Christopher R. Tudan

Inhibition of TNF-α-Induced Neutrophil Apoptosis by Crystals of Calcium Pyrophosphate Dihydrate Is Mediated by the Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-Kinase/Akt Pathways Up-Stream of Caspase 3

The role of protein kinases in the inhibition of TNF-α associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-α and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-α produced only a modest and delayed activation of Akt. In the presence of TNF-α, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-α itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 μM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-α. Furthermore, the inhibition of the Mek1/Mek2→ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-α-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.

Calcium pyrophosphate dihydrate crystals activate MAP kinase in human neutrophils: inhibition of MAP kinase, oxidase activation and degranulation responses of neutrophils by taxol.

The activation of MAP kinase in human neutrophils stimulated by both uncoated and plasma‐opsonized crystals of triclinic calcium pyrophosphate dihydrate (CPPD) was investigated. The effect of taxol on MAP kinase activation and on the responses of neutrophils stimulated by plasma‐opsonized crystals was determined. MAP kinase activation was identified and quantified in Mono Q chromatography separated fractions of neutrophils that had been incubated with CPPD crystals by measuring []> γ‐32P]adenosine triphosphate (ATP) phosphorylation of myelin basic protein and using immunoblotting techniques. Human neutrophils were incubated with taxol (0–50 μm), added to plasma‐opsonized CPPD (50 mg/ml) and MAP kinase activation, chemiluminescence, superoxide anion generation, lysozyme and myeloperoxidase release were monitored. Both uncoated and plasma coated CPPD crystals induced a large increase in MAP kinase activity in neutrophils over control levels within 1 min of incubation. Pretreament of neutrophils with taxol was able to suppress this activation of MAP kinase. Taxol produced a concentration‐dependent inhibition of opsonized CPPD‐induced neutrophil chemiluminescence, superoxide anion production and myeloperoxide release. Taxol at 28 μm also significantly inhibited chemiluminescence, superoxide anion production and myeloperoxidase release from neutrophils stimulated by opsonized zymosan. This is the first report of crystal‐induced activation of MAP kinase in neutrophils. Microtubule‐associated processes, such as signal transduction, secretion and phagocytosis are involved in particulate‐induced neutrophil responses. We have suggested that the inhibitory effect of taxol observed in this work is due to its stabilizing effect on microtubules and disruption of MAP kinase activation associated with microtubules.

Selective inhibition of protein kinase C, mitogen-activated protein kinase, and neutrophil activation in response to calcium pyrophosphate dihydrate crystals, formyl-methionyl-leucyl-phenylalanine, and phorbol ester by O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470).

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 microM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 microM, 1 min), but had no effect on PMA-treated cells (0.5 microM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46+/-9 and 54+/-3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.

The effect of inhibiting topoisomerase I and II on the antiapoptotic response associated with pro-inflammatory crystals of calcium pyrophosphate dihydrate in human neutrophils

Abstract:Objective and Design: To investigate the ability of various topoisomerase I and II inhibitors to reverse the pro-survival effects of calcium pyrophosphate dihydrate (CPPD) crystals on human neutrophils, thereby identifying potential agents that may promote the resolution of neutrophil accumulation typical of crystal associated inflammatory diseases.¶Materials and Methods: Freshly isolated human neutrophils incubated in the presence of CPPD crystals, with or without the pro-apoptotic cytokine TNF-α, were pre-incubated in the presence or absence of the topoisomerase I inhibitors camptothecin, nogalamycin or β-lapachone, or topoisomerase II inhibitors etoposide, doxorubicin or mitoxantrone. Neutrophil respiratory burst was assessed via chemiluminescence, and two quantitative methods were used for the determination of neutrophil apoptosis; cytoplasmic histone-associated-DNA fragmentation assessment, and endogenous caspase 3 substrate (Ac-DEVD-AMC) cleavage.¶Results: β-lapachone and mitoxantrone effectively repressed CPPD crystal associated respiratory burst, whereas the other topoisomerase inhibitors had no inhibitory or stimulatory effect. Camptothecin and all of the topoisomerase II inhibitors induced neutrophil apoptosis, even in the presence of the CPPD crystals that normally repress TNF-α-induced and spontaneous apoptosis.¶Conclusions: These results suggest that although topoisomerase II antagonists are distinctively effective agents at reversing the pro-survival effects of crystals on neutrophils, camptothecin was unique as a topoisomerase I inhibitor in that it was significantly more effective as a pro-apoptosis inducer than the topoisomerase II poisons without affecting normal neutrophil activation responses.

CPPD crystal-induced suppression of neutrophil apoptosis is regulated by the ERK1/2 and PI3-kinase/Akt pathways

The inflammation associated with many forms of arthritis is thought to arise from the presence of large numbers of immune cells, such as neutrophiis, in the synovial joints of humans. The extended inflammation associated with crystalinduced arthritis arises from the interaction of neutrophils with crystals in the synovial joint, which activate the neutrophils to initiate an acute inflammatory response [1]. In vitro, both plasmaor synovial fluid-coated crystals of calcium pyrophosphate dihydrate (CPPD) have been shown to induce neutrophil activation [2]. One reason for the extended duration of inflammation in this disease may be due to the inhibition of neutrophil apoptosis by crystals. This inhibition may result in the neutrophils remaining in the joint for extended periods. Intracellular mediators of neutrophil apoptosis are currently under investigation as potential pharmacological targets for anti-inflammatory drug development. The ERK family kinases and the PI3-K/Akt pathways have been shown to regulate apoptosis in other cells [3, 4]. In the present study, we investigated the effects of CPPD crystals on spontaneous and TNF-a associated neutrophil apoptosis, and the role of the ERK1/ERK2 and PI3-K/Akt signalling pathways in the regulation of neutrophil apoptosis.