James M. Justen

Postreceptor events associated with human neutrophil activation by interleukin‐8

Recombinant human monocyte–derived interleukin‐8 (IL‐8M), recombinant human endotheliumderived IL‐8 (IL‐8E), and a recombinant human truncated form of IL‐8 (IL‐8T) stimulated a time‐dependent (t1/2 ~ 2–3 s) and concentration‐dependent (0.1–100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B–treated human neutrophils (HNs) wherein IL‐8T = IL‐8M > IL‐8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL‐8T– than in IL‐8M– or IL‐8E–activated HNs, and IL‐8T was more potent than either IL‐8M or IL‐8E in sequentially desensitizing the HNs to the effects of the other IL‐8 forms. IL‐8 induced a time‐ and concentration‐dependent (0.1–100 nM) increase in the production of inositol 1,4,5‐trisphosphate (IP3) in HNs. U‐73122 (1‐[6‐[[17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐1H‐pyrrole‐2,5‐dione), a potent inhibitor of phospholipase C–catalyzed events in HNs, suppressed IL‐8–triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane‐associated activity of the α and β subtypes of protein kinase C was significantly enhanced in IL‐8–activated cells.

Human polymorphonuclear neutrophil activation with arachidonic acid

1 The capacity of arachidonic acid (AA) to stimulate granule exocytosis from human polymorphonuclear neutrophils (PMNs) was investigated. 2 AA induced the selected extracellular release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12 binding protein) granule constituents from human PMNs in a time‐and concentration‐dependent manner. 3 Cytochalasin B (CB) enhanced but was not required for PMN activation with AA. 4 Although extracellular calcium had no effect on granule exocytosis, AA did stimulate the mobilization of intracellular sequestered Ca2+ which resulted in an increase in cytosolic‐free Ca2+ ([Ca2+]i) as reflected by increased fluorescence of Fura‐2‐treated cells. 5 AA stimulated Ca2+/phospholipid‐dependent protein kinase C (PK‐C) activity in PMNs. 6 4,4′‐Diisothiocyano‐2,2′‐disulphonic acid stilbene (DIDS), an anion channel blocker, caused a concentration‐dependent inhibition of granule enzyme release. 7 Activation of PMNs with AA was unaffected by the lipoxygenase/cyclo‐oxygenase inhibitors, 5,8,11, 14‐eicosatetraynoic acid (ETYA) and benoxaprofen, a lipoxygenase inhibitor, 6, 9, deepoxy‐6,9‐(phenylimino)‐Δ6,8‐prostaglandin 11 (piriprost potassium) or a pure cyclo‐oxygenase inhibitor, flurbiprofen. 8 These data define the properties of AA as a secretory stimulus for human PMNs.

Human neutrophil activation with interleukin-1: A role for intracellular calcium and arachidonic acid lipoxygenation

Human monocyte-derived interleukin-1 (IL-1) stimulated the selective extracellular release of cytoplasmic granule-associated elastase from human neutrophils. Although extracellular calcium (Ca2+) enhances but is not required for the expression of granule exocytosis, IL-1 did induce the mobilization of previously sequestered intracellular Ca2+ as measured with the highly selective fluorescent Ca2+ indicator, Quin 2. Further, IL-1 stimulated the mobilization of cell membrane-associated Ca2+ as monitored by a decrease in fluorescence of chlorotetracycline (CTC)-loaded neutrophils. W-7, a calmodulin antagonist, and TMB-8[8(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride], an intracellular Ca2+ antagonist, inhibited the Quin 2 fluorescent response by neutrophils to IL-1. TPCK (N-alpha-p-tosyl-L-lysine chloromethylketone), a serine protease inhibitor, suppressed IL-1-induced Quin 2 and CTC fluorescence. Exposure of neutrophils to IL-1 resulted in a concentration-dependent production of the 5-lipoxygenase product, LTB4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] which was enhanced in the presence of arachidonic acid (AA). LTB4 production by IL-1-activated neutrophils was suppressed by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and piriprost potassium [6,9,deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin l1], and a cyclooxygenase/lipoxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), whereas a cyclooxygenase inhibitor, flurbiprofen, was inactive. These data indicate that cytosolic free Ca2+ ([Ca2+]i) and a metabolite(s) of AA lipoxygenation mediate granule exocytosis elicited with IL-1.

Diacylglycerols modulate human polymorphonuclear neutrophil responsiveness: effects on intracellular calcium mobilization, granule exocytosis, and superoxide anion production.

The synthetic diacylglycerols (DG), sn‐1,2‐dihexanoylglycerol (diC6), sn‐1,2‐dioctanoyl‐glycerol (diC8), and 1‐oleoyl‐2‐acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (Of) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic‐free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura‐2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor‐specific ligands, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), acetylsn‐glyceryl‐3‐phosphorylcholine (AGEPC), and 5(S),12(R)‐dihydroxy‐6,14‐cis‐8,10‐trans‐eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP‐, AGEPC‐, LTB4 and A23187‐stimulated O2 ‐ production. Activation of the respiratory burst with phorbol 12‐myristate 13‐acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG‐mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1‐(‐5‐isoquinolinesulfonyl)‐2‐methylpiperazine hydrochloride (H‐7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC‐mediated autoregulatory mode to influence PMN activation in a response‐specific manner by affecting certain components of receptor‐coupled and receptor‐independent signal transduction systems in a stimulus‐specific manner.

Effects of a protein kinase C inhibitor, K-252a, on human polymorphonuclear neutrophil responsiveness.

We investigated the capacity of K-252a, an inhibitor of rat brain protein kinase C (PKC), to influence polymorphonuclear neutrophil (PMN) PKC and PMN activation with chemically and structurally dissimilar agonists. K-252a inhibited PMN PKC (IC50 = 0.58 microM), and caused a concentration-dependent (0.1-10 microM) inhibition of degranulation elicited with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), the lipid agonists, 5(S), 12(R)-dihydroxy-5,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and phorbol 12-myristate 13-acetate. Superoxide anion (O2-) production by PMNs exposed to these stimuli as well as sn-1,2-dioctanoylglycerol (diC8) was also suppressed by K-252a. PMN PKC activity was inhibited with concentrations of K-252a which suppressed PMN responsiveness. Therefore, K-252a appears to be a useful probe for examining the role of PKC in the underlying pathway(s) of PMN activation.

Recombinant human interleukin-1α and recombinant human interleukin-1β stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis

Recombinant human interleukin-1α (rhIL-1α) and recombinant human interleukin 1β (rhIL-1β) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage expiants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1α was consistently more potent than rhIL-1β. In addition to inducing cartilage matrix resorption, rhIL-1α and rhIL-1β also inhibited the incorporation of [35SO4]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1α and rhIL-1β specifically neutralized the respective cytokines.

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: autoregulatory role for protein kinase C in signal transduction.

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1–10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands,N-formylmethionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4α-phorbol 12, 13-didecanoate (4α-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB4, and A23187-stimulated Superoxide anion (O2−) production. The mobilization of intracellular sequestered calcium (Ca2+) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca2+ concentration ([Ca2+]i) in PMNs loaded with the CA2+-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA2+ mobilization. However, another, but less potent PKC inhibitor,N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), had no effect on the inhibition of PMN activation by PMA.

Recombinant human granulocytemacrophage colony-stimulating factor induces granule exocytosis from human polymorphonuclear neutrophils

Exposure of human polymorphonuclear neutrophils (PMNs) to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in a time- and concentration-dependent (3–100 units/ml) extracellular release of a specific (vitamin B12-binding protein) but not azurophil granule constituent (myeloperoxidase). Negligible granule exocytosis occurred if PMNs were not preincubated with cytochalasin B prior to contact with GM-CSF. The extent of degranulation elicited with GM-CSF was reduced but not abolished when PMNs were incubated with EGTA in calcium-free medium. GM-CSF did not stimulate a rise in the cytosolic-free calcium concentration ([Ca2+],), and it had no effect on PMN protein kinase C (PKC) activity.

NPC 15669‐modulated human polymorphonuclear neutrophil functional responsiveness: effects on receptor‐coupled signal transduction

1 The effect of NPC 15669, N‐carboxy‐L‐leucine, N‐[(2,7‐dimethylfluoren‐9‐yl)methyl]ester), an inhibitor of human polymorphonuclear neutrophil (PMN) adhesion, on granule exocytosis and the oxidative burst was investigated in PMN activated with receptor‐specific pathophysiological stimuli. 2 NPC 15669 caused a concentration‐dependent (1–30 μm) inhibition of the extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12‐binding protein) granule constitutents from PMN exposed to N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), leukotriene B4 (LTB4), platelet activating factor (PAF), C5a and interleukin‐8 (IL‐8). 3 The receptor agonist‐triggered PMN oxidative burst, measured as superoxide anion (O‐2) production, was suppressed by NPC 15669. 4 Phorbol myristate acetate (PMA)‐stimulated degranulation and O‐2) production were unaffected by NPC 15669. 5 NPC 15669 (0.1–10 μm) inhibited receptor‐triggered inositol 1,4,5‐trisphosphate (IP3) production and the IP3‐triggered increase in cytosolic‐free calcium ([Ca2+] I) in FMLP‐activated PMN, but not in cells exposed to the other receptor agonists. 6 NPC 15669 suppressed FMLP but not PMA‐stimulated redistribution of protein kinase C (PKC) in PMN. 7 The specific binding of [3H]‐FMLP but not [125I]‐C5a to PMN was inhibited by NPC 15669. 8 NPC 15669 suppressed O‐2 production and the rise in [Ca2+]i in PMN treated with the guanine nucleotide‐binding protein (G‐protein) activators, sodium fluoride (NaF) and mastoparan, respectively. 9 The results show that NPC 15669 inhibits PMN responsiveness to various receptor agonists, and suggest interference with receptor‐coupled signal transduction in this inflammatory cell at both the receptor and post‐receptor level in a stimulus‐specific manner.

Induction of neutral proteinase and prostanoid production in bovine nasal chondrocytes by interleukin-1 and tumor necrosis factor α: Modulation of these cellular responses by interleukin-6 and platelet-derived growth factor

We have previously reported that recombinant human interleukin-1 (IL-1) stimulates matrix erosion in bovine nasal cartilage explants (R. J. Smith et al., Inflammation 13, 367-382, 1989). This action of IL-1 is believed to be caused by matrix-degrading neutral proteinases produced by activated chrondrocytes. Accordingly, we investigated the effects of recombinant human interleukin-1 alpha (IL-1 alpha), recombinant human interleukin-1 beta (IL-1 beta), and recombinant human tumor necrosis factor alpha (TNF alpha) on bovine nasal chondrocyte (BNC) responsiveness. IL-1 alpha and IL-1 beta stimulated a time (0-72 hr) and concentration-dependent (0.01-10 ng/ml) production of collagenase, gelatinase, caseinase, and prostaglandin E2 (PGE2) in BNC monolayer cultures. Neutral proteinase and PGE2 production by BNC was also induced by TNF alpha (0.2-200 ng/ml) in a time-dependent (0-72 hr) manner. Recombinant human interleukin-6 (IL-6) caused a concentration-dependent (6-200 ng/ml) potentiation of IL-1-stimulated neutral proteinase and PGE2 production by BNC. However, recombinant human platelet-derived growth factor homodimer BB suppressed BNC responsiveness to IL-1. A recombinant human IL-1 receptor antagonist protein inhibited BNC activation by IL-1 but not TNF alpha.