Search Results

You are looking at 1 - 10 of 19 items for

  • Author or Editor: Max J. Paape x
  • Refine by Access: All Content x
Clear All Modify Search

Summary

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (cl) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong cl responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the cl reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced cl reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To investigate the role of extracellular Ca2+ and Mg2+ in aggregated IgG (algG)-mediated cellular activation, and to determine how algG-induced activation is coupled to Ca2+ homeostasis in bovine neutrophils.

Sample Population

4 clinically normal, lactating Holstein cows, in their second lactation, which ranged between 60 and 150 days.

Procedure

algG was prepared by heating bovine igG, and C5a was obtained by activating fetal bovine serum with zymosan. Luminol-amplified chemiluminescence (CL) of isolated neutrophils was measured in the presence of algG or phorbol 12-myristate 13-acetate (PMA). The reaction mixture contained either Hanks’ balanced salt solution or Ca2+- and Mg2+-free Hanks’ balanced salt solution. Binding of algG to neutrophils was measured by flow cytometry after incubation with fluorescein isothiocyanate-conjugated second antibody. Intracellular-free concentration [Ca2+] was measured in a fluorescence spectrofluorometer after incubation of neutrophils, loaded with the fluorescent dye fura-2 acetoxymethyl ester, with either algG or C5a.

Results

In a Ca2+- and Mg2+-containing reaction mixture, algG induced strong CL responses, whereas removal of extracellular divalent cations almost abolished the respiratory burst activity. The CL emission on stimulation with PMA was independent of extracellular Ca2+ and Mg2+. Examination of cells by flow cytometry after incubation with algG indicated that the binding of algG was identical in the presence and absence of extracellular Ca2+ and Mg2+. No increase in [Ca2+] was seen in fura-2 acetoxymethylester-loaded neutrophils after stimulation with algG. C5a induced a typical transient increase in [Ca2+].

Conclusions

algG-induced activation of bovine neutrophils is highly dependent on presence of extracellular divalent cations. This dependency is not caused by the need of divalent cations for binding of algG by neutrophils or because the influx of Ca2+ from the extracellular space is an integral component of algG-mediated activation pathway. Because need for extracellular Ca2+ and Mg2+ could be partially circumvented by pretreating neutrophils with PMA, it is possible that this activation pathway may involve a protein kinase C, which is not directly coupled to receptors for algG. (Am J Vet Res 1996;57:1312-1316)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers.

Sample Population—Polymorphonuclear neutrophils from 3 lactating cows.

Procedure—Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino- sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots.

Results—Lipopolysaccharide, PMA, and ≤ 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, ≥ 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MACT cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or ≥ 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner.

Conclusions and Clinical Relevance—Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability. (Am J Vet Res 2001;62:286–293)

Full access
in American Journal of Veterinary Research

Abstract

Objectives—To evaluate effects of proinflammatory mediators on phagocytosis and killing of Staphylococcus aureus, the oxidative burst (OB), and expression of receptors for opsonins by bovine neutrophils.

Sample Population—Neutrophils from 10 cattle.

Procedure—Neutrophils were primed with recombinant bovine tumor necrosis factor-α (TNF-α) or the des-arginine derivative of bovine C5a (C5adesArg) and mixed with S aureus. Phagocytosis and OB were measured by use of flow cytometry. Rate of phagocytosis and intracellular killing were evaluated. Expression of receptors for immunoglobulins and the C3bi fragment of complement were estimated by use of flow cytometry.

Results—Priming of neutrophils by TNF-α improved phagocytosis of S aureus with a concentrationdependent effect. Phagocytosis of preopsonized washed bacteria was increased by activation of neutrophils with C5adesArg. Phagocytosis was optimal when neutrophils primed with TNF-α were activated with C5adesArg. The OB of phagocytizing neutrophils was highest when TNF-α and C5adesArg were used in combination. Bactericidal activity of neutrophils was stimulated by priming with TNF-α or C5adesArg. Binding of bovine IgM or IgG2 to bovine neutrophils was not stimulated by TNF-α, C5adesArg, or both, and aggregated IgG1 did not bind to neutrophils regardless of their activation state. Both TNF-α and C5adesArg increased expression of β2 integrins (CD18), with the highest expression when they were used in combination.

Conclusions and Clinical Relevance—The mediators TNF-α and C5adesArg stimulated phagocytic killing by neutrophils and potentiated each other when used at suboptimal concentrations. Bovine neutrophils have enhanced bactericidal activities at inflammatory sites when TNF-α, C5adesArg, or both are produced locally. (Am J Vet Res 2000;61:951–959)

Full access
in American Journal of Veterinary Research

Summary

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3α-6α-diphenylgly-couril, and proteins were separated by sds-page and autoradiographed.

Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5′-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for β-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 μg/ 109 bovine blood neutrophils. The sds-page of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.

Free access
in American Journal of Veterinary Research

Summary

Polymorphonuclear neutrophils (pmn) from 4 cows were preincubated (30 minutes, 37 C) in either actinomycin D (100 μg/ml) or puromycin (10 μg/ ml), inhibitors of mRNA transcription and protein translation, or in medium 199. The pmn were incubated for a further 4.5 hours in medium containing 100 U of recombinant bovine interferon-γ (rbolfn-γ). The pmn were then incubated with bovine IgG1, IgG2, IgM, or aggregated IgG (algG; 4 C, 12 hours) for flow cytometric analysis, using fluoresceinated is-otype-specific antibody. The percentage of pmn binding the ligand and the logarithmic mean fluorescent channel (lmfc), an indicator of the amount of receptor (R) expression, were recorded. Competitive inhibition of ligand binding was measured by incubating pmn with fluoresceinated IgG2 in the presence or absence of 100-fold excess of IgG1, IgG2, and aIgG. Activation with rboIfn-γ induced a 4.5-fold increase in binding of IgG1 and a fivefold increase in lmfc for IgG2. These increases were inhibited by actinomycin D and puromycin. Percentage of pmn binding aIgG decreased after activation by rboIfn-γ. Interferon-γ treatment did not affect binding or lmfc of IgM. However, binding of IgM was reduced by treatment with actinomycin D. Binding of fluoresceinated IgG2 was inhibited by unlabeled IgG1, IgG2, and aIgG. Results indicate that bovine pmn Fc receptors (FcR) for IgG1 and IgG2 were rboIfn-γ inducible, that induction required de novo transcription and translation, that a heterogeneous population of FcR exist on bovine pmn, and that IgG1 and IgG2 share a common FcR. Further, bovine pmn are capable of gene activation and are responsive to changes in their environment, thus being amenable to modulation for effective pathogen destruction.

Free access
in American Journal of Veterinary Research

Summary

Ten healthy first- and second-lactation Holstein cows were observed from 1 week before to 1 week after calving and at postpartum day 30 to determine polymorphonuclear neutrophil (pmn) functional variation and immunoglobulin binding profiles. Blood and mammary pmn were obtained 3 times weekly and within 24 hours of calving. Functional traits measured included phagocytosis of Staphylococcus aureus and in vitro chemotaxis through micropore filters in a Boyden chamber. Additionally, pmn were evaluated for endogenous binding of IgG1, IgG2, IgA, and IgM before and after in vitro chemotaxis. Exogenous binding of the same isotypes was determined after incubation in pooled colostrum, purified immunoglobulin, and pooled sera. Phagocytosis results indicated a significant and transient increase in percentage of milk pmn with associated, rather than phagocytosed, bacteria for 1 week after calving. Blood pmn phagocytosis was not significantly different during this period. Though total chemotaxis was essentially unchanged, the percentage of pmn that were unable to complete migration increased substantially on the day of calving, an effect that disappeared by postpartum day 4. A significant (P < 0.01) positive correlation (r = 0.29) between percentage of pmn migrating completely through the micropore filter and percentage of blood pmn with associated bacteria was observed. Changes were not observed in endogenous immunoglobulin binding, with the exception of a peak in relative fluorescence intensity for IgG1 on the day of calving; this disappeared within 2 days after calving. Correlations between relative intensities of IgG2 and IgM, and percentage of mammary neutrophils phagocytosing were 0.37 and 0.70. Exogenous binding of antibody to blood neutrophils before chemotaxis was generally accomplished most effectively by pooled colostrum, whereas use of pooled sera markedly reduced binding and percentage and intensity of IgM in all cases. Binding of all isotypes was slightly higher before than after calving. Incubation of blood neutrophils in isotypes G1, G2, A, and M after chemotaxis yielded lower immunoglobulin binding among all isotypes, particularly IgM. Fluctuations in neutrophil function were observed immediately around parturition, and these changes correlated strongly with endogenous immunoglobulin-binding profiles.

Free access
in American Journal of Veterinary Research

Summary

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surrace antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (mab) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by wholecell elisa. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by mab was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I.

The flow cytometric method was proven to be more sensitive and rapid than elisa to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 × 105 neutrophils and 1 µg of fluorescein-labeled F(ab′)2/assay as the second antibody. The optimal conditions for hybridoma screening by elisa were neutrophil concentration of 2.5 × 105/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2′-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromogenic substrate. Tissue culture plates as solid support and 3,3′, 5,5′- tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive.

Panel mab reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter mab reactivity.

Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with Monoclonal antibody S7G8 identified a 65-kd protein and mab S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, mab S7G8 and S8G10 are comparable to human CD15, CDl6, and CD64 molecules. The mab generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

Free access
in American Journal of Veterinary Research

SUMMARY

A flow cytometric method was developed to perform differential leukocyte counts on bovine blood. Blood specimens from 50 healthy Holstein cows were analyzed by use of a flow cytometer. The method entailed diluting blood with phosphate-buffered, hypotonic saline solution containing acridine orange, and performing a step-wise, 3-parameter analysis on the bases of cell size, cellular granularity, and granulocyte fluorescence. Initially, proportions of monocytes, granulocytes, and lymphocytes were determined by creating appropriate windows on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Eosinophils were resolved by analysis of granulocytes as dot plots of logarithms of green vs red fluorescence ascribed to acridine orange. Proportions of eosinophils and neutrophils were computed from data so generated. Microclumps of platelets spuriously affected counts of some granulocytes, particularly eosinophils.

Differential leukocyte counts determined by flow cytometry generally compared favorably with those obtained by use of the conventional microscopic method, using Wright-stained blood films. Mean neutrophil and eosinophil counts determined by the 2 methods did not differ significantly, but lymphocyte counts determined by flow cytometry were significantly higher than those determined by microscopy (P < 0.01). Correlation coefficients for counts of neutrophils, eosinophils, and lymphocytes determined by the 2 methods ranged from 0.519 to 0.833. Correlation between monocyte counts was low (r = 0.147), although mean monocyte counts determined by the 2 methods did not differ significantly. Total leukocyte counts determined by flow cytometry were significantly lower (P < 0.01) than counts determined by use of an automated cell counter; correlation between the 2 counts was low (r = 0.350).

Free access
in American Journal of Veterinary Research

SUMMARY

Sixty-three drugs, belonging to 10 chemical classes, were tested in vitro to determine effects on phagocytosis of 32P-labeled Staphylococcus aureus by neutrophils isolated from milk. Within each class, the number of antibiotics tested were: nonsteroidal anti-inflammatory drugs (nsaid; 8), peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), β-lactam antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and antibacterial quinolones (8). Percentage of phagocytosis was determined after incubating (2 hours at 37 C) 12.5 × 106 viable neutrophils, 200 × 106 32P-labeled S aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics tested were 1,000, 500, and 10 μg/ml of incubation media. When compared with nonantibiotic controls at the highest drug concentration, the nsaid acetylsalicylic acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively, and benzydamine, indomethacin, phenylbutazone, ibuprofen, and acetominophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0, and 18.2%, respectively. The peptolids novobiocin and pristinamycin decreased phagocytosis 24.5 and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and gentamicin decreased phagocytosis 21.1, 15.4, and 19.2%, respectively. For the tetracyclines and fusidic acid, minocycline and doxycycline decreased phagocytosis 39.8 and 54.2%, respectively. The β-lactam antibiotics carfecillin, cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8, and 23.8%, respectively. The secretolytic agent, bromhexin, increased phagocytosis 10.8%. These data indicate that the potential for enhanced phagocytosis exists through use of some nsaid, and for depressed phagocytosis through use of aminoglycosides, peptolids, tetracyclines, and beta-lactams, as well as certain other nsaid.

Free access
in American Journal of Veterinary Research