Georgia Schuller-Levis

Taurine chloramine inhibits the synthesis of nitric oxide and the release of tumor necrosis factor in activated RAW 264.7 cells.

Taurine is present in high concentrations in most mammalian tissues, including those that prodigiously produce oxidants. Taurine protects against bronchiolar damage induced by NO2, ozone, bleomycin, and amio‐ darone. Taurine is chlorinated to form taurine chloramine (Tau‐Cl) by the halide‐dependent myeloperoxidase system and, under physiological conditions, reduces HOC1 toxicity. Although NO and its metabolites, ΝO2− and NOs−, are thought to be major mediators of tissue damage resulting from oxidant exposure, cytokines, including tumor necrosis factor (TNF), are also involved. We examined the effects of Tau‐Cl on NO production and TNF release by using RAW 264.7 cells activated with recombinant interferon‐γ (rIFN‐γ; 50 U/ml) and lipopoly‐ saccharide (LPS; 10 μg/ml). NO was measured spec‐ trophotometrically as ΝO2− after reaction with Griess reagent and TNF was measured by FLISA. Tau‐Cl (0.5 mM) inhibits NO and TNF released into the medium by 47% and 43%, respectively. Tau‐Cl is actively transported into RAW 264.7 cells by an uptake system that is energy, temperature, and Na+ dependent. Competition experiments demonstrate that the uptake system for Tau‐ Cl is distinct from that for taurine. In addition, the NO synthase activity of cytosolic preparations from activated RAW 264.7 cells is irreversibly inhibited by pretreatment with Tau‐Cl. We demonstrate that Tau‐Cl inhibits production of NO and TNF by activated macrophages and suggest a mechanism through which taurine supplementation may protect against oxidant‐induced tissue damage.

The production of superoxide anion and nitric oxide by cultured murine leukocytes and the accumulation of TNF-α in the conditioned media is inhibited by taurine chloramine

Taurine chloramine (Tau-Cl) inhibits production of nitric oxide (NO) by activated peritoneal macrophages and attenuates accumulation of tumor necrosis factor-alpha (TNF-alpha) in the culture media, similar to that previously reported for activated RAW 264.7 cells. In addition, the effect of Tau-Cl and taurine on superoxide anion (O2-) production in murine peritoneal exudate polymorphonuclear leukocytes (PMN) was examined. Tau-Cl inhibited O2- production in a manner that was dose-dependent and reversible. Taurine also inhibited O2- production by stimulated PMN, but at higher concentrations and to a lesser extent than Tau-Cl. The effects of taurine on O2- production was attributed to the in vitro formation of Tau-Cl catalyzed by PMN associated halide-dependent myeloperoxidase. In contrast, production of NO by activated peritoneal macrophages and accumulation of TNF-alpha in the media was inhibited by Tau-Cl while taurine was without effect. These data lend support to the notion that Tau-Cl may participate in the inflammatory response by modulating production of inflammatory mediators.

Immunologic consequences of taurine deficiency in cats.

Our results show that a lack of taurine in the diet of cats results in a significant leukopenia, a shift in the percentage of polymorphonuclear and mononuclear leukocytes, an increase in the absolute count of mononuclear leukocytes, and a change in the sedimentation characteristics of white cells. Functional studies of polymorphonuclear cells isolated from cats fed taurine‐free diets show a significant decrease in the respiratory burst as measured by chemiluminescence as well as a decrease in phagocytosis of Staphylococcus epidermis compared to cats fed the same diet containing taurine. In addition, serum gamma globulin in cats fed taurine‐free diets was significantly increased compared to taurine‐supplemented cats, indicating that other immune cells may be affected by taurine deficiency. Histological examination of lymph nodes and spleen revealed regression of follicular centers with depletion of reticular cells, mature and immature lymphocytes (B cell areas), as well as mild extravascular hemolysis. These results indicate that there are profound immunologic consequences in cats with prolonged taurine deficiency.

Taurine protects against oxidant-induced lung injury: possible mechanism(s) of action.

It is thought that oxidant-induced tissue damage is not a direct effect of the oxidant per se, but rather results from the inflammatory response that occurs thereafter. As a result of inflammation following oxidant exposure, there are neutrophils, monocytes, and macrophages with myeloperoxidase-H2O2-halide activity in the lung. Leukocytes and especially neutrophils contain high intracellular concentrations (22-50mM) of taurine (6, 8, 11, 20). Taurine acts as a trap for toxic hypochlorous acid (HOCl) and forms the less reactive metabolite, N-chlorotaurine (5-6). Thus, the biological activity of halide-dependent myeloperoxidase may be regulated by endogenous taurine. Although taurine had no effect in the present study, polymorphonuclear leukocytes have an active myeloperoxidase system capable of producing N-chlorotaurine (9, 19) and would be present at the site of inflammation in oxidant-exposed lungs. Our data suggest that taurine via N-chlorotaurine formation may protect the lung from oxidant injury, at least in part, by inhibiting production of nitrite and TNF-alpha. Moreover, lavage cells isolated from rats pretreated with taurine and exposed to O3 have a significant decrease in the production of nitrite and TNF-alpha, compared with lavage cells from rats exposed to O3 without taurine supplementation (preliminary studies). Both the concentration of taurine and the effects of N-chlorotaurine strengthen the potential impact of this chlorinated amine in vivo. N-Chlorotaurine may protect against oxidant-induced lung injury by inhibiting production of nitrite and the release of TNF-alpha which are both known to be directly linked to tissue injury.

Taurine Protects Rat Bronchioles from Acute Ozone-Induced Lung Inflammation and Hyperplasia

Ozone is a potent respiratory irritant known to induce lung injury in humans and experimental animals. The present studies determined if ozone-induced lung inflammation was modified by pretreatment of animals with taurine, a detoxifying antioxidant. Rats were pretreated for 10 days with 5% taurine in their drinking water (controls received water only) prior to exposure to 2 ppm ozone for 3 h. At 2, 6, 12, 24, 48, and 72 h after ozone exposure, rats were anesthetized and the lungs were perfusion-fixed for histopathologic evaluation. An additional group of rats was used to examine bronchoalveolar lavage cell counts and hydroxyproline levels. A count of bronchoalveolar lavage cells 48 h after ozone exposure showed significantly fewer total inflammatory cells and fewer polymorphonuclear leukocytes accompanied by a reduction in hydroxyproline in the lavage fluid of ozone-exposed rats pretreated with taurine compared to rats that did not receive taurine. Light microscopy revealed an inflammatory cell infiltrate in the lungs of rats exposed to ozone. This was followed by focal hyperplasia in the terminal and respiratory bronchioles. Rats pretreated with taurine and then exposed to ozone showed none of these alterations. In addition, although there was a significant reduction in cell proliferation as measured by DNA precursor incorporation in the lungs of rats pretreated with taurine prior to ozone exposure compared to unsupplemented rats, the distribution of labeled cells was the same in taurine supplemented and unsupplemented groups. Also, significantly higher levels of taurine were found in the plasma, whole blood, and lavage fluid of rats pretreated with dietary taurine compared to rats that received water only. The results suggest that supplemental taurine protects rat lung epithelium from acute ozone-induced lung inflammation and hyperplasia.

Taurine Chloramine Inhibits the Production of Superoxide Anion, IL-6 and IL-8 in Activated Human Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes (PMN) are the initial cells recruited to the site of inflammation where foreign invaders such as microorganisms, toxic gases and chemicals elicit an inflammation reaction4,6,12. Activated PMN produce various oxygen and hydroxyl radicals which kill bacteria and fungi but may also induce indiscriminate cellular damage14. For example, HOCl/OCl- produced by the halide-dependent myeloperoxidase of PMN is highly toxic. Taurine attenuates the damage caused by HOCl/OCl- by forming taurine chloramine (Tau-Cl), a relatively non-toxic and long-lived oxidant7,9,13,15. Previously we demonstrated that Tau-Cl suppressed the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in activated murine peritoneal macrophages and/or RAW 264.7, a murine macrophage cell line8,10,11. Tau-Cl also inhibits the production of superoxide anion in activated murine peritoneal PMN5. Since human PMN produce Tau-Cl by halide-dependent myeloperoxidase, we investigated the effect of Tau-Cl on the production of various cytokines and superoxide anion by activated human PMN. In this report we demonstrate that Tau-Cl down-regulates the production of superoxide anion, IL-6 and IL-8 by activated human PMN.

Taurine chloramine inhibits prostaglandin E2 production in activated raw 264.7 cells by post-transcriptional effects on inducible cyclooxygenase expression

Taurine chloramine (Tau-Cl) was recently demonstrated to inhibit production of nitric oxide and tumor necrosis factor-alpha (TNF-alpha) by activated macrophages. Since increased production of prostaglandin E2 (PGE2), a reaction catalyzed by induction of cyclooxygenase-2 (COX-2), is also associated with the inflammatory response, we determined the effects of Tau-Cl on PGE2 production and on expression of COX-2 protein and COX-2 mRNA in activated RAW 264.7 cells, a murine macrophage-like cell line. Tau-Cl inhibited production of PGE2 in a concentration dependent manner with an IC50 of 0.4 mM. The decrease in PGE2 production was largely accounted for by decreased expression of COX-2 protein. Although the kinetics of COX-2 mRNA expression was altered in Tau-Cl treated cells, mRNA expression appeared to be quantitatively unimpaired. These results suggest that Tau-Cl affects the post-transcriptional regulation of COX-2 expression and support the idea that Tau-Cl may function as an inhibitory modulator of the inflammatory response.

Taurine Protects rat Bronchioles from Acute Ozone Exposure: A Freeze Fracture and Electron Microscopic Study

Dietary taurine has been shown to protect rat and hamster lung epithelia from acute oxidant injury. One of the earliest morphologic criteria of oxidant injury is the alteration of tight junctions of the peripheral lung airways. In the present study, we have used this criteria to evaluate whether taurine was capable of protecting rat lungs from ozone exposure. Rats were treated for 10 days with 50% taurine in their drinking water, prior to exposure to 2 ppm of ozone for 3 hours. The lungs from rats pretreated with taurine and exposed to ozone were compared to untreated rats exposed to ozone and air-exposed controls. At 2, 6, 12, 24, 48, and 72 hours after exposure to air or ozone, rats were anesthetized and the lungs perfusion-fixed through the right side of the heart with a solution of glutaraldehyde and paraformaldehyde. Light microscopy revealed the typical, mild inflammatory cell infiltrate beginning at 6 hours after ozone exposure in bronchioles, alveolar ducts, and surrounding alveoli which was absent in the lungs of animals treated with taurine. Electron microscopic analysis of thin sections indicated alterations in tight junctions which was confirmed by tracer studies using ruthenium red and lanthanum. Alterations in airway epithelium tight junctions were seen 2 and 6 hours after ozone treatment and only in the 2-hour tissues from animals pretreated with taurine prior to ozone exposure. Freeze-fracture replicas from all exposure groups by electron microscopy revealed that only the 2- and 6-hour groups showed alterations in tight junctions. The alterations were characterized by decreased number of fibrils and breaks in the fibrils. Rats treated with taurine and exposed to ozone exhibited these alterations focally at 2 hours exposure and no changes were noted at 6 hours post ozone exposure. These data confirmed previous findings that injury induced by ozone is transient and that taurine protects the bronchioles from this form of oxidant injury.

Activation of alveolar leukocytes isolated from cats fed taurine-free diets.

Taurine is a ubiquitous amino sulfonic acid in mammals, present in high concentrations in tissues, including those exposed to elevated levels of oxidants. Experiments were designed to examine the consequences of taurine deficiency on production of ROI in leukocytes isolated from the lungs and blood of cats fed taurine-deficient diets. Cats were maintained on taurine-free or taurine-supplemented diets for at least 12 months at which time taurine deficiency was evident. To analyze alveolar cells, lungs were lavaged to recover lung macrophages and PMNs. Lung lavage fluid from cats contained macrophages and PMNs, although taurine deficiency was associated with a decrease in the percentage of PMNs in the lungs. This is similar to our findings in blood that taurine deficiency reduced the proportion of PMNs. Taurine measurements revealed 2.1 +/- 1.6 mumol/g wet wt of taurine in the lungs from cats fed a taurine-deficient diet versus 8.3 +/- 2.6 in lungs from cats fed a diet supplemented with taurine (n = 16). The effects of taurine deficiency on the functional activity of lung macrophages and PMNs were analyzed including the production of ROI. Alveolar leukocytes from cats fed taurine-deficient diets produced more superoxide anion in response to phorbol myristate acetate than cats fed taurine supplemented diets. Similar results were obtained using a chemiluminescence assay. Using the highly specific H2O2 indicator dye, dichlorofluorescin, and flow cytometry we found that alveolar leukocytes made more H2O2 than cells from cats fed taurine-supplemented diets. Forty-two percent of the cells from cats fed a taurine-supplemented diet expressed class II antigens. In contrast, 72% of cells from the taurine-deficient cats expressed this antigen. We hypothesize that taurine functions to prevent terminal activation and release of cytotoxic mediators by lung macrophages. Thus, a deficiency of taurine will indeed cause an activation of leukocytes, as evidenced by our data which show an increase in ROI, as well as an increase in class II antigen.

Distribution of taurine-like immunoreactivity in cerebellum of kittens from taurine-supplemented and taurine-deficient mothers

Using an antibody prepared against taurine conjugated to bovine serum albumin with glutaraldehyde, the distribution of taurine in cerebellum of newborn and 8‐week‐old kittens from mothers fed 0, 0.02, 0.05, or 1% dietary taurine has been determined. In general, taurine‐like immunoreactivity was greater in kittens from mothers fed the greatest amounts of taurine, as was the total cerebellar taurine concentration. The most notable feature in newborn kitten cerebellum was a dense band of staining in the inner molecular layer adjacent to the Purkinje cell layer, which corresponds to the short Purkinje cell dendrites. In cerebellum of 8‐week‐old kittens, taurine‐like immunoreactivity was present in Purkinje cells and their dendrites, most granule cells, and a few interneurons in the molecular layer of the 0.02, 0.05, and 1% groups. The cerebellum of the 0% group was distinctive in that virtually no neurons were reactive, appearing as ‘ghosts’ against the background, and both white matter and the granule cell layer contained large numbers of reactive astrocytes. The presence of such large numbers of reactive astrocytes and the immunoglobulin within the brain suggests an impairment of the blood‐brain barrier in such taurine‐deficient kittens.