Cpg-Oligodeoxynucleotide-Stimulated Chicken Heterophil Degranulation Is Serum Cofactor and Cell Surface Receptor Dependent

Developmental and Comparative Immunology 29 (2005) 255–264 www.elsevier.com/locate/devcompimm

CpG-oligodeoxynucleotide-stimulated chicken heterophil degranulation is serum cofactor and cell surface receptor dependent

Haiqi Hea,*, Virginia K. Lowryb, Pamela J. Ferroc, Michael H. Koguta

aUSDA, ARS, Southern Plains Agricultural Research Center, 2881 F and B Road, College Station, TX 77845, USA bDepartment of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A and M University, College Station, TX 77843, USA cDepartment of Poultry Sciences, Texas A and M University, College Station, TX 77843, USA

Received 26 April 2004; revised 30 June 2004; accepted 12 July 2004 Available online 28 August 2004

Abstract

Synthetic oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) is immune stimulatory to chicken heterophils. Recognition of CpG-ODN by chicken heterophils leads to the mobilization and release of granules. This CpG- ODN-induced heterophil degranulation was chicken serum (CS)-dependent. Heat-denaturation and membrane ﬁltration of CS revealed that the active serum cofactor(s) was likely a protein in nature with a molecule mass within 50,000 to 100,000. This serum cofactor(s) was heat-resistant at 56 8C for 1 h. The involvement of a cell surface receptor in recognition of CpG-ODN was also demonstrated by (1) trypsin treatment of the heterophils abrogated the degranulation response and (2) CpG-ODN- induced heterophil degranulation was sensitive to the inhibition of Clathrin-dependent endocytosis. In addition, among various microbial agonists, including CpG-ODN, lipopolysaccharide, lipoteichoic acid, phorbol myristate acetate, and formalin-killed Salmonella enteritidis, CpG-ODN was the only agonist that displayed serum-dependent induction of degranulation in chicken heterophils. This is the ﬁrst report that shows serum-dependent activation of leukocytes by CpG-ODN. q 2004 Elsevier Ltd. All rights reserved.

Keywords: CpG-ODN; TLR9; Serum cofactor; Degranulation; Innate immune response; Heterophil; Neutrophil; Chicken

1. Introduction numerous cell types in circulating blood of most birds [1]. As a major component of cellular innate Heterophils are polymorphonuclear (PMN) leuko- immunity, heterophils play a critical role as the ﬁrst cytes in poultry and functionally equivalent to line of defense against microbial infection. In poultry, mammalian neutrophils. They are the second most these granulocytic phagocytes are rapidly recruited to the site of infection, where they engage in phagocytosis and killing of pathogens [2,3]. * Corresponding author. Tel.: C1-979-260-3771; fax: C1-979- 260-9332. Activation of heterophils has been shown to be E-mail address: [email protected] (H. He). critical in protecting neonatal chickens from systemic

0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2004.07.005 256 H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264 infection of Salmonella and Salmonella-induced reported. In this study, we focused on activation of mortality [3,4]. This heterophil-mediated immunity heterophil degranulation by a previously identified was associated with recruitment of large numbers of avian immune stimulatory CpG-ODN [19,20]. Our heterophils to the site of bacterial invasion and an study shows that activation of heterophil degranula- increased number of peripheral blood heterophils tion by CpG-ODN is dependent on the presence of [5,6]. Heterophils, like mammalian neutrophils, chicken serum (CS). In the presence of CS, CpG-ODN respond to microbial stimulation by producing toxic induces dose-dependent activation of heterophil reactive oxygen species and releasing bactericidal degranulation. substances and proteolytic enzymes in the process of degranulation [1–3,7–9]. DNA from bacteria, but not vertebrates, is a 2. Materials and methods powerful stimulator of the innate immune system. The immune stimulatory activities of bacterial DNA 2.1. Reagents are attributed to the presence of a high frequency of an unmethylated CpG dinucleotide motif, which is an The nuclease-resistant phosphorothioate ODNs unmethylated cytosine followed by guanosine (CpG) were purchased from Integrated DNA Technologies dinucleotide flanked by certain nucleotides [10]. It has (Coralville, IA, USA) and further purified by ethanol been well documented that bacterial DNA and precipitation. ODNs were dissolved in sterile phos- synthetic oligodeoxynucleotide (ODN) containing phate-buffered saline (PBS, pH 7.2) at a concentration unmethylated CpG-dinucleotides (CpG-ODN) of 1 mg/ml. The sequences of synthetic ODNs used in activate B-lymphocyte and innate immune cells the present study were: CpG-ODN#17, GTC GTT (macrophage, dendritic cells, and natural killer cells) GTC GTT GTC GTT; and a non-CpG containing to secret cytokines [interleukin-1b (IL-1b), IL-6, control ODN (nCpG-ODN), CCA TGG CCA TGG IL-12, IL-18, tumor necrosis factor-a (TNF-a), CCA TGG [19]. Lipopolysaccharide (LPS, from interferon-a (IFN-a), and IFN-g] and to promote Salmonella enteritidis), lipoteichoic acid (LTA, adaptive immune responses [10]. Recognition of from Staphylococcus aureus), phorbol myristate CpG-ODN and signaling of cell activation are acetate (PMA), CS and fetal bovine serum (FBS), mediated by the Toll-like receptor 9 (TLR9) [11,12]. RPMI-1640, Hank’s balanced salt solution (HBSS), Interaction of CpG-ODN with TLR9 occurs Histopaque, monodansylcadaverine (MDC), and exclusively in the intracellular compartment [13]. chloroquine were purchased from Sigma (St Louis, Internalization by endocytosis and subsequent MO, USA). endosomal maturation is therefore the prerequisite for CpG-ODN activity [14]. 2.2. Animals Despite the fact that neutrophils play an important role in innate immunity as mediators of immune Leghorn chickens (2–4 day old), obtained from a recognition and destruction of microbial pathogens, local commercial hatchery (Hy-Line International, there is a lack of research on neutrophil responses to Bryan, TX, USA), were used as blood donors for CpG-ODN stimulation [15–17]. More recently, heterophil isolation. Chickens were kept in a con- studies on immune stimulatory activities of trolled environment with ad libitum access to water CpG-ODN in avians have begun to emerge [18–22]. and feed. The diet was a balanced and un-medicated We have previously demonstrated the CpG motif-de- corn-soybean ration [23]. pendent induction of nitric oxide (NO) production in an avian macrophage cell line (HD11) by synthetic 2.3. Isolation of heterophils CpG-ODN [19]. Further studies demonstrated that induction of NO in HD11 cells by CpG-ODN requires Collection of peripheral blood and heterophil clathrin-dependent endocytosis and endosomalma- isolation were conducted as previously described turation [20]. However, immune stimulatory activity [8]. Briefly, peripheral blood from approximately of CpG-ODN in chicken heterophils has not been 50 chicks was pooled, mixed with 1% methylcellulose H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264 257

(1:1 v/v), and centrifuged at 25!g (rcf) for 15 min. 2.6. Data analysis The supernatant was removed, diluted with Ca2C- and Mg2C-free Hanks balanced salt solution, carefully Data are means and standard deviations of three layered onto a discontinuous Histopaque gradient independent experiments with three to ﬁve-replicates (speciﬁc gravity 1.077/1.119) in 50-ml conical each. Statistical difference was determined at the level centrifuge tubes, and centrifuged at 250!g for of p!0.05 by Student’s t-test using SigmaStat 60 min. The heterophils, below the Histopaque software (Jandel Corp, San Rafael, CA, USA). 1.077/1.119 interface, were collected, washed, and re-suspended in RPMI-1640. Heterophils were counted and kept on ice until used. 3. Results

3.1. Serum dependence of CpG-ODN induced 2.4. Preparation of formalin-killed S. enteritidis heterophil degranulation (FKSE) The effects of CpG-ODN on heterophil A primary poultry isolate of S. enteritidis degranulation are dependent on the presence of CS. (NVSL#97-11771, National Veterinary Services Lab- In the absence of CS, incubation of heterophils with oratory, Ames, IA, USA) was used in this study. CpG-ODN reduced release of b-glucuronidase even Bacteria were inoculated and cultured overnight in below the level of non-stimulated spontaneous tryptic soy broth. Formalin-killed bacteria were degranulation (Fig. 1A). In the presence of CS, prepared by incubation of bacteria in 1% formalin CpG-ODN stimulated dose-dependent degranulation solution at 4 8C for 24 h followed by washing four by heterophils (Fig. 1B). Likewise, increasing times with PBS to remove the formalin, and stored at concentration of CS in the medium enhanced CpG- 4 8C until used. ODN-stimulated degranulation in heterophils (Fig. 1C). Additionally, ODN containing immunosti- 2.5. Degranulation assay mulatory CpG motifs was significantly more effective than the control ODN without CpG motif (nCpG- Heterophil degranulation was measured by ODN) in the induction of heterophil degranulation quantifying b-glucuronidase activity [24] in (Fig. 1D). Pre-incubation of heterophils with culture medium following stimulation of heterophils CpG-ODN followed by washing did not retain (8!106/ml) with CpG-OND or other agonists in the CpG-ODN stimulatory activity for degranulation presence or absence of CS for 60 min on a rocker when serum was subsequently added to the reaction platform at 39 8C in a 5% CO2 incubator. In the (data not shown). This indicates that serum com- inhibition assay, heterophils (8!106/ml) were treated ponents were required to initiate CpG-ODN signal with or without an inhibitor for 30 min prior to leading to heterophil degranulation. stimulation with CpG-ODN. After incubation, the cells were pelleted by centrifugation at 250!g (rcf) 3.2. Comparison of agonist, CpG-ODN, LPS, LTA, for 10 min at 4 8C and supernatants were collected for PMA, and FKSE stimulation on heterophil the assay. An aliquot of 25 ml supernatant was degranulation incubated with 50 ml of freshly prepared substrate (10 mM 4-methylumbelliferyl-b-D-glucuronide, 0.1% To determine if this serum-dependent heterophil Triton X-100 in 0.1 M sodium acetate buffer) for 4 h degranulation is a CpG-ODN specific phenomenon, at 41 8C. The reaction was stopped by adding 200 ml degranulation activities were compared when hetero- of stop solution (0.05 M glycine and 5 mM EDTA; pH phils were stimulated with various agonists such as 10.4) to each well. Liberated 4-methylumbelliferone CpG-ODN, LPS, LTA, PMA, and FKSE (Fig. 2). The was measured fluorimetrically (355/460 nm) using an results show that LPS and LTA do not induce fmax fluorescence microplate reader (Molecular heterophil degranulation, whereas, PMA and FKSE Devices, Sunnyvale, CA). stimulate heterophil degranulation regardless of 258 H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264

Fig. 1. Serum dependence of CpG-ODN induced heterophil degranulation. (A) Heterophils (8!106/ml) were stimulated with 5 mg/ml of CpG-

ODN with or without CS (5%). Supernatants were harvested after the indicated length of incubation (at 39 8C and 5% CO2) and b-glucuronidase activity was measured. (B) Heterophil stimulated with CpG-ODN at various concentrations for 60 min. (C) Heterophil stimulated with 5 mg/ml of CpG-ODN at various concentrations (%) of CS for 60 min. (D) Comparison of heterophil degranulation stimulated by CpG-ODN or nCpG- ODN for 60 min with or without CS (5%). *p!0.05 compared to the control. the presence of serum. Only CpG-ODN showed the serum dependence in its ability to induce the heterophil degranulation.

3.3. Effects of CS ﬁltrates from different molecule- weight-cut-off membranes on CpG-ODN induced degranulation

To estimate the molecule mass of the serum active component, CS was fractionated with Centriprep with 10,000, 50,000 and 100,000 molecule weight cut-off (MWCO) membranes (Amicon, Beverly, MA) by centrifugation at 1800!g (rcf). The ﬁltrates were Fig. 2. Comparison of heterophil degranulation stimulated by tested for their ability to mediate CpG-ODN induced various agonists (CpG-ODN, LPS, LTA, PMA, and FKSE) with or degranulation. Our results indicate that serum active without CS. Heterophils were stimulated with CpG-ODN (5 mg/ml), LPS (10 mg/ml), LTA (10 mg/ml), PMA (1 mg/ml), or FKSE component resides in the molecule mass range from (equivalent to 108 CFU of live SE) for 60 min with or without CS 50,000 to 100,000, since the 100,000 MWCO ﬁltrate (5%). Heterophils in the controls were unstimulated. *p!0.05 retained 100% activity for CpG-ODN induced compared to the control. H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264 259

indicate that the CpG-ODN induced degranulation was probably mediated by protein components of the serum other than complement system.

3.5. Comparison of effectiveness of chicken and bovine sera in CpG-ODN induce heterophil degranulation

Since CS was shown to be necessary for CpG-ODN to induce heterophil degranulation, we Fig. 3. Effects of whole CS and CS filtrates from different molecule- compared the effectiveness of CS and FBS for weight-cut-off (MWCO) membranes on CpG-ODN stimulated CpG-ODN stimulated degranulation (Fig. 5). The heterophil degranulation. Heterophils were stimulated with results indicate that FBS was also able to mediate 5 mg/ml of CpG-ODN in the medium containing 5% whole CS or CpG-ODN induced degranulation, however, it was CS filtrates for 60 min. Controls were unstimulated and contained significantly less effective compared to the CS at the 5% whole CS. *p!0.05 compared to the control. same concentration. This observation indicates that there is certain cross-species activity of the degranulation, whereas, fractions between 10,000 and serum cofactor in mediating CpG-ODN induced 50,000 MWCO completely lost their activity (Fig. 3). degranulation.

3.4. Effect of heat denaturation of CS on CpG-ODN 3.6. Involvement of cell surface receptor in CpG-ODN induced degranulation induced heterophil degranulation

The heat-denaturation of CS at various Intracellular localization of TLR9 [14] suggests temperatures was used to determine if the CpG-ODN must be internalized in order to activate complement cascade was involved in mediating cellular function. This internalization of CpG-ODN is CpG-ODN induced heterophil degranulation. Heating most likely receptor mediated endocytosis. CS at 56 8C on a waterbath for 60 min, which However, a cell surface CpG-ODN binding protein inactivates the complement system, did not reduce or a receptor has not been identiﬁed. To demonstrate the activity of CS in CpG-ODN stimulated degranulation. However, activity was lost when CS was heated at 75 and 95 8C for 30 min (Fig. 4). These results

Fig. 4. Effects of heat denaturation of CS on CpG-ODN stimulated heterophil degranulation. Heterophils were stimulated for 60 min Fig. 5. Comparison of effects of chicken (CS) and fetal bovine sera with 5 mg/ml of CpG-ODN in the medium containing 5% CS which (FBA) on CpG-ODN stimulated heterophil degranulation. Hetero- was either unheated or heated at various temperatures (56 8C for phils were stimulated with 5 mg/ml of CpG-ODN with either CS 60 min or 75 and 95 8C for 30 min). Controls were unstimulated and (5%) or FBS (5%) in the medium for 60 min. *p!0.05 compared contained 5% unheated CS. *p!0.05 compared to the control. between CS and FBS treatments. 260 H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264

Fig. 6. Effects of trypsin treatment on heterophil degranulation stimulated CpG-ODN. Heterophils were treated with trypsin (500 mg/ml) at 37 8C for indicated periods. Then heterophils were washed and stimulated with 5 mg/ml of CpG-ODN with CS (5%) for 60 min. The control heterophils were neither treated with trypsin nor stimulated with CpG-ODN. *p!0.05 compared to the control. the involvement of a receptor in CpG-ODN-mediated stimulatory activities, the heterophils were incubated with 0.05% trypsin ATV solution prior to stimulation with CpG-ODN. Our results show that treating heterophils with trypsin proteinase for 30 min completely abolished CpG-ODN induced degranulation (Fig. 6). Fig. 7. Inhibition of CpG-ODN stimulated heterophil degranulation by monodansylcadaverine (MDC, an inhibitor for clathrin-dependent endocytosis) and chloroquine (an inhibitor for endosomal 3.7. Roles of clathrin-dependent endocytosis and maturation). Heterophils were preincubated with one of the endosomal maturation in CpG-ODN induced inhibitors for 30 min and then stimulated with 5 mg/ml of CpG- ODN with CS (5%) for an additional 60 min. *p!0.05 compared to heterophil degranulation the control.

Clathrin-dependent endocytosis is a well characterized mechanism and is utilized by many receptor mediated endocytic pathways [25]. MDC is a 4. Discussion selective inhibitor for clathrin-dependent endocytosis [26–28]. Pretreatment of heterophils with MDC The innate immune system has developed and been resulted in dose-dependent inhibition of CpG-ODN conserved during the course of evolution for rapid induced degranulation (Fig. 7A). These results recognition and response to invading pathogens indicate the clathrin-dependent endocytic pathway through recognition of pathogen associated molecule was involved in CpG-ODN signaling. Our results patterns (PAMPs). One example of such recognition further show that subsequent endosomal maturation of PAMPs is that the vertebrate innate immune system was critical for CpG-ODN stimulated heterophil (from fish and birds to mammals) can discriminate degranulation. Increasing concentration of chloro- both bacterial and viral DNA from self DNA by quine, an endosomal maturation inhibitor, in the recognition of unmethylated CpG motifs present in media attenuated the stimulatory effect of CpG-ODN the pathogen’s DNA [10]. CpG DNA therefore is on heterophil degranulation (Fig. 7B). This result recognized as a danger signal by the host and evokes confirms the essential role of endosomal maturation/ innate immune responses, which in turn promote the acidification in CpG-ODN mediated cellular host adaptive immune system to combat potential signaling. infection. H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264 261

Chicken heterophil degranulation plays an import- of CpG-ODN by heterophils requires facilitation of ant role in host defense and inflammatory activity. serum cofactor(s) to induce signal transduction Granule proteins, including cationic bactericidal leading to mobilization and release of granules. peptides [29], defensins [30], and proteolytic enzyme CpG-ODN stimulating leukocyte activation has been such as gelatinase B [31], a matrix metalloprotei- demonstrated in numerous species. However, this nase, have been identified in heterophils. These serum-dependent activation of leukocyte by granule proteins are prepacked in the granules and CpG-ODN is the first to be reported. In fact, released in response to certain microbial agonist serum is routinely added to cell culture media stimulation to engage bactericidal activities and aide when stimulated with CpG-ODN and its effect on in heterophils’ migration to the site of infection. CpG-ODN induced activation has not been eval- Deficiency in granule proteolytic protein has been uated [17]. Our results also show that synthetic associated with impaired bacterial killing capacity of ODN containing immune stimulatory CpG motif neutrophils [32]. was more effective (p%0.05) in stimulating hetero- The specific interaction of pathogen-associated phil degranulation than ODN containing no CpG molecules with soluble and cell-surface located motif (Fig. 1D). However, in this study, we did pattern recognition receptors is a prerequisite for observe an elevated level (p%0.05) of degranulation initiating host innate immune responses to a when heterophils were stimulated with the control pathogen. At least four soluble microbial molecule- ODN containing no CpG motif in the presence of binding proteins, including LPS-binding protein serum (Fig. 1D). Human neutrophils also have been (LBP) [33–35], soluble CD14 [33–35], mannose- shown to degranulate in response to CpG-ODN binding lectin [36], and one of the peptidoglycans stimulation, however, the response was not serum recognition proteins (PGRP) [37] have been reported dependent [16]. to play significant roles in pattern recognition and Our results indicate that CS contains cofactor(s) modulation of innate immune responses. The roles which facilitate CpG-ODN recognition by a of LBP and CD14 in activation of Toll-like heterophil cell surface receptor, leading to heterophil receptors (TLR2 and TLR4) in the innate immune activation. Although serum cofactor(s) and cells are well documented [33]. The responses of receptor(s) for CpG-ODN recognition have not both TLRs are strongly dependent on serum, where been physically identified, evidence from this study LBP and soluble CD14 are the active components supported the conclusion. First, our results show [33]. Recognition of CpG-ODN and signaling cell serum has to be present in the cell culture medium in activation are mediated by the Toll-like receptor 9 order for CpG-ODN to induce heterophil (TLR9) [11,12]. Interaction of CpG-ODN with degranulation (Fig. 1A). CpG-ODN itself failed to TLR9 has been shown to occur exclusively in the induce any degranulation activity in heterophils. intracellular compartment [13]. Internalization by Heterophils appeared unable to bind CpG-ODN endocytosis and subsequent endosomal maturation is without the presence of CS, since heterophils, therefore the precondition for CpG-ODN activity which were preincubated with CpG-ODN followed [14,20]. However, the initial recognition of by washing to remove CpG-ODN from the medium, CpG-ODN by immune cells is unknown. displayed little degranulation activity when serum Here, we demonstrated that immune recognition was added back to the culture medium. Secondly, of CpG-ODN by chicken heterophils is serum heat denaturation and fractionation of CS demon- dependent. This recognition of CpG-ODN led to strated that the active serum cofactor(s) was most the mobilization and release of heterophil granules likely proteinaceous with a molecule mass in the in the process of degranulation. The response of range of 50,000–100,000 (Fig. 3). The serum heterophil degranulation to CpG-ODN stimulation cofactor(s) was heat-resistant up to 56 8C for 1 h requires the presence of serum in the medium without any loss of activity (Fig. 4), which ruled out (Fig. 1A). Removal of CS from the cell culture the participation of complement cascade. This active rendered heterophils unresponsive to CpG-ODN serum cofacter also demonstrated cross-activity stimulation. Those results indicate that recognition between species (chicken and bovine) (Fig. 5). 262 H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264

Lastly, recognition of CpG-ODN by cell surface heterophil activation by these agonists and that receptor(s) was demonstrated by the loss of degra- effector functions may vary in response to different nulation activity when heterophils were treated with microbial agonists. trypsin for 30 min prior to CpG-ODN stimulation In summary, we have demonstrated the (Fig. 6). Together, these results suggest that uni- participation of a serum cofactor and a cell surface dentified serum cofactor(s) may act as a CpG-ODN receptor in CpG-ODN stimulated degranulation of binding factor facilitating interaction of CpG-ODN chicken heterophils. These results allow us to with cell surface receptor(s) to initiate the process of hypothesize that the serum cofactor mediates the degranulation. However, we do not know if the interaction of the CpG-ODN and possibly the TLR9 was involved in the recognition of CpG-ODN nCpG-ODN as well with the cell surface receptor, at the cell surface and mediated the signal transduc- where both CpG-ODN and nCpG-ODN are tion pathway. internalized by Clathrin-dependent endocytosis. Clathrin-mediated endocytosis is a well This endocytosis activity may contribute certain characterized mechanism which involves many degree of degranulation by heterophils in response receptor-mediated endocytic pathways [25], to both CpG- and nCpG-ODN stimulation. However, therefore, to further demonstrate the involvement recognition of the internalized CpG-ODN by the of a receptor in mediating CpG-ODN induced TLR9 leads to a greater activation of heterophils heterophil degranulation, we examined clathrin- which results in significantly higher degranulation mediated endocytosis pathway using a selective activity. Further studies will be conducted to identify inhibitor, MDC. The results indicate that the and characterize the serum cofactor and the cell clathrin-dependent endocytic pathway is involved surface receptor of the heterophils. in CpG-ODN signaling due to pretreatment of heterophils with MDC resulted in dose-dependent inhibition to CpG-ODN induced degranulation (Fig. 7A). Furthermore, we showed that subsequent Acknowledgements endosomal maturation was important for CpG-ODN stimulated heterophil degranulation. This was Mention of trade names or commercial products in demonstrated by the diminished stimulatory effect this publication is solely for the purpose of providing of CpG-ODN on heterophil degranulation as a result specific information and does not imply recommen- of increased concentration of chloroquine, an dation or endorsement by the US Department of endosomal maturation inhibitor, in the media Agriculture. (Fig. 7B). These results support previous reported findings that induction of NO by CpG-ODN in chicken HD11 macrophage cell line is mediated by References clathrin-dependent endocytosis and requires endosomal maturation [20]. [1] Maxwell MH,RobertsonGW.The avianheterophil leucocyte:a Additionally, our study demonstrated differential review. World’s Poult Sci J 1998;54:155–78. responses of heterophils to various microbial [2] Stabler JG, McCormick T, Powell K, Kogut MH. Avian agonists. At the doses examined, heterophils were heterophils and macrophages: phagocytic and bactericidal unresponsive to LPS and LTA stimulation in terms activities against Salmonella enteritidis. Vet Microbiol 1994; 38:293–305. of induction of degranulation regardless of the [3] Kogut MH, McGruder ED, Hargis BM, Corrier DE, presence of CS. On the other hand, PMA and DeLoach JR. In vivo activation of heterophil function in FKSE both induced serum-independent degranula- chickens following injection with Salmonella enteritidis- tion reaction in heterophils. CpG-ODN was the immune lymphokines. J Leukoc Biol 1995;57:56–62. only agonist examined that displayed serum-depen- [4] Kogut MH, Tellez GI, McGruder ED, Wong RA, Isibasi A, Ortiz VN, et al. Evaluation of Salmonella enteritidis- dent induction of degranulation in chicken hetero- immune lymphokines on host resistance to Salmonella phils. These results indicate that differential enteritidis ser. Gallinarum in broiler chicks. Avian Pathol signaling pathways are probably involved in 1997;25:734–47. H. He et al. / Developmental and Comparative Immunology 29 (2005) 255–264 263

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