Role of acidic mammalian chitinase and chitotriosidase in nasal polyps.

OBJECTIVE: Chitin is a recognition element for tissue infiltration by innate cells implicated in allergy and helminth immunity, and this process can be negatively regulated by vertebrate chitinases. Acidic mammalian chitinase (AMCase) and chitotriosidase (ChT) have chitinolytic activity, but little is known about their roles in nasal polyps. STUDY DESIGN: A prospective controlled study. SETTING: A tertiary referral center. SUBJECTS AND METHODS: Nineteen subjects with nasal polyps and 12 subjects with deviated nasal septums were recruited to obtain inferior turbinate mucosa samples. The expression levels of AMCase and ChT were compared in nasal polyp and inferior turbinate tissue samples. The tissue samples were analyzed by reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemical staining. RESULTS: AMCase and ChT were detected in all nasal polyps and inferior turbinate tissues. AMCase and ChT messenger RNA and protein expression were significantly higher in nasal polyps than in inferior turbinate tissues. In nasal polyps, AMCase-positive and ChT-positive cells were detected in the epithelium, inflammatory cells, and submucosal gland. CONCLUSIONS: AMCase and ChT may be important mediators in the pathogenesis of nasal polyps. Nasal polyps appear to have elevated levels of chitinases, and the presence or growth of chitin-containing pathogens might enhance chitinase expression, resulting in nasal polyp formation and growth in susceptible individuals.

Nasal mucosal expression of nitric oxide synthases in patients with allergic rhinitis and its relation to asthma.

BACKGROUND: Nitric oxide (NO) has contradictory roles in the pathophysiology of allergic inflammation in both allergic rhinitis (AR) and asthma. Small amounts of NO produced by constitutive NO synthase (NOS) is anti-inflammatory, whereas large amounts produced by inducible NOS (iNOS) are proinflammatory. OBJECTIVE: To investigate the difference in constitutive endothelial NOS (eNOS) and iNOS expression in nonallergic and allergic mucosa and the possible relation of this to the coexistence of asthma in seasonal AR. METHODS: Seventeen patients (10 women and 7 men) with seasonal AR and 9 nonallergic patients (5 women and 4 men) with nasal septum deviation were enrolled. Inferior turbinate nasal biopsy specimens were obtained in all. Levels of eNOS and iNOS expressed as immunohistochemical scores (HSCOREs) were determined immunohistochemically from the specimens. RESULTS: The mean +/- SD HSCOREs for eNOS in patients with seasonal AR were not significantly different from those of the nonallergic controls (1.85 +/- 0.78 vs 1.63 +/- 0.54; P = .12). On the other hand, the mean +/- SD HSCOREs for iNOS were significantly higher in patients with seasonal AR (1.75 +/- 0.75 vs 0.71 +/- 0.6; P = .004). Furthermore, although eNOS expression was not different between seasonal AR patients with and without asthma, the mean +/- SD HSCOREs for iNOS were significantly higher in the patients with asthma (1.93 +/- 0.78 vs 1.65 +/- 0.55; P = .01). CONCLUSION: Increased expression of iNOS might have a role in the development of allergic inflammation in upper and lower airways and in comorbidity of AR and asthma.

Inhibition of furin-like enzymes blocks interleukin-1alpha/oncostatin M-stimulated cartilage degradation.

OBJECTIVE: To investigate the role of furin-like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation. METHODS: Bovine nasal cartilage was stimulated to resorb with the addition of interleukin-1alpha (IL-1alpha)/oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec-RVKR-CH(2)Cl, or selective matrix metalloproteinase 1 (MMP-1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using (3)H-acetylated collagen and gelatin zymography, respectively. RESULTS: The addition of Dec-RVKR-CH(2)Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP-2, suggesting that furin-like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP-1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP-13 resulted in a 50% blockage of collagen release. The addition of Dec-RVKR-CH(2)Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin-activated enzymes in the pathways leading to proteoglycan degradation. CONCLUSION: Furin-like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP-13, which can be activated by furin-processed membrane-type 1 MMP-1, appears to be a major collagenase involved in collagen degradation induced by IL-1alpha/OSM. Furin-like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.

Heme oxygenase-2 and nitric oxide synthase immunoreactivity of bovine olfactory receptor neurons and a comparison with the distribution of NADPH-diaphorase staining.

It has recently been suggested that, in addition to nitric oxide (NO), carbon monoxide (CO) is an important gaseous messenger which might be involved in vertebrate olfactory transduction because its effects include activation of guanylyl cyclase and the formation of cGMP. As there is no information regarding the presence of heme oxygenase-2 -- the constitutive isoform of the heme oxygenase system -- in olfactory neurons of non-rodent species, we have investigated the distribution pattern of heme oxygenase-2 in the olfactory epithelium of the bovine, a representative of macrosmatics. Localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity of the olfactory epithelium was compared with heme oxygenase-2 and NO synthase (NOS) immunoreactivities in order to obtain possible hints at functional significance. NADPH-d activity was particularly intense in apical dendrites of receptor neurons. It was also found in Bowman glands and intraepithelial duct cells. Less intense, discrete NADPH-d activity was present also at intermediate and basal levels of the olfactory epithelium, corresponding to the layer of receptor neuron somata and basal cells. While heme oxygenase-2 activity mainly occurred in neuronal perikarya, a very intense NOS immunoreactivity, exclusively for the inducible isoform, was detected in the apical dendrites. Ultrastructurally, NADPH-d histochemistry showed distinct labelling of membranes, in particular of endoplasmic reticulum, mitochondria and nucleus. The coincident localization of the moderate NADPH-d activity and heme oxygenase-2 immunoreactivity in receptor cell perikarya suggest a functional association between NADPH-cytochrome P450 reductase and heme oxygenase-2. In contrast, dendritic localization of NADPH-d activity is topically and possibly functionally related to the presence of the inducible isoform of NOS. The results suggest that both CO and NO may be generated in bovine receptor neurons and thus involved in odorant stimulation. Based on immunocytochemical localization of synthesizing enzymes, NO might be regarded as a direct regulator of transduction related processes while CO might act as a modulator of the initial signal.

Cellular distribution of isozymes of protein kinase C in septal olfactory epithelium of mice.

To determine the presence of protein kinase C (PKC) isozymes in the septal olfactory epithelium of mice (mSOE), western blotting and immunohistochemistry were performed using antibodies against PKC isozymes. With the exception of PKC-betaI, all of the PKC isozymes were detected in the whole lysate of septal tissue layer and apparent molecular weights for each isoform were found. PKC-alpha, PKC-gamma and PKC-epsilon were detected in the olfactory glandular cells of the lamina propria, and PKC-betaI and PKC-betaII were located in the microvillar cells. Neither novel PKC nor atypical PKC was detected in olfactory glandular cells or microvillar cells, except for PKC-epsilon. PKC-lambda was localized in the mucous layer of the mSOE. Meanwhile, PKC-delta and PKC-xi were distributed in the receptor cells in the mSOE. These data demonstrate the isoform-specific expression of PKC in mSOE and suggest a role for the novel and atypical types of PKC in olfactory transduction.

Dependence of salt concentration on glycosaminoglycan-lysozyme interactions in cartilage.

The cationic protein, lysozyme, has an extracellular distribution in cartilage but its precise role in this tissue has not yet been established. This study describes the dependence of salt concentration on the binding properties of lysozyme isoforms of different cationic charges, isolated from bovine cartilage, to the two major and structurally similar glycosaminoglycans of cartilage, i.e., chondroitin sulfate and hyaluronan. The binding of most cartilage lysozyme isoforms and hen egg-white lysozyme (control) to chondroitin sulfate and hyaluronan linked to agarose supports displayed optimal levels at approximately 20 and 5-10 mM salt, respectively, but decreased at both lower and higher salt concentrations indicating the electrostatic nature of the interactions. However, optimal binding of the most cationic lysozyme isoform to chondroitin sulfate occurred at 60 mM salt, with significant binding remaining at 150 mM. This isoform also showed binding to hyaluronan up to 60 mM salt, while for the other isoforms binding was observed only up to 150 and 40 mM salt for chondroitin sulfate and hyaluronan, respectively. The low salt concentrations at which these interactions occur are likely to exist in cartilage as shown from equilibrium dialysis studies performed using solutions of chondroitin sulfate (up to 10%, a concentration likely to occur in cartilage). From Scatchard analysis, the affinity of binding of all lysozymes to chondroitin sulfate was similar (Kd = 10(-6) M) and slightly lower than their binding to hyaluronan (Kd = 10(-7) M) of similar molecular mass.

Coexistence of nitric oxide synthase and neuropeptides in the mouse vomeronasal organ demonstrated by a combination of double immunofluorescence labeling and a multiple dye filter.

Nitric oxide synthase (NOS)-immunofluorescence techniques were applied to the mouse vomeronasal organ. Immunoreactivity for NOS was found in the nerve fibers distributed in the receptor-free epithelium, and around the blood vessels and glands in the cavernous tissue. No NOS fibers were seen in the receptor area. A combination of double immunofluorescence labeling and multiple dye filter revealed that a part of the substance P (SP)-immunoreactive nerve fibers in the cavernous tissue contained NOS and that all the vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers around the blood vessels and glands in the cavernous tissue contained NOS. A few SP-immunoreactive cell bodies in the trigeminal ganglion showed coexistence with NOS, and almost all VIP-immunoreactive cell bodies in the sphenopalatine ganglion showed coexistence with NOS. Immunoreactivity for NOS without VIP in the cell bodies in the sphenopalatine ganglion was also found. These results suggest that NOS-immunoreactive nerve fibers in the mouse vomeronasal organ originate from the trigeminal and the sphenopalatine ganglia, and may modulate the vascular tone and the glandular secretion. In addition, these functions may be controlled in part by the interaction of nitric oxide and neuropeptides.

Localization of nitric oxide synthase in the mouse olfactory and vomeronasal system: a histochemical, immunological and in situ hybridization study.

The distribution of nitric oxide synthase (NOS) in the mouse olfactory bulb and olfactory epithelium, including the vomeronasal organ, was studied using an anti-NOS antibody, NADPH diaphorase histochemistry and in situ hybridization with NOS specific antisense oligonucleotide probes. Interneurons containing NOS protein and mRNA, and exhibiting NADPH diaphorase activity were detected in the plexiform layer of the main olfactory bulb and the granule cell layer of main and accessory olfactory bulbs. Periglomerular cells and granule cells in the main olfactory bulb were also NOS positive with diaphorase and immunostaining for NOS. In contrast, no evidence for NOS expression was found either in the main olfactory epithelium or in the vomeronasal organ, in spite of the strong diaphorase staining of the surface of the main olfactory epithelium. Polymerase chain reaction amplification experiments for detection of NOS gene expression further indicated that NOS is expressed in the olfactory bulb but not in either the main olfactory epithelium or vomeronasal organ. Use of an antibody raised against another enzyme, NADPH-P450 oxidoreductase, showed that this protein was strongly expressed in the olfactory epithelium. Activity of this enzyme may account for the diaphorase histochemical staining of the epithelia. An involvement of neuronal nitric oxide synthase in signalling in olfactory receptor neurons is therefore doubtful, although NOS is clearly expressed in neurons in both main and accessory olfactory bulbs.

Sensory receptor-like cells in the human foetal vomeronasal organ.

The vomeronasal organ (VNO) was studied in ten human foetuses, 12 to 36 weeks old. At 12 to 23 weeks, it was lined by a smooth pseudostratified epithelium, with neurone-specific enolase (NSE) positive cells looking like olfactory receptors. Clusters of NSE-positive cells were seen in relation with the posterosuperior end of the organ and along nerve fascicles in the nasal septum. At 36 weeks, the organ was lined by a respiratory epithelium and did not show any receptor-like cells; some pear-shaped NSE-positive cells of unknown significance were seen at the upper part of the respiratory epithelium. Our results suggest that during the early foetal period, the VNO could have some, as yet unknown, sensory function.

Stimulation of alkaline phosphatase activity by ascorbic acid and suppression by 1,25-dihydroxycholecalciferol in rabbit craniofacial chondrocytes in culture.

To clarify the role of vitamins D and C in chondrocyte hypertrophy of craniofacial cartilage, we have studied cultured chondrocytes from rabbit mandibular condylar cartilage (MCC), sphenooccipital synchondrosis (SOS) and nasal septal cartilage (NSC) under conditions in which these cells mature into hypertrophic chondrocytes. In cultures of MCC- and SOS-chondrocytes, alkaline phosphatase (ALPase) activity started to increase on day 9 at confluence and the cessation of cell division, and reached a maximum on day 18. The degree of the increase of ALPase activity on day 18 was higher in MCC-chondrocytes than in SOS-chondrocytes. ALPase activity was very low level in NSC- and CGC-chondrocytes. Ascorbic acid induced a marked increase in ALPase activity in MCC-, SOS-, NSC- and CGC-chondrocytes. The ALPase activities in MCC- and SOS-chondrocytes with 50 micrograms/ml ascorbic acid were 2.5-times those in its absence. Those in NSC- and CGC-chondrocytes were 10 times and 20 times, respectively. When chondrocytes were cultured with 10% charcoal-treated serum, ALPase activity decreased less than that in cultures with 10% normal serum in MCC-, SOS-, NSC- and CGC-chondrocytes. Treatment of 1,25-(OH)2D3 for 9 days from days 4 to 13 in MCC-chondrocytes and for 14 days from days 4 to 18 in SOS-, NSC- and CGC-chondrocytes inhibited ALPase activity dose-dependently at the concentrations of 10(-12) M to 10(-8) M in MCC- chondrocytes, 10(-10) M to 10(-8) M in SOS- and CGC-chondrocytes, 10(-11) M to 10(-9) M in NSC-chondrocytes. These findings suggest that 1,25-(OH)2D3 and ascorbic acid may be involved in the control of cartilage growth and terminal differentiation.

Immunolocalization of two cytochrome P450 isozymes in rat nasal chemosensory tissue.

The NMa and NMb isoforms of cytochrome P450 enzymes are expressed in three nasal chemosensory organs: the olfactory, septal and vomeronasal mucosae. The NMa isoform is widely distributed throughout the nasal mucosa whereas the NMb isoform is present primarily in the chemosensory mucosae. The localization of cytochromes P450 demonstrates that sustentacular cells in the olfactory and septal epithelia, the mucus of the vomeronasal organ and the acinar cells of glands in the lamina propria of all three chemosensory systems engage in xenobiotic metabolism and participate in odorant/pheromone clearance, a perireceptor process associated with chemosensory transduction.

Histochemical analysis of enzymes involved in the formation and metabolism of the nasal septal cartilage.

Formation of the nasal septal cartilage in prenatal and neonatal rats was studied histologically and by histochemistry to determine the manner, degree and participation of the nasal septal cartilage in midface growth and in bone formation of the face. Chondrogenesis of the nasal septal cartilage started at the 13th embryonic day, premaxillary and vomerin bone formation at the 14th embryonic day and endochondral bone formation of the septo-presphenoid area at the 17th embryonic day. After differentiation of the nasal septal cartilage, this cartilage supported ethmoid bone formation by endochondal ossification in the septo-presphenoid area. Nasal septal cartilage showed intense activity of lactate dehydrogenase, NADH2-diaphorase and a moderate activity of acid phosphatase, whereas premaxillary and vomerin bone showed intense activity of alkaline phosphatase. Osteoblasts showed intense activity of alkaline phosphatase, lactate dehydrogenase and NADH2-diaphorase and osteoclasts showed intense activity of acid phosphatase. During the embryonal period growth of the nasal septal cartilage could occur in an ethmoido-rostral direction supported by endochondral ossification and growth in length and height supported by apposition and interstitial growth.

Enzymatic activity and distribution of phospholipase A2 in human cartilage.

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.

Human nasal septal cartilage: analysis of intracellular enzyme activities, glycogen content, cell density and clonal proliferation of septal chondrocytes of healthy adults and acromegalic patients.

The human septal cartilage is of ectodermal origin and contributes to midfacial growth and development. Acromegaly is an endocrine disease due to growth hormone (Gh) excess originating from a somatotrophic adenoma of the pituitary gland. Excessive Gh levels lead to high insulin-like growth factor I (IGF I) concentrations, which are known to stimulate cartilage growth in vivo and in vitro. One of the salient clinical pictures is coarsening of the midface and enlargement of the septal cartilage. Septal cartilage was obtained from 8 acromegalic patients during transnasal hypophysectomy and from 10 healthy adults during septoplasty to analyse the following aspects of cartilage biochemistry, metabolism and growth. 1. Intracellular glycogen, the major source of energy of chondrocytes, was determined enzymatically and found to be drastically reduced in acromegaly. 2. Several intracellular enzymes, related to biomatrix degradation, showed a strict local pattern of distribution. Cathepsin B activity, a neutral proteinase degrading both the helical and nonhelical region of the collagen molecule was significantly increased in acromegaly, whereas alkaline phosphatase activity, an enzyme related to mineralization of the cartilage at the chondroosseous junction was depressed in acromegaly. 3. The cell density in some areas of the septal cartilage was increased in acromegaly, whereas the clonal proliferation rate of its chondrocytes in response to serum and growth factors was decreased. Chondrocytes both of healthy adults and acromegalic patients could be effectively stimulated by insulin-like growth factor I and II and to a lesser extent by epidermal growth factor.

Thymidylate synthetase and thymidine kinase activities in rat growing cartilage.

The purpose of the present study is to determine whether or not the differences in cellular proliferation in rat nasal septal cartilage and tibial cartilage are reflected by differences in the pattern of age-related changes in thymidylate synthetase (TS) and thymidine kinase (TK) activities. TS and TK are responsible for thymidine monophosphate formation via, respectively, the de novo and the salvage pathways. The present study also examines the effects of glucocorticoids, which are known to affect the proliferation of chondrocytes, on TS and TK activities in these cartilaginous tissues. Both enzyme activities declined with age, and the decline in TK activity was more rapid than that of TS. The TK/TS ratio decreased with age more rapidly in nasal septal cartilage. These findings suggest that nasal septal cartilage might mature earlier than tibial cartilage and, furthermore, that in rapidly growing tissues the salvage pathway functions predominantly. Prednisone injection (2.5 mg/100 g, at 4 days after birth) clearly disturbed the gain of body weight. TK activity significantly decreased concomitantly with this arrest in weight gain, while in contrast TS activity was not depressed. This marked difference between TS and TK activities induced by prednisone suggests that these two enzymes may be regulated independently.

Human nasal septal cartilage: local distribution of different enzyme activities in healthy adults and acromegalic patients.

Septal cartilage was obtained during septoplasty from healthy adults or during transnasal hypophysectomy from acromegalic patients. Small strips of cartilage were excised from five different areas of the septum: the anterior free end, the suprapremaxillary area, the central area, the posterior area, and the caudal prolongation of the septum. Five different enzymatic pathways were analyzed in these areas. Cathepsin D, an acid proteinase, did not show a specific local activity pattern and was not influenced by the augmented growth hormone level in acromegaly, whereas cathepsin B, a neutral proteinase, showed its highest activity in the caudal prolongation and the posterior area and was significantly increased in all areas in acromegaly. Beta-hexosaminidase activity was highest in the central and posterior area and caudal prolongation of the septum. In acromegaly, a significant increase of its activity was found in the suprapremaxillary and posterior area. Acid phosphatase activity was highest in the caudal prolongation of the septum, but its activity was significantly increased in all tested areas in acromegaly. Alkaline phosphatase activity could only be found in the posterior area and the caudal prolongation in healthy adults. However, in acromegaly this enzyme could be detected in the central area and the posterior end of the suprapremaxillary area suggesting an altered process of mineralization. A distinct local pattern of enzymes related to intercellular substance metabolism and mineralization can be demonstrated in the septal cartilage of healthy adults and acromegalic patients.