DOI 10.1007/s10517-015-2910-0 Cell Technologies in Biology and Medicine, No. 4, February, 2015
142
Effect of In Vitro Cold Exposure on Phagocytic Activity of Human Peripheral Blood Neutrophils T. V. Polezhaeva, O. O. Zaitseva, A. N. Khudyakov, and O. N. Solomina Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 29-32, January, 2015 Original article submitted June 25, 2014 Experiments on peripheral blood leukocytes from healthy donors using the biochemiluminescent method demonstrated the possibility of in vitro modulating phagocytic activity of neutrophils by cold exposure. Cold exposure at 2oC for 30 min increased phagocytic activity of cells, while slow cooling to -2oC reduced it. Key Words: leukocytes; cold exposure; phagocytosis
Long-term experience of using cold exposure for therapeutic purposes first of all implies the local application of hypothermia for pain relief, bleeding control, and treatment of inflammatory diseases [14]. In all living organisms, abrupt environmental changes trigger a stress response typical of not only the whole organism, but also organs, tissues, and cells [19]. Neutrophils and macrophages that perform bactericidal and phagocytic functions are the emergency cells of non-specific resistance in the human body. Neutrophils deserve special attention, because their activation does not require morphological rearrangement or triggering of additional mechanisms: the agent is a sufficient stimulus. Migration of phagocytes into the inflammatory focus proceeds in the following order: neutrophils migrate first, monocytes starts to migrate simultaneously with neutrophils, but their maximum content in the infiltrate is attained later; lymphocytes migrate last. The ideas about the physiological role of neutrophils are now greatly extended. Along with bactericidal activity, they produce cytotoxic and antiviral effects, contribute to manifestation of the killer effect of other cells, mediate various cellular and humoral responses, and can prevent the appearance and growth of malignant cell clones [4]. Oxygen-dependent bactericidal mechanism of neutrophils is studied in detail: it is based on generation Laboratory of Blood Cryophysiology, Institute of Physiology, Komi Research Center, Ural Division of the Russian Academy of Sciences, Syktyvkar, Russia. Address for correspondence: [email protected]. T. V. Polezhaeva
of reactive oxygen metabolites by NADPH oxidase, a multicomponent enzyme of the plasma membranes and membranes of secretory granules [1]. Superoxide anion radical, hydrogen peroxide, hydroxyl radical, and singlet oxygen can destroy intact bacterial cell walls and cell membranes, which determines special role of oxygen-dependent mechanism in comparison with hydrolytic one. Reduced activity of NADPH oxidase is one of the mechanisms of phagocytic insufficiency observed in some innate and genetically determined diseases as well as in thermal burns, radiation therapy, tumor diseases, and in premature babies. The function of NADP oxidase is routinely corrected by drug therapy. In particular, K.Pellegrin recommends the use of recombinant GM-CSF for restoring the neutrophil count and generation superoxide radical by these cells in burned patients [1]. The search for new nonaggressive means for restoring the function of NADP oxidase is now in progress. The developed methods of correction of phagocytic insufficiency are based on the use of stem cells and electromagnetic [5,18] and laser [6] radiation. The use of cold exposure for these purposes is little studied. In clinical and laboratory studies, phagocytic activity of neutrophils is now assessed by chemiluminescent method allowing measurement of the initial functional activity of cells and its changes in response to exposure to various factors. A strong correlation between killing and chemiluminescence of phagocytes has been demonstrated [15]. Chemiluminescent analysis is widely used: from diagnosis of pathologi-
0007-4888/15/15910142 © 2015 Springer Science+Business Media New York
T. V. Polezhaeva, O. O. Zaitseva, et al.
143
cal conditions of the human body to the personalized selection of pharmacological agents [10]. Since neutrophil are the main producer of reactive oxygen species producing bactericidal effect, the contribution of monocyte and lymphocyte luminescence is neglected when analyzing chemiluminescence of venous or capillary blood [9]. Here we studied the effect of cold exposure on phagocytic activity of neutrophils from healthy donors measured in vitro by the chemiluminescent method.
MATERIALS AND METHODS Leukocytes were isolated from the whole blood of volunteers (mean age 39±10 years; all donors signed informed consent) by cytapheresis (2500 rpm with cooling; 5 min, Sorvall). The volume of leukocyte concentrate was 22.0±2.0 ml. This leukocyte-rich transfusion medium (27,000-32,000 per 1 μl) contained minor admixture of erythrocytes, platelets, stem cells, and plasma. Cold exposure at near-zero temperatures -2oC and o 2 C were used [11,12]. For exposure at 2oC, the leukocyte concentrate in microtubes (2-ml aliquots) was incubated for 30 min in a Saratov 1615M refrigerator. For exposure at -2oC, the leukocyte concentrate in a plastic tube (5 ml) was placed in a Derby freezer (-20oC). The temperature of cooled cell suspension was controlled with a Checktemp 1 remote digital thermometer. The mean cooling rate was 2.3oC/min. Gradual cooling ensuring viscous state of the bioobject and excluding crystallization heat release was performed. The total cooling time was 9-10 min. The integrity of cells cooled to -2oC was verified by light microscopy (Nikon H550S) in samples with 1.0% eosin; diffuse pink staining of the cytoplasm was considered a sign of damage to the cell membrane [13]. The effect of cooling on lipid peroxidation and antioxidant activity (AOA) of leukocytes was evaluated by chemiluminescence (CL) induced by hydrogen
peroxide with ferrous sulfate on a BKhL-07 biochemiluminometer (Central Research Laboratory, Nizhny Novgorod State Medical Academy, IMBIO). In a measuring cuvette containing 0.1 ml leukocyte concentrate, 0.4 ml phosphate buffer (pH 7.5), and 0.4 ml 0.01 mM ferrous sulfate (Spectrum Chem), 0.2 ml 2% H2O2 (Khimprom) was promptly added and CL signal was recorded for 30 sec. The following parameters were analyzed: maximum intensity of fast flash (Imax, mV) reflecting free radical oxidation potential of the biological object and CL yield over 30 sec (S, mV×sec) reflecting the content of RO2 radicals. Antioxidant potential (activity of the cell enzyme systems that regulate the content of hydroperoxides) was evaluated by the slope of the curve tg(-2α) (characterizes the maximum rate of curve decline with minus sign); the higher tg(-2α), the higher antioxidant activity of the test sample. Phagocytic activity of leukocytes was analyzed before and after cold exposure on a BKL-07 biochemiluminometer. After automatic noise deduction, the following parameters were analyzed: maximum CL intensity (Imax, mV) reflecting free radical oxidation potential of the biological object, time of CL recording T (sec), and CL yield over 30 sec (S, mV×sec) measured as the area under CL curve. The leukocyte concentrate (0.1 ml) was mixed with 0.05 ml suspension of 0.08-μ latex particles (Sigma-Aldrich). Then, 0.05 ml sample, 0.95 ml Hanks solution, and 0.2 ml luminol working solution were added to the measuring cuvette. The measurements were performed for 30 min at 37oC and constant stirring. In a special experimental series, the effect of epinephrine (10–6 g/liter) on phagocytic activity of neutrophils was evaluated on BKhL-07 biochemiluminometer. To this end, 0.05 ml latex suspension was added to 0.1 ml mixture of leukocyte concentrate with epinephrine (Moscow endocrine plant). Then, 0.05 ml sample, 0.95 ml Hanks solution, and 0.2 ml luminol working solution were added to the measuring cuvette. The measurements were performed for 30 min at 37oC
TABLE 1. Effect of Cold Exposure on Parameters of LPO and AOA of Human Peripheral Blood Leukocytes (Chemiluminescent Analysis) CL parameters Series
2oC (n=12)
o
-2 C (n=12)
Imax, mV
S, mV×sec
tg(-2α)
initial
178.0±22.4
1104±220
46.9±4.7
after cooling
187.0±31.7
1147.0±277.1
50.4±8.2
initial
238.0±13.8
1705.0±257.2
49.4±6.3
after cooling
245.0±10.9
1765±260
49.0±5.8
Note. Here and in Table 2: n: number of samples.
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TABLE 2. Effect of Cold Exposure and Epinephrine on Parameters of Phagocytic Activity of Human Peripheral Blood Leukocytes (Chemiluminescent Analysis) CL parameters Series
2oC (n=10)
o
-2 C (n=10)
Epinephrine 10–6 g/liter
Imax, mV
T, sec
S, mV×sec
226.0±7.1
716.5±50.2
177,100.0±32,380.3
after cooling
308.0±25.5*
631.5±47.4*
267,900.0±37,840.5*
initial
217.0±45.3
711.5±65.8
208,300.0±67,250.5
after cooling
81.0±19.6*
150.7±30.6*
57,810.0±3630.3*
initial
288.0±27.3
683.6±56.5
310,900.0±44,230.2
after cooling
170.0±56.6*
171.3±37.4*
159,100.0±37,280.2*
initial
Note. *p
142
Effect of In Vitro Cold Exposure on Phagocytic Activity of Human Peripheral Blood Neutrophils T. V. Polezhaeva, O. O. Zaitseva, A. N. Khudyakov, and O. N. Solomina Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 29-32, January, 2015 Original article submitted June 25, 2014 Experiments on peripheral blood leukocytes from healthy donors using the biochemiluminescent method demonstrated the possibility of in vitro modulating phagocytic activity of neutrophils by cold exposure. Cold exposure at 2oC for 30 min increased phagocytic activity of cells, while slow cooling to -2oC reduced it. Key Words: leukocytes; cold exposure; phagocytosis
Long-term experience of using cold exposure for therapeutic purposes first of all implies the local application of hypothermia for pain relief, bleeding control, and treatment of inflammatory diseases [14]. In all living organisms, abrupt environmental changes trigger a stress response typical of not only the whole organism, but also organs, tissues, and cells [19]. Neutrophils and macrophages that perform bactericidal and phagocytic functions are the emergency cells of non-specific resistance in the human body. Neutrophils deserve special attention, because their activation does not require morphological rearrangement or triggering of additional mechanisms: the agent is a sufficient stimulus. Migration of phagocytes into the inflammatory focus proceeds in the following order: neutrophils migrate first, monocytes starts to migrate simultaneously with neutrophils, but their maximum content in the infiltrate is attained later; lymphocytes migrate last. The ideas about the physiological role of neutrophils are now greatly extended. Along with bactericidal activity, they produce cytotoxic and antiviral effects, contribute to manifestation of the killer effect of other cells, mediate various cellular and humoral responses, and can prevent the appearance and growth of malignant cell clones [4]. Oxygen-dependent bactericidal mechanism of neutrophils is studied in detail: it is based on generation Laboratory of Blood Cryophysiology, Institute of Physiology, Komi Research Center, Ural Division of the Russian Academy of Sciences, Syktyvkar, Russia. Address for correspondence: [email protected]. T. V. Polezhaeva
of reactive oxygen metabolites by NADPH oxidase, a multicomponent enzyme of the plasma membranes and membranes of secretory granules [1]. Superoxide anion radical, hydrogen peroxide, hydroxyl radical, and singlet oxygen can destroy intact bacterial cell walls and cell membranes, which determines special role of oxygen-dependent mechanism in comparison with hydrolytic one. Reduced activity of NADPH oxidase is one of the mechanisms of phagocytic insufficiency observed in some innate and genetically determined diseases as well as in thermal burns, radiation therapy, tumor diseases, and in premature babies. The function of NADP oxidase is routinely corrected by drug therapy. In particular, K.Pellegrin recommends the use of recombinant GM-CSF for restoring the neutrophil count and generation superoxide radical by these cells in burned patients [1]. The search for new nonaggressive means for restoring the function of NADP oxidase is now in progress. The developed methods of correction of phagocytic insufficiency are based on the use of stem cells and electromagnetic [5,18] and laser [6] radiation. The use of cold exposure for these purposes is little studied. In clinical and laboratory studies, phagocytic activity of neutrophils is now assessed by chemiluminescent method allowing measurement of the initial functional activity of cells and its changes in response to exposure to various factors. A strong correlation between killing and chemiluminescence of phagocytes has been demonstrated [15]. Chemiluminescent analysis is widely used: from diagnosis of pathologi-
0007-4888/15/15910142 © 2015 Springer Science+Business Media New York
T. V. Polezhaeva, O. O. Zaitseva, et al.
143
cal conditions of the human body to the personalized selection of pharmacological agents [10]. Since neutrophil are the main producer of reactive oxygen species producing bactericidal effect, the contribution of monocyte and lymphocyte luminescence is neglected when analyzing chemiluminescence of venous or capillary blood [9]. Here we studied the effect of cold exposure on phagocytic activity of neutrophils from healthy donors measured in vitro by the chemiluminescent method.
MATERIALS AND METHODS Leukocytes were isolated from the whole blood of volunteers (mean age 39±10 years; all donors signed informed consent) by cytapheresis (2500 rpm with cooling; 5 min, Sorvall). The volume of leukocyte concentrate was 22.0±2.0 ml. This leukocyte-rich transfusion medium (27,000-32,000 per 1 μl) contained minor admixture of erythrocytes, platelets, stem cells, and plasma. Cold exposure at near-zero temperatures -2oC and o 2 C were used [11,12]. For exposure at 2oC, the leukocyte concentrate in microtubes (2-ml aliquots) was incubated for 30 min in a Saratov 1615M refrigerator. For exposure at -2oC, the leukocyte concentrate in a plastic tube (5 ml) was placed in a Derby freezer (-20oC). The temperature of cooled cell suspension was controlled with a Checktemp 1 remote digital thermometer. The mean cooling rate was 2.3oC/min. Gradual cooling ensuring viscous state of the bioobject and excluding crystallization heat release was performed. The total cooling time was 9-10 min. The integrity of cells cooled to -2oC was verified by light microscopy (Nikon H550S) in samples with 1.0% eosin; diffuse pink staining of the cytoplasm was considered a sign of damage to the cell membrane [13]. The effect of cooling on lipid peroxidation and antioxidant activity (AOA) of leukocytes was evaluated by chemiluminescence (CL) induced by hydrogen
peroxide with ferrous sulfate on a BKhL-07 biochemiluminometer (Central Research Laboratory, Nizhny Novgorod State Medical Academy, IMBIO). In a measuring cuvette containing 0.1 ml leukocyte concentrate, 0.4 ml phosphate buffer (pH 7.5), and 0.4 ml 0.01 mM ferrous sulfate (Spectrum Chem), 0.2 ml 2% H2O2 (Khimprom) was promptly added and CL signal was recorded for 30 sec. The following parameters were analyzed: maximum intensity of fast flash (Imax, mV) reflecting free radical oxidation potential of the biological object and CL yield over 30 sec (S, mV×sec) reflecting the content of RO2 radicals. Antioxidant potential (activity of the cell enzyme systems that regulate the content of hydroperoxides) was evaluated by the slope of the curve tg(-2α) (characterizes the maximum rate of curve decline with minus sign); the higher tg(-2α), the higher antioxidant activity of the test sample. Phagocytic activity of leukocytes was analyzed before and after cold exposure on a BKL-07 biochemiluminometer. After automatic noise deduction, the following parameters were analyzed: maximum CL intensity (Imax, mV) reflecting free radical oxidation potential of the biological object, time of CL recording T (sec), and CL yield over 30 sec (S, mV×sec) measured as the area under CL curve. The leukocyte concentrate (0.1 ml) was mixed with 0.05 ml suspension of 0.08-μ latex particles (Sigma-Aldrich). Then, 0.05 ml sample, 0.95 ml Hanks solution, and 0.2 ml luminol working solution were added to the measuring cuvette. The measurements were performed for 30 min at 37oC and constant stirring. In a special experimental series, the effect of epinephrine (10–6 g/liter) on phagocytic activity of neutrophils was evaluated on BKhL-07 biochemiluminometer. To this end, 0.05 ml latex suspension was added to 0.1 ml mixture of leukocyte concentrate with epinephrine (Moscow endocrine plant). Then, 0.05 ml sample, 0.95 ml Hanks solution, and 0.2 ml luminol working solution were added to the measuring cuvette. The measurements were performed for 30 min at 37oC
TABLE 1. Effect of Cold Exposure on Parameters of LPO and AOA of Human Peripheral Blood Leukocytes (Chemiluminescent Analysis) CL parameters Series
2oC (n=12)
o
-2 C (n=12)
Imax, mV
S, mV×sec
tg(-2α)
initial
178.0±22.4
1104±220
46.9±4.7
after cooling
187.0±31.7
1147.0±277.1
50.4±8.2
initial
238.0±13.8
1705.0±257.2
49.4±6.3
after cooling
245.0±10.9
1765±260
49.0±5.8
Note. Here and in Table 2: n: number of samples.
Cell Technologies in Biology and Medicine, No. 4, February, 2015
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TABLE 2. Effect of Cold Exposure and Epinephrine on Parameters of Phagocytic Activity of Human Peripheral Blood Leukocytes (Chemiluminescent Analysis) CL parameters Series
2oC (n=10)
o
-2 C (n=10)
Epinephrine 10–6 g/liter
Imax, mV
T, sec
S, mV×sec
226.0±7.1
716.5±50.2
177,100.0±32,380.3
after cooling
308.0±25.5*
631.5±47.4*
267,900.0±37,840.5*
initial
217.0±45.3
711.5±65.8
208,300.0±67,250.5
after cooling
81.0±19.6*
150.7±30.6*
57,810.0±3630.3*
initial
288.0±27.3
683.6±56.5
310,900.0±44,230.2
after cooling
170.0±56.6*
171.3±37.4*
159,100.0±37,280.2*
initial
Note. *p
