Neutrophil chemotaxis in rhesus macaques (Macaca mulatta)

Chemotactic responses of isolated peripheral blood neutrophils from rhesus macaques (Macaca mulatta) were studied, using a micropore filter method. Cell migration toward zymosan-activated serum was similar to that of human cells, whereas the response to N-formyl methionyl leucyl phenylalanine (FMLP) was weaker than was that in human cells, requiring higher concentrations of FMLP for maximal migration. Optimal FMLP concentrations for attraction of rhesus neutrophils and human neutrophils were 5.0 X 10(-7)M and 1.0 X 10(-8)M, respectively. The chemotactic responses of the 2 neutrophils to complement (zymosan-activated serum) were similar. However, rhesus neutrophils required a higher concentration of the formyl peptide, FMLP, for maximal migratory response.     

Platelet activating factor is a mediator of equine neutrophil and eosinophil migration in vitro.

Platelet activating factor (PAF) is known to be a chemoattractant for equine neutrophils in vivo and in vitro. In this study the in vitro migratory response of equine eosinophils and neutrophils to PAF has been examined and compared with that to leukotriene (LT)B4. PAF (10(-8) to 10(-5) M), but not lyso-PAF (10(-6) M), caused dose related migration of both equine eosinophils and neutrophils, maximal responses occurring at 10(-6) M. Responses to PAF were inhibited by the receptor antagonist WEB 2086. LTB4 (10(-8) to 10(-6) M) also induced migration of both cell types, although the maximum effect was observed with a 10-fold lower concentration. Moreover, the maximum response of equine eosinophils to LTB4 was significantly greater than to PAF. It is concluded that LTB4 and PAF, if released in vivo at sites of allergic or inflammatory reactions, could mediate the recruitment of leucocytes to the involved tissue.     

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.     

CXCL8 attenuates chemoattractant-induced equine neutrophil migration

The chemokine, CXCL8, is a potent chemoattractant but it has also been shown to attenuate the migratory response of human neutrophils to the bacterial peptide, FMLP; this could lead to retention of cells in infected tissue and, potentially, to enhanced clearance of bacteria. This study has examined the effect of CXCL8 on equine neutrophil migration and adherence in response to PAF and LTB(4), chemoattractants that may play a role in non-infectious inflammatory conditions of the horse associated with neutrophil recruitment to the target tissue. The effects of CXCL8 on PAF- and LTB(4)-induced responses were determined using a ChemoTx plate migration assay and by measuring adhesion to protein-coated plastic. The CXCR1/2 antagonist, SB225002, was used to investigate whether the observed effects were receptor mediated and the role of cAMP was examined by measuring intracellular cAMP following exposure to agonists alone and in combination and by establishing the effect of dibutyryl cAMP on neutrophil migration. CXCL8, LTB(4) and PAF each induced migration and adhesion. Exposure of neutrophils to a combination of CXCL8 and PAF reduced the magnitude of the responses to that of unstimulated cells. In contrast, although the effect was less than additive, the response to co-stimulation with CXCL8 and LTB(4) were not nearly as pronounced. CXCL8 acted in a receptor mediated manner, the attenuation of PAF-induced responses being reversed by SB225002 at a concentration that blocks CXCR2. CXCL8, PAF and LTB(4) alone increased intracellular cAMP. In co-incubation studies, combination of CXCL8 with PAF led to an additive increase in cAMP whereas no increase above that obtained in response to LTB(4) alone was seen. Dibutyryl cAMP significantly reduced neutrophil migration in response to either CXCL8 or PAF alone. These results demonstrate that CXCL8, in addition to being a potent chemoattractant and pro-adhesive molecule for equine neutrophils, is able to attenuate responses to PAF and, to a much lesser extent, LTB(4). This effect, which appears to be CXCR2-mediated and cAMP dependent, could lead in vivo to trapping of cells at sites of inflammation resulting potentially in either enhanced clearance of injurious stimuli or increased local tissue damage by activated cells.     

Comparison of the response of bovine and human neutrophils to various stimuli.

Elastase release, oxidant production and cytoplasmic Ca2+ fluxes by bovine and human neutrophils were compared using sensitive kinetic assays on a photon-counting spectrofluorometer. The stimulants used were phorbol myristate acetate (PMA), cytochalasin B, zymosan opsonized with bovine complement (bOZ) or human complement (hOZ), calcium ionophore, formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The respiratory burst of bovine and human neutrophils was stimulated by PMA and OZ but not by cytochalasin B, or calcium ionophore. Con A weakly stimulated this response in human neutrophils but not bovine. FMLP stimulated the respiratory burst of human but not bovine neutrophils. For evaluation of elastase release, human neutrophils were pretreated with cytochalasin B for 5 min and then stimulated. Cytochalasin B alone did not stimulate elastase release from human neutrophils. Phorbol myristate acetate, calcium ionophore, hOZ, FMLP and Con A did stimulate human neutrophils pretreated with cytochalasin B to release elastase. Human serum OZ was also able to stimulate elastase release from human neutrophils not pretreated with cytochalasin B. Some bovine neutrophils released elastase in response to cytochalasin B alone. Those bovine neutrophils that did not release elastase in response to cytochalasin B alone released elastase when stimulated with Con A or calcium ionophore after cytochalasin B pretreatment. Bovine neutrophils did not release elastase in response to FMLP or PMA with or without cytochalasin B pretreatment, but did release elastase in response to bOZ alone. Total elastase activity of bovine neutrophils was determined to be about 50 times less than that of human neutrophils. Intracellular calcium fluxes were stimulated in human neutrophils by calcium ionophore, FMLP, hOZ and Con A but not by PMA or cytochalasin B. Bovine neutrophil calcium fluxes were stimulated by calcium ionophore, Con A and bOZ; cytochalasin B also stimulated bovine neutrophils to increase cytoplasmic calcium concentration. Cytoplasmic calcium fluxes were not stimulated in bovine neutrophils by PMA or FMLP. In summary, human and bovine neutrophils respond similarly to calcium ionophore and OZ, but differently to PMA, cytochalasin B, Con A and FMLP.     

Response of heifer mammary gland macrophages and neutrophils to interferon-gamma stimulation in vitro.

The phagocytic and killing abilities of heifer mammary gland macrophages (M phi) and neutrophils were evaluated after exposure to recombinant bovine interferon-gamma (rBoIFN-gamma) stimulation in vitro. Macrophages or neutrophils were cultured for 2 h with 0, 10(2), 10(4) and 10(5) units rBoIFN-gamma/mL. Phagocytosis assays were performed by incubation with Staphylococcus aureus at a leukocyte:bacteria ratio of 1:10. After 45 min, cells were stained with acridine-orange and phagocytic and killing abilities were determined. Although rBoIFN-gamma had no effect on M phi phagocytic activity, neutrophil phagocytic activity after incubation in 10(4) units rBoIFN-gamma (41.62%) was significantly higher than 0 (25.24%) or 10(2) units rBoIFN-gamma (24.73%). Neutrophil and M Phi killing abilities were not affected by any dose of rBoIFN-gamma. Results suggested that rBoIFN-gamma promoted neutrophil phagocytic activity, but did not affect neutrophil killing or overall M phi function in vitro.     

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.     

In vivo chemotaxis of rat leucocytes to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)

FMLP was potently chemotactic in vivo to rat leucocytes. Doses of 1 microgram attracted net leucocytes into experimental cotton pellets in 6 h. Maximal chemotactic activity occurred at 0.01 mg. Doses greater or less than 0.01 mg attracted less net leucocytes into experimental pellets. The effect of time on chemotaxis of 0.05 mg FMLP showed that between 3 and 36 h, chemotactic index remained above unity and rose maximally to over 1.5. Maximal chemotactic index occurred at 6 h followed by maximal leucocytic infiltration. FMLP showed early chemoattraction in vivo to rat leucocytes. Leucocytic migration into control pellets rose from 3 h, reached maximum levels at 24 h and remained almost at this level at 36 h. L-methionyl-L-leucyl-L-phenylalanine (MLP), studied as a negative control, showed a chemotactic index and chemotactic differential that rose and fell together between 3 and 36 h. The kinetics of migrating leucocyte populations in response to FMLP showed absolute polymorphonuclear leucocytes at 3 h and over 90% mononuclear leucocytes at 36 h.     

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.     

Suppressive effect of deoxynivalenol, a Fusarium mycotoxin, on bovine and porcine neutrophil chemiluminescence: an in vitro study

We evaluated the immunotoxicity of deoxynivalenol (DON), a Fusarium mycotoxin, on bovine and porcine neutrophils in vitro by using two function parameters, luminol-dependent chemiluminescence and random migration under agarose. A 2-hr DON treatment suppressed the chemiluminescence of bovine and porcine cells by 42% and 35% (on average) at 10(-5)M, and by 19% and 26% at 10(-6)M. Slight suppression was observed at concentrations lower than 10(-6)M. However, after an 18-hr DON treatment, random migration of neutrophils of both species remained unaffected, even at the highest concentration (10(-5)M). Although further extensive studies are needed, to our knowledge this is the first study to have revealed in vitro that DON can affect neutrophil function.     

A colorimetric assay for quantitating bovine neutrophil bactericidal activity

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.     

Modulation of equine neutrophil adherence and migration by the annexin-1 derived N-terminal peptide, Ac2-26

Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived.     

Innate immune responses of temperamental and calm cattle after transportation

The objective was to investigate measures of cellular innate immune responses among calm and temperamental Brahman bulls in response to handling and transportation. Sixteen Brahman bulls (344 ± 37 d of age; 271.6 ± 45.5 kg BW) classified as either calm (n=8) or temperamental (n=8) were loaded onto a trailer, transported for 4h to a novel facility, rested 16 h overnight, and then were returned to their original facility after a 4h transport. Blood samples were collected immediately prior to (time 0) and at 24, 48, and 96 h after initial loading for analyses of innate immune and blood parameters. Leukocyte counts did not differ (P>0.05) due to temperament before or after transportation, but neutrophil:mononuclear cell ratios were greater in temperamental bulls compared to calm bulls at 24h. At 24h, expression of peripheral neutrophil β(2)-integrin decreased among all bulls compared with 0 h (P<0.01). Temperamental bulls had greater glucose and cortisol than calm bulls (P<0.01) at 48 h; whereas calm bulls had elevated neutrophil L-selectin expression, and phagocytic and oxidative burst activity compared with temperamental bulls (P<0.10) at 48 h. The supernatant collected from endotoxin-stimulated whole blood cultures had greater TNF-α concentrations at 48 h than at the other time points (P<0.05), but no temperament effect was observed (P>0.05). In contrast, 96 h after initial loading the supernatant TNF-α concentrations were lower (P<0.05) among all cattle. Lastly, transportation increased neutrophil phagocytosis, oxidative burst, and cell adhesion molecule expression 96 h post-transportation and the effect was more pronounced among calm bulls. These data suggest that neutrophils from calm bulls are more likely to resist microbial invasion at 96 h after transportation than neutrophils from temperamental bulls.     

Linoleic acid increases adhesion,chemotaxis, granule release,intracellular calcium mobilisation,MAPK phosphorylation and gene expression in bovine neutrophils

Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 μM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 μM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2–5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2 h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.     

Preincubation of bovine blood neutrophils with bovine herpesvirus-1 does not impair neutrophil interaction with Pasteurella haemolytica A1 in vitro

In this study we examined the direct effects of bovine herpesvirus-1 on the interaction of bovine blood neutrophils with Pasteurella haemolytica A1. Preincubation of neutrophils for approximately 2 h in vitro with BHV-1 at a multiplicity of infection of 5:1 had no effect on neutrophil random migration and directed migration to zymosan-activated bovine serum. Neutrophils also were unimpaired in their ability to ingest and kill P. haemolytica A1. Preincubation of neutrophils with BHV-1 did not elicit an oxidative burst, as measured by luminol-enhanced chemiluminescence, nor did it alter neutrophil chemiluminescence in response to opsonized P. haemolytica A1. Prolonged preincubation with BHV-1 for 18-24 h similarly did not affect neutrophil chemiluminescence in response to opsonized P. haemolytica A1. The susceptibility of neutrophils to the lethal effects of crude P. haemolytica cytotoxin also was unaltered by preincubation with BHV-1. We observed no evidence of BHV-1 replication in bovine neutrophils as determined by indirect immunofluorescence and electron microscopy. Previous reports have indicated that active BHV-1 infection alters certain neutrophil functions and results in hypersusceptibility to pulmonary pasteurellosis. Our results suggest that these effects are unlikely to be mediated directly by BHV-1, but instead may reflect the action of endogenous mediators that are released during active BHV-1 infection.     

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.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.     

In vivo chemotaxis of oxidised lipids: 1. Chemotactic effects of oxidised arachidonic acid on rat leucocytes

In vivo chemotaxis to rat leucocytes was shown to mildly oxidised arachidonic acid in the migration into cotton pellet method. Amounts of oxidised acid, 0.25, 0.5 and 2.5 mg, mobilized net leucocytes (mostly neutrophils) into experimental pellets in 12 h. The 0.5 mg of oxidised acid showed maximum chemotactic activity and attracted maximum net leucocytes into the experimental pellets at 12 h. Doses lower or higher than this were less effective. Equivalent doses of pure arachidonic acid failed to show these chemotactic activities but evoked inflammatory reactions at the experimental pellet sites. Chemotactic activity seemed therefore to require mild oxidation of the lipid. Oxidation also seemed to stimulate spontaneous migratory activity into the control pellets.     

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.     

An Elevated Dietary Level of Ascorbic Acid Fails to Influence the Response of Anterior Kidney Neutrophils to Edwardsiella ictaluri in Channel Catfish

Abstract

Channel catfish Ictalurus punctatus (weight, 320–420 g) were fed diets with 100 or 1,000 mg ascorbic acid/kg of diet for 7 weeks in ponds. Anterior kidney neutrophil function was assessed by incubating the neutrophils with Edwardsiella ictaluri. Percent phagocytosis, phagocytic index (the mean number of bacteria phagocytized per cell by all neutrophils, including nonphagocytic ones), and bactericidal capabilities of the anterior kidney neutrophils were measured at 0, 90, 180, 270, and 360 min. Serum cortisol was also measured. No significant difference was observed between the two diets with respect to anterior kidney neutrophil function or serum cortisol levels. However, serum cortisol levels did affect mean phagocytic index of anterior kidney neutrophils. Catfish that had serum cortisol levels greater than 10 μg/dL displayed a significant decrease in the phagocytic index when compared with catfish that had cortisol levels less than 5 μg/dL.