Noritake Watanabe

The development of mucosal immunity in guinea pig middle ears

Recent studies have suggested to us that the middle ear is an organ capable of responding immunologically to antigenic substance. However, it is still not known when and how the middle ear starts to provide the immunologic defense system. To clarify this, we investigated immunoglobulin forming cells of different classes and secretory proteins, such as secretory component and lactoferrin, in the mucous membrane of eustachian tube and tympanic bulla of 20 developing and 5 normal adult guinea pigs by the use of a direct immunofluorescence technique. Other organs, such as nose, larynx, trachea, small intestine, spleen and lymph node were also subjected to this investigation. Changes in the middle ear mucosa were also observed after antigenic challenges directly to the tympanic cavity of 6 developing and 39 adult guinea pigs. IgA and IgM forming cells began to appear in the tubal mucosa on 7th postnatal day, wile it was scarcely possible to find IgG1 and IgG2 forming cells in developing guinea pigs. Immunoglobulin forming cells of all classes increased in the middle ear mucosa after the antigenic stimuli. Results of this study showed that local synthesis of IgA, as well as other classes, is latent in the middle ear, that the middle ear of immature animals is vulnerable to antigenic stimuli, and that the middle ear of developing animals possesses potential immune responsiveness.

Immunoglobulin E (IgE) in Middle Ear Effusions

This study was undertaken to further clarify the relation of atopic allergy to otitis media with effusion. Ninety-six specimens of middle ear effusions and corresponding sera were collected from 92 patients, and each sample was quantitatively measured by a radioactive single radial diffusion (RSRD) technique for immunoglobulin E (IgE) concentration. Specimens consisted of 70 serous effusions (38 acute and 32 chronic) and 26 mucoid effusions (10 acute and 16 chronic). The mean concentration of IgE in the serous effusions was 143.1 ± 28.7 ng/ml, and in the corresponding sera 227.5 ± 40.7 ng/ml. The mean concentration of IgE in the mucoid effusions was 221.7 ± 48.9 ng/ml and in the corresponding sera 398.0 ± 81.5 ng/ml. The difference between these values was not statistically significant. In both mucoid and serous types the mean IgE concentration was lower in the effusions than in the sera from the same patients, but this difference was not statistically significant. The mean value of the IgE concentration in mucoid effusions was higher than in the serous effusions, but this difference was not significant. Three of 70 serous effusions (4.3%) and 4 of 26 mucoid effusions (15.4%) had an IgE concentration exceeding 500 ng/ml. In only 2 of the 96 cases (2.1%) did the IgE concentration in effusion exceed both the 500 ng/ml level and the IgE concentration in the corresponding serum. Results of this study suggest that IgE in middle ear effusions, rather than being a local product, may be derived from the serum, and also that the fluid is not an atopic allergic effusion.

Substance P and Vasoactive Intestinal Peptide in Nasal Secretions and Plasma from Patients with Nasal Allergy

To clarify the role of substance P (SP) and vasoactive intestinal peptide (VIP) in nasal allergy, we measured their concentrations in the nasal secretions and plasma of normal subjects and patients with nasal allergy to house dust and Japanese cedar pollen by competitive enzyme-linked immunoassay. The mean levels of SP (224 pmol/L) and VIP (41.6 pmol/L) in the nasal secretions of normal subjects were significantly higher than those in plasma (SP 3.04 pmol/L and VIP 1.04 pmol/L; p < .01). The mean levels of SP and VIP in the nasal secretions of the pollinosis group were significantly higher than those of the control group (p < .05 and p < .01), while the levels of the house dust allergy group were not higher than those of the control group. Intranasal allergen challenge significantly reduced SP levels in the nasal secretions of the allergy groups, while it did not influence VIP levels in the nasal secretions. These findings suggest that SP and VIP are actively secreted into the nose and may play an important role in the allergic reaction on the surface of the human nasal mucosa.

Secretory Immunoglobulin a (SIgA) in Middle Ear Effusions; A Further Report

Immunochemical and immunofluorescent studies of secretory immunoglobulin A (SIgA) in otitis media with effusion were carried out to investigate the local immunologic defense system and secretory activity in the middle ear. SIgA was isolated from pooled middle ear effusions by an immunoadsorbents technique and its antigenicity and subunit structure compared with SIgA derived from other external secretions, such as saliva, nasal discharge, and colostrum. The antigenicity and subunit structure of the isolated SIgA from middle ear effusions were identical or very similar to those of SIgA obtained from other external secretions. Radioimmunodiffusion analysis revealed that SIgA was present in 43 of 47 (91.5%) cases of serous middle ear effusion, whereas the sera from only 3 of 47 (6.4%) of these patients was found to have SIgA. Immunoelectrophoresis, using anti-SIgA antiserum, and radioimmunoelectrophoresis detected free SC (secretory component) in 1 of 47 (2%) serous and 6 of 30 (20%) mucoid effusions. Apices of epithelial cells and glandular acinar cells of the middle ear mucosa obtained from patients with otitis media with effusion were stained specifically with anti-SC antibodies. Findings of the study would suggest: 1) that the middle ear maintains the local immunologic defense system, as do respiratory tracts and salivary glands, and 2) that while serous middle ear effusions are a mixture of normal middle ear secretions bathing the membrane surface and transudates from serum, mucoid effusions are the result of enhanced epithelial secretory activity and transudates.

Effects of anti-allergic drugs on substance P (SP) and vasoactive intestinal peptide (VIP) in nasal secretions.

To clarify the effects of anti-allergic drugs on substance P (SP) and vasoactive intestinal peptide (VIP) levels in nasal secretions, we employed competitive enzyme-linked immunoassays to measure concentrations of those neuropeptides in nasal secretions from 40 patients with house dust nasal allergy before and after administration of azelastine and oxatomide. One mg of azelastine and 30 mg of oxatomide were administrated twice a day for 4 weeks. Mean values of SP concentrations and ratios of SP to total protein of the nasal allergy group were significantly higher than those of the control group (p < 0.002). The VIP/total protein ratio of the allergy group was also significantly higher than that of the control group, although the VIP concentration alone was not. Mean levels of SP and VIP from patients with severe symptoms were significantly higher than those of the control group (p < 0.05), although those values were not significantly different between patients with moderate symptoms and control subjects. Azelastine and oxatomide effectively reduced SP levels in nasal secretions (p < 0.005), but they did not significantly decrease VIP levels. The reduction of SP levels was significant in patients with excellent responses to those drugs (p < 0.005), but not in patients with poor responses. These findings suggest that SP and VIP levels in nasal secretions may reflect the clinical state of nasal allergy and be one of the better parameters available for evaluating the clinical efficacy of anti-allergic drugs against nasal allergy.

Laryngeal Secretions: An Immunochemical and Immunohistological Study

This study was designed to investigate properties of laryngeal secretion and secretory activity of IgA in the larynx. Laryngeal secretions were collected by adsorption method on filter paper during laryngomicrosurgery from 20 patients having an inflammatory lesion in the larynx. Contents of IgG, IgA, IgE, secretory component (SC), and lactoferrin in the laryngeal secretions were determined and compared with results of those in nasal secretions, tracheobronchial washings, and serum samples obtained from the same subjects. The laryngeal mucosae of 8 laryngectomized materials for cancer lesion were subjected to immunofluorescence studies including the cytoplasmic SC affinity test. Results of this study indicate that laryngeal secretions are characterized by exocrine secretion, resembling nasal and tracheobronchial secretions in the electrophoretic pattern and immunoglobulins content. The immunofluorescence studies and SC affinity test found that the larynx possesses secretory activity of IgA, particularly in the ventricle and subglottis.

Quantitative Determination of Secretory Immunoglobulin a (SIgA) in Middle Ear Effusions

For the purpose of investigating the nature of middle ear effusion, quantitative analysis of secretory immunoglobulin A (SIgA) by radioactive single radial diffusion (RSRD) technique was performed on specimens of middle ear effusion and serum from the corresponding patient. Specimens consisted of 61 serous effusions (32 acute and 29 chronic type) and 29 mucoid effusions (9 acute and 20 chronic). The mean value of SIgA concentration in the serous effusions was 212.7 ± 13.5 μg/ml and that in sera from these patients was 27.0 ± 3.0 μg/ml. In the mucoid type the mean value of SIgA in effusions was 357.7 ± 13.2 μg/ml and that in sera was 24.3 ± 2.8 μg/ml. The difference in the mean value of SIgA concentration between the serous and mucoid types of effusions was statistically significant. The mean value of SIgA concentration in serous effusions was eight times that in sera from these patients and in mucoid effusions the SIgA value was approximately 14 times that in sera. The calculated percentage of SIgA in the total IgA of middle ear effusions was 11–12% in both the serous and mucoid categories. The percentage in sera was about 1%. Results of this study indicate that all middle ear effusions contain appreciable amounts of the secretory element, although the transudate from serum is the major component of effusion, and support an hypothesis that while serous middle ear effusions are a mixture of normal ear secretions bathing the membrane surface and transudates from serum, mucoid effusions are the result of enhanced epithelial secretory activity and transudates.

Radioimmunoassay of IgE in Middle Ear Effusions

Fifty-six sample pairs of middle ear effusions and sera obtained from patients with otitis media with effusion were investigated for The IgE antibody activity against mites, using radioallergosorbent test (RAST) which was recently developed as an in vitro allergy test. The IgE concentrations were also determined by radioimmunosorbent test (RIST). Results showed that the IgE antibody activity against this allergen was found in 5 middle ear effusions and 3 sera. Four of 5 patients with the positive RAST test had nasal allergy or signs suggesting allergy. Theree were two middle ear effusions which seemed to be an allergic fluid. However, findings in the present study do not suggest that middle ear effusions result from the direct allergic reaction induced by mites in the mucous membrane lining the middle ear cavity.

Induction of Antigen-Specific IgA-Forming Cells in the Upper Respiratory Mucosa

We investigated the actual processes for activating respiratory mucosal immunity. Hartley guinea pigs were immunized with particulate antigen of dinitrophenylated ovalbumin into the duodenum (group A), trachea (group B), and nasal cavity (group C) 1 week after systemic priming with the soluble antigen. The control animals (group D) received only systemic priming. Immunoglobulin A antibody titers in nasal secretions and saliva from groups A and B significantly exceeded those of groups C and D (p<.01). Many antigen-specific IgA-forming cells were detected in the respiratory and gastrointestinal mucosae of groups A and B. Following duodenal immunization, the appearance of antigen-specific IgA-forming cells in Peyers patches preceded those in mesenteric and hilar lymph nodes, while they simultaneously appeared in the lymphoid tissues after tracheal immunization. These findings suggest that intratracheal immunization mainly stimulates bronchus-associated lymphoid tissue as a source of IgA precursors, as intraduodenal immunization stimulates gut-associated lymphoid tissue.

Lymphocyte Migration to the Middle Ear Mucosa

We investigated the migration of antigen-specific IgA—forming cells to the middle ear mucosa. Antigen-specific lymphocytes of IgA and IgG classes were induced in guinea pigs according to an immunization strategy previously described. From those animals, chromium 51–labeled lymphocytes of Peyers patches and spleen were transferred to radiated chimera recipients. The radioactivity levels of the middle ears with antigenic and nonantigenic stimuli were significantly higher than those of the control ears (p < .05). Those levels of radioactivity were influenced neither by origins and subsets of transferred cells nor by antigenic stimuli to the mucosa (p > .05). Many labeled cells were observed in the middle ear effusion, while few were found in the inflamed mucosa. These findings suggest that in the early stage of inflammation, lymphocytes, including antigen-specific T and B cells, may be recruited from the blood circulation to the inflamed middle ear mucosa by nonspecific inflammatory processes that may mask antigen-specific factors in lymphocyte migration.