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.     

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.     

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.     

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.     

Elaboration of chemotactic substances by alveolar cells: possible mechanisms for the initial neutrophilic response in feline caliciviral pneumonia.

In vitro experiments established that the interaction of feline calicivirus (FCV) with alveolar macrophages and pneumocytes results in the generation of chemotactic factors that produce directed migration of neutrophils in Boyden chambers. Factors were produced independent of immune mechanisms and of discernible serum factors. Lysates of noninfected alveolar macrophages and pneumocytes did not possess chemotactic activity, indicating that chemotactic factors were not preformed in these cells. Noninfected alveolar macrophages also elaborated neutrophil chemotactic factors in culture; however, activity was greater when macrophages were infected with FCV. The results of this study suggested that the neutrophilic response in the peripheral portion of the lung of cats exposed to aerosols of FCV was the result of the elaboration of complete chemotactic factors from FCV-infected alveolar cells.     

Cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis

Both random and directional migration of blood neutrophils from 9 mares susceptible to persistent endometritis were significantly less (p less than 0.05) than neutrophils from 8 resistant mares. Serum from susceptible mares had significantly more (p less than 0.01) chemotactic activity than serum from resistant mares. Although phagocytosis of yeast blastospores by blood neutrophils from 4 resistant and 3 susceptible mares was similar, uterine neutrophils from susceptible mares were significantly worse (p less than 0.01) at phagocytosis than uterine neutrophils from resistant mares. Uterine washings from 17 susceptible mares were significantly better at opsonising yeast blastospores than washings from 14 resistant mares; however, washings from both groups had a similar ability to promote killing of S. zooepidemicus by neutrophils. When an immunologically non-specific endometritis was induced, washings from 3 susceptible mares were significantly worse at promoting bactericidal activity by 144 h than washings from 4 resistant mares (p less than 0.01). Haemolytic complement activity was significantly greater (p less than 0.001) in washings from 17 susceptible mares than from 14 resistant mares. Induction of acute endometritis resulted in high levels of haemolytic complement activity in 2 of 3 susceptible mares at 24 and 144 h, but only in small increases in 4 resistant mares. Thus, some abnormalities in neutrophil function were detected and a possible defect in promotion of neutrophil bactericidal activity by uterine secretions from susceptible mares but there was no evidence for any deficiency in haemolytic complement activity.     

Chemotactic and chemokinetic activity of Streptococcus faecalis culture supernatant for equine neutrophils

Although equine neutrophils did not respond towards formylated methionyl peptides, Streptococcus faecalis culture supernatant caused an in vitro stimulation of equine neutrophil motility when measured by an under-agarose assay. The migration of neutrophils towards the culture supernatant increased sigmoidally with the logarithmic concentration of the culture supernatant in the chemoattractant wells. The streptococcal culture supernatant was chemokinetic because it stimulated the random motility of the phagocytes. Because granulocytes migrated further towards the supernatant than could be explained by the chemokinetic activity of the bacterial products, the streptococcal culture fluid also exerted a chemotactic effect on the leukocytes. The chemotactic activity of the supernatant was further confirmed by the changes in the orientation of the migrating cells during incubation. These results indicate that bacteria produce cytotaxins other than formylmethionyl peptides which are recognized by equine neutrophils.     

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.     

Interleukin-8 concentration and neutrophil chemotactic activity in bronchoalveolar lavage fluid of horses with chronic obstructive pulmonary disease following exposure to hay

OBJECTIVE: To analyze effects of hay dust exposure on interleukin-8 (IL-8) concentration, percentage of neutrophils, and neutrophil chemotactic activity in bronchoalveolar lavage fluid (BALF) of horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 16 healthy horses and 29 horses with COPD. PROCEDURE: IL-8 concentration, percentage of neutrophils, and neutrophil chemotactic activity in BALF were measured. Values were analyzed with respect to hay dust exposure.These variables were also measured in 5 asymptomatic horses with COPD after the induction of clinical signs by changing feed from silage to hay. RESULTS: L-8 concentrations and chemotactic activity in BALF were greater in horses with COPD, compared with healthy horses, and greater in horses with COPD exposed to hay dust, compared with nonexposed affected horses. An increase in IL-8 concentration accompanied by an increase in percentage of neutrophils in BALF and development of clinical signs of COPD were induced in asymptomatic horses with COPD by changing feed from silage to hay. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of horses with COPD to hay dust components resulted in an increase in IL-8 secretion at the bronchoalveolar surface. This chemokine may play a role in the pathogenesis of COPD, because it causes neutrophil accumulation in the bronchoalveolar space. Our results underscore the importance of eliminating dust sources for the treatment and prevention of COPD in horses.     

Effect of selenium deficiency on caprine polymorphonuclear leukocyte production of leukotriene B4 and its neutrophil chemotactic activity

These studies were designed to measure leukotriene B4 (LTB4) production by polymorphonuclear leukocytes (PMN) from selenium (Se)-deficient and Se-adequate goats. Leukotriene B4 was measured by both radioimmune and chemotactic activity assays in supernatants of PMN stimulated by calcium ionophore A23187. The concentration of LTB4 produced by PMN from Se-deficient goats was significantly (P less than 0.05) lower than the concentration of LTB4 produced by PMN from Se-adequate goats. Neutrophil chemotactic activity of LTB4 was found to be directly dependent (r2 = 0.85) on the LTB4 concentration. When supernatants from Se-deficient and Se-adequate goats were adsorbed with caprine neutrophils, significant (P less than 0.05) reduction in LTB4 was found in supernatants from both groups. Furthermore, neutrophil adsorption of LTB4 from supernatants of both groups was indicated by significantly (P less than 0.05) decreased chemotaxis to LTB4 supernatants. Decrease in chemotactic response was, however, significantly (P less than 0.05) lower when neutrophils were treated with supernatants from Se-deficient goats. These results indicate that Se deficiency decreases the production of LTB4 by caprine PMN and, hence, LTB4-mediated neutrophil chemotaxis.     

Chemotaxis of porcine neutrophils under agarose.

The standardization of pig neutrophil chemotaxis under agarose is described. The mean chemotactic index for pig neutrophils from six pigs measured over four days was 1.29. Comparative studies of human and pig neutrophil chemotaxis under agarose revealed a lower chemotactic index for pig neutrophils (mean of 1.18) compared to human neutrophils (mean of 2.43). The results suggest that this is due to differences intrinsic to human and pig neutrophils. In vitro pig neutrophil chemotaxis was measured in normal pigs and in pigs following experimental Salmonella typhimurium infection. Significant alterations in chemotaxis were not evident one and seven days postinfection.     

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

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.     

Bovine neutrophils treated with chemotactic agents: morphologic changes

Neutrophils were isolated from the peripheral blood of cattle. After the neutrophils were incubated with zymosan-activated serum, the neutrophils changed from spherical to a bipolar shape. Ninety percent of the neutrophils became bipolar in 5 to 10 minutes. Bacterial cell filtrate and casein also induced bipolar shape changes in neutrophils, but N-formyl-L-methionyl-L-leucinyl-L-phenylalanine did not. The neutrophil-shape-change response was a rapid in vitro assay to evaluate early chemotactic events.     

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.     

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.     

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.     

In vitro function of bovine neutrophils against Actinomyces pyogenes

Factors that influenced the in vitro bactericidal activity of bovine neutrophils against Actinomyces pyogenes were investigated. Neutrophils and serum from 2 clinically normal donor cows were incubated with bacteria for 2 hours. To determine bactericidal activity, colony-forming units were counted after a 48-hour incubation on blood agar plates. Microscopic examination indicated that in the presence of serum, bacteria were cell associated after incubation, whereas when serum was replaced by medium, bacteria were not cell associated. Bactericidal activity of neutrophils was similar whether the sera were heat-treated at 56 C for 30 minutes or were not heated. Heating the serum at 65 C for 30 minutes significantly (P less than 0.001) reduced bactericidal activity. Bactericidal activity decreased (P less than 0.001) as serum concentration (less than 10%) decreased. More than 80% of the bacteria were killed within the 40 minutes of incubation. The opsonizing capacity of serum varied significantly (P less than 0.01) among 12 cows. Similarly, neutrophil bactericidal activity (by cow) was affected significantly (P less than 0.001). Preincubation of serum with A pyogenes significantly (P less than 0.001) reduced the opsonizing ability of the serum. Culture filtrate of A pyogenes was not chemotactic for neutrophils in vitro.     

Maturation of cellular defence in the respiratory tract of young calves

The maturation of respiratory tract defence was investigated in a longitudinal study of calves during the first 100 days of life. From day 7, the proportions of the cell types identified in bronchoalveolar lavage (BAL) fluid were similar to those found in adults, with a predominance of alveolar macrophages over polymorphonuclear neutrophils (PMNs) and lymphocytes. Functionally, bactericidal activity of BAL cells was defective and for the first 21 days they supported intracellular bacterial growth. At 24 hours of life, the movement of peripheral blood neutrophils to a chemotactic source was poor, but this increased rapidly during the first week of life. Like BAL cells, peripheral blood PMNs supported intracellular bacterial growth for the first two weeks of life. These studies suggest that cellular defence mechanisms may be compromised during the first week of life.     

Impairment of neutrophil functions in a dog with an eosinophilic dermatosis.

An eosinophilic dermatosis was diagnosed in a dog with a multifocal distribution of pruritic, pustular and erosive lesions on the trunk. Ectoparasites, fungi and bacteria were not detected in the skin of the dog. At a subsequent admission to the university clinic, bacterial conjunctivitis and superficial pyoderma had developed. At a time when the dog received no medical therapy, evaluation of phagocytosis and chemotactic migration towards a variety of chemotactic factors revealed a general suppression of patient neutrophil responsiveness, as compared to a control dog. Viability of the patient's neutrophils was normal. It was proposed that deactivation of neutrophil functions occurred following exposure to suppressive factors from mast cells, or as a consequence of surface receptor down-regulation due to prolonged cell activation by as yet unknown stimulants. The observed susceptibility to infections in the dog suffering from a primary, sterile eosinophilic dermatosis may be related to impaired host defence against opportunistic microorganisms.