Indian Journal of Experimental Bio logy Vol. 39, April 2001 , pp. 323-328

Comparative functional characterization of mouse bone marrow-delived mast cells and peritoneal mast cells in response to non-immunological stimuli

Rashmi Singh, P Kumar* & P P Gupta** Divi sion of Pharmacology, Central Drug Research Institute, Lucknow 226 00 I, India

*Department of Zoology, University of Lucknow, Lucknow 226 007, India

Received 6 April 2000; revised 19 December 2000

The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cell s in terms of their T-cell dependence and histochemical staining charac­teristics. Mast cell heterogeneity has been confirmed by functi onal characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium lonophore A23 187 on the other hand, re leased hi stamine in do e-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, di sodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced hi stamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca lonophore induced histamine re lease was blocked by Quercetin. The results indicate that response of mast cells at one anatomic si te to a g iven stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity wi th in a single species. So, it cannot be assumed that antiallergic com­pounds stabili zing mast ce lls in one ti ssue site or organ will be equally efficacious against mast cells in other sites.

Mast cell is a key cell and its activation is the promi­nent feature of many allergic diseases like allergic asthma, allergic rhinitis and anaphylaxisl -4 . Mast cells compri se a heterogeneous population of cell s found in high numbers at mucosal surfaces such as lung, intes­tine, nose, eye and in connective ti ssues, such as skin and submucosal tissues. The predominant mast cell subtypes at mucosal surfaces and in connective tissues differ from each other in their development, chemical composition and functionally in their abi lity to inter­act with their local environment. Functional differ­ences between mast cell types are illustrated by their response to different non-lgE-dependent secreta­gogues and their susceptibility to pharmacological inhibitors of activation. For example, compound 48/80 is a potent activator of rat serosal and cutaneous mast cells (CMC)5 but fails to activate mucosal mast cell (MMC) from rat intestine ill viv06 or ill vitro after enzymatic dispersion 7 .The functional properties of MMCs have been defined by investigating in vitro cultured bone marrow-derived mast cells (MBM MC) after 4 weeks of incubation at 37°C/5% CO2 air and comparing their properties to the well characterized mouse and rat peritoneal mast cells (CTMC).

**Correspondent author: Te l: 212411 , FAX : 91-0522-223405 E-mail : root@cscdri.rcn.nic.in

In particular, they also respond differently to anti­allergic agents8

.9

. Most probably, both functional and pharmacological differences exist between these cells and may prove critical in the pathogenesis and ther­apy of allergic diseases.

Materials and Methods Chemica/s-The following were used and pur­

chased from manufactureres as indicated. Calcium ionophore A23187, Concanavaline-A, Compound 48/80, Aleian blue, Safranine, Dulbecco's modified Eagle's medium (DMEM) and Quercetin were pur­chased from Sigma Chemical CO.,USA. Fetal Calf Serum from GlBCO, Grand Island, New York and Disodium cromoglycate from Rallis India Ltd., Mum­bai.

AnimaLs- Four to six weeks old male Balb/c mice (20-25) were obtained from animal house facility of Central Drug Research Institute, Lucknow and were kept in raised mesh bottom metallic cages. Every ef­fort was made to minimize animal suffering.

Purification of peritoneaL mast ceLLs-Peritoneal mast cells (PMC) were harvested from the peritoneal cavities of mice and rat and purified by 35% Ficoll in Tris-EDT A buffer (PH 7.6) and puaty of mast cells was assesed by the toluidine blue staining which ranged from 80-90%.

324 INDIAN J EXP BIOL, APRIL 2001

Cell clllture mediulIl-DMEM supplimented with 2 mM L-glutamine, 10% (v/v) Fetal Calf serum, 50 ~-2 mercaptoethanol , 0.1 mM non-essential amino acids, 20 mM HEPES buffer and NaHC03 (2.0 giL), Genta­micin 40 mglL was used in cell culture medium. pH was maintanined at 7.2 to 7.4.

Mouse splenocyte conditiolled medium-Mouse splenocyte-derived conditioned medium was used to stimulate the proliferation and differentiation of bone marrow cells into mast cells. ln brief, spleen cell s from Balb/c mice and Swiss (106 cells/ml) from each strain were cultured in 75 cm2 ti ssue culture fl asks contain­ing 2.5 ~g/ml con-A and cell culture medium. After the cells had been incubated for 42 to 48 hours at hu­midified 5%C02/95 % air, the splenocyte conditioned medium was centrifuged for 20 min at 5000rpm. The supernatant was filtered using 0.45 ~m filter and stored at -20°C until used as conditioned medium (CM).

Bone marrow cell cultures-Bone marrow cells were obtained by flushing the femurs of Balb/c mice and bone marrow cell s (BMC) (I x I 06 cells) were sus­pended in a fi nal volume of 10 ml in a 25 cm2ti ssue cu lture flask contai ning cell culture medium and sple­nocyte-derived cond itioned medium ( I: I; vol/vOI)IO. The cells were maintained at appropriate culture envi­ronment and the fresh cu lture medium was changed week ly up to 2 1 days to assay histami ne content.

Mast cell coullt- AII cultures for enumeration of cells were ex pressed as mean ± SE. umber of mast cells identified by metachromatic staining of their granules with alcian bluelsafranin or 0.1 % toluidine blue [in 50%(v/v) ethanol, pH 3.0].More than 90% of the cells were viable as judged by trypan blue dye exclusion.

Effect of secretagog lle.\· alld illhibitors Oil /tistamill e release-Compound 48/80 was dissolved in 0.9% saline and required dilution was prepared whereas Ca ionophore A23187 was dissolved in dimethyl sulfox­ide (DMSO) to give stock solution of 10-3 M and di ­luted in buffer as required. The final concentration of DMSO ($1 %) was tested and had no effect on the secretory response of the cells.

Male Balb/c mice and rats were sacrificed by cervi­cal dislocation, after inj ecting 5 ml of 0.9% saline into the peritoneal cav ity. The peritoneal ex udate was

taken out with a pasteur pipette through midline inci­sion. The cell suspension was prepared in the Tyrode solution in aliquotes of I ml in centrifuge tubes and centrifuged at 120 g for 5 min and resuspended in the 800 ~I of tyrode solution without calcium according to the experimental protocol. The cells were pre­incubated for 10 min to permit temperature equilib­cration and different concentrations of inhibitors of mas t cell degranulation and hi stamine release, like disodium cromoglycate (DSCG) were added and in­cubated at 37°C for 15-20 min before the addition of hi stamine releasing agent in the volume of 100 ~1.

Further incubation of 10 min was then allowed fo r histamine release to make final incubati on volume being I ml. MBMMC exhibited dose-dependent hi s­tamine release when incubated with different concen­trations of Ca ionophore A23187. To assess the effect of quercetin, MBMMC were incubated 2 days with 30 ~M of quercetin , a plant-derived flavonoid, before addition of secretagogues. The hi stamine release was then terminated by quenching each tube by adding 1 ml of ice- cold tyrode solution. The tubes were centri­fuged at 800 rpm for 5 min and supernatants were stored at -20°C untill hi stamine assay. The pellets were again resuspended in 5 ml of tyrode solution and heated at water bath at 100°C for 5 min to release re­sidual hi stamine from the cell pellet. Same procedure was used to estimare histamine from MBMMCs after recovery of cells at different intervals by trypsiniza­ti on and triple washing.

The released and residual hi stamine was assayed spectrophotoflurometrically after condensation with o-pthalialdehyde without extraction steps II. Histami ne release was calculated as percent of total hi stamine content in each tube. Release occurred in the absence of any agent or drug (spontaneous release) is sub­tracted from the stimulated release. The composition of tyrode solution in mM was aCi 134, KCI 2.7, glu­cose 5.6, NaH2P04 0.4, HEPES [4-(2-hydroxlythyl)-I ­piperazine sulphonic acidJ 20 mM. Calcium wherever added was a small volume of stock CaCI 2 I M.

Results After 14 days of culture, 74-80 % cells exhibited

definite metachromasia on staining with toluidine blue which suggested that these ceils were mast cells and their further characterizat ion was done by stai ning wi th Alcian bluel safranin and estimating their hi sta­mine content at different phases of their develop­ment.(Table I)

SINGH et at.: BONE MARROW-DERIV ED AND PERITONEAL MAST CELLS 325

Table I- Histamine content in MBMMCs [Values are mean ± SE of live cultures in triplicates]

Period of culture (days)

7 14 2 1

Histami ne content ng/ 106 cells

200±25 380±70 400+70

Growth characteristics and identification of MBMMC as /IIucosal type of /IIast cells-]nitial bone marrow preparations contained less than 0.1 %mast cells. By day 14 onwards, however more than 80% of the cultured cell s could be identified as mast cells. The MBMMC were typical of mas t cell s as described earl ier in that they stained with Alci an blue and tolu­idine blue at acidic pH and contain less hi stamine (0.3-0.4 Ilg/ 106 cells) which was thought to somewhat lower than the reported mouse mast cells (1-7 Ilg/l 06

ce ll s)1 2. I3. In contras t to mice and rat connective tissue type of mast cells, MBMMC granules did not stain with safranine. Electron microscopy of MBMMCs revealed a characteri stic s ingle eccentric nucleus with cytoplasm containing electron dense granules and vacuoles (Fig. I).

Response to secretagogues and antiallergic drugs­Hi stamine can also be released from mast ce ll s found at different locations by non-immunological stimuli in specific fashion . Moue peritoneal mast cells (MPMC) release histamine following stimulation with com­pound 48/80 and maximum release of 30% observed at 100 flglml and cultured mouse mast cells were al­most unresponsive to the compound 48/80 in this dose range (Fig. 2).]n marked contrast, RPMCs re leased around 80% histamine with compound 48/80 ( I flg/ml). MBMMC released hi stamine in dose­dependent manner on challenge with Ca ionophore A23 187 (~I J..LM) (Fig. 3). MBMMC also responded con-A (10 flg/ml) and maximum release was 25 % (not expressed in results).

Effect of antiallergic drugs acting as inhibitors o f hi stamine release from peritoneal mast ce ll s of rat, mice as well as from MBMMCs have been compared. The mouse cells were rather less sensitive to the diso­dium chromoglycate than those o f rat (response in mice has not been expressed in results). Disodium cromoglycate (inhibited hi stamine secretion from peritoneal mas t ce lls in dose dependent manner (Fig. 4).

Plant-deri ved fl avonoid, quercetin (Fig. I) which is structurally similar to DSCG I4 exhibited inhibitory

Fig. I-Electron microscopic picture of mouse bone marrow­deri ved mast cell (MBMMC).

- . - RPMC - e - MPMC

~ MBMMC 80

70

Q) 60 VI co Q)

W 50 ..... Q) c ·E 40 co iii I 30 :oR 0

20

10

0 0.1ug/ml 1.0 10 100

Compound 48/80 (ug/ml)

Fig. 2-Histamine release induced by compound 48/80 in iso lated mast cells obtained from vari ous sources. [Values are ± SE for Ihree experiments in triplicates].

activity (40%) against ionophore A231 87 induced histamine release in MBMMC when pre-incubated before stimulation.

Discussion Much of what we know about T-cell dependent

mast cell populations has been derived from studies

326 INDIAN J EXP 1310L, APRIL 200 1

conducted in mice.The provenance and lineage of the mast cell precursors that populate in the connective ti ssue or serous spaces have been the topic of consid­erable discussion. Presence of mast cell presursors in the bone marrow was confirmed by Kitamura el al. 14

•

Interleukin-3 (IL-3) dependent mast cells generated from mouse bone marrow cell cultures have been considered analogous to rat intestinal mucosal mast cells largely based on comparisons with rat but not mouse peritoneal connective tissue type mast cel ls. However, despite being closely related species, rat and mouse mast cells have different characteri stic features. Rat and mouse connective ti ssue type of mast cells has different characteristic features . Func­tional differences also exist among these connecti ve tissue type of mast cells which has been clearly shown by stimulation with different stimuli of degranu lation. To avoid any cross-species difference cont ributing to mast cell heterogeneity, we tried to show that mouse peritoneal mast ce ll s and cultured mast cells generated from bone marrow cell s differ in term of their hista­mine content as assesed by spectrofiuorophotometric method.

Histamine content in the mast ce ll derived from bone marrow cell s (OAO Ilgll 06 cells) is somewhat lower than that was reported from ( 1-7 Ilg/l06)I S. The lower hi stamine conten t of the cultured mast cells may be attributable to the presence o f Con-A in the

**

60

Q) en 50 ro Q)

(ij L..

'+- 40 0 c 0

:;::. ro 30 :;::. c Q) -0 (L 20 ~ 0

10

o 0 .05 0.1 0 .5 1.0

Ca lonophore A23187 (uM)

Fig. 3-DllS~-d~p~lI(kn t r~sponsc of hi stamine re lease induced by calc ium ionophore A23 187 from mouse bone marrow-derived mast cells. (*P<O.O I; **P<O.OOI)

culture medium which is known to trigger degranula­tion of mast cells 16. It is also reasonable to assume that the difference in the histamine assay systems used also may contribute to the varia ti on in the re­sults. As bone marrow-derived mast cell s are depend­ent on the T-cell derived growth faclor for the differ­entiation and development, so it is reasonable to call these cells as mucosal mast cell s and are functionall y different from connective tissue type of mast cells .

Q) en ro Q)

~ Q) c E ro -en

I -;f?

Control 10uM 20uM 30uM

DSCG + Compound 48/80

Fig. 4-Effect of different concentrations of DSCG on compou nd 48/80 induced histamine re lease from rat peritoneal mas t cells IValues are ± SE. *P<0.02; **P<O.OIJ

~ CH'-~:-CH2hl Naooc~o~ ~ 0 COONa

DISODIUM CROMO GLYCATE

OH 0

HO

OH OH

QUERCETIN

Fig. 5- Structrure of quercetin

SINGH el al.: BONE MARROW-DERIVED A D PERITONEAL MAST CELLS 327

Q)

65

60

55

50

CJ) 45 co Q)

Q) 40 L.. ..... I35 ...... o c 30 o :e 25 ..0

-§ 20

~ 15

10

5

o

*

RPMC MPMC MBMMC

Quercetin+ Ca lonophore

Fig. 6-Effect of quercet ine o n Ca ionophore A23187 induced hi stamine release from di fferent sources of peritoneal mast ce lls

IValues are ±SE. * 1'<0.02)

Both type of ce lls are responsive to IgE-directed lig­ands wh ich is not described in our experiments . The study was undertaken to observe responsiveness of these cells to non-immunological stimul i. Like muco­sal mast cells, bone malTow-derived mast cells were remain unresponsive to basic secretagogues, like compound 48/80 whereas RPMC responded to com­pound 48/80. Calcium ionophore A23187 induced mast cell degranulation in dose-dependent manner which releases hi stamine probably by transporting calcium from extracellular compartment into the cell 17 .

The effect of flavonoids on the histamine release from BMMC by the divalent cation ionophore which bypasses receptor-dependent processes and carries ci+ directly into the cytoplasm8

.19. Quercetin exhib­ited activity against non-receptor-dependent secreta­gogue, Ca ionophore A23187, but whether this "anti­ionophoric activity" represents inhibition of trans­membrane C}+ uptake or inhibition of intracellular Ca++ translocation or some other effect is unknown at

present. Therefore it can be assumed that bone mar­row-derived mast cells are dependent on Ca2+ influx as MBMMC and MPMC, di sodium cromoglycate showed its effectiveness agai nst RPMC by stab ili zing its cell membrane but it is not effective against MPMC and MBMMC.

In the present study, different secretagogues in­duced degranulation in MBMMCs indicate th at in­spite of immunological stimuli, these cells are also responsive to non-immunological st imuli and in vesti­gations suggest that mast cells obtained during culture from haematopoitic tissues of the mouse have typi cal characteristics of mast cells and may be suitable for the bioevaluat ion and the biochemical stud ies of the cells.

Acknowledgment This work was supported by the grant from the

ICMR, ew Delhi , Indi a.

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