Introduction

Adhesion molecules including integrins play a key role in cell–cell and cell–extracellular matrix interactions. These interactions are necessary for proliferation and differentiation of both eosinophils and other inflammatory cells in the bone marrow,¹ their migration into blood,² their recruitment into tissues^{3, 4} and also their activation at inflammatory sites.^{5, 6} It is pertinent, therefore, to study the expression and function of eosinophil adhesion molecules under both physiological and pathological conditions.⁷ During migration to inflammatory sites changes in expression of surface molecules occur that influence subsequent function of the emigrated cells. Eosinophils in sputum from asthmatics have increased expression of CD11b, and decreased expression of CD62L.⁸ In severe allergic reactions⁸ and parasitic infections^{9, 10} similar changes may be seen on eosinophils in peripheral blood. Intravascular activation may lead to systemic sequellae and modify the capacity of cells to migrate to extravascular sites.¹¹

Parasitism of sheep with the gastrointestinal parasite Trichostrongylus colubriformis induces tissue eosinophilia and with repeated exposure in young animals a degree of immunity develops. Peripheral blood eosinophilia is thought to coincide with the expression of immunity^{12, 13} and may be driven by release of eosinophil-activating agents including IL-5, GM-CSF¹⁴ and chemokines.¹⁵ Maturation of larvae to viable adults is not essential for the induction of immunity as irradiated larvae, which fail to develop into long-lived adults, induce strong immunity to T. colubriformis infections.¹⁶ An improved understanding of the involvement of eosinophils in the acquisition and expression of immunity may assist work towards immunoprophylaxis of trichostrongylosis. The current study investigates changes in the expression of adhesion molecules on peripheral blood eosinophils during trickle infections with irradiated or infective third stage larvae.

Materials and Methods

Sheep

Fifteen mature multiparous non-pregnant Merino ewes and castrated males that had a prior history of field exposure and were therefore likely to have developed a degree of immunity to T. colubriformis were used. Sheep were housed in a group pen in an animal house and fed 1 kg per day of a standard pelleted diet.

Parasite infections

Sheep were allocated to three groups of five with three females and two castrated males per group. These animals underwent a trickle infection of T. colubriformis larvae over a period of 8 weeks. One group (infected) received 400 T. colubriformis infective third stage larvae three times per week for 8 weeks. A second group (vaccinated) received 400 T. colubriformis irradiated third stage larvae three times per week that had been irradiated by exposure to approximately 400 mRads gamma radiation. The third group (controls) received no larvae over the 8 week period. Larvae were administered per os via a Roux drenching gun. Blood was collected weekly from weeks 1 to 8 by jugular venepuncture into 5 mL EDTA vacutainers for haematology and weeks 2–8 for flow cytometry. The infectivity of T. colubriformis larvae was confirmed in young lambs.

Mammary exudates

Eosinophils were elicited in non-lactating mammary glands of six female sheep, by aseptic intramammary infusion of 50 g of an antigen extract from T. colubriformis third stage larvae.¹⁷ Two animals were selected from each of the three different treatment groups outlined.

Twenty-four hours later, exudates were recovered by infusion of 20 mL of sterile saline for injection (Baxter Healthcare, Old Toongabbie, NSW, Australia), gently massaging the gland and expressing the liquid into sterile polypropylene tubes (Falcon, Becton Dickinson, Lane Cove, NSW, Australia). Blood was collected by jugular venepuncture into 5 mL EDTA vacutainers immediately before collection of mammary exudates. Comparative flow cytometry was then carried out on both blood and mammary exudates.

Haematology

Total and differential leucocyte counts on blood and mammary exudates were carried out using a Cell-Dyn 3500R automated haematology analyser (Abbott Diagnostics, Lane Cove, NSW, Australia) calibrated for sheep blood.

Flow cytometry

One hundred microlitre aliquots of blood or mammary exudate were stained for flow cytometry as previously described.¹⁸ A single colour staining reaction was employed using the following panel of antibodies: CD11b (Clone MM12A), CD11c (Clone BAQ153A), CD18 (BAQ30A) and CD62L (DUI-29) all supplied by VMRD Inc., Pullman, WA, USA. The antibodies CD11b (Clone F10-50), CD44 (Clone 25–32) and CD49d (Clone 218.1) were a kind gift from Dr SJ McClure, CSIRO Livestock Industries, Prospect, NSW, Australia. Phycoerythrin-labelled goat antimouse IgG (Southern Biotechnology Associates Inc., Silenius Laboratories, Vic., Australia, Cat no: 1031–09) was used to report the staining reaction. Data were acquired on a FACS Vantage flow cytometer (Becton Dickinson) and analysed on Cell Quest Software. Eosinophils were identified as cells with high side scatter that autofluoresced at 530 nm as previously described.^{19, 20}

Statistical analyses

Data were analysed by ANOVA for repeated measures in the statistical package, Systat Version 5 (Systat Inc, Evanston, IL, USA).

Results

Mammary exudates

Eosinophils comprised 40.8 10.9% of leucocytes recovered in mammary exudates 24 h after infusion of T. colubriformis larval antigens. At this time eosinophils comprised 4.5 1.2% of peripheral blood leucocytes. The percentage of neutrophils and eosinophils expressing adhesion molecules and the mean fluorescent intensity of staining is presented in Table 1. In comparison with blood, neutrophils and eosinophils in mammary exudates had elevated expression of CD18 and decreased expression of CD62L. Mammary neutrophils also had increased expression of CD11b. The mean fluorescent intensity (MFI) of CD11a was decreased on exudate neutrophils and eosinophils, MFI of CD49d was decreased on mammary eosinophils and MFI of CD44 was decreased on mammary neutrophils.

Table 1 - Percentage expression and mean fluorescent intensity (MFI) of adhesion molecules by blood and mammary neutrophils and eosinophils.

Full table

Parasite infection

Eosinophil counts in blood during the course of the experiment did not differ between groups (Figure 1). The percentage of eosinophils positive for CD11a was decreased in the infected group in comparison with controls at weeks 6–8 of infection (Figure 2). CD44⁺ eosinophils at these times were less prevalent in the infected group than in either the control or the vaccinated group, although CD44⁺ eosinophils in the vaccinated group had also declined by week 8 to a lower percentage than the control group (Figure 2).

Figure 1.

Peripheral blood eosinophil counts over 8 weeks during trickle infection of immune sheep with infective () or irradiated () third stage Trichostrongylus colubriformis larvae, and uninfected controls (). Values plotted are mean SEM for five sheep per group.

Full figure and legend (19K)

Figure 2.

Percentages of peripheral blood eosinophils bearing CD11a and CD44 adhesion molecules over eight weeks during trickle infection of immune sheep with infective () or irradiated () third stage Trichostrongylus colubriformis larvae, and un-infected controls (). Values plotted are mean SEM for five sheep per group. Week 5 samples could not be processed for CD11a.

Full figure and legend (20K)

Faecal egg counts

Faecal egg counts in sheep receiving infective larvae by trickle infection were below the threshold for reliable detection at weeks 4–8. Therefore, two sheep were killed to determine whether larvae had established. Worm burdens of 980 and 1250 worms were detected.

Discussion

The mammary gland has been extensively used for studying inflammatory processes in sheep.^{17, 21, 22} Both eosinophil- and neutrophil-rich exudates can be elicited and the exudates provide a source of cells for detailed studies of cell functions and phenotypes. Greenhalgh et al.²² observed, in comparison with peripheral blood eosinophils, a decrease in CD49d, CD62L and 1-integrin expression on mammary eosinophils elicited by Haemonchus contortus third stage larvae in ewes primed by previous intramammary infusion of larvae. Similar changes were observed in adhesion molecule expression on eosinophils in the current experiment. It is noteworthy that we have previously seen upregulation of CD18 and downregulation of CD62L on neutrophils migrating into inflammatory exudates in the mammary gland of sheep.¹⁸

Bone marrow release of eosinophils in response to IL-5 leads to increased expression of 2-integrin (CD18) and is retarded by activity of 4-integrin (CD49d).²³ CD62L expression by granulocytes is downregulated following adhesion to activated endothelium, while CD18/CD11 heterodimers are upregulated during emigration from blood vessels.^{24, 25} Involvement of CD49d in eosinophil emigration is also well recognized.^{26, 27} CD44 participates in endothelial adhesion of eosinophils to CD58 [lymphocyte function- associated antigen-3 (LFA-3)].²⁸ Changes in expression of adhesion molecules by eosinophils within the vascular compartment may modify their ability to respond to inflammatory signals at the blood vessel wall and thus affect their migration into tissues at sites of parasite invasion.

The infective dose of larvae used in the current experiment failed to induce the peripheral blood eosinophilia characteristic of expression of immunity to T. colubriformis. This was perhaps due to the age of the sheep. Nonetheless there was a decrease in expression of CD44 and CD11a on peripheral blood eosinophils in infected sheep after larvae would have developed to mature adults at approximately week 4. CD44 also decreased in the vaccinated group by week 8 of the experiment. The decreased MFI of CD11a and a trend for decreased MFI of CD44 on mammary exudate eosinophils elicited by T. colubriformis antigens suggests that systemic activation of eosinophils was occurring during the course of infection and vaccination. These findings are in accord with the observation of activation of blood eosinophils by intravascular parasites such as Schistosoma mansoni⁹ and Onchocerca volvulis.¹⁰ The importance of these changes in adhesion molecule expression by intravascular eosinophils on cell functions and immunity to T. colubriformis requires further study.

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Acknowledgements

LM Stevenson was a recipient of an OECD Fellowship during these studies. The skilled technical assistance of D. Niemeyer is gratefully acknowledged.