Alveolar Macrophages Intermediate Between Blood Monocytes And

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

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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 © 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 manufacturer’s protocol for S phase-specific staining, with modifications. Before staining with the anti-PCNA Ab, cells were incubated with indicated biotin-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). Cells were then ϩ pled anti-CD11b Ab. The percentage of M⌽ (identified as CD11c magnetically separated according to manufacturer protocol. The positive Ϫ fraction was collected and i.v. injected to recipient mice. For mixed BM CD11b cells, as indicated by gates) in the lung parenchyma and BAL of chimeras, recipient mice were irradiated with a lethal dose (950 rad) and 1 each mouse was determined. Numbers indicated the percentage of gated day later i.v. injected with 5 ϫ 106 donor BM cells. cells. B, Depletion of pulmonary M⌽ is followed by their rapid reconsti- by guest on September 28, 2021 tution. CD11c:DTR mice were i.t. treated on day 0 with 100 ng of DTx and Intratracheal instillation analyzed on days 0, 1, 2, 4, 6, and 9 for the number of CD11cϩCD11bϪ ⌽ E f PBS (80 ␮l) containing either DTx (List Biological Laboratories; catalogue M (as described in A) in their lung parenchyma ( ) and BAL ( ) fluids. 150) or without addition was applied to the tracheae of mice, as previously Graph shows absolute lung and alveolar M⌽ numbers on analyzed days. described, with modifications (32). Briefly, mice were lightly anesthetized n ϭ 3 for each time point. Data show one representative of two indepen- using isofluorane and placed vertically, and their tongues were pulled out. dent experiments. Using a long-nasal tip, liquid was placed at the top of trachea and actively aspirated by the mouse. Gasping of treated mice verified liquid application to the alveolar space.

Results treatment their amount almost reached initial levels. Lung M⌽,in Depletion of lung and alveolar M⌽ results in their rapid contrast, continued to decline, reaching their lowest value on day reconstitution 4, after which also their numbers recovered. By day 9, lung M⌽ Murine pulmonary DC and M⌽, both in the lung parenchyma and were restored to their initial levels, whereas at that time the size of ⌽ the alveolar space, express the ␤-integrin CD11c. However, the alveolar M population was significantly enlarged over steady B whereas pulmonary DC are also positive for MHC II, CD11b, and state (Fig. 1 ). CX CR1, M⌽ have been defined as low for MHC II and negative These results indicate that, following their conditional depletion, 3 both lung and alveolar M⌽ populations are rapidly reconstituted, for CD11b and CX3CR1 (5, 27, 33, 34). CD11c:DTR transgenic mice allow for the conditional ablation albeit with distinct kinetics. of pulmonary DC and M⌽ (27, 35). This ablation system exploits the fact that murine cells are generally resistant to DTx because Adoptively transferred blood monocytes can give rise to ⌽ they lack a high-affinity DTR required for toxin entry into the cell alveolar M (36). CD11c:DTR transgenic mice express a primate DTR under Elsewhere, we have shown that grafted blood monocytes give rise the control of the cd11c promoter, resulting in sensitivity of to lung M⌽ in M⌽-depleted, but not untreated, recipients (27). CD11c-expressing cells to DTx-induced cell death (29). Hence, Four days after transfer, monocyte graft-derived M⌽ could be when applied intratracheally (i.t.) to CD11c:DTR transgenic mice, readily observed in recipient lungs, whereas we failed to detect DTx causes the depletion of lung and alveolar CD11cϩ cells, in- graft descendants in the alveolar space (27). This result could sup- cluding both DC and M⌽ (27, 35) (Fig. 1A). port the reported notion that blood monocytes do not give rise to One day following DTx treatment, lung and alveolar M⌽ num- alveolar M⌽ (25). Alternatively, the kinetics of monocyte differ- bers were dramatically reduced (Fig. 1B). The decrease in alveolar entiation into alveolar M⌽ might vary from their differentiation M⌽ was followed by their rapid reconstitution, and 2 days after the into lung M⌽, precluding detection of monocyte-derived alveolar 3490 ALVEOLAR M⌽ ORIGIN

kinetics. Interestingly, our data suggest that lung M⌽ begin to be replaced by donor BM cells only after exchange of the blood monocyte compartment, and alveolar M⌽ start to be replaced only after full reconstitution of parenchymal lung M⌽ (Fig. 3B).

Alveolar M⌽ do not originate directly from blood monocytes The DTx-induced ablation of alveolar M⌽ in CD11c:DTR transgenic mice was followed by their rapid reconstitution (Fig. 1B). To investigate the origin of these newly generated alveolar M⌽,we transferred CD45.1 wt BM into irradiated CD11c:DTR transgenic recipients (CD45.2). The resulting wt3CD11c:DTR BM chimeras allow the conditional depletion of host lung and alveolar M⌽.On ⌽ FIGURE 2. Grafted blood monocytes give rise to alveolar M . DTx- day 5 after BM transfer, i.e., at a time when some blood monocytes CD11c ϩ treated :DTR mice were grafted with either CD115 monocytes are of donor BM origin, but all lung and alveolar M⌽ are still host (106 cells) isolated from blood of cx cr1gfp/ϩ;CD45.1 mice (ϩmonocytes) 3 derived (Fig. 3B), chimeras were either treated i.t. with DTx or left or with no graft (w/o graft) on day 0. Fourteen days after transfer, BAL of recipient mice were analyzed for the presence of graft-derived alveolar untreated. Mice were analyzed on day 8, 3 days after DTx treat- ⌽ ⌽ ϩ Ϫ Ϫ ment, when Ͼ90% of blood monocytes are donor derived (Figs. M . A, Alveolar M were identiﬁed as CD11c CD11b CX3CR1/GFP cells, as indicated by gates. B, Graft-derived alveolar M⌽ were further 3B and 4A). Whereas parenchymal lung M⌽ of untreated chimeras Downloaded from identiﬁed as CD45.1ϩ cells, as indicated by gate. Numbers indicate per- were still host derived, in DTx-treated mice one-half of them were centage of graft-derived cells (CD45.1ϩ) of total gated population and their replaced by donor cells, most likely of blood monocyte origin (27) absolute numbers (in parentheses). Data show one representative of three (Fig. 4A). Interestingly, in the same mice, Ͼ90% of newly gener- independent experiments. ated alveolar M⌽ were of host origin (CD45.1Ϫ) (Fig. 4A). Hence, in our experimental setting, newly generated alveolar M⌽ origi-

nated from a host-derived precursor, although all blood monocytes http://www.jimmunol.org/ M⌽ 4 days after transfer. To distinguish between these two op- were donor derived (Figs. 3B and 4A). This result argues that al- tions, we decided to extend our previous study and analyze mono- veolar M⌽ do not originate directly from blood monocytes. cyte recipients 14 days after transfer. Monocytes, defined as positive for CD115 (also known as M- gfp/ϩ Lung M⌽ serve as alveolar M⌽ precursor CSFR), were isolated from blood of cx3cr1 ;CD45.1 mice and transferred into CD11c:DTR;CD45.2 transgenic mice, which had Depletion of both CD11c:DTR transgenic lung and alveolar M⌽ been pretreated by i.t. DTx installation. Two weeks after transfer, by i.t. DTx treatment was followed by their rapid reconstitution the BAL content of recipient mice was analyzed for graft-derived (Fig. 1B). The kinetics of these processes was, however, different. ϩ ϩ alveolar M⌽. The latter were identified as CD45.1 CD11c Although alveolar M⌽ numbers arose after 1 day, the number of by guest on September 28, 2021 Ϫ Ϫ ϩ CD11b CX3CR1/GFP cells to discriminate them from CD45.1 parenchymal M⌽ continuously declined for the first 4 days fol- ϩ ϩ ϩ ϩ CD11c CD11b CX3CR1/GFP graft-derived DC and CD45.2 lowing the DTx treatment (Fig. 1B). The mean human serum t1/2 recipient’s cells (27) (Fig. 2A). As shown in Fig. 2B, 2 wk after of DTx has been estimated to be 18 h (38). Accordingly, the DTx transfer, we could detect graft-derived alveolar M⌽. This indicates treatment of CD11c:DTR transgenic mice generally induces a tran- that blood monocytes do have the capacity to give rise to sient, short-term depletion of CD11cϩ cells (29). It is therefore alveolar M⌽. unlikely that the persistent decline of lung M⌽ from day 1 to day 4 (Fig. 1B) results from ongoing toxin-induced apoptosis, but ⌽ Lung and alveolar M show distinct reconstitution kinetics rather suggests M⌽ emigration from the lung parenchyma to the following irradiation and BM transfer alveolar space. In addition, on day 5 after BM transfer, newly Together with our previous study (27), the above result shows that generated alveolar M⌽ originated from host-derived precursors, blood monocytes can give rise to both lung and alveolar M⌽, whereas at that time lung M⌽ were of host origin (Fig. 4A). Taken albeit with distinct kinetics. To further investigate the precursor/ together, our results suggest that parenchymal lung M⌽ act as progeny relation of blood monocytes and pulmonary M⌽,wein- immediate alveolar M⌽ precursors. vestigated the kinetics of their reconstitution after irradiation and To further investigate this option, we next studied the origin of total BM engraftment. To this end, we transferred syngeneic wild- alveolar M⌽ at a time when parenchymal lung M⌽ were recon- type (wt) BM cells (CD45.1) into lethally irradiated recipients stituted by donor-derived cells. To this end, wt3CD11c:DTR chi- (CD45.2) and followed the regeneration of the blood monocyte meras were treated 12 days after BM transfer with DTx i.t., and compartment, as well as lung and alveolar M⌽, by donor-derived control chimeras were left untreated. At this time, all blood mono- cells (Fig. 3A). cytes and a fraction of the parenchymal lung M⌽ are of donor As expected from the literature (20), 1 wk following BM trans- origin, but alveolar M⌽ are still host derived (Fig. 3B). We ana- fer, all blood monocytes were fully replaced by donor-derived lyzed the recipient mice 3 days later, i.e., on day 15, when all lung (CD45.1ϩ) cells (Fig. 3B). In contrast, and in agreement with their M⌽ are donor derived (Figs. 3B and 4B). Although in untreated earlier reported delayed reconstitution (14, 16, 37), alveolar M⌽ mice most alveolar M⌽ were of host origin, following their DTx- began to be replaced by donor cells only 3 wk after transfer (Fig. induced depletion all newly generated alveolar M⌽ were donor 3B). Interestingly, the exchange of lung M⌽ by donor BM-derived derived (Fig. 4B). cells significantly preceded this event and showed intermediate To conclude, the origin of newly generated alveolar M⌽ corre- kinetics, beginning at day 10, with full reconstitution being lated with the donor/host origin of lung M⌽ (Fig. 4). Taken to- reached 3 wk after transfer (Fig. 3B). gether with their differential reconstitution kinetics following de- In conclusion, all three cell types studied are reconstituted after pletion, these data support the notion that lung M⌽ act as alveolar irradiation by BM-derived cells, albeit with significantly distinct M⌽ precursors. The Journal of Immunology 3491

FIGURE 3. Different reconstitution kinetics of blood monocytes, lung M⌽, and alveolar M⌽ follow irradiation and BM transfer. CD45.1 BM cells were transferred to CD45.2 wt recipient mice on day 0, and recipients’ blood, digested lungs, and BAL ﬂuids were analyzed on days 1, 5, 7, 9, 12, 14, 16, 19, and 26 for donor-derived monocytes and M⌽. A, Identiﬁcation of donor BM-derived cells. Blood of recipient mice was stained with PE-coupled anti CD115

Ab and biotin-coupled anti CD45.1 Ab, followed by allophycocyanin-conjugated streptavidin. Cells isolated from lung parenchyma and BAL ﬂuids were Downloaded from stained with PE-coupled anti-CD11c Ab, PerCP-coupled anti-CD11b Ab, and biotin-coupled anti-CD45.1 Ab, followed by allophycocyanin-conjugated streptavidin. R1 gate indicates CD45.1ϩ donor-derived cells of total blood monocytes (CD115ϩ cells). R2 and R3 gates indicate CD45.1ϩ donor-derived cells of total lung and alveolar M⌽, respectively (identiﬁed as CD11cϩCD11bϪ cells, as indicated by gates). B, Kinetics of blood monocytes (Œ), lung M⌽ (E), and alveolar M⌽ (f) reconstitution by donor BM-derived cells. Graph shows the percentage of donor-derived cells (CD45.1ϩ) of total blood monocytes (cells gated in R1, as indicated in A), lung M⌽ (cells gated in R2, as indicated in A), and BAL M⌽ (cells gated in R3, as indicated in A). n ϭ 2 for each time point. http://www.jimmunol.org/

Proliferation of lung and alveolar M⌽ cells (40), they most likely lost the GFP label over time. Alterna- tively, the rapid loss of the GFP label can be explained by cell All blood monocytes are CX3CR1 positive, and therefore express gfp/ϩ divisions, which in the absence of GFP de novo synthesis will cytoplasmatic GFP in cx3cr1 knockin mice (30), whereas pul- ⌽ result in the dilution of the label between daughter cells. The latter monary M are CX3CR1 negative (27) (Fig. 5A). Because GFP is stable for several days (39), all monocyte descendants inherit the is in agreement with previous studies suggesting that murine al- ⌽ cytoplasmic GFP label, even though expression of CX3CR1/GFP veolar M originate from proliferating precursors in the lung pa-

might have ceased. Although they are monocyte derived and there- renchyma (13, 23, 25). by guest on September 28, 2021 ⌽ gfp/ϩ fore expected to be GFP labeled, pulmonary M of cx3cr1 To distinguish between those two options, we analyzed the GFP mice are GFP negative (27) (Fig. 5A). Because M⌽ are long-lived label of newly coming lung and alveolar M⌽. To this end, we

FIGURE 4. Alveolar M⌽ do not originate directly from blood monocytes. BM cells of CD45.1 wt mice were adoptively transferred into irradiated CD11c:DTR CD45.2 transgenic recipients on day 0. Recipient mice were either treated i.t. with DTx on indicated days or left untreated and sacrificed 3 days later. Cells were then analyzed for CD45.1 expression, as described in Fig. 3A: donor-derived (CD45.1ϩ) blood monocytes, lung M⌽, and alveolar M⌽ are cells gated in R1, R2, and R3, respectively, whereas host-derived (CD45.1Ϫ) cells are cells outside those gates. A, Recipient mice were either treated i.t. with DTx on day 5 (ϩ) or left untreated (Ϫ) and were sacrificed on day 8. Bar diagram shows percentage of donor (CD45.1ϩ, f)- and host (CD45.1Ϫ, u)-derived blood monocytes, lung M⌽, and alveolar M⌽ of the total population. n ϭ 5 for each group. One representative of three independent experiments. B, Re- cipient mice were either treated i.t. with DTx on day 12 (ϩ) or left untreated (Ϫ) and were sacrificed on day 15. Bar diagram shows percentage of donor (CD45.1ϩ, f)- and host (CD45.1Ϫ, u)-derived blood monocytes, lung M⌽, and alveolar M⌽ of the total population. n ϭ 5 for each group. One representative of three independent experiments. 3492 ALVEOLAR M⌽ ORIGIN Downloaded from http://www.jimmunol.org/

⌽ gfp/ϩ FIGURE 5. Lung and alveolar M proliferate follow depletion. A, Cx3cr1 ;CD11c:DTR double-transgenic mice were i.t. treated with PBS and sacrificed 1 day later. Blood monocytes (upper panel, identified as CD115ϩ cells), lung M⌽ (left lower panel, identified as CD11cϩCD11bϪ cells), and alveolar M⌽ (right lower panel, identified as CD11cϩCD11bϪ cells) were analyzed for green fluorescence intensity. Data show one representative of three gfp/ϩ mice. B, cx3cr1 ;CD11c:DTR mice were i.t. treated with DTx on day 0 and sacrificed on day 1, 3, 5, 7, or 10 following treatment. Lung (left) and alveolar ⌽ ϩ Ϫ ⌽ (right)M (identified as CD11c CD11b cells) were analyzed for green fluorescence intensity. Note dilution of GFP label in CX3CR1-negative M with time. Data show one representative of three mice for each time point. C, CD11c:DTR were i.t. treated with either DTx or PBS on day 0 and sacrificed on by guest on September 28, 2021 day 3, and their blood, lungs, and BAL fluids were analyzed for PCNA expression. Lungs and BAL fluid cells were stained with biotin-coupled anti-CD11c Ab, followed by allophycocyanin-conjugated streptavidin and either PE-coupled anti-PCNA Ab or isotype control (broken line histogram). Histogram shows PCNA expression by CD11cϩ cells, as gated in dot plot, of DTx (solid line)- and PBS (gray filled)-treated mice. Blood cells were stained with biotin-coupled anti-CD115 Ab, followed by allophycocyanin-conjugated streptavidin and either PE-coupled anti-PCNA Ab or isotype control (broken line histogram). Histogram shows PCNA expression by CD115ϩ cells, as gated in dot plot, of DTx (solid line)-treated mouse. Data show one representative of four mice for each group. D, CD11c:DTR;␮MT mice, which are B cell deficient, were i.t. treated with DTx on day 0 and sacrificed on day 3, and their lungs were analyzed for PCNA expression. Half of lung cells were stained with biotin-coupled anti-CD11c Ab and half with biotin-coupled anti-MHC II Ab, both followed by allophycocyanin-conjugated streptavidin and PE-coupled anti-PCNA Ab. Histogram shows PCNA expression by CD11cϩ cells (solid line, as gated in A), including both M⌽ and DC, and MHC IIϩ cells (gray filled, as gated on dot plot), including only DC. Data show one representative of four mice.

gfp/ϩ generated a cx3cr1 ;CD11c:DTR double-transgenic mouse Next, we decided to directly study the proliferation status of line, treated the mice with DTx i.t., and investigated the green lung and alveolar M⌽ by determining their PCNA expression lev- ⌽ fluorescence intensities of their lung and alveolar M on the fol- els. The amount of PCNA increases during late G1 phase at sites lowing days (Fig. 5B). During the first 5 days after depletion, of DNA replication, reaches a maximum in S phase, and declines ⌽ newly coming lung M showed various green fluorescence inten- during G2 phase (41, 42). sities, ranging from high to low (Fig. 5B). This may result from We therefore stained lung, BAL, and blood cells of CD11c:DTR differentiation of CX3CR1/GFP-positive monocytes into CX3CR1- mice 3 days after i.t. treatment with either DTx or PBS for PCNA. negative M⌽ and the division of the latter. From day 7 and on, In PBS-treated mice, CD11cϩ lung cells were PCNAint, indicating ⌽ lung M fluorescence intensity was low and comparable to that of that they were in G1 phase, and upon DTx treatment a majority of lung M⌽ from PBS-treated mice (Fig. 5, A and B). This suggests these cells became PCNAhigh (Fig. 5C), documenting S phase en- there is no more input of monocytes at this stage, but because lung try and proliferation (41). Because CD11c is expressed by both M⌽ population increases (Fig. 1B), proliferation of differentiated lung M⌽ and DC (33, 34), the analyzed population includes both lung M⌽ most likely persists. cell types. In contrast, MHC II is highly expressed by lung DC, but Throughout the experiment, alveolar M⌽ showed similar green not by M⌽, and therefore allows the discrimination of the two cell fluorescence intensities to those of PBS-treated mice (Fig. 5, com- types (34). To exclude a potential contamination with MHC II- pare A and B). This supports the notions that the differentiation of expressing B cells, we crossed CD11c:DTR mice with B cell-de- blood monocyte into alveolar M⌽ is indirect and requires a lung ficient mice (␮MT mice) (31), allowing the exclusive definition of M⌽ intermediate. Furthermore, these data also suggest that the lung MHC IIϩ cells as DC. The comparison of PCNA expression proliferation of lung M⌽ (which allows the loss of the GFP label) levels of MHC II- and CD11c-positive lung cells of DTx-treated preceded their migration into the alveolar space. CD11c:DTR;␮MT mice confirmed that the PCNAhigh cells were The Journal of Immunology 3493 lung M⌽ (CD11cϩMHC IIlow), whereas lung DC (CD11cϩMHC celerate the M⌽ turnover rate, allowing us to detect lung M⌽- IIϩ) were PCNAlow-int (Fig. 5D). This indicates that lung M⌽, but derived alveolar M⌽ in resting state. not lung DC, undergo proliferation. Under conditions other than steady state, the dependency of al- Blood monocytes were PCNAint (Fig. 5C), in agreement with veolar M⌽ population on input from the blood might differ. In- other studies showing that monocytes do not proliferate (28, 43). flammation is, for example, associated with an increase in alveolar Interestingly, following the DTx treatment, alveolar M⌽ became M⌽ levels (16, 37, 48), and i.t. LPS treatment of BM recipient PCNAhigh (Fig. 5C), supporting the notion that they can prolifer- mice accelerates the replacement of alveolar M⌽ by donor cells ate, as previously reported in humans (44). (16). Interestingly, the rate of lung M⌽ replacement by donor- To conclude, both lung and alveolar M⌽ have proliferative po- derived cells was also promoted in these studies (16). In agreement tential, which manifests itself upon their DTx-induced depletion with these findings, we have previously shown the differentiation and reconstitution. In addition, our results show that lung M⌽ of blood monocytes into lung M⌽ in i.t. LPS-treated recipients, but proliferation precedes their migration into the alveolar space. not in untreated mice (27). We, however, had failed to detect graft- derived alveolar M⌽ in these studies, which included the analysis Discussion of recipient mice 4 days following monocyte transfer (27). In ad- In this study, we provide direct evidence that alveolar M⌽ origi- dition, Adamson and Bowden’s (48) kinetic studies (when inter- nate from blood monocytes, and show that this process requires a preted as discussed above) suggest the existence of a lung inter- lung-resident intermediate. We proposed that generation of alve- mediate for alveolar M⌽ also during inflammation. We therefore olar M⌽ involves the differentiation of blood monocytes into M⌽ suggest the kinetics of both monocyte differentiation into lung M⌽ in the lung parenchyma, proliferative expansion of these cells, and and the migration of the latter to the alveolar space to accelerate Downloaded from their emigration into the alveolar space. In such a scenario, lung under inflammation. It thus remains possible that the differentia- M⌽ may serve as a local reservoir, from which alveolar M⌽ can tion of alveolar M⌽ under inflammation also requires a lung M⌽ be generated whenever needed. intermediate. This model is in accordance with results of most previous stud- The irradiation protocol used for the depletion of host BM may ies on the alveolar M⌽ origin. Our finding, suggesting an indirect also affect the kinetic of alveolar M⌽ differentiation from blood

connection of blood monocytes and alveolar M⌽, agrees, for ex- monocyte. In this respect, it is of note that different irradiation http://www.jimmunol.org/ ample, with the finding of Sawyer et al. (25), showing that the protocols before BM transfer resulted in distinct alveolar M⌽ re- alveolar M⌽ population is unaffected by experimentally induced constitution kinetics (11, 13–15). Because irradiation primarily af- blood monocyte depletion. Furthermore, the derivation of alveolar fects dividing cells, proliferating alveolar M⌽ might become sen- M⌽ from blood monocytes is in agreement with the previously sitive to irradiation. Therefore, the turnover of alveolar M⌽ can be reported replacement of these M⌽ by donor cells upon BM trans- promoted by the stress caused by tissue damage (49), by a direct fer (11, 12), and the need for a lung intermediate can further ex- effect on pulmonary M⌽ (13), or by a combination of the two. plain the observed delayed reconstitution (13, 14). Bowden and One of the serious side effects of BM transplantation in humans Adamson (23) suggested a dual origin for alveolar M⌽, either is the increased susceptibility of patients to pulmonary infections directly from blood-borne precursor or from local proliferation. (17, 18). It has been proposed that in addition to their lower num- by guest on September 28, 2021 Our results suggest the steady-state derivation of alveolar M⌽ ber, alveolar M⌽ of BM transplantation patients might be func- from local proliferating precursor, i.e., lung M⌽, and therefore tionally impaired and therefore less reactive (37). The proliferative partially agree with the mentioned study. Alveolar DC had not capacity of both lung parenchymal and alveolar M⌽ might make been identified by that time Bowden’s study was conducted (24), both these cell types sensitive to irradiation. This can affect both and our current data and previous reports suggest that they are the functionality of alveolar M⌽ and the size of their reservoir in derived from blood monocytes without local proliferation (27). We the lung parenchyma, reducing their capacity to efficiently combat would therefore like to propose that the cells shown by Bowden pathogens. The indirect route from monocytes to alveolar M⌽ and Adamson (23) to directly derive from blood monocyte are might result in a delayed replacement of damaged alveolar M⌽ alveolar DC, potentially mistaken for alveolar M⌽. To con- with new and unaffected cells, maintaining the patient susceptibil- clude, our model offers a connection between works reporting ity to pathogen. Therefore, our results suggest that a strategy aim- local precursor (13, 23, 25, 45) and those indicating blood- ing at the active depletion of pulmonary M⌽ might trigger an borne precursor (11, 12, 40) for alveolar M⌽, previously advantageous renewal from donor BM-derived monocytes, and thought to be contradictory. could potentially reduce the period of patient vulnerability to pul- We show that the pulmonary M⌽ reservoir is maintained by monary pathogens. M⌽ proliferation in situ, in both lung parenchyma and alveolar space. In situ proliferating precursors were also suggested for other Acknowledgments M⌽ population, such as splenic M⌽ and liver Kupffer cells (46, We thank our laboratory members for critical reading of the manuscript, 47). Therefore, dependency on a local self-renewal capacity seems and are grateful to Y. Chermesh and Y. Melamed for animal husbandry. to be a general feature of M⌽ populations, rather than unique to alveolar M⌽. Disclosures Because the main role of alveolar M⌽ is the maintenance of the The authors have no financial conflict of interest. alveolar space by clearing dying cells, particles, and pathogens, References there is a constant need for their presence in adequate numbers. By 1. Martin, T. R., and C. W. Frevert. 2005. Innate immunity in the lungs. Proc. Am. abolishing the dependency on recruitment of blood precursors, the Thorac. Soc. 2: 403–411. existence of local precursor as reservoir for alveolar M⌽ allows a 2. Lambrecht, B. N., and H. Hammad. 2003. Taking our breath away: dendritic cells tight control and prompt adjustment of their numbers. Conse- in the pathogenesis of asthma. Nat. Rev. Immunol. 3: 994–1003. 3. MacLean, J. A., W. Xia, C. E. Pinto, L. Zhao, H. W. Liu, and R. L. Kradin. 1996. quently, the rate of monocyte differentiation into lung M⌽ under Sequestration of inhaled particulate antigens by lung phagocytes: a mechanism steady state is likely to be low, precluding the detection of mono- for the effective inhibition of pulmonary cell-mediated immunity. Am. J. Pathol. ⌽ 148: 657–666. cyte-derived alveolar M (25, 45). In this study, we use a nonin- 4. Holt, P. G., J. Oliver, N. Bilyk, P. G. McMenamin, G. Kraal, and T. Thepen. flammatory, experimentally induced M⌽ depletion protocol to ac- 1993. Down-regulation of the antigen presenting cell function(s) of pulmonary 3494 ALVEOLAR M⌽ ORIGIN

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