Lung Macrophages Serve as Obligatory Intermediate between Blood Monocytes and Alveolar Macrophages This information is current as Limor Landsman and Steffen Jung of September 28, 2021. J Immunol 2007; 179:3488-3494; ; doi: 10.4049/jimmunol.179.6.3488 http://www.jimmunol.org/content/179/6/3488 Downloaded from References This article cites 49 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/6/3488.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Lung Macrophages Serve as Obligatory Intermediate between Blood Monocytes and Alveolar Macrophages1 Limor Landsman and Steffen Jung2 Alveolar macrophages are a unique type of mononuclear phagocytes that populate the external surface of the lung cavity. Early studies have suggested that alveolar macrophages originate from tissue-resident, local precursors, whereas others reported their derivation from blood-borne cells. However, the role of circulating monocytes as precursors of alveolar macrophages was never directly tested. In this study, we show through the combined use of conditional cell ablation and adoptive cell transfer that alveolar macrophages originate in vivo from blood monocytes. Interestingly, this process requires an obligate intermediate stage, the differentiation of blood monocytes into parenchymal lung macrophages, which subsequently migrate into the alveolar space. We also provide direct evidence for the ability of both lung and alveolar macrophages to proliferate. The Journal of Immunology, 2007, 179: 3488–3494. Downloaded from he constant exposure to environmental microbial chal- from the BM (21). As for the origin of alveolar M⌽, although they lenges renders the respiratory tract one of the major sites clearly belong to the hemopoietic lineage, a direct connection to T of primary viral and bacterial infections (1). Highlighting monocytic precursors remains to be shown (22). Moreover, several this state of permanent alert, the lung, which comprises paren- studies published during the 1970s and 1980s reported either cir- chyma and alveolar space, is seeded with numerous mononuclear culating or local precursor for alveolar M⌽, depending on the http://www.jimmunol.org/ phagocytes, including dendritic cells (DC)3 and macrophages method used (for review, see Ref. 22). Based on kinetic studies, (M⌽) (1, 2). In steady state, M⌽ are the major cell type in the Bowden and Adamson (23) suggested, for instance, a dual origin alveolar space, representing ϳ90% of its hematopoietic cellular of alveolar M⌽: from blood-borne precursor and from proliferat- content (1) and playing a key role in its clearance from pulmonary ing cells in the lung parenchyma. With the discovery of alveolar pathogens and dying cells (3). Pulmonary M⌽, however, are poor DC (24), it became, however, unclear whether this study referred T cell stimulators (4, 5), and have even been shown to suppress to two distinct cell types, i.e., alveolar DC and M⌽. In addition, lung inflammations, probably by inhibiting DC function (4, 6–10). the alveolar M⌽ population was reported to remain unaffected by Alveolar M⌽ are bone marrow (BM)-derived cells (11, 12). depletion of blood monocytes (25), leading to the conclusion that Interestingly, however, after whole body irradiation and engraft- alveolar M⌽ are not monocyte derived (26). Because alveolar M⌽ by guest on September 28, 2021 ment, their replacement by donor BM cells takes considerably are eventually replaced by BM-derived cells (11), the existence of longer than that of most other cells of the hematopoietic system circulating precursor was, however, not ruled out. Taken together, (13, 14). Depending on the irradiation protocol used, the time re- the identity of the alveolar M⌽ precursor, as well as its own origin, quired for the complete exchange of alveolar M⌽ has been re- remains to be revealed (22). ported to vary from several weeks up to 1 year (11, 13–16). This Through an adoptive monocyte transfer approach, we recently delay in alveolar M⌽ replacement by BM-derived cells is dis- showed that parenchymal lung M⌽ originate in vivo from blood cussed as one of the critical causes for the sensitivity of BM trans- monocytes (27). Furthermore, we reported that only one of the two low high Ϫ plantation patients to pulmonary infections (17, 18). major murine monocyte subsets, the Gr1 CX3CR1 CCR2 Monocytes are BM-derived circulating phagocytes that are con- cells (28), harbors the immediate potential to give rise to paren- sidered to be the in vivo M⌽ precursors (19, 20). However, spe- chymal lung M⌽ (27). In this study, we extended these studies and cific M⌽ populations, such as the brain microglia, have been investigated the in vivo origin of alveolar M⌽ using a combination shown to originate from local precursors, without persisting input of conditional cell ablation and reconstitution. We show that grafted blood monocytes can give rise to alveolar M⌽. However, we provide evidence that alveolar M⌽ do not originate directly Department of Immunology, Weizmann Institute of Science, Rehovot, Israel from blood monocytes, but require a parenchymal lung M⌽ inter- Received for publication April 17, 2007. Accepted for publication July 3, 2007. mediate. Lastly, we directly show that both lung and alveolar M⌽ The costs of publication of this article were defrayed in part by the payment of page can undergo proliferation. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the MINERVA Foundation. S.J. is the incumbent of the Pauline Recanati Career Development Chair and a scholar of the Benoziyo Center for Materials and Methods Molecular Medicine. Mice 2 Address correspondence and reprint requests to Dr. Steffen Jung, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: This study involved the use of the following C57BL/6 mouse strains: [email protected] CD11c:diphtheria toxin (DTx) receptor (DTR) transgenic mice (B6.FVB- 3 Tg(Itgax-DTR/GFP)57Lan/J) that carry a DTR transgene under the murine Abbreviations used in this paper: DC, dendritic cell; BAL, bronchoalveolar lavage; GFP cd11c promotor (29); CX3CR1 mice harboring a targeted replacement BM, bone marrow; DTR, diphtheria toxin receptor; DTx, diphtheria toxin; int, inter- ␮ mediate; i.t., intratracheal; M⌽, macrophage; PCNA, proliferating cell nuclear Ag; of the cx3cr1 gene by a GFP reporter (30); and MT mice (C57BL/6-IgH- wt, wild type. 6tmlCgn) lacking the membrane exon of the Ig ␮-chain gene, and therefore are B cell deficient (31). Mice were backcrossed with CD45.1 mice Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 (B6.SJL-Ptprca Pep3b/BoyJ) when indicated. All mice were maintained www.jimmunol.org The Journal of Immunology 3489 under specific pathogen-free conditions and handled under protocols ap- proved by the Weizmann Institute Animal Care Committee, according to international guidelines. Cell isolations Mice were sacrificed, and blood was collected from main artery and sub- jected to a Ficoll density gradient (Amersham) to remove erythrocytes and neutrophils. For bronchoalveolar lavage (BAL), the trachea was exposed to allow insertion of a catheter, through which the lung was filled and washed four times with 1 ml of PBS without Ca2ϩ/Mg2ϩ. Lung parenchyma was then collected and digested with 4 mg/ml collagenase D (Roche) for1hat 37°C, followed by incubation with ACK buffer to lyse erythrocytes. All isolated cells were suspended in PBS supplemented with 2 mM EDTA, 0.05% sodium azide, and 1% FCS. Flow cytometric analysis The following fluorochrome-labeled mAbs were purchased from eBio- science and used according to manufacturer’s protocols: PE-conjugated anti-CD11c and anti-CD115 Abs, allophycocyanin-conjugated streptavidin and anti-CD11b Ab, and biotin-conjugated anti-CD45.1, CD11c, CD115, and MHC II Abs. PerCP-conjugated anti-CD11b Ab was purchased from BD Pharmingen. PE-labeled anti-proliferating cell nuclear Ag (PCNA) Ab Downloaded from was purchased from DakoCytomation and used according to the manufac- turer’s protocol for S phase-specific staining, with modifications. Before staining with the anti-PCNA Ab, cells were incubated with indicated bi- otin-conjugated Abs, followed by methanol fixation. Fixed cells were then incubated with streptavidin-fluorochrome conjugates and stained for PCNA. Cells were analyzed on a FACSCalibur cytometer (BD Bio- sciences) using CellQuest software (BD Biosciences). http://www.jimmunol.org/ ⌽ Cell transfers FIGURE 1. In vivo depletion and reconstitution of pulmonary M . Flow cytometric analysis of collagenase-digested lung and BAL fluid of For blood monocyte transfers, ϳ20 mice were sacrificed and bled to collect CD11c:DTR mice. A, Depletion of lung and alveolar M⌽ of CD11c:DTR an average of 18 ml of blood. Erythrocytes and neutrophils were removed mice upon DTx treatment. On day 0, mice were treated i.t. with either PBS by a Ficoll density gradient, and cells were washed and exposed to biotin- or DTx (100 ng) or left untreated. BAL and lung cells were isolated on day conjugated anti-CD115 Ab (eBioscience), followed by incubation with 1 and stained with PE-coupled anti-CD11c Ab and allophycocyanin-cou- strepavidin-conjugated MACS beads (Miltenyi Biotec).