The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their 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

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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 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 , 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 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 ; 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 olar macrophages and neutrophils. Animals Total cellular RNA isolation, cDNA synthesis, and amplification ϩ/GFP Heterozygous (CX3CR1 ) mice were generated on a mixed C57BL/ ϫ ϩ/GFP Total cellular RNA was isolated from sorted and highly purified (Ͼ98%) 6 129/Ola genetic background. BALB/c CX3CR1 mice were de- rived from repeated backcrosses (N9) into the BALB/c background (10). GFP-positive mononuclear phagocytes from the various treatment groups GFP/GFP ϩ/ϩ using a commercially available RNA isolation kit (Carl Roth). RNA quan- Downloaded from Parent CX3CR1 and CX3CR1 mice were bred to yield offspring ϩ/GFP tification and purity was determined on an Agilent Bioanalyzer 2100 (Agi- with the CX3CR1 genotype, which were then used in all the exper- iments at 8–12 wk of age. All mice were bred and kept under specific lent Biosystems). Only those RNA preparations exceeding absorbance ra- Ͼ pathogen free conditions with free access to food and water. All animal tios of (A260/280 nm) 1.90 were further processed for amplification and experiments were approved in accordance with the guidelines of our In- real-time RT-PCR validation experiments. Because numbers of constitu- stitutional Animal Care and Use Committee. tively alveolar recruited GFP-positive mononuclear phagocytes contained ϩ/GFP ϳ in BALFs of untreated CX3CR1 mice amounted to only 0.3–0.5% of total BALF cellular constituents (ϳ1500 cells/mouse), RNA samples http://www.jimmunol.org/ Reagents ϳ ϩ/GFP from mononuclear phagocytes of 100 untreated group 1 CX3CR1 Atlas Mouse 1.2 II nylon array membranes, BD Supersmart mRNA am- mice (10–15 mice of group 2 ϩ 3 mice) were pooled together and subse- plification kit and BD Atlas SMART fluorescent probe amplification kit quently used for RNA amplification before gene expression profiling using were purchased from BD Biosciences. Nylon membrane processing was a nylon cDNA array (12). Experimental details regarding synthesis of com- performed according to the manufacturer’s instructions. The DNA isolation plementary DNA and real-time RT-PCR validation were performed ac- and Qiaquick PCR purification kit were purchased from Qiagen. The mu- cording to recently described experimental protocols (13–15). rine CCL2 protein, the homologue of the human MCP-1 gene product (JE/MCP-1), was purchased as a recombinant protein preparation from Statistical analysis PeproTech and was routinely ascertained to be free of endotoxin as ana- The data are presented as mean Ϯ SEM. Differences in outcome variables Ͻ lyzed with the Coatest amoebocyte lysate assay (detection limit 10 pg/ between treatment groups were analyzed by one-way ANOVA followed by ml; Chromogenix). Escherichia coli LPS (0111:B4) was purchased from Scheffe’s post hoc analysis. Values of p Ͻ 0.05 were considered to be by guest on September 28, 2021 Sigma-Aldrich. Roti-Quick kit for total RNA isolation was purchased from significant. Carl Roth. SYBR green 1 kit was purchased from Eurogentec. Random hexamer primers were purchased from Boehringer Mannheim. Moloney Results murine leukemia virus-RT and recombinant RNase inhibitor were pur- ϩ/GFP chased from Promega. AmpliTaq polymerase, 10ϫ PCR buffer containing CX3CR1 mice allow tracking of circulating blood

15 mM MgCl2, and dNTPs were purchased from Applied Biosystems. mononuclear phagocytes into the alveolar compartment under DTT was purchased from Invitrogen Life Technologies. baseline and inflammatory conditions Because of their homogeneously expressed, intrinsic GFP label, Treatment of animals ϩ/GFP peripheral blood monocytes from CX3CR1 mice were easily RNA profiling of trafficking mononuclear phagocytes was performed in detectable by their increased fluorescence 1 characteristics in flow three different treatment groups: mice in group 1 were left untreated to cytometry, which is consistent with recently published reports monitor the constitutive mononuclear phagocyte trafficking into the lungs of mice in the absence of any previous manipulation. Mice in group 2 (Ref. 10; Fig. 1A). To confirm the specificity of the chosen sorting received intratracheal applications of CCL2 (50 ␮g) alone for 24 h to study gates, the gated GFP-positive peripheral blood leukocyte popula- changes in RNA expression profiles occurring in mononuclear phagocytes tion was sorted by high-speed flow cytometry combining both FSC accumulating within the lungs of mice in response to the major monocyte area vs FITC area characteristics (Fig. 1A) and fluorescence 1 chemoattractant, CCL2. Mice in group 3 received combined intratracheal applications of CCL2 (50 ␮g) in the presence of low doses of endotoxin (FITC, GFP) vs fluorescence 2 characteristics (Fig. 1B). Subse- (10 ng) for 24 h. The combined application of CCL2ϩLPS has recently quently, flow-sorted cells were reanalyzed by FACS to assess the been shown to induce an ARDS-like acute lung inflammatory response purity of the sorted cell populations, which was found to be con- with monocytes acting as facilitators of the developing neutrophilic alve- sistently Ͼ98% (Figs. 1C and 2C). Flow-sorted cells were also olitis (7, 11). All treatment protocols were done essentially as described ϩ/GFP subjected to Pappenheim staining, which clearly identified these recently (6–9, 11). Briefly, CX3CR1 mice were anesthetized with tetrazoline hydrochloride and ketamine, and the trachea was exposed by cells to consist almost exclusively of peripheral blood monocytes, surgical resection. Intratracheal instillation of CCL2 in the absence (group as judged by their typical monocytic morphology (Fig. 1D). In 2) or presence (group 3) of endotoxin (10 ng/mouse) was performed under addition, the sorted cells clearly stained positive for unspecific stereomicroscopic control using a 29 gauge Abbocath, which was inserted esterase (data not shown). into the trachea. After instillations, wounds were closed with sterile su- tures. Mice were allowed to recover from anesthesia and subsequently FACS analysis of BALF cells collected from untreated ϩ/GFP returned to their cages with free access to food and water. CX3CR1 mice consistently revealed a small but clearly iden- tifiable GFP-expressing cell population amounting to 0.3–0.5% of Collection of blood and BAL fluid (BALF) total BAL cells that was easily distinguishable from GFP-negative Twenty-four hours after intratracheal instillations, animals were sacrificed resident alveolar macrophages (Fig. 2, A and B). High-speed cell with an overdose of isoflurane (Forene; Abbott Laboratories). Blood and sorting and subsequent Pappenheim and unspecific esterase stain- BALF were collected as described earlier (6–9, 11). ing of these cells again revealed a monocytic cell population and, 1886 GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 1. Flow cytometric identification and flow sorting of peripheral blood mononuclear phagocytes of untreated, CCL2-treated, and CCL2ϩLPS- ϩ/GFP ϩ/GFP treated CX3CR1 mice. Transgenic CX3CR1 mice were either left untreated (A–D, left column) or received intratracheal instillations of CCL2 (50 ␮g/mouse, 24 h) in the absence (A–D, middle column) or presence (A–D, right column) of LPS. Twenty-four hours later, mice were killed, and peripheral blood was collected for sorting of circulating monocytes. Gating of circulating monocytes was done using three-hierarchy sort gates, as described by guest on September 28, 2021 in Materials and Methods. Population 1 (P1) in A and B (left, middle, and right dot plot) identifies the GFP-positive circulating monocyte populations in the various treatment groups. The dot plots in C (left, middle, and right) illustrate the postsort analysis of the respective monocyte populations gated by P1. The photomicrographs in D depict Pappenheim-stained cytospin preparations of the respectively sorted mononuclear phagocyte populations. PB-Mo, peripheral blood monocyte; const., constitutive. thus, to the best of our knowledge, identifies these rare cells as cytes from untreated, CCL2 alone- and CCL2ϩLPS-treated ϩ/GFP contributors to either alveolar macrophage and/or dendritic cell CX3CR1 mice. Using nylon array technology covering a set (DC) homeostasis in the absence of lung inflammation (Fig. 2D). of 1176 spotted genes, RNA expression levels assessed in alveolar The intratracheal application of recombinant murine CCL2 in recruited mononuclear phagocytes are presented as mean fold reg- the absence (or presence) of endotoxin did not affect GFP expres- ulation relative to the corresponding RNA expression levels de- sion levels and proportions of circulating mononuclear phagocytes, tected in circulating mononuclear phagocytes of the same treat- as shown by flow cytometry (Fig. 1, A–C, middle and right col- ment groups from three independent experiments. In addition, umns) but induced a strong increase in alveolar accumulating GFP- RNA level changes for selected genes of interest were validated by positive leukocytes (Fig. 2, A–C, middle and right columns), which real-time RT-PCR. is in agreement with recently published reports (6, 8, 9). As ex- Baseline alveolar mononuclear phagocyte recruitment observed ϩ/GFP pected, sorting of these CCL2-elicited, GFP-positive mononuclear in the lungs of untreated CX3CR1 mice was associated with phagocytes and subsequent Pappenheim-staining identified these only minor changes in gene expression profiles, and most of the cells as highly purified (Ͼ98%) alveolar recruited monocytic cells differentially regulated genes (n ϭ 13) were found to belong to the (Fig. 2D, middle and right photographs). These data expand pre- growth and transcription factor gene families (Table I; Fig. 3, A vious observations and clearly demonstrate the feasibility to track and B). This finding clearly suggests that the homeostatic process and purify GFP-positive mononuclear phagocytes from both intra- of constitutive mononuclear phagocyte extravasation into the al- vascular and intra-alveolar compartments under baseline and acute veolar compartment under noninflammatory conditions does not inflammatory conditions for subsequent gene expression profiling lead to a significant activation of the recruited cells. In contrast, ϩ/GFP in CX3CR1 mice. when mice received a single intratracheal instillation of the mono- cyte chemoattractant, CCL2, which is the key factor eliciting in- Inflammatory vs constitutive trafficking of mononuclear flammatory monocyte trafficking into the lung, we observed an phagocytes into the lungs of mice is associated with drastic overall ϳ6-fold increase in the number of differentially regulated changes in their gene expression profiles genes in alveolar vs circulating mononuclear phagocytes (n ϭ 66) In the current study, we compared changes in gene expression compared with constitutively migrated mononuclear phagocytes profiles of circulating vs alveolar recruited mononuclear phago- (Table I; Fig. 3A). Interestingly, most of these genes belong to the The Journal of Immunology 1887 Downloaded from http://www.jimmunol.org/

FIGURE 2. Flow cytometric identification and flow sorting of alveolar recruited mononuclear phagocytes of untreated, CCL2-treated, and CCL2ϩLPS- ϩ/GFP ϩ/GFP treated CX3CR1 mice. Transgenic CX3CR1 mice were either left untreated (A–D, left column) or received intratracheal instillations of CCL2 (50 ␮g/mouse, 24 h) in the absence (A–D, middle column) or presence (A–D, right column) of LPS. Twenty-four hours later, mice were killed, and BAL was collected for sorting of alveolar recruited mononuclear phagocytes from the various treatment groups. Gating of alveolar mononuclear phagocytes was by guest on September 28, 2021 done using three-hierarchy sort gates, as described in Materials and Methods. Population 1 (P1) in A and B (left dot plots) identifies the GFP-positive, constitutively alveolar migrated mononuclear phagocyte populations amounting to ϳ0.3–0.5% of the total BALF cellular constituents, whereas P1 in dot plot A and B of the middle and right columns depict the CCL2- and CCL2ϩLPS-elicited alveolar-accumulating mononuclear phagocytes, respectively. The P2 populations in A and B (left, middle, and right column) illustrate the resident alveolar macrophages with lower FITC-area (FITC-A) characteristics compared with alveolar mononuclear phagocytes. The populations not gated in A and B (middle and right columns) depict the fraction of CCL2-elicited alveolar lymphocytes (middle dot plot in A and B) and alveolar-recruited neutrophils (right dot plots in A and B). The dot plots in C (left, middle, and right) illustrate the postsort reanalysis of the respective mononuclear phagocytes populations gated by P1. The photomicrographs in D show Pappenheim-stained cytospin preparations of the respectively sorted mononuclear phagocytes populations. FSC-A, FSC-area. cell adhesion molecule families, including CD63 and CD68, as terized recently to induce an ARDS-like acute lung inflammation well as the growth factor, cytokine, and chemokine families. Gene (7, 16). A total of 70 genes was found to be differentially regulated expression of GRO-1 encoding the neutrophil chemoattractant KC, within this treatment group, out of which 34 genes overlapped with monocyte chemoattractant CCL2, and platelet-derived growth fac- CCL2 alone-treated mice (Table I; Fig. 3, A and B). Importantly, tor-B were found to be up-regulated in CCL2-elicited alveolar CCL2ϩLPS elicited alveolar mononuclear phagocytes again mononuclear phagocytes compared with their circulating progen- showed strongly up-regulated mRNA levels for neutrophil che- itors. CCL2-elicited alveolar mononuclear phagocytes also dem- moattractants such as KC and inflammatory protein (IP)-10, onstrated strongly elevated gene expression levels of the matrix whereas mRNA levels of chemokine receptors CX3CR1, CXCR2, metalloproteinase MMP-10 and the lysosomal cathepsins K, L, and CXCR3 were down-regulated in CCL2ϩLPS-elicited alveolar and D (Table I). In contrast, the L-specific inhibitor, mononuclear phagocytes (Table I; Fig. 3, A and B). The lysosomal cystatin F, was found to be strongly down-regulated in alveolar cathepsins K, L, and D were strongly up-regulated in CCL2ϩLPS- compared with circulating mononuclear phagocytes of the CCL2 elicited mononuclear phagocytes, whereas the inhibitor cystatin F treatment group, demonstrating that CCL2-elicited alveolar mono- was not detectable by nylon array analysis (Table I). nuclear phagocytes exhibit a differentially regulated spectrum of To validate gene expression data by an independent method, a proteolytically active enzymes with established functions in extra- selected set of interesting genes was additionally evaluated by real- cellular matrix degradation, pathogen elimination, as well as Ag time RT-PCR without any further preamplification step. As shown processing. in Table II, differential gene expression analysis by real-time RT- As anticipated, most drastic changes in gene expression profiles PCR of alveolar vs circulating mononuclear phagocytes largely of alveolar vs circulating mononuclear phagocytes were observed matched the data obtained by nylon array analysis, albeit at a much ϩ/GFP in CX3CR1 mice cochallenged with CCL2 in the presence of higher sensitivity level when compared with the nylon array ap- low levels of endotoxin. This treatment regimen has been charac- proach. This suggests that the array technique, although accurately 1888 GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES

Table I. Nylon array gene expression profiling of alveolar mononuclear phagocytesa

Mean Fold Mean Fold Mean Fold GenBank Regulation Regulation Regulation Accession no. Gene Symbol Gene Description Constitutive CCL2 CCL2 ϩ LPS

Cell surface Ags and receptors X14951 ITGB2 Integrin ␤-2 ND Ϫ1.7 Ϫ1.4 L23108 CD36 CD36 Ag ND Ϫ3.3 Ϫ3.3 U18372 CD37 Leukocyte Ag 37 ND Ϫ2.0 Ϫ2.0 D16432 CD63 CD63 Ag ND 5.3 4.6 X68273 CD68 CD68 Ag ND 1.8 ND AF043445 CD84 CD84 Ag ND ND 1.8 X65493 ICAM2 Intercellular adhesion molecule-2 ND Ϫ2.5 ND L15435 TNFSF9 Tumor necrosis factor superfamily member 9 ND 2.9 ND M16367 FCGR2 IgG Fc R II ␤ ND 2.4 ND M14215 FCGR3 IgG Fc R III ND 1.6 ND AF074912 CX3CR1 CX3C chemokine receptor 1 ND ND Ϫ2.5 D17630 CXCR2 High-affinity interleukin-8 receptor B ND ND Ϫ3.3 AB003174 CXCR3 (CD183) CXC chemokine receptor 3 ND ND Ϫ3.3 D89571 SDC4 Syndecan-4 ND 3.5 2.3 AB007599 MD-1 MD-1 protein ND ND Ϫ2.0 AJ223765 AR1 Activating receptor 1 ND Ϫ10 Ϫ5 Downloaded from Growth factors, cytokines, and chemokines S65032 BMP4 Bone morphogenetic protein 4 ND Ϫ2.5 ND M86736 GRN Granulin ND 2.9 ND J04596 GRO1 (CXCL1) Growth-regulated protein 1 Ϫ2.5 21.9 3.3 M86829 SCYB10 (CXCL10) Small inducible cytokine B10 ND ND 2.7 X53798 MIP2 (CXCL2) Macrophage inflammatory protein 2 ND 4.2 ND J04467 MCP-1 (CCL2) Monocyte chemotactic protein-1 ND 4.8 4.1 http://www.jimmunol.org/ X57413 TGFB2 Transforming growth factor ␤-2 Ϫ2.5 ND 2.4 M84453 PDGFB Platelet-derived growth factor, B chain ND 4.5 4.9 L26349 TNFRSF1A Tumor necrosis factor receptor superfamily member 1A ND Ϫ1.7 Ϫ2.0 IL and IFNs L12120 IL10R-␣ IL-10R ␣ chain ND ND Ϫ2.5 U64199 IL12R-␤2 IL-10R ␤2 chain ND Ϫ3.3 ND X53802 IL6RA IL-6 RA IL-6R ␣ chain ND ND Ϫ3.3 X01450 IL1A IL-1␣ ND 2.4 ND M15131 IL1B IL-1␤ ND 1.6 ND K00083 IFNG IFN-␥ ND Ϫ3.3 ND

Miscellaneous by guest on September 28, 2021 M24554 ANXA1 A1 ND 2.6 1.5 AJ001633 ANX3 Annexin A3 ND 3.5 ND D63423 ANX5 ND 3.2 ND X70100 KLBP Keratinocyte lipid-binding protein ND 5.3 5.6 J05020 FCERG Low-affinity IgE Fc R 1 ␥ ND ND Ϫ1.4 X63535 AXL Tyrosine-protein kinase receptor UFO ND 1.9 2.9 X12905 PFC Properdin factor complement ND 1.7 ND X03479 SAA3 Serum amyloid A-3 ND 6.6 8 M35186 Apob Apolipoprotein ND ND Ϫ2.0 D49733 LMNA Lamin A ND 2.3 3.6 M26251 VIM Vimentin ND ND 2.3 Y07919 AP1B1 Adaptor protein complex AP-1 ␤-1 subunit 2.8 ND ND D00208 S100A4 S-100 calcium binding protein A4 ND ND Ϫ1.7 M83219 S100A9 S-100 calcium binding protein A9 ND ND Ϫ5 U65586 TRF1 Telomeric repeat binding factor 1 ND ND Ϫ2.5 X93167 FN Fibronectin ND ND 3.2 M14342 PTPRC Protein tyrosine phosphatase receptor type C ND ND Ϫ3.3 M31811 MAG Myelin-associated glycoprotein ND 2.4 3.5 M57470 LGALS1 Lectin galactose-binding soluble protein 1 ND ND 1.5 Z31554 CCT4 Chaperonin subunit 4 (␦) ND ND 1.5 U27129 HSC70 Heat shock cognate 71-kDa protein 1.4 ND ND X16834 LGALS3 Lectin galactose-binding soluble protein 3 ND 2.4 ND Transcription factors AF077742 TCFEC Transcription factor TFEC ND 4.8 8.1 X03039 EIF4A1 Eukaryotic initiation factor 4A-1 ND 1.7 1.6 X52803 PPIA Peptidyl-prolyl cis-isomerase A ND Ϫ1.4 ND M31418 IFI202A IFN-activated gene 202A 3.6 ND ND U51992 ISGF3G IFN-stimulated gene factor 3 ␥ 2.8 ND ND M32489 ICSBP1 IFN concensus sequence binding protein ND Ϫ2.5 ND X65553 PABPC1 Poly A binding protein cytoplasmic 1 ND ND Ϫ2.0 V00727 FOS Cellular oncogene fos Ϫ1.4 ND ND Metabolism enzymes J04060 THBD Thrombomodulin ND ND Ϫ3.3 U11494 SNF1LK SNF1-like kinase ND ND Ϫ5 Y07708 NDUFA1 NADH-ubiquinone oxidoreductase MWFE subunit ND ND Ϫ1.7 S80446 ALOX12 Arachidonate 12-lipoxygenase ND Ϫ3.3 ND (Table continues) The Journal of Immunology 1889

Table I. Continued

Mean Fold Mean Fold Mean Fold GenBank Regulation Regulation Regulation Accession no. Gene Symbol Gene Description Constitutive CCL2 CCL2 ϩ LPS

U44389 HPGD NADϩ-dependent 15-hydroxyprostaglandin dehydrogenase ND ND Ϫ5 M60847 LPL Lipoprotein lipase ND 1.8 ND U37091 CA4 Carbonic anhydrase IV ND 2.6 2 AF082567 HEPH Hephaestin Ϫ2.0 ND ND J04627 MTHFD2 Methenyltetrahydrofolate cyclohydrolase ND ND 2.7 U90886 ARG2 Arginase II ND 6.8 4.7 M22867 BPGM 2,3-bisphosphoglycerate mutase ND Ϫ2.0 ND X53157 COX5B Cytochrome c oxidase polypeptide V ND ND 1.5 L06465 COX6A1 Cytochrome c oxidase polypeptide VIa ND Ϫ2.0 1.4 U37721 COX8 Cytochrome c oxidase polypeptide VIII ND Ϫ1.7 ND Proteases and kinases D67076 ADAMTS1 A disintegrin-like & metalloproteinase domain with Ϫ5NDND thrombospondin type 1 motif 1 X76537 MMP10 Matrix metalloproteinase 10 ND 3 ND U96696 MMP8 Matrix metalloproteinase 8 ND 2 ND Y10656 CAPN5 5 ND ND 3.4 X94444 CTSK ND 4.7 4.6 Downloaded from X06086 CTSL ND 3.7 2.3 AJ223208 CTSS ND 1.3 ND U06119 CTSH ND 1.6 ND X52886 CTSD Cathepsin D ND 1.8 4.1 AJ000990 LGMN Legumain ND 2.5 2 AF026124 PLD3 Phospholipase D3 ND 1.9 2.1 D10445 PROC Vitamin K-dependent protein C ND ND 2.8 http://www.jimmunol.org/ X13215 KLK11 Kallikrein 11 3.8 ND ND M94541 cAMP-specific PDE4D PDE4D ND ND Ϫ3.3 inhibitors AF031825 CST7 Cystatin F ND Ϫ2.5 ND U59807 CSTB Cystatin B ND 4.8 3.3 U07425 HCF2 Heparin cofactor II ND ND 4 M65736 MUG1 Murinoglobulin 1 ND 1.9 2.2 U96700 SPI6 Serine protease inhibitor 6 ND Ϫ3.3 Ϫ3.3 Functionally unclassified proteins M74425 HTR3A 5-Hydroxytryptamine 3 receptor ND ND Ϫ3.3

D50872 PAFR Platelet-activating factor receptor ND 2.4 2.4 by guest on September 28, 2021 AF077375 GALR2 Galanin receptor 2 ND ND 4.5 U05671 ADORA1 Adenosine A1 receptor ND Ϫ5ND M64298 ATP6VOC Vacuolar ATP synthase ND 2.4 1.9 L12693 CNBP Cellular nucleic acid binding protein ND ND Ϫ2.0 AB016496 ITLN Intelectin ND Ϫ5ND AF022371 IFI203 IFN-activatable protein 203 6.1 ND ND L32974 IFI49 IFN-inducible protein 49 ND ND 3.3 U43673 IL18R1 IL-18R 1 ND Ϫ3.3 Ϫ3.3 U69172 PLUNC Palate lung and nasal epithelium clone protein ND ND 2.4 AF081947 TEKT1 Tektin 1 Ϫ3.3 ND ND AF042317 CACNA1B Voltage-dependent N type calcium channel ␣ 1B subunit ND ND 3.6 Housekeeping genes X51703 UBB Ubiquitin Ϫ1.4 Ϫ2.0 Ϫ1.4 D78647 KCIP1 Protein kinase C inhibitor protein 1 ND Ϫ1.4 ND M32599 G3PDH Glyceraldehyde-3-phosphate dehydrogenase ND Ϫ1.3 ND M12481 ACTB Beta-actin ND Ϫ2.0 ND L31609 RPS29 Ribosomal protein S29 ND Ϫ2.0 Ϫ1.4

a The table shows the summary of genes differentially expressed in alveolar mononuclear phagocytes of different treatment groups. Positive values in the columns indicate mean fold up-regulation of that particular gene in alveolar mononuclear phagocytes in comparison to peripheral blood monocytes, whereas negative values indicate mean fold down-regulation of the respective gene in alveolar mononuclear phagocytes as compared with peripheral blood monocytes of the same treatment group. Lack of a given value (indicated as ND) either indicates lack of regulation or regulation on a level too low to allow its quantification. detecting trends in altered gene expression profiles, may not be mice. In line with the findings of the nylon array analysis, real-time suitable to accurately quantify low levels of gene expression in PCR confirmed a consistent decline in the mRNA levels of che- unstimulated cells, such as constitutively alveolar recruited mono- mokine receptor CX3CR1 in alveolar compared with circulating nuclear phagocytes. In fact, real-time RT-PCR analysis confirmed mononuclear phagocytes from untreated, CCL2 alone-, and ϩ ϩ/GFP that alveolar recruited compared with circulating mononuclear CCL2 LPS-treated CX3CR1 mice. In contrast, drastically ϩ/GFP phagocytes of CCL2-treated CX3CR1 mice expressed sig- increased mRNA levels of the lysosomal cathepsins B, L, D, and nificantly elevated mRNA levels of the neutrophil chemoattrac- K together with down-regulated cystatin F mRNA levels were ob- tants KC, MIP-2 (not included in the nylon array analysis), and served only in alveolar mononuclear phagocytes of the CCL2 and IP-10. Particularly, the mRNA level of the major neutrophil che- CCL2ϩLPS treatment groups. moattractant, MIP-2, was found significantly up-regulated in alve- To gain further insight into changes in pattern recognition re- olar vs circulating mononuclear phagocytes of CCL2ϩLPS-treated ceptor (PRR) signaling molecules expression profiles of alveolar 1890 GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES

Discussion In the current study, we exploited the endogenous GFP expression ϩ/GFP characteristics of transgenic CX3CR1 mice to identify and molecularly characterize mononuclear phagocytes before and after their spontaneous, as well as inflammatory, trafficking from the circulation into the alveolar compartment of the lung. Use of this novel mouse strain enabled us to identify cells with a monocytic phenotype within the alveolar compartment under baseline condi- tions, which may be involved in maintaining either alveolar mac- rophage and/or lung DC homeostasis. Combining DiVa-assisted FACS with nylon array gene expression analysis and real-time RT-PCR validation, we found that constitutively alveolar migrated mononuclear phagocytes showed a basal gene expression profile similar to circulating monocytes, including unchanged mRNA lev- els of major neutrophil chemoattractants, cell adhesion molecules, lysosomal cysteine proteases, and PRRs. The same approach re- vealed that mononuclear phagocytes recruited into the lungs of ϩ/GFP

CX3CR1 mice in response to the major monocyte chemoat- Downloaded from tractant, CCL2 or CCL2, in the presence of low levels of endotoxin demonstrated drastic changes in their gene expression profiles compared with circulating mononuclear phagocytes, as reflected by increased mRNA levels of major neutrophil chemoattractants, LPS recognition molecules such as CD14 and TLR4, matrix met-

alloproteinases, and lysosomal cysteine proteases. The combina- http://www.jimmunol.org/ tion of flow cytometric and molecular approaches in the present study reveals that the leukocyte recruitment stimulus within the alveolar compartment strongly affects the gene expression profile of the recruited leukocyte subsets. FIGURE 3. Distribution and schematic summary of genes expressed in Various studies in the past few years have identified peripheral alveolar mononuclear phagocytes of the various treatment groups. A, Venn blood monocytes of mice not only to consist of different subpopu- diagram illustrating selective and overlapping spectra of genes expressed in lations, mainly depending on their Gr-1 expression profiles (Gr- the different treatment groups, as determined by nylon array analysis. The 1high vs Gr-1low), but also to exhibit the plasticity to differentiate nonoverlapping areas represent the numbers of genes specifically ex- into alveolar macrophages and/or pulmonary DCs upon their in- by guest on September 28, 2021 pressed in the respective treatment group. B, Distribution pattern of dif- flammatory recruitment to the lungs (6, 17–19). Because the rare ferentially regulated genes belonging to different functional categories in alveolar recruited mononuclear phagocytes of the various treatment groups, cell population of GFP-positive mononuclear phagocytes detected ϩ/GFP according to nylon array analysis. f, CCL2ϩLPS treatment; Ⅺ, CCL2 in the lungs of untreated CX3CR1 mice showed a monocytic alone treatment; and u, without treatment (constitutive (const.)). morphology similar to what has been described elsewhere to re- flect monocytes/small macrophages (18) and stained positive for unspecific esterase (data not shown), it appears that these cells might contribute to alveolar macrophage homeostasis. In contrast, it cannot be excluded that these cells also contribute to the main- vs circulating mononuclear phagocytes of the various treatment tenance of pulmonary DCs, which are derived from the pool of groups, we additionally determined mRNA expression levels of circulating monocytes as well. Because of its rarity, this alveolar TLR2, TLR4, CD14, MD-1, Ly78, and syndecan-4 in the corre- cell population was not extensively characterized with respect to sponding mononuclear phagocyte populations. Interestingly, most its immunophenotype in the current study, and clarification of drastic changes in PRR mRNA levels were noted for CD14, show- whether constitutively alveolar migrated monocytic cells primarily ing highest up-regulation in CCL2-recruited and significantly less differentiate into macrophages or DCs will be subject to future increase in CCL2ϩLPS-elicited alveolar mononuclear phagocytes investigations. when compared with circulating mononuclear phagocytes, which An important finding of the present study was that genes of the is in agreement with recent data addressing CD14 cell surface key CXC chemokines KC, CXCL2 (MIP-2), and CXCL10 (IP-10) protein expression on freshly alveolar recruited mononuclear were significantly up-regulated by a magnitude ranging from 10 to phagocytes (8). Along the same line, we also found that PRR- 33 in the CCL2ϩLPS treatment group, as compared with untreated signaling molecules other than CD14, such as TLR4 and synde- animals. These data clearly support the concept that CCL2ϩLPS can-4, were significantly up-regulated in alveolar vs circulating challenge provokes neutrophil chemotactic activities in alveolar- mononuclear phagocytes recovered from CCL2- and CCL2ϩLPS- recruited mononuclear phagocytes, enabling them to act as facili- treated mice when compared with mononuclear phagocytes col- tators in the development of neutrophilic alveolitis in acute lung lected from untreated mice. Interestingly, TLR2 mRNA levels injury and, at least in part, contribute to the strong neutrophil che- were found to be slightly down-regulated in alveolar vs peripheral motactic activities observed in the BALFs of CCL2ϩLPS-treated blood mononuclear phagocytes of all three treatment groups, mice (2, 6, 7, 16). This concept is further supported by the obser- whereas expression levels of Ly78 remained largely unchanged, vation that S-100A9, a myeloid-related protein of the indicating differential regulation of various PRR molecules asso- family known to be expressed by monocytes/macrophages that ex- ciated with the recruitment of mononuclear phagocytes. erts neutrophil chemotactic activities in inflamed tissues (20–25), The Journal of Immunology 1891

Table II. Real-time RT-PCR profiling of alveolar recruited mononuclear phagocytesa

Mean Fold Gene Up-/Down-Regulation within Fold Difference in Gene Up-/Down-Regulation between Treatment Group (Mean Ϯ SEM) Treatment Groups

Constitutive vs. Constitutive vs. CCL2 vs. Gene Name/Transcript Constitutive CCL2 CCL2ϩLPS CCL2 CCL2ϩLPS CCL2ϩLPS

Cell adhesion CD18 Ϫ2.7 Ϯ 0.9 Ϫ1.2 Ϯ 0.1 Ϫ2.5 Ϯ 1.1 2.2 1.1 Ϫ2.1 Ϫ1.3 1.5 2.0 ءϪ3.9 Ϯ 1.8 Ϫ5.0 Ϯ 1.0 ءCD36 Ϫ7.5 Ϯ 2.0 CD37 Ϫ2.8 Ϯ 0.7 1.7 Ϯ 0.2 Ϫ2.5 Ϯ 1.6 4.7 1.1 Ϫ4.2 Ϯ 2.1 2.3 Ϫ1.4 Ϫ3.1 2.0 ءCD63 2.7 Ϯ 0.8 6.2 Ϯ 0.8 ءϪ7.7 2.7 ءϮ 0.1 21.5 1.3 ءCD68 Ϫ2.1 Ϯ 0.4 10.3 Ϯ 1.9 ICAM-2 Ϫ3.1 Ϯ 1.2 Ϫ1.2 Ϯ 0.1 Ϫ1.6 Ϯ 0.1 2.6 2.0 Ϫ1.3 Cytokines, chemokines, chemokine receptors, and growth factors 2.3 ء33.3 14.3 ءϮ 1.4 10.0 Ϯ 3.7 4.3 ءGRO-1 (KC) Ϫ3.3 Ϯ 0.6 ء5.9 ءϪ1.5 Ϯ 0.1 3.9 Ϯ 1.3 1.6 9.1 ءMIP-2 Ϫ2.3 Ϯ 0.2 ء2.6 ء20.5 ء7.9 ءϮ 1.8 12.9 ءCXCL 10 (IP10) Ϫ1.6 Ϯ 0.5 5.0 Ϯ 0.6 CCR2 Ϫ2.1 Ϯ 0.6 1.7 Ϯ 0.3 Ϫ3.3 Ϯ 1.5 3.5 Ϫ1.7 Ϫ5.6 ء Ϫ ء ء Ϫ Ϯ Ϫ Ϯ ء Ϫ Ϯ CX3CR1 5.0 0.8 1.3 0.1 4.6 0.9 3.9 1.1 3.4

TGF␤1 Ϫ2.4 Ϯ 0.7 Ϫ1.9 Ϯ 0.2 Ϫ2.6 Ϯ 1.0 1.3 Ϫ1.1 Ϫ1.4 Downloaded from TGF␤2 Ϫ1.1 Ϯ 0.1 Ϫ1.7 Ϯ 0.1 Ϫ2.6 Ϯ 0.8 Ϫ1.5 Ϫ2.5 Ϫ1.6 Proteases 1.2 ء6.3 ءϮ 0.2 1.7 Ϯ 0.2 5.2 1.4 ءMMP-10 Ϫ3.7 Ϯ 0.8 ءϪ14.3 4.5 ءϮ 0.1 65.2 1.4 ءCathepsin B Ϫ3.2 Ϯ 1.3 20.2 Ϯ 3.2 Ϫ2.3 5.4 ء12.2 ءϮ 2.5 16.2 ءCathepsin L 3.0 Ϯ 1.1 36.7 Ϯ 10.7 ءϪ1.7 Ϫ4.7 ء2.8 ءϮ 0.9 4.6 ءϮ 4.4 22.1 ءCathepsin D 7.8 Ϯ 2.2 ءϪ1.7 Ϫ4.2 ءϮ 0.1 2.5 1.2 ءCathepsin S 2.0 Ϯ 0.2 5.0 Ϯ 0.7 /http://www.jimmunol.org 1.2 3.4 2.8 ءϮ 7.0 43.3 ءϮ 12.1 35.7 ءCathepsin K 12.8 Ϯ 4.2 PRR signaling Ϫ1.1 2.1 2.3 ءϮ 1.3 4.3 ءϮ 1.1 4.8 ءTLR4 2.1 Ϯ 0.1 Ϫ3.5 Ϯ 1.1 Ϫ2.0 Ϫ1.3 1.5 ءϪ5.4 Ϯ 1.5 ءTLR2 Ϫ2.6 Ϯ 0.2 ءϪ2.5 ء5.6 ء14.0 ءϮ 1.9 18.9 ءCD14 3.4 Ϯ 1.0 47.6 Ϯ 4.8 ءϪ4.8 1.6 ءMD-1 Ϫ3.6 Ϯ 1.3 2.1 Ϯ 0.7 Ϫ2.2 Ϯ 0.3 7.5 Ly Ag 78 Ϫ2.5 Ϯ 0.6 Ϫ1.6 Ϯ 0.1 Ϫ2.2 Ϯ 0.6 1.6 1.1 Ϫ1.4 ءϪ3.0 ء13.5 ء40.8 ءϮ 0.3 3.1 ءSyndecan-4 Ϫ4.4 Ϯ 1.6 9.4 Ϯ 0.4 Proinflammatory ءϪ5.6 135.8 ء770.7 ءϮ 8.3 61.1 ءSerum amyloid A3 Ϫ2.2 Ϯ 0.8 346.8 Ϯ 25.4

by guest on September 28, 2021 ءϪ2.7 1.6 ءϮ 0.2 Ϫ1.7 Ϯ 0.5 4.2 1.6 ءCyclophilin A Ϫ2.6 Ϯ 0.3 1.1 ء10.6 ء9.7 ءTNF-␣ Ϫ3.0 Ϯ 1.0 3.2 Ϯ 1.0 3.5 Ϯ 0.5 Ϫ2.4 ء7.7 ءϪ1.8 Ϯ 0.8 Ϫ4.3 Ϯ 1.5 18.7 ءS-100A9 Ϫ39.0 Ϯ 7.2 Protease inhibitors 2.8 ء4.7 1.7 ءϪ2.4 Ϯ 0.1 ءϪ6.7 Ϯ 1.7 ءCystatin F Ϫ11.0 Ϯ 1.5 Miscellaneous 1.3 ء3.6 ءϪ2.5 Ϯ 0.7 Ϫ2.0 Ϯ 0.1 2.9 ءAquaporin 1 Ϫ7.4 Ϯ 1.3

a Expression profiles of selected genes belonging to different functional categories were quantified in alveolar mononuclear phagocytes of the different treatment groups by real-time RT-PCR, as outlined in Materials and Methods. The values are given as mean fold regulation within groups, i.e., gene expression level in alveolar compared with circulating monocytes of the same treatment group set to 1 indicates no regulation, and values Ͻ 1(Ͼ 1) indicate respective down-regulation (up-regulation) of a given gene in alveolar compared with circulating monocytes, columns 2–4 from the left). In addition, values are also expressed as mean fold change between groups (comparative gene expression level in alveolar mononuclear phagocytes of different groups, columns 5–7 from the left, as indicated). Positive values in the columns indicate up-regulation of that particular gene, whereas negative values indicate the down-regulation of a given gene. The given values represent the mean Ϯ SEM from at least three independent experiments .indicates p at least Ͻ0.05 ء .performed was also found to be significantly up-regulated in alveolar mono- molecules in alveolar mononuclear phagocytes supports the con- nuclear phagocytes of CCL2 alone and CCL2ϩLPS-treated cept that freshly recruited mononuclear phagocytes may signifi- ϩ/GFP CX3CR1 mice. cantly contribute to the orchestration of acute lung inflammation. Our study also addressed changes in gene expression levels of In this regard, the observed weak changes in mononuclear cell several PRR and associated signaling molecules, including TLR2 gene expression levels of the PRR MD-1 may well reflect the fact and TLR4, CD14, MD1, and syndecan 4. Most dramatic changes that only a 24-h time point posttreatment was analyzed. were seen in mRNA levels of TLR4, syndecan-4, and CD14. The Interestingly, we found significantly increased alveolar mRNA strong up-regulation of CD14 (26, 27) by CCL2-elicited mononu- levels of lysosomal cathepsins together with decreased cystatin F clear phagocytes confirms earlier reports from our group and pos- mRNA levels in mononuclear phagocytes of the CCL2 and sibly reflects a priming of monocytes during the recruitment pro- CCL2ϩLPS treatment groups compared with untreated mice. cess with potential relevance for the pulmonary host defense (8). Cathepsins play important roles in phagolysosomal degradation, Syndecan-4, a transmembrane heparan sulfate proteoglycan of the Ag processing, and tissue remodelling by professional phagocytes, syndecan family, was even more dramatically up-regulated in al- including mononuclear phagocytes. These proteolytic enzymes veolar vs circulating mononuclear phagocytes of CCL2 and have also been suggested to assist mononuclear cell transmigration CCL2ϩLPS-treated but not untreated mice. Recently, Muramatsu (30–33). Cystatin F belongs to the superfamily of endogenous in- and colleagues (28, 29) demonstrated that syndecan-4Ϫ/Ϫ mice tracellular thiol proteinase inhibitors (34–36). The reported find- lack susceptibility to endotoxic shock. Thus, the strong up-regu- ings of reciprocal expression patterns of cathepsins vs cystatins in lation of syndecan-4 together with other important LPS-signaling alveolar recruited mononuclear phagocytes of the CCL2 and 1892 GENE EXPRESSION PROFILING OF ALVEOLAR MONOCYTES

CCL2ϩLPS treatment groups support the potentially important In summary, the relative low proportion of circulating mono- contribution of these freshly immigrated cells in lung inflamma- cytes and the lack of sophisticated purification methods in mice are tory remodeling processes. Interestingly, increased the main reasons why the proinflammatory molecular phenotype of and K mRNA levels were also found to be elevated in mononu- this important leukocyte population is only poorly defined. In the clear phagocytes in bleomycin-induced lung injury in rats and current study, we demonstrate the feasibility to track, sort to high mice (37, 38). In addition, our study shows that the genes for purity and transcriptionally profile peripheral blood and alveolar ϩ/GFP cathepsins L, D, and K were among those few genes that were mononuclear phagocytes of CX3CR1 mice without prior in up-regulated even in alveolar mononuclear phagocytes compared vivo manipulation. Based on this technique, this is the first study with peripheral blood monocytes of untreated mice, indicating that to identify and sort constitutively trafficking alveolar mononuclear constitutive trafficking of monocytic cells into the lungs to some phagocytes. Using gene array and real-time RT PCR we observed extent alters the cathepsin gene expression pattern in these cells. that the gene expression patterns of these cells differs drastically We and others recently demonstrated that F4/80-positive periph- from that observed in CCL2-driven alveolar mononuclear phago- eral blood monocytes of mice consist of two principal subpopula- cytes recruitment. These findings further support the concept that tions, Gr-1high and Gr-1low (6). Because these subsets were found constitutive mononuclear phagocyte trafficking is tightly con- to be equally recruited into the lungs of mice upon CCL2ϩLPS trolled to avoid inflammatory activation and may be linked to dif- treatment, the currently used treatment regimen to recruit mono- ferent molecular pathways enabling migration processes. In addi- ϩ/GFP tion, our gene expression profile analysis shows that inflammatory- nuclear phagocytes into the lungs of CX3CR1 mice did not allow us to specifically monitor subset-specific changes in gene elicited mononuclear phagocytes activate genetic programs that expression profiles between Gr-1high vs Gr-1low monocyte subsets. render them potent contributors to the overall acute lung inflam- Downloaded from However, a selective recruitment of Gr-1high but not Gr-1low matory response. Future studies using selective knockout mouse models and gene silencing techniques, as well as protocols to spe- CX3CR1-expressing monocyte subsets was recently observed to occur in a model of thioglycolate-induced peritonitis (19). These cifically purify monocyte subpopulations, will help to further re- data suggest that differences in experimental models and organs fine the role of distinct gene products in subsets of mononuclear investigated and/or inflammatory stimuli applied may possibly af- phagocytes and their relative contribution to the lung inflammatory fect the recruitment profiles of Gr-1high vs Gr-1low monocyte sub- response to microbial challenge. http://www.jimmunol.org/ sets into tissues, which may have implications on the overall in- flammatory response. Acknowledgments Various steps in the recruitment process may potentially con- We are grateful to the excellent technical support by Regina Maus, tribute to the activation of genetic programs in recruited mononu- Petra Janssen, and Marlene Stein in preparing samples for flow cytometric sorting of mononuclear phagocytes and in lung histology experiments. clear phagocytes, particularly 1) the CCL2-CCR2 interaction itself driving inflammatory monocyte recruitment, 2) additional molec- ular interactions during mononuclear phagocyte transendo/-epithe- Disclosures The authors have no financial conflict of interest. lial migration, and 3) exposure of the recruited cells to an inflam- by guest on September 28, 2021 matory altered alveolar microenvironment. In vitro exposure of isolated monocytes to CCL2 is known to activate genetic programs References 1. Goodman, R. B., R. M. Strieter, D. P. Martin, K. P. Steinberg, J. A. Milberg, in monocytes but to a much more limited extent than that seen after R. J. Maunder, S. L. Kunkel, A. Walz, L. D. Hudson, and T. R. Martin. 1996. CCL2-driven monocyte recruitment to the alveolar space in vivo Inflammatory cytokines in patients with persistence of the acute respiratory dis- (unpublished data). Therefore, we believe that additional activa- tress syndrome. Am. J. Respir. Crit. Care Med. 154: 602–611. 2. Rosseau, S., P. Hammerl, U. Maus, H. D. Walmrath, H. Schu¨tte, F. Grimminger, tion signals that operate during the recruitment process are likely W. Seeger, and J. Lohmeyer. 2000. Phenotypic characterization of alveolar to play a key role in determining the phenotype of alveolar re- monocyte recruitment in acute respiratory distress syndrome. Am. J. Physiol. 279: cruited mononuclear phagocytes, particularly in the CCL2 alone- L25–L35. 3. Belperio, J. A., M. P. Keane, M. D. Brudick, J. P. Lynch, III, Y. Y. Xue, treated group, which showed no significant changes in BALF cy- A. Berlin, D. J. Ross, S. L. Kunkel, I. F. Charo, and R. M. Strieter. 2001. Critical tokine profiles compared with untreated animals (16). The fact that role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis oblit- erans syndrome. J. Clin. Invest. 108: 547–556. several of the investigated genes, including cathepsins B and L, 4. Smith, R. E., R. M. Strieter, K. Zhang, S. H. Phan, T. J. Standiford, N. W. Lukacs, were found to be less fold up-regulated in alveolar mononuclear and S. L. Kunkel. 1995. A role for C-C chemokines in fibrotic lung disease. phagocytes of the CCL2ϩLPS as compared with the CCL2 alone- J. Leukocyte Biol. 57: 782–787. 5. Bhalla, K. S., and R. J. Folz. 2002. Idiopathic pneumonia syndrome after syn- treated group might indicate a specific role of the alveolar mi- geneic bone marrow transplant in mice. Am. J. Respir. Crit. Care Med. 166: cromilieu differentially modulating the expression kinetics of the 1579–1589. 6. Maus, U., K. von Grote, W. A. Kuziel, M. Mack, E. J. Miller, J. Cihak, investigated genes, including the cathepsins. Indeed, LPS activa- M. Stangassinger, R. Maus, D. Schlo¨ndorff, W. Seeger, and J. Lohmeyer. 2002. tion of alveolar mononuclear phagocytes (particularly monocytes) The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil im- may accelerate their transdifferentiation toward a “macrophage ge- migration in intact mice. Am. J. Respir. Crit. Care Med. 166: 268–273. 7. Maus, U. A., K. Waelsch, W. A. Kuziel, T. Delbeck, M. Mack, T. S. Blackwell, notype” as is evident by the drastic up-regulation of cathepsin K J. W. Christman, D. Schlo¨ndorff, W. Seeger, and J. Lohmeyer. 2003. Monocytes transcript levels in the CCL2ϩLPS treatment group. LPS activa- are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis. J. 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