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Early Inflammatory Changes of the Haemophilus influenzae-Induced Experimental Otitis Media
Auris·Nasus·Larynx (Tokyo) 22, 80-85 (1995)
Early Inflammatory Changes of the Haemophilus injluenzaeInduced Experimental Otitis Media Masahiro
KAWANA,
M.D.
Department of Otolaryngology, Niigata University School of Medicine, Niigata, Japan
Haemophilus injluenzae is one of the most frequent pathogens of acute otitis media. To determine the middle ear response during the early stage of acute inflammation, a small amount of H. injluenzae was inoculated into the bullae of guinea pigs through the tympanic membrane. The bullae were harvested at 6, 12, 24, 36, and 48 hours after H. injluenzae inoculation and washed with phosphate-buffered saline (PBS). The number of viable H. injluenzae and inflammatory cells, the concentrations of myeloperoxidase (MPO) and lysozyme in the washing suspensions were measured, and compared with those in PBS-inoculated control ears. The number of viable H. injluenzae increased very rapidly from 6 to 12 hours after inoculation and remained stationary up to 48 hours. The number of inflammatory cells and the MPO concentration were significantly higher in the H. injluenzae-inoculated ears than in the control ears from 12 to 48 hours after inoculation. The lysozyme concentration was already significantly higher at 6 hours in the H. injluenzae-inoculated ears; the lysozyme was released in the middle ear before the accumulation of inflammatory cells and degranulation of MPO from inflammatory cells. The results indicated that inflammatory reactions were present already at 6 hours after bacterial inoculation, and were rapidly accelerated during the subsequent hours. Consequently, acute middle ear inflammatory responses were seen immediately following inoculation of viable bacteria, and these responses originated in direct responses of middle ear mucosa, and oxidative and non-oxidative neutrophil metabolic products, which may cause tissue injury.
Studies of the pathogenesis of otitis media have explored inflammatory responses to Haemophi/us injluenzae because H. injluenzae is frequently the cause of acute otitis media. 1,2 and is often isolated in middle ear effusions (MEEs) of chronic otitis media with effusion (OME).2,3 Kinetic studies in an animal model using nonviable H. injluenzae demonstrated an increased number of neutrophils in middle ear fluid (MEF) and capillary endothelial destruction of the middle ear mucosa, and these inflammatory responses were determined by the influence of endotoxin. 4 In fact, we already reported that the endotoxin and the outer cell membrane of the H. injluenzae induced otitis media in guinea pigs by observing increases in the number of inflammatory cells and mucosal damage of the middle ear. 5,6 Migrating inflammatory cells, such as neutrophils, are essential elements of host defense and are abundant in MEE in humans with acute purulent otitis media. The phagocytosis of bacteria by neutrophils stimulates intracellular oxidative responses and the bacteria are killed in the phagosomes. However, the growth pattern of H. injluenzae in the middle ear, and the role of inflammatory cell accumulation in response to increases in the number of viable H. injluenzae are still unclear. We, therefore, sought to determine the growth pattern of H. injluenzae and host response with an increase in the number of viable bacteria in the early stage of H. injluenzae-induced experimental otitis media. Received 11 August 1994; accepted 17 October 1994. Correspondence should be addressed to: Masahiro Kawana, Asahimachi-l, Niigata, 951 Japan.
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Auris·Nasus·Larynx (Tokyo) Vol. 22 (1995)
MATERIALS AND METHODS
Non-typableH. injiuenzae biovar 2 (NCTC 8143) was grown on a chocolate agar plate (Eiken Chemicals, Tokyo, Japan). Two to three colonies were collected and put in a liquid culture medium containing 1% trypton (Difco Laboratories, Detroit, MI, USA), 0.5% yeast extract (Difco), 0.5% sodium chloride (Wako Pure Chemical Industries, Osaka, Japan), 0.1% glucose (Kanto Chemical, Tokyo, Japan), 100,ugjml ,B-nicotinamide-adenine dinucleotide (,B-NAD, Wako) , and loo,ugjml Hemin (Wako) as previously described. 5 The culture medium was incubated until the growth of H. injiuenzae reached the mid-log phase and was diluted to approximately 2 X 103 CFUjml in sterile 0.01 M phosphate-buffered saline (PBS), pH 7.4. Forty healthy guinea pigs weighing 350-450 g and with normal middle ears as ascertained by otoscopy were used for this study. All procedures were done with 20 mg per kg of ketamine hydrochloride intramuscular anesthesia. A volume of 0.1 ml of the H. injiuenzae suspension inoculum (200 CFUjear) was injected directly into the right side of the middle ear bulla through the tympanic membrane using a specially prepared 23 G needle. PBS was placed on the left side by the same procedure as the control. Middle ear bullae were harvested 6, 12, 24, 36, and 48 hours after inoculation, and a hole, approximately 3 mm in diameter, was opened in the tympanic bulla using an electric bar. The middle ear mucosa, tympanic membrane, and the volume of MEF were observed, and the cavity of the bulla was washed with 1oo,u1 of PBS three times through the hole. MEF and a total of 3oo,u1 of the washing suspension were collected, and adjusted to the same final volume (500 ,ul) with PBS. Quantitative culturing of the adjusted middle ear suspensions (AMES) was performed by inoculating 1O-,u1 aliquots of serial dilutions on chocolate agar plates. Inflammatory cells in AMES were counted with a hemacytometer. AMES were centrifuged at 500 X g, and the supernatants were collected for myeloperoxidase (MPO) and lysozyme quantitations. The cell pellets were stained by Wright and Giemsa stain to confirm that inflammatory cells contained more than 90% neutrophils. MPO activity was measured in triplicate samples by a colorimetric method. 7 Net MPO activity after the background (sodium azide pretreatment) subtraction was expressed as units per ml of the washing suspension. Lysozyme activity was measured using a modified 18-hour enzymatic assay as previously described and expressed as units per ml of the washing suspension. 8 The set of AMES from the right middle ear bulla of pure H. injiuenzae growth and from the corresponding left bulla of sterile was used for analysis. Data were analyzed for statistical significance using Student's t-test for paired data. RESULTS
The number of guinea pigs analyzed for this study was 7 at 6 hours, 8 at 12 hours, 7 at 24 hours, 8 at 36 hours, and 7 at 48 hours after inoculation, or 37 in total. The pinhole perforation of the tympanic membranes with the inoculations of H. injiuenzae or PBS remained in all of the analyzed bullae, but no large perforation was observed. The edemas of the middle ear mucosae in the H. injiuenzae-inoculated ears were slight at 6 hours, moderate at 12 and 24 hours, and marked at 36 and 48 hours. No edema of the middle ear mucosa was seen in PBS-inoculated ears. Serous MEF was observed in all of the H. injiuenzaeinoculated ears, but the volumes of the MEF were small and could not be measured at all measuring points. The number of viable H. injiuenzae CFU increased rapidly at 6 hours (geometric mean: 5.69 X 1Q4 CFUjml) and 12 hours (4.95 X 105 CFUjml) after inoculation, reached 1.11 X 106 Auris·Nasus·Larynx (Tokyo) Vol. 22 (1995)
81
CFV/ml at 24 hours and remained constant at 36 and 48 hours after inoculation (36 hours: 1.34 X 106 CFV/ml, 48 hours: 1.25 X 106 CFV/ml, Fig. 1). The number of inflammatory cells in H. injluenzae-inoculated ears slightly increased at 6 hours after inoculation, remarkably increased at 12 hours (1,484 cells/mm3 ) and 24 hours (10,139 cells/mm3 ) , was maximum at 36 hours (10,552 cells/mm3 ) , and slightly decreased at 48 hours after inoculation (7,81Ocells/mm3 ). The number of inflammatory cells in PBS-inoculated control ears increased slightly at 24 hours (818cells/mm3 ), but remained low at 6 hours (93 cells/mm3), 12 hours (240 cells/mm3 ), 36 hours (262 cells/mm3), and 48 hours (343 cells/mm3 ) after inoculation, which was significantly increased in H. injluenzae-inoculated ears compared with PBS-inoculated ears at 12, 24, 36, and 48 hours after inoculation (12 hours: p < 0.05; 24, 36, 48 hours: p < 0.01; Fig. 2). The MPO titer in AMES was also increased in H. injluenzae-inoculated ears at 6 hours after inoculation (164.8V/ml), remarkably increased at 12 hours (234.1 V/ml) and 24 hours (365.8 V/ml) , remained the same 36 hours (361.1 V/ml) , slightly decreased at 48 hours (338.5 V/ml), which were significantly higher in H. injluenzae-inoculated ears at 12, 24, 36, and 48 hours than those in PBS-inoculated ears (PBS-inoculated ears at 12 hours: 125.9 V/ml, 24 hours: 156.6 V/ml, 36 hours: 125.2 V/ml, 48 hours: 206.9 V/ml; 12, 24, 36 hours: p < 0.01; 48 hours:p<0.05; Fig. 3). The lysozyme titer, which was already high in H. injluenzae-inoculated ears at 6 hours after inoculation (H. injluenzae-inoculated ears: 4,264 V/ml, PBS-inoculated ears: 2,320 V/ml) , increased rapidly and continuously at 12 hours (11,928 V/ml), 24 hours (15,639 V/ml) , 36 hours (16,551 V/ml), and 48 hours (20,663 V/ml). It is noteworthy that a significant difference in the lysozyme titers between the H. injluenzae-inoculated ears and the PBS-inoculated ears was seen at all measuring points, including 6 hours after inoculation (6, 24, 48 hours: p < 0.05; 12,36 hours:p
6.5 6.0
1 ~ 5.5 U Cl
.3
5.0
4. 5
4.0
12
24
36
48
Hours after Inoculation
..
Fig. 1. Geometric mean ( ± standard error of the mean) number of viable H. injiuenzae in ears after inoculation of H. injluenzae.
Il
"e
• p
~
~ 5,000 2,500
o L..--=":-_
12
24
36
Hours after Inoculation
82
Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)
48
Fig. 2. Geometric mean ( ± standard error of the mean) number of inflammatory cells in ears after inoculation of H. injluenzae.
200
100
o
12
24
36
48
Hours after Inoculation
E 15,000 3
Fig. 3. Geometric mean ( ± standard error of the mean) concentration of MPO in MEF after inoculation of H. injluenzae.
• p
10,000
5,000
12
24
36
48
Hours after Inoculation
Fig. 4. Geometric mean ( ± standard error of the mean) concentration of lysozyme in MEF after inoculation of H. injluenzae.
DISCUSSION
H. injiuenzae is one of the most common bacteria causing acute otitis media. 1-3 They are the second most frequently cultured bacteria from MEE in cases of acute otitis media. To determine the occurrence and prolongation of acute otitis media, it is important to define the growth style of H. injiuenzae in the middle ear cavity, and the mechanism of injury of the middle ear mucosa by H. injiuenzae. Endotoxin is a component of Gram-negative rods including H. injiuenzae, the endotoxin of H. injiuenzae type b and the outer cell wall of non-typable H. injiuenzae induced acute otitis media in guinea pigs. 5,6 Staphylococcus pneumoniae is another important bacteria causing acute otitis media. When a small amount of pneumococcus was inoculated into the middle ear cavity of the chinchilla, the pneumococcus grew rapidly in the middle ear and induced acute otitis media. Penicillin treatment leads to severe inflammatory responses in the middle ear compared to the non-treated middle ear, 8 which is thought to be due to increased local responses with penicillin-induced pneumococcal lysis and cell wall accumulation. These phenomena suggested that the correlation of the bacterial components, such as endotoxin and outer cell membrane, and middle ear inflammatory responses were the most important fac;tors for the acute and prolonged otitis media. Clinical examination also demonstrated endotoxin in 80% of chronic OME samples. 3 In addition, specific IgG and IgA antibodies against the outer membrane proteins of non-typable H. injiuenzae are significantly higher in children with mucoid MEE than serous MEE in chronic OME, which suggests that bacterial infection and subsequent immune response contribute to the prolongation of OME.9 Using a guinea pig model of H. injiuenzae-induced otitis media, viable H. injiuenzae was Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)
83
found to increase logarithmically at 6 hours after inoculation, whereas the number of inflammatory cells increased later than 12 hours. Namely, viable H. injluenzae started increasing just after the inoculation of the middle ear. Later, the rapid increase of viable H. injluenzae caused a high titer of chemotactic factors in the middle ear, which induced acute inflammatory responses. The 6-hour delay may be due to the necessity to reach sufficient concentrations of chemotactic factors from H. injluenzae and middle ear mucosa, such as complement 5a or leukotriene B4, and to have migration of the neutrophils into the middle ear cavity. In recent years, it has been clarified that oxidative and non-oxidative bactericidal activities in neutrophils are important in acute inflammatory responses. In experimental pneumococcal otitis media, these inflammatory changes are observed as an increase in the numbers of neutrophils and metabolic products from neutrophils stimulated by bacteria such as oxygen radicals, MPO, lysozyme, and lactoferrin. 8,IO,lI In H. injluenzae-induced otitis media, edema, and histological damage to middle ear mucosa are directly correlated with the neutrophil number, not the bacterial count. 12 It is thus evident that oxidative or non-oxidative bactericidal effects of neutrophils kill the bacteria and also damage middle ear epithelial cells. MPO, which is an important enzyme in the oxidative metabolic pathways and is contained in the azulophil granules of the neutrophils, increased with the number of inflammatory cells. The high concentration of MPO in MEF may reflect high concentrations of oxidative metabolic products in the middle ear cavity.1O The oxidative metabolic products released from neutrophils kill the bacteria, and may also damage middle ear epithelial cells. \3 High titers of MPO were also measured in a guinea pig model of H. injluenzae-induced otitis media, and these results suggested that oxidative metabolic pathways were activated in the middle ear which may cause tissue injury. Lysozyme is an important enzyme of non-oxidative bactericidal mechanisms. It is contained in azurophil and specific granules in neutrophils, and goblet cells and glands in epithelium. 14 Consequently, an increase of lysozyme titers in MEF reflected both the neutrophil- and epithelium-oriented lysozymes. Lysozyme accumulation in MEF preceding significant inflammatory cell influx has been observed before in experimental pneumococcal otitis media. 7,ls The early release oflysozyme may be considered an indication of injury to middle ear epithelial cells by H. injluenzae, and the migrating neutrophils also released lysozyme in MEF later than 6 hours. The lysozyme titers in the middle ears increased at every measuring point including 36 to 48 hours after H. injluenzae inoculation, whereas the inflammatory cell numbers and MPO concentrations tended to decrease their numbers at the same measuring points. Since the lysozyme titers from infiltrating neutrophils in the middle ear are thought to be decreased at these measuring points, the continuous increase in the lysozyme titers at 36 and 48 hours may reflect strong and progressive damage of the middle ear epithelium. In summary, a small amount of H. injluenzae increased rapidly in the middle ear of guinea pigs and triggered the accumulation of products released by neutrophils and epithelial cells, including oxidants and lysozyme. These products are essential to host defense, but damage the middle ear mucosa. Future experiments are needed to demonstrate the relative contributions of these products to middle ear tissue injury. The author wishes to thank Prof. Yuichi Nakano, M.D., Naobumi Nonomura, M.D., and Osamu Fujioka, M.D. for their valuable suggestions. REFERENCES
1.
Wald ER: Haemophilus injluenzae as a cause of acute otitis media. Pediatr Infect Dis I 8:S28-S30,
2.
Bluestone CD, Stephenson IS, Martin LM: Ten-year review of otitis media pathogens. Pediatr
1989.
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Infect Dis J 11:S7-S11, 1992. 3. Giebink GS: The microbiology of otitis media. Pediatr Infect Dis J 8:S18-S20, 1989. 4. DeMaria TF, Briggs BR, Lim DJ, et al: Experimental otitis media with effusion following middle ear inoculation of nonviable H. injluenzae. Ann Otol Rhinol Laryngol 93:52-56, 1984. 5. Nonomura N, Nakano Y, Fujioka 0, et al: Experimentally induced otitis media with effusion following inoculation with the outer cell wall of non-typable Haemophilus injluenzae. Arch Otorhinolaryngol 244:253-257, 1987. 6. Nonomura N, Nakano Y, Satoh Y, et al: Otitis media with effusion following inoculation of Haemophilus injluenzae type b endotoxin. Arch Otorhinolaryngol 243:31-35, 1986. 7. Henson PM, Zanolari B, Schwartzman NA, et al: Intracellular control of human neutrophil secretion. I. C5a-induced stimulus-specific desensitization and the effects of cytochalasin Bl. Br J Immunol 121:851-855, 1978. 8. Kawana M, Kawana C, Giebink GS: Penicillin treatment accelerates middle ear inflammation in experimental pneumococcal otitis media. Infect Immun 60:1908-1912, 1992. 9. Fujioka 0: The immunological role of the outer membrane proteins of non-typable Haemophilus injluenzae in otitis media with effusion in children. Eur Arch Otorhinolaryngol 248:483-486, 1991. 10. Kawana M, Kawana C, Yokoo T, et al: Oxidative metabolic products released from polymorphonuclear leukocytes in middle ear fluid during experimental pneumococcal otitis media. Infect Immun 59:4084-4088, 1991. 11. Giebink GS, Carlson BA, Hetherington SV, et al: Bacterial and polymorphonuclear leukocyte contribution to middle ear inflammation in chronic otitis media with effusion. Ann Otol Rhinol Laryngol 94:398-402, 1985. 12. Patel J, Chonmaitree T, Schmalstieg F: Effect of modulation of polymorphonuclear leukocyte migration with anti-CD18 antibody on pathogenesis of experimental otitis media in guinea pigs. Infect Immun 61:1132-1135,1993. 13. Kawana M, Kawana C, Amesara R, et al: Neutrophil oxygen metabolites inhibit growth of cultured chinchilla middle ear epithelial cells. Ann Otol Rhinol Laryngol 103:812-816, 1994. 14. Hanamure Y, Lim DJ: Normal distribution of lysozyme- and lactoferrin-secreting cells in the chinchilla tUbotympanum. Am J OtolaryngoI4:410-425, 1986. 15. Nonomura N, Giebink GS, Zelterman D, et al: Middle ear fluid lysozyme source in experimental pneumococcal otitis media. Ann Otol Rhinol Laryngol 100:593-594, 1991.
Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)
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Early Inflammatory Changes of the Haemophilus injluenzaeInduced Experimental Otitis Media Masahiro
KAWANA,
M.D.
Department of Otolaryngology, Niigata University School of Medicine, Niigata, Japan
Haemophilus injluenzae is one of the most frequent pathogens of acute otitis media. To determine the middle ear response during the early stage of acute inflammation, a small amount of H. injluenzae was inoculated into the bullae of guinea pigs through the tympanic membrane. The bullae were harvested at 6, 12, 24, 36, and 48 hours after H. injluenzae inoculation and washed with phosphate-buffered saline (PBS). The number of viable H. injluenzae and inflammatory cells, the concentrations of myeloperoxidase (MPO) and lysozyme in the washing suspensions were measured, and compared with those in PBS-inoculated control ears. The number of viable H. injluenzae increased very rapidly from 6 to 12 hours after inoculation and remained stationary up to 48 hours. The number of inflammatory cells and the MPO concentration were significantly higher in the H. injluenzae-inoculated ears than in the control ears from 12 to 48 hours after inoculation. The lysozyme concentration was already significantly higher at 6 hours in the H. injluenzae-inoculated ears; the lysozyme was released in the middle ear before the accumulation of inflammatory cells and degranulation of MPO from inflammatory cells. The results indicated that inflammatory reactions were present already at 6 hours after bacterial inoculation, and were rapidly accelerated during the subsequent hours. Consequently, acute middle ear inflammatory responses were seen immediately following inoculation of viable bacteria, and these responses originated in direct responses of middle ear mucosa, and oxidative and non-oxidative neutrophil metabolic products, which may cause tissue injury.
Studies of the pathogenesis of otitis media have explored inflammatory responses to Haemophi/us injluenzae because H. injluenzae is frequently the cause of acute otitis media. 1,2 and is often isolated in middle ear effusions (MEEs) of chronic otitis media with effusion (OME).2,3 Kinetic studies in an animal model using nonviable H. injluenzae demonstrated an increased number of neutrophils in middle ear fluid (MEF) and capillary endothelial destruction of the middle ear mucosa, and these inflammatory responses were determined by the influence of endotoxin. 4 In fact, we already reported that the endotoxin and the outer cell membrane of the H. injluenzae induced otitis media in guinea pigs by observing increases in the number of inflammatory cells and mucosal damage of the middle ear. 5,6 Migrating inflammatory cells, such as neutrophils, are essential elements of host defense and are abundant in MEE in humans with acute purulent otitis media. The phagocytosis of bacteria by neutrophils stimulates intracellular oxidative responses and the bacteria are killed in the phagosomes. However, the growth pattern of H. injluenzae in the middle ear, and the role of inflammatory cell accumulation in response to increases in the number of viable H. injluenzae are still unclear. We, therefore, sought to determine the growth pattern of H. injluenzae and host response with an increase in the number of viable bacteria in the early stage of H. injluenzae-induced experimental otitis media. Received 11 August 1994; accepted 17 October 1994. Correspondence should be addressed to: Masahiro Kawana, Asahimachi-l, Niigata, 951 Japan.
80
Auris·Nasus·Larynx (Tokyo) Vol. 22 (1995)
MATERIALS AND METHODS
Non-typableH. injiuenzae biovar 2 (NCTC 8143) was grown on a chocolate agar plate (Eiken Chemicals, Tokyo, Japan). Two to three colonies were collected and put in a liquid culture medium containing 1% trypton (Difco Laboratories, Detroit, MI, USA), 0.5% yeast extract (Difco), 0.5% sodium chloride (Wako Pure Chemical Industries, Osaka, Japan), 0.1% glucose (Kanto Chemical, Tokyo, Japan), 100,ugjml ,B-nicotinamide-adenine dinucleotide (,B-NAD, Wako) , and loo,ugjml Hemin (Wako) as previously described. 5 The culture medium was incubated until the growth of H. injiuenzae reached the mid-log phase and was diluted to approximately 2 X 103 CFUjml in sterile 0.01 M phosphate-buffered saline (PBS), pH 7.4. Forty healthy guinea pigs weighing 350-450 g and with normal middle ears as ascertained by otoscopy were used for this study. All procedures were done with 20 mg per kg of ketamine hydrochloride intramuscular anesthesia. A volume of 0.1 ml of the H. injiuenzae suspension inoculum (200 CFUjear) was injected directly into the right side of the middle ear bulla through the tympanic membrane using a specially prepared 23 G needle. PBS was placed on the left side by the same procedure as the control. Middle ear bullae were harvested 6, 12, 24, 36, and 48 hours after inoculation, and a hole, approximately 3 mm in diameter, was opened in the tympanic bulla using an electric bar. The middle ear mucosa, tympanic membrane, and the volume of MEF were observed, and the cavity of the bulla was washed with 1oo,u1 of PBS three times through the hole. MEF and a total of 3oo,u1 of the washing suspension were collected, and adjusted to the same final volume (500 ,ul) with PBS. Quantitative culturing of the adjusted middle ear suspensions (AMES) was performed by inoculating 1O-,u1 aliquots of serial dilutions on chocolate agar plates. Inflammatory cells in AMES were counted with a hemacytometer. AMES were centrifuged at 500 X g, and the supernatants were collected for myeloperoxidase (MPO) and lysozyme quantitations. The cell pellets were stained by Wright and Giemsa stain to confirm that inflammatory cells contained more than 90% neutrophils. MPO activity was measured in triplicate samples by a colorimetric method. 7 Net MPO activity after the background (sodium azide pretreatment) subtraction was expressed as units per ml of the washing suspension. Lysozyme activity was measured using a modified 18-hour enzymatic assay as previously described and expressed as units per ml of the washing suspension. 8 The set of AMES from the right middle ear bulla of pure H. injiuenzae growth and from the corresponding left bulla of sterile was used for analysis. Data were analyzed for statistical significance using Student's t-test for paired data. RESULTS
The number of guinea pigs analyzed for this study was 7 at 6 hours, 8 at 12 hours, 7 at 24 hours, 8 at 36 hours, and 7 at 48 hours after inoculation, or 37 in total. The pinhole perforation of the tympanic membranes with the inoculations of H. injiuenzae or PBS remained in all of the analyzed bullae, but no large perforation was observed. The edemas of the middle ear mucosae in the H. injiuenzae-inoculated ears were slight at 6 hours, moderate at 12 and 24 hours, and marked at 36 and 48 hours. No edema of the middle ear mucosa was seen in PBS-inoculated ears. Serous MEF was observed in all of the H. injiuenzaeinoculated ears, but the volumes of the MEF were small and could not be measured at all measuring points. The number of viable H. injiuenzae CFU increased rapidly at 6 hours (geometric mean: 5.69 X 1Q4 CFUjml) and 12 hours (4.95 X 105 CFUjml) after inoculation, reached 1.11 X 106 Auris·Nasus·Larynx (Tokyo) Vol. 22 (1995)
81
CFV/ml at 24 hours and remained constant at 36 and 48 hours after inoculation (36 hours: 1.34 X 106 CFV/ml, 48 hours: 1.25 X 106 CFV/ml, Fig. 1). The number of inflammatory cells in H. injluenzae-inoculated ears slightly increased at 6 hours after inoculation, remarkably increased at 12 hours (1,484 cells/mm3 ) and 24 hours (10,139 cells/mm3 ) , was maximum at 36 hours (10,552 cells/mm3 ) , and slightly decreased at 48 hours after inoculation (7,81Ocells/mm3 ). The number of inflammatory cells in PBS-inoculated control ears increased slightly at 24 hours (818cells/mm3 ), but remained low at 6 hours (93 cells/mm3), 12 hours (240 cells/mm3 ), 36 hours (262 cells/mm3), and 48 hours (343 cells/mm3 ) after inoculation, which was significantly increased in H. injluenzae-inoculated ears compared with PBS-inoculated ears at 12, 24, 36, and 48 hours after inoculation (12 hours: p < 0.05; 24, 36, 48 hours: p < 0.01; Fig. 2). The MPO titer in AMES was also increased in H. injluenzae-inoculated ears at 6 hours after inoculation (164.8V/ml), remarkably increased at 12 hours (234.1 V/ml) and 24 hours (365.8 V/ml) , remained the same 36 hours (361.1 V/ml) , slightly decreased at 48 hours (338.5 V/ml), which were significantly higher in H. injluenzae-inoculated ears at 12, 24, 36, and 48 hours than those in PBS-inoculated ears (PBS-inoculated ears at 12 hours: 125.9 V/ml, 24 hours: 156.6 V/ml, 36 hours: 125.2 V/ml, 48 hours: 206.9 V/ml; 12, 24, 36 hours: p < 0.01; 48 hours:p<0.05; Fig. 3). The lysozyme titer, which was already high in H. injluenzae-inoculated ears at 6 hours after inoculation (H. injluenzae-inoculated ears: 4,264 V/ml, PBS-inoculated ears: 2,320 V/ml) , increased rapidly and continuously at 12 hours (11,928 V/ml), 24 hours (15,639 V/ml) , 36 hours (16,551 V/ml), and 48 hours (20,663 V/ml). It is noteworthy that a significant difference in the lysozyme titers between the H. injluenzae-inoculated ears and the PBS-inoculated ears was seen at all measuring points, including 6 hours after inoculation (6, 24, 48 hours: p < 0.05; 12,36 hours:p
6.5 6.0
1 ~ 5.5 U Cl
.3
5.0
4. 5
4.0
12
24
36
48
Hours after Inoculation
..
Fig. 1. Geometric mean ( ± standard error of the mean) number of viable H. injiuenzae in ears after inoculation of H. injluenzae.
Il
"e
• p
~
~ 5,000 2,500
o L..--=":-_
12
24
36
Hours after Inoculation
82
Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)
48
Fig. 2. Geometric mean ( ± standard error of the mean) number of inflammatory cells in ears after inoculation of H. injluenzae.
200
100
o
12
24
36
48
Hours after Inoculation
E 15,000 3
Fig. 3. Geometric mean ( ± standard error of the mean) concentration of MPO in MEF after inoculation of H. injluenzae.
• p
10,000
5,000
12
24
36
48
Hours after Inoculation
Fig. 4. Geometric mean ( ± standard error of the mean) concentration of lysozyme in MEF after inoculation of H. injluenzae.
DISCUSSION
H. injiuenzae is one of the most common bacteria causing acute otitis media. 1-3 They are the second most frequently cultured bacteria from MEE in cases of acute otitis media. To determine the occurrence and prolongation of acute otitis media, it is important to define the growth style of H. injiuenzae in the middle ear cavity, and the mechanism of injury of the middle ear mucosa by H. injiuenzae. Endotoxin is a component of Gram-negative rods including H. injiuenzae, the endotoxin of H. injiuenzae type b and the outer cell wall of non-typable H. injiuenzae induced acute otitis media in guinea pigs. 5,6 Staphylococcus pneumoniae is another important bacteria causing acute otitis media. When a small amount of pneumococcus was inoculated into the middle ear cavity of the chinchilla, the pneumococcus grew rapidly in the middle ear and induced acute otitis media. Penicillin treatment leads to severe inflammatory responses in the middle ear compared to the non-treated middle ear, 8 which is thought to be due to increased local responses with penicillin-induced pneumococcal lysis and cell wall accumulation. These phenomena suggested that the correlation of the bacterial components, such as endotoxin and outer cell membrane, and middle ear inflammatory responses were the most important fac;tors for the acute and prolonged otitis media. Clinical examination also demonstrated endotoxin in 80% of chronic OME samples. 3 In addition, specific IgG and IgA antibodies against the outer membrane proteins of non-typable H. injiuenzae are significantly higher in children with mucoid MEE than serous MEE in chronic OME, which suggests that bacterial infection and subsequent immune response contribute to the prolongation of OME.9 Using a guinea pig model of H. injiuenzae-induced otitis media, viable H. injiuenzae was Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)
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found to increase logarithmically at 6 hours after inoculation, whereas the number of inflammatory cells increased later than 12 hours. Namely, viable H. injluenzae started increasing just after the inoculation of the middle ear. Later, the rapid increase of viable H. injluenzae caused a high titer of chemotactic factors in the middle ear, which induced acute inflammatory responses. The 6-hour delay may be due to the necessity to reach sufficient concentrations of chemotactic factors from H. injluenzae and middle ear mucosa, such as complement 5a or leukotriene B4, and to have migration of the neutrophils into the middle ear cavity. In recent years, it has been clarified that oxidative and non-oxidative bactericidal activities in neutrophils are important in acute inflammatory responses. In experimental pneumococcal otitis media, these inflammatory changes are observed as an increase in the numbers of neutrophils and metabolic products from neutrophils stimulated by bacteria such as oxygen radicals, MPO, lysozyme, and lactoferrin. 8,IO,lI In H. injluenzae-induced otitis media, edema, and histological damage to middle ear mucosa are directly correlated with the neutrophil number, not the bacterial count. 12 It is thus evident that oxidative or non-oxidative bactericidal effects of neutrophils kill the bacteria and also damage middle ear epithelial cells. MPO, which is an important enzyme in the oxidative metabolic pathways and is contained in the azulophil granules of the neutrophils, increased with the number of inflammatory cells. The high concentration of MPO in MEF may reflect high concentrations of oxidative metabolic products in the middle ear cavity.1O The oxidative metabolic products released from neutrophils kill the bacteria, and may also damage middle ear epithelial cells. \3 High titers of MPO were also measured in a guinea pig model of H. injluenzae-induced otitis media, and these results suggested that oxidative metabolic pathways were activated in the middle ear which may cause tissue injury. Lysozyme is an important enzyme of non-oxidative bactericidal mechanisms. It is contained in azurophil and specific granules in neutrophils, and goblet cells and glands in epithelium. 14 Consequently, an increase of lysozyme titers in MEF reflected both the neutrophil- and epithelium-oriented lysozymes. Lysozyme accumulation in MEF preceding significant inflammatory cell influx has been observed before in experimental pneumococcal otitis media. 7,ls The early release oflysozyme may be considered an indication of injury to middle ear epithelial cells by H. injluenzae, and the migrating neutrophils also released lysozyme in MEF later than 6 hours. The lysozyme titers in the middle ears increased at every measuring point including 36 to 48 hours after H. injluenzae inoculation, whereas the inflammatory cell numbers and MPO concentrations tended to decrease their numbers at the same measuring points. Since the lysozyme titers from infiltrating neutrophils in the middle ear are thought to be decreased at these measuring points, the continuous increase in the lysozyme titers at 36 and 48 hours may reflect strong and progressive damage of the middle ear epithelium. In summary, a small amount of H. injluenzae increased rapidly in the middle ear of guinea pigs and triggered the accumulation of products released by neutrophils and epithelial cells, including oxidants and lysozyme. These products are essential to host defense, but damage the middle ear mucosa. Future experiments are needed to demonstrate the relative contributions of these products to middle ear tissue injury. The author wishes to thank Prof. Yuichi Nakano, M.D., Naobumi Nonomura, M.D., and Osamu Fujioka, M.D. for their valuable suggestions. REFERENCES
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Infect Dis J 11:S7-S11, 1992. 3. Giebink GS: The microbiology of otitis media. Pediatr Infect Dis J 8:S18-S20, 1989. 4. DeMaria TF, Briggs BR, Lim DJ, et al: Experimental otitis media with effusion following middle ear inoculation of nonviable H. injluenzae. Ann Otol Rhinol Laryngol 93:52-56, 1984. 5. Nonomura N, Nakano Y, Fujioka 0, et al: Experimentally induced otitis media with effusion following inoculation with the outer cell wall of non-typable Haemophilus injluenzae. Arch Otorhinolaryngol 244:253-257, 1987. 6. Nonomura N, Nakano Y, Satoh Y, et al: Otitis media with effusion following inoculation of Haemophilus injluenzae type b endotoxin. Arch Otorhinolaryngol 243:31-35, 1986. 7. Henson PM, Zanolari B, Schwartzman NA, et al: Intracellular control of human neutrophil secretion. I. C5a-induced stimulus-specific desensitization and the effects of cytochalasin Bl. Br J Immunol 121:851-855, 1978. 8. Kawana M, Kawana C, Giebink GS: Penicillin treatment accelerates middle ear inflammation in experimental pneumococcal otitis media. Infect Immun 60:1908-1912, 1992. 9. Fujioka 0: The immunological role of the outer membrane proteins of non-typable Haemophilus injluenzae in otitis media with effusion in children. Eur Arch Otorhinolaryngol 248:483-486, 1991. 10. Kawana M, Kawana C, Yokoo T, et al: Oxidative metabolic products released from polymorphonuclear leukocytes in middle ear fluid during experimental pneumococcal otitis media. Infect Immun 59:4084-4088, 1991. 11. Giebink GS, Carlson BA, Hetherington SV, et al: Bacterial and polymorphonuclear leukocyte contribution to middle ear inflammation in chronic otitis media with effusion. Ann Otol Rhinol Laryngol 94:398-402, 1985. 12. Patel J, Chonmaitree T, Schmalstieg F: Effect of modulation of polymorphonuclear leukocyte migration with anti-CD18 antibody on pathogenesis of experimental otitis media in guinea pigs. Infect Immun 61:1132-1135,1993. 13. Kawana M, Kawana C, Amesara R, et al: Neutrophil oxygen metabolites inhibit growth of cultured chinchilla middle ear epithelial cells. Ann Otol Rhinol Laryngol 103:812-816, 1994. 14. Hanamure Y, Lim DJ: Normal distribution of lysozyme- and lactoferrin-secreting cells in the chinchilla tUbotympanum. Am J OtolaryngoI4:410-425, 1986. 15. Nonomura N, Giebink GS, Zelterman D, et al: Middle ear fluid lysozyme source in experimental pneumococcal otitis media. Ann Otol Rhinol Laryngol 100:593-594, 1991.
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