J Clin Pathol 1984;37:1249-1255

Human mast cells detected by monoclonal antibodiesEF RIMMER, C TURBERVILLE, MA HORTON

From the Haemopoiesis Research Group, Department ofHaematology, St Bartholomew's Hospital MedicalCollege, London ECIA 7BE

SUMMARY We report the establishment of seven mouse-mouse hybridoma cell lines secretingmonoclonal antibodies with specificity for granule components of all human mast cells. Reactivityis directed against a molecule which is also found intracytoplasmically in human mature smallintestinal enterocytes, liver parenchymal cells, and kidney proximal tubule epithelial cells. Noreactivity of these antibodies was found with any other human or animal cell type examined. Inparticular, the antibodies did not react with basophils or other haemopoietic cell types. This studyshows the potential of specific monoclonal antibodies as a tool for identifying and enumeratinginfiltrating mast cells in tissues. Such antibodies should be of value in investigations into the roleof the mast cell in immunological reactions and hypersensitivity diseases.

The mast cell is the most important cell type par-ticipating in type I (reaginic) hypersensitivity reac-tions; mast cells also play a subsidiary role in thegeneration of hypersensitivity, and associated tissuedamage, in some other immune phenomena. Localinfiltration by, or proliferation of, mast cells is thusassociated, for example, with parasitic infection,'with inflammatory diseases of skin2 and gastrointes-tinal tract,3 and with some carcinomas.4 Theimmunological properties and clinical importance ofmast cells have been reviewed extensively else-where.56

Central to further understanding of the role ofmast cells in disease is the ability to identify andhence to enumerate them accurately in tissue. Mastcells show a characteristic metachromatic stainingreaction with dilute acidic toluidine blue and this isthe classic method for their demonstration. Afterfixation in standard formalin based histologicalfixatives, however, a substantial proportion ofmucosal mast cells, in particular, fail to stain meta-chromatically with toluidine blue7; this problem iscompounded in the presence of large numbers ofdegranulated mast cells, which may be difficult toidentify. It has, therefore, been suggested that thesefactors lead to underestimation of mast cell numbersin formalin fixed tissues and hence the contributionof mast cells to certain disease states may not havebeen fully appreciated. Toluidine blue staining hasthe further disadvantage that the pH values whichare optimal for demonstrating connective tissue,

Accepted for publication 8 August 1984

mucosal, and neoplastic mast cells differ consider-ably from each other4 and that basophils are alsostained metachromatically by this technique. Othermethods which have been used for demonstratinginfiltrating mast cells include the histochemicaldemonstration of chloroacetate esterase enzyme;immunohistochemical demonstration of 5-hydroxy-tryptamine, heparin, fibronectin, prostaglandin,reaginic immunoglobulin8; and distinctive stainingreactions with alcian blue and astra blue dyes.'Unfortunately, none of these methods is mast cellspecific. We thought that the use of monoclonalantibodies with specificity for mast cells would be avaluable alternative to such methods. We reporthere the establishment of seven hybridomas secret-ing monoclonal antibodies which distinguish mastcells from all other haemopoietic, connective tissue,and most epithelial cell types.

Material and methods

MONOCLONAL ANTIBODY PRODUCTIONBALB/c mice were immunised with approximately5 x 101 high density (>1-077; erythrocyte depletedcell pellet after fractionation on Histopaque(Sigma)) bone marrow cells from a patient with sys-temic mastocytosis; the preparation consisted of50% typical mast cells. Three weeks later the micewere boosted with 200 ,ul of packed, washed spleencells from the same patient, most of which were mastcells and of similar morphology to those in the bonemarrow. Details of the characterisation of mast cellsin systemic mastocytosis have been published else-

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where.9 Three days after boosting, the mouse spleencells were fused with X63.Ag8.653 myeloma cellsby a standard method and growing hybridomas wereselketed in HAT medium.'0

Supernatants from growth positive cultures werescreened for antibody against acetone fixed frozensections of the mast cell infiltrated spleen used forimmunisation using the indirect immunofluores-cence technique described below. Supematantsreactive with mast cells but not with residual spleencells were further screened against acetone fixedcytocentrifuge preparations of buffy coat cells fromperipheral blood of patients with chronic myeloidleukaemia and against acetone fixed cytocentrifugepreparations of normal human bone marrow. Cul-tures producing antibodies reactive with any other'haemopoietic cell type (granulocytes, monocytes, or

lymphocytes) were discarded. Antibodies withapparent specificity for mast cells gave a characteris-tic strong, coarse granular immunofluorescence,apparently corresponding with the distribution ofmast cell granules identified by phase microscopy.Hybridomas producing such antibodies were clonedtwice by limiting dilution, and their supernatantswere rescreened for reactivity with mast cells in sec-

tions of infiltrated spleen.The following hybridoma clones were selected for

further characterisation: MCG 3, 8, 9, 10, 34, 35,42.

PREPARATION OF TISSUES USED FOR SCREENINGSolid tissuesSpecimens were obtained from operative or surgicalbiopsy samples or from freshly killed animals, asappropriate, with the exception of human liver andkidney, which were obtained at necropsy, and fetaltissues, which were obtained from an 18 week oldfetus after suction termination of pregnancy. Piecesof roughly 0-5 cm2 were cut, placed in OCT medium(Tissue-Tek), and snap frozen in liquid nitrogen,where they were stored until use. Frozen sections(5-8 ,um) were cut on a cryostat (Cryocut E,Reichert-Jung), air dried, fixed in acetone for 10min, and stored at - 20°C for no longer than onemonth.

Blood cells and cell linesPeripheral blood leucocytes were obtained by cen-trifugation of the heparin anticoagulated blood of a

patient with chronic myeloid leukaemia and removalof the resultant buffy coat layer. Basophils were iso-lated by the same method from the blood of apatient with chronic myeloid leukaemia with 50%basophilia and from long term suspension culturesof normal human bone marrow. Peripheral bloodmononuclear cells were obtained from the low

Rimmer, Turberville, Horton

density (<1.077) interface layer after Histopaque(Sigma) fractionation of heparinised normal humanblood; granulocytes were obtained from the highdensity (> 1.077) cell pellet separated from the samesamples.

Established cell lines (HL-60, U937, K562,CEM, DAUDI, CHANG) were maintained in cul-ture in RPMI 1640 medium, supplemented with10% fetal calf serum (FCS), glutamine, and antibio-tics (all from Gibco, Paisley) at 37°C in an atmos-phere of 5% CO2 in air.

Cytocentrifuge preparations (Shandon UK,Cytospin II) were made of about 5 x 104 washed,live cells of each blood cell type or cell line. Thesewere acetone fixed, air dried, and stored at -20°Cfor not more than one month.

SCREENING PROTOCOLSupernatants from growth positive hybridomaclones, after screening for positive antimast cellreactivity, were further screened for reactivity(assessed by indirect immunofluorescence) againstthe panel of human and animal tissues detailed inTables 1-4.

Indirect immunofluorescence techniqueFrozen sections or cytospins were allowed to attainroom temperature, then rehydrated for 5 min inphosphate buffered saline (PBS)-FCS. Excessmedium was then blotted from the slide (withoutpermitting drying of the specimen) and 50 .ul of testsupernatant was placed on to the specimens. Theslides were incubated for 30 min in a humidifiedbox, then immersed in PBS-FCS for 30 min. Excessmoisture was then blotted, 50 ,ul of FITC conju-gated polyspecific goat antiserum to mouseimmunoglobulin (Nordic or Coulter-Clone) wasplaced on the specimen, and the slides were incu-bated for 30 min in a humid box, after which theywere washed in PBS-FCS for not less than 1 h.Coverslips were then mounted in a 9:1, glycerol andPBS mixture and examined by ultraviolet epi-illumination with a Zeiss microscope.

Demonstration of mast cells by toluidine blueSerial sections from the same tissues as those used inthe screening protocol were stained by toluidineblue to show the presence of mast cells. Air driedfrozen sections or cytospin specimens were fixed inMota' s basic lead acetate for 30 s, washed in distilledwater, stained with 0-1% aqueous toluidine blue(pH 4-0 or 2-0) for 5 min, washed in tap water, airdried and mounted. These sections served as refer-ence controls, allowing the enumeration and local-isation of mast cells within each specimen, thusfacilitating identification of mast cells by phase con-

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Human mast cells detected by monoclonal antibodies 1251

Table 1 Distribution ofmast cells in human tissues as defined by monoclonal antibodies

Source ofmast cells*t Reactivity ofmonoclonal antibodies with mast cellst

MCG3 MCG8 MCG9 MCGIO MCG34 MCG35 MCG42

Mast cell infiltrated spleen (1) +++ +++ +++ +++ +++ +++ +++Mast cell infiltrated bone marrow smears (4) -/+ + + ++/+++ +±/+++ +/+++ ++/+++ +±+/+++ -/+++Nasal polyp (2) ++ + + + ++ ++ ++Nasal mucosal biopsy (3) ++ + + + ++ ++ ++Lung (1) ++ + + + ++ ++ ++Skin (2) + + + + + + +Tonsil (2) ++ ++ ++ ++ ++ ++ ++Gastric mucosa (2) + + + + + + + + + + +Colonic mucosa (3) + + + + + + + + + + +Ileal mucosa (2) + + + + + + + + + + +Jejunal mucosa (2) ++ + + + ++ ++ ++Rectal mucosa (2) + + + + + + + + + + +Fetal intestine (1)§ + ND ND ND ND ND ND

*All tissue specimens were taken from macroscopically undamaged areas of surgical specimens.tNumbers in parentheses indicate the number of tissue samples studied.tAssessed by indirect immunofluorescence. Reactivity graded as: - = negative, = weakly positive, + to +++ = increasing degrees ofpositivity.§Morphologically recognisable mast cells were not seen in the mucosal layer of fetal intestine, though they were present in small numbers inthe smooth muscle layers around the gut.ND = not done.

Table 2 Distribution ofantigens defined by mast cellgranule monoclonal antibodies in non-haemopoietic tissue

Tissue Reactivity with mast cell granulemonoclonal antibodies*

ThyroidLung parenchymaGastric epitheliumJejunal epithelium + +Ileal epithelium + +Colonic epitheliumRectal epitheliumFetal gut epithelium +/+++Adult liver hepatocytes +++Fetal liver hepatocytest +++CHANG liver cell line + + +Adult kidney

GlomerulusGlomerular capsule +Proximal tubule + +/+ + +Distal tubule

Fetal kidney:S-bodyGlomerulusProximal tubules +++++Distal tubules

SkinEpitheliumDermis

SpleenEndothelium

*Assessed on the same sections used for assessment of monoclonalantibody reactivity with mast cells.tlslands of mononuclear haemopoietic cells were seen in the fetalliver, many of which were J5/BA2 positive, but which werenegative with the mast cell granule monoclonal antibodies.1The pattern of staining with mast cell granule monoclonalantibodies on fetal kidney was similar to that with J5(anti-CALLA), except that the former do not react withglomerular visceral epithelium.

Table 3 Reactivity ofmonoclonal antibodies withhaemopoietic cells other than mast cells

Haemopoietic ceUl typeltissue source Reactivity withmonoclonal antibodies

MCG3-42 Hle-l *

SpleenWhite pulp + +Red pulp - ++

Tonsillar lymphocytes - NDPeripheral blood lymphocytes - +++Peripheral blood monocytes - + +Peripheral blood granulocytes - + +Normal bone marrow cells - +/+++Leucocytes from chronic myeloidleukaemia - + +

Basophils from chronic myeloid leukaemiawith basophilia - +

Basophils in long term bone marrowculture - +

Eosinophils from eosinophilic patient - NDCEM (T lymphoid cell line) - +rAUDI (B lymphoid cell line) - +HL60 (promyelocytic leukaemia cell line)Uninduced - + +Dimethyl sulphoxide induced - + +

K562 (chronic myeloid leukaemia derivedcell line with erythroid/myeloidphenotype) - ++

U937 (myelomonocytic leukaemia cellline) ++

ND = not done.*The 2D1 monoclonal antibody defining the Hle- 1panhaemopoietic antigen was a kind gift of Dr Peter Beverley.

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Table 4 Non-reactivity ofmonoclonal antibodies with mast cells and other cell types in a panel ofanimal tissues

Species

Rat Mouse Dog

Tissues examined Kidney Colon StomachSkeletal Muscle Ileum DuodenumPeritoneal mast cells* Peritoneal mast cells* JejunumTongue P815 (mastocytoma cell line) IleumColon ColonIleumSubcutaneous connective tissue

*Obtained by peritoneal lavage.

trast microscopy in the immunostained sections.

DETERMINATION OF IMMUNOGLOBULINSUBCLASS OF MAST CELL GRANULE ANTIBODIESImmunoglobulin subclasses of the monoclonal anti-bodies MCG3, 9, 34, 35, and 42 were determined byradial immunodiffusion. The supernatants contain-ing mast cell granule monoclonal antibodies wereconcentrated by precipitation with saturatedammonium sulphate and reacted overnight withrabbit antisera to mouse IgM, IgG, IgGI, IgG2a,IgG2b, and IgG3 (Miles-Yeda, Israel). The gelswere then stained with Coomassie blue to displaylines of precipitation.

Results

REACTIVITY OF MONOCLONAL ANTIBODIESWITH HUMAN TISSUESSeven cloned hybridomas (MCG 3, 8, 9, 10, 34, 35,42) were established which secreted monoclonalantibodies reacting with granule components ofhuman mast cells. These gave a characteristic coarsegranular fluorescence pattern with mast cells in theimmunising tissue (Figure (a)) and in all normalhuman tissues screened (see Figure (b-d) and Table1). Both connective tissue mast cells and gastrointes-tinal mucosal mast cells were labelled with theseantibodies. The Figure illustrates these findings-thus, dermal connective tissue mast cells (b) andjejunal and colonic. gastrointestinal mucosal mastcells (c and d) and connective tissue mast cells (notillustrated) are visualised. The different monoclonalantibodies appeared to have equivalent specificityfor mast cells in different tissue locations, althoughMCG 8, 9, and 10 consistently gave weaker reac-tions. In general, connective tissue mast cells weremore intensely stained than gastrointestinal mucosalmast cells. This may be due to their greater granular-ity, as seen by staining of parallel sections with acidictoluidine blue and phase microscopy; it was also

noted that the highly granular infiltrating mast cellsof systemic mastocytosis stained more intensely thannormal connective tissue mast cells (Figure (a)).Furthermore, the monoclonal antibodies bound toextracellular mast cell granules and hence identifiedat least some degranulated mast cells, such as areseen in large numbers in nasal mucosal biopsies(data not shown).The seven monoclonal antibodies also stained

mature small intestinal enterocytes, but not smallintestinal crypt, colonic, gastric, or rectal epithelialcells (Table 2 and Figure (c and d)). They alsoreacted with hepatocytes (Figure (f)) and with fetaland adult renal proximal tubules and parietalglomerular epithelium (Figure (e) and Table 2). Inall these latter cell types reactivity was of a diffusetype, evenly distributed throughout the cytoplasm,and hence strikingly different from the granular pat-tern seen with mast cells. With these exceptions noreactivity was seen with any other non-haemopoieticcell type (Tables 2 and 3), although the panel oftissues tested was not exhaustive.The monoclonal antibodies did not react with any

other type of haemopoietic cell against which theywere tested (Table 3). In particular, it should benoted that the granules of other myeloid cells, and ofbasophils especially (Table 3), are not labelled withthese monoclonal antibodies.

FIXATION SENSITIVITY OF MAST CELL GRANULEANTIGENSNone of the seven monoclonal antibodies stainedmast cells, or other cell types, in tissues fixed bymethods other than acetone fixation. The antigenicdeterminants thus were not retained in conventionalformalin fixed, wax embedded histology specimens.

REACTIVITY OF MONOCLONAL ANTIBODIESWITH ANIMAL TISSUESNone of the monoclonal antibodies reacted withmast cells or other cell types in a wide range oftissues from rat, mouse, or dog (see Table 4).

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Human mast cells detected by monoclonal antibodies

e _ fImmunofluorescence micrographs offrozen sections oftissues stained with monoclonal antibody MCG3. Magnification:Figs. a-e x 264; f x 66.(a) Spleen from a patient with systemic mastocytosis showing strong granular reaction pattern ofmonoclonal antibodyMCG3 with mast cells. Residual uninvolved splenic tissue is negative.(b) Cluster ofstained mast cells (arrowed) around dermal blood vessel in section ofnormal adult skin.(c) Section ofadult jejunal viUus showing positive granular staining pattern ofmast cells (arrowed) in villus core andeven cytoplasmic staining ofmature enterocytes.(d) Mast cells in adult colonic mucosa (arrowed). Note that the colonic epithelium is not stained by monoclonal antibodyMCG3.(e) 18 wk fetal kidney showing staining ofproximal tubules and parietal glomerular epithelium.(f) 18 wk fetal liver showing staining ofhepatocytes. Unstained areas consisted offoci offetal haemopoietic cells, asidentified by routine histological stains.

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IMMUNOGLOBULIN SUBCLASS OF MAST CELLGRANULE MONOCLONAL ANTIBODIESAll of the mast cell granule monoclonal antibodieswere of the IgGI subclass. Monoclonal antibodiesMCG8 and 10 were not tested.

Discussion

Mast cells play a major part in the immediate typehypersensitivity response and participate in someother hypersensitivity reactions. Furthermore, mastcell infiltration is associated with some carcinomasand is thought to mediate tissue damage in certaininflammatory bowel and skin diseases. In many ofthese conditions mast cell infiltration is associatedwith, and thus influenced by, other cells presentwithin the inflammatory infiltrate. An understandingof the relative contributions of these different celltypes in disease pathogenesis clearly depends ontheir accurate identification in tissues. Standard his-tological techniques are not readily able to identifymast cells in tissues if they are: (a) present within theintestinal mucosa, (b) partially or completelydegranulated, or (c) part of a malignant or pre-malignant proliferation of mast cells. Our previousstudies on the cell membrane phenotype of mastcells suggested that they were antigenically dissimi-lar from other leucocytes, and especially from mor-phologically and functionally similar basophils,9"1and we therefore reasoned that the production ofmonoclonal antibodies of defined specificity for mastcells would be a valid approach towards an alterna-tive means of cell identification. Our original inten-tion was to make monoclonal antibodies to mem-brane markers of mast cells and their haemopoieticprecursors, which would aid in the distinction ofconnective tissue mast cells from gastrointestinalmucosal mast cells and thus enable their develop-ment and relative involvement in disease to bestudied. However, we actually derived a series ofmonoclonal antibodies which reacted with granulecomponent(s) of all mast cells. Nevertheless, thestudies reported here suggest that these antibodieswill be an equally useful immunohistological tool foruse in the identification of mast cells in tissues.The monoclonal antibodies possess several prop-

erties which make them a viable alternative tometachromatic dyes for the in situ identification ofmast cells in tissues. Most importantly, they arestrongly reactive with all human mast cells in bothconnective tissue and mucosal sites, but not with anyother infiltrating leucocyte or connective tissue celltype. This is a considerable advantage over toluidineblue, which is not specific for mast cells and alsolargely fails to stain mucosal mast cells except intissues fixed in non-routine fixatives such as Car-

Rimmer, Turberville, Horton

noy's or Mota' s. Furthermore, the ability to distingu-ish mast cells from basophils in certain situations-for example, in renal allograft rejection or cutane-ous basophil hypersensitivity-is an added advan-tage and should allow their relative involvement tobe further defined. The reaction of mast cell mono-clonal antibodies seen with certain other celltypes-that is, small intestinal enterocytes, hepato-cytes, and proximal renal tubules--is substantiallydifferent in character to that seen with mast cells andis thus unlikely to interfere with the interpretation ofimmunostained tissue biopsies. In fact, theseimmunological "cross reactions" may aid in theeventual identification of the molecule(s) recognisedby this series of monoclonal antibodies. All the tis-sues in question have major synthetic/secretoryfunctions and this suggests that the mast cell granulecomponent is likely to be one of the many enzymesreleased during mast cell degranulation.6 12. To datepreliminary biochemical studies, by means ofimmunoprecipitation from metabolically labelledCHANG human liver cells, have as yet failed toidentify the molecular nature of the antigen(s) con-cerned.

In summary, monoclonal antibodies specific formast cells allow the identification and hence enum-eration of mast cells compared with other cells pres-ent in inflammatory tissue infiltrates by means ofimmunohistological staining of either serial frozensections or by appropriate double labelling techni-ques. The use of the mast cell granule monoclonalantibodies described in this paper should thus proveto be a valuable adjunct to conventional histologicaltechniques in the investigation of the role of mastcells in the genesis of various skin, respiratory, andgastrointestinal diseases.

We thank the Wellcome Trust for financial support.MAH is a Wellcome Trust senior research fellow inclinical science.

References

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2 Soter NA. Mast cells in cutaneous inflammatory disorders. JInvest Dermatol 1983;80:no 6 (suppl):22-5.

3 Lemanske RF, Atkins FM, Metcalfe DD. Gastrointestinal mastcells in health and disease. Part II.JPediatr 1983; 103:343-51.

Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: areview. Histopathology 1979;3: 349-65.

5 Metcalfe DD, Kaliner M, Donlon MA. The mast cell. CRC Criti-cal Reviews in Immunology 1981;3:23-74.

6 Pepys J, Edwards AM, eds. The mast cell: its role in health anddisease. Tunbridge Wells: Pitman Medical, 1979.

Strobel S, Miller HRP, Ferguson A. Human intestinal mucosalmast cells: evaluation of fixation and staining techniques. J

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Requests for reprints to: Dr MA Horton, Department ofHaematology, St Bartholomew's Hospital, WestSmithfield, London EClA 7BE, England.

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