Neural Signaling Activated by Pro-inflammatory Cytokines Following Trauma

Specific Aims

The molecular mechanisms of response of the central nervous system (CNS) to traumatic injury involve the expression of a variety of signaling molecules including neurohormones, transcription factors, cellular stress proteins, stress and mitogen-activated protein (MAP) kinases and pro-inflammatory cytokines.  The pro-inflammatory cytokines represent a broad family of proteins primarily of hematopoetic cell origin, which regulate inflammatory and immune responses.  Expression of pro-inflammatory cytokines has been found to be a universal response to different types of systemic and immune challenges, including traumatic injury and hemorrhagic shock.  While interaction of pro-inflammatory cytokines with the CNS has been shown to be essential for modulation of the systemic inflammatory and immune responses, the mechanism by which peripheral pro-inflammatory cytokines activate central neuroendocrine response of the brain cells involved in control of the body’s homeostasis is far from clear (reviewed in Turnbull and Rivier, 1999; Rivest et al., 2000; Vitkovic et al., 2000).  Recent studies have indicated that different MAP kinase pathways, including p38 kinase, stress-activated protein kinase or Jun N-terminal kinase (SAPK/JNK) and extracellular signal regulated kinase (ERK), may mediate the effects of pro-inflammatory cytokines (Koj, 1996). 

The hypothesis to be examined is that MAP kinase signaling pathways play a major role in mediating effects of pro-inflammatory cytokines in the CNS.   Activation of MAP kinases may in turn lead to activation of transcription factors, expression of the pro-inflammatory and other cytokines and to activation or inhibition of programmed cell death in specific neuronal populations of the brain involved in control and restoration of the body’s homeostasis after injury.  The specific aims for the three-year period are:

Aim 1.  Characterize MAP kinase subfamilies involved in mediating effects of the pro-inflammatory cytokines in the brain.  The goal of this specific aim is to delineate the MAP kinase pathways specifically activated by cytokines in the regions of the mouse brain involved in control of homeostasis. We will focus on three major pathways: p38, SAPK/JNK and ERK.  This will be accomplished by two approaches: i) immunoblot analysis and kinase assay of brain tissue extracts before and after trauma; and ii) immunohistochemistry on brain sections using phospho-specific antibodies against p38, SAPK/JNK and ERK.   [Year 1]

Aim 2.  Investigate the influence of specific blockers for MAP kinase pathways and cytokine receptors on cytokine production in the CNS.  The hypothesis to be examined is that effects of the peripheral inflammatory cytokines are mediated by central cytokine receptors and MAP kinases, which in turn stimulate local production of cytokines in the brain.  This will be accomplished by using specific TNF-a, and IL1 receptor antibodies, and inhibitors of p38 and ERK pathways (SB203580 and PD98059).   The antibodies and inhibitors will be administered before traumatic injury and expression of TNF-a, IL-1, IL-6 and IL-8 will be assessed by Northern blot and immunoblot analyses, and by in situ hybridization and immunohistochemistry on brain sections. [Year 1-2]

Aim 3.  Investigate the influence of -/- mutations for pro-inflammatory cytokines or cytokine receptors on the activation of MAP kinase signaling pathways after hemorrhagic shock. The hypothesis to be examined is that mutation for specific cytokine or the cytokine receptor will disrupt activation of the corresponding MAP kinase pathway.  This will be accomplished by analyzing mutant mice for inflammatory cytokines, which are ready available from the Jackson Laboratory including mutation for TNF-a(Tnf^tm1Gkl), TNF-a receptor (Tnfrsf1a^tm1Mak and Tnfrsf1b^tm1Imx), IL-1 receptor I (Il1r1^tm1Imx), IL-6 (Il6^tm1Kopf), and double knockout for TNF-aand IL-1 receptors (Tnfrsf1a^tm1Imx Il1r1^tm1Imx).  The mutant mice will be subjected to hemorrhagic shock and activation of different MAP kinases will be assessed by immunoblot analysis and kinase assay of brain tissue extracts, and immunohistochemistry on brain sections using phospho-specific antibodies against p38, SAPK/JNK and ERK.  The recovery of mutant mice after resuscitation will be assessed by monitoring blood pressure and compared to sham operated and wild type mice. [Year 2-3]

Introduction and Background Information

Pro-inflammatory cytokines are proteins produced largely by the immune system that appear to be critical mediators of shock following trauma.  Recent studies show that effects of pro-inflammatory cytokines are mediated by different stress or mitogen-activated protein (MAP) kinases.  Three major MAP kinase pathways, p38, SAPK/JNK and ERK have been implicated in mediating effects of the pro-inflammatory cytokines in different organ systems including the brain (reviewed in Rivest et al., 2000).  These phosphorylation pathways may lead to induction or protection from apoptosis, cell growth, proliferation, and activation of different transcription factors including ATF-2 (Stein at al., 1996), Elk1 (Zhao et al., 1999), CREB (Deak et al., 1998), NF-kappaB (Craig et al., 2000) and STAT (Hierholzer et al., 1999), as well as to phosphorylation of MAPKAPK-2 kinase and heat shock protein Hsp25 (Murashov et al., 1998; Murashov et al., 2000) (Fig. 1).

One recently identified member of the MAP kinase family, protein kinase p38, has been shown to be specifically activated by a number of cytokines including TNF-a and IL-1 in cell cultures (Foltz et al., 1997; Clerk et al., 1999).  Interestingly, a specific inhibitor of p38, SB203580, blocked production of TNF-ain glial culture (Bhat et al., 1998), and IL-6 and IL-8 in fibroblasts (Suzuki et al., 2000).  In cardiac myocytes the stimulation of p38 activated the transcription factor NF-kappaB and protected cells from apoptosis (Craig et al., 2000).   Although these data were obtained in cell culture conditions, a number of other studies showed expression of these pro-inflammatory cytokines (for review see Rivest et al., 2000) and p38 kinase in the CNS (Murashov et al., 1998; Murashov et al., 2000).  ERK is activated by IL-1 and TNF-ain a variety of cultured cells including fibroblasts, endothelial cells, glial cells (Goncalves et al., 2000) and neuronal cells (Hashimoto et al., 2000).  Activation of ERK linked to protection from neuronal cell death and activation of cell growth (Xia et al., 1995).  Activation of SAPK/JNK kinase by IL-1 and TNF-ahas an opposite effect on apoptosis activating programmed cell death in a variety of cell cultures and also in vivo (reviewed in Saklatvala et al., 1996).  Moreover, p38, ERK and SAPK/JNK activate a variety of transcription factors, which may lead to local expression of pro-inflammatory and other cytokines.  This reveals a complex picture of signaling with p38, ERK and SAPK/JNK as possible central players in modulation of cytokine’s effects in the brain. 

This project is funded by the Department of Naval Research (N00014-01-1-0099)