Original Paper Int Arch Allergy Immunol 1992;97:109-114
Department of Respiratory Medicine, CHU Sart Tilman, University of Liège, Belgium
Key Words Histamine Formylmethionyl peptides Polymorphonuclear leukocytes Chemotactic deactivation
Role of Histamine in the Chemotactic Deactivation of Polymorphonuclear Leukocytes Following Incubation with Formylmethionyl Peptides Abstract To clarify the mechanism of chemotactic deactivation of polymorphonuclear neutrophils (PMN) following incubation with the synthetic dipeptide N-formylmethionyl phenylalanine (FMP), we tested the hypothesis that histamine, which is released from leukocytes during incubation with FMP, could explain this inhibition. Human PMN were incubated in the presence or absence of FMP (10-7 to 10~5 M) and histamine measured fluorometrically in the super natant. Washed PMN were then tested in Boyden chambers against FMP (10-5 M ) and other chemoattractants. Incubation of leukocytes with FMP caused a nonpreferential PMN deactivation which was proportionally and kinetically related to FMP-induced histamine release. No histamine release or chemotactic deactivation was observed in the absence of Ca2+ and Mg2+. The inhibitory effect of the peptide was significantly prevented by an H2 blocker or when using basophil-depleted PMN suspensions. Histaminase abolished the capacity of FMP-incubated leukocyte supernatants to decrease PMN chemotaxis. Preincubation of leukocytes with anti-IgE or a sensitizing allergen caused a significant PMN chemotactic deactivation. These results show that hista mine, which is released during leukocyte incubation with FMP, contributes, at least in part, to the chemotactic deactivation of PMN.
Introduction Prcincubation of human polymorphonuclear neutro phils (PMN) with any of several synthetic chemotactic formylmethionine-containing oligopeptides causes a de crease of the PMN chemotactic response towards the peptide but also towards other structurally unrelated che moattractants [1-3]. The mechanism of this heterologous (nonpreferential) deactivation of chemotaxis remains poorly understood. In addition to being chemotactic, for-
mylmethionyl peptides induce histamine release from hu man basophils [4], Since histamine is capable of inhibiting the chemotactic response of neutrophils [5-7], the present experiments were undertaken to investigate the role of histamine in the mechanism of formylmethionyl peptide induced nonpreferential chemotactic deactivation of PMN. Our results show that histamine, which is released from basophils during incubation of leukocytes with the peptide, may explain, at least in part, the chemotactic deactivation of the PMN.
Correspondence to: Dr. M. Radermecker Department of Respiratory Medicine C H U Sart Tilman University of Liège B-4000 Liège (Belgium)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Th. Bury M. Radermecker
Materials and Methods Reagents
The synthetic chemotactic dipeptide N-formylmethionyl phenyl alanine (FMP) was purchased from Sigma Chemical (St. Louis, Mo., USA). FMP was found to be 10-100 times less efficient than the tripeptide N-formylmcthionyl-lcucyl phenylalanine as chemoattractant and histamine rclcascr for human leukocytes. It was dissolved in M e,S 04 (Fisher) and diluted to 50% M e,S 04to make stock solutions. Additional reagents included zymosan (Sigma), diamine oxidase at grade II purity (Sigma), leukotriene B4 (LTB4, Merck-Frost, Cana da), histamine dihydrochloride (Fluka, Buchs, Switzerland), clemas tine (Specia, Paris, France), and cimetidinc (Smith-Kline RIT, Rixensart, Belgium). Purified allergenic extracts (house dust, grass pol len) were provided by the Pasteur Institute (Paris). Polyclonal rabbit antihuman IgE and antihuman IgG were obtained from Bchringwerke (Marburg, FRG). Preparation o f Leukocyte Suspension Peripheral venous blood (40 ml) was drawn from healthy adult volunteers into 10-ml Vacutainer tubes (Becton Dickinson), each containing 143 USP units of lithium heparin and 1 ml of Plasmagel (Bellon Laboratories, Neuilly, France). The blood was gently mixed and allowed to sediment for 45 min at 38 °C. The upper plasma layer rich in leukocytes was removed and pooled into a plastic tube. The concentration of leukocytes was adjusted with Hanks’ balanced salt solution (HBSS; Gibco) without phenol red to a final concentration of 107 cells/ml. The cell viability was above 95%, as assessed by trypan blue exclusion. Chemotactic Deactivation o f PMN A suspension of human leukocytes (107 cells/ml) was incubated for 30 min at 37°C with HBSS in the absence or presence of FMP. The leukocytes were harvested by centrifugation and washed three times in HBSS without Ca2+ and Mg2+. The leukocytes were then re suspended at 2 •106/ml in HBSS containing 2% bovine serum albumin.
Histamine Assay Histamine was measured by an automated spectrofluoromctric assay [9], Histamine release, as a percent of the histamine content of the leukocyte suspension, was calculated using the following formula: HK)x(c-b) % release - -------------c-b where e represents the fluorometric reading for the experimental su pernatant, b the fluorometric reading of the blank, and c the fluoro metric reading of the leukocyte suspension. FMP at 10-5 M and below does not interfere with the assay. Separation o f Mononuclear Cells from Neutrophils Mononuclear cells were separated from neutrophils by density gradient centrifugation of the leukocyte suspension on Lymphoprcp (Nyegaard, Oslo, Norway). Nine-milliliter aliquots of leukocyte sus pension (107 cells/ml) diluted 1/3 with HBSS without Ca2+ and Mg2+ were layered on 4.5-ml cushions of Lymphoprcp (density 1.077) and centrifuged at 400g for 40 min at 22 °C. The neutrophils were freed of red blood cells by hypotonic lysis using NH4C1. Mononuclear cells and neutrophils were separately washed twice with cold HBSS with out Ca2+ and Mg2+ containing 0.3 mg/ml human serum albumin and resuspended in HBSS to 2 -106 cells/ml. Leukocytes were counted us ing a cell counter (Coulter). Basophil and histamine contents were measured from mononuclear cell and neutrophil suspensions (> 95% purity). Basophils were stained by toluidinc blue and their percent age calculated by examining 500 cells. Statistical Analysis The unpaired Student t test was used. A p value < 0.05 was consid ered significant. Results are presented as mean values ±SD of at least three different experiments.
Results
111)
Bury/Radermecker
Figure 1 shows that prior exposure of human leukocytes to FMP at concentrations ranging from 10~13 to 10-5 M caused a rapid and dose-related inhibition of the chemo tactic response of the PMN towards the peptide (10~5 M). The viability of the PMN, as assessed by trypan blue exclu sion, was not affected by prior exposure to the peptide. As shown in table 1, preincubation of leukocytes with FMP at 10-5 M caused a nonpreferential chemotactic deactivation. The same level of chemotactic inhibition was indeed ob served for FMP and other structurally unrelated chemo attractants such as LTB4 or activated autologous serum. When leukocytes were incubated with FMP (10~5 M, 30 min, 37 °C), histamine was released in the supernatant. It was found that leukocytic histamine release and PMN chemotactic deactivation kinetics were closely parallel (fig. 2). When leukocytes were incubated in HBSS con taining FMP (10~9 to 10~5M), we found parallel dose-effect relationships between basophilic histamine release and
Histamine and Neutrophil Chemotactic Deactivation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Neutrophil Chemotaxis Neutrophil chemotaxis was studied by using a modification of the assay of Boyden [8]. Briefly, a modified Boyden chemotactic chamber (Schleicher and Schiill, Dasscl, FRG) and a 5-um MF Millipore filter (Millipore, Bedford, Mass., USA) were used. Chemotactic factors (FMP 10"5M, LTB43 x lO^/W) in HBSS or 1/3 diluted zymosan-activa ted scrum were injected (1 ml) into the lower compartment of the chamber and a 2-ml aliquot of the leukocyte suspension into the up per compartment. The chambers were incubated for 3 h at 37 °C in humidified air containing 5% C 0 2. The filters were then removed, stained with Diff-Quick (Harleco), and the migration quantified mi croscopically. The chemotaxis was measured by counting the neutro phils at high power (x 440) in ten microscopic fields on both surfaces of the filter. All samples were run in duplicate and the mean values calculated. The results were expressed as percent migration which is the number of cells on the lower side and as percentage of cells on the starting side of the filter. The random mobility of neutrophils was as sessed by determining the percentage of migration when no chemo tactic agent (HBSS alone) was present in the lower compartment of the modified chamber. Under our experimental conditions, the PMN chemotactic response was not significantly different when using a pu rified neutrophil preparation (95% purity) instead of a whole leuko cyte suspension.
Fig. 1. Effect of the time of preincubation on the ability of FMP (K)'1', 10'9, and 10"5 M) to chemotactically deactivate human PMN towards this peptide (10"5 M).
Fig. 2. Kinetics of chemotactic deactivation and of histamine re lease following incubation of human leukocytes with FMP (K)'5 M) After different incubation times, histamine was measured in the su pernatant of centrifuged leukocyte suspensions and washed leuko cytes tested for chemotactic response to FMP (10'5 M).
Table 1. Effect of prior exposure (30 min, 37 °C) of human leu kocytes to FMP (10"5M) on their chemotactic response against dif ferent chemoattractants at optimal concentrations
FMP (10"5M) LTB4 (3 x KPM) Activated serum (diluted 1/3)
Prior exposure to HBSS
HBSS + FMP (10“5M)
% inhi bi tion
441 ±98 348 ±42 442 ±21
120 ±22 142 ±34 109±46
73 59 75
PMN chemotactic deactivation. When repeating the same experiments with HBSS lacking Ca2+ and Mg2+, neither histamine release nor PMN chemotactic deactivation was observed, i.e., washed PMN responded normally towards FMP (10~5 M) when tested in the Boyden chamber using HBSS containing Ca2+ and Mg2+ (fig. 3). Incubation of leukocytes with FMP (10~7M) in the presence of an H2 his tamine receptor antagonist, cimetidine (10~7 and 1(P5 M ), decreased in a dose-related fashion the inhibitory effect of the peptide on the chemotactic response of the PMN (p < 0.01). By contrast, clemastine, an H, histamine recep tor antagonist, had no significant effect (fig. 4). Prior ex posure of human leukocytes to cimetidine or clemastine at 10~7 and 10~5 M had no significant effect on their random
Fig. 3. Dose-effect relationships and effect of divalent cations on the ability of FMP to cause histamine release from basophils and to chemotactically deactivate neutrophils. Incubation time 30 min.
I ll
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Chemoattractants
Table 2. Basophil and histamine content of mononuclear cell and neutrophil suspensions obtained following Ficoll-Hypaque cen trifugation and dextran sedimentation of whole blood
Mononuclear cells
Cimetidine + FMP ( 1 0 M)
Clemastine + FMP (1 0 '7 /W)
Fig. 4. Effect of H, and H2 histamine antagonists on the ability of FMP to cause human PMN chcmotactic deactivation. Leukocytes were incubated (30 min, 37 °C) in HBSS alone (control migration) or in HBSS containing FMP (10~7M) in the absence or presence of H, or H, histamine receptor antagonist (10~7 and 10~5 M). Washed leuko cytes were then tested in the Boyden chamber for chemotactic responsiveness to FMP (10"s M).
H I
Leukocytes or neutrophils preincubated in HBSS alone I Leukocytes preincubated with FMP
W/M Neutrophils preincubated with FMP Increasing proportions of mononuclear cells (and basophils) were added to neutrophils and preincubated with FMP
Fig. 5. Effect of basophil depletion and enrichment on the ability of FMP to cause chcmotactic deactivation of human leukocytes. Mononuclear cells and the bulk of basophils can be separated from the neutrophils by centrifugation of a whole leukocyte suspension on Lymphoprep. Whole leukocyte suspensions and pure neutrophil preparations were incubated in the presence or absence of FMP (K)'5M) and tested for chemotaxis in a Boyden chamber against FMP
(10's M ) .
112
Bury/Radermecker
Basophils (per 103 cells)
8 19 15 22
0 0 3 4
Histamine (ng/2-106 cells)
22 55 49 64
0 3 11 23
mobility or chemotactic response to FMP. Mononuclear cells and the bulk of basophils can be separated from neu trophils by centrifugation of the whole leukocyte suspen sion on Lymphoprep. Our experience has, however, shown that a ‘purified’ neutrophil suspension may contain up to 0.4% basophils and 10 ng histamine/106 cells (table 2). Comparing the ability of FMP at 10~5M to chemotactically deactivate PMN from a whole leukocyte suspension ver sus a ‘purified’ neutrophil preparation (fig. 5), we found that the deactivating effect of the peptide was significantly reduced with purified neutrophils (p < 0.05). However, the full inhibitory capacity of the peptide could be re stored by adding increased proportions of the basophilcontaining mononuclear cell fraction to the neutrophils. As expected, incubation of fresh leukocytes (30 min, 37 °C) with the supernatant of leukocytes preincubated with FMP at 10'5 M caused a significant decrease of the chemotactic response of PMN. This effect could be pre vented by treating the supernatant with histaminase (0.2%) for 2 h at 37°C(fig. 6). Antibodies to human IgE and allergens reacting with leukocytes from atopic donors sensitive to it induce hista mine release from human basophils. It was of some in terest, therefore, to investigate whether these substances, which are not chemoattractant, were capable of deactivat ing chemotactically PMN. Leukocytes were preincubated (30 min, 37 °C) with rabbit antihuman IgE (1/2.000) or a house dust extract (10"5, w/v) at concentrations precali brated for causing maximum histamine release. As con trols, rabbit antihuman IgG (1/2.000) and a grass pollen ex tract (10-5) to which cell donors were not sensitive were used. After incubation, the leukocytes were washed and tested in the chemotactic chamber against FMP (10-5 M).
Histamine and Neutrophil Chemotactic Deactivation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
FMP (1O '7 M )
Neutrophils
Prior exposure to antihuman IgE or to the sensitizing al lergen significantly (p < 0.05) depressed the chemotactic response of the washed leukocytes, whereas exposure to antihuman IgG and pollen extract did not (fig. 7). The in tensity of the deactivation, however, was weaker than that caused by FMP (p
Department of Respiratory Medicine, CHU Sart Tilman, University of Liège, Belgium
Key Words Histamine Formylmethionyl peptides Polymorphonuclear leukocytes Chemotactic deactivation
Role of Histamine in the Chemotactic Deactivation of Polymorphonuclear Leukocytes Following Incubation with Formylmethionyl Peptides Abstract To clarify the mechanism of chemotactic deactivation of polymorphonuclear neutrophils (PMN) following incubation with the synthetic dipeptide N-formylmethionyl phenylalanine (FMP), we tested the hypothesis that histamine, which is released from leukocytes during incubation with FMP, could explain this inhibition. Human PMN were incubated in the presence or absence of FMP (10-7 to 10~5 M) and histamine measured fluorometrically in the super natant. Washed PMN were then tested in Boyden chambers against FMP (10-5 M ) and other chemoattractants. Incubation of leukocytes with FMP caused a nonpreferential PMN deactivation which was proportionally and kinetically related to FMP-induced histamine release. No histamine release or chemotactic deactivation was observed in the absence of Ca2+ and Mg2+. The inhibitory effect of the peptide was significantly prevented by an H2 blocker or when using basophil-depleted PMN suspensions. Histaminase abolished the capacity of FMP-incubated leukocyte supernatants to decrease PMN chemotaxis. Preincubation of leukocytes with anti-IgE or a sensitizing allergen caused a significant PMN chemotactic deactivation. These results show that hista mine, which is released during leukocyte incubation with FMP, contributes, at least in part, to the chemotactic deactivation of PMN.
Introduction Prcincubation of human polymorphonuclear neutro phils (PMN) with any of several synthetic chemotactic formylmethionine-containing oligopeptides causes a de crease of the PMN chemotactic response towards the peptide but also towards other structurally unrelated che moattractants [1-3]. The mechanism of this heterologous (nonpreferential) deactivation of chemotaxis remains poorly understood. In addition to being chemotactic, for-
mylmethionyl peptides induce histamine release from hu man basophils [4], Since histamine is capable of inhibiting the chemotactic response of neutrophils [5-7], the present experiments were undertaken to investigate the role of histamine in the mechanism of formylmethionyl peptide induced nonpreferential chemotactic deactivation of PMN. Our results show that histamine, which is released from basophils during incubation of leukocytes with the peptide, may explain, at least in part, the chemotactic deactivation of the PMN.
Correspondence to: Dr. M. Radermecker Department of Respiratory Medicine C H U Sart Tilman University of Liège B-4000 Liège (Belgium)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Th. Bury M. Radermecker
Materials and Methods Reagents
The synthetic chemotactic dipeptide N-formylmethionyl phenyl alanine (FMP) was purchased from Sigma Chemical (St. Louis, Mo., USA). FMP was found to be 10-100 times less efficient than the tripeptide N-formylmcthionyl-lcucyl phenylalanine as chemoattractant and histamine rclcascr for human leukocytes. It was dissolved in M e,S 04 (Fisher) and diluted to 50% M e,S 04to make stock solutions. Additional reagents included zymosan (Sigma), diamine oxidase at grade II purity (Sigma), leukotriene B4 (LTB4, Merck-Frost, Cana da), histamine dihydrochloride (Fluka, Buchs, Switzerland), clemas tine (Specia, Paris, France), and cimetidinc (Smith-Kline RIT, Rixensart, Belgium). Purified allergenic extracts (house dust, grass pol len) were provided by the Pasteur Institute (Paris). Polyclonal rabbit antihuman IgE and antihuman IgG were obtained from Bchringwerke (Marburg, FRG). Preparation o f Leukocyte Suspension Peripheral venous blood (40 ml) was drawn from healthy adult volunteers into 10-ml Vacutainer tubes (Becton Dickinson), each containing 143 USP units of lithium heparin and 1 ml of Plasmagel (Bellon Laboratories, Neuilly, France). The blood was gently mixed and allowed to sediment for 45 min at 38 °C. The upper plasma layer rich in leukocytes was removed and pooled into a plastic tube. The concentration of leukocytes was adjusted with Hanks’ balanced salt solution (HBSS; Gibco) without phenol red to a final concentration of 107 cells/ml. The cell viability was above 95%, as assessed by trypan blue exclusion. Chemotactic Deactivation o f PMN A suspension of human leukocytes (107 cells/ml) was incubated for 30 min at 37°C with HBSS in the absence or presence of FMP. The leukocytes were harvested by centrifugation and washed three times in HBSS without Ca2+ and Mg2+. The leukocytes were then re suspended at 2 •106/ml in HBSS containing 2% bovine serum albumin.
Histamine Assay Histamine was measured by an automated spectrofluoromctric assay [9], Histamine release, as a percent of the histamine content of the leukocyte suspension, was calculated using the following formula: HK)x(c-b) % release - -------------c-b where e represents the fluorometric reading for the experimental su pernatant, b the fluorometric reading of the blank, and c the fluoro metric reading of the leukocyte suspension. FMP at 10-5 M and below does not interfere with the assay. Separation o f Mononuclear Cells from Neutrophils Mononuclear cells were separated from neutrophils by density gradient centrifugation of the leukocyte suspension on Lymphoprcp (Nyegaard, Oslo, Norway). Nine-milliliter aliquots of leukocyte sus pension (107 cells/ml) diluted 1/3 with HBSS without Ca2+ and Mg2+ were layered on 4.5-ml cushions of Lymphoprcp (density 1.077) and centrifuged at 400g for 40 min at 22 °C. The neutrophils were freed of red blood cells by hypotonic lysis using NH4C1. Mononuclear cells and neutrophils were separately washed twice with cold HBSS with out Ca2+ and Mg2+ containing 0.3 mg/ml human serum albumin and resuspended in HBSS to 2 -106 cells/ml. Leukocytes were counted us ing a cell counter (Coulter). Basophil and histamine contents were measured from mononuclear cell and neutrophil suspensions (> 95% purity). Basophils were stained by toluidinc blue and their percent age calculated by examining 500 cells. Statistical Analysis The unpaired Student t test was used. A p value < 0.05 was consid ered significant. Results are presented as mean values ±SD of at least three different experiments.
Results
111)
Bury/Radermecker
Figure 1 shows that prior exposure of human leukocytes to FMP at concentrations ranging from 10~13 to 10-5 M caused a rapid and dose-related inhibition of the chemo tactic response of the PMN towards the peptide (10~5 M). The viability of the PMN, as assessed by trypan blue exclu sion, was not affected by prior exposure to the peptide. As shown in table 1, preincubation of leukocytes with FMP at 10-5 M caused a nonpreferential chemotactic deactivation. The same level of chemotactic inhibition was indeed ob served for FMP and other structurally unrelated chemo attractants such as LTB4 or activated autologous serum. When leukocytes were incubated with FMP (10~5 M, 30 min, 37 °C), histamine was released in the supernatant. It was found that leukocytic histamine release and PMN chemotactic deactivation kinetics were closely parallel (fig. 2). When leukocytes were incubated in HBSS con taining FMP (10~9 to 10~5M), we found parallel dose-effect relationships between basophilic histamine release and
Histamine and Neutrophil Chemotactic Deactivation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Neutrophil Chemotaxis Neutrophil chemotaxis was studied by using a modification of the assay of Boyden [8]. Briefly, a modified Boyden chemotactic chamber (Schleicher and Schiill, Dasscl, FRG) and a 5-um MF Millipore filter (Millipore, Bedford, Mass., USA) were used. Chemotactic factors (FMP 10"5M, LTB43 x lO^/W) in HBSS or 1/3 diluted zymosan-activa ted scrum were injected (1 ml) into the lower compartment of the chamber and a 2-ml aliquot of the leukocyte suspension into the up per compartment. The chambers were incubated for 3 h at 37 °C in humidified air containing 5% C 0 2. The filters were then removed, stained with Diff-Quick (Harleco), and the migration quantified mi croscopically. The chemotaxis was measured by counting the neutro phils at high power (x 440) in ten microscopic fields on both surfaces of the filter. All samples were run in duplicate and the mean values calculated. The results were expressed as percent migration which is the number of cells on the lower side and as percentage of cells on the starting side of the filter. The random mobility of neutrophils was as sessed by determining the percentage of migration when no chemo tactic agent (HBSS alone) was present in the lower compartment of the modified chamber. Under our experimental conditions, the PMN chemotactic response was not significantly different when using a pu rified neutrophil preparation (95% purity) instead of a whole leuko cyte suspension.
Fig. 1. Effect of the time of preincubation on the ability of FMP (K)'1', 10'9, and 10"5 M) to chemotactically deactivate human PMN towards this peptide (10"5 M).
Fig. 2. Kinetics of chemotactic deactivation and of histamine re lease following incubation of human leukocytes with FMP (K)'5 M) After different incubation times, histamine was measured in the su pernatant of centrifuged leukocyte suspensions and washed leuko cytes tested for chemotactic response to FMP (10'5 M).
Table 1. Effect of prior exposure (30 min, 37 °C) of human leu kocytes to FMP (10"5M) on their chemotactic response against dif ferent chemoattractants at optimal concentrations
FMP (10"5M) LTB4 (3 x KPM) Activated serum (diluted 1/3)
Prior exposure to HBSS
HBSS + FMP (10“5M)
% inhi bi tion
441 ±98 348 ±42 442 ±21
120 ±22 142 ±34 109±46
73 59 75
PMN chemotactic deactivation. When repeating the same experiments with HBSS lacking Ca2+ and Mg2+, neither histamine release nor PMN chemotactic deactivation was observed, i.e., washed PMN responded normally towards FMP (10~5 M) when tested in the Boyden chamber using HBSS containing Ca2+ and Mg2+ (fig. 3). Incubation of leukocytes with FMP (10~7M) in the presence of an H2 his tamine receptor antagonist, cimetidine (10~7 and 1(P5 M ), decreased in a dose-related fashion the inhibitory effect of the peptide on the chemotactic response of the PMN (p < 0.01). By contrast, clemastine, an H, histamine recep tor antagonist, had no significant effect (fig. 4). Prior ex posure of human leukocytes to cimetidine or clemastine at 10~7 and 10~5 M had no significant effect on their random
Fig. 3. Dose-effect relationships and effect of divalent cations on the ability of FMP to cause histamine release from basophils and to chemotactically deactivate neutrophils. Incubation time 30 min.
I ll
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
Chemoattractants
Table 2. Basophil and histamine content of mononuclear cell and neutrophil suspensions obtained following Ficoll-Hypaque cen trifugation and dextran sedimentation of whole blood
Mononuclear cells
Cimetidine + FMP ( 1 0 M)
Clemastine + FMP (1 0 '7 /W)
Fig. 4. Effect of H, and H2 histamine antagonists on the ability of FMP to cause human PMN chcmotactic deactivation. Leukocytes were incubated (30 min, 37 °C) in HBSS alone (control migration) or in HBSS containing FMP (10~7M) in the absence or presence of H, or H, histamine receptor antagonist (10~7 and 10~5 M). Washed leuko cytes were then tested in the Boyden chamber for chemotactic responsiveness to FMP (10"s M).
H I
Leukocytes or neutrophils preincubated in HBSS alone I Leukocytes preincubated with FMP
W/M Neutrophils preincubated with FMP Increasing proportions of mononuclear cells (and basophils) were added to neutrophils and preincubated with FMP
Fig. 5. Effect of basophil depletion and enrichment on the ability of FMP to cause chcmotactic deactivation of human leukocytes. Mononuclear cells and the bulk of basophils can be separated from the neutrophils by centrifugation of a whole leukocyte suspension on Lymphoprep. Whole leukocyte suspensions and pure neutrophil preparations were incubated in the presence or absence of FMP (K)'5M) and tested for chemotaxis in a Boyden chamber against FMP
(10's M ) .
112
Bury/Radermecker
Basophils (per 103 cells)
8 19 15 22
0 0 3 4
Histamine (ng/2-106 cells)
22 55 49 64
0 3 11 23
mobility or chemotactic response to FMP. Mononuclear cells and the bulk of basophils can be separated from neu trophils by centrifugation of the whole leukocyte suspen sion on Lymphoprep. Our experience has, however, shown that a ‘purified’ neutrophil suspension may contain up to 0.4% basophils and 10 ng histamine/106 cells (table 2). Comparing the ability of FMP at 10~5M to chemotactically deactivate PMN from a whole leukocyte suspension ver sus a ‘purified’ neutrophil preparation (fig. 5), we found that the deactivating effect of the peptide was significantly reduced with purified neutrophils (p < 0.05). However, the full inhibitory capacity of the peptide could be re stored by adding increased proportions of the basophilcontaining mononuclear cell fraction to the neutrophils. As expected, incubation of fresh leukocytes (30 min, 37 °C) with the supernatant of leukocytes preincubated with FMP at 10'5 M caused a significant decrease of the chemotactic response of PMN. This effect could be pre vented by treating the supernatant with histaminase (0.2%) for 2 h at 37°C(fig. 6). Antibodies to human IgE and allergens reacting with leukocytes from atopic donors sensitive to it induce hista mine release from human basophils. It was of some in terest, therefore, to investigate whether these substances, which are not chemoattractant, were capable of deactivat ing chemotactically PMN. Leukocytes were preincubated (30 min, 37 °C) with rabbit antihuman IgE (1/2.000) or a house dust extract (10"5, w/v) at concentrations precali brated for causing maximum histamine release. As con trols, rabbit antihuman IgG (1/2.000) and a grass pollen ex tract (10-5) to which cell donors were not sensitive were used. After incubation, the leukocytes were washed and tested in the chemotactic chamber against FMP (10-5 M).
Histamine and Neutrophil Chemotactic Deactivation
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 3:12:32 AM
FMP (1O '7 M )
Neutrophils
Prior exposure to antihuman IgE or to the sensitizing al lergen significantly (p < 0.05) depressed the chemotactic response of the washed leukocytes, whereas exposure to antihuman IgG and pollen extract did not (fig. 7). The in tensity of the deactivation, however, was weaker than that caused by FMP (p
