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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

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

Objective—To evaluate the effects of cis–urocanic acid (cis-UCA) on mammary gland (MG) inflammation and injury associated with Escherichia coli–induced mastitis in dairy cows.

Animals—12 lactating dairy cows (36 MGs).

Procedures—At 7-week intervals, a different MG in each cow was experimentally inoculated with E coli. At 6-hour intervals from 6 to 36 hours after inoculation, the inoculated MG in each cow was infused with 40 mL of saline (0.9% NaCl) solution, 12.5mM cis-UCA, or 25mM cis-UCA (4 cows/group); ultimately, each cow received each treatment. Immediately prior to and at various time points after inoculation and treatment, milk samples were collected. Bacterial CFUs, somatic cell counts (SCCs), N-acetyl-beta-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) activities, and concentrations of bovine serum albumin, tumor necrosis factor-α, and cis-UCA were quantified in each milk sample.

Results—Compared with findings in saline solution–treated MGs, NAGase and LDH activities in milk samples from cis-UCA–treated MGs were lower. Cis-UCA had no effect on milk SCCs and milk concentrations of bovine serum albumin and tumor necrosis factor-α. Furthermore, cis-UCA had no adverse effect on bacterial clearance; CFUs of E coli in MGs treated with saline solution or cis-UCA were equivalent.

Conclusions and Clinical Relevance—In cows, milk NAGase and LDH activities were both lower in E coli–infected MGs infused with cis-UCA than in those infused with saline solution, which suggests that cis-UCA reduced mastitis-associated tissue damage. Furthermore, these data indicated that therapeutic concentrations of cis-UCA in milk can be achieved via intramammary infusion.

Full access
in American Journal of Veterinary Research

SUMMARY

Objective

To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils.

Animals

16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources.

Procedure

Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils.

Results

MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes.

Conclusion

MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells. (Am J Vet Res 1997;58:1392–1401)

Free access
in American Journal of Veterinary Research