key: cord-0881003-siax7m66 authors: Pitkäranta, Anne; Hovi, Tapani; Karma, Pekka title: Interferon production by leukocytes in children with otitis media with effusion date: 1996-01-31 journal: International Journal of Pediatric Otorhinolaryngology DOI: 10.1016/0165-5876(95)01231-1 sha: 925d34591db1ef0a6f6e43c96cab7df02b9990ff doc_id: 881003 cord_uid: siax7m66 Abstract We have previously shown that leukocyte cultures of children suffering from recurrent respiratory tract infections produce less interferon (IFN) than those of healthy children. In the present study this tentative marker of recurrent infections was used to study the pathogenetic background of otitis media with effusion (OME). Altogether 57 consecutive children, aged 2–11 years, who came for tympanostomy and/or adenoidectomy were divided into three subgroups: 25 of them had OME and a history of recurrent acute otitis media (rAOM/OME +), 20 had OME without an infectious background (inf- /OME +), and 12 had a history of recurrent upper respiratory infections (inf +/OME —) without OME. All the children were free of acute illness at the time of sampling. Differences between the groups were seen in IFN yields when leukocyte cultures were stimulated with adeno-, rhino-, corona-, respiratory syncytial or influenza A viruses. Leukocytes from inf- /OME + children produced more IFN than those of the other two groups. Though no sex differences in the IFN responses were seen among rAOM/OME + and inf + /OME- children, leukocytes from inf- /OME + girls produced significantly higher amounts of IFN than those of inf- /OME + boys, or rAOM/OME + and inf + /OME- children. These differences between clinically different groups of children support the view that the etiology of OME can be heterogeneous. Otitis media with effusion (OME, secretory otitis media) is a common childhood disease without a completely clarified etiology. A number of clinical studies suggest an association between OME and upper respiratory tract infections [7, 12] . However, all patients do not have a clear history of respiratory tract infections or otitis media [5, 11, 15] , and it has been suggested that OME is not a single clinical entity [S] . On the other hand, it has also been difficult to evaluate the significance of the different infection-related and other pathogenetic factors in OME. While laboratory studies on the pathogenesis or classification of OME have mainly been based on the analyses or characteristics of the middle ear effusion and middle ear mucosa, also more general immunologic factors may contribute to the genesis of OME [21, 24] . We have previously shown that leukocytes from children prone to respiratory tract infections have a deficiency in their IFN production capacity when exposed in vitro to pathogenic viruses [18] . It is not yet clear, whether the deficiency is a marker of a genetic trend to increased susceptibility to respiratory infections or is secondary to frequent infections. Anyhow, it may serve as a marker for a recent history of recurrent infections. In the present study we investigated whether children suffering from OME with or without a history of recurrent acute otitis media differ with respect to the capacity of their leukocytes to produce virus-induced IFN. The study population comprised of 57 consecutive children, aged 2-l 1 years, admitted during January/February 1993 for scheduled operation (tympanostomy and/or adenoidectomy) to the Department of Otolaryngology, Helsinki University Central Hospital, because of OME, or because of recurrent upper respiratory tract infections (acute otitis media included) without OME (inf + /OME -). In this study the OME children were subdivided into a group (N = 20) with an asymptomatic incidentally discovered OME but without an apparent infectious background that is otitis or other respiratory tract infections only occasionally or not at all (inf -/OME + ) and into a group (N= 25) with known recurrent attacks of acute otitis media (rAOM/OME +), Table 1 shows the age and sex distribution of these 57 children. The diagnosis of these children was based on the patients history, pneumatic otoscopy and otomicroscopy. The criteria for diagnosing OME includes the presence of effusion in pneumatic otoscopy, and confirmed in tympanostomy, behind an intact eardrum without sign and symptoms of acute infection. The definition of AOM was that a child had findings suggesting middle ear effusion, and at least one of the following symptoms: otalgia, tugging at or rubbing of the ear, rectal or axillary temperature of at least 38.O"C, irritability, restless sleep, acute gastrointestinal symptoms (vomiting or diarrhea) or other simultaneous respiratory infection. The criteria of rAOM/OME + group were that a child had had at least 4 AOM episodes during the last 6 months. To be included in the inf + /OME -group a child had to have experienced at least 6 episodes of upper respiratory tract infection during the last 6 months. Such infections included rhinitis, pharyngitis or tonsillitis, occasionally combined with sinusitis or otitis, but without any other complications. The infections were mostly accompanied by a moderate fever. Otherwise all the children were healthy and had grown normally. All the children with known allergy or other diseases were excluded. The patients were enrolled in the study after an informed consent of the parents. In all patients the serum levels of immunoglobulins and complement components were measured by immunoturbidometric methods, and none of the children showed any noticeable immunological aberrations. The children had been healthy for at least one week since the last infection. Blood specimens for leukocyte preparation were drawn using the needle inserted for injecting intravenous anaesthetics, but immediately before that. Leukocytes from heparinized blood were separated over a Lymphopaque gradient as described earlier [4] . The mononuclear cells at the interphase of the gradient were collected and washed 3 times with phosphate-buffered saline, adjusted to a concentration of 2 x 106/ml in tissue culture medium (RPMI-1640 supplemented with 10% foetal calf serum) and distributed as 0.1 ml aliquots into 96-well tissue culture plates, 12 wells of each cell batch. Freshly made parallel leukocyte cultures were inoculated with samples of 5 crude virus preparations, 2 wells each. A single preparation of each inducer was used throughout the study and a fresh aliquot was thawed for each experiment. The source and propagation of the inducer viruses was described previously [18] . Influenza, adeno-and rhinoviruses were added into the cultures at a calculated p2) (b R) OME = otitis media with effusion; inf -/OME + = otitis media with effusion and a history of otitis or other respiratory infections only occasionally or not at all; rAOM/OME + = recurrent acute otitis media preceding otitis media with effusion; inf + /OME -= recurrent upper respiratory tract infections without otitis media with effusion. multiplicity of infection of 1 TCID,,/cell. The titres of the stocks of coronavirus 229E and respiratory syncytial (RS) virus were too low for this and 0.001 and 0.1 TCID,,/cell, respectively, were used. The plates were sealed with adhesive tape and incubated at 36°C. Cell-free specimens from the culture medium were harvested at day 2 postinfection and stored at -20°C until assayed [16] . 2.4. IFN assay IFN concentrations were measured by a biological micromethod based on the reduction of cytopathic effect caused by vesicular stomatitis virus [13] . A continuous bovine cell line (NBL) was used in the assay. Samples from patient cultures were analysed blindly. A calibrated leukocyte IFN standard (a gift from Dr. Kari Cantell, National Public Health Institute, Helsinki) was included in all assays so that the results could be given in international units (IU). The results given are means of 2 parallel cultures, a maximum of a 2-fold difference was seen between the parallel specimens. A constant donor-specific pattern in the IFN responses has been seen [17] . The subtype of IFN was not specifically assessed in these studies. Virus-induced leukocyte IFN is mostly a mixture of different IFN-a:s and the NBL cell line used in the assays is not sensitive to human IFN-y. Therefore, we believe that the IFN yields recorded represent IFN-c(. Differences between the groups were evaluated with the Mann-Whitney U-test. 3 . Results Leukocytes from all 20 inf -/OME + ,25 rAOM/OME + and 12 inf + /OMEchildren showed an IFN production response to at least two different inducers. IFN yields from cultures stimulated by adeno-, rhino-, corona-, RS or influenza A viruses were higher for the inf-/OME + children's leukocytes than for those of the rAOM/OME + and inf + /OME -children's leukocytes (Fig. 1) . Statistically significant differences were seen between inf -/OME + and rAOM/OME + children when leukocytes were stimulated by rhino-(P < 0.01) corona-and RS viruses (P < 0.05). The results with adeno-and influenza A virus showed a similar tendency but not statistically significant differences. A statistically significant difference between inf -/OME + and inf + /OME -was seen when the leukocytes were stimulated by adeno-, rhino-or coronaviruses (P < 0.01) and by RS and influenza A viruses (P < 0.05). The difference between rAOM/OME + and inf + /OMEchildren was not significant for any inducer. Interferon production was different between the two sexes among the inf -/ OME + children but not among the rAOM/OME + or inf + /OME -children. IFN yields from cultures stimulated by rhino-, corona-or RS viruses were higher (P < 0.05) for the inf -/OME + girls than for those of the inf -/OME + boys. 0 inf-/OME+ a rAOM/OME+ n inf+/OME-A C R Fig. 1 . Geometric means of IFN yields produced by leukocyte cultures from children with inf -1 OME + , rAOM/OME + and inf + /OME -Significance of the differences: inf -/OME + vs. rAOM/ OME + with adenovirus P < 0.1, with rhinovirus P c 0.01, with coronavirus P < 0.05, and with RS virus P < 0.05: inf -,'OME + vs. inf + /OME -with adenovirus P < 0.01. with rhinovirus P < 0.01, with coronavirus P