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Mouse Mast Cells That Possess Segmented/Multi-lobular Nuclei
By Michael F. Gurish, Daniel S. Friend, Matthew Webster, Namit Ghildyal, Christopher F. Nicodemus,
and Richard L. Stevens
Because in humans mast cells and basophils tend to possess
nonsegmented and segmented/multi-lobular nuclei, respectively, nuclear morphology has been a major criterion for
assessing the lineage of metachromatic cells of hematopoietic origin. Immature metachromatic cells with mono- and
multi-lobular nuclei were both obtained when bone marrow
cells from BALB/c mice were cultured for 3 weeks in the
presence of interleukin-3. Analogous to the indigenous mature mast cells that reside in the peritoneal cavity and skin,
both populations of in vitro–derived cells expressed the surface receptor c-kit, the chymase mouse mast cell protease
(mMCP) 5, the tryptase mMCP-6, and the exopeptidase carboxypeptidase A (mMC-CPA). Immunogold electron microscopy confirmed the granule location of mMC-CPA and
mMCP-6 in both populations of cells, and cytochemical analysis confirmed the presence of chymotryptic enzymes in the
granules. Because mature mast cells possessing multi-lobular nuclei also were occasionally found in the skeletal muscle
and jejunum of the BALB/c mouse, the V3 mouse mast cell
line was used to investigate the developmental relationship
of mast cells that have very different nuclear structures.
After the adoptive transfer of V3 mast cells into BALB/c
mice, v-abl –immortalized mast cells with mono- and multilobular nuclei were detected in the lymph nodes and other
tissues of the mastocytosis mice that expressed c-kit,
mMCP-5, mMCP-6, and mMC-CPA. These studies indicate
that mouse mast cells can exhibit varied nuclear profiles.
Moreover, the nuclear morphology of this cell type gives no
insight as to its protease phenotype or stage of development.
q 1997 by The American Society of Hematology.
M
possess multi-lobular nuclei. While Galli et al12-14 concluded
that this latter population of cells in the cultures probably
are mast cells, Rottem et al15 concluded that they probably
are basophils.
Because mature human mast cells expresses substantially
more c-kit on their surfaces than mature human basophils,
the relative abundance of this plasma membrane receptor is
regularly used to distinguish the two populations of cells
in humans.16 Mouse mast cells also express c-kit on their
surfaces.17 Moreover, they store in their secretory granules
various combinations of an exopeptidase (termed mouse
mast cell carboxypeptidase A [mMC-CPA]) and at least nine
different serine proteases (termed mouse mast cell protease
[mMCP] 1 to mMCP-9).18-29 cDNAs and genes that encode
all of these granule proteases except mMCP-3 have been
cloned and sequenced, and protease-specific antibodies have
been obtained in rabbits that recognize mMCP-1, mMCP2, mMCP-5, mMCP-6, and mMCP-7.7,30-33 Along with the
cDNAs, the protease-specific antibodies have been useful
for phenotyping in vivo–differentiated mouse mast cells and
for monitoring the regulated differentiation and maturation
of hematopoietic progenitor cells into mast cells in vitro and
in vivo. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot and Nterminal amino acid analysis of resolved granular protein,
RNA blot analysis, and/or immunohistochemical analysis,
mast cells have been detected at different tissue sites in
the BALB/c mouse that express various panels of granule
proteases. For example, the mast cells that increase in number in the jejunal lamina propria of V3 mastocytosis mice
and in the jejunal epithelium of Trichinella spiralis–infected
mice at the height of the infection preferentially express
mMCP-1 and mMCP-2, whereas the mast cells in the jejunal
muscle of these mice preferentially express mMCP-4,
mMCP-5, mMCP-6, and mMC-CPA.7,19,21,30,34,35 Immunohistochemical analysis of the mastocytosis that develops in the
jejunum during helminth infection7 or in different tissues
of BALB/c mice after the adoptive transfer of the v-abl–
immortalized V3 mast cell line34 indicate that the protease
phenotype of a mast cell can be modulated and is not predetermined in the BM.
As assessed by RNA blot analysis, mBMMC cultures con-
AST CELLS, like basophils, possess high-affinity receptors for IgE (FceRI) on their cell surfaces and
contain numerous histamine-rich secretory granules that become metachromatic when stained with cationic dyes. Because antibodies to basophil-specific proteins are not available and because antibodies to mast cell–specific proteins
became available only recently, mouse mast cells and basophils have been distinguished primarily by ultrastructural
criteria.1-4 Attempts have been made to distinguish basophils
from mast cells by the number and morphology of their
granules. However, the observation that the granules in a
single rat5 or mouse6,7 intraepithelial mast cell can differ
dramatically in their size, maturation, and ultrastructure indicates that granule ultrastructure, by itself, cannot be used to
definitively phenotype a metachromatic cell. Thus, the nuclear profile of a cell thought to be in the basophil or mast
cell lineage is presently one of the major structural features
used to distinguish in vivo–differentiated mouse basophils
and mast cells.
Immature populations of FceRI//metachromatic cells are
obtained when progenitor cells from the bone marrow
(mBMMC) and other organs are cultured for 3 to 4 weeks
in the presence of purified interleukin-3 (IL-3), recombinant
IL-3, or IL-3–enriched cell-conditioned media.8-11 Most of
the cells in these different cultures are mononuclear but some
From the Departments of Medicine and Pathology, Harvard Medical School; and the Division of Rheumatology, Immunology, and
Allergy and the Department of Pathology, Brigham and Women’s
Hospital, Boston, MA.
Submitted November 18, 1996; accepted February 21, 1997.
Supported by Grants No. AI-22531, AI-23483, AI-31599, AR36308, HL-36110, and HL-48598 from the National Institutes of
Health.
Address reprint requests to Richard L. Stevens, PhD, Harvard
Medical School, Seeley G. Mudd Bldg, Room 617, 250 Longwood
Ave, Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/9001-0002$3.00/0
Blood, Vol 90, No 1 (July 1), 1997: pp 382-390
382
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MOUSE MAST CELLS WITH DIFFERENT NUCLEAR PROFILES
tain high levels of the transcripts that encode FceRI, c-kit,
mMCP-5, mMCP-6, mMCP-7, and mMC-CPA.17,18,23,24,36-38
Most of these transcripts are readily detected 1 to 2 weeks
after BM cells are exposed to IL-3–enriched medium. Because mature, cutaneous mast cells of the BALB/c mouse
express FceRI, c-kit, mMCP-5, mMCP-6, mMCP-7, and
mMC-CPA, we and others have concluded that most, if not
all, of the cells in the 3- to 4-week-old cultures belong to
the mast cell lineage. In the present study, we show that
the metachromatic cells in the 3- to 4-week-old BALB/c
mBMMC cultures that contain multi-lobular nuclei express
c-kit, mMCP-5, mMCP-6, and mMC-CPA, as do the predominant mononuclear mBMMC. We also show that mast
cells with multi-lobular nuclei can be found occasionally in
the skeletal muscle of normal mice, the jejunum of helminthinfected mice, and in various tissues of V3 mastocytosis
mice. Thus, in the mouse, mast cells can exhibit very different nuclear profiles.
MATERIALS AND METHODS
In vitro– and in vivo–derived mast cells. mBMMC were obtained by culturing BALB/c mouse BM cells for 3 to 4 weeks in
enriched medium (RPMI-1640 supplemented with 10% fetal calf
serum [FCS] [GIBCO, Grand Island, NY], 2 mmol/L L-glutamine,
0.1 mmol/L nonessential amino acids, 100 U/mL penicillin, and 100
mg/mL streptomycin) containing either 50% WEHI-3 cell (line TIB68; American Type Culture Collection [ATCC], Rockville, MD)
conditioned medium or recombinant IL-3 in a humidified atmosphere
at 377C and 5% CO2 .10 V3 mastocytosis mice were generated by
injecting É106 cultured V3 mast cells intravenously into the tail
veins of each 6-week-old BALB/c mouse (The Jackson Laboratory,
Bar Harbor, ME).34 Previous DNA blot analysis revealed that the
V3 mast cell line contains a single copy of the pGDv-abl provirus in
its genome. Because the oncogene was inserted at a site distinct
from that in the V7 mast cell line and the V8 lymphoblastic cell
line, the V3 mast cell line is of clonal origin. V3 mastocytosis mice
were used 2 to 4 weeks after their adoptive transfer. Mouse Kirsten
sarcoma virus–immortalized mast cells (KiSV-MC) were maintained in 100% enriched medium as previously described.39 Serosal
mast cells were obtained under sterile conditions from SpragueDawley rats (The Jackson Laboratory) by peritoneal lavage with
Tyrode’s buffer containing 5% FCS, 100 U/mL penicillin, 100 mg/
mL streptomycin, and 20 mg/mL gentamicin, and then were enriched
to 90% to 99% purity by density-gradient centrifugation in 25%
metrizamide.40 Swiss albino mouse 3T3 fibroblasts (line CCL-92;
ATCC) were grown in enriched medium. BALB/c mice were infected with T spiralis as described to evaluate the mast cells in the
jejunal epithelium at the height of the infection.7,30,35
Production of a mMC-CPA-specific antibody. The deduced
amino acid sequences of mMC-CPA18 and rat MC-CPA41 were compared with that of other metallocarboxypeptidases42-46 to define potential antigenic regions. As assessed by the Protein Identification
Resource database of the National Biomedical Foundation
(Bethesda, MD), residues 146 to 157 are not present in any other
carboxypeptidase or protein. Because of its novelty, hydrophilicity,
and potential antigenicity, we speculated that antibodies directed
against the peptide Asp-Val-Ser-Trp-Ser-Ser-Pro-Asn-Thr-Asp-Lys
would be specific for mMC-CPA and rat MC-CPA. Thus, a synthetic
peptide (3 mg) corresponding to this sequence was suspended in 5
mL of 10 mmol/L phosphate-buffered saline (PBS) containing 5 mg
keyhole limpet hemocyanin (Sigma Chemical Co, St Louis, MO),
and coupling was performed in the presence of 0.25% glutaraldehyde
(Sigma) at 47C. Noncoupled peptide was removed by dialyzing the
reaction mixture against PBS using a membrane with a 12-kD cutoff.
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383
Polyclonal antibodies to the coupled peptide were raised in New
Zealand White rabbits according to standard immunization protocols
in which each rabbit was injected intramuscularly (IM) with an
emulsion consisting of 0.5 mL of the coupled peptide (É500 mg)
mixed with an equal volume of TiterMax synthetic adjuvant (CyRx
Crop, Norcross, CA). The immunized animals received booster injections IM monthly, and their sera were collected at 2-week intervals over a number of months. An enzyme-linked immunosorbent
assay (ELISA) was used to evaluate the presence of specific antibodies in the sera, and antibodies that exhibited strong reactivities
against the immunizing peptide were purified with a peptide-affinity
column, as previously described for anti–mMCP-2 Ig and anti–
mMCP-5 Ig.30,31
The first 10 residues of the immunizing peptide also are found in
the corresponding region of rat MC-CPA.41 Because considerably
more MC-CPA containing mast cells can be purified from a normal
rat than from a normal mouse, the native exopeptidase was purified
from rat serosal mast cells on a potato carboxypeptidase-inhibitor
Sepharose column18,41 to evaluate whether or not mMC-CPA and rat
MC-CPA were both recognized by the antipeptide antibody. When
the wells of the microtiter plates were coated with purified rat MCCPA and incubated with the purified antibody, the antibody avidly
bound to purified rat MC-CPA in the ELISA at a greater than 1,000fold dilution (data not shown). As assessed by SDS-PAGE/immunoblot analysis, anti–mMC-CPA Ig identified an É36-kD protein in
whole cell lysates and granule-enriched preparations from mBMMC,
mouse KiSV-MC, and purified rat serosal mast cells. In contrast, it
failed to recognize any protein in the lysates of mouse fibroblasts.
The É36-kD protein was not recognized by preimmune sera, and
the reactivity of anti–mMC-CPA Ig was abolished by preincubation
with the immunizing peptide but not with the synthetic peptide that
corresponds to residues 276 to 298 of mMC-CPA.
Histochemistry, enzyme cytochemistry, and immunohistochemistry. For histological examination, cytocentrifuge preparations of
BALB/c mBMMC were air dried and stained for 20 seconds in
a 5% ethanolic solution of methylene blue.47 Serial 1.5-mm thick
glycolmethacrylate sections of lymph node tissue from V3 mastocytosis mice, tongue skeletal muscle from normal BALB/c mice,
and jejunum from T spiralis–infected BALB/c mice were air dried
and incubated sequentially with double-strength hematoxylin for 2
minutes, 1% aqueous eosin Y for 15 minutes, azure II for 1 minute,
and then with ethylene glycol monomethyl ether for 5 seconds.48
Individual mast cells containing chymotryptic activities were identified in tissue sections by the method of Leder.49 mBMMC and fixed
tissue sections were incubated at 307C for 1 hour with a solution
containing naphthol AS-D chloroacetate; the resulting preparations
were rinsed and counterstained with hematoxylin. Mast cells containing tryptic activities were identified in tissue sections by the
cytochemistry method of Osman et al,50 with Z-Ala-Ala-Lys-4-methoxy-2-naphthylamide (AAK) used as the substrate.
Light and electron microscopic immunohistochemistry were performed as described.7,30-34,51 For light microscopy, collected tissues
were fixed for 4 hours at room temperature in 4% paraformaldehyde
in 0.1 mol/L sodium phosphate, pH 7.6. Alternatively, selected samples were fixed in Methacarn or Carnoy’s solution. Preparations
were washed twice with PBS containing 2% dimethyl sulfoxide and
were suspended in 50 mmol/L NH4Cl overnight at 47C. The specimens were dehydrated and embedded in accordance with the JB-4
embedding kit instructions from Polysciences Inc (Warrington, PA).
For electron microscopy, tissues were fixed in 0.25% glutaraldehyde
and 4% paraformaldehyde and then frozen in liquid nitrogen. Tissue
sections and/or cells were stained with either rat monoclonal antimouse c-kit Ig (PharMingen, San Diego, CA) followed by goatantirat IgG (PharMingen) or with affinity-purified rabbit anti–
mMCP-2 Ig, anti–mMCP-5 Ig, anti–mMCP-6 Ig, or anti–mMCCPA Ig followed by goat-antirabbit IgG.30-32
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384
GURISH ET AL
RESULTS
Histochemistry, enzyme cytochemistry, and immunohistochemistry of mBMMC that have different nuclear profiles.
Metachromatic mBMMC were obtained that possessed different nuclear morphologies when BALB/c mouse BM cells
were cultured for greater than 3 weeks in the presence of
IL-3 (Fig 1). When ú300 BALB/c mBMMC were analyzed
in each of three experiments, 8%, 14%, and 15% of the cells
in the culture had multi-lobular nuclei. Regardless of whether
these mBMMC were heavily or lightly granulated in terms
of their metachromasia when stained with toluidine blue,
they phenotypically resembled the mononuclear cells in the
culture. Although these cells contained low levels of tryptase
activity (data not shown), they contained substantial amounts
of chloroacetate esterase (chymase) activity (Fig 1). As assessed immunohistochemically at low magnification, both
populations of mBMMC expressed c-kit, mMCP-5, mMCP6, and mMC-CPA. When the mBMMC with mono- and
multi-lobular nuclei were examined with transmission electron microscopy and immunogold cytochemistry, most of
the secretory granules in both populations of cells were relatively immature (Figs 2 and 3). Nevertheless, some cells in
both cultures possessed electron dense granules and these
granules were stained by anti–mMC-CPA Ig and anti–
mMCP-6 Ig (Fig 2). The chromatin was condensed to the
same extent on the nuclear membrane in both populations
of mBMMC. Analysis of four different levels through a
single mBMMC showed that the apparent segmented nature
of the nucleus was not a consequence of the section analyzed
(Fig 3). Further ultrastructural analysis revealed that the varied lobes of the nucleus can be joined by a thin connection.
Histochemistry, enzyme cytochemistry, and immunohistochemistry of mast cells in normal BALB/c mice and in V3
mastocytosis mice that possess multi-lobular nuclei. Because of the thinness of the sections, it is not always possible
to determine the nuclear configuration of a tissue-localized
mast cell precisely. Nevertheless, metachromatic//chloroacetate esterase/ cells that appear to possess multi-lobular nuclei were occasionally seen in the skeletal muscle of the
tongue of normal BALB/c mice and the jejunal epithelium
of T spiralis–infected BALB/c mice (Fig 4, see page 385).
The observation that the individual nuclear lobes in these
cells are smaller in size than the intact nucleus of a mononuclear mast cell in the same section suggests that the nuclear
lobes represent portions of the same nucleus rather than
distinct nuclei. Analogous to the mononuclear mast cells at
these two tissue sites, the cells with multi-lobular nuclei in
the muscle could be stained by anti–mMC-CPA Ig but not
anti–mMCP-2 Ig, whereas those in the jejunum could be
stained by anti–mMCP-2 Ig but not anti–mMC-CPA Ig
(Fig 4).
V3 mastocytosis mice were used to determine if an immature mast cell–committed line could give rise to mast cells
in vivo with very different nuclear profiles. When greater
than 100 V3 mast cells were analyzed in each of four separate experiments, 1%, 2%, 3%, and 4% of the cells in the
culture contained multi-lobular nuclei. As obtained previously,34 a prominent mastocytosis developed in a number
of tissues of the BALB/c mouse after the adoptive transfer
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of the v-abl–immortalized V3 mast cell line into this animal.
Most of the methylene blue//toluidine blue/ V3 mast cells
found in the mesenteric lymph nodes of these animals appeared to be mononuclear. However, occasionally a methylene blue/ V3 mast cell could be detected that appeared to
contain a multi-lobular nucleus (Fig 5, see page 385). Although V3 mast cells with multi-lobular nuclei were most
readily found in the mesenteric lymph nodes, similar cells
were observed in liver, spleen, and intestine of V3 mastocytosis mice (data not shown). Analysis of tissue sections
of varied organs of three different animals showed that 0.7%
of the 1,400 V3-MC examined contained multi-lobular nuclei. Because sections are analyzed rather than isolated whole
cells, this percentage of V3 mast cells in tissues that have
multi-lobular nuclei is an underestimation. Nevertheless, like
mononuclear V3 mast cells, these cells contained chloroacetate esterase and AAK (tryptase) activities. As assessed immunohistochemically, both populations of V3 mast cells in
lymph nodes expressed c-kit, mMCP-5, mMCP-6, and
mMC-CPA.
DISCUSSION
Although in vitro–differentiated mast cells possessing
multi-lobular nuclei have been found occasionally in culturederived mouse mast cells,12-15 the consensus opinion is that
in vivo–differentiated mast cells in this species are primarily
mononuclear. Thus, nuclear morphology is one of the criteria
routinely used to distinguish mast cells from basophils in
the mouse. We now show that it is possible to obtain mouse
mast cells in vitro and in vivo that possess segmented/multilobular nuclei, and that an immature mouse mast cell-committed line can give rise to mast cells in vivo with very
different nuclear morphologies.
Mast cells and basophils in humans are granule-containing
hematopoietic cells that become metachromatic when stained
with cationic dyes. mBMMC and all tissue-localized mouse
mast cells that have been analyzed so far contain large
amounts of at least one chymase in their granules, and most
of these mast cells also express at least one tryptase. Thus,
even when tissue sections are fixed, dehydrated, and embedded, mast cells are readily recognized in mouse tissues
because of their pronounced tryptic and chymotryptic activities.7,49,50 With enzyme cytochemistry and immunohistochemistry, it was discovered that the toluidine blue//methylene blue/ cells with multi-lobular nuclei in mBMMC
cultures (Fig 1) and in the mesenteric lymph nodes of V3
mastocytosis mice (Fig 5) all contain abundant levels of
mMCP-5 and mMCP-6 in their granules. Although mMCP5 and mMCP-6 are both serine proteases, they are members
of two distinct families whose genes reside on chromosomes
14 and 17, respectively. Thus, these immunohistochemical
and cytochemical findings suggested that the metachromatic
cells in the analyzed cultures with multi-lobular nuclei are
mast cells, as Galli et al12-14 concluded in their ultrastructural
analysis of comparable cultured cells. Dvorak et al52 noted
the transient appearance of metachromatic cells very early
in mBMMC cultures which morphologically resemble basophils. Because these in vitro–derived cells were not analyzed
in the present investigation, we cannot support or dispute
the concept that basophils exist in the mouse as a develop-
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MOUSE MAST CELLS WITH DIFFERENT NUCLEAR PROFILES
385
Fig 1. Histochemistry, enzyme cytochemistry, and immunohistochemistry of cells in 4-week cultures of BALB/c mBMMC that possess
mono- (a through g) and multi-lobular (h through n) nuclei. Cytocentrifuge preparations of cells were evaluated for toluidine blue reactive
granules (a, b, h, i) that contained chloroacetate esterase enzymatic activity (d, k), mMCP-5 protein (e, l), mMC-CPA protein (f, m), or mMCP6 protein (g, n). Cells also were evaluated immunohistochemically for their surface expression of c-kit (c, j).
Fig 4. Cytochemistry and immunohistochemistry of mast cells in the skeletal muscle of a normal BALB/c mouse (a, b) and the jejunal epithelium of a T spiralis –infected
BALB/c mouse (c, d). Serial sections were evaluated for chloroacetate esterase enzymatic activity (a, c), immunoreactive mMC-CPA (b), and immunoreactive mMCP-2 (d).
The arrows indicate mast cells that appear to possess multi-lobular nuclei. Panel (a)
was heavily counterstained with methyl green. Thus, the three chloroacetate esterase" mast cells depicted in this panel exhibit a blue color. The red reaction product
on the brush border of the villi (d) is due to endogenous intestinal alkaline phosphatase. The presence of this product indicates that the color substrate was active in
the immunohistochemical reaction.
Fig 5. Histochemistry, cytochemistry, and immunohistochemistry of V3 mast cells in the lymph nodes of mastocytosis mice. The V3 mast
cells in the various tissue sections that have multi-lobular nuclei were evaluated for the expression of c-kit (b) and for methylene blue reactive
granules (a) that contain chloroacetate esterase enzymatic activity (c), mMCP-5 protein (d), mMC-CPA protein (e), mMCP-6 protein (f), and
AAK enzymatic activity (g).
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386
GURISH ET AL
Fig 2. Immunoelectron microscopy of mBMMC with mono- (a) and multi-lobular (b through e) nuclei. Most cells in the culture have
numerous multi-vesicular structures in their granules (V). However, in terms of their electron density, some of the granules (D) in these
cultured cells are nearly as mature as those in tissue mast cells. Frozen thin sections of the mBMMC were stained with immunogold anti–
mMC-CPA Ig (c), anti–mMCP-6 Ig (d), or a control rabbit Ig (e). Ten-nanometer gold particles were used with the antibodies. N, nucleus.
mentally distinct cell type. However, the finding that in
vitro– and in vivo–differentiated mouse mast cells can have
different nuclear profiles now indicates that the lineage of a
metachromatic mouse cell cannot be determined conclusively by the shape of the cell’s nucleus alone.
In contrast to their granule serine proteases, only one carboxypeptidase has been identified in the granules of mouse,
rat, and human mast cells.18,41,53,54 In humans, MC-CPA protein is preferentially found in the tryptase//chymase//cathep-
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sin G/ subset of mast cells that reside in the skin and certain
other tissue sites.55 Basophils in the peripheral blood of humans do not contain detectable amounts of this exopeptidase
in their granules.55 In mice and rats,18,20,41 MC-CPA is a
major granule constituent of the serosal mast cells isolated
from the peritoneal cavity. The ear and skin of BALB/c
mice also contain high steady-state levels of the mMC-CPA
transcript,33 and in situ hybridization analysis56 has shown
that most, if not all, of the mast cells in these connective
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MOUSE MAST CELLS WITH DIFFERENT NUCLEAR PROFILES
387
Fig 3. Ultrastructural analysis of mBMMC. Four levels
through a single mBMMC are analyzed in panels (a) through
(d). The electron micrograph depicted in panel (e) shows a thin
connection joining the two lobes of the nucleus of another
mBMMC. N, lobes of the nucleus; D, relatively dense granules;
V, relatively immature granules containing predominantly
multi-vesicular structures.
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388
GURISH ET AL
tissue sites express mMC-CPA. Because high levels of the
mMC-CPA transcript are present in the BALB/c mBMMC
cultures,18 it has been presumed that the prominent mononuclear cells in these cultures store the exopeptidase in their
granules. Therefore, whether the mMCP-5/ cells in V3 mastocytosis mice and in the mBMMC cultures that contain
multi-lobular nuclei also store mMC-CPA protein in their
granules was of interest. To this end, an mMC-CPA–specific
antibody was derived with an antipeptide approach. When
immunohistochemistry was performed with anti–MC-CPA
Ig, the granules of both populations of mBMMC in the culture were strongly reactive (Figs 1 and 2), as were the granules of mast cells in the skeletal muscle of the normal BALB/
c mouse (Fig 4) and the V3 mast cells in the lymph node
(Fig 5) that possessed multi-lobular nuclei. Analogous to
mature mast cells,17 these V3 mast cells (Fig 5) and mBMMC
(Fig 1) also expressed c-kit.
Because a higher percentage of the cells in cultures of
mBMMC15 and human cord blood/BM57-61 have multi-lobular nuclei when hematopoietic progenitor cells are cultured
in the presence of IL-3 rather than in c-kit ligand, IL-3
seems to promote nuclear segmentation. What other factors
are involved and the functional significance of the nuclear
changes remain to be ascertained. Nuclear segmentation of
neutrophils and eosinophils is a late event in the maturation
processes of these granulocytes. Nevertheless, the nuclear
segmentation process of mouse mast cells does not appear
to depend on the cell being at its final stages of granule
maturation because these mast cells in the mBMMC cultures and in the varied tissues of the V3 mastocytosis mouse
are only of modest maturity in terms of their granulation.
In a number of ways (eg, presence of metachromatic
secretory granules that contain histamine, expression of
membrane-bound FceRI, FceRI-mediated metabolism of arachidonic acid to leukotrienes, and FceRI-mediated expression of IL-4 and IL-13) human mast cells are more similar
to basophils than are any other cells in the body. The discovery of an ‘‘intermediate’’ cell in the BM and peripheral
blood of patients with chronic myelogenous leukemia possessing certain ultrastructural features of normal peripheral
blood basophils and certain ultrastructural features of normal tissue mast cells62 suggested that the two populations
of metachromatic cells were developmentally related. However, because of nuclear morphology and certain biochemical differences,16,63-67 it is generally accepted that basophils
and mast cells in humans are derived from distinct progenitors. For example, in terms of granule constituents, chymase and tryptase are relatively mast cell – specific markers
at protein and mRNA levels.64,67 Nevertheless, Li et al68
recently found that a metachromatic/FceRI//tryptase//
chymase/ population of cells that resembled basophils in
terms of nuclear morphology developed when human BM
cells from normal donors were cultured for 6 weeks in the
presence of c-kit ligand and conditioned medium derived
from the HBM-M cell line.
In vivo and in vitro studies have shown that granule protease expression in a mouse mast cell is neither fixed nor
predetermined in the BM.7,30,34,35,37,69 The ability of a BALB/
c mouse mast cell to rapidly change its chymase phenotype
is regulated, in part, by a cytokine-dependent, posttranscrip-
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tional mechanism.70 The posttranscriptional findings in the
mouse, coupled with the in vitro findings of Li et al,68 now
raise the possibility that human basophils are more closely
related developmentally to human mast cells than previously
recognized. If the ultimate phenotype of a basophil is not
fixed but is instead regulated by its microenvironment (as
are tissue mast cells in the mouse), the failure of a peripheral
blood human basophil to express high levels of c-kit, tryptase, chymase, and/or MC-CPA protein may simply be a
consequence of the cell failing to localize in the relevant
tissue for an extended period of time.
Whether or not mast cells and basophils in the human are
more closely related than previously thought, it is now clear
that mouse mast cells can possess very different nuclear
profiles. Moreover, the detection in tissues of immature and
mature mouse mast cells with multi-lobular nuclei that express different panels of granule proteases indicates that the
nuclear morphology of a mouse mast cell gives no insight
as to its protease phenotype or its stage of development.
ACKNOWLEDGMENT
The technical assistance of Xuzhen Hu, Kathy M. Grattan, and
Maria Ericsson are gratefully acknowledged.
REFERENCES
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1997 90: 382-390
Mouse Mast Cells That Possess Segmented/Multi-lobular Nuclei
Michael F. Gurish, Daniel S. Friend, Matthew Webster, Namit Ghildyal, Christopher F. Nicodemus and
Richard L. Stevens
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