Kinetics of Interaction and Fate of Pasteurella hemolytica in Bovine Alveolar Macrophages

To study the role of pulmonary alveolar macrophages (PAMs) in phagocytizing Pasteurella hemolytica, we developed an in vitro cultivation method for preparing them. This procedure provided an adherent monolayer of PAMs which were nonspecific esterase-positive and phagocytized latex beads. The phagocytosis and fate of P. hemolytica (biotype A, serotype 1) by PAMs in suspension were studied. The kinetics of phagocytosis were determined by quantitatively measuring the uptake of 24-h [³H]thymidine-labeled bacteria by the PAMs in the presence of opsonins. Results showed that the uptake of P. hemolytica was enhanced in the presence of normal serum or antiserum. A total of 90% of the bacteria were phagocytized in the presence of normal adult bovine serum, and up to 95% were phagocytized in the presence of an antiserum. These studies also showed that normal serum, but not fetal calf serum, contained heat-stable natural antibodies which readily initiated the opsonization of P. hemolytica. The heat-labile complement system was also involved in the opsonization. The fate of P. hemolytica inside the PAMs was investigated by transmission electron microscopy and by the viable plate count method. Approximately 90% of the normal serum- or antiserum-opsonized P. hemolytica were phagocytized by PAMs at a bacteria/PAM ratio of 20:1 and were completely degraded after 60 min of exposure. Prolonged incubation of this mixture of bacteria and PAMs resulted in cytotoxic changes and destruction of PAMs. At a low bacteria/PAM ratio (10:1 or less), there was phagocytosis and killing of bacteria but no cytotoxic changes on the PAMs. The exact mechanism which initiated this phenomenon was not demonstrated. Perhaps toxic substance(s) released by the excess unphagocytized bacteria caused the cytotoxic changes to the PAMs.     

The Biphasic mRNA Expression Pattern of Bovine Interleukin-8 in Pasteurella haemolytica Lipopolysaccharide-Stimulated Alveolar Macrophages Is Primarily due to Tumor Necrosis Factor Alpha

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-α) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-α in the induction of IL-8 from bovine alveolar macrophages.     

Ketamine Inhibits Calcium Elevation and Hydroxyl Radical and Nitric Oxide Production in Lipopolysaccharide-Stimulated NR8383 Alveolar Macrophages

Macrophages play a critical role in mediating inflammatory processes; activated macrophages respond to endotoxin by releasing pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. Ketamine, a widely used anesthetic agent, has unequivocally anti-inflammatory effects in vivo and in vitro. However, the detailed mechanisms for the anti-inflammatory effects of ketamine in microglia have not been elucidated yet. This study aimed to evaluate the effects of ketamine on lipopolysaccharide (LPS)-induced nitric oxide (NO), hydroxyl radical (·OH) production, and intracellular calcium accumulation in macrophages. Macrophages were pretreated with ketamine at the concentrations of 10, 100, and 1,000 μM 1 h before LPS stimulation. The production of NO and ·OH in the culture supernatant of macrophages was assayed by Griess Reagent Kit. LPS enhanced NO and ·OH production and provoked a significant intracellular calcium elevation. With the concentrations higher than 100 μM, ketamine inhibited LPS-induced NO and ·OH accumulation and intracellular calcium elevation. However, a low concentration of ketamine (10 μM) did not exert anti-inflammatory effects. These results suggest that intracellular calcium elevation is, at least, partially involved in the inhibitory effect of ketamine.     

In vivo production of neuraminidase by Pasteurella haemolytica A1 in goats after transthoracic challenge.

Nine goats were injected transthoracically with Pasteurella haemolytica A1 to determine if an extracellular bacterial enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 9) each received 1 ml of 7.25 x 10(5) live P. haemolytica A1 cells in polyacrylate beads transthoracically in the left lung on days 0 and 21. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 21. Serum was obtained from all animals on days -4, 3, 7, 14, 21, 24, and 32. Preimmune serum from all animals showed no detectable antibody to P. haemolytica A1 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals possessed a significant antibody concentration in response to the P. haemolytica A1 neuraminidase. On day 32, serum samples from the nine infected animals possessed enzyme neutralizing activity that ranged from 62% to 100%. Anti-neuraminidase antibody could be detected as early as day 14 by the enzyme neutralization assay. These data demonstrate that the enzyme neuraminidase is produced in vivo during an active P. haemolytica A1 lobar infection.     

Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli.     

Effect of repeated in vitro transfer of Pasteurella haemolytica A1 on encapsulation, leukotoxin production, and virulence.

Pasteurella haemolytica serotype 1 was transferred daily for 128 serial passages on both unsupplemented brain heart infusion agar and the same basal medium supplemented with bovine blood, horse serum, and yeast extract. Repeatedly transferred cultures were shown to retain the ability to produce both capsular material and leukotoxin. Furthermore, intact organisms were found to be as toxic in vitro for bovine leukocytes and as virulent for mice as unpassaged cultures. These results indicate that the precaution of using only freshly isolated cultures in the study of this organism may not be necessary.     

Increase of glycocalyx and altered lectin agglutination profiles of Pasteurella haemolytica A1 after incubation in bovine subcutaneous tissue chambers in vivo or in ruminant serum in vitro.

Pasteurella haemolytica serotype A1 (bovine strain OK) was incubated for 2 and 6 h in bovine subcutaneous tissue chambers in vivo, and ovine strain 82-25 and bovine strain L011 were incubated in vitro for 2 h in heat-inactivated ovine or bovine serum from which gamma globulin had been depleted by protein G affinity chromatography to assess changes in morphology and lectin agglutination profiles (strains 82-25 and L101 only). Cells, removed from chambers after 2 h, were covered with an extensive, dense glycocalyx extending approximately 0.5 microm from the cell surface. In many cells, the glycocalyx was separated from the cell surface by a clear, electron-transparent area. Cells, removed at 6 h, were covered with a sparse glycocalyx of fine fibers 0.2 to 0.3 microm from the cell surface. Strains 82-25 and L101, incubated for 2 h in heat-inactivated ovine or bovine serum or in heat-inactivated ovine or bovine serum depleted of gamma globulin by protein G affinity chromatography, were also covered with a glycocalyx. The glycocalyx did not bind protein A-colloidal gold and therefore did not contain aggregates of accumulated antibody. Strains 82-25 and L101 were incubated individually for 2 h in 10 mM sodium phosphate buffer (pH 7.2) containing 0.14 M NaCl, 0.5 mM CaCl2, and 0.15 mM MgCl2 or with this buffer and either 25% heat-inactivated, gamma globulin-depleted ovine serum or 25% heat-inactivated, gamma globulin-depleted bovine serum. Agglutination profiles were then determined with 17 lectins in 10 mM HEPES-buffered saline (pH 8.4) with 0.1 mM CaCl2 and 0.08% sodium azide. Profiles did not vary with 10 of 17 lectins. However, profiles did vary with peanut agglutinin, Phaseolus vulgaris leucoagglutinin, Sophora japonica agglutinin, Maackia amurensis lectin II, Narcissus pseudonarcissus (daffodil) lectin, Griffonia simplicifolia lectin I, and Pisum sativum agglutinin. Altered profiles indicate a change in the bacterial cell surface, possibly by adsorption or alteration of surface carbohydrate moieties by serum constituents.     

Macrophages Protect against Muscle Atrophy and Promote Muscle Recovery in Vivo and in Vitro : A Mechanism Partly Dependent on the Insulin-Like Growth Factor-1 Signaling Molecule

Hindlimb unloading and reloading are characterized by a major loss of muscle force and are associated with classic leukocyte infiltration during recovery from muscle atrophy. Macrophages act as a cellular cornerstone by playing both pro- and anti-inflammatory roles during muscle recovery from atrophy. In the present study, we investigated the role of macrophages in muscle atrophy and regrowth using in vivo and in vitro models. Mice depleted in monocytes/macrophages and submitted to a hindlimb unloading and reloading protocol experienced a significant delay in muscle force recovery compared with matched placebo mice at 7 and 14 days after reloading. Furthermore, an in vitro myotube/macrophage coculture showed that anti-inflammatory macrophages, which contain apoptotic neutrophils and express low levels of cyclooxygenase-2, completely prevented the loss of protein content and the myotube atrophy observed after 2 days in low serum medium. The presence of macrophages also protected against the decrease in myosin heavy chain content in myotubes exposed to low serum medium for 1 day. Interestingly, the addition of an anti-IGF-1 antibody to the coculture significantly decreased the ability of macrophages to protect against myotube atrophy and myosin heavy chain loss after 2 days in low serum medium. These results clearly indicate that macrophages and, more precisely, the release of IGF-1 by macrophages, play an important role in recovery from muscle atrophy.Monocytes are large mononuclear cells that circulate in the blood and differentiate into macrophages in invaded tissues in response to various stimuli.¹ Macrophages have a strong phagocytic capacity and can orchestrate the inflammatory process via the release of a wide variety of cytokines and chemokines such as interleukin (IL)-1, tumor necrosis factor-α, and macrophage inflammatory protein-2.^2,3 Numerous studies have also demonstrated that macrophages play a direct role in tissue recovery through the release of the anti-inflammatory molecules and anabolic growth factors IL-10, basic fibroblast growth factor, and insulin-like growth factor-1 (IGF-1).^4,5,6The regulation of the multiple and occasionally opposing functions of macrophages is very complex and poorly understood.^7,8,9 To add to this complexity, the diversity of experimental models can also lead to different conclusions regarding the roles of each macrophage phenotype in muscle recovery from atrophy or damage. For example, in a model of eccentric contractions, in which injured muscle is basically devoid of neutrophils, macrophage invasion contributes to secondary damage to the skeletal muscle.¹⁰ On the other hand, the phagocytosis of apoptotic neutrophils and necrotic cells by macrophages induces a change in macrophage phenotype from pro- to anti-inflammatory, which has a strong modulatory effect on cytokine profiles and is essential for dampening environmental inflammatory signals.^11,12 Regarding the different subsets of macrophages, coculture experiments have shown that pro-inflammatory macrophages (Ly-6C^hi) stimulate myogenic cell proliferation, whereas anti-inflammatory macrophages (Ly-6C^lo) exert a strong differentiating influence on myogenic cells.^12,13 Nonetheless, in a model in which rodent hindlimbs are deprived of mechanical loading for 10 days followed by reloading, the high concentration of leukocytes in reloaded muscles regardless of the absence of significant muscle damage raises intriguing questions about the detrimental and beneficial roles of leukocytes in muscle dysfunction and recovery from atrophy.^14,15,16 It is thus tempting to speculate that the roles of macrophages may vary as a function of the type of insult.In the present study, mice depleted in macrophages were submitted to hindlimb unloading and reloading to evaluate the roles of macrophages in muscle atrophy and regrowth. Our results showed that macrophages neither prevent the loss in muscle force nor promote recovery during the early inflammatory phase (1 and 3 days after reloading). However, they play a key role in muscle growth and recovery at later times (7 and 14 days after reloading). In addition, an in vitro coculture model in which atrophied myotubes were combined with macrophages expressing an anti-inflammatory phenotype showed that the presence of macrophages protects myotubes from atrophy and that this protective effect is partly mediated by the release of IGF-1.     

Mendelian Disorders of Cornification Caused by Defects in Intracellular Calcium Pumps: Mutation Update and Database for Variants in ATP2A2 and ATP2C1 Associated with Darier Disease and Hailey–Hailey Disease

The two disorders of cornification associated with mutations in genes coding for intracellular calcium pumps are Darier disease (DD) and Hailey–Hailey disease (HHD). DD is caused by mutations in the ATP2A2 gene, whereas the ATP2C1 gene is associated with HHD. Both are inherited as autosomal‐dominant traits. DD is mainly defined by warty papules in seborrheic and flexural areas, whereas the major symptoms of HHD are vesicles and erosions in flexural skin. Both phenotypes are highly variable. In 12%–40% of DD patients and 12%–55% of HHD patients, no mutations in ATP2A2 or ATP2C1 are found. We provide a comprehensive review of clinical variability in DD and HHD and a review of all reported mutations in ATP2A2 and ATP2C1. Having the entire spectrum of ATP2A2 and ATP2C1 variants allows us to address the question of a genotype–phenotype correlation, which has not been settled unequivocally in DD and HHD. We created a database for all mutations in ATP2A2 and ATP2C1 using the Leiden Open Variation Database (LOVD v3.0), for variants reported in the literature and future inclusions. This data may be of use as a reference tool in further research on treatment of DD and HHD.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Human Alveolar Macrophages Cultured on Fibronectin in Vitro

We studied the phagocytosis of agarose beads by human alveolar macrophages in terms of the morphology, the receptors involved, and the cellular substrates (plastic or fibronectin) used. Beads coated with C3b (58%) and iC3b (42%) by treatment with serum, were ingested during 45 min by CR1 and CR3 on the macrophages. This ingestion was inhibited 80-90% by the presence of polyclonal F(ab')2 anti-C3 fragments. Since the phagocytosis of both C3b- and iC3b-coated beads was about threefold stronger than for C3b-coated beads (trypsinized serum-treated beads), the results indicate that the CR3 is more phagocytic than the CR1. The phagocytosis of initially complement uncoated beads, which are slowly opsonized with macrophage-produced C3b and iC3b in vitro, was also strongly inhibited (70-80%) by the presence of anti-human C3 F(ab')2 fragments. There was an increased phagocytosis (10-17%) of complement precoated beads by macrophages cultured on the fibronectin substrate versus the plastic substrate. The morphology and rapid phagocytosis of the complement precoated beads was demonstrated by SEM. The general impression was that membranous protrusions stretched towards the beads, which became increasingly enclosed by plasma membrane.