Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

TNF and IL-10 are major factors in modulation of the phagocytic cell environment in lung and lymph node in tuberculosis: A next-generation two-compartmental model

Tuberculosis (TB) is one of the earliest recorded human diseases and still one of the deadliest worldwide. Its causative agent is the bacteria Mycobacterium tuberculosis (Mtb). Cytokine-mediated macrophage activation is a necessary step in control of bacterial growth, and early immunologic events in lymph node and lung are crucial to the outcome of infection, although the factors that influence these environments and the immune response are poorly understood.Our goal is to build the next-generation two-compartmental model of the immune response to provide a gateway to more spatial and mechanistic investigations of M. tuberculosis infection in the LN and lung. Crucial immune factors emerge that affect macrophage populations and inflammation, namely TNF-dependent recruitment and apoptosis, and IL-10 levels. Surprisingly, bacterial load plays a less important role than TNF in increasing the population of infected macrophages and inflammation.Using a mathematical model, it is possible to distinguish the effects of pro-inflammatory (TNF) and anti-inflammatory (IL-10) cytokines on the spectrum of phagocyte populations (macrophages and dendritic cells) in the lung and lymph node. Our results suggest that TNF is a major mediator of recruitment of phagocytes to the lungs. In contrast, IL-10 plays a role in balancing the dominant macrophage phenotype in LN and lung.     

Pneumolysin-induced lung injury is independent of leukocyte trafficking into the alveolar space

Pneumolysin (PLY) is a major virulence factor released by Streptococcus pneumoniae and has been implicated in the pathogenesis of pneumococcal pneumonia. In this study, we evaluated the contribution of newly recruited neutrophils and monocytes and resident alveolar macrophages to the pathogenesis of PLY-induced lung injury. Mice received either adhesion-blocking Abs to inhibit alveolar leukocyte trafficking or liposomal clodronate to deplete alveolar macrophages before intratracheal application of native PLY or its noncytotoxic derivative PdB. We found that treatment with PLY but not PdB resulted in increased lung vascular permeability. In addition, PLY also induced a decrease in the resident alveolar macrophage population, and the recruitment of peripheral blood neutrophils and monocytes into the alveolar space. Blockade of PLY-induced alveolar leukocyte trafficking by pretreatment of mice with anti-CD18 plus anti-CD49d Abs or depletion of circulating neutrophils did not attenuate the increase in lung permeability observed in response to intratracheal PLY. In addition, depletion of resident alveolar macrophages with clodronated liposomes did not reduce alveolar injury developing in response to PLY. PLY-induced lung injury was associated with only a small increase in bronchoalveolar lavage concentrations of cytokines. These data indicate that PLY-induced lung injury results from direct pneumotoxic effects on the alveolar-capillary barrier and is independent of both resident and recruited phagocytic cells.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Keratinocyte growth factor augments pulmonary innate immunity through epithelium-driven, GM-CSF-dependent paracrine activation of alveolar macrophages

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of insults. In this study, we tested the hypothesis that KGF augments pulmonary host defense. We found that a single dose of intrapulmonary KGF enhanced the clearance of Escherichia coli or Pseudomonas aeruginosa instilled into the lungs 24 h later. KGF augmented the recruitment, phagocytic activity, and oxidant responses of alveolar macrophages, including lipopolysaccharide-stimulated nitric oxide release and zymosan-induced superoxide production. Less robust alveolar macrophage recruitment and activation was observed in mice treated with intraperitoneal KGF. KGF treatment was associated with increased levels of MIP1γ, LIX, VCAM, IGFBP-6, and GM-CSF in the bronchoalveolar lavage fluid. Of these, only GM-CSF recapitulated in vitro the macrophage activation phenotype seen in the KGF-treated animals. The KGF-stimulated increase in GM-CSF levels in lung tissue and alveolar lining fluid arose from the epithelium, peaked within 1 h, and was associated with STAT5 phosphorylation in alveolar macrophages, consistent with epithelium-driven paracrine activation of macrophage signaling through the KGF receptor/GM-CSF/GM-CSF receptor/JAK-STAT axis. Enhanced bacterial clearance did not occur in response to KGF administration in GM-CSF(-/-) mice, or in mice treated with a neutralizing antibody to GM-CSF. We conclude that KGF enhances alveolar host defense through GM-CSF-stimulated macrophage activation. KGF administration may constitute a promising therapeutic strategy to augment innate immune defenses in refractory pulmonary infections.     

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration

Tissue damage induces early recruitment of neutrophils through redox-regulated Src family kinase (SFK) signaling in neutrophils. Redox-SFK signaling in epithelium is also necessary for wound resolution and tissue regeneration. How neutrophil-mediated inflammation resolves remains unclear. In this paper, we studied the interactions between macrophages and neutrophils in response to tissue damage in zebrafish and found that macrophages contact neutrophils and induce resolution via neutrophil reverse migration. We found that redox-SFK signaling through p22phox and Yes-related kinase is necessary for macrophage wound attraction and the subsequent reverse migration of neutrophils. Importantly, macrophage-specific reconstitution of p22phox revealed that macrophage redox signaling is necessary for neutrophil reverse migration. Thus, redox-SFK signaling in adjacent tissues is essential for coordinated leukocyte wound attraction and repulsion through pathways that involve contact-mediated guidance.     

Role of macrophages in early host resistance to respiratory Acinetobacter baumannii infection

Acinetobacter baumannii is an emerging bacterial pathogen that causes nosocomial pneumonia and other infections. Although it is recognized as an increasing threat to immunocompromised patients, the mechanism of host defense against A. baumannii infection remains poorly understood. In this study, we examined the potential role of macrophages in host defense against A. baumannii infection using in vitro macrophage culture and the mouse model of intranasal (i.n.) infection. Large numbers of A. baumannii were taken up by alveolar macrophages in vivo as early as 4 h after i.n. inoculation. By 24 h, the infection induced significant recruitment and activation (enhanced expression of CD80, CD86 and MHC-II) of macrophages into bronchoalveolar spaces. In vitro cell culture studies showed that A. baumannii were phagocytosed by J774A.1 (J774) macrophage-like cells within 10 minutes of co-incubation, and this uptake was microfilament- and microtubule-dependent. Moreover, the viability of phagocytosed bacteria dropped significantly between 24 and 48 h after co-incubation. Infection of J774 cells by A. baumannii resulted in the production of large amounts of proinflammatory cytokines and chemokines, and moderate amounts of nitric oxide (NO). Prior treatment of J774 cells with NO inhibitors significantly suppressed their bactericidal efficacy (P<0.05). Most importantly, in vivo depletion of alveolar macrophages significantly enhanced the susceptibility of mice to i.n. A. baumannii challenge (P<0.01). These results indicate that macrophages may play an important role in early host defense against A. baumannii infection through the efficient phagocytosis and killing of A. baumannii to limit initial pathogen replication and the secretion of proinflammatory cytokines and chemokines for the rapid recruitment of other innate immune cells such as neutrophils.     

The heterogenic properties of monocytes/macrophages and neutrophils in inflammatory response in diabetes

Inflammation is a complicated biological process in response to harmful stimuli, which involves the cooperation of immune system and vascular system. Upon pathogen invasion or tissue injury, resident innate immune cells such as macrophages and dendritic cells are activated and release inflammatory mediators, which result in the vasodilation and recruitment of leukocytes, mainly monocytes and neutrophils. As two of the most important inflammation-mediating immune cells, macrophages and neutrophils have long been regarded to have a pro-inflammatory effect. However, increasing evidences suggest the role of macrophage and neutrophil in inflammation is more complicated and diversified than we thought. Differently activated macrophages and neutrophils lead to diverse even opposite activities. Precise understanding of the role of different subpopulations is critical to achieve the effective treatment for inflammatory diseases. In this review, we discuss the two potentially distinct activation routes of macrophages and neutrophils in obesity and diabetes.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

RORγt Modulates Macrophage Recruitment during a Hydrocarbon Oil-Induced Inflammation

Hydrocarbon oils are often utilized as adjuvants in vaccines. In response to naturally occurring hydrocarbon oils, inflammation is initiated and persists with the continuous recruitment of immune cells such as macrophages and neutrophils. However, the mechanism underlying the chronic inflammation in response to hydrocarbon oils is not fully defined. In this study, we revealed an essential role of retinoid-related orphan receptor gamma t (RORγt) in sustaining the recruitment of macrophages following pristane treatment. RORγt absence resulted in the incompetent formation of mesenteric oil granulomas which may associate to a reduction in the migration of macrophages into the mesentery during pristane-induced inflammation. This is at least partially dependent on the expression of the monocyte chemoattractant protein-1 (MCP-1) in the mesentery and the decrease in the macrophage reservoir in the spleen. However, the absence of RORγt had no impact on the recruitment of neutrophils to the mesentery after pristane treatment. Our data uncovered an important role of RORγt in the recruitment of macrophages during hydrocarbon oil-induced chronic inflammation.     

Delineating the roles of neutrophils and macrophages in zebrafish regeneration models

The outcome following injury can be healing, scarring or regeneration, all of which initiate within a resolving inflammatory response. Regeneration, comprising the complete anatomical and functional restoration of lost tissue with minimal residual consequence of injury, is the outcome that most holistically restores prior function. Leukocytes are recognized as playing an important role in determining the balance between fully regenerative or only partially reparative outcomes. Although macrophages have attracted considerable attention for their capacity to direct pro-regenerative outcomes, neutrophils are also key players in initiating inflammation and in influencing its ensuing outcome. In the context of prior studies investigating the role of neutrophils and macrophages in wound healing and in tissue/organ regeneration (mostly wound repair/healing models in mice), we comprehensively review the experimental possibilities that zebrafish models offer for delineating the individual and interactive contributions of neutrophils and macrophages to the regenerative process in embryos and adults. Zebrafish are a highly regenerative vertebrate and have a myeloid system very analogous to that of less-regenerative mammalian models. There are well-characterized reporter lines for imaging and distinguishing neutrophil and macrophage behaviors in vivo, and tools enabling selective, independent manipulation of these two leukocyte lineages for functional studies. Zebrafish are an attractive model for delineating neutrophil and macrophage contributions not only to regeneration, but also to many other pathological processes.This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Recent insights into the role of the innate immune system in the development of alcoholic liver disease

The innate immune system is responsible for the rapid, initial response of the organism to potentially dangerous stresses, including pathogens, tissue injury, and malignancy. Pattern-recognition receptors of the toll-like receptor (TLR) family expressed by macrophages provide a first line of defense against microbial invasion. Activation of these receptors results in a stimulus-specific expression of genes required to control the infection, including the production of inflammatory cytokines and chemokines, followed by the recruitment of neutrophils to the site of infection. The early stages in the development of alcoholic liver disease (ALD) follow a pattern characteristic of an innate immune response. Kupffer cells, the resident macrophages in the liver, are activated in response to bacterial endotoxins (lipopolysaccharide, LPS), leading to the production of inflammatory and fibrogenic cytokines, reactive oxygen species, as well as the recruitment of neutrophils to the liver. One mechanism by which chronic ethanol can turn the highly regulated innate immune response into a pathway of disease is by disrupting the signal transduction cascades mediating the innate immune response. Recent studies have identified specific modules in the TLR-4 signaling cascade that are disrupted after chronic ethanol exposure, including CD14 and the mitogen-activated protein kinase family members, ERK1/2 and p38. Enhanced activation of these TLR-4 dependent signaling pathways after chronic ethanol likely contributes to the development of alcoholic liver disease.     

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.     

Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity.

When phagocytic leukocytes, e.g. neutrophils, monocytes and macrophages, interact with soluble or particulate stimuli, the cells respond with an increased production of reactive oxygen metabolites. This production can be measured with the luminol-amplified chemiluminescence (CL) technique. In the present study, the CL reaction induced in monocyte-derived macrophages was investigated and compared to the responses of neutrophils and monocytes. In systems without additives the CL response of macrophages to soluble stimuli (FMLP, PMA and ionomycin) was very low. Addition of a peroxidase (HRP) to the reaction mixtures resulted in a pronounced increase in CL activity. The cellular CL response in macrophages is thus limited by the amount of peroxidase available. The macrophage response differs qualitatively from the responses of neutrophils and monocytes, in that the intracellular phase of the response is missing.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.