Soybean trypsin inhibitor. An IL-1-like protein?

Soybean trypsin inhibitor (SBTI) shares some structural homology with interleukin-1 (IL-1) and was tested for IL-1 bioactivity. Human T-cells proliferated maximally when stimulated with PMA and SBTI but failed to respond to either stimulus alone. This response was abrogated by neutralizing antibodies to IL-1 beta but not to IL-1 alpha. However, immunoblots showed no cross-reactivity between SBTI and anti-IL-1 antibodies. Furthermore, SBTI did not bind to IL-1 receptors on YT cells and did not activate a murine T-lymphoma or human T-hybridoma. Supernantants from monocytes stimulated with SBTI contained significant levels of IL-1 activity. The data show that SBTI has no direct IL-1 activity but can stimulate T-cells indirectly through an IL-1 dependent mechanism.

Effects of lectins and tunicamycin on IL-1 binding to YT cells.

Specific binding of iodinated-interleukin-1 alpha or beta to YT cells could be inhibited by the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A). WGA and Con A inhibition of IL-1 binding was abrogated by previous exposure of these plant proteins to the lectin-specific sugars N-acetylglucosamine (GlcNAc) and methyl glucoside (MG), respectively. Tunicamycin, an inhibitor of glycosylation, decreased interleukin-1 (IL-1) binding to YT cells, but also reduced total protein synthesis. These observations suggest that carbohydrate moieties on or near the interleukin-1 receptor may be important for optimal receptor binding of IL-1 to intact YT cells.

Requirement for cell-bound proteases in the mechanism of human neutrophil activation with various stimuli.

A cell membrane-associated protease/esterase has been implicated in the mechanism of "stimulus-secretion coupling" described for human neutrophil degranulation. In this regard, a broad spectrum of protease inhibitors were evaluated for their effects on granule enzyme release from neutrophils exposed to soluble, surface-active stimuli. The serine protease inhibitors, L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and a thiol protease inhibitor, p-hydroxymercuribenzoate (PHMB), caused a concentration-related suppression of neutrophil degranulation elicited with 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), ionophore A23187, phorbol myristate acetate (PMA), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). However, other inhibitors, such as aprotinin and p-hydroxyphenylpyruvic acid, were inactive. The maximum inhibitory effect with TPCK, TLCK, and PHMB was observed when neutrophils were exposed to these inhibitors prior to contact with the respective stimuli. In addition, the magnitude of inhibition increased in proportion to the preincubation time of protease inhibitor with stimulus. The results of these studies implicate proteases in the sequence of events underlying activation of the human neutrophil secretory process in response to structurally and chemically dissimilar stimuli.

Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils.

Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium. DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils.

A possible requirement for arachidonic acid lipoxygenation in the mechanism of phagocytic degranulation by human neutrophils stimulated with aggregated immunoglobulin G.

Aggregated immunoglobulin G (AggIgG) caused a concentration-dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Generation of the 5-lipoxygenase product of arachidonic acid (AA) metabolism, 5(S),12(R)-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)], by neutrophils is exposed to AggIgG occurred in the presence but not absence of exogenous AA. U-60,257B (piriprost potassium), an inhibitor of leukotriene synthesis, caused a dose-related suppression of LTB4 production and granule exocytosis by AggIgG-treated cells. These data suggest that a lipoxygenase product of AA metabolism may mediate AggIgG-induced phagocytic release of granule constituents from neutrophils.

Interleukin-1 stimulates granule exocytosis from human neutrophils.

The interaction of human polymorphonuclear neutrophilic leukocytes (neutrophils) with interleukin-1 (IL-1) resulted in a time- and concentration-dependent, selective, release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12-binding protein) granule constituents. Myeloperoxidase (MPO) and lysozyme secretion was markedly attenuated if neutrophils were not exposed to cytochalasin B (CB) prior to contact with IL-1. Degranulation was significantly enhanced in the presence of extracellular calcium. IL-1-elicited granule exocytosis was inhibited by the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), a calmodulin antagonist, trifluoperazine (TFP), and an anion channel blocker, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). An evaluation of the role of arachidonic acid metabolites in IL-1-induced neutrophil activation revealed a suppressive effect on enzyme release exerted by the lipoxygenase inhibitors, piriprost potassium (6,9,deepoxy-6,9-(phenylimino)-delta 6,8 -prostaglandin I1, U-60,257B) and NDGA (nordihydroguaiaretic acid), and a cyclooxygenase/lipoxygenase inhibitor, ETYA (5,8,11,14-eicosatetraynoic acid). These data describe the characteristics of IL-1 as a human neutrophil secretagogue, and enhance our insight into the mechanism of inflammatory cell activation with this monokine.

Properties of interleukin-1 as a complete secretagogue for human neutrophils.

Human monocyte-derived Interleukin-1 (IL-1) stimulated a concentration-dependent extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12-binding protein) granule constituents from cytochalasin B-treated human neutrophils. The serine protease inhibitors, L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TPCK) as well as an inhibitor of thiol protease activity, p-hydroxymercuribenzoate (PHMB), suppressed granule enzyme release from neutrophils activated with IL-1. Cycloheximide, an inhibitor of protein synthesis, had no effect on IL-1-induced neutrophil degranulation. Neutrophils pretreated with IL-1 were rendered unresponsive to subsequent exposure to this stimulus. IL-1-elicited granule exocytosis appears to be stimulus specific in that N-formyl-methionyl-leucyl-phenylalanine (FMLP), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorycholine (AGEPC), and 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) were capable of eliciting a secretory response from IL-1-pretreated cells.

Effects of soluble stimuli on human monocyte secretion.

We report here that the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and the mitogenic phorbol ester, phorbol myristate acetate (PMA) cause a time- and concentration-dependent, selective, extracellular release of N-acetyl-beta-glucosaminidase and lysozyme from freshly isolated, adherent human peripheral blood monocytes. The inability of the protein synthesis inhibitor, cycloheximide, to influence enzyme release indicates that these enzymes are constitutive secretory products. 1-O-Hexadecyl-/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine demonstrated moderate secretory activity, whereas pepstatin A, concanavalin A, and leukotriene B4 were essentially inactive. FMLP- and PMA-induced enzyme release were inhibited with the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride and the anion channel blocker, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. These results demonstrate the capacity of soluble, surface-active stimuli to activate the human monocyte secretory process.