Abstract
Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ∼0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP3 (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.
Highlights
Cells of myelomonocytic lineage constitute the first lines of defense against pathogens
LPS and other Toll-like receptors (TLRs) ligands caused severe inhibition of chemotactic potential of monocytes towards CCL2, CXCL12 (Figure 1 A) or fMLF in the Trans-well assay. To determine if this characteristic was limited to some TLR ligands, we tested monocyte chemotaxis after treatment with TLR4 specific LPS, TLR1/2 heterodimer ligand Macrophage stimulatory lipopeptide 2 (MALP2), TLR3 targeting poly I:C, TLR2/6 specific tripalmitoylated lipopeptide, Pam3CSK4; TLR5 ligand flagellin, poly(U) that binds TLR7 or 8, TLR9 ligand CpG ODN, lipid A (TLR4) and a NOD2 ligand, muramyl dipeptide
Since many downstream signaling events of TLR stimulation have been attributed to MAPK activation [4], we evaluated the LPS treatment at 20 ng/ml induced little or no inhibition of of U937 myelmonocytic cell chemotaxis towards 20 nM CCL2, n = 3. doi:10.1371/journal.pone.0030404.g002
Summary
Cells of myelomonocytic lineage constitute the first lines of defense against pathogens. Leukocytes are endowed with a diverse family of G-protein coupled receptors (GPCRs) that sense chemoattractants and regulate directed cell migration in the immune system [1]. During chemotaxis, which is governed by spatially restricted integration of diverse signaling pathways that lead to cell polarity [2], leukocytes change rapidly from a roughly spherical to a polarized morphology with distinct leading and trailing edges and F-actin accumulation at the front [3]. Toll-like receptor (TLR) family members recognize microbial products to usher in innate immune response and bridge the innate and acquired immune response to pathogens Of the TLRs, TLR4 forms hetero- and homo-dimers at the cell surface and is the sole receptor for lipoplysaccharide (LPS). TLR signaling stimulates various transcriptional pathways, which prime innate immune cells against pathogens by facilitating pro-inflammatory cytokine and chemokine secretion [4]
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