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Phagocytosis of human blood leukocytes: A simple micromethod
Immunology Letters, 5 (1982) 97-100
Elsevier BiomedicalPress
P H A G O C Y T O S I S O F H U M A N B L O O D L E U K O C Y T E S : A SIMPLE M I C R O M E T H O D V. V~TVI(~KA, L. FORNUSEK, J. KOPE~EK*, J. KAMIfNKOVA**, L. KA~PAREK*** and M. VRANOVA*** Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4, *Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6, e'Faculty of Pediatrics, Charles University, 180 81 Prague 8 and ¢**Bulovka Hospital, Clinic of Otorhinolaryngology, 180 81 Prague8, Czechoslovakia
(Received 10 June 1982) (Accepted 17 June 1982)
1. Summary A simple micromethod for testing human blood leukocyte phagocytosis employing synthetic hydrophilic particles based on 2-hydroxyethylmethacrylate is described. The normal level of phagocytosing leukocytes in healthy children was 20.1 +-2.7%, and in healthy adult donors 34.3 -+ 6.1%. The method was found suitable for routine testing in both clinical and laboratory practice.
2. Introduction Phagocytosis, the original function of which in unicellular and primitive metazoa was feeding of ceils, won a new meaning during evolution. As a process that is responsible for removal of bacteria, damaged cells and other foreign particles, it became an important mechanism of nonspecific immunity. Phagocytic potential belongs to basic parameters used for functional characterization of macro- and microphages. In circulating blood of man phagocytic activity is exhibited by neutrophils, monocytes and eosinophils. Apart from various strains of bacteria and erythrocytes, particles commonly classified as inert are employed in methods for determination of phagocytic activity. This group of particles includes silica, carbonylated metals, CdCO3 microcrystals and latex beads. However, certain types of cells avidly form nonspecific bonds with such particles in depen-
dence on their surface charge and an increasing hydrophobic character [ 1], exhibiting no ability for subsequent ingestion. None of these materials, whether opsonized or not, makes it possible to discern at the light-microscopic level between engulfed particles, particles specifically bound to the surface of phagocytosing cells and particles adhering spontaneously. These difficulties in the distinguishing ability may often hamper correct evaluation of the test for phagocytosis. There are two ways in which to overcome these problems: (i) the nonspecifically bound particles may be washed away or dissolved according to their kind by trypsin-versene [2], xylene or dioxane [3], lysozyme [4] or EDTA treatment [5]. This, however, makes the test complicated and may considerably affect results due to the high sensitivity of macrophages and microphages, particularly neutrophils, to any experimental treatment [6];(ii) that type of particle may be used that guarantees a minimal nonspecific adhesivity and the cells thus marked could be unambiguously denoted as phagocyting. This group of particles comprises synthetic hydrophilic particles on the basis of 2-hydroxyethylmethacrylate which were prepared in our laboratory. Their nonspecific adhesivity to cell surfaces (particularly those of erythrocytes and lymphocytes) is limited due to their low negative charge. Their utilization for the phagocytic micromethod seems to be very suitable for mono- and polymorphonuclear leukocytes of human peripheral blood. Other merits of this method make it suitable for a wide-scale utilization in clinical practice.
0165-2478/82/0000-0000/$2.75 © 1982 Elsevier BiomedicalPress
97
3. Materials and methods
3.1. Cells Fresh samples of complete blood with at most 5 IU of heparine per ml were used. Peripheral blood was obtained from healthy voluntary donors from our department (20-65 years of age), or from children ( 2 - 1 2 years of age, courtesy of the Surgery Clinic of the Bulovka Hospital). Blood of children ( 2 - 1 2 years of age) with chronic otitis was obtained from the Otorhinolaryngologlcal Department of the Bulovka Hospital. 3.2. Particles Particles based on 2-hydroxyethylmethacrylate copolymer [7,8] were prepared by gamma-irradiation activated (6°Co, 10 kGy) copolymerization of 100 ml of a nitrogen-purged 5% aqueous solution of a mixture consisting of 2-hydroxyethylmethacrylate (77.9 w%), ethylenedimethacrylate (0.1 w%), methylenebisacrylamide (2.0 w%) and methacrylic acid (20.0 w%). Chemicals were obtained from Serva, Heidelberg, F.R.G. All detectable monomers were removed by a 10-fold thorough washing with distilled water after irradiation. The procedure yielded a suspension of single (nonagglutinated) particles (12-15 × 10~° particles per ml, diameter 0.9/am). Before use, the stock suspension was diluted with phosphate-buffered saline to a concentration of 4--5 × 108 particles per ml. 3.3. Test for phagocytosis 0.1 ml of heparinized fresh blood was added to 0.05 ml of diluted particle suspension at 37°C with continuous agitation. After 60 rain of incubation the suspension was smeared over microscope slides. Smears were gently stained using the May-GriinwaldGiemsa procedure. At least 200 leukocytes were evaluated; leukocytes with at least 3 engulfed particles were considered as phagocytosing cells.
II la ÷
20~'
10HEALTHy ADULTS1
NON-HEALTItY 3 HEALTHY CHILDREN 2 CHILDREN z
Fig. 1. Phagocytosis o f 2 - h y d r o x y e t h y l m e t h a c r y l a t e copolymer particles by h u m a n peripheral blood leukocytes; rage 2 0 - 6 5 years, n = 14; 2age 2 - 1 2 years, n = 19; %titis media chronica.
Fig. 3). We found no phagocytosing lymphoid cells. From the blood leukocytes of healthy children phagocytosis was exhibited by 20.1% of cells (by 34.2% of monocytes and 34.4% of granulocytes). The test for phagocytosing lymphoid cells was also negative. The found lower phagocytosing activity of cells of peripheral blood of children with respect to that of ceils in adults is in keeping with earlier findings [9]. Significant differences between males and females were observed neither in children nor in adults. Because of findings of a lower phagocytic activity of neutrophils during some diseases [10] we subjected
,,~ 6 0 Ubl
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40
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MONOCYTES GRANULOCYTES
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~ 3o z II.
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ii!i !:?ill ~ i! ~!.: :~:
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4. Results
Our results are summarized in Figs. 1 and 2. From blood leukocytes of healthy adult donors phagocytosis was exhibited by 34.4% of cells, of which by 48.8% of monocytes and 57.8% granulocytes (these were usually neutrophils, eosinophils only exceptionally, 98
HEALTHY ADULTS 1
HEALTHY NON-HEALTI~Y 3 CHILDREN 2 CHILDREN L
Fig. 2. Phagocytosis of 2 - h y d r o x y e t h y l m e t h a c r y l a t e copolymer particles by h u m a n m o n o c y t e s and granulocytes; t age 2 0 - 6 5 , n = 14; 2age 2 - 1 2 , n = 19; %titis media chronica.
to our test a group of children afflicted with chronic middle-ear inflammations (otitis media chronica). We found only 8.2% of phagocytosing blood leukocytes ( 19.3% were monocytes and only 14.4% granulocytes). Exceptionally we found activated lymphoid particlescontaining cells. Of some significance could be a finding that lowering of phagocytic potential is particularly marked with granulocytes (it decreased below 43% of the potential in the group of healthy children). Phagocytic indices (i.e. an average number of phagocytosed particles per phagocytosing ceil) varied in all analyzed groups between 5 and 6.
be due to a low negative charge of these particles and a hydrophilic character of their surfaces [ 1]. Overcoming of the repulsive forces and thus also of the labeling of cells by these particles therefore requires an active participation of the cell surface, i.e. participation of processes that take place during phagocytosis. To verify that the particles really were phagocytosed we screened serial sections with an electron microscope (unpublished results). For a full confidence of positive cells only those cells that were labeled with at least 3 particles were taken. Among other merits of the method are the need for a minimal blood sample and no need for sophisticated laboratory equipment. The latter merit renders possible introduction of this method into nonspecialized laboratories and a broad standardization, that was unthinkable with some earlier methods based on phagocytosis of bacteria and yeasts and on the use of latex particles of a high nonspecific adsorption to cell surfaces. Furthermore, samples prepared for this method could be used for an approximative evaluation of the blood count. The particles could be dyed with common histological dyes: this fact could be appraised in the clinical practice. Similarly to other methods, 2 disadvantages of the described method are: sensitivity to higher concentrations of heparin; ancl upon the use of older blood samples no standard results are obtained. The described method could be used for determination of phagocytic activity of peripheral blood cells as well as of other phagocytosing cells [8]. It therefore represents a suitable test for phagocytosis in both the laboratory and clinical practice.
5. Discussion
References
: f: i i
Fig. 3. Neutrophil phagocytosing 2-hydroxyethyhnethacrylate copolymer particles. Smear of complete blood stain according to May-Grtinwald-Giemsa (× 1600).
Phagocytosis is one of basic elements of a nonspecific resistance of organisms against infection. An important role in this nonspecific resistance is played by monocytes and granulocytes of peripheral blood [11,12, for review, see 6]. Determination of phagocytic activity of leukocytes from peripheral blood was performed by a simple method that proved competent in our laboratory. One of its merits is a negligible spontaneous adherence of the used particles to the tested cells which could
[1] Van Oss, C. J. (1978) Ann. Rev. Microbiol. 32, 19-39. [2] lto, M., Ralph, P. and Moore, M. A. S. (1979) Cell. hnmunol. 46, 48-56. [3] van Furth, R. and Diesselhoff-Den Dulk, M. M. C.
(1980) Scand. J. Immunol. 12,265-269. [4] Vray, B., Hoebeke, J., Saint-GuiUain, M., Leloup, R. and Strosberg, A. D. (1980) Scand. J. lmmunol. 11, 147-153. [5] Palisa, V. (1978) lmunol, zpravodaj 9, 42-45 (in Czech). [6] Lisiewicz, J. (1980) Human Neutxophils, 1st Edn., The Charles Press, Bowie. 99
[7] Rembaum, A., Yen, S. P. S., Cheong, E., Wallace, S., Molday, R. S., Gordon, I. L. and Dreyer, W. J. (1976) Macromolcculcs 9,328-336. [8] Forniisck, L., V~tvi~ka, V. and Kope~ek, J. (1981) Expcrientia 37,418-420. [9] Miyamoto, K. (1965) J. Med. Sci. 14, 19-26.
I00
[ 10 ] lvady, G., Dux, E. and lllyes, M. (1961 ) Z. Kinderheilkd. 85,378-386. [ 11 ] Rabinovitch, M. (1968) Semin. Hematol. 5, 134-155. [ 12] Sbarra, A. J., Selvaraj, R.J., Paul, B. P., Paskitt, P. K. F., Zgliczyfisky, J. M., Mitchell, G. W. and Louis, F. (1976) Int. Rev. exp. Pathol. 16, 249-271.
Elsevier BiomedicalPress
P H A G O C Y T O S I S O F H U M A N B L O O D L E U K O C Y T E S : A SIMPLE M I C R O M E T H O D V. V~TVI(~KA, L. FORNUSEK, J. KOPE~EK*, J. KAMIfNKOVA**, L. KA~PAREK*** and M. VRANOVA*** Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4, *Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6, e'Faculty of Pediatrics, Charles University, 180 81 Prague 8 and ¢**Bulovka Hospital, Clinic of Otorhinolaryngology, 180 81 Prague8, Czechoslovakia
(Received 10 June 1982) (Accepted 17 June 1982)
1. Summary A simple micromethod for testing human blood leukocyte phagocytosis employing synthetic hydrophilic particles based on 2-hydroxyethylmethacrylate is described. The normal level of phagocytosing leukocytes in healthy children was 20.1 +-2.7%, and in healthy adult donors 34.3 -+ 6.1%. The method was found suitable for routine testing in both clinical and laboratory practice.
2. Introduction Phagocytosis, the original function of which in unicellular and primitive metazoa was feeding of ceils, won a new meaning during evolution. As a process that is responsible for removal of bacteria, damaged cells and other foreign particles, it became an important mechanism of nonspecific immunity. Phagocytic potential belongs to basic parameters used for functional characterization of macro- and microphages. In circulating blood of man phagocytic activity is exhibited by neutrophils, monocytes and eosinophils. Apart from various strains of bacteria and erythrocytes, particles commonly classified as inert are employed in methods for determination of phagocytic activity. This group of particles includes silica, carbonylated metals, CdCO3 microcrystals and latex beads. However, certain types of cells avidly form nonspecific bonds with such particles in depen-
dence on their surface charge and an increasing hydrophobic character [ 1], exhibiting no ability for subsequent ingestion. None of these materials, whether opsonized or not, makes it possible to discern at the light-microscopic level between engulfed particles, particles specifically bound to the surface of phagocytosing cells and particles adhering spontaneously. These difficulties in the distinguishing ability may often hamper correct evaluation of the test for phagocytosis. There are two ways in which to overcome these problems: (i) the nonspecifically bound particles may be washed away or dissolved according to their kind by trypsin-versene [2], xylene or dioxane [3], lysozyme [4] or EDTA treatment [5]. This, however, makes the test complicated and may considerably affect results due to the high sensitivity of macrophages and microphages, particularly neutrophils, to any experimental treatment [6];(ii) that type of particle may be used that guarantees a minimal nonspecific adhesivity and the cells thus marked could be unambiguously denoted as phagocyting. This group of particles comprises synthetic hydrophilic particles on the basis of 2-hydroxyethylmethacrylate which were prepared in our laboratory. Their nonspecific adhesivity to cell surfaces (particularly those of erythrocytes and lymphocytes) is limited due to their low negative charge. Their utilization for the phagocytic micromethod seems to be very suitable for mono- and polymorphonuclear leukocytes of human peripheral blood. Other merits of this method make it suitable for a wide-scale utilization in clinical practice.
0165-2478/82/0000-0000/$2.75 © 1982 Elsevier BiomedicalPress
97
3. Materials and methods
3.1. Cells Fresh samples of complete blood with at most 5 IU of heparine per ml were used. Peripheral blood was obtained from healthy voluntary donors from our department (20-65 years of age), or from children ( 2 - 1 2 years of age, courtesy of the Surgery Clinic of the Bulovka Hospital). Blood of children ( 2 - 1 2 years of age) with chronic otitis was obtained from the Otorhinolaryngologlcal Department of the Bulovka Hospital. 3.2. Particles Particles based on 2-hydroxyethylmethacrylate copolymer [7,8] were prepared by gamma-irradiation activated (6°Co, 10 kGy) copolymerization of 100 ml of a nitrogen-purged 5% aqueous solution of a mixture consisting of 2-hydroxyethylmethacrylate (77.9 w%), ethylenedimethacrylate (0.1 w%), methylenebisacrylamide (2.0 w%) and methacrylic acid (20.0 w%). Chemicals were obtained from Serva, Heidelberg, F.R.G. All detectable monomers were removed by a 10-fold thorough washing with distilled water after irradiation. The procedure yielded a suspension of single (nonagglutinated) particles (12-15 × 10~° particles per ml, diameter 0.9/am). Before use, the stock suspension was diluted with phosphate-buffered saline to a concentration of 4--5 × 108 particles per ml. 3.3. Test for phagocytosis 0.1 ml of heparinized fresh blood was added to 0.05 ml of diluted particle suspension at 37°C with continuous agitation. After 60 rain of incubation the suspension was smeared over microscope slides. Smears were gently stained using the May-GriinwaldGiemsa procedure. At least 200 leukocytes were evaluated; leukocytes with at least 3 engulfed particles were considered as phagocytosing cells.
II la ÷
20~'
10HEALTHy ADULTS1
NON-HEALTItY 3 HEALTHY CHILDREN 2 CHILDREN z
Fig. 1. Phagocytosis o f 2 - h y d r o x y e t h y l m e t h a c r y l a t e copolymer particles by h u m a n peripheral blood leukocytes; rage 2 0 - 6 5 years, n = 14; 2age 2 - 1 2 years, n = 19; %titis media chronica.
Fig. 3). We found no phagocytosing lymphoid cells. From the blood leukocytes of healthy children phagocytosis was exhibited by 20.1% of cells (by 34.2% of monocytes and 34.4% of granulocytes). The test for phagocytosing lymphoid cells was also negative. The found lower phagocytosing activity of cells of peripheral blood of children with respect to that of ceils in adults is in keeping with earlier findings [9]. Significant differences between males and females were observed neither in children nor in adults. Because of findings of a lower phagocytic activity of neutrophils during some diseases [10] we subjected
,,~ 6 0 Ubl
$OE o
40
T
MONOCYTES GRANULOCYTES
T
~ 3o z II.
i~i!iiiiii ii~i!i!i!i
ii!i !:?ill ~ i! ~!.: :~:
!ii~iii~i!iil~ ii!i!iii~i
4. Results
Our results are summarized in Figs. 1 and 2. From blood leukocytes of healthy adult donors phagocytosis was exhibited by 34.4% of cells, of which by 48.8% of monocytes and 57.8% granulocytes (these were usually neutrophils, eosinophils only exceptionally, 98
HEALTHY ADULTS 1
HEALTHY NON-HEALTI~Y 3 CHILDREN 2 CHILDREN L
Fig. 2. Phagocytosis of 2 - h y d r o x y e t h y l m e t h a c r y l a t e copolymer particles by h u m a n m o n o c y t e s and granulocytes; t age 2 0 - 6 5 , n = 14; 2age 2 - 1 2 , n = 19; %titis media chronica.
to our test a group of children afflicted with chronic middle-ear inflammations (otitis media chronica). We found only 8.2% of phagocytosing blood leukocytes ( 19.3% were monocytes and only 14.4% granulocytes). Exceptionally we found activated lymphoid particlescontaining cells. Of some significance could be a finding that lowering of phagocytic potential is particularly marked with granulocytes (it decreased below 43% of the potential in the group of healthy children). Phagocytic indices (i.e. an average number of phagocytosed particles per phagocytosing ceil) varied in all analyzed groups between 5 and 6.
be due to a low negative charge of these particles and a hydrophilic character of their surfaces [ 1]. Overcoming of the repulsive forces and thus also of the labeling of cells by these particles therefore requires an active participation of the cell surface, i.e. participation of processes that take place during phagocytosis. To verify that the particles really were phagocytosed we screened serial sections with an electron microscope (unpublished results). For a full confidence of positive cells only those cells that were labeled with at least 3 particles were taken. Among other merits of the method are the need for a minimal blood sample and no need for sophisticated laboratory equipment. The latter merit renders possible introduction of this method into nonspecialized laboratories and a broad standardization, that was unthinkable with some earlier methods based on phagocytosis of bacteria and yeasts and on the use of latex particles of a high nonspecific adsorption to cell surfaces. Furthermore, samples prepared for this method could be used for an approximative evaluation of the blood count. The particles could be dyed with common histological dyes: this fact could be appraised in the clinical practice. Similarly to other methods, 2 disadvantages of the described method are: sensitivity to higher concentrations of heparin; ancl upon the use of older blood samples no standard results are obtained. The described method could be used for determination of phagocytic activity of peripheral blood cells as well as of other phagocytosing cells [8]. It therefore represents a suitable test for phagocytosis in both the laboratory and clinical practice.
5. Discussion
References
: f: i i
Fig. 3. Neutrophil phagocytosing 2-hydroxyethyhnethacrylate copolymer particles. Smear of complete blood stain according to May-Grtinwald-Giemsa (× 1600).
Phagocytosis is one of basic elements of a nonspecific resistance of organisms against infection. An important role in this nonspecific resistance is played by monocytes and granulocytes of peripheral blood [11,12, for review, see 6]. Determination of phagocytic activity of leukocytes from peripheral blood was performed by a simple method that proved competent in our laboratory. One of its merits is a negligible spontaneous adherence of the used particles to the tested cells which could
[1] Van Oss, C. J. (1978) Ann. Rev. Microbiol. 32, 19-39. [2] lto, M., Ralph, P. and Moore, M. A. S. (1979) Cell. hnmunol. 46, 48-56. [3] van Furth, R. and Diesselhoff-Den Dulk, M. M. C.
(1980) Scand. J. Immunol. 12,265-269. [4] Vray, B., Hoebeke, J., Saint-GuiUain, M., Leloup, R. and Strosberg, A. D. (1980) Scand. J. lmmunol. 11, 147-153. [5] Palisa, V. (1978) lmunol, zpravodaj 9, 42-45 (in Czech). [6] Lisiewicz, J. (1980) Human Neutxophils, 1st Edn., The Charles Press, Bowie. 99
[7] Rembaum, A., Yen, S. P. S., Cheong, E., Wallace, S., Molday, R. S., Gordon, I. L. and Dreyer, W. J. (1976) Macromolcculcs 9,328-336. [8] Forniisck, L., V~tvi~ka, V. and Kope~ek, J. (1981) Expcrientia 37,418-420. [9] Miyamoto, K. (1965) J. Med. Sci. 14, 19-26.
I00
[ 10 ] lvady, G., Dux, E. and lllyes, M. (1961 ) Z. Kinderheilkd. 85,378-386. [ 11 ] Rabinovitch, M. (1968) Semin. Hematol. 5, 134-155. [ 12] Sbarra, A. J., Selvaraj, R.J., Paul, B. P., Paskitt, P. K. F., Zgliczyfisky, J. M., Mitchell, G. W. and Louis, F. (1976) Int. Rev. exp. Pathol. 16, 249-271.