Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

Abstract

SummaryRickettsia rickettsii , a Gram-negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c-jun Nterminal kinases (JNK) mitogen-activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose-dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre-treatment of host EC with cytochalasin D, or pre-incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38-specific inhibitor, had no effect on infection-induced activation of the ubiquitous transcriptional regulator nuclear factor-kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 by R. rickettsii -infected EC. Selective inhibition of p38 activity may be exploited as an anti-inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.

Rickettsia rickettsiiInfection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

“…Correspondingly, infected HMECs were also found to secrete the largest amounts of IL-8 and MCP-1. These results are in agreement with our earlier findings from in vitro infection of primary cultures of HUVECs (8,23) and further implicate an important role for signaling through NF-B and p38 MAP kinase pathways in inflammatory responses to R. rickettsii. Because HMECs, known to display nearly all important functional, phenotypic, and morphological characteristics of human microvasculature (2,19), respond most efficiently to in vitro infection, they likely represent a relevant and wellsuited culture model system to define rickettsial interactions with their preferred target cell type during human disease.…”

“…2). Thus, the intensities and dynamics of p38 activation were apparently similar among different cell types and resembled the kinetics of p38 activation in HUVECs (23). (Fig.…”

“…p38 is an important stress-activated MAP kinase, the inhibitors of which have promising efficacy in several models of disease, including inflammatory disorders, septic shock, and other diseases (9). Our previous studies have revealed the evidence for increased phosphorylation (a marker of activation) and enzymatic activity of p38 MAP kinase in HUVECs infected with R. rickettsii and a role for p38-mediated signaling in rickettsial invasion into host cells and consequent chemokine response (23). Because all MAP kinases are activated following dual phosphorylation of the threonine-X-tyrosine motif, where X is glycine in p38 kinases, we investigated the activation of p38 in ECs from different organs after R. rickettsii infection by determining its phosphorylation status.…”

Infection of Human Endothelial Cells with Spotted Fever Group Rickettsiae Stimulates Cyclooxygenase 2 Expression and Release of Vasoactive Prostaglandins

“…Although several protein kinases with unique specificities for serine or threonine residues are expressed in human vascular EC, only selective isoforms of protein kinase C and p38 MAP kinase have so far been established to play a crucial role in the regulation of cellular functions (32,34). Our results suggesting attenuation of R. rickettsii-induced COX-2 expression by a specific inhibitor of p38 MAP kinase (SB 203580) extend our recent findings that signaling through this particular MAP kinase module promotes changes in the level of chemo- b Ϫ, negative staining; positive staining was rated on a scale of 1 (ϩ ͓weakly positive͔) to 4 (ϩϩϩϩ ͓strongly positive͔).…”

“…Human umbilical vein EC, an established model cell type that has been used to investigate in vitro rickettsiaendothelium interactions (39) and a number of different host cell responses (8,9,13,15,22,30,32,34,(37)(38)(39)(40)(41)(42), were isolated from freshly collected umbilical cords by collagenase digestion and then seeded on gelatin (2% [wt/vol])-coated cell culture plates as described previously (33). Primary cultures were allowed to grow to confluence in McCoy's medium supplemented with 20% fetal bovine serum, heparin (100 g/ml), and endothelial cell growth supplement (50 g/ml), at which point they were routinely split at a ratio of 1:3.…”