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*
Section of Pulmonary and Critical Care Medicine, Department of Medicine and
Department of Neurobiology, Pharmacological and Physiology, Pediatrics, Anesthesia and Critical Care, and Committees on Clinical Pharmacology and Cell Physiology, Division of Biological Sciences, University of Chicago, Chicago, IL 60637; and
Akita University College of Allied Medical Science, Akita, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin CD49d (very late Ag
(VLA)-4)/CD29 (5, 6). Interaction between eosinophils and
FN has been reported to augment eosinophil effector and metabolic
functions such as degranulation (6), superoxide release
(7), leukotriene synthesis (6), and
prolongation of cell survival due to cytokine production (8, 9). Prior reports have demonstrated that ligation of integrins to FN in inflammatory cells causes intracellular signaling responses that lead to rearrangement of the cytoskeleton, up-regulation of adhesion molecule affinity, and cell spreading (10, 11). These intracellular signaling responses include increases in the tyrosine phosphorylation of a subset of proteins, activation of serine/threonine kinases, and alterations in cellular phospholipid and calcium influx (12). Among these, mitogen-activated protein kinase (MAPK), a known serine/threonine protein kinase (13, 14), can be rapidly activated by adhesion to FN (15, 16).
Several subgroups of MAPK exist in mammalian cells: extracellular signal-regulated kinase (ERK)1 and 2, c-Jun N-terminal kinase (JNK), and p38. These subgroups are distinguished both by the sequence of the tripeptide dual phosphorylation motif required for MAPK activation and by the distinct subgroups of MAPK kinases that activate the ERK group (MAPK kinase (MEK)1 and 2) and the JNK and p38 groups (MKK). Activated MAPK can phosphorylate downstream cytoplasmic targets, such as cytosolic phospholipase A2 (cPLA2) in a variety of cells (17, 18).
cPLA2 is an enzyme involved in the conversion of
membrane phospholipids to arachidonic acid and lysophospholipids
(19, 20). We have reported previously that
cPLA2 also serves as a signaling protein for both
1 and 2
integrin-dependent adhesion of eosinophils to VCAM-1 and ICAM-1,
respectively (21). However, signaling pathways mediating
eosinophil adhesion to the matrix protein, FN, are not completely
defined.
The objective of this study was to examine the role of MAPK isoforms and cPLA2 in eosinophil adhesion to FN, as well as the relationship between MAPK and cPLA2 activation. Our data define the relationship between upstream phosphorylation events and the essential role of cPLA2 in mediating integrin adhesion. We find that ERK1/2-mediated phosphorylation of cPLA2 is both induced by and essential for adhesion of human eosinophils to FN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SB203580 was purchased from Upstate Biotechnology (Lake Placid, NY). U0126 and phosphorylation-specific ERK1/2 Ab were obtained from Promega (Madison, WI). PD98059, phosphorylation-specific and -nonspecific p38 MAPK mAb, and ERK1/2 mAb were purchased from New England Biolabs (Beverly, MA). FN was obtained from Chemicon International (Temecula, CA). Okadaic acid (OA) was purchased from Calbiochem-Novabiochem (San Diego, CA). Eosinophil isolation materials were obtained from Miltenyi Biotec (Sunnyvale, CA). Anti-CD18 Ab (clone 7E4) and anti-CD49d Ab (clone HP2/1) were purchased from Immunotech (Westbrook, ME). Anti-CD29 Ab (4B4) was purchased from Coulter (Miami, FL). FITC-conjugated phalloidin was purchased from Sigma (St. Louis, MO). The cPLA2 inhibitor, arachidonic trifluoromethyl ketone (TFMK), was purchased from Biomol (Plymouth Meeting, PA). Polyclonal rabbit anti-cPLA2 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Isolation of human peripheral blood eosinophils
Eosinophils were isolated from previously characterized mildly
atopic donors (21) by a modification of the negative
selection immunomagnetic separation technique of Hansel et al.
(22). The method is based on Percoll centrifugation
(density 1.089 g/ml) to isolate granulocytes, hypotonic lysis of RBC,
and finally, immunomagnetic depletion of neutrophils by the magnetic
cell separation system using anti-CD16-coated MACS particles.
Eosinophil purity of 
98% was routinely obtained. Cells were kept on
ice until use.
Eosinophil adhesion assay
The eosinophil adhesion assay was modified from the method of Nagata et al. (21, 23). Eosinophil adherence was assessed as residual eosinophil peroxidase activity of adherent cells. Briefly, 96-well microplates were coated with human plasma FN and blocked with heat-inactivated FBS. Eosinophils (1 x 104/100 µl HBSS/0.1% gelatin) were preincubated with different concentrations of U0126, PD98059, SB203580, OA, anti-CD18 mAb (7E4), anti-CD29 mAb (4B4), anti-VLA-4 mAb (HP2/1), or isotype control for 30 min at 37°C. Cells then were added to FN-coated wells and allowed to settle for 10 min on ice. Plates were rapidly warmed to 37°C and incubated for the indicated times. After washing three times with HBSS, 100 µl of HBSS/0.1% gelatin was added to the reaction wells, and serial dilutions of original cell suspension were added to the empty wells to generate a standard curve. A total of 100 µl eosinophil peroxidase substrate (1 mM H2O2, 1 mM o-phenylenediamine, and 0.1% Triton X-100 in Tris buffer, pH 8.0) was then added to the wells. After 30 min incubation at room temperature, 50 µl of 4 M H2SO4 was added to stop the reaction. Absorbance was measured at 490 nm in a microplate reader (Thermomax; Molecular Devices, Menlo Park, CA).
Western blot analysis of MAPK and cPLA2 phosphorylation
Eosinophils (3 x 106/group) were preincubated in the presence or absence of U0126 (24, 25), SB203580, or TFMK for 30 min and added to 10 µg/ml FN-coated six-well plates for various times. The reaction was stopped by adding 4°C buffer (2 mM EDTA in PBS without calcium). Eosinophils were then collected and centrifuged at 400 x g for 10 min. The pellets were lysed in 80 µl of lysis buffer (20 mM Tris-HCl, 30 mM Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, pH 7.4) containing 1% Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 2 mM Na3VO4, and 0.5% deoxycholic acid. After 15 min on ice, samples were centrifuged at 12,000 x g for 20 min to remove nuclear and cellular debris. Protein concentrations of supernatants then were measured by a Bio-Rad (Richmond, CA) protein assay kit. Afterward, 14 µl of 6x loading buffer was added to the collected supernatants, boiled for 5 min, and then saved at -70°C. Samples were subjected to SDS-PAGE using 10% acrylamide gels under reducing condition (15 mA/gel). For MAPK, electrophoresis was stopped when the 30-kDa rainbow mark reached bottom; for cPLA2, it was stopped when the 66-kDa rainbow marker reached bottom. Electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semidry system (400 mA, 60 min). The membrane was blocked with 1% BSA for 60 min, then incubated with 1:5000 anti-phosphorylated ERK1/2 Ab, anti-phosphorylated p38 MAPK Ab, 1 µg/ml anti-ERK1/2 mAb, 1 µg/ml anti-p38 MAPK Ab, or 1 µg/ml anti-cPLA2 mAb diluted in TBST for 60 min. The membranes were then washed three times for 20 min with TBST. Goat anti-rabbit Ig conjugated with HRP was diluted 1:3000 in TBST and incubated with polyvinylidene fluoride membrane for 60 min. The membranes were again washed three times with TBST and assayed by an ECL system (Amersham, Arlington Heights, IL).
Fluorescent staining of F-actin
Eosinophils were preincubated with buffer, U0126 (30 µM), SB203580 (30 µM), or OA (1 µM) for 30 min and allowed to adhere to 10 µg/ml FN-coated LAB-TEK chamber slides (Nalge Nunc International, Naperville, IL) for 30 min at 37°C. After supernatants were removed, cells were fixed with 3.7% formalin in PBS (pH 7.4) for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. After three washes, the cells were incubated with 1% BSA in PBS for 30 min to reduce nonspecific binding and stained by adding 1 µg/ml FITC-phalloidin for 30 min at 4°C. The cells then were washed three times with PBS, mounted, and observed by fluorescent microscopy.
Evaluation of eosinophil survival after incubation with MEK inhibitors, p38 MAPK inhibitor, or OA
Trypan blue exclusion analysis was performed to assess whether MEK inhibitors, p38 inhibitor, or OA affected eosinophil viability during these experiments. Briefly, aliquots of 104 eosinophils were incubated with various concentrations of MEK inhibitors, p38 MAPK inhibitor, or OA at 37°C for various times. Eosinophils then were centrifuged at 400 x g, and pellets were resuspended in 10 µl HBSS. An equal volume of 0.01% trypan blue was added, and the percentage of viable eosinophils was counted in a hemocytometer.
Statistical analysis
All data were expressed as mean ± SEM. The significance of differences between groups was assessed by the nonparametric Mann-Whitney unpaired t test. Statistical significance was claimed when p < 0.05. Statistical analysis was performed using the StatView 4.1 statistics package (Abacus Concepts, San Francisco, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed to determine the effect of FN coating
concentration on eosinophil adhesion. Nonstimulated eosinophils adhered
to plate-coated FN in a concentration-dependent manner (Fig. 1
A). At 10 µg/ml FN,
eosinophil adhesion was near maximal at 24.9 ± 1.8% vs 8.1
± 1.1% adhesion for buffer-coated control wells
(p < 0.001); this concentration of FN was used
in all subsequent experiments. Eosinophil adhesion to FN was also time
dependent; at 15 min, adhesion was 15.3 ± 1.3% vs 8.3 ±
2.7% adhesion for buffer-coated control wells
(p < 0.001). Maximal adhesion occurred at 30
min (27.9 ± 1.5% adhesion) (Fig. 1B). Eosinophil
adhesion then decreased gradually to 16.1% at 90 min.
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4/1 integrin
dependent and minimally CD18 dependent (Fig. 1
C). Adhesion
was blocked by anti-VLA-4 mAb (clone HP2/1) from 29.9 ± 1.2
to 16.4 ± 2.1% and to 16.6 ± 2.3% by the common
1-chain mAb, anti-CD29 (clone 4B4)
(p < 0.001 for both comparisons). The common
2-chain mAb anti-CD18 (clone 7E4) had no
blocking effect on eosinophil adhesion to FN. However, a combination of
anti-CD18 with anti-CD29 and anti-CD49d completely blocked
eosinophil adhesion to FN. Effect of MEK inhibitors, p38 inhibitor, or cPLA2 inhibitor on eosinophil adhesion to FN
Experiments were performed next to examine the intermediate
kinases involved in eosinophil adhesion to FN using specific
pharmacological inhibitors. U0126, a direct inhibitor of MEK1 and 2
that has been shown previously to inhibit ERK1/2 phosphorylation
(24, 25), decreased eosinophil adhesion in a
concentration-dependent manner (IC50 = 3.1 µM)
(Fig. 2). At 30 µM U0126, eosinophil
adhesion was reduced from 26.0 ± 3.1 to 12.2 ± 1.2%
(p < 0.01). Similarly, PD98059, a structurally
unrelated MEK1/2 inhibitor, also attenuated eosinophil adhesion in a
concentration-dependent manner (Fig. 2). By contrast, inhibition of p38
MAPK activity by SB203580 had no effect on eosinophil adhesion to FN.
Trifluoromethyl ketone (TFMK), a selective inhibitor of
cPLA2, also blocked eosinophil adhesion to FN in
a manner comparable to MEK inhibition with U0126
(IC50 = 8.2 µM). Maximal inhibition was
achieved at 30 µM (9.0 ± 3.5% vs 28.8 ± 2.3% adhesion
for nontreated control; p < 0.01).
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To determine whether MAPK or cPLA2 was
phosphorylated during adhesion, eosinophils were adhered to
plate-coated FN for various times, and cell lysates were subjected to
immunoblotting. ERK1/2 and p38 phosphorylations were detected by mAbs
specific for the phosphorylated forms of ERK1/2 and p38, whereas
cPLA2 phosphorylation was detected by
gel-shifting assay as described previously (21). Equal
loading of proteins in different lanes was confirmed by staining with
ERK1/2 or p38 MAPK Ab (Fig. 3, A and B, lower panels). ERK1/2
phosphorylation was observed at 5 min, and plateaued after 15 min (Fig. 3A). By contrast, p38 MAPK phosphorylation was constitutive
in nontreated eosinophils and did not change during adhesion to FN
(Fig. 3B). As demonstrated by the shift into the upper
phosphorylated band, cPLA2 phosphorylation was
observed at 15–90 min, which followed ERK1/2 phosphorylation (Fig. 3C).
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). By contrast, neither SB 203580, a p38
MAPK-inhibitor, nor TFMK, a selective inhibitor of
cPLA2 activity, blocked FN-induced ERK1/2
phosphorylation (Fig. 4). Equivalent loading in all lanes was
established by restaining of the membrane with ERK1/2 Ab (Fig. 4, lower panel).
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OA, a serine/threonine phosphatase inhibitor, caused a
concentration-dependent increase in eosinophil adhesion to FN (Fig. 5A); maximal adhesion
increased to 36.6 ± 1.9% after treatment with 1 µM OA vs
25.3 ± 1.3% for nontreated control (p <
0.01). OA also augmented the magnitude and sustained the time of
eosinophil adhesion to FN (Fig. 5B); OA-stimulated
eosinophil adhesion was maximal at 36.3 ± 1.7% at 30 min vs
26.9 ± 1.0% adhesion for nontreated control, and was sustained
at this level for > 60 min (vs 30 min for controls). (For times
>90 min and concentrations of OA >1 µM, adhesion was decreased
nonspecifically due to cytotoxicity (Fig. 5, C and
D).
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A) and was maximal after
60–120 min with 1.0 µM OA (Fig. 6B).
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Because actin polymerization provides the contractile forces
necessary for focal adhesion, we examined FN-induced ERK1/2
phosphorylation on actin polymerization. Adhesion to FN (Fig. 7B) or pretreatment with OA
followed by adhesion to FN (Fig. 7C) changed both the actin
distribution and polymerization into bundles after 30 min (compare with
Fig. 7A, which shows suspended cells receiving no
treatment). This effect of FN adhesion on intracellular actin
rearrangement was blocked by the MEK inhibitor, U0126 (Fig. 7D), but not by the p38 MAPK inhibitor, SB203580 (Fig. 7E).
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Because prior studies have suggested that ERK1/2 can directly
phosphorylate cPLA2 both in vivo and in vitro
(26, 27), we examined the causal relationship between
ERK1/2 and cPLA2 activation in eosinophils during
adhesion to FN. In these studies, the supernatant first was removed
before cell lysis. This allowed for easy identification of
phosphorylated cPLA2 as a single upper band
because nonadhered cells expressing nonphosphorylated
cPLA2 (lower band) were
partially removed. As shown in Fig. 8, U0126 at the concentration that completely inhibited ERK1/2
phosphorylation prevented cPLA2 phosphorylation
(lane C). OA at concentrations that
enhanced ERK1/2 phosphorylation also caused cPLA2
phosphorylation (lane E). By contrast,
SB203580, a p38 MAPK inhibitor, had no effect on
cPLA2 phosphorylation (lane
D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The objective of this study was to assess the role of MAPK isoforms and
cPLA2 in the regulation of eosinophil adhesion to
matrix protein FN and in the potential pathway leading to
cPLA2 phosphorylation, which is essential to
integrin adhesion. We first confirmed that eosinophils’ adherence to
FN was dependent on
41 integrin (6, 28) and minimally dependent on 2
integrin in our in vitro system (29, 30). These results,
together with our previous finding (6, 21) that
nonstimulated eosinophils have the ability to adhere to VCAM-1 via
41, clearly
demonstrated that VLA-4/CD29 expressed on eosinophil surface was
partially active and has the ability to make the initial contact with
its counterligands, VCAM-1 or FN. The paradoxical role of CD18
enhancement of adhesion to FN remains unexplained, but has been
observed by others in similar systems (29). We further
demonstrated that ERK1/2 phosphorylation and consequent
cPLA2 activation was necessary for maintaining
the firm adhesion after initial contact between eosinophils and FN.
Finally, we demonstrated that ERK1/2 but not p38 MAPK is essential for
cPLA2 phosphorylation.
Several lines of evidence support the role of ERK1/2 in eosinophil adhesion to FN. Two structurally unrelated and mechanistically different MEK inhibitors at the concentrations preventing ERK1/2 activation blocked eosinophil adhesion to FN. U0126 inhibits MEK1 and MEK2 catalytic activity, whereas PD98059, which binds to the inactive forms of MEK1, prevents its activation by upstream activators such as c-Raf or MEKK1 (24, 25). Inhibitors at the concentrations used in this study have been shown previously to be specific and do not inhibit the related MAPK family members MKK3, MKK4, MKK6, JNK1, or p38 (25). Inhibition of adhesion was not caused by toxicity of either inhibitor, and cell viability was always >95% as assessed by trypan blue exclusion (data not shown). Western blot analysis also showed that ERK1/2 but not p38 MAPK were phosphorylated during eosinophil adhesion to FN. Finally, OA, a serine/threonine phosphotase inhibitor, at the concentration that prevented ERK1/2 dephosphorylation sustained eosinophil adhesion to FN. Accordingly, we believe that ERK1/2 activation is necessary for maintenance of eosinophil adhesion to FN.
ERK activation has been observed after integrin engagement with FN in a variety of cells (31, 32, 33). However, the precise mechanism by which ERK contributes to adhesion is not clear. It has been suggested that actin cytoskeleton redistribution was necessary for cell adhesion in a variety of cells (34, 35, 36, 37). FN binding also induced redistribution of actin in eosinophils. This was ERK1/2 dependent, because U0126 blocked F-actin rearrangement. A recent finding that purified diphosphorylated ERK2 can bind to purified rabbit skeletal muscle actin under native conditions (38) suggests that actin may be a direct target of ERK phosphorylation. ERK1/2 activation also regulates eotaxin-induced F-actin polymerization in eosinophils (39). Other investigators have shown that phosphorylated ERK is translocated to newly forming focal adhesion sites after integrin engagement with FN in cultured fibroblast cells (40). In contrast to the role of ERK in eosinophil adhesion to FN, p38 MAPK was not involved. At a concentration that completely inhibited p38 activity (41), SB203580 had no effect on eosinophil adhesion to FN. This is somewhat surprising, because p38 MAPK activation has recently been reported to result in the activation of MAPK activated protein kinase 2, which phosphorylates hsp27 and lymphocyte-specific protein 1, both of which are F-actin-binding proteins (42, 43).
We previously reported that TFMK, a selective inhibitor of cPLA2, inhibited eosinophil adhesion to ICAM-1 and VCAM-1, whereas arachidonic acid and its metabolites played no role in eosinophil adhesion (21). No mechanism has been defined for this messenger protein activity of phosphorylated cPLA2; however, preliminary results suggest that cPLA2 catalysis of phosphotidylcholine to lysophospholipid could mediate an autocrine proadherence factor in eosinophils and other inflammatory cells (21). In this study, we extended our examination of the role of cPLA2 in mediating adhesion to extracellular matrix protein FN. We demonstrated that cPLA2 phosphorylation correlated to eosinophil adhesion to FN; and the selective cPLA2 inhibitor, TFMK, also blocked eosinophil adhesion to FN. Accordingly, our data suggest that cPLA2 activation is a common event in integrin-mediated eosinophil adhesion to all of its counterligands (VCAM-1, FN, ICAM-1). Our data suggest that lysophospholipid metabolites synthesized by active cPLA2 may be involved in eosinophil adhesion to FN because platelet-activating factor receptor antagonist CV-6209 blocked eosinophil adhesion to FN (data not shown). The mechanism by which platelet-activating factor receptor activation causes maintenance of adhesion remains to be discerned. However, upstream regulation of this process appears to be ERK1/2 dependent.
cPLA2 can be phosphorylated by protein kinase C, ERK1/2, or protein kinase A in vitro, but only phosphorylation by ERK results in a significant increase in cPLA2 activity and induces a cPLA2 gel shift (26, 27). Because both ERK1/2 and cPLA2 were phosphorylated during eosinophil adhesion to FN, we questioned whether ERK1/2 were the upstream kinases for cPLA2 activation. U0126 at the concentrations that inhibited ERK1/2 phosphorylation prevented cPLA2 phosphorylation, whereas OA at the concentrations that induced ERK1/2 phosphorylation caused cPLA2 phosphorylation. Our results clearly demonstrated that ERK1/2 activation was required for cPLA2 phosphorylation during adhesion to FN. Our results concurred with a previous observation in NIH 3T3 cells during adhesion to FN (31), but contrast with the finding in HeLa cells during adhesion to gelatin (44). Cell-specific or ligand-specific pathways may explain these different results.
It is important to consider some potential implications and limitations
to our work. Although data obtained from these studies define an
upstream pathway for phosphorylation of cPLA2
that leads to eosinophil adhesion, all experiments were performed in
vitro using soluble plated ligand. Hence, it was not possible to
determine whether these same mechanisms apply identically in vivo.
However, we have reported previously that cPLA2
is essential for integrin adhesion of eosinophils to ICAM-1 and VCAM-1.
Our current data suggest that this is a common mechanism and that
cPLA2 may be an essential messenger protein for
all integrin adhesion. Because neutrophils possess the same
2 integrin as eosinophils, the upstream
regulation of both 1 and
2 integrin adhesion leading to phosphorylation
may well apply to other leukocytes, and the final pathway leading to
integrin adhesion for all integrins appears to be the same. Recent
findings that cPLA2 knockout mice have reduced
neutrophil infiltration and pulmonary edema caused by LPS
administration support this notion (45).
cPLA2 knockout mice also do not develop either
the airway remodeling or airway hyperresponsiveness that is associated
with eosinophilic infiltration in Ag-sensitized wild-type controls
(46).
We conclude that ERK1/2 but not p38 MAPK are the upstream kinases for cPLA2 phosphorylation, which is essential for sustained adhesion of human eosinophils to FN. These findings suggest a pathway for cPLA2 phosphorylation that appears to be essential to the maintenance of eosinophil adhesion to FN as well as to other ligands (21). The implications of these findings for future therapeutic targets for prevention of chronic inflammatory cell migration remain to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637.
3 Abbreviations used in this paper: FN, fibronectin; cPLA2, cytosolic phospholipase A2; TFMK, arachidonic trifluoromethyl ketone; MAPK, mitogen-activated protein kinase; MEK/MKK, MAPK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; OA, okadaic acid.
Received for publication February 10, 2000. Accepted for publication December 18, 2000.
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References |
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47 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro. J. Immunol. 163:6217.1 integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J. 17:4391.[Medline]
1 and 2 integrin-dependent adhesion of human eosinophils. J. Immunol. 163:3423.41 (VLA-4). Immunology 82:222.[Medline]
4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1. J. Allergy Clin. Immunol. 99:648.[Medline]
1 and 2 integrin avidity by chemoattractants in eosinophils. Proc. Natl. Acad. Sci. USA 93:10939.This article has been cited by other articles:
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), PD98059 (•), SB203580 (
), and TFMK (
) on
eosinophil adhesion to FN. Eosinophil adhesion was assessed as detailed
in Materials and Methods in the presence of these
inhibitors. The results are shown as mean ± SEM
(n = 3).
) to plate-coated FN. C and
D, Effect of OA on eosinophil viability as assessed by
trypan blue exclusion. All data are expressed as mean ± SEM from
four separate experiments.
