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Arch Dermatol Res (1988) 280:358- 362

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9 Springer-Verlag1988

Purification of rat cutaneous mast cells with Percoll density centrifugation


H. Hachisuka, M. Kusuhara, M. Higuchi, K. Okubo, and Y. Sasai Cutaneous Biology Unit, Department of Dermatology, Kurume University School of Medicine, 67, Asahimachi, Kurume, 830, Japan

Summary. The skin is the major site on anaphylaxis, and cutaneous mast cells have an important role in its reactions. The isolation and purification of rat cutaneous mast ceils are described here. Rat abdominal skin was digested with collagenase and hyaluronidase, and centrifuged with Percoll. The buoyant density of cutaneous mast cells was high, and relatively pure mast cells were obtained. The purity of cutaneous mast cells was 7.4% -I- 2.4% before and 50.0% 6.4% after Percoll density centrifugation; peritoneal mast cells revealed 5.8% 1.3% purity before and 61.0% 10.6% purity after the same procedure. The isolated cutaneous cells released 21.3% 3.8% histamine and the peritoneal mast cells released 55.5% 3.8% histamine upon stimulation with 10 ~g/ml compound 48/80. These findings suggest that there are functional subsets of connective tissue mast ceils. Key words: Mast cell isolation - Skin - Pereoll

immunohistochemical characteristics, rat mast cell proteinase I ( R M C P I) from connective tissue mast cells and R M C P II f r o m mucosal m a s t cells are mast cell subsets feasibly distinguishable by their serine proteinase content [6]. To examine the function of mast cells, m a n y investigators have attempted to isolate and purify them f r o m various organs, including peritoneum [3, 5, 9, 17, 20], intestinal mucosa [17], lung [16], and skin [2]. R a t peritoneal mast cells have been used for evaluation of antianaphylactic agents [8, 1 1 - 1 3 ] . Since skin is the m a j o r site on anaphylaxis, it has been assumed that it purifies m a s t cells. The present study was undertaken to isolate cutaneous m a s t cells f r o m rat abdominal skin.

Materials and methods


Enzyme dispersion of rat cutaneous mast cells

R a t mast cells have been divided into two m a j o r subpopulations according to the cytochemically detectable presence or absence o f glycosaminoglycans: connective tissue m a s t cells and mucosal m a s t cells [4]. The former are found in skin, muscle and the peritoneal cavity, while the latter, which are devoid of glycosaminoglycans, are found in m u c o s a o f the gastrointestinal tract. Differences in function between them m a y be reflected in differences in their stimuli for ontogeny and maturation, cytochemistry, and ability to respond to activators and modulators of mediator secretion [4, 14]. To identify the rat m a s t cell subsets, an alternative a p p r o a c h is to examine the distribution of granule proteinases. According to
Offprint requests to: Dr. Hiroshi Hachisuka (address see above)

Wistar strain rats of both sexes weighing about 200 g each were used for the experiment. Rat cutaneous mast cells were dispersed enzymically in a manner similar to that previously described for dispersion of cells from rat skin [1]. The animals were anesthetized with ether and killed by exsanguination. The abdominal skin weighing about 5 g was dissected and dermal fat was removed with scissors. After washing in Tyrodc's HEPES buffer, the tissue was then finely chopped into fragments of about 1 mm 3. It was incubated (4 h, 37~ in a Hanks solution containing 1 mg/ml collagenase (Type I, Sigma, St. Louis, Mo.) and 1 mg/ml hyaluronidase (Type I, Sigma) buffered with 10 mM HEPES pH 7.3, and supplemented with 10% fetal calf sertlrn. The incubation mixture was gently gassed throughout with a mixture of air (95 %) and CO2 (5%). After 4 h incubation, the tissue was stirred with a magnetic stirrer (45 min, 37~C). The cell and tissue suspension was filtered through gauze, then the cells were recovered by centrifugation (5 rain, 150 g, 4~C) and washed once in ice-cold Tyrode's buffer. Finally, the cells were resuspended in Tyrode's buffer and passed through a nylon mesh to remove debris.
Isolation of rat peritoneal cells

The animals were anesthetized with ether and killed by exsanguination. Hanks solution containing 0.1% bovine serum

H. Hachisuka et al.: Purification of cutaneous mast cells


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Fig. 1. Buoyant density of nucleated cells, red blood cells, and mast cells on rat skin. Rat abdominal skin was digested with collagenase and hyaluronidase, and cell suspension was centrifuged with Percoll discontinuous gradient. Cells were identified with Blutstan stain. Straight line shows the buoyant density of each fraction. Buoyant density was monitored by density marker beads

albumin and 10 units/ml heparin was injected into the peritoneal cavity. After 90 s, the exudate was collected by pipette, and washed twice with Tyrode's solution by centrifugation at 200 g for 5 min at 4~ Then, pellets were resuspended in Tyrode's solution. Viability of cutaneous and peritoneal mast cells was always greater than 95%, as determined by trypan blue dye exclusion. Percoll centrifugal elutriation of mast cells Isolation of mast cells was done using a method similar to that previously described [5]. Percoll (colloidal silica polyvinylpyrrolidone) and density marker beads were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Isotonic Percoll solutions were prepared by dissolving nine parts of Percoll with one part of a tenfold concentrated Hanks solution. Colored density marker beads were used for calibration of Percoll gradients and for checking the equilibrium conditions. An aliquot of 0.75 ml of the cell suspension in Hanks solution was added to 3.5 ml Percoll isotonic solution, resulting in a final density of 1.110. Then, 0.5 ml Hanks buffer was layered on top of the solution and centrifuged at 125 g for 15 rain. The two uppermost milliliter were removed together with 0.5 ml Hanks solution for washing the wall of the test tube. The remaining volume containing the mast cells was washed twice in Hanks solution. Histamine concentration assay Compound 48/80-induced histamine release was studied using the following procedure. Aliquots of 0.9 ml of peritoneal cell suspension (ca. 1 x l04 mast cells) were preincubated for 10 min at 37~ t00 gg/mt compound 48/80 (N-methyl-p-methoxy-

phenylamine with formaldehyde; Sigma) was added in a volume of 0.1 ml, followed by incubation at 37~ for 10 rain. The reaction was stopped by dipping into ice-cold water. The cells were sedimented by centrifugation at 400 g for ~0 min at 4~ and the supernatants were collected. The pellets were resuspended in 1 ml Tyrode's solution, heated at 100~ for 5 rain to release their residual histamine and centrifuged at 400 g for 10 rain. The aliquots from these supernatants were assayed for their histamine content using reverse phase high-performance liquid chromatography (HPLC) [19J. The HPLC system (Toyo Soda, Tokyo, Japan) consists of a CCPM multipump, an FS-8000 spectrophotofluorometer, a TSKgel ODS-120T reversed phase chromatography column, an AS-48 auto-sampler, and a Chromatocorder 11. The fluorescence intensity was monitored at the emission wavelength of 450 nm with the excitat!on wavelength set at 350 nm. The results were expressed as ~ percentage of released histamine by the total histamine, and each histamine release represents the average of the means _+SD (n = 6). Statistical analyses were performed using Student's t-test. Microscopic examination Slides for the microscopical examination of the dispersed cells were prepared using Cytospin 2 (Shoadon, Cheshire, England); they were fixed in absolute methanol and stained with Giemsa solution.
Results

T h e digestion o f o n e rat a b d o m i n a l skin with eollagenase a n d h y a l u r o n i d a s e yielded a p p r o x i m a t e l y 7 x ]06 n u c l e a t e d cells.

360
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H. Hachisuka et al.: Purification of cutaneous mast cells Table 1. Compound 48/80-induced histamine release from peritoneal and cutaneous mast cells
Histamine release (%)

50

Unpurified
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Purified 55.5 + 3.8 21.3 +_3.8

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Peritoneal mast cells Cutaneous mast cells

61.0 _+8.3 28.0 + 4.5

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Each mast cell was purified by Percoll and stimulated with 10 gg/ ml compound 48/80. The histamine release was expressed as a percentage of released histamine by the total histamine. None of the differencesbetween the treatment groups was statistically significant

1.124

1,110

1.100

1.090

.080

Bouyant Density
Fig.

2. Buoyant density of rat cutaneous mast ceils. Rat cutaneous celt suspension was layered on each density of Percoll,

and cells were recovered from the lower portion of the tubes. Most of the contaminated cells were red blood cells. Cells were identified with Blutstan stain

The buoyant density of cutaneous mast cells was studied in Percoll continuous centrifugation. At 20,000 g for 90 rain 8 ml isotonic PercoU solution was centrifuged with density marker beads: Then 1 ml of enzymically dispersed cutaneous Cell suspension was layered and centrifuged a t 400 g for 30 rain at 4~ Aliquots of 0.5 ml were aspirated and washed three times with Hanks solution. Since identification of unstained cells is difficult, t0 I-tl of cell suspension was smeared in a glass slide precoated with methylene blue and cresyl violet (Blutstan, Daiichi Pure Chemical, Tokyo, Japan). Mast cells, nucleated cells, and red blood cells were easily differentiated and cell numbers were counted microscopically within a high-power field (40 x 10). The buoyant density of rat cutaneous mast cells is shown in Fig. 1. The upper fraction, which was of low buoyant density, showed increased numbers of nucleated cells and red blood cells. Seven or later fractions, higher than 1.100, revealed relatively pure mast cells in each fraction. Similarly, the buoyant density of cutaneous mast cells was monitored using Percoll discontinuous centrifugation. Cutaneous cells were mixed with 5 ml Percoll-Hanks solution of varying densities and layered with 0.5 ml Hanks solution. After centrifugation at 125 g for 15 min at 4~ the upper I ml of the solutions was discarded and the remainder washed three times with Hanks solution. Each fraction was stained with Blutstan, and the purity of mast cells counted. As shown in Fig. 2, the higher density of

Percoll-Hanks showed a higher purity of mast cells. Contaminated cells were mostly red blood cells. Since the total numbers of mast cells were reduced during the purification process, the buoyant density of Percoll was set at 1.110. The procedure for purification of rat cutaneous mast cells was carried out as described in Materials and methods. Cutaneous and peritoneal mast cells were isolated and purified from the same animals, and the purity of mast cells was compared with each tissue. Before Percoll fractionation, 7.4% __+2.4% of nucleated cells were regarded as cutaneous mast cells, while 5.8% +_ 1.3% were considered peritoneal to be mast cells. After Percoll centrifugation, among the nucleated cells the purity of cutaneous mast cells reached 50.0% + 6.4%, while the purity reading for peritoneal mast cells was 61.0% + 10.6%. For the study of mast cell secretion, histamineliberating compound 48/80 was used as a secretagogue in unpurified and purified mast cell suspensions. Table I summarizes the results. Compound 48/80-induced histamine release from unpurified and purified mast cells was slightly reduced during Percoll fractionation. Release of histamine from peritoneal mast cells was about twofold higher than that of cutaneous mast cells. Discussion Collagenase and hyaluronidase digestion of rat abdominal skin has been shown to disperse intradermal nucleated cells, including fibroblasts, capillary endothelial cells, and mast cells. This procedure has an almost negligible effect on histamine release by peritoneal mast cells, and the mast cells obtained responded well to various stimuli [1]. Therefore, we attempted to further purify rat cutaneous mast cells. For the isolation of mast cells, several media have been introduced: bovine serum albumin [18], Ficoll solution [10, 20], Metrizamide [21], and Percoll [5].

H. Hachisuka et al.: Purification of cutaneous mast cells Percoll (colloidal silica polyvinylpyrrolidone) is a unique density gradient m e d i u m designed to maintain physiological conditions throughout centrifugation experiments. It has low osmolarity and viscosity, and can be mixed with various physiological solutions. In addition, density gradients are self-generated during high-speed centrifugation. Using this medium, we have previously reported that guinea pig epidermal cells successfully separate into three fractions which correspond relatively to their arrangement in vivo [7, 15]. With Percotl density gradient centrifugation, the rat cutaneous mast cells locate at higher density fractions, especially at b u o y a n t densities of 1.100 or above. In those fractions, contaminated cells are mainly red blood cells. Discontinuous fractionation with Percoll centrifugation shows that purity of m a s t cells increases at higher fractions. Since contaminated cells are mostly red blood cells, assumed to have no influence on mast cells, we t o o k the b u o y a n t Percoll density at 1.100 for the dicontinuous gradient. This density for rat cutaneous m a s t cells is the same as that for peritoneal mast cells [5]. Blutstan or Giemsa staining of cytocentrifuged smears shows the purified cutaneous mast cells to be morphologically intact T r y p a n blue dye exclusion reveals a viability greater than 95%. Recently, Benyon et al. [2] have reported the isolation and purification of cutaneous m a s t cells f r o m h u m a n foreskin. They used collagenase and hyaluronidase digestion to disperse of cutaneous m a s t cells, as Barrett et al. [1] and we have done. Pretreatment with these enzymes showed negligible effects for viability and histamine release from various stimuli [1]. Benyon et al. [2] purified h u m a n cutaneous m a s t cells to 80%, however, only 16% o f the total mast cells were recovered, and 50% purity was recovered from 53% of total mast cells. Thus, the purity and recovery o f the rat cutaneous m a s t cells in our experiment are almost the same as reported by Benyon et al. [2]. F o r comparison of rat cutaneous and peritoneal m a s t cells, peritoneal mast cells were also purified according to Enerb/ick and Sevensson [5]. C o m p o u n d 48/80 was used to c o m p a r e the histamine-releasing activity of these mast cells. Our results indicate that cutaneous m a s t cells are less reactive to c o m p o u n d 48/80, and this observation is consistent with a previous report [1]. These findings suggests that there are functional subsets of connective tissue mast cells. In summary, the present study provides a simple method for the isolation and purification o f rat cutaneous mast cells. Because of the apparent heterogeneity of m a s t cells from different sites, it m a y be important to use cutaneous m a s t cells in the evaluation of antianaphylactic drugs. Also, the availability of

361 purified cutaneous mast cells m a y facilitate further study of the role of m a s t cells in skin allergy.

References

1. Barrett KE, Ali H, Pearce FL (1985) Studies on ihistamine secretion from enzymically dispersed cutaneous mast cells of the rat. J Invest Dermatol 84:22-25 2. Benyon RC, Lowman MA, Church MK (1;987) Human skin mast cells: their dispersion, purification, and secretory characterization. J Immunol 138:861-867 3. Coutts SM, Nchring RE Jr, Jariwala NU (1980) Purification of rat peritoneal mast cells: occupation of IgE receptors by IgE prevents loss of the receptors. J Immunol 124: 2309 - 2315 4. Enerb/ick L (1981) The gut mucosal mast cell. Monogr Allergy 17 :222 5. Enerbfick L, Sevensson I (1980) Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll. J Immunol Methods 39:135 - 145 6. Gibson S, Miller HRP (1986) Mast cell subsets in the rat distinguished immunohistochemically by their content of serine proteinases. Immunology 58 : 101 - 104 7. Hachisuka H, Nomura H, Sasai Y, Shiotsuki K, Yokoyama MM (1986) Changes of intracellular fluidky during keratinocyte differentiation measured by: fluorescence polarization. Cell Tissue Res 246 : 557- 560 8. Hachisuka H, Nomura H, Sakamoto F, Mori O, Okubo K, Sasai Y (1988) Effect of antianaphylactic agents on substance-P induced histamine release from rat peritoneal mast cells. Arch Dermatol Res 280:158 - 167 9. Heiman AS, Crews FT (1985) Characterization of the effects of phorbol esters on rat mast cell secretion. J Immunol 134: 548- 555 10. Ishizaka T, K6nig W, Kurata M, Mauser L, Ishizaka K (1975) Immunologicproperties of mast cells from rats infected with Nippostrongylus brasiliensis. J Irnmunol 115: 1078-1083 11. Koda A, Nagai H, Watanabe S, Yanagihar a Y, Sakamoto K (1976) Inhibition of hypersensitivity reactions by a new drug, N(3",4"-dimethoxycinnamoyl) anthran~lic acid (N-5'). J Allergy Clin Immunol 57:396-407 12. Kusner EJ, Dubnick B, Herzig DJ (1973) The inhibition by disodium cromoglycate in vitro of anaphylaCtically induced histamine release from rat peritoneal mast cells. J Pharmacol Exp Ther 184:41-46 13. Martin U, R6mer D (1978) The pharmacological properties on a new, orally active antianaphylacfic compound: ketotifen, a benzocycloheptathiophene. Arzneimittelforsch 28: 770- 782 14. Newlands GF, Huntley JF, Miller HR (1984) Concomitant detection ofmucosal mast cells and eosinophils in the intestines of normal and Nippostrongylus-immune rats. A reevaluation of histochemical and immunocyt0chemical techniques. Histochemistry 81:585-589 15. Sasai Y, Hachisuka H, Mori O, Nomura H (1984) Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry. Histochemistry 80:133--136 16. Schleimer RP, Macglashan DW Jr, Peters SP, Pinckard RN, Adkinson NF Jr, Lichtenstein LM (1986) Characterization of inflammatory mediator release from purified human lung mast cells. Am Rev Respir Dis 133 : 614- 617 17. Shanahan F, Denburg JA, Fox J, Bienenst,ock J, Befus D (1985) Mast cell heterogeneity: effects Of neuroenteric peptides on histamine release. J Immunol 135:1331-1337

362 18. Sullivan TJ, Parker KL, Stenson W, Parker CW (1975) Modulation of cyclic AMP in purified rat mast cells. J hnmunol 114:1473-1479 19. Tsuruta Y, Kohashi K, Ohkura Y (1978) Determination of histamine in plasma by high-speed liquid chromatography. J Chromatogr 146: 4 9 0 - 493 20. Uvn/is B, Thon I-L (1959) Isolation of"biologically intact" mast cells. Exp Cell Res 18: 512- 520

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Received December 24, 1987