Substance P represents a novel first-line defense mechanism in the nose.

BACKGROUND: Neuropeptides, such as substance P (SP), have long been seen as mediators of widespread continuous airway inflammation, a process known as neurogenic inflammation. However, this has been difficult to demonstrate clinically, suggesting an alternative role for these signaling molecules. OBJECTIVES: We sought to examine the role of SP in nasal infection by assessing the release of SP in response to viral stimulation and characterizing the effects of SP on innate immunity, with the latter reflected in changes in local Toll-like receptor (TLR) expression. METHODS: The distribution of SP and TLRs in the nasal mucosa and local airway neurons was assessed with immunohistochemistry. The TLR7 agonists R-837 and R-848 were used to mimic a viral insult in the upper airways represented by primary human nasal epithelial cells (HNECs) and murine nasal epithelial cells (MNECs) and isolated murine trigeminal ganglial neurons. SP release from HNECs, MNECs, and trigeminal ganglial neurons was quantified with EIA. The effects of SP on TLR expression on HNECs were determined by using flow cytometry and confocal microscopy. RESULTS: SP was released from the sensory neurons, MNECs, and HNECs within 15 minutes of local TLR7 stimulation. Subsequently, stimulation with SP induced upregulation of TLR expression in HNECs within 30 minutes through induction of TLR movement within HNECs. Upregulation of TLR expression was not evident when cells were treated with the neurokinin 1 receptor antagonist aprepitant before SP stimulation. CONCLUSIONS: This highlights a novel role for sensory neuropeptides as acute and local mediators of pathogen-driven inflammation, rapidly priming innate immune defenses in the airway.

NET-producing CD16high CD62Ldim neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC.

The concept of functional neutrophil subsets is new and their clinical significance in malignancies is unknown. Our study investigated the role of CD16dim CD62Lhigh , CD16high CD62Lhigh and CD16high CD62Ldim neutrophil subsets in head and neck squamous cell carcinoma (HNSCC) patients. These neutrophil subsets may play different roles in immune-related activity in cancer, based on their profile, activation state and migration ability within a tumor site, which may be important in predicting cancer prognoses. Tumor biopsies and blood were obtained from newly diagnosed untreated HNSCC patients and healthy controls. Neutrophil subsets and their phenotype were characterized using flow cytometry. Isolated granulocytes were assessed for anti-tumor immune functions. Compared to controls HNSCC patients exhibited increased CD16high CD62Ldim neutrophils in blood; this subset displayed a distinct phenotypes with high expression of CD11b and CD18. This subset was prone to migrate into the tumor facilitated by tumor-derived IL-8. Furthermore, IL-8 was also found to activate neutrophils and thereby promoting subset transition. Various assays demonstrated that activated CD16high CD62Ldim neutrophils inhibited migration, proliferation and induced apoptosis of FaDu cancer cells. Neutrophil elastase detected in activated CD16high CD62Ldim neutrophils and tumor biopsies suggested that CD16high CD62Ldim neutrophils impart anti-tumoral activity via neutrophil extracellular traps. Furthermore, increased fraction of CD16high CD62Ldim neutrophils was shown to correlate with an increased survival rate. Our study demonstrates the clinical relevance of the CD16high CD62Ldim neutrophil subset, providing evidence for its increased migration capacity, its anti-tumor activity including increased NET formation and finally its correlation with increased survival in HNSCC patients.

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines.

BACKGROUND: S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR). METHODS: Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC). RESULTS: Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC. CONCLUSIONS: The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.

TLR3 in human eosinophils: functional effects and decreased expression during allergic rhinitis.

BACKGROUND/AIM: Viral respiratory infections are increasingly implicated in allergic exacerbations. Virus-induced activation of eosinophils through Toll-like receptors (TLRs) could be involved. The present study was designed to examine TLR3 expression in eosinophils from bone marrow (BM) and peripheral blood (PB) during symptomatic allergic rhinitis, and to evaluate the functional responsiveness of TLR3 in purified eosinophils. METHODS: BM and PB samples were obtained from healthy volunteers and patients with seasonal allergic rhinitis outside and during the pollen season. Eosinophils were analyzed for TLR3 expression by flow cytometry. Polyinosinic:polycytidylic acid [poly(I:C)], an agonist for TLR3, was used to assess its functional role in purified eosinophils and the intracellular signaling pathways involved. RESULTS: TLR3 expression was demonstrated in BM and PB eosinophils. It was higher in BM-derived than in circulating cells and it was downregulated in both compartments during symptomatic allergic rhinitis. TLR3 expression was also downregulated in the presence of interleukin (IL)-4 and IL- 5. Stimulation with poly(I:C) increased the percentage of CD11b+ cells and enhanced the secretion of IL-8, effects mediated via the p38 mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways. Moreover, pretreatment with IL-5 augmented the poly(I:C)-induced IL-8 release. CONCLUSIONS: Eosinophils activated via TLR3 might be more able to home and recruit leukocytes to sites of inflammation. The decreased TLR3 expression during symptomatic allergic rhinitis and in the presence of Th2 cytokines indicates a role in allergic airway inflammation. Thus, eosinophils might function as a link between viral infections and exacerbations of allergic disease.

Reduced iNOS expression in adenoids from children with otitis media with effusion.

Nitric oxide (NO) is a key mediator in the local immune response of human airways. Inducible NO-synthases (iNOS), and endothelial NO-synthases (eNOS) are two enzymes known to regulate its production. The role of NO in middle ear disease is not fully known. Previous studies suggest that NO might have a dual role, both promoting and suppressing middle ear inflammation. The aim of the present study was to compare the eNOS and iNOS expression in adenoids obtained from children with otitis media with effusion (OME) with the expression seen in adenoids derived from children without middle ear disease. In addition, the expression of IL-1ß and TNF-α were analyzed, because of their role in the iNOS-induction pathway. The iNOS and eNOS expression were analyzed with real-time PCR in 8 OME and 11 control adenoids. The corresponding proteins were demonstrated by immunohistochemical staining of adenoid tissue. A Luminex(®) assay was performed to analyze IL-1ß and TNF-α in nasopharyngeal secretion in 10 OME and 8 controls, and immunohistochemistry was performed on adenoid tissue and imprints from the adenoid surface. Children with OME exhibited lower levels of iNOS than controls without middle ear disease. No such difference was seen for eNOS. The corresponding proteins were found mainly in conjunction with surface epithelium. No significant changes were seen among the cytokines tested. The present results indicate that local induction of iNOS in adenoids might be of importance for preventing development of OME.

Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas.

Toll-like receptors (TLRs) are increasingly implicated in the pathogenesis of cancer. The present study describes TLR expression and function in healthy and malignant airway epithelial cells. The squamous cell carcinoma cell line Detroit-562 was compared with the healthy bronchial epithelial cell line NL-20 and primary human nasal epithelial cells (HNECs). TLR2, TLR3 and TLR5 were present in primary head and neck squamous cell carcinomas (HNSCCs). Consistent with this, Detroit-562 expressed TLR2, TLR3 and TLR5, whereas NL-20 expressed mainly TLR3 and HNECs expressed TLR2-5. In Detroit-562, Pam(3)CSK(4), poly(I:C) and flagellin, ligands for TLR2, TLR3 and TLR5, respectively, induced an up-regulation of intercellular adhesion molecule 1 (ICAM-1), an increase in interleukin (IL)-6 and IL-8 secretion and a decrease in cell viability. Additionally, poly(I:C) affected IL-1beta production and the migratory behaviour of Detroit-562. NL-20 responded with a slight increase in IL-8 secretion upon poly(I:C) stimulation. Poly(I:C) induced a small increase in IL-1beta, IL-6 and IL-8 production in HNECs, while Pam(3)CSK(4) increased viability. The TLR signalling was transcription-dependent, but the pathways involved differed among TLRs as well as cells. In Detroit-562, TLR2 and TLR5 activation was mediated via c-jun N-terminal kinase (JNK)-, p38-, phosphatidylinositol 3-kinase (PI3K)- and nuclear factor (NF)-kappaB-related pathways, while TLR3 was dependent on NF-kappaB. In NL-20, TLR3 signalled via p38, and in HNECs, NF-kappaB, JNK and extracellular signal-regulated kinase (ERK) appeared to be involved. We found that TLR agonists induced a robust response in HNSCCs, characterized by generation of inflammation and cell death. A similar response was not seen in normal epithelial cells. Thus, the TLR system should be considered an important target in future antitumour immunotherapy.

Expression of Toll-like receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis.

BACKGROUND: Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis. METHODS: The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively. RESULTS: TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis. CONCLUSION: The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.

Enhanced expression of CGRP in rat trigeminal ganglion neurons during cell and organ culture.

The sensory innervation of intracranial vessels originates in the trigeminal ganglion with calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase activating peptide (PACAP) as frequent neuronal messengers. The present study was designed to study the expression of these neuropeptides (a) in primary culture of adult rat trigeminal ganglion neuronal cells and (b) in organ culture of sections of the trigeminal ganglion. In cell culture, axons grow in the peripheral direction for up to 48 h. Immunocytochemistry revealed that the cell bodies showed increased expression of CGRP at 24 h and SP at 24-48 h (p<0.05), whereas cell culture did not increase the expression of PACAP at 24 h (p>0.05), but at 48 h (p<0.05). A significant elevation of CGRP mRNA was seen at 12 h, i.e. before the increased CGRP immunoreaction was observed. In organ culture of sections of trigeminal ganglia, the number of CGRP immunoreactive (-ir) cells and the mRNA expression were significantly increased at 24 and 48 h of incubation as compared to control (p<0.05), while the number of SP-ir cells was not altered (p>0.05). In conclusion, neurons of rat trigeminal ganglia alter their expression of neuropeptides during cell and organ culture differently, but it is mainly the CGRP system that is up-regulated. We have compared two methods for future studies of underlying molecular mechanisms responsible for regulation of neuropeptide expression in the trigeminal system.

Neurotransmitter candidates in the vomeronasal organ of the rat.

CONCLUSION: The rich supply of nerve fibres containing neurotransmitters, particularly those containing SP and CGRP, is suggested to be a prerequisite for the recognition of chemical irritants as part of a chemical sense. OBJECTIVE: The present study was designed to examine the distribution of different neurotransmitter candidates in the vomeronasal organ (VNO) of rats. MATERIALS AND METHODS: The distribution of neurotransmitter candidates was studied in the vomeronasal organ of the rat using immunocytochemistry. RESULTS: The neuronal marker protein gene product 9.5 revealed a very rich supply of nerve fibres within and beneath the sensory epithelium, around blood vessels and glands. A moderate supply of nerve fibres containing tyrosine hydroxylase and neuropeptide Y was mostly seen close to blood vessels. Numerous nerve fibres containing nitric oxide synthase and vasoactive intestinal peptide were seen around blood vessels and in the subepithelial layer, with occasional fibres within the epithelium. Only few fibres located in the subepithelial layer contained pituitary adenylate cyclase activating peptide. Nerve fibres containing substance P and in particular calcitonin gene-related peptide were abundant in and beneath the epithelium and scattered in the submucosal layers around blood vessels.

Lipopolysaccharide-induced down-regulation of uteroglobin in the human nose.

CONCLUSION: Lipopolysaccharide (LPS) challenge of the human nose has the capacity to reduce the amount of natural anti-inflammatory proteins, such as uteroglobin. OBJECTIVES: Nasal challenge with LPS, an activator of innate immunity, has been shown to increase the amount of pro-inflammatory mediators in nasal lavage fluid. Uteroglobin is a newly described anti-inflammatory mediator that is secreted in the nose. This study examined the effect of nasal LPS application on the level of uteroglobin in nasal lavage fluid as well as on the expression of uteroglobin in nasal mucosa. MATERIALS AND METHODS: Thirty-eight volunteers were challenged nasally with either 50 microg LPS or vehicle; 6 h later, nasal lavage fluid was collected and a nasal biopsy was obtained. Levels of uteroglobin, albumin and the pro-inflammatory mediators interleukin (IL)-6 and IL-8 were analysed in the lavage fluids using enzyme-linked immunosorbent assays (ELISAs). Biopsies were used for either quantification of uteroglobin mRNA by real-time PCR or for localization of the corresponding protein with immunohistochemistry. RESULTS: The uteroglobin level decreased in nasal lavage fluid following LPS challenge, whereas the levels of IL-6 and albumin increased. Uteroglobin was mainly seen in the respiratory epithelium and its mRNA expression decreased as a consequence of the LPS challenge.

Endothelin ETA and ETB receptor expression in the human trigeminal ganglion.

OBJECTIVES: Endothelin is a potent peptide mediator that is synthesized by a number of cells. Previous studies have revealed the occurrence of endothelin in nerve cell bodies of some peripheral ganglia. Endothelin mediates its effects via two distinct receptor subtypes ETA and ETB. The present study was designed to investigate the presence of these two receptors in the human trigeminal ganglion. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show the presence of mRNA encoding ETA and ETB receptors in the human trigeminal ganglion. To localize the protein immunocytochemistry with antibodies against the endothelin receptors was used. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed mRNA for both receptor subtypes in the human trigeminal ganglion. Immunocytochemistry revealed numerous cell bodies containing the ETA and the ETB receptor proteins. CONCLUSIONS: The expression of ETA and ETB receptors in the human trigeminal ganglion suggests a role for endothelin in autonomic and sensory neural transmission.

Topical steroids do not downregulate expression of growth-related oncogene-alpha in nasal polyps.

CONCLUSIONS: Topical steroids did not affect expression of growth-related oncogene-alpha (GRO-alpha) in nasal polyps. The results of this study suggest roles for steroid-resistant gene expression in the pathogenesis of nasal polyps and point to the need for additional pharmacological strategies. OBJECTIVE: Infiltration of inflammatory cells is believed to play a role in the development of nasal polyps. GRO-alpha is a chemokine that recruits and activates neutrophils and also possesses growth stimulatory and angiogenetic properties. An increased presence of GRO-alpha has been demonstrated in nasal polyps compared with normal nasal tissue. In this study we evaluate the presence and expression levels of GRO-alpha in nasal polyps before and after glucocorticoid treatment. MATERIAL AND METHODS: Nasal polyps were surgically removed in patients before and 6 weeks after treatment with topically applied fluticasone. GRO-alpha gene expression and the presence of GRO-alpha peptide were detected in polyp tissue by means of in situ hybridization, quantitative real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: Strong GRO-alpha gene expression and the presence of GRO-alpha peptide were seen in both the epithelium and stromal inflammatory cells of nasal polyps. No differences in gene expression levels in tissue homogenates were found when untreated polyp tissue was compared with polyps treated for 6 weeks with topically applied steroids.

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) alpha, betadelta and gamma are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. METHODS: Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. RESULTS: mRNA expression of PPARalpha, PPARbetadelta, PPARgamma was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARalpha and PPARgamma than normal nasal mucosa and these levels were, for PPARgamma, further reduced following steroid treatment. PPARgamma immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARgamma immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. CONCLUSION: The down-regulation of PPARgamma, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARgamma might be of importance in long standing inflammations.

Up-regulation of Toll-like receptors 2, 3 and 4 in allergic rhinitis.

BACKGROUND: Toll-like receptors enable the host to recognize a large number of pathogen-associated molecular patterns such as bacterial lipopolysaccharide, viral RNA, CpG-containing DNA and flagellin. Toll-like receptors have also been shown to play a pivotal role in both innate and adaptive immune responses. The role of Toll-like receptors as a primary part of our microbe defense system has been shown in several studies, but their possible function as mediators in allergy and asthma remains to be established. The present study was designed to examine the expression of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with intermittent allergic rhinitis, focusing on changes induced by exposure to pollen. METHODS: 27 healthy controls and 42 patients with seasonal allergic rhinitis volunteered for the study. Nasal biopsies were obtained before and during pollen season as well as before and after allergen challenge. The seasonal material was used for mRNA quantification of Toll-like receptors 2, 3 and 4 with real-time polymerase chain reaction, whereas specimens achieved in conjunction with allergen challenge were used for immunohistochemical localization and quantification of corresponding proteins. RESULTS: mRNA and protein representing Toll-like receptors 2, 3 and 4 could be demonstrated in all specimens. An increase in protein expression for all three receptors could be seen following allergen challenge, whereas a significant increase of mRNA only could be obtained for Toll-like receptor 3 during pollen season. CONCLUSION: The up-regulation of Toll-like receptors 2, 3 and 4 in the nasal mucosa of patients with symptomatic allergic rhinitis supports the idea of a role for Toll-like receptors in allergic airway inflammation.

Prolonged exposure to NT-3 attenuates cholinergic nerve-mediated contractions in cultured murine airways.

Chronic airway inflammation may induce subsequent airway hyper-responsiveness (AHR) including pathological alteration of neural activity. Asthmatic airways contain elevated levels of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) albeit, their effect on neural activity is unclear. This study evaluates the effects of NT-3 and BDNF on nerve mediated airway contractions in vitro. Tracheal segments from BALB/c J mice were cultured for 4 days with NT-3 or BDNF. Responsiveness to electric field stimulation (EFS) was evaluated in organ-bath and innervation patterns were examined by quantitative immunohistochemistry. In cultured segments the EFS-induced contractions were inhibited by tetrodotoxin or atropine. NT-3 reduced the EFS contractions in a concentration-dependent manner whereas BDNF had no effect. The amount of nerve fibers, found in conjunction with the tracheal smooth muscle, was similar in NT-3 treated and control segments. In conclusion, NT-3 attenuates cholinergic nerve-mediated contractions of airway in vitro. Considering the elevated levels of NT-3 found in asthmatic airways, the findings imply a protective role of NT-3 in AHR.

Peptide-containing Neurons Projecting to the Tongue of the Rat: Retrograde Tracing and Immunocytochemistry.

The origin and neuropeptide content of nerve fibres in the rat circumvallate papilla was studied by retrograde tracing in combination with immunocytochemistry. An injection of the retrograde tracer True Blue into the circumvallate papilla resulted in the appearance of labelled nerve cell bodies in the superior cervical, the stellate, the thyroid, the nodose, the jugular, the petrosal, the otic, the trigeminal and the dorsal root ganglia at level C2. Most of the True Blue-labelled nerve cells in the superior cervical ganglia contained neuropeptide Y. The majority of labelled cell bodies in the thyroid ganglia contained vasoactive intestinal peptide. In the jugular and trigeminal ganglia, the majority of the labelled nerve cell bodies stored calcitonin gene-related peptide. A small number of neurons in the medial reticular formation of the central nervous system was labelled. Tracer injections deep into the tongue tissue beneath the circumvallate papilla gave rise to True Blue-labelled neurons in the hypoglossal nucleus.

Peptide-containing nerve fibres in human extracranial tissue: a morphological basis for neuropeptide involvement in extracranial pain?

It has been suggested that a number of peptides may be involved in the transmission of pain. In order to evaluate the possible role of peptides in the development of headache, we have, in the present study, examined the presence of nerve fibres containing neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) in human temporal and occipital tissues. In the skin, delicate VIP, SP and CGRP fibres occur beneath the epidermis, sometimes running into the folds of the dermal ridges. In deeper layers of the dermis, small blood vessels are occasionally surrounded by single nerve fibres containing NPY, VIP, SP and CGRP. Large temporal and occipital arteries are surrounded by a meshwork of such fibres. In addition, NPY and VIP fibres are seen around sweat glands and hair follicles. Smooth muscle bundles in the dermis are surrounded by VIP fibres, whereas the temporal muscle per se is devoid of such fibres.

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFalpha up-regulates 5-HT(2A)-mediated contraction.

1. Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of inflammatory mediators that affect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1-32 days, and investigated contractile responses mediated by muscarinic and 5-hydroxytryptamine (5-HT) receptors following long-term exposure to tumour necrosis factor-alpha (TNFalpha). 2. Following culture, in the absence of TNFalpha, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not affected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 3. 5-HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFalpha produced a time- and concentration-dependent increase in the maximal contraction to 5-HT, evidently mediated by 5-HT(2A) receptors, whereas, the potency for carbachol was reduced. 4. In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the first days of culture. The time-dependent effect of TNFalpha indicates the importance of studying the long-term effect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

PACAP enhances the expression of CD11b, CD66b and CD63 in human neutrophils.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.