry is discussed, and it is suggested that it acts as a special mechanism to protect the thymus from undesirable, and possible tolerigizing antigen exposure before immunocompetence is attained.

The ontogenetic development of the reticulo‐endothelial system in the rainbow trout, Salmo gairdneri Richardson

Abstract. The phagocytosis of colloidal carbon in the adults and fry of Salmo gairdneri Richardson was investigated. Adult fish accumulated trapped carbon in the spleen, kidney and heart. Fry, as early as 4 days post‐hatch, had an efficient phagocytic system, the carbon being engulfed by free macrophages which accumulated in the connective tissue, under the skin, in the gut and in the gills. By 18 days post‐hatch the situation was similar to that found in the adult. The trapping of carbon in the gills of young fry is discussed, and it is suggested that it acts as a special mechanism to protect the thymus from undesirable, and possible tolerigizing antigen exposure, before immunocompetence is attained.

Phagocytosis of colloidal carbon in perfused liver of rats and rabbits.

A constant-flow, nonrecirculating system was used to study the extraction efficiency of colloidal carbon in the perfused liver of rats and rabbits. In both species, a diminution in perfusate flow produced an increased extraction efficiency. The extraction efficiency also increased with a decrease in the carbon concentration of the perfusate. In identical conditions regarding the flow rate and carbon concentration, the perfused liver of the rabbits showed a relatively lower phagocytic efficiency than that of the rats.

Establishment of heteroploid cell lines from mouse peritoneal exudate cells.

Proliferation was observed during in vitro cultivation of peritoneal exudate cells that had been educed from a C3H mouse with Freund's incomplete adjuvant. These cells were successfully subcultured by release with trypsin-EDTA solution and are now at passage 108 after 22 months in culture. Using this technique, 12 other rapidly growing peritoneal exudate cultures were obtained, whereas 10 cultures not educed with adjuvant did not proliferate. Characteristics of four adjuvant-induced cell lines established in culture include: rapid attachment to glass, doubling time in culture of 18 to 19 hr, phagocytosis of colloidal carbon, enhanced phagocytosis of specifically sensitized bacteria, epithelium-like morphology, and retention of C3H histocompatible specificities. These cell lines had widely varying chromosome distributions with modes from 37.3 +/- 2.4 to 82.6 +/- 2.30, but inoculation of 10(7) cultured cells into syngeneic animals did not produce tumors. Procedures described for the reproducible establishment of peritoneal exudate cell lines did not require use of conditioned media or exogenous viral infection.

Phagocytosis of Colloidal Carbon by the Fixed Tissue and Peritoneal Macrophages of New Zealand Mice

The peritoneal cellular response to colloidal carbon has been studied in NZB, NZW, BWF1, BALB/c, CBA and C3Hf mice aged 8 to 10 weeks. The overall patterns of total and differential peritoneal cell counts was similar in all 6 strains of mice. No inter-strain differences were noted in either the removal of carbon from the peritoneal cavity or the distribution of carbon in the fixed tissue macrophages. The results show that phagocytosis of colloidal carbon, an inert particle, is normal in New Zealand mice. Intravenous carbon clearance studies performed on NZB, BALB/c and CBA mice showed that phagocytosis of carbon was identical in each strain when correction was made for variations in liver and spleen weight.

Phagocytosis of colloidal carbon by the reticuloendothelial system during hepatocarcinogenesis in rats.

SummaryAn early stimulation in the clearance of colloidal carbon by the reticuloendothelial system was noted in rats fed 3′-methyl-4-dimethylaminozobenzene or DL-ethionine. No stimulation was observed in animals receiving the basal diet or 4′-methyl-4-dimethylaminozobenzene. Elimination of the carcinogenic potency of the azo dye by hypophysectomy and of ethionine by methionine abolished the stimulation of phagocytic activity by these carcinogens. Administration of ACTH and insulin restored phagocytic activity in hypophysectomized rats fed diets containing 3′-methyl-4-dimethylamino-zobenzene.