Evaluation of bovine polymorphonuclear leukocyte function

Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of cattle. Five in vitro procedures were utilized to evaluate PMN function: 1) Random migration under agarose, 2) Ingestion of ¹²⁵I-iododeoxyuridine labeled $\text{Staphylococcus aureus}$, 3) Quantitative nitroblue tetrazolium reduction, 4) Chemiluminescence and 5) Iodination. Normal values for bovine PMNs are reported and interpretation of results is discussed. The PMN function tests were designed so that all 5 procedures may be performed in a short period of time on the same cell preparation. This allows for the detection and partial characterization of a potential PMN dysfunction.     

Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes

Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions.     

Association of increased estradiol and progesterone blood values with altered bovine polymorphonuclear leukocyte function

Polymorphonuclear leukocyte (PMN) function and serum concentrations of estradiol, progesterone, and cortisol (hydrocortisone) were monitored concurrently in clinically normal cows during the estrous cycle. Five parameters were used to evaluate PMN function: (i) random migration under agarose, (ii) ingestion of 125I-labeled Staphylococcus aureus, (iii) nitroblue tetrazolium (NBT) reduction, (iv) iodination, and (v) antibody-dependent cell-mediated cytotoxicity. Increased serum estradiol concentrations were associated with enhanced random migration, but had no apparent effect on NBT reduction, iodination, or ingestion of S aureus by bovine PMN. Increased serum estradiol was also associated with increased serum cortisol. Increased serum progesterone values were associated with a depression of NBT reduction and iodination by PMN, but with enhanced random migration and antibody-dependent cell-mediated cytotoxicity. These results indicate that physiologic changes in steroid hormone values during the normal estrous cycle of the cow are associated with alterations in PMN function.     

Effects of bovine respiratory disease viruses and isoprinosine on bovine leukocyte function in vitro

Peripheral blood mononuclear cells obtained from 4- to 6-month-old-calves were inoculated in vitro with bovine herpesvirus-1, parainfluenza-3, or bovine virus diarrhea viruses. No increase in infectious virus progeny was observed; however, the viruses were detected in the cells for at least 96 h post-infection without any significant reduction in cell viability. The three viruses, either alone or in combination, suppressed phytohemagglutinin-induced proliferation of the mononuclear cells. The greatest suppression was observed in cultures inoculated with bovine virus diarrhea virus. Addition of isoprinosine partially restored this viral-induced suppression of proliferative response, and the efficiency of reversal was greater in bovine virus diarrhea virus-infected cells. Interleukin-2 activity was higher in cultures of virus-infected mononuclear cells than in cultures of non-infected cells.     

Effects of peripheral blood polymorphonuclear leukocyte function and blood components in Japanese black steers administered ACTH in a cold environment.

An examination of the effects of artificial stress induced by adrenocorticotropin (ACTH) on total and differential leukocyte counts, plasma cortisol levels, metabolic profiles and peripheral blood polymorphonuclear leukocyte (PMN) function was performed on Japanese Black steers kept in a cold environment, with the following regimes; 1) -5 degrees C x ACTH (100 IU/day for 3 days), 2) 0 degrees C x ACTH, 3) 15 degrees C x ACTH and 4) 15 degrees C x PBS. Blood samples were collected before and at 1, 2, 24, 48, 72 and 96 hr prior to the application of the stressor. The plasma cortisol level was found to greatly increase at one hr after the first treatment of ACTH, particularly so in animals exposed to -5 degrees C. Total leukocytes (-5 degrees C and 0 degrees C experiments, respectively), the monocytes (-5 degrees C), neutrophils and eosinophils (-5 degrees C, 0 degrees C and 15 degrees C, respectively) obviously increased just after the first administration, although lymphocyte counts at -5 degrees C were inversely related to those described above. All of these tendencies were augmented by the cold environment except for eosinophils. The chemiluminescent (CL) response of PMN decreased in the ACTH-administered steers at an early stage of post-administration, however, it tended to recover from the lower-than-base value in the cold-affected steers. ACTH administration resulted in higher plasma glucose (Glu) compared to a control, although only steers housed at -5 degrees C evidently showed lower plasma inorganic phosphorus (IP). No abnormal serum acute phase protein, or immunosuppressor, was noted. ACTH thus appears not only to promote physiological reactions but also to temporarily suppress PMN cellular immune function in Japanese Black steers. Although, a cold environment rapidly restored the CL activity to over the pre-administrational value, suggesting that a vital response was activated by crymo-stimuli.     

Effect of antimicrobial agents and corticosteroids on bovine polymorphonuclear leukocyte chemotaxis

The effect of certain antimicrobial agents and corticosteroids on bovine polymorphonuclear leukocyte chemotaxis was investigated. Peripheral blood was fractioned by density-gradient centrifugation, using Ficoll-Hypaque. The chemotactic assay was performed in modified Boyden chambers, using Micropore filters, and the chemotactic response was measured by the leading-front technique. Tetracyclines, streptomycin, and penicillin had no effect on chemotaxis at concentrations normally achieved in blood during systemic treatment. However, higher concentrations that were achievable with local therapy, such as intramammary injection or topical application, inhibited the chemotactic response. This inhibition was eliminated by serum. Dexamethasone stimulated polymorphonuclear leukocyte chemotaxis with the effect being manifested after the cells were incubated with the drug for 3 hours. Hydrocortisone caused slight inhibition of chemotaxis, whereas prednisone and prednisolone had no effect.     

Effect of levamisole on lymphocyte blastogenesis and neutrophil function in dexamethasone-treated cattle

Levamisole was evaluated at 6 dose levels for its ability to prevent the dexamethasone-induced suppression of in vitro lymphocyte blastogenesis or neutrophil function in cattle. Dexamethasone (0.4 mg/kg of body weight, IM) and levamisole hydrochloride (0.5, 1.0, 2.0, 4.0, or 8.0 mg/kg orally) were administered to groups of 4 cattle daily for 3 days. Another group of 4 cattle were given the 3-day dexamethasone treatment and 6.0 mg/kg of levamisole (the recommended anthelmintic dose) was given only once on the 1st day that dexamethasone was given. Results obtained from the dexamethasone-levamisole-treated cattle were compared with results obtained from cattle that were given only dexamethasone. Levamisole had no apparent consistent ability to enhance lymphocyte blastogenic responsiveness (to the mitogens phytohemagglutinin, concanavalin A, or pokeweed mitogen or in a 1-way mixed lymphocyte reaction) or to enhance neutrophil function (random migration, nitroblue tetrazolium reduction, iodination, or antibody-dependent cell-mediated cytotoxicity) in dexamethasone-treated cattle.     

穿心莲对肉鸡淋巴细胞活性及白细胞吞噬功能影响的研究

本试验研究了不同剂型穿心莲对肉鸡细胞免疫的影响.将1日龄肉鸡随机分为七个试验组,处理如下:A组为1%超微粉;B组为2%超微粉;C组为0.2%浓缩颗粒(1g浓缩颗粒相当于5g生药);D组为0.4%浓缩颗粒;E组为1%浓缩颗粒;F组为2%细粉;G组为不加中药的空白对照.在试验的第14、21,28、35、42 d分别采血,进行淋巴细胞转化率、白细胞吞噬率及吞噬指数测定.试验结果表明,穿心莲对肉鸡淋巴细胞活性及白细胞活性(吞噬率和吞噬指数)有显著的提高作用(P＜0.05).同时,穿心莲超微粉和浓缩颗粒提高细胞免疫的效果优于细粉;相同剂量下,超微粉的效果有优于浓缩颗粒的趋势.     

Effects of steroids on bovine T-lymphocyte blastogenesis in vitro

Day 13 to 24 bovine conceptuses convert C-19 and C-21 neutral steroids to 5 beta-reduced steroids with great efficiency. Pregnancy steroids have been reported to be immunomodulatory in several species. This study examined the possibility that 5 beta-reduced products of bovine conceptus steroid metabolism, precursors, related steroids, progesterone or cortisol might affect bovine T-cell blastogenesis. The steroids tested were 5 alpha-androstan-17 beta-ol-3-one, androstene-3, 17-dione, 5 beta-pregnane-3,20-dione and 5 beta-pregnane-3 alpha,20 alpha-diol. The ability of each steroid, over a dose range of 10(-2) to 10(4) nM, to affect phytohemagglutinin (PHA)-induced or concanavalin A (Con A)-induced bovine T cell blastogenesis, or the mixed lymphocyte reaction (MLR) was evaluated. Five lactating Holstein cows served as lymphocyte donors for mitogen studies and two, that exhibited strong MLR, were donors for the MLR evaluation. Of the seven steroids tested none affected blastogenesis in a dose-related manner except for cortisol, which suppressed Con A-induced lymphocyte transformation as well as the MLR. Cortisol did not affect PHA-induced blastogenesis. Thus, of the pregnancy steroids tested at the concentrations noted, none had significant immunomodulatory effects.     

Suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with and without the administration of ACTH

Effects of a modified live vaccine (MLV) strain of bovine viral diarrhea virus (BVD) on lymphocyte and neutrophil function were determined in cattle with and without increased plasma cortisol (hydrocortisone) concentrations. Cattle were given MLV-BVD vaccine IM and intranasally. Cattle given ACTH received 200 IU every 12 hours for 10 doses. The MLV-BVD virus when administered alone caused no apparent clinical signs or body temperature response. Of 4 MLV-BVD-treated calves that were also given ACTH, 2 developed increased body temperature and respiratory distress. The MLV-BVD virus caused a decrease in circulating lymphocytes and neutrophils, whereas administration of ACTH and MLV-BVD induced a neutrophilia and lymphopenia. The MLV-BVD virus and ACTH when administered separately or in combination caused a depression of lymphocyte blastogenesis in response to selected mitogens. Neutrophils were separated from the peripheral blood and their function was evaluated, using the following procedures: (i) random migration under agarose, (ii) ingestion of 125I-labeled Staphylococcus aureus, (iii) quantitative nitroblue tetrazolium reduction, (iv) iodination, and (v) antibody-dependent cell-mediated cytotoxicity (ADCC). The MLV-BVD virus produced a significant (P less than 0.05) suppression of neutrophil iodination and ADCC. Neutrophils from cattle given MLV-BVD virus and ACTH had enhanced random migration, enhanced S aureus ingestion, suppressed iodination, and suppressed ADCC activity.     

Effect of Rhodococcus equi on equine polymorphonuclear leukocyte function

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.     

Enhancement of lymphocyte blastogenesis and neutrophil function by avridine in dexamethasone-treated and nontreated cattle

The lipoidal amine, N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine (avridine or CP 20,961), formulated in liposomes, was evaluated for its effect on leukocyte kinetics, lymphocyte blastogenesis, and polymorphonuclear leukocyte (PMN) function in dexamethasone-treated and nontreated cattle. In the 1st experiment, cattle were given avridine in a single IM injection of 0.1, 1.0, or 10 mg/kg of body weight. All doses induced swelling at the injection site, a febrile response, and a leukocytosis due to a neutrophilia. Mononuclear cell numbers were normal. All 3 groups of avridine-treated animals had a higher mean lymphocyte blastogenic response to mitogens on the 4 days after administration than did the control nontreated animals. Avridine administration was associated with an enhanced ability of PMN to ingest Staphylococcus aureus and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The highest dose (10 mg/kg) was associated with a depression of the ability of PMN to iodinate protein. An effect of avridine on PMN random migration under agarose or nitroblue tetrazolium (NBT) reduction was not observed. In a 2nd experiment, cattle were given no treatment, 0.04 mg of dexamethasone/kg IM, or 10 mg of avridine/kg IM followed 24 hours later by 0.04 mg of dexamethasone/kg. Dexamethasone administration caused a leukocytosis due to a neutrophilia with normal mononuclear cell numbers, an enhancement of PMN random migration under agarose, and an inhibition of NBT reduction, iodination, and ADCC activity of PMN. Dexamethasone did not have a detectable effect on lymphocyte blastogenesis or on ingestion of S aureus by PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of lymphocyte blastogenesis by whey

Bovine whey samples were evaluated by use of lymphocyte-transformation tests to determine their effect on lymphocyte blastogenesis. Whey samples from mammary glands with clinical mastitis strongly inhibited DNA synthesis and blastogenesis in lymphocytes stimulated with mitogens or dividing because of bovine leukemia virus infection. Whey samples from apparently healthy glands either did not inhibit lymphocyte DNA synthesis or inhibited it to a lesser degree than did whey from mastitic glands. Degree of inhibition was dose-dependent. The molecules causing inhibition were noncytotoxic and underwent minimal binding to the lymphocytes. Inhibitory molecules were susceptible to various proteolytic and glycolytic enzymes, indicating a glycoprotein-like structure. Whey inhibited incorporation of thymidine if it was in the cell cultures during the early stages of stimulation. Incubation of lymphocytes in whey that inhibited thymidine incorporation did not affect DNA synthesis in subsequent culturing of the same cells without whey. Degree of inhibition was affected by the method of whey preparation.     

Effects of nitric oxide on bovine polymorphonuclear functions.

The effects of nitric oxide (NO) on the functionality of polymorphonuclear neutrophils (PMNs) in bovine milk or blood were investigated. In 2 experiments, mastitis was induced by infusing both hind quarters with saline containing Escherichia coli endotoxins. In addition, the left hind quarter was infused with aminoguanidine, an inhibitor of the inducible form of NO synthase (iNOS). At various times after infusion, somatic cells were isolated from milk samples, and superoxide (O2-) production induced by phorbol myristate acetate was evaluated. In both experiments, the addition of aminoguanidine had no inhibitory effect on the number of milk somatic cells or on their O2- production. The effect of NO and iNOS inhibitors on the functionality of bovine PMNs isolated from blood was investigated in vitro. The neutrophils did not produce NO. A neutrophil:monocyte co-culture system was used to study the effect of NO derived from monocytes on O2- production by bovine neutrophils. Neither NO derived from activated monocytes nor the iNOS inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine had an effect on the ability of bovine neutrophils to release O2-. Moreover, aminoguanidine did not affect the ability of bovine neutrophils to phagocytose bacteria. These results suggest that inhibition of NO release during inflammation does not interfere with the migration of immune cells to the site of infection or the ability of these cells to destroy pathogens. Thus, NO does not appear to play a major role in the control of the functions of bovine neutrophils.     

Effects of bovine leukemia virus infection on milk neutrophil function and the milk lymphocyte profile

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138⁺ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5⁻/CD11b⁻ B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.     

Modulation of canine lymphocyte blastogenesis via histamine

The effect of histamine on in vitro T cell blastogenic responses of canine peripheral blood lymphocytes to phytohemagglutinin-P (PHA-P) was investigated. A dose dependent inhibition of blastogenesis was observed; an effect which could be blocked by cimetidine, a type II histamine receptor antagonist, but not by diphenhydramine, a type I receptor antagonist, suggesting that histamine's inhibitory effect is mediated through a type II histamine receptor. The inhibitory effect of histamine on blastogenesis was also reversible by indomethacin, a prostaglandin synthetase inhibitor, implicating prostaglandin involvement in histamine suppression. Histamine release at sites of inflammation may result in down regulation of local immune responses by activation of specific immunoregulatory cells. This could permit the escape of certain neoplasia from local immunosurveillance mechanisms. Cimetidine may block activation of histamine responsive regulatory cells bearing type II receptors, which may help explain the beneficial effect cimetidine therapy has on regression of certain human tumors (i.e., malignant melanomas).     

Effect of ammonia on viability and blastogenesis of bovine lymphocytes

The effect of addition of ammonia into the tissue culture on viability and functions of bovine lymphocytes was studied. The concentrations of ammonia in the tissue cultures represented toxic, subtoxic, and normal concentrations of ammonia in the bovine blood during clinical and subclinical urea toxicosis. Lymphocytes separated from peripheral bovine blood were incubated in control medium and test medium with various concentrations of ammonia and/or PHA or Con A. Viability of the lymphocytes was measured by trypan blue exclusion test and their mitogenic reactivity by incorporation of ³H thymidine into DNA of lymphocytes. Approximately 30% bovine lymphocytes were killed by ammonia in medium during 72 hours of incubation. Ammonia also affected the response of lymphocytes to stimulation with PHA or Con A as well as mixed lymphocyte culture reaction. The mitogenic response of lymphocytes was also reduced when lymphocytes were preincubated with ammonia for even 1 hour. The mitogenic response was not restored when the number of lymphocytes preincubated with ammonia was reconstituted to the initial concentration to compensate for the killed lymphocytes before stimulation with PHA. Therefore, addition of ammonia to the culture either killed lymphocytes or permanently impaired their functions.     

Effects of bovine mammary secretion during the early nonlactating period and antibiotics on polymorphonuclear neutrophil function and morphology.

The effect of bovine mammary secretion during the early nonlactating period and of antibiotic preparations on bovine polymorphonuclear neutrophil (PMN) phagocytic function and morphology were evaluated in a series of in vitro multifactorial experiments. Benzathine cloxacillin (CL), benzathine cephapirin (CE), sodium novobiocin (NO), and a combination of dihydrostreptomycin with procaine penicillin G (DP) were prepared in the presence and absence of a peanut oil aluminum monostearate vehicle. The PMN were isolated from bovine blood, and the effect of each antibiotic preparation on PMN function and morphology was evaluated in a buffer, fat, skin, and a combination of fat with skim from bovine mammary secretion during the nonlactating period. The fat and skim were diluted with buffer to approximate their concentration in mammary secretion. Phagocytic functions of PMN were monitored by fluorescent microscopy, which made it possible to estimate both ingestion and intracellular killing of bacteria by PMN. Changes in PMN morphology were monitored by transmission electron microscopy. The ability of PMN to ingest and kill Staphylococcus aureus ATCC 25923 was significantly decreased by fat, skim, CL, CE, NO, and DP. Effects of some antibiotics on ingestion and killing of bacteria by PMN were influenced by the addition of vehicle and by interactions with mammary secretion. Neutrophil morphology was altered by fat, skim, CL, CE, NO, and DP. The detrimental effects of CL, CE, NO, and DP on PMN morphology were influenced (some significantly) by the presence of vehicle and interactions with mammary secretion. There were significant correlations among secretion- and antibiotic-induced changes in PMN ingestion of bacteria, PMN killing of bacteria, and PMN morphology.     

Lymphocyte blastogenesis and neutrophil function in cattle persistently infected with bovine viral diarrhea virus

Neutrophil function and mononuclear cell proliferative responses to mitogens were determined in healthy cattle and in cattle persistently infected with bovine viral diarrhea (BVD) virus. Uptake of [3H]thymidine by resting and mitogen-stimulated peripheral blood mononuclear cells was significantly lower in cattle persistently infected with BVD virus than in healthy cattle. Neutrophils from cattle persistently infected with BVD virus had significantly impaired capability to ingest Staphylococcus aureus, but were normal in respect to random migration under agarose, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Impairment of neutrophil function in cattle persistently infected with BVD virus differs from impairment of neutrophil function reported in healthy cattle mounting an immune response to recent BVD virus infection.