No abstract

Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of “semimature” 5-day human monocyte-derived macrophages (Mφ) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day Mφ ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day Mφ uptake of alternative apoptotic “targets” (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine Mφ elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived Mφ, with greater increases in 5-day Mφ uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into Mφ. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases.

Eosinophils and neutrophils are closely related, terminally differentiated cells that in vitro undergo constitutive cell death by apoptosis. The onset of apoptosis in both cell types can be delayed by hemopoietins and inflammatory mediators. Although there have been a number of reports demonstrating that glucocorticoids (in particular dexamethasone) antagonize the eosinophil life-prolonging effects of hemopoietins, direct effects of dexamethasone on eosinophil apoptosis have not been documented. In this study we examined the direct effects of glucocorticoids on eosinophil and neutrophil apoptosis in light of their common therapeutic use as anti-inflammatory and anti-allergic/hypereosinophilic agents. We found that treatment with dexamethasone induced eosinophil apoptosis. In contrast, dexamethasone was a potent inhibitor of neutrophil apoptosis. The effect of dexamethasone on both cell types was mediated through the glucocorticoid receptor, i.e., it was abolished by the glucocorticoid receptor antagonist RU38486. This is the first description of an agent that promotes eosinophil apoptosis while inhibiting neutrophil apoptosis, and thus presents a novel approach to the study of control of apoptosis in these closely related cell types as well as increases our understanding of the clinical action of glucocorticoids in inflammation.

Neutrophilic 111 and eosinophilic [2] granulocytes undergo rapid and spontaneous cell death by apoptosis, resulting in their recognition and phagocytosis by macrophages. This clearance mechanism is thought to be important in the resolution of the inflammatory response, thereby limiting tissue injury and promoting wound repair [3]. However the underlying intracellular regulatory signalling mechanisms governing granulocyte apoptosis are unknown. In this study, we examined the constitutive rate of granulocyte apoptosis in the absence and presence of agents which (1) inhibit protein kinase C, using staurosporine (0.1 pM -10 pM) and Ro-31-8220 (0.1 pM -10 pM) and (2) elevate [CaZ+]i using thapsigargin, which increases [CaP+]i by a mechanism involving the mobilization of intracellular calcium stores 141, and A23187, a calcium ionophore.Human neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers, as described previously [1,2]. Harvested cells of both types were resuspended in Iscove's DMEM supplemented with 5% autologous serum and 50 U/ml penicillin and streptomycin, and finally cultured at 37 OC in a humidified 5% COz-rich atmosphere. Granulocytes (3 x 105) were incubated in the presence of various concentrations of staurosporine (0.1 pM -10 pM), Ro-31-8220 (0.1 pM -10 IM), thapsigargin (2 pM) and A23187 (0.1 vM), as indicated in the results, in a final volume of 150 p1 in flatbottomed 96 well flexiplates. After 20h in culture, the granulocytes were harvested for assessment of morphology, viability (trypan blue exclusion) and recovery. Cytocentrifuge preparations of duplicate cell samples in each experiment were prepared, fixed in methanol, and stained with Diff-Quick. Slides were examined microscopically to determine the number of cells with apoptotic morphology in five fields of view (at least 500 cells were counted) on each preparation. Results were expressed as mean f SEM for each preparation and analysed by analysis of variance followed by Student-Newman-Keuls post test. When p<0.05 (' ), the results were considered to be significant. Figure 1 demonstrates that inhibitors of PKC induced a pro-apoptotic effect upon both neutrophils and eosinophils, as illustrated in graph A and B respectively.

The onset of constitutive cell death by apoptosis in human neutrophils can be delayed by treatment with glucocorticosteroids such as dexamethasone [ 1-31. The mechanisms underlying this steroid-mediated prolongation of neutrophil survival are however unknown. Okadaic acid and calyculin A, specific and potent inhibitors of protein phosphatases 1 and 2A, have been shown to influence the rate of apoptosis in different cell types [4, 51. Moreover, protein dephosphorylation has been implicated as an essential step for glucocorticoid-mediated apoptosis in T-cell hybridomas [6]. In this study, okadaic acid and calyculin A were used to investigate whether phosphorylation/dephosphorylation plays a role in the intracellular signalling pathways controlling dexamethasone-mediated inhibition of neutrophil apoptosis.Human neutrophils were isolated from the peripheral blood of healthy volunteers, as described previously [7]. Harvested neutrophils were resuspended in Iscove's DMEM supplemented with 5% autologous serum and 50 U/ml penicillin and streptomycin, and finally cultured at 37 OC in a humidified 5% C02-rich atmosphere. Neutrophils (3 x 10s) were incubated in the presence of various concentrations of okadaic acid (0.5 nM -500 nM) and calyculin A (1 nM -100 nM) both alone and in combination with dexamethasone (1 pM), in a final volume of 150 pl in flat-bottomed 96 well flexiplates. After 20h in culture, neutrophils were harvested for assessment of morphology, viability (trypan blue exclusion) and recovery. Cytocentrifuge preparations of duplicate cell samples in each experiment were prepared, fixed in methanol, and stained with Diff-Quick. Slides were examined microscopically to determine the number of cells with apoptotic morphology in five fields of view (at least 500 cells were counted) on each preparation. Results were expressed as mean f SEM for each preparation and analysed by analysis of variance followed by Student-Newman-Keuls post test. When p<0.05 (*), the results were considered to be significant. Figure 1 demonstrates that okadaic acid (100 nM) had no . . 2 e d i a ted Neutrophils were cultured for 20 h in the presence of dexamethasone (1 pM), okadaic acid (100 nM) or both. Data represent mean f SEM of 4 experiments, each performed in duplicate. significant effect upon the control rate of neutrophil apoptosis while reversing dexamethasone-mediated inhibition of neutrophil apoptosis at the concentration stated. We have previously shown that over a range of concentrations (0.5 nM-500 nM) okadaic acid has a bi-phasic effect; low concentrations inhibited whereas high concentrations promoted the rate of neutrophil apoptosis [8]. ' 0°1 80 -Y) . I 5 60 9 40 ae 20 n 3 w e 2 . Effect o f calvculin A uDon dexamethasone-medw Neutrophils were cultured for 20h in the presence of dexamethasone (1 pM), calyculin A (10 nM) or both. Data represent mean k SEM of 3 -4 experiments, each performed in duplicate.Calyculin A (10 nM) also had no significant effect upon the control rate of neutrophil apoptosis while reversing dexametha...