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Thymulin Inhibits Monocrotaline-Induced Pulmonary Hypertension Modulating Interleukin-6 Expression and Suppressing p38 Pathway

Department of Physiology (T.H.-C., S.M.O., R.R.-A., A.L.N., M.S., F.L.F.C., A.B.-N., A.F.L.-M.), Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal; and Life and Health Sciences Research Institute (ICVS) (R.S.M., C.N.-S., J.C.-P.), School of Health Sciences, University of Minho, 4704-553 Braga, Portugal

Address all correspondence and requests for reprints to: Professor Adelino F. Leite-Moreira, Department of Physiology, Faculty of Medicine, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail: amoreira{at}med.up.pt.

	Abstract

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The pathogenesis of pulmonary hypertension (PH) includes an inflammatory response. Thymulin, a zinc-dependent thymic hormone, has important immunobiological effects by inhibiting various proinflammatory cytokines and chemokines. We investigated morphological and hemodynamic effects of thymulin administration in a rat model of monocrotaline (MCT)-induced PH, as well as the pattern of proinflammatory cytokine gene expression and the intracellular pathways involved. Adult Wistar rats received an injection of MCT (60 mg/kg, sc) or an equal volume of saline. One day after, the animals randomly received during 3 wk an injection of saline, vehicle (zinc plus carboxymethyl cellulose), or thymulin (100 ng/kg, sc, daily). At d 23–25, the animals were anesthetized for hemodynamic recordings, whereas heart and lungs were collected for morphometric and molecular analysis. Thymulin prevented morphological, hemodynamic, and inflammatory cardiopulmonary profile characteristic of MCT-induced PH, whereas part of these effects were also observed in MCT-treated animals injected with the thymulin’s vehicle containing zinc. The pulmonary thymulin effect was likely mediated through suppression of p38 pathway.

	Introduction

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INFLAMMATION SEEMS to play a significant role in the genesis or progression of some forms of pulmonary arterial hypertension (PAH), namely those associated with connective tissue diseases and HIV infection (1, 2). There are several reports that demonstrated the presence of inflammatory cells, including macrophages, polymorphonuclear neutrophils, lymphocytes, and mast cells, in the vicinity of pulmonary vessels and plexiform lesions (3, 4). Elevated levels of the proinflammatory cytokines IL-1 and -6 (5), fractalkine (6), regulated upon activation normal T cell expressed and secreted (7), macrophage inflammatory protein-1a (8), among others, have also been observed in patients with PAH. The role of inflammation in pulmonary hypertension (PH) is further supported by the experimental (9) and clinical (10) improvements observed after steroid treatment or immunosuppressor administration, such as rapamycin (11, 12) or mycophenolate mofetil (13).

Monocrotaline (MCT) and hypoxia models of experimental PH share the involvement of inflammation on its pathophysiology (14, 15). MCT causes endothelial cell damage and mononuclear infiltration into the perivascular regions of arterioles and muscular arteries. By its turn, IL-1 and IL-6, strong proinflammatory cytokines, are excessively produced in animals treated with MCT, and the administration of an IL-1 receptor antagonist reduced PH and right ventricle hypertrophy (9, 16). Further evidence implicating the inflammatory response in the genesis of PH is given by an animal model in which PH was induced by IL-6 administration (17, 18). Multiple studies using serum from patients with PAH have demonstrated increased circulating IL-6 level (5). Moreover, the genome of human herpes virus type 8, which encodes a particularly potent viral IL-6 analog, is associated with PAH in the setting of several diseases, namely HIV/AIDS (19).

Thymulin, also known as "serum thymic factor" (20, 21, 22, 23), is a nonapeptide, coupled to ion zinc (24), exclusively secreted by thymic epithelial cells (25, 26). Thymulin enhances an antiinflammatory cytoprotective response and depresses inflammatory cascades (27). Regarding its role in lung diseases, thymulin reduced the cellular inflammatory response and fibrotic changes in an experimental model of lung fibrosis (28). These pulmonary effects were related to an inhibition of the local synthesis of various proinflammatory cytokines and chemokines. Thymulin’s beneficial effects in other inflammatory disease models were attributed to modulation of MAPK family members (29, 30). Thymulin action is strictly dependent on the presence of the metal zinc because it induces the conformational changes within the molecule that are necessary for the full expression of its biological activity (24). Besides being an integral part of this thymic hormone, zinc is essential for many enzymes (31), and plays a role as an antiinflammatory and antioxidant agent (32). The potential role of thymulin in PH has not been studied before. Knowing that MCT induces an early and potent inflammatory response that culminates in severe PH, and that thymulin is an immunoregulatory peptide, the rationale of the present study was to evaluate cardiac and pulmonary effects of thymulin in this experimental model, and exploit the intracellular signaling pathways involved.

	Materials and Methods

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Study design
Animal experiments were performed according to the Portuguese law for animal welfare and conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996). Adult male Wistar rats (Charles River, Barcelona, Spain) weighing 180–200 g were housed in groups of five per cage in a controlled environment under a 12-h light, 12-h dark cycle at a room temperature of 22 C, with a free supply of food and water. Rats randomly received a sc injection of MCT (60 mg/kg body weight; Sigma, Barcelona, Spain) or an equal volume of vehicle [control (Ctrl) groups]. One day after, animals from both groups were randomly assigned to receive saline, vehicle, or thymulin, which was injected sc once a day during 3 wk. Vehicle consisted in a solution of carboxymethyl cellulose and zinc without thymulin. Thymulin (100 ng/kg; Sigma) was prepared using carboxymethyl cellulose (0.5%) as a carrier and zinc (ZnCl₂; 3.8 µg/ml) as a cofactor (33, 34).

The protocol resulted in six groups: Ctrl rats injected with saline (Ctrl plus S) (n = 6), Ctrl rats injected with vehicle (Ctrl plus V) (n = 6), Ctrl rats injected with thymulin (Ctrl plus T) (n = 6), MCT rats injected with saline (MCT plus S) (n = 8), MCT rats injected with vehicle (MCT plus V) (n = 8), and MCT rats injected with thymulin (MCT plus T) (n = 8). During d 23–25 after MCT or saline injection, the animals were anesthetized and submitted to hemodynamic instrumentation.

Hemodynamic studies
Animals were anesthetized with pentobarbital (60 mg/kg, ip; CEVA), placed over a heating pad, and tracheostomized for mechanical ventilation with room air (Harvard Small Animal Ventilator, Model 683; Harvard Apparatus, Holliston, MA). Anesthesia was maintained with additional bolus of pentobarbital (2 mg/100 g) as needed. Under binocular surgical microscopy (Wild M651.MS-D; Leica, Herbrugg, Switzerland), the right jugular vein was cannulated for fluid administration (prewarmed 0.9% NaCl solution) to compensate for perioperative fluid losses. The heart was exposed through a median sternotomy and the pericardium widely opened. Right and left ventricular (RV and LV, respectively) pressures were measured with a 2F high-fidelity micromanometer (SPR-324; Millar Instruments, Houston, TX) inserted into RV and LV cavities, respectively. After complete instrumentation the animal was allowed to stabilize for 15 min. Hemodynamic recordings were made with ventilation suspended at end expiration. Parameters were converted on line to digital data with a sampling frequency of 1000 Hz. RV and LV pressures were measured at end diastole and peak systole. Peak rates of RV and LV pressure increase (dP/dt_max) and pressure decrease (dP/dt_min) were measured as well. Relaxation rate was estimated with the time constant () by fitting isovolumetric pressure decrease to a monoexponential function.

After hemodynamic evaluation was completed, cardiac arrest was induced by injection of KCl 7.5%. Heart and lungs were excised and weighed. Under binocular magnification (x3.5), the RV free wall was dissected from the left ventricle and septum, and weighed separately. Heart, lung, RV, and left ventricle plus septum (LVS) weights were normalized to body weight, whereas RV weight was normalized to LVS weight. Samples from lung and right ventricle were collected for morphology and molecular analysis.

mRNA quantification
After collection, samples were quickly immersed in RNAlater (QIAGEN, Inc., Valencia, CA) and frozen (–80 C). Total mRNA was extracted through the guanidium-thiocyanate selective silica-gel membrane-binding method (QIAGEN 74124) according to the manufacturer’s instructions. Concentration and purity were assayed by spectrophotometry (Eppendorf 6131000.012; Eppendorf International, Hamburg, Germany). Two-step real-time RT-PCR was used to perform relative quantification of mRNA. For each studied mRNA molecule and for each tissue, standard curves were generated from the correlation between the amount of starting total mRNA and PCR threshold cycle (second derivative maximum method) of graded dilutions from a randomly selected tissue sample (r > 0.97). For relative quantification of specific mRNA levels, 50 ng total mRNA from each sample underwent two-step real-time RT-PCR. A melt curve analysis of each real-time PCR and 2% agarose gels (0.5 µg/ml ethidium bromide) were performed to exclude primer-dimer formation and assess the purity of the amplification product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were similar in all experimental groups, which enabled the use of this gene as an internal Ctrl. Results of mRNA quantification are expressed in an arbitrary unit (AU) set as the average value of the Ctrl group (1 AU) after normalization for GAPDH. Reverse transcription (20 µl; 10 min at 22 C, 50 min at 50 C, and 10 min at 95 C) was performed in a standard thermocycler (Whatman Biometra 050–901; Goettingen, Germany): 40 U/reaction of reverse transcriptase (Invitrogen 18064-014; Invitrogen Corp., Carlsbad, CA); 20 U/reaction of ribonuclease inhibitor (Promega N2515; Promega Corp., Madison, WI); 30 ng/ml random primers (Invitrogen 48190-011), 0.5 mM nucleotide mix (MBI Fermentas R0192; MBI Fermentas GmbH, St. Leon-Rot, Germany); 1.9 mM MgCl₂; and 10 mM dithiothreitol. Of the cDNA yield, 10% was used as a template for real-time PCR (LightCycler II; Roche Diagnostics Corp., Roche Applied Science, Indianapolis, IN) using SYBR green (QIAGEN 204143) according to the manufacturer’s instructions. Specific PCR primer pairs for the studied genes were: GAPDH, forward 5'-TGG CCT TCC GTG TTC CTA CCC-3' and reverse 5'-CCG CCT GCT TCA CCA CCT TCT-3'; IL-6, forward 5'-CCG TTT CTA CCT GGA GTT TG-3' and reverse 5'-GAA GTT GGG GTA GGA AGG AC-3'; IL-1β, forward 5'-ATG GCA ACT GTC CCT GAA CTC-3' and reverse 5'-AAT CCT TAA TCT TTT GGG GTC TG-3'; and fractalkine, forward 5'-TGT GTA CTC TGC TGG CGG GTC-3' and reverse 5'-GTC TCC AGG ATG ATG GCG CGC T-3'.

Immunohistochemistry (IHC) studies
IL-6 immunostainings were performed on formalin-fixed and paraffin-embedded lung. Sections (5 µm) were placed on SuperFrostPlus slides (Menzel Glaser GmbH, Braunschweig, Germany). The primary antibody, a polyclonal goat anti IL-6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), was used in a 1:50 dilution. After dewaxing in xylene and rehydration in ethanol, antigen retrieval was achieved by boiling in 10 mM citrate buffer followed by cool down at room temperature. Incubation with the goat ImmunoCruz Staining System (Santa Cruz Biotechnology) was performed according to the manufacturer’s instructions. Incubation of the primary antibody occurred at 4 C overnight. Negative Ctrl reactions included omission of the primary antibody. To visualize the peroxidase activities in sections, diaminobenzidine tetrahydrochloride was used. Sections were counterstained with hematoxylin. The slides were observed and photographed with the Olympus BX61 microscope (Olympus, Hamburg, Germany).

Western blot analysis
Lung tissue samples were processed for Western blot analysis. Proteins were obtained according to Kling et al. (35). Ten micrograms of protein were loaded onto 10% acrylamide minigels, electrophoresed at 100V at room temperature, and then transferred to Hybond-C Extra (GE Healthcare Life Sciences, Uppsala, Sweden). Blots were probed with polyclonal p38, c-Jun N-terminal kinase (JNK1/2) and p44/42 (ERK1/2), and phospho-p38 (dp-p38), phospho-JNK (dp-JNK1/2), and phospho-p44/42 (dp-ERK1/2), antibodies (Cell Signaling Technology, Inc., Danvers, MA) according to the manufacturer’s instructions. For loading Ctrl, blots were probed with β-tubulin rabbit polyclonal antibody (1:100,000) (Abcam plc, Cambridge, UK). Afterwards, blots were incubated with a secondary horseradish peroxidase conjugate (1:2000) (Cell Signaling Technology). Membranes probed simultaneously with p38 and JNK, as well as dp-p38 and dp-JNK, were stripped [62.5 mM Tris-HCl (pH 6.7), 2% sodium dodecyl sulfate, and 100 mM 2-mercaptoethanol] for 30 min at 50 C and then incubated with β-tubulin. Membranes were developed with Super Signal West Femto Substrate (Pierce, Rockford, IL) and ChemiDoc XRS System (Bio-Rad Laboratories, Inc., Hercules, CA). Quantitative analysis was performed with Quantity One 4.6.5 1-D Analysis Software (Bio-Rad Laboratories).

Statistical analysis
The results were presented as mean ± SEM and were compared using two-way ANOVA. When treatments were significantly different, the Holm-Sidak test was selected to perform pairwise multiple comparisons. Statistical significance was set at P < 0.05.

	Results

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Morphometric data
Cardiac and lung morphometric data are summarized in Table 1. Administration of vehicle or thymulin to Ctrl animals did not affect any of the studied morphometric parameters.

Biventricular hemodynamics
Hemodynamic data are summarized in Table 2. In Ctrl groups, neither vehicle nor thymulin modified the studied hemodynamic parameters. MCT induced a significant increase in peak systolic RV pressure (RVP_max), a parameter used to estimate PH, as well as in RV dP/dt_max, an index of RV contractility. Regarding RV diastolic function, relaxation rate as estimated by the was prolonged in MCT-treated animals. MCT-treated animals that received vehicle presented less PH when compared with the MCT plus S group, whereas MCT-treated animals that received thymulin did not demonstrate any hemodynamic evidence of PH. Indeed, there was no increase in RVP_max or changes in dP/dt_max. Concerning RV diastolic function, was not different from Ctrl animals.

Pro-inflammatory cytokines
Pro-inflammatory cytokines IL-1β, IL-6, and fractalkine mRNA levels were measured in lung and RV myocardial samples by real-time PCR (Fig. 1).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Lung and right ventricle IL-1β, IL-6, and fractalkine mRNA levels, normalized for GAPDH (housekeeping gene) and expressed as AUs, in the six studied groups: Ctrl plus S, Ctrl plus V, Ctrl plus T, MCT plus S, MCT plus V, and MCT plus T. P < 0.05: , vs. Ctrl plus S; , vs. Ctrl plus T; , vs. MCT plus S.

IHC studies were performed to identify the pattern of cellular expression of IL-6 in the lung. In Ctrl animals (Fig. 2, A–C), IL-6 is mainly expressed in smooth muscle cells both in arterial and bronchial walls, and weakly in bronchial epithelial cells and vascular endothelium. In MCT-treated animals (Fig. 2, D–F), a similar pattern was observed. However, the intensity of this staining seems to be increased in the MCT plus S group. In contrast to the Ctrl group, the MCT groups presented an infiltration of mononuclear inflammatory cells expressing IL-6 protein.

View larger version (96K): [in this window] [in a new window]   FIG. 2. Pulmonary IL-6 protein expression in different experimental groups by IHC. A, Ctrl plus S animals. B, Ctrl plus V animals. C, Ctrl plus T animals. D, MCT plus S animals. E, MCT plus V animals. F, MCT plus T animals. All images are at the same magnification (x200). Arrows displayed IL-6 positive inflammatory cells.

MAPK signaling pathway
Pulmonary protein levels of p38, JNK1/2, and ERK1/2 (both unphosphorylated and phosphorylated forms) were analyzed by Western blot and quantified by densitometric analysis (Fig. 3).

View larger version (42K): [in this window] [in a new window]   FIG. 3. MAPK activities at pulmonary level in Ctrl plus S animals (1 ), Ctrl plus V animals (2 ), Ctrl plus T animals (3 ), MCT plus S animals (4 ), MCT plus V animals (5 ), and MCT plus T animals (6 ). Western blot analysis of MAPK with antibodies to p38, ERK1/2, and JNK1/2 (A), and to diphosphorylated forms of p38 (dp-p38), ERK1/2 (dp-ERK1/2), and SAPK/JNK (dp-JNK1/2) (B). Ctrl loading was performed using β-tubulin (55 kDa). ERK1 and 2 correspond to 44 and 42 kDa, respectively. JNK1 and 2 correspond to 46 and 54 kDa, respectively. p38 corresponds to 38 kDa. Quantitative analysis for dp-p38, dp-ERK1/2, and dp-JNK1/2 are presented as AUs in the six studied groups (C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that thymulin prevented morphological, hemodynamic, and inflammatory cardiopulmonary profile characteristics of MCT-induced PH, whereas part of these effects were also observed in MCT-treated animals injected with the thymulin’s vehicle containing zinc. The pulmonary thymulin effect was likely mediated through suppression of p38 pathway.

The pathogenesis of PH involves vasoconstriction, pulmonary vascular remodeling, in situ thrombosis, and inflammation. The inflammatory hypothesis of PH is supported by both experimental and human studies (3, 4, 5, 6, 7, 8). PH induced by MCT is an experimental model with some similarities with human PAH, such as hemodynamic repercussions, histological changes, and high mortality (36). On the other hand, it diverges from human PAH in the precocious loss of endothelial barrier and in the inflammatory adventitial proliferation (15).

Inflammation is a main feature of the MCT model, as demonstrated by early inflammatory cells recruitment (37) and cytokine activation (9). To clarify the underlying mechanisms of MCT-induced PH, inflammatory cytokine expression and MAPK activation were examined. We observed an increase in IL-6 gene expression not only in the lungs but also in the right ventricle in the fourth week after MCT injection. These findings confirm previous results from Bhargava et al. (9) that demonstrated elevated levels of IL-6 at 48 h, 1 and 2 wk after MCT injection. We also evaluated IL-1β and fractalkine, other cytokines typically involved in human PH pathophysiology, but in our study MCT did not increase these cytokines in the lung tissue and had a very mild effect in the myocardium.

MAPKs are important mediators of signal transduction processes, and are activated in a variety of physiological and pathological conditions. Lu et al. (38) found an increase in p38 MAPK activity in lungs from rats treated with MCT and prevented the progression to PH through the administration of a selective p38 inhibitor. On other hand, Morty et al. (39) only observed a slight reduction in ERK1/2 phosphorylation, and no change in p38 MAPK phosphorylation. In our study we demonstrated an increase in both p38 and JNK phosphorylation in the MCT-treated animals, confirming the involvement MAPK pathways in PH pathophysiology. On the other hand, zinc exposure has activated several MAPKs in airway epithelial cells (40, 41). Levels of MAPK phosphorylation are regulated in opposite directions by MAPK kinases and phosphatases (42), and zinc seems to act through inhibition MAPK phosphatase activity (40). In fact, our data also suggest that in Ctrl animals, zinc has an important role in modulating MAPK, namely p38 phosphorylation. However, in MCT-treated animals, zinc exposure did not change the pattern of p38 activation, whereas thymulin treatment resulted in a significant decrease in p38 phosphorylation.

Thymulin has a potent immunomodulator effect, in addition to its action as a thymic hormone. Previous reports have shown that thymulin prevents bleomycin-induced pulmonary fibrosis (28), alloxan- and streptozotocin-induced diabetes (43), myocarditis caused by the encephalomyocarditis virus (44), and cisplatin- (29) and cephaloridine-induced nephrotoxicity (30). In the setting of PH, our data revealed that thymulin administration prevented biventricular hemodynamic changes typically found in the MCT model. Part of these effects had been also observed in animals treated with vehicle containing zinc. These beneficial effects could be partially explained by IL-6 suppression at pulmonary and myocardium levels, reinforcing the pivotal role of IL-6 in PH pathogenesis. In fact, delivery of recombinant IL-6 protein reproduced the main hemodynamic and histological features of PH in a rat model (17), and inhibition of IL-6 with dexamethasone (9) or serotonin antagonist (18) prevented the development of PH. IL-6 exerts its effects by activation of the janus kinase/signal transducers and activators of transcription-signaling pathway and induction of the MAPK cascade (45). In vitro, IL-6 stimulated lung branching through activation of p38-MAPK intracellular pathway (46). Recently, Hagen et al. (47) demonstrated the presence of a negative feedback loop between IL-6 and the bone morphogenetic protein pathway mediated through p38 MAPK activity, and proposed that IL-6 activation can be the inflammatory "second hit" in patients with familial PAH with bone morphogenetic protein receptor 2 mutations.

In conclusion, we demonstrated that thymulin inhibited the development of PH in the MCT model. This effect could be related to the inhibition of proinflammatory IL-6 expression and suppression of p38 phosphorylation.

	Acknowledgments

 
We thank Antónia Teles for her invaluable technical assistance in this study.

	Footnotes

 
This work was supported by grants from the Portuguese Foundation for Science and Technology (No. POCI/SAVFCF/60803/2004; POCTI/SAV-MMO/61547/2004 and PTDC/SAV-FCF/65793/2006) through Cardiovascular R&D Unit (FCT No. 51/94). R.S.M. was supported by Fundação para a Ciência e a Tecnologia (reference SFRH/BPD/15408/2005).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2008

Abbreviations: AU, Arbitrary unit; Ctrl, control; Ctrl plus S, control rats injected with saline; Ctrl plus T, control rats injected with thymulin; Ctrl plus V, control rats injected with vehicle; dP/dt_max, peak rate of right ventricular and left ventricular pressure increase; dP/dt_min, peak rate of right ventricular and left ventricular pressure decrease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; JNK, c-Jun N-terminal kinase; LV, left ventricular; LVS, left ventricle plus septum; MCT, monocrotaline; MCT plus S, monocrotaline rats injected with saline; MCT plus T, monocrotaline rats injected with thymulin; MCT plus V, monocrotaline rats injected with vehicle; PAH, pulmonary arterial hypertension; PH, pulmonary hypertension; RV, right ventricular; RVP_max, peak systolic right ventricular pressure; , time constant .

Received January 4, 2008.

Accepted for publication May 21, 2008.

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