A macrophage subpopulation recruited by CC chemokine ligand-2 clears apoptotic cells in noninfectious lung injury

CC chemokine ligand-2 (CCL2)/monocyte chemoattractant protein (MCP)-1 expression is upregulated during pulmonary inflammation, and the CCL2-CCR2 axis plays a critical role in leukocyte recruitment and promotion of host defense against infection. The role of CCL2 in mediating macrophage subpopulations in the pathobiology of noninfectious lung injury is unknown. The goal of this study was to examine the role of CCL2 in noninfectious acute lung injury. Our results show that lung-specific overexpression of CCL2 protected mice from bleomycin-induced lung injury, characterized by significantly reduced mortality, reduced neutrophil accumulation, and decreased accumulation of the inflammatory mediators IL-6, CXCL2 (macrophage inflammatory protein-2), and CXCL1 (keratinocyte-derived chemokine). There were dramatic increases in the recruitment of myosin heavy chain (MHC) II IA/IE(int)CD11c(int) cells, exudative macrophages, and dendritic cells in Ccl2 transgenic mouse lungs both at baseline and after bleomycin treatment compared with levels in wild-type mice. We further demonstrated that MHCII IA/IE(int)CD11c(int) cells engulfed apoptotic cells during acute lung injury. Our data suggested a previously undiscovered role for MHCII IA/IE(int)CD11c(int) cells in apoptotic cell clearance and inflammation resolution.     

Inflammation and ischemia-induced lung angiogenesis

A role for inflammation in modulating the extent of angiogenesis has been shown for a number of organs. The present study was undertaken to evaluate the importance of leukocyte subpopulations for systemic angiogenesis of the lung after left pulmonary artery ligation (LPAL) in a mouse model of chronic pulmonary thromboembolism. Since we (24) previously showed that depletion of neutrophils did not alter the angiogenic outcome, we focused on the effects of dexamethasone pretreatment (general anti-inflammatory) and gadolinium chloride treatment (macrophage inactivator) and studied Rag-1(-/-) mice (T/B lymphocyte deficient). We measured inflammatory cells in bronchoalveolar lavage fluid and lung homogenate macrophage inflammatory protein-2 (MIP-2) and IL-6 protein levels within 24 h after LPAL and systemic blood flow to the lung 14 days after LPAL with labeled microspheres as a measure of angiogenesis. Blood flow to the left lung was significantly reduced after dexamethasone treatment compared with untreated control LPAL mice (66% decrease; P < 0.05) and significantly increased in T/B lymphocyte-deficient mice (88% increase; P < 0.05). Adoptive transfer of splenocytes (T/B lymphocytes) significantly reversed the degree of angiogenesis observed in the Rag-1(-/-) mice back to the level of control LPAL. Average number of lavaged macrophages for each group significantly correlated with average blood flow in the study groups (r(2) = 0.9181; P = 0.01 different from 0). Despite differences in angiogenesis, left lung homogenate MIP-2 and IL-6 did not differ among study groups. We conclude that inflammatory cells modulate the degree of angiogenesis in this lung model where lymphocytes appear to limit the degree of neovascularization, whereas monocytes/macrophages likely promote angiogenesis.     

The role of CXCR2 in systemic neovascularization of the mouse lung.

We previously showed increased expression of the ELR+, CXC chemokines in the lung after left pulmonary artery obstruction. These chemokines have been shown in other systems to bind their G protein-coupled receptor, CXCR(2), and promote systemic endothelial cell proliferation, migration, and capillary tube formation. In the present study, we blocked CXCR(2) in vivo using a neutralizing antibody and also studied mice that were homozygous null for CXCR(2). To estimate the extent of neovascularization in this model, we measured systemic blood flow to the left lung 14 days after left pulmonary artery ligation (LPAL). We found blood flow significantly reduced (67% decrease) with neutralizing antibody treatment compared with controls. However, blood flow was not altered in the CXCR(2)-deficient mice compared with wild-type controls after LPAL. To test for ligand availability, we measured macrophage inflammatory protein (MIP)-2 in lung homogenates after LPAL, because this is the predominant CXC chemokine previously shown to be increased after LPAL (22). MIP-2 protein was two- to fourfold higher in the left lung relative to the right lung in all treatment groups 4 h after LPAL and this increase did not differ among groups. We speculate that the CXCR(2)-deficient mice have compensatory mechanisms that mitigate their lack of gene expression and conclude that CXCR(2) contributes to chemokine-induced systemic angiogenesis after pulmonary artery obstruction.     

Pulmonary ischemia induces lung remodeling and angiogenesis.

Cellular remodeling during angiogenesis in the lung is poorly described. Furthermore, it is the systemic vasculature of the lung and surrounding the lung that is proangiogenic when the pulmonary circulation becomes impaired. In a mouse model of chronic pulmonary thromboembolism, after left pulmonary artery ligation (LPAL), the intercostal vasculature, in proximity to the ischemic lung, proliferates and invades the lung (12). In the present study, we performed a detailed investigation of the kinetics of remodeling using histological sections of the left lung of C57Bl/6J mice after LPAL (4 h to 20 days) or after sham surgery. New vessels were seen within the thickened visceral pleura 4 days after LPAL predominantly in the upper portion of the left lung. Connections between new vessels within the pleura and pulmonary capillaries were clearly discerned by 7 days after LPAL. The visceral pleura and the lung parenchyma showed intense tissue remodeling, as evidenced by markedly elevated levels of both proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells. Rapidly dividing cells were predominantly macrophages and type II pneumocytes. The increased apoptotic activity was further quantified by caspase-3 activity, which showed a sixfold increase relative to naive lungs, by 24 h after LPAL. Because sham surgeries had little effect on measured parameters, we conclude that both thoracic wound healing and pulmonary ischemia are required for systemic neovascularization.     

Chemokine Localization in Bronchial Angiogenesis

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0–24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.     

Blockade of secondary lymphoid tissue chemokine exacerbates Propionibacterium acnes-induced acute lung inflammation

Chemokine-chemokine receptor interaction plays an essential role in leukocyte/dendritic cell (DC) trafficking in inflammation and immune responses. We investigated the pathophysiological roles of secondary lymphoid tissue chemokine (SLC; CCL21) and macrophage inflammatory protein-2 (MIP-2) in the development of acute pulmonary inflammation induced by an intratracheal injection of Propionibacterium acnes in mice. Immunohistochemical studies revealed that SLC was constitutively expressed in the peribronchial areas and perivascular lymphatics in normal mice. MIP-2-positive cells were observed in alveolar spaces in mice challenged with P. acnes. Both neutralization Abs against MIP-2 and CXC chemokine receptor 2 alleviated the P. acnes-induced pulmonary inflammation when injected before P. acnes Ag challenge. On the other hand, polyclonal anti-SLC Abs (pAbs) exacerbated the pulmonary inflammation. The numbers of mature DCs (MHC class II +, CD11c+, and CD86+) as well as macrophages and neutrophils in the P. acnes Ag-challenged lungs were increased, whereas the number of CD4+ T cells, including memory T cells, was decreased. The numbers of mature and proliferating CD4+ T cells (bromodeoxyuridine(+)CD4+) in regional lymph nodes were decreased in mice injected with anti-SLC pAbs compared with those in mice treated with control Abs. An in vitro proliferation assay confirmed the impairment of the Ag-specific T cell response in regional lymph nodes of mice treated with anti-SLC pAbs. These results indicate for the first time a regulatory role for SLC-recruited mature DCs in bridging an acute inflammatory response (innate immunity) and acquired immunity in the lung.     

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.     

Changes in lung permeability after chronic pulmonary artery obstruction.

We have shown that left pulmonary artery ligation (LPAL) in mice causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 6 days. Because angiogenic vessels in other organs have been shown to exhibit increased permeability, we studied vascular permeability (Evans blue dye extravasation, lung wet weight-to-dry weight ratio, and lavaged protein) in naive C57BL/6 mice and 4 h, and 14 and 21 days after LPAL (4-6 mice/time point). We also measured radiolabel clearance as an index of functional perfusion after LPAL. Tracer clearance from the left lung was maximal by 6 days after LPAL and not different from right lungs. Thus a functional vasculature is established before 6 days of LPAL that results in normal tracer clearance. By 21 days after LPAL, Evans blue-albumin was significantly increased in the left lung relative to both 4 h (no vasculature) and 14 days after LPAL. Only after 21 days of LPAL was left lung wet weight-to-dry weight ratio significantly different from naive lungs. Additionally, lavaged protein was significantly increased both 4 h and 21 days after LPAL relative to control mice. Thus, using three different methods, results consistently demonstrated increased permeability to protein and water 21 days after LPAL. Although changes in surface area of perfusion might affect the interpretation of these results, blood flow measured with labeled microspheres indicated no change in left lung perfusion between 14 and 21 days of LPAL. Thus the lung vasculature, remodeled as a consequence of chronic pulmonary artery obstruction, demonstrates increased water and protein permeability.     

Effects of pulmonary ischemia on lung morphology

Pulmonary ischemia resulting from chronic pulmonary embolism leads to proliferation of the systemic circulation within and surrounding the lung. However, it is not clear how well alveolar tissue is sustained during the time of complete pulmonary ischemia. In the present study, we investigated how pulmonary ischemia after left pulmonary artery ligation (LPAL) would alter lung mechanical properties and morphology. In this established mouse model of lung angiogenesis after chronic LPAL (10), we evaluated lung function and structure before (3 days) and after (14 days) a functional systemic circulation to the left lung is established. Age-matched na?ve and sham-operated C57Bl/6 mice and mice undergoing chronic LPAL were studied. Left and right lung pressure-volume relationships were determined. Next, lungs were inflated in situ with warmed agarose (25-30 cmH(2)O) and fixed, and mean chord lengths (MCL) of histological sections were quantified. MCL of na?ve mice averaged 43.9 +/- 1.8 mum. No significant changes in MCL were observed at either time point after LPAL. Left lung volumes and specific compliances were significantly reduced 3 days after LPAL. However, by 14 days after LPAL, lung pressure-volume relationships were not different from controls. These results suggest that severe pulmonary ischemia causes changes in lung mechanics early after LPAL that are reversed by the time a new systemic vasculature is known to perfuse pulmonary capillaries. The LPAL model thus affords a unique opportunity to study lung functional responses to tissue ischemia and subsequent recovery.     

Decreased alveolar oxygen induces lung inflammation

Molecular mechanisms of the inflammatory reaction in hypoxia-induced lung injury are not well defined. Therefore, effects of alveolar hypoxia were studied in rat lungs, exposing rats to 10% oxygen over periods of 1, 2, 4, 6, and 8 h. An increase in the number of macrophages in bronchoalveolar lavage fluid of hypoxic animals was shown between 1 and 8 h. Extravasation of albumin was enhanced after 1 h and remained increased throughout the study period. NF-kappaB-binding activity as well as mRNA for TNF-alpha, macrophage inflammatory protein (MIP)-1beta, and monocyte chemoattractant protein (MCP)-1 were increased within the first 2 h of exposure to hypoxia. Hypoxia-inducible factor (HIF)-1alpha and intercellular adhesion molecule (ICAM)-1 mRNA were upregulated between 1 and 6 h. Elimination of alveolar macrophages by intratracheal application of liposome-encapsulated clodronate led to a decreased expression of NF-kappaB binding activity, HIF-1alpha, TNF-alpha, ICAM-1, and MIP-1beta. In summary, alveolar hypoxia induced macrophage recruitment, an increase in albumin leakage, and enhanced expression of inflammatory mediators, which were mainly macrophage dependent. Alveolar macrophages appear to have a prominent role in the inflammatory response in hypoxia-induced lung injury and the related upregulation of inflammatory mediators.     

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.     

CD1d-restricted IFN-γ-secreting NKT cells promote immune complex-induced acute lung injury by regulating macrophage-inflammatory protein-1α production and activation of macrophages and dendritic cells

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d(-/-) and Jα18(-/-) mice contained few Ly6G(+)CD11b(+) granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d(-/-) mice. Adoptive transfer of wild type (WT) but not IFN-γ-, MIP-1α-, or FcγR-deficient NKT cells into CD1d(-/-) mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR-deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d(-/-) or CD1d(-/-)IFN-γ(-/-) mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ-producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ-deficient NKT cells induced less IL-1β and TNF-α production by alveolar macrophages and dendritic cells in CD1d(-/-) mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ-producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.     

Macrophage CD74 contributes to MIF-induced pulmonary inflammation

Background

MIF is a critical mediator of the host defense, and is involved in both acute and chronic responses in the lung. Neutralization of MIF reduces neutrophil accumulation into the lung in animal models. We hypothesized that MIF, in the alveolar space, promotes neutrophil accumulation via activation of the CD74 receptor on macrophages.

Methods

To determine whether macrophage CD74 surface expression contributes MIF-induced neutrophil accumulation, we instilled recombinant MIF (r-MIF) into the trachea of mice in the presence or absence of anti-CD74 antibody or the MIF specific inhibitor, ISO-1. Using macrophage culture, we examined the downstream pathways of MIF-induced activation that lead to neutrophil accumulation.

Results

Intratracheal instillation of r-MIF increased the number of neutrophils as well as the concentration of macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived chemokine (KC) in BAL fluids. CD74 was found to be expressed on the surface of alveolar macrophages, and MIF-induced MIP-2 accumulation was dependent on p44/p42 MAPK in macrophages. Anti-CD74 antibody inhibited MIF-induced p44/p42 MAPK phosphorylation and MIP-2 release by macrophages. Furthermore, we show that anti-CD74 antibody inhibits MIF-induced alveolar accumulation of MIP-2 (control IgG vs. CD74 Ab; 477.1 ± 136.7 vs. 242.2 ± 102.2 pg/ml, p < 0.05), KC (1796.2 ± 436.1 vs. 1138.2 ± 310.2 pg/ml, p < 0.05) and neutrophils (total number of neutrophils, 3.33 ± 0.93 × 10⁴ vs. 1.90 ± 0.61 × 10⁴, p < 0.05) in our mouse model.

Conclusion

MIF-induced neutrophil accumulation in the alveolar space results from interaction with CD74 expressed on the surface of alveolar macrophage cells. This interaction induces p44/p42 MAPK activation and chemokine release. The data suggest that MIF and its receptor, CD74, may be useful targets to reduce neutrophilic lung inflammation, and acute lung injury.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.     

Role of IL-6 in systemic angiogenesis of the lung.

The multifunctional cytokine interleukin (IL)-6 has been shown to modulate inflammation and angiogenesis. In a mouse model of lung angiogenesis induced by chronic left pulmonary artery ligation (LPAL), we previously showed increased expression of IL-6 mRNA in lung homogenates 4 h after the onset of pulmonary ischemia. To determine whether IL-6 influences both new vessel growth and inflammatory cell influx, we studied wild-type (WT) and IL-6-deficient C57Bl/6J (KO) mice after LPAL (4 h and 1, 7, 14 days). We measured IL-6 protein of the lung by ELISA, the lavage cell profile of the left lung, and new systemic vessel growth with radiolabeled microspheres (14 days after LPAL) in WT and KO mice. We confirmed a 2.4-fold increase in IL-6 protein in the left lung of WT mice compared with right lung 4 h after LPAL. A significant increase in lavaged neutrophils (7.5% of total cells) was observed only in WT mice 4 h after LPAL. New vessel growth was significantly attenuated in KO relative to WT (0.7 vs. 1.9% cardiac output). In an additional series, treatment of WT mice with anti-neutrophil antibody demonstrated a reduction in lavaged neutrophils 4 h after LPAL; however, IL-6 protein remained elevated and neovascularization to the left lung (2.3% cardiac output) was not altered. These results demonstrate that IL-6 plays an important modulatory role in lung angiogenesis, but the changes are not dependent on trapped neutrophils.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Early interaction of Yersinia pestis with APCs in the lung

Despite the importance of pneumonic plague, little is known of the early pulmonary immune responses that occur following inhalation of Yersinia pestis. Therefore, we conducted studies to identify the early target cells for uptake of Y. pestis in the lungs following intratracheal or i.v. inoculation. Following intratracheal inoculation, Y. pestis was rapidly internalized primarily by a distinctive population of CD11c+DEC-205+CD11b- cells in the airways, whereas i.v. inoculation resulted in uptake primarily by CD11b+CD11c- macrophages and granulocytes in lung tissues. The airway cells internalized and were infected by Y. pestis, but did not support active replication of the organism. Intratracheal inoculation of Y. pestis resulted in rapid activation of airway CD11c+ cells, followed within 24 h by the selective disappearance of these cells from the airways and lungs and the accumulation of apoptotic CD11c+ cells in draining lymph nodes. When CD11c+ cells in the airways were depleted using liposomal clodronate before infection, this resulted in a significantly increased replication of Y. pestis in the lungs and dissemination to the spleen and draining lymph nodes. These findings suggest that CD11c+ cells in the airways play an important role in suppressing the initial replication and dissemination of inhaled Y. pestis, although these results will also require confirmation using fully virulent strains of Y. pestis. Depletion of these airway cells by Y. pestis may therefore be one strategy the organism uses to overcome pulmonary defenses following inhalation of the organism.     

Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus

Unlike their role in bacterial infection, less is known about the role of neutrophils during pulmonary viral infection. Exposure to pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) results in excess neutrophils in the lungs of mice infected with influenza A virus. TCDD is the most potent agonist for the aryl hydrocarbon receptor (AhR), and exposure to AhR ligands has been correlated with exacerbated inflammatory lung diseases. However, knowledge of the effects of AhR agonists on neutrophils is limited. Likewise, the factors regulating neutrophil responses during respiratory viral infections are not well characterized. To address these knowledge gaps, we determined the in vivo levels of KC, MIP-1alpha, MIP-2, LIX, IL-6, and C5a in infected mouse lungs. Our data show that these neutrophil chemoattractants are generally produced transiently in the lung within 12-24 h of infection. We also report that expression of CD11a, CD11b, CD49d, CD31, and CD38 is increased on pulmonary neutrophils in response to influenza virus. Using AhR-deficient mice, we demonstrate that excess neutrophilia in the lung is mediated by activation of the AhR and that this enhanced neutrophilia correlates directly with decreased survival in TCDD-exposed mice. Although AhR activation results in more neutrophils in the lungs, we show that this is not mediated by deregulation in levels of common neutrophil chemoattractants, expression of adhesion molecules on pulmonary neutrophils, or delayed death of neutrophils. Likewise, exposure to TCDD did not enhance pulmonary neutrophil function. This study provides an important first step in elucidating the mechanisms by which AhR agonists exacerbate pulmonary inflammatory responses.