Loxosceles deserta spider venom induces NF-kappaB-dependent chemokine production by endothelial cells.
BACKGROUND
Loxosceles spider evenomation in man frequently results in disfiguring necrotic skin lesions. Recent studies suggest that several proinflammatory mediators participate in lesion development. We have observed that Loxosceles deserta venom induces production of the chemokines interleukin-8, growth-related oncogene alpha, and monocyte chemoattractant protein-I by human umbilical vein endothelial cells. Members of the Rel/Nuclear factor (NF)-kappaB family of transcription factors are important regulators of many genes involved in immune and inflammatory responses. We hypothesized that Loxosceles-venom-induced chemokine expression in human umbilical vein endothelial cells is mediated by NF-kappaB.


METHODS
Human umbilical vein endothelial cell monolayers were exposed to activating concentrations of Loxosceles deserta venom. Nuclear extracts of these monolayers were analyzed by electrophoretic mobility shift assay. A direct cause and effect linkage between NF-kappaB activation and chemokine expression by Loxosceles venom was established through examination of the effect of SN50 on interleukin-8 and monocyte chemoattractant protein-1 production using a whole-cell enzyme immunoassay. SN50 is a cell-permeable peptide that specifically blocks cytosolic to nuclear translocation of NF-kappaB. Furthermore, the venom-induced synthesis of chemokine mRNAs was investigated by RNase protection assays.


RESULTS
Loxosceles deserta venom induces the activation of NF-kappaB in human umbilical vein endothelial cells. Antibodies to p50 and p65, but not to p52, c-Rel, or Rel B, induce supershifts of the DNA-protein complexes formed by oligonucleotide probes and nuclear extracts from venom-activated human umbilical vein endothelial cells. SN50 peptide inhibits NF-kappaB translocation and interleukin-8 and monocyte chemoattractant protein-1 production in activated human umbilical vein endothelial cells.


CONCLUSIONS
Loxosceles deserta venom induces synthesis of interleukin8 and monocyte chemoattractant protein-1 mRNAs in human umbilical vein endothelial cells. The expression of chemokines occurs via an NF-kappaB-dependent pathway.
Desai
A
A
University of Michigan Medical School, Ann Arbor 48109-0602, USA.
Miller
M J
MJ
Gomez
H F
HF
Warren
J S
JS
D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism*; D004586:Electrophoresis; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry; D016209:Interleukin-8 / Q000378:metabolism; D016328:NF-kappa B / Q000145:classification / Q000502:physiology*; D009687:Nuclear Proteins / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000187:drug effects; D013111:Spider Venoms / Q000494:pharmacology*; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism
D018932:Chemokine CCL2; D018925:Chemokines; D016209:Interleukin-8; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D013111:Spider Venoms; D012260:Ribonucleases