G Model VETPAR-7119; No. of Pages 5
ARTICLE IN PRESS Veterinary Parasitology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro Adriano F. Vatta a,b , Michael Dzimianski a , Bob E. Storey a , Melinda S. Camus c , Andrew R. Moorhead a , Ray M. Kaplan a , Adrian J. Wolstenholme a,b,∗ a b c
Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA Center for Tropical & Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA Department of Pathology, University of Georgia, Athens, GA 30602, USA
a r t i c l e
i n f o
Keywords: Dirofilaria immitis Macrocyclic lactones Neutrophil Peripheral blood mononuclear cell
a b s t r a c t The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host’s innate immune system. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The macrocyclic lactone (ML) class of anthelmintics, which includes ivermectin, moxidectin, selamectin and the milbemycins, are the only drugs currently used for the prevention of heartworm disease caused by Dirofilaria immitis. Ivermectin is also used for removing microfilariae following adulticide treatment (Bowman and Mannella, 2011).
∗ Corresponding author at: University of Georgia, College of Veterinary Medicine, Dept of Infectious Diseases, 501 DW Brooks Drive, Athens, GA 30602, USA. Tel.: +1 706 542 2404. E-mail address: [email protected] (A.J. Wolstenholme).
However, despite the importance and widespread use of ML drugs for preventing disease with D. immitis, the mechanism of action of these drugs against D. immitis is unknown. There is very strong evidence from other species of parasitic nematodes and Caenorhabditis elegans that the pharmacologically relevant target of the MLs is the glutamate-gated chloride channel (GluCl) (Cully et al., 1994; Wolstenholme and Rogers, 2005), and an ivermectinsensitive GluCl subunit has been identified in D. immitis (Yates and Wolstenholme, 2004). The GluCl gene family is less complex in D. immitis and other filarioid species than in C. elegans (Ghedin et al., 2007; Godel et al., 2012; Williamson et al., 2007) and it is likely that only a single gene, Dii-avr-14, (nomenclature according to Beech et al.,
http://dx.doi.org/10.1016/j.vetpar.2014.02.004 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
G Model VETPAR-7119; No. of Pages 5
ARTICLE IN PRESS A.F. Vatta et al. / Veterinary Parasitology xxx (2014) xxx–xxx
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2010), encodes ML-sensitive GluCl subunits. GluCls control nematode movement, feeding and sensory processes (Wolstenholme, 2012) and the anthelmintic effects of the MLs and other anthelmintics are frequently assessed by measuring their effects on locomotion (Marcellino et al., 2012; Smout et al., 2010). In vitro migration assays are frequently used to detect ML-resistance in gastrointestinal nematodes (Demeler et al., 2012; Kotze et al., 2006; Wagland et al., 1992), and a similar assay has been developed for measuring the sensitivity of D. immitis L3 to the ML drugs (Evans et al., 2013). Reduced drug-induced paralysis of D. immitis microfilariae in vitro has also been taken as an indication of potential ML-resistant isolates (Bourguinat et al., 2011a, 2011b). However, the concentrations needed to produce 50% inhibition of L3 migration (Evans et al., 2013) or 95% paralysis of microfilariae (Bourguinat et al., 2011a) were approximately three orders of magnitude higher than those reported to be present in the plasma of dogs receiving either the preventative (6 g/kg) or microfilaricidal (100 g/kg) doses of the drugs (Daurio et al., 1992; McKellar and Gokbulut, 2012). This suggests that paralysis is unlikely to be the major mechanism of ML action against D. immitis L3 or microfilariae. This suggestion is supported by data from the human filarioid parasite Brugia malayi, where the GluCl were expressed solely around the excretory/secretory apparatus of the microfilariae (Moreno et al., 2010) and similarly high drug concentrations are required to paralyze B. malayi microfilariae in vitro (Tompkins et al., 2010). Consistent with this finding, in vitro treatment of B. malayi Mf with ivermectin reduced protein secretion (Moreno et al., 2010). Filarioid nematodes are well known to secrete immunomodulatory molecules (Hewitson et al., 2008), and there have been previous reports that the killing of microfilariae by host granulocytes or peripheral blood mononuclear cells is enhanced, in vitro, by ivermectin (Rao et al., 1987; Zahner et al., 1997). This is consistent with evidence showing that clearance of microfilariae from dogs with occult D. immitis infections (i.e. infections where an adult nematode(s) are present but microfilariae are absent from the circulation) is mediated by neutrophils (Elsadr et al., 1983; Rzepczyk and Bishop, 1984). Since it has not been possible to reproduce the in vivo effectiveness of low concentrations of the MLs against D. immitis microfilariae by measuring their effects on motility in vitro (Bourguinat et al., 2011b), we examined the effect of ivermectin on the adherence of peripheral blood mononuclear cells (PBMC) and neutrophils to the parasites in vitro. 2. Materials and methods 2.1. Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Georgia. 2.2. Cell-free microfilariae Microfilaraemic blood was drawn by jugular venipuncture into heparin-coated blood collection tubes (BD
Vacutainer, BD, Franklin Lakes, NJ) the day before an assay was set up. Blood utilized was from two different D. immitis-infected dogs. The blood cells were lysed with 0.2% saponin (Tokyo Industry Chemical Co., Ltd., Tokyo, Japan) in 0.85% NaCl. Briefly, 1 or 2 mL blood were diluted in a ratio of 1:11 with the saponin in 15 mL centrifuge tubes and placed in a water bath at 37 ◦ C for 15 min. The tubes were then centrifuged at room temperature at 850 × g for 5 min. The supernatant was siphoned off using a plastic Pasteur pipette, and the pellet was rinsed once with phosphate buffered saline (10 mM PBS, Boston BioProducts, Ashland, MA) and centrifuged again at 850 × g for 5 min. Adapting the methods from Galal et al. (1989), the pellet was resuspended in 2 mL RPMI (BioWhittaker® RPMI-1640 with l-glutamine, Lonza, Walkersville, MD) and placed in SephadexTM -containing columns (PD-10 Columns, GE Healthcare, UK Limited, Little Chalfont, Buckinghamshire, UK) equilibrated with RPMI. The liquid was allowed to move through the column and the filtrate from the column was collected in a centrifuge tube. When the top portion of the column had emptied itself, this was topped up with RPMI and the next fraction of filtrate collected in a separate tube. This procedure was repeated approximately 11 times. Each fraction was examined under a standard compound microscope and the filtrate was qualitatively scored for cellular content and number of microfilariae. Fractions that did not contain cells or that contained microfilariae but negligible numbers of cells (estimated at less than 5 cells per microfilariae) were pooled and centrifuged at 850 × g for 10 min. The supernatant was siphoned off, the microfilariae rinsed with sterile RPMI and the tube centrifuged again at the same speed and time. This process was carried out once. The numbers of microfilariae in the suspension were estimated by counting three aliquots of 10 L and stored in a tissue-culture incubator at 37 ◦ C overnight. 2.3. Leukocytes Blood leukocytes were prepared from blood from three specified pathogen-free beagles maintained in an insectfree indoor facility. For each assay, 6 mL of blood was drawn from an uninfected donor dog in EDTA-coated tubes (BD Vacutainer, BD, Franklin Lakes, NJ) and was allowed to cool to room temperature. Following the methods of Brinkmann et al. (2010), Histopaque® -1119 (SigmaAldrich, St. Louis, MO) at room temperature was used to separate the blood into mononuclear and polymorphonuclear cells, which were collected in separate centrifuge tubes. The cells were washed with PBS/1 mM EDTA, and each cell population was resuspended in 2 mL of PBS (Alam et al., 2006). The resuspended polymorphonuclear cells were centrifuged on a PercollTM (GE Healthcare BioSciences AB, Uppsala, Sweden) gradient (Brinkmann et al., 2010), and the contaminating mononuclear cells were separated into a new tube, washed with PBS/1 mM EDTA and combined with the mononuclear cells collected previously. The combined mononuclear cells were washed two more times. The polymorphonuclear cells derived from the PercollTM gradient were washed once with PBS containing 0.2% (w/v) glucose. The erythrocytes remaining in this suspension were lyzed in ice-cold 0.2% (w/v)
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
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NaCl as described by Alam et al. (2006). The suspension was returned to isosmolarity, and the cells were washed in PBS containing 0.2% (w/v) glucose. The concentration of cells per population was estimated in an aliquot of cells stained with 0.4% trypan blue (Gibco, Life Technologies Corporation, Grand Island, NY) in a disposable hemocytometer (C-Chip Neubauer Improved Disposable Hemocytometer, Incyto, SKC, Korea) following standard procedures. All populations used were estimated at 85% or greater viability. Confirmation of cell population was performed intermittently by the University of Georgia Clinical Pathology Laboratory using a hematology analyzer (Heska HemaTrueTM , Heska Corporation, Loveland, Colorado) and a differential count of 100 cells. For the latter, direct smears were prepared and stained with Modified Wright’s stain with a Wescor Aerospray Stainer (ELITech Biomedical Systems, Logan, Utah). The leukocytes were differentially counted by a licensed medical technologist. 2.4. Sera Four milliliters of blood from naïve dogs, not infected with D. immitis, were collected by jugular venipuncture in serum tubes (BD Vacutainer, BD, Franklin Lakes, NJ). After clotting, the blood was centrifuged at 800 × g, and the serum was pipetted off into a centrifuge tube. This process was repeated once. 2.5. Assay design The assays were set up in 96-well tissue culture plates (Costar® , Corning Incorporated, Corning, NY). Plates with wells containing cells and serum or no serum in increasing concentrations of ivermectin (Sigma) in 1% DMSO were prepared. There were four basic components to each well: cells, serum, ivermectin dissolved in 1% DMSO, and microfilariae. These were included in 50-L volumes each to give a total volume of 200 l. To create the respective controls, 50 L RPMI was substituted for the relevant component. The controls we used included wells without any drug, without serum and without drug or serum. Cells were included at a ratio of approximately 900 mononuclear cells per microfilariae (range: 432–1599) and 750 polymorphonuclear cells per microfiariae (range: 362–1444). These ratios were based on previous studies that had included cells at ratios of 1:100 (Zahner et al., 1997) and 1:1000 (Rao et al., 1987). The number of cells included was sufficiently high so that the number of cells available to adhere to the microfilariae was not a limiting factor in the experiments. Concentrations of ivermectin evaluated were 0.001 M, 0.01 M, 0.1 M, 1 M and 10 M in addition to a vehicle control (0.1% DMSO (v/v)). Approximately 100 microfilariae were included per well. 2.6. Effects on cell attachment The tissue culture plates were incubated at 37 ◦ C and examined at 16 h, 40 h, and 64 h after the start of incubation. Attachment of cells to microfilariae was assessed by examining 100 microfilariae per well (or all the
3
microfilariae if there were fewer than 100 in a well). Microfilariae with at least one cell attached were included. On occasion, preparations were made of the parasites in culture using a Cytospin® centrifuge [Cytospin® 3 Cell Preparation System (Shandon Scientific Limited, Astmoor, Runcorn, Cheshire, England)] and stained with Modified Wright’s stain. 3. Results & discussion Microfilariae isolated from the blood of infected dogs did not have any host leukocytes attached to them. When the isolated microfilariae were placed in the wells for the assays, the microfilariae exhibited S-shaped body bends, but they tended to remain in the same spot, allowing for counting of the individual microfilariae with relative ease. When microfilariae were incubated in ivermectin for 16 h we found that, as reported previously, very high drug concentrations were required to paralyze the parasites, and in fact we did not observe complete paralysis at the drug concentrations we could achieve in 1% (v/v) DMSO. At 10 M ivermectin nearly all of the microfilariae were still motile, although they were moving more slowly than those incubated in lower concentrations or in no drug. By contrast, the maximum plasma concentration of ivermectin following oral administration to dogs was 3.39 nM in dogs given 6 g/kg, 50.6 nM in those given 100 g/kg (Daurio et al., 1992) and 130–150 nM in dogs given ivermectin at 200 g/kg (Al-Azzam et al., 2007; Gokbulut et al., 2006). We examined the ability of PBMC and granulocytes isolated from uninfected dogs to bind to the microfilariae. In the absence of any ivermectin concentration, only limited binding of either cell population was detected, but this was increased in the presence of the drug. The binding was dependent on the presence of serum. Binding of PBMC to the microfiariae increased for at least 40 h of incubation, and increased with drug concentration up until ∼1 M, when >50% of the parasites had cells attached (Fig. 1B). The numbers of PBMC attached to individual microfiariae varied, and occasionally, individual parasites were observed to have large numbers of cells attached (Fig. 1C and D). The proportion of microfilariae with adherent neutrophils was less than that seen with the PBMC and maximal adherence was observed after 16 h of incubation rather than after 40 h, when approximately 20% of the microfilariae had at least one neutrophil attached (Fig. 2B). Binding of granulocytes, mainly neutrophils, to the microfilariae was more sensitive to the presence of ivermectin, showing increased levels at concentrations of 10 nM and higher (Fig. 2A). As with the PBMC, the number of neutrophils attached to individual microfilariae varied greatly and could be considerable. Staining of the adhered cells shows the characteristic change in nuclear morphology that accompanies neutrophil activation, indicating that the cells were attacking the parasites (Fig. 2C and D). The adherence of both leukocyte populations was dependent on the presence of serum from uninfected dogs. Similar observations on the effects of ivermectin on PBMC and neutrophil adhesion to, and killing of, filarioid parasites have been made previously. The concentrations at which ivermectin stimulates the adhesion of leukocytes to
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
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Fig. 1. Ivermectin-dependent adhesion of canine peripheral blood mononuclear cells (PBMC) to Dirofilaria immitis microfilariae (mf). (A) The proportion of D. immitis mf with adherent PBMC after various times of incubation. The bars represent the average values (±SE) for 4 assays. The Mf were incubated in 1 M ivermectin and 25% normal canine serum at 37 ◦ C for the times indicated. PBMC adherence was assessed visually. (B) The effect of ivermectin concentration on PBMC adherence to Mf. The parasites and cells were incubated together at 37 ◦ C for 40 h. The bars represent the average values (±SE) for 4 assays. ** P =
ARTICLE IN PRESS Veterinary Parasitology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro Adriano F. Vatta a,b , Michael Dzimianski a , Bob E. Storey a , Melinda S. Camus c , Andrew R. Moorhead a , Ray M. Kaplan a , Adrian J. Wolstenholme a,b,∗ a b c
Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA Center for Tropical & Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA Department of Pathology, University of Georgia, Athens, GA 30602, USA
a r t i c l e
i n f o
Keywords: Dirofilaria immitis Macrocyclic lactones Neutrophil Peripheral blood mononuclear cell
a b s t r a c t The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host’s innate immune system. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The macrocyclic lactone (ML) class of anthelmintics, which includes ivermectin, moxidectin, selamectin and the milbemycins, are the only drugs currently used for the prevention of heartworm disease caused by Dirofilaria immitis. Ivermectin is also used for removing microfilariae following adulticide treatment (Bowman and Mannella, 2011).
∗ Corresponding author at: University of Georgia, College of Veterinary Medicine, Dept of Infectious Diseases, 501 DW Brooks Drive, Athens, GA 30602, USA. Tel.: +1 706 542 2404. E-mail address: [email protected] (A.J. Wolstenholme).
However, despite the importance and widespread use of ML drugs for preventing disease with D. immitis, the mechanism of action of these drugs against D. immitis is unknown. There is very strong evidence from other species of parasitic nematodes and Caenorhabditis elegans that the pharmacologically relevant target of the MLs is the glutamate-gated chloride channel (GluCl) (Cully et al., 1994; Wolstenholme and Rogers, 2005), and an ivermectinsensitive GluCl subunit has been identified in D. immitis (Yates and Wolstenholme, 2004). The GluCl gene family is less complex in D. immitis and other filarioid species than in C. elegans (Ghedin et al., 2007; Godel et al., 2012; Williamson et al., 2007) and it is likely that only a single gene, Dii-avr-14, (nomenclature according to Beech et al.,
http://dx.doi.org/10.1016/j.vetpar.2014.02.004 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
G Model VETPAR-7119; No. of Pages 5
ARTICLE IN PRESS A.F. Vatta et al. / Veterinary Parasitology xxx (2014) xxx–xxx
2
2010), encodes ML-sensitive GluCl subunits. GluCls control nematode movement, feeding and sensory processes (Wolstenholme, 2012) and the anthelmintic effects of the MLs and other anthelmintics are frequently assessed by measuring their effects on locomotion (Marcellino et al., 2012; Smout et al., 2010). In vitro migration assays are frequently used to detect ML-resistance in gastrointestinal nematodes (Demeler et al., 2012; Kotze et al., 2006; Wagland et al., 1992), and a similar assay has been developed for measuring the sensitivity of D. immitis L3 to the ML drugs (Evans et al., 2013). Reduced drug-induced paralysis of D. immitis microfilariae in vitro has also been taken as an indication of potential ML-resistant isolates (Bourguinat et al., 2011a, 2011b). However, the concentrations needed to produce 50% inhibition of L3 migration (Evans et al., 2013) or 95% paralysis of microfilariae (Bourguinat et al., 2011a) were approximately three orders of magnitude higher than those reported to be present in the plasma of dogs receiving either the preventative (6 g/kg) or microfilaricidal (100 g/kg) doses of the drugs (Daurio et al., 1992; McKellar and Gokbulut, 2012). This suggests that paralysis is unlikely to be the major mechanism of ML action against D. immitis L3 or microfilariae. This suggestion is supported by data from the human filarioid parasite Brugia malayi, where the GluCl were expressed solely around the excretory/secretory apparatus of the microfilariae (Moreno et al., 2010) and similarly high drug concentrations are required to paralyze B. malayi microfilariae in vitro (Tompkins et al., 2010). Consistent with this finding, in vitro treatment of B. malayi Mf with ivermectin reduced protein secretion (Moreno et al., 2010). Filarioid nematodes are well known to secrete immunomodulatory molecules (Hewitson et al., 2008), and there have been previous reports that the killing of microfilariae by host granulocytes or peripheral blood mononuclear cells is enhanced, in vitro, by ivermectin (Rao et al., 1987; Zahner et al., 1997). This is consistent with evidence showing that clearance of microfilariae from dogs with occult D. immitis infections (i.e. infections where an adult nematode(s) are present but microfilariae are absent from the circulation) is mediated by neutrophils (Elsadr et al., 1983; Rzepczyk and Bishop, 1984). Since it has not been possible to reproduce the in vivo effectiveness of low concentrations of the MLs against D. immitis microfilariae by measuring their effects on motility in vitro (Bourguinat et al., 2011b), we examined the effect of ivermectin on the adherence of peripheral blood mononuclear cells (PBMC) and neutrophils to the parasites in vitro. 2. Materials and methods 2.1. Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Georgia. 2.2. Cell-free microfilariae Microfilaraemic blood was drawn by jugular venipuncture into heparin-coated blood collection tubes (BD
Vacutainer, BD, Franklin Lakes, NJ) the day before an assay was set up. Blood utilized was from two different D. immitis-infected dogs. The blood cells were lysed with 0.2% saponin (Tokyo Industry Chemical Co., Ltd., Tokyo, Japan) in 0.85% NaCl. Briefly, 1 or 2 mL blood were diluted in a ratio of 1:11 with the saponin in 15 mL centrifuge tubes and placed in a water bath at 37 ◦ C for 15 min. The tubes were then centrifuged at room temperature at 850 × g for 5 min. The supernatant was siphoned off using a plastic Pasteur pipette, and the pellet was rinsed once with phosphate buffered saline (10 mM PBS, Boston BioProducts, Ashland, MA) and centrifuged again at 850 × g for 5 min. Adapting the methods from Galal et al. (1989), the pellet was resuspended in 2 mL RPMI (BioWhittaker® RPMI-1640 with l-glutamine, Lonza, Walkersville, MD) and placed in SephadexTM -containing columns (PD-10 Columns, GE Healthcare, UK Limited, Little Chalfont, Buckinghamshire, UK) equilibrated with RPMI. The liquid was allowed to move through the column and the filtrate from the column was collected in a centrifuge tube. When the top portion of the column had emptied itself, this was topped up with RPMI and the next fraction of filtrate collected in a separate tube. This procedure was repeated approximately 11 times. Each fraction was examined under a standard compound microscope and the filtrate was qualitatively scored for cellular content and number of microfilariae. Fractions that did not contain cells or that contained microfilariae but negligible numbers of cells (estimated at less than 5 cells per microfilariae) were pooled and centrifuged at 850 × g for 10 min. The supernatant was siphoned off, the microfilariae rinsed with sterile RPMI and the tube centrifuged again at the same speed and time. This process was carried out once. The numbers of microfilariae in the suspension were estimated by counting three aliquots of 10 L and stored in a tissue-culture incubator at 37 ◦ C overnight. 2.3. Leukocytes Blood leukocytes were prepared from blood from three specified pathogen-free beagles maintained in an insectfree indoor facility. For each assay, 6 mL of blood was drawn from an uninfected donor dog in EDTA-coated tubes (BD Vacutainer, BD, Franklin Lakes, NJ) and was allowed to cool to room temperature. Following the methods of Brinkmann et al. (2010), Histopaque® -1119 (SigmaAldrich, St. Louis, MO) at room temperature was used to separate the blood into mononuclear and polymorphonuclear cells, which were collected in separate centrifuge tubes. The cells were washed with PBS/1 mM EDTA, and each cell population was resuspended in 2 mL of PBS (Alam et al., 2006). The resuspended polymorphonuclear cells were centrifuged on a PercollTM (GE Healthcare BioSciences AB, Uppsala, Sweden) gradient (Brinkmann et al., 2010), and the contaminating mononuclear cells were separated into a new tube, washed with PBS/1 mM EDTA and combined with the mononuclear cells collected previously. The combined mononuclear cells were washed two more times. The polymorphonuclear cells derived from the PercollTM gradient were washed once with PBS containing 0.2% (w/v) glucose. The erythrocytes remaining in this suspension were lyzed in ice-cold 0.2% (w/v)
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
G Model VETPAR-7119; No. of Pages 5
ARTICLE IN PRESS A.F. Vatta et al. / Veterinary Parasitology xxx (2014) xxx–xxx
NaCl as described by Alam et al. (2006). The suspension was returned to isosmolarity, and the cells were washed in PBS containing 0.2% (w/v) glucose. The concentration of cells per population was estimated in an aliquot of cells stained with 0.4% trypan blue (Gibco, Life Technologies Corporation, Grand Island, NY) in a disposable hemocytometer (C-Chip Neubauer Improved Disposable Hemocytometer, Incyto, SKC, Korea) following standard procedures. All populations used were estimated at 85% or greater viability. Confirmation of cell population was performed intermittently by the University of Georgia Clinical Pathology Laboratory using a hematology analyzer (Heska HemaTrueTM , Heska Corporation, Loveland, Colorado) and a differential count of 100 cells. For the latter, direct smears were prepared and stained with Modified Wright’s stain with a Wescor Aerospray Stainer (ELITech Biomedical Systems, Logan, Utah). The leukocytes were differentially counted by a licensed medical technologist. 2.4. Sera Four milliliters of blood from naïve dogs, not infected with D. immitis, were collected by jugular venipuncture in serum tubes (BD Vacutainer, BD, Franklin Lakes, NJ). After clotting, the blood was centrifuged at 800 × g, and the serum was pipetted off into a centrifuge tube. This process was repeated once. 2.5. Assay design The assays were set up in 96-well tissue culture plates (Costar® , Corning Incorporated, Corning, NY). Plates with wells containing cells and serum or no serum in increasing concentrations of ivermectin (Sigma) in 1% DMSO were prepared. There were four basic components to each well: cells, serum, ivermectin dissolved in 1% DMSO, and microfilariae. These were included in 50-L volumes each to give a total volume of 200 l. To create the respective controls, 50 L RPMI was substituted for the relevant component. The controls we used included wells without any drug, without serum and without drug or serum. Cells were included at a ratio of approximately 900 mononuclear cells per microfilariae (range: 432–1599) and 750 polymorphonuclear cells per microfiariae (range: 362–1444). These ratios were based on previous studies that had included cells at ratios of 1:100 (Zahner et al., 1997) and 1:1000 (Rao et al., 1987). The number of cells included was sufficiently high so that the number of cells available to adhere to the microfilariae was not a limiting factor in the experiments. Concentrations of ivermectin evaluated were 0.001 M, 0.01 M, 0.1 M, 1 M and 10 M in addition to a vehicle control (0.1% DMSO (v/v)). Approximately 100 microfilariae were included per well. 2.6. Effects on cell attachment The tissue culture plates were incubated at 37 ◦ C and examined at 16 h, 40 h, and 64 h after the start of incubation. Attachment of cells to microfilariae was assessed by examining 100 microfilariae per well (or all the
3
microfilariae if there were fewer than 100 in a well). Microfilariae with at least one cell attached were included. On occasion, preparations were made of the parasites in culture using a Cytospin® centrifuge [Cytospin® 3 Cell Preparation System (Shandon Scientific Limited, Astmoor, Runcorn, Cheshire, England)] and stained with Modified Wright’s stain. 3. Results & discussion Microfilariae isolated from the blood of infected dogs did not have any host leukocytes attached to them. When the isolated microfilariae were placed in the wells for the assays, the microfilariae exhibited S-shaped body bends, but they tended to remain in the same spot, allowing for counting of the individual microfilariae with relative ease. When microfilariae were incubated in ivermectin for 16 h we found that, as reported previously, very high drug concentrations were required to paralyze the parasites, and in fact we did not observe complete paralysis at the drug concentrations we could achieve in 1% (v/v) DMSO. At 10 M ivermectin nearly all of the microfilariae were still motile, although they were moving more slowly than those incubated in lower concentrations or in no drug. By contrast, the maximum plasma concentration of ivermectin following oral administration to dogs was 3.39 nM in dogs given 6 g/kg, 50.6 nM in those given 100 g/kg (Daurio et al., 1992) and 130–150 nM in dogs given ivermectin at 200 g/kg (Al-Azzam et al., 2007; Gokbulut et al., 2006). We examined the ability of PBMC and granulocytes isolated from uninfected dogs to bind to the microfilariae. In the absence of any ivermectin concentration, only limited binding of either cell population was detected, but this was increased in the presence of the drug. The binding was dependent on the presence of serum. Binding of PBMC to the microfiariae increased for at least 40 h of incubation, and increased with drug concentration up until ∼1 M, when >50% of the parasites had cells attached (Fig. 1B). The numbers of PBMC attached to individual microfiariae varied, and occasionally, individual parasites were observed to have large numbers of cells attached (Fig. 1C and D). The proportion of microfilariae with adherent neutrophils was less than that seen with the PBMC and maximal adherence was observed after 16 h of incubation rather than after 40 h, when approximately 20% of the microfilariae had at least one neutrophil attached (Fig. 2B). Binding of granulocytes, mainly neutrophils, to the microfilariae was more sensitive to the presence of ivermectin, showing increased levels at concentrations of 10 nM and higher (Fig. 2A). As with the PBMC, the number of neutrophils attached to individual microfilariae varied greatly and could be considerable. Staining of the adhered cells shows the characteristic change in nuclear morphology that accompanies neutrophil activation, indicating that the cells were attacking the parasites (Fig. 2C and D). The adherence of both leukocyte populations was dependent on the presence of serum from uninfected dogs. Similar observations on the effects of ivermectin on PBMC and neutrophil adhesion to, and killing of, filarioid parasites have been made previously. The concentrations at which ivermectin stimulates the adhesion of leukocytes to
Please cite this article in press as: Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.004
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ARTICLE IN PRESS A.F. Vatta et al. / Veterinary Parasitology xxx (2014) xxx–xxx
Fig. 1. Ivermectin-dependent adhesion of canine peripheral blood mononuclear cells (PBMC) to Dirofilaria immitis microfilariae (mf). (A) The proportion of D. immitis mf with adherent PBMC after various times of incubation. The bars represent the average values (±SE) for 4 assays. The Mf were incubated in 1 M ivermectin and 25% normal canine serum at 37 ◦ C for the times indicated. PBMC adherence was assessed visually. (B) The effect of ivermectin concentration on PBMC adherence to Mf. The parasites and cells were incubated together at 37 ◦ C for 40 h. The bars represent the average values (±SE) for 4 assays. ** P =
