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Profiles Drastic Changes in Their Gene Expression the Alveolar The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression This information is current as Profiles of September 28, 2021. Mrigank Srivastava, Steffen Jung, Jochen Wilhelm, Ludger Fink, Frank Bühling, Tobias Welte, Rainer M. Bohle, Werner Seeger, Jürgen Lohmeyer and Ulrich A. Maus J Immunol 2005; 175:1884-1893; ; Downloaded from doi: 10.4049/jimmunol.175.3.1884 http://www.jimmunol.org/content/175/3/1884 http://www.jimmunol.org/ References This article cites 39 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/175/3/1884.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression Profiles1 Mrigank Srivastava,*¶ Steffen Jung,‡ Jochen Wilhelm,† Ludger Fink,† Frank Bu¨hling,§ Tobias Welte,¶ Rainer M. Bohle,† Werner Seeger,* Ju¨rgen Lohmeyer,* and Ulrich A. Maus2*¶ Mononuclear phagocytes enter the lungs both constitutively to maintain alveolar macrophage and dendritic cell homeostasis, as /؉ well as during lung inflammation, where the role of these cells is less well defined. We used a transgenic mouse strain (CX3CR1 Downloaded from GFP) that harbors a GFP label in circulating monocytes to identify and sort these cells from the vascular and alveolar compart- ments under both constitutive and acute lung inflammatory conditions. Using nylon arrays combined with real-time RT-PCR for gene expression profiling, we found that flow-sorted, highly purified mononuclear phagocytes recruited to acutely inflamed mouse lungs showed strongly up-regulated mRNA levels of the neutrophil chemoattractants KC, MIP-2, and IP-10, which contrasted with alveolar mononuclear phagocytes that immigrated in steady state. Similar observations were made for the lysosomal cathepsins B, L, and K being strongly up-regulated in mononuclear phagocytes upon recruitment to inflamed lungs but not during consti- tutive alveolar immigration. Inflammatory elicited mononuclear phagocytes also demonstrated significantly increased mRNA http://www.jimmunol.org/ levels of the cytokine TNF-␣ and the PRR-associated molecules CD14, TLR4, and syndecan-4. Together, inflammatory elicited mononuclear phagocytes exhibit strongly increased neutrophil chemoattractants, lysosomal proteases, and LPS signaling mRNA transcripts, suggesting that these cells may play a major role in acute lung inflammatory processes. The Journal of Immunology, 2005, 175: 1884–1893. ononuclear phagocytes are known to contribute to in gene expression profiles of these cells transmigrating from the both acute and chronic inflammatory diseases of the vascular into the alveolar compartment under baseline vs inflam- lung, including acute respiratory distress syndrome matory conditions. by guest on September 28, 2021 M3 (ARDS) , bronchiolitis obliterans, and idiopathic pneumonia syn- Previous studies from our laboratory made use of the lipophilic drome (1–5). In addition, we recently demonstrated that mono- intravital dye, PKH26-PCL, to discriminate resident alveolar mac- cytes may act as regulators of the neutrophilic response in a mouse rophages (strongly PKH26 positive) from newly alveolar-recruited model of acute lung inflammation (6, 7). However, the molecular monocytic cells (PKH26 dull) to study their recruitment pathways mechanisms and potential candidate genes that might regulate the during lung inflammation (6, 8, 9). However, this method did not accessory function of mononuclear phagocytes in acute lung in- stain circulating blood monocytes to allow their subsequent puri- flammation have not been characterized. The lack of potent puri- fication for molecular and functional characterization. Therefore, fication protocols allowing the isolation of monocytic cells from in the current study, we made use of a novel transgenic mouse both peripheral blood and alveolar compartments may partially ϩ strain (CX CR1 /GFP) that allows the identification and subse- explain the lack of currently available data addressing the changes 3 quent FACS of both circulating and alveolar recruited mononu- clear phagocytes to determine changes in their gene expression *Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine profiles during their recruitment into the alveolar compartment un- and †Department of Pathology, Justus-Liebig-University, Giessen, Germany; ‡De- der both baseline and acute lung inflammatory conditions. In partment of Immunology, The Weizmann Institute of Science, Rehovot, Israel; ϩ/GFP §Institute of Immunology, Otto-von-Guericke University, Magdeburg, Germany; CX3CR1 mice, one allele for the gene encoding CX3CR1, and ¶Department of Pulmonary Medicine, Hannover School of Medicine, Hannover, the receptor for the membrane-tethered chemokine fractalkine Germany (CX3CL1, Fkn) is replaced by the gene encoding GFP (10). Be- Received for publication March 24, 2005. Accepted for publication May 9, 2005. cause the transgene GFP is under the control of the CX3CR1 gene The costs of publication of this article were defrayed in part by the payment of page promoter and because CX CR1 is homogeneously expressed on charges. This article must therefore be hereby marked advertisement in accordance 3 with 18 U.S.C. Section 1734 solely to indicate this fact. circulating monocytes but not on resident alveolar macrophages, ϩ/GFP 1 This work was supported by German Research Foundation Grant 547 “Cardiopul- CX3CR1 mice were used in the current study to track, sort, monary Vascular System” and the National Network on Community-Acquired Pneu- and genotype mononuclear phagocytes during their migration into monia (CAPNETZ). S.J. is a Scholar of the Benoziyo Center for Biomolecular Medicine. the alveolar air space and, at the same time, allowing their clearcut 2 Address correspondence and reprint requests to Dr. Ulrich A. Maus, Department of discrimination from differentiated, resident alveolar macrophages Pulmonary Medicine, Hannover School of Medicine, Feodor-Lynen Strasse 21, Han- and most other inflammatory elicited leukocyte subsets. nover 30625, Germany. E-mail address: [email protected] DiVa-assisted FACS analysis of bronchoalveolar lavage (BAL) 3 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; BAL, fluid cells collected from CX CR1ϩ/GFP mice enabled us for the bronchoalveolar lavage; BALF, BAL fluid; FSC, forward scatter; DC, dendritic cell; 3 IP, inflammatory protein; PRR, pattern recognition receptor. first time to detect, sort, and transcriptionally profile constitutively Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 1885 migrated alveolar mononuclear phagocytes under noninflamma- Flow cytometry tory conditions. In addition, we demonstrate that alveolar mono- A high-throughput FACSVantage SE flow cytometer (BD Biosciences) nuclear phagocyte recruitment in response to the monocyte che- equipped with a DiVa sort option and an argon ion laser operating at 488 moattractant, CCL2, is associated with profound changes in their nm excitation wavelength and a laser output of 200 mW were used for the gene expression profiles. Finally, we provide evidence that mono- sorting of peripheral blood and BALF mononuclear phagocytes. Blood and ␮ nuclear phagocytes recruited into the lungs of mice in response to BALF specimen were filtered through a 40- m cell strainer (BD Bio- sciences) before cell sorting. Flow cytometric data of GFP-positive periph- CCL2 in the presence of low endotoxin challenge exhibit an acti- eral blood and alveolar mononuclear phagocytes from the various treat- vated, highly proinflammatory genotype, characterizing these cells ment groups were acquired on five-decade log-scale dot plots displaying as powerful cellular contributors of the lung inflammatory re- forward scatter (FSC) area vs side scatter area and fluorescence 1 area vs sponse. The current technical approach may help to identify can- fluorescence 2 area characteristics, respectively. First, hierarchy sort gates were set in FSC vs side scatter dot plots to exclude lymphocytes; second, didate genes regulating the functional role of mononuclear phago- hierarchy sort gates specific for GFP-expressing mononuclear phagocytes cytes in both acute and chronic lung inflammatory conditions. were set according to FSC area vs FL1 (F525 Ϯ 15 nm; FITC/GFP) char- acteristics; and third, hierarchy sort gates were set according to FL1 vs FL2 characteristics (F575 Ϯ 25 nm), thus allowing the exclusion of both alve- Materials and Methods
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