Granulocyte Apoptosis Research Articles

Rexinoid-Triggered Differentiation and Tumor-Selective Apoptosis of Acute Myeloid Leukemia by Protein Kinase A–Mediated Desubordination of Retinoid X Receptor

Apart from PML-retinoic acid receptor-alpha (RARalpha) acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists ("rexinoids") the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cells and patients' blasts. Protein kinase A activation leads to corepressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in "desubordination" of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARbeta is blunted in mouse embryo fibroblasts lacking RARs, but reintroduction of exogenous RARalpha reestablishes responsiveness, thus confirming that the RARalpha-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, tumor necrosis factor-related apoptosis inducing ligand, both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of tumor necrosis factor-related apoptosis inducing ligand and DR5 in AML patient blasts cultured ex vivo. AML patients' blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and French-American-British classification status. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Our results suggest that despite the genetic, morphologic, and clinical variability of this disease, the combination of rexinoids and cAMP-elevating drugs, such as phosphodiesterase inhibitors, might lead to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

Novel Therapies for Asthma - Advances and Problems

It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma. While corticosteroids remain the most important anti-inflammatory treatment for asthma they are rather non-specific in their actions. Their use also raises concerns over side effects and compliance issues, particularly in children and adolescents. There is therefore much effort being made to develop novel more specific and safer therapy for asthma. Efforts are being made to improve existing drugs together with the use of combination therapy with anti-histamines and leukotriene antagonists. An important area for potential advances in glucocorticoid (GC) development include the elucidation of the crystal structure of the GC receptor ligand-binding domain that may provide vital information in dissociating the anti-inflammatory effects of GCs from unwanted side-effects. Other areas include the development of humanised monoclonal antibodies for asthma therapy including those against IgE, IL-4 and IL-5 together with the inhibition of adhesion pathways and/or chemokines responsible for inflammatory cell accumulation in the asthmatic lung. The potential for immunotherapy using T cell peptide epitopes or DNA-based vaccines and the use of anti-inflammatory cytokines such as IL-10 or IFN-gamma are discussed. Several avenues of research are currently underway in an attempt to define mechanisms by which pro-inflammatory cells such as eosinophils can be safely removed from the asthmatic lung through apoptosis induction and their subsequent ingestion by phagocytes. Novel strategies include elucidation of the intracellular pathways controlling granulocyte apoptosis and the receptor mediated events employed by macrophages and bronchial epithelial cells in the recognition and removal of apoptotic cellular corpses. This paper will provide an overview of both the potential and shortcomings of these diverse approaches to drug development for asthma.

CEACAM1 (CD66a) mediates delay of spontaneous and Fas ligand-induced apoptosis in granulocytes

Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro-apoptotic and pro-inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin V staining revealed that binding of CEACAM1-specific antibodies, Fab fragments and soluble CEACAM1-Fc constructs to cell surface-expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)-induced apoptosis. Tyrosine phosphorylation of CEACAM1-L, its association with SHP-1, the activation of Erk1/2 and caspase-3 appeared to be crucial for the CEACAM1-mediated anti-apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes.

Higher Spontaneous and TNFα-Induced Apoptosis of Neonatal Blood Granulocytes

Granulocytes play an important role in inflammatory diseases. Neonates tend to develop granulocytopenia under sepsis and stress. It remains unclear whether apoptosis of neonatal granulocytes is different from that of adult granulocytes. In this study, we analyzed the discrepancy of granulocyte apoptosis between cord blood (CB) and adult blood (AB). We found that spontaneous and tumor necrosis factor-alpha (TNFalpha)-induced granulocyte apoptosis as determined by phosphatidylserine expression and DNA fragmentation were more prominent in CB granulocytes than AB granulocytes. CD95 ligand and TNFalpha levels were significantly higher in CB than in AB (p = 0.001 and p < 0.001, respectively). TNF receptor-2 and CD95 expression on CB granulocytes were not different from those on AB granulocytes. However, the TNF receptor-1 (TNFR1) expression was lower on CB granulocytes than that on AB granulocytes (69.98 +/- 7.32 versus 89.04 +/- 3.73%, p = 0.029). This decrease of TNFR1 expression on neonatal granulocytes might be related to a higher plasma TNFalpha level associated with an intrinsic defect on the dynamic change of membrane TNFR1 expression in neonatal granulocytes. The expression of anti-apoptotic molecule Bcl-2 in neonatal granulocytes was also lower than that in adult granulocytes (4.64 +/- 0.51 versus 7.24 +/- 1.17 unit/mg protein, p = 0.032). Moreover, another anti-apoptotic signal, nuclear factor-kappaB nuclear translocation, was also lower in CB than AB granulocytes. Results from this study suggest that higher plasma death ligands associated with lower anti-apoptotic molecules in granulocytes may act an important role in triggering neonatal granulocyte apoptosis.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Ranasevosa

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog ( Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions.

THE EFFECTS OF TEMPERATURE AND SALINITY ON APOPTOSIS OF PERKINSUS MARINUS

Oyster culture, which is a large part of the North American aquaculture industry, is greatly hindered by the parasite Perkinsus marinus. The different oyster defense mechanisms and how the parasite seems to evade them are not fully understood, and the role of apoptosis as an oyster defense mechanism remains unclear. Apoptosis of eastern oyster (Crassostrea virginica) hemocyte subpopulations (granulocytes and hyalinocytes) was quantified at the single cell level using flow cytometric Annexin-V and TUNEL assays. The influences of habitat salinity (540 mOsm [18.9‰] and 910 mOsm [31.9‰] for oysters and 488 mOsm [17.1‰] and 810 mOsm [28.4‰] for P. marinus) and temperature (14.5°C and 25°C for oysters, 14.4°C and 22.5°C for P. marinus) on apoptosis frequencies were evaluated experimentally on the host hemocytes and the parasite. Apoptosis frequency was higher in granulocytes than hyalinocytes, regardless of temperature or salinity. Salinity affected apoptosis of granulocytes and hyalinocytes, with more apoptosis at 910 mOsm than at 540 mOsm (at 14.5°C and 25°C). Parasite apoptosis frequency was significantly higher on culture at the low salinity (488 mOsm) at both low and high temperatures (14.5°C and 22.5°C). Temperature did not significantly affect either hemocyte or parasite apoptosis frequencies. Investigation of apoptosis as an oyster defense mechanism and how temperature and salinity affect C. virginica and P. marinus apoptosis may lead to a better understanding of host/parasite interactions and the mechanisms leading to disease susceptibility or resistance. Such information could lead to better management strategies that may result in reduced disease morbidity and mortality for oyster producers.

Loss of GM1 surface expression precedes annexin V‐phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging

Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.

Endotoxin reduces CD95-induced neutrophil apoptosis by cIAP-2-mediated caspase-3 degradation.

Reduced apoptosis of neutrophil granulocytes (PMN) contributes to pathogenesis of systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction syndrome. The intracellular inhibitor of apoptosis proteins has been shown to inhibit activated caspase-3. We investigated the turnover dynamics of cIAP-2 mRNA and caspase-3 protein in a neutrophil ex vivo model of sepsis. PMN (1 x 10(6)/mL) from 7 healthy volunteers were preincubated with endotoxin (lipopolysaccharide [LPS], 1 microg/mL) for 5 hours, followed by an additional hour with or without the proteasome inhibitor (30 microM), before incubation with or without agonistic CD95 antibody (100 ng/mL) for another 16 hours. Apoptosis was quantified by Annexin-V and propidium iodide staining by flow cytometry (using a fluorescence-activated cell sorter). Caspase-3 activity was determined by DEVD-afc-cleavage assay. Expression of ubiquitinated caspase-3 and cIAP-2 protein was detected by Western blot analysis and cIAP-2 mRNA by reverse transcriptase-polymerase chain reaction. Within 2 hours LPS induced cIAP-2 mRNA and protein. In addition, LPS increased ubiquitination of activated caspase-3. LPS significantly (p < 0.05) reduced spontaneous (66.1 +/- 2.3% to 24.8 +/- 4.8%) and CD95-induced (90.8 +/- 0.9% to 64.3 +/- 4.2%) apoptosis and caspase-3 activation. Inhibition of the proteasome completely abolished the antiapoptotic effect of LPS on spontaneous (52.6 +/- 2.4%) and CD95-induced (88.7 +/- 2.6%) apoptosis and degradation of caspase-3. Induction of cIAP-2 by endotoxins and accelerated degradation of activated caspase-3 by the proteasome might be responsible for reduced apoptosis in PMN during sepsis.

CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes.

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. We now show that a novel signal for delaying neutrophil apoptosis is unmethylated CpG motifs prevalent in bacterial DNA (CpG- DNA). Human neutrophils express toll-like receptor 9 that recognizes these motifs. CpG-DNA, but not mammalian DNA or methylated bacterial DNA, markedly enhanced neutrophil viability by delaying spontaneous apoptosis. Endosomal maturation of CpG-DNA is prerequisite for these actions and was coupled to concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of decreases in mitochondrial transmembrane potential, cytochrome c release and caspase-3 activation. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3-kinase partially reversed these actions of CpG-DNA; however, they did not produce additive inhibition. Furthermore, intravenous injection of CpG-DNA (200 microg/kg) into rats evoked slight decreases in blood pressure and induced a modest leukocytosis, whereas it effectively suppressed neutrophil apoptosis as assayed ex vivo. Our results indicate that unmethylated CpG motifs in bacterial DNA promote neutrophil survival by suppressing the apoptotic machinery and may therefore contribute to prolongation and amplification of inflammation.

Regulation of granulocyte apoptosis by NF-kappaB.

Granulocyte apoptosis is a crucial part of the successful resolution of inflammation. In vitro results show that activation of NF-kappaB (nuclear factor kappaB) in granulocytes is a survival mechanism. NF-kappaB inhibitors increase the rate of constitutive apoptosis in neutrophils and eosinophils and cause these cells to respond to the pro-apoptotic effects of TNF-alpha (tumour necrosis factor-alpha). Results from both in vivo and in vitro experiments suggest that there are at least two important waves of NF-kappaB activation in inflammatory loci, which increase the expression of COX-2 (cyclooxygenase-2), itself an NF-kappaB controlled gene. The first wave causes the production of inflammatory mediators such as PGE2 (prostaglandin E2), allowing the establishment of inflammation. The second wave causes the synthesis of PGD2 and its metabolites that induce granulocyte apoptosis by inhibiting NF-kappaB activation. These metabolites may therefore be important physiological mediators controlling the resolution of inflammation. Although NF-kappaB is an important target for anti-inflammatory therapy, the timing of inhibition in vivo may be crucial, to ensure that production of PGD2 and its sequential metabolites can occur.

Granulocyte apoptosis: who would work with a ‘real’ inflammatory cell?

The neutrophil granulocyte is a key factor in cellular innate defence mechanisms against infection or tissue damage. Granulocyte apoptosis is now acknowledged to have a critical role in progression of inflammatory responses. Granulocytes are preprogrammed to die with important physiological mechanisms for non-inflammatory clearance. Shutdown of secretory capacity represents an important aspect of the programme of biochemical events that accompany neutrophil apoptosis together with surface molecular changes that serve to identify apoptotic cells as targets for phagocytic removal. Defining the underlying regulatory mechanisms together with the changes in patterns of gene/protein expression associated with granulocyte death remains a challenge. Use of novel strategies for inducing cell death will allow biochemical approaches to dissect the underlying pathways. Although study of granulocyte cell death has especial difficulties when compared with other cell types, there are clearly potential benefits for new therapeutic approaches to treat inflammatory diseases.

Characterization of the survival effect of tumour necrosis factor-alpha in human neutrophils.

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFalpha (tumour necrosis factor-alpha) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-kappaB (nuclear factor-kappaB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFalpha increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFalpha addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFalpha, implying a TNFR1-dependent process. Finally, I-kappaBalpha (inhibitory kappaB-alpha) and NF-kappaB time-course studies demonstrated that the survival effects of both GM-CSF and TNFalpha could be explained by maintenance of functional NF-kappaB.

Getting to grips with the granulocyte: manipulation of granulocyte behaviour and apoptosis by protein transduction methods.

Human granulocytes clearly play an important role in host defence against invading organisms, however they have also been implicated in the pathogenesis and progression of many chronic inflammatory diseases. In addition, these cells have been paramount in gaining a better understanding of many key-signalling pathways regulating fundamental biological processes. Since granulocytes are terminally differentiated and undergo relatively rapid constitutive apoptosis it has been difficult to manipulate intracellular events by transfection or transduction procedures. It has been shown in recent years that the HIV-TAT protein transduction system can be successfully used in granulocytes to manipulate key signalling mechanisms regulating functional responsiveness and survival. In this paper, we review recent literature highlighting important developments using this system in isolated human granulocytes and in inflammatory process in vivo where these cells play a prominent role.

Regulation of granulocyte apoptosis by phosphatidylinositol 3-kinase.

Granulocytes are critical components of the innate immune system whose lifespan is limited by an intrinsic, constitutive, apoptotic pathway. However, the lifespan of these cells can be extended at an inflammatory locus through interaction with survival factors. Although a wide variety of factors can modulate granulocyte survival, they often utilize a common subset of intracellular signal transduction pathways. Over the last decade, evidence has accumulated that the PI3K (phosphatidylinositol 3-kinase) family of lipid kinases may be critical in regulating the ability of granulocytes to survive at inflammatory loci. Studies utilizing both pharmacological inhibitors of PI3K and isoform-specific knockout mice have demonstrated that this enzyme is needed for the anti-apoptotic effects of granulocyte survival factors. More recently, a serine/threonine protein kinase, termed protein kinase B (also known as c-akt), has been demonstrated to be important in modulating the prosurvival effects of PI3K activation. This can occur through modulation of the expression or phosphorylation of members of the Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) family of apoptosis regulators. This review summarizes recent results that have implicated a role for PI3K in regulating granulocyte survival.

Tissue inhibitor of metalloproteinase 1 activates normal human granulocytes, protects them from apoptosis, and blocks their transmigration during inflammation.

Urinary levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) higher than those of matrix metalloproteinase 9 (MMP-9) during acute pyelonephritis have previously been associated with a higher degree of acute inflammation and of postinfective renal scarring. The aim of the present study was to evaluate possible mechanisms by which TIMP-1 could affect the scarring process already during the acute phase of inflammation. The growth of Escherichia coli, bactericidal activity of fresh human blood, and respiratory burst, spontaneous apoptosis, and trans-basement membrane migration of normal human granulocytes were studied in vitro in the presence of different concentrations of recombinant human TIMP-1. To imitate the "normal" environment during inflammation in the kidney, granulocytes were also incubated with a conditioned medium from E. coli-stimulated renal epithelial cells. In order to compare our data with the in vivo situation, blood and urinary leukocyte levels were analyzed for 40 children with acute pyelonephritis, together with urinary MMP-9 and TIMP-1 levels. TIMP-1 at a concentration of 500 ng/ml increased the bactericidal activity of blood, increased the respiratory burst of granulocytes, decreased phosphatidylserine exposure and caspase 3 activity, which are features of spontaneous apoptosis, and inhibited granulocyte transmigration. Moreover, in the patients with pyelonephritis, MMP-9/TIMP-1 ratios in urine correlated with the degree of leukocyte transmigration. Thus, our data suggest that TIMP-1 specifically blocks the transmigration of granulocytes into urine. Entrapped and activated granulocytes, protected from apoptosis, might excessively destroy renal parenchyma and thus contribute to the pathogenesis of renal scarring following acute pyelonephritis.

Hemopoiesis and Allergy, An Introduction to the Special Issue

Recent research from many laboratories highlighted important connections between hemopoiesis and allergy / asthma. Hemopoiesis in an allergic inflammation site differs greatly from steady-state (homeostatic) hemopoiesis in the bone-marrow, due to differences in the hemopoietic factors present, as well as on their targets. One major aspect concerns the role of GM-CSF, which seems unnecessary for steady-state hemopoiesis in the absence of infection, but plays a central role in the immunobiology of the lungs, especially in the instructive phase of imunity, by its ability to influence dendritic cell differentiation and function. Allergens, alone or in combination with other factors, can induce GM-CSF production in the airways, and therefore bypass the requirement for microbial products in the activation of dendritic cells. Hemopoietic progenitors and dendritic cell precursors recruited to the lung provide additional potential targets for GM-CSF. Stem Cell Factor also plays a major role, by acting on mast cells to promote their maturation and functional activation, and on additional cell types from the lung to promote allergic inflammation, in concert with IgE antibodies and a variety of chemokines. Hemopoietic cells isolated from bone-marrow, blood and allergic lung may differ in their responses to hemopoietic factors and drugs. This is changing our perception of the effects of glucocorticoids on hemopoiesis, and especially on eosinophil differentiation. Manipulation of the process of granulocyte (eosinophil and neutrophil) apoptosis, as well as engineering modified versions of hemopoietic cytokines, such as IL-5, are current approaches to developing valuable tools for intervention in the context of hemopoiesis in allergy. Keywords: hemopoiesis, allergy, corticosteroids, asthma, allergic rhinitis, gm-csf, Ige, stem cell factor, interleukin- 5

Regulation of granulocyte apoptosis by hemopoietic growth factors, cytokines and drugs: potential relevance to allergic inflammation.

It has become apparent that the resolution of inflammation depends on the removal of unwanted inflammatory cells, a process governed by physiological apoptosis and non-inflammatory clearance of apoptotic cells. Granulocytes are central to many of the pathophysiological consequences of uncontrolled inflammatory reactions. Hemopoietic factors and cytokines play a critical role in regulating the longevity of these cells in vitro and in vivo. Here we review the progress that has been made in the understanding of granulocyte apoptosis and the implications for immunotherapy and pharmacological strategies in the treatment of allergic inflammatory diseases for therapeutic gain.

Lymphocyte Subpopulations, Oxidative Burst and Apoptosis in Peripheral Blood Cells of Patients with Multiple Sclerosis–Effect of Interferon-β

At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by IFN-β. However, its in vivo effects remain incompletely understood. If applied parenterally, the hydrophobic IFN-β acts primarily on blood cells with probable selectivity for functionally different lymphocyte subpopulations, monocytes and granulocytes. We have investigated the expression of the activation marker interleukin-2 receptor-α (CD25) on CD3+ T cells, CD19+ B cells, foetal-type γδ+CD3+ T cells and foetal-type CD5+CD19+ B cells of the peripheral blood. In addition, the oxidative burst activity and apoptosis have been determined in mononuclear and polymorphonuclear blood cells, respectively. The study accompanied a phase III trial with IFN-β1b (BETAFERON®, Schering). Two groups of MS patients with relapsing-remitting course of the disease have been investigated at 8 time points (days 0, 5, 15, 31, 60, 90, 180 and 270 after starting therapy): (1) verum group (n=8) with application of 8 Mill. units IFN-β1b every other day, and (2) placebo group (n=4) with application of placebo for 3 months and therapy as in (1) from day 90 onward. The main results were: (1) Activated T cells decreased until day 180 in the verum group and return thereafter to pre-treatment values, whereas in the placebo group the values remained relatively stable over the whole observation period. (2) Activated B cells increased between days 90 and 270 in both groups, i.e. after verum application in both groups. (3) Foetal-type B cells were more activated than total B and T cells with increase over time in both groups. (4) Foetal-type T cells exerted relatively stable intra-individual levels with generally low CD25 expression, but punctual CD25 peaks in both groups. (5) The spontaneous oxidative burst was higher in lymphocytes, more variable in monocytes and faster increasing in granulocytes in the verum group than in the placebo group. (6) Apoptosis of mononuclear cells and granulocytes showed similar variations in the verum and placebo groups with the exception of a selective increase over time of the proportion of granulocytes undergoing induced apoptosis in the verum group. It is concluded that IFN-β has the following main effects on the immune system of MS patients: (1) the T cell immunity is systemically and reversibly suppressed, (2) the foetal-type lymphocytes, which are responsible for the first line of defence of infections, are stimulated in the long range, (3) the oxidative burst activity is increased in lymphocytes and granulocytes and instable in monocytes, and (4) the inducibility of apoptosis in granulocytes is increased. Re-examination of the altered blood cell parameters after long-term IFN-β therapy is warranted.

Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation.

Glucocorticoids represent one of the most effective clinical treatments for a range of inflammatory conditions, including severe acute inflammation. Although glucocorticoids are known to affect processes involved in the initiation of inflammation, the influence of glucocorticoids on the mechanisms by which acute inflammation normally resolves have received less attention. Apoptosis of granulocytes present at inflamed sites leads to their rapid recognition and internalisation by macrophages, a process which may be important for resolution of inflammation. However, if clearance of either eosinophils or neutrophils is impaired, these cells rapidly undergo secondary necrosis leading to release of pro-inflammatory mediators from the phagocyte, potentially prolonging inflammatory responses. Physiologically relevant concentrations of glucocorticoids accelerate eosinophil apoptosis whilst delaying neutrophil apoptosis during in vitro culture. Here we discuss key pathways regulating the granulocyte apoptotic programme and summarise the effects of glucocorticoids on monocyte differentiation and the consequent changes to apoptotic cell clearance capacity. Definition of the mechanisms underlying resolution of inflammatory responses following glucocorticoid treatment may unveil new targets for modulation of inflammatory disease, allowing co-ordinated augmentation of granulocyte apoptosis together with increased macrophage capacity for clearance of apoptotic cells.

Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis.

1 Since most inflammatory mediators that stimulate granulocyte responsiveness also delay apoptosis, it is often assumed that activation and longevity are causally related. Using isolated human peripheral blood neutrophils and eosinophils, we examined this association by exploiting the proinflammatory lipid mediators, the leukotrienes (LTs), and investigated granulocyte function and apoptosis. 2 LTB(4) induced elevation of intracellular free Ca(2+) concentration ([Ca(2+)](i)), cell polarisation and retardation of neutrophil apoptosis, although the antiapoptotic effect occurred only at concentrations > or =300 nM. LTB(4)-induced activation was attenuated by CP-105,696, a BLT1-specific antagonist suggesting classical LTB(4) receptor BLT1 involvement. 3 Despite demonstrating the presence of the neutrophil intracellular LTB(4) receptor peroxisome-proliferator activator receptor-alpha (PPARalpha) in neutrophils, the selective PPARalpha agonist WY-14,643 did not mimic LTB(4)-induced prosurvival effects. 4 LTB(4)-induced survival, however, also appeared to be mediated by BLT1 since CP-105,696 inhibited the LTB(4)-mediated antiapoptotic effect. Furthermore, based on studies with CP-105,696 and 5-lipoxygenase inhibitors, lipopolysaccharide (LPS)-, granulocyte-macrophage colony-stimulating factor (GM-CSF)-, dexamethasone- and dibutyryl-cAMP (db-cAMP)-induced delay of neutrophil apoptosis did not involve autocrine production of LTB(4). 5 Although LTB(4) and LTD(4) induced human eosinophil [Ca(2+)](i) elevation and polarization, these LTs did not influence eosinophil apoptosis. Furthermore, LTB(4)- and LTD(4)-induced eosinophil activation was attenuated by CP-105,696 and the Cys-LT(1) receptor antagonist montelukast, respectively, highlighting specific receptor dependency. 6 Thus, mediator-triggered granulocyte activation and antiapoptotic pathways are distinct events that can be differentially regulated.