Neuropeptide Y (NPY) in human nasal mucosa.

Neuropeptide Y (NPY), a potent vasoconstrictor peptide found in sympathetic neurons, was analyzed in human inferior turbinate nasal mucosal tissue. NPY content determined by radioimmunoassay was 3.13 +/- 0.79 pmol/g tissue (n = 6) in mucosa extracted with ethanol-acetic acid. NPY-immunoreactive nerves were found around small muscular arteries, arterioles, arteriovenous anastomoses, and as free fibers near arteriolar and venous vessels. They formed a plexus around the arterial vessels, and were also present between vascular smooth muscle cells. Few NPY fibers were present near glands or the epithelium. [125I]NPY binding sites were localized by autoradiography to small muscular arteries, arterioles, and a few venous sinusoids. In explant culture experiments, 4 microM NPY did not stimulate release of [3H]glucosamine-labeled glycoconjugates or lactoferrin (a product of serous cells) from nasal mucosal fragments. Degradation of NPY by a tissue homogenate was rapid (t1/2 = 13.5 +/- 2.3 min). The degradation was inhibited by thiorphan and phosphoramidon, inhibitors of neutral endopeptidase activity. NPY released from sympathetic neurons may play a role as a constrictor of arterial vessels and regulate vasomotor tone in the human nasal mucosa.

Gastrin-releasing peptide in human nasal mucosa.

Gastrin-releasing peptide (GRP), the 27 amino acid mammalian form of bombesin, was studied in human inferior turbinate nasal mucosa. The GRP content of the mucosa measured by radioimmunoassay was 0.60 +/- 0.25 pmol/g tissue (n = 9 patients; mean +/- SEM). GRP-immunoreactive nerves detected by the immunogold method of indirect immunohistochemistry were found predominantly in small muscular arteries, arterioles, venous sinusoids, and between submucosal gland acini. 125I-GRP binding sites determined by autoradiography were exclusively and specifically localized to nasal epithelium and submucosal glands. There was no binding to vessels. The effects of GRP on submucosal gland product release were studied in short-term explant culture. GRP (10 microM) significantly stimulated the release of the serous cell-specific product lactoferrin, and [3H]glucosamine-labeled glycoconjugates which are products of epithelial goblet cells and submucosal gland cells. These observations indicate that GRP released from nerve fibers probably acts on glandular GRP receptors to induce glycoconjugate release from submucosal glands and epithelium and lactoferrin release from serous cells, but that GRP would probably not affect vascular permeability.

Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb.

Two unique forms of cytochrome P-450 (P-450), designated NMa and NMb, were recently isolated in this laboratory from nasal microsomes of rabbits. In the present study, polyclonal antibodies to the purified nasal cytochromes were prepared. Immunochemical analysis with specific rabbit anti-NMa and sheep anti-NMb antibodies indicated that P-450 isozymes identical to or having a high structural homology with NMa are present in both olfactory and respiratory mucosa, as well as in liver, but NMb was detected only in the olfactory mucosa. Neither form was detected in other tissues examined, including brain, esophageal mucosa, heart, intestinal mucosa, kidney, and lung. The specific occurrence of NMb in the olfactory mucosa was further substantiated by the detection and specific inhibition by anti-NMb of the formation of unique NMb-dependent metabolites of testosterone in olfactory microsomes but not in microsomes from liver or respiratory mucosa. Similar experiments with antibodies to previously purified rabbit hepatic P-450 isozymes indicated that not all of the hepatic cytochromes are expressed in the nasal tissues. Thus, P-450 isozymes structurally homologous to hepatic forms 2, 3a, and 4, but not 3b and 6, were found in the olfactory mucosa. On the other hand, only form 2 was detected in the respiratory mucosa. Immunoquantitation experiments revealed that NMa and NMb are the major P-450 forms in olfactory microsomes, whereas NMa and P-450 form 2 (or its homolog) constitute the major portion of the respiratory nasal microsomal P-450. The level of NMa in the liver is relatively low, accounting for less than 3% of total microsomal P-450 in this tissue. In addition, evidence is provided that NMa is the major catalyst in the dealkylation of two nasal carcinogens, hexamethylphosphoramide and phenacetin, in both olfactory and respiratory nasal microsomes.

Calcitonin gene-related peptide in human nasal mucosa.

To explore the potential range of functions for calcitonin gene-related peptide (CGRP) in human mucosa, we quantified human inferior turbinate nasal mucosal CGRP content by radioimmunoassay, localized CGRP-immunoreactivity by immunohistochemistry, detected 125I-CGRP binding sites by autoradiography, and tested the ability of CGRP to induce submucosal gland secretion in short-term explant culture of human nasal mucosa. Nasal mucosa contained 0.45-0.54 pmol CGRP/g wet wt (n = 18). Immunoreactive CGRP was found in nerve fibers that densely innervated the walls of small muscular arteries arterioles. Venules and venous sinusoids were innervated by individual CGRP staining fibers. Occasional CGRP-containing nerve fibers were also noted adjacent to submucosal gland acini, near the epithelial basement membrane, and between epithelial cells. Specific 125I-CGRP binding sites were concentrated on small muscular arteries and arterioles. CGRP (4 microM) did not stimulate glycoconjugate or lactoferrin release from mucosal explants. These results indicate that in the human nasal mucosa, CGRP is present in nerve fibers, which most likely represent nociceptive sensorimotor nerves that innervate vascular structures (muscular arteries, arterioles, veins and venous sinusoids). It is likely that CGRP release from sensory neurons may play a role in the regulation of vasomotor responses, but no evidence for a role of CGRP in glandular secretion was found.

Glandular secretion of lactoferrin in a patient with neutrophil lactoferrin deficiency.

Patients with specific granule deficiency (SGD) develop recurrent severe bacterial skin infections. Neutrophils from patients with SGD are deficient in lactoferrin (Lf), an antimicrobial protein commonly found in many mucosal secretions. Unstimulated and stimulated nasal secretions, saliva, and tears were collected from a patient with SGD and from normal control subjects and were analyzed for Lf. The secretions from the patient contained normal values of Lf, suggesting that the glands secrete Lf from a source other than neutrophils. Immunohistochemical staining of normal nasal mucosa demonstrated that Lf is localized within serous submucosal gland cells and that neutrophils are not normally observed in the nasal mucosa. These findings suggest that glandular tissues produce and locally secrete Lf by processes that are independent of neutrophil degranulation.

Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa.

The sequence is reported of the pyrazine-binding protein from cow olfactory/respiratory mucosa. The protein consists of 159 amino acids and clearly belongs to the retinol-binding protein family. It is most closely related to the urinary proteins from mice and rats and to the odour-binding protein from rat nasal epithelium. It is unique however, in that only one of the otherwise conserved features of the family is still present--namely a single tryptophan. Most surprisingly the protein contains no cysteine and, therefore, does not rely for its structural stability on the disulphide bond(s) present in other members of this group. A model for the protein has been constructed based on the co-ordinates of beta-lactoglobulin. From this, it is possible to identify residues which may line the binding site. The impression gained is of a much larger pocket than occurs with retinol-binding protein or beta-lactoglobulin. The character of the binding pocket remains essentially hydrophobic but with a significant reduction in its aromatic content and an increase in H-bonding side chains.

Airway secretion electrolytes: reflection of water and salt states of the body.

Water and electrolyte content influences the rheology of respiratory mucus. Nasal secretions can be obtained from almost all patients and may be regarded as a possibly useful model of the electrolyte composition of lower airway secretions that are difficult to collect in most patients. Na, K, and Cl were determined from nasal secretions in 35 ICU patients. We studied the relationship of those values to the patients' water and salt states. Our study indicates that: a) lower K and Cl levels and higher Na levels than those found in plasma are common to both nasal and bronchial secretions; b) variations of electrolyte levels in nasal secretions are interrelated; c) patients with lower values of free-water clearance show lower Na and higher Cl levels in nasal secretions, possibly due to increased epithelial transport; d) the amount of K in nasal secretions appears correlated with its urinary fractional excretion (this could be explained by the variations in intracellular K levels); and e) in hyperchloremic patients, plasma/secretion differences of Na are decreased, possibly due to decreased epithelial transport.

Transport of serum IgA into murine respiratory secretions and its implications for immunization strategies.

Both nonlabeled and radiolabeled IgA mAb with specificity toward Sendai virus, a respiratory pathogen, were used to investigate the transport of serum polymeric and monomeric IgA into murine upper and lower respiratory secretions as well as into the gut. After purification by affinity chromatography, IgA mAb were fractionated into monomers and polymers by gel filtration and radiolabeled with 125I. Mice were injected i.v. with either 125I-monomer and 131I-albumin or 125I-polymer and 161I-albumin. At various times after injection, serum and gut, nasal and bronchoalveolar lavage samples were collected. The TCA precipitable radioactivities were determined and the selective transport indices calculated. The results indicated selective transport of polymeric IgA but not monomeric IgA from serum into upper respiratory and intestinal secretions. The degree of TCA precipitability in nasal lavage and to a lesser extent gut secretions suggested significant degradation of the antibody during or after transport. To investigate further the integrity of the IgA in mucosal secretions, ELISA viral binding activity of nonradiolabeled IgA was determined for both IgA incubated with nasal secretions in vitro and polymeric IgA recovered by nasal lavage 4 h after i.v. injection. Although reconstitution experiments indicated no significant loss of antibody binding activity after incubation of antibody with lavage fluid in vitro, only negligible ELISA binding activity was detected in nasal washes after i.v. injection of antibody. The data overall suggest that although there is a quantitatively small, but selective transport of polymeric IgA into the upper respiratory tract, this transport results in minimal functional antibody activity. Implications of these and other findings for strategies of oral immunization in prophylaxis against respiratory infections are discussed.

[Electron microscopic study of glycoconjugate in nasal mucosa of nasal allergy by lectin histochemistry].

Glycoconjugates in glandular and goblet cells of nasal mucosa were compared between normal and nasal allergy by using various horseradish peroxidase (HRP)-conjugated lectins; WGA, PNA, UEA-I and RCA-I. Specific sugar residues of glycoconjugates could be identified under electron microscope. Golgi's complex of the serous secretory cells in nasal allergic mucosa had positive staining in UEA-I. Goblet cells of nasal allergic mucosa were stained strongly in PNA, compared to normal mucosa, however, they were stained weakly in WGA. In conclusion, glycoconjugates in glandular and goblet cells seem to be changed in nasal allergy.

[Electron microscopic study of intercellular junction in nasal mucosa of nasal allergy by lectin histochemistry].

To explain of the mechanism of the enhanced nasal epithelial permeability to HRP in patients with nasal allergy, the inferior turbinate mucosa was removed from 6 normal adults and 7 adults with nasal allergy. Difference of the fine structure of the intercellular junction was compared between normal mucosa and mucosa of nasal allergy by electron microscope. Staining pattern of four kinds of HRP-conjugated lectin (HRP-WGA, PNA, UEA-I and RCA-I) was also studied by electron microscope. There was no significant difference in the intercellular space of the mucosa between the normal mucosa and mucosa of nasal allergy. In the epithelial cell membrane, pattern of HRP-lectin staining was almost similar in both groups. In normal nasal epithelium, the intercellular junction consisted of junctional complex; adherent junction, desmosome and gap junction. The intercellular space was approximately 150-250 A in width. The tight junction was located beneath the luminal surface of the epithelium, and belt-like continuation connecting the adjacent cells. It was concluded that enhanced permeability to HRP in nasal allergy was not morphologic changes of the intercellular junction and component and distribution of the glycoconjugates in epithelial cellular membrane, but this may be based on functional changes.

CD-1 (T6), HLA-DR-expressing cells, presumably Langerhans cells, in nasal mucosa.

In the skin, epidermal Langerhans cells (LC) constitute a major population of antigen-presenting cells. These cells are characterized by the expression of both CD-1 (T6) and HLA-DR on the cell membrane. We wanted to know whether similar CD-1/HLA-DR-positive cells occur in the nasal mucosa of patients with an isolated grass pollen allergy and in non-allergic controls. CD-1/HLA-DR-positive dendritic cells were found in columnar and cuboidal epithelium and the lamina propria of the nasal mucosa. These CD-1/HLA-DR-positive cells presumably correspond with LC in the skin. We also found significantly more CD-1-positive cells in nasal biopsy samples of allergic than in those of the non-allergic controls. In the allergic patients some of the CD-1-positive cells were found to be surface IgE-positive, possibly due to passive adherence of IgE to Fc receptors.

Immunoglobulin lambda-light-chain-derived amyloidosis (A lambda) in two horses.

Tumorous amyloid deposits in the nasal mucosa of two horses differed from generalized AA-amyloidosis with respect to clinical features, organ distribution, and resistance to KMnO4 treatment. Using a panel of antibodies directed against different human amyloid fibril proteins and employing the peroxidase-anti-peroxidase (PAP) technique, we showed the described equine amyloid to be A lambda-type, as demonstrated by immunohistochemical cross-reactivity. Consequently, we identified a second amyloid class in horses and showed that immunoglobulin light-chain-derived amyloid may also be present in animals.

[The relationship between the number of the autonomic nerve receptors and degree of hyperreactive nasal symptoms in patients with hyperesthetic rhinitis].

It is generally accepted that abnormal autonomic responsiveness may contribute to the pathogenesis of hyperesthetic rhinitis. Histologically, in the nasal mucosa, cholinergic fibers are found close to blood vessels, but are particularly numerous around the glands. Adrenergic fibers are found mainly around the vascular structures. Physiological and pharmacological studies demonstrate that parasympathetic hypersensitivity causes hypersecretion, and sympathetic hyposensitivity causes vasodilatation. alpha 1-adrenergic receptor function is dominant for this vasodilatation. Using radioligand binding techniques, it has been found that there is an increased number of muscarinic cholinergic receptors and a decreased number of alpha 1- and beta-adrenergic receptors in patients with nasal allergy, while the binding affinities do not change. In this report, using radioligand binding techniques, we investigated the relationships between the number of receptors and the degree of the hyperreactive nasal symptoms in patients with hyperesthetic rhinitis. The results are as follows. 1. The number of muscarinic cholinergic receptors of human nasal mucosa in patients with hyperesthetic rhinitis was related significantly (P less than 0.01) to the degree of hypersecretion induced by methacholine and frequency of blowing nose estimated from allergy diary. 2. There was no relationship between frequency of sneezing and the number of muscarinic cholinergic receptors. 3. The number of alpha 1-adrenergic receptors was related significantly (P less than 0.05) to the degree of swelling of nasal mucosa induced by methoxyamine. Judging from these results, it was assumed that pathogenesis of hyperreactive nasal symptoms may be associated at least partially to the changes of number of autonomic nerve receptors in the nasal mucosa.

Intra-epithelial lymphocytes and non-lymphoid cells in the human nasal mucosa.

Immunohistochemical staining of biopsy specimens was used to investigate the occurrence of lymphocyte subsets and non-lymphoid cells within the epithelial layer of the human nasal mucosa. The CD19 (B cell) marker was not expressed on the intra-epithelial lymphocytes, whereas the pan T cell marker CD2 was varyingly detected. The HLA-Dr antigen was abundantly present on epithelial cells, lymphocytes, and non-lymphoid cells. The latter are probably dendritic or Langerhans' cells. The findings stated above were the same in patient and control samples. In biopsy sections of 9 ear, nose, and throat patients, many CD8-positive (T suppressor/cytotoxic) cells and very few weakly stained CD4-expressing (T helper/inducer) cells were present. Quantification on single-cell preparations showed an average of 67% of the lymphocytes to be CD2 positive, 73% to be CD8 positive, while only 12% of the lymphocytes expressed the CD4 antigen. In control sections CD8 was similarly present as in patient sections, and, in addition, some clearly stained CD4-positive cells were seen.

Demonstration of alpha 1-adrenoceptors in rat nasal mucosa.

3H-Prazosin was used to demonstrate alpha 1-adrenoceptors in rat nasal mucosa. Specific binding is saturable and occurs to a homogeneous class of binding sites with high affinity (Kd = 0.07 +/- 0.01 nmol/l and with a receptor density of 0.36 +/- 0.02 pmol/g tissue or 14 +/- 1 fmol/mg protein. Kinetic experiments resulted in a Kd-value of 0.03 nmol/l. The binding is stereoselectively inhibited by epinephrine enantiomers. The antagonist prazosin inhibits 3H-Prazosin binding with high affinity, in contrast to yohimbine, classifying the binding sites as alpha 1-adrenoceptors. Inhibition experiments with WB4101 indicated the presence of alpha 1a- (31 +/- 9%) and alpha 1b-adrenoceptor subtypes in the rat nasal mucosa. The order of potencies of agonists determined in competition experiments was (-)epinephrine greater than (+)epinephrine greater than (-)phenylephrine.

[The diffuse lymphatic system of the nasal mucosa in allergic rhinitis]. / Le système lymphatique diffus de la muqueuse nasale dans la rhinopathie allergique.

Morphologic and immunologic study were performed on the mucosa associated lymphoid tissue (MALT) of patients with allergic rhinitis. Scraping from 14 healthy subjects and 36 allergic ones were used. Besides ordinary hystological methods immunohistochemical ones wilk polyclonal antibodies were employed to study IgG, IgA, IgM, IgE and monoclonal antibodies used for B and T lymphocyte and subset T-suppressor cell identification. In a comparison between normal and allergic mucosa, the morphopathology show an accentuated edema and a slight fibrosis. Among the IgG, IgA and IgM do not show any substantial differences in the samples in normal or in allergic subjects; white traces of IgE are found in allergic patients, but are totally absent in normal ones. In the lymphocyte populations does not show any substantial changes between the two groups. Was also analyzed the mechanism and the majority of the factors by which were obtained the results.

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors.

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.

Nasal secretory response to allergen provocation.

Nasal provocation with ragweed pollen extract was performed on ragweed-sensitive and non-atopic subjects. Nasal lavage fluids were collected 15 min after saline and allergen challenges, and assayed for total protein, albumin, potassium, lysozyme activity and peroxidase activity. There was no statistically significant increase in any of these lavage fluid constituents in non-atopic subjects after allergen provocation, compared with after saline provocation. The lavage fluids of ragweed-sensitive subjects had significant rises in each of the constituents following allergen provocation. This method provides a simple mechanism for quantitating the nasal secretory response to allergen provocation.

Phenotypic expression of proteoglycan in mast cells of the human nasal mucosa.

The phenotypic expression of the proteoglycan of human mast cells in the nasal mucosa and normal skin was analysed using histochemical techniques. Nasal mucosa was obtained from normal subjects, from patients with seasonal allergic rhinitis before and during the pollen season and from patients with nasal polyps. In the latter groups, specimens were taken from both polyp tissue and adjacent nasal mucosa. Formaldehyde treatment blocked the cationic dye binding in 75-84% of the mast cells located in the nasal mucosa, as compared to the optimum fixation with IFAA (iso-osmotic formaldehyde-acetic acid). A significantly lower degree of blocking of dye binding was obtained in the human skin where 45% of the mast cells were susceptible to formaldehyde treatment (P less than 0.01). The mast cells of the polyp tissue also showed a relatively low degree of blocking (54%), which was significantly lower than the blocking of mast cells of the nasal mucosa taken from the same individuals (P less than 0.05). Staining of serial tissue sections in Alcian Blue containing graded concentrations of MgCl2 was used to determine the critical electrolyte concentration (CEC) of the dye binding, defined as the salt concentration at which the staining of 50% of the mast cells is extinguished. The CEC of the skin mast cells was 0.64M MgCl2 which is significantly higher than that of the mast cells of the nasal mucosa of normal subjects [0.49M (P less than 0.05)], allergic subjects [0.52M (P less than 0.01)], patients with polyp disease [0.52M (P less than 0.01)] and the polyp tissue proper [0.57M (P less than 0.05)].(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular ketone reductase distribution and its role in the metabolism of ocularly applied levobunolol in the pigmented rabbit.

The distribution of ketone reductase activity in the anterior segment tissues of the pigmented rabbit eye and its influence on the ocular metabolism of topically applied levobunolol were studied. A reversed phase high-performance liquid chromatography procedure was used to assay for this drug and its metabolite, dihydrolevobunolol. Ocular ketone reductase activity was 3 to 4 times more dependent on NADPH than on NADH. The rank order of activity was corneal epithelium greater than iris-ciliary body greater than conjunctiva greater than lens. No activity was detected in the tears, corneal stroma, sclera or aqueous humor. Ketone reductase activity was entirely cytosolic. The pigmented rabbit was significantly less active than the albino rabbit in ketone reductase. Its activity in the corneal epithelium, iris-ciliary body and lens was most sensitive to inhibition by quercetin, whereas that in the conjunctiva was most sensitive to metyrapone. The ketone reductase in the corneal epithelium contributed more to the metabolism of topically applied levobunolol than its counterpart in the iris-ciliary body and lens. Moreover, the extent of levobunolol metabolism both during and after corneal penetration was dose-dependent. Overall, these findings indicate that ocularly applied drugs containing the ketone functional group are subject to varying degrees of metabolism by NADPH-dependent ketone reductases in the corneal epithelium, iris-ciliary body and lens.