Effects of L-arginine on the proliferation of T lymphocyte subpopulations.

BACKGROUND: Dietary supplementation of L-arginine as a mechanism to enhance cellular immune response (T lymphocytes), has slowly gained approval, and appears especially important during critical illness. Despite its clinical use, little is known as to the direct effects of L-arginine on the different T lymphocyte subpopulations. METHODS: Lymphocytes were harvested from spleens of C57 B1/6 mice, and proliferation was induced with anti-CD3 in the presence of different concentrations of L-arginine ranging from 0 to 1000 micromol/L. Flow cytometry was used to evaluate the effect of L-arginine on T lymphocyte subpopulations. Interleukin-2 production was measured by ELISA and gene expression by RT-PCR. RESULTS: L-Arginine at or greater than 100 micromol/L significantly enhanced anti-CD3 stimulated T lymphocyte proliferation (p = .01). L-Arginine was essential for adequate T lymphocyte (CD3+) cellular maturation (p = .01). Proliferation of Helper T cells (CD4+) was not dependent on L-arginine. In contrast, Cytotoxic T cells (CD8+) showed a dose dependent proliferation in response to L-arginine (p = .01). Of the CD8+ cells, an increase in the CD45RA negative CD8 positive (memory) T cell subpopulation was observed with the addition of L-arginine. In addition, the number of cell surface CD8 receptors (CD8R) and CD3 receptors (CD3R) increased in the presence of L-arginine (p = .01, p = .04). Interleukin-2 receptor (IL-2R) expression was not up-regulated by L-arginine. L-Arginine modestly increased IL-2 production and had pronounced effects on its disappearance from the culture media (p < .0001). Interleukin-2 mRNA expression was not dependent on L-arginine. CONCLUSIONS: The requirements for L-arginine for the proliferation of CD3 stimulated T lymphocytes vary widely, and have to be taken into account when studying the mechanism of how L-arginine enhances cellular proliferation. L-Arginine may increase cellular proliferation by increasing specific receptor expression and the utilization of interleukin-2.

Beta adrenoceptor regulation of macrophage arginase activity.

BACKGROUND: Arginase, which metabolizes L-arginine within the urea cycle, is essential for production of polyamines and affects production of nitric oxide by depletion of L-arginine, the common substrate for both arginase and nitric oxide synthase. Having shown that trauma increases splenic macrophage arginase activity, we seek to define the mechanisms for this. RAW macrophage arginase activity and expression are increased by 8-bromo-cAMP in vitro. We hypothesize that since catecholamines increase cAMP, trauma-induced splenic arginase activity may be mediated by post-injury catecholamine release. METHODS: RAW 264.7 macrophage arginase activity was measured in vitro in response to 4 catecholamines with or without propranolol or lipopolysaccharide (LPS). C57BL/6 mice underwent laparotomy as a model of moderate trauma after propranolol treatment, with and without intraperitoneal Escherichia coli LPS administration as a simulated pro-inflammatory stimulus. RESULTS: Macrophage arginase activity increased in vitro in response to catecholamines or LPS (P < .05). Propranolol pretreatment blocked macrophage arginase activity induced by epinephrine (10 mumol/L) in vitro (P < .05). Trauma or LPS alone increased splenic arginase activity in vivo (P < .05). Propranolol did not alter LPS-induced splenic arginase activity but did significantly reduce trauma-induced splenic arginase activity (P < .05). CONCLUSIONS: Catecholamines alone increase macrophage arginase activity through beta-adrenoceptor activation. Increased splenic arginase activity induced by moderate trauma is decreased by beta-adrenoceptor blockade, suggesting that trauma-induced arginase activity is partly mediated by endogenous catecholamines.

Regional brain polyamine levels in permanent focal cerebral ischemia.

Transient global cerebral ischemia has been shown to induce marked changes in the polyamine pathway with a significant increase in putrescine, the product of the ornithine decarboxylase reaction. This study examined the relationship between tissue and extracellular polyamines and regional cerebral blood flow and brain edema. Six hours of focal ischemia in cats (n = 10) was produced by permanent middle cerebral artery occlusion. Extracellular polyamines were measured in extracellular fluid obtained by microdialysis. Regional cerebral blood flow using laser Doppler flowmetry and specific gravity, an indicator of brain edema, were measured in contralateral (non-ischemic), penumbra and densely ischemic brain regions. A significant increase in the tissue putrescine level was found in the penumbra but there was no difference in the putrescine levels between contralateral and densely ischemic regions. There was no significant change in the spermidine and spermine levels in the three regions. Extracellular levels of putrescine and spermidine were found to be significantly lower than the tissue levels and no change in polyamines was observed in any region. Significant edema formation was observed in densely ischemic and penumbra regions. This is the first demonstration that tissue putrescine is increased in the penumbra region, an area of incomplete ischemia that is developing brain edema.

Regulation of polyamine synthesis and transport by fibroblast growth factor in aortic smooth muscle cells.

Basic-FGF (FGF2) is implicated as a regulator of smooth muscle cell proliferation that develops after arterial injury. Polyamines are essential for cell growth and differentiation and may mediate some of the FGF2-elicited responses. To examine this possibility, the effect of FGF2 on polyamine synthesis and uptake was tested on rat arterial smooth muscle cells. Exposure of cells to FGF2 for 24 and 48 h resulted in increased intracellular polyamine content. Ornithine decarboxylase (ODC) activity increased in FGF2-treated cells after 6 h of treatment, whereas no increases were detected in ODC mRNA steady-state levels. Basic-FGF increased maximal polyamine transport rate without changes in Km. Treatment with actinomycin D decreased polyamine transport. The effect of cyclohexamide on polyamine uptake was dose dependent. These studies indicate that treatment of vascular smooth muscle cells with FGF2 results in increases in intracellular polyamine content, polyamine synthetic activity, and polyamine transport.

Effect of cardiogenic shock on pharmacokinetics and tissue distribution of ampicillin-sulbactam.

A reversible cardiogenic shock model in pigs investigated shock-induced changes in the pharmacokinetics and tissue distribution of ampicillin-sulbactam and the efficacy of this antibiotic regimen in eliminating enteric bacterial translocation. Sixteen pigs were randomly allocated to 3 groups: group I (shock, ampicillin-sulbactam, n = 6), group II (no shock, ampicillin-sulbactam, n = 6), and group III (shock, no ampicillin-sulbactam, n = 4). Nalidixic acid-resistant E. coli (60 x 10(6) CFU) were instilled into a jejunal loop created in each pig, and bacterial cultures were taken from thoracic duct lymph, periportal, and mesenteric lymph nodes. Ampicillin-sulbactam was administered intravenously at a standard dose of 3 g. Results showed that 1) ampicillin and sulbactam concentrations generally increase during cardiogenic shock; 2) cardiogenic shock does not increase ampicillin concentrations in jejunum and liver; 3) during resuscitation, thoracic duct lymph ampicillin concentrations decrease; and 4) during and immediately after cardiogenic shock, standard doses of ampicillin-sulbactam appear efficacious in eliminating translocated bacteria.

Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure.

The antibiotic resistance patterns of gram-negative fecal bacteria from pigs in three herds with different histories of antibiotic exposure were examined. In general, smaller proportions of antibiotic-resistant or multiply resistant fecal isolates (P less than 0.05) were obtained from pigs in a herd not exposed to antimicrobial agents for 154 months than from pigs in a herd continuously exposed to antimicrobial agents at subtherapeutic doses or from pigs in a herd exposed only to therapeutic doses of antimicrobial agents. The proportions of antibiotic-resistant and multiply resistant strains were greater among isolates from pigs in the therapeutic herd than in the non-antibiotic-exposed herd (P less than 0.05). The proportion of antibiotic-resistant isolates in the non-lactose-fermenting population was greater than that in the lactose-fermenting population, regardless of herd. The results suggest that any form of antimicrobial exposure will increase the prevalence of antimicrobial resistance and multiple resistance of fecal bacteria.

Stimulation of pheomelanogenesis in cultured B16 melanoma cells by 4-tertiary butylcatechol.

Intermediates of pheomelanin in tissue cultured B16 melanoma cells were analyzed by high performance liquid chromatography, and reduced glutathione (GSH), L-dopa, 2-[(L)-S-cysteinyl]-L-dopa (2-SCD) and 5-[(L)-S-cysteinyl]-L-dopa (5-SCD) were quantified. The effects of 4-tertiary butylcatechol (TBC), an antioxidant which causes skin depigmentation, on the levels of the intermediate were then examined. A concentration of 10(-4) M TBC increased the intracellular levels of GSH, 2-SCD and 5-SCD, whereas the L-dopa level was unchanged. The time-course of the increased intermediates corresponded to the elevation of glutathione-metabolizing enzyme activities previously reported by Kawashima et al. [J. invest. Derm. 82, 53 (1984)] in the same cell line exposed to 10(-4) M TBC. The findings establish chemical evidence that TBC stimulates pheomelanogenesis in melanocytes.

Dermatoses acquired by solid-waste handlers.

A clinical evaluation of the skin changes and injuries among refuse (waste) collectors in San Francisco was conducted in 1983. Almost 75% had palmar calluses--a result of repeated pressure and friction. Those workers who protected their hands with gloves had practically normal skin, with minor or absent calluses. The majority of waste collectors sustained work-related injuries each year. These consisted mainly of sprains, abrasions and lacerations, fractures, and eye injuries. Based on the injury rate, waste collection is a hazardous occupation. The skin is invariably traumatized.

Effects of 4-tertiary butyl catechol on glutathione-metabolizing enzymes in vivo and in vitro.

4-Tertiary butyl catechol (TBC) causes depigmentation in humans and animals and stimulates formation of pheomelanosomes. In this study, we investigated the effects of noncytotoxic doses of TBC on glutathione S-transferase (GST) activity in the skin of Uscd strain mice and B16 murine melanoma cells in culture, in relation to changes in activities of glutathione reductase (GR) and gamma-glutamyl transpeptidase (GGT) reported to be involved in pheomelanogenesis. Occurrence of pheomelanosomes in skin melanocytes was demonstrated by electron microscopy and reduction (25%) of eumelanin content in melanoma cells was shown by spectrophotometry. Topical application of 1 M TBC-DMSO-acetone solution on the ear skin elevated GST activity about 27%, and activities of GGT and GR to 35% and 19%, respectively, within 1 week. Melanoma cells cultured in 10(-4) M TBC-containing medium for 2 h showed no changes in GST and GGT activities, but 12% increase of GR activity during the first 12 h. Activities of all 3 enzymes was elevated (11-17%) 24 h later. The elevation detected by 48 h was 25% for GST, 26% for GGT, and 14% for GR. The findings were interpreted to show that depigmentation produced by the antioxidant results from stimulated pheomelanogenesis through activation of glutathione-metabolizing enzymes and suppressed oxidation of eumelanin intermediates.

Reduction in eumelanin by the activation of glutathione reductase and gamma-glutamyl transpeptidase after exposure to a depigmenting chemical.

Topical application of 4-tertiary butyl catechol (TBC) causes vitiligo in the skin of man and animals, and previous electron microscopic studies showed pheomelanin formation in the affected areas. In the present study, we investigated changes of enzyme activities, eumelanin content and amount of sulfur in tissue cultured human melanoma cells exposed to the depigmenting chemical. TBC enhanced glutathione reductase activity without changing the eumelanin content by 24 hr after exposure and subsequently (by 42 hr) increased gamma-glutamyl transpeptidase activity and sulfur content in the cells with a decrease in eumelanin content. It is suggested that this chemical alters the types of melanin formed by modulation of these enzyme activities.

Glutathione reductase activity in skin exposed to 4-tertiary butyl catechol.

The effect of 4-tertiary butyl catechol (TBC), a potent depigmenting chemical, on glutathione reductase (GR) in pigmented ear skin of hairless mice was investigated. Three topical applications of TBC, which cause neither skin color changes nor melanocyte degeneration, induced an increase in enzyme activity. Since the same treatment resulted in pheomelanin formation as evidenced by electron microscopy, it is suggested that the GR increase correlates at least in part with changes in melanocyte metabolism. This enzyme generates a reduced form of glutathione which may be involved in the formation of glutathionedopas, substrates for hydrolytic enzymes to produce cysteinyldopas, and pheomelanin. Elevation of GR may be an indication of melanogenesis before clinical skin color changes due to TBC appear. It may be used for the early detection of occupational leukoderma.

Ultrastructural demonstration of chemical modification of melanogenesis in hairless mouse skin.

We investigated chemical and physical modifications of the genetically determined ultrastructure of melanosomes. The flank skin of hairless mice was treated with ultraviolet energy (UV) shorter than 320 nm or with a combination of a photosensitizer and UV (PUVA treatment). All melanosomes in the induced melanocytes and those in resident melanocytes in the ear skin showed eumelanogenesis, although the degree of melanin deposition differed considerably according to the induction process. Eumelanogenesis was most advanced in the resident melanocytes while PUVA-induced melanocytes showed more immature premelanosomes. We then topically applied 4-tertiary butyl catechol on the skin. The depigmenting agent caused an appearance of pheomelanosomes. The alteration in melanogenesis was seen most distinctly in premelanosomes of the PUVA-induced cells. Altered ultrastructure was also observed in matured melanosomes; this change was most apparent in the resident melanocytes. These findings indicate that cells with eumelanogenesis may undergo pheomelanogenesis. The present study demonstrated effects of chemicals on genetically determined function of melanocytes by quantitative analysis of melanosome ultrastructure.

Effects of 4-tertiary butyl catechol on melanocytes of hairless mice.

Depigmentary effects of 4-tertiary butyl catechol (TBC) on UV-stimulated melanocytes on the flanks and naturally active melanocytes of ears were compared in Uscd strain hairless mice. UVB irradiation, twice a week for 1 or 2 mo, induced brown-black pigmentation on the flanks. A 1 M TBC application twice a week for 1 mo not only prevented the UV-stimulated pigmentation, but also promoted fading of the post-UV tanning. Dopa-stained split epidermal sheets showed a decrease in the number of melanocytes to less than one-half during the first month of TBC treatment. Melanocytes were often enlarged or lost their dendrites, and both premelanosomes and melanosomes showed ultrastructural changes. However, TBC application on the ears for 1 mo showed neither color change nor a decrease in the number of melanocytes. There were ultrastructural changes in melanocytes but the degree of abnormality was much less than those seen in UV-stimulated melanocytes. Continuation of TBC application for 2 mo with the UV irradiation on the flanks or the nonirradiated ears caused an increase in the number of melanocytes. These results suggest that the initial effect of TBC may be primarily cytotoxicity to melanocytes, and may correlate with their cellular functions. The stimulatory effects of TBC on melanocytes seen during the longer period of time requires further investigation.

Inhibition of tyrosinase activity by 4-tert-butylcatechol and other depigmenting agents.

4-tert-Butylcatechol (TBC) is an antioxidant widely used in industry and a potent depigmenting agent to the skin of the workers. In this study, tyrosinase was extracted from tissue-cultured human melanoma cells and purified by polyacrylamide gel electrophoresis. T1 and T2 tyrosinase, which migrated differently on the gels, were treated with TBC as well as other depigmenting agents and natural substrates of tyrosinase. Changes in the enzyme activity on dopa oxidation were quantified by photometric and radiometric analysis. The enzyme activity was inhibited by TBC and hydroquinone, but only at a high concentration that is toxic to tissue-cultured melanocytes. The activity was also decreased by tyrosine, but was increased by dopa, particularly at a lower concentration. The results indicated that the reported methodology is useful for testing the effects of chemicals with depigmenting or pigmenting potential. TBC and hydroquinone are inhibitors of tyrosinase at concentrations higher than 1 x 10(-3) M. Dopa and tyrosine alter tyrosinase activity in the second step of melanogenesis in the same manner that has been reported to occur in the first step-conversion of tyrosine to dopa.

Detection of environmental depigmenting substances.

We systematically screened the depigmenting capacity of several phenols, catechols and organic antioxidants. Clear-cut depigmentation was achieved with monomethyl ether of hydroquinone (MMH) and tertiary butyl catechol (TBC) using black guinea pigs and black mice as animal models. A goal was to establish a reliable in vivo method to demonstrate or to predict the depigmenting action of chemicals on mammalian melanocytes. There was no universal solvent or optimal body site, although all tested areas could be depigmented. Irritation induced by some vehicles and test materials produced false positive responses. False negative responses with known depigmenting chemicals were observed. Utilizing these observations, we propose a model for screening medicinal and industrial chemicals for depigmenting capacity.

Effects of 4-tertiary butyl catechol on tissue cultured melanocytes.

Depigmenting effects of 4-tertiary butyl catechol (TBC) were investigated using tissue cultured melanocytes of adult guinea pig. Black guinea pig ear epidermis was trypsinized, suspended in BME Eagle media (5 X 10(6) cells) supplemented with 10% calf serum, and seeded in Petri dishes (2 ml/ea). On the 5th day of culture, TBC dissolved in DMSO was added to the media, and the central area of Petri dishes with large numbers of melanocytes (but not keratinocytes) present were examined by light and electron microscopy. After 6 hr of treatment with 1.5 X 10(-3) mg TBC per ml of media, about 15 to 30% of melanocytes detached and about half of the remaining cells showed reversible changes in the cell shapes. An increase of TBC concentration to more than 2 X 10(-3) mg per ml of media resulted in larger numbers of melanocyte deaths. The surviving cells contained numerous, but ultrastructurally unusual premelanosomes and melanosomes. In addition, microfilaments showed a wavy appearance.

Is the antioxidant butylated hydroxytoluene a depigmenting agent in man?

Because of the allegation that butylated hydroxytoluene produced depigmentation in man, a prospective study was performed. Sixteen adult darkly pigmented males received daily occlusive applications for 60 days. Depigmentation was not observed.