Philip N. Bochsler

Identification and characterization of a bovine lipopolysaccharide-binding protein.

Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccharide (LPS, endotoxin) stemming from gram‐negative bacterial infections. Serum proteins and cell surface receptors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreactive, thiol‐cleavable, radiolabeled derivative of E. coli 0111:B4 LPS [LPS‐(p‐azidosalicylamido)‐1,3′‐dithiopro‐pionamide; 125I‐ASD‐LPS], to identify the presence of LPS‐binding proteins (LBPs) in bovine serum. Ion exchange chromatography was used to fractionate bovine serum, and eluted protein was subsequently photoaffinity labeled using 125I‐ASD‐LPS. LBPs were identified by autoradiography of sodium dodecyl sulfate‐polyacrylamide gels. Several LBPs including three with apparent molecular masses of 65, 60, and 50 kDa were variably present within the chromatography pools. A 22‐residue NH2‐terminal amino acid sequence of the 60‐kDa protein showed 77% homology with human LBP and 68% with rabbit LBP within this region. Further purification utilizing high‐performance liquid chromatography yielded a protein fraction that contained the 60‐kDa protein and was distinctly more active than whole bovine serum in LPS‐dependent macrophage activation assays (up to 1600‐fold on a weight/volume basis). The LPS‐mediated macrophage activation in concert with chromatographically purified serum protein in tissue factor assays was inhibitable using anti‐CD14 monoclonal antibodies. The results indicate that an LPS‐binding protein exists in samples of pooled bovine serum and that this protein has features in common with human and rabbit LBP. J. Leukoc. Biol. 56: 784–791; 1994.

Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharide-mediated cytotoxicity and activation of bovine vascular endothelial cells in vitro.

Bacterial endotoxin (lipopolysaccharide, LPS) has potent proinflammatory properties toward many cell types, including vascular endothelial cells. Bovine endothelial cells are often used for investigations involving the vascular endothelium in vitro, and other bovine products such as fetal bovine serum are also widely utilized in research laboratories. Evidence is presented that soluble CD14 (sCD14) is present in bovine serum and that LPS‐mediated activation and cytotoxicity to bovine endothelial cells in vitro are dependent on sCD14. LPS‐mediated activation of endothelial cells was quantitated by measuring tissue factor expression using an activated factor X–related chromogenic assay. Concentrations of 0.1–5.0% fetal bovine serum in the culture medium promoted LPS‐induced tissue factor expression on bovine endothelial cells, and anti‐CD14 monoclonal antibody (mAb) (20 μg/ml) inhibited tissue factor expression, whereas control antibodies did not. LPS‐mediated damage to endothelial cells was assayed using the MTT tetrazolium assay. We found that either serum or recombinant human soluble CD14 (rsCD14, 20–2000 ng/ml) was required for LPS‐related endothelial cell damage and that anti‐CD14 mAb inhibited cytotoxicity. In addition, bovine LPS‐binding protein (LBP, 20 ng/ml) purified from bovine serum had no effect on LPS‐mediated cytotoxicity, but bovine LBP greatly enhanced the cytotoxic effect of LPS plus rsCD14. Western blot analysis performed on fractionated bovine serum samples with anti‐CD14 mAb revealed immunoreactivity with a 50–55‐kd protein, a size consistent with sCD14. Evidence of endothelial cell‐associated CD14 was not detected using an immunofluorescence technique on cell preparations, nor by Northern blot analysis. These results indicate the existence of sCD14 in bovine serum and that soluble bovine serum factors including sCD14 and LBP facilitate presentation of LPS to receptive cells.

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor α (TNF‐α) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion‐exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 μg/ml NBS, 192 μg/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than1 whole NBS and 300 times more potent than whole FBS. We similarly found that TNF‐α secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF‐α secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 μg/ml NBS, 320.0 μg/ml FBS, and 3.2 μg/ml fraction 2 serum protein induced near‐maximal TNF‐α secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 μg/ml) inhibited tissue factor expression and TNF‐α secretion by the macrophages. J. Leukoc. Biol. 55: 483–488; 1994.

Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages

Alveolar macrophages play a central role in host defense in the lower respiratory tract. Production of the reactive intermediate nitric oxide (NO), via expression of inducible nitric oxide synthase (iNOS) is an important microbicidal effector mechanism possessed by macrophages. In this study, cytokine regulation of NO production by bovine alveolar macrophages (bAM) was evaluated. Bovine alveolar macrophages were exposed to one or more of the following: recombinant human (rh) and recombinant bovine (rb) IFN gamma, rh- and rbIL-1 beta, rbGM-CSF, rhTNF alpha, rhIL-4, endotoxin (LPS), fetal bovine serum (FBS), mitogen-stimulated bovine splenic supernatant (SS), and purified human TGF beta-1. LPS alone, or in combination with SS, rbIFN gamma, or rbIL-1 beta stimulated production of NO in a time and dose dependent fashion. Recombinant bovine IFN gamma, rbIL-1 beta, and rhTNF alpha in combination produced maximal stimulation which was not further enhanced by LPS. Recombinant human IFN gamma, rhIL-1 beta, and rbGM-CSF had minimal effect either as single stimuli, or in combination with LPS, rbIFN gamma, rbIL-1 beta, or rhTNF alpha. Nitric oxide production was inhibited by rhIL-4, and the L-arginine analogue antagonists of iNOS, N-G-monomethyl-L-arginine (NGMMA) and aminoguanidine (AG). Purified human TGF beta-1 did not inhibit NO production. Messenger RNA for iNOS was maximally expressed by 8 h and remained detectable for at least 48 h. Expression of iNOS mRNA induced by cytokines and LPS varied with strength of the stimulus as determined by nitrite production in culture supernatant.

Pulmonary Carcinoma with Metastases in a Moluccan Cockatoo (Cacatua moluccensis)

Abstract Pulmonary carcinoma was diagnosed in a 14-year-old male Moluccan cockatoo (Cacatua moluccensis) presented for sudden onset of ataxia of the pelvic limbs lasting several hours. On physical examination, the cockatoo had paraparesis of both legs and a reduction in pectoral muscle mass. The bird responded to initial supportive therapy; however, its clinical condition deteriorated over a 3-week period. Histopathologic examination revealed pulmonary carcinoma with intrapulmonary metastases as well as metastases to the vertebral column and right humerus.

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.

Characterization of Uptake of 2-Deoxy-2-[18F] Fluoro-D-Glucose by Fungal-Associated Inflammation: The Standardized Uptake Value is Greater for Lesions of Blastomycosis than for Lymphoma in Dogs with Naturally Occurring Disease

PURPOSE Based on limited reports, fungal lesions can have remarkably high intensity uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) on positron emission tomography (PET) images. The purpose of this investigation was to compare the standardized uptake value (SUV) of naturally occurring lesions of blastomycosis with the SUV of naturally occurring lymphoma in a series of dogs. PROCEDURES Five dogs with naturally occurring blastomycosis and three dogs with lymphoma underwent whole-body FDG-PET prior to receiving any treatment for their disease. RESULTS The (mean +/- SD) SUV for 13 blastomycosis lesions was 7.7 +/- 2.0 versus a mean for 17 lymphomas of 4.8 +/- 1.8. These values were significantly different (P = 0.0537). There was overlap between the SUV of Blastomyces-associated lesions versus lymphomas, but a cut-off SUV of 7.0 was 100% specific for Blastomyces lesions. Numerous sites of disease were detected on the FDG-PET images that were not detected clinically. CONCLUSIONS FDG-PET is useful for determining the extent of disease in dogs with blastomycosis. The SUV for Blastomyces-associated lesions are as high or higher than for malignant lymphoma. Due to the similarities in canine and human blastomycosis and lymphomas, similar results would be predicted in human patients. In regions where blastomycosis is endemic, Blastomyces granulomas should be considered a differential diagnosis for lesions with high intensity uptake of FDG.

Signal transduction pathways of bacterial lipopolysaccharide-stimulated bovine vascular endothelial cells

Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS) —stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00μM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.

Purification of lipopolysaccharide-binding protein from bovine serum

Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transen‐dothelial migration of neonatal neutrophils (N‐PMNs) and adult bovine peripheral‐blood PMNs (A‐PMNs) in vitro using monolayers of endothelium and a two‐chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0‐μm‐diameter pores. 51Cr‐labeled PMNs were added to the upper chamber, with or without an anti‐CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin‐8 (rhIL‐8; 75 ng/ml), rhC5a (10‐7 M), and zymosan‐activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL‐8, greater numbers (P < .01) of N‐PMNs (24.70 ± 5.95%) than of A‐PMNs (15.77 ± 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N‐PMNs and A‐PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti‐CD18 monoclonal antibody significantly decreased migration (P < .01) of both N‐PMNs and A‐PMNs to low levels when IL‐8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL‐8, rhC5a, ZAS). J. Leukoc. Biol. 55: 43–49; 1994.