CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.     

Initial recruitment of neutrophils to alveolar structures in acute lung injury

The adult respiratory distress syndrome and bacterial pneumonia are both characterized by an influx of neutrophils into the lung. The neutrophil has been implicated as having a "pathological" role in adult respiratory distress syndrome, in contrast to its role in bacterial pneumonia. We hypothesized that processes resulting in neutrophil recruitment to the lung are distinct, depending on whether the inflammatory stimulus arises in the intravascular or the alveolar compartment of the lung. Anesthetized sheep with lung lymph fistulas were utilized to access the three compartments of the lung relevant to studies of transpulmonary neutrophil migration. Serum, lung lymph, and bronchoalveolar lavage fluid were studied for neutrophil influx and chemotactic activity before and after administration of endotoxin by either an intravascular or inhaled alveolar route. Both groups developed significant neutrophil influx into the lymph and bronchoalveolar lavage fluid by 3 h postendotoxin. Those animals receiving intravascular endotoxin developed chemotactic gradients opposing neutrophil migration into the lung in contrast to animals receiving alveolar endotoxin, suggesting that neutrophil influx into the lung occurs by random migration.     

IL-12, but not IL-18, is critical to neutrophil activation and resistance to polymicrobial sepsis induced by cecal ligation and puncture

Sepsis is a systemic inflammatory response resulting from local infection due, at least in part, to impaired neutrophil migration. IL-12 and IL-18 play an important role in neutrophil migration. We have investigated the mechanism and relative role of IL-12 and IL-18 in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. Wild-type (WT) and IL-18(-/-) mice were resistant to sublethal CLP (SL-CLP) sepsis. In contrast, IL-12(-/-) mice were susceptible to SL-CLP sepsis with high bacteria load in peritoneal cavity and systemic inflammation (serum TNF-alpha and lung neutrophil infiltration). The magnitude of these events was similar to those observed in WT mice with lethal CLP sepsis. The inability of IL-12(-/-) mice to restrict the infection was not due to impairment of neutrophil migration, but correlated with decrease of phagocytosis, NO production, and microbicidal activities of their neutrophils, and with reduction of systemic IFN-gamma synthesis. Consistent with this observation, IFN-gamma(-/-) mice were as susceptible to SL-CLP as IL-12(-/-) mice. Moreover, addition of IFN-gamma to cultures of neutrophils from IL-12(-/-) mice restored their phagocytic, microbicidal activities and NO production. Mortality of IL-12(-/-) mice to SL-CLP was prevented by treatment with IFN-gamma. Thus we show that IL-12, but not IL-18, is critical to an efficient host defense in polymicrobial sepsis. IL-12 acts through induction of IFN-gamma and stimulation of phagocytic and microbicidal activities of neutrophils, rather than neutrophil migration per se. Our data therefore provide further insight into the defense mechanism against this critical area of infectious disease.     

Leukocyte-epithelial interactions

As a 'double-edged sword', neutrophil (polymorphonuclear leukocyte) migration across epithelial-lined organs is an important component of host defense, but it also results in epithelial pathophysiology and disease symptoms. There have been significant advances in better understanding the mechanisms of how leukocytes cross the vascular endothelium to exit the bloodstream; however, many of the mechanisms that govern polymorphonuclear leukocyte transepithelial migration are different and we are only just beginning to understand them. Recent findings include new junctional adhesion molecules and carbohydrate moieties as receptors for migrating neutrophils. In addition, new insights into leukocyte-epithelial signaling events have emerged that are beginning to shed light on the role of SIRP-CD47 interactions in regulating the rate of neutrophil transepithelial migration and how neutrophils modulate epithelial barrier function.     

Effects of an anti-inflammatory VAP-1/SSAO inhibitor,PXS-4728A,on pulmonary neutrophil migration

Background and purpose

The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A.

Methods

Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A.

Results

Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity.

Conclusions and implications

This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.     

Macrophage dysfunction and susceptibility to pulmonary Pseudomonas aeruginosa infection in surfactant protein C-deficient mice

To determine the role of surfactant protein C (SP-C) in host defense, SP-C-deficient (Sftpc-/-) mice were infected with the pulmonary pathogen Pseudomonas aeruginosa by intratracheal injection. Survival of young, postnatal day 14 Sftpc-/- mice was decreased in comparison to Sftpc+/+ mice. The sensitivity to Pseudomonas bacteria was specific to the 129S6 strain of Sftpc-/- mice, a strain that spontaneously develops interstitial lung disease-like lung pathology with age. Pulmonary bacterial load and leukocyte infiltration were increased in the lungs of Sftpc-/- mice 24 h after infection. Early influx of polymorphonuclear leukocytes in the lungs of uninfected newborn Sftpc-/- mice relative to Sftpc+/+ mice indicate that the lack of SP-C promotes proinflammatory responses in the lung. Mucin expression, as indicated by Alcian blue staining, was increased in the airways of Sftpc-/- mice following infection. Phagocytic activity of alveolar macrophages from Sftpc-/- mice was reduced. The uptake of fluorescent beads in vitro and the number of bacteria phagocytosed by alveolar macrophages in vivo was decreased in the Sftpc-/- mice. Alveolar macrophages from Sftpc-/- mice expressed markers of alternative activation that are associated with diminished pathogen response and advancing pulmonary fibrosis. These findings implicate SP-C as a modifier of alveolar homeostasis. SP-C plays an important role in innate host defense of the lung, enhancing macrophage-mediated Pseudomonas phagocytosis, clearance and limiting pulmonary inflammatory responses.     

The effects of Escherichia coli capsule, O-antigen, host neutrophils, and complement in a rat model of Gram-negative pneumonia

Gram-negative enteric bacilli are agents of life-threatening pneumonia. The role of the bacterial capsule and O-antigen moiety of lipopolysaccharide in the pathogenesis of Gram-negative pneumonia was assessed. In a rat model of pneumonia the LD(50) of a wild-type extraintestinal pathogenic Escherichia coli strain (CP9) was significantly less than its isogenic derivatives deficient in capsule (CP9.137), O-antigen (CP921) or both capsule and O-antigen (CP923) (P< or =0.003). Studies using complement depleted or neutropenic animals established that both neutrophils and complement are important for the pulmonary clearance of E. coli. Data from these studies also support that capsule and O-antigen serve, at least in part, to counter the complement and neutrophil components of the pulmonary host defense response. Lastly, the contribution of E. coli versus neutrophils in causing lung injury was examined. Findings suggest that E. coli virulence factors and/or non-neutrophil host factors are more important mediators of lung injury than neutrophils. These findings extend our understanding of Gram-negative pneumonia and have treatment implications.     

Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury.

The use of positive pressure mechanical ventilation can cause ventilator-induced lung injury (VILI). We hypothesized that hyperoxia in combination with large tidal volumes (VT) would accentuate noncardiogenic edema and neutrophil infiltration in VILI and be dependent on stretch-induced macrophage inflammatory protein-2 (MIP-2) production. In rats ventilated with VT 20 ml/kg, there was pulmonary edema formation that was significantly increased by hyperoxia. Total lung neutrophil infiltration and MIP-2 in bronchoalveolar lavage (BAL) fluid were significantly elevated, in animals exposed to high VT both on room air (RA) and with hyperoxia. Hyperoxia markedly augmented the migration of neutrophils into the alveoli. Anti-MIP-2 antibody blocked migration of neutrophils into the alveoli in RA by 51% and with hyperoxia by 65%. We concluded that neutrophil migration into the alveoli was dependent on stretch-induced MIP-2 production. Hyperoxia significantly increased edema formation and neutrophil migration into the alveoli with VT 20 ml/kg, although BAL MIP-2 levels were nearly identical to VT 20 ml/kg with RA, suggesting that other mechanisms may be involved in hyperoxia-augmented neutrophil alveolar content in VILI.     

LL-37 and its analog FF/CAP18 attenuate neutrophil migration in sepsis-induced acute lung injury

Introduction: Sepsis can result in acute lung injury. LL-37 is a small cationic host defense peptide involved in anti-inflammatory. In the current study, it was hypothesized that antimicrobial peptide LL-37 could play a protective role in attenuating the progression of sepsis-induced acute lung injury. Methods: Forty male C57BL/6 mice were induced into sepsis using cecal ligation and puncture, and subsequently administered with recombinant mouse osteopontin. Peptides LL-37, the LL-37 analog (FF/CAP18, called sLL-37), or normal saline was intravenously administered into septic mice for 20 hours. Then, proinflammatory cytokines (IL-6 and IL-1β), acute lung injury markers (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]), the neutrophil infiltration marker (myeloperoxidase [MPO]), and neutrophil infiltration were detected. Furthermore, the neutrophil migration and expression of migration-related factors (focal adhesion kinase [FAK], ERK, and P38) in differentiated HL-60 cells were detected. Results: Septic mice had upregulated IL-6, IL-1β, ALT, AST, LDH, MPO, p-FAK, p-ERK, and p-P38, infiltrated neutrophils, and migrated neutrophil-like HL-60 cells. In contrast, the administration of peptide LL-37 and sLL-37 inhibited all these changes. Compared with septic mice, it was found that proinflammatory cytokines, lung injury markers, MPO, and infiltrated neutrophils decreased in mice treated with LL-37 and sLL-37. In addition, the migrated neutrophil-like HL-60 cells and activated p-FAK, p-ERK, and p-P38 proteins were suppressed by LL-37 and sLL-37 treatments. Conclusions: Peptide LL-37 and its analog sLL-37 attenuated the progression of sepsis-induced acute lung injury by inhibiting neutrophil infiltration and migration through the FAK, ERK, and P38 pathways.     

High susceptibility to respiratory Acinetobacter baumannii infection in A/J mice is associated with a delay in early pulmonary recruitment of neutrophils

Acinetobacter baumannii is an important cause of both community-associated and nosocomial pneumonia, which have become increasingly difficult to treat because of the rapid development of resistance to multiple antibiotics. Despite its clinical importance, the pathogenesis of and host defense against respiratory A. baumannii infection remains largely unknown. To examine host factors that could contribute to the defense, we compared the susceptibilities of A/J and C57BL/6 mice to intranasal (i.n.) inoculation with A. baumannii. We found that A/J mice were significantly more susceptible to infection with higher mortality (P < 0.05) and tissue bacterial burdens (P < 0.01) as well as greater histopathology in the lung and spleen than C57BL/6 mice. More importantly, the high susceptibility of A/J mice was associated with a reduced local proinflammatory cytokine/chemokine (particularly IL-1β, MIP-2 and TNF-α) responses and a significant delay and reduction in the early influx of neutrophils in the lung (P < 0.05). Intranasal administration of neutrophil-inducing chemokine MIP-2 to A/J mice enhanced pulmonary neutrophil influx and partially restored host resistance to A. baumannii to a level comparable to the more resistant C57BL/6 mice. Our results imply that the early recruitment of neutrophils into the lung is critical for initiating an efficient host defense against respiratory A. baumannii infection.     

Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus ("normal" model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated ("thick") mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.     

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.     

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.     

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.     

Inhibitory role of neutrophils on influenza virus multiplication in the lungs of mice.

The protective role of neutrophils on intranasal infection of influenza virus was investigated in 3 strains of tumor-bearing mice with neutrophilic leukocytosis. In vitro multiplication of influenza virus was inhibited by neutrophils from both normal and tumor-bearing mice, and the inhibitory effect of neutrophils was augmented by an addition of fMLP to the culture. Pulmonary virus infectivities in the early phase after infection decreased in such ICR and BALB/c mice, and virus elimination in the late phase was accelerated in the ICR mice. However, no decrease in pulmonary virus infectivity was observed in tumor-bearing C57BL/6 mice. Intranasal administration of fMLP into normal and tumor-bearing C57BL/6 mice after infection significantly inhibited the virus propagation in the lungs. The decrease in neutrophil infiltration into the lung in tumor-bearing C57BL/6 mice was confirmed from histological observations of the lung and lung lavage after infection and from analysis of the neutrophil chemotactic activity induced by fMLP. This might be responsible for the high level of pulmonary virus titer in tumor-bearing C57BL/6 mice. Phagocytic activities of alveolar macrophages and productions of neutralizing antibody were suppressed in the 3 strains of tumor-bearing mice. These observations indicated that neutrophils could be significant effector cells as a host defense mechanism against influenza virus infection in vivo, and infiltration and functional activation of neutrophils could play a significant role in virus elimination from the infected site. Furthermore, the inhibition of virus propagation by neutrophils in vitro was almost completely abrogated by an addition of ZnSO4, suggesting that calprotectin could inhibit influenza virus multiplication.     

Role of alveolar macrophages in endotoxin-induced neutrophilic alveolitis in rats

Although bacterial endotoxins have potent effects on blood monocytes and tissue macrophages, the role of alveolar macrophages in regulating intrapulmonary neutrophil traffic following endotoxemia has not been studied previously. We have previously reported that a single intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 causes acute lung inflammation by neutrophils (PMN) in rats. The factors which influence the migration of PMN in the lung in this model are unknown. To determine whether macrophage-derived products could play a role in directing migration, we enumerated neutrophils in histologic sections and employed electron microscopy to document the location of neutrophils in the lung in vivo following endotoxin. We also cultured the alveolar macrophages recovered by lung lavage to measure the effect of their culture supernatants on neutrophil migration in vitro. In the first 6 hr following endotoxin, and also 24 hr later, there was an increase in the number of PMN enumerated in the lung parenchyma by light microscopy. Electron microscopy showed the location of the neutrophils to be exclusively intravascular at 6 hr. By contrast, neutrophils were observed in both interstitial and bronchoalveolar spaces at 24 hr, confirming that transvascular migration was active at that time. The pulmonary macrophages which were recovered by lung lavage from groups of rats sacrificed at 4 and at 15 hr following the administration of endotoxin were assayed for the release into culture media of migration-stimulatory activity for neutrophils. Macrophages from animals sacrificed 4 hr following endotoxin released less migration-stimulating activity into media than macrophages from controls. These macrophages could be stimulated to release migration-stimulating activity into culture media at levels comparable to macrophages from controls by the addition of opsonized Zymosan to the culture media. By contrast, macrophages from animals sacrificed 15 hr after endotoxin spontaneously released more migration-stimulating activity for neutrophils than did macrophages from controls. Thus, in this model, a specific increase in the synthesis or release by alveolar macrophages of factors which stimulate the migration of neutrophils in vitro coincided with a transition from intravascular to extravascular alveolar inflammation by neutrophils in vivo. These observations are consistent with the hypothesis that pulmonary alveolar macrophages may contribute to the regulation of alveolar inflammation following endotoxemia by releasing factors which influence the migration of neutrophils.     

Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.     

Selective transport of cytokine-induced neutrophil chemoattractant from the lung to the blood facilitates pulmonary neutrophil recruitment

The CXC chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) are potent neutrophil chemoattractants in rats. We have previously shown that CINC, unlike MIP-2 and most other proinflammatory cytokines, is elevated in the systemic circulation in response to an intratracheal (IT) challenge. Therefore, we hypothesized that CINC generated within the lung selectively enters the vascular compartment to facilitate pulmonary neutrophil recruitment. Rats were administered IT LPS, and plasma CINC and MIP-2 levels were measured 90 min and 4 h after injection, along with mRNA expression in lung, spleen, liver, and kidney. Ninety minutes and 4 h after IT LPS, CINC and MIP-2 mRNA expression were largely confined to lung homogenate, but of the two chemokines, only CINC was present in plasma. In separate experiments, rats received IT injections of recombinant CINC and/or MIP-2. Here, plasma levels of CINC, but not MIP-2, were significantly increased throughout the 4-h observation period. This finding was verified by individually administering (125)I-labeled forms of each chemokine. Instillation of recombinant MIP-2 or CINC into the lung increased the number of neutrophils recovered in bronchoalveolar lavage fluid at 4 h, and this effect was enhanced when both chemokines were administered together. In addition, intravenous (IV) CINC, but not IV MIP-2, increased pulmonary neutrophil recruitment in response to IT MIP-2. Our results show that CINC, in contrast to MIP-2, is selectively transported from the lung to the systemic circulation, where it promotes neutrophil migration into the lung in response to a chemotactic stimulus.