Vasoactive intestinal peptide (VIP) receptor expression in ...

Abstract

Vasoactive intestinal peptide (VIP) is one of the most abundant molecules found in the respiratory tract. Due to its anti-inflammatory and bronchodilatatory properties, it has been proposed as a novel treatment for chronic obstructive pulmonary disease (COPD). The actions of VIP are mediated via three different G-protein-coupled receptors (VPAC1, VPAC2 and PAC1) which are expressed in the respiratory tract and on immunocompetent cells including macrophages. Alveolar macrophages (AM) are key players in the pathogenesis of COPD and contribute to the severity and progression of the disease. While VPAC1 has been reported to be elevated in subepithelial cells in smokers with chronic bronchitis, little is known about VPAC expression of AM in COPD patients. AM from COPD patients show a strong VPAC1 expression which exceeds VPAC2. A similar receptor expression pattern was also observed in lipopolysaccharide (LPS)-activated monocyte-derived macrophages (MDM) from healthy volunteers and COPD patients. VIP has been shown to down-regulate interleukin 8 (IL-8) secretion significantly in MDM after LPS stimulation. The response to VIP was similar in MDM from COPD patients and healthy volunteers. Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism in response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is dominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling. The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lung of COPD patients.

Introduction

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide of the secretin/glucagon family [37]. It has a broad spectrum of biological functions, including anti-inflammatory, bronchodilatatory, vasodilatatory and anti-proliferative effects. VIP is produced and secreted by inhibitory non-adrenergic non-cholinergic nervous fibers [19], [37]. The peptide acts via three different G-protein coupled receptors (VPAC1, VPAC1 and PAC1) that have been identified in various human tissues including lung, brain, kidney, gastrointestinal tract and also on immunocompetent cells like macrophages and lymphocytes [5], [11], [17], [22], [44]. The known anti-inflammatory effects of VIP are in part achieved by the down-regulation of several pro-inflammatory cytokines such as interleukin 8 (IL-8), IL-6 and IL-1. It was therefore already used experimentally to treat various inflammation-driven diseases such as rheumatoid arthritis [2], [6], [10], [46], sepsis [7], [30] and chronic inflammatory bowel disease [1], [23] in animals.

Chronic obstructive pulmonary disease (COPD) is an increasingly important pro-inflammatory disease which is expected to become the third leading cause of death worldwide by 2020 [33]. The disease is attributed to long-term exposure to toxic gases and particles, such as particulate air pollutants from cigarette, industrial or car exhaust smoke which lead to an unbalanced inflammatory response in airways. Bronchodilators and inhalative steroids are frequently used for symptomatic relief [13], but they are insufficient to stop the progression of the disease.

Macrophages play a major role in the pathophysiology of COPD as they release key chemotactic and pro-inflammatory cytokines such as IL-8. IL-8 has been shown to be positively correlated with disease severity and progression [25], [43].

Macrophages promote tissue destruction and airway remodeling [9] mediated via growth factors and matrixmetalloproteinases [3], [4], [9], [21], [40], [42], thereby accelerating the normal decline in pulmonary function.

In human airways, VIP is one of the most abundantly expressed molecules with several physiological functions. VIP-ergic nerve fibers form a network around bronchi [15], bronchioles, submucosal glands and are co-localized to cholinergic fibers [24]. It is known that VIP plays an important role in the regulation of bronchial tone [26] as it is one of the most potent bronchodilators known so far [35]. It also acts on bronchial mucus secretion [12], [17].

Several animal models corroborate the role of VIP in controlling airway inflammation. For example, it has been shown that the administration of VIP decreases neutrophilic inflammation and prevents alveolar damage in the lungs of rats exposed to cigarette smoke [27], [34], [39]. In line with this notion, mice with target deletion of VIP gene spontaneously exhibit peribronchiolar and perivascular inflammation with increased levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid; this can be reversed by VIP substitution [41].

Recently, it has been demonstrated that human smokers with bronchitis show an increased expression of VPAC1 in lung tissue and on subepithelial mononuclear cells. This is accompanied by an increased expression of VIP-immunoreactive nerve fibers around bronchi [28], [31]. Since macrophages are key players in the pathogenesis of COPD, we assessed the VPAC expression in alveolar macrophages (AM) from patients with COPD. Additionally, we analyzed the VPAC regulation in response to LPS and investigated the effect of VIP on IL-8 secretion in monocyte-derived macrophages (MDM) from non-smoking healthy volunteers and COPD patients.

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Section snippets

Alveolar macrophage receptor staining

Human lung tissue was obtained from lung tumor patients with COPD (n = 8) subjected to lobectomy or pneumectomy. We used COPD-affected but tumor-free peripheral lung tissue which was immediately fixed in formaldehyde and embedded in paraffin for further immunohistochemical analysis [32]. Immunostaining was done with antibodies for human VPAC1 and VPAC2 (Antibody Solutions, CA, USA, Clone AS 58 and AS 59, diluted 1:200), and with biotinylated secondary antibodies, peroxidase-conjugated

Statistical analysis

The results of immunostaining and ELISA were statistically analyzed by Student's t-test or Wilcoxon signed rank test, as appropriate. Data are presented as mean ± standard error of the mean (SEM).

Receptor expression on alveolar macrophages

In paraffin-embedded lung tissue from COPD patients, we observed that macrophages accumulated in the alveolar space. Irrespective of whether the macrophages were observed in clusters or as singles, they were immunoreactive for VPAC1 and VPAC2. The immunoreactivity was more prominent for VPAC1 than for VPAC2 (Fig. 1).

Receptor expression on in vitro differentiated macrophages (MDM)

MDM from healthy non-smoking volunteers show the same VPAC receptor expression and regulation in response to LPS as MDM from patients with COPD. Typical examples of

Discussion

We have demonstrated that alveolar macrophages (AM) from COPD patients show a high VPAC1 expression which is dominant over VPAC2. The receptor expression pattern is comparable to that of LPS-stimulated MDM from healthy non-smoking volunteers and patients with moderate COPD. The high expression of VPAC1 on AM may reflect the chronic inflammatory process which is known to occur in the lung of COPD patients. The up-regulation of VPAC1 is not specific for COPD as it can also be observed in MDM from

Conclusion

Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism of response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is predominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling.

The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lungs of COPD patients.

Acknowledgements

Supported by the European Commission (Grant No QLRT-2001-02031), and by the Austrian National Bank (grant no 9673). The Austrian Nano-Initiative co-financed this work as part of the Nano-Health project (no. 0200), the sub-projects NANO-Breath being financed by the Austrian FWF (Fonds zur Förderung der Wissenschaftlichen Forschung, Project no. N206-NAN).

Glossary

AMalveolar macrophages

AUarbitrary units

BSAbovine serum albumin

COPDchronic obstructive pulmonary disease

FCSfetal calf serum

GOLDglobal initiative for chronic obstructive lung disease

HRPhorseradish peroxidase

IL-1interleukin 1

IL-6interleukin 6

IL-8interleukin 8

LPSlipopolysaccharide

MDMmonocyte-derived macrophages

NFκBnuclear factor kappa B

NP-40nonyl phenoxylpolyethoxylethanol

PAC1pituitary adenylate cyclase activating polypeptide receptor 1

PBMCperipheral blood mononuclear cells

PBSphosphate buffered

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