Auris' Nasus' LarYllx (Tokyo) 18, 133-143 (1991)
HYDROGEN PEROXIDE GENERATION BY EOSINOPHILS IN ALLERGIC RHINITIS Hiroshi OGASAWARA, M.D., Shiro YOSHIMURA, M.D., and Takeo KUMOI, M.D. Departmellt of OtolarYllgology, Hyogo College of Medicille, Nishillomiya, Japall
It was the aim to study the hydrogen peroxide (H 20 2) generation by eosinophils in allergic rhinitis caused by house dust which was examined in nasal secretion and peripheral blood. The concentration of H 2 0 2 in nasal secretions was increased after nasal challenge with house dust, and subsided gradually by the increase of peroxidase activity. The population of eosinophils and H 2 0 2 generation which was morphologically detected on the plasma membrane of eosinophils in nasal secretion, were increased with the release of eosinophil chemotactic activity after nasal challenge. Also, in peripheral blood, the number and phagocytic activity of eosinophils in extremely high density 1.102 glml were increased after nasal challenge. A high number of eosinophils was found in a density of 1.097 glml in the high IgE group, but showed less phagocytic activity than in the lower IgE group. Considering from these findings, H 20 2 generation by eosinophils appeared to be an important event in tissue injury and augmentation of allergic reaction.
Eosinophils have destructive effects on tissue in the reactions of allergy which are thought to be dependent on release of several distinctive proteins including major basic protein (MBP) (GLEICH, LOEGERING, and MALDoNAD, 1973), eosinophil-derived neurotoxin (DURACK, ACKERMAN, LOEGERING, and GLEICH, 1981), eosinophil peroxidase (EPO) (BuYS, WEVER, and RUITENBERG, 1984) and eosinophil cationic protein (ECP) (VENGE, DAHL, HALLGREN, and OLSOON, 1980; BISGAARD, GRONBORG, MYGIND, DAHL, LIDQVIST, and VENGE, 1990), and generation of oxygen-derived free radicals. Myeloperoxidase and EPO change oxidize chloride to the more powerful oxidant hypochlorous acid (Buys et al., 1984), however, human EPO did not preferentially oxidize chloride under physiologic conditions, as EPO did oxidize bromide to hypobromous acid (WEISS, TEST, ECKMANN, Roos, and REGIANI, 1986). When EPO was supplemented with hydrogen peroxide Received for publication
June 13, 1990 133
134
H. OGASAWARA et al.
(H 20 2) and a halide, degranulation of rat mast cell was induced (HENDERSON, CHI, and KLEBAN OFF, 1980). Since H 20 Z alone induced a weak histamine release from human basophils, and low concentrations of H 20 2 appeared to augment, and high concentrations to inhibit antigen induced histamine release (OGASA WARA, FUJITANI, DRZEWIECKI, and MIDDLETON, 1986), we sought to determine whether eosinophils were associated with H 20 2 generation in patients with nasal allergy. MATERIALS AND METHODS
Chemicals. Percoll and Dextran 250 were obtained from Pharmacia Fine Chemicals, Piscataway, N. J. Scopoletin, horseradish peroxidase, zymosan-A, human albumin, and 3-amino-2,4-triazole (A TZ) were obtained from Nakarai Chemical Co., Kyoto, Japan. Guaiacol, cerium chloride, and 1,4-benzoquinone were obtained from Wako Chemical Ind., Osaka, Japan. Dermatophagoides farinae (D. farinae) allergen extract was obtained from Torii Co., Tokyo, Japan. Patients. Thirty patients with allergic rhinitis ranging in age from 8 to 56 years with a mean age 27.6± 12.0 years, 15 male and 15 female. All of the patients had positive intradermal immediate skin test to house dust and D. farinae, and positive provocation test by the method of disk (OKUDA, 1977). The 7 members of the control group had no symptoms of allergic rhinitis and had negative intradermal immediate skin test. Isolation of cell and nasal secretion. Nasal secretions were aspirated gently to avoid irritating the mucosa into a test tube which contained I ml of saline, then the patients were challenged with house dust, and 5, 15, and 30 min later, additional nasal secretions were collected. Nasal secretions were not treated with enzymes, such as trypsin, pronase E, and hyaluronidase, because in preliminary studies they inhibited HzO z production from leukocytes. They were pi petted till secretions became homogenous at 4°C, and filtered with a number 50 mesh, then the cells were separated by centrifugation. The cells were washed once and suspended in phosphate-buffered saline (PBS), pH 7.4, containing CaH , Mg2-l, glucose, and human serum albumin. Supernatants were ultracentrifuged and stored at -20°C until the day of analysis. Heparinized venous blood was mixed with 6 % dextran in 0.15 mol/liter of NaCI and left at room temperature for 40 min. The dextran-plasma-leukocyte suspension was collected, and washed once with saline and re-suspended in Perc 011 solution. The leukocyte suspension and five different densities (1.074, 1.082, 1.092, 1.097, 1.102) of Percoll solution were overlaid sequentially from the bottom to the top of a tube, and centrifuged at 1,600 X g for 20 min at room temperature. The cells were collected from each layer, contaminating erythrocytes were lysed by hypotonic shock, and the cells were suspended in PBS. Cell viability was more than 95 % as determined by try pan blue dye exclusion. Measurement of H 2 0 2 generation. HzO z generation was measured using
HYDROGEN PEROXIDE IN ALLERGIC RHINITIS
135
horseradish peroxidase mediated extinction of scopoletin fluorescence during its oxidation (ROOT and METCALF, 1977). Cell suspensions were incubated without stimulation for 10 min at 37°C and another 10 min incubated with opsonized zymosan. The results were calculated as the maximal rate of H 20 2 release (pmol per I million per min). Zymosan was suspended in saline at a concentration of 10 mg/ml and boiled for 5 min. The opsonization was obtained by incubation of 10 mg of zymosan with 1 ml of fresh serum from the same patients. They were stained with Giemsa stain and phagocytic cells were counted. The ultrastructural localization of H 2 0 2 production in suspended leukocytes from nasal secretions was studied using CeCla technique (ROBINSON, BRIGGS, and KARNOVSKY, 1978). Cells were washed in Tris-maleate buffer (0.1 M, pH 7.5) with 5 % sucrose and then preincubated for 10 min at 37°C in Tris-maleate buffer with 5 % sucrose containing I mM ATZ. Cells were incubated finally in Tris-maleate buffer with 5 % sucrose, 10 mM ATZ, and 1 mM CeCla for 20 min at 37°C. Following cytochemical reaction, cells were fixed in 2 % of glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 60 min. After washing, cells were postfixed in 1 % OsO. in the same buffer at 4°C for 60 min. Enzyme assay. Peroxidase activity was assayed using guaiacol and H 20 2 by the method of JERMYN and THOMAS (1954). Activity was expressed as micrograms of horseradish peroxidase (Sigma; 46 U /mg). Arylsulfatase activity was assayed using 2-hydroxy-5-nitrophenyl sulfate. Activity was expressed as micrograms of 4-nitrocathechol liberated per hour at 37°C (TANAKA, VLENTINE, and FREDRICKS, 1962). Measurement of eosinophil chemotactic activity (ECA). ECA in nasal secretions was measured in vitro by modification of Boyden's method using 5 ,ltm pore sized Millipore filters (HIRASHIMA and HAYASHI, 1976). Nasal secretions diluted 30 times in PBS were placed in the lower compartment, and eosinophil-rich cell suspension (80 %) poured into the upper compartment. The chambers were incubated for 3 hrs at 3rC under 5 % CO 2 atmosphere. The cells that had migrated through the filter to the lower surface of the filter were stained by Giemsa stain. The chemotactic counts were expressed as mean count of migrated eosinophils in 20 high power fields (10 X 40), randomly selected. Statistical analysis. Data are expressed as mean±SE. Significance was determined by the paired Student's t test or unpaired Student's t test. RESULTS
Figure 1 illustrates the concentration of H 20 2 in cell free nasal secretion. H 2 0 2 was measured at 532±143 before challenge, and with 5, 15, and 30 min elapsed at 739±230, 728±185, and 697±193 pmol/g respectively after nasal challenge. Maximum peroxidase activity was observed in the secretion 15 min after challenge, and also, maximum arylsulfatase activity was observed 15 min
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later but showed minimum change. Figure 2 illustrates the ECA in cell free nasal secretion. This activity was detected as the migration of 20.5 eosinophils per 10 fields in 2 out of 5 cases in secretion at 5 min after challenge, even if the secretion were diluted 30 times finally. The cells were not homogenous except in one case, and were contaminated with a certain number of neutrophils. The concentration of eosinophils was increased 47.2 ± 12.0 to 63.6±9.9 % (p
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Fig. 4. Release of peroxidase (.) and aryl sulfatase ( ... ) from cells in nasal secretion stimulated with zymosan. Activity of peroxidase (0 ) and arylsulfatase ( .6) in nasal secretion were shown converted into cell number.
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Fig. 5. The H 2 0 z location on non-stimulated eosinophils from nasal secretions. Occasional cerium deposits (arrows) were seen on the external surface of the plasma membrane, but not on cytoplasmic organelle in secretion guttered before nasal challenge (A). Numerous cerium deposits were seen in secretion guttered 15 min after nasal challenge (8).
ophil concentration to 44.S ± 11.7 % increased after challenge in a density of 1.097, and 30.7±10.3% the earlier value was increased to S2.8±14.1 % in a density of J.102. The phagocytic percent of eosinophil in a density of 1.097 increased singificantly 8.2±S.0 to 16.7 ± 9.8 % (p
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high IgE group, and showed less phagocytic activity than in the low IgE group and in the control group. This indicates that perhaps in the high IgE group phagocytosis by eosinophils becomes fatigued, and that their function becomes lowered. A certain number of neutrophils had always been found in nasal secretions, however, neutrophils chemotactic activity (NCA) in nasal secretions was not demonstrated in this study. This is an expression of the physiological inflammation of nasal mucosa, which is continuously exposed to microoganisms and inhaled particles (MYGIND and THOMSEN, 1973). However, NCA was generated in mite sensitive subjects with asthma after mite challenge (SASTRE, BANKS, LOPEZ, BARKMAN, and SALVAGGIO, 1990). Also, neutrophils generated H 20 2 , and probably aggravated the allergic reaction less than eosinophils. The concentration of H 2 0 2 was increased after nasal challenge, and fell to the initial level after 15 min later with an increase of peroxidase activity. The increase of peroxidase mainly seemed to be caused by the release from eosinophils by reason of the following evidence: The population of eosinophils in nasal secretion was increased after nasal challenge, the presence of ECA, but the absence of NCA in the early phase of reaction, and the increase of aryl sulfatase which is a specific enzyme of eosinophils. The retardation of H 2 0 2 concentration by the release of peroxidase was suited to protect the organ from direct tissue injury, but augmented antigen mediated histamine release from human basophils (OGASAWARA et al., 1986). Some of the experiments have demonstrated that ECP, MBP, and other eosinophil products are found in toxic concentrations in sputa from subjects with asthma, in bronchoalveolar lavage fluid, and nasal lavage fluid. These products and H 2 0 2 probably contribute in nasal allergic reaction by damage of epithelial cells and by damage of nervous tissue. In conclusion, eosinophils in nasal allergic patients were abnormal in both
142
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peripheral blood and nasal secretions as the eosinophil influx, the increases of H 20 2 generation and the increases of phagocytic activity which might be due to ECA. H 20 2 can be scavent by release of peroxidase and controlled as stationary concentration. H 2 0 2 generation appears to be an important event in tissue injury and augmentation of allergic reaction in the patients with nasal allergy. REFERENCES BASS, D. A., GROVER, W. H., LEWIS, J. C., SZEDJA, P., DECHATELET, L. R., and MCCALL, C. E.: Comparison of human eosinophils from normals and patients with eosinophilia. 1. Clill. In)lest. 66: 1265-1273, 1980. BISGAARD, H., GRONBORG, H., MYGIND, N., DAHL, R., LIDQVIST, N., and VENGE, P.: Allergeninduced increase of eosinophil cationic protein in nasal lavage fluid: Effect of the glucocorticoid budesonide. 1. Allergy elill. Imll1l/llol. 85: 891-895, 1990. Buys, J., WEVER, R., and RUlTENBERG, E. J.: Myeloperoxidase is more efficient than eosinophil peroxidase in the ill )litro killing of newborn larvae of Trichinells spiralis. Immullology 51: 601-607, 1984. DURACK, D. T., ACKERMAN, S. J., LOEGERING, D. A., and GLEICH, G. J.: Purification of human eosinophil-derived neurotoxin. Proc. NaIl. A cad. Sci. U.S.A . 78: 5165-5169, 1981. GLEICH, G. J., LOEGERING, D . A., and MALDONAD, J. E. : Identification of major basic protein in guinea pig eosinophil granules. 1. Exp. Med. 137: 1459- 1471,1973. HENDERSON, W. R ., CHI, E. Y., and KLEBANOFF, S. J.: Eosinophil peroxidase-induced mast cell secretion. 1. Exp. Med. 152: 265-279, 1980. HIRASHlMA, M., and HAYASHI, H.: The mediation of tissue eosinophilia in hypersensitivity reaction: 1. Isolation of two different chemotactic factors from DNP-ascaris extract-induced skin lesion on guinea-pig. Immunology 30: 203-212, 1976. JERMYN, M. A., and THOMAS, R.: Multiple components in horse-radish peroxidase. Biochem. 1.56: 631-639, 1954. METZGER, W. J., RICHERSON, H. B., and WASSERMAN, S. 1.: Generation and partial characterization of eosinophil chemotactic activity and neutrophil chemotactic activity during early and late phase asthmatic response. 1. Allergy Clill. llI1l1ulllol. 78 : 282-290, 1986. MYGIND, N., and THOMSEN, J.: Cytology of nasal mucosa. A/'ch. Klill. Exp. Olll'ell Nasell Kehlkop/heilkd. 204: 123-129, 1973. OGASAWARA, H., FUJITANI, T., DRZEWIECKI, G., and MIDDLETON, F.: The role of hydrogen peroxide in basophil histamine release and the effect of selected flavonoids. .T. Allel'gy Clin. 111Il1Iunol. 78: 321-328, 1986. OKUDA, M.: Basic study of nasal provocation test, first report: site of the nose, size of site and allergen amount. Arch. Otol'hillolal'Yl1gol. 214: 241-246, 1977. , ROBINSON, J. M., BRIGGS, R. T., and KARNOVSKY, M. J.: Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes. 1. Cell. BioI. 77: 59-71, 1978. ROOT, R. K., and METCALF, J. A . : H 20 2 release from human granulocytes during phagocytosis: Relationship to superoxide anion formation and cellular catabolism of H 20 2 : studies with normal and cytochalasin B-treated cells. 1. elill. IIIYest. 60 : 1266-1279, 1977. SASTRE, J., BANKS, D . E., LOPEZ, M., BARKMAN, H. W ., and SALVAGGIO, J. E.: Neutrophil chemotactic activity in toluene diisocyanate (TDI)-induced asthma. 1. Allergy Clill. Im1111/1/01. 85: 567-572, 1990. SHULT, P. A., GRAZIANO, F. M., and Bussw, W. W.: Enhanced eosinophilluminol-dependent chemiluminescence in allergic rhinitis. 1. Allergy Clill. 11/111111110/. 77: 702-708, 1986. TANAKA, K. R., VLENTlNE, W. N., and FREDRICKS, R. E.: Human leukocyte arylsulphatase
HYDROGEN PEROXIDE IN ALLERGIC RHINITIS
143
activity. B/,. J. Haematol. 8: 86-92, 1962. VENGE, P., DAHL, R., HALLGREN, R., and OLSOON, 1.: Cationic proteins of human eosinophils and their role in the inflammatory reaction. In Eosinophil in Health and Disease (Mahmoud, A. A. F., and Austen, K. F., eds.), pp. 131-144, Grune & Stratton, New York, 1980. VENGE, P., DAHL, R., HAKANSSON, L., and PETERSON, c.: Generation of heat-labile chemotactic activity in blood after inhalation challenge and its relationship to neutrophil and monocyte/ macrophage turn over and activity. Allergy 37: 55-62, 1982. WEISS, S. J., TEST, S. T., ECKMANN, C. M., Roos, D., and REGIANI, S.: Brominating oxidants generate human eosinophils. Science 234: 200-203, 1986.
Request reprints to:
Dr. H. Ogasawara, Department of Otolaryngology, Hyogo College of Medicine, \-\ Mukogawacho, Nishinomiya 663, Japan
HYDROGEN PEROXIDE GENERATION BY EOSINOPHILS IN ALLERGIC RHINITIS Hiroshi OGASAWARA, M.D., Shiro YOSHIMURA, M.D., and Takeo KUMOI, M.D. Departmellt of OtolarYllgology, Hyogo College of Medicille, Nishillomiya, Japall
It was the aim to study the hydrogen peroxide (H 20 2) generation by eosinophils in allergic rhinitis caused by house dust which was examined in nasal secretion and peripheral blood. The concentration of H 2 0 2 in nasal secretions was increased after nasal challenge with house dust, and subsided gradually by the increase of peroxidase activity. The population of eosinophils and H 2 0 2 generation which was morphologically detected on the plasma membrane of eosinophils in nasal secretion, were increased with the release of eosinophil chemotactic activity after nasal challenge. Also, in peripheral blood, the number and phagocytic activity of eosinophils in extremely high density 1.102 glml were increased after nasal challenge. A high number of eosinophils was found in a density of 1.097 glml in the high IgE group, but showed less phagocytic activity than in the lower IgE group. Considering from these findings, H 20 2 generation by eosinophils appeared to be an important event in tissue injury and augmentation of allergic reaction.
Eosinophils have destructive effects on tissue in the reactions of allergy which are thought to be dependent on release of several distinctive proteins including major basic protein (MBP) (GLEICH, LOEGERING, and MALDoNAD, 1973), eosinophil-derived neurotoxin (DURACK, ACKERMAN, LOEGERING, and GLEICH, 1981), eosinophil peroxidase (EPO) (BuYS, WEVER, and RUITENBERG, 1984) and eosinophil cationic protein (ECP) (VENGE, DAHL, HALLGREN, and OLSOON, 1980; BISGAARD, GRONBORG, MYGIND, DAHL, LIDQVIST, and VENGE, 1990), and generation of oxygen-derived free radicals. Myeloperoxidase and EPO change oxidize chloride to the more powerful oxidant hypochlorous acid (Buys et al., 1984), however, human EPO did not preferentially oxidize chloride under physiologic conditions, as EPO did oxidize bromide to hypobromous acid (WEISS, TEST, ECKMANN, Roos, and REGIANI, 1986). When EPO was supplemented with hydrogen peroxide Received for publication
June 13, 1990 133
134
H. OGASAWARA et al.
(H 20 2) and a halide, degranulation of rat mast cell was induced (HENDERSON, CHI, and KLEBAN OFF, 1980). Since H 20 Z alone induced a weak histamine release from human basophils, and low concentrations of H 20 2 appeared to augment, and high concentrations to inhibit antigen induced histamine release (OGASA WARA, FUJITANI, DRZEWIECKI, and MIDDLETON, 1986), we sought to determine whether eosinophils were associated with H 20 2 generation in patients with nasal allergy. MATERIALS AND METHODS
Chemicals. Percoll and Dextran 250 were obtained from Pharmacia Fine Chemicals, Piscataway, N. J. Scopoletin, horseradish peroxidase, zymosan-A, human albumin, and 3-amino-2,4-triazole (A TZ) were obtained from Nakarai Chemical Co., Kyoto, Japan. Guaiacol, cerium chloride, and 1,4-benzoquinone were obtained from Wako Chemical Ind., Osaka, Japan. Dermatophagoides farinae (D. farinae) allergen extract was obtained from Torii Co., Tokyo, Japan. Patients. Thirty patients with allergic rhinitis ranging in age from 8 to 56 years with a mean age 27.6± 12.0 years, 15 male and 15 female. All of the patients had positive intradermal immediate skin test to house dust and D. farinae, and positive provocation test by the method of disk (OKUDA, 1977). The 7 members of the control group had no symptoms of allergic rhinitis and had negative intradermal immediate skin test. Isolation of cell and nasal secretion. Nasal secretions were aspirated gently to avoid irritating the mucosa into a test tube which contained I ml of saline, then the patients were challenged with house dust, and 5, 15, and 30 min later, additional nasal secretions were collected. Nasal secretions were not treated with enzymes, such as trypsin, pronase E, and hyaluronidase, because in preliminary studies they inhibited HzO z production from leukocytes. They were pi petted till secretions became homogenous at 4°C, and filtered with a number 50 mesh, then the cells were separated by centrifugation. The cells were washed once and suspended in phosphate-buffered saline (PBS), pH 7.4, containing CaH , Mg2-l, glucose, and human serum albumin. Supernatants were ultracentrifuged and stored at -20°C until the day of analysis. Heparinized venous blood was mixed with 6 % dextran in 0.15 mol/liter of NaCI and left at room temperature for 40 min. The dextran-plasma-leukocyte suspension was collected, and washed once with saline and re-suspended in Perc 011 solution. The leukocyte suspension and five different densities (1.074, 1.082, 1.092, 1.097, 1.102) of Percoll solution were overlaid sequentially from the bottom to the top of a tube, and centrifuged at 1,600 X g for 20 min at room temperature. The cells were collected from each layer, contaminating erythrocytes were lysed by hypotonic shock, and the cells were suspended in PBS. Cell viability was more than 95 % as determined by try pan blue dye exclusion. Measurement of H 2 0 2 generation. HzO z generation was measured using
HYDROGEN PEROXIDE IN ALLERGIC RHINITIS
135
horseradish peroxidase mediated extinction of scopoletin fluorescence during its oxidation (ROOT and METCALF, 1977). Cell suspensions were incubated without stimulation for 10 min at 37°C and another 10 min incubated with opsonized zymosan. The results were calculated as the maximal rate of H 20 2 release (pmol per I million per min). Zymosan was suspended in saline at a concentration of 10 mg/ml and boiled for 5 min. The opsonization was obtained by incubation of 10 mg of zymosan with 1 ml of fresh serum from the same patients. They were stained with Giemsa stain and phagocytic cells were counted. The ultrastructural localization of H 2 0 2 production in suspended leukocytes from nasal secretions was studied using CeCla technique (ROBINSON, BRIGGS, and KARNOVSKY, 1978). Cells were washed in Tris-maleate buffer (0.1 M, pH 7.5) with 5 % sucrose and then preincubated for 10 min at 37°C in Tris-maleate buffer with 5 % sucrose containing I mM ATZ. Cells were incubated finally in Tris-maleate buffer with 5 % sucrose, 10 mM ATZ, and 1 mM CeCla for 20 min at 37°C. Following cytochemical reaction, cells were fixed in 2 % of glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 60 min. After washing, cells were postfixed in 1 % OsO. in the same buffer at 4°C for 60 min. Enzyme assay. Peroxidase activity was assayed using guaiacol and H 20 2 by the method of JERMYN and THOMAS (1954). Activity was expressed as micrograms of horseradish peroxidase (Sigma; 46 U /mg). Arylsulfatase activity was assayed using 2-hydroxy-5-nitrophenyl sulfate. Activity was expressed as micrograms of 4-nitrocathechol liberated per hour at 37°C (TANAKA, VLENTINE, and FREDRICKS, 1962). Measurement of eosinophil chemotactic activity (ECA). ECA in nasal secretions was measured in vitro by modification of Boyden's method using 5 ,ltm pore sized Millipore filters (HIRASHIMA and HAYASHI, 1976). Nasal secretions diluted 30 times in PBS were placed in the lower compartment, and eosinophil-rich cell suspension (80 %) poured into the upper compartment. The chambers were incubated for 3 hrs at 3rC under 5 % CO 2 atmosphere. The cells that had migrated through the filter to the lower surface of the filter were stained by Giemsa stain. The chemotactic counts were expressed as mean count of migrated eosinophils in 20 high power fields (10 X 40), randomly selected. Statistical analysis. Data are expressed as mean±SE. Significance was determined by the paired Student's t test or unpaired Student's t test. RESULTS
Figure 1 illustrates the concentration of H 20 2 in cell free nasal secretion. H 2 0 2 was measured at 532±143 before challenge, and with 5, 15, and 30 min elapsed at 739±230, 728±185, and 697±193 pmol/g respectively after nasal challenge. Maximum peroxidase activity was observed in the secretion 15 min after challenge, and also, maximum arylsulfatase activity was observed 15 min
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later but showed minimum change. Figure 2 illustrates the ECA in cell free nasal secretion. This activity was detected as the migration of 20.5 eosinophils per 10 fields in 2 out of 5 cases in secretion at 5 min after challenge, even if the secretion were diluted 30 times finally. The cells were not homogenous except in one case, and were contaminated with a certain number of neutrophils. The concentration of eosinophils was increased 47.2 ± 12.0 to 63.6±9.9 % (p
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Fig. 5. The H 2 0 z location on non-stimulated eosinophils from nasal secretions. Occasional cerium deposits (arrows) were seen on the external surface of the plasma membrane, but not on cytoplasmic organelle in secretion guttered before nasal challenge (A). Numerous cerium deposits were seen in secretion guttered 15 min after nasal challenge (8).
ophil concentration to 44.S ± 11.7 % increased after challenge in a density of 1.097, and 30.7±10.3% the earlier value was increased to S2.8±14.1 % in a density of J.102. The phagocytic percent of eosinophil in a density of 1.097 increased singificantly 8.2±S.0 to 16.7 ± 9.8 % (p
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Fig. 6. The percentage of eosinophils, percentage of phagocytosis by eosinophils and H 2 0 2 generation by eosinophils and neutrophils from peripheral blood before and after nasal challenge with D. /arinae. The percentage of phagocytosis by eosinophils in the density of 1.102 gjdl of Percoll fraction was increased significantly after nasal challenge (p Z
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Fig. 9. Percentage of phagocytosis of zymosan by eosinophils in IgE over 400 U Iml patients (., 11 = 10), IgE under 400 UIml patients (A, 11=22), and control (0, 11=7). Values represent mean±SE.
high IgE group, and showed less phagocytic activity than in the low IgE group and in the control group. This indicates that perhaps in the high IgE group phagocytosis by eosinophils becomes fatigued, and that their function becomes lowered. A certain number of neutrophils had always been found in nasal secretions, however, neutrophils chemotactic activity (NCA) in nasal secretions was not demonstrated in this study. This is an expression of the physiological inflammation of nasal mucosa, which is continuously exposed to microoganisms and inhaled particles (MYGIND and THOMSEN, 1973). However, NCA was generated in mite sensitive subjects with asthma after mite challenge (SASTRE, BANKS, LOPEZ, BARKMAN, and SALVAGGIO, 1990). Also, neutrophils generated H 20 2 , and probably aggravated the allergic reaction less than eosinophils. The concentration of H 2 0 2 was increased after nasal challenge, and fell to the initial level after 15 min later with an increase of peroxidase activity. The increase of peroxidase mainly seemed to be caused by the release from eosinophils by reason of the following evidence: The population of eosinophils in nasal secretion was increased after nasal challenge, the presence of ECA, but the absence of NCA in the early phase of reaction, and the increase of aryl sulfatase which is a specific enzyme of eosinophils. The retardation of H 2 0 2 concentration by the release of peroxidase was suited to protect the organ from direct tissue injury, but augmented antigen mediated histamine release from human basophils (OGASAWARA et al., 1986). Some of the experiments have demonstrated that ECP, MBP, and other eosinophil products are found in toxic concentrations in sputa from subjects with asthma, in bronchoalveolar lavage fluid, and nasal lavage fluid. These products and H 2 0 2 probably contribute in nasal allergic reaction by damage of epithelial cells and by damage of nervous tissue. In conclusion, eosinophils in nasal allergic patients were abnormal in both
142
H. OGASA WARA
el
al.
peripheral blood and nasal secretions as the eosinophil influx, the increases of H 20 2 generation and the increases of phagocytic activity which might be due to ECA. H 20 2 can be scavent by release of peroxidase and controlled as stationary concentration. H 2 0 2 generation appears to be an important event in tissue injury and augmentation of allergic reaction in the patients with nasal allergy. REFERENCES BASS, D. A., GROVER, W. H., LEWIS, J. C., SZEDJA, P., DECHATELET, L. R., and MCCALL, C. E.: Comparison of human eosinophils from normals and patients with eosinophilia. 1. Clill. In)lest. 66: 1265-1273, 1980. BISGAARD, H., GRONBORG, H., MYGIND, N., DAHL, R., LIDQVIST, N., and VENGE, P.: Allergeninduced increase of eosinophil cationic protein in nasal lavage fluid: Effect of the glucocorticoid budesonide. 1. Allergy elill. Imll1l/llol. 85: 891-895, 1990. Buys, J., WEVER, R., and RUlTENBERG, E. J.: Myeloperoxidase is more efficient than eosinophil peroxidase in the ill )litro killing of newborn larvae of Trichinells spiralis. Immullology 51: 601-607, 1984. DURACK, D. T., ACKERMAN, S. J., LOEGERING, D. A., and GLEICH, G. J.: Purification of human eosinophil-derived neurotoxin. Proc. NaIl. A cad. Sci. U.S.A . 78: 5165-5169, 1981. GLEICH, G. J., LOEGERING, D . A., and MALDONAD, J. E. : Identification of major basic protein in guinea pig eosinophil granules. 1. Exp. Med. 137: 1459- 1471,1973. HENDERSON, W. R ., CHI, E. Y., and KLEBANOFF, S. J.: Eosinophil peroxidase-induced mast cell secretion. 1. Exp. Med. 152: 265-279, 1980. HIRASHlMA, M., and HAYASHI, H.: The mediation of tissue eosinophilia in hypersensitivity reaction: 1. Isolation of two different chemotactic factors from DNP-ascaris extract-induced skin lesion on guinea-pig. Immunology 30: 203-212, 1976. JERMYN, M. A., and THOMAS, R.: Multiple components in horse-radish peroxidase. Biochem. 1.56: 631-639, 1954. METZGER, W. J., RICHERSON, H. B., and WASSERMAN, S. 1.: Generation and partial characterization of eosinophil chemotactic activity and neutrophil chemotactic activity during early and late phase asthmatic response. 1. Allergy Clill. llI1l1ulllol. 78 : 282-290, 1986. MYGIND, N., and THOMSEN, J.: Cytology of nasal mucosa. A/'ch. Klill. Exp. Olll'ell Nasell Kehlkop/heilkd. 204: 123-129, 1973. OGASAWARA, H., FUJITANI, T., DRZEWIECKI, G., and MIDDLETON, F.: The role of hydrogen peroxide in basophil histamine release and the effect of selected flavonoids. .T. Allel'gy Clin. 111Il1Iunol. 78: 321-328, 1986. OKUDA, M.: Basic study of nasal provocation test, first report: site of the nose, size of site and allergen amount. Arch. Otol'hillolal'Yl1gol. 214: 241-246, 1977. , ROBINSON, J. M., BRIGGS, R. T., and KARNOVSKY, M. J.: Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes. 1. Cell. BioI. 77: 59-71, 1978. ROOT, R. K., and METCALF, J. A . : H 20 2 release from human granulocytes during phagocytosis: Relationship to superoxide anion formation and cellular catabolism of H 20 2 : studies with normal and cytochalasin B-treated cells. 1. elill. IIIYest. 60 : 1266-1279, 1977. SASTRE, J., BANKS, D . E., LOPEZ, M., BARKMAN, H. W ., and SALVAGGIO, J. E.: Neutrophil chemotactic activity in toluene diisocyanate (TDI)-induced asthma. 1. Allergy Clill. Im1111/1/01. 85: 567-572, 1990. SHULT, P. A., GRAZIANO, F. M., and Bussw, W. W.: Enhanced eosinophilluminol-dependent chemiluminescence in allergic rhinitis. 1. Allergy Clill. 11/111111110/. 77: 702-708, 1986. TANAKA, K. R., VLENTlNE, W. N., and FREDRICKS, R. E.: Human leukocyte arylsulphatase
HYDROGEN PEROXIDE IN ALLERGIC RHINITIS
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activity. B/,. J. Haematol. 8: 86-92, 1962. VENGE, P., DAHL, R., HALLGREN, R., and OLSOON, 1.: Cationic proteins of human eosinophils and their role in the inflammatory reaction. In Eosinophil in Health and Disease (Mahmoud, A. A. F., and Austen, K. F., eds.), pp. 131-144, Grune & Stratton, New York, 1980. VENGE, P., DAHL, R., HAKANSSON, L., and PETERSON, c.: Generation of heat-labile chemotactic activity in blood after inhalation challenge and its relationship to neutrophil and monocyte/ macrophage turn over and activity. Allergy 37: 55-62, 1982. WEISS, S. J., TEST, S. T., ECKMANN, C. M., Roos, D., and REGIANI, S.: Brominating oxidants generate human eosinophils. Science 234: 200-203, 1986.
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Dr. H. Ogasawara, Department of Otolaryngology, Hyogo College of Medicine, \-\ Mukogawacho, Nishinomiya 663, Japan
