Nonphagocytic clearance of Staphylococcus aureus from murine lungs.

Several investigations of host and bacterial factors critical to staphylococcal clearance from lungs suggest that alveolar macrophages may not provide the principal defense against inhaled staphylococci. We evaluated possible contributions of extracellular bactericidal activities in lungs with a standard aerosol challenge model by using methods which allowed recovery of macrophages for in vitro bactericidal assays and recovery of intrapulmonary staphylococci for clearance studies. Macrophages recovered by a gentle lavage technique immediately after aerosol exposure contained 6.0 +/- 2.3 colony-forming units of viable staphylococci per 100 glass-adherent macrophages. These intracellular staphylococci were killed in vitro with a half-life of 10.8 +/- 2.1 h, which is identical to our results with a completely in vitro system for ingestion and killing. However, 99.4 +/- 0.2% and 94.9 +/- 1.5% of the viable cocci recovered in bronchoalveolar lavage at 0.5 and 6.0 h after aerosol exposure were sensitive to lysostaphin, a rapidly bactericidal enzyme with no demonstrable activity against intracellular organisms and therefore, presumably extracellular. Photomicrographs from lavage pellets obtained 0.5, 1.5, 3.0, and 5.5 h after aerosol exposure confirmed the presence of numerous extracellular cocci. These extracellular cocci were eliminated at the same rate as whole lung cocci (half-life = 3.07 and 3.14 h, respectively) and at a much faster rate than intracellular cocci. In summary, we found large numbers of extracellular staphylococci in bronchoalveolar spaces during the first 6 h after aerosol exposure that are inactivated at the same rate as the whole lung bacterial population. Since only a small number of staphylococci are ingested by macrophages and intracellular bactericidal activity appears too slow to explain intrapulmonary killing, we conclude that an as yet unidentified extracellular killing process contributed to staphylococcal clearance.     

Role of C5 and recruited neutrophils in early clearance of nontypable Haemophilus influenzae from murine lungs.

We used a mouse model system to investigate the pulmonary defense mechanisms involved in clearance of nontypable Haemophilus influenzae from the lower respiratory tract. The importance of the C5 complement protein molecule in polymorphonuclear leukocyte (PMN) recruitment was studied by using congenic C5-sufficient B10.D2/nSn (C5+) and C5-deficient B10.D2/oSn (C5-) mice. The C5+ and C5- mice were inoculated with saline or nontypable H. influenzae via an endobronchial catheter. Clearance of bacteria was studied by using quantitative lung cultures. Bronchoalveolar lavage was performed at several time intervals. The number of cells in the lavage fluid were counted, and chemotactic activity was assayed in lavage fluid by the leading front technique, using human PMN in modified Boyden chambers. Pulmonary clearance of bacteria was significantly impaired in the absence of C5 (P less than 0.05). The C5+ mice recruited significantly more PMN after challenge with nontypable H. influenzae than C5- mice did (P less than 0.05), but significant PMN recruitment occurred in C5- mice. Similarly, although chemotactic activity was present in both C5+ and C5- mice, significantly more intraalveolar chemotactic activity was noted in C5+ mice than in C5- mice (P less than 0.05). The C5 molecule yields important chemotaxins during this early time period, but other chemotaxins are also present within the alveoli, demonstrating the redundancy of the inflammatory response after pulmonary challenge with nontypable H. influenzae. Nitrogen mustard-induced neutropenic animals were studied to evaluate the functional importance of PMN in pulmonary clearance of nontypable H. influenzae. Pulmonary clearance of nontypable H. influenzae was significantly impaired in neutropenic animals (P less than 0.05). Our results indicate that the prompt appearance of PMN in lungs is crucial for early clearance of nontypable H. influenzae.     

Early clearance of pneumococci from the lungs of decomplemented rats.

Pneumococcal types which exhibited varying degrees of interaction with the complement system in vitro wee aerosolized into normal and decomplemented rats, and the rate of killing of pneumococci was monitored by serial cultures of whole lung homogenates. The clearance of pneumococci from the alveoli did not correlate with the ability of the pneumococci to be opsonized by complement in vitro. Similarly, rats depleted of complement in the serum and the bronchoalveolar lavage fluid maintained their ability to rapidly inactivate aerosolized pneumococci. These results indicate that the early phase of pneumococcal killing in the alveoli does not require complement and suggest that in the nonimmune host, phagocytosis by alveolar macrophages can be achieved without complement-dependent opsonization or, alternatively, that extracellular factors rapidly inactivate inhaled pneumococci.     

Enhancement of clearance of bacteria from murine lungs by immunization with detoxified lipooligosaccharide from Moraxella catarrhalis conjugated to proteins

Moraxella catarrhalis strain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance of M. catarrhalis in an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS-high-molecular-weight proteins (dLOS-HMP) from nontypeable Haemophilus influenzae (NTHi), or nonconjugated materials in Ribi adjuvant and then challenged with M. catarrhalis strain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P<0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers against M. catarrhalis and bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P<0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains of M. catarrhalis in mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine against M. catarrhalis and NTHi infections.     

TLR4 inhibition impairs bacterial clearance in a therapeutic setting in murine abdominal sepsis

Objective and design

To investigate the therapeutic effect of E5564 (a clinically used TLR4 inhibitor) in murine abdominal sepsis elicited by intraperitoneal infection with a highly virulent Escherichia coli in the context of concurrent antibiotic therapy.

Methods

Mice were infected with different doses (~2 × 10⁴–2 × 10⁶ CFU) of E. coli O18:K1 and treated after 8 h with ceftriaxone 20 mg/kg i.p. combined with either E5564 10 mg/kg i.v. or vehicle. For survival studies this treatment was repeated every 12 h. Bacterial loads and inflammatory parameters were determined after 20 h in peritoneal lavage fluid, blood, liver and lung tissue. Plasma creatinin, AST, ALT and LDH were determined to assess organ injury.

Results

E5564 impaired bacterial clearance under the antibiotic regime after infection with a low dose E. coli (1.7 × 10⁴ CFU) while renal function was slightly preserved. No differences were observed in bacterial load and organ damage after infection with a tenfold higher (1.7 × 10⁵ E. coli) bacterial dose. While treatment with E5564 slightly attenuated inflammatory markers provoked by the sublethal doses of 104–105 E. coli under the antibiotic regime, it did not affect lethality evoked by infection with 1.7 × 106 E. coli.

Conclusions

The impact of TLR4 inhibition during abdominal sepsis by virulent E. coli bacteria is only beneficial at low infection grade at cost of bactericidal activity.     

Fibrogenic response in murine lungs to asbestos.

Pulmonary fibrogenic response was investigated in mice following intratracheal inoculation of amosite, anthophylite and tremolite varieties of Indian asbestos and studies were made over a period of 150 days. At early periods all the varieties produced acute inflammatory reaction in the lungs. Thick reticulum fibers were encountered at later periods with amosite, while only thin reticulum fibers developed with anthophyllite or tremolite variety. The formation of asbestos bodies did not take place with any of the asbestos varieties even at 150 days. The deviation in the pulmonary fibrogenic response in mice has been attributed to species difference.     

Chronic staphylococcal osteomyelitis: a new experimental rat model.

A rat model of chronic staphylococcal osteomyelitis was developed. Fibrin glue (5 microliters) and Staphylococcus aureus (2 x 10(6) CFU/5 microliters) were inoculated into the proximal metaphysis of the tibia. The rats were killed at intervals of between 1 and 6 months, and the tibias were removed. Induced lesions were evaluated by radiographic, macroscopic, and histological examinations and bacterial counts. Roentgenograms revealed osteomyelitis in more than 90% of the tibias. Gross bone pathology revealed skeletal deformation, new bone formation, abscesses, and draining skin fistulas in more than 80% of cases. Histological examination revealed osteomyelitis in more than 90% of cases, and bacterial counts were positive in 86% of cases. Only fibrin glue (5 microliters) was inoculated into controls. Controls showed no osteomyelitic lesions, and counts were negative in seven of eight control tibias. The main feature of this model is the use of fibrin glue instead of the sclerosing agents and foreign bodies used in other models. The model reproduces lesions similar to those of human posttraumatic osteomyelitis and can be reliably used in pathophysiological and therapeutic studies.     

Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin binding was sensitive to temperature and pH and was directly proportional to the concentration of spleen cells in the incubation mixture. We estimated that there was a single class of toxin-binding sites, which had an apparent equilibrium dissociation constant (Kd) of 8 x 10(-7) M and numbered 3,600 sites per cell. SEA and the antigenically distinct compounds staphylococcal enterotoxins B and E in excess competitively inhibited binding of [125I]SEA to mouse spleen cells. Our data suggest a common class of binding sites for the three staphylococcal enterotoxins.     

Susceptibility to staphylococcal alpha-toxin of Friend virus-infected murine erythroblasts during differentiation.

Splenic erythroblasts obtained from BALB/c mice infected with the anemia strain of Friend virus were compared with "matured" cells and adult erythrocytes for their sensitivity to staphylococcal alpha-toxin. Matured cells were obtained by treating erythroblasts in culture with erythropoietin for 48 h. Sensitivity to staphylococcal alpha-toxin, measured both by release of 86Rb and by cell lysis, failed to demonstrate significant differences among the cell types. Since maturation of erythroblasts to matured cells or erythrocytes is associated with synthesis of band 3, hemoglobin, and spectrin and the loss of transferrin receptors, we conclude that none of these compounds serves as the specific receptor for staphylococcal alpha-toxin in BALB/c mice.     

Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection

Virus respiratory infections often precede bacterial pneumonia in healthy individuals. In order to determine the potential role of respiratory syncytial virus (RSV) in bacterial secondary infections, a mouse sequential pulmonary infection model was developed. Mice were exposed to RSV then challenged with Streptococcus pneumoniae (StPn). Exposure of BALB/c mice to 10(6)-10(7) plaque forming units (pfu) of virus of RSV significantly decreased StPn clearance 1-7 days following RSV exposure. This finding was not restricted to StPn alone: exposure to RSV followed by Staphylococcus aureus (SA) or Pseudomonas aeruginosa(PA) resulted in similar decreases in bacterial clearance. Both bronchoalveolar lavage (BAL) cell counts and pulmonary histopathology demonstrated that RSV-StPn exposed mice had increased lung cellular inflammation compared to mice receiving StPn or RSV alone. The effect of RSV infection on bacterial clearance was dependent on the mouse genetic background: C57BL/6J mice (relatively resistant to RSV infection) demonstrated a modest change in StPn clearance following RSV exposure, whereas FVBN/J mice (similar to the BALB/cJ mice in RSV susceptibility) demonstrated a similar degree of RSV-associated decrease in StPn clearance 7 days following RSV exposure. Neutrophils from the RSV-StPn sequentially exposed BALB/cJ mice were functionally altered-produced greater levels of peroxide production but less myeloperoxidase (MPO) compared to mice receiving StPn alone. These data demonstrate that RSV infection decreases bacterial clearance, potentially predisposing to secondary bacterial pneumonia despite increased lung cellular inflammation, and suggest that functional changes occur in the recruited neutrophils that may contribute to the decreased bacterial clearance.     

Tumor necrosis factor-alpha blockage therapy impairs hepatitis B viral clearance and enhances T-cell exhaustion in a mouse model

Hepatitis B virus (HBV) reactivation and recurrence are common in patients undergoing immunosuppression therapy. Tumor necrosis factor (TNF) blockage therapy is effective for the treatment of many autoimmune inflammatory diseases. However, the role of TNF-α blockage therapy in the innate and adaptive immune responses against HBV is still not clear. A detailed analysis of HBV infection under TNF-α blockage therapy is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV clearance and T-cell responses were analyzed in a HBV-transfected mouse model under anti-TNF blockage therapy. Our results demonstrated that under TNF-α blockage therapy, HBV viral clearance was impaired with persistent elevated HBV viral load in a dose- and temporal-dependent manner. The impairment of HBV clearance under anti-TNF-α blockage therapy occurred at early time points after HBV infection. In addition, TNF-α blockade maintained a higher serum HBV viral load and increased the number of intrahepatic programmed cell death (PD)-1^highCD127^low exhausted T cells. Furthermore, TNF-α blockade abolished Toll-like receptor 9 (TLR9) ligand-induced facilitation of HBV viral clearance. Taken together, TNF-α blockade impairs HBV clearance and enhances viral load, and these effects depend on early administration after HBV infection. Our results here demonstrate that early TNF-α blockade reduces viral clearance and persistently maintains elevated HBV viral load in a mouse model, suggesting that HBV may reactivate during therapy with TNF-α-blocking agents.     

Intranasal interleukin-12 therapy inhibits Mycoplasma pneumoniae clearance and sustains airway obstruction in murine pneumonia

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentrations in respiratory secretions. We evaluated the effects of IL-12 therapy on microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in mice. BALB/c mice were inoculated with M. pneumoniae or SP4 broth. Mice were treated with intranasal IL-12 or placebo daily for 8 days, starting on day 1 after inoculation. Mice were evaluated at baseline and on days 1, 3, 6, and 8 after therapy. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic score (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and granulocyte-macrophage colony-stimulating factor), and plethysmography, both before and after methacholine treatment. M. pneumoniae-infected mice treated with IL-12 (MpIL12 mice) were found to have significantly higher BAL M. pneumoniae concentrations than those of M. pneumoniae-infected mice treated with placebo (MpP mice) (P < 0.001). MpIL12 mice had higher BAL concentrations of IL-12, IFN-γ, TNF-α, and IL-6, with differences in IL-12 and IFN-γ concentrations reaching statistical significance (P < 0.001). Airway obstruction was statistically elevated in MpIL12 mice compared to that in MpP mice (P = 0.048), while airway hyperreactivity was also elevated in MpIL12 mice but did not reach statistical significance (P = 0.081). Lung parenchymal pneumonia subscores were significantly higher in MpIL12 mice (P < 0.001), but no difference was found for overall HPS, even though a strong trend was noticed (P = 0.051). Treatment of experimental M. pneumoniae pneumonia with intranasal IL-12 was associated with more severe pulmonary disease and less rapid microbiologic and histological resolution.     

Induction of L-phase variants of Nocardia caviae within intact murine lungs.

The data presented show that cells of Nocardia caviae 112 were converted to cell wall-deficient microbial variants within the intact murine lung after intranasal administration. At the time that these L-phase variants were recovered in large numbers from the lung, there was a correspondingly enhanced inflammation leading to alveolar consolidation and animal death. During the peak of this response (at 1 week after infection), normal nocardial cells were neither isolated from nor seen within the lung. It is suggested that the conversion of these normal nocardial cells to their L-phase variant leads to this extensive pulmonary damage. Furthermore, the L-phase organisms appear to play an active role in this pathological effect since introduction of similar amounts of killed nocardial cells into the lungs of the mice failed to produce a similar response.     

Activation of murine T-suppressor lymphocytes by group A streptococcal and staphylococcal pyurogenic exotoxins.

The effect of group A streptococcal pyrogenic exotoxin (PE) type C and staphylococcal PE on the in vitro antibody response to sheep erythrocytes was studied in cultures of mouse spleen cells. Both exotoxins suppressed the day 4 direct plaque-forming cell response when added to the cultures. The maximum suppression was obtained with 1.0 or 0.1 ng of toxin per culture, and the suppressive effect was reversed by addition of gangliosides to the cultures at the same time as the exotoxins. Preincubation of T lymphocytes for 4 days with either exotoxin resulted in the generation of a suppressor cell population, which produced dose-dependent suppression of the direct plaque-forming cell response when added to fresh sheep eyrthrocyte-activated splenocytes. The suppression obtained was not reversed by gangliosides indicating toxin carry-over was not responsible for the effect. B cells, preincubated with exotoxin, failed to suppress the direct plaque-forming cell response of fresh erythrocyte-activated spleen cells.     

High mass clearance of autoantibodies from a murine model of lupus nephritis by immunoadsorption using star-configured polyethylene glycols.

The extracorporeal immunoadsorption of antibodies as part of the therapy for human autoimmune diseases has been limited by technology with inadequate and nonselective mass clearance or problems with bioincompatibility. To overcome these shortcomings, we designed a method utilizing star-configured polyethylene glycols (star-PEGs) having up to 63 free arms with immunoreactive (tresylate ester) end-groups for each arm immobilized to a polymer support substrate. The flexibility and length of the arms are thought to allow optimization of epitope presentation and to permit interaction with immunoligands on adjacent arms. To demonstrate efficacy we used an in vitro murine antibody model of human lupus nephritis, wherein we could study the kinetics and mass clearance of hybridoma derived antihistone antibodies from human plasma. Histones were covalently bound to the star-PEG end-groups and the kinetics of antibody adsorption were assessed using a surface plasmon resonance technique. The equilibrium constants of antihistone antibody binding to histone-star-PEGs that were linked to a support grid demonstrated high affinity with a KA of 3.56E + 07 and a KD of 2.81E - 08. The optimum reaction conditions were determined to accomplish the hydrophilization of polysulfone (PS; by an aqueous nitration method) and polymethylmethacrylate substrates (PMMA; by hydrazine), using sheet casts of both polymer substances. Hollow fiber devices of these polymers (commercial hemodialyzers) were modified so that histone-bound star-PEGs were linked to their intracapillary luminal surfaces, using a process which we have shown retains their immunoadsorption properties for antihistone antibodies. A closed loop recirculating model was constructed to measure mass clearance of antibodies from a reservoir. After optimizing conditions using extraction from saline solutions, the removal of antibody from human plasma by control and surface-modified devices was assessed over 4 h. There was no measurable antibody clearance by the control fibers over this time interval. The 2.1 m2 luminal surface area PMMA devices removed 5.0 +/- 1.1 mg, with a maximum of 7.0 mg. The 1.8 m2 PS device cleared 11.3 +/- 6.2 mg with a maximum of 17.5 mg. In summary, star-PEG immunoadsorption is a promising technique for the treatment of human autoimmune disease because it can achieve very high-mass clearance of autoantibodies using modified biocompatible hollow-fiber polymer devices.     

Ecology of the murine alphaherpesvirus and its isolation from lungs of rodents in cell culture

Together 381 sera of murine rodents (Apodemus flavicollis, Clethrionomys glareolus, Microtus arvalis and Mus musculus) trapped in different localities of Czechoslovakia were examined for the presence of antibodies to a murine alphaherpesvirus (MHV) and to murine cytomegalovirus (CMV). Positivity of rodent sera to the two MHV and one murine CMV strains varied from none to 12.5% and from none up to 11.3%, respectively, depending on the locality under study. From the lungs of A. flavicollis showing serum antibodies to MHV, a further murine herpes virus strain was isolated in rabbit embryo fibroblasts (REF). The latter MHV reached a titre of 10(10) TCID50/ml in the 13th passage, causing typical cytopathic effect from the 2nd passage on.