Influence of technical parameters on the in vitro motility of equine neutrophils in the presence of streptococcal culture supernatant

To identify the influence of technical factors on the in vitro motility of equine neutrophils towards streptococcus culture supernatant in an under-agarose assay, we studied the changes in eight cell migration parameters. The distances the phagocytes travelled by directed, random and spontaneous migration increased with incubation time, cell concentration and the gelatin and serum contents of the migration plates. The contribution of chemotaxis to the phagocyte migrations, however, decreased simultaneously. The directed and random, though not the spontaneous, migrations of the phagocytes increased also when the chemoattractant wells were placed closer to the cell wells but so did the influence of the chemokinetic activity of the bacterial culture supernatant on phagocyte motility. In contrast, preincubation of migration plates with the chemoattractant, the agarose content of the migration plates and contamination of the granulocytes with non-migrating, mononuclear cells did not substantially affect the in vitro migrations of the neutrophils. The changes in the in vitro motility of the equine neutrophils by these technical factors were, in general, comparable to those reported for human cells attracted by a variety of host-and bacteria-derived chemoattractants.     

Migration of fish leucocytes in vitro: the effect of factors which may be involved in mediating inflammation

Plaice (Pleuronectes platessa L.) neutrophils were isolated from the kidney on a discontinuous Percoll gradient and from the peritoneal cavity at the peak of a glycogen-elicited inflammatory response. The migratory ability of neutrophils was assessed using a 48-well microchemotaxis chamber, with an incubation of 1.5 h at 12 degrees C. The two neutrophil populations showed different responses to N-formylmethionyl-leucyl-phenylalanine (FMLP). Whereas kidney neutrophils only showed a significant enhancement of migration at 10(-7) M, inflammatory neutrophils exhibited a bimodal response, with one peak of migratory activity at 10(-9) M and a second at greater than 10(-6) M. Kidney neutrophils showed a consistent response with various concentrations of a 24 h culture supernatant of Vibrio alginolyticus. In every case increased migration was observed with 5-, 10- and 100-fold dilutions, with the latter two conditions producing a significant enhancement (p less than 0.01 and p less than 0.05 respectively). The undiluted and 2-fold diluted supernatant caused a decreased cell migration compared with control values. The supernatant from kidney neutrophils cultured with serum-opsonized, heat-killed V. alginolyticus produced greater migratory activity than neutrophils or the treated bacteria incubated alone (the controls). In each case, the enhanced activity of the supernatant was detectable by 1 h of incubation. By 4 h, the activity of the neutrophil/bacteria supernatant was significantly higher than that of the controls (p less than 0.01), but by 24 h had fallen to control levels. There was no evidence for a chemotactic response with FMLP, the bacterial supernatant or the neutrophil-derived factor and the responses were therefore assumed to be chemokinetic.     

In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections

Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophils and neutrophils from S. vulgaris-naive ponies without eosinophilia (noneosinophilic ponies; nonactivated eosinophils and neutrophils). Differences in cell density following centrifugation in a continuous Percoll gradient were used to further characterize the heterogeneity of activated eosinophils and neutrophils. Activated and nonactivated eosinophils demonstrated similar chemotactic responses to ZAS while activated and nonactivated neutrophils demonstrated similar chemokinetic responses to ZAS. A higher percentage of activated eosinophils and neutrophils expressed Fc and C receptors compared with nonactivated cells (P less than 0.05). Generally, higher percentages of eosinophils and neutrophils expressed C than Fc receptors. However, the percentage of neutrophils with both receptors was higher than that of eosinophils. Phagocytosis and killing of E. coli by either type of eosinophil were not consistently observed. Both activated and nonactivated neutrophils phagocytized E. coli and significant differences between the two cell types were not observed. The bacterial activity, however, of activated neutrophils was significantly greater than that obtained using nonactivated neutrophils (P less than 0.05). Activated eosinophils and neutrophils were both separated into two distinct fractions based on differences in cell densities. A higher percentage of band 2 eosinophils (density of 1.106) expressed C receptors than did band 1 eosinophils (density of 1.049) (P less than 0.05). A higher percentage of band 1 neutrophils (density of 1.072) expressed both Fc and C receptors and these neutrophils were more phagocytic and bactericidal than were band 2 neutrophils (density of 1.082) (P less than 0.05). These data suggest that equine eosinophils and neutrophils are activated by chronic S. vulgaris infections.     

Defective in vitro motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak-Higashi cats.

The in vitro migratory responses of neutrophils of homozygote and heterozygote Chediak-Higashi cats were defective in an under-agarose assay when compared to the behavior of phagocytes of control cats. The linear distances traversed by the leading front of migrating Chediak-Higashi neutrophils toward streptococcal culture supernatant, zymosan-activated serum or buffer were reduced and smaller numbers of Chediak-Higashi phagocytes populated the resulting migration areas than did cells of control animals. The relative migration parameters of the Chediak-Higashi phagocytes, however, did not differ from the corresponding parameters of control neutrophils in the presence of streptococcal culture supernatant. Therefore, phagocytes of homozygote and heterozygote Chediak-Higashi cats recognized and responded equally well to the bacterial stimuli as did cells of control animals but traveled shorter distances primarily because of a reduced inherent motility. Similar results were also obtained when the feline phagocytes were attracted by zymosan-activated serum. In addition the relative migration parameters of the neutrophils of homozygote Chediak-Higashi cats were reduced and the normalized spatial distributions of their migrating cells were significantly different in the presence of 100% and 20% zymosan-activated serum when compared to the corresponding migration parameters of carrier and control animals. Defective recognition or responses to the higher concentrations of these host-derived attractants complicated, therefore, the already reduced inherent motility of the phagocytes of homozygote Chediak-Higashi cats.     

Chemotactic responses of neutrophils in cats with spontaneous feline infectious peritonitis

The culture supernatant of peritoneal exudate cells (PEC) from cats with effusive feline infectious peritonitis (FIP) was chemotactic for peripheral blood neutrophils (PBN) from healthy cats, magnitude of the chemotactic activity being approximately 10-fold lower than that in zymosan-activated fresh serum of healthy cats (ZAS). The migration profile of PBN from healthy cats was slightly different between the PEC culture supernatant and ZAS. These findings suggest that the chemotactic activity detected in the PEC culture supernatant is distinct from that in ZAS. The chemotactic responses of PBN from FIP cats to ZAS were reduced, as compared with that from healthy controls. In contrast, the neutrophil chemotactic response and sensitivity to the PEC culture supernatant in FIP cats were not remarkably different from those in healthy controls. Furthermore, the chemotactic responsiveness of PEC from FIP cats to ZAS was slightly different from that of PEC to the PEC culture supernatant. These results suggest that neutrophils from FIP cats have altered reactivities against these chemoattractants.     

Chemotactic response of bovine neutrophils to Pasteurella haemolytica culture fluid

Bovine neutrophil chemotactic activity was detected in the supernatant fluid of logarithmic phase cultures of P. haemolytica serotype 1. The chemoattractant was produced under culture conditions suitable for P. haemolytica leukotoxin production. An inverse correlation existed between the leukotoxin LC50 and the chemotactic activity in the culture fluid. Elimination of leukotoxin activity by heating, dilution or ultrafiltration, exposed the chemotactic activity in the culture fluid. The chemoattractant was partially resistant to heating (60 degrees C, 30 min), and had an apparent molecular weight greater than 100,000. Detection of chemotactic activity in both the concentrate and filtrate after XM300 filtration suggested that there might be more than one component with chemotactic activity or else that polymerization was occurring. Production of a potent neutrophil chemoattractant by P. haemolytica may explain the rapid infiltration of neutrophils that occurs during the early stages of bovine pneumonic pasteurellosis.     

Generation of neutrophil chemoattractants by phagocytosing bovine mammary macrophages

Macrophages from bovine mammary gland were cultured in vitro and the growth medium collected at intervals. Using an in vitro system in which neutrophils migrated under agarose, both chemotactic and chemokinetic activity for bovine neutrophils was detected in supernatants of macrophage cultures to which heat killed preopsonised Staphylococcus aureus had been added. The suspensions of killed bacteria were not themselves chemotactic for neutrophils and no chemotactic activity was present either in supernatants from unstimulated macrophage cultures or in sonicated macrophages. The chemotaxin(s) was generated within two hours of the addition of staphylococci to the cultures and was largely stable to heating at 56 degrees C for 45 minutes, although its activity was reduced by boiling for 15 minutes. Traces of proteolytic activity were also detected in some supernatants. Substantial proteolytic activity was found in lysates of neutrophils. Unlike chemotaxis, proteolytic activity was suppressed by addition of milk from early lactation and containing high natural levels of protease inhibitors. Proteolytic activity was destroyed by boiling for 15 minutes.     

Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.     

Influence of arachidonic acid metabolites in vitro and in uterine washings on migration of equine neutrophils under agarose

The influence of arachidonic acid metabolites on migration of equine neutrophils under agarose was investigated. Leukotriene B4 (LTB4) was chemotactic at concentrations between 0.1 and 1000 ng ml-1 and prostaglandin E2 (PGE2) at 1 and 10 ng ml-1 but not at higher or lower concentrations. Prostaglandin F2 alpha (PGF2 alpha) was not chemotactic for equine neutrophils at any concentration. Random migration was significantly inhibited (P less than 0.05) by suspension of neutrophils in LTB4 (0.1 to 1000 ng ml-1) and PGF2 alpha (0.1 ng ml-1) but not at high concentrations. There was a significant positive correlation between random migration of neutrophils suspended in uterine washings from persistently endometritic mares and concentrations of endogenous PGF (P less than 0.002) and PGE2 (P less than 0.05) in washings. Thus certain metabolites of arachidonic acid affect migration of equine neutrophils and may play a significant role in recruitment of neutrophils to sites of inflammation in the horse.     

Detection of chemotactic factors in preovulatory follicular fluid from mares

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.     

Neutrophil migration induced by equine respiratory secretions, bronchoalveolar lavage fluids and culture supernatants of pulmonary lavage cells

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells.     

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.     

Neutrophilic nodules in the intestinal walls of Japanese monkeys associated with the neutrophil chemotactic activity of larval extracts and secretions of Oesophagostomum aculeatum

High neutrophil chemotactic activity was detected in the culture medium from Oesophagostomum aculeatum larvae in vitro using blind-well chambers with Millipore filters, and guinea pig leucocytes as indicator cells. Neutrophil chemotactic activity was also detected in the extract from larval worms in a dose dependent fashion. This activity was detected in the low molecular weight fractions adjacent to a sodium chloride marker by gel filtration on Sephadex G200. These results were further confirmed with monkey neutrophils. The possible role of this activity in the formation of granulomatous lesions rich in neutrophils found in O aculeatum infections in the Japanese monkey is discussed.     

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Streptococcal products and leukocyte activities.

Various streptococcal species are directly responsible for udder infections which should normally be countered by polymorphonuclear neutrophils (PMNs). In order to detect a putative inhibition of streptococcal products on the activities of bovine PMNs, we used a combination of four tests which permits an adequate evaluation of PMNs functions, e.g. PMN adherence on endothelial cells, chemotactic assay, phagocytosis of bacteria labelled with fluorescein isothiocyanate (FITC) and measurement of anion superoxide production. The conclusion is that neither of the two pathogenic streptococcal species isolated from mastitis appeared to produce in vitro factors affecting PMN activities.     

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.     

Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.     

Partial characterization of phagocytic activity in neutrophils of the nine-banded armadillo Dasypus novemcinctus

Though the armadillo is important as a research model in leprosy studies, the activity of armadillo's neutrophils is an aspect of little research. The aim of this study was carried out to partially characterize the chemotaxis, endocytosis and bacteriocidal ability of the neutrophils found in the nine-banded armadillo (Dasypus novemcinctus). Results showed that the chemotactic activity of the neutrophils, evaluated by the movement of the neutrophils through a nitrocellulose membrane (5 microm) in response to a chemo-attractive substance, was greater towards the armadillo serum (5.16+/-1.35 migration index, p<0.05) than towards the formil methionyl leucil phenylalanine (fMLP, 1.43+/-0.18 migration index) or human serum (0.56+/-0.18 migration index). Regarding endocytic capacity of the neutrophils and the monocytes against Escherichia coli was evaluated by a flow cytometry and using opsonized and non-opsonized E. coli-FITC at the following incubation times: 5, 10, 20, 30 and 60 min. The largest percentage of endocytosis by the neutrophils was 92.32+/-0.12% with opsonized bacteria and 77.73+/-14.33% with non-opsonized bacteria at 10 min incubation time, while the largest percentage of endocytosis by monocytes was 89.94+/-1.40% with opsonized bacteria and 73.07+/-15.6% with non-opsonized bacteria at 20 min incubation time. Evaluation of the bacteriocidal capacity of neutrophils using the methyl-thiazol-tetrazolium salts (MTT) reduction color-measurement assay showed an 89.0+/-10% mortality rate of non-opsonized E. coli and 89.0+10% of opsonized E. coli. In conclusion, the armadillo neutrophils show a good phagocytosis and bacteriocidal activity; however, a deficiency in the migration towards the fMLP was observed. This deficiency could be a cause so that the armadillo neutrophils do not respond quickly to invading microorganism.