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Poult Sci 2008. 87:61-63. doi:10.3382/ps.2007-00356
© 2008 Poultry Science Association
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IMMUNOLOGY, HEALTH AND DISEASE: Research Note

Broiler Chick Thrombocyte Response to Lipopolysaccharide

F. Ferdous, D. Maurice and T. Scott1

Clemson University, Animal and Veterinary Science Department, 123 P&A Building, Clemson, SC 29634-0311

1 Corresponding author: trscott{at}clemson.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The effect of lipopolysaccharide (LPS) on the expression of proinflammatory cytokines, interleukin (IL)-1β, IL-6, and IL-12 in broiler chick thrombocytes was investigated. At 4 wk of age, blood samples were collected, and isolated thrombocytes were incubated with LPS for 1 h. Ribonucleic acid was extracted from the cells to examine the expression of the proinflammatory cytokines using real-time reverse transcription PCR. It was found that expressions of IL-1β, IL-6, and IL-12 in thrombocytes were unaffected by diets containing corticosterone and vitamin C fed to chicks. However, LPS exposure did increase the expressions of these cytokines. The fact that thrombocytes are so abundant and can be stimulated by LPS makes them primary effector cells in innate host defenses against bacterial infections in chickens.

Key Words: broiler chick • thrombocyte • proinflammatory cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Chicken thrombocytes are nucleated blood leukocytes and represent the most abundant white blood cell types in chicken blood (Chang and Hamilton, 1979a). Because the avian bone marrow lacks megakaryocytes, thrombocytes arise from the antecedent mononucleated cells (Lucas and Jamroz, 1961). Thrombocytes are homologous in function to mammalian platelets. Platelets are the smallest blood cell, being only fragments of the megakaryocyte cytoplasm (Klinger, 1997). Platelets have become intriguing subjects for immunological research in recent years because of their role in natural host defense mechanisms (Meseguer et al., 2002). Platelets are mostly known for the process of hemostasis and the initiation of wound repair (George, 2000). Besides their role in hemostasis and thrombus formation after endothelial injury, platelets also take part in the processes of inflammation and tissue repair that follows (Klinger, 1997). Platelets release a vast array of bioreactive molecules such as chemotactic factors for other cell types, cationic proteins, histamine, serotonin, prostaglandin E2, and prostaglandin D2 (Klinger, 1997; Klinger and Jelkmann, 2002; Elzey et al., 2005).

Chicken thrombocytes have been used in this study to analyze gene expression of certain proinflammatory cytokines. Commercial broilers can be exposed to a variety of stressors and are raised in an environment where airborne microorganisms and microbial components challenge the health of the chicks. Therefore, isolated thrombocytes were cultured with lipopolysaccharide (LPS) to determine if broiler chicks have the capacity to respond to this microbial component via this unique cell type and whether the response is affected by oxidative stress and ascorbic acid.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The 54 broiler chicks used for this study were obtained from a local hatchery and received a starter diet until 2 wk of age. For the 2 replicate experiments, 18 and 36 chicks, respectively, were randomly assigned to 3 groups that received different diets from 2 to 4 wk of age and were housed at the Clemson University Morgan Poultry Center, Clemson, South Carolina, which is an Association for Assessment and Accreditation of Laboratory Animal Care approved facility at which Institutional Animal Care and Use Committee approved research protocols are monitored for assurance of animal care and well-being. The dietary groups were the control diet (standard cornsoy diet), the control diet with 30 mg/kg of corticosterone to stimulate oxidative stress (Maurice et al., 2007), and the control diet with 30 mg/kg of corticosterone plus 500 mg/kg of vitamin C.

Blood samples were collected into syringes from the wing vein with 10% EDTA as anticoagulant for thrombocyte isolation. The collected blood was stored on ice for transport to the laboratory. The thrombocyte isolation protocol used here was modified from the protocol used by Horiuchi et al. (1990). Blood samples were diluted (1:1) with Ca2+- and Mg2+-free Hanks’ balanced salt solution. Diluted blood samples were layered on a lymphocyte separation medium (1.077 to 1.080 g/mL) and centrifuged at 1,700 x g for 30 min at room temperature. The band containing the thrombocytes was collected, washed twice, and resuspended in Hanks’ balanced salt solution. Try-pan blue exclusion stain was used for quantification of viable cell numbers on a hemacytometer. Cell concentrations were adjusted to 1 x 107/mL for in vitro culture. Thrombocytes (107) were incubated with 10 µg of Ultra Pure Salmonella Minnesota LPS (InvivoGen, San Diego, CA) on a rocking platform at 40°C (5% CO2) for 1 h.

After thrombocyte stimulation, cells were centrifuged at 5,000 x g for 2 min to pellet. Pellets were stored in 100 µL of RNAlater (Qiagen Inc., Valencia, CA) overnight at 4°C. After 24 h, cells were centrifuged again to remove the supernatant and stored at –20°C for later use. The RNeasy Kit (Qiagen Inc.) was used, and the protocol of the manufacturer was followed to isolate the total RNA from these samples with on-column DNase treatment to remove any genomic DNA.

Real-time reverse transcription PCR (RT-PCR) was performed using a QuantiTech SYBR Green RT-PCR Kit (Qiagen Inc.) using an Eppendorf Mastercycler ep realplex4 (Eppendorf North America, Westbury, NY). Primers used for glyceraldehyde 3-phosphate dehydrogenase (NM_204305 [GenBank] , F: 5'-ATGCCATCACAGCCACACAGAA GA-3',R: 5'-ATGCCATCACAGCCACACAGAAGA-3'), interleukin (IL)-1β (Y15006, F: 5'-GCTCTACATGTCGTG-TGTGATGAG-3', R: 5'-TGTCGATGTCCCGCATGA-3'), IL-6 (AJ309540, F: 5'-ATGTGCAAGAAGTTCACCGTG TGC-3', R: 5'-TTCCAGGTAGGTCTGAAAGGCGAA-3'), and IL-12 (NM_213571, F: 5'-TGTCTCACCTGCTATTTG-CCTTAC-3', R: 5'-CATACACATTCTCTCTAAGTTTC-CACTGT-3') amplifications were obtained from Integrated DNA Technologies Inc. (Coralville, IA). The RT-PCR mixture consisted of 0.25 µL of QuantitTect RT Mix, 12.5 µL of 2x QuantitTect SYBR Green RT-PCR Master Mix, 0.5 µL (0.5 µM) of each specific primer, 10 µL (1 ng/µL) of template RNA, and 1.25 µL of RNase-free water to make the final reaction volume of 25 µL. The cycling profile used for all the reactions is 1 cycle of 50°C for 30 min, 95°C for 15 min, and 40 cycles of 94°C for 15 s, 57°C for 20 s, and 72°C for 20 s. Relative quantification of mRNA levels was determined through the use of the relative fold change calculation according to Pfaffl (2001).

The factorial experiments were conducted using split-plot designs with chicks (whole plots) randomly assigned to provide replications for each of the 3 diets in replicated experiments. The blood sample collected from each of the chicks was split into 2 parts (subplots) with 1 part serving as a control (no LPS) and the other part stimulated with LPS. Analysis of variance was performed with the GLM procedure of SAS 9.1 (SAS, 2003), and hypothesis testing was conducted using {alpha} = 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Diets did not affect the expression of proinflammatory cytokines, but LPS stimulation increased the level of expression compared with untreated control thrombocytes (Figure 1Go). It is evident from this figure that the relative fold expression increased for IL-1β, IL-6, and IL-12. The highest expression was observed in the relative fold change of IL-6. Interleukin-1β had significant expression but at a lower degree of change than seen for IL-6. In-terleukin-12 was moderately induced compared with the 2 other proinflammatory cytokines, but its fold change was also significant.


Figure 1
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  		Figure 1. Lipopolysaccharide (LPS, 10 µg/mL) of Salmonella Minnesota induces mRNA expression for interleukin (IL)-1β, IL-6, and IL-12 in thrombocytes of 4-wk-old broiler chickens. Open bars ({square}) are mean responses of untreated thrombocytes, and black bars ({blacksquare}) are mean responses of LPS-treated thrombocytes within each respective cytokine. a,bMean differences for each cytokine (P ≤ 0.01).

 
Lipopolysaccharide affects cell function and induces gene expression in chicken monocytes, macrophages, and heterophils (Hussain and Qureshi, 1997, 1998; Dil and Qureshi., 2002a,b). In fact, we have demonstrated in this study that LPS also induces expression of the proinflammatory cytokines IL-1β, IL-6, and IL-12 in thrombocytes similar to heterophils, the assumed primary effector cells of the innate host defenses to bacterial infections in poultry. Heterophils are the avian equivalent of neutrophils and function as professional phagocytes to aid in regulation of innate host defenses (Kogut et al., 2002). Chicken heterophils constitutively express Toll-like receptor (TLR) 1/6/10, TLR2 type 1, TLR2 type 2, TLR3, TLR4, TLR5, and TLR7 mRNA (Kogut et al., 2005). Our laboratory has recently determined that thrombocytes express TLR2, TLR3, TLR4, TLR5, and TLR7 (T. Scott and M. Dimmick Owens, Clemson University, unpublished data). Therefore, the observed LPS effects on broiler chick thrombocytes are mediated through TLR4 with activation of nuclear factor {kappa}B and mitogen-activated protein kinases (Scott and Dimmick Owens, 2008).

Since the first demonstration of avian thrombocytes as phagocytes by Glick et al. (1964), there has been extensive work done in the area of phagocytic ability of thrombocytes (Carlson et al., 1968; Carlson and Allen, 1969; Chang and Hamilton, 1976, 1979a, Chang and Hamilton, b). There are also some reports in which avian thrombocytes have been shown to play a major role in hemostasis like mammalian platelets by aggregating to form a hemostatic plug (Stalsberg and Prydz, 1963; Hodges, 1979). Thrombocytes have also been shown to secrete antiheparin proteins (Wachowicz et al., 1981). But to date, there is no study done with thrombocytes that shows their ability to be major effector immune cells by being stimulated by LPS or other pathogenic components. The findings of this study, along with other data from our laboratory, indicate that thrombocytes express TLR, which potentially place thrombocytes as primary effector cells.

Thrombocytes are the most abundant white blood cells in the avian blood (Chang and Hamilton, 1979a), and there are almost 6 times more thrombocytes than heterophils in circulation (Glick 1958; Lucas and Jamroz, 1961). This demonstrates that although thrombocytes and heterophils both have similar effector cell functions in the innate host defenses to bacterial infections, thrombocytes by shear number could be characterized as being the primary innate effector cells in chickens.

Received for publication August 24, 2007. Accepted for publication October 3, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Carlson, H. C., and J. R. Allen. 1969. The acute inflammatory reaction in chicken skin: Blood cellular response. Avian Dis. 13:817–833.[CrossRef][Web of Science][Medline]

Carlson, H. C., P. R. Sweeny, and J. M. Tokaryk. 1968. Demonstration of phagocytic and trephocytic activities of chicken thrombocytes by microscopy and vital staining techniques. Avian Dis. 12:700–715.[CrossRef][Web of Science][Medline]

Chang, C. F., and P. B. Hamilton. 1976. Phagocytic properties of chicken thrombocytes. Poult. Sci. 55:2018. (Abstr.)

Chang, C. F., and P. B. Hamilton. 1979a. The thrombocyte as the primary circulating phagocyte in chickens. J. Reticuloendothel. Soc. 25:585–590.[Web of Science][Medline]

Chang, C. F., and P. B. Hamilton. 1979b. Refractory phagocytosis by chicken thrombocytes during aflatoxicosis. Poult. Sci. 58:559–561.[Web of Science][Medline]

Dil, N., and M. A. Qureshi. 2002a. Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds. Vet. Immunol. Immunopathol. 84:191–207.[CrossRef][Web of Science][Medline]

Dil, N., and M. A. Qureshi. 2002b. Involvement of lipopolysaccharide related receptors and nuclear factor {kappa}B in differential expression of inducible nitric oxide synthase in chicken macrophages from different genetic backgrounds. Vet. Immunol. Immunopathol. 88:149–161.[CrossRef][Web of Science][Medline]

Elzey, B. D., D. L. Sprague, and T. L. Ratliff. 2005. The emerging role of platelets in adaptive immunity. Cell. Immunol. 238:1–9.[CrossRef][Web of Science][Medline]

George, J. N. 2000. Platelets. Lancet 355:1531–1539.[CrossRef][Web of Science][Medline]

Glick, B. 1958. The effect of cortisone acetate on the leukocytes of young chickens. Poult. Sci. 37:1446–1452.[Web of Science]

Glick, B., K. Sato, and F. Cohenour. 1964. Comparison of phagocytic ability of normal and bursectomized birds. J. Reticuloendothel. Soc. 1:442–449.[Web of Science]

Hodges, R. D. 1979. The blood cells. Pages 361–379 in Form and Function in Birds. Vol. 1. A. S. King and J. McLelland, ed. Acad. Press, New York, NY.

Horiuchi, H., H. Matsuda, and M. Murata. 1990. Preliminary evidence of growth factor(s) from chicken thrombocytes –growth effects on chicken embryo fibroblasts culture. Nippon Juigaku Zasshi 52:559–565.[Medline]

Hussain, I., and M. A. Qureshi. 1997. Nitric oxide synthase activity and mRNA expression in chicken macrophages. Poult. Sci. 76:1524–1530.[Abstract/Free Full Text]

Hussain, I., and M. A. Qureshi. 1998. The expression and regulation of inducible nitric oxide synthase gene differ in macrophages from chickens of different backgrounds. Vet. Immunol. Immunopathol. 61:317–329.[CrossRef][Web of Science][Medline]

Klinger, M. H. 1997. Platelets and inflammation. Anat. Embryol. (Berl.) 196:1–11.[CrossRef][Medline]

Klinger, M. H., and W. Jelkmann. 2002. Role of blood platelets in infection and inflammation. J. Interferon Cytokine Res. 22:913–922.[CrossRef][Web of Science][Medline]

Kogut, M. H., M. Iqbal, H. He, V. Philbin, P. Kaiser, and A. Smith. 2005. Expression and function of Toll-like receptors in chicken heterophils. Dev. Comp. Immunol. 29:791–807.[CrossRef][Web of Science][Medline]

Kogut, M., L. Rothwell, and P. Kaiser. 2002. Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2. Dev. Comp. Immunol. 26:817–830.[CrossRef][Web of Science][Medline]

Lucas, A. M., and C. Jamroz. 1961. Atlas of Avian Hematology. Agriculture Monograph 25, Washington, DC. USDA, US Gov. Print. Off., Washington, DC.

Maurice, D., S. F. Lightsey, J. E. Toler, and S. Canty. 2007. Effect of chronic oxidative/corticosterone-induced stress on ascorbic acid metabolism and total antioxidant capacity in chickens (Gallus gallus domesticus). J. Anim. Physiol. Anim. Nutr. (Berl.) 91:355–360.[Medline]

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SAS. 2003. SAS 9.1. SAS Inst. Inc., Cary, NC.

Scott, T., and M. Dimmick Owens. 2008. Thrombocytes respond to lipopolysaccharide through Toll-like receptor-4, and MAP kinase and NF-{kappa}B pathways leading to expression of interleukin-6 and cyclooxygenase-2 with production of prostaglandin E2. Mol. Immunol. 45:1001–1008.[CrossRef][Medline]

Stalsberg, H., and H. Prydz. 1963. Studies on chick embryo thrombocytes II. Function in primary homeostatsis. Thromb. Diath. Haemorrh. 9:291–299.[Web of Science]

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