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Further characterization of dispersed human sinus mast cells
Life Sciences, Vol. 53, pp. 775-782 Printed in the USA
FURTHER
Pergamon Press
CHARACTERIZATION
Haruhisa
Mira,
OF DISPERSED
HUMAN
SINUS
T o y o t a Ishii, T e t s u o Y a m a d a , and Takao Shida
Clinical Research 18-1 S a k u r a d a i ,
Center, N a t i o n a l S a g a m i h a r a City,
MAST
CELLS
Kazuo Akiyama,
Sagamihara Hospital, K a n a g a w a 228, J a p a n
(Received in final form June 22, 1993) Summary We have examined biochemical and functional characteristics of dispersed sinus mast cells and compared them with those of mast cells dispersed from other tissues. This experiment yielded the following results, i) Although no difference was observed in h i s t a m i n e content, tryptase content in sinus mast cells was significantly l o w e r t h a n that of s k i n a n d l u n g m a s t cells. 2) In c o n t r a s t w i t h the situation in foreskin mast cells, anti-IgE-induced histamine release from sinus and lung mast cells was potentiated with lower concentrations of adenosine. 3) Similar to lung mast cells, sinus mast cells did not respond to compound 48/80 or s u b s t a n c e P, w h e r e a s s k i n m a s t c e l l s w e r e s t i m u l a t e d to release histamine with either i0 ~ g/ml of compound 48/80 (14.0%) or 10-4M of substance P (23.4%). 4) S i n u s mast cells are similar to l u n g m a s t c e l l s in t e r m s of r e l e a s e of a r a c h i d o n i c acid metabolites. Anti-IgE challenge of s i n u s m a s t c e l l s caused the generation of both prostaglandin D2 (89.5 ± 33.7 ng/10 ~ mast cells, n=14) and i-leukotriene D4 (78.7 ± 46.8 ng/108 mast cells, n=10).
The concept of m a s t cell h e t e r o g e n e i t y is w e l l e s t a b l i s h e d in rodent, and two subpopulations of mast cells, which are referred to as m u c o s a l and connective t i s s u e types, have been defined on the basis of morphological and functional differences [i]. Similar heterogeneity has been reported in human mast cells and, as a n e x a m p l e of c l a s s i f i c a t i o n , human mast cells can be divided into two types depending on the differences in n e u t r a l p r o t e a s e c o n t e n t s [2, 3, 4]. T - m a s t c e l l (MCr), which contains only tryptase, is the predominant type f o u n d in the l u n g a n d i n t e s t i n a l m u c o s a , a n d T C - m a s t cell (MCrc), which contains tryptase together with chymase, is the most common type observed in the skin and intestinal submucosa. MCT a n d MCTc have been considered to c o r r e s p o n d to the r o d e n t mucosal mast cell and connective tissue mast cell, respectively. Besides neutral protease contents, mast cells 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Characterization of Sinus Mast Ceils
f r o m the l u n g a r e in v a r i o u s a s p e c t s .
considerably
different
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from
skin
mast
cells
W e h a v e r e c e n t l y r e p o r t e d a m e t h o d for p r o d u c i n g s i n g l e - c e l l suspensions of m a s t c e l l s f r o m h u m a n s i n u s m u c o s a [5], b e c a u s e sinus mucosa is readily available for investigation of m a s t cells. However, it is u n c l e a r w h e t h e r s i n u s m a s t c e l l s b e l o n g to e i t h e r of the t y p e s w i t h r e s p e c t to m a s t cell h e t e r o g e n e i t y . In t h e p r e s e n t study, w e h a v e a d d r e s s e d t h r e e a s p e c t s in o r d e r to characterize dispersed s i n u s m a s t cells: h i s t a m i n e r e l e a s e w i t h some secretagogues; e f f e c t of a d e n o s i n e o n m e d i a t o r release; a n d g e n e r a t i o n of a r a c h i d o n i c a c i d m e t a b o l i t e s . Materials
and Methods
Chemicals: Reagents were purchased from the following suppliers: collagenase from Chlostridium histolycum ( c o l l a g e n a s e A, B o e h r i n g e r Mannheim, W. G e r m a n y ) ; hyaluronidase ( t y p e l-S, Sigma, U.S.A.); deoxyribonuclease (type ~ , Sigma, U.S.A.); affinity-purified goat antibody to human IgE (~) (anti-IgE, K i r k e g a a d & P e r r y Lab. Inc., U . S . A ) ; fatty-acid-free human serum albumin (HSA, S i g m a ) ; s u b s t a n c e P (Sigma, U.S.A.); compound 48/80 (Sigma, U.S.A.); adenosine (Tokyo Kasei Kogyo, Japan); prostaglandin Dz (pGDz) radioimmunoassay kit (Amersham, UK); peptidyl-leukotriene (LT) radioimmunoassay kit (New England Nuclear, U.S.A.), The manufacturer suggested that the a m o u n t s r e q u i r e d for 50% i n h i b i t i o n of 3H-LTE4 b i n d i n g is 0 . 0 7 5 n g for LTC4, 0 . 0 7 0 n g for LTD4, a n d 0 . 1 6 0 ng for LTE4. S i n c e the standard curve was constructed b y u s i n g LTD4, the r e s u l t s w e r e e x p r e s s e d as i m m u n o r e a c t i v e - L T D 4 (i-LTD4). Preparation of dispersed cells: Human sinus mucosa was obtained at the time of surgery in patients with chronic sinusitis as described previously [5]. None of the patients suffered with atopic diseases or asthma and the cause could not be disclosed. F o r e s k i n w a s o b t a i n e d f r o m c i r c u m c i s i o n s of 4- to 16-year old subjects (mean 6.9 y e a r s old) a n d h u m a n lung m a s t cells were isolated from normal-appearing areas of lungs removed for lung carcinoma or pneumothorax. The procedure of enzymatic dispersion of the t i s s u e w a s s l i g h t l y m o d i f i e d from the previous experiment. Briefly, the tissue was dipped into 0.05% chlorhexidine gluconate for a few seconds and then processed by enzymatic dispersion with collagenase, hyaluronidase and deoxyribonuclease in calcium-free Tyrode's buffer containing gentamicin ( 5 0 ~ g/ml). The cells obtained from the two-step digestion were suspended in RPMI 1640 containing fetal calf serum (10%) and cultured in h u m i d i f i e d 95% air, 5% CO2 at 37°C. About 20% of the total histamine present in the t i s s u e f r a g m e n t s w a s r e c o v e r e d in the d i s p e r s e d cells. The overnight survival rate was 78.9 ± 24.0% (n=19), a n d the m a s t c e l l p u r i t i e s r a n g e d f r o m 1 to 2 3 . 8 % (5.0 ± 5.5%, n=17). Mast cell activation: After overnight culture, cells were harvested and washed twice with Tyrode's buffer containing 0.1% HSA. T h e c e l l s w e r e s u s p e n d e d at a b o u t 5 x i 0 3 ~ 1 x 104 m a s t cells in 1 ml of the same buffer and then challenged with various concentrations of a n t i - I g E (5, i, 0.i or 0.01 ~ g/ml), substance P (10-4M) o r c o m p o u n d 4 8 / 8 0 (I0 ~ g/ml) for 20 m i n at 37°C. H i s t a m i n e w a s a l w a y s r e l e a s e d f r o m m a s t c e l l s b y a n t i - l g E
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777
challenge without sensitizing the cells with atopic serum. i-LTD4 and pGD2 in the supernatant were measured using radioimmunoassays without further purification. In p h a r m a c o l o g i c studies, a g o n i s t s w e r e a d d e d 5 m i n b e f o r e a d d i t i o n of a n t i - l g E (i ~ g/ml). T h e t o t a l h i s t a m i n e c o n t e n t w a s d e t e r m i n e d b y c e l l l y s i s in 0.4 N p e r c h l o r i c acid. Spontaneous histamine release, which was assessed with addition of buffer instead of stimulants, was always below 10% of total histamine content. The results were expressed as p e r c e n t a g e r e l e a s e of the t o t a l histamine after correction of the spontaneous release. Histamine was measured with a Technicon autoanalyzer and tryptase was measured with a commercial radioimmunoassay kit (Pharmacia, Sweden) after sonication of the cells with the B r a n s o n cell d i s r u p t e r . Metabolism of ~H-LTC4: Metabolism of LTC4 was studied by i n c u b a t i o n of d i s p e r s e d c e l l s (1.52 ± 0.57 x l0 B cells, 1.82 ± 0 . 2 4 x 104 m a s t cells) w i t h 3H-LTC4 (18.5 kBq, 7.8 ng) for 20 min in the p r e s e n c e of a n t i - l g E (5~ g/ml). After addition of authentic LTC4, LTD4 a n d LTE4 as c a r r i e r s , the supernatant was analyzed using a NOVA-PAK C18 column (Waters) with a solvent mixture of acetonitrile:methanol:distilled water:acetic acid= 336:54:610:i0, v / v ( a d j u s t e d to pH 5.6 w i t h t r i e t h y l a m i n e ) [6]. Elutions were collected in 0.5 ml fractions and the radioactivity of each fraction was measured by a liquid scintillation spectrometer. S t a t i s t i c a l a n a l y s i s : V a l u e s in the text a r e m e a n ± S.D., a n d the statistical analysis of the data was performed with S t u d e n t ' s t - t e s t u s i n g S t a t V i e w ( A b a c u s C o n c e p t s , Inc. U . S . A . ) . Results
and Discussion
H i s t a m i n e c o n t e n t in s i n u s m a s t c e l l s w a s 5.3 ± 2.1 p g / m a s t c e l l (n=17), w h i c h is s i m i l a r to t h a t of s k i n m a s t c e l l s (3.8 ± 1.6, n=7), and lung mast cells (4.5 ± 0.6, n=4). Tryptase c o n t e n t of sinus, s k i n a n d l u n g m a s t c e l l s w a s 14.4 ± 5.4, 3 8 . 7 ± 18.3 and 60.0 ± 37.2 pg/mast cell, respectively, and the c o n t e n t in s i n u s m a s t c e l l s w a s s i g n i f i c a n t l y l o w e r t h a n that of s k i n a n d l u n g m a s t cell ( p < 0 . 0 0 1 ) . T r y p t a s e c o n t e n t in l u n g m a s t cells was higher than that of previous data reporting that tryptase was contained in l u n g m a s t c e l l s at a level of 10.8 p g / m a s t c e l l s [3]. T h e r e a s o n for t h e d i f f e r e n c e s is not clear. The observations that skin mast cells differ from mast cells isolated from other human tissues in their reactivity to secretagogues and their susceptibility to drugs inhibiting histamine release prompted us to e x a m i n e the c h a r a c t e r i z a t i o n o f m a s t c e l l s o b t a i n e d f r o m d i f f e r e n t a n a t o m i c a l s i t e s [7-13]. We evaluated the effect of adenosine on anti-IgE-induced histamine release from dispersed sinus mast cells. Adenosine possesses a unique property of modulating the release of histamine in a h e t e r o g e n e o u s fashion by interacting with the receptors. At least two types of cell surface adenosine r e c e p t o r s h a v e b e e n i d e n t i f i e d o n the b a s i s of p h a r m a c o l o g i c a l and biochemical criteria; that is, h i g h - a f f i n i t y receptors and low affinity-receptors. High-affinity receptors for adenosine are inhibitory to adenylate cyclase and thereby enhance immunologically stimulated histamine release. Low-affinity
778
Characterization of Sinus Mast Cells
Vol. 53, No. 10, 1993
receptors activate adenylate cyclase and increase cyclic AMP levels. The exact mechanisms of a c t i o n in m o d u l a t i n g histamine release have not been fully defined and studies with adenosine uptake blockers revealed that adenosine may inhibit histamine release, at least in part, by intracellular mechanisms. Although adenosine inhibits IgE-mediated histamine release from human basophils through the activation of low-affinity receptors, modulation of a d e n o s i n e on histamine release varies among mast cells obtained from different anatomical sites. In contrast to t h e f i n d i n g s in b a s o p h i l s , a biphasic response to adenosine is observed in human lung mast cells in which histamine release was attenuated with higher concentrations of adenosine and potentiated with low concentrations [14, 15, 16]. However, contradictory f i n d i n g s h a v e b e e n r e p o r t e d b y M a r o n e et al. w h o f o u n d that a d e n o s i n e p r o d u c e d a b e l l - s h a p e d i n c r e a s e of anti-IgE-induced histamine release in human lung mast cells without inhibitory effect [17]. On the o t h e r hand, adenosine exerts only inhibitory effects on histamine release from foreskin mast cells. In 3 different preparations, adenosine inhibited anti-IgE induced histamine release from foreskin mast cells and the c o n t r o l h i s t a m i n e r e l e a s e (27.7 ± 15.5%, n=3) w a s s i g n i f i c a n t l y diminished by 60.5 ± 22.6% (p=0.043) after pretreating the cells with 10-3M adenosine, results which were consistent with t h o s e of M a r o n e a n d c o - w o r k e r s [17]. P r e i n c u b a t i o n of l u n g m a s t cells with 10-SM or 10-SM adenosine resulted in 13.4 ± 5.0% (p=0.013) and 14.8 ± 10.6% (p=0.068) increase of histamine release over the control release (25.1 ± 14.2%, n=4), respectively. In c o n t r a s t , a d e n o s i n e at c o n c e n t r a t i o n s of 1 0 - 3 M inhibited the r e l e a s e of h i s t a m i n e b y 39.7 ± 2 3 . 1 % (p=0.041). O u r r e s u l t s c o n f i r m t h e f i n d i n g s o f P e t e r s et al. [14] but a r e in c o n f l i c t w i t h t h e r e p o r t of M a r o n e et al. s u g g e s t i n g that adenosine did not inhibit histamine r e l e a s e in l u n g m a s t c e l l s [17]. In s i n u s m a s t cells, a b i p h a s i c r e s p o n s e to a d e n o s i n e w a s always observed. Thus, at low concentrations of adenosine, potentiation o f the r e l e a s e o f h i s t a m i n e w a s o b s e r v e d a n d the enhancement over the control release (40.9 ± 22.0, n=6) was 16.9 ± 13.4% at 10-SM (p=0.027) in 6 preparations from different donors, whereas at higher concentrations, a counteractive inhibitory process was evident (17.2 ± 15.2% at 10-4M, p=0.040 and 80.2 ± 13.2% at 10-3M, p=0.0001). These observations suggest that sinus mast cells resemble lung mast cells r a t h e r t h a n f o r e s k i n m a s t c e l l s w i t h r e g a r d to r e s p o n s e to a d e n o s i n e . S k i n m a s t c e l l s a r e q u i t e r e s p o n s i v e to s e v e r a l s t i m u l i a n d a r e s t i m u l a t e d to r e l e a s e h i s t a m i n e b y n e u r o p e p t i d e substance P or by other basic secretagogues s u c h as m o r p h i n e and compound 48/80. H o w e v e r , t h e o t h e r d i s p e r s e d m a s t c e l l s s u c h as of lung, adenoids, tonsils, intestine and uterus differ markedly in their susceptibility to t h e s e s e c r e t a g o g u e s [7, 8, 9, 18, 19]. This provides clear evidence of functional heterogeneity between human mast cell subpopulations. In 16 d i f f e r e n t e x p e r i m e n t s u s i n g d i s p e r s e d s i n u s m a s t cells, anti-lgE induced a concentration-dependent r e l e a s e of h i s t a m i n e t h a t r e a c h e d a m a x i m u m of 38.8 ± 2 1 . 8 % at 1 o r 5 ~ g/ml o f anti-IgE. When secretagogues t h a t a r e k n o w n to s t i m u l a t e skin m a s t c e l l s w e r e used, s u b s t a n c e P (I0 4M) o r c o m p o u n d 4 8 / 8 0 (I0
Vol. 53, No. 10, 1993
Characterization of Sinus Mast Cells
~(D
Compound 48/80
Substance P
Anti-lgE 80 ~" 7060. (n 504
779
p
p < 0.001
I
I
I
|
I
p < 0.05
p < 0.05
I
|
I
I
40-
~- 30"~ 20l-J ~ 100
| | | i
Sinus
i
Lung
Histamine foreskin anti-IgE,
i
Foreskin
Sinus
Lung
Foreskin
Sinus
Lung
Foreskin
FIG. I. r e l e a s e f r o m d i s p e r s e d sinus, l u n g a n d mast cells by stimulation with s u b s t a n c e P o r c o m p o u n d 48/80.
g/ml) r e l e a s e d 3.6 ± 3.8% a n d 2.2 ± 1.3% of h i s t a m i n e a b o v e the spontaneous release, respectively. Lung mast cells showed s l i g h t h i s t a m i n e r e l e a s e w i t h s u b s t a n c e P (4.5 ± 3.5%, n=3) a n d compound 48/80 (0.4 ± 0.5%). By contrast, skin mast cells showed significantly higher histamine release with either 10-4M o f s u b s t a n c e P (23.4 ± 12.9%, n=5, p < 0 . 0 0 1 ) o r w i t h 1 0 u g/ml of compound 48/80 (14.0 ± 8.5%, n=7, p<0.01) as compared with s i n u s m a s t cells. S k i n m a s t c e l l s a n d l u n g m a s t c e l l s r e l e a s e d 23.5 ± 17.8% and 32.5 ± 9.3% of histamine with anti-IgE challenge, respectively, which did not significantly differ f r o m t h a t of s i n u s m a s t cells. In addition to differences to secretory response to nonimmunological stimulation, lung mast cells and skin mast cells also differ in their ability to generate eicosanoids after immunological stimulation. Mast cells respond to anti-IgE with histamine r e l e a s e a n d the c o n c o m i t a n t generation of pGD,. PGD2 is the most abundant cyclooxygenase product g e n e r a t e d a n d r e l e a s e d a f t e r s t i m u l a t i o n of h u m a n m a s t cells. It w a s s u g g e s t e d t h a t pGD, g e n e r a t i o n p e r 108 m a s t c e l l s w a s 3 9 . 5 o r 114.8 ng in l u n g [20, 21], 4 4 . 6 n g in s y n o v i a l [22], 89 n g in uterine [23] a n d 43 n g in s k i n [24], w h i l e a m a r k e d d i f f e r e n c e was observed between skin mast cells and other mast cells in the production o f i-LTC4. A f t e r s t i m u l a t i o n with anti-lgE, the a m o u n t o f i-LTC4 in l u n g [21], synovial [22] a n d u t e r i n e [23] mast cells was 64.6, 20.4 a n d 45 ng/106 cells, respectively, w h i l e s k i n m a s t c e l l s h a v e a v e r y l i m i t e d a b i l i t y to m e t a b o l i z e arachidonic a c i d to LTC4 a n d n o d e t e c t a b l e g e n e r a t i o n of i-LTC4 was observed in s k i n m a s t cells [8]. Thus, experiments were carried out to measure eicosanoid production in sinus mast cells following anti-IgE challenge. First, we demonstrated w h a t t y p e o f LTs e x i s t e d at the e n d of the incubation period, b e c a u s e the c o n v e r s i o n of LTC4 to D4 and E4 might take place rapidly during incubation. In 3 different experiments, the radioactivity was eluted with LTC4 (10.7, 32.9, 35.4%; m e a n 26.3%), LTD4 (65.5, 43.8, 48.1%; m e a n
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Characterization of Sinus Mast Cells
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52.5%) and LTE4 (13.8, 7.5, 8.2%; mean 9.8%). These results suggest that the conversion of LTC4 to LTD4 was the major metabolic pathway, and a little LTE4 was found in the supernatant in m o s t cases, although different specimens showed a variable pattern of c o n v e r s i o n . Since it h a s b e e n reported t h a t LTC4 g e n e r a t e d is not f u r t h e r m e t a b o l i z e d b y p u r i f i e d m a s t cells [24, 25], the m e t a b o l i s m of LTC4 m a y be d u e to o t h e r cells rather than mast cells. Peptidyl-LTs can readily adsorb to various surfaces from an aqueous solution. However, more t h a n 85% of 3 H - r a d i o a c t i v i t y was recovered in the media, thus excluding the p o s s i b i l i t y of a d s o r p t i o n of LTs. In a d d i t i o n , peptidyl-LTs a r e k n o w n to be u n s t a b l e m o l e c u l e s that undergo a variety of oxidative reactions [26]. However, most of the radioactivity r e c o v e r e d in the s u p e r n a t a n t w a s e l u c i d a t e d to be LTC4, LTD4 a n d LTE4, s u g g e s t i n g t h a t d e c o m p o s i t i o n of LTC4 into p r o d u c t s o t h e r t h a n LTD4 a n d LTE4 is n e g l i g i b l y s m a l l u n d e r the experimental conditions. To determine the kinetics of m e d i a t o r release by anti-IgE c h a l l e n g e , d i s p e r s e d s i n u s m a s t c e l l s w e r e i n c u b a t e d w i t h 5 ~ g/ ml of a n t i - I g E for v a r i o u s d u r a t i o n s (2, 5, i0, 20, 30, 40 min), and the products released in the supernatants were measured. The kinetics of m e d i a t o r release after stimulation were quite similar to t h o s e of l u n g m a s t cells [27], in w h i c h h i s t a m i n e release occurred rapidly and reached a maximum within 15 min. The time to reach 50% of the m a x i m u n histamine release was between 1 and 2 min. Generation of pGD2 and i-LTD4 occurred more slowly and the half-maximal generation was achieved b e t w e e n 3 a n d 5 min, r e s p e c t i v e l y . The generation was completed w i t h i n 20 min. In 3 of 7 d i f f e r e n t e x p e r i m e n t s , the d o s e - r e s p o n s e c u r v e for histamine release could not be distinguished from that for pGD2 a n d i-LTD4 generation. O n t h e o t h e r hand, in the o t h e r 4 cases, the o p t i m a l concentration of a n t i - l g E for pGD2 release w a s a b o u t 1 0 - f o l d l o w e r t h a n t h a t r e q u i r e d for the r e l e a s e of histamine a n d i-LTD4, w h i c h is in a c c o r d a n c e w i t h the f i n d i n g s of S c h l e i m e r a n d c o - w o r k e r s [27]. Based on these results, dispersed cells were challenged with several concentrations of anti-IgE (5 ~ 0.01 ~ g/ml) for 20 min., and the amounts of histamine, pGD2 and i-LTD4 were measured in t h e supernatant. Table I summarizes the results. S i n u s m a s t c e l l s r e s u l t e d in 52.0 ± 20.0% of h i s t a m i n e r e l e a s e w i t h a r a n g e of 13.4 ~ 8 8 . 3 % (n=14). A n t i - I g E a l s o c a u s e d the generation of 8 9 . 5 ± 33.7 n g o f pGD2/106 mast cells (ranging f r o m 34.1 to 150.8) w h i c h w a s a l e v e l s i m i l a r to t h a t o b s e r v e d in uterine mast cells [23]. Although there was a slight detectable rise in spontaneous pGD2 release in 6 of 14 specimens (2.8 ng/106 mast cells), anti-lgE enhanced the generation by 7.8 ~ 1 8 8 . 5 - f o l d . A n t i - I g E a l s o i n d u c e d i-LTD4 generation from sinus mast cells and the amount ranged from 19.2 to 182.3 ng/106 m a s t c e l l s (78.7 ± 46.8, n = 1 0 ) . T h e r e w a s a large variation in the capacity of sinus mast cells from different specimens to generate i-LTD4. Spontaneous LTD4 production was not detected in a n y of the i0 s p e c i m e n s . Sinus mast cells produced pGD2 and i-LTD4 in q u a n t i t i e s similar to those of lung mast cells. These mast cells obtained from different anatomical sites differed with respect to i-LTD4 release. Unlike l u n g a n d s i n u s m a s t cells, a small a m o u n t of
Vol. 53, No. 10, 1993
Characterization of Sinus Mast Cells
TABLE
781
I
A n t i - I g E - i n d u c e d h i s t a m i n e , p r o s t a g l a n d i n D~ a n d i - l e u k o t r i e n e D4 r e l e a s e f r o m d i s p e r s e d sinus, l u n g a n d f o r e s k i n m a s t cells. Histamine (%) Sinus
Lung
Foreskin
52.0 (3.5 n=14 32.5 (3.6 n=5 32.0 (3.6 n=5
Prostaglandin (ng/108
± 20 _+ 4.2)
Dz i-Leukotriene m a s t cells)
8 9 . 5 ± 33.7 (< 4.8) n=14 126.5 ± 72.7 (< 3.7) n=5 1 0 4 . 4 ± 84.3 (< 4.9) n=4
± 8.0 ± 2.0) ± 15.1 ± 1.7)
F i g u r e s in p a r e n t h e s i s each mediator. i-LTD4 could be detected s p e c i m e n s (8.5, 4.6, <3, <3;
show
in 4.8
spontaneous
D4
78.7 ± 4 6 . 8 (< 4.0) n=10 78.0 ± 52.8 (< 3.7) n=5 4.8 ± 2.6 (< 3.5) n=4 release
skin mast cells ± 2.6 ng/106 m a s t
of
in two cells).
of
4
When sinus mast cells were stimulated at optimal concentrations of anti-IgE, the release of histamine was significantly correlated with both pGD2 release (r=0.70, p<0.001, n=13) and i-LTD4 release (r=0.85, p<0.02, n=9). The maximal r e l e a s e of pGDz w a s a l s o s i g n i f i c a n t l y correlated with that of i-LTD4 (r=0.82, p<0.05, n=13). The observations that there were significant correlations among the release of histamine, pGD2 and i-LTD4 agrees with previous reports [18, 27, 28], s u g g e s t i n g t h a t t h e s e m e d i a t o r s are derived from mast cells. In s u m m a r y , sinus mast cells dispersed f r o m s i n u s m u c o s a of chronic sinusitis patients differed from skin and lung mast cells with respect to t r y p t a s e content. Sinus mast cells are s i m i l a r to l u n g m a s t c e l l s in t e r m s of p h a r m a c o l o g i c m o d u l a t i o n of h i s t a m i n e release with adenosine, response to s e c r e t a g o g u e s such as compound 48/80 and substance P, and release of arachidonic acid metabolites. References i. L. E N E R B A C K , Int. A r c h s A l l e r g y A p p l . Immunol. 82 2 4 9 - 2 5 5 (1987). 2. L.B. S C H W A R T Z , Ann. A l l e r g y 58 2 2 6 - 2 3 5 (1987). 3. L.B. S C H W A R T Z , T.R. B R A D F O R D , A - M . A . IRANI, G. D E B L O I S a n d S.S. CRAIG, Am. Rev. R e s p i r . Dis. 135 1 1 8 6 - 1 1 8 9 (1987). 4. L.B. S C H W A R T Z , A - M . A . IRANI, K. R O L L E R , M.C. C A S T E L L S and N.M. S C H E C H T E R , J. Immunol. 138 2 6 1 1 - 2 6 1 5 (1987). 5. H. MITA, T. ISHII, K. A K I Y A M A , T. T A M U R A , K. T A D O K O R O , H. SAITO, H. Y A S U E D A a n d T. SHIDA, L i f e Sci. 49 1 4 7 7 - 1 4 8 4 (1991). 6. M. ABE, Y. K A W A Z O E a n d N. S H I G E M A T S U , Anal. B i o c h e m . 144 4 1 7 - 4 2 2 (1985). 7. R~C. BENYON, M.A. L O W M A N a n d M.K. CHURCH, J. Immunol. 138
782
8.
9. i0. ii. 12. 13. 14.
15. 16. 17. 18. 19. 20.
21. 22.
23.
24. 25.
26. 27.
28.
Characterization of Sinus Mast Cells
Vol. 53, No. 10, 1993
8 6 1 - 8 6 7 (1987). I.D. L A W R E N C E a n d J.A. WARNET, V.L. COHAN, W.C. HUBBARD, A. K A G E Y - S O B O T K A a n d L.M. L I C H T E N S T E I N , J. Immunol. 139 3 0 6 2 - 3 0 6 9 (1987). M.K. CHURCH, S. E I - L A T I and J.P. C A U L F I E L D , Int. A r c h s A l l e r g y Appl. Immunol. 94 3 1 0 - 3 1 8 (1991). A - M . A . IRANI a n d L.B. S C H W A R T Z , Clin. Exp. A l l e r g y 19 1 4 3 - 1 5 5 (1989). M.K. CHURCH, S. EL-LATI, a n d Y. O K A Y A M A , Clin. Exp. A l l e r g y 21 (suppl 3) 1-9 (1991). R.C. BENYON, M.K. CHURCH, L.S. C L E G G and S.T. H O L G A T E , Int. A r c h s A l l e r g y Appl. Immunol. 79 3 3 2 - 3 3 4 (1986). R.C. BENYON, Clin. Exp. A l l e r g y 19 3 7 5 - 3 8 7 (1989). S.P. PETERS, E.S. S C H U L M A N , R.P. S C H L E I M E R , D.W. M A C G L A S H A N JR., H.H. N E W B A L L a n d L.M. L I C H T E N S T E I N , Am. Rev. Respir. Dis. 126 1 0 3 4 - 1 0 3 9 (1982). P.T. P E A C H E L L , L.M. L I C H T E N S T E I N a n d R.P. S C H L E I M E R , B i o c h e m . P h a r m a c o l . 38 1 7 1 7 - 1 7 2 5 (1989). P.T. P E A C H E L L , L.M. L I C H T E N S T E I N a n d R.P. S C H L E I M E R , J. P h a r m a c o l . Exp. Ther. 256 7 1 7 - 7 2 6 (1991). G. MARONE, R. C I R I L L O , A. G E N O V E S E , O. M A R I N O a n d S. Q U A T T R I N , Life Sci. 45 1 7 4 5 - 1 7 5 4 (1989). R.C. BENYON, C. R O B I N S O N a n d M.K. CHURCH, Br. J. P h a r m a c o l . 97 8 9 8 - 9 0 4 (1989). M.K. CHURCH, G.J-K. P A O and S.T. HOLGATE, J. Immunol. 129 2 1 1 6 - 2 1 2 1 (1982). R.A. LEWIS, N.A. SOTER, P.T. D I A M O N D , K.F. AUSTEN, J.A. O A T E S a n d L.J. R O B E R T S II, J. Immunol. 129 1627-1631 (1982). E.S. S C H U L M A N , R.B. POLLACK, T.J. P O S T and S.P. PETERS, J. Immunol. 144 4 1 9 5 - 4 2 0 1 (1990). J.A. K O P I C K Y - B U R D , A. K A G E Y - S O B O T K A , S.P. PETERS, A.M. DVORAK, D.W. LENNOX, L.M. L I C H T E N S T E I N a n d F.M. WIGLEY, J. R h e u m a t o l . 15 1 3 2 6 - 1 3 3 3 (1988). W.A. MASSAY, C-B. GUO, A.M. DVORAK, W.C. H U B B A R D , B.S. B H A G A V A N , V.L. COHAN, J.A. W A R N E R , A. K A G E Y - S O B O T K A a n d L.M. L I C H T E N S T E I N , J. Immunol. 147 1 6 2 1 - 1 6 2 7 (1991). E.S. S C H U L M A N , R.B. POLLACK, T.J. P O S T and S.P. PETERS, J. Immunol. 144 4 1 9 5 - 4 2 0 1 (1990). S.P. PETERS, D.W. M A C G L A S H A N JR., E.S. SCHULMAN, R.P. S C H L E I M E R , E.C. HAYES, J. ROKASH, N.F. A D K I N S O N JR. a n d L.M. L I C H T E N S T E I N , J. Immunol. 132 1 9 7 2 - 1 9 7 9 (1984). J.Y. W E S C O T T , K.L. C L A Y a n d R.C. MURPHY, J. A l l e r g y Clin. Immunol. 74 3 6 3 - 3 6 8 (1984). R.P. S C H L E I M E R , D.W. M A C G L A S H A N JR., S.P. PETERS, R.N. PINCKARD, N.F. A D K I N S O N JR. a n d L.M. L I C H T E N S T E I N , Am. Rev. R e s p i r . Dis. 133 6 1 4 - 6 1 7 (1986). S.T. H O L G A T E , G.B. BURNS, C. R O B I N S O N and M.K. CHURCH, J. I~maunol. 133 2 1 3 8 - 2 1 4 4 (1984).
FURTHER
Pergamon Press
CHARACTERIZATION
Haruhisa
Mira,
OF DISPERSED
HUMAN
SINUS
T o y o t a Ishii, T e t s u o Y a m a d a , and Takao Shida
Clinical Research 18-1 S a k u r a d a i ,
Center, N a t i o n a l S a g a m i h a r a City,
MAST
CELLS
Kazuo Akiyama,
Sagamihara Hospital, K a n a g a w a 228, J a p a n
(Received in final form June 22, 1993) Summary We have examined biochemical and functional characteristics of dispersed sinus mast cells and compared them with those of mast cells dispersed from other tissues. This experiment yielded the following results, i) Although no difference was observed in h i s t a m i n e content, tryptase content in sinus mast cells was significantly l o w e r t h a n that of s k i n a n d l u n g m a s t cells. 2) In c o n t r a s t w i t h the situation in foreskin mast cells, anti-IgE-induced histamine release from sinus and lung mast cells was potentiated with lower concentrations of adenosine. 3) Similar to lung mast cells, sinus mast cells did not respond to compound 48/80 or s u b s t a n c e P, w h e r e a s s k i n m a s t c e l l s w e r e s t i m u l a t e d to release histamine with either i0 ~ g/ml of compound 48/80 (14.0%) or 10-4M of substance P (23.4%). 4) S i n u s mast cells are similar to l u n g m a s t c e l l s in t e r m s of r e l e a s e of a r a c h i d o n i c acid metabolites. Anti-IgE challenge of s i n u s m a s t c e l l s caused the generation of both prostaglandin D2 (89.5 ± 33.7 ng/10 ~ mast cells, n=14) and i-leukotriene D4 (78.7 ± 46.8 ng/108 mast cells, n=10).
The concept of m a s t cell h e t e r o g e n e i t y is w e l l e s t a b l i s h e d in rodent, and two subpopulations of mast cells, which are referred to as m u c o s a l and connective t i s s u e types, have been defined on the basis of morphological and functional differences [i]. Similar heterogeneity has been reported in human mast cells and, as a n e x a m p l e of c l a s s i f i c a t i o n , human mast cells can be divided into two types depending on the differences in n e u t r a l p r o t e a s e c o n t e n t s [2, 3, 4]. T - m a s t c e l l (MCr), which contains only tryptase, is the predominant type f o u n d in the l u n g a n d i n t e s t i n a l m u c o s a , a n d T C - m a s t cell (MCrc), which contains tryptase together with chymase, is the most common type observed in the skin and intestinal submucosa. MCT a n d MCTc have been considered to c o r r e s p o n d to the r o d e n t mucosal mast cell and connective tissue mast cell, respectively. Besides neutral protease contents, mast cells 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
776
Characterization of Sinus Mast Ceils
f r o m the l u n g a r e in v a r i o u s a s p e c t s .
considerably
different
Vol. 53, No. 10, 1993
from
skin
mast
cells
W e h a v e r e c e n t l y r e p o r t e d a m e t h o d for p r o d u c i n g s i n g l e - c e l l suspensions of m a s t c e l l s f r o m h u m a n s i n u s m u c o s a [5], b e c a u s e sinus mucosa is readily available for investigation of m a s t cells. However, it is u n c l e a r w h e t h e r s i n u s m a s t c e l l s b e l o n g to e i t h e r of the t y p e s w i t h r e s p e c t to m a s t cell h e t e r o g e n e i t y . In t h e p r e s e n t study, w e h a v e a d d r e s s e d t h r e e a s p e c t s in o r d e r to characterize dispersed s i n u s m a s t cells: h i s t a m i n e r e l e a s e w i t h some secretagogues; e f f e c t of a d e n o s i n e o n m e d i a t o r release; a n d g e n e r a t i o n of a r a c h i d o n i c a c i d m e t a b o l i t e s . Materials
and Methods
Chemicals: Reagents were purchased from the following suppliers: collagenase from Chlostridium histolycum ( c o l l a g e n a s e A, B o e h r i n g e r Mannheim, W. G e r m a n y ) ; hyaluronidase ( t y p e l-S, Sigma, U.S.A.); deoxyribonuclease (type ~ , Sigma, U.S.A.); affinity-purified goat antibody to human IgE (~) (anti-IgE, K i r k e g a a d & P e r r y Lab. Inc., U . S . A ) ; fatty-acid-free human serum albumin (HSA, S i g m a ) ; s u b s t a n c e P (Sigma, U.S.A.); compound 48/80 (Sigma, U.S.A.); adenosine (Tokyo Kasei Kogyo, Japan); prostaglandin Dz (pGDz) radioimmunoassay kit (Amersham, UK); peptidyl-leukotriene (LT) radioimmunoassay kit (New England Nuclear, U.S.A.), The manufacturer suggested that the a m o u n t s r e q u i r e d for 50% i n h i b i t i o n of 3H-LTE4 b i n d i n g is 0 . 0 7 5 n g for LTC4, 0 . 0 7 0 n g for LTD4, a n d 0 . 1 6 0 ng for LTE4. S i n c e the standard curve was constructed b y u s i n g LTD4, the r e s u l t s w e r e e x p r e s s e d as i m m u n o r e a c t i v e - L T D 4 (i-LTD4). Preparation of dispersed cells: Human sinus mucosa was obtained at the time of surgery in patients with chronic sinusitis as described previously [5]. None of the patients suffered with atopic diseases or asthma and the cause could not be disclosed. F o r e s k i n w a s o b t a i n e d f r o m c i r c u m c i s i o n s of 4- to 16-year old subjects (mean 6.9 y e a r s old) a n d h u m a n lung m a s t cells were isolated from normal-appearing areas of lungs removed for lung carcinoma or pneumothorax. The procedure of enzymatic dispersion of the t i s s u e w a s s l i g h t l y m o d i f i e d from the previous experiment. Briefly, the tissue was dipped into 0.05% chlorhexidine gluconate for a few seconds and then processed by enzymatic dispersion with collagenase, hyaluronidase and deoxyribonuclease in calcium-free Tyrode's buffer containing gentamicin ( 5 0 ~ g/ml). The cells obtained from the two-step digestion were suspended in RPMI 1640 containing fetal calf serum (10%) and cultured in h u m i d i f i e d 95% air, 5% CO2 at 37°C. About 20% of the total histamine present in the t i s s u e f r a g m e n t s w a s r e c o v e r e d in the d i s p e r s e d cells. The overnight survival rate was 78.9 ± 24.0% (n=19), a n d the m a s t c e l l p u r i t i e s r a n g e d f r o m 1 to 2 3 . 8 % (5.0 ± 5.5%, n=17). Mast cell activation: After overnight culture, cells were harvested and washed twice with Tyrode's buffer containing 0.1% HSA. T h e c e l l s w e r e s u s p e n d e d at a b o u t 5 x i 0 3 ~ 1 x 104 m a s t cells in 1 ml of the same buffer and then challenged with various concentrations of a n t i - I g E (5, i, 0.i or 0.01 ~ g/ml), substance P (10-4M) o r c o m p o u n d 4 8 / 8 0 (I0 ~ g/ml) for 20 m i n at 37°C. H i s t a m i n e w a s a l w a y s r e l e a s e d f r o m m a s t c e l l s b y a n t i - l g E
Vol. 53, No. 10, 1993
Characterization of Sinus Mast Ceils
777
challenge without sensitizing the cells with atopic serum. i-LTD4 and pGD2 in the supernatant were measured using radioimmunoassays without further purification. In p h a r m a c o l o g i c studies, a g o n i s t s w e r e a d d e d 5 m i n b e f o r e a d d i t i o n of a n t i - l g E (i ~ g/ml). T h e t o t a l h i s t a m i n e c o n t e n t w a s d e t e r m i n e d b y c e l l l y s i s in 0.4 N p e r c h l o r i c acid. Spontaneous histamine release, which was assessed with addition of buffer instead of stimulants, was always below 10% of total histamine content. The results were expressed as p e r c e n t a g e r e l e a s e of the t o t a l histamine after correction of the spontaneous release. Histamine was measured with a Technicon autoanalyzer and tryptase was measured with a commercial radioimmunoassay kit (Pharmacia, Sweden) after sonication of the cells with the B r a n s o n cell d i s r u p t e r . Metabolism of ~H-LTC4: Metabolism of LTC4 was studied by i n c u b a t i o n of d i s p e r s e d c e l l s (1.52 ± 0.57 x l0 B cells, 1.82 ± 0 . 2 4 x 104 m a s t cells) w i t h 3H-LTC4 (18.5 kBq, 7.8 ng) for 20 min in the p r e s e n c e of a n t i - l g E (5~ g/ml). After addition of authentic LTC4, LTD4 a n d LTE4 as c a r r i e r s , the supernatant was analyzed using a NOVA-PAK C18 column (Waters) with a solvent mixture of acetonitrile:methanol:distilled water:acetic acid= 336:54:610:i0, v / v ( a d j u s t e d to pH 5.6 w i t h t r i e t h y l a m i n e ) [6]. Elutions were collected in 0.5 ml fractions and the radioactivity of each fraction was measured by a liquid scintillation spectrometer. S t a t i s t i c a l a n a l y s i s : V a l u e s in the text a r e m e a n ± S.D., a n d the statistical analysis of the data was performed with S t u d e n t ' s t - t e s t u s i n g S t a t V i e w ( A b a c u s C o n c e p t s , Inc. U . S . A . ) . Results
and Discussion
H i s t a m i n e c o n t e n t in s i n u s m a s t c e l l s w a s 5.3 ± 2.1 p g / m a s t c e l l (n=17), w h i c h is s i m i l a r to t h a t of s k i n m a s t c e l l s (3.8 ± 1.6, n=7), and lung mast cells (4.5 ± 0.6, n=4). Tryptase c o n t e n t of sinus, s k i n a n d l u n g m a s t c e l l s w a s 14.4 ± 5.4, 3 8 . 7 ± 18.3 and 60.0 ± 37.2 pg/mast cell, respectively, and the c o n t e n t in s i n u s m a s t c e l l s w a s s i g n i f i c a n t l y l o w e r t h a n that of s k i n a n d l u n g m a s t cell ( p < 0 . 0 0 1 ) . T r y p t a s e c o n t e n t in l u n g m a s t cells was higher than that of previous data reporting that tryptase was contained in l u n g m a s t c e l l s at a level of 10.8 p g / m a s t c e l l s [3]. T h e r e a s o n for t h e d i f f e r e n c e s is not clear. The observations that skin mast cells differ from mast cells isolated from other human tissues in their reactivity to secretagogues and their susceptibility to drugs inhibiting histamine release prompted us to e x a m i n e the c h a r a c t e r i z a t i o n o f m a s t c e l l s o b t a i n e d f r o m d i f f e r e n t a n a t o m i c a l s i t e s [7-13]. We evaluated the effect of adenosine on anti-IgE-induced histamine release from dispersed sinus mast cells. Adenosine possesses a unique property of modulating the release of histamine in a h e t e r o g e n e o u s fashion by interacting with the receptors. At least two types of cell surface adenosine r e c e p t o r s h a v e b e e n i d e n t i f i e d o n the b a s i s of p h a r m a c o l o g i c a l and biochemical criteria; that is, h i g h - a f f i n i t y receptors and low affinity-receptors. High-affinity receptors for adenosine are inhibitory to adenylate cyclase and thereby enhance immunologically stimulated histamine release. Low-affinity
778
Characterization of Sinus Mast Cells
Vol. 53, No. 10, 1993
receptors activate adenylate cyclase and increase cyclic AMP levels. The exact mechanisms of a c t i o n in m o d u l a t i n g histamine release have not been fully defined and studies with adenosine uptake blockers revealed that adenosine may inhibit histamine release, at least in part, by intracellular mechanisms. Although adenosine inhibits IgE-mediated histamine release from human basophils through the activation of low-affinity receptors, modulation of a d e n o s i n e on histamine release varies among mast cells obtained from different anatomical sites. In contrast to t h e f i n d i n g s in b a s o p h i l s , a biphasic response to adenosine is observed in human lung mast cells in which histamine release was attenuated with higher concentrations of adenosine and potentiated with low concentrations [14, 15, 16]. However, contradictory f i n d i n g s h a v e b e e n r e p o r t e d b y M a r o n e et al. w h o f o u n d that a d e n o s i n e p r o d u c e d a b e l l - s h a p e d i n c r e a s e of anti-IgE-induced histamine release in human lung mast cells without inhibitory effect [17]. On the o t h e r hand, adenosine exerts only inhibitory effects on histamine release from foreskin mast cells. In 3 different preparations, adenosine inhibited anti-IgE induced histamine release from foreskin mast cells and the c o n t r o l h i s t a m i n e r e l e a s e (27.7 ± 15.5%, n=3) w a s s i g n i f i c a n t l y diminished by 60.5 ± 22.6% (p=0.043) after pretreating the cells with 10-3M adenosine, results which were consistent with t h o s e of M a r o n e a n d c o - w o r k e r s [17]. P r e i n c u b a t i o n of l u n g m a s t cells with 10-SM or 10-SM adenosine resulted in 13.4 ± 5.0% (p=0.013) and 14.8 ± 10.6% (p=0.068) increase of histamine release over the control release (25.1 ± 14.2%, n=4), respectively. In c o n t r a s t , a d e n o s i n e at c o n c e n t r a t i o n s of 1 0 - 3 M inhibited the r e l e a s e of h i s t a m i n e b y 39.7 ± 2 3 . 1 % (p=0.041). O u r r e s u l t s c o n f i r m t h e f i n d i n g s o f P e t e r s et al. [14] but a r e in c o n f l i c t w i t h t h e r e p o r t of M a r o n e et al. s u g g e s t i n g that adenosine did not inhibit histamine r e l e a s e in l u n g m a s t c e l l s [17]. In s i n u s m a s t cells, a b i p h a s i c r e s p o n s e to a d e n o s i n e w a s always observed. Thus, at low concentrations of adenosine, potentiation o f the r e l e a s e o f h i s t a m i n e w a s o b s e r v e d a n d the enhancement over the control release (40.9 ± 22.0, n=6) was 16.9 ± 13.4% at 10-SM (p=0.027) in 6 preparations from different donors, whereas at higher concentrations, a counteractive inhibitory process was evident (17.2 ± 15.2% at 10-4M, p=0.040 and 80.2 ± 13.2% at 10-3M, p=0.0001). These observations suggest that sinus mast cells resemble lung mast cells r a t h e r t h a n f o r e s k i n m a s t c e l l s w i t h r e g a r d to r e s p o n s e to a d e n o s i n e . S k i n m a s t c e l l s a r e q u i t e r e s p o n s i v e to s e v e r a l s t i m u l i a n d a r e s t i m u l a t e d to r e l e a s e h i s t a m i n e b y n e u r o p e p t i d e substance P or by other basic secretagogues s u c h as m o r p h i n e and compound 48/80. H o w e v e r , t h e o t h e r d i s p e r s e d m a s t c e l l s s u c h as of lung, adenoids, tonsils, intestine and uterus differ markedly in their susceptibility to t h e s e s e c r e t a g o g u e s [7, 8, 9, 18, 19]. This provides clear evidence of functional heterogeneity between human mast cell subpopulations. In 16 d i f f e r e n t e x p e r i m e n t s u s i n g d i s p e r s e d s i n u s m a s t cells, anti-lgE induced a concentration-dependent r e l e a s e of h i s t a m i n e t h a t r e a c h e d a m a x i m u m of 38.8 ± 2 1 . 8 % at 1 o r 5 ~ g/ml o f anti-IgE. When secretagogues t h a t a r e k n o w n to s t i m u l a t e skin m a s t c e l l s w e r e used, s u b s t a n c e P (I0 4M) o r c o m p o u n d 4 8 / 8 0 (I0
Vol. 53, No. 10, 1993
Characterization of Sinus Mast Cells
~(D
Compound 48/80
Substance P
Anti-lgE 80 ~" 7060. (n 504
779
p
p < 0.001
I
I
I
|
I
p < 0.05
p < 0.05
I
|
I
I
40-
~- 30"~ 20l-J ~ 100
| | | i
Sinus
i
Lung
Histamine foreskin anti-IgE,
i
Foreskin
Sinus
Lung
Foreskin
Sinus
Lung
Foreskin
FIG. I. r e l e a s e f r o m d i s p e r s e d sinus, l u n g a n d mast cells by stimulation with s u b s t a n c e P o r c o m p o u n d 48/80.
g/ml) r e l e a s e d 3.6 ± 3.8% a n d 2.2 ± 1.3% of h i s t a m i n e a b o v e the spontaneous release, respectively. Lung mast cells showed s l i g h t h i s t a m i n e r e l e a s e w i t h s u b s t a n c e P (4.5 ± 3.5%, n=3) a n d compound 48/80 (0.4 ± 0.5%). By contrast, skin mast cells showed significantly higher histamine release with either 10-4M o f s u b s t a n c e P (23.4 ± 12.9%, n=5, p < 0 . 0 0 1 ) o r w i t h 1 0 u g/ml of compound 48/80 (14.0 ± 8.5%, n=7, p<0.01) as compared with s i n u s m a s t cells. S k i n m a s t c e l l s a n d l u n g m a s t c e l l s r e l e a s e d 23.5 ± 17.8% and 32.5 ± 9.3% of histamine with anti-IgE challenge, respectively, which did not significantly differ f r o m t h a t of s i n u s m a s t cells. In addition to differences to secretory response to nonimmunological stimulation, lung mast cells and skin mast cells also differ in their ability to generate eicosanoids after immunological stimulation. Mast cells respond to anti-IgE with histamine r e l e a s e a n d the c o n c o m i t a n t generation of pGD,. PGD2 is the most abundant cyclooxygenase product g e n e r a t e d a n d r e l e a s e d a f t e r s t i m u l a t i o n of h u m a n m a s t cells. It w a s s u g g e s t e d t h a t pGD, g e n e r a t i o n p e r 108 m a s t c e l l s w a s 3 9 . 5 o r 114.8 ng in l u n g [20, 21], 4 4 . 6 n g in s y n o v i a l [22], 89 n g in uterine [23] a n d 43 n g in s k i n [24], w h i l e a m a r k e d d i f f e r e n c e was observed between skin mast cells and other mast cells in the production o f i-LTC4. A f t e r s t i m u l a t i o n with anti-lgE, the a m o u n t o f i-LTC4 in l u n g [21], synovial [22] a n d u t e r i n e [23] mast cells was 64.6, 20.4 a n d 45 ng/106 cells, respectively, w h i l e s k i n m a s t c e l l s h a v e a v e r y l i m i t e d a b i l i t y to m e t a b o l i z e arachidonic a c i d to LTC4 a n d n o d e t e c t a b l e g e n e r a t i o n of i-LTC4 was observed in s k i n m a s t cells [8]. Thus, experiments were carried out to measure eicosanoid production in sinus mast cells following anti-IgE challenge. First, we demonstrated w h a t t y p e o f LTs e x i s t e d at the e n d of the incubation period, b e c a u s e the c o n v e r s i o n of LTC4 to D4 and E4 might take place rapidly during incubation. In 3 different experiments, the radioactivity was eluted with LTC4 (10.7, 32.9, 35.4%; m e a n 26.3%), LTD4 (65.5, 43.8, 48.1%; m e a n
780
Characterization of Sinus Mast Cells
Vol. 53, No. 10, 1993
52.5%) and LTE4 (13.8, 7.5, 8.2%; mean 9.8%). These results suggest that the conversion of LTC4 to LTD4 was the major metabolic pathway, and a little LTE4 was found in the supernatant in m o s t cases, although different specimens showed a variable pattern of c o n v e r s i o n . Since it h a s b e e n reported t h a t LTC4 g e n e r a t e d is not f u r t h e r m e t a b o l i z e d b y p u r i f i e d m a s t cells [24, 25], the m e t a b o l i s m of LTC4 m a y be d u e to o t h e r cells rather than mast cells. Peptidyl-LTs can readily adsorb to various surfaces from an aqueous solution. However, more t h a n 85% of 3 H - r a d i o a c t i v i t y was recovered in the media, thus excluding the p o s s i b i l i t y of a d s o r p t i o n of LTs. In a d d i t i o n , peptidyl-LTs a r e k n o w n to be u n s t a b l e m o l e c u l e s that undergo a variety of oxidative reactions [26]. However, most of the radioactivity r e c o v e r e d in the s u p e r n a t a n t w a s e l u c i d a t e d to be LTC4, LTD4 a n d LTE4, s u g g e s t i n g t h a t d e c o m p o s i t i o n of LTC4 into p r o d u c t s o t h e r t h a n LTD4 a n d LTE4 is n e g l i g i b l y s m a l l u n d e r the experimental conditions. To determine the kinetics of m e d i a t o r release by anti-IgE c h a l l e n g e , d i s p e r s e d s i n u s m a s t c e l l s w e r e i n c u b a t e d w i t h 5 ~ g/ ml of a n t i - I g E for v a r i o u s d u r a t i o n s (2, 5, i0, 20, 30, 40 min), and the products released in the supernatants were measured. The kinetics of m e d i a t o r release after stimulation were quite similar to t h o s e of l u n g m a s t cells [27], in w h i c h h i s t a m i n e release occurred rapidly and reached a maximum within 15 min. The time to reach 50% of the m a x i m u n histamine release was between 1 and 2 min. Generation of pGD2 and i-LTD4 occurred more slowly and the half-maximal generation was achieved b e t w e e n 3 a n d 5 min, r e s p e c t i v e l y . The generation was completed w i t h i n 20 min. In 3 of 7 d i f f e r e n t e x p e r i m e n t s , the d o s e - r e s p o n s e c u r v e for histamine release could not be distinguished from that for pGD2 a n d i-LTD4 generation. O n t h e o t h e r hand, in the o t h e r 4 cases, the o p t i m a l concentration of a n t i - l g E for pGD2 release w a s a b o u t 1 0 - f o l d l o w e r t h a n t h a t r e q u i r e d for the r e l e a s e of histamine a n d i-LTD4, w h i c h is in a c c o r d a n c e w i t h the f i n d i n g s of S c h l e i m e r a n d c o - w o r k e r s [27]. Based on these results, dispersed cells were challenged with several concentrations of anti-IgE (5 ~ 0.01 ~ g/ml) for 20 min., and the amounts of histamine, pGD2 and i-LTD4 were measured in t h e supernatant. Table I summarizes the results. S i n u s m a s t c e l l s r e s u l t e d in 52.0 ± 20.0% of h i s t a m i n e r e l e a s e w i t h a r a n g e of 13.4 ~ 8 8 . 3 % (n=14). A n t i - I g E a l s o c a u s e d the generation of 8 9 . 5 ± 33.7 n g o f pGD2/106 mast cells (ranging f r o m 34.1 to 150.8) w h i c h w a s a l e v e l s i m i l a r to t h a t o b s e r v e d in uterine mast cells [23]. Although there was a slight detectable rise in spontaneous pGD2 release in 6 of 14 specimens (2.8 ng/106 mast cells), anti-lgE enhanced the generation by 7.8 ~ 1 8 8 . 5 - f o l d . A n t i - I g E a l s o i n d u c e d i-LTD4 generation from sinus mast cells and the amount ranged from 19.2 to 182.3 ng/106 m a s t c e l l s (78.7 ± 46.8, n = 1 0 ) . T h e r e w a s a large variation in the capacity of sinus mast cells from different specimens to generate i-LTD4. Spontaneous LTD4 production was not detected in a n y of the i0 s p e c i m e n s . Sinus mast cells produced pGD2 and i-LTD4 in q u a n t i t i e s similar to those of lung mast cells. These mast cells obtained from different anatomical sites differed with respect to i-LTD4 release. Unlike l u n g a n d s i n u s m a s t cells, a small a m o u n t of
Vol. 53, No. 10, 1993
Characterization of Sinus Mast Cells
TABLE
781
I
A n t i - I g E - i n d u c e d h i s t a m i n e , p r o s t a g l a n d i n D~ a n d i - l e u k o t r i e n e D4 r e l e a s e f r o m d i s p e r s e d sinus, l u n g a n d f o r e s k i n m a s t cells. Histamine (%) Sinus
Lung
Foreskin
52.0 (3.5 n=14 32.5 (3.6 n=5 32.0 (3.6 n=5
Prostaglandin (ng/108
± 20 _+ 4.2)
Dz i-Leukotriene m a s t cells)
8 9 . 5 ± 33.7 (< 4.8) n=14 126.5 ± 72.7 (< 3.7) n=5 1 0 4 . 4 ± 84.3 (< 4.9) n=4
± 8.0 ± 2.0) ± 15.1 ± 1.7)
F i g u r e s in p a r e n t h e s i s each mediator. i-LTD4 could be detected s p e c i m e n s (8.5, 4.6, <3, <3;
show
in 4.8
spontaneous
D4
78.7 ± 4 6 . 8 (< 4.0) n=10 78.0 ± 52.8 (< 3.7) n=5 4.8 ± 2.6 (< 3.5) n=4 release
skin mast cells ± 2.6 ng/106 m a s t
of
in two cells).
of
4
When sinus mast cells were stimulated at optimal concentrations of anti-IgE, the release of histamine was significantly correlated with both pGD2 release (r=0.70, p<0.001, n=13) and i-LTD4 release (r=0.85, p<0.02, n=9). The maximal r e l e a s e of pGDz w a s a l s o s i g n i f i c a n t l y correlated with that of i-LTD4 (r=0.82, p<0.05, n=13). The observations that there were significant correlations among the release of histamine, pGD2 and i-LTD4 agrees with previous reports [18, 27, 28], s u g g e s t i n g t h a t t h e s e m e d i a t o r s are derived from mast cells. In s u m m a r y , sinus mast cells dispersed f r o m s i n u s m u c o s a of chronic sinusitis patients differed from skin and lung mast cells with respect to t r y p t a s e content. Sinus mast cells are s i m i l a r to l u n g m a s t c e l l s in t e r m s of p h a r m a c o l o g i c m o d u l a t i o n of h i s t a m i n e release with adenosine, response to s e c r e t a g o g u e s such as compound 48/80 and substance P, and release of arachidonic acid metabolites. References i. L. E N E R B A C K , Int. A r c h s A l l e r g y A p p l . Immunol. 82 2 4 9 - 2 5 5 (1987). 2. L.B. S C H W A R T Z , Ann. A l l e r g y 58 2 2 6 - 2 3 5 (1987). 3. L.B. S C H W A R T Z , T.R. B R A D F O R D , A - M . A . IRANI, G. D E B L O I S a n d S.S. CRAIG, Am. Rev. R e s p i r . Dis. 135 1 1 8 6 - 1 1 8 9 (1987). 4. L.B. S C H W A R T Z , A - M . A . IRANI, K. R O L L E R , M.C. C A S T E L L S and N.M. S C H E C H T E R , J. Immunol. 138 2 6 1 1 - 2 6 1 5 (1987). 5. H. MITA, T. ISHII, K. A K I Y A M A , T. T A M U R A , K. T A D O K O R O , H. SAITO, H. Y A S U E D A a n d T. SHIDA, L i f e Sci. 49 1 4 7 7 - 1 4 8 4 (1991). 6. M. ABE, Y. K A W A Z O E a n d N. S H I G E M A T S U , Anal. B i o c h e m . 144 4 1 7 - 4 2 2 (1985). 7. R~C. BENYON, M.A. L O W M A N a n d M.K. CHURCH, J. Immunol. 138
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Characterization of Sinus Mast Cells
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