Abstract
Rationale: Resident alveolar macrophages have been attributed a crucial role in host defense toward pulmonary infection. Their contribution to alveolar repair processes, however, remains elusive.
Objectives: We investigated whether activated resident alveolar macrophages contribute to alveolar epithelial repair on lipopolysaccharide (LPS) challenge in vitro and in vivo and analyzed the molecular interaction pathways involved.
Methods: We evaluated macrophage–epithelial cross-talk mediators for epithelial cell proliferation in an in vitro coculture system and an in vivo model of LPS-induced acute lung injury comparing wild-type, granulocyte–macrophage colony-stimulating factor (GM-CSF)–deficient (GM−/−), and human SPC–GM mice (GM−/− mice expressing an SPC-promotor–regulated GM-CSF transgene).
Measurements and Main Results: Using reverse transcription-polymerase chain reaction and ELISA we showed that LPS-activated alveolar macrophages stimulated alveolar epithelial cells (AEC) to express growth factors, particularly GM-CSF, in coculture. Antibody neutralization experiments revealed epithelial GM-CSF expression to be macrophage tumor necrosis factor (TNF)–α dependent. GM-CSF elicited proliferative signaling in AEC via autocrine stimulation. Notably, macrophage TNF-α induced epithelial proliferation in wild-type but not in GM-CSF–deficient AEC as shown by [3H]-thymidine incorporation and cell counting. Moreover, intraalveolar TNF-α neutralization impaired AEC proliferation in LPS-injured mice, as investigated by flow cytometric Ki-67 staining. Additionally, GM-CSF–deficient mice displayed reduced AEC proliferation and sustained alveolar barrier dysfunction on LPS treatment compared with wild-type mice.
Conclusions: Collectively, these findings indicate that TNF-α released from activated resident alveolar macrophages induces epithelial GM-CSF expression, which in turn initiates AEC proliferation and contributes to restoring alveolar barrier function.
Little is known about the contribution of activated alveolar macrophages to alveolar epithelial repair processes in inflammatory lung injury.
This study demonstrates that alveolar macrophage tumor necrosis factor-α initiates alveolar epithelial repair on LPS challenge by induction of autocrine epithelial granulocyte–macrophage colony-stimulating factor signaling. Macrophage tumor necrosis factor-α thereby contributes to epithelial renewal and restoration of alveolar barrier function.
Granulocyte–macrophage colony-stimulating factor (GM-CSF) has widely been known as a growth factor for myeloid cells, affecting their survival, proliferation, maturation, and differentiation. Several authors demonstrated GM-CSF importance for alveolar macrophage functions during bacterial pneumonia (10, 11). Moreover, GM-CSF has been attributed a role in homeostasis of normal lung tissue metabolites (12). In addition to its known effects on hematopoietic cells, GM-CSF has lately been recognized as potent growth factor for AEC in vitro and in vivo (13, 14).
Several authors suggested that alveolar macrophages or their inflammatory mediators might contribute to alveolar epithelial cell turnover (15, 16). Hence, Morimoto and colleagues showed that alveolar macrophages release the epithelial mitogen hepatocyte growth factor during the resolution/repair phase of bacterial pneumonia on phagocytosis of apoptotic neutrophils (17). However, the potential of early activated AMϕ to initiate epithelial repair mechanisms after lung inflammation and the molecular signals underlying macrophage–epithelial crosstalk during these processes remain largely elusive.
In the following study we show that AMϕ activated on lipopolysaccharide (LPS) may contribute to alveolar repair by inducing the release of epithelial GM-CSF in a TNF-α−dependent manner, resulting in AEC II proliferation. Additionally, in an in vivo model of LPS-induced lung injury using GM-CSF–deficient mice or transgenic mice selectively overexpressing GM-CSF in AEC II, we show that epithelial GM-CSF largely contributes to the resolution of inflammation, AEC II renewal, and restoration of alveolar barrier function. Some of the results of this study have been previously reported as abstracts (18, 19).
Materials and reagents used for flow cytometry, Western blot, and in vitro and in vivo applications are listed in the online supplement.
Wild-type C57BL/6 mice (weight 18–21 g) were purchased from Charles River (Sulzfeld, Germany). GM-CSF–deficient mice (GM−/−) were produced by gene targeting on a C57BL/6 background, as previously described (12). Transgenic mice overexpressing GM-CSF in AEC II were generated in GM−/− mice by expression of a chimeric gene containing GM-CSF under control of the human SP-C promoter (SPC-GM) (20). Both GM−/− and SPC-GM mice were a kind gift from Dr. Jeffrey Whitsett (University of Cincinnati, OH). CCR2−/− mice were obtained from W. A. Kuziel (1). Animals were kept under special pathogen-free conditions and used at 8 to 11 weeks of age. All animal experiments were approved by the local government committee.
AEC were isolated by the method developed by Corti and colleagues (see the online data supplement). AMϕ were gained by bronchoalveolar lavage from mouse lungs. For coculture experiments, AEC were seeded on the lower side of Transwell inserts or on Matrigel:collagen matrix, and subsequently cocultured with AMϕ in a 24-well plate. Coculture was maintained for the next 48 hours (see online supplement for details).
TNF-α and GM-CSF levels in cell culture supernatants and bronchoalveolar lavage fluid (BALF) were measured using commercially available ELISA kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions.
RNA isolation, cDNA synthesis, and real-time reverse transcription-polymerase chain reaction analysis were performed as outlined in the online supplement. Hydroxymethylbilane synthase served as the reference gene. The ΔCt (cycle threshold) values for each target gene were calculated using the reference gene hydroxymethylbilane synthase and the formula ΔCt = Ctreference − Cttarget.
For fluorescence-activated cell sorter (FACS) analysis, cells were processed as described in the online supplement. Flow cytometric analysis was performed using a FACSCanto flow cytometer (BD Biosciences) equipped with a FACSDiva (BD Biosciences, Heidelberg, Germany) and WinMDI 2.8 (Scripps Institute, La Jolla, CA) software packages.
To assess Stat5 activation, AEC were treated with recombinant GM-CSF (500 pg/ml) as indicated, lysed, and processed as described in the online supplement.
AEC proliferation in vitro was assessed by [3H]-thymidine incorporation and cell counting as outlined in detail in the online supplement.
Mice were treated intratracheally with ultra-pure LPS (10 μg/mouse in sterile phosphate-buffered saline in a total volume of 70 μl) or LPS and anti–TNF-α antibodies (or respective IgG, 10 μg/mouse), as described previously in detail (21) and killed at the indicated time points. Processing of BALF cells and lung homogenates and analysis of alveolar leakage are outlined in the online supplement.
AEC were methanol/acetone fixed and costained with pro–SP-C and T1-α antibodies. Lung cryosections were prepared from perfused and lavaged lungs, and costained with pro–SP-C and Ki-67 antibodies (see online supplement).
Data analysis and statistics were performed using the R statistical program. All data are presented as means ± SD. Statistical significance was estimated using one-way analysis of variance and Tukey-HSD post hoc test; paired samples were analyzed using the two-tailed paired Student t test. A value of P less than 0.05 was considered as significant.
Keratinocyte growth factor, vascular endothelial growth factor, platelet-derived growth factor, GM-CSF, and fibroblast growth factor 2 (FGF2) have all been described as potent epithelial mitogens (14, 22–27). To investigate whether AMϕ are capable of inducing expression of these growth factors in alveolar epithelial cells under inflammatory conditions, AEC were either mono- or cocultured with AMϕ for 48 hours and treated with LPS (1 μg/ml for 48 h), or left untreated. Analysis of mRNA expression of the aforementioned growth factors in AEC revealed a significant up-regulation of keratinocyte growth factor, vascular endothelial growth factor, platelet-derived growth factor–a, and, most prominent, GM-CSF message in AEC cocultured with LPS-stimulated AMϕ compared with AEC in monoculture. A slight, yet not significant, up-regulation of epithelial FGF2 was detected in coculture on LPS treatment. Of note, LPS stimulation of monocultured AEC or unstimulated AEC/AMϕ coculture revealed no significant up-regulation of any of the analyzed growth factors in AEC (Figure 1A). In contrast to the findings in AEC, AMϕ, did not show any significant regulation of the gene products named above, nor did coculture with AEC influence their expression in absence or presence of LPS (Figure 1B). FGF2 gene expression in AMϕ was below the detection level. Given that among the growth factors analyzed GM-CSF mRNA up-regulation was most pronounced, we further investigated GM-CSF protein release in mono- and coculture on LPS stimulation. As demonstrated in Figure 1C, AMϕ alone did not release significant amounts of GM-CSF into the supernatant, irrespectively of the presence or absence of LPS. AEC alone showed significantly higher release of GM-CSF, which was not enhanced in the presence of LPS. Supernatants from LPS-stimulated cocultures, however, contained significantly higher amounts of GM-CSF than supernatants from AEC monocultures or from unstimulated cocultures. Of note, presence of AMϕ reduced GM-CSF levels observed in AEC monoculture (Figure 1C, lanes 3–5), most likely due to macrophage GM-CSF consumption. Collectively, these data indicate that AEC are the primary alveolar source of epithelial growth factors and that AMϕ have the potential to significantly amplify epithelial expression of various growth factors, in particular of epithelial GM-CSF, on inflammatory stimulation.
Figure 1. Lipopolysaccharide (LPS)–stimulated resident alveolar macrophages enhance the expression of growth factors in cocultured alveolar epithelial cells (AEC). Freshly isolated AEC cultured for 60 hours on Transwell inserts were either monocultured and left unstimulated or were stimulated with LPS (1 μg/ml), or were cocultured with unstimulated or LPS-stimulated resident alveolar macrophages (AMϕ) for 48 hours. Subsequently, relative gene expression of different growth factors was analyzed in both cell types. (A) Relative mRNA expression of keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)–a, granulocyte–macrophage colony-stimulating factor (GM-CSF), and fibroblast growth factor 2 (FGF2) in unstimulated or LPS-treated monocultured AEC (lanes 1 and 2) or cocultured AEC (lanes 3 and 4). (B) Relative mRNA expression of KGF, VEGF, PDGFa, and GM-CSF in unstimulated or LPS-treated monocultured AMϕ (lanes 1 and 2) or cocultured AMϕ (lanes 3 and 4). (C) GM-CSF release in the supernatants from monocultured AMϕ (lanes 1 and 2), monocultured AEC (lanes 3 and 4), and AEC/AMϕ cocultures (lanes 5 and 6) in the presence or absence of LPS was analyzed by ELISA. All given values are means ± SD from at least n = 3 (in A and B) or n = 5 (in C) different experiments, each of which was performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001. ns = not significant.
Given that LPS-stimulated AMϕ induced GM-CSF expression in AEC, most likely by a soluble mediator, we speculated that the proinflammatory TNF-α might mediate these effects. AMϕ secrete significant amounts of TNF-α on LPS treatment and in the early phase of gram-negative infections (28, 29), and AEC are known to respond to TNF-α (1, 6). Accordingly, TNF-α solely originated from AMϕ in the LPS-treated cultures, whereas AEC alone did not release any detectable levels of TNF-α on LPS stimulation (Figure 2A, lanes 2, 6, and 4, respectively). Of note, both TNF-α receptors 1 and 2 (TNFR1/2) were expressed on cultured AEC on the mRNA level (data not shown). To evaluate whether macrophage TNF-α induced GM-CSF production in AEC, we applied neutralizing anti–TNF-α antibodies in our model. Indeed, anti–TNF-α treatment significantly decreased epithelial GM-CSF expression in LPS-treated cocultures, both on mRNA and protein level (Figure 2B). Moreover, stimulation of monocultured AEC with recombinant TNF-α resulted in increased expression of GM-CSF, both on mRNA and protein level (Figure 2C). Taken together, these data demonstrate that macrophage TNF-α, released on LPS recognition, induces GM-CSF expression in cocultured AEC, indicating that AMϕ might contribute to the initiation of epithelial repair processes yet in the early phase of inflammation.
Figure 2. Alveolar macrophage tumor necrosis factor (TNF)–α mediates epithelial granulocyte–macrophage colony-stimulating factor (GM-CSF) production. (A) TNF-α levels in supernatants taken from unstimulated and lipopolysaccharide (LPS)–stimulated monocultured and cocultured alveolar epithelial cells (AEC) and resident alveolar macrophages (AMϕ) were determined by ELISA. (B) Anti–TNF-α (a-TNF-α) antibodies or isotype IgG (Iso IgG) were added to the medium of LPS-stimulated AEC/AMϕ cocultures (1 μg/ml at 2, 12, and 20 h post LPS treatment), and after 48 hours GM-CSF mRNA expression in AEC (left panel) and GM-CSF protein in AEC/AMϕ coculture supernatants (right panel) were determined. (C) AEC monocultures were stimulated with recombinant TNF-α (100 ng/ml, 48 h) and GM-CSF mRNA and protein were measured (left and right panel, respectively). Untreated AEC were used as a control. All given values are presented as means ± SD from n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. untr = Untreated.
To evaluate whether GM-CSF signaling in epithelial cells would result in enhanced proliferation, we first investigated the expression of both GM-CSF receptor subunits (α and β) over the 5 days of in vitro culture. As shown in Figure 3A, freshly isolated AEC expressed both subunits of the GM-CSF receptor, but their expression decreased during 5 days of culture. Given that rat and human AEC II cultured on plastic have been shown to change their phenotype during in vitro culture, thereby acquiring features of AEC I (30, 31), we examined whether GM-CSF receptor subunit expression might be related to the AEC II phenotype. Indeed, mRNA expression of the AEC II specific marker pro–SP-C (32) was pronounced at Day 0 and rapidly declined during 5 days of culture, whereas mRNA levels of the AEC I marker T1-α (33) increased (Figure 3B). In addition, immunolabeling of these markers revealed corresponding results on protein level, as demonstrated in representative flow cytometric dot plots in Figure 3C (Day 0, 91 ± 1.64% pro–SP-C+ AEC; Day 5, 90.5 ± 5.8% T1α+ AEC) and immunofluorescent staining in Figure 3D. Of note, GM-CSF stimulation of AEC (Figure 3C) as well as primary AMϕ when cocultured with AEC (data not shown) did not influence AEC phenotype changes. Together, these data indicate that GM-CSF receptor subunits α and β are expressed on AEC II and down-regulated during transdifferentiation into AEC I-like cells.
Figure 3. Granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor expression is associated with the alveolar epithelial cells (AEC) II phenotype. (A) mRNA expression analysis of GM-CSF receptor subunits α (left panel) and β (right panel) in AEC at Day 0, 1, 3, and 5 of monoculture. Values are means ± SD from n = 4 independent experiments. (B) Relative mRNA expression of pro-surfactant protein C (pro–SP-C) and T1-α in freshly isolated AEC (Day 0) or AEC cultured for 1, 3, or 5 days. Values are presented as means ± SD from at least three independent experiments. (C) Representative flow cytometric dot plots of pro–SP-C and T1-α expression of AEC at Day 0, 1, 3, and 5 post isolation, untreated (untr) or GM-CSF stimulated, including staining with respective isotype controls; P1, AEC II; P2, AEC I. (D) Pro–SP-C (red) and T1-α (green) expression between Day 0 and Day 5 of Transwell culture was analyzed by immunofluorescence and representative stainings are presented. *P < 0.05, **P < 0.01, ***P < 0.001. iso IgG = isotype IgG control.
GM-CSF receptor downstream signaling has been described to be mediated by various intracellular pathways involving Stats, MAPK, and PI3K/Akt (34). Stimulation of AEC at Day 1 of culture with recombinant murine GM-CSF induced rapid and transient phosphorylation of Stat5 and increased expression of Cyclin D1 mRNA after 24 hours (Figures 4A and 4B). Cyclin D1 is known to be activated by the Stat5 pathway and to modulate local chromatin structure and transcription of genes involved in proliferation (35, 36). In contrast, GM-CSF did not induce MAPK or Akt activation in Day 1 AEC (data not shown). Moreover, GM-CSF stimulation of Day 1 but not Day 3 AEC resulted in increased proliferation, as assessed by [3H]-thymidine incorporation and cell counting (Figure 4C). These data demonstrate that a Stat5-dependent intracellular signal is induced on GM-CSF receptor binding in day 1 AEC, resulting in Cyclin D1 expression and likely mediating the subsequent proliferation. Of note, Day 1 AEC, despite acquisition of the type I phenotype in terms of marker expression, were still capable of responding to the proliferative GM-CSF signal as opposed to Day 3 AEC, indicating that Day 1 AEC might functionally still possess type II characteristics during transition toward the type I phenotype. Likewise, AEC grown on Matrigel:collagen matrix, thereby maintained in the “classical” type II phenotype until Day 3 of culture (see Figure E1 in the online supplement), similarly expressed and released GM-CSF on LPS stimulation in the coculture and responded to GM-CSF stimulation with enhanced proliferation (Figure E2), further supporting the concept that the proliferative response to GM-CSF is related to the type II AEC phenotype.
Figure 4. Granulocyte–macrophage colony-stimulating factor (GM-CSF) induces proliferative signaling in alveolar epithelial cells (AEC). (A) Representative Western blot analysis of Stat5 phosphorylation in AEC stimulated with GM-CSF (500 pg/ml) at Day 1 of culture for the indicated time point; total Stat5 was used as loading control. Three independent experiments were performed. (B) Analysis of Cyclin D1 mRNA levels in Day 1 AEC stimulated with 100 ng/ml GM-CSF for 24 hours; untreated AEC were used as control. Values are means ± SD from n = 3 independent experiments. (C) Freshly isolated AEC were left to adhere for 16 hours, were then serum starved for 10 hours, and immediately thereafter stimulated with GM-CSF for 48 hours. Likewise, AEC at Day 3 of culture were stimulated with GM-CSF for 48 hours in serum-starving medium. Subsequently, AEC proliferation was measured by [3H]-thymidine incorporation (left panel) and cell counting (right panel). Data are presented as fold induction of untreated control; values are means ± SD from n = 4 independent experiments performed in quadruplicates for [3H]-thymidine incorporation, and n = 3 for cell counting. *P < 0.05, **P < 0.01. untr = untreated.
Given that GM-CSF induced proliferative signaling in AEC at Day 1 of culture, and macrophage TNF-α stimulated GM-CSF expression in epithelial cells, we hypothesized that AEC proliferation might be induced by TNF-α in a GM-CSF–dependent manner. Therefore, AEC isolated from GM-CSF–deficient mice (GM−/− AEC) were stimulated with recombinant TNF-α at Day 1 of culture, and compared with wild-type AEC (wt AEC) for proliferation, assessed by [3H]-thymidine incorporation and AEC cell counting. As shown in Figure 5A, GM−/− AEC did not show enhanced proliferation on TNF-α stimulation, whereas TNF-α–stimulated wt AEC demonstrated significantly increased proliferation, compared with untreated control (gray bars). Similarly, to evaluate whether LPS-stimulated AMϕ may induce proliferation of AEC via GM-CSF, GM−/− AEC grown on Transwell plates for 1 day were cocultured with LPS-stimulated wt AMϕ and compared with cocultured wt AEC for proliferation. Interestingly, wt AEC cocultured with AMϕ in the presence of LPS showed remarkably increased [3H]-thymidine incorporation and AEC counts compared with unstimulated wt AEC in monoculture, whereas LPS-stimulated AMϕ could not influence the proliferation of GM−/− AEC (solid bars). These data clearly demonstrate that macrophage TNF-α released on LPS stimulation triggers GM-CSF secretion in alveolar epithelial cells, which in turn induces AEC proliferation by an autocrine stimulation loop in vitro.
Figure 5. Alveolar epithelial cell (AEC) proliferation is induced by macrophage tumor necrosis factor (TNF)-α and mediated by granulocyte–macrophage colony-stimulating factor (GM-CSF). (A) AEC monocultures from wild-type (wt) and GM−/− mice at Day 1 of culture were stimulated with recombinant TNF-α (100 ng/ml) for 48 hours or left untreated (untr) and proliferation was assayed by [3H]-thymidine incorporation and cell counting (shaded bars). For proliferation analysis of cocultured epithelial cells, wt AEC or GM−/− AEC were plated on Transwells and cocultured at Day 1 of culture with lipopolysaccharide (LPS)–stimulated alveolar macrophages for 48 hours before proliferation analysis (solid bars). Data are presented as fold induction of monocultured untreated AEC (open bars). Values are means ± SD from at least n = 4 independent experiments performed in quadruplicates for [3H]-thymidine incorporation, and n = 3 for cell counting. (B) Top panel: Flow cytometric analysis of AEC II proliferation after LPS-induced lung injury. Lung homogenates from wt mice treated intratracheally with 10 μg LPS with either anti–TNF-α (aTNF-α) antibodies or isotype IgG, respectively, were analyzed for CD45, pro–SP-C, and Ki-67 expression. Representative dot plots show coexpression of pro–SP-C and Ki-67 (or respective iso IgG) of CD45−/pro–SP-C+ cells (P1). Bottom panel: Quantification of fluorescence-activated cell sorter analysis of proliferating AEC II of n = 3 mice per group; bar graphs show the percentage of Ki-67+ of total AEC II (pro–SP-C+/CD45−) in lung homogenates. (C) Wt mice were treated either with LPS plus IgG or LPS plus anti–TNF-α for 96 hours and immunofluorescence staining of pro–SP-C (red) and nuclear Ki-67 (green), or respective IgGs, was performed on cryosections from lavaged lungs. Arrows depict proliferating AEC II; magnification ×20. (D) Analysis of GM-CSF levels after 6 hours of LPS in bronchoalveolar lavage fluid from the various treatment groups was performed by ELISA. Values are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. IgG = isotype IgG antibodies.
To evaluate the potential of macrophage TNF-α to mediate AEC II proliferation in LPS-induced acute lung injury in vivo, AEC II proliferation at 96 hours post LPS instillation was analyzed in wt mice treated intratracheally with either anti–TNF-α or isotype control antibodies. Proliferating AEC II in lung homogenate samples were defined as CD45−/pro-SP-C+/Ki-67+, as demonstrated in the representative FACS plots in Figure 5B. A remarkable increase in the percentage of proliferating AEC II was observed in LPS-injured mice after 96 hours post LPS instillation compared with untreated mice. Interestingly, anti–TNF-α treatment significantly reduced the proliferating proportion of AEC II in LPS-challenged wt mice compared to treatment with isotype control antibodies (Figure 5B). This finding was additionally supported by immunofluorescence analysis of whole lung tissue slices, demonstrating that anti–TNF-α treatment markedly reduced the proportion of proliferating AEC II after LPS challenge (pro–SP-C+/Ki-67+ cells, Figure 5C). Significant differences in the total number of AEC II in the lungs due to the anti–TNF-α treatment were not observed (data not shown). Analysis of GM-CSF levels in the BAL fluid after 6 hours of LPS-induced injury in mice revealed significantly lower GM-CSF release from alveolar cells when mice were treated with anti–TNF-α antibodies as compared with isotype-treated mice (Figure 5D). These data provide evidence that indeed TNF-α mediates alveolar epithelial cell proliferation in vivo after LPS-induced lung injury via induction of GM-CSF.
To analyze the influence of GM-CSF on alveolar repair after LPS-induced lung injury in vivo, three groups of mice (wt, GM−/− and SPC-GM mice, constitutively overexpressing GM-CSF in AEC II in a GM-CSF–deficient genetic background) were treated intratracheally with LPS for various lengths of time and subjected to BAL for evaluation of amount and composition of alveolar leukocyte infiltration. As shown in Figure 6A, we observed a pronounced accumulation of leukocytes in the alveolar air spaces of all treatment groups between 12 and 48 hours post LPS treatment. Leukocyte subpopulation analysis in the time course revealed that this correlated with the neutrophil recruitment in the alveolar air space. Alveolar neutrophil peaks reached in wt, GM−/−, and SPC-GM mice were virtually identical. Recruitment of ExMϕ and lymphocytes was comparable in all treatment groups and peaked after 48 and 96 hours of LPS treatment, respectively (data not shown).
Figure 6. Granulocyte–macrophage colony-stimulating factor (GM-CSF) enhances alveolar epithelial cell (AEC) II proliferation and alveolar barrier renewal after lipopolysaccharide (LPS)–induced acute lung injury. (A) Bronchoalveolar lavage (BAL) was performed in wild-type (wt) (open bars), GM−/− (shaded bars), and SPC-GM (solid bars) mice at various time intervals post intratracheal administration of 10 μg LPS. Total BAL fluid (BALF) cell counts were determined and leukocyte differential counts were determined from Pappenheim-stained cytocentrifuge preparations. Data are given as total cells and represent means ± SD from n = 3 animals per group. (B) Tumor necrosis factor (TNF)–α and GM-CSF levels in BALF from LPS-treated wt (open bars), GM−/− (shaded bars) and SPC-GM (solid bars) were quantified by ELISA. Data are presented as means ± SD from n = 3 mice per group. (C) Flow cytometric quantification of the proliferating proportion of AEC II in LPS-treated wt (open bars), GM−/− (shaded bars), and SPC-GM mice (solid bars). Left panel: bar graphs represent the percentage of Ki-67+ of total AEC II (proSP-C+/CD45−); right panel: AEC II proportion (pro–SP-C+) of lung homogenate cells from lavaged lungs of n = 3 mice per group. Values and are means ± SD. (D) Immunofluorescence of lung cryosections obtained from untreated or 96-hour LPS-treated wt, GM−/−, and SPC-GM mice. Arrows depict pro–SP-C+ cells (red) expressing Ki-67 (green nuclei); magnification ×20. Iso IgG = isotype IgG control. (E) Flow cytometric analysis of AEC I (T1-α+) percentage in lung homogenates of untreated and LPS-treated wt (open bars), GM−/− (shaded bars), and SPC-GM (solid bars). Data are given as means ± SD from n = 3 mice per group. (F) Alveolar leakage in wt (open bars), GM−/− (shaded bars), and SPC-GM (solid bars) was determined at various time intervals post LPS administration and is given as the ratio between fluorescein isothiocyanate fluorescence in BALF and serum (arbitrary units, AU). Data are presented as means ± SD from n = 3 animals per group; *P < 0.05, **P < 0.01, ***P < 0.001. untr = untreated. (G) Proposed model of alveolar macrophages (AMϕ)/AEC cross-talk in alveolar barrier repair: TNF-α, released from LPS-activated AMΦ, induces expression of GM-CSF in AEC, which in turn mediates AEC II proliferation and barrier function via a Stat5-dependent autocrine signaling loop.
GM−/− mice had similar AMϕ counts to wt mice in the early stages of LPS-induced inflammation, but substantially lower AMϕ numbers during the later stages (48–240 h). Total BALF AMϕ numbers were significantly higher in untreated SPC-GM mice as well as at all time intervals after LPS treatment compared with wt mice, an observation that has been described before (13, 14). Analysis of BALF TNF-α levels on intratracheal LPS administration in the three different treatment groups demonstrated that TNF-α was alveolarly released in wt, GM−/−, and SPC-GM mice, most prominent at 6 hours (Figure 6B). GM-CSF was released into the BALF of LPS-treated wt mice at 6 hours post LPS treatment and undetectable in GM−/− mice. SPC-GM mice produced significantly higher amounts of GM-CSF at baseline conditions (0 h) and at 6 and 12 hours post LPS treatment compared with wt mice (Figure 6B). Of note, GM-CSF levels in constitutively overexpressing SPC-GM mice decreased between 12 and 24 hours post LPS administration, most likely due to consumption by alveolar neutrophils and macrophages. Alveolar GM-CSF levels at 48 to 240 hours reached increased values observed in untreated SPC-GM mice (data not shown).
To investigate the role of GM-CSF in alveolar epithelial repair processes after LPS-induced acute lung injury, AEC II proliferation in the various treatment groups was determined by flow cytometry and lung immunofluorescence staining after 96 hours post LPS instillation, a time point at which recruited inflammatory leukocytes were virtually resolved from the air spaces and alveolar repair processes should likewise be initiated. As shown in Figure E3A and Figures 6C and 6D, proliferation of AEC II peaked at 96 hours post LPS and was significantly higher in LPS-treated compared with untreated wt mice. Of note, the proliferating proportion of type II AEC was lower in GM−/− mice at 96 hours post LPS administration, whereas in SPC-GM mice with selective GM-CSF overexpression in AEC II, proliferation was comparable to wt mice. Likewise, the AEC II proportion declining until 96 hours after LPS challenge in wt lung homogenates (Figure E3B) was significantly lower in GM−/− mice and higher in SPC-GM mice compared with wt mice at this time point (Figure 6C). The percentage of AEC I (T1α+ cells) in lung homogenates, which decreased shortly after LPS challenge in wt mice probably due to enhanced apoptosis (Figures E3B and E3C), recovered and was virtually identical in all of the treatment groups after 96 hours of LPS (Figure 6E). These findings suggest that epithelial GM-CSF induces AEC type II proliferation and that lack of epithelial GM-CSF is associated with impaired AEC II renewal in LPS-induced lung injury.
Given that epithelial GM-CSF contributed to AEC II proliferation in the resolution phase of LPS-induced lung injury, we questioned whether epithelial GM-CSF might also enhance restoration of alveolar barrier function in our model. Therefore, alveolar leakage was assessed in LPS-injured mice of the three treatment groups in the time course after LPS administration. We detected prominent induction of alveolar leakage in wt and SPC-GM mice after 6 hours of LPS instillation, which was found to be reduced to baseline levels after 96 hours. GM−/− mice, however, showed sustained alveolar barrier dysfunction until 240 hours post LPS administration, suggesting that GM-CSF enhances AEC II proliferation and renewal and contributes to restoration of alveolar barrier function severely disturbed in LPS-induced acute lung injury (Figure 6F). Altogether, these data demonstrate that LPS-activated AMϕ, via TNF-α release, induce epithelial GM-CSF expression, which in turn initiates AEC II proliferation and contributes to restore alveolar barrier function (Figure 6G).
Acute lung injury on bacterial infection is associated with severe damage of the alveolar epithelial barrier, followed by edema formation and impaired gas exchange (37). Consequently, epithelial repair processes are initiated to restore the normal lung homeostasis. The contribution of resident AMϕ to alveolar repair processes has been shown to occur during the later phase of infection and is usually associated with a transition from a proinflammatory into an antiinflammatory macrophage phenotype (5, 6). However, the potential of early-activated, proinflammatory AMϕ to influence epithelial repair processes remains elusive.
We hypothesized that proinflammatory AMϕ may contribute to effective epithelial repair after LPS-induced lung inflammation. Hence, in vitro experiments revealed that alveolar epithelial cells cocultured with LPS-stimulated AMϕ express significantly higher amounts of growth factors, particularly of GM-CSF. Macrophage TNF-α released on LPS stimulation was identified as a mediator inducing GM-CSF expression in epithelial cells, which in turn elicited autocrine proliferative signaling in alveolar epithelial cells. Genetic deletion of GM-CSF resulted in absence of AMϕ-induced epithelial cell proliferation. Similarly, in vivo TNF-α neutralization after LPS-induced lung injury impaired alveolar epithelial type II proliferation. Finally, GM-CSF–deficient mice displayed reduced AEC II proliferation and sustained alveolar leakage after LPS challenge. Together, these data reveal that alveolar repair processes are initiated early in the inflammatory course of LPS-induced acute lung injury and are mediated by macrophage TNF-α and epithelial GM-CSF.
Interestingly, our group recently showed that ExMϕ massively recruited during influenza virus pneumonia induce alveolar epithelial apoptosis via TNF-related apoptosis-inducing ligand, thereby contributing to loss of barrier function (38). In contrast, in the LPS model with very limited ExMϕ accumulation in the airspaces (0.0565 ± 0.02 × 106 after 96 h, Figure E4A), we observe opposing reparative effects of AMϕ toward alveolar epithelium and comparable AEC II proliferation in CCR2−/− mice lacking ExMϕ recruitment (Figure E4B). Such a divergent role of lung macrophages emerges most likely from the macrophage phenotype analyzed (resident versus recruited) and the different inflammatory models applied. Previous reports demonstrated that lung macrophages actively contribute to the resolution of inflammation once they have been signaled to acquire an antiinflammatory state by lipid mediators, such as resolvins, lipoxins, or prostaglandins (39). These signaling events result in enhanced macrophage phagocytosis activity and finally in clearance of apoptotic neutrophils and debris from the site of infection, restoring normal organ function (4). Moreover, antiinflammatory alveolar macrophages were noted to directly release epithelial mitogens, thereby inducing alveolar epithelial cell proliferation (17, 22, 40). Our data add to the aforementioned concept of macrophage–epithelial cross-talk during alveolar reparative events. We furthermore provide data demonstrating that alveolar epithelial cell proliferation is dependent on macrophage TNF-α in vitro and in vivo, whereas LPS-activated AMϕ did not express any of the epithelial mitogens analyzed. These data suggest that epithelial repair processes are implemented yet in the acute phase of alveolar inflammation and highlight the complexity of intercellular communication in lung inflammation and repair.
TNF-α is an early proinflammatory cytokine, known to be primarily released from activated AMϕ and to stimulate alveolar cell populations for chemokine release and adhesion molecule expression, thereby initiating and maintaining innate host defense (1). Besides its predominant proinflammatory, tissue-destructive role, several reports suggested TNF-α to exert resolution- and repair-enhancing effects by different mechanisms. In this line, TNF-α was shown to induce urokinase-type plasminogen activator in alveolar epithelial cells followed by lysis of alveolar fibrin and resolution of inflammation (41). Similar to the data presented in our study, proliferation of gastric epithelium revealed to be TNF-α dependent (16). Moreover, TNF-α has been previously reported to induce expression of GM-CSF in endothelial cells and fibroblasts via involvement of the transcription factor NF-κB, shown to be nuclearly translocated on TNF receptor binding in primary human lung epithelial cells (42–44). Besides its widely known proinflammatory function, NF-κB has recently been associated with signaling events mediating the resolution of inflammation (45), strengthening the concept of a dichotomic role of TNF-α–induced signaling events in acute inflammation.
We furthermore demonstrated that TNF-α–mediated alveolar epithelial cell proliferation was largely mediated by the epithelial growth factor GM-CSF in vitro and in vivo. GM-CSF is a well-known growth factor for phagocytes, but also stimulates maturation of eosinophils, erythrocytes, megakaryocytes, and dendritic cells. Apart from its effects on progenitor cells, GM-CSF improves host defense functions of mature hematopoietic cells, such as alveolar macrophages (46). More recent reports suggested a role of GM-CSF in the proliferation of alveolar type II cells (13, 14); however, the contribution of GM-CSF to epithelial repair and restoration of alveolar barrier function on LPS-induced acute lung injury has not previously been addressed.
AEC type II have been shown to express both α and β GM-CSF receptor subunits in rodents (14, 47). We found both subunits expressed in cultured alveolar epithelial cells and down-regulated during transdifferentiation into type I–like cells, which was associated with pronounced GM-CSF–induced proliferation of AEC at Day 1 but not at Day 3 of culture. Similarly, AEC grown on Matrigel:collagen thereby maintained in the type II phenotype as previously described (48) expressed GM-CSF on coculture with LPS-stimulated AMϕ and proliferated in response to GM-CSF, confirming the concept that type II cells as opposed to differentiated type I cells represent the proliferating subpopulation (9). However, in vitro AEC transdifferentiation analyzed by marker expression may be influenced by several factors, including culture conditions, and does not imperatively reflect AEC phenotypes or differentiation processes occurring in vivo. We provide additional evidence that GM-CSF–induced AEC proliferation was signaled by Stat5 phosphorylation resulting in increased expression of Cyclin D1. The JAK2-Stat5-Cyclin D1 pathway has been shown to be the underlying mechanism in prolactin-stimulated proliferation of mammary epithelial cells (35). Additionally, GM-CSF was reported to activate MAPK and PI3K in myeloid cells (34); however, we could not detect activation of these pathways in alveolar epithelial cells.
In accordance with data obtained from a rat model (49), we demonstrated that alveolar repair processes in terms of epithelial cell type II proliferation were initiated 4 days after LPS instillation, when alveolar inflammation decreased virtually to baseline levels. In contrast, we observed significantly reduced epithelial proliferation and sustained loss of barrier function throughout Day 10 post LPS challenge in GM-CSF–deficient mice in vivo, confirming our in vitro findings with GM-CSF–deficient alveolar epithelial cells lacking a TNF-α–induced proliferative response. Interestingly, AEC II proliferation after LPS challenge was completely rescued in SPC-GM mice, and epithelial GM-CSF release was widely reduced on alveolar TNF-α neutralization in wt mice in vivo. These data indicate that the alveolar epithelium itself is the primary source of GM-CSF, which is in turn released in the presence of TNF-α, emphasizing the central role of alveolar type II epithelial cells in perpetuating self-renewal once they have received an initial macrophage signal. Of note, alveolar leakage was associated with AEC I apoptosis and declined on recovery of the AEC I pool in wt mice, suggesting that transdifferentiation of existing AEC II into AEC I might occur fast, as observed in vitro, and preceded AEC II proliferation peaking at 48 to 96 hours post injury. Although we do not provide direct evidence of AEC II to AEC I transdifferentiation in our model, it is a well-described phenomenon (31) and might occur in a GM-CSF–independent way as demonstrated in vitro, supported by the fact that GM−/− mice display a recovered AEC I pool after 96 hours (Figure 6E). However, sustained barrier dysfunction observed in GM−/− mice clearly indicates that GM-CSF is indispensable for successful barrier restoration after injury.
Interestingly, the neutrophilic response in GM−/− mice was more pronounced at 6 to 24 hours after LPS treatment as compared with wt mice, correlating with the previous findings that GM−/− neutrophils are fully functional and their recruitment at the onset of inflammation is successfully (over)compensated (11, 50). Prolonged alveolar neutrophil presence was observed at 96 and 148 hours post LPS instillation and is most likely due to alveolar macrophage dysfunction in GM−/− mice, with decreased phagocytosis of apoptotic neutrophils and delayed resolution of alveolar inflammation (50). Alveolar barrier dysfunction in various models of acute lung injury has been attributed to neutrophil release of tissue-damaging agents, such as proapoptotic factors, proteases, or reactive oxygen/nitrogen species (51, 52). However, sustained lung leakage in GM−/− mice was observed beyond the neutrophil decrease (240 h) indicating that the inflammatory injured epithelial barrier lacked an adequate proliferation stimulus in absence of GM-CSF. In contrast, neutrophil clearance was enhanced in GM-CSF–overexpressing SPC-GM mice, most likely correlated to enhanced macrophage phagocytotic function. SPC-GM mice also displayed faster alveolar neutrophilic influx than wt mice, probably due to the chemotactic activity of GM-CSF taking effect when present in excessive amounts (53). Alveolar TNF-α levels peaked at 6 hours post LPS instillation in all treatment groups; however, they were significantly increased in SPC-GM mice and decreased in GM-CSF–deficient compared with wt mice, indicating that, apart from its reparative effects on epithelial cells, GM-CSF may enhance macrophage host defense functions. A recent report suggested that GM-CSF regulates TLR4-dependent signaling events such as TNF-α release from LPS-treated alveolar macrophages via activation of the transcription factor PU.1 (54). Therefore, GM-CSF might promote alveolar repair on bacterial pneumonia in two ways: first, due to its direct proliferative effects on alveolar epithelium, and second, by enhancing macrophage TNF-α release, which in turn mediates further epithelial GM-CSF expression. TNF-α inhibition as therapeutic strategy to attenuate acute or chronic pulmonary inflammation might therefore hold the risk of insufficient tissue repair.
Our study demonstrates that epithelial repair processes may be primed already in the proinflammatory phase of acute lung injury. We provide evidence for the key role of macrophage TNF-α inducing alveolar repair via epithelial GM-CSF. Thus, detection of distinct intercellular cross-talk mechanisms mediating tissue repair in the course of severe pneumonia may identify therapeutic targets allowing timed and compartment-specific intervention strategies promoting regeneration of the injured alveolar barrier.
The authors thank P. Janssen, D. Hensel, and M. Lohmeyer for excellent technical assistance.
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