Americanjournal of Pathology, Vol.
136, No. 3, March 1990
Copyright C) American Association ofPathologists
Protection Against Membrane-mediated Cytotoxicity by Calcium and Zinc
Daruka Mahadevan,* Amelia Ndirika,* Jennifer Vincent,* Lindsay Bashford,* Timothy Chambers,t and Charles Pasternak* From the Departmenits of Cellular atd Molecular Sciences* and Histopathology,rt St George s Hospital Medical School, Cranimer Terrace, Lonidon, Eniglanid
Injection of S. aureus a toxin or ultraviolet-inactivated Sendai virus into the mammary gland of lactating mice leads to cytopathic changes in the alveolar cells compatible with a breakdown oftheirpermeability barrier. Simultaneous administration of Ca2+ or Zn2+ prevents such changes, as it does when added to alveolar cells in vitro. It is concluded that increased levels of Zn2+ (and under certain conditions of Ca2+) may exert a protective role that is clinically significant. (Am J Pathol 1990, 136: 513-520)
Extracellular Ca2+ is known to play a role in physiologic processes such as blood coagulation1 and complement activation,2 and its concentration in plasma is under tight endocrine control.3 Its protective role on cell membranes, first described for plant cells in 19144 and extended to animal (neuronal) cells in 1957,5 has recently been shown to include the prevention of cytotoxicity caused by hemolytic agents as diverse as certain viruses,67 bacterial,69 and animal10 toxins, the membrane attack complex of complement11 and synthetic compounds such as detergents12 and cationic proteins.13 Zn2+ protects animals against bacterial infections14,15 and is said to shorten the duration of the common cold in humans.1617 The demonstration that Zn2+, like Ca2+, inhibits the leakage of ions and intracellular metabolites across the plasma membrane of cells affected in vitro by hemolytic agents10-13-1819 supports the hypothesis that one of the physiologic roles of Zn2+,2012 as of Ca2-,22 is to prevent membrane damage. As yet no experiments to test this hypothesis with intact animals have been carried out. The mouse mammary gland is an anatomically appropriate system for studying the cytotoxic effects of injected pathogens and drugs, and has been used to assess the
pathology of S. aureus a toxin, a common causative agent of bovine mastitis as well as of respiratory disease in humans, in particular.23 Here we show that Ca2+ and Zn2+ directly protect alveolar cells against the effects of purified a toxin. The demonstration that hemolytic (but not nonhemolytic) Sendai virus causes similar damages as toxin, that are inhibited by Ca2+ or Zn2+, supports the proposal24-26 that certain viruses exert a toxinlike action in vivo and widens the scope for the therapeutic use of zinc in human and animal diseases.
Materials and Methods
Chemicals [Methyl-3H]choline, 45CaCI2, 65ZnSO4, and methyl (-D[U-
14C]gluco)pyranoside were obtained from Amersham International, United Kingdom. Sendai virus was grown in the laboratory as previously described.27 It was either of the early-harvest nonhemolytic but infectious12,28 type, or of the late-harvest hemolytic type; in the latter case it was ultraviolet (UV)-irradiated (30 minutes on ice) before use to render it noninfective. Unless stated otherwise, virus was of the late-harvest type. S. aureus a and 6 toxins were partially purified samples donated by Dr. Joyce de Azavedo. Polylysine (Type IV, molecular weight 4000 to 15,000) was from Sigma Chemical Co. (St. Louis, MO). Hemagglutination and hemolysis assays were carried out by conventional techniques with human (Sendai virus) or rabbit (S. aureus a and 6 toxin) blood, and are expressed as hemagglutination units (HAU) and hemolysis units (HU), respectively.
Animals Lactating MFI mice (30 to 40 g) 4 to 5 days after parturition were used. Supported by the Cell Surface Research Fund. Accepted for publication April 25, 1989. Address reprint requests to Dr. C. A. Pasternak, Department of Cellular and Molecular Sciences, St George's Hospital Medical School, Cranmer Terrace, London SW1 7 ORE.
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Experiments With Intact Animals Mice were lightly anesthetized with ether (cotton wool pad placed over nostril). Toxin, virus, Ca2", Zn2+, or buffer in a total volume of 0.01 to 0.1 ml was injected through the nipple into one of the pair of the abdominal (fourth) mammary glands; the other abdominal gland received a relevant control (eg, buffer versus toxin, toxin plus Ca2' versus toxin, and so on). For experiments with radioisotopes, the same solution (10 molar [mmol] 45Ca2+; 2 X i07 cpm/ ml, or 10 mmol/l 65Zn2+; 107 cpm/ml or 0.1 mmol/l methyl[14C]glucoside; 2.5 X 1i07 cpm/ml) was injected into each of the two abdominal glands. After varying periods of time the animals were killed by cervical dislocation. For some experiments, a 5% aqueous solution of procion yellow (Derma fast yellow MX 4R, Sandoz, Inc.) was injected into each abdominal gland 10 minutes before death. The abdominal glands were removed and processed for paraffin sectioning or placed on a drop of Tissue-tek on cork and immersed in isopentane cooled in liquid nitrogen. Paraffin sections were stained with hematoxylin and eosin. Frozen sections were cut as previously described' and photographed under a Leitz ultraviolet microscope (absorption 500 mm). The exposure time was the same in each case (as a result of which some sections are under exposed and others overexposed), as well as details of development, to allow the extent of procion yellow uptake to be assessed under standard conditions. For experiments with radioisotopes, mice were stunned, decapitated, and 0.2 to 0.4 ml blood collected into a heparinized beaker. The blood was spun and 0.1 ml plasma assayed for 14C, 45Ca or 65Zn by scintillation counting; the cell pellet was extracted with 14% cold trichloroacetic acid (TCA), spun, and a 0.1 ml sample of the supernatant assayed for 14C, 45Ca, or 65Zn; the TCAinsoluble pellet contained essentially no radioactivity in any sample. The two abdominal mammary glands were removed and processed as follows: either six thin sections (each 20 to 30 mg wet weight) were cut from each gland at intervals from the point of injection, or the entire gland was homogenized in 2 ml of 5% TCA and 0.1 ml samples assayed. Duplicate sections (20 to 30 mg wet weight) were cut from one of the other (noninjected) glands (L3) and from liver and from each kidney. In the case of the abdominal mammary glands from 65Zn- and 45Ca-injected mice there was a sharp decrease in radioactivity away from the point of injection: sections near the nipple contained up to 20 times as much radioactivity as those further away.
Experiments With Mammary Gland Cells The two abdominal (L4 and R4) and the two inguinal (L5 and R5) mammary glands of a lactating mouse were re-
moved and washed with an ice-cold solution of tricinebuffered saline (147 mmol/l NaCI, 3.7 mmol/l KCI, 1 mmol/l MgCI2, and 20 mmol/l tricine pH 7.4). Fatty tissue was removed, the glands finely chopped, and placed in 5 ml of collagenase (C2139, Sigma; 5 mg/ml of tricinebuffered saline) for 30 minutes at 37 C with occasional shaking. The suspension was diluted with an equal volume of Eagle's medium, filtered through gauze to remove residual clumps of cells, and the filtrate spun at 200g for 5 minutes. The cell pellet was suspended in 1.5 ml Eagle's medium and incubated with 10, of [3H]choline (1 mC/ml) for 30 minutes at 37 C. The cells were washed in hepesbuffered saline (150 mmol/l NaCI, 5 mmol/l KCI, 1 mmol/l MgCl2, and 5 mmol/l tris, pH 7.4; HBS) and resuspended in 0.75 ml of HBS containing 0.1% albumin. Most of the radioactivity in such cells is in the form of phosphoryl[3H]choline, which only leaks out of damaged cells.27 Labeled cells (0.02 ml in a final volume of 0.5 ml of HBS containing 0.1% albumin; 1 to 2 X 105/ml) were incubated with or without added agent (toxin, virus, or polylysine) at 37 C, in the absence or presence of CaCl2 or ZnSO4, as indicated. Samples were removed at intervals, spun, and the radioactivity in supernatant and pellet was measured. Leakage is expressed as a percentage of the radioactivity leaked into the supernatant. In the experiment illustrated in Figure 2, leakage from control cells was 34% during 30 minutes; in other experiments this value varied between 30% and 50%. An alternate procedure for assaying leakage was as follows. Alveolar cells prepared as above were suspended (approximately 1 06/ml) in HBS containing 1% albumin and incubated with or without an added agent in the absence or presence of CaC12 or ZnSO4 for varying periods of time at 37 C. Samples were removed at intervals, added to ethidium bromide (final concentration 1 mM) at room temperature, and spun and washed in HBS 1 minute later. The cell pellets were resuspended in HBS and the number of stained and unstained cells determined by fluorescence microscopy. Several randomly chosen fields, total more than 20 cells per condition, were assessed. Only well-defined single cells were counted; small debris or clumped cells were excluded.
Results Retention of 45Ca2+ and 65Zn2+ in Mammary Glands In Vivo Of the five pairs of mouse mammary glands, the fourth (abdominal) pair forms two discrete organs that retain material injected through the nipple for some time and are then easy to manipulate and section. Thus toxin can be injected into one gland, and saline, or toxin plus Ca2' or
Membrane Protection by Calcium and Zinc
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AJPMarch 1990, Vol. 136, No. 3
Zn2+, can be injected into the other gland for comparison. Because it was thought that Ca2+ and Zn2+ might be eliminated very rapidly from such glands, preliminary experiments were carried out to determine how well glands retain Ca2+ or Zn2+. 45Ca2+ or 65Zn2+ were injected and the tissues were assayed later for radioactivity at various times; because extracellular Ca2+ and Zn2+ might be expected to be removed through binding to plasma proteins, a soluble, nonmetabolizable marker that does not bind to proteins, namely a-methyl[14C]glucoside, was injected for comparison. The results show that 45Ca and 65Zn are retained in the injected glands at least as well as a-methyl[14C]glucoside (8% and 2% of injected 45Ca; 14% and 18% of injected 65Zn retained at 20 minutes and 3 hours after injection, compared with 5% and 4% of injected [14C] at the same times). An uninjected mammary gland (L3) of an animal injected with 45Ca, 65Zn, or 14C assayed for comparison had much less radioactivity present. In the case of 65Zn there was little loss from the abdominal (L4 and R4) mammary glands between 20 minutes (14%) and 9.5 hours (1 1%), with much of it probably bound to protein; 2% was in blood at 20 minutes, falling to
136, No. 3, March 1990
Copyright C) American Association ofPathologists
Protection Against Membrane-mediated Cytotoxicity by Calcium and Zinc
Daruka Mahadevan,* Amelia Ndirika,* Jennifer Vincent,* Lindsay Bashford,* Timothy Chambers,t and Charles Pasternak* From the Departmenits of Cellular atd Molecular Sciences* and Histopathology,rt St George s Hospital Medical School, Cranimer Terrace, Lonidon, Eniglanid
Injection of S. aureus a toxin or ultraviolet-inactivated Sendai virus into the mammary gland of lactating mice leads to cytopathic changes in the alveolar cells compatible with a breakdown oftheirpermeability barrier. Simultaneous administration of Ca2+ or Zn2+ prevents such changes, as it does when added to alveolar cells in vitro. It is concluded that increased levels of Zn2+ (and under certain conditions of Ca2+) may exert a protective role that is clinically significant. (Am J Pathol 1990, 136: 513-520)
Extracellular Ca2+ is known to play a role in physiologic processes such as blood coagulation1 and complement activation,2 and its concentration in plasma is under tight endocrine control.3 Its protective role on cell membranes, first described for plant cells in 19144 and extended to animal (neuronal) cells in 1957,5 has recently been shown to include the prevention of cytotoxicity caused by hemolytic agents as diverse as certain viruses,67 bacterial,69 and animal10 toxins, the membrane attack complex of complement11 and synthetic compounds such as detergents12 and cationic proteins.13 Zn2+ protects animals against bacterial infections14,15 and is said to shorten the duration of the common cold in humans.1617 The demonstration that Zn2+, like Ca2+, inhibits the leakage of ions and intracellular metabolites across the plasma membrane of cells affected in vitro by hemolytic agents10-13-1819 supports the hypothesis that one of the physiologic roles of Zn2+,2012 as of Ca2-,22 is to prevent membrane damage. As yet no experiments to test this hypothesis with intact animals have been carried out. The mouse mammary gland is an anatomically appropriate system for studying the cytotoxic effects of injected pathogens and drugs, and has been used to assess the
pathology of S. aureus a toxin, a common causative agent of bovine mastitis as well as of respiratory disease in humans, in particular.23 Here we show that Ca2+ and Zn2+ directly protect alveolar cells against the effects of purified a toxin. The demonstration that hemolytic (but not nonhemolytic) Sendai virus causes similar damages as toxin, that are inhibited by Ca2+ or Zn2+, supports the proposal24-26 that certain viruses exert a toxinlike action in vivo and widens the scope for the therapeutic use of zinc in human and animal diseases.
Materials and Methods
Chemicals [Methyl-3H]choline, 45CaCI2, 65ZnSO4, and methyl (-D[U-
14C]gluco)pyranoside were obtained from Amersham International, United Kingdom. Sendai virus was grown in the laboratory as previously described.27 It was either of the early-harvest nonhemolytic but infectious12,28 type, or of the late-harvest hemolytic type; in the latter case it was ultraviolet (UV)-irradiated (30 minutes on ice) before use to render it noninfective. Unless stated otherwise, virus was of the late-harvest type. S. aureus a and 6 toxins were partially purified samples donated by Dr. Joyce de Azavedo. Polylysine (Type IV, molecular weight 4000 to 15,000) was from Sigma Chemical Co. (St. Louis, MO). Hemagglutination and hemolysis assays were carried out by conventional techniques with human (Sendai virus) or rabbit (S. aureus a and 6 toxin) blood, and are expressed as hemagglutination units (HAU) and hemolysis units (HU), respectively.
Animals Lactating MFI mice (30 to 40 g) 4 to 5 days after parturition were used. Supported by the Cell Surface Research Fund. Accepted for publication April 25, 1989. Address reprint requests to Dr. C. A. Pasternak, Department of Cellular and Molecular Sciences, St George's Hospital Medical School, Cranmer Terrace, London SW1 7 ORE.
513
514
Mahadevan et al
AlPMarch 1990, Vol. 136, No. 3
Experiments With Intact Animals Mice were lightly anesthetized with ether (cotton wool pad placed over nostril). Toxin, virus, Ca2", Zn2+, or buffer in a total volume of 0.01 to 0.1 ml was injected through the nipple into one of the pair of the abdominal (fourth) mammary glands; the other abdominal gland received a relevant control (eg, buffer versus toxin, toxin plus Ca2' versus toxin, and so on). For experiments with radioisotopes, the same solution (10 molar [mmol] 45Ca2+; 2 X i07 cpm/ ml, or 10 mmol/l 65Zn2+; 107 cpm/ml or 0.1 mmol/l methyl[14C]glucoside; 2.5 X 1i07 cpm/ml) was injected into each of the two abdominal glands. After varying periods of time the animals were killed by cervical dislocation. For some experiments, a 5% aqueous solution of procion yellow (Derma fast yellow MX 4R, Sandoz, Inc.) was injected into each abdominal gland 10 minutes before death. The abdominal glands were removed and processed for paraffin sectioning or placed on a drop of Tissue-tek on cork and immersed in isopentane cooled in liquid nitrogen. Paraffin sections were stained with hematoxylin and eosin. Frozen sections were cut as previously described' and photographed under a Leitz ultraviolet microscope (absorption 500 mm). The exposure time was the same in each case (as a result of which some sections are under exposed and others overexposed), as well as details of development, to allow the extent of procion yellow uptake to be assessed under standard conditions. For experiments with radioisotopes, mice were stunned, decapitated, and 0.2 to 0.4 ml blood collected into a heparinized beaker. The blood was spun and 0.1 ml plasma assayed for 14C, 45Ca or 65Zn by scintillation counting; the cell pellet was extracted with 14% cold trichloroacetic acid (TCA), spun, and a 0.1 ml sample of the supernatant assayed for 14C, 45Ca, or 65Zn; the TCAinsoluble pellet contained essentially no radioactivity in any sample. The two abdominal mammary glands were removed and processed as follows: either six thin sections (each 20 to 30 mg wet weight) were cut from each gland at intervals from the point of injection, or the entire gland was homogenized in 2 ml of 5% TCA and 0.1 ml samples assayed. Duplicate sections (20 to 30 mg wet weight) were cut from one of the other (noninjected) glands (L3) and from liver and from each kidney. In the case of the abdominal mammary glands from 65Zn- and 45Ca-injected mice there was a sharp decrease in radioactivity away from the point of injection: sections near the nipple contained up to 20 times as much radioactivity as those further away.
Experiments With Mammary Gland Cells The two abdominal (L4 and R4) and the two inguinal (L5 and R5) mammary glands of a lactating mouse were re-
moved and washed with an ice-cold solution of tricinebuffered saline (147 mmol/l NaCI, 3.7 mmol/l KCI, 1 mmol/l MgCI2, and 20 mmol/l tricine pH 7.4). Fatty tissue was removed, the glands finely chopped, and placed in 5 ml of collagenase (C2139, Sigma; 5 mg/ml of tricinebuffered saline) for 30 minutes at 37 C with occasional shaking. The suspension was diluted with an equal volume of Eagle's medium, filtered through gauze to remove residual clumps of cells, and the filtrate spun at 200g for 5 minutes. The cell pellet was suspended in 1.5 ml Eagle's medium and incubated with 10, of [3H]choline (1 mC/ml) for 30 minutes at 37 C. The cells were washed in hepesbuffered saline (150 mmol/l NaCI, 5 mmol/l KCI, 1 mmol/l MgCl2, and 5 mmol/l tris, pH 7.4; HBS) and resuspended in 0.75 ml of HBS containing 0.1% albumin. Most of the radioactivity in such cells is in the form of phosphoryl[3H]choline, which only leaks out of damaged cells.27 Labeled cells (0.02 ml in a final volume of 0.5 ml of HBS containing 0.1% albumin; 1 to 2 X 105/ml) were incubated with or without added agent (toxin, virus, or polylysine) at 37 C, in the absence or presence of CaCl2 or ZnSO4, as indicated. Samples were removed at intervals, spun, and the radioactivity in supernatant and pellet was measured. Leakage is expressed as a percentage of the radioactivity leaked into the supernatant. In the experiment illustrated in Figure 2, leakage from control cells was 34% during 30 minutes; in other experiments this value varied between 30% and 50%. An alternate procedure for assaying leakage was as follows. Alveolar cells prepared as above were suspended (approximately 1 06/ml) in HBS containing 1% albumin and incubated with or without an added agent in the absence or presence of CaC12 or ZnSO4 for varying periods of time at 37 C. Samples were removed at intervals, added to ethidium bromide (final concentration 1 mM) at room temperature, and spun and washed in HBS 1 minute later. The cell pellets were resuspended in HBS and the number of stained and unstained cells determined by fluorescence microscopy. Several randomly chosen fields, total more than 20 cells per condition, were assessed. Only well-defined single cells were counted; small debris or clumped cells were excluded.
Results Retention of 45Ca2+ and 65Zn2+ in Mammary Glands In Vivo Of the five pairs of mouse mammary glands, the fourth (abdominal) pair forms two discrete organs that retain material injected through the nipple for some time and are then easy to manipulate and section. Thus toxin can be injected into one gland, and saline, or toxin plus Ca2' or
Membrane Protection by Calcium and Zinc
515
AJPMarch 1990, Vol. 136, No. 3
Zn2+, can be injected into the other gland for comparison. Because it was thought that Ca2+ and Zn2+ might be eliminated very rapidly from such glands, preliminary experiments were carried out to determine how well glands retain Ca2+ or Zn2+. 45Ca2+ or 65Zn2+ were injected and the tissues were assayed later for radioactivity at various times; because extracellular Ca2+ and Zn2+ might be expected to be removed through binding to plasma proteins, a soluble, nonmetabolizable marker that does not bind to proteins, namely a-methyl[14C]glucoside, was injected for comparison. The results show that 45Ca and 65Zn are retained in the injected glands at least as well as a-methyl[14C]glucoside (8% and 2% of injected 45Ca; 14% and 18% of injected 65Zn retained at 20 minutes and 3 hours after injection, compared with 5% and 4% of injected [14C] at the same times). An uninjected mammary gland (L3) of an animal injected with 45Ca, 65Zn, or 14C assayed for comparison had much less radioactivity present. In the case of 65Zn there was little loss from the abdominal (L4 and R4) mammary glands between 20 minutes (14%) and 9.5 hours (1 1%), with much of it probably bound to protein; 2% was in blood at 20 minutes, falling to
