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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 623-631
Regulation and Processing of a Precursor Form of Eosinophil Granule
Major Basic Protein (ProMBP) in Differentiating Eosinophils
By
Pamela Popken-Harris,
James Checkel,
David Loegering,
Benjamin Madden,
Margaret Springett,
Gail Kephart, and
Gerald J. Gleich
From the Departments of Immunology, Internal Medicine, and
Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation,
Rochester, MN.
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ABSTRACT |
The cDNA for eosinophil granule major basic protein (MBP) encodes a
prepromolecule with a total length of 222 amino acids (preproMBP).
PreproMBP includes a secretory leader of 15 amino acids, an acidic
propiece of 90 amino acids, and a basic MBP portion of 117 amino acids.
The function of the propiece, which has a predicted pI of 3.9, is
unknown, but it gives proMBP an overall acidic charge. Because proMBP
is not found in mature eosinophils, we analyzed eosinophil
differentiation in interleukin-5 (IL-5)-stimulated umbilical cord stem
cells cultured for 24 days. By immunofluorescence, proMBP appeared by
day 6 and peaked on day 18, whereas MBP was prominent at days 12 to 24. By day 6, Western blots detected heterogeneous glycosylated 33-kD
proMBP; its peak expression occurred on day 12. Western blots showed
sequential processing of 33-kD proMBP to an 18-kD intermediate form and
finally to 14-kD MBP. By dual label immunoelectron microscopy, proMBP
was localized primarily to large uncondensed eosinophil granules,
whereas MBP was localized to granules containing a condensed central
area. Thus, proMBP is likely expressed and processed as the granule
condenses in a multistep process to 14-kD MBP in differentiating
eosinophils.
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INTRODUCTION |
UNDER THE INFLUENCE of key
hematopoietins, eosinophils develop from stem cells in the bone marrow
and, from there, are released into the peripheral
circulation.1-6 Once in the periphery, they are believed to
play a key role in defense against invading helminths and serve as an
important mediator cell in hypersensitivity diseases.7-9 Eosinophil granules contain four principal cationic
proteins8; their avidity for and binding of the acidic dye
eosin imparts a characteristic orange-red coloration. Among these
cationic proteins is the 14-kD major basic protein (MBP).8
Named for its relative abundance and extreme cationicity (pI of 11),
MBP forms the electron-dense crystalline core of the specific granules
of mature peripheral blood eosinophils.8,10,11
Eosinophil granule MBP is a known toxin,8,9 and application
of physiological amounts of MBP to tumor cells,12,13
parasites,9 and bacteria14 causes direct
toxicity. MBP may mediate effects on targets by its ability to react
with acidic lipid bilayers,15 or alternatively, MBP may
stimulate cells by binding to a specific receptor on the target. For
example, application of physiological amounts of MBP to neutrophils
caused both activation and degranulation, and this may involve G
proteins as critical mediators.16,17 Application of MBP to
platelets causes activation and aggregation,18 whereas
application of MBP to basophils causes histamine
release.17,19 MBP is deposited directly on affected tissue
in allergic diseases, including asthma,7-9 eosinophilic
gastroenteritis,20 and conjunctivitis.21 Therefore, in areas of inflammation where eosinophil degranulation occurs, MBP most likely exerts an effect that is both toxic and proinflammatory in nature.
Therapeutic intervention aimed at halting inimical effects of MBP might
include inhibition of the conversion of the proform of MBP (proMBP) to
MBP. However, we have been unable to detect proMBP in mature
eosinophils.22 Therefore, to study the processing of proMBP
to MBP, we analyzed differentiating eosinophils in vitro by stimulating
umbilical cord stem cells (UCC) with interleukin-5 (IL-5).23-25 By using a combination of antibodies to proMBP
and MBP,22,26-28 we determined the kinetics of proMBP and
MBP expression in developing eosinophils and localized the site of
proMBP to MBP conversion. Our results show that conversion of proMBP to MBP occurs in the granule and lend support to the hypothesis that the
function of the propiece of proMBP is to protect maturing eosinophils
from toxic MBP during granule processing.12,22,28-30
| |
MATERIALS AND METHODS |
Preparation and evaluation of UCC cultures.
Cord blood cells were collected in heparin and layered over Histopaque
1077 (Sigma Chemical Co, St Louis, MO). After centrifugation at
400g for 30 minutes, mononuclear fractions were harvested. The
cells were washed in phosphate-buffered saline (PBS) and incubated at 1 × 106 cells/mL in RPMI 1640 with 10% fetal calf
serum (FCS; HyClone, Logan, UT) with or without recombinant human IL-5
(a generous gift from Schering-Plough Research Institute, Kenilworth,
NJ) at 0 to 10 ng/mL for 0 to 24 days. Every 6 days, half of the media was harvested and replaced. At this time, 1 to 2 mL of cells were harvested, and cell counts and viabilities were determined. Cytospins of cell suspensions were prepared by centrifugation at 400g for 5 minutes onto clean glass slides that had been prespun for 2 minutes
with RPMI containing 10% FCS. The slides were allowed to air dry and
were stained with Wright-Giemsa. Poststaining, the slides were briefly
destained in 95% ethanol for approximately 30 seconds, examined under
oil with a ×100 objective on a Zeiss Axiophot microscope (Zeiss,
Oberkochen, Germany), and photographed with Kodak Ektachrome 64 slide
film (Eastman Kodak, Rochester, NY).
Specificity of antibodies to proMBP and MBP.
One antibody to proMBP and two antibodies to MBP were used. First, a
murine monoclonal antibody, J163-15E10, was used for detection of
proMBP in the immunofluorescence, Western blot, and immunoelectron
microscopy analyses. J163-15E10 reacts with proMBP, but not MBP, by
Western blot and immunoassay.26-28 Second, a murine monoclonal antibody for MBP, J6-8A4, was used for the detection of MBP
in the Western blot analyses. J6-8A4 reacts intensely with 14 kD MBP
and faintly with proMBP by Western blotting.22,26-28 Third,
a polyclonal rabbit antibody, rabbit #14, was used for detection of MBP
in the immunofluorescence and electron microscopy analyses. The
specificity of rabbit #14 anti-MBP for 14 kD MBP was analyzed by
radioimmunoassay (RIA) and by Western blotting. A competitive binding
RIA was performed as described previously31 with
modifications. Briefly, native proMBP and reduced and alkylated MBP
standards (final concentrations of 0 to 128 ng/mL) were incubated with
a 1:5000 dilution of rabbit #14 anti-MBP. 125I-radiolabeled
reduced and alkylated MBP (1 ng/sample) was subsequently added, and the
resulting immune complexes were precipitated by addition of normal
rabbit serum and burro antirabbit IgG. After the samples were
centrifuged, supernatants were discarded and sediments were counted.
For Western blotting, proMBP and purified MBP (1.25 µg/lane) were
analyzed as described below (Western blot analyses) by using a 1:200
dilution of rabbit #14 anti-MBP.
Immunofluorescence.
Cytospins obtained as described were fixed in 100% methanol for 10 minutes and air dried. The immunofluorescence assays for MBP and proMBP
were performed as described previously7 with modifications.
The slides were blocked overnight at 4°C in PBS containing 10%
normal goat serum. After blocking, the slides were incubated with the
primary antibody, either mouse monoclonal antihuman proMBP
(J163-15E10),28 normal mouse Ig (control for J163-15E10), polyclonal rabbit #14 antihuman MBP, or normal rabbit serum (control for rabbit #14). Both the antibody to proMBP and the normal mouse Ig
were purified by protein-A affinity chromatography (Pierce Chemical Co,
Rockford, IL) according to the manufacturer's specifications and used
at 20 µg/mL, and the antibody to MBP and the normal rabbit serum were
used at a 1:40 dilution. After incubation in the primary antibody for 1 hour at 37°C in a humidified atmosphere, the slides were washed in
PBS and incubated with a solution of 1% chromotrope 2R (Sigma) in
distilled water for 1 hour. After the chromotrope 2R treatment, the
slides were incubated with the secondary antibody, either goat
antimouse Ig or goat antirabbit Ig conjugated to fluorescein isothiocyanate (Cappel, Durham, NC), also diluted 1:40, for 1 hour at
37°C. After incubation, slides were washed in PBS, mounted in
glycerol containing 0.1% p-phenylenediamine, and coverslipped. Slides
were examined and photographed as described above with Kodak Ektachrome
200 slide film. Replicates of a minimum of two cultures, and in most
cases three or more cultures, were scored for the number of cells
positive (minimum of 100 cells counted) at each time point and dose.
Western blot analysis.
Cells (1.0 × 106) induced with IL-5, 5 ng/mL, from
each time point were harvested, pelleted, and resuspended in 100 µL 1 × sodium dodecyl sulfate (SDS) sample buffer followed by
denaturation at 100°C for 3 to 5 minutes. Twenty-five microliters
of each denatured sample, corresponding to 250,000 cells, was loaded
onto a 15% Laemmli gel and electrophoresed. Postelectrophoresis, gels
were transblotted onto Millipore Immobilon-P membranes (Millipore, Bedford, MA). Posttransfer, blots were blocked in Blotto/0.3% Tween25 for 1 hour at room temperature and incubated
overnight with a 1:1,000 dilution of protein-A purified primary
antibody against either proMBP (J163-15E10) or MBP
(J6-8A4).22,26-28 Blots were then washed with Blotto/0.3%
Tween followed by incubation for a minimum of 2 hours at room
temperature with a 1:1,000 dilution of sheep antimouse Ig conjugated to
alkaline phosphatase (Sigma). After washing, development was as
described.28
Amino acid sequence analyses.
UCC lysates suspended in 1 × SDS sample buffer were
electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gels and transferred to ProBlot (Applied
Biosystems, Foster City, CA). The blots were stained and destained
according to the manufacturer's specifications and bands for analysis
were excised. N-terminal amino acid sequencing was performed on a
Porton 2090E Integrated Microsequencing System (Porton Instruments Inc, Tarzana, CA).
Immunoelectron microscopy.
UCC induced with 5 ng/mL IL-5 for 24 days were harvested and fixed
overnight in Trump's fixative.32 After fixation, cells were pelleted, rinsed in 0.1 mol/L phosphate buffer, dehydrated in
graded acetone, embedded in Quetol 651, and polymerized at 42°C.
Thin (0.12 µm) sections for labeling were mounted on nickel grids and
dried at 40 to 50°C. Sections were etched in
NaIO4,33 rinsed with water, and blocked in Tris
buffered saline containing 0.1% Tween 20 and 1% bovine serum albumin
(TBST). As primary antibodies, protein A purified antibodies to proMBP
(J163-15E10) and MBP (rabbit #14) were diluted in TBST and applied to
sections for 4 hours at 22°C at the following working dilutions:
mouse anti-proMBP or normal mouse IgG at 20 µg/mL and rabbit anti-MBP
or normal rabbit IgG at 17 µg/mL. After incubation in the primary
antibodies, sections were rinsed in TBST and incubated in a secondary
antibody (either goat antimouse or goat antirabbit IgG) conjugated to
15 nm colloidal gold (Amersham, Arlington Heights, IL). Sections were
thoroughly rinsed in TBST, followed by water, and stained with uranyl
acetate and lead citrate.
For double labeling, the sections were labeled first with anti-proMBP
and then with anti-MBP. Between the two labeling procedures, the
sections were fixed in Trump's so that the first primary antibody could no longer react with the protein A gold Fc binding
receptors.34 Additionally, after rinsing in TBST, sections
were treated with TBST/1% bovine serum albumin/0.2 mol/L glycine to
block free aldehydes, which may have been generated by the fixation
procedure. The proMBP label was localized with a 15-nm gold conjugate,
and the MBP with a 30-nm gold conjugate. After labeling, sections were
rinsed thoroughly with TBST and water, stained with uranyl acetate and
lead citrate, and examined by transmission electron microscopy.
Micrographs were taken on a JEOL 1200 EXII (Japanese Electronic Optical
Laboratories, Peabody, MA) operating at 60 KV.
| |
RESULTS |
Morphologic evaluation of IL-5 stimulated UCC.
UCC were placed in complete medium for 24 days with and without IL-5.
Morphological observation of cells from these cultures stained with
Wright-Giemsa confirmed that cells characteristic of eosinophil lineage
developed only in the presence of IL-5 (results not shown). Eosinophils
at all phases of maturation were present, including eosinophil
myelocytes, metamyelocytes, and even mature bilobed forms. In contrast,
only partially degenerated mononuclear cells usually persisted in
cultures maintained without IL-5.
Specificity of antibodies to proMBP and MBP.
Prior studies by Western blotting had shown that the monoclonal
antibody for proMBP (J163-15E10) reacts specifically with proMBP, but
not MBP, and that the monoclonal antibody for MBP (J6-8A4) reacts
intensely with MBP and weakly with proMBP.22,26-28 The
specificity of rabbit #14 was analyzed by RIA and by Western blotting.
The RIA results (Fig 1) showed that MBP,
but not proMBP, inhibits the binding of rabbit #14 antibody to
radiolabeled MBP, showing that rabbit #14 antibody is specific for MBP
under nondenaturing conditions. The Western blotting experiment (data
not shown) showed that rabbit #14 anti-MBP has approximately equal
reactivity with both proMBP and MBP under denaturing conditions.
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| Fig 1.
Analysis of the specificity of rabbit #14 anti-MBP by
RIA. Native proMBP and reduced and alkylated MBP standards (0 to 128 ng/mL) were incubated with a 1:5000 dilution of rabbit #14 anti-MBP, followed by addition of 125I-radiolabeled MBP. Immune
complexes were precipitated with burro antirabbit IgG and normal rabbit
serum, and radioactivity in the precipitates was counted.
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Immunofluorescence.
We tested by immunofluorescence the ability of varying IL-5
concentrations to induce proMBP production during eosinophil
maturation. Representative photomicrographs of UCC cultured in the
presence of 5 ng/mL IL-5 and stained with anti-proMBP
(Fig 2a-e) or anti-MBP (Fig 2f-j) on
culture days 0, 6, 12, 18, and 24 are shown. At each time point and
dose, the absolute cell number and overall viability were also
calculated (Fig 3). At every IL-5
concentration tested (0.5 to 10 ng/mL), both proMBP and MBP appeared by
day 6 (Figs 2b, 2g, 3a, and 3b). In the presence of IL-5, the
percentage of cells positive for proMBP increased until day 18, but by
day 24 declined to approximately half of that seen at the day-18
maximum (Figs 2a-e and 3a). In contrast, the percentages of cells
positive for MBP rose to nearly 100% by day 18 and remained at 100%
throughout the remaining culture period (Figs 2f-j and 3b). When
control UCC were cultured without IL-5, proMBP and MBP were not
detected (Fig 3a and 3b) and absolute cell numbers fell by days 18 and 24 (Fig 3d). Even in the presence of IL-5, an initial decrease in
absolute cell number occurred on days 0 to 12 (Fig 3d). This was
followed by an absolute increase in the number of cells on days 12 to
24 with a final approximate doubling time of 6 days. Interestingly,
overall cell viability did not change appreciably even in the absence
of IL-5 (Fig 3c).
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| Fig 2.
Immunofluorescent localization of proMBP and MBP in IL-5
induced UCC. UCC were stimulated for 0 to 24 days with 5 ng/mL IL-5. Panels (a to e) show examples of proMBP immunofluorescence on days 0, 6, 12, 18, and 24, respectively. Panels (f to j) show examples of MBP
immunofluorescence on days 0, 6, 12, 18, and 24, respectively. Bar (a
and f) = 10 µm; bar (b to e) and (g to j) = 10 µm.
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| Fig 3.
Analyses of UCC stimulated for 0 to 24 days with 0 to 10 ng/mL of IL-5. Cells from different donors at days 0, 6, 12, 18, and 24 were analyzed, and the mean for each separate point was determined.
Each point represents the mean from at least two separate donors. (a)
The percentage of immunofluorescent cells positive for proMBP
determined by staining with anti-proMBP J163-15E10. (b) The percentage
of cells positive for MBP determined by staining with rabbit #14
anti-MBP. (c) The percentage of viable cells determined by trypan blue
uptake. (d) The absolute number of cells ×106/mL.
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Western blot analysis.
Western blots of duplicate gels containing equivalent UCC lysates from
days 0, 6, 12, 18, and 24 and a lysate of purified mature eosinophils
were analyzed with two murine monoclonal antibodies, J163-15E10
(specific for proMBP)22,26-28 and J6-8A4 (strongly reactive
with 14 kD MBP and weakly reactive with proMBP).22,27 Analyses with anti-proMBP (J163-15E10) showed that proMBP reactivity is
maximal on day 12 (Fig 4a) and that
UCC-derived proMBP is highly heterogeneous, with a spectrum of
molecular weights from approximately 29 kD to greater than 43.8 kD.
However, one predominant form with a band at an estimated 33 kD was
consistently present. No proMBP was present in the purified mature
eosinophil lysate. In contrast, analyses with anti-MBP (J6-8A4) showed
that MBP reactivity was prominent at days 12, 18, and 24 (Fig 4b). Both
an intermediate form of proMBP migrating at approximately 18 kD and
mature MBP migrating at 14 kD were detected. Similar to the 33-kD
unprocessed proMBP, the level of intermediate 18-kD proMBP peaked on
day 12 and decreased until day 24. To verify these observations, the N-terminal sequences of UCC-derived 18-kD intermediate proMBP and the
14-kD mature MBP were obtained. Figure 5
shows the amino acid sequences of UCC-derived preproMBP, the 18-kD
intermediate, and 14-kD MBP.
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| Fig 4.
Western blotting of proMBP and MBP in IL-5-induced UCC
and peripheral blood eosinophils. (a) ProMBP with a major band at 33 kD
detected with an anti-proMBP specific monoclonal antibody, J163-15E10.
(b) MBP and a processed form of proMBP at 18 kD detected with the
anti-MBP specific monoclonal antibody, J6-8A4. Lanes 1 to 5 were loaded
with equivalent cell lysates from UCC at days 0, 6, 12, 18, and 24, respectively. Lane 6 was loaded with an equivalent cell lysate from
purified peripheral eosinophils, and lane 7 contained 5 µg
recombinant proMBP.28
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| Fig 5.
Sequence analysis of recombinant and UCC-derived proMBP.
The complete amino acid sequence of preproMBP28-30 is
shown. N-terminal sequences were obtained as described in Materials and
Methods. Underlined sequences correspond to (1) the N-terminus of
purified 33-kD recombinant proMBP, (2) the N-terminus of the
corresponding 18-kD intermediate form of proMBP in day-12-induced UCC,
and (3) the N-terminus of 14-kD MBP in day-24-induced UCC. Amino acid residues corresponding to the leader sequence are in plain font, the
propiece residues are in italics, and mature MBP residues are in
bold.
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Immunoelectron microscopy.
Morphologic analyses of both mature eosinophils and day-24
IL-5-induced UCC by immunoelectron microscopy were performed with proMBP-specific monoclonal antibody (J163-15E10) and with polyclonal antibody to MBP (rabbit #14). No proMBP staining was observed in
peripheral blood eosinophils (Fig 6a); in
contrast, MBP was localized specifically to the granule crystalline
core (Fig 6b). Day-24 IL-5-induced UCC showed marked proMBP labeling
of large uncondensed granules and minimal labeling of small condensed
granules (Fig 6c); remarkably, proMBP was often seen as a ring around
what appeared to be a granule in the process of condensing (black
arrow). MBP labeling was confined primarily to condensing granules (Fig 6d). Figure 7 shows results from double
immunogold labeling of both proMBP (small gold) and MBP (large gold).
ProMBP was concentrated in large uncondensed granules, and MBP was
concentrated in condensed granules. Some granules (black arrow in Fig
7b) contained labeling for MBP (primarily in the center) and proMBP (at
the periphery). Controls using either normal mouse immunoglobulin or
normal rabbit immunoglobulin as the primary label were negative
(results not shown).
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| Fig 6.
Localization of proMBP and MBP by immunoelectron
microscopy. (a) A peripheral blood eosinophil labeled with anti-proMBP
J163-15E10. Note the absence of specific labeling. (b) A peripheral
blood eosinophil labeled with rabbit anti-MBP. Note that MBP is
specifically localized to the granule crystalline core (black arrows).
(c) Day-24 IL-5-induced UCC labeled with anti-proMBP. The black arrow shows localization of proMBP predominantly to the periphery of a
condensing granule; very little proMBP is present in the interior of
that granule. The white arrow points to a condensed granule, which
lacks proMBP label. Also, note the presence of a large uncondensed granule densely labeled with proMBP proximal to the nucleus (open white
arrowhead). (d) Day-24 IL-5-induced UCC labeled with anti-MBP. MBP is
localized primarily to the interior of condensing granules (black
arrows). (a, c, and d) Original magnification ×11,000; (b)
×15,000.
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| Fig 7.
Localization of proMBP and MBP in day-24 IL-5-induced
UCC by immunoelectron microscopy using double labeling. ProMBP was
localized with 15-nm colloidal gold particles and MBP with 30-nm gold
particles. (a) Although proMBP labeling (small gold) is seen throughout
the Golgi, it is concentrated predominantly in the large uncondensed granules (white arrow); only minimal MBP labeling (large gold) is
evident in the large granules. In contrast, the small condensed granules show marked MBP labeling in the interior and minimal proMBP
labeling at the periphery (black arrow). (b) At higher magnification,
dense labeling for proMBP (small gold) and minimal labeling for MBP
(large gold) is evident in a large uncondensed granule (white arrow).
The black arrow identifies a small condensed granule that contains
predominantly MBP, along with minimal proMBP at the periphery. (a)
Original magnification ×11,000; (b) ×20,000.
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DISCUSSION |
Before this report, the existence of proMBP in developing eosinophils
had only been deduced from cDNA analysis.29,30 Here, with
the proMBP-specific monoclonal antibody, J163-15E10, we show by
immunofluorescence, Western blot analyses, and immunoelectron microscopy that proMBP is developmentally regulated in eosinophils and
that processing to its mature MBP form occurs in condensing eosinophil
granules. We found that the addition of recombinant human IL-5 to human
UCC is sufficient not only to cause production of proMBP, but also
results in the formation of mature MBP as well. By Western blot
analyses, we showed that both recombinant Chinese hamster ovary (CHO)
cell-derived proMBP and eosinophil-derived UCC proMBP are similarly
heterogeneous with molecular masses ranging from approximately 33 to 84 kD.28 This marked heterogeneity is likely accounted for by
differential carbohydrate addition. By sequence analysis, the propiece
of proMBP contains one potential N-linked (Asn-X-Thr), one potential
glycosaminoglycan (Gly-Ser-Gly), and 15 potential O-linked (Ser or Thr)
attachment sites. Analyses of placentally derived proMBP, which has a
similar molecular weight profile, indicate that the extreme molecular
weight heterogeneity is accounted for by differential carbohydrate
addition onto the propiece.27,35
By immunoelectron microscopy, proMBP was expressed in promyelocytes,
myelocytes, and metamyelocytes of the eosinophilic lineage that were
actively forming specific granules. Consistent with the electron
microscopic description by Bainton and Farquhar1 of
peroxidase, arylsulfatase, and acid phosphatase processing in the
specific granules of eosinophil myelocytes, heavy proMBP labeling was
seen throughout the Golgi, in secretory vesicles, and in uncondensed
granules. Double immunostaining of proMBP and MBP showed that granules
in the process of condensing have proMBP around the periphery of the
central area and MBP in the central area, consistent with the view that
conversion of proMBP to MBP is occurring in the granule.
Detailed examination by immunoelectron microscopy of cultured UCC
labeled singly for proMBP or dually for both proMBP and MBP indicates
that one or both of these forms are present in every granule. This is
in accordance with the model for eosinophil development proposed by
Hardin and Spicer36 and Parmley et al37;
namely, that primary or azurophilic granules do not exist separately from mature specific granules. We extend their work by proposing that
azurophilically or metachromatically staining "primary granules" are either immature granules containing unprocessed proMBP or condensing granules containing both proMBP and MBP.1,36
Binding of acidic proMBP by the blue cationic elements of the dye would result in the characteristic azurophilic and metachromatic staining. Prior studies indicated that immature differentiating eosinophils contain granules with large amounts of "sulfated acid
muco-substance,"36 which in retrospect may be proMBP.
After immature proMBP-containing granules have undergone proteolytic
processing and condensation, cationic MBP would become available, and
the maturing granules would take up the acidic dye eosin. This, plus
the contribution of dye binding by the other three cationic proteins in
the granular matrix (eosinophil peroxidase, eosinophil cationic
protein, and eosinophil-derived
neurotoxin),8,11 would result in the appearance of bright
red granules characteristic of mature eosinophils.
Additionally, our studies describing the localization of proMBP to
immature granules parallel the description by Parmley et al37 of chondroitin sulfate localization and traffickingin
differentiating eosinophils derived from bone marrow. Initially,
staining for chondroitin sulfate was present in the Golgi. In the next
phase, staining was present in small transfer vesicles that appeared to
deposit their contents into large immature granules. In the following
phase of granule maturation, somewhat distal from the Golgi,
chondroitin sulfates formed rings around the periphery of what appeared
to be granules in the process of condensing. The final phase of granule
maturation was characterized by the total disappearance of chondroitin
sulfate moieties and the appearance of condensed granules containing
crystalline cores. Our studies on proMBP closely parallel their
findings. We found that proMBP was initially present in large
uncondensed granules. As granule maturation and eosinophil
differentiation proceeded, proMBP ringed the periphery of condensing
granules. During the final phase of granule condensation, peripheral
reactivity for proMBP was lost and reactivity for mature MBP was gained
(summarized in Fig 8). What remains
enigmatic in our system, but is characteristic of eosinophils processed
in cell culture2 is the observation that MBP fails to form
crystalline cores in induced UCC.
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| Fig 8.
Schematic representation of eosinophil granule maturation
with proposed staining patterns. ProMBP is deposited into large vacuoles just proximal to the Golgi. From here, proMBP containing vacuoles appear to move toward the periphery where granule condensation begins to occur. As the granule condenses, proMBP can be seen around
the periphery, and MBP can be seen in the interior. During the final
stage of granule maturation, proMBP reactivity is lost, and only MBP
reactivity remains.
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The signal or trigger that mediates the cleavage of proMBP to MBP is
unknown. It may be that a certain proMBP concentration has to be
reached or that the converting enzyme must translocate to the granule
before processing can occur. Palade38 has suggested that
secretory granule condensation occurs as a function of sulfated polyanion aggregation and that sulfated polyanions bind to cationic proteins. Binding would result in an osmotic loss of water from the
granule, thereby causing granule condensation. We have recently shown
by isoelectric focusing and Western blotting that recombinant proMBP
derived from bulk CHO supernatants, despite its predicted slightly acid
pI of 6.2,29 is in reality quite anionic.22,28 The predominant 33-kD form of proMBP possessed a pI of approximately 4.5, and higher molecular mass forms of proMBP (presumably more heavily
glycosylated) possessed progressively more anionic isoelectric points.22,28 Also, by Western blot analyses, proMBP in
IL-5-induced UCC appears electrophoretically identical to unprocessed
recombinant proMBP secreted into CHO supernatants.22,28
These observations suggest that eosinophil-derived proMBP, like
recombinant proMBP, has excess anionic charge available for binding and
aggregation. A test of this concept showed that proMBP was able to
neutralize up to a twofold molar excess of 14-kD MBP.28
Therefore, as suggested by Palade,38 the process of
sulfated polyanionic aggregation with subsequent cationic protein
binding may play an important role in eosinophil granule condensation,
except that the acidic proportion of proMBP would substitute for a
sulfated peptidoglycan.
In summary, to better understand how eosinophils develop and how
granule processing of proMBP to MBP occurs, we studied differentiating eosinophils by the induction of UCC with IL-5. IL-5, first described as
B-cell growth factor II,39,40 is now known to play a
pivotal role in both eosinophil differentiation and
activation.2,6,23,41 Our finding that IL-5 is sufficient to
drive eosinophil differentiation is in contrast with the report by
Warren and Moore,41 which cited the necessary role of
additional cytokines in inducing eosinophil differentiation, but is in
agreement with reports by Clutterbuck et al2 and Saito et
al.23 However, Saito et al describe the decrease of
absolute cell numbers and maturation only to myelocytes by week 3 of
culture,23 whereas we report that precursor cells are still
actively dividing and that maturation to bilobed forms has occurred by
day 24. Finally, we propose that IL-5 induction of eosinophil
differentiation can be used as a model for the study of protein
processing in the pathway for stored secretory granule products.
| |
FOOTNOTES |
Received July 15, 1997; accepted March 18, 1998.
Supported by grants from the National Institutes of Health, AI 09728 and AI 07047, and by Mayo Foundation.
Address reprint requests to Gerald J. Gleich, MD, Department of
Immunology, Mayo Clinic, 200 1st St SW, Rochester, MN 55905.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Lester Harris, PhD, from the Abbott Northwestern Cancer
Research Laboratory for proofreading of the manuscript and helpful
suggestions; Diane Squillace, BS, for technical assistance; Cheryl
Adolphson, MS, for editorial assistance; and Linda H. Arneson for
secretarial assistance from the Department of Immunology, Mayo Clinic
and Foundation.
| |
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Abstract
Introduction
Abstract