Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.

Fas deficiency delays the resolution of airway hyperresponsiveness after allergen sensitization and challenge.

BACKGROUND: In asthma, persistent inflammation might be the result of (1) an impaired ability to clear inflammatory cells from the airways and/or (2) impaired apoptotic responses. OBJECTIVE: In a mouse model, we investigated the regulatory role of Fas (CD95)-induced apoptosis in the development and resolution of airway inflammation and airway hyperresponsiveness (AHR). METHODS: Mice that were either Fas-sufficient (wild-type; WT) or Fas-deficient (lpr ) were sensitized by intraperitoneal injections of ovalbumin (OVA) and challenged once intranasally with OVA (IP-IN mice). Control (IN) mice were challenged only. RESULTS: IP-IN WT mice developed AHR at 48 hours; changes in airway resistance resolved by 96 hours. Airway responsiveness at 48 hours in IP-IN lpr mice was similar to that in IP-IN WT mice. However, in contrast to WT mice, IP-IN lpr mice sustained significant AHR at 96 hours in comparison with IN lpr mice; the AHR resolved by 6 days. Bronchoalveolar lavage fluid cell composition was similar in all of the different groups at 48 hours and 96 hours. Both IP-IN WT mice and lpr mice exhibited similar tissue eosinophilia, whereas IP-IN lpr mice had significantly lower numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells in comparison with IP-IN WT mice at 48 hours. Anti-IL-5 antibody given to IP-IN lpr mice 48 hours and 72 hours after the challenge significantly decreased AHR and eosinophilic inflammation and increased TUNEL-positive cell numbers at 96 hours. CONCLUSION: These results suggest that Fas expression can regulate the onset and resolution of AHR through an increase in eosinophil apoptosis.

Apoptotic cell removal.

Ingestion by professional or amateur phagocytes is the fate of most cells that undergo apoptosis. Studies in both Caenorhabditis elegans and mammals are now converging to reveal some of the key mechanisms and consequences of this removal process. At least seven corpse removal genes in nematodes have mammalian equivalents, and represent elements of signaling pathways involved in uptake. In mammals, a wide variety of apoptotic cell recognition receptors has been implicated and appears to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. Apoptotic cell removal is normally efficient and non-inflammatory. By contrast, the process may become subverted by parasites to yield a more favorable growth environment, or in other cases lead to fibrosis. Removal may also clinch the apoptotic process itself in cells not yet completely committed to death.

Montelukast, a leukotriene receptor antagonist, for the treatment of persistent asthma in children aged 2 to 5 years.

BACKGROUND: The greatest prevalence of asthma is in preschool children; however, the clinical utility of asthma therapy for this age group is limited by a narrow therapeutic index, long-term tolerability, and frequency and/or difficulty of administration. Inhaled corticosteroids and inhaled cromolyn are the most commonly prescribed controller therapies for young children with persistent asthma, although very young patients may have difficulty using inhalers, and dose delivery can be variable. Moreover, reduced compliance with inhaled therapy relative to orally administered therapy has been reported. One potential advantage of montelukast is the ease of administering a once-daily chewable tablet; additionally, no tachyphylaxis or change in the safety profile has been evidenced after up to 140 and 80 weeks of montelukast therapy in adults and pediatric patients aged 6 to 14 years, respectively. To our knowledge, this represents the first large, multicenter study to address the effects of a leukotriene receptor antagonist in children younger than 5 years of age with persistent asthma, as well as one of the few asthma studies that incorporated end points validated for use in preschool children. OBJECTIVE: Our primary objective was to determine the safety profile of montelukast, an oral leukotriene receptor antagonist, in preschool children with persistent asthma. Secondarily, the effect of montelukast on exploratory measures of asthma control was also studied. DESIGN AND STATISTICAL ANALYSIS: We conducted a double-blind, multicenter, multinational study at 93 centers worldwide: including 56 in the United States, and 21 in countries in Africa, Australia, Europe, North America, and South America. In this study, we randomly assigned 689 patients (aged 2-5 years) to 12 weeks of treatment with placebo (228 patients) or 4 mg of montelukast as a chewable tablet (461 patients) after a 2-week placebo baseline period. Patients had a history of physician-diagnosed asthma requiring use of beta-agonist and a predefined level of daytime asthma symptoms. Caregivers answered questions twice daily on a validated, asthma-specific diary card and, at specified times during the study, completed a validated asthma-specific quality-of-life questionnaire. Physicians and caregivers completed a global evaluation of asthma control at the end of the study. Efficacy end points included: daytime and overnight asthma symptoms, daily use of beta-agonist, days without asthma, frequency of asthma attacks, number of patients discontinued because of asthma, need for rescue medication, physician and caregiver global evaluations of change, asthma-specific caregiver quality of life, and peripheral blood eosinophil counts. Although exploratory, the efficacy end points were predefined and their analyses were written in a data analysis plan before study unblinding. At screening and at study completion, a complete physical examination was performed. Routine laboratory tests were drawn at screening and weeks 6 and 12, and submitted to a central laboratory for analysis. Adverse effects were collected from caregivers at each clinic visit. An intention-to-treat approach, including all patients with a baseline measurement and at least 1 postrandomization measurement, was performed for all efficacy end points. An analysis-of-variance model with terms for treatment, study center and stratum (inhaled/nebulized corticosteroid use, cromolyn use, or none) was used to estimate treatment group means and between-group differences and to construct 95% confidence intervals. Treatment-by-age, -sex, -race, -radioallergosorbent test, -stratum, and -study center interactions were evaluated by including each term separately. Fisher's exact test was used for between-group comparisons of the frequency of asthma attacks, discontinuations from the study because of worsening asthma, need for rescue medication, and the frequencies of adverse effects. Because of an imbalance in baseline values for eosinophil counts for the 2 treatment groups, an analysis of covariance was performed on the eosinophil change from baseline with the patient's baseline as covariate. STUDY PARTICIPANTS: Of the 689 patients enrolled, approximately 60% were boys and 60% were white. Patients were relatively evenly divided by age: 21%, 24%, 30%, and 23% were aged 2, 3, 4, and 5 years, respectively. For 77% of the patients, asthma symptoms first developed during the first 3 years of life. During the placebo baseline period, patients had asthma symptoms on 6.1 days/week and used beta-agonist on 6.0 days/week. RESULTS: In over 12 weeks of treatment of patients aged 2 to 5 years, montelukast administered as a 4-mg chewable tablet produced significant improvements compared with placebo in multiple parameters of asthma control including: daytime asthma symptoms (cough, wheeze, trouble breathing, and activity limitation); overnight asthma symptoms (cough); the percentage of days with asthma symptoms; the percentage of days without asthma; the need for beta-agonist or oral corticosteroids; physician global evaluations; and peripheral blood eosinophils. The clinical benefit of montelukast was evident within 1 day of starting therapy. Improvements in asthma control were consistent across age, sex, race, and study center, and whether or not patients had a positive radioallergosorbent test. Montelukast demonstrated a consistent effect regardless of concomitant use of inhaled/nebulized corticosteroid or cromolyn therapy. Caregiver global evaluations, the percentage of patients experiencing asthma attacks, and improvements in quality-of-life scores favored montelukast, but were not significantly different from placebo. There were no clinically meaningful differences between treatment groups in overall frequency of adverse effects or of individual adverse effects, with the exception of asthma, which occurred significantly more frequently in the placebo group. There were no significant differences between treatment groups in the frequency of laboratory adverse effects or in the frequency of elevated serum transaminase levels. Approximately 90% of the patients completed the study. CONCLUSIONS: Oral montelukast (4-mg chewable tablet) administered once daily is effective therapy for asthma in children aged 2 to 5 years and is generally well tolerated without clinically important adverse effects. Similarly, in adults and children aged 6 to 14 years, montelukast improves multiple parameters of asthma control. Thus, this study confirms and extends the benefit of montelukast to younger children with persistent asthma.

The phosphatidylserine receptor: a crucial molecular switch?

The uptake and removal of necrotic or lysed cells involves inflammation and an immune response, due in part to processes that involve members of the collectin family, surface calreticulin and CD91. Clearance of apoptotic cells, by contrast, does not induce either inflammation or immunity. Could the phosphatidylserine receptor be the molecular switch that determines what the outcome will be?

Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases.

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases and other toxic intracellular contents into tissues can create significant damage, prolonging the inflammatory response. Binding and/or uptake of apoptotic cells by macrophages inhibits release of proinflammatory cytokines by mechanisms that involve anti-inflammatory mediators, including TGF-beta. To model the direct effects of necrotic cells on macrophage cytokine production, we added lysed or apoptotic neutrophils and lymphocytes to mouse and human macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-alpha, and IL-10. Concomitantly, induction of TGF-beta1 by lysed neutrophils was significantly lower than that observed for apoptotic cells. The addition of selected serine protease inhibitors and anti-human elastase Ab markedly reduced the proinflammatory effects, the lysed neutrophils then behaving as an anti-inflammatory stimulus similar to intact apoptotic cells. Separation of lysed neutrophils into membrane and soluble fractions showed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils was largely due to liberated proteases. Therefore, we suggest that anti-inflammatory signals can be given by PtdSer-containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during lysis. Therefore, the outcome of an inflammatory reaction and the potential immunogenicity of Ags within the damaged cell will be determined by which signals predominate.

Impact of a multidisciplinary day program on disease and healthcare costs in children and adolescents with severe asthma: a two-year follow-up study.

For patients whose asthma remains in poor control necessitating high utilization of medical services, a referral to a specialized "center of excellence" is often considered. A decade ago, we evaluated our pediatric asthma program of long-term hospitalization (median stay of 75 days) and found significant decreases in subjects' medical utilization following this intervention. In an effort to contain treatment costs, the former program was markedly altered to one of abbreviated stay with emphasis on family management of asthma. The purpose of the present study was to determine the outcome of children treated in the revised program with regard to disease severity, quality of life, and subsequent utilization of medical resources. Children with severe asthma who were admitted to the program and fulfilled study criteria were consecutively enrolled. Data was obtained concerning disease characteristics, treatment, and quality of life at admission, and at 1 and 2 years following discharge. Medical records for the year prior to program admission and for the 2 years following discharge were coded for medical care encounters. Ninety-eight children, aged 9 months to 18 years (mean age, 10.9 years), were enrolled. They participated in the program for a mean of 15.6 ( +/- 8 SD), median of 15.0, and range of 2-51 treatment days. The group showed significant improvement (P < 0.0001) from admission to 1- and 2-year follow-up in median corticosteroid use, asthma functional severity, perceived competence in asthma management, and quality of life for both caregiver and child. Medical record data showed significant improvement (P < 0.0001) at both 1- and 2-year follow-up in median number of corticosteroid bursts, emergency department visits, hospital days, and overall utilization of medical care encounters. A median total medical encounter cost/patient of $16,250 ($6,972-$25,714 interquartile range (IQR)) for the year prior to program participation was reduced to $1,902 ($505-$6,524 IQR) at 1-year and $690 ($185-$3,550 IQR) at 2- year follow-up (P < 0.0001). We conclude that multidisciplinary care in a short-term, outpatient, day treatment program can significantly contribute to improvement in asthma severity, quality of life, and reduction in healthcare costs.

Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.

A receptor for phosphatidylserine-specific clearance of apoptotic cells.

cytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta.

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.

Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study.

Exhaled nitric oxide (ENO) is a surrogate marker of airway inflammation in asthma. In 12 children aged 6-11 years with mild to moderate persistent asthma, ENO concentrations were measured before and after 4 weeks of treatment with montelukast sodium, a leukotriene receptor antagonist, and 2 weeks after withdrawal of therapy. Baseline ENO levels (mean and 95% confidence interval) were significantly elevated in patients with asthma compared to age-matched nonasthmatic control subjects, with levels of 83 (42-123) vs. 13 (11-15) ppb (P < 0.001). After treatment with montelukast sodium, there was a significant (P < 0.01) reduction in ENO to 58 (27-89) ppb which again rose to 69 (38-99) ppb 2 weeks after treatment was withdrawn. During treatment, the fall in ENO was accompanied by nonsignificant improvements in prebronchodilator forced expiratory volume in 1 s (FEV(1)) from 81-85% predicted or reductions in use of albuterol from a mean of 2.5 to 1.6 puffs/day. Individual ENO measurements and change in ENO concentrations with treatment did not correlate with either pulmonary function changes or use of bronchodilator. These data show that ENO is elevated in children with relatively mild asthma treated with bronchodilator alone, and that treatment with montelukast sodium for 4 weeks results in a significant reduction in ENO concentrations, even in the absence of significant changes in pulmonary function. These findings suggest an anti-inflammatory role for leukotriene D(4) receptor antagonism in the treatment of children with mild to moderate asthma.

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.

Staphylococcal toxic shock syndrome toxin-1 inhibits monocyte apoptosis.

BACKGROUND: Chronic atopic dermatitis (AD) lesions are associated with colonization by exotoxin-producing Staphylococcus aureus. Evidence suggests that cytokine production in AD, particularly of GM-CSF, prolongs survival of both peripheral blood monocytes and dermal monocyte-macrophages, the predominate inflammatory cell in lesions caused by chronic AD. OBJECTIVE: We sought to determine whether the staphylococcal exotoxin, toxic shock syndrome toxin-1 (TSST-1), could stimulate prosurvival cytokine production in monocytes and thereby inhibit apoptosis. METHODS: Cultures of peripheral blood monocytes from normal donors and subjects with AD were incubated with various concentrations of TSST-1, and the incidence of apoptosis was assessed by examining cytospin preparations and the appearance of hypodiploid DNA in the flow cytometer. Culture supernatants were analyzed for GM-CSF, IL-1beta, and TNF-alpha by ELISA. RESULTS: TSST-1, in a concentration-dependent manner starting at 0.1 pg/mL, significantly inhibited monocyte apoptosis and resulted in the production of the prosurvival cytokines GM-CSF, IL-1beta, and TNF-alpha. In coculture conditions with conditioned media from TSST-1-stimulated monocytes, with or without neutralizing antibody to the various cytokines, the data show GM-CSF production was responsible for the inhibition of apoptosis. CONCLUSIONS: The data strongly suggest that staphylococcal exotoxins known to colonize skin lesions on patients with chronic AD may induce the production of GM-CSF, resulting in inhibition of monocyte-macrophage apoptosis, and thereby contribute to the chronicity of this inflammatory disease.

CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (alpha v beta 3).

In vivo, apoptotic cells are efficiently removed by professional or nonprofessional phagocytes, a process thought to be essential for tissue remodeling and resolution of inflammation. Macrophages recognize apoptotic cells by several mechanisms, including recognition of exposed phosphatidylserine (PS); however, PS recognition on apoptotic cells has not been identified as a feature of human macrophages. The purpose of this study was to determine whether human monocyte-derived macrophages could be stimulated to recognize PS, defined as inhibition of phagocytosis by PS-containing liposomes. We also assessed the potential roles for scavenger receptors, CD14, and lectins. Uptake of apoptotic neutrophils into unstimulated macrophages was blocked about 50% by Arg-Gly-Asp-Ser and anti-alpha(v), and up to 20% by oxidized low density lipoprotein and N-acetylglucosamine, implying a major role for integrin and minor roles for scavenger and lectin receptors. Uptake into macrophages stimulated with beta-1,3-glucan was blocked 50% by PS liposomes and 40% by oxidized low density lipoprotein, suggesting that the macrophages had switched from using integrin to recognition of PS. MEM-18 and 61D3 (anti-CD14 mAbs) were poor inhibitors of apoptotic neutrophil uptake, but good inhibitors of apoptotic lymphocyte uptake. The switch to PS recognition was accompanied by down-regulation of alpha(v)beta3 expression and function. Anti-CD36 blocked uptake into unstimulated or stimulated macrophages, suggesting CD36 involvement not only with the alpha(v)beta3 integrin mechanism (as previously reported) but also with PS recognition. A maximum of 70% inhibition was achieved by combining anti-CD36 with either anti-a(v) or PS liposomes.

p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils.

Human neutrophils undergo apoptosis spontaneously when cultured in vitro; however, the signal transduction pathways involved remain largely unknown. In some cell types, c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase (MAPK) have been implicated in the pathways leading to stress-induced apoptosis. In this study, we begin to define two pathways leading to apoptosis in the neutrophil induced either by stress stimuli (UV, hyperosmolarity, sphingosine) or by anti-Fas antibody or overnight culture in vitro (spontaneous apoptosis). Apoptosis induced by stress stimuli activated p38 MAPK, and apoptosis was inhibited by the specific p38 MAPK inhibitor, 6-(4-Fluorophenyl)-2.3-dihydro-5-(4-puridinyl)imidazo(2, 1-beta)thiazole dihydrochloride. Furthermore, differentiation of HL-60 cells toward the neutrophil phenotype resulted in a loss in c-Jun NH2-terminal kinase activation with concomitant acquisition of formylmethionylleucylphenylalanine-stimulatable and stress-inducible p38 MAPK activity as well as apoptosis blockade by the p38 MAPK inhibitor. In contrast, anti-Fas-induced or spontaneous apoptosis occurred independent of p38 MAPK activation and was not blocked by the inhibitor. Both pathways appear to utilize member(s) of the caspase family, since pretreatment with either Val-Ala-Asp-fluoromethyl ketone or Asp-Glu-Val-Asp-fluoromethyl ketone inhibited apoptosis induced by each of the stimuli. We propose the presence of at least two pathways leading to apoptosis in human neutrophils, a stress-activated pathway that is dependent on p38 MAPK activation and an anti-FAS/spontaneous pathway that is p38 MAPK-independent.

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.

Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells, a critical process in tissue remodeling, regulation of the immune response, or resolution of inflammation. Phagocytosis of apoptotic cells by macrophages has been suggested to be a quiet process that does not lead to production of inflammatory mediators. Here we show that phagocytosis of apoptotic neutrophils (in contrast to immunoglobulin G-opsonized apoptotic cells) actively inhibited the production of interleukin (IL)-1beta, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha, as well as leukotriene C4 and thromboxane B2, by human monocyte-derived macrophages. In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2, and platelet-activating factor (PAF) was increased. The latter appeared to be involved in the inhibition of proinflammatory cytokine production because addition of exogenous TGF-beta1, prostaglandin E2, or PAF resulted in inhibition of lipopolysaccharide-stimulated cytokine production. Furthermore, anti-TGF-beta antibody, indomethacin, or PAF receptor antagonists restored cytokine production in lipopolysaccharide-stimulated macrophages that had phagocytosed apoptotic cells. These results suggest that binding and/or phagocytosis of apoptotic cells induces active antiinflammatory or suppressive properties in human macrophages. Therefore, it is likely that resolution of inflammation depends not only on the removal of apoptotic cells but on active suppression of inflammatory mediator production. Disorders in either could result in chronic inflammatory diseases.

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes.

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.