Mobilization and Margination of Bone Marrow Gr-1^high Monocytes during Sub-clinical Endotoxemia Predisposes the Lungs towards Acute Injury

Abstract

The specialized role of mouse Gr-1^high monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during sub-clinical endotoxemia in mice, and investigated if marginated monocytes contribute to lung injury in response to further septic stimuli. Sub-clinical low-dose i.v. LPS induced a rapid (within 2h), large scale mobilization of bone marrow Gr-1^high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these pre-marginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the pre-administration of low-dose LPS. The LPS-zymosan induced permeability increases were effectively abrogated by pre-treatment (30 min before zymosan challenge) with the platelet-activating factor (PAF) antagonist WEB 2086, in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of PAF/ceramide mediated pathways in this model. Depletion of monocytes (at 18h post clodronate-liposome treatment) significantly attenuated the LPS-zymosan induced permeability increase. However, restoration of normal LPS-induced Gr-1^high monocyte margination to the lungs (at 48h post clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1^high monocytes during sub-clinical endotoxemia primes the lungs toward further septic stimuli, and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.

INTRODUCTION

Accumulation of leukocytes within the microcirculation in contact with the endothelium is a central feature of the systemic inflammatory response during sepsis (1, 2). Margination of leukocytes is particularly pronounced in the lungs due in part to the combined effects of narrow capillary diameter of the pulmonary vasculature and reduced leukocyte deformability upon exposure to inflammatory stimuli (3, 4). Marginated leukocytes produce a highly localized and focussed release of inflammatory and injurious mediators, and hence pulmonary margination of leukocytes, in particular neutrophils, is considered to play a major role in the pathogenesis of acute lung injury (ALI) of extrapulmonary origin, related to sepsis and other causes of systemic inflammation (5-8). Monocytes also marginate to the lungs during systemic inflammation (9, 10) and have the capability to produce a wide range of pro-inflammatory mediators. We have previously shown that in response to high-dose i.v. LPS challenge, rapid monocyte margination to the pulmonary circulation produces activation of endothelial cells via cell contact-dependent TNF signaling mechanisms (11). This finding supports the notion that marginated monocytes can directly modify the course of pulmonary microvascular inflammation, but whether they make a direct or substantial contribution to the pathophysiology of ALI remains to be determined.

Monocytes are a pluripotent and heterogeneous population, consisting of at least two phenotypically distinct subsets in mice (12-14). Monocytes possessing the Gr-1^high/CCR2+/CX3CR1^low phenotype migrate into local sites of inflammation and injury (13, 15-20), including atherosclerotic lesions (21, 22), and have been termed the ‘inflammatory’ subset. A second subset with the phenotype Gr-1^low/CCR2-/CX3CR1^high is capable of entering tissues under resting as well as local inflammatory conditions, albeit in smaller numbers than the inflammatory subset (13, 23, 24), where they can differentiate into tissue macrophages (13, 25). Gr-1^low monocytes have also been shown to patrol mesenteric post-capillary venules through crawling on the resting endothelium and rapidly extravasate in response to bacterial peritonitis, suggesting a unique role for this subset in orchestrating the early response in certain vascular beds (24). Based on transient monocyte depletion and repopulation studies, it has been proposed that circulating Gr-1^high (Ly-6C^high) monocytes are derived directly from the bone marrow, and give rise to the Gr-1^low (Ly-6C^low) subset in a maturation process lasting several days (23, 26, 27). In contrast to their recruitment and participation in local inflammatory responses, little is known of the kinetic behavior of these monocyte subsets among the bone marrow, circulating and marginated pools during acute systemic inflammation such as sepsis. ALI is a frequent complication of sepsis, but the margination profiles of the monocyte subsets to pulmonary vascular beds and their potential roles in the progression of ALI have not been investigated.

Certain species, including livestock animals, possess a resident lung macrophage population attached to the pulmonary endothelium, known as pulmonary intravascular macrophages (PIM) (28, 29). The localized and vigorous production of vasoactive mediators and proinflammatory cytokines by PIM seems to be directly responsible for the unique susceptibility of sheep and pigs to experimental ALI of extrapulmonary origin induced by i.v. administration of LPS, particulate antigens or bacteria (30-32). In species lacking PIM, which include humans and laboratory animals such as mice and rats, it has been suggested that monocyte accumulation within the lungs under sustained systemic inflammatory conditions could eventually give rise to a population with a PIM-like phenotype (10, 33, 34).

In this study we hypothesized that monocytes marginated within the pulmonary microcirculation during sub-clinical endotoxemia, in particular those monocytes of the Gr-1^high inflammatory subset, are responsive to secondary septic stimuli in situ and therefore contribute to the development of extrapulmonary ALI. We show that during sub-clinical endotoxemia, the rapid and substantial mobilization of Gr-1^high bone marrow monocytes and their prolonged margination within the pulmonary vasculature produces a state of latent ‘priming’ in the lungs. By assessing the pro-inflammatory response of individual monocyte subsets to secondary LPS challenge and using a two-hit LPS-zymosan model of ALI, we provide direct in vivo evidence that suggests a central role for inflammatory monocytes in the evolution of ALI.

MATERIALS AND METHODS

Animals

All protocols were reviewed and approved by the UK Home Office, in accordance with the Animals (Scientific Procedures) Act 1986, UK. Experiments were performed using male C57BL6 mice (Charles River, Margate, UK) aged 8-12 weeks (22-26 g).

Induction of endotoxemia

Mice received a single i.v. (via tail vein) injection of 0.2 ng - 20 μg of LPS (Ultra Pure Escherichia coli O111:B4; Autogen Bioclear, Calne, UK). At various times after challenge, mice were heparinized i.v. and sacrificed by isoflurane overdose. Blood was obtained by cardiac puncture, and lungs excised with care taken to avoid the hilum connective tissues. In some experiments, single femur and tibia were removed for analysis of bone marrow cells.

Preparation of single cell suspensions

Lung cell suspensions were prepared from the excised lungs by mechanical disruption to release intravascular cells, as described previously (11). For quantitation and characterization of lung monocytes and neutrophils, finely minced lung tissue was homogenized on a 40-μm nylon mesh sieve (BD Falcon, Oxford, UK) with a syringe plunger and flushed through sieves with FACS medium (PBS with 2% FCS, 0.1% sodium azide and 5 mM EDTA). Homogenization and flushing of cells was then repeated in order to ensure maximal recovery of intravascular cells. For determination of in situ membrane-bound TNF (memTNF) levels on lung monocytes, minced lung tissues were resuspended with 10 μM BB94 (British Biotech, Abingdon, UK), a hydroxamate-based TNF-α converting enzyme inhibitor, before tissue homogenization and all subsequent procedures performed at 4°C or on ice, to minimize changes in memTNF levels prior to flow cytometry analysis (11). Lavage of the lungs for recovery of intra-alveolar cells was performed using 750 μl of saline, as described previously (35). Bone marrow cells were obtained by insertion of a 23 gauge needle into dissected femurs and tibias and flushing with 2 ml of FACS medium. Cells were then dispersed by pipetting and passed though a 40-μm nylon mesh sieve. After centrifugation, lung and bone marrow cells were resuspended in FACS medium supplemented with 20% goat serum (Invitrogen, Paisely, UK).

Flow cytometric analysis

Cells were stained in the dark at 4°C for 30 min with fluorophore-conjugated anti-mouse antibodies for CD11b (clone M1/70), Gr-1 (RB6-8C5), Ly-6G (1A8), Ly-6C (AL-21), NK-1.1 (PK136), TNF (MP6-XT22) (BD Pharmingen, Oxford, UK), F4/80 (CI:A3-1), CD115 (604B5 2E11), 7/4 (AbD Serotec, Oxford, UK), or appropriate isotype-matched control antibodies. In experiments involving blood or bone marrow, RBC lysis and fixation were performed using FACS-lyse solution (BD Pharmingen). Samples were acquired (a minimum of 1000 gated monocytes per sample) using a FACSCalibur flow cytometer and Cell Quest software (BD, Oxford, UK), and the data analyzed with Flowjo software (Ashland, OR, USA). Absolute cell counts in samples were determined using microspheres beads (Catlag Medsystems, Towcester, UK), as previously described (11, 36).

Immunohistochemistry

Mice were heparinised i.v., exsanguinated via the brachial artery and the lungs instilled with 750μl of 4% paraformaldehyde. Lungs were then excised and fixed for 18h before paraffin embedding and preparation of 5μm thick sections. Prior to antibody incubation, antigen recovery was performed by treatment with proteinase K (Sigma) at 20μg/ml in Tris-EDTA for 10min at room temperature. Lung tissues were then incubated with the Gr-1 antibody or isotype-matched control (2μg/ml, 30min room temperature) followed by detection with the Vectastain ABC rat IgG peroxidase Kit (Vector Laboratories, Peterborough, UK) according to manufacturer’s instructions. After 3, 3′-diaminobenzidine substrate (Vector Laboratories) development, sections were counterstained with methylene green.

Localization of monocytes within lungs

Localization of monocyte subsets within the lungs was determined by ex vivo perfusion of the pulmonary vessels and in vivo intravascular cell labelling. For washout and quantification of intravascular monocytes, we used a mouse isolated perfused lung system (IL-1, Hugo-Sachs Elektronik, Germany) as previously described (37, 38). Briefly, at 2h after LPS challenge, mice were anesthetized, tracheostomized and ventilated with air using a mouse ventilator (35). After midline thoracotomy and laparotomy, heparin (100 IU) was administered via the inferior vena cava, which was then cut for exsanguination. The pulmonary artery and left atrium were cannulated, and the lungs were perfused at a constant flow of 50 ml/kg and left atrial pressure of 2.5 mmHg, and ventilated with 21% O₂ and 5% CO₂ in N₂. The perfusate used was RPMI-1640 without phenol red (Invitrogen) with pH adjusted to 7.35-7.45, supplemented with 4% BSA (Sigma, Poole, UK) and 5 mM EDTA to facilitate release of marginated monocytes (39). After perfusion for 40 min at 37°C, the lungs were harvested and single cells suspensions prepared for analysis by flow cytometry. For in vivo staining of lung-marginated monocytes, we used a modified version of a labelling technique of intravascular cells previously reported in the literature (40). At 2h after LPS challenge, mice received i.v. injection of 2 μg of anti-CD45 PE-conjugated antibody (clone 30-F11, BD Pharmingen) just before they were sacrificed. A 10 min pulmonary arterial perfusion using PBS was performed to wash out residual antibody within the pulmonary vasculature, prior to analysis of lung single cell suspensions by flow cytometry.

BrdU labeling of dividing monocytes in vivo

Mice received a single i.p. injection of 0.2 ml of BrdU (10 mg/ml in saline), and after 6h injected i.v. with saline or LPS. Margination of recently divided bone marrow cells to the lungs was determined by flow cytometry using a BrdU ‘Flow Kit’ according to the manufacturer’s instructions (BD Pharmingen). Single cell suspensions from lungs were stained with appropriate marker antibodies to identify monocyte subsets, and then washed, fixed, permeabilised and treated with DNAse to reveal BrdU epitopes. BrdU incorporation was determined by staining with a FITC anti-BrdU monoclonal antibody.

Depletion of monocytes in vivo

Clodronate (dichloromethylene-bisphosphonate) was a gift from Roche Diagnostics GmbH (Mannheim, Germany) and was incorporated into liposomes as previously described (41). Intravascular monocytes and macrophages were depleted by i.v. injection of 0.2 ml of clodronate as described previously (23, 42). Mice were used for LPS or LPS-zymosan challenge experiments at 18h or 48h after clodronate-liposome treatment, before significant repopulation of splenic macrophages (43) and liver Kupffer cells (44).

Zymosan challenge and measurement of pulmonary vascular permeability

Zymosan-induced pulmonary vascular permeability changes were assessed using an adaptation of previously reported fluorescence-based method for measuring vascular leak into the alveolar space (45, 46). Zymosan particles (Invitrogen) were suspended in saline and mixed with Alexa-Fluor 594-conjugated BSA (Invitrogen) (1 mg/mouse) and immediately injected i.v. in a total volume of 0.2 ml. To exclude the influence of intravascular BSA in the residual blood left in the lung samples, mice were injected i.v. with 0.2 ml of Alexa-Fluor 488-conjugated BSA (Invitrogen) (2 mg/mouse), together with heparin (100 U/ml) 5 min before sacrifice. Lungs were excised, rinsed briefly in saline and swabbed gently with tissue to remove surplus blood. Lungs were then homogenized (Kinematica Polytron PTA 7, Philip Harris Scientific, Lichfield, UK) and incubated with collagenase type IV (Sigma) for 30 mins at 37°C to ensure release of tissue-sequestered BSA. After further homogenization, lung suspensions were centrifuged and supernatants collected. Fluorescence levels were measured in lung homogenate supernatants and diluted whole blood samples (1/100) using fluorescence plate reader (Flx-800, Bio-tek Instruments Inc., UK). An index of vascular permeability was calculated by subtracting the lung:blood ratio of BSA-Alexa-Fluor 488nm (intravascular content) from the lung:blood ratio of BSA-Alexa-Fluor 594nm (total lung content).

Pharmacological inhibition of LPS-zymosan induced ALI

30 min before zymosan challenge in the above LPS-zymosan model, mice were injected (i.p., 0.2ml) with a platelet-activating factor (PAF) receptor antagonist WEB 2086 (5mg/kg in 1.25% DMSO in PBS) alone, or in combination with a non-selective cyclo-oxygenase inhibitor indomethacin (10mg/kg in DMSO/PBS) or a phosphatidylcholine-phospholipase C (PC-PLC) inhibitor D609 (40mg/kg in PBS). DMSO in PBS solution served as a vehicle control.

Statistical analysis

Data are expressed as mean ± SD. Statistical comparisons were made by t-tests or ANOVA with Bonferroni tests. Statistical significance was defined as p< 0.05.

RESULTS

Sub-clinical endotoxemia produces prolonged margination of Gr-1^high ‘inflammatory’ monocytes to the lungs

Monocyte recruitment to the pulmonary circulation in mice during endotoxemia was evaluated by flow cytometric analysis of lung single cell suspensions prepared from the excised lungs of mice, as described previously (11). Monocytes were identified as CD11b+, F4/80+ cells, and differentiation between individual monocyte subsets was performed by staining for Gr-1 (Ly-6C and Ly-6G specific). In lung cell suspensions from mice injected i.v. with saline or low-dose LPS (20 ng), two main monocyte populations were defined; Gr-1^high and Gr-1^low monocytes (Figure 1A). The Gr-1^high monocytes (CD11b+, F4/80+, Gr-1 high) expressed uniformly high levels of 7/4 antigen and were negative for NK-1.1 expression (Figure 1B), excluding the possibility of eosinophil (CD11b+, F4/80+, Gr-1 high, 7/4 low) or NK cell (CD11b+, F4/80-ve, Gr-1 low to medium) contamination (47, 48). The CD11b+, F4/80+ cells were found to be negative for Ly-6G expression, indicating that neutrophil (CD11b+, F4/80−, Gr-1 very high, Ly-6G high) contamination was minimal in the gated Gr-1^high monocyte population. This gating strategy for identification of monocytes was confirmed appropriate by staining with another monocyte/macrophage lineage marker, the M-CSF receptor, CD115 (data not shown), however, we found partial downregulation of CD115 expression on blood and lung monocytes after LPS challenge, which precluded the routine use of CD115 as a marker in this study.

Figure 1. Flow cytometric analysis of monocyte subsets in the lungs of normal and LPS challenged mice.

A. Mice were injected i.v. with saline or 20 ng of LPS, and after 2h the lungs were processed for flow cytometric analysis. Monocytes were identified as CD11b+, F4/80+ events, and their subsets were defined as either Gr-1 low (R1) or high (R2), differentiated from neutrophils as CD11b+, F4/80−, Gr-1 very high events (R3). F4/80+ events did not express high levels of Ly-6G (R4), thereby ruling out contamination of R1 and R2 gates with neutrophils. Percentage frequencies among total CD11b+ events are depicted for each gate. B. Staining with 7/4 or anti-NK-1.1 antibodies (red line) compared to isotype-matched control antibody (blue fill) indicated that gate R2 did not contain eosinophils (7/4 low) or NK cells.

To characterize monocyte subset recruitment to the lungs in response to various degrees of endotoxemia, LPS dose-response experiments were performed (Figure 2). At 2h after i.v. LPS challenge (0.2 ng - 20 μg/mouse), numbers of Gr-1^high monocytes within the lungs were substantially increased at LPS doses > 0.2 ng (n=3-4/each dose, p<0.05), while Gr-1^low monocytes did not show significant increases. Although there were large increases in both lung-associated and circulating neutrophils, their mean numbers in the lungs did not exceed those of Gr-1^high monocytes. Doses of LPS less than 2 μg/mouse did not produce obvious symptoms such as lethargy or piloerection, and were therefore termed sub-clinical. At the sub-clinical dose of 20 ng, the recruitment of Gr-1^high monocytes reached the maximum, i.e. ~20-fold increases compared to saline-treated controls (1.58 vs. 0.08 × 10⁶ cells), but elevated numbers (6-fold increase), although not statistically significant, were already observed even at the lowest LPS dose of 0.2 ng. In contrast to the increases seen in the lungs, circulating Gr-1^high monocyte numbers were not markedly altered compared to saline-treated mice, except with the lowest (0.2 ng) and highest (20 μg) LPS doses, where there were tendencies towards monocytophilia and monocytopenia. Because the LPS dose of 20 ng produced maximal monocyte recruitment while being substantially lower than the clinical threshold dose of 2 μg, it was chosen for all subsequent sub-clinical LPS challenge experiments.

Figure 2. Gr-1^high monocyte margination to the lungs during clinical and sub-clinical endotoxemia.

Mice were injected i.v. with different doses of LPS (0.2 ng - 20 μg) and after 2h numbers of Gr-1^low monocytes, Gr-1^high monocytes and neutrophils in lungs and blood were quantified by flow cytometry. Mice receiving less than the 2 μg dose of LPS appeared normal, displaying no clinical effects of the treatment. n=3-4 for each LPS dose, *p < 0.05, **p < 0.01, different from saline-treated mice of the same cell type. The differences in dose-response curves among the cell types were confirmed by a significant interaction p-value (p<0.01) by two-way ANOVA.

To determine whether the LPS-induced elevation of lung monocyte numbers is maintained for prolonged periods and therefore likely to have an influence on pulmonary physiology, time course experiments were performed (Figure 3, n=3-4/each time point). At 1h after i.v. LPS challenge, numbers of lung-associated Gr-1^high monocytes were similar to baseline, suggesting that unlike the acute LPS challenge protocol with high bolus doses (20 - 200 μg) used in our previous study (11), sub-clinical LPS challenge did not induce an immediate relocation of circulating monocytes to the lungs. Numbers of lung-associated Gr-1^high monocytes then increased and were maximal at 2h, but the decline thereafter was gradual, i.e. Gr-1^high monocytes remained elevated up to 8h (p<0.01), with an 8.5-fold higher than baseline, albeit non-significant increase even at 18h after LPS challenge. This gradual decrease in the lung Gr-1^high monocytes was accompanied by an increase in the circulating Gr-1^high monocytes (p<0.05), and tendencies towards increases in the lung and circulating Gr-1^low monocytes. Significantly, the kinetics of the neutrophil response was quite distinct from that of monocytes, with increases in circulating and lung-associated cells as early as 1h, but then a more rapid decline after 2h, with numbers returning to normal by 18h after LPS challenge.

Figure 3. Time course of Gr-1^high monocyte margination to the lungs during sub-clinical endotoxemia.

Mice were injected i.v. with the sub-clinical 20 ng LPS dose and sacrificed for quantification of the lung and blood cell at the time points shown. n=3-4 for each time point, *p < 0.05, **p < 0.01, different from saline-treated mice of the same cell type. The differences in the time course among the cell types were confirmed by a significant interaction p-value (p<0.01) by two-way ANOVA.

Localization of these monocytes within the lungs, i.e. whether they still reside in the capillaries (i.e. marginated) or have extravasated into the lung tissue, was assessed by both immunohistochemical and flow cytometric analysis. Gr-1 staining of lung sections from untreated and LPS treated (20ng i.v., 2h) mice (Figure 4) indicated that Gr-1^high monocytes and neutrophils were confined to the alveolar septa and therefore located within capillaries or interstitial spaces. To determine the proportion of monocytes residing within the vascular space after LPS challenge, ex vivo perfusion of the lungs was performed with 3-colour quantitative flow cytometric analysis of monocyte subsets as described above. Perfusion of lungs under stable physiological conditions for 40 min resulted in a marked reduction of both monocyte subsets (~70% for Gr-1^low and ~85% for Gr-1^high) recovered from the lungs, compared to the non-perfused lungs from control, surgery-only LPS-challenged mice (Figure 5A). Consistent with these data from lung cell suspensions, substantial numbers of monocytes (0.59±0.14 × 10⁶ cells for Gr-1^low and 1.61±0.30 × 10⁶ cells for Gr-1^high) were found to be washed out and recovered in perfusates. Alveolar macrophages (high forward scatter, F4/80+) were not detected in perfusates, indicating that the alveolar compartment cell barrier remained intact during the procedure. Further confirmation of monocyte subset location within the lungs was established by the in vivo intravascular cell labeling technique using fluorescence-conjugated anti-CD45 antibody injected i.v. just before sacrificing mice. Following a short pulmonary arterial perfusion to recover loosely adhered cells as well as wash out residual antibodies, the CD11b+ F4/80+ monocytes remaining in the lungs were found to be uniformly stained for CD45 (and therefore considered to be “exposed” in vivo to the intravascular space), and the level of this staining was similar to that of the washed-out monocytes in the perfusates (Figure 5B). Alveolar macrophages were negative for CD45 (data not shown), indicating that the in vivo labeling was restricted to cells within the intravascular compartment. Taken together, these results demonstrated that the majority, if not all, of the monocytes in lung cell suspensions harvested from LPS-challenged mice still reside within the pulmonary vasculature, and therefore represent a ‘marginated’ cell population.

Figure 4. Immunohistochemical analysis of Gr-1 positive cell recruitment to the lungs during sub-clinical endotoxemia.

Lung sections from untreated (a and c) and low-dose LPS treated (20ng i.v., 2h) (b and d) mice were stained with the Gr-1 mAb (RB6-8C5) or an isotype-matched antibody control (not shown). Antibody binding was detected using the 3, 3′-diaminobenzidine substrate followed by counterstaining with methylene green. Increased densities of Gr-1 positive cells were evident after LPS challenge (magnification ×200: a and c), with the majority of positive cells associated with the alveolar septa (magnification ×1000: b and d, arrows).

Figure 5. Monocytes recruited to the lungs during sub-clinical endotoxemia are marginated within the pulmonary vasculature.

A. The proportion of monocytes residing within the pulmonary vasculature was determined by pulmonary artery perfusion. At 2h after LPS (20ng, i.v.) challenge, mice were anaesthetised and surgery/instrumentation performed for isolated perfused lung preparation. Following exsanguination, the lungs were perfused at a constant flow using RPMI with 4% BSA and 5 mM EDTA for 40min. Perfused lungs were analysed by flow cytometry. n=4/each group, **p < 0.01 compared to non-perfused, surgery-only controls. B. Localization of monocyte subsets within the lungs after LPS challenge was confirmed by i.v. injection of PE-labelled anti-CD45 antibody just before sacrificing the mice, followed by a short (10 min) pulmonary artery perfusion step to remove excess antibody. Lung cells suspensions were stained in vitro for CD11b, F4/80 and Gr-1 and analyzed by flow cytometry. Panels on the right represent the in vivo CD45 staining of monocyte (CD11b+, F4/80+) subsets in the lungs after perfusion (top right) and the perfusate (bottom right), indicating that the majority of cells were exposed to intravascular space. For comparison, the left panel shows isotype control antibody staining of monocytes in non-perfused lungs of LPS pre-treated mice.

Lung-marginated Gr-1^high monocytes are derived from the bone marrow reservoir during sub-clinical endotoxemia

The normal size of the total monocyte circulating pool in mice has been estimated at 6 × 10⁵ (49). Given that the numbers of the Gr-1^high subset in the lungs increased by approximately 1.0 × 10⁶ cells, and that this increase was not accompanied by any marked change in circulating monocyte numbers, we investigated the possibility that the bone marrow is a major source of newly-marginated monocytes in the lungs during sub-clinical endotoxemia. Mobilization of bone marrow monocytes was quantified by flow cytometry using anti-Ly-6C (the target of anti-Gr-1 binding to monocytes) and anti-CD115 antibodies. This staining combination, despite reduced expression of CD115 on monocytes after LPS, produced clearer separation of monocyte subsets in bone marrow (Figure 6A) than the CD11b, F4/80 and Gr-1 combination, due to higher F4/80 staining on neutrophils in bone marrow than those seen in blood and lung. At 2h after i.v. LPS challenge, the Ly-6C^high and CD115+ cells in bone marrow were significantly decreased, compared with untreated control mice (from 3.0±0.4 to 1.8±0.2 × 10⁶ cells per single femur and tibia of a mouse, n=5/each group, p<0.01). To confirm that these Ly-6C^high (Gr-1^high) monocytes released from bone marrow actually marginate to the lungs, the thymidine analogue BrdU was administered i.p. 6h prior to LPS challenge, to pulse-label dividing cells in vivo. At 2h after i.v. LPS challenge, BrdU-labelled Gr-1^high monocytes in the lung cell suspensions showed a marked (~12-fold) increase compared to the control non-LPS challenged mice (Figure 6B, n=3/each group, p<0.01), indicating that sub-clinical endotoxemia induced substantial margination of recently-divided bone marrow Gr-1^high monocytes to the lungs.

Figure 6. Bone marrow mobilisation and margination of immature Ly-6C^high (Gr-1^high) monocytes to the lungs during sub-clinical endotoxemia.

A. Mice were injected i.v with 20 ng of LPS, and after 2h bone marrow (femur and tibia) and lungs processed for flow cytometry. Bone marrow monocytes were identified using the combination of anti-Ly-6C and anti-CD115 (M-CSF receptor) antibodies (top panel). Quantification of total immature monocytes (Ly-6C^high, CD115+) per single femur and tibia were performed in saline and LPS treated mice (bottom panel). n=5/each group, **p<0.01 compared to control saline-treated mice. B. To determine the origin of lung-marginated monocytes, mice were injected i.p. with 2 mg of BrdU to label dividing bone marrow precursor cells in vivo. At 6h post BrdU treatment, mice were injected with saline or LPS and after 2h lungs were analysed by staining of permeabilized cells with FITC-conjugated anti-BrdU antibody. Dots plots of Gr-1 vs anti-BrdU labelling in gated monocytes (CD11b+, F4/80+, top panel) and the mean percentage of BrdU positive cells among lung-marginated Gr-1^high monocytes (bottom panel) are depicted. n=3/each group, **p<0.01 compared to control saline-treated mice.

Extrapolation of the above data using single femur and tibia to the whole body bone marrow pool suggests that the numbers of Ly-6C^high (Gr-1^high) monocytes released from bone marrow in response to sub-clinical endotoxemia were far in excess of those found in the lungs and blood. Distribution of Gr-1^high monocytes amongst major body organs was therefore evaluated in control and LPS-treated mice, using flow cytometric analysis of cell suspensions derived from excised brain, heart, liver, spleen and kidney. In addition to the lungs, large increases in the marginated pool of Gr-1^high monocytes were observed in the liver (0.068±0.011 to 3.4±2.4 × 10⁶ cells) and the spleen (0.53±0.24 to 2.7±2.2 × 10⁶ cells) (n=3-4/each group).

Lung-marginated Gr-1^high monocytes are primed and respond vigorously to further intravascular LPS challenge

Accumulation of monocytes in the pulmonary vasculature in contact with endothelial cells provides the conditions necessary for cell-mediated and highly focused inflammatory signaling, potentially leading to active local monocyte-endothelial interactions in response to further intravascular septic stimuli. To evaluate the responsiveness of lung-marginated monocytes in such two-hit models of pulmonary microvascular inflammation, mice pre-treated with low dose i.v. LPS (to induce monocyte margination) were re-challenged at 2h with low (20 ng) or high dose (20 μg) i.v. LPS, and the monocyte pro-inflammatory response assessed by measuring levels of surface memTNF expression at 30min after the secondary LPS challenge. Primary LPS followed by secondary low-dose LPS challenge produced a dramatic increase in memTNF expression on Gr-1^high but not Gr-1^low monocytes (n=3-4/each group, p<0.01), whereas memTNF levels after a single, low dose LPS injection (at either the primary or secondary challenge time-points) were negligible (Figure 7). Thus, sub-clinical, low-dose LPS induced margination and ‘priming’ (enhanced responsiveness to secondary septic stimuli) of Gr-1^high monocytes in the lungs. When a high LPS dose was used for the secondary LPS challenge, this priming effect of low-dose LPS pre-treatment on the Gr-1^high subset was still evident despite the relatively high levels of memTNF expressed on monocytes in non-primed mice. Numbers of lung-marginated Gr-1^high monocytes were unchanged at 30 min after secondary low-dose LPS challenge (1.0±0.28 × 10⁶ cells) when compared with mice receiving only the priming low dose of LPS (0.95±0.11 × 10⁶ cells), but increased after the high dose of LPS (1.7±0.43 × 10⁶cells, n=4/each group, p<0.05), with a substantial reduction in circulating Gr-1^high monocytes compared to the single LPS challenge (0.13±0.088 vs.1.5 ±0.58 × 10⁵ cells/ml blood, n=4/each group, p<0.01). Thus, depending on the dose of the secondary LPS challenge, the lung-marginated monocyte population may expand further, enhancing the total cell-mediated pulmonary vascular inflammatory response. Only mice receiving the high secondary LPS dose developed symptoms over a 4h observation period, with a tendency to increase the severity of symptoms in primed as compared to non-primed mice.

Figure 7. Gr-1^high monocytes marginated to the lungs during sub-clinical endotoxemia are primed towards further LPS stimulation.

Mice were injected i.v. with saline or LPS (20ng) and after 2h received a secondary challenge of either saline, high dose (20 μg) or low dose (20 ng or 40 ng) LPS. At 30 min after the second injection, lungs were processed and monocyte subsets analysed for expression of memTNF. A. Representative histograms showing levels of memTNF expression (black line), compared to isotype-matched control antibody binding (gray fill), on individual monocyte subsets after single or two-hit low-dose LPS challenge. B. Levels of memTNF on individual monocyte subsets expressed as the geometric mean of fluorescent intensity (MFI) with values of isotype control subtracted. n=3-4/each group. ^†p < 0.01, different from each other.

Lung-marginated Gr-1^high monocytes contribute directly to pulmonary vascular leak in a model of zymosan-induced extrapulmonary ALI

To investigate whether monocyte margination and priming induced by sub-clinical LPS challenge would contribute to the progression of ALI of extrapulmonary origin, we developed a modification of the previously characterized LPS-zymosan two-hit model of ALI (50-52). In contrast to the model described in the literature in which mice are pre-treated with a high clinical dose of LPS to enhance zymosan-induced ALI changes, we pre-treated mice with the low sub-clinical dose of i.v. LPS (20 ng/mouse), to induce monocyte priming of the lungs in the absence of pronounced physiological effects. Secondary i.v. zymosan challenge (150 μg/mouse) at 2h after LPS resulted in the rapid (within minutes) development of severe but transient shock-like symptoms, including crawling and prostration followed by a state of prolonged lethargy with piloerection. At a higher dose of zymosan, i.e. 250 μg/mouse similar to the highest dose used in previous studies (50-52), early mortality within 20-30 min was observed in more than half of LPS-primed mice, while the lower dose of zymosan produced overt clinical symptoms but no mortality. At the lower dose of zymosan without LPS priming, clinical symptoms were completely absent with mice appearing visibly unaffected. Zymosan-induced ALI was assessed by measuring changes in pulmonary vascular permeability, an early and direct cause of lung dysfunction in this model, at 1h after zymosan challenge (Figure 8A). Priming of mice with LPS, which on its own had no effect on lung permeability, led to a significant enhancement of zymosan-induced pulmonary vascular leak (n=4-6/each group, p<0.01), i.e. ~6-fold increases in the vascular permeability index as compared to control saline-treated mice, as assessed by accumulation of labeled BSA in lung tissue. At 1h post-zymosan in LPS pre-treated mice, neither monocytes nor neutrophils were present in appreciable numbers in lung lavage fluid (<100 gated cells, n=3), indicating that migration of leukocytes across the endothelial-epithelial barrier into the alveolar space is not a feature of this model.

Figure 8. Sub-clinical endotoxemia enhances zymosan-induced pulmonary vascular leak.

A. Mice were injected i.v. with low-dose LPS (20ng) and then challenged i.v at 2h with zymosan (150 μg/mouse) and fluorescence-labeled BSA. Changes in pulmonary vascular permeability were determined at 1h after zymosan challenge by measurement of extravascular BSA and expressed as a permeability index. n=4-6/each group. **p < 0.01, different from control non-treated mice. B. Attenuation of LPS-zymosan-induced pulmonary vascular leak by administration (i.p.) of WEB 2086 (5mg/kg), or WEB 2086 with D609 (40mg/kg), but not indomethacin (10mg/kg), 30min before challenge of LPS pre-treated mice with zymosan. n=4-6/each group. *p < 0.05, **p < 0.01 different from control non-treated mice. †p < 0.01, different from each other.

To gain an insight into the mechanisms responsible for increased vascular permeability in this model, we performed in vivo pharmacological inhibition studies. Contrasting with the response to the LPS-LPS two-hit model, memTNF expression on monocytes at 30 or 60 min post-zymosan challenge were negligible (data not shown), suggesting that TNF signalling is unlikely to be a significant component in this LPS-zymosan model of ALI. As PAF has been implicated as the major mediator in the previously described high-dose LPS-zymosan model (50), low-dose LPS (20ng/mouse) pre-treated mice were administered (i.p.) the PAF receptor antagonist WEB 2086 at 30 min before zymosan challenge. Treatment with 5 mg/kg WEB 2086 resulted in a dramatic reduction in the severity of clinical symptoms described above and a partial reduction (~30%) in the lung permeability index (Figure 8B). Because higher doses of WEB 2086 did not produce any further decrease in permeability (data not shown), we then examined combined effects of WEB 2086 with inhibitors of two major downstream pathways implicated in PAF-induced ALI, i.e. cyclo-oxygenase-mediated pathway and PC-PLC-mediated activation of spyngomyelinase/ceramide pathway (53). Treatment with WEB 2086 and indomethacin (cyclo-oxygenase inhibitor) did not result in any further attenuation, whereas treatment with WEB 2086 and D609 (PC-PLC inhibitor) produced a substantial decrease (~70%) in permeability. This treatment produced no significant effect on numbers of lung-marginated monocytes and neutrophils at 2h post LPS (Gr-1^low monocytes, 0.25±0.082 × 10⁶ cells; Gr-1^high monocytes, 1.6±0.35 × 10⁶ cells; neutrophils, 1.3±0.20 × 10⁶ cells; n=4 for each group) compared with the data in Figure 2, indicating that the protective effect was not due to a reversal of margination process.

The contribution of LPS-pre-marginated Gr-1^high monocytes to zymosan-induced pulmonary vascular leak was assessed by i.v. treatment with liposome-encapsulated clodronate (clodronate-liposomes), a standard method used for depletion of monocytes and resident intravascular macrophages (41). To confirm the ability of clodronate-liposome treatment to reduce monocyte margination to the lungs during sub-clinical endotoxemia, mice were injected i.v. with clodronate-liposomes, and then challenged with low-dose LPS (20ng/mouse) 18h and 48h later. At 18h post-clodronate-liposomes, LPS-induced margination of both Gr-1^high and Gr-1^low monocytes was effectively ablated, with circulating numbers also decreased (Gr-1^low monocytes, 1.2±0.41 × 10⁴ cells; Gr-1^high monocytes, 2.5±0.097 × 10⁴ cells/ml blood, n=3) when compared with data in Figure 2, whereas no apparent alteration to LPS-induced neutrophil margination was observed (Figure 9A). At 48h post-clodronate-liposomes, normal Gr-1^high monocyte margination levels in response to low-dose LPS were restored, presumably due to the replenishment of the bone marrow reservoir of Gr-1^high monocytes at this time (27). Thus it was possible to compare the effects of LPS-zymosan challenge in mice lacking both monocytes and macrophages (18h post-clodronate-liposomes) with mice in which only the Gr-1^high monocyte population was present (48h post-clodronate-liposomes). Mice at 18h post-clodronate-liposomes displayed markedly less severe symptoms and substantially reduced permeability increases compared to non-clodronate-liposome treated animals after challenge with the lower zymosan dose of 150 μg (Figure 9B, n=4-6/each group, p<0.01), and no mortality at the higher dose of 250 μg (n=5). By contrast at 48h post-clodronate-liposomes, severity of clinical symptoms, permeability increases and mortality at higher zymosan dose returned to levels seen in non-clodronate-liposome treated mice, indicating a direct role for lung-marginated Gr-1^high monocytes in promoting lung permeability in zymosan-induced ALI.

Figure 9. Gr-1^high monocytes mediate zymosan-induced pulmonary vascular leak in low dose LPS-pre-treated mice.

A. To deplete monocytes, mice were injected i.v. with clodronate-liposomes (200 μl). The efficacy and specificity of depletion was assessed at 18h and 48h after clodronate-liposome treatment by injection of LPS and after a further 2h, determining numbers of lung-marginated monocytes and neutrophils. (n=3/each group). B. Attenuation of LPS-zymosan-induced pulmonary vascular leak by pre-treatment with clodronate-liposomes 18h before LPS challenge and its reversal at 48h post clodronate-liposome pre-treatment. n=4-6/each group. ^†p < 0.01, different from each other; **p < 0.01, different from control non-treated mice (displayed in Figure 6). Clod: clodronate-liposomes.

DISCUSSION

Although monocyte accumulation within the pulmonary circulation in response to systemic septic stimuli is well documented, there is little evidence to suggest that lung-marginated monocytes are directly involved in the pathophysiology of sepsis-related ALI. In this study, we evaluated monocyte subset margination to the lungs during sub-clinical endotoxemia, the responsiveness of the marginated monocytes to secondary stimuli, and their impact on pulmonary vascular permeability in the two-hit LPS-zymosan model of ALI in mice. Our results provide the first in vivo evidence that margination of bone marrow-derived Gr-1^high monocytes during sub-clinical endotoxemia transforms the lungs into a latent primed state, thereby playing a crucial role in the evolution of sepsis-related ALI.

We previously investigated margination of total monocytes to the lungs at early time points during acute overwhelming high-dose endotoxemia, and demonstrated that marginated monocytes can produce pulmonary endothelial activation through local and cell-contact dependent signaling mechanisms (11). Here we extended our analysis to the more long-term consequences of monocyte margination during sub-acute endotoxemia, in particular with sub-clinical low-dose LPS challenge. We studied monocyte margination at the level of individual monocyte subsets, namely Gr-1^high (inflammatory) and Gr-1^low (resident) monocytes, in view of their recently-described distinct migratory and inflammatory properties (13, 54). Contrasting with the previously described early margination event at 45 min after high dose LPS challenge (11), sub-clinical endotoxemia induced a delayed (apparent at 2h) but long-lasting (up to 8h), and very substantial (>1.0 × 10⁶ cells, exceeding neutrophil numbers) accumulation of monocytes within the pulmonary circulation, predominantly due to margination of the Gr-1^high subset. This margination event was very sensitive to intravascular endotoxin, occurring at LPS doses 3-4 orders of magnitude below the threshold dose that induces clinical septic symptoms. Based on the size of the normal circulating monocyte pool and the absence of monocytopenia, this margination could not be explained by subset-specific entrapment of circulating monocytes. Instead, it was shown to be a result of the large scale mobilization of immature Gr-1^high monocytes from the bone marrow reservoir, and their preferential accumulation within the marginated, as opposed to circulating pool.

Mobilization of immature monocytes from bone marrow represents an important regulatory mechanism to expand the total monocyte pools within the vasculature during systemic or local infection and inflammation. Recent studies have identified CCR2 and its ligands, MCP-1 and MCP-3, as the key signalling molecules required for monocyte release from bone marrow to local sites under normal conditions, Listeria infection and sterile peritonitis (18, 55). However, previous analyses of monocyte recruitment from bone marrow have focused mainly on the resultant blood monocytosis, without paying much attention to the dynamic equilibrium that exists between the circulating and marginated pools. In the present study, during the first 2-3h after LPS challenge, we did not detect monocytosis in blood despite massive mobilization of bone marrow monocytes, instead, the newly-released Gr-1^high monocytes appeared to marginate directly to the peripheral organs including the lungs, liver and spleen. In contrast, neutrophils displayed increases in both the circulating and marginated pools immediately after LPS challenge, but the increases did not last as long as those in monocytes. Thus, our findings indicate that monocyte trafficking profile from the bone marrow to the vasculature during endotoxemia is different from neutrophils, with a greater propensity of monocytes to reside within the microcirculation. Circulating blood monocytes may represent only the tip of the iceberg of total (circulating + marginated) monocyte pools within the vasculature, highlighting potential limitations of monocyte analyses based on blood samples in sepsis or other inflammatory conditions.

The predominant margination of Gr-1^high, as compared to Gr-1^low, monocytes within the lungs could be attributed to the massive, LPS-induced influx of this subset from the bone marrow into the blood. The differences in cell adhesion and migration between the two subsets could also contribute to this Gr-1^high predominance, i.e. L-selectin or CCR2 which are expressed on Gr-1^high but not Gr-1^low monocytes (13). The mechanism of margination of monocytes as well as neutrophils to the lungs during endotoxemia, as compared to other body organs, has been generally ascribed to the unique anatomical structure of the pulmonary microvascular bed. Leukocytes must deform to pass through the narrow pulmonary capillaries, and LPS-induced physical stiffening of leukocytes as well as recruitment of stiffer immature bone marrow cells (56, 57) would lead to enhanced physical entrapment by the capillaries (9, 58). It has been suggested that there are some differences in the lung margination mechanism between monocytes and neutrophils. Sustained margination of monocytes with LPS has been shown to be CD18-dependent (9), while neutrophil margination is not affected by CD18 blocking in vivo (58). Compared to neutrophils, much lower concentrations of LPS seem to be required to induce monocyte margination in vivo (9, 59) and adherence to endothelial cells in vitro (60, 61), which were not investigated here but could be relevant to the different margination behaviour of monocytes and neutrophils during sub-clinical endotoxemia.

To assess the proinflammatory capacity of lung-marginated monocytes during sub-clinical endotoxemia, we evaluated their responsiveness to additional septic stimuli by measuring in vivo memTNF expression, which was previously shown to be involved in monocyte-mediated pulmonary endothelial activation (11). The results demonstrated the ‘priming’ effect of low-dose LPS on lung Gr-1^high monocytes. Gr-1^high monocytes in the lungs pre-treated by low-dose LPS showed substantial enhancement of memTNF expression in response to both low and high-dose secondary LPS, as compared to Gr-1^high monocytes in the lungs of mice not pre-treated with LPS. This implies that low-dose LPS pre-treatment increased the sensitivity of these cells to secondary stimuli as well as the magnitude of their response to saturating stimuli. In contrast, lung Gr-1^low monocytes displayed much less responsiveness than Gr-1^high monocytes. This higher TNF producing capacity of Gr-1^high as compared to Gr-1^low monocytes has recently been demonstrated in cells recruited to infracted cardiac tissue (20). Thus, the increased Gr-1^high monocyte margination and their enhanced responsiveness to secondary stimuli during sub-clinical endotoxemia produce a monocyte-mediated latent inflammatory priming of the lungs.

To explore the physiological effects of low-dose LPS-induced monocyte margination and priming on the development of ALI, we adapted a previously described two-hit ALI model comprised of high-dose i.v. LPS followed by i.v. zymosan challenge (51, 52). In our study we modified the original model to use a much lower, sub-clinical primary LPS dose, but despite this, mice developed clear signs of ALI following zymosan challenge, associated with a large increase in pulmonary vascular permeability. Conversely, mice administered the same doses of zymosan alone (without preceding low-dose LPS) appeared outwardly normal and showed only marginal permeability increases. Thus, the model enabled us to dissect out the impact of the LPS pre-treatment on the development of ALI. We found a role for PAF signalling in induction of pulmonary vascular leak in this model, but unlike the previous high-dose LPS- zymosan model (50), treatment with WEB 2086 at doses expected to ablate PAF signalling in vivo (62) resulted in only partial attenuation of vascular permeability. A recent report provided evidence of a role for acid sphingomyelinase/ceramide pathway in PAF-induced pulmonary vascular leak (63), in addition to the previously described role for prostaglandins (64). Consistent with this, we observed that WEB 2086 with D609 markedly attenuated permeability increases, but addition of indomethacin had no effect. These results indicate that lipid mediators including PAF and ceramide are at least in part responsible in the development of pulmonary edema in the low-dose LPS-zymosan model of ALI.

To directly address the role of marginated monocytes in this model, we chose a monocyte/macrophage depletion approach using i.v. clodronate-liposomes (65, 66). An adoptive transfer approach, using purified bone marrow CD115+, Gr-1^high monocytes was also attempted, but after transfer of cells with either prior ex vivo or in vivo low dose LPS treatment, only a fraction of the cells injected remained detectable in the blood, lungs or other tissues (data not shown) for prolonged periods (>30mins), suggesting differences in monocyte trafficking behavior between endogenously recruited cells from bone marrow vs. exogenously purified and administered cells. Using the monocyte depletion strategy, we found a substantial attenuation of LPS-zymosan induced symptoms and permeability increases at the 18h time point after clodronate-liposome treatment. In contrast, no protective effects were observed at 48h post clodronate-liposome treatment, the time point when bone marrow monocyte repopulation (27) and restoration of LPS-induced Gr-1high subset margination to the lungs had occurred, but before the recovery from any depleting effects of i.v. clodronate-liposomes on tissue macrophages would have taken place (43, 67, 68). Taken together, these results strongly suggest that Gr-1^high monocyte margination and priming within the lungs are important in the evolution of zymosan-induced ALI, presumably through a pathway involving both PAF and ceramide.

We speculate that the LPS-primed monocytes are likely to be the direct cell sources for PAF and ceramide production in response to zymosan in this model. In vitro studies have previously shown that PAF is produced by human and rabbit monocytes in response to zymosan stimulation (69, 70). Consistent with our in vivo findings of LPS priming towards zymosan-induced pulmonary vascular leak, LPS pre-treatment in vitro, although on its own it is a weak stimulus for lipid mediator production, enhances arachidonic acid metabolite and PAF production in response to stimuli such as zymosan (71-74). Furthermore, optimal PAF-mediated production of lipid mediators, including ceramide, is also dependent on LPS pre-treatment, as shown in the mouse macrophage cell line P388D₁ (75, 76). However, the precise molecular mechanisms whereby marginated and primed Gr-1^high monocytes contribute to pulmonary edema in response to septic stimuli such as zymosan remains to be further elucidated.

The critical role attributed to neutrophils in the evolution of ALI during systemic inflammation is based on a number of experimental observations, including neutrophil depletion studies using anti-neutrophil antibodies or anti-cancer drugs (8, 77). However, caution should be taken when interpreting these depletion studies, as recently suggested by Rydstrom and Wick (78). The anti-neutrophil antibody RB6-C5, widely used to demonstrate neutrophil function in mice including models of ALI (79, 80), is essentially an anti-Gr-1 antibody, and relatively small doses of this antibody may deplete circulating Gr-1^high monocytes in addition to neutrophils (81, 82). The tendency for cross-reactivity of neutrophil depleting antibodies with monocytes, and the lack of specificity of pharmacological depletion methods using anti-cancer myelosupressing drugs may lead to inappropriate over-attribution of immunological and pathophysiological functions to neutrophils. Identification of a distinct ‘inflammatory’ subset of mouse monocytes now allows measurements to be focused on the relevant population, and it has been described that the Gr-1^high subset and neutrophils have a similar role in innate immunity in certain conditions. Gr-1^high monocytes are required for the control of local Toxoplasma (83) and Listeria (84) infection, while during oral Salmonella infection Gr-1^high monocytes in the gut lymphoid tissues showed increased TNF and iNOS production and were capable of phagocytosis and killing bacteria in vitro (78). In addition to enhanced TNF producing capacity, Gr-1^high monocytes recruited to injured myocardium also possess higher protease activity (20). Evaluation of the mediator production by resting and primed Gr-1^high monocytes in vivo and in vitro, particularly early, short-lived locally acting mediators such as reactive oxygen species and lipid mediators, will enhance our understanding of their role in microvascular pathophysiology during ALI.

The results of the present study confirm our previous notion (11) that the role of monocytes in acute systemic inflammation is concerned primarily with local cell-mediated effects within the microvasculature, as opposed to augmentation of systemic mediator release within the central circulation. The substantial accumulation of Gr-1^high monocytes in the lungs, and their vigorous response to secondary septic stimuli, supports the hypothesis that without the need for extensive pre-stimulation or conditioning, these monocytes have the potential to fulfil a similar role to PIM observed in livestock animals, replicating the functions of PIM in species lacking this population. This view is further supported by our findings that clodronate-liposome-mediated depletion of marginated monocytes attenuates pulmonary vascular leak in the LPS-zymosan induced ALI, analogous to the observation in the literature that the depletion of PIM reduces the degree of various forms of ALI in livestock animals (31, 85-89). Although the role of neutrophils in ALI, with some exceptions (77), is generally not disputed, our findings and pre-existing evidence in the literature showing that immature monocyte subsets possess a specialised pro-inflammatory phenotype point to a complementary or independent role for monocytes in ALI and the potential for interactions between the two cell types during systemic inflammation. Further studies will be required to ascertain whether our results with sub-acute endotoxemia and the 2-hit model of ALI are applicable to other more complex pathophysiological process of clinical ALI of extrapulmonary origin.