Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils.
We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.
Hébert
C A
CA
Genentech, Inc., South San Francisco, CA 94080.
Luscinskas
F W
FW
Kiely
J M
JM
Luis
E A
EA
Darbonne
W C
WC
Bennett
G L
GL
Liu
C C
CC
Obin
M S
MS
Gimbrone
M A
MA
Baker
J B
JB
D002448:Cell Adhesion; D015550:Cell Degranulation / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007962:Leukocytes / Q000502:physiology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000502:physiology*; D010196:Pancreatic Elastase / Q000378:metabolism; D011971:Receptors, Immunologic / Q000502:physiology; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
D007375:Interleukin-1; D016209:Interleukin-8; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010196:Pancreatic Elastase; D013917:Thrombin