The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Triggiani, M.
Right arrow Articles by Marone, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Triggiani, M.
Right arrow Articles by Marone, G.
The Journal of Immunology, 2000, 164: 4908-4915.
Copyright © 2000 by The American Association of Immunologists

Secretory Phospholipases A2 Induce ß-Glucuronidase Release and IL-6 Production from Human Lung Macrophages1

Massimo Triggiani2, Francescopaolo Granata, Alfonso Oriente, Valeria De Marino, Marco Gentile, Cecilia Calabrese, Cristiana Palumbo and Gianni Marone

Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of ß-glucuronidase (16.2 ± 2.4% and 13.1 ± 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of ß-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in ß-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on ß-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced ß-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phospholipases A2 (PLA2s)3 are a family of enzymes that hydrolyze fatty acids at the sn-2 position of phospholipids (1, 2). Different PLA2s have been isolated from snake and bee venoms as well as from mammalian cells and tissues (3). PLA2s differ in their site of action in secretory PLA2s (sPLA2s) and cytosolic PLA2s (cPLA2s). The sPLA2s are low m.w. (14–18 kDa) enzymes, active in the extracellular environment. This class includes the PLA2 isolated from snake venoms (group IA) and from pancreas (group IB) (4, 5). Group IIA includes the sPLA2 initially isolated from human platelets (6) and that released in inflammatory fluids, such as the synovial fluid of patients with rheumatoid arthritis (6, 7) and the bronchoalveolar lavage fluid of patients with bronchial asthma (8).

The sPLA2s are considered digestive, neurotoxic, myotoxic, and anticoagulant enzymes (9, 10). However, more recently, sPLA2s have been reported to exert also proinflammatory and antibacterial activities (11, 12, 13). The recovery of large quantities of group IIA sPLA2 from inflammatory fluids and increased levels of this enzyme in plasma of patients with inflammatory diseases such as septic shock, adult respiratory distress syndrome, and acute pancreatitis led to the hypothesis that sPLA2s may play a role in inflammation and tissue damage (14). One mechanism by which sPLA2s may participate in inflammatory reactions is by generating arachidonic acid, the precursor of eicosanoids (15, 16). These molecules are potent mediators of inflammation by influencing vascular and bronchial responses and promoting inflammatory cell recruitment (17). However, it is becoming increasingly evident that many effects of sPLA2s are unrelated to their enzymatic activity; rather, they can be attributed to the engagement of specific receptors on target cells (18, 19, 20). One of the receptors for sPLA2s, the M-type, displays a high degree of homology with the mannose receptor, a member of the lectin-binding family of receptors, constitutively expressed on macrophages (21). Binding of the mannose receptor on macrophages activates phagocytosis (22) and the production of proinflammatory cytokines (23). These observations raised the question of whether sPLA2s activate human macrophages by binding to specific membrane receptors. Macrophages play a central role in inflammatory and immune responses by releasing a variety of mediators and cytokines (24). In addition, macrophages are abundant at sites where group IIA sPLA2 is released in vivo, such as the synovial fluid (25) or the alveolar space (26). Our hypothesis was that macrophages may be activated by sPLA2s to produce different proinflammatory mediators. To test this hypothesis we explored the abilities of two different sPLA2s (group IA and IIA) to induce the release of the lysosomal enzyme ß-glucuronidase and the production of IL-6 from human lung macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and buffers

Group IA sPLA2 (from Naja mossambica mossambica venom), p-aminophenyl-mannopyranoside-BSA (mp-BSA), LPS, Percoll, L-glutamine, antibiotic-antimycotic solution (10,000 IU/ml penicillin, 10 mg/ml streptomycin, and 25 µg/ml amphotericin B), fatty acid-free HSA, Triton X-100, and phenolphthalein glucuronide were purchased from Sigma (St. Louis, MO). RPMI and FCS were purchased from ICN (Costa Mesa, CA). Arachidonoyl trifluoromethyl ketone (AACOCF3) was purchased from Biomol (Plymouth Meeting, PA). The mAb anti-human synovial PLA2 was purchased from Cayman Chemical (Ann Arbor, MI). Group IIA (recombinant human synovial) PLA2 was a gift from Dr. James Winkler (SmithKline and Beecham, King of Prussia, PA). LY311727 was a gift from Dr. Jerome H. Fleisch (Lilly Research Laboratories, Indianapolis, IN). The anti-mannose receptor Ab (PAM-1) was a gift from Dr. Silvano Sozzani (Istituto Mario Negri, Milan, Italy). A 100-bp DNA ladder was purchased from Life Technologies (Gaithersburg, MD). IL-6 and ß-actin primers were designed by Dr. David Essayan (Johns Hopkins University, Baltimore, MD) and were produced and purified by The Johns Hopkins DNA Core Facility. All other reagents were obtained from Carlo Erba (Milan, Italy).

PIPES buffer was composed of 25 mM PIPES (Sigma), 110 mM NaCl, and 5 mM KCl, pH 7.4. Glycine buffer was composed of 400 mM glycine and 400 mM NaCl, pH 10.3 (27).

Isolation and purification of human lung macrophages

Macrophages were obtained from the lung parenchyma of patients undergoing thoracic surgery for lung carcinoma as previously reported (28). Macroscopically normal tissue was minced finely with scissors and washed extensively with PIPES buffer over Nytex cloth (120 µm pore size; Tetko, Elmsford, NY). The macrophage suspension was enriched (75–85%) by flotation over Percoll density gradients as previously described (28), and the cells were resuspended (106 cells/ml) in RPMI containing 5% FCS, 2 mM L-glutamine, and 1% antibiotic-antimycotic solution. In selected experiments we used macrophages isolated from the bronchoalveolar lavage of patients undergoing diagnostic bronchoscopy for lung carcinoma. The bronchoalveolar lavage procedure and the isolation of macrophages were previously described (29). The cells were then incubated in 24-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 12 h the medium was removed, and the plates were gently washed three times with RPMI. Adherent cells were >95% macrophages as assessed by {alpha}-naftylacetate esterase staining (30).

Cell incubations

Macrophages adherent to 24-well plates were incubated (37°C, 15 min to 18 h) in RPMI containing various concentrations of group IA PLA2 (from N. mossambica mossambica), group IIA sPLA2 (recombinant human synovial PLA2) or mp-BSA. The sPLA2 preparations were frequently checked and were found to be free of LPS contamination. FCS was not used in these incubations, because preliminary experiments indicated that it significantly reduced macrophage responses to sPLA2s. In selected experiments, preincubation with LY311727 (10 µM), AACOCF3 (10 µM), or a blocking Ab anti-group IIA sPLA2 (10 µg/ml) was conducted for 45 min at 37°C before the addition of sPLA2s. In the experiments with the anti-mannose receptor the cells were incubated (4°C, 20 min) with PAM-1 (10 µg/ml), washed three times with RPMI, and subsequently incubated with mp-BSA or group IIA sPLA2. At the end of the experiment the supernatant was removed, centrifuged twice (1000 x g, 4°C, 5 min), and stored up to 72 h at -80°C for subsequent determination of ß-glucuronidase and IL-6 release. At the end of each experiment, an aliquot of cells was stained with trypan blue to determine cell viability. The cells remaining in the plates were lysed with 0.1% Triton X-100 for determination of the total cellular content of proteins and ß-glucuronidase.

ß-Glucuronidase assay

ß-Glucuronidase release in cell-free supernatants or in cell lysates was measured by a colorimetric assay (31). Briefly, 200 µl of supernatants or 100 µl of cell lysates were incubated (37°C, 18 h) with 400 µl of 0.1 M acetate buffer, pH 4.5, containing 1 µmol of phenolphthalein glucuronide. At the end of the incubation, 2 ml of glycine buffer were added to each tube, and the mixture was transferred into a 3-ml cuvette for OD reading at 540 nm. ß-Glucuronidase release was expressed as a percentage of the total cellular content determined in cells lysed with 0.1% Triton X-100. All experiments were conducted in duplicate determinations. Controls were performed to exclude the presence of contaminating ß-glucuronidase activity in the sPLA2s used in these experiments.

IL-6 ELISA assay

IL-6 release in the culture supernatant of macrophages was measured in duplicate determinations by a commercially available ELISA (Euro Clone, Torquay, U.K.) according to the manufacturer’s instructions. The linearity range of the assay was between 5–150 pg/ml. Because the number of adherent macrophages can vary in each well and in different experiments, the results were normalized for the total protein content in each well, determined in the cell lysates (0.1% Triton X-100) by the Lowry method (32).

IL-6 gene expression assay

Macrophages (5 x 106/2 ml) were incubated (37°C, 12 h) in RPMI containing 5% FCS in six-well plates. The cells were then washed and incubated in FCS-free medium alone or with group IA sPLA2 (1 µg/ml) or LPS (10 µg/ml; 37°C, 3 h). At the end of the incubation RNA was isolated by the RNAzol B technique (Tel-Test, Friendswood, TX), according to the manufacturer’s instructions. Diethylpyrocarbonate-treated water without SDS was used for the final resuspension step; RNA was stored at -80°C. RT was performed with 5 mM MgCl2, oligo(dt)16 primer, and Moloney leukemia virus reverse transcriptase according to the manufacturer’s instructions (Perkin-Elmer, Norwalk, CT) on a thermocycler (GeneAmp PCR System 2400, Perkin-Elmer). PCR was performed using Taq polymerase (1–2.5 U/reaction) at the annealing temperature of 60°C with target-specific primers for IL-6 (5'-ATGAACTCCTTCTCCACAAGCGC-3' and 3'-GAAGAGCCCTCAGGCTGGACTG-5' at 0.2–1 µM/primer) at subsaturating cycle number (30 cycle). Normalization of RNA was achieved by RT-PCR for the constitutive marker gene ß-actin at subsaturating cycle numbers. Strict RNase-free conditions were maintained throughout the procedure (33). All PCR products were visualized by ethidium bromide-stained gel electrophoresis and photographed.

Determination of the enzymatic activity of sPLA2s

The enzymatic activity of group IA and IIA sPLA2 was determined as previously described (20), using [3H]arachidonate-labeled Escherichia coli membranes (New England Nuclear, Boston, MA). The data are expressed as nanomoles of arachidonate released per hour.

LDH assay

LDH release at the end of the incubations was determined as an index of cytotoxicity. LDH was measured in cell-free supernatants using a commercially available kit (Sigma).

Statistical analysis

The data are expressed as the mean ± SE of the indicated number of experiments. p values were determined with t test for unpaired samples with Bonferroni’s correction (34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of group IA and IIA sPLA2 on ß-glucuronidase release from macrophages

Initial experiments were performed to determine whether group IA and IIA sPLA2s induced the release of the lysosomal enzyme ß-glucuronidase from human lung macrophages. The cells were incubated (37°C, 2 h) with various concentrations of snake venom sPLA2 (group IA) or human synovial sPLA2 (group IIA). Fig. 1Go shows that both sPLA2s induce the release of ß-glucuronidase from macrophages in a concentration-dependent fashion. Group IA sPLA2 was ~10-fold more potent than group IIA sPLA2. In addition, the maximum response with group IA sPLA2 (16.2 ± 2.4%) was higher than that obtained with group IIA sPLA2 (13.1 ± 1.5%). Concentrations of group IA sPLA2 >10 µg/ml or of group IIA sPLA2 >20 µg/ml did not induce further release of ß-glucuronidase. It is important to note that the group IA and IIA sPLA2s used in these experiments have an equivalent enzymatic activity as assessed by their ability to release arachidonate from labeled E. coli membranes (3.1 and 3.4 nmol/h with 1 µg of group IA and IIA, respectively).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 1. Effects of increasing concentrations of group IA and IIA sPLA2s on the release of ß-glucuronidase from human lung macrophages. The cells were incubated (37°C, 2 h) with the indicated concentrations of group IA (•) and group IIA ({circ}) sPLA2s. At the end of the incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). ß-Glucuronidase release was determined using a colorimetric technique (30 ). The values are expressed as the percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. Control (unstimulated) release was 3.4 ± 0.3%. The data are the mean ± SE of six experiments. *, p < 0.05; **, p < 0.01 (vs control).

 
In the next group of experiments we studied the kinetics of ß-glucuronidase release from macrophages incubated with optimal concentrations of sPLA2s, 1 µg/ml of group IA or 10 µg/ml of group IIA (Fig. 2Go). The release of ß-glucuronidase induced by both sPLA2s was relatively rapid, reaching a maximum between 30 min and 2 h after addition of the stimulus.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. Kinetics of ß-glucuronidase release induced by group IA and IIA sPLA2s from human lung macrophages. The cells were incubated with RPMI (spontaneous release; {blacksquare}), group IA sPLA2 (1 µg/ml; •) or group IIA sPLA2 (10 µg/ml; {circ}). At each time point, supernatants were collected and centrifuged (1000 x g, 4°C, 5 min). ß-Glucuronidase release in the supernatants was determined using a colorimetric technique (30 ). The values are expressed as the percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of six experiments. *, p < 0.05; **, p < 0.01 (vs control).

 
The release of ß-glucuronidase induced by group IA and IIA sPLA2s from human macrophages was Ca2+ and temperature dependent. Table IGo shows that the release of ß-glucuronidase from macrophages incubated with group IA or IIA sPLA2 does not occur when the cells are incubated with EDTA to block extracellular Ca2+ or at 4°C. In these experiments the release of LDH, used as an indicator of cytotoxicity, was always <5% of the total cellular content. Furthermore, the viability of macrophages, as assessed by trypan blue exclusion at the end of the experiment, was always >97%. Taken together, these data indicate that the release of ß-glucuronidase induced by the sPLA2s is not due to a cytotoxic effect of the enzymes.


View this table:
[in this window]
[in a new window]
  		Table I. Effect of EDTA and incubation temperature on sPLA2-induced ß-glucuronidase release from human lung macrophages1

 
The macrophage population isolated from lung tissue contains a variable proportion of alveolar and parenchymal macrophages. These two cell populations often react differently in response to various stimuli (35). We therefore compared the release of ß-glucuronidase induced by group IA and IIA sPLA2s from alveolar macrophages (isolated from bronchoalveolar lavage) vs that from parenchymal macrophages. Table IIGo shows that the two cell populations release comparable amounts of ß-glucuronidase when stimulated with optimal concentrations of group IA and IIA sPLA2s. Therefore, sPLA2s are equally effective in inducing exocytosis from alveolar and parenchymal macrophages.


View this table:
[in this window]
[in a new window]
  		Table II. Effect of group IA and IIA sPLA2s on ß-glucuronidase release from human lung macrophage populations1

 
Effect of group IA and IIA sPLA2 on IL-6 production from macrophages

To explore further the activation of macrophages induced by sPLA2s, we determined whether, in addition to a preformed mediator such as ß-glucuronidase, group IA and IIA sPLA2s were able to induce the production of IL-6, a major cytokine produced by human macrophages (36). In these experiments the cells were incubated with increasing concentrations of sPLA2s for 6 h. Both group IA and group IIA sPLA2 significantly increased the basal secretion of IL-6 from macrophages (Fig. 3Go). As for ß-glucuronidase, group IA sPLA2 was ~10-fold more potent than group IIA sPLA2 in inducing the release of IL-6. The maximum increases in IL-6 release over basal production were 275 and 220% with group IA and IIA sPLA2, respectively. Kinetic analysis of IL-6 secretion induced by sPLA2s revealed that this event required 4–6 h of incubation to become evident (Fig. 4Go). In addition, there was no difference in the kinetics of IL-6 release induced by optimal concentrations of group IA (1 µg/ml) and group IIA (10 µg/ml) sPLA2s.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. Effects of increasing concentrations of group IA (•) and IIA ({circ}) sPLA2s on the release of IL-6 from human lung macrophages. The cells were incubated (37°C, 6 h) with the indicated concentrations of group IA and group IIA sPLA2s. At the end of the incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). IL-6 release was determined by ELISA. The values are expressed as nanograms of IL-6 per milligrams of protein. Control release was 0.52 ± 0.15 ng/mg of protein. The data are the mean ± SE of five experiments. *, p < 0.05; **, p < 0.01 (vs control).

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 4. Kinetics of IL-6 release induced by group IA and IIA sPLA2s from human lung macrophages. The cells were incubated with RPMI (spontaneous release; {blacksquare}), group IA sPLA2 (1 µg/ml; •), or group IIA sPLA2 (10 µg/ml; {circ}). At each time point supernatants were collected and centrifuged (1000 x g, 4°C, 5 min). IL-6 release in the supernatants was determined by ELISA. The values are expressed as nanograms of IL-6 per milligrams of protein. The data are the mean ± SE of five experiments. *, p < 0.01 (vs control).

 
The latency of the effect of group IA and IIA sPLA2s on IL-6 release suggested that this may be mediated by the induction of IL-6 gene expression. To test this hypothesis we evaluated IL-6 mRNA expression in macrophages exposed to group IA sPLA2 (1 µg/ml) or LPS (10 µg/ml), used as positive control, in an FCS-free medium. Fig. 5Go depicts the ß-actin- and IL-6-specific RT-PCR amplification products from a representative experiment in which macrophages were cultured for 3 h with medium alone, group IA sPLA2 (1 µg/ml) or LPS (10 µg/ml). Adequate normalization of RNA for each sample was confirmed by the equality of RT-PCR amplification products for the constitutive marker gene of ß-actin at subsaturating cycle number (first row). Exposure to group IA sPLA2 (1 µg/ml) increased IL-6 mRNA compared with that in unstimulated cells (control), and this enhancement was quantitatively similar to that induced by LPS (10 µg/ml; second row). These findings suggest that group IA sPLA2 may induce IL-6 generation through the activation of IL-6 gene expression.



View larger version (70K):
[in this window]
[in a new window]
  		FIGURE 5. Stimulation of IL-6 gene expression from human lung macrophages by group IA sPLA2 and LPS. The figure depicts ß-actin- and IL-6-specific RT-PCR amplification products from a representative experiment in which macrophages were cultured for 3 h with RPMI alone (control), group IA sPLA2 (1 µg/ml), or LPS (10 µg/ml). A 100-bp DNA ladder was used as the standard. Adequate normalization of RNA for each sample was confirmed by the equality of RT-PCR amplification products for ß-actin gene expression at subsaturating cycle number.

 
Effect of inhibitors of PLA2 hydrolytic activity on ß-glucuronidase release induced by PLA2

The sPLA2s are able to mobilize arachidonic acid and to induce the production of eicosanoids in a variety of inflammatory cells, including macrophages (37). This effect may be due either to a direct enzymatic effect of sPLA2 or to the generation of membrane signals inducing the activation of the cytosolic (group IV) PLA2 (20, 37, 38). Therefore, ß-glucuronidase and IL-6 release may be secondary to sPLA2s-mediated activation of arachidonate metabolism and leukotriene B4 production. Leukotriene B4 is the major eicosanoid produced by human macrophages (30), and it induces enzyme and cytokine release from these cells (39). To evaluate the possibility that ß-glucuronidase and IL-6 release may be secondary to arachidonate mobilization, we preincubated (37°C, 45 min) macrophages with LY311727, an inhibitor of the enzymatic activity of group IIA sPLA2 (37), with AACOCF3, an inhibitor of cytosolic PLA2 (40) or vehicle (0.1% DMSO), and subsequently stimulated the cells with group IIA sPLA2. At the concentrations used in these experiments (10 µM), LY 311727 inhibited by 92% the hydrolytic activity of sPLA2s. Fig. 6Go shows that incubation with LY311727 or AACOCF3 did not influence the release of ß-glucuronidase from macrophages stimulated with group IIA sPLA2. In contrast, the release of ß-glucuronidase was completely inhibited when macrophages were preincubated with a blocking mAb anti-IIA sPLA2, used as a positive control.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of LY311727, an inhibitor of the hydrolytic activity of sPLA2, and AACOCF3, an inhibitor of cytosolic PLA2 on group IIA sPLA2-induced release of ß-glucuronidase from human lung macrophages. The cells were preincubated (37°C, 45 min) with LY311727 (10 µM), AACOCF3 (10 µM), or an mAb anti-group IIA sPLA2 (10 µg/ml), used as a positive control, and were then incubated (2 h, 37°C) with group IIA sPLA2 (10 µg/ml). At the end of the incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). ß-Glucuronidase release was determined by a colorimetric technique (30 ). The values are expressed as the percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of three experiments. *, p < 0.05 (vs medium alone); **, p < 0.01 (vs IIA-sPLA2 alone).

 
Effect of mp-BSA on sPLA2-induced ß-glucuronidase release

The latter group of experiments showed that the release of ß-glucuronidase and the de novo synthesis of IL-6 from human macrophages were due neither to the hydrolytic activity of sPLA2s nor to their ability to activate a cytosolic PLA2. Increasing evidence indicates that at least some of the biological activities of low m.w. PLA2s are mediated by the activation of specific membrane receptors (M-type and N-type) (15, 18, 19, 41, 42, 43). The group IA sPLA2 used in our experiments (from N. mossambica mossambica) is reported to recognize both receptors (43), whereas group IIA sPLA2 binds only to the M-type (21). Furthermore, the M-type receptor is homologous to the mannose receptor (21), and it is activated by mannose-containing proteoglycans (20). To test the hypothesis that activation of the mannose receptor was involved in the release of ß-glucuronidase and IL-6 induced by the sPLA2s, we incubated the macrophages with mp-BSA, a known ligand of the mannose receptor (20, 21, 44, 45). In addition, we explored whether there was interference between the three ligands (group IA and IIA sPLA2 and mp-BSA), so as to obtain initial information on the types of receptors expressed on human macrophages.

Fig. 7Go shows that mp-BSA alone induced a significant release of ß-glucuronidase from macrophages (7.6 ± 1.3% of the total cellular content). The maximal concentration of mp-BSA used was 30 µg/ml, because higher concentrations were found to be cytotoxic for macrophages. Similar concentrations of nonglycosylated BSA had no effect on ß-glucuronidase release (data not shown), indicating that the release of ß-glucuronidase induced by mp-BSA was not due to a nonspecific effect of proteins. Preincubation with mp-BSA followed by group IA sPLA2 induced a greater release of ß-glucuronidase than that induced by the two stimuli alone (additive effect). Similarly, incubation with group IIA sPLA2 followed by the addition of group IA sPLA2 significantly enhanced the release of ß-glucuronidase induced by group IA sPLA2 alone. In contrast, incubation with mp-BSA followed by group IIA sPLA2 had no additive effect. These data are compatible with the hypothesis that mp-BSA and group IIA sPLA2 activate the same receptor, i.e., either the mannose or the M-type receptor. To understand the role of the mannose receptor, macrophages were incubated with a blocking Ab anti-mannose receptor (PAM-1) and subsequently stimulated with mp-BSA or group IIA sPLA2 at approximately equimolar concentrations (~0.3 µM). Fig. 8Go shows that preincubation with PAM-1 significantly inhibits the release of ß-glucuronidase induced by group IIA sPLA2. As expected, PAM-1 also inhibited ß-glucuronidase release induced by mp-BSA, a known ligand of the mannose receptor (44, 45). Taken together, these data suggest that mp-BSA and group IIA sPLA2 are acting on the same receptor (presumably the mannose receptor) and that the group IA sPLA2 may be acting on a different receptor (presumably the N-type) or on both receptors (N-type and mannose/M-type).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of various ligands of sPLA2 receptors on ß-glucuronidase release from human lung macrophages. The cells were preincubated (37°C, 10 min) as indicated under First Incubation and were then incubated (37°C, 2 h) as indicated under Second Incubation. At the end of the second incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). ß-Glucuronidase release was determined using a colorimetric technique (30 ). The values are expressed as a percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of three experiments. *, p < 0.05 (vs medium alone); **, p < 0.01 (vs medium alone); §, p < 0.05 (vs group IA sPLA2 alone).

 


View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of a blocking anti-mannose receptor Ab (PAM-1) on ß-glucuronidase release from human lung macrophages induced by group IIA sPLA2. The cells were preincubated (37°C, 10 min) as indicated under First Incubation and were then incubated (37°C, 2 h) as indicated under Second Incubation. At the end of the second incubation, the supernatant was collected and centrifuged (1000 x g, 4°C, 5 min). ß-Glucuronidase release was determined by a colorimetric technique (30 ). The values are expressed as a percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of four experiments. *, p < 0.01 (vs medium alone); §, p < 0.01 (vs the corresponding group pretreated with PAM-1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A large body of evidence points to extracellular PLA2s as mediators of inflammatory reactions and tissue damage (14). The occurrence of large quantities of extracellular PLA2s in plasma and biological fluids from patients with inflammatory diseases strongly suggests that these enzymes play a role in inflammation. In this study we have investigated the effects of two different sPLA2s on human lung macrophages. The macrophage is a pivotal cell in the modulation of immune and inflammatory responses because it acts as an APC and a phagocytic cell (46) and is a major source of mediators and cytokines (24). Our results indicate that a group IA PLA2 (from N. mossambica mossambica venom) and a group IIA PLA2 (human synovial) induce the release of ß-glucuronidase and the production of IL-6. These effects of sPLA2 are concentration, Ca2+, and temperature dependent, and they are not cytotoxic. The release of IL-6 induced by sPLA2s is associated with increased mRNA expression.

These data indicate that sPLA2s generate an intracellular signal that activates both exocytosis and cytokine gene transcription in macrophages. The simultaneous effect of sPLA2s on degranulation and gene expression suggests that these molecules may influence not only the proinflammatory potential but also other functions of macrophages, including the interaction with T and B cells, phagocytosis, and bacterial killing (47, 48).

ß-Glucuronidase is a lysosomal enzyme whose release is generally used as a marker of exocytosis in phagocytic cells (49). This enzyme is involved in Ag processing and in the killing of intracellular pathogens. When released, ß-glucuronidase plays a role in tissue damage and remodeling, two key features of inflammation (49).

IL-6 is a pleiotropic cytokine involved in hemopoietic cell differentiation and in B-cell proliferation and activation (36). It is also a hepatocyte-stimulating factor, and it induces the expression and release of acute-phase proteins (50). Group IIA sPLA2 shares many features with acute-phase proteins: it is released in vivo during systemic inflammation, and it can be secreted in vitro by hepatocytes stimulated with proinflammatory cytokines such as IL-1, IL-6, and GM-CSF (51). Although many studies have documented that cytokines, particularly IL-6, may induce the expression and release of group IIA sPLA2 (51, 52, 53), our study provides the first evidence that sPLA2 is a potent stimulus for cytokine production by human macrophages. Therefore, sPLA2 and IL-6 may synergistically cooperate to the development of the inflammatory response by potentiating each other’s synthesis.

In vivo administration of exogenous sPLA2 in experimental animals elicits a severe inflammatory reaction. For example, intratracheal administration of group IA sPLA2 in rats induces interstitial and alveolar edema associated with neutrophil influx and impaired gas exchange and high mortality (12). These changes strongly resemble those observed in patients with adult respiratory distress syndrome. Similarly, the intra-articular injection of group IIA sPLA2 induces inflammation with a predominantly neutrophilic infiltrate and extensive tissue damage (11). Although sPLA2 can directly activate neutrophils (16), this effect may be at least in part induced by the activation of resident macrophages. Studies are currently ongoing to determine whether sPLA2 can stimulate human macrophages to release, in addition to IL-6, other cytokines active on neutrophils, such as IL-1, IL-8, TNF-{alpha}, and GM-CSF.

The concentrations of sPLA2s sufficient to elicit exocytosis and IL-6 production from macrophages are comparable with those detectable in vivo. For example, plasma concentrations of sPLA2 in conditions such as septic shock or extensive burns increase to between 0.1 and 1 µg/ml (54), and levels as high as 4 µg/ml are reported in acute pancreatitis (55). Even higher concentrations can be reached in inflammatory fluids, such as the synovial, bronchoalveolar lavage, or peritoneal fluid. Therefore, activation of resident macrophages clearly occurs at sites of sPLA2 release in vivo.

Like other effects of sPLA2 increasingly reported in the literature (18, 19, 20, 42, 53, 56), the stimulation of ß-glucuronidase and IL-6 release from human macrophages is apparently independent from its direct or indirect (via cPLA2) arachidonate-mobilizing activity. This concept is supported by at least three lines of evidence. First, exocytosis induced by sPLA2 is not blocked by LY311727 and AACOCF3, two previously characterized inhibitors of sPLA2 (37) and cPLA2 (40), respectively. Second, there is no correlation between the enzymatic activities of the sPLA2s, which are similar for groups IA and IIA, and their capacities to release ß-glucuronidase and IL-6, with group IA being 10-fold more potent than group IIA. Third, mp-BSA, a molecule that has no direct arachidonate-releasing capacity, also induces the release of ß-glucuronidase. The observation that removal of extracellular Ca2+, which is necessary for the hydrolytic activity of sPLA2, also inhibits ß-glucuronidase release can be explained by the fact that Ca2+ is required for activation of exocytosis in macrophages (24). In fact, initial experiments indicate that sPLA2s generate a cytosolic Ca2+ signal in human macrophages (M. Triggiani et al., unpublished observation). Taken together, these observations suggest the existence of specific binding sites for sPLA2s on human macrophages.

Several sPLA2 receptors have been demonstrated in cells, including mast cells (57), vascular smooth muscle cells (56), platelets (58), neutrophils (16), chondrocytes (59), fibroblasts (60), hepatocytes (51), and mesangial cells (53), and in tissues such as brain (41), lung (18), and skeletal muscle (42). The two best characterized receptors for sPLA2 are the M-type and the N-type. The different dose-response curves of group IA sPLA2, active on both the M-type and the N-type, and group IIA sPLA2, active only on the M-type, suggest that both receptors may be present on human macrophages. The M-type receptor has been included in a family of lectin-binding receptors (61), including the mannose receptor, uniquely expressed on macrophages (44), and the DEC-205, expressed on dendritic cells (62). mp-BSA, which is a ligand of the mannose receptor, induces ß-glucuronidase release quantitatively similar to that induced by group IIA sPLA2. In addition, the simultaneous exposure of macrophages to both mp-BSA and group IIA sPLA2 has no additive effect suggesting that mp-BSA and group IIA sPLA2 share a common receptor. This hypothesis is further supported by the experiments showing that blockade of the mannose receptor results in a significant inhibition of exocytosis induced by both mp-BSA and group IIA sPLA2. Although these data do not exclude the presence of the M-type receptor, they strongly implicate the mannose receptor as the binding site activated by group IIA sPLA2 on human macrophages. Although the binding of sPLA2s to the mannose receptor has been shown previously (46, 63), our data provide the first evidence that this interaction is functionally coupled with exocytosis in human macrophages. In contrast, the effect of group IA sPLA2 is significantly enhanced when macrophages are preincubated with either mp-BSA or group IIA sPLA2. Taken together, these data are compatible with the hypothesis that at least two sPLA2 receptors may be present on human macrophages, one of which may be the mannose receptor.

In conclusion, our data provide the first demonstration that sPLA2s are potent stimuli of exocytosis and IL-6 production in human macrophages in vitro via activation of the mannose receptor and probably another specific receptor(s). This effect may play a major role in the proinflammatory activity of sPLA2s, and it may concur, along with the arachidonate-mobilizing capacity of these enzymes, to the mechanism of sPLA2-induced inflammatory responses and tissue damage. The relative abundance of macrophages at sites where sPLA2 is released during inflammatory diseases, such as the joints, the airways, and the peritoneal cavity, strengthens the in vivo relevance of this finding.


   Footnotes
 
1 This work was supported in part by grants from the Consiglio Nazionale delle Ricerche (Target Project Biotechnology Grants 99.00216.PF31 and 99.00401.PF49), from the Istituto Superiore di Sanità (AIDS Project 9403-70 and Project Tuberculosis 96/D/T35) (Rome, Italy), and from the Associazione Italiana per la Ricerca sul Cancro (Milan, Italy). Back

2 Address correspondence and reprint requests to Dr. Massimo Triggiani, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy. Back

3 Abbreviations used in this paper: PLA2, phospholipase A2; sPLA2, secretory PLA2; AACOCF3, arachidonoyl trifluoromethyl ketone; HSA, human serum albumin; mp-BSA, p-aminophenyl-mannopyranoside-BSA; LDH, lactate dehydrogenase. Back

Received for publication October 25, 1999. Accepted for publication February 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Waite, M.. 1990. Biochemistry, molecular biology, and physiology of phospholipase A2 and its regulatory factors: phospholipases, enzymes that share a substrate class. Adv. Exp. Med. Biol. 279:1.[Medline]
  2. Mayer, R. J., L. A. Marshall. 1993. New insights on mammalian phospholipase A2(s): comparison of arachidonoyl-selective and -nonselective enzymes. FASEB J. 7:339.[Abstract]
  3. Dennis, E. A.. 1994. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269:13057.[Free Full Text]
  4. Tischfield, J. A.. 1997. A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J. Biol. Chem. 272:17247.[Free Full Text]
  5. Kudo, I., M. Murakami, S. Hara, K. Inoue. 1993. Mammalian non-pancreatic phospholipase A2. Biochim. Biophys. Acta 117:217.
  6. Kramer, R. M., C. Hession, B. Johansen, G. Hayes, P. McGray, E. P. Chow, R. Tizard, R. B. Pepinsky. 1988. Structure and properties of a human non-pancreatic phospholipase A2. J. Biol. Chem. 264:5768.[Abstract/Free Full Text]
  7. Seilhamer, J. J., W. Pruzansky, P. Vadas, S. Plant, J. A. Miller, J. Kloss, L. K. Johnson. 1989. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J. Biol. Chem. 264:5335.[Abstract/Free Full Text]
  8. Chilton, F. H., F. J. Averill, W. C. Hubbard, A. N. Fonteh, M. Triggiani, M. C. Liu. 1996. Antigen-induced generation of lyso-phospholipids in human airways. J. Exp. Med. 183:2235.[Abstract/Free Full Text]
  9. Vadas, P., S. Wasi, H. Z. Movat, J. B. Hay. 1981. Extracellular phospholipase A2 mediates inflammatory hyperaemia. Nature 293:583.[Medline]
  10. Kini, R. M., H. J. Evans. 1989. A model to explain the pharmacological effects of snake venom phospholipase A2. Toxicon 27:613.[Medline]
  11. Bomalaski, J. S., P. Lowton, J. L. Browning. 1991. Human extracellular recombinant phospholipase A2 induces an inflammatory response in rabbit joints. J. Immunol. 146:3904.[Abstract]
  12. Edelson, J. D., P. Vadas, J. Villar, J. B. M. Mullen, W. Pruzanski. 1991. Acute lung injury induced by phospholipase A2: structural and functional changes. Am. Rev. Respir. Dis. 143:1102.[Medline]
  13. Foreman-Wykert, A. K., Y. Weinrauch, P. Elsbach, J. Weiss. 1999. Cell-wall determinants of the bactericidal action of group IIA phospholipase A2 against Gram-positive bacteria. J. Clin. Invest. 103:715.[Medline]
  14. Vadas, P., W. Pruzansky. 1986. Biology of disease: role of secretory phospholipase A2 in the pathobiology of disease. Lab. Invest. 55:391.[Medline]
  15. Fonteh, A. N., D. A. Bass, L. A. Marshall, M. Seeds, J. M. Samet, F. H. Chilton. 1994. Evidence that secretory phospholipase A2 plays a role in arachidonic acid release and eicosanoid biosynthesis by mast cells. J. Immunol. 152:5438.[Abstract]
  16. Takasaki, J., Y. Kawauchi, Y. Masuho. 1998. Synergistic effect of type II phospholipase A2 and platelet-activating factor on Mac-1 surface expression and exocytosis of gelatinase granules in human neutrophils: evidence for the 5-lipoxygenase-dependent mechanism. J. Immunol. 160:5066.[Abstract/Free Full Text]
  17. Serafin, W. E., K. F. Austen. 1987. Mediators of immediate hypersensitivity reactions. N. Engl. J. Med. 317:30.[Medline]
  18. Kanemasa, T., A. Arimura, J. Kishino, M. Ohtani, H. Arita. 1992. Contraction of guinea pig lung parenchyma by pancreatic type phospholipase A2 via its specific binding site. FEBS Lett. 303:217.[Medline]
  19. Kanemasa, T., K. Hanasaki, H. Arita. 1992. Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites. Biochim. Biophys. Acta 1125:210.[Medline]
  20. Fonteh, A. N., J. M. Samet, M. Surette, W. Reed, F. H. Chilton. 1998. Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase A2. Biochim. Biophys. Acta 1393:253.[Medline]
  21. Lambeau, G., P. Ancian, J. Barhanin, M. Lazdunski. 1994. Cloning and expression of a membrane receptor for secretory phospholipase A2. J. Biol. Chem. 269:1575.[Abstract/Free Full Text]
  22. Ezekowitz, R. A., K. Sastry, P. Bailly, A. Warner. 1990. Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells. J. Exp. Med. 172:1785.[Abstract/Free Full Text]
  23. Yamamoto, Y., T. W. Klein, H. Friedman. 1997. Involvement of mannose receptor in cytokine interleukin-1ß (IL-1ß), IL-6, granulocyte-macrophage colony-stimulating factor responses, but not in chemokine macrophage inflammatory protein 1ß (MIP-1ß), MIP-2, KC responses, caused by attachment of Candida albicans to macrophages. Infect. Immun. 65:1077.[Abstract]
  24. Nathan, C. F.. 1987. Secretory products of macrophages. J. Clin. Invest. 79:319.
  25. Koch, A. E., S. L. Kunkel, J. C. Burrows, H. L. Evanoff, G. K. Haines, R. M. Pope, R. M. Strieter. 1991. Synovial tissue macrophage as a source of the chemotactic cytokine IL-8. J. Immunol. 147:2187.[Abstract]
  26. Chanez, P., I. Bousquet, L. Couret, G. Cornillac, G. Barneon, P. Vic, F. B. Michel, P. Godard. 1991. Increased numbers of hypodense alveolar macrophages in patients with bronchial asthma. Am. Rev. Respir. Dis. 144:923.[Medline]
  27. Marinò, I., M. Columbo, G. Marone. 1996. The releasability of lysosomal enzymes from neutrophil leukocytes in patients with rheumatoid arthritis. Clin. Exp. Rheum. 14:387.[Medline]
  28. Triggiani, M., A. Oriente, M. C. Seeds, D. A. Bass, G. Marone, F. H. Chilton. 1995. Migration of human inflammatory cells into the lung results in the remodelling of arachidonic acid into a triglyceride pool. J. Exp. Med. 182:1181.[Abstract/Free Full Text]
  29. Triggiani, M., V. De Marino, M. Sofia, S. Faraone, G. Ambrosio, L. Carratù, G. Marone. 1997. Characterization of platelet-activating factor acetylhydrolase in human bronchoalveolar lavage. Am. J. Respir. Crit. Care Med. 156:94.[Abstract/Free Full Text]
  30. Triggiani, M., A. Oriente, G. Marone. 1994. Differential roles for triglyceride and phospholipid pools of arachidonic acid in human lung macrophages. J. Immunol. 152:1394.[Abstract]
  31. Triggiani, M., D. W. Goldman, F. H. Chilton. 1991. Biological effects of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil. Biochim. Biophys. Acta 1084:41.[Medline]
  32. Lowry, O., A. Rosebrough, A. Farr, R. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265.[Free Full Text]
  33. Essayan, D. M., G. Krishnaswamy, S. K. Huang. 1997. Immunologic investigations of T-cell regulation of human IgE antibody secretion and allergic responses. Methods 13:69.[Medline]
  34. Snedecor, G. W.. 1980. Statistical Methods Iowa State University Press, Ames.
  35. Weissler, J. C., C. R. Lyons, M. F. Lipscomb, G. B. Toews. 1986. Human pulmonary macrophages: functional comparison of cells obtained from whole lung and by bronchoalveolar lavage. Am. Rev. Respir. Dis. 133:473.[Medline]
  36. Kishimoto, T.. 1989. The biology of interleukin-6. Blood 74:1.[Free Full Text]
  37. Fleisch, J. H., C. T. Armstrong, C. R. Roman, E. D. Mihelich, S. M. Spaethe, W. T. Jackson, J. L. Bobbit, S. Draheim, N. J. Bach, L. D. Dillard, et al 1996. Recombinant human secretory phospholipase A2 released thromboxane from guinea pig bronchoalveolar lavage cells: in vitro and ex vivo evaluation of a novel secretory phospholipase A2 inhibitor. J. Pharmacol. Exp. Ther. 278:252.[Abstract/Free Full Text]
  38. Murakami, M., S. Shimbara, T. Kambe, H. Kuwata, M. V. Winstead, J. A. Tischfield, I. Kudo. 1998. The functions of five distinct phospholipase A2s in regulating arachidonic acid release: type IIA and type V secretory phospholipase A2s are functionally redundant and act in concert with cytosolic phospholipase A2. J. Biol. Chem. 273:14411.[Abstract/Free Full Text]
  39. Dubois, C., E. Bissonnette, M. Rola-Pleszczynsky. 1989. Asbestos fibers and silica particles stimulates rat alveolar macrophages to release tumor necrosis factor: autoregulatory role of leukotriene B4. Am. Rev. Respir. Dis. 139:1257.[Medline]
  40. Bartoli, F., H. K. Lin, F. Ghomaschi, M. H. Gelb, M. K. Jain, R. Apiz-Castro. 1994. Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets. J. Biol. Chem. 269:15625.[Abstract/Free Full Text]
  41. Lambeau, G., J. Barhanin, H. Schweitz, J. Quar, M. Lazdunski. 1989. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom. J. Biol. Chem. 264:11503.[Abstract/Free Full Text]
  42. Lambeau, G., A. Schmid-Alliana, M. Lazdunski, J. Barhanin. 1990. Identification of a very high affinity binding protein for toxic phospholipase A2 in skeletal muscle. J. Biol. Chem. 265:9526.[Abstract/Free Full Text]
  43. Lambeau, G., J. Barhanin, M. Lazdunski. 1991. Identification of different receptor types for toxic phospholipase A2 in rabbit skeletal muscle. FEBS Lett. 293:29.[Medline]
  44. McNally, A. K., K. M. DeFife, J. M. Anderson. 1996. Interleukin-4-induced macrophage fusion is prevented by inhibitors of mannose receptor activity. Am. J. Pathol. 149:975.[Abstract]
  45. Shepherd, V. L., Y. C. Lee, H. Schlesinger, P. D. Stahl. 1981. L-fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages. Proc. Natl. Acad. Sci. USA 78:1019.[Abstract/Free Full Text]
  46. Aderem, A., D. M. Underhill. 1999. Mechanism of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593.[Medline]
  47. Murakami, M., Y. Nakatami, G. Atsumi, K. Inoue, I. Kudo. 1997. Regulatory functions of phospholipase A2. Crit. Rev. Immunol. 17:225.[Medline]
  48. Hack, C. E., G. J. Wolbink, C. Schalkwijk, H. Speijer, W. T. Hermens, H. van den Bosch. 1997. A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells. Immunol. Today 18:111.[Medline]
  49. Borregaard, N., J. B. Cowland. 1997. Granules of the neutrophilic polymorphonuclear leukocyte. Blood 89:3503.[Free Full Text]
  50. Gauldie, J., C. Richards, D. Harnish, P. Lansdorp, H. Baumann. 1987. Interferon ß2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc. Natl. Acad. Sci. USA 84:7251.[Abstract/Free Full Text]
  51. Crowl, R. M., T. J. Stoller, R. R. Conroy, C. R. Stoner. 1991. Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J. Biol. Chem. 266:2647.[Abstract/Free Full Text]
  52. Gilman, S. C., J. Chang, P. R. Zeigler, J. Uhl, E. Mochan. 1988. Interleukin-1 activates phospholipase A2 in human synovial cells. Arthritis Rheum. 31:126.[Medline]
  53. Wada, A., H. Tojo, T. Sugiura, Y. Fujiwara, T. Kamada, N. Ueda, M. Okamoto. 1996. Group II phospholipase A2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells. Biochim. Biophys. Acta 1345:99.
  54. Nakae, H., S. Endo, K. Inada, H. Yamashita, Y. Yamada, T. Takakuwa, T. Kasai, M. Ogawa, K. Uchida. 1995. Plasma concentrations of type II phospholipase A2, cytokines and eicosanoids in patients with burns. Burns 21:422.[Medline]
  55. Nisijima, J., M. Okamoto, M. Ogawa, G. Kosaki, T. Yamano. 1983. Purification and characterization of human pancreatic phospholipase A2 and development of a radioimmunoassay. J. Biochem. 94:137.[Abstract/Free Full Text]
  56. Nakajima, M., K. Hanasaki, M. Ueda, H. Arita. 1992. Effect of pancreatic phospholipase A2 on isolated porcine cerebral arteries via its specific binding sites. FEBS Lett. 309:261.[Medline]
  57. Murakami, M., N. Hara, I. Kudo, K. Inoue. 1993. Triggering of degranulation in mast cells by exogenous type II phospholipase A2. J. Immunol. 151:5675.[Abstract]
  58. Mourier, C., B. B. Vargaftig, P. A. Franken, H. M. Verheij, C. Bon, L. Touqui. 1994. Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2. Biochim. Biophys. Acta 1214:88.[Medline]
  59. Kishino, J., T. Masahiro, H. Arita. 1992. Proliferative effect of phospholipase A2 in rat chondrocyte via its specific binding sites. Biochem. Biophys. Res. Commun. 186:1025.[Medline]
  60. Reddy, S. T., H. R. Herschman. 1996. Transcellular prostaglandin production following mast cell activation is mediated by proximal secretory phospholipase A2 and distal prostaglandin synthase 1. J. Biol. Chem. 271:186.[Abstract/Free Full Text]
  61. Nicolas, J. P., G. Lambeau, M. Lazdunski. 1995. Identification of the binding domain for secretory phospholipases A2 on their M-type 180-kDa membrane receptor. J. Biol. Chem. 270:28869.[Abstract/Free Full Text]
  62. Jiang, W., W. J. Swiggard, C. Heufler, Y. Peng, A. Mirza, R. M. Steinman, M. C. Nussenzweig. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375:151.[Medline]
  63. Mukhopadhyay, A., P. Stahl. 1995. Bee venom phospholipase A2 is recognized by the macrophage mannose receptor. Arch. Biochem. Biophys. 324:78.[Medline]



This article has been cited by other articles:


Home page
 FASEB J.Home page
L. Servillo, C. Balestrieri, A. Giovane, P. Pari, D. Palma, G. Giannattasio, M. Triggiani, and M. L. Balestrieri
Lysophospholipid Transacetylase in the Regulation of Paf Levels in Human Monocytes and Macrophages
FASEB J, May 1, 2006; 20(7): 1015 - 1017.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. Jaulmes, B. Janvier, M. Andreani, and M. Raymondjean
Autocrine and Paracrine Transcriptional Regulation of Type IIA Secretory Phospholipase A2 Gene in Vascular Smooth Muscle Cells
Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1161 - 1167.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Granata, A. Petraroli, E. Boilard, S. Bezzine, J. Bollinger, L. Del Vecchio, M. H. Gelb, G. Lambeau, G. Marone, and M. Triggiani
Activation of Cytokine Production by Secreted Phospholipase A2 in Human Lung Macrophages Expressing the M-Type Receptor
J. Immunol., January 1, 2005; 174(1): 464 - 474.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
H. W.M Niessen, P. A.J Krijnen, C. A Visser, C. J.L.M Meijer, and C Erik Hack
Type II secretory phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and ischemic damage to cardiomyocytes?
Cardiovasc Res, October 15, 2003; 60(1): 68 - 77.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Beck, G. Lambeau, K. Scholz-Pedretti, M. H. Gelb, M. J. W. Janssen, S. H. Edwards, D. C. Wilton, J. Pfeilschifter, and M. Kaszkin
Potentiation of Tumor Necrosis Factor {alpha}-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor {alpha} Activation
J. Biol. Chem., August 8, 2003; 278(32): 29799 - 29812.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Triggiani, F. Granata, B. Balestrieri, A. Petraroli, G. Scalia, L. Del Vecchio, and G. Marone
Secretory Phospholipases A2 Activate Selective Functions in Human Eosinophils
J. Immunol., March 15, 2003; 170(6): 3279 - 3288.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D.-W. Park, J.-R. Kim, S.-Y. Kim, J.-K. Sonn, O.-S. Bang, S.-S. Kang, J.-H. Kim, and S.-H. Baek
Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression
J. Immunol., February 15, 2003; 170(4): 2093 - 2099.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. de Paulis, G. Florio, N. Prevete, M. Triggiani, I. Fiorentino, A. Genovese, and G. Marone
HIV-1 Envelope gp41 Peptides Promote Migration of Human Fc{epsilon}RI+ Cells and Inhibit IL-13 Synthesis Through Interaction with Formyl Peptide Receptors
J. Immunol., October 15, 2002; 169(8): 4559 - 4567.
[Abstract] [Full Text] [PDF]

Home page