Immunopharmacology and Immunotoxicology
ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: http://www.tandfonline.com/loi/iipi20
Mercuric Chloride Inhibition of Leucocyte Migration K. Nordlind & S. Lidén To cite this article: K. Nordlind & S. Lidén (1990) Mercuric Chloride Inhibition of Leucocyte Migration, Immunopharmacology and Immunotoxicology, 12:4, 715-721 To link to this article: http://dx.doi.org/10.3109/08923979009019686
Published online: 28 Sep 2008.
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Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iipi20 Download by: [University of California Santa Barbara]
Date: 05 November 2015, At: 23:04
IMMUNOPHARMACOLOGY A N D IMMUNOTOXICOLOGY, 12(4), 715-721 (1990)
MERCURIC CHLORIDE INHIBITION OF LEUCOCYTE MIGRATION
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
K. Nordlind* and S. Liden
Department of Dermatology, Karolinska Hospital, S-104 01 Stockholm, Sweden
ABSTRACT The effects of mercuric chloride, at different concentrations, were tested on polymorphonuclear and mononuclear leucocyte migration by using the sealed capillary migration technique, and these effects were compared to influence on cell number and dye exclusion. A migration inhibition was obtained at 1.~ x ~ O when ~ M tested on polymorphonuclear cells, while a concentration of 6 . 6 ~ 1 0 - ~ inhibited the migration of mononuclear cells. Polymorphonuclear cells seemed to be more sensitive (were inhibited at a lower dose) to mercuric chloride as regards migration compared to cell number and dye exclusion, while mononuclear cells showed migration inhibition at about the same dose range as that which had effect on dye exclusion and cell number.
INTRODUCTION Through interactions with different chemical groups, like sulphur groups, mercury is a very reactive substance and may modify both membrane structure and function, and enzyme activity (1). Mercuric chloride has been shown to
*
To whom correspondence should be addressed 715
Copyright 0 1991 by Marcel Dekker, Inc.
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
716
NORDLIND AND LIDEN
influence a variety of in vitro and in vivo functions of the human and animal immune system (for refs see, e.g. , 1,2) , higher concentrations give a toxic effect. At a high concentration of mercuric chloride (3.~ x ~ O - ,~ Man) inhibited proliferation (3) and a cell degeneration, as judged by dye exclusion and fine structure (4), were found in guinea pig thymocytes. In order to further investigate the toxicity of mercuric chloride on leucocytes, the present work was designed to study its influence on the capacity for polymorphonuclear and mononuclear leucocyte migration and this effect was related to effect on methylene blue dye exclusion (5) and on cell number. MATERIALS AND METHODS Test Substance The final concentrations of mercuric chloride in the capillaries were 2.6~10-~-2. ~ x ~ O - ~Physiological M. saline was added to the control capillaries. Micrration Test The sealed capillary migration test was carried out as described by Ibrahim and Vyas (6), Jones (7) and Sandberg ( 8 ) . 30 ml of heparinized blood was obtained from healthy control subjects. Mononuclear leucocytes were obtained by allowing 10 ml blood to sediment in plastic tubes for 30 min at 37OC in the presence of 2 ml 1% Methocel MC 25 (Fluka AG, Switzerland) solution. The leucocyte-rich supernatant was diluted in Gey's solution pH 7.2 and centrifuged at 500 g for 5 min (10). The cell pellets were washed three times with phosphate buffered saline and finally resuspended in RPMI 1640 medium with 10% heat-inactivated human AB serum. The cell concentrations were adjusted so that lx107 cells/ml were
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
INHIBITION OF LEUCOCYTE MIGRATION
717
obtained after addition of the mercuric chloride doses. The cell suspensions were divided into groups of 50 p 1 and different amounts of mercuric chloride dissolved in saline, or saline, were added in a volume of 25 p1. The mixture was allowed to spontaneously fill glass capillaries (length 50 mm, volume 25 pl: Kebo, Sweden) to a given fraction of their length, usually 35 mm. Five capillaries were filled with cells from each group. The dry end was closed by melting in fire, and the capillaries were centrifuged for 5 min at 170 g. The open ends were then sealed with Seale-easeR (Clay Adams, N. J. , USA) , and the capillaries were attached to microscopic slides. The starting position of the cell-fluid interphase was determined using a microscope with an ocular scale (grade from 0-100) at a magnification of 25X. The capillaries were incubated in a horizontal position at 37OC and the linear migration of the cells was determined after 1 and 24 h. The migration was expressed as migration units (=scale fractions). Vital Dve Test and Cell Number At 2 4 h incubation time, absolute cell counts were determined by counting in a light microscope (Burker chamber). For the vital dye test one drop of 1% methylene blue solution was added to 0.25 ml of a concentrated suspension of mononuclear or polymorphonuclear leucocytes at 24 h of culture. After 1 min the suspension was poured onto slide, covered with a coverslip and the frequency of stained cells determined in a light microscope (1,000~). Statistical Analysis The Mann-Whitney test was used for statistical evaluation of results.
718
NORDLIND AND LIDEN
TABLE 1 Effect of Mercuric Chloride on the Migration of Human Polymorphonuclear Leucocytes.
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
~
~~
~~
~
Mercuric chloride, M
lh
24h
2. 6x10m4
10+4a
17+3a
1.3~10-~
21+7a
25+6a
6.6~10-~
27+5a
38+ba
1.1x10-~
39+6
64k16b
2.4~10-~
4 1+5
83+2 1
Control
43+5
91+2 1
Results are given as the mean+S.E.M. of triplicate experiments with cells from different donors. a p
ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: http://www.tandfonline.com/loi/iipi20
Mercuric Chloride Inhibition of Leucocyte Migration K. Nordlind & S. Lidén To cite this article: K. Nordlind & S. Lidén (1990) Mercuric Chloride Inhibition of Leucocyte Migration, Immunopharmacology and Immunotoxicology, 12:4, 715-721 To link to this article: http://dx.doi.org/10.3109/08923979009019686
Published online: 28 Sep 2008.
Submit your article to this journal
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iipi20 Download by: [University of California Santa Barbara]
Date: 05 November 2015, At: 23:04
IMMUNOPHARMACOLOGY A N D IMMUNOTOXICOLOGY, 12(4), 715-721 (1990)
MERCURIC CHLORIDE INHIBITION OF LEUCOCYTE MIGRATION
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
K. Nordlind* and S. Liden
Department of Dermatology, Karolinska Hospital, S-104 01 Stockholm, Sweden
ABSTRACT The effects of mercuric chloride, at different concentrations, were tested on polymorphonuclear and mononuclear leucocyte migration by using the sealed capillary migration technique, and these effects were compared to influence on cell number and dye exclusion. A migration inhibition was obtained at 1.~ x ~ O when ~ M tested on polymorphonuclear cells, while a concentration of 6 . 6 ~ 1 0 - ~ inhibited the migration of mononuclear cells. Polymorphonuclear cells seemed to be more sensitive (were inhibited at a lower dose) to mercuric chloride as regards migration compared to cell number and dye exclusion, while mononuclear cells showed migration inhibition at about the same dose range as that which had effect on dye exclusion and cell number.
INTRODUCTION Through interactions with different chemical groups, like sulphur groups, mercury is a very reactive substance and may modify both membrane structure and function, and enzyme activity (1). Mercuric chloride has been shown to
*
To whom correspondence should be addressed 715
Copyright 0 1991 by Marcel Dekker, Inc.
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
716
NORDLIND AND LIDEN
influence a variety of in vitro and in vivo functions of the human and animal immune system (for refs see, e.g. , 1,2) , higher concentrations give a toxic effect. At a high concentration of mercuric chloride (3.~ x ~ O - ,~ Man) inhibited proliferation (3) and a cell degeneration, as judged by dye exclusion and fine structure (4), were found in guinea pig thymocytes. In order to further investigate the toxicity of mercuric chloride on leucocytes, the present work was designed to study its influence on the capacity for polymorphonuclear and mononuclear leucocyte migration and this effect was related to effect on methylene blue dye exclusion (5) and on cell number. MATERIALS AND METHODS Test Substance The final concentrations of mercuric chloride in the capillaries were 2.6~10-~-2. ~ x ~ O - ~Physiological M. saline was added to the control capillaries. Micrration Test The sealed capillary migration test was carried out as described by Ibrahim and Vyas (6), Jones (7) and Sandberg ( 8 ) . 30 ml of heparinized blood was obtained from healthy control subjects. Mononuclear leucocytes were obtained by allowing 10 ml blood to sediment in plastic tubes for 30 min at 37OC in the presence of 2 ml 1% Methocel MC 25 (Fluka AG, Switzerland) solution. The leucocyte-rich supernatant was diluted in Gey's solution pH 7.2 and centrifuged at 500 g for 5 min (10). The cell pellets were washed three times with phosphate buffered saline and finally resuspended in RPMI 1640 medium with 10% heat-inactivated human AB serum. The cell concentrations were adjusted so that lx107 cells/ml were
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
INHIBITION OF LEUCOCYTE MIGRATION
717
obtained after addition of the mercuric chloride doses. The cell suspensions were divided into groups of 50 p 1 and different amounts of mercuric chloride dissolved in saline, or saline, were added in a volume of 25 p1. The mixture was allowed to spontaneously fill glass capillaries (length 50 mm, volume 25 pl: Kebo, Sweden) to a given fraction of their length, usually 35 mm. Five capillaries were filled with cells from each group. The dry end was closed by melting in fire, and the capillaries were centrifuged for 5 min at 170 g. The open ends were then sealed with Seale-easeR (Clay Adams, N. J. , USA) , and the capillaries were attached to microscopic slides. The starting position of the cell-fluid interphase was determined using a microscope with an ocular scale (grade from 0-100) at a magnification of 25X. The capillaries were incubated in a horizontal position at 37OC and the linear migration of the cells was determined after 1 and 24 h. The migration was expressed as migration units (=scale fractions). Vital Dve Test and Cell Number At 2 4 h incubation time, absolute cell counts were determined by counting in a light microscope (Burker chamber). For the vital dye test one drop of 1% methylene blue solution was added to 0.25 ml of a concentrated suspension of mononuclear or polymorphonuclear leucocytes at 24 h of culture. After 1 min the suspension was poured onto slide, covered with a coverslip and the frequency of stained cells determined in a light microscope (1,000~). Statistical Analysis The Mann-Whitney test was used for statistical evaluation of results.
718
NORDLIND AND LIDEN
TABLE 1 Effect of Mercuric Chloride on the Migration of Human Polymorphonuclear Leucocytes.
Downloaded by [University of California Santa Barbara] at 23:04 05 November 2015
~
~~
~~
~
Mercuric chloride, M
lh
24h
2. 6x10m4
10+4a
17+3a
1.3~10-~
21+7a
25+6a
6.6~10-~
27+5a
38+ba
1.1x10-~
39+6
64k16b
2.4~10-~
4 1+5
83+2 1
Control
43+5
91+2 1
Results are given as the mean+S.E.M. of triplicate experiments with cells from different donors. a p
