David O. Slauson

Alveolar macrophage phagocytic kinetics following pulmonary parainfluenza-3 virus infection.

Qualitative and quantitative evaluations of the cellular components of bronchoalveolar washings of calves with experimental parainfluenza‐3 virus pneumonitis and control calves were made. Calves were exposed to 109 TCID50 of PI‐3 by intranasal aerosol exposure and bronchoalveolar cells obtained 7 days after infection by volume‐controlled bronchopulmonary lavage. Transient tachypnea and pyrexia occurred in all infected calves, and virus was recoverable at 7 days from nasal swabs and lung tissue. Pulmonary lesions were typical of viral pneumonitis, characterized by patchy alveolitis and bronchiolitis with accumulations of cells and inflammatory debris. The mean total lavage cell yield was elevated in the virus‐infected calves, and the percentage of neutrophils was elevated (P < 0.05). Increased numbers of pulmonary alveolar macrophages (PAM) were also recovered but the difference was not significant. Linear regression equations showed that a decreased proportion of PAM from virus‐infected animals were phagocytic. The mean initial phagocytic rates of macrophages from calves with viral pneumonitis were significantly decreased (P < 0.05) over controls. This difference was concentration dependent and required a phagocytic stimulus in excess of 12.5 × 106beads/ml. Studies of phagocytic kinetics showed that PAM from calves with viral pneumonitis had a lower Vmax than PAM from control calves, but that Km values were comparable. No differences in PAM β‐glucuronidase and acid phosphatase activity were observed. These results indicate depressed phagocytic function in PI‐3 virus‐inflamed lungs relative to controls. In concert with virus‐induced airway lesions, such in vivo depression of PAM phagocytic functions would be expected to depress pulmonary particulate clearance and lung defense mechanisms.

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.

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.

Neonatal tolerant immunity for vaccination against autoimmunity.

Autoimmunity arises when the immune system no longer tolerates self and precipitates lymphocyte reactivity against our own antigens. Although the developing T cell repertoire is constantly purging, self-recognition events do exist when such tight control is evaded and autoreactive lymphocytes escape the thymus (the sites of T cell development) and migrate to the periphery. Upon activation these autoreactive cells may exert aggressive behavior toward ones own tissues and organs leading to autoimmune disease. Multiple sclerosis, Rheumatoid arthritis, and type I diabetes are autoimmune diseases mediated by autoreactive T cells. A logical approach to prevent such autoimmunity would be to reprogram those lymphocytes to tolerate the self antigen. Injection of antigen at the neonatal stage promotes a state of tolerance such that successive encounter with antigen does not precipitate aggressive reactions. The mechanism underlying neonatal tolerance involves priming of T cells whose effector functions do not cause inflammatory reactions upon recognition of antigen but rather induce protective immunity. This form of tolerant immunity provides an attractive strategy for vaccination against autoimmunity. Herein, it is shown that neonatal exposure to a self-peptide-immunoglobulin chimera drives a tolerant immunity toward the self-peptide and protects against the autoimmune disease, experimental allergic encephalomyelitis.

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.

Increased adhesiveness of complement-stimulated neonatal calf neutrophils and its pharmacologic inhibition.

Several in vitro functions of neonatal neutrophils (N‐PMN) have been reported to be deficient and may be functionally related to the increased susceptibility of the newborn to infection. To evaluate an in vitro event corresponding to one of the early steps in the sequence of inflammation, we used zymosan‐activated plasma as a source of activated complement fragments (Cf) and measured adherence of normal and Cf‐stimulated bovine N‐PMN to columns of Sephadex G‐25. Adherence of control N‐PMN and adult PMN (A‐PMN) was comparable. When N‐PMN and A‐PMN were stimulated with a subaggregating dose of Cf, both responded with similar increases in adhesiveness. The stimulatory effect of Cf on N‐PMN adhesiveness could be inhibited by pre‐incubation of the N‐PMN with either steroidal (0.05 mM dexamethasone) or non‐steroidal (32 mM phenylbutazone) anti‐inflammatory drugs. Ultrastructural observations correlated well with the results of the adhesiveness assays, and morphometric evaluation revealed an increase in the sectional circumference of Cf‐stimulated N‐PMN. Control cells were round with few short cytoplasmic projections, whereas Cf‐stimulated cells exhibited marked shape irregularity, polarity, and prominent organelle‐free lamellipodia development. There was a highly significant (P < 0.001) increase in the measured circumference of Cf‐stimulated cells. Thus, N‐PMN were highly responsive to Cf stimulation, developed morphologic and functional changes indistinguishable from Cf‐stimulated A‐PMN, and were sensitive to pharmacologic inhibition.

Induction of procoagulant activity in virus infected bovine alveolar macrophages and the effect of lipopolysaccharide

Three viruses known to be associated with the bovine respiratory disease complex were evaluated in vitro for potential impact upon the procoagulant activity (PCA) of bovine alveolar macrophages (bAM). Cultures of bAM were inoculated with bovine parainfluenza virus Type 3 (PI-3), cytopathic bovine viral diarrhea virus (cpBVDV), non-cytopathic BVDV (ncpBVDV), or bovine herpes virus Type 1 (BHV-1) and incubated for several time periods (24, 48, 72, 96 h). BAM were then exposed to E. coli lipopolysaccharide (LPS), or LPS with bovine serum. The amount of PCA expressed was quantified using a chromogenic assay. Viral inoculation increased bAM expression of PCA (P < 0.01). The increase in PCA expression was larger at higher rates of viral inoculation (P < 0.01). LPS enhanced PCA expression by bAM at low rates of viral inoculation (P < 0.01). The effect of LPS-serum treatment was greater than the LPS alone (P < 0.01). At high rates of viral inoculation, LPS had no enhancing effect on PCA expression. The effect of LPS on virus inoculated bAM varied with virus type, rate of inoculation, and duration of virus exposure (P < 0.01). The results suggest that these four viruses initiate the production of PCA by bAM independently of LPS. In the field situation, an initial viral infection may induce fibrin deposition in the pulmonary alveoli prior to the establishment of a secondary gram negative bacterial infection.

Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide, serum proteins, and modulators of signal transduction

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gramnegative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055 ∶ B5, 10 ng/ml-1μg/ml) was a weak stimulus for generation of superoxide anion (O2−) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4–1.0% vol/vol, equivalent to 128–320μg protein/ml), O2− generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25–1.0μg/ ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O2− generation was observed. Stimulation of macrophages for generation of O2− either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor herbimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12μM), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5μg/ml protein). Essentially complete inhibition of O2− synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1μg/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256μg/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O2−. Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca++-dependent, but do not relay on G-protein-mediated signaling.

Stimulus-dependent actin polymerization in bovine neutrophils

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10−6 M), and rhC5a (10−7 M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane raffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.

Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro

Interleukin-6 (IL-6) is a pluripotent cytokine that may play a role in pulmonary defense against bacterial pathogens. We have quantitated the response of bovine alveolar macrophages (bAM) to bacterial lipopolysaccharide (LPS; E. coli 055: B5) in vitro using the IL-6 sensitive 7TD1 cell line. Bacteria LPS in the absence of serum induced IL-6 secretion from bAM (1 x 10(6) ml-1) over a range of LPS concentrations from 10 ng ml-1 to 10 micrograms ml-1. This resulted in IL-6 levels ranging from approximately 5 to over 200 U ml-1.IL-6 secretion by from approximately 5 to over 200 U ml-1.IL-6 secretion by LPS-stimulated bAM was increased by 24 h poststimulation, and continued to increase up to 72 h after stimulation. Fetal bovine serum (FBS, 1% vol/vol; 320 micrograms ml-1) enhanced IL-6 secretion from macrophages in the presence of LPS by approximately 10-fold compared with LPS alone. A bovine serum fraction (1 microgram ml-1 protein) prepared using ion-exchange chromatography also markedly enhanced IL-6 secretion versus LPS alone. The stimulatory effect of IL-6-like activity in the bAM supernatants was neutralized by an anti-human IL-6 polyclonal antibody. Northern blot analysis revealed increased IL-6 mRNA at 2 h poststimulation with LPS + FBS, peak levels at 4 h, and levels were decreased by 6 h poststimulation. Results suggest that IL-6 is secreted by bovine alveolar macrophages, and that bacterial LPS and serum components synergize to produce this response.