Inhaled Vasoactive Intestinal Peptide Exerts ... - ATS Journals

Inhaled Vasoactive Intestinal Peptide Exerts Immunoregulatory Effects in Sarcoidosis ¨ tzen1, Jonas Schupp1, Rene Schmiedlin1, Elena Gonzalez-Rey2, Antje Prasse1, Gernot Zissel1, Niklas Lu ¨ ller-Quernheim1* Anne Rensing-Ehl3, Gerald Bacher4, Vera Cavalli4, Dorian Bevec4, Mario Delgado2*, and Joachim Mu 1 Department of Pneumology and 3Allergy Research Group, University Hospital, Freiburg, Germany; 2Institute of Parasitology and Biomedicine, CSIC, Granada, Spain; and 4mondoBIOTECH, Basel, Switzerland

Rationale: Previous studies suggest an important immunoregulatory role of vasoactive intestinal peptide (VIP) in experimental models of chronic noninfectious inflammation. Sarcoidosis is characterized by noncaseating epitheloid cell granulomas, where excessive tumor necrosis factor-a production by pulmonary macrophages plays a critical role in granuloma formation and disease progression, which may lead to fatal organ dysfunction. Objectives: To test whether inhaled VIP has an immunoregulatory role. Sarcoid alveolitis was used as a prototype of immune-mediated chronic lung inflammation. Methods: In an open clinical phase II study, we treated 20 patients with histologically proved sarcoidosis and active disease with nebulized VIP for 4 weeks. Measurements and Main Results: VIP inhalation was safe, well-tolerated, and significantly reduced the production of tumor necrosis factor-a by cells isolated from bronchoalveolar lavage fluids of these patients. VIP treatment significantly increased the numbers of bronchoalveolar lavage CD41CD1272CD251 T cells, which showed regulatory activities on conventional effector T cells. In vitro experiments demonstrated the capacity of VIP to convert naive CD41CD252 T cells into CD41CD251FoxP31 regulatory T cells, suggesting the generation of peripheral regulatory T cells by VIP treatment. Conclusions: This study is the first to show the immunoregulatory effect of VIP in humans, and supports the notion of inhaled VIP as an attractive future therapy to dampen exaggerated immune responses in lung disorders. Thus, the inhalation of neuropeptides may be developed into a new therapeutic principle for chronic inflammatory lung disorders in humans. Keywords: sarcoidosis; vasoactive intestinal peptide; TNF-a; regulatory T cells; treatment

Sarcoidosis is a systemic disease characterized by granuloma formation that mainly affects the lung (1, 2). Although the exact etiology still remains obscure, evidence indicates that a strong interaction of alveolar macrophages and TH1 cells within the granuloma is a central element in the development of sarcoidosis (1–7). The intense inflammation observed in patients with sarcoidosis is a consequence of excessive local activity of TH1 cells and the uncontrolled production of proinflammatory factors, especially tumor necrosis factor (TNF)-a (5–8). On the other hand, a critical role of T regulatory cells (Tregs) in the

(Received in original form September 26, 2009; accepted in final form April 28, 2010) Supported in part by the Federal Ministery of Education and Research (BMBF 01 EO 0803) and by the Spanish Ministry of Science and Innovation and Junta de Andalucia (M.D. and E.G-R.). * These authors contributed equally to the article. Correspondence and requests for reprints should be addressed to Antje Prasse, M.D., Department of Pneumology, University Hospital, Killianstrasse 5, 79106 Freiburg, Germany. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 182. pp 540–548, 2010 Originally Published in Press as DOI: 10.1164/rccm.200909-1451OC on May 4, 2010 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Previous studies suggest an important immunoregulatory role of vasoactive intestinal peptide (VIP) in experimental models of chronic noninfectious inflammation, but its efficiency in humans has not been addressed. What This Study Adds to the Field

This study shows the immunoregulatory effect of VIP in humans and supports the notion of inhaled VIP as a therapeutic option to dampen exaggerated immune responses in lung disorders.

maintenance of immune tolerance and prevention of autoimmune disorders has been established (9, 10). The role played by Tregs in sarcoidosis is not fully understood. As has been recently shown, circulatory Tregs exert profound immunosuppressive actions on sarcoid effector T cells, but are unable to totally suppress TNF-a production in sarcoid inflammation (11, 12). Decreased Treg counts and their dysfunction in affected organs have also been described in autoimmune and graftversus-host diseases, and therapeutic strategies increasing peripheral Treg counts would be highly anticipated. In principle, there is little knowledge regarding the generation of Tregs in human organs. However, evidence suggests that neuropeptides are involved in the maintenance of immune homeostasis in peripheral organs (13, 14). Vasoactive intestinal peptide (VIP) is a 28-aa neuropeptide released from neural cells and by TH2 cells in response to antigen stimulation. VIP is a potent antiinflammatory agent that affects both innate and adaptive immunity (13) and has been proved effective in the treatment of various experimental models of inflammatory and autoimmune diseases (14, 15). The therapeutic effect of VIP was initially attributed to the down-regulation of a wide panel of inflammatory mediators and to the inhibition of autoreactive TH1 cells (13, 14). Furthermore, recent evidence has demonstrated the involvement of Tregs in the beneficial effect of VIP and other neuropeptides on experimental immune disorders and their capacity to induce the emergence of antigenspecific Tregs in vivo with suppressive activity on effector T cells (16, 17). Although there is mounting evidence for a beneficial role of VIP in animal models of chronic inflammation, the immunoregulatory activity of VIP in the human system has never been addressed. Some of the results of these studies have been previously reported in the form of an abstract (18).

METHODS Patients with Sarcoidosis Twenty patients with histologically proved active pulmonary sarcoidosis were included in the study evaluating the inhalative synthetic VIP.

Prasse, Zissel, Lu ¨ tzen, et al.: VIP Inhalation in Sarcoidosis

Because of severe nasal hemorrhage during bronchoalveolar lavage (BAL) (Day 0) and resultant contamination of BAL, the BAL cell culture data of two patients were excluded from analysis. Additionally, 35 patients with sarcoidosis were included in the analysis of BAL Treg counts. Their activity was assessed at the 1-year follow-up. All patients with chronic active disease had a need for treatment with prednisolone. Patients with nonactive disease did not have a need for immunosuppressive treatment. Diagnosis was made according to the American Thoracic Society/European Respiratory Society criteria (19). The study evaluating VIP inhalation was approved by the local ethics committee and the competent German federal authority (BfARM), and designed as a clinical phase II study. All patients provided informed consent in writing before study entry.

VIP Treatment The patients received 50 mg synthetic VIP (Aviptadil; Bachem, Basel, Switzerland) four times daily by inhalation by way of an ultrasonic nebulizer (Optineb; Nebu-Tec, Elsenfeld, Germany) for 28 days. At Day 1, before the first inhalation of VIP, vital signs, pulmonary function, CO diffusion capacity, laboratory chemistry, and BAL were obtained. After that, the patients were advised in detail about the inhalation method and received the study medication. The baseline procedures of Day 0 were repeated in all the patients at the end of the treatment period. TNF-a production by cultured BAL cells was defined as the primary end point of the study. Pulmonary function tests were routinely performed, according to the American Thoracic Society recommendations (20) using a bodyplethysmograph (Ja¨ger, Ho¨chberg, Germany).

BAL Cell Isolation and Culture BALs were obtained using a standard technique (21). After isolation, differential BAL cell counts were performed as described in the online supplement. Moreover, BAL cells (106 cells/ml) were immediately cultured in 24-well plates in RPMI 1640 medium supplemented with 2% human serum and 1% penicillin/streptomycin. When indicated, BAL cells were stimulated with phorbol 12-myristate 13-acetate (SigmaAldrich, Deisenhofen, Germany) plus phytohemagglutinin (SigmaAldrich). Culture supernatants were harvested after 24 hours culture and the cytokine levels determined by using commercially available ELISA systems following manufacturers’ instructions: TNF-a (Immunotools, Germany); IFN-g (Thermopierce, Germany); IP (interferoninducible protein)-10 (BD Bioscience, Friesoythe, Germany); and for macrophage inflammatory protein (MIP)-1a, IL-17, transforming growth factor-b, and IP-10 all R&D (Wiesbaden-Nordenstadt, Germany).

Flow Cytometry Analysis Isolated BAL cells were stained by antibodies using antihuman CD11b (clone ICRF44), CD3 (clone HIT3a), CD4 (clone RPA-T4), CD11a (clone G43–25B), CD11c (clone B-ly6), CD54 (clone 28), HLADR (clone G46–6), CD80 (clone L307.4), CD86 (clone 2331), CD25 (clone M-A251), CD127 (clone hIL-7R-M21) antibodies (all BD Bioscience, Heidelberg, Germany), FoxP3 (clone PCH101; eBioscience, San Diego, CA) and analyzed by flow cytometry (FACSCalibur; BD Bioscience) as previously described (22). Lymphocyte and macrophage population were defined by forward and sideward scatter, in accordance of cell granularity and size. The percentage of positive cells was determined using isotype-matched biotinylated antibodies as controls.

Functional Analysis of BAL CD41CD1272CD251 T Cells CD41CD1272CD251 T cells were sorted by a high-speed MoFlo cell sorter (Beckman Coulter, Krefeld, Germany) from BALs of patients with sarcoidosis at the beginning and at the end of the VIP treatment. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and healthy control subjects by Ficoll-Hypaque density gradient. CD41CD252 T cells were isolated from the PBMCs derived from patients with sarcoidosis by magnetic cell sorting using MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). PBMCs from healthy volunteers were g-irradiated. CD41CD252 T cells (2 3 104 Teff) were stimulated with 5 3 104 g-irradiated allogeneic PBMCs with or without 2 3 104 BAL CD41CD1272D251 T cells (Treg). After 7 days T-cell proliferation was analyzed by looking at [3H]-thymidine incorporation within the last 18 hours of the experiment. Plates were

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harvested with a Filter-Mate and [3H]-thymidine incorporation was determined by liquid scintillation spectroscopy using a TopCount. In vitro effect of VIP on generation of Treg was assessed as described in the online supplement.

Determination of Costimulatory Activity of Monocytes Monocytes isolated from PBMCs of healthy subjects as described previously were incubated with complete medium and stimulated with LPS (1 mg/ml) in the absence or presence of VIP (0.1 mM). After 24 hours, the expression of different costimulatory molecules in monocytes was determined by flow cytometry and the cytokine content in culture supernatants was determined by ELISA as described. To determine the costimulatory capacity of monocytes on T cells, untreated and VIP-treated monocytes were fixed with 0.5% paraformaldehyde and incubated at different ratios with syngeneic T cells (4 3 105 cells) stimulated with anti-CD3 antibodies (100 ng/ml), and the proliferative response was determined after 4-day culture by [3H]-thymidine incorporation as described previously.

RESULTS Inhalation of VIP Is Safe and Well Tolerated by Patients with Sarcoidosis

In this proof of concept study, we investigated the immunologic effects of short-term VIP inhalation in 20 patients (13 males, 7 females; mean age, 49.6 6 13.1 yr) with histologically proved active sarcoidosis. The patients received 50 mg synthetic VIP four times daily by inhalation by way of a nebulizer for 28 days. According to Scaddings chest radiograph classification (23), 14 patients had sarcoidosis chest radiograph type II, 7 had sarcoidosis chest radiograph type III, and one patient had chest radiograph type IV. None of the patients had Lo¨fgren syndrome. Further characteristics, such as pulmonary function data, CO-diffusion capacity, and PaO2 at rest for Day 0 and after the treatment period are listed in Table 1. There was no statistical difference between pulmonary function data comparing Day 0 with Day 28. The study was not designed to quantify cough and dyspnea by a symptom score. Nevertheless, in our cohort 12 out of 20 patients suffered from cough and 14 out of 20 patients suffered from dyspnea on exertion. After VIP treatment 9 of 12 patients who suffered from chronic cough and 6 of 14 patients with dyspnea reported an amelioration of their symptoms. None over 20 patients reported any deterioration in either cough or dyspnea. We did not observe any severe adverse event during the VIP treatment period. Particularly, we did not observe any episode of a significant drop in blood pressure or an episode of tachycardia. In total, 19 adverse events were recorded in 12 patients. Although a large proportion of the adverse events could be directly related to the invasive diagnostic procedures (i.e., dry throat, hoarseness, syncope, cough, and hemoptysis), none of them could unequivocally be attributed to the study medication. Concerning severity, they were all rated as ‘‘mild’’ with the exception of a case of syncope and one case of common cold, both rated as ‘‘moderate’’ and directly after bronchoscopy. Most of the adverse events disappeared spontaneously. In two cases flatulence and diarrhea were reported. Laboratory chemistry revealed no influence of the study medication on blood differential cell counts or liver enzymes. The increase in creatinine was statistically significant (P , 0.048), although clinically ‘‘nonsignificant’’ (Table 1). Increase in BAL CD81 T Cells after VIP Inhalation Period

Neither total nor differential BAL cell counts were significantly altered after the VIP treatment period (see Figure E1 in the online supplement, Table 1). However, we observed an increase in BAL CD81 T cells after the inhalation of VIP (P 5 0.014)

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TABLE 1. CHARACTERISTICS OF PATIENTS WITH SARCOIDOSIS INCLUDED IN THE STUDY AT BASELINE (DAY 0) AND AT THE END OF THE TREATMENT PERIOD (DAY 28) Mean (SD) Day 0 Pulmonary function test Residual volume, L Vital capacity, L Total lung capacity, L FEV1, L FEV1/VC MAX PO2, mm Hg PCO2, mm Hg DLCO BAL differential cell count Cell count, 106/100 ml BAL AM, % BAL L, % BAL N, % BAL E, % BAL-cell cytokine production TNF-a, pg/ml TGF-b1, pg/ml IL-13, pg/ml IP-10, pg/ml MIP-1a, pg/ml IFN-g, pg/ml IL-17, pg/ml Laboratory chemistry CRP, mg/L sIL-2R, U/ml Neopterin, nmol/L ACE, U/L Safety data Creatinine, mg/dl AST, U/L ALT, U/L LDH, U/L CK, U/L Leukocytes, 103/ml Platelets, 103/ml Systolic BP, mm Hg Diastolic BP, mm Hg Heart rate, bpm

Mean (SD) Day 28

n

P Value

1.93 3.71 5.64 2.71 72.93 76.4 38.75 7.65

(0.54) (1.03) (1.14) (0.81) (8.56) (8.35) (3.14) (2.42)

1.95 3.69 5.64 2.73 73.75 77.2 38.95 7.27

(0.53) (1.01) (1.17) (0.83) (9.43) (7.58) (3.57) (2.21)

20 20 20 20 20 20 20 19

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

13.9 53 44 2 1

(11.4) (21) (20) (2) (1)

14.3 56 39 3 1

(8.3) (19) (18) (3) (2)

20 20 20 20 20

n.s. n.s. n.s. n.s. n.s.

2,869 979 14 144 2,081 522 448

(6,506) (312) (19) (241) (2,703) (937) (415)

559 1,040 11 120 952 146 259

(831) (139) (20) (160) (747) (249) (257)

18 18 16 18 18 18 18

0.04 n.s. n.s. n.s. 0.04 0.063 0.079

7.2 1,700 15.5 62.2

(7.3) (1,582) (9.4) (33.5)

8.7 1,780 16.5 71.5

(9) (1,724) (11) (40.3)

18 20 20 20

n.s. n.s. n.s. n.s.

0.96 31.5 28.2 218 94 6.04 246 122 83 74

(0.13) (9) (18.4) (31) (47) (1.56) (37) (15) (11) (9)

1.02 30.5 27.5 216 93 6.01 256 122 80 74

(0.15) (7.4) (19.6) (26) (75) (1.89) (43) (14) (10) (11)

19 19 19 18 19 20 20 20 20 20

0.048 n.s. n.s. n.s. n.s. n.s. 0.08 n.s. n.s. n.s.

Definition of Abbreviations: ACE 5 angiotensin-converting enzyme; AM 5 alveolar macrophages; AST 5 aspartate aminotransferase; ALT 5 alanine aminotransferase; BAL 5 bronchoalveolar lavage; BP 5 blood pressure; CK 5 creatine kinase; CRP 5 C-reactive protein; DLCO 5 diffusion capacity for carbon monoxide; E 5 eosinophils; IP 5 ; L 5 lymphocytes; LDH 5 lactate dehydrogenase; MIP 5 macrophage inflammatory protein; N 5 neutrophils; sIL-2R 5 soluble interleukin2 receptor; TGF 5 transforming growth factor; TNF 5 tumor necrosis factor.

(Figure 1A). CD41 T cell counts were not significantly influenced. The increase in CD81 T cells was mirrored by a decrease in the CD4/CD8 ratio (P 5 0.02). Influence of VIP Inhalation on Cytokine Production by BAL Cells

After the treatment period we observed a decrease in spontaneous TNF-a production by cultured BAL cells (P 5 0.04) (Figure 1B). Thereby the study achieved its primary end point. Not all included patients had an elevated spontaneous TNF-a production by BAL cells. Our previous studies showed that the upper threshold of the normal spontaneous TNF-a production is 600 pg/ml, defined by the mean level 1 2 3 SD of spontaneous TNF-a production in healthy volunteers (8). We observed a decrease in TNF-a production at the end of the study period in all patients with elevated spontaneous TNF-a release above this threshold ( Figure E2). In addition, there was a significant decrease in spontaneous MIP-1a production by BAL cells (Table 1) (P 5 0.04) and a

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nonsignificant trend toward a reduction in spontaneous IFN-g and phorbol 12-myristate 13-acetate/phytohemagglutinin–induced IL-17 production (Figure 1B). Spontaneous IL-13, IP-10, and transforming growth factor-b production were not altered at the end of the study period (Table 1). We did not observe any systemic effects of VIP inhalation neither regarding cytokine production by PBMCs nor serum sIL-2R, angiotensin-converting enzyme, and neopterin concentrations (Table 1). BAL Tregs Are Decreased in Patients with Chronic Active Disease

To test the role of pulmonary Tregs in sarcoidosis, we analyzed BAL Treg counts in 35 patients with sarcoidosis and 10 healthy volunteers (Figures 2A and 2B). BAL Tregs were defined by their CD41CD1272CD251 expression. Additional experiments revealed that most BAL CD41CD251 T cells also expressed FoxP3 (Figure 2C). There was a close correlation between the percentage of BAL CD41CD1272CD251 T cells and BAL CD41FoxP31 T cells (r 5 0.70; P , 0.001). Patients with chronic active disease had a significantly lower BAL CD41CD1272CD251 T-cell ratio than patients with spontaneous resolution (P , 0.001); nonactive disease (P 5 0.003); and healthy volunteers (P , 0.001) (Figures 2A and 2B). Patients with spontaneous resolution of their disease had a higher BAL CD41CD1272CD251/CD41 T-cell ratio than patients with nonactive disease (P 5 0.043). Patients with high spontaneous TNF-a production by their BAL cells had low BAL Treg counts, whereas patients with TNF-a secretion within the normal range had the highest BAL Treg counts (P 5 0.002; r 5 0.55). Increase in BAL Tregs after VIP Inhalation

Because several studies in mice have shown an increase in Tregs by VIP administration, we analyzed the percentage of BAL CD41CD251CD1272 T cells. There was a significant increase in the percentage of BAL CD41CD251CD1272 T cells after the treatment period (Figure 3A). To test the regulatory function of BAL CD41CD1272CD251 T cells, we sorted them by a high-speed cell sorter (n 5 7). The amount of recovered BAL CD41CD1272CD251 T cells by sorting 20 million BAL cells was significantly higher after VIP inhalation than at study entry (270 3 103 cells vs. 115 3 103 cells, respectively; P 5 0.029). Coculture experiments with conventional peripheral blood CD41CD252 T cells (Teff) and stimulation by irradiated allogeneic PBMCs were performed. BAL CD41CD1272CD251 T cells did efficiently inhibit Teff proliferation analyzed by [3H]thymidine incorporation in a 1:1 ratio. There was no significant difference in the capacity to suppress Teff proliferation between BAL CD41CD1272CD251 T cells sorted at study entry compared with BAL CD41CD1272CD251 T cells sorted at study end point in a ratio of 1:1 (Figure 3B). However, BAL CD41CD1272CD251 T cells sorted after VIP treatment suppressed Teff proliferation more efficiently in a ratio 1:2 than BAL CD41CD1272CD251 T cells sorted at study entry. De Novo Generation of Human Tregs by VIP

In several mouse models of chronic inflammatory diseases, we showed that VIP treatment induces the generation of Treg from the CD41CD252 T-cell compartment (16). However, it remains unknown whether VIP is able to induce human Treg with suppressive functions in a similar way. To address this issue, human naive CD41 T cells were activated through TCR/CD3 and CD28 costimulation by using anti–CD3/CD28-coated beads in the absence (Tcontrol) or presence (TVIP) of VIP. TVIP showed a significant increase in the percentage of CD41CD251 T cells expressing the Treg markers FoxP3 and CTLA4, compared with


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Figure 1. Vasoactive intestinal peptide (VIP) inhalation increased the percentage of bronchoalveolar lavage (BAL) CD81 T cells and decreased spontaneous tumor necrosis factor (TNF)-a production by BAL cells. Patients with active sarcoidosis were treated with inhaled VIP for 28 days. BAL cells were isolated before (baseline, open bars) and at the end of the VIP treatment (shaded bars), and the cell counts and spontaneous cytokine production were determined. (A) Total and differential cell count. (B) CD4/CD8 ratios and percentage of CD41 and CD81 BAL cells. BAL cells were cultured for 24 hours and the production of (C ) TNF-a, (D) IFN-g, and (E ) IL-17 were determined in the culture supernatants by ELISA. For IL-17 production, BAL cells were stimulated with phorbol myristate acetate and phytohemagglutinin. * P , 0.05.

Tcontrol (Figure 4A). In addition, VIP-induced CD41CD251 cells showed an effector-memory phenotype characterized by expression of CD45RO, but not CD62L and CD27, making these cells suitable to act at a site of inflammation. To determine the suppressive activity of TVIP on effector T cells, TVIP were cocultured with syngeneic T cells in the presence of allogeneic mature dendritic cells. Whereas Tcontrol contributed to the activation of effector T cells, TVIP dose-dependently suppressed their clonal expansion and IFN-g production (Figure 4B). We obtained similar regulatory activities with purified CD41CD252 T cells primed with VIP (Figure 4C). This indicates that the induction of regulatory activity by VIP occurs within the naive CD41CD252 T cell fraction, irrespective of the presence and expansion of naturally occurring CD41CD251

Tregs. To confirm this conclusion, CD41CD25high T cells isolated from healthy subjects were labeled with CFSE and stimulated with anti–CD3/CD28-coated beads in the absence or presence of VIP, and their proliferative response determined. In contrast to the treatment with IL-2 plus IL-15, two cytokines widely used to expand naturally occurring Tregs, VIP failed to promote proliferation of CD41CD251 Tregs in vitro (Figure 4D). Down-Regulation of HLA-DR and Costimulatory Molecule Expression by VIP Inhalation

In vitro data have suggested an influence of VIP on the expression of costimulatory molecules by macrophages (13). Therefore, the expression of several surface molecules by alveolar macrophages was tested. Our analysis revealed a sig-

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Figure 2. The number of bronchoalveolar lavage (BAL) regulatory T cells (Tregs) is associated with disease course in patients with sarcoidosis. (A) Patients with chronic active sarcoidosis have a deficiency in BAL Tregs. BALs were isolated from healthy subjects (n 5 9) or from patients with chronic active sarcoidosis (n 5 12), with nonactive sarcoidosis (n 5 13), or with spontaneous remission (n 5 10). The percentages of CD1272CD251 in the CD41 population were determined by flow cytometry. Numbers and horizontal lines indicate the mean percentage. * P , 0.05; ** P , 0.001. (B) Representative flow cytometry plots for each group analyzing the BAL CD41CD251 T-cell population. (C) BAL CD41CD251 T cells also expressed FoxP3.

nificant decrease in the percentage of alveolar macrophages, which expressed HLA-DR molecules and a decrease in the mean fluorescence intensity of HLA-DR expression after VIP inhalation (P 5 0.002) (Figure 5A). Furthermore, VIP inhalation down-regulated the expression of the costimulatory molecules CD86 and CD80 (P 5 0.01 and P 5 0.006) (Figures 5B and 5C). The expression of CD11b, CD11c, CD11a, CD54, and CD68 was not influenced by VIP treatment. In Vitro Effects of VIP Stimulation on Antigen-presenting Cells

Studies in murine models demonstrated that VIP mediates its immunomodulatory effect in part by regulating the costimulatory activity of antigen-presenting cells (13, 14). We investigated the capacity of VIP to deactivate human monocytes stimulated with the bacterial endotoxin LPS. VIP decreased the LPS-induced expression of HLA-DR, CD40, CD80, and CD86 and the production of the inflammatory mediators TNF-a and IL-12 by human monocytes from healthy subjects (Figure

5D). Identical results were obtained with human monocytes stimulated with TNF-a. These effects correlated with the fact that VIP-treated monocytes showed impaired costimulatory activity for allogeneic T cells (Figure 5E).

DISCUSSION The antiinflammatory effects of neuropeptides are well known. In mice, ample evidence points to an immunoregulatory role of VIP in multiple disease models (13–15). However, so far no study has tested the immunoregulatory capacity of VIP in humans. We tested the immunologic effects of short-term inhaled synthetic VIP, given the generic name Aviptadil, in 20 patients with active sarcoidosis. We demonstrated a significant reduction in TNF-a production by BAL cells after the 4-week treatment period and an increase in BAL Tregs. In vitro studies suggested de novo generation of pulmonary Tregs by VIP. These findings suggest that local VIP administration exerts an immunoregulatory role in humans.


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Figure 3. Vasoactive intestinal peptide (VIP) inhalation increased the number of bronchoalveolar lavage (BAL) regulatory T cells (Tregs). (A) VIP treatment increased the percentage of Tregs in lungs of patients with sarcoidosis. Patients with active sarcoidosis were treated with VIP by inhalation for 28 days and the percentage of BAL CD41CD1272CD251 T cells was determined at baseline and after the treatment period. * P , 0.05 (B) VIP-induced Tregs showed suppressive activity. BAL CD41CD1272CD251 T cells (Treg) were isolated from patients with sarcoidosis both before and at the end of the VIP treatment and cocultured with effector T cells (Teff). The suppressive activity on the proliferative response of Teff by BAL CD41CD1272CD251 T cells was measured by [3H]-thymidine incorporation (mean 6 SD of quintuplicate experiments, shown is one out of seven). In a ratio of 2:1 VIP-induced Tregs suppressed efficiently Teff proliferation in contrast to BAL CD41CD1272D251 T cells sorted at study entry.

A previous study demonstrated that, after intravenous administration of radiolabeled VIP, more than 30% of the peptide was rapidly found in the lung (24) and points to inhalation of VIP as an attractive application route (25). VIP inhalation was safe and well tolerated by all patients. We did not observe any severe adverse event, including effects on blood pressure and heart rate or on bone marrow, liver, and kidney functions. Moreover, we did not observe any infectious complications during or after the study period. To test the immunoregulatory role, we isolated BAL cells 1 day before the first VIP inhalation and again at the end of the VIP treatment period. Disease activity is increased by BAL cell TNF-a production, and neutralizing TNF-a is an efficient treatment strategy in sarcoidosis (5, 9, 26–28). We found that VIP treatment significantly down-regulated spontaneous TNF-a production by BAL cells. Not all patients included in this study had TNF-a production above background levels. However, all patients with elevated initial TNF-a production responded to VIP treatment by a reduction of their BAL cell TNF-a release. In addition to excessive TNF-a release, sarcoid alveolitis is characterized by the increased production of TH1-type cytokines and other macrophage-derived cytokines, such as IP-10 and MIP-1a (1, 2, 4, 29). We found a trend, although not statistically significant, toward reduced production of the TH1-cytokine IFN-g, and a significant reduction in the chemokine MIP-1a, which attracts CCR51 TH1-cells, macrophages, and immature dendritic cells. Inflammatory disease activity is also strongly influenced by Tregs (8, 9). In sarcoidosis, the number of BAL Tregs is decreased in patients with chronic active disease compared with healthy volunteers and patients with stable disease. Conversely, patients with sarcoidosis with spontaneous resolution had increased Treg counts. VIP inhalation resulted in significant increases in BAL CD41CD1272CD251 Treg counts. The VIP-induced BAL CD41CD1272CD251 T cells showed potent regulatory activities because their addition to cocultures of conventional CD41CD252 T (Teff) cells isolated from

peripheral blood stimulated with irradiated allogeneic PBMCs efficiently inhibited Teff cell proliferation. No differences were found between the suppressive capacities of BAL CD41CD1272CD251 T cells isolated at the beginning and at the end of the study, if BAL Tregs were cocultured with Teff cells in a ratio 1:1. However, whereas BAL Treg isolated at the beginning of the study failed to suppress effector T cells at a lower 1:2 ratio, VIP-induced BAL CD41CD1272CD251 T cells were still efficient Tregs. These findings indicate that VIP inhalation changed the balance between conventional T cells and Tregs in the lung and increased the efficiency of Tregs. To the best of our knowledge, inhaled synthetic VIP is the first therapeutic strategy that documents an increase in pulmonary Treg counts in humans. In several mouse models of chronic inflammatory diseases, it was shown that VIP treatment induces the generation of Tregs from the CD41CD252 T-cell compartment (16); however, it remained unknown whether VIP is able to induce human Tregs with suppressive functions in a similar way. This study demonstrates that VIP treatment of naive T cells resulted in a significant emergence of CD41CD251 T cells expressing the Treg markers FoxP3 and CTLA4, which suppressed clonal expansion of Teff cells. Similar regulatory activities were obtained using purified CD41CD252 T cells primed with VIP. This indicates that the induction of regulatory activity by VIP occurs within the naive CD41CD252 T cell fraction, independent of the presence and expansion of naturally occurring CD41CD251 Tregs. Previous studies have suggested a link between therapeutic strategies blocking systemic TNF-a and a consecutive increase in Treg counts in humans (30–32). We did not observe a clear correlation between the decrease in spontaneous TNF-a production and the increase in BAL Tregs after the VIP treatment period. However, patients with high TNF-a production had low BAL Treg counts, whereas patients with TNF-a secretion within the normal range had the highest BAL Treg counts. Interestingly, all patients with initially high TNF-a production

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Figure 4. Vasoactive intestinal peptide (VIP) induced the generation of regulatory functions in CD41 T cells in vitro. (A) Naive CD41 T cells isolated from healthy subjects were activated with anti–CD3/CD28 mAb-coated beads in the absence (control) or presence of VIP (0.1 mM). After 4 days, CD25 expression was analyzed in the CD41 T-cell fraction by flow cytometry. The CD41CD251 T-cell population was additionally analyzed for FOXP3 and CTLA4 expression. (B) Naive CD41 T cells isolated from peripheral blood mononuclear cells donor A were activated with anti–CD3/ CD28 mAb-coated beads in the absence (Tcontrol) or presence of VIP (TVIP) for 4 days. After the 2-day rest period, the suppressive potential of the recovered cells (suppressor Tcontrol or TVIP cells) was determined by adding increasing numbers of Tcontrol or TVIP cells to cocultures of responder T cells (from donor A) and allogeneic mature dendritic cells (from donor B). The proliferation of responder T cells was determined after 4 days of coculture. IFN-g levels in supernatants were determined after 48 hours of culture. (C) VIP induces the new generation of regulatory T cells (Treg) from the CD41CD252 repertory. CD41CD252 T cells isolated from donor A were activated with anti-CD3/CD28 mAb-coated beads in the absence (Tcontrol) or presence of VIP (TVIP) for 4 days. After a 2-day rest period, the suppressive potential of the recovered cells was determined by adding increasing numbers of Tcontrol or TVIP cells to cocultures of carboxyfluorescein succinimidyl ester (CFSE)-labeled responder T cells (from donor A) and allogeneic mature dendritic cells (from donor B). The proliferation of responder T cells was determined after 4 days of coculture by measuring the percentage of CFSE1 divided T cells. IFN-g and IL-2 levels in supernatants were determined after 48 hours culture. (D) VIP does not expand natural occurring T. CD41CD25high T cells isolated from healthy subjects were labeled with CFSE and stimulated with anti-CD3/CD28 mAb-coated beads in the absence (control) or presence of VIP (0.1 mM) for 4 days. Activation with anti-CD3/CD28 mAb-coated beads in the presence of IL-2 plus IL-15 was used as a positive control for Treg expansion. The percentage of CFSE1 divided CD41CD251 T cells was determined by flow cytometry. The proliferative response was assayed by [3H]-thymidine incorporation. Fold cell expansion was determined by counting CD41CD251 Treg numbers at the end of culture and comparing this with Treg numbers at initial set up. Results are mean 6 SD of three experiments performed in duplicate.

responded to VIP inhalation by reducing the release of this cytokine and increasing BAL Treg counts at the end the treatment period. Moreover, the only two patients who responded to VIP inhalation with a decrease in BAL Treg counts had normal spontaneous TNF-a release. Valencia and coworkers (33) showed that TNF-a down-modulates Treg function. However, the underlying mechanism that explains how the blockade of TNF-a influences Treg counts in humans remains obscure. In mice, a balance between TH17 and Treg exists (17, 34), which could be influenced by TNF-a. We observed a trend toward a decrease in induced IL-17 production by BAL cells after the VIP treatment period.

It is widely known that the close interaction of alveolar macrophages with T cells is a pivotal mechanism in sarcoidosis. Contrary to normal alveolar macrophages, alveolar macrophages from patients with sarcoidosis exert strong antigenpresenting capacities (4, 35), which are partially mediated by increased expression levels of HLA molecules and the costimulatory molecules CD86 and CD80 (36). Our data show that the expression of HLA-DR and of the costimulatory molecules CD86 and CD80 by alveolar macrophages was significantly down-regulated after the VIP treatment period. Studies in murine models demonstrated that VIP mediates its immunoregulatory effect in part by regulating the costimulatory activity


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Figure 5. Vasoactive intestinal peptide (VIP) inhalation decreased HLA-DR, CD86, and CD80 expression by alveolar macrophages. (A–C ) Patients with sarcoidosis were treated with VIP inhalation for 28 days and the expression of HLA-DR (A), CD80 (B), and CD86 (C) were determined by flow cytometry in macrophages isolated from bronchoalveolar lavage (BAL) before and at the end of VIP treatment. Results are expressed as fluorescence intensity. MCF 5 mean channel fluorescence. * P , 0.05. (D and E ) VIP inhibits the costimulatory activity of human monocytes. Monocytes were incubated with medium (unstimulated), with LPS, or with LPS plus VIP (0.1 mM). After 24 hours, the expression of different costimulatory molecules in monocytes was determined by flow cytometry, and the cytokine content in culture supernatants was determined by ELISA (D). Monocytes were fixed and incubated at different ratios with syngeneic T cells stimulated with anti-CD3 antibodies, and their proliferative response determined after 4 days culture (E). Results are the mean 6 SD of five experiments performed in duplicate.

of antigen-presenting cells (13, 14). Here, we found that VIP decreases the expression of HLA-DR, CD40, CD80, and CD86, and the production of the inflammatory mediators TNF-a and IL-12 by activated human monocytes. These effects correlated with the fact that VIP-treated monocytes showed impaired costimulatory activity for T cells. These data suggest that VIP inhalation may alter the antigen-presenting capacity of alveolar macrophages and thereby could so influence the interaction of alveolar macrophages and T cells in sarcoidosis. This investigation was designed as a proof-of-principle study and therefore we selected end points of immunopathologic relevance, which we expected to change within a period of 4 weeks. TNF-a release is a pathomechanism widely regarded as pivotal in sarcoidosis and here we demonstrate VIP inhalation as a new therapeutic approach to dampen TNF-a. This shortterm study did not show any significant effect on pulmonary function data. It could be speculated that the treatment period was too short to demonstrate an improvement of pulmonary function. Recently, a double-blinded, placebo-controlled, mul-

ticenter study testing 6 months TNF-a blockade by infliximab in 138 patients with sarcoidosis with the primary end point of change in pulmonary function was published (26). In view of the fact that this study yielded an only minor but significant gain in pulmonary function, it might not be surprising that we could not observe any significant change in physiologic parameters. On the other hand patients reported an amelioration of both cough and dyspnea with VIP treatment. Thus, placebo-controlled VIP inhalation studies with relevant clinical end points are indicated as a next step. Conclusion

Our study is the first to report that the inhalation of VIP has clear immunoregulatory effects in humans. VIP inhalation might be an attractive therapeutic option for patients with chronic inflammatory lung diseases, particularly those with pulmonary sarcoidosis. A new immunoregulatory treatment strategy that increases Treg counts without any obvious side effects and without systemic immunosuppression would be highly

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desirable. However, further multicentric controlled studies are needed to evaluate the clinical efficiency of this new treatment option in sarcoidosis. Author Disclosure: A.P. received $1,001–$5,000 from Sanofi-Aventis in lecture fees for IPF and up to $1,000 from Novartis in lecture fees for CME. G.Z. received $1,001–$5,000 from Siemens GmbH in lecture fees and $50,001–$100,000 from Nycomed Germany in industry-sponsored grants. N.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.G-R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.R-E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.B. is an employee of mondoBIOTECH AG. V.C. is an employee of Mondobiotech Group and served on an advisory board for mondoBIOTECH holding AG. D.B. is an employee of mondoBIOTECH AG and holds a patent from mondoBIOTECH AG for VIP in sarcoidosis. M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.M-Q. received $1,001–$5,000 from MondoBiotec in consultancy fees for Phase II studies, $1,001–$5,000 from Talecris for lectures on asthma, and $1,001–$5,000 from Boehringer Ingelheim for lectures on COPD. Acknowledgment: MondoBIOTECH holds the orphan drug status for Aviptadil in sarcoidosis, adopted by the European Commission. The Department of Pneumology of the University of Freiburg, Freiburg, Germany, collected fees from mondoBIOTECH to pursue the clinical part of the study.

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