Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

The main objective of the study reported here was to generate a panel of monoclonal antibodies (MAB) to bovine neutrophil surface antigens, and to identify MAB that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study MAB reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules. A panel of 14 MAB was generated by producing murine hybridomas. Neutrophils incubated with MAB at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-1,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The MAB S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The MAB S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas MAB S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The MAB S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with MAB served as controls for comparison. The MAB binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of MAB S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C. Binding of these MAB was further increased after incubation with phorbol myristate acetate (100 ng/ml) at 37 C, indicating recognition of activation antigens by MAB. The MAB generated in this study appeared to be potential candidates for studying mechanisms of neutrophil function and for enhancing neutrophil function in vitro and in vivo.

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P < 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 x 10(5) neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.

The chemotactic activity of turkey peritoneal macrophages in response to an atherosclerotic plaque extract from a hypertensive strain of turkeys was determined. Atherosclerotic plaque extract stimulated macrophage chemotaxis, whereas normal aortic extract did not stimulate macrophage chemotaxis. However, differences were not revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of extracts of atherosclerotic plaque and normal aorta. Chemotactic activity was diminished with pronase treatment, suggesting the chemoattractant is a protein. Seemingly, atherosclerotic plaque of turkeys contains a macrophage chemotaxin.

Binding of endogenous and exogenous homologous IgG, 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 IgG, 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.

Ovulation has been likened to an inflammatory process. Inflammatory cells accumulate in the ovulating follicle, presumably because of chemotactic factors. Chemotactic activity was measured in fluid aspirated from follicles of estrous mares 0, 12, 24, and 36 hours after ultrasonographic detection of a 35-mm follicle and IV treatment with 2,500 IU of human chorionic gonadotropin. Chemotaxis was assessed by measuring directional migration of equine neutrophils under agarose. Follicular fluid acted as a chemoattractant for neutrophils, but there was no significant difference in chemotactic activity among different time intervals after administration of human chorionic gonadotropin. On the basis of results of various treatments, chemotactic properties of serum and follicular fluid were similar. Chemotactic activity was significantly reduced by heating (56 C for 30 minutes) and by trypsinization and was virtually removed by charcoal treatment. Dialyzing the follicular fluid (3,500 and 8,000 molecular weight cut-off) significantly reduced the chemotactic activity of follicular fluid and serum. The importance of chemotactic factors in the process of ovulation in the mare is yet to be established.

Chemotaxis under agarose was evaluated to establish an assay system and to characterize chemotacticresponses of canine neutrophils. A method for the measurement of canine neutrophil chemotaxis was established, with optimal responses obtained with agarose containing 10% pooled canine serum, a concentration of 5 X 10(5) cells/well, zymosan-activated serum (ZAS), or autologous serum or plasma as the chemoattractants, and a 120-minute incubation period. Canine neutrophils responded well to ZAS, heat-inactivated ZAS, autologous serum and plasma, and heat-inactivated pooled serum. Chemotactic activity was proportional to the concentration of serum used as the chemoattractant. Mean (+/- SD) random migration, chemotaxis, chemotactic index, and chemotactic differential of neutrophils from 9 healthy Greyhounds were 1.09 (+/- 0.23), 1.95 (+/- 0.38), 1.82 (+/- 0.31), and 0.86 (+/- 0.32) mm,respectively.

Quantitative evaluation of neutrophil chemotaxis was performed on cells obtained by hypotonic-lysis techniques from heparinized blood samples from clinically normal dogs. The techniques resulted in neutrophil recovery rates between 60 and 80%. Chemotaxis comparisons were based on cellular migration in microchambers equipped with polycarbonate membranes with 5-micrometer pores. Chemo-attractant comparisons were based on neutrophil migration to medium, normal canine plasma, zymosan-activated plasma, and xanthine oxidase. Cellular migration to zymosan-activated plasma in buffer (1:100 dilution) was significantly (P less than 0.001) enhanced over random baseline medium migration. Neutrophil migrations to normal canine plasma and xanthine oxidase were quantitatively less than to zymosan-activated plasma, but were equivalent to each other and significantly greater than for random migration. Migration to xanthine oxidase was maximal at concentrations near 1 U/ml within 30 minutes.

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 Stapbylococcus 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.

A suction blister technique was used in 10 healthy dogs to remove the epidermis from the dermis in a standardized way. Collection chambers were attached to these skin windows and filled with autologous serum to attract exudative neutrophils. The chambers were emptied by fine-needle aspiration at 4-hour intervals and were refilled with serum for 24 hours after the Int aspiration. The collected cells were counted, differentiated, and stained, using the trypan blue dye-exclusion method to determine cell viability. Multiple skin biopsy specimens obtained during the procedure were examined histologically. The chamber fluid collected after 24 hours was cultured for bacteria. Increasing numbers of viable neutrophils were collected during the 24-hour period from the induced skin windows. In all but 1 dog, sufficient viable neutrophils could be collected to perform further functional tests in vitro. Our conclusion is that this technique might be useful to study chemotaxis in vivo and to perform functional tests on exudative neutrophils.