Thrombin is a serine proteinase best known for its role in blood coagulation 1. In addition to its hemostatic effects, thrombin can induce numerous effects on neural cells through activation of proteinase-activated receptors (PARs) 2. PARs are a family (PAR1–PAR4) of G-protein coupled receptors whose activation can trigger multiple intracellular signaling pathways 1. Picomolar concentrations of thrombin cause process retraction in neurons and astrocytes, whereas higher concentrations induce astrocyte proliferation 2. Thrombin/PAR signaling can modulate the viability of both astrocytes and neurons 2 and has been directly implicated in the pathophysiology of a number of central nervous system (CNS) diseases including stroke 3 and traumatic brain injury 4.
Astrocytes are the most numerous cell type in the brain and play a critical role in CNS function 5. Astrocytes are central actors in both neuroinflammation and the response to cerebral ischemia 6. Astrocytes, along with microglia, are the primary sources of chemokine production after CNS injury 7. Chemokines are chemotactic cytokines that direct the movement of circulating leukocytes to sites of inflammation or injury 8. Chemokines regulate cell–cell interactions in both the immune and nervous systems and have broad function in both development and disease 9. In the brain, chemokines initiate inflammatory responses through their chemoattractive properties; however, they can also exert direct effects on neural cells 7.
Rodent astrocytes express all four PAR subtypes 10; however, only PAR1 and PAR4 are capable of independent thrombin-induced signaling in these cells 1. Several studies on rat astrocytes 11,12 have demonstrated that thrombin can induce expression and release of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1); a chemokine with structural and functional similarities to human IL-8 13. Immunohistochemical studies on human autopsy tissue demonstrated that PAR1 expression is most robust in astrocytes where it colocalized with GFAP in all brain regions 14. In the human glioblastoma cell line U178MG, thrombin-induced dose-dependent phosphoinositide hydrolysis and Ca2+ mobilization in a PAR1-dependent manner 14. Several studies using the human astrocytoma cell line 1321N1 have also demonstrated thrombin-induced responses including Ca2+-dependent morphological changes 15 and nucleoside release 16. However, the ability of thrombin to induce responses in nontransformed primary human astrocytes remains unknown. We therefore investigated the ability of thrombin to induce proliferation/metabolic activity and chemokine release in cultured human fetal astrocytes.
Pharmaceutical grade recombinant human a-thrombin (Recothrom) was obtained from ZymoGenetics Inc. (Seattle, Washington, USA). Thrombin-specific proteolytic activity inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK), was obtained from Calbiochem (La Jolla, California, USA). PAR agonist and scrambled peptides (mouse sequence, >98% purity) were purchased from Genscript Corporation (Scotch Plains, New Jersey, USA). PAR peptide amino acid sequences were as follows: PAR1 selective activating peptide, TFLLR; PAR1 scrambled peptide, FSLLR; PAR4 selective activating peptide, GYPGQV.
Human fetal tissue was obtained from Birth Defects Research Laboratories at the University of Washington (UW) in accordance with federal and UW institutional guidelines (E4 exempt). Cultures of primary human fetal astrocytes were prepared according to previously described protocol 17. For experiments, astrocytes were washed twice with PBS without calcium and magnesium (Gibco/Life Technologies, Grand Island, New York, USA) and detached in 0.05% Tris-EDTA containing 10 μg/ml DNase. Cells were gently triturated and plated at 3×104 cells per well in a 96 well plate. Cells were washed 8–12 h later with PBS containing calcium and magnesium (Gibco) and then incubated for 5–7 days in fresh Dulbecco’s modified Eagle’s Medium (DMEM)+5% horse serum+G5 supplement (Gibco). For all experiments, cells were washed three times in DMEM and serum starved in DMEM for 24 h before stimulation. Cells were stimulated with indicated concentrations of thrombin or PAR activating peptides for 24 h. All experiments with PAR peptides were carried out in the presence of polymyxin B (10 μg/ml). For proteolysis inhibition experiments, 5 μl of thrombin (1130 U/ml) was preincubated with 1.3 μl PPACK (5 mg/ml) for 30 min and the reaction mix was diluted to the indicated thrombin concentrations in DMEM before being added to cells.
Proliferation/metabolic activity and chemokine release assays
Quantification of proliferation/metabolic activity was determined with the WST-1 reagent (Boehringer Mannheim, Indianapolis, Indiana, USA) as described 18. Quantification of IL-8, IP-10, IL-6, and RANTES was assessed using the Luminex multibead array system and the Luminex IS100 analyzer according to manufacturer’s instructions (Upstate/Millipore, Billerica, Massachusetts, USA).
Statistical evaluation was carried out using PRISM software (GraphPad, San Diego, California, USA). Comparisons were made using one-way analysis of variance with Bonferroni’s post-test. P value less than 0.05 was considered to be significant. Data are given as mean±SEM.
Thrombin induces proliferation/metabolic activity in human fetal astrocytes
Human fetal astrocytes were treated with increasing concentrations of thrombin for 24 h. Results from WST-1 assay showed that thrombin induced a dose-dependent increase in proliferation/metabolic activity starting at 0.01 U/ml (Fig. 1). This effect reached 2.15-fold baseline control levels at a dose of 1 U/ml (∼7.7 nM) (Fig. 1). The thrombin-induced increase was reduced 84% (down to 1.19-fold baseline) by preincubation with PPACK (Fig. 1).
Thrombin induces release of chemokines IL-8 and IP-10 in human fetal astrocytes
In the human fetal astrocytes, treatment with thrombin also induced a dose-dependent increase in release of IL-8 and IP-10 starting at 0.1 U/ml (Fig. 2a) and 0.01 U/ml (Fig. 2b), respectively. The concentrations of IL-8 and IP-10 reached peak levels of 955±157 and 707±133 pg/ml after treatment with 1.0 or 0.3 U/ml thrombin, respectively. The thrombin-induced effects on both chemokines were completely inhibited by PPACK (Fig. 2a and b). Neither IL-6 nor RANTES were consistently detected above background levels after thrombin stimulation (data not shown).
PAR1, but not PAR4, activating peptide induces IL-8 and IP-10 release
PAR1 selective agonist peptide TFLLR induced a dose-dependent release of both IL-8 (Fig. 3a) and IP-10 (Fig. 3b) from human fetal astrocytes. Following treatment with 100 μM TFLLR, the concentrations of IL-8 and IP-10 reached peak levels of 86±16 and 118±33 pg/ml, respectively (Fig. 3a and b). Neither 100 μM PAR1 scrambled peptide FSLLR nor 100 μM PAR4 activating peptide GYPGQV induced chemokine release (Fig. 3a and b). PAR1 selective agonist peptide TFLLR (100 μM) did not induce release of either IL-6 or RANTES (data not shown).
The central findings in this report are that thrombin induces a dose-dependent increase in proliferation/metabolic activity as well as release of chemokines IL-8 (CXCL8) and IP-10 (CXCL10) from nontransformed cultured human fetal astrocytes. Our studies using PAR subtype selective agonist peptides suggest that the above effects are mediated by PAR1. These findings are consistent with, and expand upon, previous work on rat astrocytes demonstrating both the proliferative 2 and chemokine-inducing 11,12 effects of thrombin/PAR signaling. In particular, these results add to emerging evidence that astrocytes are a primary and functionally important cellular target for thrombin-induced PAR activation in the human brain 14–16.
In this study, the PAR1 selective peptide agonist induced a dose-dependent chemokine release profile similar to thrombin, whereas equimolar concentrations of PAR4 activating peptide failed to induce a response (Fig. 3). Although these results clearly implicate PAR1 in the regulation of astrocytic chemokine release, we cannot completely exclude a contribution from PAR4 as PAR activating peptides have variable potencies 1 and very high doses are necessary to reproduce some thrombin-like responses 2. Nonetheless, the PPACK inhibition (Fig. 2) and PAR activating peptide (Fig. 3) results confirm the chemokine release response as a bona fide thrombin/PAR-mediated effect. Similarly, we cannot entirely exclude the possibility that a small number of microglia could exist in our human fetal astrocyte cultures and contribute to these chemokine release responses. However, this possibility is unlikely given that prior studies on thrombin-induced chemokine release in microglia have failed to detect either IL-8 or IP-10 19,20. Finally, our use of pharmaceutical grade thrombin 18 in these studies along with the results seen with our PPACK and polymyxin B controls effectively exclude the possibility that LPS contamination 21 might be contributing to the thrombin-induced and/or PAR1 activating peptide-induced effects seen.
The thrombin-induced release of IL-8 from human fetal astrocytes is interesting in light of previous findings demonstrating thrombin-induced release of GRO/CINC-1 from rat astrocytes 11,12. GRO/CINC-1 (homologous to GRO-α or CXCL1 in humans) shares structural and functional similarities to human IL-8 13. Both GRO/CINC-1 and IL-8 belong to the glutamic acid-leucine-arginine (ELR)+ subclass of CXC chemokines. ELR+ chemokines tend to be regulated in tandem by inflammatory or immune stimuli such as IL-1, tumor necrosis factor-α, and lipopolysaccharide 8. The latter three stimuli can all induce IL-8 expression specifically in human fetal astrocytes 22. The IL-8 promoter contains binding sites for proinflammatory transcription factors such as NFκB. The latter has recently been implicated in thrombin-induced expression of IL-8 in human lung epithelial cells 23.
The ability of thrombin to induce both GRO/CINC-1 in rat astrocytes and IL-8 in human fetal astrocytes suggest a common functional consequence of thrombin exposure to these two distinct but related chemokines. GRO/CINC-1 and IL-8 share a common receptor (CXCR2) and actively participate in inflammatory reactions by inducing leukocyte (particularly neutrophil) recruitment and the generation of reactive oxygen species 8. Thrombin is generated immediately at sites of vascular injury in stroke 2, whereas neutrophil recruitment into the ischemic hemisphere typically follows 1–2 days later 24. Thrombin-induced release of IL-8 from astrocytes in the ischemic penumbra could be a possible mechanism for poststroke neutrophil recruitment to the CNS. In addition, CXCR2 is present on neurons and astrocytes and its activation by GRO/CINC-1 protects both cell types against C2-ceramide-induced apoptotic cell death 11,12. Thus, thrombin-induced release of GRO/CINC-1 from rat astrocytes has been suggested as a key step in a novel CXCR2-mediated neuroprotective pathway 11. Although it is uncertain whether or not IL-8 has the same capability as GRO/CINC-1 to alter neural cell viability, thrombin-induced astrocytic release of IL-8 could potentially function in a neuroprotective manner analogous to GRO/CINC-1.
This is the first report of thrombin-induced expression of IP-10 in any species or cell type. IP-10, which activates the proinflammatory chemokine receptor CXCR3, has been implicated in the pathophysiology of multiple sclerosis as well as other neurological disorders 25. From a functional standpoint, IP-10 is critical in the recruitment of effector T lymphocytes, natural killer cells, macrophages, and dendritic cells 25. IP-10 also induces inhibition of both angiogenesis and fibrosis and helps promote recovery of neural tissue after injury 24. The direct effects of IP-10 on neural cell viability and function are largely unknown. Thrombin-induced IP-10 release from astrocytes may play an important and complementary role to IL-8 in directing the brain’s neuroinflammatory response to various CNS injuries. In addition, thrombin-induced IP-10 release from astrocytes could facilitate both glial–glial and glial–neuronal cell interactions by activating CXCR3 expressed on nearby microglia and neurons, respectively 25.
Thrombin induces a dose-dependent increase in proliferation/metabolic activity as well as release of chemokines IL-8 (CXCL8) and IP-10 (CXCL10) from human fetal astrocytes. These effects are mediated by PAR1. The selective thrombin-induced chemokine release profile observed (IL-8 and IP-10, but not IL-6 or RANTES) could help determine the character and kinetics of immune cell infiltration into the CNS under pathological conditions.
This study was funded by the National Institute of Health.
Conflicts of interest
There are no conflicts of interest.
1. Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost. 2005;3:1800–1814
2. Grand RJ, Turnell AS, Grabham PW. Cellular consequences of thrombin
-receptor activation. Biochem J. 1996;313(Pt 2):353–368
3. Junge CE, Sugawara T, Mannaioni G, Alagarsamy S, Conn PJ, Brat DJ, et al. The contribution of protease-activated receptor 1 to neuronal damage caused by transient focal cerebral ischemia. Proc Natl Acad Sci USA. 2003;100:13019–13024
4. Nicole O, Goldshmidt A, Hamill CE, Sorensen SD, Sastre A, Lyuboslavsky P, et al. Activation of protease-activated receptor-1 triggers astrogliosis after brain injury. J Neurosci. 2005;25:4319–4329
5. Kettenmann H, Ransom BR Neuroglia. 2005;Vol. 12nd ed. New York, NY Oxford University Press
6. Nedergaard M, Dirnagl U. Role of glial cells in cerebral ischemia. Glia. 2005;50:281–286
7. Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia. 2005;50:307–320
8. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine
receptors in inflammation. N Engl J Med. 2006;354:610–621
9. Ransohoff RM. Chemokines and chemokine
receptors: standing at the crossroads of immunobiology and neurobiology. Immunity. 2009;31:711–721
10. Wang H, Ubl JJ, Reiser G. Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia. 2002;37:53–63
11. Wang Y, Luo W, Reiser G. Activation of protease-activated receptors in astrocytes evokes a novel neuroprotective pathway through release of chemokines of the growth-regulated oncogene/cytokine-induced neutrophil chemoattractant family. Eur J Neurosci. 2007;26:3159–3168
12. Wang Y, Luo W, Stricker R, Reiser G. Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine
GRO/CINC-1. J Neurochem. 2006;98:1046–1060
13. Ramos CD, Heluy-Neto NE, Ribeiro RA, Ferreira SH, Cunha FQ. Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1. Cytokine. 2003;21:214–223
14. Junge CE, Lee CJ, Hubbard KB, Zhang Z, Olson JJ, Hepler JR, et al. Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes. Exp Neurol. 2004;188:94–103
15. Nakao K, Shirakawa H, Sugishita A, Matsutani I, Niidome T, Nakagawa T, et al. Ca2+
mobilization mediated by transient receptor potential canonical 3 is associated with thrombin
-induced morphological changes in 1321N1 human astrocytoma cells. J Neurosci Res. 2008;86:2722–2732
16. Kreda SM, Seminario-Vidal L, Heusden C, Lazarowski ER. Thrombin
-promoted release of UDP-glucose from human astrocytoma cells. Br J Pharmacol. 2008;153:1528–1537
17. Yagle K, Lu H, Guizzetti M, Moller T, Costa LG. Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: role in DNA synthesis and effect of ethanol. Glia. 2001;35:111–120
18. Weinstein JR, Hong S, Kulman JD, Bishop C, Kuniyoshi J, Andersen H, et al. Unraveling thrombin
’s true microglia-activating potential: markedly disparate profiles of pharmaceutical-grade and commercial-grade thrombin
preparations. J Neurochem. 2005;95:1177–1187
19. Hanisch UK, van Rossum D, Xie Y, Gast K, Misselwitz R, Auriola S, et al. The microglia-activating potential of thrombin
: the protease is not involved in the induction of proinflammatory cytokines and chemokines. J Biol Chem. 2004;279:51880–51887
20. Watanabe Y, Miura I, Ohgami Y, Fujiwara M. Extracellular presence of IL-8 in the astrocyte
-rich cultured cerebellar granule cells under acidosis. Life Sci. 1998;63:1037–1046
21. Weinstein JR, Swarts S, Bishop C, Hanisch UK, Moller T. Lipopolysaccharide is a frequent and significant contaminant in microglia-activating factors. Glia. 2008;56:16–26
22. Hua LL, Lee SC. Distinct patterns of stimulus-inducible chemokine
mRNA accumulation in human fetal astrocytes and microglia. Glia. 2000;30:74–81
23. Lin CH, Cheng HW, Ma HP, Wu CH, Hong CY, Chen BC. Thrombin
induces NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells by a Rac1-dependent PI3K/Akt pathway. J Biol Chem. 2011;286:10483–10494
24. Frangogiannis NG. Chemokines in ischemia and reperfusion. Thromb Haemost. 2007;97:738–747
25. Groom JR, Luster AD. CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol Cell Biol. 2011;89:207–215