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Expression of the chemokine PARC mRNA in bronchoalveolar cells Frantisek Mrazek a, Veronika Sekerova a, Jiri Drabek a, Vitezslav Kolek b, Roland a Department of Immunology, Palacky´ University, I. P. Pavlova str. 6, Olomouc CZ-775 20, Czech Republic b Department of Respiratory Medicine, Palacky´ University, I. P. Pavlova str. 6, Olomouc CZ-775 20, Czech Republic c Interstitial Lung Disease Unit, Royal Brompton Hospital, London, UK Received 2 April 2002; accepted 22 May 2002 Background: Chemotactic cytokines (chemokines) have been recently implicated in the pathogenesis of interstitial lung diseases. A novel chemokine called pulmonary and activation-regulated chemokine (PARC/CCL18), which attracts lymphocytes in vitro, hasbeen detected in the human lung. We have, therefore, investigated PARC mRNA expression in bronchoalveolar lavage fluid(BALF) cells of patients with pulmonary sarcoidosis */a disease characterised by a lymphocytic infiltrate. Further, because severalimmunomodulators are used in the treatment of sarcoidosis, we have determined the effects of selected drugs on PARC mRNAexpression in vitro. Subjects and methods: BALF cells were obtained by standard bronchoalveolar lavage (BAL) from 30 patientswith pulmonary sarcoidosis (S) and 16 control subjects (C). BALF cells from seven subjects were cultured in the presence ofdexamethasone (Dx), cyclosporin A (CyA) and pentoxifylline (Px). PARC mRNA expression was semiquantitated by reverse-transcription polymerase chain reaction (RT-PCR) using normalisation to the expression of the b-actin gene. Results: PARCmRNA transcripts were detected in 87% of all investigated BALF samples. The expression (ODR PARC/b-actin; median, the first tothe third quartile range) was similar in both groups tested (S, 0.60 (0.50 Á/0.95); C, 0.59 (0.36 Á/0.93); S vs. C: P /0.05). PARCmRNA expression was not associated with the number of lymphocytes in bronchoalveolar space. PARC mRNA expression wassignificantly suppressed by Dx (P 0/0.02); CyA and Px showed a moderate inhibitory effect which did not attain significance.
Conclusion: mRNA for the chemokine PARC is expressed in the lower respiratory tract in both healthy subjects and patients withpulmonary sarcoidosis. Out of the three immunomodulatory drugs tested, Dx downregulates PARC mRNA expression in BALFcells in vitro. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: CCL18; DC-CK-1; AMAC-1; In vitro regulation; Dexamethasone; Cyclosporin A; Pentoxifylline infiltrate. Therapeutic approaches to this disorder areusually based on corticosteroids. However, other drugs Chemokines play a fundamental role in the activation with immunomodulatory effects (e.g. cyclosporin A and migration of different leukocyte subtypes from (CyA) and pentoxifylline (Px)) have also been tested vessels to inflamed tissues They have been implicated in the complex pathogenesis of interstitial The chemokine pulmonary and activation-regulated lung diseases (ILD) which are characterised by an chemokine (PARC) has been described during search for chemokine homologous sequences in the database of sarcoidosis, the most frequent representative of the ILD expressed sequence tags This chemokine is known group, is often accompanied by a CD4' lymphocytic under the synonymous names DC-CK-1 , AMAC-1; in recent chemokine nomenclature it was desig-nated as CCL18 . PARC mRNA expression hasbeen detected in several tissues: at high levels in the lung; * Corresponding author. Tel.: '/420-68-585-2285; fax: '/420-68- at low levels in lymph nodes, thymus and appendix; and at marginal levels in small intestine and bone marrow 0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 1 3 0 - X F. Mrazek et al. / Immunology Letters 84 (2002) 17 Á/22 . PARC has been shown to be a selective chemoat- 0.1% diethyl pyrocarbonate in phosphate buffered saline tractant for lymphocytes in vitro; it did not recruit monocytes or granulocytes . The specific cellularreceptor for PARC ligand has not been identified to 2.3. PARC in vitro regulation experiments Considering chemotactic properties of PARC and its BALF cells from seven individuals were cultured (at a expression patterns, we hypothesised that this chemo- concentration of 1.106/ml) in RPMI medium (U kine may participate in the chemoattraction of lympho- Prague, Czech Republic) with 8% fetal calf serum (Flow cytes to the respiratory tract of patients with pulmonary Labs, UK) and 10 ng/ml of tumor necrosis factor-a sarcoidosis. We here, therefore, investigated PARC (TNF-a, NIBSC, Potters Bar, UK) at 37 8C, 5% CO2 mRNA expression in bronchoalveolar lavage fluid for 24 h in 24-well tissue-culture plates (Nunc, Roskilde, (BALF) cells of sarcoidosis patients in comparison to Denmark). Four types of cultures were set up: (a) with healthy subjects. Further, we wished to determine if 10(6 M dexamethasone (Dx) (Dexamethazon, Le´cˇiva, selected drugs used or clinically tested for the treatment Czech Republic), (b) with 10 ng/ml CyA (Sandimmun, of sarcoidosis can modulate PARC mRNA expression Sandoz, Switzerland), (c) with 20 ng/m Px (Pentoxifylli- num, Biotika, Slovakia) or (d) with medium containingonly TNF-a. The concentrations of tested drugs weredetermined to reflect usual therapeutic levels in tissues.
After 21 h of culture, the supernatants were aspired andthe cells were pelleted and washed twice in PBS Á/DEPC.
2.4. Semiquantification of PARC mRNA expression Bronchoalveolar lavage (BAL) was performed ac- cording to the standard protocol in 30 patients with pulmonary sarcoidosis and 16 control subjects. The Total cellular RNA was isolated from unseparated control subjects did not show clinical or laboratory signs BALF cells from patients using the single-step acid Á/ of lung inflammation at the time of BAL and did not GITC (guanidinium isothiocyanate-phenol-chloroform) have lung disease in their medical history. Individuals with abnormal BALF cytology, immunology or micro- directly extracted from the cultured cells by biomagnetic biology were not included in the control group.
separation (Dynal, Oslo, Norway) Extracted total The diagnosis of pulmonary sarcoidosis was based on RNA was stored in 50 ml of sterile deionised water with typical clinical features, together with granulomas on 40 U of human placental RNAse inhibitor (Promega, lung biopsies and supported by BALF cellular profile Madison, WI) at (/80 8C until reverse transcription; (CD4' lymphocytosis), and was compatible with the mRNA from cultured cells was reverse-transcribed criteria contained in International Statement on Sarcoi- dosis . Based on chest radiography, patients weredivided into stages (Stage 1, n 0/19; Stage 2, n 0/9; Stage 2.4.2. Reverse transcriptase-polymerase chain reaction RNA obtained from BALF cells was reverse-tran- An additional subdivision of patients with sarcoidosis scribed to complementary DNA (cDNA), primed by was established to provide an approximate index of the oligo (dT)15 (Promega) Each cDNA sample was disease course: patients requiring corticosteroid treat- amplified in PCR reactions with oligonucleotide primers ment (n 0/18) and patient with spontaneous disease specific for the sequence of PARC and b-actin genes resolution (n 0/12). Patients did not receive corticoster- . For reaction conditions of b-actin-specific PCR see oid treatment before BAL. Detailed clinical character- isation of the study groups is shown in .
The reaction mix for PARC-specific PCR contained The study was performed with the approval of the 0.5 mM of specific primers (sense 5?TCT GAC CAC Ethics Committee of Medical Faculty of Palacky´ Uni- versity and Faculty Hospital Olomouc.
AGG ATG A), 0.2 mM each dNTP, 10 mM Tris Á/HCl(pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 0.1 mg/ml gelatine, 0.3 U Taq DNA polymerase (Promega) and5 ml cDNA in a total reaction volume of 12.5 ml.
The cells were recovered from BALF by centrifuga- Amplification was performed in an MJR-Tetrad ther- tion (200g , 4 8C, 10 min). Absolute and differential mocycler (MJR, Waltham, MA, USA). PARC-specific numbers of each cell type were counted on May Á/ PCR was carried out using a modification of a versatile Gru˝nwald Á/Giemsa-stained cytocentrifuge preparations.
amplification protocol ; an optimum number of The cells were washed twice before RNA extraction in amplification cycles was ascertained after determination F. Mrazek et al. / Immunology Letters 84 (2002) 17 Á/22 Table 1Clinical and laboratory data of investigated patients and control subjects n.e., not evaluated. Data are shown as median values with interquartile range (first to third) in parentheses.
a Number of individuals in whom particular data were available.
b Treatment initiated only after BAL.
of the exponential phase of amplification The reaction resulted in one amplification product of pre- PARC mRNA was semiquantitated by normalising to the expression of the housekeeping b-actin gene. The The specificity of the PARC amplification was tested optical density ratio (ODR) of PARC/b-actin was in three randomly selected cDNA samples by seminested determined for each investigated sample. This approach PCR of 1000-fold diluted PCR product using the has been validated in the determination of mRNA antisense primer as above and an additional internal sense primer (5?GTC CCA TCT GCT ATG CC). In allthree cases, one PCR product of predicted size (158 bp) Using the statistical package STAT-100 (Biosoft, Fer- guson, MO, USA), the expression of PARC mRNA wascompared between the study groups by the Mann Á/Whitney U -test. Correlation between PARC mRNAexpression and the numbers of leukocyte subsets inBALF was tested by Spearman’s rank correlation.
Student’s paired t -test was used to investigate differ-ences in PARC mRNA expression in cultures modu-lated in vitro. A P -value less than 0.05 was consideredsignificant.
3.1. PARC mRNA expression in bronchoalveolar cells Fig. 1. Verification of the PARC RT-PCR specificity by seminestedPCR: (a) Amplification of three randomly selected cDNA samples with In order to evaluate the expression of mRNA for the PARC-specific primers (lanes 1 Á/3); (b) reamplification of 1000-fold chemokine PARC, total RNA was extracted from diluted amplicons obtained in the initial PCR (a) with additional lavage cell pellets, and reverse-transcription polymerase internal primer (lanes 1 Á/3). NC */negative control (cDNA replaced by chain reaction (RT-PCR) was performed. PARC sterile H2O). M */PCR marker (Promega): bands are from the top1000, 750, 500, 300, 150 and 50 bp.
mRNA was detected in BALF cells from 25 of 30 F. Mrazek et al. / Immunology Letters 84 (2002) 17 Á/22 (83%) patients with sarcoidosis and from 15 of 16 (94%)control subjects. The presence of PARC mRNA was notassociated with gender, and it was characterised by wideinterindividual variability. The normalised expression(ODR PARC/b-actin; median, the first to the thirdquartile range) was similar in both groups tested (S, 0.60(0.50 Á/0.95); C, 0.59 (0.36 Á/0.93)) PARC mRNAexpression did not differ between the subgroups ofpatients with sarcoidosis that required treatment andthose with spontaneous disease resolution. There wasalso no association of PARC mRNA expression withchest radiography stages of sarcoidosis (data notshown).
3.2. Correlation of PARC mRNA expression withbronchoalveolar cell numbers Fig. 3. Effect of Dx, CyA and Px on TNF-a-induced PARC mRNAexpression in cultured bronchoalveolar cells. The data are based on Because PARC is a lymphocyte attractant in vitro, we results of seven (Dx), five (CyA) and six (Px) in vitro experiments.
investigated a possible relationship of PARC mRNA PARC mRNA expression in cultures with TNF-a alone is used as a expression with BALF lymphocyte numbers. We did not reference value (normalised to 1.00). Index of inhibition for drugs (Dx, find any correlation between ODR PARC/b-actin and CyA and Px) is given as mean9/standard error of the mean. *PARCmRNA expression in cultures with TNF-a and Dx compared with the absolute or relative numbers of lymphocytes in BALF.
expression in TNF-a alone stimulated cultures, P 0/0.02.
PARC mRNA expression also did not correlate with thenumber of other BALF cell types (macrophages, neu- sion was significantly decreased in cultures treated with Dx. On average, Dx reduced PARC mRNA expressionby 49% (P 0/0.02); the level of suppression achieved in 3.3. Effect of immunomodulators on PARC mRNA individual subjects is shown in CyA and Px also induced the downregulation of PARC mRNA expres-sion in BALF cells culture: average reduction of the In order to assess whether immunomodulatory ther- expression was by 35% for CyA and 42% for Px.
apy has an effect on the expression of PARC mRNA, However, these differences did not reach statistical BALF cells were cultured in the presence of usual therapeutic levels of Dx, CyA, and Px. All culturescontained TNF-a as a potent inducer of chemokineexpression. PARC mRNA expression in cultures treatedwith TNF-a alone was used as a reference value. Asshown in TNF-a-induced PARC mRNA expres- Fig. 4. In vitro regulation of PARC mRNA expression in BALF cellsby Dx. BALF cells samples from seven individuals were cultured in the Fig. 2. Expression of PARC mRNA in bronchoalveolar cells of presence of TNF-a alone or TNF-a combined with Dx (TNF Á/ Dx).
patients with sarcoidosis (n 0/30) compared with healthy subjects Expression of PARC mRNA (ODR PARC/beta-actin) was then (n 0/16). PARC mRNA expression is given as ODR of PARC/beta- determined for each BALF cells culture. ODR values of paired actin-specific RT-PCR products. Lines, median values.
F. Mrazek et al. / Immunology Letters 84 (2002) 17 Á/22 mRNA expression in peripheral blood monocytes(PBMC) in vitro No data are available on drug In this study, we have shown that mRNA for the regulation of PARC in lung cells. Therefore, we were chemokine PARC is expressed in BALF cells of patients interested whether corticosteroids and other drugs used with sarcoidosis and healthy control subjects. PARC for the treatment of sarcoidosis can affect PARC mRNA expression was similar in patients compared to mRNA expression in BALF cells in vitro. Interestingly, control subjects without lung inflammation. Further, we we found that dexamethasone downregulates TNF-a- have demonstrated that PARC mRNA was downregu- induced PARC mRNA expression in BALF cells lated in vitro by immunomodulatory drugs that are used culture. Because non-activated PBMC do not express in the treatment of sarcoidosis, namely by dexametha- PARC in contrast to BALF cells, we suggest that different regulation of PARC in these cell compartments There have been several reports on PARC in human may explain the opposite in vitro effect of corticoster- disease to date. PARC mRNA has been identified in oids on PARC expression. A trend towards reduction of atherosclerotic plaques and, also, in macrophages PARC mRNA expression in BALF cells was also obtained from BALF of healthy smokers and of patients observed after co-culture with two other investigated with bronchial asthma . Kusano et al. found immunomodulatory drugs (CyA and Px).
increased PARC mRNA expression in the liver of PARC is induced in vitro by cytokines associated with patients with active hepatitis C infection Another a Th2-type of immune response, and inhibited by a Th1 study showed that PARC mRNA is present in homo- cytokine INF-g These regulation patterns are quite genates of human lung tissue and, also, in the alveolar different to those of another CC chemokine RANTES macrophages of healthy subjects Recently, Pardo et (CCL-5) which has been previously shown to be a major al. reported the upregulation of PARC in chronic lung attractant of lymphocytes to the lung in patients with inflammatory diseases, namely in hypersensitivity pneu- sarcoidosis We, therefore, speculate that the monitis (HP) To our knowledge, there have been cytokine response, which accompanies sarcoidosis and no data on the expression of PARC in pulmonary is able to induce RANTES, is not an adequate stimulus for the activation of PARC. In addition, PARC has The detection of PARC mRNA in the great majority been shown to exhibit an antagonistic effect on the of BALF samples investigated in our study is consistent chemokine receptor CCR3, which is one of the receptors with the concept of constitutive PARC expression in the human lung Importantly, a considerable interindi- Pardo et al. found recently increased levels of PARC vidual variability in PARC mRNA expression was in the lung of patients with HP The authors found in all groups tested. The similarity between the propose extrapolation of their results also to other median expression of PARC mRNA in patients with ILD, characterised by a lymphocyte infiltrate, particu- sarcoidosis and in control subjects suggests that this larly to sarcoidosis. In our study, we cannot confirm this chemokine does not differentially attract lymphocytes to speculation. However, there is a limitation that prevents the lung in health or disease. Our conclusion that PARC comparison of our data with the results obtained in the is not a key lymphocyte attractant in sarcoidosis is also HP study : PARC expression in HP was assessed in supported by the absence of any relationship between its samples of whole lung tissue in contrast to our data mRNA expression and the BALF lymphocyte numbers.
obtained on BALF cells. This is important in the context Though lymphocyte alveolitis typically accompanies of previous findings in inflamed liver describing site- sarcoidosis, the predominant infiltrative cells are acti- specific overexpression of PARC in mononuclear cells from lymphoid follicles of the portal area, together with Adema et al. described PARC as a selective the accumulation of naive T lymphocytes .
chemoattractant for naive (CD45RA') cells. It is, In conclusion, PARC mRNA is expressed with great therefore, possible that not PARC, but chemokines variability in unseparated BALF cells from patients with with chemotactic preference for activated T cells play pulmonary sarcoidosis and healthy subjects. Its mRNA essential role for the development of a lymphocyte expression in BALF cells is downregulated in vitro by infiltrate in pulmonary sarcoidosis.
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