Isolation and functional analysis of normal canine blood monocytes and resident alveolar macrophages

The percentage of mononuclear phagocytes bearing the Fc receptor for immunoglobulin G, the percentage of cells phagocytic for Candida albicans and latex particles, and the phagocytic index for blood monocytes and alveolar macrophages from healthy dogs are reported. Blood monocytes were concentrated by density-gradient centrifugation, whereas alveolar macrophages were obtained in high yield by bronchoalveolar lavage. Adherent populations of those cells were used for functional assays after repeated washing to remove nonadherent cells. A greater percentage of adherent alveolar macrophages than adherent blood monocytes showed evidence of the Fc receptor for immunoglobulin G. Similarly, adherent alveolar macrophages showed significantly greater phagocytic ability, as measured by percent phagocytic cells and phagocytic index, using C albicans and latex particles, than did adherent canine blood monocytes.     

In vitro killing of Pasteurella multocida: the effect of rabbit granulocyte and specific antibody source.

In vitro granulocyte-killing assays were performed to examine the ability of granulocytes from pasteurella-free or immunized rabbits, the in combination with specific immune serum, to kill Pasteurella multocida. Granulocytes from healthy rabbits and from rabbits with P multocida infections were equally competent. Granulocyte source, serum source, and specific antibody titer had no effect on granulocyte phagocytic activity. Moreover, serum containing specific antibody and complement supported the growth of the bacterium. These data suggest that chronic P multocida infections are not attributable to defective granulocytes or lack of serum antibody production.     

Cultured bovine monocytes exhibit decreased release of superoxide anion and increased levels of lysosomal enzymes but do not secrete detectable lysozyme activity

In this study we determined the effects of in vitro maturation on the phagocytic activity, lysosomal enzyme content and oxidative response of bovine monocytes. Intracellular levels of the lysosomal enzymes acid phosphatase, beta glucuronidase, and beta glucosaminidase increased as bovine monocytes matured in vitro. However, in marked contrast to the mononuclear phagocytes of other mammalian species, lysozyme activity was undetectable in the culture supernatants and cell lysates of adherent bovine blood monocytes cultured for one to fifteen days. In vitro maturation of bovine monocytes also increased their phagocytic activity as determined by the ingestion of opsonized yeast. A greater percentage of monocyte-derived macrophages that were stimulated with opsonized yeast and phorbol myristic acetate (PMA) reduced nitroblue tetrazolium than did similarly treated monocytes. Monocyte-derived macrophages stimulated with PMA released significantly less superoxide anion than did PMA-stimulated monocytes. Bovine monocytes and macrophages also failed to bind the monoclonal antibody Mac-1, which binds to human and mouse macrophages. Bovine monocytes demonstrated both similarities and differences with other mammalian mononuclear phagocytes, thus making them a useful model for further study of the comparative and developmental biology of mononuclear phagocytes.     

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10⁷ cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.     

The effect of fungal and yeast glucan and levamisole on the level of the cellular immune response in vivo and leukocyte phagocytic activity in mice]

The level of cell-mediated immune response in vivo was investigated using the test of delayed hypersensitive reaction (DHR) to DNFB, along with the phagocytic activity (PA) of blood leucocytes in mice after subcutaneous implantation of fungal and yeast glucan and levamisol in dependence on the dose and administration schedule. The soluble form of fungal glucan (Pleurotus ostreatus) potentiated the DHR significantly at a dose of 10 mg/kg (but not at a dose of 50 mg/kg) while it was administered during DNFB sensitization (P < 0.05)-Tab. I and when its pre-medication effect was investigated (days -7 and -14; P < 0.05) with regard to the time of sensitization (Tab. II). The identical dose of glucan also had a positive effect (P < 0.05 or 0.01) on the percentual proportion of phagocytic cells (PC) reaching the maximum in the 2nd and 3rd week of investigation, as well as on the phagocytic activity index (P < 0.05; 3rd week) and percentage of neutrophil granulocytes (P < 0.05; 2nd week)-Tab. III. Yeast glucan (Saccharomyces cerevisiae) showed a potentiating effect on the DHR to DNFB only in the case of its pre-medication use; its soluble form was effective at both doses (10 mg and 50 mg/kg) in days -7 and -14 (P < 0.05), and its corpuscular form at a dose of 50 mg/kg on days -7, -14 and -21 (P < 0.05 or 0.01)-Tab. II. PA parameters of blood leucocytes displayed a stimulative effect only on the PC percentage. The most significant effects in this case were observed in the soluble form (both doses) in the 2nd and 3rd week (P < 0.01 and 0.05, resp.) and in the insoluble form (both doses) in the 3rd and 4th week of observation (P < 0.05 and 0.01, resp.). An increase in the number of neutrophil granulocytes was significant in the 2nd (P < 0.05 or 0.01; corpuscular form) and 3rd week of the experiment (P < 0.01; soluble form)-Tab. III. Levamisol affected both investigated parameters (DHR and PA) only at a dose 20 mg/kg (10 mg/kg-no effect). Its potentiating effect on the DHR level was observed both for its administration at the time of sensitization (P < 0.05) and for its administration on days 7 (P < 0.05) and 14 (P < 0.01) before DNFB sensitization (Tabs. I and II). A statistically significant increase in PC was recorded in weeks 2, 3 and 4 (P < 0.05 or 0.01), a statistically significant increase in the number of neutrophil granulocytes in the 3rd week of investigation (P < 0.05). The phagocytic activity index was not affected.     

Induction of functional defects in macrophages by a poult enteritis and mortality syndrome-associated turkey astrovirus.

The interaction of a poult enteritis and mortality syndrome (PEMS)-turkey astrovirus-Ohio State University (TAst-OSU) with the mononuclear phagocytic system cells, namely macrophages, was examined after in vitro and in vivo exposure. In vitro exposures were performed by incubating adherent turkey macrophages with various volumes of 10(6) 50% embryo infective dose (EID50)/ml TAst-OSU stock, whereas for in vivo challenge, poults were given a 200 microl inoculum of 10(6) EID50/ml TAst-OSU stock at 7 days of age. Results show that TAst-OSU in vitro exposure reduced macrophage viability relative to controls (P < 0.05) and decreased phagocytosis (P < 0.05) and intracytoplasmic killing of Escherichia coli (P < 0.05) after a 42-48-hr exposure. Poults challenged with TAst-OSU in vivo recruited almost 50% fewer Sephadex-elicited inflammatory cells in the abdominal cavity (P < 0.05) as compared with the sham controls. Similar to in vitro exposure, macrophages isolated from in vivo TAst-OSU-challenged poults exhibited reduced percentage of phagocytic macrophages (P < 0.05) as well as fewer intracytoplasmic E. coli per phagocytic macrophage (P < 0.05). TAst-OSU-challenged poults had a greater number of viable E. coli in their spleens (P < 0.05) after an intravenous E. coli challenge as compared with the non-TAst-OSU-challenged control poults. Macrophage-mediated cytokines and metabolites were also examined during this study. Both in vitro and in vivo TAst-OSU challenge resulted in reduced interleukin (IL)-1 and IL-6 activity. On the contrary, nitrite levels in macrophage culture supernatant fraction of TAst-OSU-challenged macrophages were significantly higher (P < or = 0.05). The findings of these studies indicated that TAst-OSU challenge induced defects in macrophage effector functions, implying that PEMS-turkey astrovirus can potentially impair the immune response of turkeys, thereby leading to enhanced susceptibility of turkeys to secondary, perhaps even fatal, bacterial infections.     

Phagocytosis induced by yeast cells in canine granulocytes: a methodological study

A phagocytic function assay of canine granulocytes was established. This method allows the proportion of active granulocytes to be estimated as well as the number of adhered and ingested yeast cells. The influence of different factors on phagocytosis was studied. Temperature variation within the interval 36-41 degrees C did not affect phagocytosis. The incubation time for optimal phagocytosis of yeast cells was 35 min. The opsonization procedure giving the optimal phagocytosis was purified IgG and serum together.     

Supernatants from leucocytes treated with melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) have a stimulatory effect on rainbow trout (Oncorhynchus mykiss) phagocytes in vitro

Melanin-concentrating hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH) are widespread vertebrate neuropeptides. In teleost fish the peptides are involved in the hormonal control of skin pigmentation, but they have also been shown to modulate corticosteroid secretion in both fish and mammals. alpha-MSH has additional potent anti-inflammatory actions in mammals and both peptides stimulate leucocyte phagocytosis in rainbow trout in vitro. The effects of these peptides on phagocytosis and the release of immunomodulatory factors by rainbow trout head kidney leucocytes were investigated in vitro. Neither MCH nor alpha-MSH had any effect on the adherence of phagocytes to glass slides or the activity of isolated phagocytes. When added to mixed leucocyte suspensions, however, MCH (50 and 100nM) and alpha-MSH (1 and 10nM) significantly increased the percentage of cells undergoing phagocytosis and MCH (50nM), but not alpha-MSH, stimulated the phagocytic index. In subsequent experiments, isolated phagocytes were exposed to supernatants derived from mixed leucocyte suspensions exposed to MCH (50 and 100nM) and alpha-MSH (1 and 10nM). Supernatants from leucocytes exposed to all doses of the peptides significantly increased the percentage phagocytosis and those from cells stimulated with MCH (100nM) and alpha-MSH (1 and 10nM) increased the phagocytic index of the phagocytes. The results suggest that cells other than phagocytes are required for MCH and alpha-MSH to exert their stimulatory effects on trout phagocytic cells through the release of one or more macrophage-activating factors.     

Non-specific responses of turbot (Scophthalmus maximus L.) adherent cells to microsporidian spores

We investigated non-specific responses of turbot spleen- and pronephros-resident adherent cells to spores of fish microsporidians, and the effects of the glucocorticoid dexamethasone (DX) on these responses. On average, 65% of adherent cells from the spleen and pronephros showed esterase activity (as characteristic of macrophages); 32% showed peroxidase activity (as characteristic of neutrophils), and 19% of peroxidase-positive cells were capable of phagocytosing microsporidian spores. A significantly higher proportion of adherent cells showed phagocytic activity when viable spores were the target than when non-viable spores were the target. Microsporidian spores stimulated adherent cells to produce reactive oxygen and nitrogen intermediates (ROIs and RNIs), though less effectively than the other stimulants tested. Adherent cells exposed to viable spores produced significantly less intracellular superoxide than adherent cells exposed to non-viable spores. Daily injection of fish with DX over 6 days significantly inhibited both phagocytosis of microsporidian spores and spore-induced ROI production, and similar effects were observed when adherent cells were exposed to DX in vitro.     

Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and un-quenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.     

Leukocyte profile of cattle persistently infected with bovine viral diarrhea virus

Animals acutely infected with bovine viral diarrhea virus (BVDV) exhibit transient immunosuppression as a result of the virus' predilection for cells that play critical roles in the host immune system. Acute BVDV infections have major effects on thymic and follicular T-lymphocytes, as well as follicular B-lymphocytes, often resulting in severe reduction in circulating numbers of lymphocytes and suppression of functional activities of these cells. Granulocytes and monocytes are equally susceptible to BVDV infections with reduction in numbers and suppression functions. However, there is limited information on the leukocyte profile of cattle persistently infected (PI) with BVDV. This study reports on phagocytic activities of granulocytes and monocytes as well as immunophenotyping by flow cytometric analysis of leukocytes isolated from healthy non-PI (NPI) and PI animals. No significant differences were found between the leukocyte profiles and the phagocytic activities of PI animals when compared to a group of healthy NPI animals.     

Immunomodulatory effects of alpha melanocyte stimulating hormone on common carp (Cyprinus carpio L.)

The immunomodulatory effects of phagocytic cells in common carp Cyprinus carpio L., by alpha melanocyte stimulating hormone (alpha-MSH) was analyzed in vitro. Carp head kidney leucocytes were cultured in RPMI 1640 medium containing 0.05, 0.1, 1 and 10 nM alpha-MSH and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with alpha-MSH showed an increase in the production of superoxide anion in comparison to control macrophages cultured without hormone. Phagocytic cells treated with alpha-MSH also displayed increased phagocytosis. Furthermore, carp lymphocytes treated with alpha-MSH increased the mitogenic responses to phytohaemoagglutinin (PHA-P). These results show that alpha-MSH directly influences fish immune responses.     

Expression of procoagulant activity by equine lung macrophages: stimulation by blood lymphocytes

Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses.     

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.)

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.     

Comparison of binding and effects of Escherichia coli Shiga toxin 1 on bovine and ovine granulocytes

Granulocytes play a pivotal role in the pathogenesis of Shiga toxin (Stx)-producing Escherichia coli (STEC) related diseases in humans. Granulocytes are attracted and activated by Stxs in the enteric mucosa and are believed to thereby contribute to the intestinal inflammation. Mature ruminants, the main reservoir hosts of STEC, do not develop pathological changes that can be attributed to the Stxs. To prove whether the latter phenomenon correlates with the inability of the Stxs to affect granulocytes of ruminants, we investigated the ability of Stx1 to bind to granulocytes of cattle and sheep and analysed the effects of Stx1 on viability, phagocytosis, and oxidative burst activity. Bovine granulocytes from blood and milk did not express Stx1-binding sites even after activation of the cells and also were resistant to Stx1. In contrast to bovine granulocytes, granulocytes of sheep constitutively expressed Stx1-receptors of the Gb(3)/CD77 type ex vivo and bound the recombinant B-subunit of Stx1 (rStxB1). Stx1 holotoxin induced apoptosis in ovine granulocytes after prolonged incubation (18h) but Stx1 only slightly altered the phagocytosis and oxidative burst activities. The rStxB1 had no effect on granulocytes of either species. While arguing in favour of our initial hypothesis, that granulocytes of both, cattle and sheep are not activated by Stxs, the results of our study are the first evidences for differences in the cellular distribution of Stx-receptors in species equally regarded as STEC carriers.     

Duck lymphocytes. VI. Requirement for phagocytic and adherent cells in lymphocyte transformation.

Preparations of duck (Anas platyrhynchos) spleen and blood lymphocytes depleted of cells capable of phagocytosing carbonyl iron gave lower transformation responses to the mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), lentil lectin (LL) and phorbol ester (PMA) than intact cell preparations. When cell populations were fractionated on the basis of their adherence to plastic, it was found that the adherent cells were responsive to PHA, Con A, BSS, WGA and PMA, while the non-adherent cells responded to LL. These observations confirm the expected requirement for phagocytic accessory cells in the induction of in vitro mitogen-driven duck lymphocyte responses. The responses of plastic-adherent populations of cells to most mitogens are believed to reflect the generally close physical relationship between the adherent accessory cells and the lymphocytes, although it remains possible that duck monocytes respond to some of the mitogens employed. The data also suggest that LL stimulates a population of cells different to those responding to other mitogens.     

Establishment and characterisation of ovine blood monocyte-derived cell lines

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.     

Morphology of blood cells in carp (Cyprinus carpio L.)

The morphology of blood cells in the carp was investigated by light and electron microscopy. Erythrocytes, thrombocytes, lymphocytes, granulocytes and monocytes were identified as the peripheral blood cells. Thrombocytes were round to long oval, each containing vesicular and microtubular structures and an oval nucleus with abundant heterochromatins. Lymphocytes were divided into three types in size, small, medium and large. Some of the small and medium lymphocytes were alpha-naphthyl-acetate esterase (ANAE) positive, while large lymphocytes were pyroninophilic. Granulocytes were distinguished into three types (type I, type II and type III) according to the morphology of the nucleus and granules. Type I granulocytes possessed lobulated nuclei and a large number of cytoplasmic granules, each of which was oval and contained electron-dense materials and a crystalloid. Type II granulocytes had small eccentric nuclei and were subdivided into IIa and IIb granulocytes by electron microscopic analysis. Granules of type IIa granulocytes were furnished with an electron-dense rim. Granules of type IIb granulocytes were larger than those of type IIa, containing randomly distributed electron-dense and electron-lucent materials. Type III granulocytes possessed round nuclei and a few large granules. The granules were filled with regularly arranged fibriform materials and some needle-like structures. Monocytes were morphologically similar to those of mammals.     

Interaction between Salmonella typhimurium and phagocytic cells in pigs. Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes

Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related. Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC). When opsonized in normal serum the Salmonella bacteria, in the range of 2-5 x 10(7) cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD). Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs. Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer. Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response. In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.     

Isolation and cryopreservation of functionally competent equine leucocytes

Sufficient numbers of functionally competent polymorphonuclear neutrophil granulocytes (PMN) seem to be of major importance during the course of equine endometritis. In this study, we wanted to establish a method for cryopreservation of functionally competent neutrophils for an intended local endometritis therapy in mares. The separation of leucocytes by hypotonic lysis of whole blood from clinically healthy mares was superior to the separation by dextrose sedimentation. After suspension of the cells in the cryoprotective solution [equine plasma with 5% (v/v) dimethyl sulphoxide (DMSO)], the leucocytes were frozen in liquid nitrogen. A temperature gradient with low cooling velocity (1 degree C/min between 4 and -70 degrees C) resulted in highest numbers of viable cells after thawing. Thawed PMN had a high phagocytic capacity for opsonized streptococci. Their ability to generate reactive oxygen species (ROS) after stimulation with a phorbol ester was even higher than that of freshly isolated PMN and was preserved up to 6 h after thawing. The results of this study indicate that cryopreservation of PMN may provide viable and functionally competent neutrophils for therapeutic use in mares susceptible to endometritis.