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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

Binding of endogenous and exogenous homologous IgG2 and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry. Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated. Before and after in vitro migration, neutrophils with endogenously bound IgG2 and IgM averaged 1 and 2% and 23 and 7%, respectively. Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively. Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively. Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively. When serum was used as a source of exogenous immunoglobulins, binding of total IgG after in vitro migration increased from 5% to 28% and of IgM from 4% to 20%. After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively. Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature. Endogenous IgG2 was unaffected by temperature; however, binding of IgM decreased from 23% at 4 C to 2% at 37 C. These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo. Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM. Source and concentration of ligand and serum components, other than immunoglobulins, appeared to contribute to receptor expression and availability. Neutrophils that were exposed to endotoxin and migrated into milk expressed more receptors than did unstimulated and nonmigrating neutrophils. The association of IgM with its receptor was temperature-dependent.

Free access
in American Journal of Veterinary Research

SUMMARY

Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence- related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (lad) on ligand binding and receptor expression.

We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from lad, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (cl) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of cl was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1.

A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from lad- affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and lad-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between lad-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from lad-affected calves. Receptor expression for aIgG was greater on neutrophils from lad-affected calves than on those from normal calves. Luminol-enhanced cl of neutrophils in response to IgG2 opsonized zymosan was not different between lad-affected and normal calves.

Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with lad. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

Free access
in American Journal of Veterinary Research