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 granules. Because mature mast cells possessing multi-lobu-nonsegmented and segmented/multi-lobular nuclei, respec- lar nuclei also were occasionally found in the skeletal muscletively, nuclear morphology has been a major criterion for and jejunum of the BALB/c mouse, the V3 mouse mast cellassessing the lineage of metachromatic cells of hematopoi- line was used to investigate the developmental relationshipetic origin. Immature metachromatic cells with mono- and of mast cells that have very different nuclear structures.multi-lobular nuclei were both obtained when bone marrow After the adoptive transfer of V3 mast cells into BALB/ccells from BALB/c mice were cultured for 3 weeks in the mice, v-abl–immortalized mast cells with mono- and multi-presence of interleukin-3. Analogous to the indigenous ma- lobular nuclei were detected in the lymph nodes and otherture mast cells that reside in the peritoneal cavity and skin, tissues of the mastocytosis mice that expressed c-kit,both populations of in vitro–derived cells expressed the sur- mMCP-5, mMCP-6, and mMC-CPA. These studies indicateface receptor c-kit, the chymase mouse mast cell protease that mouse mast cells can exhibit varied nuclear profiles.(mMCP) 5, the tryptase mMCP-6, and the exopeptidase car- Moreover, the nuclear morphology of this cell type gives noboxypeptidase A (mMC-CPA). Immunogold electron micros- insight as to its protease phenotype or stage of develop-copy confirmed the granule location of mMC-CPA and ment.mMCP-6 in both populations of cells, and cytochemical anal-

q 1997 by The American Society of Hematology.ysis confirmed the presence of chymotryptic enzymes in the

M possess multi-lobular nuclei. While Galli et al12-14 concludedthat this latter population of cells in the cultures probably

AST CELLS, like basophils, possess high-affinity re-ceptors for IgE (FceRI) on their cell surfaces and

contain numerous histamine-rich secretory granules that be- are mast cells, Rottem et al15 concluded that they probablyare basophils.come metachromatic when stained with cationic dyes. Be-

cause antibodies to basophil-specific proteins are not avail- Because mature human mast cells expresses substantiallymore c-kit on their surfaces than mature human basophils,able and because antibodies to mast cell–specific proteins

became available only recently, mouse mast cells and baso- the relative abundance of this plasma membrane receptor isregularly used to distinguish the two populations of cellsphils have been distinguished primarily by ultrastructural

criteria.1-4 Attempts have been made to distinguish basophils in humans.16 Mouse mast cells also express c-kit on theirsurfaces.17 Moreover, they store in their secretory granulesfrom mast cells by the number and morphology of their

granules. However, the observation that the granules in a various combinations of an exopeptidase (termed mousemast cell carboxypeptidase A [mMC-CPA]) and at least ninesingle rat5 or mouse6,7 intraepithelial mast cell can differ

dramatically in their size, maturation, and ultrastructure indi- different serine proteases (termed mouse mast cell protease[mMCP] 1 to mMCP-9).18-29 cDNAs and genes that encodecates that granule ultrastructure, by itself, cannot be used to

definitively phenotype a metachromatic cell. Thus, the nu- all of these granule proteases except mMCP-3 have beencloned and sequenced, and protease-specific antibodies haveclear profile of a cell thought to be in the basophil or mast

cell lineage is presently one of the major structural features been obtained in rabbits that recognize mMCP-1, mMCP-2, mMCP-5, mMCP-6, and mMCP-7.7,30-33 Along with theused to distinguish in vivo–differentiated mouse basophils

and mast cells. cDNAs, the protease-specific antibodies have been usefulfor phenotyping in vivo–differentiated mouse mast cells andImmature populations of FceRI//metachromatic cells are

obtained when progenitor cells from the bone marrow for monitoring the regulated differentiation and maturationof hematopoietic progenitor cells into mast cells in vitro and(mBMMC) and other organs are cultured for 3 to 4 weeks

in the presence of purified interleukin-3 (IL-3), recombinant in vivo. As assessed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE)/immunoblot and N-IL-3, or IL-3–enriched cell-conditioned media.8-11 Most of

the cells in these different cultures are mononuclear but some terminal amino acid analysis of resolved granular protein,RNA blot analysis, and/or immunohistochemical analysis,mast cells have been detected at different tissue sites in

From the Departments of Medicine and Pathology, Harvard Medi- the BALB/c mouse that express various panels of granulecal School; and the Division of Rheumatology, Immunology, and

proteases. For example, the mast cells that increase in num-Allergy and the Department of Pathology, Brigham and Women’sber in the jejunal lamina propria of V3 mastocytosis miceHospital, Boston, MA.and in the jejunal epithelium of Trichinella spiralis–infectedSubmitted November 18, 1996; accepted February 21, 1997.mice at the height of the infection preferentially expressSupported by Grants No. AI-22531, AI-23483, AI-31599, AR-

36308, HL-36110, and HL-48598 from the National Institutes of mMCP-1 and mMCP-2, whereas the mast cells in the jejunalHealth. muscle of these mice preferentially express mMCP-4,

Address reprint requests to Richard L. Stevens, PhD, Harvard mMCP-5, mMCP-6, and mMC-CPA.7,19,21,30,34,35 Immunohis-Medical School, Seeley G. Mudd Bldg, Room 617, 250 Longwood tochemical analysis of the mastocytosis that develops in theAve, Boston, MA 02115. jejunum during helminth infection7 or in different tissues

The publication costs of this article were defrayed in part by pageof BALB/c mice after the adoptive transfer of the v-abl–charge payment. This article must therefore be hereby markedimmortalized V3 mast cell line34 indicate that the protease‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely tophenotype of a mast cell can be modulated and is not prede-indicate this fact.termined in the BM.q 1997 by The American Society of Hematology.

0006-4971/97/9001-0002$3.00/0 As assessed by RNA blot analysis, mBMMC cultures con-

382 Blood, Vol 90, No 1 (July 1), 1997: pp 382-390

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MOUSE MAST CELLS WITH DIFFERENT NUCLEAR PROFILES 383

Polyclonal antibodies to the coupled peptide were raised in Newtain high levels of the transcripts that encode FceRI, c-kit,Zealand White rabbits according to standard immunization protocolsmMCP-5, mMCP-6, mMCP-7, and mMC-CPA.17,18,23,24,36-38

in which each rabbit was injected intramuscularly (IM) with anMost of these transcripts are readily detected 1 to 2 weeksemulsion consisting of 0.5 mL of the coupled peptide (É500 mg)after BM cells are exposed to IL-3–enriched medium. Be-mixed with an equal volume of TiterMax synthetic adjuvant (CyRxcause mature, cutaneous mast cells of the BALB/c mouseCrop, Norcross, CA). The immunized animals received booster in-

express FceRI, c-kit, mMCP-5, mMCP-6, mMCP-7, and jections IM monthly, and their sera were collected at 2-week inter-mMC-CPA, we and others have concluded that most, if not vals over a number of months. An enzyme-linked immunosorbentall, of the cells in the 3- to 4-week-old cultures belong to assay (ELISA) was used to evaluate the presence of specific antibod-the mast cell lineage. In the present study, we show that ies in the sera, and antibodies that exhibited strong reactivities

against the immunizing peptide were purified with a peptide-affinitythe metachromatic cells in the 3- to 4-week-old BALB/ccolumn, as previously described for anti–mMCP-2 Ig and anti–mBMMC cultures that contain multi-lobular nuclei expressmMCP-5 Ig.30,31

c-kit, mMCP-5, mMCP-6, and mMC-CPA, as do the pre-The first 10 residues of the immunizing peptide also are found indominant mononuclear mBMMC. We also show that mast

the corresponding region of rat MC-CPA.41 Because considerablycells with multi-lobular nuclei can be found occasionally inmore MC-CPA containing mast cells can be purified from a normalthe skeletal muscle of normal mice, the jejunum of helminth-rat than from a normal mouse, the native exopeptidase was purified

infected mice, and in various tissues of V3 mastocytosis from rat serosal mast cells on a potato carboxypeptidase-inhibitormice. Thus, in the mouse, mast cells can exhibit very differ- Sepharose column18,41 to evaluate whether or not mMC-CPA and ratent nuclear profiles. MC-CPA were both recognized by the antipeptide antibody. When

the wells of the microtiter plates were coated with purified rat MC-MATERIALS AND METHODS CPA and incubated with the purified antibody, the antibody avidly

bound to purified rat MC-CPA in the ELISA at a greater than 1,000-In vitro– and in vivo–derived mast cells. mBMMC were ob-fold dilution (data not shown). As assessed by SDS-PAGE/immu-tained by culturing BALB/c mouse BM cells for 3 to 4 weeks innoblot analysis, anti–mMC-CPA Ig identified an É36-kD protein inenriched medium (RPMI-1640 supplemented with 10% fetal calfwhole cell lysates and granule-enriched preparations from mBMMC,serum [FCS] [GIBCO, Grand Island, NY], 2 mmol/L L-glutamine,mouse KiSV-MC, and purified rat serosal mast cells. In contrast, it0.1 mmol/L nonessential amino acids, 100 U/mL penicillin, and 100failed to recognize any protein in the lysates of mouse fibroblasts.mg/mL streptomycin) containing either 50% WEHI-3 cell (line TIB-The É36-kD protein was not recognized by preimmune sera, and68; American Type Culture Collection [ATCC], Rockville, MD)the reactivity of anti–mMC-CPA Ig was abolished by preincubationconditioned medium or recombinant IL-3 in a humidified atmospherewith the immunizing peptide but not with the synthetic peptide thatat 377C and 5% CO2.10 V3 mastocytosis mice were generated bycorresponds to residues 276 to 298 of mMC-CPA.injecting É106 cultured V3 mast cells intravenously into the tail

Histochemistry, enzyme cytochemistry, and immunohistochemis-veins of each 6-week-old BALB/c mouse (The Jackson Laboratory,try. For histological examination, cytocentrifuge preparations ofBar Harbor, ME).34 Previous DNA blot analysis revealed that theBALB/c mBMMC were air dried and stained for 20 seconds inV3 mast cell line contains a single copy of the pGDv-abl provirus ina 5% ethanolic solution of methylene blue.47 Serial 1.5-mm thickits genome. Because the oncogene was inserted at a site distinctglycolmethacrylate sections of lymph node tissue from V3 masto-from that in the V7 mast cell line and the V8 lymphoblastic cellcytosis mice, tongue skeletal muscle from normal BALB/c mice,line, the V3 mast cell line is of clonal origin. V3 mastocytosis miceand jejunum from T spiralis–infected BALB/c mice were air driedwere used 2 to 4 weeks after their adoptive transfer. Mouse Kirstenand incubated sequentially with double-strength hematoxylin for 2sarcoma virus–immortalized mast cells (KiSV-MC) were main-minutes, 1% aqueous eosin Y for 15 minutes, azure II for 1 minute,tained in 100% enriched medium as previously described.39 Serosaland then with ethylene glycol monomethyl ether for 5 seconds.48

mast cells were obtained under sterile conditions from Sprague-Individual mast cells containing chymotryptic activities were identi-Dawley rats (The Jackson Laboratory) by peritoneal lavage withfied in tissue sections by the method of Leder.49 mBMMC and fixedTyrode’s buffer containing 5% FCS, 100 U/mL penicillin, 100 mg/tissue sections were incubated at 307C for 1 hour with a solutionmL streptomycin, and 20 mg/mL gentamicin, and then were enrichedcontaining naphthol AS-D chloroacetate; the resulting preparationsto 90% to 99% purity by density-gradient centrifugation in 25%were rinsed and counterstained with hematoxylin. Mast cells con-metrizamide.40 Swiss albino mouse 3T3 fibroblasts (line CCL-92;taining tryptic activities were identified in tissue sections by theATCC) were grown in enriched medium. BALB/c mice were in-cytochemistry method of Osman et al,50 with Z-Ala-Ala-Lys-4-me-fected with T spiralis as described to evaluate the mast cells in thethoxy-2-naphthylamide (AAK) used as the substrate.jejunal epithelium at the height of the infection.7,30,35

Light and electron microscopic immunohistochemistry were per-Production of a mMC-CPA-specific antibody. The deducedformed as described.7,30-34,51 For light microscopy, collected tissuesamino acid sequences of mMC-CPA18 and rat MC-CPA41 were com-were fixed for 4 hours at room temperature in 4% paraformaldehydepared with that of other metallocarboxypeptidases42-46 to define po-in 0.1 mol/L sodium phosphate, pH 7.6. Alternatively, selected sam-tential antigenic regions. As assessed by the Protein Identificationples were fixed in Methacarn or Carnoy’s solution. PreparationsResource database of the National Biomedical Foundationwere washed twice with PBS containing 2% dimethyl sulfoxide and(Bethesda, MD), residues 146 to 157 are not present in any otherwere suspended in 50 mmol/L NH4Cl overnight at 47C. The speci-carboxypeptidase or protein. Because of its novelty, hydrophilicity,mens were dehydrated and embedded in accordance with the JB-4and potential antigenicity, we speculated that antibodies directedembedding kit instructions from Polysciences Inc (Warrington, PA).against the peptide Asp-Val-Ser-Trp-Ser-Ser-Pro-Asn-Thr-Asp-LysFor electron microscopy, tissues were fixed in 0.25% glutaraldehydewould be specific for mMC-CPA and rat MC-CPA. Thus, a syntheticand 4% paraformaldehyde and then frozen in liquid nitrogen. Tissuepeptide (3 mg) corresponding to this sequence was suspended in 5sections and/or cells were stained with either rat monoclonal anti-mL of 10 mmol/L phosphate-buffered saline (PBS) containing 5 mgmouse c-kit Ig (PharMingen, San Diego, CA) followed by goat-keyhole limpet hemocyanin (Sigma Chemical Co, St Louis, MO),antirat IgG (PharMingen) or with affinity-purified rabbit anti–and coupling was performed in the presence of 0.25% glutaraldehydemMCP-2 Ig, anti–mMCP-5 Ig, anti–mMCP-6 Ig, or anti–mMC-(Sigma) at 47C. Noncoupled peptide was removed by dialyzing the

reaction mixture against PBS using a membrane with a 12-kD cutoff. CPA Ig followed by goat-antirabbit IgG.30-32

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GURISH ET AL384

RESULTS of the v-abl–immortalized V3 mast cell line into this animal.Most of the methylene blue//toluidine blue/ V3 mast cellsHistochemistry, enzyme cytochemistry, and immunohisto-found in the mesenteric lymph nodes of these animals ap-chemistry of mBMMC that have different nuclear profiles.peared to be mononuclear. However, occasionally a methyl-Metachromatic mBMMC were obtained that possessed dif-ene blue/ V3 mast cell could be detected that appeared toferent nuclear morphologies when BALB/c mouse BM cellscontain a multi-lobular nucleus (Fig 5, see page 385). Al-were cultured for greater than 3 weeks in the presence ofthough V3 mast cells with multi-lobular nuclei were mostIL-3 (Fig 1). When ú300 BALB/c mBMMC were analyzedreadily found in the mesenteric lymph nodes, similar cellsin each of three experiments, 8%, 14%, and 15% of the cellswere observed in liver, spleen, and intestine of V3 masto-in the culture had multi-lobular nuclei. Regardless of whethercytosis mice (data not shown). Analysis of tissue sectionsthese mBMMC were heavily or lightly granulated in termsof varied organs of three different animals showed that 0.7%of their metachromasia when stained with toluidine blue,of the 1,400 V3-MC examined contained multi-lobular nu-

they phenotypically resembled the mononuclear cells in theclei. Because sections are analyzed rather than isolated whole

culture. Although these cells contained low levels of tryptasecells, this percentage of V3 mast cells in tissues that have

activity (data not shown), they contained substantial amountsmulti-lobular nuclei is an underestimation. Nevertheless, like

of chloroacetate esterase (chymase) activity (Fig 1). As as-mononuclear V3 mast cells, these cells contained chloroace-

sessed immunohistochemically at low magnification, bothtate esterase and AAK (tryptase) activities. As assessed im-

populations of mBMMC expressed c-kit, mMCP-5, mMCP-munohistochemically, both populations of V3 mast cells in

6, and mMC-CPA. When the mBMMC with mono- andlymph nodes expressed c-kit, mMCP-5, mMCP-6, and

multi-lobular nuclei were examined with transmission elec- mMC-CPA.tron microscopy and immunogold cytochemistry, most ofthe secretory granules in both populations of cells were rela- DISCUSSIONtively immature (Figs 2 and 3). Nevertheless, some cells in

Although in vitro–differentiated mast cells possessingboth cultures possessed electron dense granules and thesemulti-lobular nuclei have been found occasionally in culture-granules were stained by anti–mMC-CPA Ig and anti–derived mouse mast cells,12-15 the consensus opinion is thatmMCP-6 Ig (Fig 2). The chromatin was condensed to thein vivo–differentiated mast cells in this species are primarilysame extent on the nuclear membrane in both populationsmononuclear. Thus, nuclear morphology is one of the criteriaof mBMMC. Analysis of four different levels through aroutinely used to distinguish mast cells from basophils insingle mBMMC showed that the apparent segmented naturethe mouse. We now show that it is possible to obtain mouseof the nucleus was not a consequence of the section analyzedmast cells in vitro and in vivo that possess segmented/multi-

(Fig 3). Further ultrastructural analysis revealed that the var-lobular nuclei, and that an immature mouse mast cell-com-

ied lobes of the nucleus can be joined by a thin connection.mitted line can give rise to mast cells in vivo with very

Histochemistry, enzyme cytochemistry, and immunohisto-different nuclear morphologies.

chemistry of mast cells in normal BALB/c mice and in V3Mast cells and basophils in humans are granule-containing

mastocytosis mice that possess multi-lobular nuclei. Be- hematopoietic cells that become metachromatic when stainedcause of the thinness of the sections, it is not always possible with cationic dyes. mBMMC and all tissue-localized mouseto determine the nuclear configuration of a tissue-localized mast cells that have been analyzed so far contain largemast cell precisely. Nevertheless, metachromatic//chloroac- amounts of at least one chymase in their granules, and mostetate esterase/ cells that appear to possess multi-lobular nu- of these mast cells also express at least one tryptase. Thus,clei were occasionally seen in the skeletal muscle of the even when tissue sections are fixed, dehydrated, and embed-tongue of normal BALB/c mice and the jejunal epithelium ded, mast cells are readily recognized in mouse tissuesof T spiralis–infected BALB/c mice (Fig 4, see page 385). because of their pronounced tryptic and chymotryptic activi-The observation that the individual nuclear lobes in these ties.7,49,50 With enzyme cytochemistry and immunohisto-cells are smaller in size than the intact nucleus of a mononu- chemistry, it was discovered that the toluidine blue//methyl-clear mast cell in the same section suggests that the nuclear ene blue/ cells with multi-lobular nuclei in mBMMClobes represent portions of the same nucleus rather than cultures (Fig 1) and in the mesenteric lymph nodes of V3distinct nuclei. Analogous to the mononuclear mast cells at mastocytosis mice (Fig 5) all contain abundant levels ofthese two tissue sites, the cells with multi-lobular nuclei in mMCP-5 and mMCP-6 in their granules. Although mMCP-the muscle could be stained by anti–mMC-CPA Ig but not 5 and mMCP-6 are both serine proteases, they are membersanti–mMCP-2 Ig, whereas those in the jejunum could be of two distinct families whose genes reside on chromosomesstained by anti–mMCP-2 Ig but not anti–mMC-CPA Ig 14 and 17, respectively. Thus, these immunohistochemical(Fig 4). and cytochemical findings suggested that the metachromatic

V3 mastocytosis mice were used to determine if an imma- cells in the analyzed cultures with multi-lobular nuclei areture mast cell–committed line could give rise to mast cells mast cells, as Galli et al12-14 concluded in their ultrastructuralin vivo with very different nuclear profiles. When greater analysis of comparable cultured cells. Dvorak et al52 notedthan 100 V3 mast cells were analyzed in each of four sepa- the transient appearance of metachromatic cells very earlyrate experiments, 1%, 2%, 3%, and 4% of the cells in the in mBMMC cultures which morphologically resemble baso-culture contained multi-lobular nuclei. As obtained pre- phils. Because these in vitro–derived cells were not analyzedviously,34 a prominent mastocytosis developed in a number in the present investigation, we cannot support or dispute

the concept that basophils exist in the mouse as a develop-of tissues of the BALB/c mouse after the adoptive transfer

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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 possessmono- (a through g) and multi-lobular (h through n) nuclei. Cytocentrifuge preparations of cells were evaluated for toluidine blue reactivegranules (a, b, h, i) that contained chloroacetate esterase enzymatic activity (d, k), mMCP-5 protein (e, l), mMC-CPA protein (f, m), or mMCP-6 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 mus-cle of a normal BALB/c mouse (a, b) and the jejunal epithelium of a T spiralis–infectedBALB/c mouse (c, d). Serial sections were evaluated for chloroacetate esterase enzy-matic 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 ester-ase" mast cells depicted in this panel exhibit a blue color. The red reaction producton the brush border of the villi (d) is due to endogenous intestinal alkaline phospha-tase. The presence of this product indicates that the color substrate was active inthe immunohistochemical reaction.

Fig 5. Histochemistry, cytochemistry, and immunohistochemistry of V3 mast cells in the lymph nodes of mastocytosis mice. The V3 mastcells in the various tissue sections that have multi-lobular nuclei were evaluated for the expression of c-kit (b) and for methylene blue reactivegranules (a) that contain chloroacetate esterase enzymatic activity (c), mMCP-5 protein (d), mMC-CPA protein (e), mMCP-6 protein (f), andAAK enzymatic activity (g).

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GURISH ET AL386

Fig 2. Immunoelectron microscopy of mBMMC with mono- (a) and multi-lobular (b through e) nuclei. Most cells in the culture havenumerous multi-vesicular structures in their granules (V). However, in terms of their electron density, some of the granules (D) in thesecultured 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 sin G/ subset of mast cells that reside in the skin and certainother tissue sites.55 Basophils in the peripheral blood of hu-vitro– and in vivo–differentiated mouse mast cells can have

different nuclear profiles now indicates that the lineage of a mans do not contain detectable amounts of this exopeptidasein their granules.55 In mice and rats,18,20,41 MC-CPA is ametachromatic mouse cell cannot be determined conclu-

sively by the shape of the cell’s nucleus alone. major granule constituent of the serosal mast cells isolatedfrom the peritoneal cavity. The ear and skin of BALB/cIn contrast to their granule serine proteases, only one car-

boxypeptidase has been identified in the granules of mouse, mice also contain high steady-state levels of the mMC-CPAtranscript,33 and in situ hybridization analysis56 has shownrat, and human mast cells.18,41,53,54 In humans, MC-CPA pro-

tein is preferentially found in the tryptase//chymase//cathep- 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 levelsthrough a single mBMMC are analyzed in panels (a) through(d). The electron micrograph depicted in panel (e) shows a thinconnection joining the two lobes of the nucleus of anothermBMMC. N, lobes of the nucleus; D, relatively dense granules;V, relatively immature granules containing predominantlymulti-vesicular structures.

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GURISH ET AL388

tissue sites express mMC-CPA. Because high levels of the tional mechanism.70 The posttranscriptional findings in themouse, coupled with the in vitro findings of Li et al,68 nowmMC-CPA transcript are present in the BALB/c mBMMC

cultures,18 it has been presumed that the prominent mononu- raise the possibility that human basophils are more closelyrelated developmentally to human mast cells than previouslyclear cells in these cultures store the exopeptidase in their

granules. Therefore, whether the mMCP-5/ cells in V3 mas- recognized. If the ultimate phenotype of a basophil is notfixed but is instead regulated by its microenvironment (astocytosis mice and in the mBMMC cultures that contain

multi-lobular nuclei also store mMC-CPA protein in their are tissue mast cells in the mouse), the failure of a peripheralblood human basophil to express high levels of c-kit, tryp-granules was of interest. To this end, an mMC-CPA–specific

antibody was derived with an antipeptide approach. When tase, chymase, and/or MC-CPA protein may simply be aconsequence of the cell failing to localize in the relevantimmunohistochemistry was performed with anti–MC-CPA

Ig, the granules of both populations of mBMMC in the cul- tissue for an extended period of time.Whether or not mast cells and basophils in the human areture were strongly reactive (Figs 1 and 2), as were the gran-

ules of mast cells in the skeletal muscle of the normal BALB/ more closely related than previously thought, it is now clearthat mouse mast cells can possess very different nuclearc mouse (Fig 4) and the V3 mast cells in the lymph node

(Fig 5) that possessed multi-lobular nuclei. Analogous to profiles. Moreover, the detection in tissues of immature andmature mouse mast cells with multi-lobular nuclei that ex-mature mast cells,17 these V3 mast cells (Fig 5) and mBMMC

(Fig 1) also expressed c-kit. press different panels of granule proteases indicates that thenuclear morphology of a mouse mast cell gives no insightBecause a higher percentage of the cells in cultures of

mBMMC15 and human cord blood/BM57-61 have multi-lobu- as to its protease phenotype or its stage of development.lar nuclei when hematopoietic progenitor cells are culturedin the presence of IL-3 rather than in c-kit ligand, IL-3 ACKNOWLEDGMENTseems to promote nuclear segmentation. What other factors The technical assistance of Xuzhen Hu, Kathy M. Grattan, andare involved and the functional significance of the nuclear Maria Ericsson are gratefully acknowledged.changes remain to be ascertained. Nuclear segmentation ofneutrophils and eosinophils is a late event in the maturation REFERENCESprocesses of these granulocytes. Nevertheless, the nuclear

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factor-dependent culture technique. J Immunol 127:788, 1981recently found that a metachromatic/FceRI//tryptase// 9. Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD,

Fitch F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynskichymase/ population of cells that resembled basophils inE, Dy M, Lebel B: Biologic properties of homogeneous interleukinterms of nuclear morphology developed when human BM3. I. Demonstration of WEHI-3 growth factor activity, mast cellcells from normal donors were cultured for 6 weeks in thegrowth factor activity, P cell-stimulating factor activity, colony-stim-presence of c-kit ligand and conditioned medium derivedulating factor activity, and histamine-producing cell-stimulating fac-from the HBM-M cell line.tor activity. J Immunol 131:282, 1983

In vivo and in vitro studies have shown that granule prote- 10. Razin E, Ihle JN, Seldin D, Mencia-Huerta J-M, Katz HR,ase expression in a mouse mast cell is neither fixed nor LeBlanc PA, Hein A, Caulfield JP, Austen KF, Stevens RL: Interleu-predetermined in the BM.7,30,34,35,37,69 The ability of a BALB/ kin 3: A differentiation and growth factor for the mouse mast cellc mouse mast cell to rapidly change its chymase phenotype that contains chondroitin sulfate E proteoglycan. J Immunol

132:1479, 1984is regulated, in part, by a cytokine-dependent, posttranscrip-

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11. Nabel G, Galli SJ, Dvorak AM, Dvorak HF, Cantor H: In- unusual characteristics by multiple sequence alignment and molecu-lar modeling. Protein Sci 1:370, 1992ducer T lymphocytes synthesize a factor that stimulates proliferation

of cloned mast cells. Nature 291:332, 1981 28. Chu W, Johnson DA, Musich PR: Molecular cloning andcharacterization of mouse mast cell chymases. Biochim Biophys12. Galli SJ, Dvorak AM, Marcum JA, Ishizaka T, Nabel G, Der

Simonian H, Pyne K, Goldin JM, Rosenberg RD, Cantor H, Dvorak Acta 1121:83, 199229. Hunt JE, Matsumoto R, Austen KF, Stechshulte S, GhildyalHF: Mast cell clones: A model for the analysis of cellular maturation.

J Cell Biol 95:435, 1982 N, Gurish MF, Webster M, Stevens RL: Molecular cloning of anovel mouse gene that resides at the chromosome 14 serine protease13. Nakano T, Sonoda T, Hayashi C, Yamatodani A, Kanayama

Y, Yamamura T, Asai H, Yonezawa T, Kitamura Y, Galli SJ: Fate complex: Its expression during helminth infection. FASEB J 9:A802,1995 (abstr)of bone marrow-derived cultured mast cells after intracutaneous,

intraperitoneal, and intravenous transfer into genetically mast cell- 30. Ghildyal N, Friend DS, Nicodemus CF, Austen KF, StevensRL: Reversible expression of mouse mast cell protease 2 mRNA anddeficient W/Wv mice. J Exp Med 162:1025, 1985

14. Dvorak AM, Wiberg L, Monahan-Earley RA, Galli SJ: A protein in cultured mast cells exposed to interleukin 10. J Immunol151:3206, 1993simple technique to facilitate the ultrastructural analysis of cells in

soft agar cultures systems: Demonstration of development in vitro 31. McNeil HP, Frenkel DP, Austen KF, Friend DS, Stevens RL:Translation and granule localization of mouse mast cell protease-5:of morphologically mature mast cells and phagocytic macrophages

from the bone marrow cells of genetically mast cell-deficient W/Wv Immunodetection with specific antipeptide Ig. J Immunol 149:2466,1992or congenic normal mice. Lab Invest 62:774, 1990

15. Rottem M, Goff JP, Albert JP, Metcalfe DD: The effects of 32. Ghildyal N, Friend DS, Stevens RL, Austen KF, Huang C,Penrose JF, Sali A, Gurish MF: Fate of two mast cell tryptasesstem cell factor on the ultrastructure of FceRI/ cells developing in

IL-3-dependent murine bone marrow-derived cell cultures. J Immu- in V3 mastocytosis and normal BALB/c mice undergoing passivesystemic anaphylaxis. Prolonged retention of exocytosed mMCP-6nol 151:4950, 1993

16. Valent P, Ashman LK, Hinterberger W, Eckersberger F, Maj- in connective tissues and rapid accumulation of enzymatically activemMCP-7 in the blood. J Exp Med 184:1061, 1996dic O, Lechner K, Bettelheim P: Mast cell typing: Demonstration

of a distinct hematopoietic cell type and evidence for immunopheno- 33. Ghildyal N, Friend DS, Freeland R, Austen KF, McNeil HP,Schiller V, Stevens RL: Lack of expression of the tryptase mousetypic relationship to mononuclear phagocytes. Blood 73:1778, 1989

17. Galli SJ, Zsebo KM, Geissler EN: The kit ligand, stem cell mast cell protease 7 in mast cells of the C57BL/6J mouse. J Immunol153:2624, 1994factor. Adv Immunol 55:1, 1994

18. Reynolds DS, Stevens RL, Gurley DS, Lane WS, Austen KF, 34. Gurish MF, Pear WS, Stevens RL, Scott ML, Sokol K, Ghil-dyal N, Webster MJ, Xu X, Austen KF, Baltimore D, Friend DS:Serafin WE: Isolation and molecular cloning of mast cell carboxy-

peptidase A: A novel member of the carboxypeptidase gene family. Tissue-regulated differentiation and maturation of a v-abl-immortal-ized mast cell-committed progenitor. Immunity 3:175, 1995J Biol Chem 264:20094, 1989

19. Le Trong H, Newlands GFJ, Miller HRP, Charbonneau H, 35. Ghildyal N, McNeil HP, Stechschulte S, Austen KF, Sil-berstein D, Gurish MF, Somerville LL, Stevens RL: IL-10 inducesNeurath H, Woodbury RG: Amino acid sequence of a mouse muco-

sal mast cell protease. Biochemistry 28:391, 1989 transcription of the gene for mouse mast cell protease-1, a serineprotease preferentially expressed in mucosal mast cells of Trichinella20. Reynolds DS, Stevens RL, Lane WS, Carr MH, Austen KF,

Serafin WE: Different mouse mast cell populations express various spiralis-infected mice. J Immunol 149:2123, 199236. Thompson HL, Metcalfe DD, Kinet J-P: Early expression ofcombinations of at least six distinct mast cell serine proteases. Proc

Natl Acad Sci USA 87:3230, 1990 high-affinity receptor for immunoglobulin E (FceRI) during differen-tiation of mouse mast cells and human basophils. J Clin Invest21. Serafin WE, Reynolds DS, Rogelj S, Lane WS, Conder GA,

Johnson SS, Austen KF, Stevens RL: Identification and molecular 85:1227, 199037. Gurish MF, Ghildyal N, McNeil HP, Austen KF, Gillis S,cloning of a novel mouse mucosal mast cell serine protease. J Biol

Chem 265:423, 1990 Stevens RL: Differential expression of secretory granule proteasesin mouse mast cells exposed to interleukin 3 and c-kit ligand. J Exp22. Serafin WE, Sullivan TP, Conder GA, Ebrahimi A, Marcham

P, Johnson SS, Austen KF, Reynolds, DS: Cloning of the cDNA Med 175:1003, 199238. Welham MJ, Schrader JW: Modulation of c-kit mRNA andand gene for mouse mast cell protease 4. Demonstration of its late

transcription in mast cell subclasses and analysis of its homology to protein by hemopoietic growth factors. Mol Cell Biol 11:2901, 199139. Reynolds DS, Serafin WE, Faller DV, Wall DA, Abbas AK,subclass-specific neutral proteases of the mouse and rat. J Biol Chem

266:1934, 1991 Dvorak AM, Austen KF, Stevens RL: Immortalization of murineconnective tissue-type mast cells at multiple stages of their differen-23. McNeil HP, Austen KF, Somerville LL, Gurish MF, Stevens

RL: Molecular cloning of the mouse mast cell protease-5 gene. A tiation by coculture of splenocytes with fibroblasts that produce Kir-sten sarcoma virus. J Biol Chem 263:12783, 1988novel secretory granule protease expressed early in the differentia-

tion of serosal mast cells. J Biol Chem 266:20316, 1991 40. Yurt RW, Leid RW Jr, Austen KF, Silbert JE: Native heparinfrom rat peritoneal mast cells. J Biol Chem 252:518, 197724. Reynolds DS, Gurley DS, Austen KF, Serafin WE: Cloning

of the cDNA and gene of mouse mast cell protease-6. Transcription 41. Cole KR, Kumar S, LeTrong H, Woodbury RG, Walsh KA,Neurath H: Rat mast cell carboxypeptidase: Amino acid sequenceby progenitor mast cells and mast cells of the connective tissue

subclass. J Biol Chem 266:3847, 1991 and evidence of enzyme activity within mast cell granules. Biochem-istry 30:648, 199125. Huang R, Blom T, Hellman L: Cloning and structural analysis

of mMCP-1, mMCP-4 and mMCP-5, three mouse mast cell-specific 42. Quinto C, Quiroga M, Swain WF, Nikovits WC Jr, StandringDN, Pictet RL, Valenzuela P, Rutter WJ: Rat preprocarboxypepti-serine proteases. Eur J Immunol 21:1611, 1991

26. McNeil HP, Reynolds DS, Schiller V, Ghildyal N, Gurley dase A: cDNA sequence and preliminary characterization of thegene. Proc Natl Acad Sci USA 79:31, 1982DS, Austen KF, Stevens RL: Isolation, characterization, and tran-

scription of the gene encoding mouse mast cell protease 7. Proc Natl 43. Gardell SJ, Craik CS, Clauser E, Goldsmith EJ, Stewart C-B, Graf M, Rutter WJ: A novel rat carboxypeptidase, CA2: Charac-Acad Sci USA 89:11174, 1992

27. Johnson DA, Barton GJ: Mast cell tryptases: Examination of terization, molecular cloning, and evolutionary implications on sub-

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44. Clauser E, Gardell SJ, Craik CS, MacDonald RJ, Rutter WJ: 198359. Valent P, Schmidt G, Besemer J, Mayer P, Zenke G, LiehlStructural characterization of the rat carboxypeptidase A1 and B

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preprocarboxypeptidase H: Cloning, characterization, and sequence 198960. Kirshenbaum AS, Goff JP, Dreskin SC, Irani A-M, Schwartzof the cDNA and regulation of the mRNA by corticotropin-releasing

factor. J Biol Chem 264:5988, 1989 LB, Metcalfe DD: IL-3-dependent growth of basophil-like cells andmast-like cells from human bone marrow. J Immunol 142:2424,46. Fricker LD, Adelman JP, Douglass J, Thompson RC, von

Strandmann RP, Hutton J: Isolation and sequence analysis of cDNA 198961. Dvorak AM, Saito H, Estrella P, Kissell S, Arai N, Ishizakafor rat carboxypeptidase E [EC 3.4.17.10], a neuropeptide processing

enzyme. Mol Endocrinol 3:666, 1989 T: Ultrastructure of eosinophils and basophils stimulated to developin human cord blood mononuclear cell cultures containing recombi-47. Matin R, Tam EK, Nadel JA, Caughey GH: Distribution of

chymase-containing mast cells in human bronchi. J Histochem Cyto- nant human interleukin-5 or interleukin-3. Lab Invest 61:116, 198962. Zucker-Franklin D: Ultrastructural evidence for the commonchem 40:781, 1992

48. Beckstead JH, Halverson PS, Ries CA, Bainton DF: Enzyme origin of human mast cells and basophils. Blood 56:534, 198063. Leiferman KM, Gleich GJ, Kephart GM, Haugen HS, Hisa-histochemistry and immunohistochemistry on biopsy specimens of

pathologic human bone marrow. Blood 57:1088, 1981 matsu K-I, Proud D, Lichtenstein LM, Ackerman SJ: Differencesbetween basophils and mast cells: Failure to detect Charcot-Leyden49. Leder LD: The chloroacetate esterase reaction. A useful

means of histological diagnosis of hematological disorders from crystal protein (lysophospholipase) and eosinophil granule majorbasic protein in human mast cells. J Immunol 136:852, 1986paraffin sections of skin. Am J Dermatopathol 1:39, 1979

50. Osman IA, Garrett JR, Smith RE: Enzyme histochemical dis- 64. Castells MC, Irani AM, Schwartz LB: Evaluation of humanperipheral blood leukocytes for mast cell tryptase. J Immunolcrimination between tryptase and chymase in mast cells of human

gut. J Histochem Cytochem 37:415, 1989 138:2184, 198765. Stain C, Stockinger H, Scharf M, Jager U, Gossinger H,51. Boenisch T, Farmilo AJ, Stead RH: in Naish SJ (ed): Immu-

nochemical Staining Methods Handbook. Carpinteria, CA, DAKO, Lechner K, Bettelheim P: Human blood basophils display a uniquephenotype including activation linked membrane structures. Blood1989, p 11

52. Dvorak AM, Seder RA, Paul WE, Morgan ES, Galli SJ: 70:1872, 198766. Kepley CL, Craig SS, Schwartz LB: Identification and partialEffects of interleukin-3, with or without the c-kit ligand stem cell

factor, on the survival and cytoplasmic granule formation of mouse characterization of a unique marker for human basophils. J Immunol154:6548, 1995basophils and mast cells in vitro. Am J Pathol 144:160, 1994

53. Goldstein SM, Kaempfer CE, Kealey JT, Wintroub BU: Hu- 67. Xia H-Z, Kepley CL, Sakai K, Chelliah J, Irani AMA,Schwartz LB: Quantitation of tryptase, chymase, FceRIa, andman mast cell carboxypeptidase. Purification and characterization. J

Clin Invest 83:1630, 1989 FceRIg mRNAs in human mast cells and basophils by competitivereverse transcription polymerase chain reaction. J Immunol54. Reynolds DS, Gurley DS, Stevens RL, Sugarbaker DJ, Austen

KF, Serafin WE: Cloning of cDNAs that encode human mast cell 154:5472, 199568. Li L, Macpherson JL, Adelstein S, Bunn CL, Atkinson K,carboxypeptidase A, and comparison of the protein with mouse mast

cell carboxypeptidase A and rat pancreatic carboxypeptidases. Proc Phadke K, Krilis SA: Conditioned medium from a cell stain derivedfrom a patient with mastocytosis induces preferential developmentNatl Acad Sci USA 86:9480, 1989

55. Irani AMA, Goldstein SM, Wintroub BU, Bradford T, of cells that possess high affinity IgE receptors and the granuleprotease phenotype of mature cutaneous mast cells. J Biol ChemSchwartz LB: Human mast cell carboxypeptidase. Selective localiza-

tion to MCTC cells. J Immunol 147:247, 1991 270:2258, 199569. Eklund KK, Ghildyal N, Austen KF, Stevens RL: Induction56. Isozaki K, Tsujimura T, Nomura S, Morii E, Koshimizu U,

Nishimune Y, Kitamura Y: Cell type-specific deficiency of c-kit by IL-9 and suppression by IL-3 and IL-4 of the levels of chromo-some 14-derived transcripts that encode late-expressed mouse mastgene expression in mutant mice of mi/mi genotype. Am J Pathol

145:827, 1994 cell proteases. J Immunol 151:4266, 199370. Xia Z, Ghildyal N, Austen KF, Stevens RL: Post-transcrip-57. Razin E, Rifkind AB, Cordon-Cardo C, Good RA: Selective

growth of a population of human basophil cells in vitro. Proc Natl tional regulation of chymase expression in mast cells. A cytokine-dependent mechanism for controlling the expression of granule neu-Acad Sci USA 78:5793, 1981

58. Ogawa M, Nakahata T, Leary AG, Sterk AR, Ishizaka K, tral proteases of hematopoietic cells. J Biol Chem 271:8747, 1996

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1997 90: 382-390  

Richard L. StevensMichael F. Gurish, Daniel S. Friend, Matthew Webster, Namit Ghildyal, Christopher F. Nicodemus and Mouse Mast Cells That Possess Segmented/Multi-lobular Nuclei 

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