Coli-induced Pneumonia Research Articles

Aerosol Delivery of a Novel Recombinant Modified Superoxide Dismutase Protein Reduces Oxidant Injury and Attenuates Escherichia coli Induced Lung Injury in Rats.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome with diverse etiologies characterized by increased permeability of alveolar-capillary membranes, pulmonary edema, and acute onset hypoxemia. During the ARDS acute phase, neutrophil infiltration into the alveolar space results in uncontrolled release of reactive oxygen species (ROS) and proteases, overwhelming antioxidant defenses and causing alveolar epithelial and lung endothelial injury. Objectives: To investigate the therapeutic potential of a novel recombinant human Cu-Zn-superoxide dismutase (SOD) fusion protein in protecting against ROS injury and for aerosolized SOD delivery to treat Escherichia coli induced ARDS. Methods: Fusion proteins incorporating human Cu-Zn-SOD (hSOD1), with (pep1-hSOD1-his) and without (hSOD1-his) a fused hyaluronic acid-binding peptide, were expressed in E. coli. Purified proteins were evaluated in in vitro assays with human bronchial epithelial cells and through aerosolized delivery to the lung of an E. coli-induced ARDS rat model. Results: SOD proteins exhibited high SOD activity in vitro and protected bronchial epithelial cells from oxidative damage. hSOD1-his and pep1-hSOD1-his retained SOD activity postnebulization and exhibited no adverse effects in the rat. Pep1-hSOD1-his administered through instillation or nebulization to the lung of an E. coli-induced pneumonia rat improved arterial oxygenation and lactate levels compared to vehicle after 48 hours. Static lung compliance was improved when the pep1-hSOD1-his protein was delivered by instillation. White cell infiltration to the lung was significantly reduced by aerosolized delivery of protein, and reduction of cytokine-induced neutrophil chemoattractant-1, interferon-gamma, and interleukin 6 pro-inflammatory cytokine concentrations in bronchoalveolar lavage was observed. Conclusions: Aerosol delivery of a novel recombinant modified SOD protein reduces oxidant injury and attenuates E. coli induced lung injury in rats. The results provide a strong basis for further investigation of the therapeutic potential of hSOD1 in the treatment of ARDS.

Nebulised mesenchymal stem cell derived extracellular vesicles ameliorate E. coli induced pneumonia in a rodent model

BackgroundMesenchymal stem cell (MSC) derived extracellular vesicles (EVs) have been proposed as an alternative to cell therapy, creating new possible delivery modalities such as nebulisation. We wished to investigate the therapeutic potential of directly nebulised MSC-EVs in the mitigation of Escherichiacoli-induced pneumonia.MethodsEV size, surface markers and miRNA content were assessed pre- and post-nebulisation. BEAS2B and A459 lung cells were exposed to lipopolysaccharide (LPS) and treated with nebulised bone marrow (BM) or umbilical cord (UC) MSC-EVs. Viability assays (MTT) and inflammatory cytokine assays were performed. THP-1 monocytes were stimulated with LPS and nebulised BM- or UC-EVs and phagocytosis activity was measured. For in vivo experiments, mice received LPS intratracheally (IT) followed by BM- or UC-EVs intravenously (IV) and injury markers assessed at 24 h. Rats were instilled with E. coli bacteria IT and BM- or UC-EVs delivered IV or by direct nebulisation. At 48 h, lung damage was assessed by physiological parameters, histology and inflammatory marker presence.ResultsMSC-EVs retained their immunomodulatory and wound healing capacity after nebulisation in vitro. EV integrity and content were also preserved. Therapy with IV or nebulised MSC-EVs reduced the severity of LPS-induced lung injury and E. coli-induced pneumonia by reducing bacterial load and oedema, increasing blood oxygenation and improving lung histological scores. MSC-EV treated animals also showed lower levels of inflammatory cytokines and inflammatory-related markers.ConclusionsMSC-EVs given IV attenuated LPS-induced lung injury, and nebulisation of MSC-EVs did not affect their capacity to attenuate lung injury caused by E. coli pneumonia, as evidenced by reduction in bacterial load and improved lung physiology.

Alveolar macrophage-derived NRP2 curtails lung injury while boosting host defense in bacterial pneumonia.

Clearance of airway intruders by immune cells is required to resolve infectious pneumonia. However, the molecular mechanisms underlying this process remain elusive. Here, we demonstrated that alveolar macrophage (AM)-derived neuropilin 2 (NRP2) plays an essential role in controlling severe pneumonia by enhancing microbial clearance. Mice with conditional deletion of the NRP2 gene in AM had persistent bacteria, uncontrolled neutrophil influx, and decreased survival during Escherichia coli-induced pneumonia. In vitro assays demonstrated that NRP2 could bind to CD11b+ Ly6Glo/+ neutrophils and promote their capacities in phagocytosis and killing of bacteria, which is partially contributed to the increased expression of TLR4 and TNF-a. These findings collectively revealed that AM-derived NRP2 protects the lungs from unwanted injury by promoting the clearance of invading pathogens. This study might provide a promising diagnostic biomarker and therapeutic target for severe pneumonia.

Umbilical cord-derived CD362+ mesenchymal stromal cells for E. coli pneumonia: impact of dose regimen, passage, cryopreservation, and antibiotic therapy

BackgroundMesenchymal stromal cells (MSCs) demonstrate considerable promise for acute respiratory distress syndrome (ARDS) and sepsis. However, standard approaches to MSC isolation generate highly heterogeneous cell populations, while bone marrow (BM) constitutes a limited and difficult to access MSC source. Furthermore, a range of cell manufacturing considerations and clinical setting practicalities remain to be explored.MethodsAdult male rats were subject to E. coli-induced pneumonia and administered CD362+ umbilical cord (UC)-hMSCs using a variety of cell production and clinical relevance considerations. In series 1, animals were instilled with E. coli and randomized to receive heterogeneous BM or UC-hMSCs or CD362+ UC-hMSCs. Subsequent series examined the impact of concomitant antibiotic therapy, MSC therapeutic cryopreservation (cryopreserved vs fresh CD362+ UC-hMSCs), impact of cell passage on efficacy (passages 3 vs 5 vs 7 vs 10), and delay of administration of cell therapy (0 h vs 6 h post-injury vs 6 h + 12 h) following E. coli installation.ResultsCD362+ UC-hMSCs were as effective as heterogonous MSCs in reducing E. coli-induced acute lung injury, improving oxygenation, decreasing bacterial load, reducing histologic injury, and ameliorating inflammatory marker levels. Cryopreserved CD362+ UC-hMSCs recapitulated this efficacy, attenuating E. coli-induced injury, but therapeutic relevance did not extend beyond passage 3 for all indices. CD362+ UC-hMSCs maintained efficacy in the presence of antibiotic therapy and rescued the animal from E. coli injury when delivered at 6 h + 12 h, following E. coli instillation.ConclusionsThese translational studies demonstrated the efficacy of CD362+ UC-hMSCs, where they decreased the severity of E. coli-induced pneumonia, maintained efficacy following cryopreservation, were more effective at early passage, were effective in the presence of antibiotic therapy, and could continue to provide benefit at later time points following E. coli injury.

Hepatic histopathology and apoptosis in diet-induced-obese mice under Escherichia coli pneumonia.

This research was to investigate the difference of hepatic histopathology and apoptosis between the diet-induced obesity (DIO) and normal (lean) mice after Escherichia coli (E. coli) pneumonia. A total of 128 ICR mice were selected to be challenged intranasally with phosphate-buffered saline (PBS) or 4×109CFUs/mL of E. coli, and the liver histopathology and apoptosis were examined pre- and post-infection. Results showed that the liver index, levels of lipid droplets, cytokines, adipocytokines, oxidative stress, apoptotic percentage, and apoptotic related factors in the E. coli-infected mice were generally higher than those in the uninfected mice, whereas the hepatic glycogen and Bcl-2 were the opposite. Interestingly, after E. coli infection, the DIO-E. coli mice exhibited decreased liver index and apoptotic percentages, and reduced levels of TNF-α, IL-6, resistin, MDA, GSH, CAT, Caspase-3, Caspase-9, Bax as well as Bax/Bcl-2 ratio in comparison to the lean-E. coli mice. Our results indicated that E. coli-induced pneumonia caused hepatic histopathological damage, increased hepatic apoptosis, oxidative damages, and higher levels of cytokines and adipocytokines. However, such changes showed less severely in the DIO mice than in the lean mice following E. coli pneumonia.

Caspase 11 Negatively Regulates Carbapenem-resistant Klebsiella Pneumoniae (CRKP)-induced Pneumonia

Abstract Background CRKP remains one of the major causes of health-care associated infections worldwide. Due to its multi-drug resistance property, host-targeted immunotherapy is a promising alternative to treat CRKP-infections. Caspase11, a non-canonical inflammasome, has been shown to protect against cytosolic bacterial pathogens. However, its role in CRKP-induced pneumonia remains elusive. Methods We used C57BL/6 (WT) and Caspase 11 knock out (KO) mice to induce pneumonia by intratracheal inoculation of 108 colony forming units (CFUs) of CRKP per mouse. In E coli-induced pneumonia model, 106 CFUs per mouse was used. Results Using a mouse model of CRKP-induced pneumonia, we found that Caspase 11 KO mice were protected from intratracheal CRKP infection. The KO mice had significantly less bacterial burden in lungs and BALF compared to their wild-type counterparts. Total leukocytes and neutrophil recruitment into the air spaces were significantly higher in KO mice. To determine whether the phenotype is specific to CRKP infection, we induced pneumonia in WT and KO mice using E. coli through intratracheal route. Consistent with CRKP model, the KO mice displayed protective phenotype during pulmonary E coli challenge suggesting that the phenotype observed in the KO mice is not specific to single bacterium. Further, the KO mice had less IL-1β, CXCL1, and CXCL2 but higher amount of IL-17A secreted in the lungs suggesting that IL-17A could mediate neutrophil recruitment in the KO mice during pulmonary CRKP infection. Conclusions Our findings together demonstrate that Caspase 11 negatively regulates CRKP-induced pneumonia and blocking Caspase 11 could be a potential therapeutic intervention for treating CRKP-infected pneumonic patients.

Novel R41Q- and R46Q-PAR1-Modified Mice Enable Proof-of-Concept Studies for In Vivo Protective Mechanisms of Action for Activated Protein C (APC) in Sepsis and Stroke

▪Introduction: Although much is known about in vitro mechanisms for Activated Protein C (APC)'s beneficial actions on endothelial, neuronal, and epithelial cells, much less is known about its in vivo mechanism(s) of action. EPCR-bound APC can cleave protease activated receptor (PAR) 1 at Arg41 or Arg46 to initiate cell signaling. Notably, cleavage at Arg46 can initiate arrestin-dependent biased signaling whereas thrombin's canonical cleavage occurs at Arg41. Which PAR1 cleavage may mediate APC's in vivo benefits? Inferences often come from using PAR1 knockout mice. But knocking out PAR1 disrupts PAR1 interactomes, thereby potentially disrupting many protein-protein interactions, e.g., PAR heterodimers, etc. To elucidate PAR1-dependent aspects of APC's in vivo mechanism of action, we generated C57BL/6 strains carrying either Arg41Gln (R41Q) or Arg46Gln (R46Q) PAR1 point mutations. Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against stroke when mice carried PAR1 mutations that prevent PAR1 cleavages at either Arg41 or Arg46.Methods:Standard homologous recombination methods and C57BL/6-ES cells were used to make C57BL/6 strains carrying PAR1 mutations. Brain microvascular endothelial cells (BECs) were isolated from mice by published protocols. Bioassays using BECs and APC, thrombin, or a thrombin receptor activating peptide (TRAP) included: (1) endothelial barrier stabilization or disruption that was monitored using Trans-Endothelial Resistance (TER) (iCelligence, Acea, San Diego) and (2) induction of cell signaling that was quantified using in-cell Western blotting. The ability of 5A-APC (mutations: KKK191-193AAA+RR229-230AA) (0.2 mg/kg, i.v. at 0h and 6 h) to reduce death due to live E. coli-induced pneumonia was determined using standard protocols. The ability of 3K3A-APC (mutations: KKK191-193AAA) (0.04 mg/kg, i.v. at 4 h) to reduce brain damage caused by transient distal middle cerebral artery occlusion (tdMCAO) (1 h) was determined at 24 h, as described.Results: R46Q mice were normal in breeding whereas R41Q mice, like PAR1 KO, gave less than half the expected QQ or null homozygotes. PAR1 knockout-derived BECs showed no TER decrease for either TRAP or thrombin, proving PAR1 was needed. TER assays showed that TRAP disrupted endothelial barriers of BECs from QQ41-PAR1 and QQ46-PAR1 mice similar to BECs from WT-mice, proving the expression of normally functional PAR1 in each PAR1-mutated strain. As expected, thrombin decreased TER for BECs from WT mice and QQ46-PAR1 mice but not for BECs from QQ41-PAR1 mice. APC inhibited thrombin-induced TER decreases for BECs from WT mice but not for BECs from QQ46-PAR1 mice, showing R46 is required for APC’s barrier stabilization. The QQ46-PAR1 BECs showed significantly reduced APC-induced phosphorylation of Akt whereas thrombin-induced phosphorylation of ERK was not significantly affected. Thus, in vitro studies showed the predicted retention or loss of responses to TRAP, thrombin or APC for each PAR1 mutation. In sepsis studies, 5A-APC reduced mortality from 50 % to 10 % in E. coli-induced pneumonia for WT-PAR1 and 41QQ-PAR1 mice (p < 0.01) but had no benefit for 46QQ-PAR1 mice. In tdMCAO stroke studies, 3K3A-APC significantly reduced infarct size, edema and neuronal apoptosis for WT mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb asymmetry and foot fault tests at 24 h after tdMCAO, 3K3A-APC was beneficial for mice with QQ41-PAR1 but not for QQ46-PAR1.Conclusions: Genetically altered mice carrying 41QQ-PAR1 and 46QQ-PAR1 provide unique, powerful tools to explore in vivo requirements for Arg41 or Arg46 cleavages by APC or other proteases that initiate PAR1-dependent signaling. The failures of 5A-APC to reduce death following intratracheal E. coli in 46QQ-PAR1 mice and of 3K3A-APC to reduce brain damage following ischemia in 46QQ-PAR1 mice provide very clear in vivo proof-of-concept data for the hypothesis that APC's cleavage of PAR1 at Arg46 is central to its in vivo mechanism of action in these two very different pathologies. These results strongly support the concept that APC's biased PAR1-dependent signaling following Arg46 cleavage is central to APC's in vivo benefits. [Display omitted] DisclosuresMosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose.

Novel R41Q-PAR1-Modified Mice Enable Proof-of-Concept Studies for in Vivo Mechanisms of Action for Thrombin (IIa) and Activated Protein C (APC)

Introduction: Thrombin (IIa) and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses that affect many cell types in the body. IIa employs the GPCR, protease activated receptor 1 (PAR1), to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC requires PAR1 for its opposing actions to stabilize endothelial barriers and to provide anti-inflammatory and anti-apoptotic activities. Studies of murine in vivo injury models using PAR1 knockout mice show that APC requires PAR1 to reduce sepsis-induced mortality and to provide robust neuroprotection following ischemic stroke. Extensive in vitro studies support the hypothesis that IIa’s cleavage at R41 in PAR1 initiates its signaling. Although APC was long thought, paradoxically, to act also via cleavage at R41, we recently proposed that APC’s cleavage at R46 initiates its endothelial barrier-protective and cytoprotective signaling via biased signaling. Since PAR1 knock-out mice cannot provide mechanistic data for testing these hypotheses in vivo, mice carrying the point mutation of R41 to Q in PAR1 were generated to enable mechanistic studies to test whether or not IIa and APC require Arg41 for their PAR1-dependent effects.Methods: Using C57BL/6-derived embryonic stem cells and standard gene targeting methods, we prepared C57BL/6 mice carrying the PAR1 mutation of R41 to Q. IIa-induced and APC-induced signaling, detected as phosphorylation of ERK1/2 or Akt in Brain Microvascular Endothelial Cells (BECs), was quantified using immunoblotting. BECs were obtained from homozygous 41QQ-PAR1 mice and wild type 41RR-littermates. Endothelial barrier disruption of cultured BECs was assayed using Trans-Endothelial Resistance (TER) assays (iCelligence, Acea, San Diego). Mortality of wild type and 41QQ-PAR1 mutant mice that was caused by live E. coli-induced pneumonia and to endotoxin was determined using standard methods. The ability of a cytoprotective-selective murine APC mutant (5A-APC) to reduce mortality of E.coli-challenged wild type and homozygous mutant mice was determined.Results: Upon breeding of R41Q-PAR1 heterozygous mice, the progeny did not fit a Mendelian pattern and yielded only 14% rather than 25% homozygous 41QQ mice. This reduced yield of homozygous mutant mice was similar to the previously reported low yield of homozygous PAR1 knockout mice. Homozygous 41QQ-PAR1 mice showed normal protein expression in BECs for PAR1 and endothelial cell protein C receptor (EPCR) antigens. When BECs from homozygous mutant mice were compared to those from wild type littermates, the IIa-induced vascular disruption in TER assays was greatly reduced by the mutation. Intracellular Ca2+ release, a hallmark of IIa-induced signaling, was greatly impaired (>90%) in BECs from homozygous mutant mice compared to wild type controls. IIa-induced phosphorylation of ERK1/2 in BECs was also significantly reduced by the mutation whereas APC-induced phosphorylation of Akt was not significantly affected. In murine sepsis-induced mortality studies, homozygosity for the R41Q-PAR1 mutation conveyed considerable resistance to death induced by either E. coli pneumonia or endotoxin in female mice but not in male mice. Tests to determine whether 5A-APC rescued male mice from sepsis-induced lethality showed that homozygous 41QQ-PAR1 mice were entirely responsive to 5A-APC therapy because 5A-APC treatment reduced mortality from 50 % to 0 % (see Figure). Wild type control mice also showed a beneficial response with reduced mortality in response to 5A-APC therapy, as previously described.Conclusions: These studies show that mutation of Arg41 to Gln in murine PAR1 diminishes or eliminates signaling induced by IIa but not by APC. Moreover, the ability of cytoprotective-selective 5A-APC to reduce bacteria-induced septic mortality in 41QQ-PAR1 mutant mice provides strong in vivo proof-of-concept data for PAR1 activation caused by non-canonical cleavage by APC. In summary, the 41QQ-PAR1 mutant mouse provides a unique and powerful tool to define in vivo requirements for cleavage sites that enable PAR1 signaling activities induced by IIa, APC or other proteases. [Display omitted] DisclosuresMosnier:The Scripps Research Institute : The Scripps Research Institute Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties.

Myeloid Differentiation Protein-2–Dependent and –Independent Neutrophil Accumulation duringEscherichia coliPneumonia

Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense.

CD44 Deficiency Leads to Enhanced Neutrophil Migration and Lung Injury in Escherichia coli Pneumonia in Mice

CD44 is a major cell-surface receptor for hyaluronic acid (HA), a glycosaminoglycan component of extracellular matrix. HA-CD44 interactions have been implicated in leukocyte extravasation into an inflammatory site. This study examined the role of CD44 in acute inflammatory responses during pneumonias induced by Escherichia coli and Streptococcus pneumoniae using CD44-deficient mice. In E. coli-induced pneumonia, neutrophil accumulation in the lungs and edema formation was increased by 84% and 88%, respectively, in CD44-deficient mice compared to wild-type mice. In contrast, no difference was observed between these genotypes in S. pneumoniae-induced pneumonia, and the HA content in the lungs decreased after instillation of S. pneumoniae, but not E. coli, in both genotypes. Studies to determine the mechanisms for this enhanced response showed that: 1) neutrophil apoptosis was not different between these two genotypes in either type of pneumonia; 2) CD44 deficiency resulted in enhanced mRNA expression of several inflammatory genes; and 3) CD44-deficient neutrophils migrated through Matrigel in response to chemoattractants faster and in greater numbers than wild-type neutrophils in vitro and this increase was in part dependent on HA content in the Matrigel. These data demonstrate that CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a previously unrecognized role for CD44 in limiting the inflammatory response to E. coli.

Neutrophil margination, sequestration, and emigration in the lungs of L-selectin-deficient mice.

These studies tested the hypothesis that L-selectin plays a role in neutrophil traffic in the lungs, particularly in neutrophil margination, sequestration, and emigration, using L-selectin-deficient mice. No defect in neutrophil margination within either capillaries or arterioles and venules was observed in uninflamed lungs of L-selectin-deficient mice. The initial rapid sequestration of neutrophils within the pulmonary capillaries 1 min after intravascular injection of complement fragments was not prevented. In contrast, L-selectin did contribute to the prolonged neutrophil sequestration (> or = 5 min). Interestingly, neutrophil accumulation within noncapillary microvessels required L-selectin at both 1 and 5 min after complement injection. During bacterial pneumonias, L-selectin played a role in neutrophil accumulation within noncapillary microvessels in response to either Escherichia coli or Streptococcus pneumoniae and within capillaries in response to E. coli but not S. pneumoniae. However, L-selectin was not required for emigration of neutrophils or edema in response to either organism. These studies demonstrate a role for L-selectin in the prolonged sequestration of neutrophils in response to intravascular complement fragments, in the intracapillary accumulation of neutrophils during E. coli-induced pneumonia, and in the accumulation of neutrophils within noncapillary microvessels when induced by either intravascular complement fragments or