J. Vet. Med. A 46, 613–619 (1999)© 1999 Blackwell Wissenschafts-Verlag, BerlinISSN 0931–184X

Istituto di Patologia Generale Veterinaria, Milan, Italy

Non-specific Immunity and Ketone Bodies. I: In Vitro Studies

on Chemotaxis and Phagocytosis in Ovine Neutrophils

P. SARTORELLI1, S. PALTRINIERI and F. AGNES

Address of authors: Istituto di Patologia Generale Veterinaria, Via Celoria 10, 20133 Milan, Italy;1Corresponding author: E-mail: Paola.Sartorelli@unimi.it

With 2 figures and 1 table

(Received for publication May 18, 1999)

Summary

The in vitro effects of the ketone bodies b-OH-butyrate (2.4 or 4.8mmol/l) and acetoacetate (2.4 or4.8mmol/l) on the uptake of latex particles (1.09 mm) and chemotaxis were investigated in ovine neutro-phils. Because the acetoacetate used was a lithium salt, the effect of 2.4 or 4.8mmol/l lithium chloride wasalso tested. Neutrophils from eight non-lactating, non-pregnant ewes were studied. The uptake of latexparticles, as measured by a spectrophotometric method, showed wide individual variation. The phagocytoticactivity was unaffected by 2.4mmol/l ketone bodies and LiCl, but it was significantly inhibited by4.8mmol/l b-OH-butyrate and activated by 4.8mmol/l LiCl. The latter result could be masking aninhibitory effect of acetoacetate. Chemotactic movements of neutrophils, as evaluated in a modifiedBoyden chamber using homologous zymosan-activated serum (ZAS) as a chemoattractant, were slightlybut significantly reduced by a 2.4 mmolar concentration of the ketone bodies, administered singly orsimultaneously, and by LiCl. We conclude therefore that the inhibitory effect of lithium-acetoacetate couldbe due to its lithium component. The 4.8mmol/l dose of acetoacetate and b-OH butyrate significantlydecreased chemotaxis only when both compounds were added simultaneously. No effect of 4.8mmol/lLiCl was observed. These results suggest that ketone bodies, in particular b-OH butyrate, could directlyinfluence particle uptake and chemotaxis in neutrophils. Although other factors could decrease theefficiency of the immune system in ketotic ruminants, the effects of the ketone bodies on neutrophilsfunctions may explain the high frequency of infectious disease during ‘ketotic syndrome’. The immuno-modulatory effect of lithium needs to be evaluated further and it should be considered when testing lithiumcompounds.

Introduction

Genetic selection and intensive husbandry and feeding have led to a breakdown in theability of ruminants to regulate their metabolic pathways. An increase in production togetherwith the peculiar anatomy and physiology of the alimentary tract of ruminants are responsiblefor input/output imbalances during critical steps of the productive cycle (late pregnancy andlactation). Energy metabolism becomes overstressed when glucose requirements are increasedby the energy demands of the developing foetus or by glucose uptake from a lactose-synthesizingudder. Both glyconeogenesis and lipolysis are stimulated, with a subsequent increase in bloodketone bodies, considered a para-physiological condition. When energy and glucose require-ments are excessive, as in high-production dairy cows or in twin-pregnant sheep, ketone bodiesincrease beyond physiological levels, leading to a disease known as ‘ketosis’ in cows or ‘pregnancytoxaemia’ in sheep (Kaneko et al., 1997)

Ketosis and pregnancy toxaemia are characterized by an increase in ketone bodies (b-OH-butyrate ×1 mmol/l; acetoacetate ×0.35 mmol/l) in blood, urine and milk (Grohn et al.,

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614 SARTORELLI et al.

1983; Kauppinen, 1983). Other haematochemical abnormalities include inconstant hypo-glycaemia (³3 mmol/l) (West, 1990), a decrease in serum tryglicerides, cholesterol and lipo-proteins, and an increase in serum non-esterified fatty acids (NEFA) and hepatic enzymes. Thishaematochemical pattern reflects the involvement of the liver in the pathogenesis of the diseaseas a consequence of lipolysis. From a clinical point of view, genuine symptoms are detectablein only a few cases (Payne, 1989). More frequently (× 80 %) bovine ketosis has a subclinicalcourse, the only evidence being a decrease in milk production (4–6 %) that greatly shortens theanimal’s productive life (Doho and Martin, 1984; Klug and Franz, 1991). In sheep, clinicalnervous symptoms are common, but many sheep recover after abortion of the fetus: completerecovery depends on complicating factors such as cerebral or renal damage due to dehydrationand acidosis. However, even when hyperketonaemia is subclinical, leukopenia (Kuzma et al.,1997) and a high prevalence of infectious disease (especially mastitis and metritis) have beenreported (Kaneko et al., 1997). This could be due to an immunodepressive state (Payne, 1977),since functional alterations of lymphocytes (Franklin et al., 1991) and granulocytes (Klucinskiet al., 1988) have been reported.

In order to study the role of non-specific immunity in ketosis, in this study we haveevaluated the in vitro effects of ketone bodies, at concentrations corresponding to those seen inmild (2.4 mmol/l) and severe (4.8 mmol/l) ketosis, on the functional activities, such as chemo-taxis and uptake ability, of sheep neutrophils (PMNs).

Materials and Methods

Blood was sampled from the jugular vein of eight non-lactating, non-pregnant, clinically healthy ewes,which were housed in a barn, given a diet of hay and concentrates and unlimited access to water. Animalswere initially accustomed to blood sample collection, in order to avoid the influence of stress on PMNs.

The number of white blood cells was evaluated by an automatic counter (Coulter counter) and thedifferential leucocyte count was made on a blood smear stained with May–Grunwald–Giemsa.

Neutrophil separation

Blood PMNs were separated by the method of Carlson and Kaneko (1973): briefly, 40ml of bloodwas added to 4ml of phosphate-buffered saline (PBS) containing 40mmol/l ethylene diaminetetraaceticacid (EDTA) as an anticoagulant and was centrifuged (1000 g for 15min); the plasma, the buffy coat andthe upper layer of red blood cells were discarded. The remaining red blood cells, including sedimentedPMNs, were lysed with distilled water and isotonicity was regained by adding a hypertonic solution (NaCl0.39mol/l). As suggested by Buchta (1990), two hypotonic lyses were carried out. After centrifugation(200 g for 10min), the resulting pellet was resuspended and washed twice with PBS; cells were resuspendedin 1ml Hanks’ balanced salt solution (HBSS), their number was evaluated and adjusted to a finalconcentration of 2× 107 cells/ml. At the end of the separation procedure, cell viability was evaluated bythe trypan blue exclusion test (Metcalf et al., 1986). To evaluate the differential leucocyte count, 100ml ofthe cell suspension (106 cells/ml) was cytocentrifuged (50 g for 1min) in a multiwell settling and adherencechamber (Neuroprobe, Cabin John, MD, USA), the slide was stained with May–Grunwald–Giemsa andobserved at ×1000 magnification. To minimize stimulation of PMNs from environmental microorganisms,all solutions were sterilized by filtration, and sterile plastic tubes were used.

Phagocytosis

PMNs (final concentration 107 cells/ml) were incubated at 37°C for 30min with polystyrene latexbeads (1.09mm diameter). The PMN : latex bead ratio was 1 : 20 (Tater et al., 1987). To test the effect ofketone bodies, 2.4 or 4.8mmol/l b-OH-butyrate (Sigma, St Louis, MO, USA) and/or 2.4 or 4.8mmol/lacetoacetate (Sigma) were added to the incubation medium. Since the acetoacetate used was a lithium salt,the effect of 2.4 or 4.8mmol/l LiCl (Sigma) was also tested. After incubation, the phagocytosis wasterminated with PBS containing 40mmol/l EDTA and cells were washed twice to eliminate the non-phagocytosed latex beads. The ingested latex was dissolved by incubating with 2ml of undiluted 1-4 dioxan(Merck, Whitehouse Station, NJ, USA) for 20 h at 23°C and the optical density (OD) was estimated in aspectrophotometer against a blank containing dioxan at 255.5 nm (Kvarstein, 1969).

615Chemotaxis and Phagocytosis in Ovine Neutrophils

Chemotaxis

Chemotaxis was evaluated in a 12-well modified Boyden chamber (Neuroprobe) using a cellulose–nitrate filter (thickness, 150 mm; pores, 3mm). As a chemotactic stimulus, 150 ml of pooled sheep zymosan-activated serum (ZAS; 50% in HBSS with 20mmol/l HEPES and 10mg/ml albumin) (Gray et al., 1982)was placed in the lower well; the upper well was filled with 120ml of cell suspension (2× 106 cells/ml).In the negative control ZAS was omitted to evaluate the spontaneous movements. To test the effect ofketone bodies, 2.4 or 4.8mmol/l b-OH-butyrate and/or 2.4 or 4.8mmol/l acetoacetate was added bothin the lower and in the upper well, in both chemotaxis and in negative control tests. The effect of 2.4 and4.8 LiCl mmol/l was also tested. Each test was carried out in triplicate. After incubation at 37°C in 5%CO2 for 1 h (Heraeus BB16, Heraeus Instruments, Hanau, Germany), the filter was removed, fixed in70% ethanol, rehydrated and stained for 2min with Mayer Hematoxylin (Merck); it was then dehydratedin alcohol (70%, 95%, absolute ethanol), clarified in xylene, put upsidedown on a slide and mounted witha non-aqueous medium (Eukitt – Kindler, Freiburg, Germany). The evaluation of chemotaxis was per-formed by the Leading Front method (Zigmond and Hirsh, 1973): using the micrometer fine adjustment,the distance between the focal plane corresponding to the top of the filter, where a large part of the cellswas detected, and the leading front, where the two last cells were in focus, was measured and expressedin mm.

To test for a possible effect of ketone bodies or lithium on cell viability, PMNs were incubated for1 h in HBSS containing 2.4 or 4.8mmol/l acetoacetate, b-OH butyrate, or LiCl and the trypan blueexclusion test was performed.

Statistical analysis

Results were analysed by ANOVA. using Statistica 5.1 (Statsoft Inc., Tulsa, OK, USA). To evaluatethe possible influence of different white cells on PMN activity, a Spearman correlation test between theresults and the absolute and relative number of other leucocyte populations in whole blood and afterisolation was carried out.

Results

Mean values of PMNs purity (68.8 % 2 13 %), viability (96.7 % 2 1.3 %) and recovery(37 % 2 11 %) were similar to those obtained in previous studies (Sartorelli and Paltrinieri,1991). The most contaminating cells were eosinophils (17.1 % 2 8.9 %) and mononuclear cells(lymphocytes and monocytes) (14.5 % 2 13.2 %). A positive correlation between the numberof PMNs in the blood and those of the isolated PMNs (P ³ 0.001; r = 0.66) was observed. Nocorrelation was found between the cell populations in the blood and the recovery rate. Incu-bation for 1 h of isolated PMN in HBSS containing 2.4 or 4.8 mmol/l ketone bodies and LiCldid not alter cell viability.

Phagocytosis

Latex beads uptake was unaffected by 2.4 mmol/l acetoacetate or by 2.4 mmol/l b-OH-butyrate, administered singly or simultaneously, or by 2.4 mmol/l LiCl. Uptake was significantlydepressed when 4.8 mmol/l b-OH-butyrate was added in vitro to the cell suspension (P ³ 0.05).The 4.8 mmol/l acetoacetate administered by itself or together with b-OH-butyrate had noeffect, while 4.8 mmol/l LiCl significantly stimulated latex ingestion (P ³ 0.05) (Table 1).

Chemotaxis

Chemotactic movements induced by ZAS were significantly greater than spontaneousmovements (P ³ 0.001). No effect of ketone bodies or LiCl at 2.4 or 4.8 mmol/l on negativecontrol was observed. A small but statistically significant reduction of neutrophil chemotaxis(P ³ 0.05) was seen when 2.4 mmol/l ketone bodies were added singly or simultaneously;2.4 mmol/l LiCl induced a similar reduction of chemotaxis (Fig. 1). Both 4.8 mmol/l aceto-acetate and LiCl were ineffective while b-OH butyrate alone or with acetoacetate decreasedchemotaxis (Fig. 2); the percentage of inhibition was similar at both concentrations (about 5 %and 8 %, respectively).

616 SARTORELLI et al.

Table 1. In vitro ingestion of latex particles by sheep PMNs in basal conditions (C) and in the presenceof 2.4 or 4.8 mmol/l acetoacetate (A), b-OH butyrate (B), acetoacetate plus b-OH butyrate (AB),lithium chloride (Li). Ingested latex was solubilized by 1-4 dioxan and results were expressed as OD

(mean 2 SD) at 255.5 nm

C A B AB Li t-test

2.4 0.3142 0.233 0.2812 0.208 – – – n.s.0.350 2 0.224 – 0.419 2 0.384 – – n.s.0.333 2 0.453 – – 0.453 2 0.344 – n.s.0.235 2 0.183 – – – 0.209 2 0.172 n.s.

4.8 0.2152 0.219 0.2142 0.165 – – – n.s.0.215 2 0.219 – 0.122 2 0.127 – – *0.300 2 0.209 – – 0.274 2 0.241 – n.s.0.313 2 0.209 – – 0.464 2 0.247 *

t-test n.s. n.s. n.s. n.s. n.s.

* P ³ 0.05 versus C.

140

120

100

80

60

40

20

0

µm

§§§ ✽ ✽✽ ✽

C ZAS LiABBA

Fig. 1. Negative control (C) and ZAS-stimulated chemotaxis of sheep PMNs, alone (ZAS) or in thepresence of 2.4 mmol/l acetoacetate (A), b-OH butyrate (B), acetoacetate plus b-OH butyrate(AB), lithium chloride (Li). Results were expressed as mm run by PMNs. *P ³ 0.05 versus ZAS;

§§§ = P ³ 0.001 versus C.

Chemotaxis and negative control were negatively correlated with the number of mono-nuclear blood cells (P ³ 0.01; r = 0.61). A similar negative correlation with the number and thepercentage of mononuclear blood cells was seen for chemotaxis in the presence of 2.4 and4.8 mmol/l ketone bodies.

Discussion

The method employed for PMNs isolation is widely used for ruminants neutrophils anddoes not employ substances (Dextran, Fycoll, etc.) that could alter cell function (Venaille et al.,1994). For sheep PMNs, unlike bovine PMNs, two osmotic lyses are required to eliminate redblood cells (Buchta, 1990), but cell viability at the end of the isolation procedure is high. Inaddition, osmotic lysis does not alter the integrity of PMNs in other species (Gruber et al.,1990). The separation method does not allow a good separation of eosinophils but these cellsshould not interfere with those PMN activities we have tested, because they were never seen

617Chemotaxis and Phagocytosis in Ovine Neutrophils

140

120

100

80

60

40

20

0

µm

§§✽

✽

C ZAS LiABBA

Fig. 2. Negative control (C) and ZAS-stimulated chemotaxis of sheep PMNs, alone (ZAS) or in thepresence of 4.8 mmol/l acetoacetate (A), b-OH butyrate (B), acetoacetate plus b-OH butyrate(AB), lithium chloride (Li). Results were expressed as mm run by PMNs. *P ³ 0.05 versus ZAS;

§§ = P ³ 0.01 versus C.

in the cytocentrifugate to phagocytose latex beads, and do not migrate into the filter, probablybecause they respond to different chemotactic stimuli (Tizard, 1994). Contaminating mono-nuclear cells were generally lymphocytes, with a few monocytes. Monocytes cannot passthrough small pores (3 mm), but may phagocytose latex beads and cause uptake activity to beoverestimated. Moreover, both lymphocytes and monocytes could influence PMNs activitiesby producing cytokines such as interleukin-2 (IL-2) or IL-8 (Mulder and Colditz, 1993; Salak etal., 1993). However, we found that contaminating mononuclear cells were in most cases veryscarce.

Mean values of latex uptake and chemotaxis were similar to those previously recorded insheep (Sartorelli and Paltrinieri, 1991). Both cell activities showed wide individual variation, asalready noted for humans and other animal species (Paape and Pearson, 1979). This variabilitycould partly be due to the frequent stimulation of non-specific immunity in vivo by environmentalmicroorganisms. PMNs can also become stimulated in vitro by the separation procedure, butthis should be minimized by using filter-sterilized solutions and sterile tubes.

Our results indicate that ketone bodies, at concentrations seen in mild and severe ketosis,are able to affect some functional activities of ovine PMNs.

Ingestion of latex beads was only decreased by 4.8 mmol/l b-OH butyrate. The lack ofeffect of 4.8 mmol/acetoacetate is probably due to the activating effect of lithium chloride atthe same concentration, that completely overcomes the decreasing effect of acetoacetatealone or together with b-OH butyrate. Klucinski et al. (1988) reported that ketone bodies, atconcentrations from 0.1 mmol/l to 4.8 mmol/l, were able to decrease the percentage of bovineblood PMNs that phagocytose Staphylococcus aureus by immunological receptors, while phago-cytosis by non-immunological receptors was inhibited only at the highest concentrations. Latexbeads, used in the present work, are ingested because they are highly hydrophobic, without theuse of immunological receptors involvement; inhibition by ketone bodies was seen only at4.8 mmol/l and could be due to a change of membrane electrical charge or to damage of thelipid layer. Klucinski et al. (1988), who also used lithium acetoacetate, did not test a possibleeffect of lithium on percentage of phagocytosing bovine blood PMNs.

Our chemotaxis results confirm previous findings (Mulder and Colditz, 1993; Paltrinieriet al., 1996) that ZAS is a good chemoattractant for ovine PMNs, which, like those of otherungulates, are not sensitive to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine(f-MLP), employed for human cells (Gray et al., 1982). The apparent inhibitory effect of2.4 mmol/l acetoacetate on ZAS-stimulated chemotaxis was probably due the effect of lithium,

618 SARTORELLI et al.

so that only 2.4 mmol/l b-OH butyrate could decrease chemotaxis; also at 4.8 mmol/l only b-OH butyrate alone or with acetoacetate decreased chemotaxis. b-OH butyrate is the only ketonebody that reaches such a high concentration in severe ketosis in vivo. Short-chain carboxylicacids, at concentrations of 5 mmol/l or greater, were reported to alter pH and/or cytoskeletalF-actin of human PMNs, which could impair cell movements (Brunkorst et al., 1992). This wasunlikely for sheep PMNs, because in our experiment negative control was not affected byketone bodies at both concentrations; instead, an interference with the response to activatingstimuli could be involved.

Mononuclear blood cells are negatively correlated with the chemotaxis of PMNs, and thiscould be due to the release of lymphokines or monokines (Mulder and Colditz, 1993).

Lithium effects were first studied in its role as an antidepressant in humans, and it hasbeen reported to inhibit the inositol phospholipid second messenger generating system inhuman PMNs (Forstner et al., 1994). Lithium exhibits immunomodulatory activity in severalspecies, by altering the number and proportion of B and T lymphocytes (Murphy et al., 1971)and T-lymphocyte proliferation in response to mitogens in vitro (Franklin et al., 1991). In dogs,lithium stimulates in vivo neutrophil proliferation in bone marrow by increasing granulocyte–macrophage colony-stimulating factor (GM-CSF) synthesis (Hammond et al., 1987), but it canbe toxic for cats (Dieringer et al., 1992). Our results indicate that lithium can have differenteffects on sheep PMNs depending on doses and on tested activity. Since lithium effectswere reported to depend on an animal’s immunological status and to be more marked inimmunodepressed animals, further studies on immunodepressed animals are needed.

In conclusion our data confirm that ketone bodies, in particular b-OH butyrate, are ableto depress in vitro two steps of the phagocytic process at concentrations similar to those observedduring ketosis. However, chemotaxis and particle uptake showed different sensitivity to thetwo ketone bodies. Impaired chemotaxis, by interfering with PMNs ability to leave the bloodvessels and reach foreign particles, together with impaired uptake, may have a dramatic effecton animal defences. To understand better the impairment of the functions of PMNs, we havetested the effect of ketone bodies at the same concentrations on adherence and superoxiderelease (P. Sartorelli et al., 1999).

Acknowledgements

This work was supported by MURST (40% and 60%).

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