Overexpression of c-Ras in hyperplasia and adenomas of the feline thyroid gland: an immunohistochemical analysis of 34 cases.

Formalin-fixed, paraffin-embedded thyroid glands from 18 cats diagnosed with hyperthyroidism were evaluated immunohistochemically for overexpression of the products of oncogenes c-ras and bcl2 and the tumor suppressor gene p53. Fourteen thyroid glands from euthyroid cats without histologically detectable thyroid lesions were examined similarly as controls. Results from these investigations showed that all cases of nodular follicular hyperplasia/adenomas stained positively for overexpression of c-Ras protein using a mouse monoclonal anti-human pan-Ras antibody. The most intensely positively staining regions were in luminal cells surrounding abortive follicles. Subjacent thyroid and parathyroid glands from euthyroid cats did not stain immunohistochemically for pan-Ras. There was no detectable staining for either Bc12 or p53 in any of the cats. These results indicated that overexpression of c-ras was highly associated with areas of nodular follicular hyperplasia/adenomas of feline thyroid glands, and mutations in this oncogene may play a role in the etiopathogenesis of hyperthyroidism in cats.

Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages.

OBJECTIVE: To investigate receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by bacterial lipopolysaccharide (LPS), using tissue factor (TF) expression as the measurable functional endpoint. SAMPLE POPULATION: Pulmonary AM harvested from 1- to 4-month-old male Holstein calves. PROCEDURE: Alveolar macrophages, acquired by use of volume-controlled bronchopulmonary lavage, were treated with CD14 monoclonal antibody (20 microg/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 microM chelerythrin, 100 microM H-7, or 50 nM staurosporin), then were stimulated with LPS alone (0.01, 0.10, 1.0, 10.0 microg/ml) or LPS (0.25, 0.5, 1.0 ng/ml) in combination with concentrated bovine serum fraction 2 (500 ng/ml). Tissue factor expression was quantified by use of a colorimetric assay. Changes in intracellular Ca2+ concentration and pH were monitored, using Ca2+- and pH-sensitive fluorescent dyes, with changes in fluorescent intensity after incubation with LPS measured by spectrophotometry. RESULTS: Treatment of AM with a CD14 monoclonal antibody caused profound inhibition of TF expression (P < 0.0001) after stimulation by LPS combined with bovine serum fraction 2. Pertussis toxin had a significant (P < 0.0319) inhibitory effect on TF expression when cells were stimulated by LPS alone. Treatment with all 3 PKC inhibitors caused marked reduction in TF expression of cells stimulated with LPS alone or with phorbol myristate acetate. Stimulation of cells by LPS failed to mobilize intracellular Ca2+ stores or to alter cytosolic pH. CONCLUSION: LPS combined with serum factors binds to CD14 on the surface of AM, and PKC is an important signaling kinase in the pathway utilized by LPS, resulting in enhanced TF expression; a pertussis toxin-sensitive G protein is involved in the signaling pathway utilized by LPS alone; and mobilization of Ca2+ does not have a role in the signal transduction pathway utilized by LPS nor does LPS affect cytosolic pH of AM.

Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transendothelial 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-micron-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).

Differential binding of bacterial lipopolysaccharide to bovine peripheral-blood leukocytes.

The response of mammalian monocytes and macrophages to bacterial lipopolysaccharide (LPS) may be influenced by serum factors that increase or decrease the propensity of LPS to bind to cell surfaces. We used fluorescence flow cytometric analysis to investigate the capability of bovine peripheral-blood leukocytes to bind LPS in the presence or absence of bovine serum. At all concentrations of FITC-LPS tested (LPS from E. coli 0111:B4; 10 ng/ml, 100 ng/ml, 1 micrograms/ml), monocytes, lymphocytes, and granulocytes bound more FITC-LPS in the presence of 10% bovine serum than in serum-free conditions (P < 0.01). At the intermediate concentration tested (100 ng/ml), monocytes displayed a relative fluorescence intensity (RFI) of 2.27 +/- 1.24 units without serum and 17.48 +/- 8.05 with 10% serum. Values for granulocytes were similar to those of monocytes, 3.55 +/- 1.31 without and 19.24 +/- 6.93 with serum, and values for lymphocytes were 1.89 +/- 0.47 RFI units without serum and 6.27 +/- 2.61 RFI units with serum. At 10 ng/ml and 1 microgram/ml FITC-LPS the RFI of monocytes and granulocytes were also similar and not significantly different, and both bound significantly more LPS than lymphocytes (P < 0.01). When 100 ng/ml FITC-LPS was coincubated with leukocytes, 10% serum, and a 100-fold excess of unlabeled LPS, the amount of FITC-LPS bound to monocytes was reduced from 23.99 to 7.23 RFI units (P < 0.01), reduced from 22.00 to 7.30 RFI units with granulocytes (P < 0.05), and reduced from 7.51 to 2.29 RFI units (P < 0.10) with lymphocytes. These data demonstrate that factors in bovine serum significantly amplify the association of LPS with peripheral-blood leukocytes and that increased binding of LPS is greatest with monocytes and granulocytes.

Comparative actin polymerization in neonatal and adult bovine neutrophils in vitro.

Neutrophils rely on active reorganization of the cytoskeleton during movement, and functional deficiencies in the cytoskeletal elements may result in impaired neutrophil-mediated host defense. We have compared and quantitated actin polymerization in neonatal (< or = 48 h old) and adult bovine peripheral-blood polymorphonuclear leukocytes (PMN) using fluorescence flow cytometry. Baseline filamentous actin (F-actin) content of neonatal and adult PMN at time zero differed slightly but were not statistically different (p > 0.05). F-actin content of recombinant human C5a (10(-7) M)-stimulated neonatal PMN increased rapidly within 10 s of stimulation to 59.0% over baseline, then declined. F-actin in adult recombinant human C5a-stimulated PMN continued to increase for 30 s and was elevated 87.3% over baseline before subsequently declining. When stimulated with zymosan-activated bovine serum (10%), neonatal (120.7% increase) and adult PMN (115.1% increase) had similar profiles with no significant differences, and both groups reached peak F-actin levels at 30 s after stimulation. Neonatal PMN stimulated with platelet-activating factor (10(-6) M) attained peak F-actin values at 10 s (72.0% increase over baseline), but actin rapidly depolymerized by 30 s poststimulation (reduced to 29.0% increase). Adult PMN stimulated by platelet-activating factor also attained peak values by 10 s (97.6% increase over baseline), but in contrast to neonatal PMN the F-actin remained elevated at 30 s in the adult PMN (still increased 79.5%; p < 0.0.5 compared to neonatal F-actin).(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase-C activity in phorbol myristate acetate-stimulated neutrophils from newborn and adult cattle.

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2-) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This compound activates the O(2-)-generating enzyme of bovine neutrophils through a pathway involving protein kinase C (PKC). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme PKC in nonstimulated and PMA-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of PMA (0, 10, 100, and 500 ng/ml) for 3 minutes, and PKC activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most PKC activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of PKC in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated PKC in nonstimulated cells did not have such dependence. Significant differences in PKC activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with PMA caused redistribution of PKC activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic PKC activity and increase in membrane-associated PKC activity. Similar to that in nonstimulated cells, PKC activity in cytosolic fractions from PMA-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas PKC activity in the membrane did not have such requirement.(ABSTRACT TRUNCATED AT 250 WORDS)

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 ruffles) 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.

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.

Expression and kinetics of induced procoagulant activity in bovine pulmonary alveolar macrophages.

Leukocytes, especially macrophages, are important cellular mediators of fibrin deposition and removal at tissue sites of inflammation. Pulmonary fibrin deposition is a prominent feature of bovine acute lung injury; therefore, we studied the resting and stimulated procoagulant responses of bovine pulmonary alveolar macrophages (PAM) and peripheral blood neutrophils (PMN). Freshly isolated normal PAM and PMN expressed negligible procoagulant activity. PAM stimulated with endotoxin lipopolysaccharide (LPS), 4 beta-phorbol 12-myristate 13-acetate (PMA) and bovine recombinant interleukin-1 beta (rBIL-1 beta) exhibited protein synthesis- and dose-dependent enhancement of procoagulant activity in 8-h cultures. Bovine recombinant granulocyte macrophage-colony stimulating factor (rBGM-CSF) and recombinant human gamma-interferon (rHIFN-gamma) did not induce procoagulant activity. The kinetics of LPS- and PMA-enhanced PAM procoagulant activity differed: LPS-induced enhancement developed earlier and more rapidly than PMA-induced enhancement. Pasteurella haemolytica LPS was more potent than Escherichia coli LPS in enhancing PAM procoagulant activity, while dexamethasone decreased both baseline and LPS- or PMA-stimulated activity by approximately 50%. PAM procoagulant activity resulted from tissue factor expression. Bovine PMN produced negligible procoagulant activity when stimulated, and are thus unlikely to be major contributors to procoagulant activity in bovine lung. Activity inhibitory to bovine tissue factor was present in both calf and adult sera, and was partly dependent on the presence of factor X for activity. Rapid induction of bovine PAM procoagulant activity by inflammatory mediators, and subsequent resistance to degradation, may thus combine to promote an alveolar microenvironment permissive to fibrin deposition in bovine acute lung injury.

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.

Membrane NADPH oxidase activity and cell size in bovine neonatal and adult neutrophils.

Neutrophils (PMN) from newborn calves generate significantly less superoxide anion (O2-) than do their adult counterparts after stimulation with direct protein kinase C agonists. To better understand this observation, we compared the activity and kinetics of NADPH oxidase in membrane fractions from phorbol 12-myristate 13-acetate-stimulated adult and newborn PMN. After phorbol 12-myristate 13-acetate stimulation, PMN were sonicated and the membranes assayed for O2- production with increasing concentrations of NADPH. O2- production was calculated 1 and 2 min after the beginning of the reaction. At all concentrations of NADPH used, there was no difference (p greater than 0.05) in O2- production between adult (n = 8) and newborn (n = 9) PMN membrane preparations. Enzyme kinetics calculations revealed no differences (p greater than 0.05) between age groups in Km and Vmax or in the velocity of the reactions. Determination of the protein content in the membrane pellet, however, showed that adult PMN yielded significantly (p less than 0.01) higher amounts of protein (2.82 +/- 0.14 mg/mL) than did newborn PMN (1.78 +/- 0.07 mg/mL). This difference could be partly attributed to cell size; flow cytometric analysis showed that newborn PMN had a significantly (p less than 0.01) smaller diameter (10.94 +/- 0.07 microns) than did adult PMN (11.65 +/- 0.06 microns), and calculated cell volume and surface area were also both significantly less (p less than 0.01) in newborn PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal-antibody-defined adhesion-related antigen on bovine neutrophils is required for neutrophil aggregation.

Surface adhesion molecules present on human leukocytes are known to regulate certain adhesion-related events, such as adhesion to endothelium, extravasation, and aggregation. We have used a mouse anti-human monoclonal antibody designated 60.3 (MAb 60.3) and indirect immunofluorescence technique to identify an antigen on bovine neutrophils (PMNs). MAb 60.3 bound to resting and stimulated bovine PMN in a surface-oriented pattern. Immunofluorescence flow cytometric analysis indicated that warming the PMNs from 4 degrees C to 37 degrees C slightly increased (13.9%) expression of the antigen recognized by MAb 60.3. Zymosan-activated serum (ZAS, 10%) increased antigen expression by 12.4% over those PMNs in buffer alone, and phorbol 12-myristate 13-acetate (PMA; 100 ng/ml) by 65.6%. Bacterial lipopolysaccharide (LPS; 1 micrograms/ml) from E. coli 0111:B4 did not enhance antigen expression. The functional nature of this antigen was demonstrated by use of MAb 60.3 and PMN aggregation. Preincubation of bovine PMN with MAb 60.3 for 10 min resulted in nearly complete inhibition of PMN-PMN aggregation upon subsequent stimulation with PMA (100 ng/ml); preincubation with a control antibody did not inhibit aggregation. These results indicate that bovine PMNs possess surface molecule(s) that may function in adhesion-related events, and surface expression may be enhanced by PMN stimulation.

Calcium mobilization in C5a-stimulated adult and newborn bovine neutrophils.

Calcium (Ca2+) is an important second messenger central to many neutrophil (PMN) functional activities. Impaired Ca2+ mobilization in newborn PMNs following membrane perturbation could be one of the mechanisms underlying observed abnormalities in neonatal PMN function. To compare Ca2+ mobilization in bovine newborn and adult PMNs, cytosolic Ca2+ responses after stimulation with recombinant human C5a (rHC5a) were measured. PMNs from normal newborn calves (N = 6) and adult cows (N = 5) were loaded with fura-2/AM for 60 min at room temperature and the fluorescence changes monitored following stimulation with 0.1, 1, 10, or 50 nM rHC5a in Ca2(+)-containing buffer. Resting levels of Ca2+ in both newborn (54.6 +/- 1.9 nM) and adult (57.3 +/- 1.8 nM) bovine PMNs were comparable. After stimulation with rHC5a, a rapid rise of cytosolic Ca2+ was observed, which peaked within 20 sec and was followed by a sustained phase of elevated Ca2+ lasting up to 20 min. There were no significant differences (P greater than 0.05) in peak levels of cytosolic Ca2+ obtained by newborn and adult PMNs at 0.1, 10, and 50 nM rHC5a. At 1 nM rHC5a, newborn PMNs reached significantly (P less than 0.05) higher levels of cytosolic Ca2+ (217.9 +/- 21.7 nM) than did adult PMNs (158.7 +/- 7.9 nM). At 1 nM rHC5a, newborn PMNs also sustained higher levels of cytosolic Ca2+ for 3 min following the peak. At all concentrations of rHC5a tested, the time required to reach the peak and the duration of the peak were comparable in both populations. In the absence of extracellular Ca2+ (Ca2(+)-free buffer with 1 mM EGTA), resting levels of cytosolic Ca2+ were lower in both newborn (33.3 +/- 2.9 nM) and adult PMNs (27.9 +/- 2.4 nM) and the magnitude of the peak response to rHC5a was diminished at all concentrations of agonist. Additionally, in the absence of extracellular Ca2+, the return to basal cytosolic Ca2+ levels occurred rapidly and the sustained phase of increased cytosolic Ca2+ seen with rHC5a-stimulated PMNs in Ca2(+)-containing buffer was virtually eliminated. These results indicate that bovine PMNs respond well to rHC5a, that stimulated newborn bovine PMNs can mobilize Ca2+ as efficiently as adult PMNs, and that the sustained cytosolic Ca2+ response to rHC5a in both age groups requires both release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+. Such data suggest that observed functional deficits in newborn bovine PMNs are probably not related to improper mobilization of Ca2+ following stimulation.

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils.

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67-85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P less than 0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8 +/- 3.2% of newborn PMNs exhibited such Cf-induced shape changes by 120 sec. which was significantly greater (P less than 0.01) than the maximum stimulation (67.7 +/- 4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O2-) generation as a measure of respiratory burst activity after incubation of newborn and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O2- (9.3 +/- 0.5 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (12.4 +/- 0.3 nmol O2-/10(6) PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O2- (7.7 +/- 0.4 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (5.5 +/- 0.5 nmol O2-/10(6) PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulus-specific O2- generation and Cf-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.

Influence of acute pulmonary interstitial inflammation on kinetics of phagocytosis by alveolar macrophages.

The pulmonary alveolar macrophage (PAM) is central to lung cellular defenses and is a potential participant in lung injury, but little is known about the influence of the nature and anatomic pattern of acute lung injury on PAM function. To assess the relationship between ongoing pulmonary inflammation and PAM function, we evaluated PAM phagocytic kinetics in a model system of experimental interstitial adjuvant pneumonitis (EIAP) in calves. PAMs were obtained from lung one and seven days postinduction (dpi) of EIAP. Lesions were typical of EIAP, characterized by acute multifocal to coalescing exudative interstitial pneumonitis at 1 dpi, which progressed to granulomatous interstitial pneumonitis by 7 dpi. The total recoverable lung cells and percentage of neutrophils (PMNs) were elevated (P less than 0.01) from animals with EIAP at both 1 and 7 dpi, and there was a four-fold increase (P less than 0.01) in the PAM yield by 7 dpi. Linear regression equations revealed that a larger proportion of control PAMs were phagocytic than were PAMs from animals with EIAP. The mean initial phagocytic rates of PAM following acute lung injury were significantly elevated (P less than 0.05) over controls; this difference was concentration dependent and required a phagocytic bead stimulus concentration in excess of 12.5 x 10(6) beads/ml. PAMs from animals with EIAP had a greater maximum rate of phagocytosis (Vmax) and Km than control PAMs. PAMs from animals with EIAP had a slightly higher proportion of cells which phagocytosed multiple beads. Levels of beta-glucuronidase were elevated (P less than 0.02) in PAM from animals with EIAP at 7 dpi. The results document enhanced PAM phagocytic function in EIAP and differ from our previous experiments in which depressed PAM phagocytic indices were obtained in a model of virus-induced acute bronchiolitis and alveolitis. The functional activities of the PAMs thus appear to be modified by injury-specific events in the lung microenvironment which may, in part, reflect the nature and anatomic pattern of developing pulmonary inflammatory reactions.

Acute inflammatory lung injury retards pulmonary particle clearance.

The deposition, retention, and clearance of inhaled cobalt oxide particles from the lungs of calves with acute inflammatory lung injury induced by parainfluenza-3 virus (PI-3) were examined. Acute pulmonary inflammation was induced by nebulization with 10(9) TCID50 of PI-3 virus on two successive days, and animals were subsequently exposed to an aerosol of particulate cobalt oxide (geometric mean diameter 0.54-0.65 microns) seven days post-virus infection (dpi). Pulmonary lesions at 7 dpi were typical of PI-3 pneumonitis and were characterized by patchy aveolitis and bronchiolitis with accumulations of neutrophils, macrophages, fibrin, and inflammatory debris. Calves were killed at 0, 7, and 21 days post-aerosol exposure (dpe) to evaluate particle clearance and retention by assay for cobalt in lung tissues, bronchoalveolar washings, and tracheobronchial lymph nodes. Control animals had a typically biphasic clearance pattern with rapid initial clearance of 50% of the initial lung burden (ILB) by 7 dpe followed by slower prolonged clearance. Clearance was significantly retarded (P less than 0.05) in calves with viral-induced acute inflammatory lung injury; 90% of the ILB was retained at 7 dpe. Essentially all particles recoverable by bronchoalveolar lavage were intracellular within pulmonary alveolar macrophages (PAM) in both experimental and control groups, but interstitial sequestration of particles within PAM was commonly observed only in the lungs of calves with viral pneumonitis. Pneumonic calves also exhibited retarded translocation of particles to regional lymph nodes. The results document impaired particulate clearance from acutely inflamed lungs, and implicate decreased mucociliary clearance and interstitial sequestration within PAM as the major contributing factors. These functional alterations would be expected to enhance the progression of virus-induced acute pulmonary inflammatory injury.

Ontogeny of inflammatory cell responsiveness: superoxide anion generation by phorbol ester-stimulated fetal, neonatal, and adult bovine neutrophils.

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7-10 days of age), fetal PMNs (210-220 days gestational age), and adult PMNs to generate superoxide anion (O2-) as an indicator of respiratory burst activity. Citrated blood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O2- generation was measured as the superoxide dismutase-inhibitable (10 micrograms/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 micrograms/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37 degrees C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O2- generation was measured for 5 min after the initial lag period and the total nanomoles of O2- generated calculated using the extinction coefficient for ferricytochrome c. Newborn PMNs (N = 10) generated significantly less O2- (5.7 +/- 0.8 nmol O2-/10(6) cells/5 min, P less than 0.01) than did adult PMNs (N = 14) (9.6 +/- 2.1 nmol O2-/10(6) cells/5 min) or fetal PMNs (N = 4) (10.7 +/- 0.7 nmol O2-/10(6) cells/5 min). PMNs from 7- to 10-day-old calves (N = 9) generated almost identical amounts of O2- as newborn PMNs (5.7 +/- 1.6 nmol O2-/10(6) cells/5 min). There was no difference in measured lag time period between newborn and adult PMNs, but fetal PMNs had significantly reduced (P less than 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O2- in response to PMA stimulation, which persists for at least 7-10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O2- as well as adult PMNs.