Hidehisa Suzumura

Quantitation and Immunocytological Identification of lnterleukin-1 in Nasal Polyps from Patients with Chronic Sinusitis

Immunoreactive interleukin-1 (IL-1) alpha and beta levels in nasal polyp (NP) extracts were measured from 22 adult patients with chronic sinusitis (CS) in order to determine their significance in the pathogenesis of NP. The NP sample was separated into mononuclear and polymorphonuclear fractions. In the mononuclear fraction, the mean value of IL-1 beta was 21.5 pg/ml/g, which was significantly higher than that of IL-1 alpha (8.4 pg/ml/g, p < 0.01). In younger adults, the IL-1 alpha and IL-1 beta levels were 21.2 and 59.4 pg/ml/g, which were significantly higher than those in older CS patients (p < 0.01). There was a significant reverse correlation between patients age and IL-1 beta levels in the mononuclear fraction (r = -0.509, p < 0.01). Immunoreactive IL-1, mainly IL-1 beta, was identified in the cytoplasmic area of monocytes. A certain amount of immunoreactive IL-1 is produced in mononuclear leukocytes, particularly activated monocytes, and IL-1 beta production is greater than IL-1 alpha. In younger adult CS patients, NPs contain larger amounts of IL-1 beta in monocytes, compared to those of older patients.

Lysosomal proteases and protease inhibitors in nasal allergy and non-atopic sinusitis

Patterns of protease activity and levels of protease inhibitors were analyzed in both nasal secretions and tissue extracts from patients with nasal allergy and non-atopic sinusitis to investigate the role of proteases in the inflammatory reaction. Protease activity was measured using specific methyl-coumaryl-7-amide substrates. The pattern of protease activity in the nasal secretions of chronic sinusitis patients was similar to that in neutrophil lysate and quite different from that in plasma. Both gluthatione activation testing and inhibition testing using synthetic inhibitors revealed that the majority of proteases in both secretions and tissues are lysosomal thiol proteases such as cathepsins B and L. Neutrophilic elastase is also a major protease in nasal secretions. In acute sinusitis, both protease activity and inhibitor levels were very high, suggesting an interaction between proteases and inhibitors. Cathepsin B and B-like thiol proteases appear to play a key role in prolonging chronic inflammation against the healing process, due to their resistance to plasma inhibitors and the shortage of thiol protease inhibitors. Protease activity in the secretions of nasal allergy patients was very weak, and the reaction between proteases and inhibitors appeared to be weak.

The relationship between proteases activity and glycoprotein levels in middle ear effusions from experimental otitis media in cats.

The relationship between lysosomal proteases activity (elastase and cathepsin B) and levels of mucous glycoproteins in middle ear effusions (MEEs) was studied using a cat model of otitis media with effusion (OME) induced by Eustachian tube obstruction (ETO). The ratio of cathepsin B activity to total protein concentration (TPC) in MEE was 25.6 +/- 19.4 RFU/g x dl-1 at 1 week after ETO, and increased with the duration of OME. The ratio of elastase activity to TPC had a significant correlation to total leukocyte count. The ratio of fucose levels to TPC, which is one of the parameters reflecting levels of mucous glycoprotein, at 1 week after ETO was significantly higher than that at both 2 and 4 weeks after ETO. The percentage of glycoprotein levels absorbed to wheat germ lectin was highest at 1 week after ETO, and decreased with the duration of OME. In conclusion, mucous glycoproteins in cat occupy a larger portion of glycoproteins in MEE at the early stage of OME, and elastase and other lysosomal proteases may play a role in both stimulation of mucin release from goblet cells and mucin degradation. The balance of these processes seems to be a key factor determining mucin levels in MEEs.

ELISA for Determination of Immunoreactive Free Elastase and Elastase in Complex with α1-antitrypsin in Nasal Secretions with Sinusitis

Sensitive double antibody sandwich ELISA methods was developed in order to quantify immunoreactive neutrophil elastase (NE) levels in nasal secretions with chronic sinusitis (CS). Microwell plate as a solid phase was coated with anti-NE antibody. Two different horseradish peroxidase (HRP) labelled antibodies used as the second antibody were anti-NE-HRP for measuring total (free + complexed) NE level and anti-alpha 1-antitrypsin (AT)-HRP for complexed NE level. Mean value of total NE was 31.0 +/- 20.7 micrograms/ml in nasal secretions from adult patients with CS, and the percentage of complexed NE in total NE was 33.7 +/- 21.4%. This sandwich ELISA is a useful method for measuring both total and complexed NE levels in nasal secretions.

Neutrophil Elastase and Its Complex with α1-antitrypsin in Soluble and Insoluble Fractions of Nasal Secretions of Chronic Sinusitis

Immunoreactive neutrophil elastase (NE) and its complex with alpha 1-antitrypsin (AT) was measured by double antibody enzyme linked immunosorbent assay (ELISA) in nasal secretions of chronic sinusitis (CS). Nasal secretions were separated into two fractions: PBS-soluble and insoluble fractions. Elastolytic activity was also examined. Mean value of total NE level was 31.0 micrograms/ml in the soluble fraction, which was significantly lower than that in the insoluble fraction (71.9 micrograms/ml, p less than 0.01). On the other hand, the percentage of complexed NE in total NE in the soluble fraction (33.7%) was significantly higher than that in the insoluble fraction (12.1%, p less than 0.01). Elastolytic activity in the soluble fraction (23.4 RFU) was significantly lower than that in the insoluble fraction (170.5 RFU, p less than 0.01). NE with elastolytic activity exists in nasal secretions of CS, and active-free NE in the insoluble fraction could be a major source of enhancement and continuation of mucosal inflammation.

Immunohistochemical Detection of Serous Cells in Nasal Mucosa With Monoclonal Antibody Against a Component in Human Nasal Secretion

Secreting mechanisms of secretory cells in nasal mucosa and the changes of nasal secretions in chronic inflammatory sinusitis have been studied by the biochemical and histochemical methods. These methods could not clarify the changes of quality and quantity of nasal secretions and secretory cells. In order to obtain the specific marker for the secretions in different cells, we have produced monoclonal antibodies against a component in human nasal discharge. One antibody was selected for further characterization, because it stained submucosal serous cells specifically. This antibody stained the components of serous cells with molecular weight of 14 kD specifically, and was sensitive to periodate oxidation treatment. This antibody will be useful for detecting the subpopulation in secretory cells of human nasal mucosa, and may be serve as a biochemical probe for secretory activity of particular secretory cell types.