Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases.

Human neutrophil migratory responses to Toll-like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which preceded neutrophil migration. TLR-mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP- or PAF-induced neutrophil chemotaxis is not affected by the pre-exposure of cells to TLR agonists.

Signaling pathways regulating interleukin-13-stimulated chemokine release from airway smooth muscle.

Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13-stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 +/- 4 and 75 +/- 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1beta was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4-dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 microM) and p38 (SB202190, 10 microM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase-dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Cross-Reactivity studies of gutta-percha, gutta-balata, and natural rubber latex (Hevea brasiliensis).

Gutta-percha and gutta-balata are derived from the Paliquium gutta and Mimusops globsa trees, respectively, that are in the same botanical family as the rubber tree Hevea brasiliensis. For this reason the potential for immunological cross-reactivity between the gutta-percha and gutta-balata used in endodontics and natural rubber latex (NRL) has been the subject of some controversy, because these products may be used in latex-allergic individuals. The objective of this study was to investigate the potential cross-reactivity between gutta-percha, gutta-balata, and NRL. Physiological extracts of seven commercially available gutta-percha products, raw gutta-percha, raw gutta-balata, and synthetic transpolyisoprene were each analyzed for cross-reactivity with NRL in a competitive radioallergosorbent test inhibition assay. No detectable cross-reactivity was observed with any of the raw or clinically used gutta-percha products. In contrast the raw gutta-balata released proteins that were cross-reactive with Hevea latex. We conclude that the absence of gutta-percha proteins that can react with Hevea latex-specific IgE antibody supports the minimal potential for commercially available gutta-percha to induce allergic symptoms in individuals sensitized to NRL. Because gutta-balata is sometimes added to commercial gutta-percha products caution should be exercised if products containing gutta-balata are used in endodontic care of latex-allergic individuals.

Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.

Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.